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Combined analytical techniques for the analysis of complex consumer productsand bio-samples

Chen, G.

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Citation for published version (APA):Chen, G. (2019). Combined analytical techniques for the analysis of complex consumerproducts and bio-samples.

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Combined analytical techniques for the analysis of complex consumer products and bio-samples

Guoqiang (Leon) ChenC

ombined analytical techniques for the analysis of com

plex consumer products and bio-sam

ples G

uoqiang (Leon) Chen

Invitation

For attending the public defence of the thesis

Combined analytical techniques for the analysis

of complex consumer products and bio-samples

On Wednesday 5th June 2019

at 14.00

In theAgnietenkapel,

Oudezijds Voorburgwal 229,Amsterdam

[email protected]

Paranymphs

Randy ZhaoBoudewijn Hollebrands

Combined analytical techniques for the analysis of

complex consumer products and bio-samples

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus

prof. dr. ir. K.I.J. Maex

ten overstaan van een door het College voor Promoties ingestelde

commissie,

in het openbaar te verdedigen in de Agnietenkapel

op woensdag 5 juni 2019, te 14:00 uur

door

Guoqiang Chen geboren te Shanghai

Promotiecommissie:

Promotor:

- prof. dr. ir. J.G.M. Janssen Universiteit van Amsterdam

Co-promotor:

- prof. dr. ir. P.J. Schoenmakers Universiteit van Amsterdam

Overige leden:

- prof. dr. A.C. van Asten Universiteit van Amsterdam

- prof. dr. W.P. de Voogt Universiteit van Amsterdam

- prof. dr. R.A.H. Peters Universiteit van Amsterdam

- prof. dr. J.P.M. van Duynhoven Wageningen University & Research

- dr. J.G.J. Mol Wageningen University & Research

Faculteit der Natuurwetenschappen, Wiskunde en Informatica

3

Table of Contents Chapter 1 ................................................................................................................................. 5 General Introduction

1.1 Needs and challenges of analytical sciences in the industry of foods and HPC ...... 6

1.2 Analytical techniques in the industry of foods and HPC ...................................... 8

1.3 Scope of the thesis ......................................................................................... 10

References .......................................................................................................... 14 Chapter 2 ............................................................................................................................... 19 A multi-residue method for fast determination of pesticides in tea

2.1 Introduction ................................................................................................... 20

2.2 Experimental ................................................................................................. 21

2.3 Results and discussion .................................................................................... 24

2.4 Conclusions ................................................................................................... 29

References .......................................................................................................... 34 Chapter 3 ............................................................................................................................... 37 Rapid and selective quantification of L-theanine in ready-to-drink teas from Chinese market

3.1 Introduction ................................................................................................... 38

3.2 Materials and methods .................................................................................... 39


3.4 Conclusions ................................................................................................... 48

References .......................................................................................................... 49 Chapter 4 ............................................................................................................................... 53 A method for measuring the noncovalent interaction between EGCG and β-CD

4.1 Introduction ................................................................................................... 54



4.4 Conclusions ................................................................................................... 63

References .......................................................................................................... 64

Appendix ............................................................................................................ 68 Chapter 5 ............................................................................................................................... 71 Quantification of climbazole deposition from shampoo onto artificial skin and human scalp

5.1 Introduction ................................................................................................... 72

5.2 Experiments .................................................................................................. 73


4

5.4 Conclusions ................................................................................................... 81

References .......................................................................................................... 82

Chapter 6 ............................................................................................................................... 85 Sensitive and simultaneous quantification of zinc pyrithione and climbazole in scalp buffer scrub samples

6.1 Introduction ................................................................................................... 86



6.4 Conclusion .................................................................................................... 95

References ......................................................................................................... 96

Chapter 7 ................................................................................................................... 99 Ex-vivo measurement of scalp follicular delivery of zinc pyrithione and climbazole from hair care products

7.1 Introduction ............................................................................................... 100

7.2 Materials and methods .............................................................................. 101

7.3 Results and discussion .............................................................................. 105

7.4 Conclusions ............................................................................................... 110

References ....................................................................................................... 111

Chapter 8 ................................................................................................................. 113 Visualization of zinc pyrithione particles deposited on the scalp from hair care products

8.1 Introduction ............................................................................................... 114

8.2 Materials and Methods .............................................................................. 115

8.3 Results and discussion .............................................................................. 116

8.4 Conclusion ................................................................................................ 118

References ....................................................................................................... 119

List of abbreviations ............................................................................................... 121 Summary ................................................................................................................. 123 Samenvatting........................................................................................................... 127 总结......................................................................................................................... 132 List of Publications ................................................................................................. 135 Overview of author’s contributions ........................................................................ 136 Acknowledgements ................................................................................................. 139

5

Chapter 1

General Introduction

Analytical chemistry is essentially the branch of chemistry that looks into molecular

compositions of complex mixtures. It involves sampling, sample treatment, instrumental

analysis, data processing and interpretation, and has the end goal of identifying and

quantifying specific matter or compounds. The science of analytical chemistry has

continuously evolved with the sustaining innovations in selective materials, computer science,

laser technology and instrumentation. In addition to these ‘technology push’ drivers, the

enormous need for analytical chemistry from new challenges in various areas has generated

a strong ‘market pull’ as well. To date there is a huge variety of analytical techniques

including titrimetry, gravimetry, potentiometry, voltammetry, spectroscopy, microscopy,

chromatography, etc. Combinations of two or more of these techniques enable the analysis of

ever more complex samples at ever increasing levels of detail, sensitivity and reliability.

The importance of analytical chemistry is without any doubt. Analytical measurements have

a tremendous impact everywhere. Not only in fundamental research, but also in industrial

settings and social applications. Without analytical sciences, other science fields including

life sciences, material sciences, space technology, etc. would come to a standstill. In industry,

quality control of raw materials and product development rely heavily on analytical sciences,

techniques and measurements. For monitoring and safeguarding public health and safety,

analytical chemistry plays a critical role in food safety assessment, environment protection

issues, forensic analysis and clinical diagnosis. In hospitals clinical analyses form the basis

of treatment strategies and in the pharmaceutical field analytical methods are indispensable

in the development and production of new drugs.

In this thesis we will demonstrate the large versatility of analytical chemistry by applying it

to two different application fields within the broader area of the life sciences. The thesis

focusses on the development and application of analytical tools and technologies for the

characterization of functional foods, and on methods to assess and underpin the efficacy of

6

personal care products, including the interaction of the functional actives with the human

body.

In this introductory chapter, the needs and challenges of analytical sciences in the industry of

foods and home and personal care (HPC) are summarized. Next, the analytical techniques

most widely used in the development and manufacturing of foods and HPC products are

introduced. Finally, the scope of this thesis is discussed by briefly introducing the successive

chapters of the thesis.

Figure 1.1. Generic applications of analytical sciences in new product development: from

raw materials to finished products on the market. MOA = mode of action.

1.1 Needs and challenges of analytical sciences in the industry of foods and HPC

High quality and safety demands are placed on products that people consume (foods) or apply

onto the body for body care or cosmetic reasons. This explains the large need for analytical

measurements from industries active in these areas. Analytical chemistry is crucial in all

phases of product development and production. As shown in Figure 1.1, analytical science is

vital at many places in the production and life-cycle of the finished products. Without high

quality analytical support, it is nearly impossible to perform the required detailed

characterization of raw materials, establish insights into the mode of action of key actives in

7

products, perform evaluation of prototypes, control the quality of products, build evidence for

claim substantiation, support patents and detect patent infringement, and eventually develop

new, improved products.

The analytical challenges in the foods and HPC industry are largely comparable to those in

other industries and in academic research. In some cases, the challenges are even more severe

because of the complicated matrix of foods and HPC products, in particular related to the high

levels of fats, salts and proteins in the products. A high sensitivity and a high degree of

selectivity are always demanded since the contents of many analytes are very low, and the

sample matrix is complex. As an example, the analysis of contaminants in foods, or of

biomarkers in in vitro and in vivo samples, requires methods with detection limits at the ppb

level that are tolerant to significant matrix variations. Sample treatment techniques like

ultrasound-assisted extraction, microwave-assisted extraction and solid phase extraction

(SPE), can be used to achieve clean-up and pre-concentration, and meet such challenges at

least to some extent in food analysis [1, 2]. However, most of the sample pretreatment

methods are time consuming and rendering automation necessary. Consumables for sample

treatment methods like solid phase extraction (SPE) are expensive and large volumes of toxic

and expensive solvents are often needed. Cost efficiency and sustainability have become

increasingly important, especially for routine analysis of large numbers of samples such as in

bioavailability studies or efficacy tests. A final important factor is speed. The world of foods

and HPC is a rapidly changing world where consumer wishes can quickly come and go.

Analysis time, manual labor and cost of ownership and operation are key features in the

development of methods for use in this industry.

In addition to having to deal with the qualitative and quantitative determination of ever more

complex chemical compositions, analytical scientists are now more than ever also requested

to determine where the components are and how they interact with each other in foods, HPC

products and even on human substrates like hair, scalp, skin, teeth, etc. There is a growing

need for methods that enable localized, spatially resolved analysis and visualization, in the

steady state or in dynamic situations, especially in in vivo studies aiming to understand the

functions and actions of key actives in personal care products.

8

To demonstrate the product benefits in the foods and HPC areas, there is a growing need to

develop methods that provide undisputable evidence on levels of actives at specific target

locations. Simple and easy-to-understand analytical methods are preferred over those that rely

on complicated processes and instrumentation. This is especially the case when the product

evaluations are used to engage consumers.

1.2 Analytical techniques in the industry of foods and HPC

To meet the above challenges and address the articulated needs, there is a massive number of

analytical techniques available to select from. Moreover, every year new analytical

instruments are launched with impressive innovations in performance. The analytical scientist

responsible for the development of novel methods will require excellent knowledge of all

analytical core capabilities including sampling, sample treatment, instrumental analysis, data

processing and interpretation. Next to this, he or she will require sufficient product knowledge

and application-understanding to be able to generate correct data and interpret these in a way

they help in building knowledge and understanding.

In clinical studies, reliable and efficient sampling methods are demanded for the analysis of

actives deposited from personal care products onto human substrates (e.g. hair, scalp, skin,

etc.) as well as for measuring their conversion products and biomarkers of efficacy in- or on

human substrates. A variety of methods for this purpose has been published in scientific

literature, each with its own application fields. To collect biomarkers or actives from human

skin, for example, various sampling methods have been proposed including buffer scrub

extraction [3], tape strip sampling [4], cyanoacrylate biopsy [5], Sebutape [6], and sorptive

tape extraction [7].

Sample treatment prior to instrumental analysis is a crucial part of the analytical process,

contributing to target enrichment and background depletion, and it strongly affects

reproducibility and accuracy of the final results. In sample preparation for food analysis, for

example, procedures as simple as weighing, liquid-liquid extraction, vortexing, filtration,

centrifugation, concentration, etc. are used, next to complicated methods like SPE [8], static

and dynamic headspace [9], solid phase microextraction [10], derivatization [11], matrix solid

9

phase dispersion [12, 13], gel permeation chromatography clean-up [14], etc. Derivatization

is necessary for stabilizing the target compounds in the analytical process, improving the

separation of target compounds from interfering species through an enhanced selectivity and

for increasing the sensitivity. Automation of sample treatment is becoming more important

because of the increasing need for a higher sample throughput. On-line extraction and analysis

is the ultimate in automation that has proven its usefulness in many applications [15-21].

Chromatographic separation techniques like high performance liquid chromatography (HPLC)

and gas chromatography (GC) coupled to a wide range of detection technologies are widely

applied for sensitive and selective quantitative analysis of compounds of interest in raw

materials and finished products. With continuous evolution, comprehensive chromatography

(GC×GC, LC×LC) has been successfully applied in both academic and industrial research

and development (R&D) laboratories [22-26].

Mass spectrometry is now a mature analytical tool that is increasingly being used, not only in

industrial applications but also in fundamental research [27-34]. In combination with HPLC

or GC, it is the most powerful method for the identification and quantitative determination of

compounds in raw materials, formulated systems, finished products, in vitro assays and

clinical samples. Especially in clinical research studies it is widely used. Matrix-assisted laser

desorption/ionization mass spectrometry imaging (MALDI MSI) has emerged as a chemical

imaging technology [35], enabling the visualization of the spatial distribution of target

molecules in various sample matrix like tea leaves, human tissues, etc.

Compared with MALDI MSI, Raman and Laser Confocal Scanning can offer higher spatial

resolution when taking chemical images. They are powerful tools in skin research [36,37], for

example to map spatial delivery of actives onto the skin, to study skin permeability, or to

monitor temporal or spatial changes in biomarkers in skin.

As an optical imaging technology, optical coherence tomography (OCT) has been

successfully applied in diagnostic medicine studies as well as in several industrial fields and

academic research projects [38-40]. In efficacy studies for skin care products, it enables in

vivo visualization of the internal microstructure of human tissues like skin and enamel.

10

Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) are

used to provide detailed microstructural information on human samples or other solid samples

at the nm to sub-mm range. They can be applied to bulk, sectioned and thin film systems,

spanning from solids to liquids through application of a wide range of preparatory techniques.

Both SEM and TEM have been employed to investigate the microstructure and nanostructure

of foods [41, 42].

Molecular spectroscopy including Infra-red (IR), Raman, UV and fluorescence is widely used

for molecular characterization of raw materials, interactions of ingredients in complex

formulations as well as for quantification of components of interest in many types of samples.

Surface enhanced Raman scattering and near Infra-red spectroscopy have been successfully

applied for the detection of adulterants and contaminants in foods and numerous other

samples [43-45].

Nuclear magnetic resonance (NMR) spectroscopy is used to provide structural information

on unknown molecules and characteristic fingerprints of complex mixtures [46]. Additionally,

it can provide quantitative information [47] or it can be used to study interactions between

different components [48].

From the above it is clear that a wide diversity of analytical techniques exists. Selection of

the most appropriate method for a given application is generally a tradeoff between

performance on the one hand, and complexity, cost and speed on the other. In the R&D

environment in the foods and HPC industry, short analysis times, a high data quality, low

costs and meeting all externally dictated safety compliance requirements are the key factors

to consider when selecting an analytical technique for a specific application.

1.3 Scope of the thesis

Foods and HPC companies must continuously launch new products to keep up with the

continuously changing demands of their customers. This high rate of innovation requires very

fast product innovation trajectories. The aim of this thesis is to develop new analytical

methods to meet the business needs for safety and quality control, performance evaluation,

11

claim substantiation and mode-of-action understanding in the case of consumer preferred tea

products and shampoos.

Following water, tea is the most widely consumed drink in the world. Pesticide residues in

tea can be a concern for tea drinkers. For reliable identification and quantification of the target

pesticide residues at trace levels, analytical laboratories are increasingly interested in finding

new analytical methods with shorter analysis times, improved sensitivities and higher sample

throughputs. In Chapter 2, a multi-residue method was developed and validated for rapid

determination of pesticide residues in tea using ultra-high-performance liquid

chromatography-electrospray tandem mass spectrometry (UHPLC-MS/MS) combined with a

modified quick, easy, cheap, effective, rugged and safe (QuEChERS) sample preparation

procedure. In order to minimize the matrix effects from tea, an SPE cartridge layered with

graphite carbon/aminopropylsilanized silica gel was applied to complement the QuEChERS

method. Representative matrix-matched calibration curves were applied for quantification to

compensate for matrix effects. The efficiency and reliability of this method were investigated

by the analysis of both fermented and unfermented Chinese tea samples.

Ready-to-drink (RTD) teas are becoming increasingly popular as a healthier alternative to

carbonated drinks. One of the nutritionally-relevant compounds in tea is the amino acid L-

theanine. This amino acid is almost solely found in tea plants. In tea it only exists in the free

(non-protein) form and it is the predominant free amino acid. In Chapter 3, a UHPLC-UV

method combined with SPE sample pre-treatment was developed and validated for the rapid

quantification of L-theanine in RTD teas. The method was applied to determine the L-

theanine content in twenty-seven RTD teas from the Chinese market. The ratio of total

polyphenols content to L-theanine content was studied as a parameter for differentiating RTD

teas.

Reducing the bitter and astringent taste of green tea will engage the consumers who are used

to the mild taste of black tea. β-cyclodextrin (CD) is used as an effective bitterness and

astringency masker for catechins in green tea, especially for epigallocatechin gallate (EGCG).

Chapter 4 investigates the noncovalent interaction between β-CD and EGCG by electrospray

ionization mass spectrometry (ESI-MS) and NMR. The stoichiometry of the β-CD-EGCG

12

complexation product was determined using Job’s method. NMR experiments are performed

to provide independent evidence on the formation of an inclusion complex of β-CD and

EGCG. The topology of the complex was derived from ROESY spectra and from chemical

shift differences of the various protons of β-CD and EGCG in the free versus complexed state.

A mechanism explaining the β-CD reduction of the bitterness and astringency of green tea

EGCG was proposed.

The efficacy of anti-dandruff (AD) shampoos depends on the deposition properties of the AD

actives and the amount retained on the human scalp in the process of shampoo application and

rinse-off. To support in vitro and in vivo studies for the performance evaluation of AD

shampoos, robust and sensitive analytical methods for in vitro and ex vivo measurement of AD

active deposition on artificial skin and human scalp are required. In Chapter 5, a sensitive and

specific UHPLC-MS/MS method was developed and validated for the measurement of

climbazole (CBZ) deposition from hair care products onto artificial skin and human scalp. A

buffer scrub method using a surfactant-modified phosphate buffered saline (PBS) solution was

selected for the sampling of CBZ from human scalp. Deuterated CBZ was used as the internal

standard. Atmospheric pressure chemical ionization (APCI) in positive mode was applied for

the detection of CBZ. Using this method, CBZ deposition from several shampoos was

compared.

Chapter 6 proposed a sensitive UHPLC-MS/MS method for the simultaneous quantification

of two AD actives, zinc pyrithione (ZPT) and CBZ, deposited onto the human scalp from AD

shampoos. Scrubbing with a buffer solution was used as the sampling method for the

extraction of ZPT and CBZ from scalp. A method for ZPT derivatization prior to UHPLC-

MS/MS analysis was developed. The identification of ZPT and CBZ was performed by

examining ratios of selected MRM transitions in combination with UHPLC retention times.

The method was applied for the analysis of scalp buffer scrub samples from an in vivo study.

The levels of ZPT and CBZ deposited on the scalp at different time points after application

of the AD shampoo were measured and the efficacy of different shampoos was compared.

In Chapter 7, several new methods for studying the location- and depth specific deposition

of AD actives were developed. A method involving scalp cyanoacrylate biopsy sampling, a

13

tailor-made cutting device, and methods for UHPLC-MS/MS analysis, SEM measurement

and Raman imaging were developed for the measurement of delivery of ZPT and CBZ from

an AD shampoo into the scalp follicular infundibulum. Using this method, ZPT and CBZ

were simultaneously quantified and visualized within the scalp follicular infundibulum, after

scalp washing with a dual-active AD shampoo.

Finally, Chapter 8 proposed an ex vivo method that combines tape strip sampling and SEM

and energy dispersive X-ray spectroscopy (EDX) for measuring and visualizing the particle

size, morphology and composition of ZPT deposited on the scalp from an AD shampoo

containing ZPT and zinc carbonate. The possibilities for distinguishing ZPT from zinc

carbonate particles were evaluated. Moreover, the ability of the new method to study the

microstructures of ZPT and other zinc particles deposited onto the scalp was assessed.

14

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18

19

Chapter 2

A multi-residue method for fast determination of pesticides in tea

Abstract

A multi-residue method was developed for rapid determination of pesticide residues in tea by

ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry

(UHPLC-MS/MS). The quick, easy, cheap, effective, rugged and safe (QuEChERS) method

was used for sample preparation. In order to minimize the matrix effects from tea, a solid phase

extraction (SPE) cartridge layered with graphite carbon/aminopropylsilanized silica gel was

applied as complementary to the QuEChERS method. For accurate quantification,

representative matrix-matched calibration curves were applied for quantification to

compensate matrix effects. Limits of quantification varied with different pesticides, but all can

be measured at 0.01 mg/kg level in a 5 g tea sample except dichlorvos (0.02 mg/kg).

Recoveries ranged from 70% to 120% and relative standard deviations (RSD) met the

European United Quality Control guideline. Efficiency and reliability of this method were

investigated by the analysis of both fermented and unfermented Chinese tea samples.

This chapter was originally published as:

G. Chen, P. Cao, R. Liu, A multi-residue method for fast determination of pesticides in tea by ultra

performance liquid chromatography-electrospray tandem mass spectrometry combined with

modified QuEChERS sample preparation procedure, Food Chem. 125 (2011) 1406-1411.

20

2.1 Introduction

Tea farming is vulnerable to a great multitude of pests, especially mites, leaf-eating beetles

and caterpillars. Weeds and diseases can also be a problem. To minimize these problems, the

most common practice in tea crop production is to use pesticides. However, similar to other

raw agriculture commodities (RACs), unsafe pesticide residues in tea have been associated

with neurological dysfunction and disease [1]. Consequently, determination of pesticide

residues is at the forefront among preventive measures in public health safety. Furthermore,

there are potential international trade barriers due to maximum residue limits (MRLs) in tea

established by most countries and several international organizations, e.g. United States

Environmental Protection Agency, Food and Agriculture organization of the United Nations,

and European United, etc. The existing MRLs for some pesticides in many RACs including

tea are periodically revised and become stricter and more comprehensive. Moreover, there is

a trend for regulators to temporarily reduce the MRLs if new data unexpectedly indicate

certain risk to human or animal health. For reliable identification and confirmation of the

target pesticide residues at trace levels, food analytical laboratories are increasingly interested

in finding new analytical methods with shorter analysis time and higher sample throughput

[2].

Gas chromatography (GC) seems to be the technical choice for analysis of pesticides in food

commodities. However, many pesticides which are thermally unstable or non-volatile such as

carbamates and benzimidazoles are difficult to be analysed with GC. High performance liquid

chromatography (HPLC) offers an alternative and powerful tool for the determination of such

compounds, as complementary to GC [3]. Moreover, ultra-high-performance liquid

chromatography (UHPLC) with columns packed with small particles (1.7 μm) and high linear

velocities (accompanied by maximum back pressures up to 15,000 psi) have been shown to

give superior chromatographic resolution, reduce analysis time, consume less solvent and

increase sensitivity [3-7].

For pesticide residue analysis in tea, there are published methods available in literature

including official methods [8-13]. Traditional analytical methods cannot provide effective

solutions to minimizing matrix effects [11]. Solid phase extraction (SPE) is a common choice

21

for clean-up. Japanese official methods for residual compositional substances of agricultural

chemicals, feed additives and veterinary drugs in food apply SPE cartridges packed with

graphite carbon/aminopropylsilanized silica gel to clean-up tea matrix [14]. Gel permeation

chromatography (GPC) is also an effective clean-up method which has been widely applied

[15]. There is a trend to shift from labour intensive traditional methods to fast and simple

approaches, such as the quick, easy, cheap, effective, rugged and safe (QuEChERS) method

[16], which symbolizes a new milestone for pesticide residue analysis. Some laboratories [17,

18] have applied this clean-up method in fruits and vegetables.

Pesticide multi-residue methods applying gas chromatography-mass spectrometry

(GC/MS/MS or GC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS)

are increasingly popular, which enable us to analyse more pesticides in one injection and with

higher sensitivity. For the confirmation of legal substances and banned substances, a

minimum of 3 and 4 identification points, respectively, are required according to EU Directive

(2002/657/EC, 2002) [19]. UHPLC coupled with triple quadruple MS/MS can improve the

speed of analysis and provide higher sensitivity and accuracy. To the best of our knowledge,

until now, there has been no literature or publications reporting about multi-residue methods

for the determination of pesticides in tea by UHPLC-MS/MS.

The purpose of this paper is to develop a multi-residue method based on the application of

UHPLC-MS/MS combined with a modified QuEChERS sample preparation procedure for

rapid determination of 65 selected pesticide residues in tea.

2.2 Experimental

2.2.1 Reagents, chemicals and materials

Pesticide reference standards, all 95% or higher purity, were obtained from Dr. Ehrenstorfer

(Augsburg, Germany), Chemservice (USA) and Accustandard (USA). Stock solutions of

mixture pesticides were prepared in acetonitrile or acetone stored in the freezer (-18 ℃). The

working solutions were prepared daily.

22

All chemicals used in the experiment were analytical grade or better: HPLC grade acetonitrile,

HPLC grade formic acid and acetic acid. A.R. Anhydrous CH3COONa and anhydrous MgSO4

were baked at 650 ℃ for 3 hours to remove phthalates and any residual water. Graphite

carbon/aminopropylsilanized silica gel layered SPE cartridges (Sep-Pak Carbon NH2, 6 cc)

were purchased from Waters Corporation (P.N. 186003369).

Analytically confirmed pesticides-free green tea from Shiru Tea Company in Guangxi

province and black tea from Unilever UK were used as blank samples for matrix-matched

calibration and recovery evaluations.

2.2.2 Apparatus

A Waters ACQUITY UPLC System (Waters, UK) was employed. An AQUITY UPLC BEH

Shield RP18, 2.1 mm (I.D.) × 150 mm, 1.7 μm column (Waters, Ireland) was applied in this

method. The mobile phase was constituted by 0.02% formic acid in acetonitrile (A) and 0.02%

formic acid in water (B) in a gradient mode [time 0 min, 10% A; 12 min, 98% A; 12.5 min,

10% A] and total analysis time of 18 min. The flow rate was 0.3 mL/min and injection volume

was 2 μL. The temperatures of column and sample room were set at 30 ℃ and 8 ℃,

respectively.

