©2011 Waters Corporation 1
Novel and Emerging Technologies in Food Analysis
Daniel McMillan
Waters UK
©2011 Waters Corporation 9
RADAR Simultaneous full scan & MRM
Rapid electronics allow instrument to switch between MRM and full scan (FS takes 100ms)
No loss of MRM data quality
Added information can be gained from full scan
— Monitor matrix
— Assess for possibility of matrix effects
— Added information for sample prep method development
— Search for significant non-targeted compounds
©2011 Waters Corporation 10
MRM
Full Scan
Example - Sewage Sludge Analysis
Spectra from humics and fulvics elution region
Suspected anionic surfactant LAS
Estriol
©2011 Waters Corporation 11
Diethylstilbestrol
17β estradiol & 17α estradiol
Ethinyl estradiol
Dual Scan-MRM Matrix Monitoring TIC
Elution of matrix components including C11 LAS 311 m/z
Possible matrix effects
Example - Sewage Sludge Analysis
©2011 Waters Corporation 14
Impact of sensitivity RADAR data for complex samples
Original Sample
x10 dilution
x100 dilution
Acquisitions allow levels of matrix to
be monitored during MRM analysis
©2011 Waters Corporation 15
PFHxA
x100
x10
x 2.
5
decr
ease
x
10.2
de
crea
se
802,268
318,971
31,270
Peak Area
Impact of sensitivity Diluting Away Matrix Effects
Expected RT
Reducing ion suppression
-75% suppression
©2011 Waters Corporation 16
Grape
Avocado
Marjoram
Ginger
What is in the samples? Ultra trace analysis
©2011 Waters Corporation 17
Ultra trace analysis Matrix effects
DisQuE Ginger QC extract
pesticides @ 0.01 mg/kg (10 ppb)
Cyromazine
Simetryn
Atrazine
Azinphos-ethyl
Poor peak shape
Low calculated recoveries
High Matrix background
©2011 Waters Corporation 18
Ultra trace analysis Reduced matrix effects
Cyromazine
Simetryn
Atrazine
Azinphos-ethyl
10 x dilution of Ginger extract
All peaks detected, recoveries good
Reduced Matrix background
Improved peak shape
-54%
-58%
-47%
Reduced ion suppression
©2012 Waters Corporation 19
Rapid Analysis of Bisphenols (BP) A, B & E
in Baby Food and Infant Formula
using ACQUITY UPLC-Xevo TQD
Food Packaging Seminar Module 1 of 4
©2012 Waters Corporation 20
BPA Background
What is BPA? – First synthesised in 1905 by Thomas Zincke of the University of Marburg, Germany
– In 1953, Dr. Hermann Schnell of Bayer in Germany and Dr. Dan Fox of General Electric in the United States independently developed manufacturing processes for a new plastic material, polycarbonate, using BPA as the starting material.
– It has been an important industrial chemical not only to make polycarbonate plastic but also epoxy resins; both of which are used in a wide variety of applications.
o Polycarbonate plastics are used in baby bottles, tableware and other food containers
• (Eyeglass lenses, medical equipment, digital media (e.g., CDs and DVDs), cell phones, consumer electronics, computers and other electrical equipment, …)
o Epoxy resins are used in can coatings, industrial floorings, adhesives, industrial protective coatings, powder coatings, and printed circuit boards
©2012 Waters Corporation 21
BPA Background
What are the potential health issues?
– Can leach into food from the epoxy resin lining of cans and from consumer products such as polycarbonate tableware, food storage containers, water bottles, and baby bottles.
