Journal Separation Science

Volume 1 / Issue 1

January 2009www.sepscience.com

Developing new LC-MS

Comparing characteristics of RP columns

Analytical approaches to food supplement analysisAnalytical approaches to food supplement analysis

methods

2 section name www.sepscience.com

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3contentsseparation science — volume 1 issue 1

contentsVolume 1 / Issue 1

January 2009www.sepscience.com

Developing new LC-MS

Comparing characteristics of RP columns

Analytical approaches to food supplement analysisAnalytical approaches to food supplement analysis

methods

Two analytical approaches to the evaluation of chondroitin sulfate in european food supplements

Nicola Volpi and Francessa Maccari

22

feature

Tl

separationdriving analytical chemistry forwardsscience

Volume 1 / Issue 1 January 2009

30

34

research round-up

Comparing separation characteristics of reversed-phase columns

The effect of pressure on small molecule retention using RP-UPLC

Rapid and direct determination of pesticides in water using anion-exchange chromatography with coulometric detection

Computer-assisted solution for multicomponent sample identification

SPE and GC–microEDC multiresidue analysis of royal jelly

Affinity partitioning of plasmid DNA with a zinc finger protein

FD-LC-MS/MS in breast cancer studies

Improved liquid chromatography — Online radioactivity detection for metabolite profiling

Fast GC–MS pesticide multiresidue analysis of apples

Sequential injection methodologies for environmental analyses

Simultaneous HPLC and GC analysis of lignans in Forsythia leaves

Extraction of amphetamines from urine using a monolithic silica disc-packed spin column and HPLC–diode array detection

Development of immobilized enzyme reactors for phase I drug metabolism studies

Bioactive compound fishing with DNA-based bioseparations and chemical analysis

MSPD extraction of carbadox and olaquindox in feed followed by hydrophilic interaction ultra-high-pressure liquid chromatographic analysis

Rr

Cd

Tu

chrom doctor In this months column, the chrom doctor examines best practice when developing new methods for LC-MS analysis.

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4 from the editor www.sepscience.com

New Year, new ideas…

David Hills

– Scientific Director

[email protected]

So it’s now 2009 and Separation Science is finally off and

running on a regular basis. Before going any further

I’d just like to thank all of you who took the time to

contact me with your thoughts on the launch issue,

whether that was in person at the ISC meeting in Münster

or subsequently by telephone or email. Thankfully, the vast

majority of these comments were positive and I’ve done my

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functionality – as well as including over 4,000 application

notes in PDF format, it is now searchable by multiple keywords

in both AND and OR logic.

the New Year why don’t you try it out.

Finally, I’d just like to pique your interest by mentioning our

upcoming scientific conference – Separation Science Singapore.

It will take place between 26-28 August this year at the state-

of-the art Biopolis Park in Singapore, and features an array of

high-quality speakers from around the world, as well as from

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pharma, enviro, bioscience and energy markets. I’ll bring you

more information on this exciting event in later issues but for

more of a teaser go to page 29.

Enjoy the issue, and as always if you have any questions or

comments just send me an email.

David Hills

[email protected]

scientific advisory

councilPeter Myers

– Chief Scientific Officer

[email protected]

David Barrow

University of Cardiff, UK

Zongwei Cai

Hong Kong Baptist University

Yi Chen

Chinese Academy of Sciences,

Beijing, China

Gert Desmet

Vrije Universiteit Brussel, Belgium

C. Bor Fuh

National Chi Nan University, Taiwan

Y.S. Fung

Hong Kong University

Xindu Geng

Northwest University, Xi’an, China

Luigi Mondello

University of Messina, Italy

Paul Haddad

University of Tasmania, Australia

Hian Kee Lee

National University of Singapore,

Singapore

Melissa Hanna-Brown

Pfizer, UK

Tuulia Hyötyläinen

University of Helsinki, Finland

Gongke Li

Sun Yat-Sen University, Guangzhou,

China

Yong-Chien Ling

National Tsing Hua University,

Taiwan

Klara Valko,

GSK, UK

Jean-Luc Veuthey

University of Geneva, Switzerland

Claudio Villani

Universita’ degli Studi di Roma “La

Sapienza”, Italy

Cheing- Tong Yan

Center of Environmental Safety and

Hygene, Taiwan

Edward Browne

GSK, Singapore

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5separation science — volume 1 issue 1

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Comment

USASilica-based chemically bonded stationary

phases continue to dominate the practice of

reversed-phase liquid chromatography, in spite

of efforts to replace them with polymeric or

other inorganic oxide-based materials.

Dr Colin Poole from the Department of

Chemistry at Wayne State University, USA,

looked at the differences in the system

constants of the solvation parameter model

and retention factor correlation plots for varied

solutes to study the retention mechanism

on XBridge C8, XBridge Phenyl and XTerra

Phenyl stationary phases (all Waters Corp.,

Milford, Massachusetts, USA) with acetonitrile–

water and methanol–water mobile phases

containing from 10 to 70% (v/v) organic

solvent. Published in Chromatographia [68

(7-8) 491-500 (2008)], these stationary phases

are compared with XBridge C18 and XBridge

Shield RP18 characterized in an earlier report

using the same protocol.

Dr Poole explained that improvements

in chemically bonded stationary phases

have resulted in new products being much

more alike than in the past. “Large changes

in retention are often due to differences in

the phase ratio, while changes in selectivity

(relative retention) are quite small. Even

changes in the structure of the bonded ligands

seems to have little effect on selectivity when

pure silica particles and high bonding densities

RrResearchround-up

are used for alkyl and aromatic ligands,” he

said.

The key findings of the study are that

for the XBridge C8, C18, C18 Shield, and

phenyl chemically bonded stationary

phases, changes in selectivity in going

from one phase to the other are small

and largely independent of the mobile

phase composition used. “Larger changes

are associated with weak electrostatic

interactions and steric repulsion for a few

specific compounds,” he added.

“There is a road block in method

development for those mixtures that are

difficult to separate, in that changing the

column type or supplier, will rarely result

in significant progress as columns become

more alike. This may largely be a waste

of effort and should be no more than a

last resort. Older column types are more

variable and may offer a greater chance of

success,” he said. The use of models such as

the solvation parameter provides a means

to select columns with different separation

characteristics and to know in advance what

chemical basis for a separation is being

exploited.

Comparing separation characteristics of reversed-phase columns

6 research round-up www.sepscience.com

The effect of pressure on small molecule retention using RP-UPLC

UKThe effect of inlet pressure on the retention of a series of low molecular

weight acids, bases and neutrals, was investigated at constant

temperature in reversed-phase liquid chromatography using a

commercial ultra-high-pressure system (Waters UPLC instrument) in the

Journal of Chromatography A [1209 (1-2), 195-205 (2008)].

“Changes in retention with pressure, can be considerable for large

molecules such as proteins, but are they significant for lower molecular

weight compounds? The study is of importance as some commercial

HPLC instruments (e.g., Waters Acquity instrument) are now capable

of solvent delivery at pressures up to 1000 bar,” explained Dr David

McCalley, Reader in Separation Science at the School of Life Sciences,

University of the West of England in Bristol, UK, who conducted the

study with graduate research student Morgane Fallas, in conjunction

with Mark Hadley at AstraZeneca in Macclesfield, UK, and Uwe Neue

from Waters Corporation in Milford, USA.

“Increases in retention with increasing pressure are normally observed,

because of changes in the molar volume of the solute in the stationary

and the mobile phase. For neutral non-polar molecules such as toluene,

increases in retention factor for an increase in the average column

pressure of 500 bar were only a few per cent. However, for ionized acidic

and basic compounds, increases of more than 50% were observed over

the same pressure range,” Dr McCalley said. These selective increases in

retention with pressure even caused reversals in the order of elution of

peaks for mixtures containing different types of compound. “We believe

that these previously unobserved and surprising differences arise from

differences in the loss of solvation for compounds of various types when

they enter the stationary phase,” McCalley remarked.

According to him, it is possible that differences in selectivity might

be observed between otherwise identical analyses performed on

conventional 5 µm and on newer sub-2 µm particles, because of the

differences in operating pressure required. Could pressure even be used

as a tool to change selectivity deliberately?



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7research round-upseparation science — volume 1 issue 1

Computer-assisted solution for multicomponent sample identification

HungaryIdentification of multicomponent

samples is one of the most common

tasks for chromatographers, and one

of the most difficult. Dr Janos Harangi,

from the Department of Biochemistry at

the University of Debrecen in Hungary,

developed a method by which the results of

chromatographic analysis multicomponent

samples can be stored in a database, such

as a fingerprint. The database records were

designed to be as independent of the

analysis environment as possible, for inter-

laboratory use. In a paper published in

Chromatographia [68 (supplement 1), 77-83

(2008)], he discussed this computer-assisted

solution and the application of this method

is tested by identification of several pine

species by recognition of their needle oils and

by identification of wine samples from their

aroma composition.

“The original need arrived from the

food industry to identify changes of

essential oil production from different

suppliers. Those kinds of samples have

hundreds of components, and the manual

evaluation of the analysis results was almost

impossible,” he explained. The computer

Rapid and direct determination of pesticides in water using anion-exchange chromatography with coulometric detection

BrazilA simple, rapid, and low-cost coulometric method for direct detection of

glyphosate and aminomethylphosphonic acid (AMPA) in water samples

using anion-exchange chromatography and coulometric detection

with copper electrode is presented in the Journal of Chromatography

A [1208 (1-2), 246-249 (2008)]. Led by Dr Cláudia Coutinho from the

Institute for Chemistry at the University of São Paulo, Brazil, conducted

the research because most analysis methodologies of glyphosate,

a common pesticide in Brazil, apply derivation reactions, which are

frequently complex, time-consuming and use expensive reagents. “The

main purpose of this work was the development of a new methodology

for glyphosate analysis that was direct, simple and low cost, when

comparing with traditional ones commonly used for the analysis of this

herbicide,” Dr Coutinho said.