A Waters Quattro Micro API mass spectrometer (Waters, UK) equipped with electrospray

source was used for all experiments. MassLynx software (version 4.1) was used for

instrument control and data acquisition. The capillary voltage was 3.50 kV and the source

temperature was 100 ℃. The desolvation gas temperature was set at 350 ℃ with a nitrogen

flow of 300 L/hr. The collision gas (argon) pressure was set at 3×10-3 mbar. The multiple

reaction monitoring (MRM) mode was operated for each pesticide. All the parameters for

MRM transitions, cone voltage and collision energy were optimized in order to obtain highest

sensitivity and resolution (Table 2.1).

23

2.2.3 Sample preparation

For both unfermented and fermented tea, weigh about 50 g and comminute in a small dis-

integrator for 1 min. Transfer 5 g comminuted sample to a 50 mL centrifugal tube. Add 10

mL H2O and 10 mL acetonitrile (containing 1% acetic acid), vortex for 3 min and then allow

to settle for 1 h. Add 1.5 g anhydrous CH3COONa and 4 g anhydrous MgSO4, vortex for 1

min. Cool the tube in an ice-water bath immediately, for 5 min. Centrifuge for 5 min at 5000

r/min. The samples were then subjected to SPE clean-up. The SPE column was conditioned

with 10 mL acetonitrile/toluene (3:1, 1% acetic acid). Transfer 1 mL extracted solution to the

column. Elute the column with 20 mL acetonitrile/toluene (3:1, 1% acetic acid). Concentrate

the effluent to 1 mL or less by evaporating under a weak nitrogen stream at 40 ℃. The residue

was reconstituted in 1 mL acetonitrile (1% acetic acid) and filtered over a 0.2 μm organic

filter (Millipore), ready for injection into UHPLC-MS/MS.

2.2.4 Method performance

The sensitivity and precision of the method were evaluated by analyzing spiked blank tea

samples. Recoveries and RSD were determined for five replicates at two concentration levels

(0.050 and 0.010 mg/kg).

The accuracy of this method was evaluated externally by participating in a 31- laboratories-

proficiency-test for pesticide residue analysis in tea which was organized by FAPAS

(http://www.fapas.com) in 2008. All the target compounds (Acetamiprid, Bifenthrin,

Cypermethrin, DDT [four homologues], Ethion, Fenpropathrin, Fenvalerate, lamda-

Cyhalothrin, Propargite, S-421) covered by the proficiency test were properly identified and

the respective z-score values obtained were satisfactory (|z|<2).

24

2.3 Results and discussion

2.3.1 Optimization of UHPLC-MS/MS conditions

Application of UHPLC in this method provides superior chromatographic resolution, shorter

analysis time and higher sensitivity. The total analytical time for instrumentation was only 18

min including 6 min for column equilibrium. At the initial developmental stage, 5 mM

ammonium acetate was used in LC mobile phase. However, with this mobile phase the

UHPLC column was found to get clogged rapidly. It was proven that 0.02% formic acid

provided the same sensitivity and resolution as 5 mM acetic ammonium. Formic acid was

then selected as the replacement for ammonium acetate.

Some pesticides including fenxoycarb, indoxacarb,clethodim and flufenoxuron showed poor

peak shapes when they were dissolved into the initial gradient of mobile phase

(acetonitrile/H2O = 1:9). For other pesticides, there were no differences in sensitivity when

acetonitrile/H2O (1:9 & 5:5) or pure acetonitrile was used as solvent. Consequently, the

residue was reconstituted in 1 mL acetonitrile (1% acetic acid) for better peak formation and

sensitivity.

The tea matrix is very “dirty”, containing high levels of caffeine, sugars, organic acids and

other interferences. A tandem mass detector, which has high selectivity and sensitivity,

provides an effective solution. MRM parameters including ion transition, collision energy and

cone voltage of UHPLC-MS/MS were listed in Table 2.1 Each pesticide was tuned using a

single standard solution at 1 μg/mL which was infused into the MS detector at a flow rate of

0.3 mL/ min. Product ion mass spectra for the pesticides were obtained in electrospray

ionization using collision induced dissociation (CID). Variations in collision energy influence

both sensitivity and fragmentation. The collision energy was optimized for two selective ion

transitions for every pesticide. Both pairs of the MRM transitions were used for confirmation

analysis, which can meet the EU Decision (2002/657/EC, 2002) [19], and the most sensitive

transitions were selected for quantification analysis. Dwell times for different transitions were

optimized to achieve higher sensitivities, as well. Some compounds like bromoxynil and

ioxynil were analyzed in negative ESI mode ([M-H]-) while other pesticides were determined

25

in positive ESI mode ([M+H]+). In this investigation, a total of 65 pesticides were determined

in tea. A combined MRM chromatogram of fortified tea sample by 15 representative LC

amenable pesticides at 0.1 mg/kg is shown in Figure 2.1.

Figure 2.1. Combined UHPLC-MS/MS chromatogram of fortified green teas at 0.1 mg/kg

1. Propamocarb; 2. Pirimicarb; 3. Carbofuran-3-hydroxy; 4. Mevinphos; 5. Acetamiprid; 6.

Thiofanox-sulfon; 7. Spiroxamine; 8. Triasulfuron; 9. Bromoxynil; 10. Promecarb; 11.

Triadimefon; 12. Fenhexamid; 13. Fenoxycarb; 14. Clethodim; 15. Flufenoxuron.

2.3.2 Sample preparation

In the process of pesticide residue analysis, sample pretreatment and preparation are the most

time-consuming, labor intensive and complicated procedures. According to the characteristics

of pesticides, several solvents can be selected as the extraction solvent, e.g. acetone [9, 18],

ethyl acetate [2] and acetonitrile [20]. In comparison to other solvents, acetonitrile shows

more advantages such as higher recoveries, less interference from lipids and proteins, better

compatibility with LC and GC, and less co-extracted matrix components. For these reasons,

acetonitrile was chosen as the extraction solvent in the QuEChERS method [16]. Application

of MgSO4 for partitioning could yield a significant volume of the upper layer and give high

recoveries. Acetic acid with CH3COONa makes up of a buffer (pH 4-5), which could give

26

adequately high recoveries for acephate and imazalil etc. Moreover, the usage of buffer could

improve stability of the base-sensitive pesticides for their analysis [21].

Several sorbents can be used in the clean-up method for pesticide residue analysis, e.g.

primary secondary amine (PSA), -NH2, graphitized carbon black (GCB) and ODS SPE

cartridges. The use of PSA+GCB SPE [14] could remove more matrix materials. The

mechanism of PSA (or -NH2) sorbent is based on the weak ion exchange. It removes fatty

acids, sugars and other components that form hydrogen bonds. The use of GCB is attractive

to remove pigment especially chlorophyll. However, GCB strongly retains planar pesticides,

as well. In this research, QuEChERS method (dispersive-SPE) was proven to be unable to

remove the pigment from tea effectively. An SPE step (NH2+ GCB) was used for further

clean-up as complementary to the QuEChERS method. Acetic acid (1%) in the elution solvent

was proven to be able to reduce the absorption of planar pesticides in GCB and thus improve

the recoveries of pesticides residues, with the exception of pymetrozin, diflubenzuron and

thiabendazole (Figure 2.2).

Figure 2.2. Comparison of the recoveries of sulphonyl and urea pesticides with and without

1% acetic acid in SPE elution (n= 5, spiked concentration= 0.05 mg/kg). Each value is

expressed as mean ± standard deviation.

27

2.3.3 Matrix effects

The matrix effects may differ for different teas, e.g. green, black, Oolong and Puer.

Consequently, it is required to compensate the matrix effects by a matrix-matched calibration

[22], or by use of an isotope labeled standard, or ECHO technique [23] in LC/MS system.

In this study, analytically confirmed pesticides-free organic green tea from Shiru Tea

Company and black tea from Unilever UK were used as blank matrix. Organic green tea was

selected as the representative matrix for green and Puer tea samples while organic black tea

was selected as the representative matrix for black and Oolong tea samples. For the

determination of matrix effects, the responses of the standard solutions prepared in solvent

were compared with the responses of the standard solutions prepared in pesticides-free blank

tea sample. Matrix enhancement or suppression effects were observed for many tea samples.

The consequences of these abnormalities were considerable, causing significant and variable

errors in the quantification of the different pesticides. Hence, the matrix-matched calibration

method was applied in the quantitative analyses.

In addition, the complex matrix from tea could have a negative impact on the separation

performance of the UHPLC column. Moreover, the UHPLC column was prone to being

blocked if the clean-up of tea samples was not complete.

2.3.4 Validation of the method

Linearity, sensitivity, accuracy and precision of the multi-residue method were validated. The

mixed matrix-matched pesticides standard solutions of 5, 10, 20, 50, 100 μg/L were injected

into the UHPLC-MS/MS system. Relative coefficients (r) are listed in Table 2.1. Limits of

quantification (LOQ) of mixed pesticides standards were determined by injecting a series of

different matrix-matched pesticides standard solutions. Parameters are listed in Table 2.1

Although the LOQ of the method varies with different pesticides, all can be measured at 0.01

mg/kg level in a 5 g tea sample except for dichlorvos (0.02 mg/kg). The mixed standard

solution was added into the pesticides-free blank tea samples to make up the concentrations

of 10 and 50 μg/kg, and then the method was carried out as described in Part 2.3. The majority

28

of recoveries for these pesticides were in the range from 70% to 120%. But some recoveries

(60% - 70% or 120% - 130%) could also be accepted (European Council N°

SANCO/2007/3131, 2007). Reproducibility of this method is shown in Table 2.1 as RSD.

2.3.5 Pesticide residues in tea samples

In order to obtain accurate results, a two-step analytical strategy was applied. The first step

was a screening method, which monitored only one MRM transition for each compound. In

this way, negative and positive samples were separated. The second step is a confirmation

method, in which at least two MRM transitions for each compound were monitored. The most

sensitive MRM transition was selected for quantification.

This strategy was applied to pesticide residue analysis of 18 tea samples from different regions.

Among the pesticide residues detected in these 18 tea samples, acetamiprid had the highest

detection frequency (61.1%), followed by imidacloprid (56.8%), carbendazim (56.6%),

triazophos (44.4%), dimethoate & methomyl & uprofezin (33.3%), and triadimenol (22.2%).

For some pesticide residues, the detected levels varied greatly. The minimum value of

acetamiprid, for example, was 0.02 mg/kg in a green tea from Anhui province while the

maximum value of 1.03 mg/kg was found in an Oolong tea from Fujian province. However,

all the residue levels of these pesticides in these 18 tea samples were below the MRL required

by Chinese government, EU and Japanese government, except for dimethoate and methomyl

in 5 tea samples resulted higher than the Chinese and EU MRL.

The patterns of pesticide usage are different from one tea plantation to another because they

have different pest problems. The fact that we found multiple residues in a number of samples

may be due to the fact that some teas are custom blend to produce distinct finished products.

For instance, there were 5 tea samples contaminated by 8 pesticides simultaneously and there

were 6 tea samples in which dimethoate, methomyl and uprofezin were detected

simultaneously. The detected frequencies and detected levels of the pesticide residues in these

18 tea samples are shown in Figure 2.3.

29

In literature, some GC amenable pesticides like DDT, HCH and some pyrethroid could be

detected easily in tea. In light of the results of tea samples investigated in this paper, easily-

detectable LC amenable pesticides in tea were found to be acetamiprid, imidacloprid,

carbendazim, triazophos, dimethoate, methomyl, uprofezin, and triadimenol.

Figure 2.3. Detection frequencies and levels of the pesticide residues in 18 tea samples. The

error bars indicated the ranges of pesticide residue levels detected in tea samples.

2.4 Conclusions

A very quick, easy, effective, rugged, reliable and accurate multi-residue method based on

modified QuEChERS method was developed for determination of pesticides in tea by ultra-

performance liquid chromatography with tandem mass spectrometry. The performance of the

method was very satisfactory with results meeting validation criteria. The method has been

successfully applied for the determination of tea samples and ostensibly has further

application opportunities, including the analysis of e.g. dry vegetable and herb extracts.

30

Tabl

e 2.

1. P

aram

eter

s for

65

pesti

cide

resid

ue a

naly

sis b

y U

HPL

C-M

S/M

S.

Nam

e R

T (m

in)

MR

M T

rans

ition

*

Con

e

(v)

CE

(e

v)

LOQ

(μg/kg

) Li

near

ity

r

Rec

over

y /%

**

50 μ

g/kg

RS

D

10 μ

g/kg

RS

D

Carb

enda

zim

1.

76

192.

1>16

0.2

192.

1>13

2.1

25

18

30

5 0.

9918

85

.06

19.2

10

9.57

9.

1

Prop

amoc

arb

1.78

18

9.3>

102.

1 18

9.3>

144.

2 25

16

12

5

0.99

92

71.2

3 9.

2 81

.49

3.7

Met

ham

idop

hos

2.07

14

2.1>

94.1

142.

1>11

2.1

21

11

11

5 0.

9959

80

.86

7.5

61.8

6 23

.9

Ace

phat

e 2.

31

184.

1>14

3.1

18

4.1>

125.

1 15

7

17

10

0.

9932

84

.96

17.8

77

.57

28.9

Om

etho

ate

2.56

21

4.0>

155.

1

214.

0>18

3.2

20

15

10

5 0.

9979

76

.73

9.8

78.4

9 11

.4

Ald

oxyc

arb

3.56

22

3.1>

148.

1

223.

1>16

6.2

23

8

6 5

0.99

92

95.1

2 5.

9 78

.49

17.7

Mon

ocro

toph

os

3.63

22

4.1>

127.

0

224.

1>19

3.1

17

16

8 5

0.99

98

78.7

9 6.

1 70

.94

8.3

Pirim

icar

b 3.

86

239.

2>72

23

9.2>

182.

3 25

18

16

5

0.99

99

91.6

4 8.

6 76

.60

5.7

Met

hom

yl

3.88

16

2.9>

105.

9

162.

9>87

.8

15

10

8

5 0.

9997

70

.64

10.3

81

.91

20.1

Mev

inph

os

4.80

22

5.1>

127.

1

225.

1>67

.0

17

18

20

5 0.

9996

10

0.17

8.

4 82

.60

14.4

Met

amitr

on

4.84

20

3>17

5.1

2

03>1

45.1

28

16

14

5

0.99

77

78.5

5 18

.2

78.4

9 30

.2

Carb

ofur

an-3

-hyd

roxy

5.

06

220.

1>16

3.1

22

0.1>

107.

0 25

10

23

5

0.99

88

98.5

5 16

.6

60.8

1 17

.5

Imid

aclo

prid

5.

16

256.

1>17

5.1

25

6.1>

209.

1 22

20

16

5

0.99

56

84.7

7 15

.4

73.7

0 10

.2

Thio

fano

x-su

lfon

5.31

26

8.1>

76.0

26

8.1>

161.

2 10

10

16

5

0.99

85

84.3

2 14

.3

64.9

8 18

.2

Dim

etho

ate

5.33

23

0.0>

199.

1

230.

0>12

5 11

9

20

5

0.99

97

88.4

2 11

.5

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1

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in)

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ity

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y /%

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g/kg

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200.

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11

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134.

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24

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164.

2 15

15

8

5 0.

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az

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308.

1

376.

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17

10

16

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9997

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81

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31.6

32

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T (m

in)

MR

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rans

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Con

e

(v)

CE

(e

v)

LOQ

(μg/kg

) Li

near

ity

r

Rec

over

y /%

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50 μ

g/kg

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D

10 μ

g/kg

RS

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Atra

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93

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146.

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16

19

5

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27

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30

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30

26

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17

10

17

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14

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20

26

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0.99

99

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306.

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12

30

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7 65

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29

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155.

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13

20

5

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98

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79.4

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308.

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28

18

21

5

0.99

93

96.9

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85.4

9 6.

2

Ioxy

nil

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36

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127.

0

369.

8>21

4.9

35

29

34

5 0.

9994

77

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8.9

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1 9.

3

Met

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47

284.

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28

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176.

3 20

15

26

5

0.99

98

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66.5

3 5.

0

33

Nam

e R

T (m

in)

MR

M T

rans

ition

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Con

e

(v)

CE

(e

v)

LOQ

(μg/kg

) Li

near

ity

r

Rec

over

y /%

**

50 μ

g/kg

RS

D

10 μ

g/kg

RS

D

Fenh

exam

id

9.57

30

2.1>

97.1

30

2.1>

55.0

31

21

33

5

0.99

97

88.6

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7 87

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10.2

Tria

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31

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162.

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314.

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21

15

33

5

0.99

97

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4.53

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116.

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302.

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20

11

14

5

0.99

96

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Azi

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34

6.0>

132.

0

346.

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16

15

6

5 0.

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11

4.55

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Difl

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82

311.

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14

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9888

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1

Tebu

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133.

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353.

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13

19

7

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9999

92

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12.9

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150.

2

528.

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25

23

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10

7.33

14

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88.0

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6

Qui

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37

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271.

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18

18

5

0.99

99

88.3

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164.

1

360.

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6.3

19

19

18

5

0.99

98

72.2

3 13

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60.0

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Fura

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11

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383.

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38

3.1>

252.

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17

11

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9992

10

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5 11

5.80

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Flua

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17

5

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99

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98

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2

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e un

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MRM

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sitio

ns a

re u

sed

for q

ualit

ativ

e an

alys

is.

** n

=5

34

References

[1] F. Kamel, J.A. Hoppin, Association of pesticide exposure with neurologic dysfunction and disease. Enviro. Health Persp., 112 (2004) 950-958.

Doi.org/10.1289/ehp.7135. [2] A.G. Frenich, M.J. Gonzalez-Rodrıguez, F.J. Arrebola, J.M. Vidal, Potentiality of Gas Chromatography-

Triple Quadrupole Mass Spectrometry in Vanguard and Rearguard Methods of Pesticide Residues in Vegetables. Anal. Chem., 77 (2005) 4640-4648.

Doi: 10.1021/ac050252o. [3] G. Gervais, S. Brosillon, A. Laplanche, C. Helen, Ultra-pressure liquid chromatography-electrospray

tandem mass spectrometry for multiresidue determination of pesticides in water. J. Chromatogr. A, 1202 (2008) 163-172.

Doi.org/10.1016/j.chroma.2008.07.006. [4] C.C. Leandro, P. Hancock, R.J. Fussell, B.J. Keely, Comparison of ultra-performance liquid

chromatography and high-performance liquid chromatography for the determination of priority pesticides in baby foods by tandem quadrupole mass spectrometry. J. Chromatogr. A, 1103 (2006) 94-101.

Doi.org/10.1016/j.chroma.2005.10.077. [5] C.C. Leandro, P. Hancock, R.J. Fussell, B.J. Keely, Ultra-performance liquid chromatography for the

determination of pesticide residues in foods by tandem quadrupole mass spectrometry with polarity switching. J. Chromatogr. A, 1144 (2007) 161-169.

Doi.org/10.1016/j.chroma.2007.01.030. [6] D.T.T. Nguyen, D. Guillarme, S. Rudaz, J.L. Veuthey, Fast analysis in liquid chromatography using small

particle size and high pressure. J. Sep. Sci., 29 (2006) 1836-1848. Doi.org/10.1002/jssc.200600189. [7] Y. Picó, M. Farré, C. Soler, D. Barceló, Identification of unknown pesticides in fruits using ultra-

performance liquid chromatography-quadrupole time-of-flight mass spectrometry Imazalil as a case study of quantification. J. Chromatogr. A, 1176 (2007) 123-134.

Doi.org/10.1016/j.chroma.2007.10.071. [8] CIQ (custom, immigration and quarantine) People’s Republic of China SN/T 1747-2006 (2006).

Inspection of carbamate insecticide multi-residues in tea for export-Gas Chromatographic method. [9] Germany DFG method S 19 (1999). Modular Multiple Analytical Method for the Determination of

Pesticide. L 0-34. [10] B. Hu, W. Song, L. Xie, T. Shao, Determination of 33 pesticides in tea using accelerated solvent

extraction/gel permeation chromatography and solid phase extraction/gas chromatography-mass spectrometry. Chin. J. Chromatogr., 26 (2008) 22-28.

Doi.org/10.1016/S1872-2059(08)60009-7. [11] Z. Huang, Y. Li, B. Chen, S. Yao, Simultaneous determination of 102 pesticide residues in Chinese teas

by gas chromatography-mass spectrometry. J. Chromatogr. B, 853 (2007) 154-162. Doi.org/10.1016/j.jchromb.2007.03.013. [12] J. Ji, C. Deng, H. Zhang, Y. Wu, X. Zhang, Microwave-assisted steam distillation for the determination

of organochlorine pesticides and pyrethroids in Chinese teas. Talanta, 71 (2007) 1068-1074. Doi.org/10.1016/j.talanta.2006.05.087.

35

[13] J. Schurek, T. Portolés, J. Hajslova, K. Riddellova, F. Hernández, Application of head-space solid-phase microextraction coupled to comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry for the determination of multiple pesticide residues in tea samples. Anal. Chim. Acta., 611 (2008) 163-172.

Doi.org/10.1016/j.aca.2008.01.007. [14] Department of Food Safety Ministry of Health, Labour and Welfare, Japan (2006). Analytical methods

for residual compositional substances of agricultural chemicals, feed additives, and veterinary drugs in food.

[15] H. Kerkdijk, H.G.J. Mol, B.V.D. Nagel, Volume overload cleanup: An approach for on-Line SPE-GC,

GPC-GC, and GPC-SPE-GC. Anal. Chem., 79 (2007) 7975-7983. Doi: 10.1021/ac0701536. [16] M. Anastassiades, S.J. Lehotay, D. Stajnbaher, Quick, easy, cheap, effective, rugged, and safe

(QuEChERS) approached for the determination of pesticide Residues. 18th Annual Waste Testing and Quality Assurance Symposium Proceedings. (2002) 231-241.

[17] C. Lesueur, P. Knittl, M. Gartner, A. Mentler, M. Fuerhacker, Analysis of 140 pesticides from

conventional farming foodstuff samples after extraction with the modified QuEChERS method. Food control, 19 (2008) 906-914.

Doi.org/10.1016/j.foodcont.2007.09.002. [18] P. Payá, M. Anastassiades, D. MackIrina, I. Sigalova, B. Tasdelen, J. Oliva, A. Barba, Analysis of

pesticide residues using the Quick Easy Cheap Effective Rugged and Safe (QuEChERS) pesticide multiresidue method in combination with gas and liquid chromatography and tandem mass spectrometric detection. Anal. Bioanal. Chem., 389 (2007) 1697-1714.

Doi.org/10.1007/s00216-007-1610-7. [19] European Council 2002/657/EC (2002). Implementing Council Directive 96/23/EC concerning the

performance of analytical methods and the interpretation of results. [20] R.M.K. Hajou, F.U. Afifi, A.H. Battah, Comparative determination of multi-pesticide residues in

Pimpinella anisum using two different AOAC methods. Food Chem., 88 (2004) 469-478. Doi.org/10.1016/j.foodchem.2004.03.051. [21] M. Hiemstra, A. de Kok, Comprehensive multi-residue method for the target analysis of pesticides in

crops using liquid chromatography-tandem mass spectrometry. J. Chromatogr. A, 1154 (2007) 3-26. Doi.org/10.1016/j.chroma.2007.03.123. [22] European Council N° SANCO/2007/3131 (2007). Method validation and quanlity control procedure for

pesticide residues analysis in food and feed. [23] L. Aldera, S. Luderitz, K. Lindtner, H. Stan, The ECHO technique-the more effective way of data

evaluation in liquid chromatography-tandem mass spectrometry analysis. J. Chromatogr. A, 1058 (2004) 67-79.

Doi.org/10.1016/j.chroma.2004.08.120.

36

37

Chapter 3

Rapid and selective quantification of L-theanine in ready-to-drink teas

from Chinese market

Abstract

An ultra-high-performance liquid chromatography (UHPLC) method combined with solid

phase extraction (SPE) sample pre-treatment was developed and validated for the rapid

quantification of L-theanine in ready-to-drink (RTD) teas. UHPLC analysis of twenty-seven

RTD teas from the Chinese market revealed that the L-theanine levels in various types of RTD

teas were significantly different. RTD green teas were found to contain highest mean L-

theanine level (37.85 ± 20.54 mg/L), followed by jasmine teas (36.60 ± 12.08 mg/L),

Tieguanying teas (18.54 ± 3.46 mg/L), black teas (16.89 ± 6.56), Pu-erh teas (11.31 ± 0.90

mg/L) and Oolong teas (3.85 ± 2.27 mg/L). The ratio of total polyphenols content to L-

theanine content could be used as a featured parameter for differentiating RTD teas. L-theanine

in RTD teas could be a reliable quality parameter that is complementary to total polyphenols.


G. Chen, Y. Wang, W. Song, B. Zhao, Y. Dou, Rapid and selective quantification of l-theanine in

ready-to-drink teas from Chinese market using SPE and UPLC-UV. Food Chem. 135 (2012) 402-

407.

38

3.1 Introduction

RTD teas are increasingly popular as a healthy alternative to carbonate drinks and bottled

water. In China, the RTD tea market has become the most dynamic category in the soft drinks

industry. It is required by Chinese national standards for tea beverages (GB/T 21733-2008)

that the content of total polyphenols in RTD black tea, green tea, Oolong tea, and other tea

should be no less than 300, 500, 400 and 300 mg/kg, respectively. Those containing total

polyphenols below this requirement are defined as tea flavoured beverages. However,

polyphenols in RTD teas are prone to oxidation during storage, which could result in

underestimation of tea extracts in RTD teas. The establishment of a reliable quality parameter

would help the RTD tea market in setting standards, creating objective price criteria, and

improving the image of RTD teas.