– BPA is a weak "estrogen like" endocrine disruptor which can mimic circulatory endogenous hormones and is suspected to be linked to a higher incidence of certain cancers, reproductive disorders, diabetes and heart disease in humans
©2012 Waters Corporation 22
International Regulations
Canada - First country to take action, listed it as a toxic substance
US-FDA - Released a report in 2010 expressing some concern regarding exposure of fetuses, infants and young children to BPA. Report encourages further studies into the safety of BPA
European Union - EU directive (2011/8/EU) has banned the use of BPA in infant feeding bottles
o France Bans Bisphenol A in All Food Contact Materials: French National Assembly has been voting to ban BPA from 2014 on. Concerning baby nutrition products the ban will step into force in 2013
Japan - Between 1998-2003, canning industry voluntarily replaced its BPA-containing epoxy resin can liners with BPA-free polyethylene terephthalate (PET) in many of its products
©2012 Waters Corporation 24
ACQUITY UPLC-Xevo TQD Conditions
UPLC system ACQUITY UPLC System
Column: ACQUITY UPLC BEH C18 2.1 x 50 mm, 1.7 µm
Column temp 40 °C
Mobile phase A 0.5 % NH4OH in H2O
Mobile phase B 0.5 % NH4OH in MeOH
Elution 3 minute linear gradient from 5% (B) to 95% (B)
Flow Rate 0.5 mL/min
Injection volume
50 µL
MS System XEVO TQD
Ionization mode ESI Negative
Capillary voltage 3.5 kV
Cone voltage 30.0 V
Source temp 140 °C
Desolvation temp 350 °C
Desolvation gas 550 L/hr
Cone gas 50 L/hr
Routine targeted quantification
with information rich qualitative data
©2012 Waters Corporation 25
• Automatically sets up instrument, • Optimises MRMs and performs system suitability
IntelliStart
• Methods generated automatically by QuanPedia, • Clear flagging when data is out of regulatory tolerance
• Reduced risk of errors
TargetLynx
• Long term system monitoring, Lab management tool
TrendPlot
• Generates optimised methods, time scheduled MRMs
Quanpedia
• Real time QC, Intelligent ‘on the fly’ decision making
QCMonitor
Waters QUAN Workflow
©2012 Waters Corporation 26
Other Related Compounds…?
BPB ( 2,2 Bis(4-hydroxyphenyl)butane)
BPA ( 2,2-Bis(4-hydroxyphenyl)propane)
BPE (1,1-Bis(4-hydroxyphenyl)ethane)
©2012 Waters Corporation 27
Developing a Suitable Method
Analysis of food samples can be challenging – need to develop a sample preparation that can:
o Reduce matrix effects (e.g. peak distortion, ion suppression…)
o Extract relevant compounds
o Provide a robust, repeatable method
Method development to investigate different extraction techniques can be time intensive
Useful to have tools that support this process and help improve lab efficiency RADAR Matrix Calculator
LC-MS/MS Analysis
Modify analytical method
LC-MS/MS Analysis
Modify sample
preparation
©2012 Waters Corporation 28
The advantage of RADAR Full scan and MRM in one analysis
1. Protein precipitation 2. SPE – OASIS HLB
1. Protein precipitation 2. DisQUE (QuEChERS) 3. SPE – OASIS HLB
©2012 Waters Corporation 29
oReduce matrix effects
oDevelop sample prep procedure
oExtract relevant compounds
oProvide a robust, repeatable method
Use results from … to Modify Sample Prep Procedure
©2012 Waters Corporation 30
Part 3 – OASIS HLB
Part 1 – Protein precipitation Part 2 - DisQUE
Optimised Sample Prep Protocol
Precipitation (10g sample, 10mL CH3CN) Centrifuge and collect supernatant
Add contents from DisQuE tube 1. Shake.
Centrifuge & collect 10 mL supernatant
Add contents from DisQuE tube 2. Shake.
Centrifuge & collect supernatant.