Under optimized conditions, the limits of detection (LODs) (S/N

= 3) were 0.038 μg/mL for glyphosate and 0.24 μg/mL for AMPA,

without any preconcentration method. The calibration curves were

linear and presented an excellent correlation coefficient. The method

was successfully applied to the determination of glyphosate and

AMPA in water samples without any kind of extraction, clean-up or

preconcentration step. No interferent was found in the water like this

and the recovery was nearly 100%.

According to Coutinho, glyphosate was directly determined with

a cooper electrode, so no derivation reaction was necessary. “This

electrode can be used for the determination of many complexant

substances. These substances, as well as glyphosate, interact with the

cooper oxide layer on the electrode surface. This interaction dissolves

the cooper oxide layer and displaces the reaction equilibrium, which

increases the electrode anodic current. The stronger the complexant

is, the bigger the sensitivity of the technique. As glyphosate is a

tridentate complexant, an excellent detection limit was obtained, which

is in accordance with many international and national organizations.

Besides the possibility of analysis without the necessity of a derivation

reaction, the developed methodology is fast, simple and uses cheaper

instrumentation,” she added.

In the future, the team will apply this technique to the determination

of other compounds of environmental and pharmaceutical importance,

such as bisphosphonates, which showed good results in preliminary

tests.


aided data evaluation is similar to when a

chromatographer overlays two (or more)

chromatograms to find similarities.

“First, the conversion of the chromatogram

to a chromatographic fingerprint had to be

performed. The conversion means that the

retention times are converted to retention

indices (Kovats indices) and the peak areas

are converted to relative intensities. These

data are easy to store and compare, similar to

mass spectra. The only difference is that the

retention indices may vary. The mathematical

comparison of the chromatographic

fingerprint counts both variables, and results

in a method for fast multicomponent sample

identification,” explained Harangi.

The fingerprinting method was tested at

a wine company to match the contents of a

wine bottle with the label. Wine producers

use several additives during production,

some of which can modify the taste and

smell of wine. “Non-expert consumers cannot

distinguish between such modified wines

and original wines. With the aroma analysis

of the wines and use the laboratory made

database of the chromatographic fingerprint

helped to find the modified wines.”

He continued, “The applied technique is

a simple chromatographic analysis with a

postrun data evaluation. I believe that this

method is one of the simplest and rather

reliable. Possible applications are one-

dimensional chromatographic analyses of

any kind of multicomponent samples, such

as perfumes, aromas, essential oils, refinery

products (distillate fragments), and many

others.”

The next step is for the method to be

tested in cooperation with laboratories in

the food industry for possible application in

quality control or proof of origin.

SPE and GC–microEDC multiresidue analysis of royal jellyGreeceRoyal jelly, one of the most important products produced by bees, can

be contaminated with pesticides and/or antibiotic residues resulting

from treatments applied either inside beehives or in the agricultural

environment. A new multiresidue method, published in the Journal

of Chromatography A [1209 (1-2), 17-21 (2008)] was developed and

validated for the analysis of nine pesticides in royal jelly.

According to lead author, Professor Urania Menkissoglu-Spiroudi from

the Pesticide Science Laboratory at Aristotle University of Thessaloniki,

Greece, EC legislation requires that all active substances used in

veterinary medicines for food-producing animals must be assessed

so that a Maximum Residue Limit (MRL) can be set. “Commission

Decision 2002/657/EC introduced the concept of Minimum Required

Performance Limits (MRPLs), which recognizes the need for EU

harmonization of analytical detection limits and corresponds to the

concentration at which regulatory laboratories should be able to detect

and confirm the presence of particular substances. As MRLs for royal jelly

have not been established either at EU or national level, the presence

of any acaricide or pesticide residues could result in a non-compliant

product. Consequently, proper analytical methods developed and

validated according to Analytical Quality Control (AQC) performance

criteria established by EC are necessary for control purposes,” Professor

Menkissoglu-Spiroudi explained.

Solid-phase extraction RP-C18 cartridges were used for sample

purification and isolation of analytes and the final solution was analysed

with GC and micro-electron-capture detection. Four synthetic acaricides

used by beekeepers (bromopropylate, coumaphos, malathion and

τ-fluvalinate), as well as one pyrethroid, two organochlorine, and two

organophosphate insecticides were tested.

“The method proved to be linear in the range of interest and the

coefficient correlation was >0.99846 for each analyte. The accuracy and

precision are good enough to make the procedure applicable to routine


Affinity partitioning of plasmid DNA with a zinc finger protein

use in the analysis of residual levels of the tested pesticides and especially the residues of the

synthetic acaricides used for apicultural purposes,” she said.

In the future, she believes this method can be used by analytical laboratories in order to

determine the presence of acaricide and other pesticide residues in royal jelly samples. “The

method has already been used for assessment of residues detected in royal jelly after the

application of four registered in apiculture synthetic acaricides,” she said. The next step is to assess

the presence of pesticide residues in royal jelly in vivo. “Royal jelly is a rather polar matrix with a

high content of water, proteins and lipids so it is possible for pesticide residues to be present,” she

concluded.

PortugalA study, published in the Journal of

Chromatography A [1206 (2), 105-112 (2008)],

investigated the possibility of using a DNA

binding protein [in this case a zinc finger

protein (ZnFP)], as an affinity ligand in aqueous

two-phase extraction of plasmid DNA (pDNA).

“Aqueous two-phase extraction has already

proved to be very promising for future

utilization in the large-scale preparation of

pDNA for DNA vaccines and gene therapy.

However, it lacks selectivity that could be

improved by addition of affinity ligands,” said

lead researcher, Dr João Marcos from the

Department of Chemistry at the University of

Minho in Braga, Portugal.

Previously, small molecules have been used

as affinity ligands in aqueous two-phase

extraction of proteins with excellent results.

However it was not known if a similar principle

could be used for pDNA given its structural

and size differences. Therefore, in this study

the affinity isolation of prepurified pDNA

from model buffer solutions using native and

poly(ethylene glycol) (PEG) derivatized zinc

finger–GST (glutathione-S-transferase) fusion

protein was examined in PEG–dextran (DEX)

aqueous two-phase systems (ATPSs).

In the absence of pDNA, partitioning of

unbound PEGylated fusion protein into the

PEG-rich phase was confirmed with 97.5% of

the PEGylated fusion protein being detected

in the PEG phase of a PEG 600–DEX 40 ATPS.

This represents a 1322-fold increase in the

protein partition coefficient in comparison

with the non-PEGylated protein (Kc = 0.013).

In the presence of pDNA containing a specific

oligonucleotide recognition sequence, the

zinc finger moiety of the PEGylated fusion

protein bound to the plasmid and steered the

complex to the PEG-rich phase.

“We found that, in systems where isolated

ZnFP and pDNA accumulate in different

phases, after mixing them together the

partition of pDNA shifts to the phase where

the ZnFP accumulates. This was true both for

the case where a native and a PEGylated ZnFP

were used. The latter case is more promising as

it selectively accumulates pDNA in the phase

where less protein accumulates,” Dr Marcos

explained.

According to him, the results presented in

this paper show it is possible to increase the

selectivity of aqueous two-phase systems for

pDNA extraction using a DNA binding protein.

“After this proof-of-concept paper we are

now applying this knowledge to design and

develop a purification process of pDNA based

on aqueous two-phase systems,” he concluded.


FD-LC-MS/MS in breast cancer studiesJapanKazuhiro Imai and colleagues from Musashino

University and the Japanese Foundation

for Cancer Research (both Tokyo, Japan),

have published the results of a proteomics

study on human breast cancer cell lines

using fluorogenic derivatization-liquid

chromatography/tandem mass spectrometry

[Biomedical Chromatography, 22 (11), 1304-

1314 (2008)].

“Previously, we developed fluorogenic

reagents possessing a benzofurazan skeleton,

such as NBD-F, which are now available

as reagents for amines and amino acids1,”

began Imai, before expanding, “The current

article relates to works combining the use of

these reagents with the HPLC separation of

fluorescent derivatives.”

He continued, “Although several molecular

markers for diagnosis and therapy of breast

cancer exist, their versatility is limited because

of the lack of direct information on dynamic

cellular processes of proteins in tissues. To

distinguish and identify minute changes

in the expressed proteins between cancer

and normal tissue, highly sensitive and

reproducible proteomic analysis are required. It

is our opinion that such an

FD-LC-MS/MS method could clarify the

dynamic cellular processes of proteins in

tissues and demonstrate other candidates for

molecular markers in breast cancer diagnosis.”

“Previous proteomic studies have reported

individual changes in expressed proteins.

In contrast, our work has demonstrated

the dynamic flow of signal proteins in both

normal and cancer cells, and thus highlighted

presumptive mechanisms leading to invasion,

metastasis, proliferation and inhibition of

cancer cells, as well as the new biomarker

candidates. Furthermore, we have suggested

that tropomyosin-1 could play a key role in

counteracting tumour progression,” explained

Imai.

The research team feel that the

FD-LC-MS/MS method is a highly reliable,

reproducible, sensitive and easy-to-handle

method for proteomics analysis and, in fact,

is superior in terms of understanding the

performance of the dynamic events in tissues.

The method should be further applied to

understanding the complex cellular events in

disease states.

1 Biomedical Chromatography, 9, 106-109 (1995).

Improved liquid chromatography — Online radioactivity detection for metabolite profiling

Belgium

“Up until this point, radiotracer technology (14C or 3H) has been the method of choice to study the in vivo disposition of

a new drug as it enables the quantitative detection of the parent drug and all of its metabolites in complex matrices.

Metabolites are structurally related entities and structure differences can be minor. Therefore, a complete separation

between all drug-related compounds is, although necessary for quantification, difficult to achieve,” said Dr Filip Cuyckens

from Global Preclinical Development at Johnson & Johnson Pharmaceutical R&D in Beerse, Belgium.

In a paper he published in the Journal of Chromatography A [1209 (1-2), 128-135 (2008)], Dr Cuyckens describes the

successful combination of very-high-pressure liquid chromatography (VHPLC) with online radioactivity detection (RAD)

facilitated by improvements in online radioactivity detection, as well as in column loading and peak capacity.