L-theanine is an amino acid almost solely found in tea plants [1]. It only exists in the free

(non-protein) form and is the predominant free amino acid in tea [2]. L-theanine in RTD teas

can be a reliable quality parameter for RTD teas. A number of analytical methods has been

developed to determine L-theanine individually, or with other amino acids both in tea

compositions and simultaneously in different matrices. These include capillary

electrophoretic [3-6] and chromatographic methods. L-theanine can be analysed

simultaneously with other amino acids by high performance liquid chromatographic (HPLC)

methods involving precolumn derivatization with o-phthaladehyde, phenylisothiocyanate or

dabsyl chloride, and fluorescence and diode array UV detection [7-12]. Other HPLC methods

employing different columns and detectors were reported for the quantitative and qualitative

analysis of L-theanine in different teas without derivatization [13-19].

In recent years, UHPLC has been shown to give superior chromatographic resolution, reduced

analysis time, reduced solvent consumption and increased sensitivity when employed for tea

related analyses [20-24]. However, to the best of our knowledge, there have been no

publications or literature reporting UHPLC methods for rapid analysis of L-theanine in RTD

teas until now.

The aim of this chapter is to develop a rapid method for the analysis of L-theanine in RTD

teas using UHPLC-UV and solid phase extraction (SPE). Furthermore, using this method will

39

enable us to assess the qualities of RTD teas from the China market by quantification of L-

theanine. It is the first time to report L-theanine level in RTD teas.

3.2 Materials and methods

3.2.1 Reagents and solvents

All reagents and solvents used in the experiments were analytical grade or above. L-theanine,

gallic acid and Folin-Ciocalteu phenol reagent were purchased from Sigma-Aldrich (St. Louis,

MO, USA). Acetonitrile and methanol were purchased from Merck (Darmastadt, Germany).

Formic acid was from Fluka (Steinheim, Germany). Pure water used to prepare standard

solutions and UHPLC mobile phase was produced by a Milli-Q system (Bedford, MA, USA).

Oasis MCX 3cc (6 mg) extraction cartridges were purchased from Waters (Milford, MA,

USA). All other reagents and solvents were purchased from SCRC (Shanghai, China).

3.2.2 Ready-to-drink tea samples

A total of twenty-seven RTD tea samples of different types, flavours and producers (listed in

Table 3.1) were analysed, which were purchased from a supermarket in Shanghai and

estimated to cover at least 90% of the Chinese market for RTD teas.

3.2.3 UHPLC-UV method for the analysis of L-theanine in RTD teas

UHPLC analysis was performed on a Waters ACQUITY UPLC System equipped with

photodiode array (PDA) detector. Different ACQUITY UPLC columns were employed for

the separation of L-theanine, including a BEH phenyl column (2.1 x 100 mm, 1.7 µm particle

size), BEH C18 column (2.1 x 100 mm, 1.7 µm particle size), BEH shield RP 18 column (2.1

x 100 mm, 1.7 µm particle size), BEH HILIC column (2.1 x 100 mm, 1.7 µm particle size)

and HSS T3 column (2.1 x 100 mm, 1.8 µm particle size). Mobile phase A was pure water

with formic acid (v/v: 0.05%, 0.1%, 0.5%) and prepared freshly for every analysis series.

Mobile phase B was acetonitrile. The analytes were monitored by UV detection at 195 ± 2

40

nm. Injection volume was 2.5 µL. The optimised conditions were: BEH phenyl column, pure

water with 0.1% formic acid for mobile phase A and a flow rate of 0.4 mL/min.

3.2.4 Estimation of total polyphenols

Total polyphenol contents of RTD teas were determined by a colorimetric assay using Folin-

Ciocalteu phenol reagent with gallic acid as standard according to a method specified by the

International Standardisation Organisation (ISO 14502-1).

3.2.5 Sample pre-treatment

For UHPLC analysis of L-theanine, all RTD samples were acidified to pH=2 with formic acid

before an optimized sample pre-treatment using Waters Oasis MCX extraction cartridges. The

cartridge was conditioned with 3 mL methanol and equilibrated with 3 mL pure water, prior

to loading 1 mL acidified RTD tea sample. Then the cartridge was washed with 2 mL pure

water containing 2% formic acid and 2 mL methanol. After that, the cartridge was eluted with

2 mL of a 5% ammonia solution in water. Finally, the fraction was evaporated to dryness and

then reconstituted in 1 mL pure water with 0.1% formic acid.

41

Tabl

e 3.

1. In

form

atio

n of

27

anal

ysed

RTD

teas

: sam

ple

ID, t

ea ty

pe, p

acka

ging

mat

eria

l, sa

mpl

e siz

e, p

rodu

cer,

flavo

ur, c

onte

nts o

f L-

thea

nine

and

tota

l pol

yphe

nols

(TP)

.

Sam

ple

ID

Tea

type

Pa

ckag

ing

mat

eria

l

Sam

ple

size

(mL)

Pr

oduc

er

Flav

our

L-th

eani

ne (m

g/L)

TP

(mg/

L)

UH

PLC

-UV

a R

P-H

PLC

-UV

b

RTD

-1

Blac

k te

a PE

T 50

0 P-

4 O

rigin

al, l

ow e

nerg

y 14

.25

± 0.

16

14.5

0 42

0.57

RTD

-2

Blac

k te

a PE

T 50

0 P-

4 Le

mon

19

.27

± 0.

14

19.1

9 50

8.54

RTD

-3

Blac

k te

a PE

T 50

0 P-

7 Ch

erry

9.

79 ±

0.1

6 N

/A

387.

83

RTD

-4

Blac

k te

a PE

T 45

0 P-

8 Le

mon

14

.16

± 0.

19

N/A

32

6.03

RTD

-5

Blac

k te

a PE

T 45

0 P-

8 O

rigin

al

26.9

8 ±

0.20

N

/A

506.

18

RTD

-6

Gre

en te

a PE

T 55

0 P-

1 O

rigin

al, l

ow su

gar

30.9

1 ±

0.18

30

.81

674.

12

RTD

-7

Gre

en te

a PE

T 50

0 P-

1 O

rigin

al, n

o su

gar

39.9

2 ±

0.10

39

.99

503.

05

RTD

-8

Gre

en te

a

PET

500

P-2

Orig

inal

, low

suga

r 63

.42

± 0.

60

N/A

65

7.67

RTD

-9c

Gre

en te

a

PET

500

P-2

Orig

inal

, zer

o en

ergy

50

.76

± 0.

28

N/A

45

5.84

RTD

-10

Gre

en te

a

PET

560

P-2

Orig

inal

, no

suga

r 59

.69

± 0.

21

N/A

64

4.15

RTD

-11

Gre

en te

a

PET

480

P-3

Orig

inal

, low

suga

r 39

.03

± 0.

13

N/A

64

8.56

RTD

-12c

Gre

en te

a

PET

500

P-5

Lem

on

8.92

± 0

.16

N/A

18

4.65

RTD

-13c

Gre

en te

a

PET

500

P-5

Hon

ey a

nd p

umel

o 10

.16

± 0.

15

N/A

21

2.33

RTD

-14

Jasm

ine

tea

PET

500

P-1

Orig

inal

, low

suga

r 31

.09

± 0.

13

32.0

0 74

4.34

RTD

-15

Jasm

ine

tea

PET

500

P-2

Orig

inal

, low

suga

r 22

.53

± 0.

17

21.1

8 51

2.35

RTD

-16

Jasm

ine

tea

PET

480

P-3

Orig

inal

43

.44

± 0.

32

N/A

75

3.09

RTD

-17

Jasm

ine

tea

PET

480

P-3

Hon

ey

49.3

4 ±

0.45

N

/A

822.

15

RTD

-18

Ool

ong

tea

PET

500

P-1

Orig

inal

7.

91 ±

0.1

2 7.

12

619.

45

RTD

-19

Ool

ong

tea

PET

350

P-5

Orig

inal

, no

suga

r 4.

51 ±

0.3

1 4.

65

577.

02

42

Sam

ple

ID

Tea

type

Pa

ckag

ing

mat

eria

l

Sam

ple

size

(mL)

Pr

oduc

er

Flav

our

L-th

eani

ne (m

g/L)

TP

(mg/

L)

UH

PLC

-UV

a R

P-H

PLC

-UV

b

RTD

-20c

Ool

ong

tea

PET

350

P-6

Orig

inal

, no

suga

r 2.

52 ±

0.8

1 N

/A

382.

62

RTD

-21

Ool

ong

tea

PET

350

P-6

Orig

inal

, no

suga

r 2.

03 ±

1.2

4 N

/A

863.

63

RTD

-22

Ool

ong

tea

PET

500

P-6

Orig

inal

, zer

o en

ergy

1.

96 ±

1.0

1 N

/A

564.

37

RTD

-23

Ool

ong

tea

PET

500

P-6

Orig

inal

, low

suga

r 4.

18 ±

0.9

7 N

/A

466.

05

RTD

-24

Pu-e

rh te

a PE

T 50

0 P-

5 O

rigin

al, n

o su

gar

11.9

5 ±

0.36

11

.99

440.

56

RTD

-25

Pu-e

rh te

a PE

T 35

0 P-

5 O

rigin

al, n

o su

gar

10.6

8 ±

0.19

N

/A

642.

87

RTD

-26

Tieg

uany

ing

Tea

PET

500

P-1

Orig

inal

, low

suga

r 16

.10

± 0.

19

16.0

3 60

4.5

RTD

-27

Tieg

uany

ing

Tea

PET

350

P-5

Orig

inal

, no

suga

r 20

.99

± 0.

61

N/A

43

4.56

a Ave

rage

± S

D (n

=3).

b Afte

r SPE

pre

-trea

tmen

t, se

lect

ed R

TD te

as w

ere

sent

to a

con

tract

lab

for t

he a

naly

sis o

f L-th

eani

ne, u

sing

a pr

e-co

lum

n O

PA d

eriv

ativ

e

RP-H

PLC

met

hod.

c A

ccor

ding

to G

B/T

2173

3-20

08, t

hese

RTD

teas

wer

e de

fined

as t

ea fl

avou

red

beve

rage

s.

43

3.2.6 Reference solutions

L-theanine standard stock solution was prepared in a mixture of pure water and acetonitrile

(9/1, v/v) at a concentration of 1 mg/mL and stored at -20 °C. The shelf life of the stock

solution is suggested to be six months. L-theanine standard solutions at 0.2, 1, 2, 5, 10, 20,

50, 100, 150, and 200 mg/L were prepared by diluting suitable amounts of stock solution in

pure water with 0.1% formic acid and stored at 4 °C. The shelf life of the calibration solutions

was suggested to be one month.

3.2.7 Method validation for the analysis of L-theanine in RTD teas

Experiments to validate the method were carried out. The precision was evaluated by running

sample analysis including UHPLC analysis and SPE pre-treatment with three replicates. To

evaluate the accuracy, the results of the L-theanine analyses of the RTD teas obtained using

UHPLC-UV and RP-HPLC-UV following derivatisation with OPA were compared.

Moreover, recovery experiments were done. The specificity was evaluated by control samples

(L-theanine standard and spiked RTD tea samples). A calibration curve was constructed from

the results of nine different concentrations. The linearity of the L-theanine response was

accessed by regression of the peak area against the corresponding concentration. Limit of

detection (LOD) and limit of quantification (LOQ) were determined as the UHPLC-UV

giving a signal equal to three and ten times the noise, respectively.


3.3.1 Method development and validation

Chromatographic conditions were optimized by selection of acidic mobile phase, column and

flow rate. The overall time required for chromatographic separation did not exceed 8 min,

including 5 min for column equilibration. The mean L-theanine retention time was 1.156 ±

0.015 min. Representative chromatograms for RTD tea samples are shown in Figure 3.1.

44

Figure 3.1. UHPLC-UV chromatograms of L-theanine standard, 100 mg/L (A) and L-

theanine in representative RTD teas, including Tieguanying without SPE pre-treatment (B),

green tea without SPE pre-treatment (C), jasmine tea without SPE pre-treatment (D), Pu-erh

tea without/after SPE pre-treatment (E/F), black tea without/after SPE pre-treatment (G/H),

Oolong tea without/after SPE pre-treatment (I/J).

thea

nine

- 1.

162

AU

0.00

0.50

1.00

Minutes0.00 1.00 2.00 3.00 4.00

thea

nine

- 1.

157

AU

0.00

0.50

1.00

Minutes0.00 1.00 2.00 3.00 4.00

thea

nine

- 1.1

54

AU

0.00

0.50

1.00

Minutes0.00 1.00 2.00 3.00 4.00

thea

nine

- 1.

152

AU

0.00

0.50

1.00

Minutes0.00 1.00 2.00 3.00 4.00

thea

nine

- 1.

145

AU

0.00

0.20

0.40

Minutes0.00 1.00 2.00 3.00 4.00

thea

nine

- 1.

170

AU

0.00

0.20

0.40

Minutes0.00 1.00 2.00 3.00 4.00

thea

nine

- 1.

152

AU

0.00

0.20

0.40

Minutes0.00 1.00 2.00 3.00 4.00

thea

nine

- 1.

168

AU

0.00

0.20

0.40

Minutes0.00 1.00 2.00 3.00 4.00

1.15

7AU

0.00

0.20

0.40

Minutes0.00 1.00 2.00 3.00 4.00

thea

nine

- 1.

170

AU

0.00

0.20

0.40

Minutes0.00 1.00 2.00 3.00 4.00

A B

C D

FE

G H

I J

45

Formic acid was selected to prepare acidic mobile phase in this study. A mobile phase of 0.1%

formic acid in water presented better retention and peak shape of L-theanine than those of

0.05% and 0.5% formic acid in water. Different columns (ACQUITY UPLC HSS T3, BEH

C18, C8, Shield RP 18, HILIC and phenyl) were investigated. The BEH phenyl column

offered the best separation efficiency. The phenyl stationary phase affords a higher level of

hydrophilicity than the C18 and C8 column, which makes L-theanine to have more retention.

Moreover, the pi-pi interaction between the phenyl stationary phase and analytes containing

pi electrons affords additional retention power. Optimization of flow rate was carried out to

shorten the analysis time while affording better separation efficiency. The optimum flow rate

was 0.4 mL/min based on the optimization of peak shape, separation and analysis time.

The method was validated with regard to precision, accuracy, specificity, linearity, LOD and

LOQ. Triplicate analyses for each sample were completed and the content of L-theanine was

expressed as the average ± SD (Table 3.1). The recovery results of L-theanine in different

RTD teas are shown in Table 3.2. Analysis of L-theanine in selected RTD teas was performed

using a pre-column OPA derivative RP-HPLC method. The L-theanine contents measured

using the UHPLC-UV method were consistent with those measured using the OPA/ derivative

RP-HPLC method (Table 3.1). The calibration curve was plotted using peak area versus

concentration of the L-theanine standard and it was fitted to a linear equation (Y = 1350 X -

242, R2=0.9999). The linear range covered from 1 to 200 mg/L, the LOD and LOQ were 0.1

and 1 mg/L respectively.

3.3.2 Minimization of matrix interference by SPE pre-treatment

MCX SPE cartridges were used for sample clean-up before UHPLC analysis. The effect of

MCX SPE clean-up on the L-theanine separation in different RTD teas varied for the different

RTD teas. For RTD black teas, Oolong teas and Pu-erh teas, MCX SPE clean-up significantly

minimized the matrix interference (Figure 3.1). For RTD green teas, jasmine teas and

Tieguanying teas, co-elution of L-theanine and matrix interference were not observed and

MCX SPE clean-up did not offer an additional benefit. The recovery study using spiked RTD

46

teas showed that MCX SPE clean-up increased the recovery of L-theanine and reduced the

variation (Table 3.2).

Table 3.2. Recovery of L-theanine in various RTD teas.

Spiked sample

ID* Tea type

Recovery (%), mean (n=5) ± RSD

Without SPE clean-up After SPE clean-up

RTD-1 Black tea 85.6 ± 20.1 95.5 ± 3.8

RTD-2 Black tea 89.3 ± 15.9 94.2 ± 2.1

RTD-6 Green tea 94.5 ± 3.2 95.1 ± 3.0

RTD-7 Green tea 96.7 ± 2.4 95.8 ± 3.1

RTD-14

RTD-18

Jasmine tea

Oolong tea

95.5 ± 4.2

88.5 ± 14.6

94.5 ± 3.0

97.0 ± 2.8

RTD-19

RTD-24

RTD-26

Oolong tea

Pu-erh tea

Tieguanying tea

85.5 ± 17.6

76.4 ± 30.3

93.6 ± 4.4

96.2 ±3.5

96.3 ± 3.9

94.2 ± 4.8

*The spiked L-theanine concentration was 50 mg/L.

3.3.3 L-theanine in various RTD teas

Many studies have reported the contents of L-theanine in different teas [4, 7-10, 12, 15-19,

25]. However, there are no literature reports for the L-theanine level in RTD teas.

In this study, twenty-seven RTD teas of various types from the Chinese market were analysed

using UHPLC-UV. The L-theanine contents of these RTD teas are shown in Table 3.1. L-

theanine was detected in all RTD teas, but the contents varied. The mean L-theanine levels in

RTD green tea, jasmine tea, black tea, Oolong tea, Pu-erh tea and Tieguanying tea were 37.85

± 20.54, 36.60 ± 12.08, 16.89 ± 6.56, 3.85 ± 2.27, 11.31 ± 0.90 and 18.54 ± 3.46 mg/L,

respectively. It was revealed that the highest L-theanine level was detected at 63.42 mg/L in

a green RTD tea and the lowest L-theanine level was detected at 1.96 mg/L in an Oolong

RTD tea.

47

3.3.4 Quality evaluation of RTD teas by contents of L-theanine and total polyphenols

The analysis of total polyphenols of twenty-seven RTD teas from the Chinese market revealed

that three of eight RTD green teas and one of six RTD Oolong teas, according to the official

GB/T 21733-2008 regulations, were tea flavoured beverages rather than RTD teas. The scatter

plot of L-theanine contents vs total polyphenols content in various RTD teas (Figure 3.2)

shows that as L-theanine content increased in RTD black and jasmine teas, total polyphenols

content increased as well. The ratio of total polyphenols content to L-theanine content (Figure

3.2) could be used as a featured parameter for differentiating RTD teas. The ratio for RTD

Oolong teas has both the biggest variation and was the highest (78- 425), followed by RTD

Pu-erh teas (37-60), RTD Tieguanying & black teas (19-40), RTD jasmine teas (16-24) and

RTD green teas (9-22).

Figure 3.2. Scatter plot of contents of L-theanine vs total polyphenols in various RTD teas: 8

green teas, 4 jasmine teas, 5 black teas, 6 Oolong teas, 2 Pu-erh teas and 2 Tieguanying teas

from Chinese market.

0

10

20

30

40

50

60

70

0 200 400 600 800 1000

Cont

ent o

f L-th

eani

ne, m

g/L

Content of Total polyphenols, mg/L

RTD balck teas RTD green teas

RTD jasmine teas RTD oolong teas

RTD Pu-erh teas RTD Tieguanying teas

48

Figure 3.3. The ratio of total polyphenols content to L-theanine content in different RTD

teas.

3.4 Conclusions

A UHPLC-UV method was proposed for the rapid quantification of L-theanine in RTD teas.

The applicability and reliability of this analytical approach was confirmed by method

validation and successful analysis of twenty-seven real samples of RTD teas. It was revealed

that the L-theanine levels in the different RTD teas were significantly different. The scatter

plot of L-theanine content vs total polyphenol content in various RTD teas suggested that

there was a positive correlation between L-theanine content and total polyphenol content in

RTD black and jasmine teas. The ratio of total polyphenols content to L-theanine content can

be used as a featured parameter for differentiating RTD teas. Quantification of L-theanine in

RTD teas can be a reliable quality parameter for RTD teas which is complementary to total

polyphenols.

49

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for comprehensive analysis of Japanese green tea quality assessment using Ultra-performance liquid chromatography with time-of-flight mass spectrometry (UPLC/TOF MS). J. Agric. Food Chem., 56 (2008) 10705-10708.

Doi: 10.1021/jf8018003.

51

[24] Y. Zhao, P. Chen, L. Lin, J.M. Harnly, L. Yu, Z. Li, Tentative identification, quantitation, and principal component analysis of green pu-erh, green, and white teas using UPLC/DAD/MS. Food Chem., 126 (2011) 1269-1277.

Doi: 10.1016/j.foodchem.2010.11.055. [25] W. Feldheim, P. Yongvanit, P.H. Cummings, Investigation of the presence and significance of theanine

in the tea plant. J. Sci. Food Agr., 37 (1986) 527-534. Doi.org/10.1002/jsfa.2740370604.

52

53

Chapter 4

A method for measuring the noncovalent interaction between EGCG

and β-CD

Abstract

Reducing the bitter and astringent taste of green tea will engage the consumers who are used

to the mild taste of black tea. β-cyclodextrin (CD) is used as an effective bitterness and

astringency masker for catechins in green tea, especially for epigallocatechin gallate (EGCG).

The present study aims to reveal the underlying mechanism of the noncovalent interaction

between β-CD and EGCG. The complex of β-CD and EGCG was directly studied by

combined electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic

resonance spectroscopy (NMR). The stoichiometry of the β-CD-EGCG complexation product

was determined using Job’s method, which showed a 1:1 stoichiometry of the β-CD-EGCG

complex. NMR experiments confirm the formation of an inclusion complex of β-CD and

EGCG. The topology of the complex was derived from ROESY spectra and chemical shift

differences of the various protons of β-CD and EGCG in the free versus complexed state. The

direct observation of noncovalent interactions using ESI-MS and NMR enables fast screening

of molecular maskers for reducing bitterness and astringency of catechins in green tea as a

potential alternative to a tasting panel.

This chapter was submitted for publication as:

G. Chen, H-G. Janssen, the use of ESI-MS & 2D NMR to reveal the reduction of bitterness and

astringency of EGCG by β-CD inclusive complexation. Food Res. Int. manuscript submitted.

54

4.1 Introduction

Consumption of green tea is associated with several health benefits like anti-oxidant effects,

anti-carcinogenity, and suppression of cardiovascular diseases and obesity [1-7]. Green tea is

rich in a series of flavanols, more specifically catechins including (+)-gallocatechin (GC), (-)-

epigallocatechin (EGC), (-)-epicatechin (EC), (+)-catechin (CA), (+)-gallocatechin gallate

(GCG), (-)-epigallocatechin gallate (EGCG), (+)-catechin gallate (CG) and (-)-epicatechin

gallate (ECG) [8]. These compounds are thought to be responsible for the above-mentioned

health benefits, yet unfortunately they also cause green tea to taste bitter and astringent [9-

12]. This bitterness and astringency can be a barrier to consumers who are used to the mild

taste of black tea. For reducing the undesired taste characteristics of green tea, β-cyclodextrin

(CD) was shown to be a very effective taste masker [13-17]. The natural, sweet taste of β-CD

might help to mask the bitterness of catechins: a 0.5% β-CD solution is as sweet as sucrose

[18]. However, it is unclear whether the sweet taste of β-CD is the only mechanism for this

bitterness reduction, or that complexation mechanisms also play a role in the masking of green

tea bitterness and astringency.

β-CD has been described many times as a scavenging molecule which is able to complex with

bitter molecules [17,19-20]. β-CD was even used to develop a detection system for sensing

the bitterness/astringency of green tea catechins [21]. However, the exact consequences of

this complexation for taste are unknown. Investigation of the interactions between β-CD and

catechins can be helpful to reveal to which extent and how β-CD masks the bitter/astringent

taste of green tea catechins.

A number of analytical techniques has been applied to study the noncovalent interactions

between β-CD and bitter variant molecules. The use of proton nuclear magnetic resonance

(NMR), UV, and circular dichroism spectroscopy were reported to examine the inclusion

complex of CA with CDs [22]. The probable structures of the inclusion complexes of β-CD

with CA and EC were investigated using NMR [23-25]. The complexation of EC and CA

with hydroxypropyl-β-CD was investigated by using isothermal titration calorimetry,

fluorescence and proton NMR [26]. Recently, Aree and his team reported the structure-

antioxidant property relationship of the CD inclusion complexes with tea non-epicatechins by

55

means of single-crystal X-ray diffraction, density functional theory calculation and radical

scavenging activity assay [27].

Green tea catechins are well known as antioxidant actives [28]. Inclusion complexes of

catechins with CDs were investigated for the enhancement of antioxidant activity [27, 29-32].

Meanwhile, EGCG is the most bitter and astringent catechin in green tea [10,33].

Understanding the noncovalent interaction between EGCG and β-CD at the molecular level

is crucial to reveal the mechanism of β-CD bitterness reduction. In addition, the use of mass

spectrometry (MS) to characterize the inclusion complexes of β-CD with catechins has not

been reported, although MS technologies have been extensively shown to be fast and reliable

tools to determine noncovalent binding interactions and complexation [34-35].

In this chapter, the noncovalent interactions between β-CD and green tea catechins were

studied by electrospray ionization-mass spectrometry (ESI-MS). The inclusion complex of β-

CD and EGCG was characterized by the combined use of ESI-MS and 2D NMR. A

mechanism of β-CD reducing the bitterness and astringency of EGCG was proposed based

on the understanding of the noncovalent interaction of β-CD and EGCG.