Dilute supernatant with 70 ml H2O. Load on Oasis HLB (3cc)
Wash – 2 mL 40 % MeOH Elute – 1 mL 100 % MeOH
Dilute eluate with 1 mL H2O Inject 50 µL for LC-MS/MS
©2012 Waters Corporation 32
oReduce matrix effects
oDevelop sample prep procedure
oExtract relevant compounds
oProvide a robust, repeatable method
Calculating the Matrix effect with the New Sample Prep Method
©2012 Waters Corporation 33
Correlation coef f icient: r = 0.998164, r^2 = 0.996332
Response type: Internal Std ( Ref 4 ), Area * ( IS Conc. / IS Area )
Curve type: Linear, Origin: Exclude, Weighting: 1/x^2,
Conc-0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0
Resp
onse
-0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10Bisphenol Infant Formula
(10 pg/μL spike) BPA 102% (3.2%) BPB 95% (5.5%) BPE 81% (4.6%)
Linearity and Repeatability
Bisphenol Baby food (20 pg/μL spike)
BPA 110% (7.8%) BPB 112% (6.7%) BPE 99% (6.1%)
©2012 Waters Corporation 35
Conclusions
BPA – WW very topical compound
Analysis of BPA in food and beverage samples require a robust method
Waters System Solution is rapid and robust
o Sample preparation- DisQUE and OASIS HLB
o Reliable chromatography - ACQUITY UPLC & ACQUITY BEH C18 columns
o Robust and Repeatable results - Xevo TQD
o Matrix monitoring tools - RADAR, Matrix Calculator
o Waters QUAN Workflow - TargetLynx, TrendPlot
©2011 Waters Corporation
Using UPLC-QTof and UPLC-Ion Mobility-Tof
for Food and Environmental Research
©2012 Waters Corporation 47
Food & Environmental Research
Studies involve the analysis of high and trace level compounds, and transformation products / break-down / metabolised compounds
Target compounds
Quantify compounds
Discover new compounds
©2012 Waters Corporation 48
Achieving these aims: Key Analytical LC-MS Challenges
Ionise compounds
Separate compounds / Maximise peak capacity / Minimise matrix effect
Confidently detect compounds at high and low levels
Analyse & interpret data acquired
Obtain repeatable results
©2012 Waters Corporation 49
Food & Environmental Research High Resolution Chromatography
From HPLC separations…. …to ACQUITY UPLC separations
©2012 Waters Corporation 50
Food & Environmental Research High Resolution MS – Xevo G2 QTof MS
Ionisation flexibility – ESI, APCI, APPI, APGC, MALDI, DESI, DART…
Ability to detect ionised compounds at high and trace level compounds in complex matrix
– Compatible with high resolution UPLC – In spectrum dynamic range in complex matrices – Comprehensive data acquisition with accurate mass data for precursor
and product ions) – MSE – Confidence in mass accuracy and stability
Analyse and interpret data – Variety of apps managers within MassLynx to support targeted and
research-based workflows
Obtain repeatable results – IntelliStart, QuanTof technology
TRIZAIC™ Source with nanoTile Technology. Plug & Play nanoFlow
APGC – Atmospheric Pressure Gas Chromatography
ASAP – Atmospheric Pressure Solids Analysis Probe
MALDI nanoFlow ESI APCi ESCi APPI TRIZAIC ASAP APGC
©2012 Waters Corporation 51
Ability to detect extracted & ionised compounds - APGC
51
MBT
DOcT
TPhT
TOcT
DPhT
DBT
MOcT
TPrT TBT
MPhT
Organotin family at
100 ppt level
©2012 Waters Corporation 52
m/z202.050
%
0
100 202.0434
Detection of trace level compounds in complex matrices – Xevo G2 QTof MS
Scan No. Measured Mass ∆M (mDa) ∆M (ppm)
505 202.0434 -0.50 -2.47506 202.0439 0.00 0.00507 202.0436 -0.30 -1.48508 202.0442 0.30 1.48509 202.0437 -0.20 -0.99510 202.0440 0.10 0.49511 202.0439 0.00 0.00512 202.0435 -0.40 -1.98513 202.0441 0.20 0.99514 202.0435 -0.40 -1.98515 202.0433 -0.60 -2.97516 202.0442 0.30 1.48
RMS = 0.30 1.50
m/z202.050
%
0
100 202.0434
m/z202.050
%
0
100 202.0439
m/z202.050
%
0
100 202.0436
m/z202.050
%
0
100 202.0442
m/z202.050
%
0
100 202.0437
m/z202.050
%