“Many researchers in our field were hoping for an efficient coupling of ultra-performance liquid chromatography

(UPLC) separation with online radioactivity detection. The major issue is that the narrow peak widths obtained in 11research round-upseparation science — volume 1 issue 1

Fast GC–MS pesticide multiresidue analysis of applesSlovakiaA fast gas chromatographic–mass

spectrometric (GC–MS) method is proposed

for pesticide multiresidue analysis of apples

in Chromatographia [68 (supplement 1),

49-55 (2008)]. Professor Eva Matisová from

the Institute of Analytical Chemistry at the

Faculty of Chemical and Food Technology of

the Slovak University of Technology, Slovak

Republic, used the QuEChERS method for

sample preparation and GC–MS analysis was

performed with a PTV, an autoinjector, and a

quadrupole benchtop MS detector.

“Speeding up GC analysis provides

unquestionable benefits towards conventional

GC, such as higher laboratory throughput,

reduced GC operating costs, and better

analytical precision by making possible

more replicate analyses. Nowadays, fast

GC can be performed on commercial gas

chromatographs, which are equipped with

high-speed injection systems, electronic gas

pressure control, rapid oven heating/cooling

and fast detection,” Professor Matisová said.

In the study, compounds were separated

under temperature-programmed conditions

on a narrow-bore diphenyldimethylsiloxane

column. In one chromatographic run 61

pesticides of different chemical classes, and

triphenyl phosphate as internal standard,

were determined in 11 min. Calibration was

performed with matrix-matched standard

solutions and response to the pesticides was

a linear function of concentration in the range

1–500 ng/mL (equivalent to 1–500 μg/kg in

real samples). High values of the determination

UPLC separations appear intrinsically incompatible with the

relatively high counting times needed to reach adequate sensitivity

in radiochemical detection,” he explained. Traditionally 500 to 1000

μL flow-through cells are used in radioactive detection of drug

metabolism samples because the radioactivity concentrations in

these (especially in vivo samples) are rather low. According to him,

these large cell volumes are detrimental for the peak width and

high-resolution separations obtained in UPLC. In order to keep a

good resolution, the cell volume needs to be decreased, which in

turn is detrimental to sensitivity.

“In our laboratory, we developed an efficient combination of

UPLC with a conventional radiodetector. This combination was

made possible by improving the on-line radioactivity detection, as

well as increasing the column loading and peak capacity for ultra-

high-pressure separations,” he said.

In the study, the sensitivity of 14C detection was improved

by the use of a variable scintillation flow achieved via a simple

modification to the classic online radiochemical detection set-

up. A modification of the flow-through cell design in which

internal diameter of the tubing was reduced further increased

the sensitivity and resolution by decreasing peak tailing. The

injection of relatively large volumes was made possible by the use

of columns packed at ultra-high-pressure with 2.2 μm particles.

Because of the reduced back pressure using these larger particles,

two 3 x 150 mm columns could be coupled, allowing four fold

larger injection volumes and a 50% improved separation at a

similar backpressure compared with a standard 2.1 x 150 mm UPLC

column.

“This is the first UPLC ¬ on-line radioactivity detection set-up that

works in practice for samples with limited radioactivity, not only

for standards. We are now using the approach in our lab for real

projects and are replacing our existing radio-HPLC systems with

radio-UPLC systems. The use of the larger and coupled columns

allowing higher loadability and plate numbers is also very useful for

other applications; for example, chemical impurity quantification,”

he concluded.


BrazilPublished in Analytical Chimica Acta [628(2), 123-132 (2008)] is an article by Jorge Masini

and colleagues (Insituto de Quimica, Universidade de São Paulo, Brazil), describing the

implementation of a stepwise solvent elution in sequential injection chromatography (SIC) for

fluorimetric determination of intracellular free amino acids in the microalgae Tetraselmis gracilis.

“Part of my research work focuses on the development of sequential injection or flow injection

methodologies for environmental analyses,” explained Masini, adding “These techniques are

suitable for precise liquid handling and control of reaction times, but are limited to only a few

analytes per sample injection, such that multi-analyte determinations have been a target for

practitioners of flow/sequential injection techniques.”

“Coupling low pressure flow systems to monolithic stationary phases has allowed us to

improve the capabilities of sequential injection or flow injection techniques toward multi-analyte

determinations. We were originally interested in the on-line determination of amino acids that are

excreted by marine microalgae in batch cultivation. This was a goal that required multi-analyte

determinations and precolumn derivatization, so sequential injection chromatography was our

choice to start the method development,” he continued.

The concept of sequential injection chromatography was exploited to automate the

fluorimetric determination of amino acids after precolumn derivatization with o-phthaldialdehyde

in the presence of 2-mercaptoethanol using a reverse-phase monolithic C18

stationary phase.

Additional to the on-line precolumn derivatization, the proposed method involved five steps of

isocratic elutions allowing the use of a wide range of polarities during the elution.

“To our best knowledge, this simple approach had not yet been described in SIC

methodologies, which had previously used only a single eluting solution (isocratic), being limited

to separate components of simple mixtures, and being especially applied to the determination

of pharmaceutical products. In a mix of 18 amino acids, the proposed procedure allowed us to

coefficients (R2; 0.9900–1.0000) were obtained for most of the pesticides.

Limits of detection and quantification were determined.

According to Matisová, the key outcome was the development and

validation of the method for multiresidue analysis of pesticides in non-

fatty food (apples) by fast GC-MS with PTV injection in solvent mode

and using a narrow-bore capillary column.

Quadrupole MS detectors have been most widely utilized in analytical

practice. “From the results obtained it can be concluded that the used

quadrupole detector is fast enough for proper reconstruction of the

analytes peaks and that the method can be used for multiresidue

analysis,” she said. The method is applicable to pesticide residues analysis

at ultra-trace concentration levels in real samples in non-fatty food. “Our

future work will be directed to endocrine disrupting pesticides analysis

in environmental matrices,” she added.

Sequential injection methodologies for environmental analyses


separate 14 analytes with resolutiongreater than1.5,” Masini explained.

He concluded, “We believe that the methodology described in the referred article can extend the application of SIC to

the separation of complex mixtures with potential application to physiological studies of amino acid metabolism in algae,

as well as in environmental studies related to carbon and nitrogen assimilation by phytoplankton. Development and

application of SIC procedures to perform on-line monitoring for adsorption/desorption of herbicides and their metabolites

onto/from soil particles is another on-going project in our laboratory.”

Simultaneous HPLC and GC analysis of lignans in Forsythia leaves

HungaryThe biological activities of lignans are of pharmaceutical importance.

Among butyrolacton lignans, arctigenin and its glucoside, arctiin occur

in Forsythia species in high quantities. Éva Sedlák from the Department

of Plant Anatomy at Eötvös Loránd University in Budapest, Hungary

conducted a study to find the most suitable extraction method for

these lignans, and identify and quantify the lignan constituents present

in leaf samples of different Forsythia species and cultivars by high-

performance liquid chromatography (HPLC) and gas chromatography,

(GC), simultaneously.

Published in Chromatographia [68 (supplement 1), 35-41 (2008)]

lignans, carboxylic acids and sugars were determined by gas

chromatography (GC) with mass selective detection, the lignans also

by high-performance liquid chromatography with UV detection in

the leaf extracts of four species and four cultivars of Forsythia plant.

Three methods were used to optimize the extraction of the main

lignan constituents (arctiin, arctigenin), which possess significant

pharmaceutical effects.

“Our results suggested that the supercritical fluid extraction applying

CO2+60% methyl alcohol was the best method for obtaining the highest

extraction yield of lignans,” said Sedlák. Regarding the identification and

quantification of aglycone lignans, both HPLC and GC-MS proved to

be efficient. To quantitate lignan glycosides, HPLC was the method of

choice. According to her, from a pharmaceutical point of view the most

relevant, arctigenin, was found in the highest quantity in the cultivar

‘Robusta’ of Forsythia ovata.

“We want to utilize our optimized extraction and analytical techniques

in further basic research and technical applications. Our final goal would

be to prepare medicinal product from the highest lignan containing

Forsythia ovata species,” she concluded.


To overcome the limitations of solid-phase

extraction, Dr Akira Namera and colleagues

from the Department of Forensic Medicine

at Hiroshima University, Japan, developed

a device comprising a spin column packed

with octadecyl silane-bonded monolithic

silica for extracting amphetamines and

methylenedioxyamphetamines from

urine. A paper published in the Journal of

Chromatography A [1208 (1-2), 71-75 (2008)]

states that the proposed method is not only

useful for drugs from biological materials but

also highly reproducible for the analysis of

these drugs in urine.

Liquid-liquid or solid-phase extraction

(SPE) methods are widely used for extracting,

purifying, and enriching drugs and medicines

from biological materials. However, these

methods involve laborious, intensive and

expensive preparatory procedures. “GL

Sciences informed me that monolithic silica

has been investigated as a new type of

separation material for high-performance

liquid chromatography (HPLC). Although

the conventional materials used for SPE

were similar to those used for HPLC, we

think monolithic silica has potential for drug

extraction and purification from biological

materials,” said Dr Namera.

Urine (0.5 mL), buffer (0.4 mL) and

methoxyphenamine (internal standard) were

directly added to the preactivated column. The

column was centrifuged (3000 rpm, 5 min) for

sample loading and washed. The adsorbed

analytes were eluted and analysed by HPLC,

without evaporation. The results were as

follows: linear curves (drug concentrations of

0.2–20 μg/mL); correlation coefficients >0.99;

detection limit, 0.1 μg/mL.

