©2007 Waters Corporation

Food and Environmental residue analysisFood and Environmental residue analysis

44rdrd Austrian Technology SymposiumAustrian Technology Symposium

Vienna, 26 April 2007Vienna, 26 April 2007

Dr Sandra Rontree Dr Sandra Rontree

©2007 Waters Corporation 2

OverviewOverview

Application of ACQUITY TQD for the analysis of pesticide residues in baby food

The application of UPLC-Travelling Wave Tandem Mass Spectrometry for multiresidue steroid analysis- But now what do we do with all the data generated.

Application of Biomarkers in the Food Industry

Multi residue analysis of EU black list priority pollutants in drinking and surface water using solid phase extraction and GC Tandem Quadrupole MSMS

©2007 Waters Corporation

Application of ACQUITY TQD Application of ACQUITY TQD for the analysis of pesticide for the analysis of pesticide

residues in baby foodresidues in baby food

©2007 Waters Corporation 4

EU LegislationEU Legislation

369/05/EC amending 91/414/EC — Harmonised for all pesticide/product combinations

— Range from “not detectable” to approximately 50mg/kg

— “Not detectable” defaults to 0.01 mg/kg if no other level is assigned

Recommendation 2006/26/EC of 18th January 2006— Coordinated monitoring programme to ensure compliance with

maximum levels of pesticide residues in or on cereals and certain other products of plant origin

EU Residue Monitoring Programme 2005-2007*— 55 active ingredients in various foods, including potatoes,

oranges and baby foods

*AGROW 468 (2005) 9

©2007 Waters Corporation 5

Quantitative Trace AnalysisQuantitative Trace AnalysisRequirementsRequirements

High selectivity — Reduce or eliminate matrix interferences

High sensitivity— Low default reporting limit of 0.01mg/kg

— Reproducibility, stability and dynamic range

— Accurate quantification of targets at low levels in matrix

Confirmation of identity— 2 MRM transitions per compound

Ruggedness— Complex sample matrices, reduced or no sample clean-up

Sample throughput— Multiple targets in one run

©2007 Waters Corporation 6

Confirmatory Analysis by UPLCConfirmatory Analysis by UPLC--TQDTQD

ACQUITY UPLCTM

Tandem quadrupole detector TQD

Multiple Reaction Monitoring (MRM) transitions— Precursor to product ion transition

— Argon collision induced reaction that is structure specific

— Two transitions were chosen for each pesticide residue

Confirmation criteria— Quality control procedures for pesticide residue analysis

(SANCO/10232/2006) of 24th March 2006

— Dependent on relative abundance of the two transitions

o >0.5 ± 20% >0.2 < 0.5 ± 25%

o >0.1 < 0.2 ± 30% < 0.1 ± 50%

— Ratio used to confirm/reject analytes in the extracts

©2007 Waters Corporation 7

UPLCUPLC--TQDTQDMassLynx v 4.1 with TargetLynx

©2007 Waters Corporation 8

The promise of the van Deemter plot

UPLC UPLC –– Using Smaller ParticlesUsing Smaller ParticlesThe enabler of productivityThe enabler of productivity

©2007 Waters Corporation 9

Waters ACQUITY UPLCWaters ACQUITY UPLCTMTM SystemSystem

Designed for rapid analysis

Columns: sub 2μm particles

System pressure: 15,000 psi

Reduced peak width

Increased peak height

Improved sensitivity

Reduced spectral overlap in complex mixtures

Improved MS spectral data

Encased 16mm

Microchip

Holder

eCordTM Technology

©2007 Waters Corporation 10

Why UPLCWhy UPLC--MS?MS?

UPLC provides the following benefits for MS:

— Improved resolution for complex mixture analysis

— Improved resolution can reduce MS ion suppression by separating species that co-elute in conventional HPLC – improved MRM detection limits

— Shorter analytical run times without compromising chromatographic resolution – increasing sample throughput

— Narrower chromatographic peaks effectively increase concentration of analytes entering the MS source – increasing signal intensity and improving detection limits.

©2007 Waters Corporation 11

UPLCUPLC--MS/MS MS/MS vsvs HPLCHPLC--MS/MSMS/MS

Peak height increase 3.7 times.