0
100 202.0440
m/z202.050
%
0
100 202.0439
m/z202.050
%
0
100 202.0435
m/z202.050
%
0
100 202.0441
m/z202.050
%
0
100 202.0435
m/z202.050
%
0
100 202.0433
m/z202.050
%
0
100 202.0442
202.0757
Thiabendazole 20 ppb
“Confidence in mass accuracy and stability”
Samples provided by SASA, UK
Excellent mass accuracy across peak
©2012 Waters Corporation 53
Ability to detect extracted & ionised compounds - QTof MSE
• “Comprehensive data acquisition”
Simetryn m/z 96.0559
Desmetryn m/z 172.0650
Time2.40 2.50 2.60 2.70
%
0
100
Simetryn m/z 214.1126
Desmetryn m/z 214.1126
Selective MSE fragments for each compound
m/z50 100 150 200 250
%0
100 172.0650
82.0401124.0617
214.1124
m/z50 100 150 200 250
%
0
100 214.1128
124.0874
96.0559144.0597
215.1148
Desmetryn
Simetryn or
Desmetryn?
©2012 Waters Corporation 54
However…
Even the highest resolution instruments struggle to find and confirm the closely eluting isobaric compounds
An additional separation dimension would be a plus – On the chromatography side: 2D-LC… – On the MS side: Ion mobility MS…
©2012 Waters Corporation 55
SYNAPT G2 HDMS Ion mobility MS Food & Environmental Research
SYNAPT G2 HDMS brings an additional dimension for the characterization of complex mixtures:
• Cleaner MS spectra - data interpretation easier
• Isobaric compounds, isomers, conformers to be separated by ion mobility
Studies have shown that utilising the ion mobility data, compounds not previously separated and recorded with other LC-MS approaches can be distinguished
Ionisation flexibility – ESI, APCI, APPI, APGC, MALDI, DESI, DART…
Ability to detect ionised compounds at high and trace level compounds in complex matrix
– Compatible with high resolution UPLC – In spectrum dynamic range in complex matrices – Comprehensive data acquisition with accurate mass data for precursor
and product ions) – MSE – Confidence in mass accuracy and stability
Analyse and interpret data – Variety of apps managers within MassLynx to support targeted and
research-based workflows
Obtain repeatable results – IntelliStart, QuanTof technology
©2012 Waters Corporation 56
Schematic of the instrument
SYNAPT G2-S High Definition MS (HDMS)
Size
Shape
Charge
©2012 Waters Corporation 57
Provided As Part of A Study of Environmental Pollutants In Water *
* Study for the ICRA
Analysis of a Unknown Mixture of Pharmaceutical Standards
UPLC MSE
Ion Mobility-MS Drift Time
©2012 Waters Corporation 58
Experimental Aims ACQUITY UPLC & SYNAPT G2 HDMS
AIMS Identify any pharmaceutical compounds found in environmental samples Quantify the compounds detected in the influent, waste and natural water
– A dilution series of 0.1 to 200 ppb of the standard pharmaceuticals was prepared.
SYSTEM SOLUTION ACQUITY UPLC to produce high resolution chromatography
SYNAPT G2 HDMS offers: – MSE data (high resolution exact mass precursor and fragment ion m/z) – Ion mobility: provides extra dimension of separation to separate out matrix
interferences and isobaric environmental compounds
Process data using – Automatic screen using data base – Use standards analysed to quantify positively detected components
©2012 Waters Corporation 59
Extracted Mass Chromatogram @ m/z 332.1410
Conventional extracted mass chromatogram m/z 332.1410 shows one peak at 1.92 minutes
But with ion mobility…. (dt = drift time)
©2012 Waters Corporation 60
There are Two Peaks!! Can only be seen with Ion Mobility Data
4947_214.raw:1
4947_214.raw : 1
XIC of 332.1407 from the LC run
Ion Mobility Data Two components are detected
@ m/z 332.1407
Different Drift Times for the two isobaric compounds at R.T 1.92
TWO COMPONENTS - SAME EXACT MASS!! m/z 332.1407
©2012 Waters Corporation 61
3D View Mobility Data Co-eluting Isobaric Masses at 1.92 mins
Ciprofloxacin (M+H=m/z 332.1410)
???