4.2.1 Reagents and solvents

All reagents and solvents used in the experiment were analytical grade or better. β-CD was

purchased from SCRC (Shanghai, China). Milli-Q pure water (>18.2 MΩ, Bedford, MA, USA)

was used to prepare solutions. D2O and EGCG (>95%) were purchased from Sigma-Aldrich

(St. Louis, MO, USA). Formic acid was from Fluka (Steinheim, Germany). HPLC grade

acetonitrile was purchased from Merck (Darmstadt, Germany). Green tea powder (batch

number: xs090805-02) was supplied by Novanat (Shanghai, China). The contents of some

selected catechins in this batch of green tea powder are shown in Table 4.1.

56

Table 4.1. Contents of catechins in the green tea powder studied.

GC EGC CA EC EGCG GCG ECG CG

MW* 306 306 290 290 458 458 442 442

w/w %** 1.31 14.28 1.09 5.68 34.52 0.76 8.47 Not detected

* MW: molecular weight

** Details of the method (ISO14502-2) for the analysis of the catechins in the green tea powder

are given in Appendix.

4.2.2 ESI-MS analysis

The ESI-MS experiments were performed with a Micromass® Quattro MicroTM API

instrument (Waters, Wilmslow, UK) equipped with an ESI source. MassLynx software

(version 4.1) was used for instrument control and data acquisition. The sample solutions were

directly infused into the ESI source with a flow rate of 30 µL/min by a syringe pump and

analysed in negative ion mode. All MS parameters were optimized to obtain highest

sensitivity and resolution as well as to minimize disturbance of the complexes. Briefly, the

capillary voltage was 3.50 kV, the cone voltage was 25 V, the extractor voltage was 3 V and

the source temperature was 100 ℃. The desolvation gas temperature was 350 ℃ with a

nitrogen flow rate of 300 L/hr. The ESI-MS scan range was set from m/z 200 to 2000.

4.2.3 NMR analysis

All 1H NMR experiments (1D and 2D) were performed at 400 MHz on a Bruker Avance-

AV400 spectrometer (9.4 T) (Bruker, Rheinstetten, Germany) at 25 ℃. The resonance at 4.8

ppm, originating from residual H2O in the D2O, was used as internal reference. Complexation

was assessed by applying the rotating frame Overhauser effect spectroscopy (ROESY)

method using the ROESYPHPR pulse sequence. The parameters of the ROESY experiment

were set as follows: 16 scans, acquisition time 0.150 s, pulse delay 2.3 s and time domain size

1224.

57

4.2.4 Preparation of inclusive complexes

Stock solutions of β-CD (1 mM), EGCG (1 mM) and green tea powder (1 mg/mL) were

prepared in Milli-Q demineralised water. The inclusion complexes of β-CD and green tea

catechins were prepared by mixing 100 µL of each stock solution into 1 mL Milli-Q water at

room temperature. The work solutions of β-CD-EGCG inclusion complexes were prepared in

molar host-to-guest ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. All complex

solutions were placed at room temperature for 24 hr before ESI-MS analysis. For NMR

analysis, equimolar mixtures of β-CD and EGCG (10 mM) were prepared in D2O.


4.3.1 Direct observation of complexation of β-CD and green tea catechins using ESI-

MS

To detect CD inclusion complexes, mixtures of β-CD and green tea powder in pure water

were analysed by ESI-MS in negative ion mode using parameter settings similar to those

employed by others in the study of CD complexation of other small organic molecules [36-

39]. As GC and EGC, CA and EC, EGCG and GCG, and EGC and GC are isomer pairs, they

have the same molecular weights (Table 4.1) and yield identical m/z fragments. A

representative ESI-MS spectrum obtained for a mixture of β-CD and green tea powder in

water is shown in Figure 4.1. Ions at m/z 289.1, 305.1, 441.1, 457.0, and 493.1 correspond to

[EC/CA-H]-, [EGC/GC-H]-, [ECG-H]-, [EGCG/GCG-H]- and [EGCG/GCG+Cl]-,

respectively. The ion at m/z 915.1 corresponds to [2EGCG-H]-, an EGCG dimer adduct ion

which possibly resulted from the high concentration of EGCG in the green tea powder. Ions

at m/z 1133.7 and 1169.7 correspond to [β-CD-H]- and [β-CD+Cl]-, respectively. Ions at m/z

1423.6, 1439.6, 1575.7, 1591.9 and 1609.0 originate from [β-CD+EC]-, [β-CD+EGC-H]-, [β-

CD+ECG]-, [β-CD+EGCG-H]- and [β-CD+EGCG+Cl]-, respectively. These observed ions

clearly demonstrate the extensive complexation of β-CD and green tea catechins.

58

Figure 4.1. ESI-MS spectrum of a mixture of β-CD (100 µL stock solution) and green tea

powder (100 µL stock solution) in 1 mL pure water.

4.3.2 Direct observation of complexation of β-CD and EGCG using 1H NMR

Mass spectrometry of non-covalent complexes holds a risk of incorrect conclusions because

complexes can either be formed, or destroyed, in the ion source. It is for this reason that MS

analyses of non-covalent complexes should, if possible, be confirmed by e.g. NMR or other

spectroscopic methods. The chemical shift differences of the protons of β-CD and EGCG in

the mixture versus that in the free solutions clearly indicate that complexes are present and

justify the conclusions from the MS noncovalent interaction studies. The exact chemical shifts

are listed in Table 4.2. Comparing the chemical shifts of specific protons allows to identify

which protons from both the β-CD and EGCG are involved in the complexation reaction.

Table 4.2 clearly shows the largest chemical shift differences for the EGCG protons a and b,

and the β-CD protons 2, 3 and 5 (Figure 4.2). This would mean that mainly the B & D rings

of the EGCG molecule are involved in the complexation process. Figure 4.3 shows 1H NMR

spectra of free β-CD, free EGCG and of a mixture of the two.

59

OHO

OH

O

OH

OH

OH

OOH

OH

OH

c

c

f;ge

d

b

b

a

a

A C

B

D

O

OHHO

OH

O

O

OH

HOOH

O

OOH

OH

OH

O

O

OHOH

OH

OO

OH

OH

HO

O

OOH

OHHO

O

OOH

HO

HO

O

12

34

5 6

Figure 4.2. Chemical structures and protons of EGCG and β-CD.

Table 4.2. 1H chemical shifts of EGCG and β-CD, in free and complex status in D2O

solution.

Protons δ Free δ Complex Δδ (PPM)

EGCG a 6.94 7.10 -0.16 b 6.52 6.79 -0.27 c 6.08 6.03 0.05 d 5.48 5.43 0.05 e 4.91 4.95 -0.04 f 2.91 2.98 -0.07 g 2.87 2.91 -0.04

β-CD 1 5.07 5.02 -0.05

2,3 3.87 3.73 -0.14 4 3.59 3.55 -0.04 5 3.97 3.81 -0.16 6 3.65 3.60 -0.05

60

Figure 4.3. 1H NMR spectra of free β-CD, free EGCG and a β-CD / EGCG mixture (mole

ratio =1:1, 10 mM in D2O).

4.3.3 Stoichiometry of the β-CD-EGCG complex

The stoichiometry of the β-CD-EGCG complex was determined using Job’s method [40]. In

this method, the total molar concentration of β-CD and EGCG is held constant while varying

their mole ratios. Information on the stoichiometry, i.e. the number of EGCG molecules

present in a complex, can then be obtained from the molar β-CD/EGCG ratio at which the

strongest complexation occurs. For the measurements, a quantitative parameter that is

proportional to complex formation has to be plotted against the mole fractions of β-CD and

EGCG. Here the absolute intensity of the β-CD-EGCG complex in the ESI MS measurements

was used. The MS absolute intensity of the β-CD-EGCG complex (m/z 1591.9) was obtained

by averaging the MS signals of 15 scans. The Job-plot showed a maximum at 0.5, indicating

a 1:1 stoichiometry of the β-CD-EGCG complex (Figure 4.4).

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

2.91

22.

980

3.27

43.

306

3.55

13.

603

3.67

73.

729

3.78

83.

815

4.79

84.

949

5.01

7

5.42

9

6.03

5

6.79

3

7.09

6

2.86

82.

913

4.79

24.

908

5.48

2

6.08

5

6.51

6

6.93

6

3.58

83.

652

3.87

23.

969

4.79

9

5.06

9

CD

CD+EGCG

EGCG

61

Figure 4.4. Job-plot for the β-CD-EGCG complex.

4.3.4 Suggested topology of the β-CD-EGCG complex

Advanced 2D NMR techniques (ROESY) were successfully used to provide unambiguous

assignment of the structures of complexes of β-CD with several small organic molecules,

including for example amino acids and peptides [41]. Figure 4.5 presents the ROESY

spectrum of the β-CD-EGCG complex with cross peaks between the hydrogen protons of β-

CD and EGCG. Intermolecular correlations between the internal H3 and H5 protons of β-CD

and aromatic hydrogen protons (Ha and Hb) of EGCG are seen and aligned with the findings

reported by Cai and his team [42]. These correlations show that parts of the EGCG molecule

enter the β-CD cavity resulting in an inclusion complex of β-CD and EGCG. Based on the

ROESY spectrum and the chemical shift differences of β-CD and EGCG protons in the free

and complexed state, the topology of β-CD-EGCG complex could be that suggested in Figure

4.6, showing inclusion of the B-ring of EGCG in the β-CD cavity.

0.0 0.2 0.4 0.6 0.8 1.01x105

2x105

3x105

4x105

5x105

6x105

Inte

nsity

Mole Fraction of EGCG/(EGCG+beta-CD)

62

Figure 4.5. Rotating frame Overhauser effect spectrum containing cross peaks between β-

CD and EGCG protons.

Figure 4.6. Suggested topology of β-CD-EGCG complex.

EGCGHa, b β-CD

H3, 5

OHO

OH

O

OH

OH

OH

OOH

OH

OH

c

c

f;ge

d

b

b

a

a

A C

B

D

OHO

OH

O

OH

OH

OH

OOH

OH

OH

c

c

f;ge

d

b

b

a

a

A C

B

D

OHO

OH

O

OH

OH

OH

OOH

OH

OH

c

c

f;ge

d

b

b

a

a

A C

B

D

+

Beta-CD

H3

H5

H3

H5

63

4.3.5 Mechanism of β-CD reducing the bitterness and astringency of EGCG

Several studies in literature have indicated that the bitter, astringent taste of green tea is caused

by catechins, with galloylated catechins being considerably more bitter than their non-

galloylated counterparts [43]. Especially the presence of a gallic acid group at the D ring of

the catechin increase bitterness significantly. Removal of the gallate moiety by means of the

enzyme tannase or the use of physical barriers to exclude the gallate moiety from the taste

receptors have been used to reduce bitterness [10,12,15,33]. Based on the ESI-MS and NMR

observations of the noncovalent interaction between β-CD and EGCG presented here, it can

be concluded that inclusion complexes of β-CD and EGCG are formed in aqueous solutions.

The D ring, representing the gallate moiety, and B ring are ‘trapped’ by the β-CD molecule

upon complexation. This molecular encapsulation could ‘hide’ the gallate from the taste

receptor, and therefore reduce the perception of bitterness and astringency of EGCG.

4.4 Conclusions

The noncovalent interaction between β-CD and EGCG was studied at the molecular level by

ESI-MS and NMR. Inclusion complexation of β-CD and green tea catechins was directly

observed by ESI-MS. The stoichiometry of the β-CD-EGCG complex was determined using

Job’s method, which showed a maximum at 0.5, indicating a 1:1 stoichiometry of the β-CD-

EGCG complex. NMR experiments indicated that inclusion complexes of β-CD and EGCG

were formed and that the D ring or B ring of EGCG were present inside the β-CD cavity. This

molecular encapsulation could prevent the gallate moiety from binding to the taste receptor,

in that way reducing the perception of a bitter-astringent taste of EGCG. The direct

observation of noncovalent interactions makes the combined deployment of ESI-MS and

NMR a valuable chemical vehicle for fast screening of molecular maskers for reducing

bitterness and astringency of green tea catechins as an alternative to a tasting panel.

64

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spectroscopic study of the structure and thermodynamic parameters of EGCG/ b-cyclodextrin inclusion complexes with potential antioxidant activity. J. Incl. Phenom. Macrocycl. Chem., 78 (2014) 287-298.

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Activates TRPA1 in an Intestinal Enteroendocrine Cell Line, STC-1. Chem. Senses, 37 (2012) 167-177. Doi.org/10.1093/chemse/bjr087. [34] J.M. Daniel, S.D. Friess, S. Rajagopalan, S. Wendt, R. Zenobi, Quantitative determination of noncovalent

binding interactions using soft ionization mass spectrometry. Int. J. Mass Spectrom., 216 (2002) 1-27. Doi.org/10.1016/S1387-3806(02)00585-7. [35] J.A. Loo, Electrospray Ionization Mass Spectrometry: A Technology for Studying Noncovalent

Macromolecular Complexes. Int. J. Mass. Spectrom., 200 (2000) 175-186. Doi.org/10.1016/S1387-3806(00)00298-0. [36] S. Lee, S. Kwon, H.J. Shin, E. Cho, K.R. Lee, S. Jung, Electrospray Ionization Mass Spectrometric

Analysis of Noncovalent Complexes of Hydroxypropyl-β-cyclodextrin and β-Cyclodextrin with Progesterone. Bull. Korean Chem. Soc., 30 (2009) 1864-1866.

[37] W. Sun, M. Cui, S. Liu, F. Song, Y.N. Elkin, Electrospray ionization mass spectrometry of cyclodextrin

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[39] H. Zhang, G. Chen, L. Wang, L. Ding, Y. Tian, W. Jin, H. Zhang, Study on the inclusion complexes of cyclodextrin and sulphonated azo dyes by electrospray ionization mass spectrometry. Int. J. Mass Spectrom., 252 (2006) 1-10.

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Part 4. Model studies with caffeine and cyclodextrins. J. Chem. Soc. Perkin Trans. 2, 0 (1990) 2197-2209. Doi: 10.1039/P29900002197. [43] S. Scharbert, T. Hofmann, Molecular definition of black tea taste by mans of quantitative studies, taste

reconstitution, and omission experiments. J. Agric. Food Chem., 53 (2005) 5377-5384. Doi: 10.1021/jf050294d.

68

Appendix

Appendix 4.1. UHPLC conditions for green tea catechin analysis.

Column: ACQUITY UPLC@BEH phenyl 1.7 µm (2.1 mm×150 mm)

UV length: 278 nm

Mobile phase: ACN with 0.2% FA., water with 0.2% FA

Column temperature: 30 ℃

Gradient table:

Time(min) flow A(0.2%formatic/ACN) B(0.2%formatic/water)

0 0.4 10 90

2 0.4 15 85

5 0.4 15 85

6 0.4 20 80

8 0.4 20 80

10 0.4 70 30

10.2 0.4 10 90

12 0.4 10 90

69

Appendix 4.2. Representative chromatogram of catechins in green tea powder.

1. Gallic acid; 2. Theobromine; 3. EGC; 4. CA; 5. Caffeine; 6. EC; 7. EGCG; 8. GCG; 9. ECG.

1

2 3

5

6

7

8

9

Minutes 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

4

70

71

Chapter 5

Quantification of climbazole deposition from shampoo onto artificial

skin and human scalp

Abstract

A sensitive and specific ultra-high-performance liquid chromatography-tandem mass

spectrometry (UHPLC-MS/MS) method was developed and validated for the measurement of

climbazole deposition from hair care products onto artificial skin and human scalp. Deuterated

climbazole was used as the internal standard. Atmospheric pressure chemical ionization (APCI)

in positive mode was applied for the detection of climbazole. For quantification, multiple

reaction monitoring (MRM) transition 293.0 > 69.0 was monitored for climbazole, and MRM

transition 296.0 > 225.1 for the deuterated climbazole. The linear range ran from 4 to 2000

ng/mL. The limit of detection (LOD) and the limit of quantification (LOQ) were 1 ng/mL and

4 ng/mL, respectively, which enabled quantification of climbazole on artificial skin and human

scalp at ppb level (corresponding to 16 ng/cm2). For the sampling of climbazole from human

scalp the buffer scrub method using a surfactant-modified phosphate buffered saline (PBS)

solution was selected based on a performance comparison of tape stripping, the buffer scrub

method and solvent extraction in in vitro studies. Using this method, climbazole deposition in

in vitro and in vivo studies was successfully quantified.


G. Chen, M. Hoptroff, X. Fei, Y. Su, H-G. Janssen, Ultra-high-performance liquid

chromatography-tandem mass spectrometry measurement of climbazole deposition from hair care

products onto artificial skin and human scalp. J. Chromatogr. A. 1317 (2013) 155-158.

72

5.1 Introduction

Climbazole (1-4-Chlorophenoxy)-1-(1H-Imidazonyl)-3, 3-Dimethylbutan-2-one) is an

imidazole anti-fungal agent widely used in marketed anti-dandruff (AD) shampoos. It claims

to offer benefits of inhibiting microbe growth and improving the skin barrier on the scalp.

Climbazole is either used as a single anti-fungal agent or applied in combination with other

AD actives like zinc pyrithione or piroctone olamine to enhance its efficacy [1, 2]. The

efficacy of climbazole as an AD agent depends on its deposition behaviour and the amount

retained on the human scalp in the process of shampoo application and rinse-off. To support

in vitro and in vivo studies for the performance evaluation of AD shampoos containing

climbazole, a robust and sensitive analytical method for the measurement of climbazole

deposition on artificial skin and human scalp is required.

Besides being used as AD active in shampoo, climbazole has been widely used in daily life

as fungicide in pharmaceutical products and personal care products like lotions, conditioners,

etc. A number of analytical methods using HPLC-UV have been published for the

determination of climbazole in shampoo [3, 4]. Although the high levels of surfactants in the

shampoo can cause problems in the analysis, these methods are not extremely complex

because of the rather high level of the active. With increasing concern on the potentially

negative impact of climbazole to the aquatic environment additional methods have been

developed for the determination of climbazole in environmental matrices [5-6]. To the best

of our knowledge, only one method appeared in the literature for the analysis of climbazole

in in vitro skin studies. Schmidt-Rose [2] proposed a reversed phase HPLC-MS method

following methanol extraction for the quantification of climbazole in an in vitro study. The

method proved to be successful for the determination of skin substantivity of climbazole using

pig skin as model substrate. However, the application of this method to human scalp samples

could be challenged by the more complicated sample matrix and lower climbazole levels.

In this chapter, an ultra-high-performance liquid chromatography-tandem mass spectrometry

(UHPLC-MS/MS) method was developed and validated for the measurement of climbazole

deposition on artificial skin (for in vitro studies) and human scalp (for in vivo studies).

Deuterium labelled climbazole (Figure 5.1) was applied as the internal standard for the

73

quantification. Tape stripping and buffer scrub extractions were evaluated and compared as

sampling methods for climbazole extraction from artificial skin and human scalp.

Figure 5.1. Chemical structure of (A) climbazole and (B) deuterated climbazole.

5.2 Experiments

5.2.1 Chemicals and reagents

All reagents and solvents used in this experiment were analytical grade or better. Climbazole

(99.9%) and PBS tablets were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Ammonium acetate, glycerol, Triton X-100 and Tween 80 were purchased from SCRC

(Shanghai, China). HPLC grade methanol and acetonitrile were purchased from Merck

(Darmstadt, Germany). Milli-Q pure water (18.2 MΩ, Millipore, Bedford, MA, USA) was

used to prepare samples, standard solutions and UHPLC mobile phases. Deuterated

climbazole (>95%) was custom synthesized and supplied by Qinba Chemical (Shanghai,

China). A surfactant-modified phosphate buffered saline (PBS) solution for the buffer scrub

sampling method was prepared by adding four PBS tablets, 1.0 g of Triton X-100, and 5.0 g

of Tween 80 to 1 L of pure water.

A B

74

5.2.2 Test shampoos

Two different shampoos were used: AD shampoo and beauty shampoo. The AD shampoo

contained 0.5% (w/w) climbazole. The beauty shampoo was a commercially available

shampoo without climbazole. The application of beauty shampoo in in vitro and in vivo study

was to obtain blank samples.

5.2.3 In vitro study (Artificial skin samples)

Artificial skin (VITRO-SKIN®) was purchased from IMS testing group (ME, USA,

www.ims-usa.com). The sheet of artificial skin was cut into 6 × 6 cm pieces and laid over one

side of the smaller diameter X-ray fluorescence spectroscopy (XRF) ring. The bigger XRF

ring was then placed on top of the smaller one and pressed down to snugly combine together

in that way yielding an XRF ‘cup’ (5 cm diameter) with rough topography of the artificial

skin inside. Pure water (1.8 mL) and test shampoo (0.2 g) were added into the cup and mixed

on the surface of the artificial skin. The mixture was stirred for 30 s with a teflon stirring rod

which remained in contact with the surface of the artificial skin. The shampoo solution was

then removed with a pipette and the artificial skin was rinsed twice with 4 mL of pure water

with stirring for 30 s (as before). The rinsing water was removed with a pipette. After the cup

was allowed to dry overnight under ambient conditions, the artificial skin was subjected to

climbazole extraction.

Three sampling methods for climbazole extraction from artificial skin were investigated: tape

stripping, buffer scrub and solvent extraction. For tape stripping, tapes of Sellotape® (Se),

Leuoflex® (Le), and Standard D-Squame® (DS) were evaluated in terms of sampling

efficiency and matrix influence. After stripping climbazole from artificial skin, the tapes were

extracted by methanol in an ultrasonic bath. In the buffer scrub experiments, surfactant-

modified PBS solution and 50% aqueous glycerol solutions were evaluated as extraction

buffers. Two milliliter of extraction buffer was added into the XRF cups where the artificial

skin had been treated by test shampoos and dried naturally. A teflon stirring rod was employed

to scrub the artificial skin for 30 s. The buffer scrub sampling was repeated five times using

2 mL extraction buffer each time. All the extraction liquid was transferred into a 10 mL

75

volumetric flask using a plastic pipette and made to volume using methanol. For solvent

extraction, the artificial skin was cut out from the XRF rings and submersed in the methanol

for ultrasonic extraction. Every method for climbazole extraction from artificial skin

described above was performed in triplicate. In vitro blank samples were prepared by applying

climbazole-free beauty shampoo onto the artificial skin. As the internal standard 0.2 µg/L

deuterated climbazole was added to the extraction solution of every artificial skin sample. All

sample solutions were centrifuged prior to UHPLC-MS/MS analysis of the supernatant.

5.2.4 In vivo study (human scalp samples)

The in vivo study was designed as a single centre, whole head, and single gender (male) study.

Healthy male subjects were screened using the so-called Total Weighted Head Score (TWHS)

system [7]. Six samples were collected for each subject (three per each half head). The study

lasted for 3 days with 2 visits. The buffer scrub sampling method using modified PBS as the

extraction fluid was selected for extracting climbazole from human scalp. This selection was

based on the performance comparison of tape stripping, buffer scrub and solvent extraction

(see below) taking into consideration also the safety characteristics of the three methods. In

the buffer scrub method, a sterile plastic ring of 18 mm internal diameter and 6 cm height was

placed and held steady on the sample site of the human scalp where 2.0 mL of modified PBS

solution was applied. The scalp was gently massaged with a teflon rod for 1 min. This was

repeated with a further 2.0 mL buffer for a further 1 min. Suspensions were pooled in a single

vial using a sterile plastic pipette. The first visit was a screening session. Five subjects were

recruited for the next phase after this screening. During the second visit, subjects firstly had

their baseline sample (in vivo blank sample) collected using the buffer scrub sampling method

and then had their hair washed using a whole head hair wash procedure in which 8 gram of

AD shampoo was applied and the scalp was massaged. After rinse-off, their hair was blown-

dried and scalp samples were collected using the buffer scrub method.

As the internal standard 0.2 µg/L deuterated climbazole was added into the extraction solution

of every human scalp sample. Further analysis was performed as described for the in vitro

artificial skin samples.

76

5.2.5 Instrumentation

All sample analyses were carried out on a Waters ACQUITY UPLC System coupled to a

Quattro Micro API mass spectrometer (Waters, Manchester, UK). MassLynx software

(version 4.1) was used for instrument control and data acquisition. The samples were

separated on an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm particle size) supplied

by Waters. The mobile phase was composed of 20 mM ammonium acetate in water (A) and

methanol (B) programmed in the linear gradient mode [time 0 min, 50% A; time 5 min,

decrease immediately to 10% A; time 8 min, increase immediately to 50% A]. Total analysis

time was 8 min including 3 min for re-equilibration. The flow rate was 0.2 mL/min and the

injection volume was 5 µL. The temperatures of the column and sample compartment were

set at 30 ℃ and 4 ℃, respectively. Based on the comparison of the performance of

atmospheric pressure chemical ionization (APCI) and electro spray ionization (ESI), APCI

under positive polarity was used for all experiments. The optimized APCI conditions were

achieved by a corona current of 3.6 µA, a cone voltage of 32 V, an extractor voltage of 3 V,

a source temperature of 100 °C, an APCI probe temperature of 300 °C, a desolvation gas flow

of 450 L/hr and a cone gas flow of 25 L/hr. The multiple reaction monitoring (MRM) mode

was used for the determination of climbazole. The collision gas (argon) pressure was set at 3

× 10-3 mbar. The dwell time for each MRM transition was 0.30 s. Climbazole was analysed

using the transitions of m/z 293.0 > 69.0 (collision energy 20 V) and 293.0 > 197.1 (collision

energy 18 V) signals while deuterated climbazole was measured using the transition of m/z

296.0 > 225.1 (collision energy 14 V). These transitions were found to be the optima in the

MRM optimization experiments.