“In our previous study, we packed monolithic

silica into a capillary glass tube (i.d. = 0.2 mm)

and the extraction device was created by

connecting a microsyringe to the capillary

column. We demonstrated that amphetamines

in urine could be extracted using this device,”

he explained. However, sample filtration was

required to avoid blockage within the tube,

and thus, only one sample could be obtained

through a batch processing cycle. In order to

overcome these problems, a monolithic silica

disc was packed into a spin column, wherein

the structure of monolithic silica combined

the support body and the surface area for

each unit volume is wide by comparing

with a particle-type silica. “The handling

procedures such as sample loading, washing

and elution of the target drugs were only

exhibited by a centrifugation of the column.

In addition, many samples can be processed

simultaneously,” he added.

According to him, this spin column has many

advantages: its operation procedure is simple,

requires a low eluate volume and does not

involve solvent evaporation. “It is expected

to take the place of conventional solid-phase

extraction cartridges in many fields,” he

concluded.

Extracting amphetamines from urine using monolithic silica disc-packed spin columnsJapan


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Development of immobilized enzyme reactors for phase I drug metabolism studiesSwitzerlandCytochrome P450 enzymes (CYP450) are the

major drug-metabolizing enzyme systems in

the human liver responsible for the oxidative

conversion of approximately 90% of marketed

drugs.

In order to determine the CYP450 isozyme

involved in a specific drug metabolism, in vitro

screening is usually performed by incubating

in-solution human liver microsomes or

recombinantly expressed human CYP450

isozymes with drugs (alone or with a potential

inductor or inhibitor). These incubations

require relatively large amounts of expensive

recombinant enzymes. Prof. Jean-Luc

Veuthey and colleagues from the School of

Pharmaceutical Sciences at the University of

Geneva, Switzerland have developed CYP450-

based immobilized enzyme reactors (IMERs)

to perform automated on-line phase I drug

metabolism studies.

Published in the Journal of Chromatography

A [1206 (1), 2-10 (2008)], the major aim of this

study was to prepare two IMERs containing

CYP2D6 and CYP3A4 for performing

automatically phase I drug metabolism.

“CYP450 is a very complex enzymatic system

constituted by three enzymes embedded in

a phospholipidic membrane and requiring

NADPH as cofactor. It was, therefore, not

possible to use the covalent immobilization

procedure already developed in our laboratory

with trypsin,” Prof. Veuthey explained.

Using an original strategy (NeutrAvidin

covalent immobilization on monolithic disc;

biotinylation of CYP450; coupling of the

biotinylated CYP450 on the NeutrA-disc), it was

possible for the team to obtain two discs

(2 x 6 mm i.d.) containing immobilized CYP2D6

and CYP3A4 to perform automatically drug

metabolism studies by LC-ESI-MS/MS. The

method was tested with probe substrates and

inhibition studies were also reported.

In comparison with in-solution assays, in

which CYP450 enzymes are known to be

rapidly inactivated, the system was stable

for performing ca. 15 experiments in 2 days,

according to him.

“This technique can be applied in drug

discovery for the evaluation of drug-drug

interactions and assessment of metabolism

profiles of novel chemical entities. Thanks

to the possibility to reuse the immobilized

enzyme for several analyses, this strategy is

cost-effective in comparison with conventional

in vitro tests,” he said. To date, the team is trying

to improve the stability of the IMERs, and is

working also on a different approach which

will be published in the near future.


Bioactive compound fishing with DNA-based bioseparations and chemical analysis ChinaAccording to a recent article in Biomedical Chromatography [22 (10), 1164-1172 (2008)], Professor Ping Li and researchers

from the China Pharmaceutical University, Nanjing, China, have performed a screening and mechanism study of

components targeting DNA from the Chinese herb Lonicera japonica by liquid chromatography/mass spectrometry and

fluorescence spectroscopy.

DNA is the potential molecular target of many antimicrobial, antiviral and antitumour active drugs. The screening and

identification of DNA-targeting agents from natural products extracts are attractive, and the interaction of DNA with small

molecules has been an intensive topic which has fascinated scientists for decades as it provides insights into rational

design of drugs targeting to DNA.

“As the biological separation system, DNA can be used to selectively fish for bioactive compounds from multicomponent

samples. By ultrafiltration sampling and LC-MS analysis, DNA-bound compounds can be easily identified from their

chromatographic fingerprints before and after interaction with DNA,” explained Li, adding “Four flavonoids were fished and

identified from Lonicera japonica extract using this method. The binding mechanism of these flavonoids with DNA was

then evaluated to be groove binding by fluorescence spectroscopy. For L. japonica, the flavonoids binding to DNA may be

one of the mechanisms of its antimicrobial, antiviral or antitumour actions.”

“Our future work will extend our bioseparation and chemical analysis strategy to other biological separation systems;

for example, target protein, cell etc., for rapid and effective screening of potential bioactive compounds from complex

multicomponent samples,” Li concluded.

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Phenomenex provides complete solutions including SPE tubes & 96-well plates, protein precipitation plates, syringe filters, vials and more.

Quality products

Responsive customer service

Complete technical support

Worldwide availability with fast delivery

Request a free sample pack of Phennex™ syringe filters visit www.phenomenex.com/samplee

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Lithuania A new method involving matrix solid-phase dispersion

(MSPD) extraction and hydrophilic interaction ultra-

performance liquid chromatography (HILIC-UHPLC)

with photodiode array detection was developed for

the determination of carbadox and olaquindox in feed.

Described in the Journal of Chromatography A [1209 (1-2),

83-87 (2008)], the separation of carbadox and olaquindox

was achieved within 1 min on the 1.7 μm Acquity UPLC

BEH HILIC column using isocratic elution with a mobile

phase consisting of

10 mmol/L ammonium acetate in acetonitrile-water (95:5,

v/v) at a flow rate of 0.5 mL/min.

“In recent years our group has collaborated closely with

the National Food and Veterinary Risk Assessment Institute

of Lithuania in developing new analytical methods for the

determination of veterinary drug residues in food and feed,”

said main author, Professor Audrius Padarauskas from the

Department of Analytical and Environmental Chemistry

at Vilnius University in Lithuania. The institute tasked his

group with the development of a simple, fast and reliable

method for the routine monitoring of carbadox and

MSPD extraction of carbadox and olaquindox in feed followed by hydrophilic interaction ultra-high-pressure liquid chromatographic analysis

rationdriving analyticalsciencd i ing anallyttiiic


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olaquindox in feeds.

Optimization of MSPD extraction parameters, such as

type of solid sorbent and elution solvent were performed.

Optimal conditions selected for MSPD extraction were:

0.25 g of feed sample, 0.5 g of octadecylsilica as solid

sorbent and 10 mL of acetonitrile-methanol (8:2, v/v) as

eluting solvent. Both analytes provided average recoveries

from spiked feed samples ranging from 89.1 to 98.4% with

relative standard deviations less than 10%.

“In my opinion, the main strength of the present work

is the combination of matrix solid-phase dispersion

extraction with hydrophilic interaction (HILIC) ultra

performance liquid chromatographic analysis.

Direct injection, without evaporation/reconstitution,

of the extract onto the separation column is therefore

possible, which saves time and prevents sample losses.

Another significant advantage is that less polar interfering

compounds requiring gradient elution by conventional

reversed-phase HPLC are eluted early in an isocratic HILIC

system, thereby simplifying and accelerating the overall

analysis,” Professor Padarauskas explained.

He believes this combination should be very promising

for the analysis of polar compounds in complex biological

matrices such as food, feed, plants etc.


22 feature article — SAX-HPLC analysis of food supplements www.sepscience.com

23feature article — SAX-HPLC analysis of food supplementsseparation science — volume 1 issue 1

and various nutraceutical preparations

[9, 10], and their purity and quality

were generally found to be

inconsistent with the specifications

claimed on the product labels. In this

article, we determine the CS amount,

quality and origin in several Czech

Republic nutraceutical samples using

the same analytical methodology

applied to a recent CS evaluation in US

food supplements [11].

Introduction

Chondroitin sulfate (CS) (Figure 1), a

very complex, polydisperse, natural

glycosaminoglycan (GAG) highly

heterogeneous for relative molecular

mass, charge density, structure, and

biological and pharmacological

activities [1, 2], is recommended

by EULAR [3, 4] as a SYSADOA

[Symptomatic Slow Acting Drug for

osteoarthritis (OA)] drug in Europe for

the treatment of knee, hip and

hand (OA) [5-7]. Moreover, CS alone

or in combination with glucosamine,

is utilized as a dietary supplement [7,

8]. As a consequence, the number

of pharmaceuticals containing CS

has increased, as well as the types of

formulations, including tablets and

caplets, capsules, ophthalmic solutions

and liquid preparations [9-11].

Previous studies reported a

determination of CS in raw materials

Two analytical approaches to the evaluation of chondroitin sulfate in european food supplementsNicola Volpi and Francesca Maccari

Department of Biologia Animale, Biological Chemistry Section, University of Modena and Reggio Emilia, Italy

The amount and quality of chondroitin sulfate (CS) from several Czech Republic food supplement/nutraceutical

preparations was determined. To quantify CS, two different analytical approaches were validated [11] and

applied: specific and sensitive agarose-gel electrophoresis and strong anion exchange (SAX)-high performance

liquid chromatography (HPLC) determination of the constituent disaccharides after treatment with specific

chondroitin lyases.

The CS content in food supplement products were found to conform to the label specifications in only four of

the ten analysed samples. Four of the food supplement preparations were found to contain approximately 0-1%

CS in comparison with 47, 17, 12 and 6% declared on the label. Two products were found to have approximately

30-45% of the declared CS, and one preparation was found to contain approximately 2% hyaluronic acid. SAX-

HPLC separation of unsaturated disaccharides for the nutraceutical CS was also used to evaluate its quality and

possible origin. The CS contained in eight food supplements resulted to be of bovine or porcine origin, one from

cartilagineous fish and in one case it was not possible to determine the origin because of very low CS content.

Based on these analytical results, quality of the sampled Czech Republic food supplement formulations was

poor and strict regulations for quality control should be mandatory in order to guarantee the manufacture of

high-quality products. Furthermore, specific and accurate analytical procedures should be enforced for the

control of high-quality products and applied by quality control laboratories to confirm the purity and label claim

of CS in raw materials and nutraceuticals.