©2012 Waters Corporation 62
CAERULEA EDULIS
INCARNATA ALATA
In collaboration with: C. A. M. Pereira, J. H. Yariwake Universidade de Sao Paulo, Brazil
Utilising the Increased Peak Capacity of
UPLC and SYNAPT HDMS to Overcome Sample
Complexity -
Passiflora Samples UPLC MSE
Ion Mobility-MS (IM-MS)
©2012 Waters Corporation 63
Marker Profiling of Passiflora Edulis EXTRACT PE 10:1
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
%
0
100EXTRACTTEST RLMM0230410_028 Sm (Mn, 1x2) 2: TOF MS ES+
BPI5.44e4
8.06
7.59
7.141.570.30
0.721.13
2.68
2.541.94
4.403.23
3.464.97
6.896.235.72 6.66
7.84
8.77
8.17
8.9713.63
9.63
9.60
9.12
9.20
10.709.73
10.39
9.95
11.1111.32 13.47
12.3711.97 12.79
13.73
14.9514.28
14.36 15.1715.40
EXTRACTTEST RLMM0230410_028 1: TOF MS ES+BPI
1.77e52.68
1.220.451.13
0.650.72
1.57
2.552.14
7.607.58
2.717.14
2.92
3.75
3.71
3.45
4.39
3.96 6.744.96 5.07 6.636.22
7.16
7.17
8.077.61
8.04
8.38
8.52
8.77
10.399.73
8.78
9.60
9.01
9.74
10.31
10.70
12.70
11.11
11.9711.18
11.89
12.72
13.63
13.14
14.9414.07 14.90 15.17
15.23
15.40
MS
MSe
©2012 Waters Corporation 64
Co-eluting compounds in a complex data set?
Use ion mobility to help clean up mass spectra
©2012 Waters Corporation 65
UPLC-SYNAPT HDMS data Without ion mobility
Orientin at 8.3 min region
HDMS not activated
OOH
OHOH
O
OH
OH
OH O
OH
OH
©2012 Waters Corporation 66
UPLC-SYNAPT HDMS data Ion mobility Activated
Mobility resolved orientin at 8.3 min region
dt 113 ms HDMS activated
©2012 Waters Corporation 67
Separation of isomers in complex matrices:
Profiling of Super Fruit: Tangerine Juice UPLC
MSE Ion Mobility-MS with TAP -
(Time Aligned Parallel) fragmentation Drift Time
Collisional Cross Section
©2012 Waters Corporation 68
UPLC/Qtof MS - Tangerine Juice
Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50
%
0
100Exract Mobility MM060710_105 1: TOF MS ES-
BPI2.51e5
BPI for tangerine juice profiling
Conventional BPI plot of sample comprised of flavonoids,
polyphenols, polysaccharides.
©2012 Waters Corporation 69
UPLC/Mobility profiling of tangerine juice
Conventional MS data indicates one component
present at 3.25 mins
Extracted ion chromatogram
m/z 609
©2012 Waters Corporation 70
Are there any isomers, conformers, stereoisomers present?