5.2.6 Quantification of climbazole

Standard stock solutions of climbazole and deuterated climbazole were prepared in methanol

at a concentration of 2 mg/mL. The working solutions of different climbazole concentrations

with 0.2 µg/L deuterated climbazole as the internal standard were prepared freshly by the

appropriate dilution of stock solutions with a mixture of 50% of mobile phase A and 50%

mobile phase B. All stock and working solutions were stored at 4 ℃ in the dark. For

77

quantification, MRM transition 293.0 > 69.0 was monitored for climbazole, and MRM

transition 296.0 > 225.1 for the deuterated climbazole. The response ratio (climbazole to

deuterated climbazole) versus climbazole concentration was plotted as the calibration curve.


5.3.1 Optimization of the UHPLC-MS/MS conditions

In order to shorten the analysis time, a UHPLC instrument equipped with a column packed

with small particles (1.7 µm, Acquity UPLC BEH C18 column) was applied. The mobile

phase composition, flow rate and gradient program were optimized to obtain both good peak

shape and high chromatographic resolution at a short total analysis time. The retention time

of climbazole was 3.36 ± 0.02 min (mean ± RSD, n=10). The total analysis time was 8 min

including 3 min for system re-equilibration. The application of MS/MS guaranteed the

method selectivity and allowed simple sample preparation. ESI and APCI were compared in

terms of signal intensity and signal suppression or enhancement. When the APCI source was

applied, no signal suppression or enhancement was observed neither within one run nor after

10 injections of artificial samples and human scalp samples. Signal suppression on the other

hand, was significant when the ESI source was applied. This phenomenon was found in many

other studies as well [e.g. 8-14]. Therefore, the APCI ionization mode was selected for the

analysis of all samples. Figure 5.2 shows the representative chromatogram obtained from

climbazole standard (20 ng/mL), an artificial skin sample and a human scalp sample.

78

Figure 5.2. Representative chromatogram of climbazole in standard solution of 20 ng/mL

(a), extraction solution of artificial skin using tape stripping and buffer scrub (b & c) and

extraction solution of human scalp using buffer scrub (d).

5.3.2 Method validation

The UHPLC-MS/MS method for climbazole analysis was validated with regard to precision,

accuracy, specificity, linearity, LOD and LOQ. In the precision study, triplicate analysis of

selected artificial skin samples and human scalp samples obtained using the buffer scrub

sampling method was carried out. The mean climbazole concentrations and standard

deviations of these two samples were 386.8 ± 33.2 and 246.3 ± 16.6 ng/cm2 for selected

artificial skin samples and human scalp samples, respectively. The recoveries of climbazole

in extraction solutions of artificial skin and human scalp samples are shown in Table 5.1. The

recoveries seen, and the repeatability found are good, meaning that the method can be applied

in studies with real hair washing. In the experiments climbazole was identified by the ratio of

a

b

c

d

79

the MRM transitions m/z 293.0 > 69.0 and 293.0 > 197.1 signals, which should be 1.4 ± 0.1,

as established from the analysis of the pure compound. The calibration curve was plotted

using the peak area ratio (climbazole to deuterated climbazole) versus concentrations of

climbazole. The linear range ran from 4 to 2000 ng/mL. The LOD and LOQ were 1 ng/mL

(signal-to-noise ratio > 3) and 4 ng/mL (signal-to-noise ratio >10), respectively. The LOQ

allowed the quantification of ppb level (corresponding to 16 ng/cm2) of climbazole on

artificial skin and human scalp.

Table. 5.1. Recovery of CBZ in extraction solutions of artificial skin samples and human

scalp samples.

Spiked sample Spiked CBZ concentration

(ng/mL)

Recovery (%)

mean (n=3) ± SD

Artificial skin 5 92.8 ± 6.0 100 96.7 ± 3.6

Human scalp 5 89.8 ± 5.5 100 94.5 ± 3.0

5.3.3 Comparison of sampling method for climbazole extraction from artificial skin

For climbazole extraction from artificial skin three sampling methods were investigated: tape

stripping, buffer scrub and solvent extraction. Figure 5.3 compares the performance of these

methods. Methanol extraction was the most efficient method for climbazole extraction from

artificial skin, followed by buffer scrub using the surfactant-modified PBS solution, buffer

scrub using 50% aqueous glycerol solution, tape stripping using DS, tape stripping using Le,

and tape stripping using Se in descending order. Compared with the tape stripping sampling

methods, buffer scrub sampling methods using the surfactant-modified PBS solution, or a 50%

aqueous glycerol solution were found to be much more efficient. Climbazole is a lipophilic

compound. Apparently, the surfactants (Triton X-100 and Tween 80) in the surfactant-

modified PBS solution improved the efficiency of extracting climbazole from (artificial) skin.

There are two possible causes to explain the extremely low efficiency of tape stripping method.

The first one can be the uneven topography of artificial skin which is designed to mimic the

80

surface properties of human skin. The second one can be the glue on the tapes which is not

able to efficiently extract climbazole from artificial skin.

Figure 5.3. Comparison of sampling methods for climbazole extraction from artificial skin.

5.3.4 Climbazole deposition levels on human scalp

Although methanol extraction showed the highest efficiency for climbazole extraction from

artificial skin, it is not allowed to apply this method to human scalps due to health and safety

reasons. Giving the second highest efficiency method, the buffer scrub method using the

surfactant-modified PBS solution was selected as the sampling method for human scalp

samples. Three samples from each side of the subject’s head were taken. The mean climbazole

deposition levels for each side of a subject’s head are presented in Figure 5.4. The error bars

(standard deviation) reflect the variability between the three samples from the same side of

the same subject’s head. Significant variations of climbazole deposition levels were observed

in the samples of the two sides of one subject as well as between the samples of different

subjects. These are most likely due to varying scalp status (e.g. sebum content) and the (local)

irreproducibility of the washing and rinsing process. Despite the rather large variability in the

81

results in real hair washing experiments, the new method presented here can be useful in

studies aimed at maximizing the deposition of the climbazole active from hair care products.

Although not studied here, our method most likely can also be applied in environmental

analysis.

Figure 5.4. Climbazole deposition on human scalp (subject # left/right side of the head).

5.4 Conclusions

A sensitive and specific UHPLC-MS/MS method was developed and validated for the

measurement of climbazole deposition on artificial skin (for in vitro studies) and human scalp

(for in vivo studies). APCI in the positive mode was applied for detection because this

ionization mode gave less signal enhancement or suppression than ESI. Deuterated

climbazole was used as the internal standard. For the extraction of climbazole from human

scalp, the buffer scrub method using a surfactant-modified PBS solution was selected based

on the performance comparison of tape stripping, buffer scrub and solvent extraction in in

vitro experiments. The sensitivity and selectivity of the developed method enabled the

quantification of ppb level (corresponding to 16 ng/cm2) of climbazole on artificial skin and

0

200

400

600

800

1000

1200

002#R 002#L 003#R 003#L 005#R 005#L 006#R 006#L 007#R 007#L

Dep

ositi

on le

vel,

ng/c

m2

scalp samples

82

human scalp. Using this method, climbazole deposition in vitro and in vivo studies was

successfully measured.

References

[1] G.A. Turner, J.R. Matheson, G.-Z. Li, X.-Q. Fei, D. Zhu, F.L. Baines, Enhanced efficacy and sensory properties of an anti-dandruff shampoo containing zinc pyrithione and climbazole. Int. J. Cosmet. Sci., 35 (2012) 78-83.

Doi.org/10.1111/ics.12007. [2] T. Schmidt-Rose, S. Braren, H. Fölster, T. Hillemann, B. Pltrogge, P. Philipp, G. Weets, S. Fey, Efficacy

of a piroctone olamine/climbazol shampoo in comparison with a zinc pyrithione shampoo in subjects with moderate to severe dandruff. Int. J. Cosmet. Sci., 33 (2011) 276-282.

Doi.org/10.1111/j.1468-2494.2010.00623.x. [3] L. Chao, Simultaneous determination of four anti‐dandruff agents including octopirox in shampoo

products by reversed‐phase liquid chromatography. Int. J. Cosmet. Sci., 23 (2001) 183-188. Doi.org/10.1046/j.1467-2494.2001.00090.x.

[4] L. Gagliardi, L. Turchetto, A. Amato, Determination of climbazole in shampoos by reversed-phase liquid

chromatography. Anal. Chim. Acta., 235 (1990) 465-468. Doi.org/10.1016/S0003-2670(00)82110-8. [5] A. Wick, G. Fink, T.A. Ternes, Comparison of electrospray ionization and atmospheric pressure chemical

ionization for multi-residue analysis of biocides, UV-filters and benzothiazoles in aqueous matrices and activated sludge by liquid chromatography-tandem mass spectrometry. J. Chromatogr. A, 1217 (2010) 2088-2013.

Doi.org/10.1016/j.chroma.2010.01.079. [6] Z.-F. Chen, G.-G. Ying, H.-J. Lai, F. Chen, H.-C. Su, Y.-S. Liu, F.-Q. Peng, J.-L. Zhao, Determination

of biocides in different environmental matrices by use of ultra-high-performance liquid chromatography-tandem mass spectrometry. Anal. Bioanal. Chem., 404 (2012) 3175-3188.

Doi.org/10.1007/s00216-012-6444-2. [7] C.R. Harding, A.E. Moore, J.S. Rogers, H. Meldrum, A.E. Scott, F.P. McGlone, Dandruff: a condition

characterized by decreased levels of intercellular lipids in scalp stratum corneum and impaired barrier function. Arch. Dermatol. Res., 294 (2002) 221-230.

Doi.org/10.1007/s00403-002-0323-1. [8] A. van Eeckhaut, K. Lanckman, S. Sarre, I. Smolders, Y. Michotte, Validation of bioanalytical LC-

MS/MS assays: Evaluation of matrix effects. J. Chromatogr. B, 877 (2009) 2198-2207. Doi.org/10.1016/j.jchromb.2009.01.003. [9] B.K. Matuszewski, M.L. Constanzer, C.M. Chaves-Eng, Strategies for the Assessment of Matrix Effect

in Quantitative Bioanalytical Methods Based on HPLC−MS/MS. Anal. Chem., 75 (2003) 3019-3030. Doi: 10.1021/ac020361s. [10] R. King, R. Bonfiglio, C. Fernandez-Metzler, C. Miller-Stein, T. Olah, Mechanistic investigation of

ionization suppression in electrospray ionization. J. Am. Soc. Mass Spectrom., 11 (2000) 942-950. Doi.org/10.1016/S1044-0305(00)00163-X.

83

[11] R. Dams, M.A. Huestis, W.E. Lambert, C.M. Murphy, Matrix effect in bio-analysis of illicit drugs with LC-MS/MS: Influence of ionization type, sample preparation, and biofluid. J. Am. Soc. Mass Spectrom., 14 (2003) 1290-1294.

Doi.org/10.1016/S1044-0305(03)00574-9. [12] S. Souverain, S. Rudaz, J. Veuthey, Matrix effect in LC-ESI-MS and LC-APCI-MS with off-line and

on-line extraction procedures. J. Chromatogr. A, 1058 (2004) 61-66. Doi.org/10.1016/j.chroma.2004.08.118. [13] B.K. Matuszewski, Standard line slopes as a measure of a relative matrix effect in quantitative HPLC-

MS bioanalysis. J. Chromatogr. B, 830 (2006) 293-300. Doi.org/10.1016/j.jchromb.2005.11.009. [14] W.M.A. Niessen, P. Manini, R. Andreoli, Matrix effects in quantitative pesticide analysis using liquid

chromatography-mass spectrometry. Mass Spectrom. Rev., 25 (2006) 881-899. Doi.org/10.1002/mas.20097.

84

85

Chapter 6

Sensitive and simultaneous quantification of zinc pyrithione and

climbazole in scalp buffer scrub samples

Abstract A sensitive ultrahigh performance liquid chromatography-tandem mass spectrometry

(UHPLC-MS/MS) method has been developed and validated for simultaneous quantification

of zinc pyrithione (ZPT) and climbazole (CBZ) deposited onto human scalp from anti-

dandruff (AD) shampoos. Scrubbing with a buffer solution was used as the sampling method

for the extraction of ZPT and CBZ from scalp. Derivatization of ZPT was carried out prior to

UHPLC-MS/MS analysis. The identification of ZPT and CBZ was performed by examining

ratios of selected multiple reaction monitoring (MRM) transitions in combination with

UHPLC retention times. The limit of detection for ZPT and CBZ was established to be 1 and

2 ng/mL, respectively. This sensitivity enables the quantification of ZPT and CBZ at

deposition levels in the low ng/cm2 range. The method was successfully applied for the

analysis of scalp buffer scrub samples from an in vivo study. The levels of ZPT and CBZ

deposited on the scalp at different time points after application of the AD shampoo were

measured. The results revealed that dual-active AD shampoo delivered more ZPT onto the

scalp in a single wash than single active shampoo did. The amount of ZPT and CBZ retained

on the scalp after AD shampoo application declined over 72 hours.


G. Chen, M. Miao, M. Hoptroff, X. Fei, L.Z. Collins, A. Jones, H-G. Janssen, Sensitive and

simultaneous quantification of zinc pyrithione and climbazole deposition from anti-dandruff

shampoos onto human scalp. J. Chromatogr. B. 1003 (2015) 22-26.

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6.1 Introduction

The use of an anti-dandruff (AD) shampoo is one of the most applied home remedies for the

treatment of dandruff. The active ingredients of AD shampoos are anti-fungals, the two most

common being zinc pyrithione (ZPT) and climbazole (CBZ). The combination of ZPT and

CBZ, as dual actives in a shampoo formulation has been proven to be able to deliver superior

antidandruff efficacy and desired end sensory benefits [1]. The amount of AD actives

deposited onto the human scalp in the process of shampoo application and rinse-off is

considered as one of the crucial factors which determine the AD shampoo efficacy [2, 3].

Hence, methods for the measurement of AD actives deposited onto the scalp are required.

There are many analytical methods for the determination of ZPT and CBZ in shampoos [4-

10]. The complicated matrices of shampoos can make the analysis challenging, but high

sensitivities for the detection of ZPT and CBZ are not required due to their rather high levels

in the AD shampoos. A number of more sensitive methods were developed for the

determination of ZPT and CBZ in environmental matrices [11-14]. For in vivo studies

monitoring ZPT and CBZ deposition onto the scalp, besides sensitive detection, efficient

sampling and sample pre-treatment techniques are required. In Chapter 5, we developed a

sensitive method for the determination of CBZ deposited on artificial skin and human scalp

from AD shampoos. For ZPT analysis, unfortunately, sensitive and easy-to-use methods are

still lacking. Due to the unwanted interactions of the compound with the silanol groups from

silica-based liquid chromatography (LC) stationary phases [13] and the trans-chelation with

other cations (e.g. Fe, Cu) [6, 13] during the analysis, poor peak shapes are often obtained,

especially at ppb levels. A liquid chromatography-mass spectrometry (LC-MS) method for

the direct analysis of ZPT was developed by Yamaguchi et al. in 2006 [15]. Unfortunately,

the sensitivity of this method was insufficient to ensure detection of ZPT at all relevant levels.

Derivatization of the ZPT with fluorescing groups was reported to stabilize the pyrithione

complex and improve the detection limits [16], but for the complex scalp samples the

selectivity of fluorescence detection is not sufficient. Mass spectral confirmation is needed,

while maintaining the excellent peak shape and sensitivity of the derivatization/fluorescence

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route. Moreover, simultaneous determination of ZPT and CBZ deposited on human scalp

would be needed, for which, however, no method has been reported till now.

In the present contribution, a method is described and validated for simultaneous

quantification of ZPT and CBZ deposited on human scalp. Scrubbing the scalp with a buffer

solution was applied as the sampling method for extraction of ZPT and CBZ from scalp and

an in vivo study was designed for method development and validation. An ultrahigh

performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method

was employed for the separation and detection of ZPT (after derivatization) and CBZ in scalp

buffer scrub samples.



All reagents and solvents used in the experiment were analytical grade or better.

Demineralised water (>18.2 MΩ) prepared using a Milli-Q® Advantage A10 Water

Purification System (Millipore, Bedford, MA, USA) was used for preparation of solutions

and dilutions. ZPT (95%), CBZ (99%), EDTA-2Na, 2, 2-Dipyridyl disulfide (DPS) and

phosphate buffered saline (PBS) tablets were supplied by Sigma Aldrich (St. Louis, MO,

USA). HPLC grade dimethyl sulfoxide (DMSO), acetonitrile and methanol were supplied by

Merck (Darmstadt, Germany). Ammonium acetate, Triton X-100 and Tween 80 were

purchased from SCRC (Shanghai, China). A surfactant-modified PBS solution for the buffer

scrub sampling method was prepared by adding four PBS tablets, 1.0 g of Triton X-100 and

5.0 g of Tween 80 to 1 L of Milli-Q water. Saturated EDTA-2Na solution was prepared by

dissolving 25 g EDTA-2Na in 100 mL Milli-Q water, stirring at 60 ℃ for 1 hour. DPS

derivatization solution was prepared by dissolving 0.6 g DPS in 50 mL acetonitrile.

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6.2.2 Test Shampoos

The three test shampoos used in this study were purchased at a local supermarket in Shanghai,

China. They were one dual-active AD shampoo (1% ZPT, 0.5% CBZ), one single active AD

shampoo (1% ZPT) and one beauty shampoo (no AD actives).

6.2.3 In vivo study

The in vivo study was conducted in Shanghai, China at the Unilever internal facility. It was

cleared by the Joint Research Ethics Committees, Shanghai, and all subjects gave their

informed consent to participate. This was a single centre, randomized, half head, double-blind

and single gender study. Each subject tested two products. Healthy male subjects aged 18-65

years (inclusive) not using anti-dandruff shampoo during the past 3 months were recruited

and screened by the so-called Total Weighted Head Score (TWHS) system [17]. The study

lasted for one week with five visits. A total of 27 subjects completed the whole study. The

buffer scrub sampling method was applied for extraction of ZPT and CBZ during the whole

study. More details on this method can be found in Chapter 5. A surfactant-modified PBS

solution was used as extraction fluid for extracting ZPT and CBZ after the scalp was gently

massaged with a Teflon rod. For each sampling site, about 3.5 mL of the buffer solution was

collected for quantitative analysis of ZPT and CBZ.

During the first visit the subjects washed their hair using the commercial beauty shampoo and

they were reminded that no hair washing, or wetting was allowed except during the visits.

The second visit was three days later. During this visit the subjects washed their hair using

the commercial AD Shampoos. Sampling was carried out before and after shampoo

application to obtain baseline samples and ‘right after wash’ samples. During the third visit

(twenty-four hours after AD shampoo application), sampling was performed to obtain 24 h

after washing samples. During the fourth visit (forty-eight hours after AD shampoo

application), sampling was performed to obtain the 48 h after washing samples. During the

fifth visit (72 h after AD shampoo application), sampling was performed to obtain 72 h after

washing samples. All the scalp buffer scrub samples were collected in Nunc 15 mL centrifuge

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tubes (Thermo Scientific, Waltham, Massachusetts USA) and were stored at -20 ℃ prior to

analysis.

6.2.4 Sample treatment

The scalp buffer scrub samples (about 3.5 mL per sample) were first diluted with methanol

(two-fold) to break the emulsion and precipitate proteins. For the DPS derivatization, 5 mL

of the methanol extract of the sample was transferred into a 15-mL centrifuge tube. Next, 80

μL of a saturated solution of EDTA-2Na and 200 μL of DPS solution were added; the sample

was thoroughly mixed by vortex and then placed in the dark for 1 hour. After the

derivatization, the sample solutions were filtered over a 0.45 μm Nylon filter supplied by

SCRC (Shanghai, China) prior to UHPLC-MS/MS analysis.

6.2.5 Standard solutions

A CBZ and ZPT mixed stock solution was prepared in DMSO at a concentration of 1000

mg/L for each analyte. It was stored at 4 ℃ in the dark prior to use. The working solutions

were prepared freshly by the appropriate dilution of this stock solution with a mixture of 50%

of mobile phase A and 50% mobile phase B. Prior to UHPLC-MS/MS analysis, the working

solutions were subjected to the same DPS derivatization procedure as the buffer scrub samples.

6.2.6 UHPLC-MS/MS analysis

A Waters ACQUITY UPLC system coupled to a Quattro Micro API mass spectrometer

(Waters, Manchester, UK) was used for the sample analysis. Separation was carried out on a

Waters ACQUITY UPLC BEH C18 column (2.1 mm x 50 mm x 1.7 μm). The mobile phase

was composed of 20 mM ammonium acetate in water (A) and methanol (B) programmed in

the linear gradient mode [time 0 min, 80% A, maintain 1 min; time 3.5 min, decrease

immediately to 50% A; time 5.5 min, decrease immediately to 10% A; time 6.5 min, maintain

10% A; time 8 min, increase immediately to 80%]. Atmospheric pressure chemical ionization

(APCI) in positive mode was used for all experiments. Optimum APCI performance was

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obtained at a corona current of 3.6 µA, a cone voltage of 32 V, an extractor voltage of 3 V, a

source temperature of 100 °C, an APCI probe temperature of 300 °C, a desolvation gas flow

(nitrogen) of 450 L/hr and a cone gas flow of 25 L/hr (nitrogen). The multiple reaction

monitoring (MRM) mode was used for the determination of CBZ and ZPT. The collision gas

(argon) pressure was set at 3 × 10-3 mbar. The dwell time for each MRM transition was 0.20

s. ZPT derivative was analysed using the transitions of m/z 237.0 > 126.0 (collision energy

17 V) and 237.0 > 111.0 (collision energy 17 V) signals while CBZ was measured using the

transition of m/z 293.0 > 69.0 (collision energy 20 V) and 293.0 > 197.1 (collision energy 18

V).

6.2.7 Method validation

The sensitivity and precision of the method were evaluated by analysing spiked blank samples

(analytically confirmed ZPT- and CBZ-free scalp buffer scrub samples). The specificity was

evaluated by the ratio of the MRM transitions. Calibration curves for ZPT and CBZ were

constructed using standards of five different concentrations. The linearity was assessed by the

regression of the peak area against the corresponding concentration. Limits of detection (LOD)

and limits of quantification (LOQ) were determined as the concentrations where the signal-

to-noise (S/N) ratios were 3:1 and 10:1, respectively.


6.3.1 Optimization of DPS derivatization

To circumvent the problems with direct analysis of ZPT we opted for the use of a

derivatization step between extraction and UHPLC-MS/MS quantification. In the current

study, PDS was applied for the derivatization of ZPT. EDTA-2Na was used to chelate zinc

and dissociate ZPT into free pyrithione which reacts with the PDS as illustrated in Figure 6.1.

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Figure 6.1. Reaction of ZPT and PDS.

Table 6.1. Effect of dosages of statured EDTA-2Na and DPS on the derivatization of ZPT.

No. Dosage of

EDTA-2Na

Dosage of

DPS

Analytical results

Mean ± SD, n=3

Peak area of ZPT

derivative

Peak area of

CBZ

1 Low Low 2011 ± 14 1023 ± 10

2 Low Medium 1887 ± 21 1125 ± 24

3 Low High 1765 ± 37 956 ± 25

4 Medium Low 1971 ± 22 1170 ± 20

5* Medium Medium 1972 ± 9 1057 ± 12

6 Medium High 1798 ± 31 961 ± 12

7 High Low 1949 ± 23 1158 ± 38

8 High Medium 1833 ± 60 1173 ± 35

9 High High 1672 ± 10 979 ± 9

*The optimum reaction conditions for the derivatization of ZPT was using 80 μL of

EDTA-2Na saturated solution, 200 μL of DPS solution and a reaction time of 1 h in the

dark at room temperature. More details are given in the text.

The PDS derivatization was carried out at room temperature. The levels of EDTA-2Na and

PDS and the reaction conditions were optimized. The results of these experiments are

summarized in Table 6.1. To obtain complete derivation of ZPT, excess DPS reagent and

EDTA-2Na salt can be used. However, this can cause interferences during the

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chromatographic separation and the mass spectrometric detection. Three levels (low, medium

and high) were selected for the concentrations of DPS reagent and the saturated EDTA-2Na

solution, respectively: 40, 80 and 400 μL for the EDTA-2Na solution; and 100, 200 and 1000

μL for DPS reagent. Peak areas of the ZPT derivative and the repeatability were used as to

establish the derivatization efficiency under different conditions. It was proven that

derivatization was most efficient when using 80 μL of EDTA-2Na saturated solution, 200 μL

of DPS solution and a reaction time of 1 h in the dark. CBZ in standard solutions and samples

was not influenced by the derivatization, evidenced by the good recovery of this compound

under all conditions.

6.3.2 Quantification of ZPT derivative and CBZ

In order to simultaneously determine ZPT (as its derivative) and CBZ in one run, the mobile

phase gradient was optimized. Total analysis time was 8 min including 2.5 min for re-

equilibration. Figure 6.2 shows a representative chromatogram obtained for a standard

solution and two real samples. The retention times for the ZPT derivative and CBZ are 2.15

± 0.02 min and 4.40 ± 0.02 min, respectively. Quantification of the two analytes was

performed in MRM mode. For the ZPT derivative, [M+H]+ ion m/z 237.0 was selected as

parent ion and fragment ions m/z 111.0 and 126.0 were selected as daughter ions. For CBZ,

[M+H]+ ion m/z 293.0 was selected as parent ion and fragment ions m/z 69.0 and 197.1 were

selected as daughter ions. For quantification, MRM transition 237.0 > 111.0 was monitored

for the ZPT derivative and MRM transition 293.0 > 69.0 for CBZ.