Sample Declared CS Content Formulation

A 250 mg/capsule Glucosamine, Collagen, Saccharides

B 250 mg/tablet Glucosamine, MSM

C 25 mg/tablet Glucosamine, Collagen, MSM

D 200 mg/tablet Glucosamine, MSM

E 200 mg/tablet Glucosamine

F 67 mg/capsule Glucosamine, Collagen, MSM, Vitamin C, Vitamin E

G 200 mg/tablet Glucosamine, MSM

H 400 mg/tablet Glucosamine

I 25 mg/tablet Glucosamine, Collagen, MSM

L 75 mg/tablet Glucosamine, Collagen

6S: ΔUA-[13]-GalNAc-6S, ΔDi-4S:

ΔUA-[13]-GalNAc-4S, ΔDi-2,6diS:

ΔUA-2S-[13]-GalNAc-6S, ΔDi-4,6diS:

ΔUA-[13]-GalNAc-4S,6S, ΔDi-2,4diS

ΔUA-2S-[13]-GalNAc-4S) were from

Seikagaku. High resolution agarose,

certified for molecular biology, was

from Sigma. All other reagents were of

analytical grade.

Sample preparation: All the used

nutraceuticals contained various other

ingredients in different amounts, in

particular glucosamine, collagen,

vitamins and/or MSM (Table 1).

Stock solutions (10 mg/mL) of all the

products were carefully prepared.

No sample pretreatment was

performed apart from centrifugation

at 10,000 RPM for 10 min to remove

insoluble material, mainly derived

from excipients and salts present in

the preparations, and a proteolytic

treatment to remove possible

interfering proteins. The EPCS

reference standard was dissolved in

water to make authentic CS solution

(10 mg/mL).

Analytical procedures: The

analytical techniques used for

the quantitative and qualitative

evaluation of CS in food supplements

Materials and Methods

Materials: Various food supplement

samples in the form of tablets or

capsules containing CS in different

formulations and amounts

(Table 1) were obtained from the

Czech Republic market. The European

Pharmacopeia CS (EPCS) reference

standard, manufactured by Bioiberica

(www.bioiberica.com/) and approved

in 2004 as the Chemical Reference

Substance (CRS) by the European

Pharmacopeia Commission was

utilized as a standard in these analyses.

This CS standard is produced from

bovine cartilage, as evaluated by HPLC

analyses [12], and has a claimed CS

content greater than 98% [12]. CS

fractions having different molecular

mass values for high-performance

size-exclusion chromatography

(HPSEC) analysis, were prepared by

gel-permeation and evaluated by

means of analytical ultracentrifugation

[13]. Chondroitinase ABC, chondroitin

ABC lyase, from Proteus vulgaris

(EC 4.2.2.4), 0.5-2 units/mg, and

chondroitinase ACII, chondroitin

AC lyase, from Arthrobacter aurescens

#(EC 4.2.2.5), were from Sigma.

Unsaturated CS/DS disaccharides

(ΔDi-0S: ΔUA-[13]-GalNAc, ΔDi-

Figure1

HH

H

HH H

H

H

HH

O

OO

O OCOO

OH

OR 3

1OR

CH OR2 2

NHCOCH3

R = R = R = H: nonsulfated chonroitinR = SO and R = R = H: chondroitin-4-sulfate, CSAR = SO and R = R = H: chondroitin-6-sulfate, CSCR = R = SO and R = H: chondroitin-2, 6-disulfate, CSDR = R = SO and R = H: chondroitin-4, 6-disulfate, CSER = R = SO and R = H: chondroitin-2, 4-disulfate, CSBR = R = R = SO : trisulfated chondroitin

1 2 3

1 3 2 3

2 3 1 3

2 3 3 1

1 2 3 3

1 3 3 2

1 2 3 3

Table1

Figure 1. Structures of disaccharides forming chondroitin sulfate.


have been previously published in

detail [11]. In particular, agarose-

gel electrophoresis and HPLC

separations of the unsaturated CS

disaccharides produced by the action

of chondroitinases ABC and ACII were

used for quantitative purposes [11,

14–17]. These methods have been

validated according to the Guidance

for Industry, Bioanalytical Method

Validation from US Department of

Health and Human Services, Food

and Drug Administration, Center for

Drug Evaluation and Research (CDER),

Center for Veterinary Medicine (CVM)

published in May 2001 [18], including

specificity, linearity, detection (LOD)

and quantitation (LOQ) limit, precision,

accuracy, recovery, and robustness

tests (see [11]). Furthermore, HPLC

of the CS disaccharides was used to

evaluate the possible origin of CS in

nutraceuticals [11, 12, 19] and HPSEC

was applied for the determination of

the CS molecular mass parameters

[11–13].

Results

First, an accurate determination

of the theoretical CS content was

performed by evaluating real weight

of tablets/capsules, repeated several

times to have a statistical variation,

and to calculate the theoretical

CS percentage in each of the food

supplements. The declared CS

percentage was found to be from 1.5

up to 47.2 (Table 2).

The CS present in the ten

nutraceuticals was separated

Parameters Food Supplements

A B C D E F G H I L

Declared CS % 47.2% 16.9% 1.5% 12.4% 24.1% 12.3% 12.3% 30.7% 1.6% 6.0%

CS content %

Agarose-gel 0.12% 0.34% 1.54% 0.62% 10.20% 4.87% 10.12% 28.50% 1.36% 0.75%

SAX-HPLC <0.20% 0.74% 2.71% 0.67% 11.42% 3.00% 12.85% 28.56% 1.09% 1.93%

Mean * <0.2% 0.9% 2.1% 0.9% 10.7% 3.9% 11.8% 29.1% 1.4% 0.8%

Molecular mass

MWn nd nd nd nd 18,400 nd 12,990 18,980 29,400 191,370

MWw nd nd nd nd 52,600 nd 26,250 45,160 117,670 726,260

MWz nd nd nd nd 161,520 nd 76,240 174,650 868,120 nd

Dispersity nd nd nd nd 2.8577 nd 2.0212 2.3796 4.0029 nd

Disaccharides

ΔDi-0s nd nd nd nd nd nd nd 6.9 7.4 nd

ΔDi-6s nd 27.1 17.7 38.0 44.5 33.0 21.8 20.9 16.0 20.3

ΔDi-4s nd 72.9 82.3 62.0 33.0 67.0 78.2 72.2 76.6 79.7

ΔDi-2,6dis nd nd nd nd 19.4 nd nd nd nd nd



R nd 1.00 1.00 1.00 1.22 1.00 1.00 0.93 0.94 1.0

4s/6s ratio nd 2.69 4.65 1.63 0.74 2.03 3.59 3.45 4.79 3.93

Origin nd Bovine/

Porcine

Porcine Bovine Cartil.

Fishes

Bovine Porcine Porcine Porcine Porcine

Table2

nd = not detected, * Mean of analyses performed under different experimental conditions, i.e. agarose-gel and HPLC of disaccharides without and in the presence of proteolytic treatment.

Table 2. CS content, molecular mass parameters (Mn, number of average molecular weight; Mw, weight average molecular weight; Mz, Z average molecular weight, polydispersity index, Mw/Mn) and unsaturated disaccharide percentages, with the charge density values (as sulfate groups number per disaccharide unit) and the 4-sulfated/6-sulfated ratio (4s/6s ratio as the ratio between the sulfated groups located in position 4 or 6 on N-acetyl-galactosamine), of CS samples from the various Czech Republic nutraceutical samples analysed in this study. The percentage of each disac-charide was calculated by considering the total absorbance %age corresponding to each peak. The data are means of three different analyses.


preparation. SAX-HPLC was also used

for the quantitative determination

(Table 2) of the CS constituent

disaccharides [11]. The relevant results

are fairly consistent with the data

obtained by electrophoresis.

Finally, the molecular mass

parameters, in particular Mn, M

w, M

z

and polydispersity, were determined

for just a few of the ten nutraceutical

samples (Figure 4 and Table 2) because

of the very low CS content. However,

this analytical approach indicates the

possible presence of depolymerized

or degraded CS. In fact, the molecular

mass values of nutraceutical CS are

similar to those of CS samples purified

from porcine or bovine cartilages, and

cartilagineous fishes [12] confirming

that these polymers have not been

degraded during the food supplement

preparations.

It is worth mentioning, that

in sample L the presence of

approximately 2% high molecular

mass hyaluronic acid, was detected by

agarose-gel [Figure 5(a)], disaccharide

analysis [Figure 5(b)] and HPSEC

evaluation [Figure 5(c)].

Discussion

In a previous study [11], we performed

the quantification and characterization

of CS present in several nutraceutical

preparations available as caplets,

tablets and capsules in the US market

by using the specific and sensitive

agarose-gel electrophoresis and HPLC

analyses. The electrophoresis and

SAX-HPLC methods were validated

and confirmed to be applicable to

the quantification of CS in finished

products even in the presence of

various additives and ingredients.

The CS content in ten Czech

and quantified by agarose-gel

electrophoresis (Figure 2) without

and in the presence of digestion

with proteases, and centrifugation

to remove insoluble material. The

quantitative results obtained using

this analytical approach are illustrated

in Table 2.

The food supplement CS samples

were treated with chondroitinases ABC

and ACII producing virtually the same

disaccharides confirming the absence

of iduronic acid (and dermatan sulfate)

as previously reported by agarose-

gel electrophoresis. The pattern of

unsaturated disaccharides (illustrated

in Figure 3) for nutraceutical CS

samples is reported in Table 2. As

can be seen, CS of various origin

— bovine, porcine and one from

cartilagineous fishes [11, 12, 19] —

have been used for the nutraceutical

Sample E

Sample G

Disulfateddisaccharides

Di-4s

Di-6s

Di-0s

Di-0sDi-6s

aADi-4s

Abs

orba

nce

at 2

32 n

mA

bsor

banc

e at

232

nm

Figure 3. Strong-anion exchange (SAX)-HPLC separation of the disaccharides from the chondroitin sulfate samples of two food supplements (Sample E and G) produced by the action of chondroitinase ABC. ΔDi-0S, ΔHexA-GalNAc; ΔDi-6S, ΔHexA-GalNAc (6-OSO3); ΔDi-4S, ΔHexA-GalNAc (4-OSO3). Various unsaturated disulfated disaccharides are also separated in sample E.