Use additional dimension of ion mobility with MSE Data Viewer
©2012 Waters Corporation 71
MSE Data Viewer Software: Hesperidin Isomers - 1
MSE Data Viewer filters out components with same mass
& different drift times
MSE data viewer filters out components with same mass retention time and different
drift times
m/z 609.1842
RT 3.25
Drift Time 161.805
©2012 Waters Corporation 72
MSE Data Viewer Software: Hesperidin Isomers - 2
MSE Data Viewer filters out components with same mass
& different drift times
MSE data viewer filters out components with same mass retention time and different
drift times
m/z 609.1843
RT 3.25
Drift Time 176.204
©2012 Waters Corporation 73
tangerine conc
m/z50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150
%
0
100
m/z50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150
%
0
100
Exract Mobility MM060710_105_dt_01 175 (9.396) AM2 (Ar,20000.0,554.26,0.00,LS 3); Cm (162:185) 1: TOF MS ES-1.01e5301.0714
286.0471
302.0740
609.1801325.0710
Exract Mobility MM060710_105_dt_02 161 (8.640) AM2 (Ar,20000.0,554.26,0.00,LS 3); Cm (148:173) 1: TOF MS ES-1.86e5301.0711
302.0744
609.1826325.0716
CH3 H
H
OO O CH3
OHOH
OHOHOH
OHO
Fragment= C16H13O6Mass=301.0712Error ppm= 0.7
Fragment= C16H13O6Mass=301.0712Error ppm= -0.3
M-H=C28H33O15Mass=609.1819Error ppm= -3.0
M-H=C28H33O15Mass=609.1819Error ppm= 1.1
CH3 H
H
OO O CH3
OHOH
OHOHOH
OHO
MSE fragmentation spectra of mobility separated Hesperidin isomers
Fragment: C16H13O6 Theoretical Mass: 301. 0712
Error (ppm): 0.7
M-H: C28H33O15 Theoretical Mass: 609. 1819
Error (ppm): 1.1
Fragment: C16H13O6 Theoretical Mass: 301. 0712
Error (ppm): -0.3
M-H: C28H33O15 Theoretical Mass: 609. 1819
Error (ppm): -3.0
©2012 Waters Corporation 74
m/z50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150
%
0
100Exract Mobility MM060710_109 348 (3.246) AM2 (Ar,20000.0,554.26,0.00,LS 1); Cm (348:350) 2: TOF MS ES-
7.67e4301.0703
164.0101
151.0023
136.0152
134.0361
108.0200
107.0125
242.0566
201.0172
609.1812302.0725
325.0697
603.2435610.1832
693.2762
”TAP” Fragmentation for Hesperidin m/z 609.1812
TAP (time aligned parallel) fragmentation: 609 has fragmented ( ) in trap to produce m/z 301 fragment and then fragmented again in the transfer region to break up the aglycone
©2012 Waters Corporation 75
3D view TAP fragmentation Hesperidin isomers
Mobility Separated Hi Energy Trap fragments (+ precursor)
Mobility Separated Hi Energy Transfer fragments of fragments
m/z 609.1812
m/z 301.0703
m/z 164.0101
©2012 Waters Corporation 77
One step further with Ion Mobility: Determining collision cross sections
MS Structural Confirmation can now be based on:
– Retention time - High resolution UPLC separation
– High resolution MSE data
o Exact mass precursor
o Time aligned exact mass fragments
– Ion mobility separation
– Collision cross section
– TAP fragmentation
©2012 Waters Corporation 78
Benefits of SYNAPT HDMS
High resolution ACQUITY UPLC data with high quality QuanTof MS technology
MSE provides comprehensive record of all precursor & fragment ions
Ion mobility separation – Cleaner MS spectra - data interpretation easier – Isobaric compounds, isomers, conformers to be separated by ion
mobility
Advanced software tools – MSE data viewer: automatically filters the Tof and IMS data for the
analyst – MassFragment: compares theoretical and observed fragmentations to
confirm compound structure – MarkerLynx: statistical data comparison – ChromaLynx find unknowns peaks in a complex chromatogram – Metabolynx: metabolite ID in a complex sample, based on exact
mass information
©2011 Waters Corporation 87
Summary
Emerging analytical challenges require new technologies
New technologies developed — Separation (UPLC), Limits of detection (MS), Other new ideas (e.g.
RADAR)
Extended capabilities of new instruments
— Complimentary techniques for more information
Working together can provide the best knowledge of — Market requirements (food and environmental issues) — Technology solutions / requirements