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Figure 6.2. Representative chromatogram of ZPT derivative and CBZ detected in a standard

solution of 10 ng/mL (A), in a buffer scrub sample from a subject who had applied dual-

active AD shampoo (B) and in a buffer scrub sample from a subject who had applied single

active AD shampoo (C).

6.3.3 Validation of the method

The method was validated with regard to precision, accuracy, specificity, linearity, LOD and

LOQ. In the precision study, triplicate analyses of ZPT and CBZ standards of 100 ng/mL and

1000 ng/mL were performed. For ZPT, the mean concentration and standard deviation of

these standards were 98.9 ± 2.3 and 997.8 ± 3.4 ng/mL. For CBZ, the mean concentration and

standard deviation were 106.3 ± 1.8 and 1014.3 ± 2.7 ng/mL. In the specificity study, ZPT

was identified by the ratios of the MRM transitions m/z 237.0 > 111.0 and 237.0 > 126.0

signals, which should be 1.5 ± 0.1, as established from the analysis of the pure compound.

CBZ was identified by the ratio of the MRM transitions m/z 293.0 > 69.0 and 293.0 > 197.1

signals, which should be 1.4 ± 0.1. In the recovery study, 100 ng/mL spiked to analytically-

A

B

C

94

confirmed ZPT- and CBZ-free scalp buffer scrub samples were used. The recoveries of ZPT

and CBZ in these samples were 100.0 ± 0.4% and 116.3± 3.5%, respectively (n=3). The

calibration curve was linear in the range from 5 to 5000 ng/mL. The LOD and LOQ were

found to be 1 ng/mL and 4 ng/mL for CBZ analysis, and 2 ng/mL and 5 ng/mL were found

for ZPT analysis, respectively. The LOQ allowed the quantification of ppb levels in the buffer

scrub solution, corresponding to approximately 5 ng/cm2 of CBZ and ZPT on the scalp.

6.3.4 ZPT and CBZ deposition on scalp from dual-active and single active shampoos

The mean levels of ZPT and CBZ deposited onto the scalp from dual-active and single active

AD shampoos are presented in Figure 6.3. The vertical bars reflect the variability resulting

from the analysis as well as inter-subject differences. Dual-active AD shampoo delivered

much more ZPT onto the scalp after one wash than the single active AD shampoo did. This

can most likely be attributed to the different compositions of the shampoos. The amount of

ZPT retained on scalp declined over 72 h, irrespective of whether dual-active AD shampoo

or single active AD shampoo was used. The amount of CBZ retained on the scalp declined

over 72 h as well. Because the study participants were not allowed to wash or wet their hair

in this 72-h time interval, the decrease of the actives is most likely caused by losses from the

skin during the normal process of skin desquamation and/or by Malassezia consumption [18].

The ZPT and CBZ delivered from dual-active AD shampoo can still be detected 72 h after

application. But in some samples in which single active AD shampoo was applied, ZPT could

no longer be detected already after some 48 h. This finding indicated that the dual-active AD

shampoo delivers longer lasting AD efficacy than single-active AD shampoo. Although not

studied here, this method can also be applied for other (in vitro or in vivo) matrices such as

for measuring ZPT and CBZ deposition on artificial skins.

95

Figure 6.3. Deposition levels of ZPT (A) and CBZ (B) on scalp from dual-active and single

active shampoos.

6.4 Conclusion

A sensitive UHPLC-MS/MS method was developed and validated for simultaneous

quantification of ZPT and CBZ deposited on human scalp from AD shampoos. The method

demonstrated high sensitivity, enabling the quantification of ng to μg amounts of the two AD

actives on human scalp. It was successfully applied for the analysis of scalp buffer scrub

samples from an in vivo study. The levels of ZPT and CBZ deposited on scalp at different

96

time points after AD shampoo application were measured. The results revealed that dual-

active AD shampoo delivered more ZPT onto the scalp after one wash than single active

shampoo did. The amount of ZPT and CBZ retained on the scalp after AD shampoo

application declined over 72 hours. The method is also applicable in other studies e.g.

artificial skin studies to improve shampoo formulations to maximize ZPT and CBZ deposition.

References

[1] G.A. Turner, J.R. Matheson, G.-Z. Li, X.-Q. Fei, D. Zhu, F.L. Baines, Enhanced sensory properties of an anti-dandruff shampoo containing zinc pyrithione and climbazole, Int. J. Cosmet. Sci., 35 (2012) 78-83.

Doi.org/10.1111/ics.12007. [2] J.R. Schwartz, R. Shah, H. Krigbaum, J. Sacha, A. Vogt, U. Blume-Peytavi, New insights on

dandruff/seborrhoeic dermatitis: the role of the scalp follicular infundibulum in effective treatment strategies, Br. J. Dermatol., 165 (2011) 18-23.

Doi.org/10.1111/j.1365-2133.2011.10573.x. [3] J.R. Schwartz, R. A. Bacon, R. Shah, H. Mizoguchi, A. Tosti, Therapeutic efficacy of anti-dandruff

shampoos: A randomized clinical trial comparing products based on potentiated zinc pyrithione and zinc pyrithione/climbazole, Int. J. Cosmet. Sci., 35 (2013) 381-387. Doi.org/10.1111/ics.12055.

[4] L. Chao, Simultaneous determination of four anti-drandruff agents including octopirox in shampoo

products by reversed liquid chromatography, Int. J. Cosmet. Sci., 23 (2001) 183-188. Doi.org/10.1046/j.1467-2494.2001.00090.x. [5] L. Gagliardi, L. Turchetto, A. Amato, Determination of climbazol in shampoos by reversed-phase liquid

chromatography, Anal. Chim. Acta., 235 (1990) 465-468. Doi.org/10.1016/S0003-2670(00)82110-8. [6] R.J. Fenn, M.T. Alexander, Determination of zinc pyrithione in hair care products by normal phase liquid

chromatography, J. Liq. Chrom., 11 (1988) 3403-3413. Doi.org/10.1080/01483918808082263.

[7] Y. Shih, J. Zen, A.S. Kumar, P. Chen, Flow injection analysis of zinc pyrithione in hair care products on

a cobalt phthalocyanine modified screen-printed carbon electrode, Talanta, 62 (2004) 912-917. Doi.org/10.1016/j.talanta.2003.10.039. [8] H. Cheng, R.R. Gadde, Analysis of zinc pyrithione in shampoos by reversed-phase high-performance

liquid chromatography, J Chromatogr., 291 (1984) 434-438. Doi.org/10.1016/S0021-9673(00)95055-6. [9] E.M. Peña-Méndez, J. Havel, J. Malecek, High-performance capillary electrophoresis determination of

pyrithione in antidandruff preparations and shampoos, J. Capillary Electrophor., 4 (1997) 269-272. [10] K. Nakajima, T. Yasuda, H. Nakazawa, High-performance liquid chromatographic determination of zinc

pyrithione in antidandruff preparations based on copper chelate formation, J. Chromatogr. A, 502 (1990) 379-384. Doi.org/10.1016/S0021-9673(01)89602-3.

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[11] A. Wick, G. Fink, T.A. Ternes, Comparison of electrospray ionization and atmospheric pressure chemical ionization for multi-residue analysis of biocides, UV-filters and benzothiazoles in aqueous matrices and activated sludge by liquid chromatography-tandem mass spectrometry, J. Chromatogr. A, 1217 (2010) 2088-2103.

Doi.org/10.1016/j.chroma.2010.01.079. [12] Z.-F. Chen, G.-G. Ying, H.-J. Lai, F. Chen, H.-C. Su, Y.-S. Liu, F.-Q. Peng, J.-L. Zhao, Determination

of biocides in different environmental matrices by use of ultra-high-performance liquid chromatography-tandem mass spectrometry, Anal. Bioanal. Chem., 404 (2012) 3175-3188.

Doi.org/10.1007/s00216-012-6444-2. [13] J. Bones, K.V. Thomas, B. Paull, Improved method for the determination of zinc pyrithione in

environmental water samples incorporating on-line extraction and preconcentration coupled with liquid chromatography atmospheric pressure chemical ionisation mass spectrometry, J. Chromatogr. A, 1132 (2006) 157-164.

Doi.org/10.1016/j.chroma.2006.07.068. [14] V.A. Sakkas, K. Shibata, Y. Yamaguchi, S. Sugasawa, T. Albanis, Aqueous phototransformation of zinc

pyrithione degradation kinetics and byproduct identification by liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry, J. Chromatogr. A, 1144 (2007) 175-182.

Doi.org/10.1016/j.chroma.2007.01.049. [15] Y. Yamaguchi, A. Kumakura, S. Sugasawa, H. Harino, Y. Yamada, K. Shibata, T. Senda, Direct analysis

of zinc pyrithione using LC-MS, Intern. J. Environ. Anal. Chem., 86 (2006) 83-89. Doi.org/10.1080/03067310500249930. [16] N. Voulvoulis, M.D. Scrimshaw, J.N. Lester, Analytical methods for the detection of 9 antifouling paint

booster biocides in estuarine water samples, Chemosphere, 38 (1999) 3503-3516. Doi.org/10.1016/S0045-6535(98)00580-3. [17] C.R. Harding, A.E. Moore, J.S. Rogers, H. Meldrum, A.E. Scott, F.P. McGlone, Dandruff: a condition

characterized by decreased levels of intercellular lipids in stratum corneum and impaired barrier function, Arch. Dermatol. Res., 294 (2002) 221-230.

Doi.org/10.1007/s00403-002-0323-1. [18] R.J. Hay, Malassezia, dandruff and seborrhoeic dermatitis: an overview, Br. J. Dermatol., 165 (2011) 2-

8. Doi.org/10.1111/j.1365-2133.2011.10570.x.

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Chapter 7

Ex-vivo measurement of scalp follicular delivery of zinc pyrithione and

climbazole from hair care products

Abstract

Efficient delivery of anti-dandruff (AD) actives into the scalp follicular infundibulum as well

as onto the scalp surface is critical for the efficacy of AD shampoos. A method involving scalp

cyanoacrylate (CA) biopsy sampling, a tailor-made cutting device, ultra-high-performance

liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis, scanning

electron microscopy (SEM) measurement and Raman imaging has been developed for the

measurement of delivery of zinc pyrithione (ZPT) and climbazole (CBZ) from an AD shampoo

into the scalp follicular infundibulum. Scalp CA biopsy enables the sampling of ZPT and CBZ

delivered into the scalp follicular infundibulum as well as onto the scalp surface. Raman

imaging of scalp CA biopsy samples allows the visualization of the spatial distribution of ZPT

and CBZ deposited on the scalp. A tailor-made cutting device enables the separation of the

scalp follicular infundibulum sample (20 µm below the scalp surface) from the scalp surface

samples (including top 20 μm of infundibula). UHPLC-MS/MS was used as a sensitive and

specific methodology enabling the quantification of ZPT and CBZ without interferences.

Using this method, ZPT and CBZ were successfully quantified and visualized within the scalp

follicular infundibulum, after the scalp was washed with an AD shampoo.


G. Chen, C. Ji, M. Miao, K. Yang, Y. Luo, M. Hoptroff, L.Z. Collins, H.-G. Janssen, Ex-vivo

measurement of scalp follicular infundibulum delivery of zinc pyrithione and climbazole from an

anti-dandruff shampoo. J. Pharma. Biomed. Anal. 143 (2017) 26-31.

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7.1 Introduction

Dandruff is a common scalp issue which affects approximately 50% of the global population

and is characterized by flakes and itch on the scalp without visible inflammation. [1-3].

Overgrowth of Malassezia, a genus of lipophilic yeasts was reported to be associated with

dandruff [4-7]. Hence, one of the most common treatments of dandruff is the use of an anti-

dandruff (AD) shampoo containing antifungal agents like zinc pyrithione (ZPT) and

climbazole (CBZ) [8, 9].

It is known that Malassezia yeasts live on human skin around the opening of the hair follicles

(also called infundibulum) as well as on the scalp surface [5, 10 and 11]. Schwartz and his

team claimed that efficient delivery of AD actives into the scalp follicular infundibulum as

well as onto the scalp surface is critical for the efficacy of AD shampoos [12, 13]. Reliable

analytical methods are required for the measurement of spatial delivery of AD actives on the

scalp. However, besides buffer scrub, cyanoacrylate (CA) biopsy, tape stripping and hair

plucking, very limited sampling methods have been developed for detecting AD actives on

the scalp after the application of AD shampoos.

In Chapter 6, we developed a method for the measurement of ZPT and CBZ deposition on

scalp from AD shampoo using buffer scrub as the sampling method. However, the method is

not able to distinguish between scalp surface delivery and follicular infundibulum delivery.

An in vivo imaging method using confocal microscopy was reported to enable the

measurement of ZPT spatial distribution in the scalp follicular infundibula [13]. Confocal

microscopy imaging can detect ZPT particles optically but not chemically. When other similar

particles are delivered together with ZPT, chemical imaging tools like Raman imaging are

demanded to offer chemical specificity, so as to eliminate the impact of other particles.

Since Marks and Dawber first used CA for skin surface biopsy in 1971 [14], CA based

sampling methods have been further developed and proven to enable both skin surface and

follicle biopsies. Combined with imaging technologies, cyanoacrylate skin surface stripping

(CSSS) has been widely applied in diagnostic dermatopathology and cosmetology, as well as

in experimental dermatology settings [15]. To investigate the penetration of topically applied

101

substances into hair follicles, Teichmann and his team developed a differential stripping

method which was based on CSSS [16, 17]. For measuring ZPT delivery into the scalp

follicular infundibula, a sampling method which combined CA biopsy and hair pluck was

reported [13]. In this method, a drop of CA glue was applied to the infundibulum of a hair

follicle. After drying, the CA glue together with hair(s) was removed, obtaining a follicular

cast containing cell debris, sebum, hair(s), ZPT etc. To make the analysis be representative

for the whole hair follicles, the sampling should cover multiple follicles.

The aim of this study was to develop a method to measure scalp follicular infundibulum

delivery of ZPT and CBZ are delivered onto the scalp from a dual-active AD shampoo. A CA

based in vivo sampling method was developed to harvest ZPT and CBZ delivered on both the

scalp surface and in follicular infundibula. After the sampling, scalp CA biopsy samples (casts)

were subjected to Raman and scanning electron microscopy (SEM) imaging. A tailor-made

cutting device was developed to efficiently and accurately separate the scalp surface casts

(SCs) from scalp follicular infundibulum casts (FICs) at a defined z-distance. After separation

of the layers, the contents of ZPT and CBZ in scalp SCs and FICs were determined by an

ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)

method.



All reagents and solvents used in the experiment were analytical grade or above. CBZ (99.9%),

ZPT (95%), and 2, 2-dipyridyl disulfide (DPS), were purchased from Sigma-Aldrich (St.

Louis, MO, USA). Ammonium acetate and EDTA-2Na were purchased from SCRC

(Shanghai, China). HPLC grade acetone, methanol and acetonitrile were purchased from

Merck (Darmstadt, Germany). Milli-Q pure water (>18.2 MΩ, Millipore, Bedford, MA, USA)

was used to prepare samples, standard solutions and UHPLC mobile phases. Model sebum

was prepared by mixing the lipid compounds purchased from Sigma-Aldrich (St. Louis, MO,

USA).

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7.2.2 Test shampoos

A commercial beauty shampoo without AD actives was used as run-in shampoo. The test

shampoo used in this study was a commercial dual-active AD shampoo containing ZPT and

CBZ.

7.2.3 In vivo clinical study and CA biopsy sampling

The in vivo clinical study was a randomized, double blind and controlled study. The study

was conducted in China and was reviewed and approved by the ethical committee of the

Shanghai Clinical Research Center. Informed consent was obtained from all subjects before

participation. Healthy subjects of both gender aged 18-60 years were screened and had their

scalp condition visually assessed using Unilever Total Weighted Head Score (TWHS) system

[3]. Subjects with scalp condition of whole head TWHS adherent flake (AF) ≤ 8, with no

flake grade B or above, were accepted. Subjects who had used shampoo containing anti-

dandruff actives within the last 4 weeks were excluded. Forty-five subjects were accepted and

43 subjects (24 females + 19 males) completed the whole study. Two subjects were withdrawn

for personal reason. No products or procedure related adverse event was reported.

All subjects had their hair washed using beauty shampoo 2 days before the test shampoo wash.

Then subjects had their hair washed using the same dose of the dual-active AD Shampoo and

blow-dried following standard hair wash operation. Square regions of 1 cm×1 cm were

identified as sampling sites for scalp CA biopsy. Hair in this region was carefully cut from

the root as close to the scalp skin as possible and operation contact with scalp skin was

minimum.

A small amount of CA glue (Loctite Medical grade CA glue 4061) was applied onto the rough

side of the Melinex® sampling strip and spread evenly to give a thin film. Afterwards, the

glue-coated strip was applied to the sampling area and pressed firmly onto the scalp. The strip

sample was peeled off when dry. All CA scalp biopsy samples were stored in an air-tight

container before further analysis. Figure 7.1 shows how CA scalp biopsy is used to obtain the

FICs and SCs consisting of cell detritus, lipids, microbes and AD actives.

103

Figure.7.1. Principle of scalp CA biopsy sampling.

7.2.4 SEM measurement

The scalp CA biopsy samples before and after cutting were sputter-coated with platinum using

an ion sputter coater (E-1045, Hitachi, Tokyo, Japan) for 90 seconds with a current of 15 mA.

The SEM images were taken by using a SEM (Hitachi S-4800) to measure the size of FIC.

Imaging conditions were set as follows: high voltage = 5 or 10 kV; beam current = ~10 μA;

working distance = 5 – 10 mm; magnification = 30 × – 20,000 ×.

7.2.5 Raman measurement

A Raman microspectrometer (Horiba Jobin Yvon, HR Evolution, Kyoto Japan) was employed

to visualize the spatial distribution of CBZ and ZPT on scalp CA biopsy samples before

cutting. Pure materials of CBZ, ZPT, CA glue and model sebum were used as reference

104

materials to assign signals in the obtained Raman spectra. On each sample, one region in size

of ~ 2 mm × 2 mm (containing at least 3 follicles) was scanned under a 10x air objective with

a 532 nm laser source. The step size was 10 µm (~ 40, 000 pixels), and spectra in each pixel

were taken from 400 cm-1 to 2000 cm-1, with 0.5 second laser exposure with 2 times

accumulation to remove cosmic rays. The resultant spectra matrix was analysed by the built-

in direct classical least square (CLS) fitting algorithm to demonstrate the relative distribution

of each active.

7.2.6 Separation of scalp FICs from scalp SCs

After sampling, the scalp CA biopsy samples were cut by a tailor-made cutting device which

was developed in house. The principle of cutting down the follicular infundibulum cast is

shown in Figure 7.2. Before cutting, the blade height of the cutting device was carefully

adjusted to 20 µm, so as to only cut down the FICs without SCs. Scalp CA biopsy samples

are placed on a weighing paper. Then the cutting device was pushed forward horizontally to

sweep over the sampling area. This was repeated several times. The obtained FICs were so

tiny that they should be carefully collected from the tape as well as from the blade. After

cutting, the white ash was transferred into a tube pre-marked “scalp infundibulum sample”.

The residual CA biopsy sample was collected as “scalp surface sample”, which included the

top 20 µm infundibulum.

Figure.7.2. Principle of cutting down the follicular infundibulum cast.

105

7.2.7 Quantification of ZPT and CBZ by UHPLC-MS/MS analysis

A Waters ACQUITY UPLC system coupled to a Quattro Micro API mass spectrometer

(Waters, Manchester, UK) was used for the quantitative analysis of ZPT and CBZ. Both

“scalp infundibulum samples” and “scalp surface samples” were subjected to acetone

extraction. A standard stock solution of ZPT and CBZ was prepared in methanol at a

concentration of 2 mg/mL each. The working solutions were prepared freshly by the

appropriate dilution of the stock solution with a mixture of 50% of mobile phase A (20 mM

ammonium acetate in water) and 50% mobile phase B (methanol). Following the previously

developed method in Chapter 6, both sample extraction and standard solutions were subjected

to DPS derivatization prior to UHPLC-MS/MS analysis. The delivery levels of ZPT and CBZ

were expressed as ng/cm2.


7.3.1 SEM imaging of scalp CA biopsy samples

The infundibulum is the upper segment of the hair follicle, extending from the surface to the

sebaceous gland. The total length of the infundibulum is approximately 500 µm [18]. SEM

images (Figure 7.3) of scalp CA biopsy samples visualize the morphology of a scalp FIC

which look like hollow cones without tips. The hollow cone was caused by residual hair shafts

in follicles, although hair was clipped to scalp level prior to sampling. The dimensions of the

FICs (height and diameter) were measured by the build-in ruler function of the SEM. The

FICs heights were in the 50-200 µm range, which indicates that scalp CA biopsy enables a

sampling depth of 200 µm in the follicular infundibula. The diameter at the base of FICs

ranged from 100 to 250 µm. All scalp CA biopsy samples were measured by SEM and images

were studied individually to verify that infundibulum casts had been successfully collected.

106

Figure.7.3. SEM images of scalp CA biopsy samples.

A and B: top view images of a representative scalp CA biopsy sample; C and D: cross-

sectional view of a representative scalp CA biopsy sample at different magnifications.

7.3.2 Spatial distribution of ZPT and CBZ on scalp CA biopsy samples

Pure CA glue, CBZ, ZPT and model sebum were used as reference materials to obtain

standard Raman spectra (Figure 7.4). A multivariate curve resolution (MCR) algorithm (CLS)

[19] was used for spectra interpretation and image reconstruction. For each image, at least

three FICs were measured. Figure 7.5 shows a typical Raman image of a scalp CA biopsy

sample, which indicates that both ZPT and CBZ were delivered more into the follicular

107

infundibula than onto the scalp surface when applying the dual-active AD shampoo. Most

likely during rinse-off, the ZPT and CBZ on the scalp surface were more easily rinsed-off

than that present in the follicular infundibula. Current Raman images enable the visualization

of ZPT and CBZ distribution on scalp.

Figure.7.4. Raman spectra of CA glue, CBZ, ZPT and sebum.

Figure. 7.5. Raman images of a representative scalp CA biopsy sample. (Scale bars indicate

40 µm). A: Mapping of CA glue (in grey scale); B: Mapping of CBZ; C: Mapping of ZPT

(Red colour shows higher intensity and blue colour shows lower intensity).

108

7.3.3 Separation of scalp FICs from scalp SCs by a cutting device

A tailor-made cutting device was designed and made for cutting down the scalp FICs. It is

constructed of a vernier caliper and a blade. The vernier caliper enables easy tuning of the

blade height with a minimum tuning step size of 1 µm. The blade is circular, which ensures

the blade edge always faces the casts after adjusting the blade height. Efficiency of the follicle

cutting device was verified by SEM images of the scalp CA biopsy samples before and after

cutting (Figure 7.6). The blade height away from surface is critical for complete separation of

FICs from SCs. Based on the surface morphology of CA biopsy samples, the blade height

was set at 20 µm in this study. This setting ensured pure FICs were cut off without any SCs.

As a result, however, some FICs (less than 20 µm height) remained on the CA tape and were

treated as scalp surface samples. After cutting, the scalp CA biopsy samples were divided into

scalp infundibulum samples (deeper than 20 μm) and scalp surface samples (including top 20

μm of infundibula).

Fig.7.6. SEM images of scalp CA FICs before cutting (left) and after cutting (right).

7.3.4 Deposition levels of ZPT & CBZ into scalp follicular infundibula

The contents of ZPT and CBZ in scalp infundibulum samples and scalp surface samples

(shown in Table 7.1) were quantified by the UHPLC-MS/MS method. The method is sensitive

enough to detect ZPT and CBZ at ppb level (corresponding to 5 ng/cm2). More ZPT and CBZ

109

were detected in scalp surface samples (ZPT, 2770 ± 2540 ng/cm2 and CBZ, 550.1 ± 270.5

ng/cm2) than in scalp infundibulum samples (ZPT, 11.0 ± 9.0 ng/cm2 and CBZ, 10.3 ± 9.5

ng/cm2). This finding looks inconsistent with the Raman imaging results in which more ZPT

and CBZ were observed in the follicular infundibula than on scalp surface. A study on the

depth profile of scalp infundibular ZPT [13] observed a sharp drop-off of the amount of ZPT

as the depth increases with the majority of ZPT (>80%) being present in the upper part of the

infundibula (<20 µm). Due to the limitation of the cutting in the current method, the scalp

surface samples included the top 20 µm of infundibula. Consequently, analysis of the

harvested FIC underestimates the total ZPT delivery to the entire infundibulum. Another

reason for the difference in findings between Raman imaging results and deposition levels is

that the combined area of scalp follicular infundibula is dramatically smaller than that of the

scalp surface [20-22].

Table 7.1. Scalp surface and follicular infundibulum delivery of ZPT and CBZ from the dual-active shampoo.

AD active deposition from the dual-active AD shampoo

ZPT CBZ

Scalp surface samples, ng/cm2 (including top 20 µm infundibulum) *

2770.0 ± 2540.0 550.1 ± 270.5

Scalp infundibulum samples, ng/cm2 (20 µm deeper than surface) *

11.0 ± 9.0 10.3 ± 9.5

Total deposition, ng/cm2 2781 ± 2547.0 560.4 ± 277.0 Ratio of infundibulum delivery vs total deposition

0.40% 1.80%

* Mean ± Standard Deviation, n=60.