Figure2

Figure 2. Agarose-gel electrophoresis of chondroitin sulfate (CS) of several Czech Republic nutraceutical samples (from A to L, see Table 1). CS was separated on an agarose-gel having a thickness of about 4-5 mm and, after migration, the plate was stained with toluidine blue. The migration of the European Standard CS reference standard (St) is also illustrated.

Figure3


Republic dietary supplements was

determined without any treatment

or in the presence of a proteolytic

treatment to remove proteins possibly

interfering with the CS content

evaluation, and the results were

quite similar. In particular, sample A,

claimed to contain 47% CS by the

manufacturer, contained almost no CS,

lower than 0.2%. Sample B was found

to contain approximately 1% (17%

declared), sample D approximately

1% (12% declared), sample E

approximately 11% (24% declared),

sample F approximately 4% (12%

declared), and sample L approximately

1% (6% declared). In this last sample,

2% hyaluronic acid was also found.

Samples C, G, H and I were found

to conform to label specifications.

As a consequence, four of the food

supplement preparations were found

to contain approximately 0-1% CS

in comparison with 47, 17, 12 and

6% declared on the labels, and two

products were found to have only

30-45% of the declared CS.

The pattern of disaccharides to

determine the CS origin and the

molecular mass parameters to

evaluate the absence of degradation,

were evaluated in samples in which CS

was detected. The CS was found to be

of high molecular mass showing that

no degradation occurred during the

food supplement preparations and it

was evaluated to be of bovine, porcine

or cartilagineous fish origin. One

sample was not determined becaues

of very low CS content.

CS, like other natural

polysaccharides, is derived from

animal sources by extraction and

purification processes [1, 2, 12] and it is

well known that its structure changes

according to the tissue, organ and

species [1, 2, 12]. As a consequence,

source material, manufacturing

processes, the presence of

contaminants, and many other factors

contribute to the overall biological

and pharmacological actions of these

agents. On the basis of these analytical

results, the quality (in particular the

CS content), of several Czech Republic

dietary supplements was found poor

and it is worthy of mention that

clinical studies and CS efficacy have

been evaluated by using a very pure

product having specific properties

and physico-chemical characteristics

as approved by various National

Institutes of Health [1, 3, 4]. However,

this is not the first report on the quality

of CS in food supplements. The quality

of raw material and finished products

was found to be poor in Korea [10]

and a great variability in the CS origin

was found in 12 products from Japan,

generally not corresponding to the

label claim when declared [20]. This

is a key point as the biological and

pharmacological properties are known

to vary with the structure, and the oral

absorption, and bioavailability and

pharmacokinetic parameters may be

influenced by the different structural

characteristics and origin [21, 22], and,

more importantly, there is a possible

risk of species specific diseases. In fact,

there are strict regulations ensuring

that material from mammals used

for drugs are prepared from healthy

mammals because of the risk of

bovine spongiform encephalopathy

(BSE), foot-and-mouth disease,

influenza spread in birds, and other

animal diseases. However, there are

no definite regulations on the origin

of ingredients in nutraceuticals. The

origin of the ingredients in natural

products is the most important factor

ensuring quality, and thus safety and

efficacy.

CS is widely used as a nutraceutical.

As the number of products containing

CS increases, stricter and more

Sample H

Sample E

Abs

orba

nce

at 2

14 n

mA

bsor

banc

e at

214

nm

Log MLog M

min

min

20000

4000

8000

12000

16000

4000

8000

12000

16000

-163

00.00 5.00 10.00 15.00 25.00 35.0020.00 30.00

0.00 5.00 10.00 15.00 25.00 34.9920.00 30.00

37.84

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

Figure4

Figure 4. High-performance size-exclusion chromatography (HPSEC) separation of the chondroitin sulfate from two nutraceuti-cal samples (E and H). 100 μg of each food supplement sample was injected in the column. The third grade polynomial curve having the formula y(fx) = – ax3 + bx2 – cx + d performed by CS fractions of different molecular mass is also illustrated.


accurate evaluation and more strict

regulation for quality control should

be required for the production of

high-quality products. Furthermore,

the regulatory requirements should be

implemented on the manufacturers

so that the ingredients and their

origin are correctly listed, and specific

and accurate analytical procedures

should be enforced for the control of

high-quality products and applied by

quality control laboratories to confirm

the purity and label claim of CS in

raw materials and nutraceuticals. At

the moment, the quality of Czech

Republic food supplements is very

poor with no possibility for the

customer to verify the content and

origin of CS. While we are waiting

for more strict regulations for quality

control, pharmaceutical formulations

approved by local National Institutes

of Health is strongly recommended

instead of general food supplements.

References

1. N. Volpi, Chondroitin sulfate:

structure, role and pharmacological

activity. Amsterdam, Boston,

Heidelberg, London, New York,

Oxford, Paris, San Diego, San Francisco,

Singapore, Sydney, Tokyo, Academic

Press (2006).

2. K. Sugahara, et al., Curr. Opin. Struct.

Biol., 13, 612-20 (2003).

3. K.M. Jordan, et al., Ann. Rheum. Dis.,

62, 1145-55 (2003).

4. W. Zhang, et al., Osteoarthritis

Cartilage, 15, 981-1000 (2007).

5. N. Volpi, Curr. Drug. Targets Immune

Endocr. Metabol. Disord, 4, 119-27

(2004).

6. N. Volpi, Curr. Med. Chem., 13, 1799-

810 (2006).

7.P. Sarzi-Puttini, et al., Semin. Arthritis.

Rheum., 35(1 Suppl 1), 1-10 (2005).

8. T.E. McAlindon, et al., JAMA, 283,

1469-75 (2000).

9. J.S. Sim, J, et al., Chromatogr. B Analyt.

Technol. Biomed. Life Sci., 818, 133-9

(2005).

10. J.S. Sim, et al., Food Chem., 101,

532–9 (2007).

11. N. Volpi & F. Maccari, Food Anal.

Chem., 1, 195-204 (2008).

12. N. Volpi, J. Pharm. Sc., 96, 3168-80

(2007).

13. N. Volpi, L. Bolognani, J.

Chromatogr., 630:390-6 (1993).

14. N. Volpi. Anal. Biochem., 273, 229-39

(1999).

15. N. Volpi, F. Maccari, J. Chrom. B, 834,

1-13 (2006).

16. N. Volpi, et al., J. Chrom. B, 820,

131-5 (2005).

17. N. Volpi, F. Maccari, Electrophoresis,

23, 4060-6 (2002).

18. Biopharmaceutics Coordinating

Committee in the Center for Drug

Evaluation and Research (CDER)

in cooperation with the Center for

Veterinary Medicine (CVM) at the Food

and Drug Administration. Guidance

for Industry, Bioanalytical Method

Validation, (May 2001).

19. N. Volpi, Carbohydr. Polym., 55, 273-

81(2004).

20. S. Sakai, et al., Chem. Pharm. Bull.

(Tokyo), 55, 299-303 (2007).

21. N. Volpi, Osteoarthritis Cart., 10, 768-

77 (2002).

22. N. Volpi, Osteoarthritis Cart., 11:433-

41(2003).

Abs

orba

nce

at 2

14 n

m

Sample L

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

min0.00 5.00 10.00 15.00 25.00 34.9920.00 30.00

Log M

HA

9000

7000

5000

3000

1000

-1586

Di-HA

Di-6sDi-6s

Abs

orba

nce

at 2

32nm

min

Figure5

Figure 5. Agarose-gel electrophoresis (A), disaccharide analysis by means of strong-anion exchange (SAX)-HPLC (B), and high-performance size-exclusion chromatography (HPSEC) evaluation (C) of sample L showing the presence of high molecular mass hyaluronic acid. ΔDi-HA is the unsaturated hyaluronic acid disaccharide produced by the action of lyase. HA = hyaluronic acid. CS = chondroitin sulfate.

Separation Science Singapore 2009 is a cross-discipline, cross-culture chromatography meeting aimed at

scientists, engineers, business and technical experts from the diverse analytical instrumentation

industries. Presentations will cover issues of vital importance to application chromatographers working

in drug discovery and development, molecular diagnostics, food and agriculture, traditional Chinese

medicine, forensics and security, process analytics and energy science industries.

Confirmed speakers include:

Professor Gert Desmet “Current and Future Approaches to Speed

Up HPLC Separations”

Professor Ping Li “HPLC and Hyphenated Techniques for

Analysis of Constituents in Herbal

Medicines”

Professor Alastair Lewis “Trace Pollutant Detection in Challenging

Environments”

Milos Novotny, (Indiana University, USA), Philip Marriott, (RMIT, Australia), Edward Browne, (GSK R&D Singapore), Peter Myers, (University of

Liverpool, UK), Hian Kee Lee, (National University of Singapore), Yi Chen, (Chinese Academy of Sciences, Beijing, China), C. Bor Fuh, (National Chi Nan University,

Taiwan), Y.S. Fung, (Hong Kong University), Yizeng Liang, (Research Center for Modernization of TCM, Changsha, China), Tung-Hu Tsai, (National Yang-Ming

University, Taiwan), Yong-Chien Ling, (National Tsing Hua University, Taiwan), Paul Haddad, (University of Tasmania, Australia), Siu Kwan Sze, (Nanyang

Technical University, Singapore), Eric Chan, (National University of Singapore), Thomas Walczyk, (National University of Singapore), Manfred Raida, (Singapore).

Singapore

ofessor Gert Desm P f Pi Li fessor Alastair Lew

Submitted contributions are invited on all aspects of separation science pertinent to the theme, and

specifically on the following topics:

Call for Papers

www.sepscience.com

Deadline for oral contributions: 15 May

Deadline for poster contributions: 12 June

Instructions for submissions and further

information can be found at

proteomics, lipidomics, metabolomics, biomarker research, human/equine

doping control, forensics

high-resolution separation methods for petrochemical, hydrocarbon and

biodiesel applications

air, soil and water testing, portable field analysis, sample preparation

protocols, industrial emissions

contaminants, traceability, ingredients testing, flavours and fragrances,

marine toxins

high-throughput separations, chiral separations, ADME/PKPD studies,

biopharmaceutical analysis

Pharma

Food

Energy

Enviro

Bioscience

TMCTCM chemistry, fingerprinting, metabolism, quality control

Confirmed sponsors:

For all programme enquiries

email David Hills.For all delegate enquiries

email Jackie Tan.

26–28 AugustBiopolis Science Park, Singapore

CdThe Chrom

DoctorDeveloping new methods for LC-MS analysis

LC-MS methods employed today must cover

a much wider range of sample types and

complexities than ever before. Compared

with isocratic elution, gradient elution

chromatography allows much faster elution

of strongly retained analytes without loss of

resolution of the more polar analytes.

Gradient elution chromatography forms the

cornerstone for the majority of LC-MS methods

developed to date. The choice of buffer, its

concentration and type of organic modifier

used are important considerations when using

MS as the detection system, as does the choice

of HPLC column.

The LC-MS interface

The basic principle of MS is the production

of ions which are subsequently separated

according to their mass-to-charge ratio

(m/z) and detected. The technique provides

information that is both wide ranging and

accurate.

Atmospheric pressure ionization (API) has

greatly expanded the range of compounds

that can be analysed by LC/MS. The analyte is

brought to the interface via a solvent typically

at a rate of 1.0mL/min, depending on column

diameter and method requirements. The API

interface must separate the analytes from

the solvent, ionize the analyte molecules and

maintain a vacuum in the mass detector. The

method by which this is done differentiates

the API processes. Electrospray (ES) gives the

widest range of application, both in terms

of molecular mass and analyte polarity.

Atmospheric pressure chemical ionization

(APCI) may be the method of choice where

slightly more hydrophobic analytes need to be

analysed (Figure 1).

Figure 2 shows basic compounds analysed

in positive ion mode. The data allows for

highly specific identification of analytes and

is more sensitive than traditional UV methods

of detection. The total ion current (TIC) gives

MolecularWeight

100,000

1000

Non-Polar Polar

LC/MSElectrospray

LC/MSAPCIGC/MS

EI & CI

Figure 1

Figure 1: The suitability of different techniques for compounds of different molecular weight.

(A) TIC

(B) m/z=311

(C) m/z=317

LC-MS13 LC-MS14

9 0 6 3

Min

200 240 280 320 340 380

M/Z

312

348

311

Figure 2

Figure 2: A comparison of different mass spectral information.

30 chrom doctor www.sepscience.com

relative abundance of all ions detected.

Those that co-elute can still be isolated and

quantified by plotting their relative abundance

as a function of time (mass chromatograms).

Proportion of organic solvent in the

mobile phase

The ratio of aqueous-to-organic solvent

is particularly important in electrospray

ionization. The efficiency of the electrospray

process depends on the conductivity and

surface tension of the liquid being nebulized.

When the conductivity and/or the surface

tension are too high (i.e., highly aqueous), it

is difficult to produce a stable spray and it is

difficult to vaporize the droplets formed by

the action of the high voltage and nebulizing

gas. The percentage of water used should

not be too high because surface tension

of water is much higher than the surface

tension of methanol or acetonitrile. Additive

concentration will have considerable effect

on conductivity of the nebulized liquid and

consequently should be kept low. These

factors are most important when working at

high flow rates because there is more solvent

to be nebulized and vaporized. One alternative

is to use a sheath liquid which is highly organic

(e.g., IPA) to help the spray and vaporization.

However, this involves a more complex setup,

and may not be suitable for high-throughput

applications. In general, it helps the LC-MS

sensitivity to have at least 20-30% organic.

100% or very high organic content may also

lower the sensitivity, especially where no

additive is used. This is primarily because the

conductivity of organic solvent is too low. A

small percentage of water in the mobile phase

helps the droplet formation.

Buffers/additives

Buffers serve two purposes in LC-MS:

to act as a buffer for the chromatographic

process in the traditional way (i.e. control and

maintain the pH of the mobile phase in order

to keep the ionization state of an analyte

constant), and to adjust the pH of the carrier

solvent (mobile phase) in such a way as to

present the analytes to the MS already in ionic

form.

The most compatible buffers are ammonium

formate, ammonium acetate and ammonium

hydroxyde at concentrations of 10 to 50mM.

The preferred additives are formic and acetic

acids (0.01 to 1%v/v) because they improve

protonation of basic samples in positive

ionization. Other additives occasionally used

include trifluoroacetic acid and trialkylamine

type bases, but these need to be used at low

concentrations (<0.1% v/v) because they may

cause ionization suppression. Non-volatile salts,

such as phosphates and borates, ion pairing

Figure 3

Figure 3: A comparison of the sensitivity of an LC-MS separation with different mobile phase additives.

31chrom doctorseparation science — volume 1 issue 1

agents and inorganic acids should be used

with precaution.

Modern orthogonal (off-axis) sources are

more robust, and have been designed to

operate with nonvolatile buffers/additives and

minimal sample clean-up. In order to achieve

the best response in LC/API-MS, mobile phase

composition must be such that the solution

and gas phase chemistries are optimized

to maximize ionization and eliminate any

components which will cause ion supression.

Additives for optimum MS sensitivity

Some additives provide more sensitivity than

others when used with MS (Figure 3). The

additive used can greatly effect the sensitivity

of the MS ionization source and hence the

sensitivity of the method. Careful consideration

is therefore required on which additive will

give the greatest MS sensitivity. A recent study

of volatile additives showed formic acid to give

the greatest sensitivity

Reducing the concentration of each additive

gave significant increase in sensitivity. For

example, improved sensitivity was observed

when the TFA concentration was reduced

from 0.1% to 0.01% TFA. Chromatographic

performance conditions are shown in

Figure 4. In this example, the additive

employed was 0.01% TFA. High levels of

additive concentration lead to ion suppression

that in turn will lead to loss in sensitivity for the

method.

Balancing UV detection with MS sensitivity

requirements

In the same study diode array detection was

used in series with the MS system. The aim

was to use the chromatographic data from

the diode array to help identify analytes

that either gave rise to poor MS detection

or which were present only as minor peaks

barely visible in the noise of the total ion

chromatogram. Diode array is ideal for this

purpose because it can operate across a wide

wavelength range. Sensitivity of each additive

system towards diode array detection was,

therefore, investigated in the 190-280 nm

wavelength region. The chromatograms in

Figure 4 show clearly the difficulties that can

arise at low wavelength where an unstable

baseline can often lead to loss in sensitivity.

In this case, TFA was found to be a more

attractive additive than formic acid as it

gives better UV transparency, especially at

the lower wavelengths. A compromise was

made whereby the final method used TFA as

the additive in place of formic acid in order

to maximize UV sensitivity, but used at low

concentrations (i.e. 0.01%), to maximize MS

sensitivity with TFA.

Figure 5 shows how the method was used

to analyze some in vitro incubation samples.

Note how the UV diode array chromatogram

can be used to help identify regions of the

total ion chromatogram where sensitivity is

poor. Identification of metabolites can often

be a time consuming process when the peaks

in the total ion chromatogram are lost amidst

the noise of the baseline. On these occasions

identification can often be aided by the

diode array chromatogram where a sensitive

LC method may allow for increased sample

Figure 4

Figure 4: Comparison of two additives with UV detection.

32 chrom doctor www.sepscience.com

detection at low UV wavelength as shown for pethidine.

In this example, the diode array provides a clear

indication for the presence of a metabolite. The lambda

max (not shown) can provide further information in

support of MS data that can aid in structural elucidation

and peak location on the MS total ion chromatogram.

As mentioned earlier, low additive concentrations offer

the reward of increased sensitivity for LC-MS. This is an

important consideration when trying to identify trace

quantities of a drug compound or impurity that may

normally disappear into the noise of the baseline.

The choice of HPLC column used is of key importance

as the quality of the bonded phase and underlying silica

can strongly influence the concentration of additive

required.

This Chrom Doctor article was written by Harold Ritchie,

Thermo Fisher Scientific, Runcorn, Ceshire, UK.

Figure 5

Figure 5: A UV (A) and MS (B) comparison of pethadine and its metabolites.

analytica Anacon India SEPT. 29–OCT. 01, 2009HYDERABAD

LOOK TOTOMORROW

analytica MARCH 23–26, 2010MUNICH

analytica VietnamMARCH 18–20, 2009HANOI

analytica ChinaSEPT. 21–23, 2010SHANGHAI

analytica Anacon India is part of the exhibitionnetwork analytica worldwide—the most impor-tant marketplace in the world for marketableproducts and solutions in the analysis, laboratory-technology and biotechnology sectors. It is abusiness and networking platform and a drivingforce behind future trends and innovations.

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33chrom doctorseparation science — volume 1 issue 1

TuTechnologyupdate

34 technology update www.sepscience.com

6500 Series Accurate-Mass Quadrupole Time-of-Flight

(Q-TOF) LC/MS

Manufacturer: Agilent

Manufacturer’s Description: Using proprietary True Hi-Def TOF technology, Agilent’s 6520 and 6530

bring an unrivaled combination of mass accuracy and resolution, sensitivity, acquisition speed,

and in-spectrum dynamic range to research challenges. The Agilent 6530 Accurate-Mass Q-TOF

LC/MS with Jet Stream thermal gradient focusing technology increases sensitivity by a further

fivefold for even greater analytical confidence. All 6500 Series instruments feature the powerful,

compound-centric MassHunter Workstation software with its data mining and qualitative and

quantitative data analysis capabilities.