The dual-active AD shampoo delivered almost the same levels of ZPT (11.0 ± 9.0 ng/cm2)

and CBZ (10.3 ± 9.5 ng/cm2) into the infundibulum. However, a comparison of the ratio of

infundibulum delivery (20 µm deeper than surface) vs total deposition between CBZ (1.80%)

and ZPT (0.40%) suggests that CBZ penetrates deeper into follicular infundibulum than ZPT,

suggesting that CBZ may be up to 4x more efficient at “targeting” the follicular infundibulum.

The reason for this difference is likely due to CBZ having a higher solubility in sebum than

ZPT.

110

7.4 Conclusions

For the measurements of follicular infundibulum delivery of ZPT and CBZ from a dual-active

AD shampoo, a method involving scalp CA biopsy sampling, FICs cutting, UHPLC-MS/MS

analysis SEM measurement and Raman imaging has been developed. Scalp CA biopsy

enables the sampling of ZPT and CBZ delivered into the scalp follicular infundibulum. Raman

imaging of scalp CA biopsy samples allows the visualization of the spatial distribution of ZPT

and CBZ deposition on the scalp. The cutting device enables the separation of scalp FICs

from scalp SCs and the contents of ZPT and CBZ can be quantitated by the sensitive UHPLC-

MS/MS analysis. The method detection limit allows the quantification of ppb levels of CBZ

and ZPT delivered onto the scalp surface and into the follicular infundibulum (corresponding

to 5 ng/cm2).

Using this method, ZPT and CBZ delivered into the scalp follicular infundibulum (20 µm

lower than scalp surface) from the dual-active AD shampoo was successfully visualized and

quantified. Due to the lipophilic nature of CBZ and subsequent increased solubility in sebum,

CBZ possesses the ability to penetrate further into the sebum-rich infundibulum whereas ZPT

remains within the upper 20 µm of infundibula. This differential distribution of actives allows

for the effective targeting of Malassezia species throughout the depth of the scalp follicular

infundibulum.

111

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Kerr, J.P. Henry, R.C. Rust, M.K. Robinson. A comprehensive pathophysiology of dandruff and seborrheic dermatitis - towards a more precise definition of scalp health. Acta. Derm. Venereol., 93 (2013) 131-137.

Doi: 10.2340/00015555-1382. [5] R.J. Hay. Malassezia, dandruff and seborrhoeic dermatitis: an overview. Br J Dermatol., 165 (2011) 2-8. Doi.org/10.1111/j.1365-2133.2011.10570.x. [6] T.L. Dawson Jr. Malassezia globosa and restricta: breakthrough understanding of the etiology and

treatment of dandruff and seborrheic dermatitis through whole-genome analysis. J. Investig. Dermatol. Symp. Proc., 12 (2007) 15-19.

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etiologic facets of dandruff and seborrheic dermatitis: Malassezia fungi, sebaceous lipids, and individual sensitivity. J. Investig. Dermatol. Symp. Proc., 10 (2005) 295-297.

Doi.org/10.1111/j.1087-0024.2005.10119.x. [8] G.A. Turner, J.R. Matheson, G.Z. Li, X.Q. Fei, D. Zhu, F. Baines. Enhanced efficacy and sensory

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human skin using confocal laser scanning microscopy. Laser Phys. Lett., 2 (2005) 148-152. Doi.org/10.1002/lapl.200410156. [11] M.K. Hill, M.J. Goodfield, F.G. Rodgers, J.L. Crowley, E.M. Saihan. Skin surface electron microscopy

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113

Chapter 8

Visualization of zinc pyrithione particles deposited on the scalp from

hair care products

Abstract

An ex vivo method that combines tape strip sampling and SEM/EDX has been developed for

measuring and visualizing the particle size, morphology and composition of zinc pyrithione

(ZPT) deposited on the scalp from AD shampoos. Hair was washed with a commercially

available AD shampoo containing ZPT and zinc carbonate (ZnCO3). Tape strips were applied

to collect the deposited particles from the scalp after AD shampoo application and rinse-off.

The scalp tape strip samples were subjected to scanning electron microscopy/energy

dispersive X-ray spectroscopy (SEM/EDX) measurement. The morphology of the ZPT

particles was visualized by SEM imaging and identification of ZPT particles was confirmed

by EDX analysis. For the commercial shampoo studied it was observed that two zinc-

containing particulates with different morphologies and composition remained on the scalp

after shampoo application and rinse-off. As indicated by the EDX spectra, the ZPT particles

deposited onto the scalp surface had polygonal crystal structures. ZnCO3 was also deposited

onto the scalp surface, mainly presenting as aggregated particulates. The new method allows

the microstructures of both ZPT and other zinc particles on the scalp to be imaged.


G. Chen, C. Ji, L.Z. Collins, M, Hoptroff, H-G. Janssen, Visualization of zinc pyrithione particles

deposited on the scalp from a shampoo by tape strip sampling and scanning electron

microscopy/energy dispersive X-ray spectroscopy measurement, Int. J. Cosmet. Sci. 40 (2018)

530-533.

114

8.1 Introduction

The anti-dandruff (AD) efficacy of an AD shampoo is highly dependent on the deposition of

the AD actives onto the human scalp during the process of shampoo application and the

amounts remaining after rinse-off. Zinc pyrithione (ZPT) is the most common anti-fungal

active formulated in commercially available (AD) shampoos [1, 2]. There are two types of

ZPT materials regularly used in commercially available AD shampoos, which are different in

particle size and morphology, and as a consequence of that, in deposition mechanism [3].

Robust and reliable methods for studying the ZPT deposition behaviour onto the scalp are

required for developing a better understanding of the difference in AD efficacy among

different formulations and products.

In Chapter 6 and 7, we developed methods for the quantification of ZPT deposition onto the

scalp surface and into the hair follicles. To measure the particle size and morphology of ZPT

deposited on the scalp, imaging methods are demanded. Schwartz et al. reported a reflectance

confocal microscopy (RCM) imaging method for in vivo optical detection of ZPT particles

deposited on the scalp [4]. Due to limitations in the resolution of the RCM method,

information on particle size and morphology of the ZPT particles could not be obtained. An

additional drawback of confocal microscopy is that when other similar particles are delivered

together with the ZPT, RCM imaging cannot distinguish between ZPT and these other

particles. Chemical imaging methods using stimulated Raman scattering (SRS) were

developed to eliminate this drawback and allow mapping the distribution of the ZPT active

on intact skin [5]. Several other imaging technologies have been developed, e.g. to understand

the effects of nanoparticles and microparticles in living tissues [6] and on human substrates

like zinc oxide and titanium dioxide on skin [7-10], and silica in dentine [11]. However,

methods that offer sufficient chemical selectivity, sensitivity and resolution to enable in vivo

or ex vivo visualization of ZPT particles deposited on the scalp so far have not been reported.

The aim of this chapter was to develop an ex vivo measurement method for the visualization

of the particle size and morphology of ZPT deposited on the scalp from AD shampoos. A

previously developed tape-strip sampling method [12] was evaluated for its ability to capture

the ZPT deposits remaining on the scalp after hair wash. Scanning electron microscopy (SEM)

115

was studied as a means to visualize particle size and morphology. Finally, energy dispersive

X-ray spectroscopy (EDX) was used to confirm the presence of the ZPT active.

8.2 Materials and Methods

8.2.1 AD Shampoo

A commercially-available AD shampoo was purchased in a local supermarket in Shanghai,

China and used in this study. The shampoo was a potentiated ZPT shampoo containing ZPT

and zinc carbonate (ZnCO3) [3, 13].

8.2.2 Shampoo wash and specimen collection

The study protocol was reviewed and approved by the Joint Research Ethics Committees

(Shanghai, China). All recruited subjects of both genders aged 18-60 years were medically

healthy and gave their informed consent prior to participating in this study. Forty-five subjects

were accepted and 43 subjects (24 females + 19 males) completed the whole study. Two

subjects were withdrawn for personal reason.

A hair wash procedure was applied to the participating subjects, as previously described in

Chapter 7. After hair wash, a standardised tape strip sampling method was used to collect

deposited actives. In brief, the sampling sites on the scalp of each subject were exposed by

parting the hair. The length of parting line was about 10 cm. A commercially-available sticky

tape (J-LAR, Permacel, Wisconsin, United States) was placed along the parting line. The tape

was pressed onto the scalp surface by applying a uniform pressure using a roller. This roller

was passed 20 timed, unidirectionally along the length of the tape. The tape was then removed

and mounted in the suitable holders for the SEM/EDX instruments (vide infra).

116

8.2.3 SEM/EDX measurement

The tape strip specimens were mounted on an aluminium sample holder, sputter-coated with

platinum (Hitachi, E-1045, Tokyo, Japan), and transferred into the SEM chamber for

visualisation (Hitachi, S-4800, Tokyo, Japan). The working distance was 15 mm, and a

voltage of 10 kV was applied. The deposition site was identified by EDX examination (Horiba,

X-max, Kyoto, Japan) through the detection of zinc (6 seconds X-ray exposure on each spot).

Molar ratios of sulphur and zinc (S/Zn) were calculated using the instrument’s built-in

software. Studies with blank samples showed that there was no detectable zinc and sulphur

on new tape nor in the scalp flakes that were captured on the tape from untreated skin.

Therefore, it can be confirmed that zinc and sulphur only came from the shampoo used.


In the SEM images particles of different size and morphology were seen. EDX analysis of

these particles showed two main characteristic types of morphology for the zinc-containing

deposits on the scalp surface. As shown in Figure 8.1A, distinct ZPT crystalline particles with

an average size of approximately 2 µm (n=129) were dispersed across the SEM image. These

particles exhibited a well-recognised polygonal shape. Another characteristic zinc-containing

deposit, consisting of aggregated sub-micron particles, was also abundantly present (Figure

8.1B). These deposits were generally of an irregular shape.

EDX signals of sulphur and the S/Zn ratios were used to differentiate ZPT from other Zn

containing materials (e.g. ZnCO3) that had deposited on the scalp from the shampoo after hair

wash. The polygonal crystals showed a typical EDX spectrum (Figure 8.1C), indicating the

presence of both zinc and sulphur. The sulphur to zinc molar ratio for these crystals is close

to its theoretical ratio for ZPT (2:1). These results demonstrate that the polygonal crystals are

ZPT. On the other hand, there was no detectable sulphur signal in the EDX spectra for the

aggregated zinc deposits (Figure 8.1D). These results indicate that these aggregates were

mainly composed of ZnCO3.

117

Figure 8.1. Typical microstructures of zinc-containing deposits (A and B).

The corresponding EDX spectra are shown accordingly (C for panel A, and D for panel B).

The white asterisks indicate the positions of the EDX analysis.

Previous studies showed that deposited ZPT can be visualised using RCM [1, 4]. In these

studies, all bright white dots in RCM images of scalp skin were assumed to be ZPT deposits.

However, based on the findings in the current study it is concluded that it is not possible to

differentiate ZPT from other particles in RCM analysis. In addition, the low resolution of

RCM does not allow for differentiating ZPT from ZnCO3 on the basis of microstructural

features. RCM hence is not an appropriate tool assess ZPT deposition on the scalp if other

crystalline materials are present in the formulation at a significant concentration, such as e.g.

ZnCO3. Clearly, under these conditions, using RCM ZPT deposition would be overestimated.

The current method with EDX verification eliminates this drawback.

118

8.4 Conclusion

An ex vivo method combining tape strip sampling and SEM/EDX characterisation has been

developed for measuring and visualizing the particle size and morphology of ZPT deposited

on the scalp from a commercially available AD shampoo. This ex vivo measurement method

provides higher imaging resolution and more chemical specificity than RCM. To the best of

our knowledge, this is the first time that ZPT particles could be distinguished from other zinc-

containing particles deposited onto the scalp. The combined SEM/EDX method also enabled

us to characterise the microstructures of both ZPT and other zinc particles deposited onto the

scalp.

119

References

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Doi.org/10.1111/ics.12055. [2] G. Turner, J. Matheson, G. Li, X. Fei, D. Zhu, L. Baines, Enhanced efficacy and sensory properties of an

anti-dandruff shampoo containing zinc pyrithione and climbazole. Int. J. Cosmet. Sci., 35 (2013) 78-83. Doi.org/10.1111/ics.12007. [3] J.R. Schwartz, Zinc Pyrithione: A Topical Antimicrobial with Complex Pharmaceutics. J. Drugs Dermatol.,

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[12] R.R. Warner, J.R. Schwartz, Y. Boissy, T.L. Dawson, Dandruff has an altered stratum corneum ultrastructure that is improved with zinc pyrithione shampoo. J. Am. Acad. Dermatol., 45 (2001) 897-903.

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List of abbreviations

AD Anti-dandruff APCI Atmospheric pressure chemical ionization CA (+)-catechin (chapter 4)

Cyanoacrylate (chapter 7) CBZ Climbazole CD Cyclodextrin CG (+)-catechin gallate CID Collision induced dissociation CLS Classical least square CSSS Cyanoacrylate skin surface stripping DPS 2, 2-Dipyridyl disulfide EC (-)-epicatechin ECG (-)-epicatechin gallate EGC (-)-epigallocatechin EGCG (-)-epigallocatechin gallate ESI Electro spray ionization ESI-MS Electrospray ionization mass spectrometry FICs Follicular infundibulum casts GC Gas chromatography

(+)-gallocatechin (in chapter 4) GCB Graphitized carbon black GCG (+)-gallocatechin gallate GC/MS Gas chromatography-mass spectrometry GPC Gel permeation chromatography HPC Home and personal care HPLC High performance liquid chromatography IR Infra-red LC Liquid chromatography LC/MS/MS Liquid chromatography-tandem mass spectrometry LOD Limit of detection LOQ Limit of quantification MALDI MSI Matrix-assisted laser-desorption ionization mass

spectrometry imaging MRLs Maximum residue limits MRM Multiple reaction monitoring MS Mass spectrometry NMR Nuclear magnetic resonance spectroscopy OCT Optical coherence tomography PBS Phosphate buffered saline

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PDA Photodiode array PSA Primary secondary amine QuEChERS Quick, easy, cheap, effective, rugged and safe RACs Raw agriculture commodities RCM Reflectance confocal microscopy R&D Research and development RSD Relative standard deviation RTD Ready-to-drink SEM Scanning electron microscopy SEM/EDX Scanning electron microscopy/energy dispersive X-ray

spectroscopy SCs Surface casts S/N Signal-to-noise SPE Solid phase extraction SRS Stimulated Raman scattering TP Total polyphenols TEM Transmission electron microscopy TWHS Total weighted head score UHPLC-MS/MS Ultra-high-performance liquid chromatography-tandem

mass spectrometry UHPLC-UV Ultra-high-performance liquid chromatography-UV XRF X-ray fluorescence spectroscopy ZnCO3 Zinc carbonate ZPT Zinc pyrithione

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Summary

Analytical chemistry plays a critical role in many fields ranging from fundamental research

to life sciences, industrial analysis and social applications. The aim of this thesis is to develop

new analytical methods to meet the ever-increasing need for safety and quality control,

performance evaluation, claim substantiation and mode-of-action understanding in the area

of consumer products for foods, and home and personal care. In Chapter 1, the needs and

challenges of analytical sciences in the industry of foods and HPC are summarized. The most

widely used analytical techniques in these product fields are introduced. Finally, the scope of

this thesis is discussed with a brief introduction of the subsequent chapters.

In Chapter 2, a multi-residue method was developed for rapid determination of pesticide

residues in tea by UHPLC-MS/MS. An adapted QuEChERS method was used for sample

preparation. In order to minimize the matrix effects from tea, an SPE cartridge layered with

graphite carbon/aminopropylsilanized silica gel was applied as complementary to the original

QuEChERS method. Representative matrix-matched calibration curves were applied for

quantification to compensate for matrix effects. Limits of quantification varied for the

different pesticides. Except for dichlorvos, that has a quantification limit of 0.02 mg/kg, all

others can be measured at 0.01 mg/kg level or better in a 5 g tea sample. Recoveries ranged

from 70% to 120% and the method RSD met the European Union Quality Control guideline.

Efficiency and reliability of this method were investigated by the analysis of both fermented

and unfermented Chinese tea samples. The method has further application opportunities,

including the analysis of e.g. dried vegetables and herb extracts.

In Chapter 3, a UHPLC-UV method combined with SPE sample pre-treatment was

developed and validated for the rapid quantification of L-theanine in ready-to-drink (RTD)

teas. UHPLC-UV analysis of twenty-seven RTD teas from the Chinese market revealed that

the L-theanine levels in various types of RTD teas were significantly different. RTD green

teas were found to contain the highest mean L-theanine level (37.85 ± 20.54 mg/L), followed

by jasmine teas (36.60 ± 12.08 mg/L), Tieguanying teas (18.54 ± 3.46 mg/L) black teas (16.89

± 6.56), Pu-erh teas (11.31 ± 0.90 mg/L) and Oolong teas (3.85 ± 2.27 mg/L). The ratio of the

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total polyphenols to L-theanine content could be used as a characteristic parameter for

differentiating RTD teas. L-theanine in RTD teas could be a reliable quality parameter that is

complementary to total polyphenols.

In Chapter 4, the noncovalent interaction between β-CD and EGCG was studied at the

molecular level by ESI-MS and NMR. Inclusion complexation of β-CD and green tea

catechins was observed by ESI-MS. The stoichiometry of the β-CD-EGCG complex was

determined using Job’s method, which showed a maximum at 0.5, indicating a 1:1

stoichiometry of the β-CD-EGCG complex. NMR experiments indicated that inclusion

complexes of β-CD and EGCG were formed and that the D ring or B ring of the EGCG

molecule penetrated into the β-CD cavity. This molecular encapsulation could prevent the

gallate moiety from binding to the human taste receptors, in that way reducing the bitter,

astringent taste of EGCG. The direct observation of non-covalent interactions makes the

combined deployment of ESI-MS and NMR a valuable chemical vehicle for fast screening of

molecular maskers for reducing bitterness and astringency of green tea catechins as an

alternative to a tasting panel.

In Chapter 5, a sensitive and specific UHPLC-MS/MS method was developed and validated

for the measurement of climbazole (CBZ) deposition from hair care products onto artificial

skin and human scalp. Deuterated CBZ was used as the internal standard. APCI in positive

mode was applied for the detection of CBZ. For quantification, MRM transition 293.0 > 69.0

was monitored for CBZ, and MRM transition 296.0 > 225.1 for the deuterated CBZ. The linear

range ran from 4 to 2000 ng/mL. The LOD and the LOQ were 1 ng/mL and 4 ng/mL,

respectively, which enabled quantification of CBZ on artificial skin and human scalp at ppb

level (corresponding to 16 ng/cm2). For the sampling of CBZ from human scalp the buffer

scrub method using a surfactant-modified PBS solution was selected based on a performance

comparison of tape stripping, the buffer scrub method and solvent extraction in in vitro studies.

Using this method, CBZ deposition in in vitro and in vivo studies was successfully quantified.

In Chapter 6, a sensitive UHPLC-MS/MS method has been developed and validated for

simultaneous quantification of Zinc pyrithione (ZPT) and CBZ deposited onto human scalp

from AD shampoos. Scrubbing with a buffer solution was used as the sampling method for

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the extraction of ZPT and CBZ from scalp. Derivatization of ZPT was carried out prior to

UHPLC-MS/MS analysis. The identification of ZPT and CBZ was performed by examining

ratios of selected MRM transitions in combination with UHPLC retention times. The limit of

detection for ZPT and CBZ was established to be 1 and 2 ng/mL, respectively. This sensitivity

enables the quantification of ZPT and CBZ at deposition levels in the low ng/cm2 range. The

method was successfully applied for the analysis of scalp buffer scrub samples from an in

vivo study. The levels of ZPT and CBZ remaining on the scalp at different time intervals after

application of the AD shampoo were measured. The results revealed that dual-active AD

shampoo delivered more ZPT onto the scalp in a single wash than a single active shampoo

did. The amount of ZPT and CBZ remained on the scalp after AD shampoo application

declined over 72 hours. The method is also applicable in other studies, e.g. in artificial skin

studies to improve shampoo formulations to maximize ZPT and CBZ deposition.

In Chapter 7, a method involving scalp cyanoacrylate biopsy sampling, a tailor-made cutting

device, UHPLC-MS/MS analysis, SEM measurement and Raman imaging are described for

the measurement of delivery of ZPT and CBZ from an AD shampoo into the scalp follicular

infundibulum. Scalp cyanoacrylate biopsy enables the sampling of ZPT and CBZ delivered

into the scalp follicular infundibulum as well as onto the scalp surface. Raman imaging of

scalp cyanoacrylate biopsy samples allows the visualization of the spatial distribution of ZPT

and CBZ deposited on the scalp. A tailor-made cutting device enables the separation of the

scalp follicular infundibulum sample (20 µm below the scalp surface) from the scalp surface

samples (including the top 20 μm of the infundibula). UHPLC-MS/MS was used as a sensitive

and specific methodology enabling the quantification of ZPT and CBZ without interferences.

Using this method, ZPT and CBZ delivered into the scalp follicular infundibulum from the

dual-active AD shampoo was successfully visualized and quantified. Due to the lipophilic

nature of CBZ and hence the increased solubility in sebum, CBZ has the ability to penetrate

further into the sebum-rich infundibulum whereas ZPT remains within the upper 20 µm of

infundibula. This differential distribution of actives allows for the effective targeting of

Malassezia species throughout the depth of the scalp follicular infundibulum.

Finally, Chapter 8 proposes an ex vivo method that combines tape strip sampling and

scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDX) for

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measuring and visualizing the particle size, morphology and composition of ZPT deposited

onto the scalp from an AD shampoo containing ZPT and zinc carbonate. Hair was washed

with a commercially available AD shampoo containing ZPT and zinc carbonate (ZnCO3).

Tape strips were applied to collect the deposited particles from the scalp after AD shampoo

application and rinse-off. The scalp tape strip samples were subjected to scanning SEM/EDX

measurement. The morphology of the ZPT particles was visualized by SEM imaging and

identification of ZPT particles was confirmed by EDX analysis. For the commercial shampoo

studied it was observed that two types of zinc-containing particles with different

morphologies and composition remained on the scalp after shampoo application and rinse-off.

As indicated by the EDX spectra, the ZPT particles deposited onto the scalp surface had

polygonal crystal structures. ZnCO3 was also deposited onto the scalp surface. This material

was mainly present as aggregated particles. This ex vivo measurement method provides higher

imaging resolution and more chemical specificity than reflectance confocal microscopy. To

the best of our knowledge, this is the first time that ZPT particles could be distinguished from

other zinc-containing particles deposited onto the scalp. The new method allows the

microstructures of both ZPT and other zinc particles on the scalp to be imaged.

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Samenvatting

Analytische chemie speelt een cruciale rol op vele terreinen. De discipline wordt veelvuldig

ingezet in gebieden variërend van fundamenteel onderzoek in de levenswetenschappen tot

industriële analyse en sociale toepassingen. Het doel van dit proefschrift is om nieuwe

analysemethoden te ontwikkelen om tegemoet te komen aan de steeds toenemende behoefte

aan veiligheids- en kwaliteitscontrole, prestatie-evaluatie, claim-onderbouwing en de opbouw

van kennis van werkingsmechanismen op het gebied van consumentenproducten voor

voeding, en huishoudelijke- of persoonlijke verzorging. In Hoofdstuk 1 worden de behoeften

en uitdagingen van de analytische chemie in de voedingsmiddelenindustrie en bij de

ontwikkeling en productie van huishoudelijke- en persoonlijke verzorgingsproducten (HPC)

samengevat. De meest gebruikte analytische technieken in deze toepassingsgebieden worden

kort besproken. Ten slotte wordt de scope van dit proefschrift besproken met een korte

introductie van elk van de volgende hoofdstukken.

In Hoofdstuk 2 werd een multi-residu methode ontwikkeld voor snelle bepaling van

bestrijdingsmiddelen en residuen daarvan in thee met behulp van UHPLC-MS/MS. Een

aangepaste QuEChERS-methode werd gebruikt voor monstervoorbewerking. Om de

matrixeffecten van thee te minimaliseren, werd een SPE-cartridge gevuld met een

combinatiebed van grafiet-koolstof en aminopropyl gemodificeerde silicagel gebruikt als

aanvulling op de SPE stap in de originele QuEChERS-methode. Representatieve matrix-

gecorrigeerde kalibratiecurves werden gebruikt voor kwantificering om te compenseren voor

matrixeffecten. De kwantificeringslimieten varieerden voor de verschillende

bestrijdingsmiddelen. Met uitzondering van dichloorvos, waarvoor een kwantificeringslimiet

van 0,02 mg/kg gevonden werd, kunnen alle andere componenten gemeten worden op 0,01

mg/kg niveau of beter, in een 5 g thee monster. De recoveries varieerden van 70% tot 120%

en de RSD van de methode voldeed aan de kwaliteitsrichtlijnen van de Europese Unie. De

efficiëntie en betrouwbaarheid van de nieuw ontwikkelde methode werd onderzocht door de

analyse van zowel gefermenteerde, als niet-gefermenteerde Chinese thee monsters. De

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werkwijze heeft verdere toepassingsmogelijkheden, bijvoorbeeld in de analyse van

gedroogde groenten of kruidenextracten.