Features include typical mass accuracy – sub-1-ppm MS and 2-ppm MS/MS rivals or exceeds

that of much more expensive FTMS and orbital trapping instruments; enhanced mass resolution

– up to 20,000 resolving power without sacrificing spectral acquisition rates; in-spectrum

dynamic range – up to five orders reveal trace-level targets even in the presence of vastly more

abundant compounds; spectral acquisition rates – up to 20 MS or 10 MS/MS spectra per second

for compatibility with high-throughput, rapid-resolution chromatography; time-of-flight mass

range – m/z 20 – 20,000; automated tuning that is unavailable from or unreliable on competitive

instruments, Agilent Jet Stream technology provides 5x greater on-column sensitivity in the 6530,

MassHunter Workstation software is compound-centric with powerful data mining and data

analysis functionality, supplemented by powerful, application-specific software packages that

maximize productivity and result quality for specialized analyses.

Key

Email the company

Product information

Applications

Additional Information

35Technology update separation science — volume 1 issue 1

Accela High-Speed LCManufacturer: Thermo Fisher Scientific

Manufacturer’s Description: The Thermo Scientific Accela high-speed chromatographic system

provides fast, efficient chromatographic separations over an expansive range of flow rates and

pressures. The Accela optimizes the performance of sub-two micron particle columns, providing

seamless operation spanning conventional LC pressures from short LC columns, up to 15,000 psi

for long-column separations of complex bio mixtures.

The Accela Pump assures rapid and reproducible transfer of even complex and aggressive

gradients. This quaternary pump is capable of handling pressures up to 15,000 psi with a delay

volume of only 65 μL, enabling high-speed chromatographic separations.

The Accela Autosampler has a specialized diamond-coated, high-pressure valve that can

handle the rigors of constant high pressure injections while the unique sampling

design enables 30 second injection cycles. This autosampler integrates isothermal

injection and separation to provide reproducibility by eliminating external

environmental influences to the chromatography.

The Accela PDA has been optimized for the detection of high-speed

chromatographic separations. The short 1 cm flow cell pathlength is combined

with a minimized flow cell volume of 2 μL. The low level of dispersion in this

LightPipe flow cell retains peak shape and chromatographic resolution from the

column.

Accela, coupled with the sub-two micron particle columns, provides fast,

controlled separations with high efficiency and resolution, accelerating LC and LC/

MS applications. It offers optimized system delay volumes for fast separations, a

flexible sample format that accepts vials or plates, a quaternary pump capable of

conventional and ultra-high pressures, a patented LightPipe technology providing

maximum sensitivity and resolution and full compatibility with Thermo Scientific

mass spectrometers.

Atlas 8.2 provides digital instrument communication and control of the Accela

pump, PDA detector, and autosampler for injection, data acquisition, and CDS

data processing and reporting as an enterprise-wide client/server CDS. Accela data collected is

processed by Atlas peak detection, integration, reporting and data presentation.


ÄKTAready Liquid Chromatography System

Manufacturer: GE Healthcare

Manufacturer’s Description: GE Healthcare has launched ÄKTAready, a liquid

chromatography system designed for process scale-up and production

for Phase I-III drug development and full-scale production to GLP and

cGMP standards. It has simplified system handling and reduced downtime

between products and batches improves cost efficiency and productivity

by saving time and expenditure for start-up, labour and consumables.

According to GE Healthcare, ÄKTAready operates with ready-to-use,

disposable flow paths, eliminating the risk of cross-contamination and the

need for cleaning and validation of cleaning procedures.

ÄKTAready comprises the chromatography unit, Unicorn software,

and a disposable ReadyToProcess Flow Kit including sensors and

detection flow cells. Unicorn includes an installation wizard that provides

instructions and reports for column installation and ensures correct functionality of

the Flow Kit. All ÄKTA systems use the same software, which enables easy process scale-up and quick

transfer to ÄKTAprocess for use in full cGMP production.

The ÄKTAready system is supported by extensive regulatory product documentation and services, including validation

documentation, product documentation with information about materials used, USP Class VI CFR 177 and AOF certificates

and a Regulatory Support File (RSF).

ÄKTAready is part of GE Healthcare’s portfolio of ReadyToProcess solutions that help increasing the efficiency in

biopharmaceutical production. ReadyToProcess enables lean production schemes by eliminating waste activities in

daily routines of upstream and downstream processing. Products include the WAVE Bioreactor and WAVE Mixer with the

disposable CellBag, chromatography columns as well as normal flow capsules and the ReadyMate disposable aseptic

connectors. The ReadyToProcess portfolio provides maximum flexibility, simplifies and speeds up bioprocessing and is

designed for scalable and smooth operations from fermentation through to purification.


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Automated Liner Exchange – ALEX

Manufacturer: Gerstel

Manufacturer’s Description: Matrix material from samples can contaminate

a GC-liner, leading to peak broadening and loss of analytes through

adsorption in the liner within a few GC runs. Under such conditions,

the analyst is forced to perform frequent liner replacement, an operation which normally requires manual intervention.

Running large series of samples will represent a serious challenge.

The Gerstel Automated Liner EXchance (ALEX) technology automates the task of replacing liners in the Gerstel Cooled

Injection System CIS 4. The ALEX system enables fully automated GC analysis of samples with a high matrix load as well

as ‘dirty’ extracts, which can be directly injected into the CIS 4 using the Gerstel MultiPurpose Sampler MPS. Specially

designed trays can hold up to 14 or 98 CIS 4 liners in individually sealed, contamination-free storage compartments.

In connection with the MPS, the ALEX system enables automated CIS 4 liner exchange at user defined intervals in a

sequence. The Gerstel MAESTRO-Software provides integrated control of the MPS, ALEX system and CIS 4. In combination

with the Agilent ChemStation, a single method and sequence table control the entire system including GC and MS.

Multichannel nanoelectrospray emitters

Manufacturer: Parteq Innovations

Manufacturer’s Description: Researchers at Queen’s University in Ontario, Canada, have

developed a multichannel nanoelectrospray (MCN) emitter that uses a micro-structured

silica fibre as a ‘shower head’ to split the fluidic flow. Electrospray ionization (ESI) has

become the preferred method of coupling liquid separation techniques to a mass

spectrometry (MS) and pulled-glass capillaries are widely employed to improve electrospray

performance at nL/min flow rates. While effective for stabilizing low flow rates, pulled-tip emitters have technical

limitations such as susceptibility to clogging, limited range of possible flow rates and poor tip-to-tip reproducibility.

This new class of ESI-MS emitters offers end-users the same signal sensitivities at 1000-300 nL/min flow rates they have

come expect from currently available high-performance emitters, but with significant improvements in tip robustness. By

virtue of their multichannel construction, MCN emitters last longer and produce almost no fluidic backpressure. Resistant

to clogging, MCN emitters allow the user to plug in the emitter, begin the measurement and walk away. The MCN emitters

can be purchased in packages of 5 or 20.


Lux Chiral HPLC/SFC columns

Manufacturer: Phenomenex

Manufacturer’s Description: Lux is a line of polysaccharide-based columns for the identification and resolution of

enantiomers. Lux columns are offered with two chiral stationary phases (CSPs), both of which use coated derivatized

cellulose as the chiral selector. The two phases combine to create a dependable screening set with a wide range of

selectivity.

Lux Cellulose-1 uses cellulose tris (3, 5-dimethylphenylcarbamate) as the chiral selector and has been demonstrated to

provide better resolution than the current market-leading column in a number of applications. LuxCellulose-2 introduces

a new chiral selector, cellulose tris (3-chloro-4-methylphenylcarbamate), providing a unique stationary phase. This column

offers complementary selectivity to Lux Cellulose-1.

These two phases combine to create a dependable screening set with a wide range of selectivity. Lux columns are

offered in 3 μm and 5 μm particle sizes, packed for analytical-scale use or Axia-packed for preparative applications.

Enantiomers of chiral compounds may have different pharmacological effects in biological systems. Phenomenex

states that the demand for chiral separations is on the rise with FDA mandates that enantiomers of all chiral drugs

(in development) must be screened separately for their pharmacodynamic and pharmacokinetic properties. Other

applications include toxicology, flavour analysis, and chemical and pesticide analysis.


www.sepscience.com


UltiMate 3000 Rapid Separation LC (RSLC) system

Manufacturer: Dionex

Manufacturer’s Description: The UltiMate 3000 Rapid Separation LC (RSLC) system provides

ultrafast LC separations using higher flow rates for increased throughput and small particle sizes

for resolving peaks efficiently. Its combination of high flow rate, high pressure and ultrafast data

collection rate facilitates high peak capacity in short run times. Even separations of 10 peaks in 10

seconds are easily achieved. RSLC system highlights include ultrafast separations at flow rates up

to 5 mL/min and pressure up to 800 bar (11,600 psi) at the same time, in-line split-loop injections

for 15 seconds, no-sample loss injections, reduced backpressure with high temperature column

compartment operating at up to 110 °C and high data collection rate UV detectors (100 Hz),

with variable wavelength, multiple wavelength or diode array (with full

spectral scan). It also features Acclaim RSLC columns (2.2 μm) in

various column formats, convenient method speed-up with

automated calculation and validation tools, significantly

reduced solvent consumption (typically by 70% or more)

and instant results with Chromeleon dynamic data

processing.

As well as supporting UHPLC methods, the UltiMate

3000 RSLC runs conventional LC methods with its flexible

components. The RS Wellplate Autosampler’s patent-

pending injection valve can robustly inject 100 μL at 800

bar pressure. It works with a multitude of different sample

formats, supporting a maximum of 1,167 samples. Column

compartments are available with integrated column

switching valves to use up to 12 columns (of up to 30 cm

length) at the same time. UV detectors come with a selection

of micro and analytical flow cells, available in stainless

steel or PEEK. Integration of any module of the UltiMate

3000 product line into the RSLC system is possible. Each

component stacks and operates together seamlessly, with

full support by Chromeleon chromatography data system.

Date post:	08-Dec-2016
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Journal Separation Science

Documents