In Hoofdstuk 3 werd een UHPLC-UV-methode met SPE-monstervoorbehandeling

ontwikkeld en gevalideerd voor de snelle bepaling van L-theanine in ‘ready to drink’ (RTD)

thee. UHPLC-UV-analyse van zevenentwintig Chinese RTD-thee monsters gaven significant

verschillende L-theanine gehalten te zien in de diverse soorten RTD-thee. Groene RTD-thee

monsters bevatten het hoogste gemiddelde L-theanineniveau (37,85 ± 20,54 mg/L), gevolgd

door jasmijnthee (36,60 ± 12,08 mg/L), Tieguanying thee (18,54 ± 3,46 mg / L), zwarte thee

(16,89) ± 6,56), Pu erh thee (11,31 ± 0,90 mg / L) en Oolong-thee (3,85 ± 2,27 mg / L). De

verhouding van het totale gehalte aan polyfenolen versus L-theanine zou kunnen worden

gebruikt als een kenmerkende parameter voor het onderscheiden van de diverse RTD-

theesoorten. L-theanine in RTD-thee kan een betrouwbare kwaliteitsparameter zijn die

complementair is aan het gehalte totaal polyphenolen.

In Hoofdstuk 4 werd de niet-covalente interactie tussen β-cyclodextrine (β-CD) en

epigallocatechinegallaat (EGCG) op moleculair niveau bestudeerd met behulp van ESI-MS

en NMR. Inclusiecomplexatie van β-CD en groene thee catechines kon direct bestudeerd

worden met ESI-MS. De stoichiometrie van het β-CD/EGCG complex werd bepaald met

behulp van de Job’s-methode, die een maximum bij 0,5 vertoonde, hetgeen duidt op een 1:1

stoichiometrie van het β-CD/EGCG complex. NMR-experimenten bevestigden dat

inclusiecomplexen van β-CD en EGCG werden gevormd en dat de D-ring of B-ring van het

EGCG molecuul in de holte van het β-CD molecuul kon binnendringen. Deze moleculaire

inkapseling zou kunnen voorkomen dat de gallaten aan menselijke smaakreceptoren binden,

waardoor de bittere smaak van EGCG wordt verminderd. De directe waarneming van niet-

covalente interacties maakt de combinatie van ESI-MS en NMR een waardevol methode voor

snelle screening van moleculaire methoden voor het maskeren van bitterheid en astringentie

van groene thee catechines als een alternatief voor een menselijk testpanel.

In Hoofdstuk 5 werd een gevoelige en specifieke UHPLC-MS/MS-methode ontwikkeld en

gevalideerd voor het meten van de depositie van climbazole (CBZ) uit

haarverzorgingsproducten zoals anti-roos shampoos op kunsthuid en op echte hoofdhuid.

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APCI in positieve mode werd toegepast voor de detectie van CBZ. Gedeutereerd CBZ werd

gebruikt als de interne standaard. Voor kwantificering werd de MRM-overgang 293,0> 69,0

gevolgd voor CBZ, en de MRM-overgang 296,0> 225,1 voor het gedeutereerde CBZ. Het

lineaire bereik van de methode liep van 4 tot 2000 ng/mL. De LOD en de LOQ waren

respectievelijk 1 ng/mL en 4 ng/mL, wat kwantificering van CBZ op kunsthuid en op de

menselijke hoofdhuid op ppb-niveau mogelijk maakte (overeenkomend met 16 ng/cm2). Voor

de bemonstering van CBZ op de menselijke hoofdhuid werd de ’buffer scrub’-methode

gevolgd waarbij een PBS-oplossing met daarin opgenomen een oppervlakte actieve stof

gebruikt werd. Deze methode was gekozen op basis van een vergelijking met het strippen met

een plakstrip, de ‘buffer scrub’-methode en vloeistofextractie in in vitro studies. Met behulp

van deze methode werd de CBZ-depositie in in vitro en in vivo studies met succes gemeten.

Hoofdstuk 6 beschrijft de ontwikkeling en validatie van een gevoelige UHPLC-MS/MS-

methode voor de gelijktijdige kwantificering van zinkpyrithione (ZPT) en CBZ deposities op

menselijke hoofdhuid vanuit anti-roos shampoos. Wassen met een bufferoplossing werd

gebruikt als de bemonsteringsmethode voor de extractie van ZPT en CBZ van de hoofdhuid.

ZPT werd gederivatiseerd voorafgaand aan de UHPLC-MS/MS-analyse. De identificatie van

ZPT en CBZ werd uitgevoerd op basis van verhoudingen van geselecteerde MRM-

overgangen in combinatie met UHPLC retentietijden. De detectiegrens voor ZPT en CBZ

werd vastgesteld op respectievelijk 1 en 2 ng/mL. Deze gevoeligheid maakt de kwantificering

van ZPT en CBZ mogelijk op depositieniveaus in het lage ng/cm2 bereik. De methode werd

met succes toegepast voor de analyse van monsters uit een in vivo onderzoek. De

hoeveelheden van ZPT en CBZ die op de hoofdhuid achtergebleven waren op verschillende

tijden na toepassing van de anti-roos shampoo werden gemeten. De resultaten toonden aan

dat ‘dual-active’ anti-roos shampoo meer ZPT op de hoofdhuid afleverde in een enkele

wasbeurt dan een anti-roos shampoo met slechts één actief bestanddeel. De hoeveelheid ZPT

en CBZ die op de hoofdhuid terug gevonden werd na toepassing van de anti-roos shampoo

daalde in 72 uur naar nul. De methode is ook toepasbaar in andere studies, b.v. in studies ter

verbetering van shampooformuleringen met als doel de ZPT- en CBZ-depositie te

maximaliseren.

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In Hoofdstuk 7 wordt een methode beschreven voor het nemen van biopsiemonsters van de

hoofdhuid. Verder wordt een nieuwe snij-inrichting voor het snijden van zeer dunne plakjes,

een UHPLC-MS/MS-analyse methode, SEM-meting en tot slot een methode voor Raman-

beeldvorming voor het meten van de overdracht van ZPT en CBZ uit een anti-roos shampoo

naar het zogenaamde folliculaire infundibulum (het haarfollikel), van de hoofdhuid

beschreven. Toepassing van de cyanoacrylaat biopt methode maakt het mogelijk de opname

van ZPT en CBZ in het folliculaire infundibulum van de hoofdhuid en depositie op het

hoofdhuidoppervlak separaat te bestuderen. Met Raman-beeldvorming van de cyanoacrylaat

biopsiemonsters kunnen vervolgens de ruimtelijke verdeling van ZPT en CBZ op- en in de

hoofdhuid gevisualiseerd worden. Met behulp van een nieuw ontworpen snij-mechanisme

kunnen separaat monsters van het folliculaire infundibulum (20 μm onder het

hoofdhuidoppervlak) en van de totale hoofdhuid (inclusief de bovenste 20 μm van de

infundibula) gemaakt worden. UHPLC-MS/MS werd gebruikt als een gevoelige en specifieke

methodologie die de kwantificering van ZPT en CBZ zonder interferenties mogelijk maakt.

Met behulp van de hier beschreven methoden werd de opname van ZPT en CBZ in het

folliculaire infundibulum van de hoofdhuid vanuit een ‘dual-active’ anti-roos shampoo met

succes gevisualiseerd en gekwantificeerd. Door het lipofiele karakter van CBZ, en daardoor

de verhoogde oplosbaarheid in talg, kan CBZ verder doordringen in het talgrijke

infundibulum terwijl ZPT in de bovenste 20 μm van het infundibulum blijft. Dit verschil in

verdeling van actieve stoffen zorgt voor een effectieve aanpak van Malassezia bacteriën

dieper in het folliculaire infundibulum van de hoofdhuid.

Tot slot wordt in Hoofdstuk 8 een ex-vivo methode voorgesteld die de plakstrip-bemonstering

en elektronenmicroscopie/energiedispersieve röntgenspectroscopie (SEM/EDX) combineert

voor het meten en visualiseren van de deeltjesgrootte, morfologie en samenstelling van ZPT

op de hoofdhuid na wassen met een anti-roos shampoo met ZPT en zinkcarbonaat. Het haar

werd gewassen met een in de handel verkrijgbare anti-roos shampoo die ZPT en

zinkcarbonaat (ZnCO3) bevatte. Plakstrips werden toegepast om de neergeslagen deeltjes van

de hoofdhuid te verzamelen na het wassen van het haar met de anti-roos shampoo en

uitspoelen. De plakstrip met daarop de hoofdhuidmonsters werden bestudeerd met scanning

SEM/EDX-meting. De morfologie van de ZPT-deeltjes werd gevisualiseerd door SEM-

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beeldvorming en verdere identificatie van de ZPT-deeltjes werd bevestigd door EDX-analyse.

Voor de bestudeerde commerciële shampoo werd waargenomen dat twee soorten

zinkbevattende deeltjes met verschillende morfologieën en samenstelling op de hoofdhuid

achterbleven na het toepassen en uitspoelen van de shampoo. Zoals aangegeven door de EDX-

spectra, hadden de ZPT-deeltjes afgezet op het hoofdhuidoppervlak veelhoekige

kristalstructuren. ZnCO3 werd ook op het hoofdhuidoppervlak afgezet. Dit materiaal was

voornamelijk aanwezig als geaggregeerde deeltjes. Deze ex-vivo meetmethode biedt een

hogere beeldresolutie en meer chemische specificiteit dan reflectie confocale microscopie.

Voor zover ons bekend, is dit de eerste keer dat ZPT-deeltjes kunnen worden onderscheiden

van andere zinkbevattende deeltjes achtergebleven op de hoofdhuid. Met de nieuwe methode

kunnen de microstructuren van zowel ZPT als andere zinkdeeltjes op de hoofdhuid bepaald

worden.

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总结

分析化学在基础研究、生命科学、工业分析和社会应用等领域都发挥着重要作用。本

文的目的是开发新的分析方法和技术，以满足食品、家庭和个人护理领域中各种新产

品开发需求，包括安全和质量控制、功能性评估、功效证明、机理理解等。第一章概

述了分析科学在食品、家庭和个人护理行业的需求和挑战，介绍了该领域中应用最广

泛的分析技术。然后，简要介绍了本论文的研究范围。

第二章介绍了一个基于超高效液相色谱和串联质谱联用的分析方法，用于快速分析茶

叶中农药残留。为了最小化茶叶的基体效应，作者使用石墨碳/氨基丙基硅烷化硅胶固

相萃取小柱结合 QuEChERS 方法的样品前处理技术，有效降低了基质干扰。另外，采

用了基质匹配的标准溶液，使得定量结果更加准确。除敌敌畏的定量限为 0.02 mg/kg，

其他所有农残的定量限皆在 0.01 mg/kg 水平或更高灵敏度。该方法的回收率在 70%-

120%之间，相对标准偏差符合欧盟质量控制准则。通过分析发酵茶和未发酵茶样品中

的农残，验证了该方法的有效性和可靠性。并且，该方法具有进一步应用的机会，比

如分析干蔬菜或草本提取物中的农残。

在第三章中，作者建立了一种固相萃取为预处理和超高效液相色谱紫外光检测器相结

合的分析方法，用于快速测定即饮茶中茶氨酸的含量。采用该方法，对中国市场上的

27 种即饮茶中茶氨酸进行了定量分析。结果表明，不同类型即饮茶中的茶氨酸水平存

在显著差异。即饮绿茶中茶氨酸含量最高(37.85 ± 20.54 mg/L),其次为即饮茉莉花茶

(36.60 ± 12.08 mg/L)、即饮铁即饮观音茶(18.54 ± 3.46 mg/L)、即饮黑茶(16.89 ± 6.56

mg/L)、即饮普洱茶(11.31 ± 0.90 mg/L)和即饮乌龙茶(3.85 ± 2.27 mg/L)。作者发现，总

多酚与茶氨酸含量的比值可以作为鉴别即饮茶的特征参数。并且，茶氨酸的含量可以

作为一种与总多酚互补的可靠质量参数。

第四章介绍了作者利用电喷雾质谱和核磁共振从分子水平上，研究了 β 环糊精（β-

CD）与表没食子儿茶素没食子酸酯（EGCG）的非共价相互作用。β-CD 与绿茶儿茶素

133

在水溶液中的相互作用可以直接被电喷雾质谱观察到。用 Job 法测定了 β-CD 和 EGCG

复合物的化学计量比，在 0.5 处达到最大值，表明 β-CD 和 EGCG 复合物的化学计量

比为 1:1。核磁共振实验表明，β-CD 与 EGCG 形成包合物，EGCG 分子的 D 环或 B 环

进入 β-CD 空腔。这种分子包埋可以防止没食子酸酯部分与味觉受体结合，从而减少

EGCG 的苦涩味道。采用电喷雾质谱和核磁共振研究分子间的非共价相互作用，可以

成为快速筛选分子掩蔽剂用于降低绿茶儿茶素的苦味和收敛性，并作为感官评测的替

代方案。

第五章，作者建立了一种灵敏的超高效液相色谱和串联质谱联用的分析方法，用于检

测使用洗发水后，在人工假皮和人的头皮上沉积的甘宝素。为了能有效的从人的头皮

上采集沉积的甘宝素，不同采样方法包括胶带剥离、缓冲液洗涤和溶剂萃取，在人工

假皮上进行的测试和比较，最后选定表面活性剂改良的磷酸盐缓冲液。大气压化学电

离阳离子模式用于甘宝素的检测。定量时，氘代甘宝素被用做内标。线性范围从 4 到

2000 ng/mL。检测限和定量限分别为 1 ng/mL 和 4 ng/mL，从而使得人工假皮和人的头

皮上，低至 16 ng/cm2的甘宝素都能被定量。使用这种方法，在人的头皮上沉积的甘宝

素成功的提取到和定量。

第六章，作者建立了一种灵敏的超高效液相色谱和串联质谱联用的分析方法，用于同

时测定使用去屑洗发香波后，沉积在人的头皮上的吡硫翁锌和甘宝素。采用表面活性

剂改良的磷酸盐缓冲液擦洗作为提取头皮中吡硫翁锌和甘宝素的采样方法。为了提高

灵敏度和稳定性，在仪器分析之前，吡硫翁锌被进行了衍生化。吡硫翁锌和甘宝素的

检测限分别为 1 和 2 ng/mL。这样的种灵敏度使得头皮上 ng/cm2范围内的吡硫翁锌和

甘宝素也能够被检测和定量。该方法已成功地应用于临床样品分析。测定了试用去屑

洗发水后，不同时间点，头皮上的吡硫翁锌和甘宝素的含量。结果表明，在一次洗涤

后，双活性去屑洗发香波比单活性去屑洗发香波在头皮上沉积更多的吡硫翁锌。使用

去屑洗发水后，头皮上残留的吡硫翁锌和甘宝素量下降随时间下降。该方法可应用于

测定使用其它个人护理产品后，皮肤上沉积的活性功效成分。

134

在第七章中，作者描述了一种方法用于测量使用去屑洗发水后，沉积在头皮毛囊漏斗

中的的吡硫翁锌和甘宝素。该方法包含了头皮氰基丙烯酸盐粘合剂活检取样（CA

biopsy）、特制的切割装置、超高效液相色谱和串联质谱联用分析、扫描电镜测量和拉

曼成像的方法。头皮 CA biopsy 能够取到头皮毛囊漏斗中以及头皮表面的吡硫翁锌和

甘宝素。对 CA biopsy 样品的拉曼成像可以显示头皮上的吡硫翁锌和甘宝素的空间分

布。一种特制的切割装置能够将头皮毛囊漏斗样品（在头皮表面下 20 微米）与头皮表

面样品（包括漏斗的顶部 20 微米）分离。超高效液相色谱和串联质谱联用分析是一种

灵敏、特异的方法，能够不受干扰地定量吡硫翁锌和甘宝素。该方法成功地应用于临

床样品分析，在使用双活性去屑洗发水后，输送到头皮毛囊漏斗中的吡硫翁锌和甘宝

素被测定。由于甘宝素的亲脂性质和随后在皮脂中的高溶解度，甘宝素具有快速扩散

到富含皮脂的毛囊漏斗区的能力，并进到更深处。而吡硫翁锌则主要保留在漏斗的上

部 20 微米区域内。这种差异性的分布使得甘宝素对于处在头皮毛囊深处的马拉色菌有

更好的抑制效果。

最后的第八章中，作者提出了一种方法用于测量使用含有吡硫翁锌和其它锌盐的去屑

香波后，沉积到头皮上的含锌颗粒，并对相关颗粒的形态和元素组成进行分析。该方

法包括胶带取样与电子显微镜/ X 射线能谱检测。在使用市售的含有吡硫翁锌和碳酸锌

去屑洗发水后，用胶带从头皮上收集沉积的颗粒物。对头皮胶带样品进行扫描电镜成

像，观察了吡硫翁锌颗粒的形貌，并通过元素分析证实了吡硫翁锌颗粒的鉴定。对于

所研究的市售洗发水，观察到两种不同形态和组成的含锌颗粒。元素分析表明，沉积

在头皮表面的吡硫翁锌颗粒具有多边形晶体结构。碳酸锌也被沉积在头皮表面上，这

种材料主要以聚集颗粒的形式存在。该方法提供了比反射共聚焦显微镜更高的成像分

辨率和更高的化学特异性。据我们所知，这是首次将沉积在头皮上的吡硫翁锌颗粒与

其他含锌颗粒有效区分开来。并且该方法允许对头皮上的吡硫翁锌和其他锌颗粒的微

观结构进行成像分析。

135

List of Publications

- van der Pijl, P.C., Chen, L., Mulder, T.P.J. (2010). Human disposition of L-theanine intea or aqueous solution. Journal of Functional Foods, 2, pp. 239-244.

- Chen, G., Cao, P., Liu, R. (2011). A multi-residue method for fast determination ofpesticides in tea by ultra performance liquid chromatography-electrospray tandem massspectrometry combined with modified QuEChERS sample preparation procedure. FoodChemistry, 125(4), pp. 1406-1411.

- Chen, G., Wang, Y., Song, W., Zhao, B., Dou, Y. (2012). Rapid and selectivequantification of l-theanine in ready-to-drink teas from Chinese market using SPE andUPLC-UV. Food Chemistry, 135(2), pp. 402-407.

- Chen, G., Hoptroff, M., Fei, X., Su, Y., Janssen, H.-G. (2013). Ultra-high-performanceliquid chromatography-tandem mass spectrometry measurement of climbazoledeposition from hair care products onto artificial skin and human scalp. Journal ofChromatography A, 1317, pp. 155-158.

- Chen, G., Miao, M., Hoptroff, M., Fei, X., Collins, L.Z., Jones, A., Janssen, H.-G. (2015).Sensitive and simultaneous quantification of zinc pyrithione and climbazole depositionfrom anti-dandruff shampoos onto human scalp. Journal of Chromatography B:Analytical Technologies in the Biomedical and Life Sciences, 1003, pp. 22-26.

- Wu, Y., Chen, G., Ji, C., Hoptroff, M., Jones, A., Collins, L.Z., Janssen, H.-G. (2016).Gas chromatography-mass spectrometry and Raman imaging measurement of squalenecontent and distribution in human hair. Analytical and Bioanalytical Chemistry, 408(9),pp. 2357-2362.

- Chen, G., Ji, C., Miao, M., Yang, K., Luo, Y., Hoptroff, M., Collins, L.Z., Janssen, H.-G. (2017). Ex-vivo measurement of scalp follicular infundibulum delivery of zincpyrithione and climbazole from an anti-dandruff shampoo. Journal of Pharmaceuticaland Biomedical Analysis, 143, pp. 26-31.

- Chen, G., Ji, C., Collins, L.Z., Hoptroff, M., Janssen, H.-G. (2018) Visualization of zincpyrithione particles deposited on the scalp from a shampoo by tape strip sampling andscanning electron microscopy/energy dispersive X-ray spectroscopy measurement.International Journal of Cosmetic Science, 40, pp.530-533.

- Chen, G., Janssen, H.-G. The use of ESI-MS & 2D NMR to reveal the reduction ofbitterness and astringency of EGCG by β-CD inclusive complexation. Food ResearchInternational, manuscript submitted.

136

Overview of author’s contributions

Chapter 1: General introduction

Guoqiang Chen: wrote the manuscript.

Hans-Gerd Janssen: reviewed the manuscript and gave suggestions for improvement.

Chapter 2: A multi-residue method for fast determination of pesticides in tea Guoqiang Chen: developed the ideas and experimental set-up, conducted the method

development and validation, interpreted the data and wrote the manuscript.

Pengying Cao: performed the sample treatment and instrumental analysis.

Renjiang Liu: gave suggestions for the optimization of the sample treatment procedure.


Chapter 3: Rapid and selective quantification of L-theanine in ready-to-drink

teas from Chinese market Guoqiang Chen: developed the idea and experiment design, conducted the method

development, optimisation and validation, interpreted the data and wrote the manuscript.

Yun Wang: performed the sample treatment and UHPLC analyses for part of the samples.

Weiqi Song: performed the sample preparation and UHPLC analyses for part of the

samples.

Bo Zhao: contributed to the experiment design and method validation.

Yuling Dou: contributed to the experiment design and method validation.


Chapter 4: A method for measuring the noncovalent interaction between

EGCG and β-CD Guoqiang Chen: developed the idea, designed the experimental plan, performed the

experiments, interpreted the results and wrote the manuscript.

Hans-Gerd Janssen: supervised the project, reviewed the manuscript and gave suggestions

for improvement.

137

Chapter 5: Quantification of climbazole deposition from shampoos onto

artificial skin and human scalp Guoqiang Chen: developed the idea and experimental set-up, conducted the method

validation, performed the sample analysis, interpreted the data and wrote the manuscript.

Michael Hoptroff: supervised the project, reviewed and revised the manuscript.

Xiaoqing Fei: contributed to the design of the in vivo study.

Ya Su: contributed to the design of the in vitro study.

Hans-Gerd Janssen: co-supervised the project, reviewed the manuscript and gave

suggestions for improvement.

Chapter 6: Sensitive and simultaneous quantification of zinc pyrithione and

climbazole in scalp buffer scrub samples Guoqing Chen: developed the idea and experimental set-up, conducted the method

validation, interpreted the data and wrote the manuscript.

Maio Miao: co-developed the idea, performed the sample analysis.

Michael Hoptroff: co-supervised the project, reviewed and revised the manuscript.

Xiaoqing Fei: contributed to the design of the in vivo study.

Luisa Z. Collins: co-supervised the project, reviewed the manuscript and gave suggestions

for improvement.

Andrew Jones: co-supervised the project, reviewed the manuscript and gave suggestions for

improvement.


for improvement.

138

Chapter 7: Ex-vivo measurement of scalp follicular delivery of zinc pyrithione

and climbazole from hair care products Guoqiang Chen: developed the idea, developed the experimental plan, interpreted the data

and wrote the manuscript.

Chengdong Ji: performed the Raman imaging analyses.

Miao Miao: performed the quantitative analysis of zinc pyrithione and climbazole by

UHPLC-MS/MS.

Kang Yang: co-developed the idea of the cutting device.

Yajun Luo: contributed to the design of in vivo study.

Michael Hoptroff: co-supervised the project, reviewed the manuscript and gave suggestions

for improvement.


for improvement.


for improvement.

Chapter 8: Visualization of zinc pyrithione particles deposited on the scalp

from hair care products Guoqiang Chen: developed the idea, compiled the experimental plan, interpreted the data

and wrote the manuscript.

Chengdong Ji: co-developed the idea, performed the SEM measurement


for improvement.

Michael Hoptroff: reviewed the manuscript and gave suggestions for improvement.


for improvement.

139

Acknowledgements

Throughout the whole procedure of my PhD study and the writing of this thesis, I have

received a great deal of assistance and support.

First and foremost, I would like to express my very great appreciation to my promotor,

Professor Hans-Gerd Janssen who gave me the invaluable opportunity to pursue the PhD

degree. His patient guidance, enthusiastic encouragement, constructive suggestions and

expertise are indispensable to my PhD study and thesis preparation.

I am particularly grateful to my co-promotor, Professor Peter Schoenmakers whose kind

support is critical for my application for the PhD study and the preparation of my defense.

The board of Unilever Research Shanghai, U66, in particular Mason Wang, Jin-Fang Wang

and Manfred Aben, I kindly acknowledge for giving me the opportunity to perform this

exciting research and allowing me to perform this PhD study.

I would also like to extend my thanks to my (former) colleagues in Unilever: Jin-Fang Wang

as my current line manage for his great mentoring, support and aid; Axel Ekani and Yumo

Zhang as my former line managers for their kind support and enthusiastic encouragement;

Renjiang Liu and Pengying Cao for their contributions to Chapter 2; Yun Wang, Yuling Dou,

and Weiqi Song for their contributions to Chapter 3; Bo Zhao for his contributions to Chapter

3 & 4; Michael Hoptroff for his contributions to Chapter 5-8; Xiaoqing Fei and Ya Su for

their contributions to Chapter 5; Andrew Jones for his contributions to Chapter 6; Luisa

Collins for contributions to Chapter 6-8; Chengdong Ji for his contributions to Chapter 7 &

8; Miao Miao for her contributions to Chapter 6 & 7; Yajun Luo and Kang Yang for their

contributions to Chapter 7.

Lastly but not least, I would like to thank my family for their unconditional support.

感谢所有帮助过我的人；感恩父母的养育；感激老婆的全力支持，尤其是为我的论文

设计了封面；希望我的博士学习对女儿的成长有所启发，活到老，学到老；我爱你们。

Combined analytical techniques for the analysis of complex consumer products and bio-samples

Guoqiang (Leon) Chen

Com

bined analytical techniques for the analysis of complex consum

er products and bio-samples

Guoqiang (Leon) C

hen

Invitation

For attending the public defence of the thesis

Combined analytical techniques for the analysis

of complex consumer products and bio-samples

On Wednesday 5th June 2019

at 14.00

In theAgnietenkapel,

Oudezijds Voorburgwal 229,Amsterdam

[email protected]

Paranymphs

Randy ZhaoBoudewijn Hollebrands

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