Signal to noise ratio 4.7 times increase

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

%

0

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

%

0

QM_HPLC_ESI+_4Compound_041205_010 MRM of 4 Channels ES+ 309 > 280.84

1.06e5Height

0.9928719

QM_UPLC_ESI+_2Compound_042605_010 MRM of 2 Channels ES+ 309 > 280.84

1.06e5Height

0.54105629

UPLC-MS/MS

HPLC-MS/MS

ESI+, Alprazolam

©2007 Waters Corporation 12

100 ms Dwell Time, 10 ms Delay

0

%

0.63

100 105 110 115 120 125 130 Scan0

%

0.63 Peak Width = 1.8 s

Points Across Peak = 7

0.25 0.75 1.25 1.75Time

Convert the x axis to scan number

UPLC Compatibility:UPLC Compatibility:Narrow peaks = less data points?Narrow peaks = less data points?

©2007 Waters Corporation 13

Peak Width 1.8 s

100 ms Dwell Time, 10 ms Delay

0

%

0.63

100 105 110 115 120 125 130 Scan0

%

0.63 Peak Width = 1.8 s

Points Across Peak = 7

Time

Convert the x axis to scan number

0.25 0.75 1.25 1.75

Points Across Peak = 60

0.25 0.75 1.25 1.75Time0

%

0.63

1100 1200 1300 1400 Scan0

%

0.63

5 ms Dwell Time, 5 ms Delay

Convert the x axis to scan number

UPLC Compatibility:UPLC Compatibility:Narrow peaks = less data points?Narrow peaks = less data points?

©2007 Waters Corporation 14

Do you need a high MRM acquisition rate? Do you need a high MRM acquisition rate?

The cycle time per MRM transition is composed of two parameters:

— a dwell period where ions are monitored

— an inter-channel delay period between successive MRM transitions to allow ions to be cleared from the collision cell.

Following the inter-channel delay the collision cell must be rapidly filled with ions of the next transition.

If this does not occur rapidly enough (i.e., when the dwell time is very short) the signal will fall in intensity.

©2007 Waters Corporation 15

TT--WaveWave™™ TechnologyTechnology

Fast MRM acquisitions— 100 data points/second

— Maintains sensitivity

— Minimises cross-talk

Improved scanning data— Precursor ion mode— Neutral loss mode

The travelling wave device described here is similar to that described by Kirchner in US Patent 5,206,506 (1993).

time

ions

Travelling Wave Pulse

©2007 Waters Corporation 16

Dwell Delay Polarity Dwell Polarity Points+ve -ve +ve ion Switch -ve ion Switch Total 3 sec

1 0.050 0.020 0.070 432 0.100 0.040 0.140 213 0.150 0.060 0.210 144 0.200 0.080 0.280 115 0.250 0.100 0.350 91 1 0.050 0.020 0.100 0.050 0.100 0.320 92 1 0.100 0.040 0.100 0.050 0.100 0.390 83 1 0.150 0.060 0.100 0.050 0.100 0.460 74 1 0.200 0.080 0.100 0.050 0.100 0.530 65 1 0.250 0.100 0.100 0.050 0.100 0.600 5

Dwell Delay Polarity Dwell Polarity Points+ve -ve +ve ion Switch -ve ion Switch Total 3 sec

1 0.005 0.005 0.010 3002 0.010 0.010 0.020 1503 0.015 0.015 0.030 1004 0.020 0.020 0.040 755 0.025 0.025 0.050 601 1 0.005 0.005 0.020 0.005 0.020 0.055 552 1 0.010 0.010 0.020 0.005 0.020 0.065 463 1 0.015 0.015 0.020 0.005 0.020 0.075 404 1 0.020 0.020 0.020 0.005 0.020 0.085 355 1 0.025 0.025 0.020 0.005 0.020 0.095 32

Compounds per run

Compounds per run

Quattro Instrument

MRM Dwell = 50ms

Delay = 20ms

Polarity switch = 100ms

TQD

MRM Dwell = 5ms

Delay = 5ms

Polarity switch = 20ms

©2007 Waters Corporation 17

Dwell Delay Polarity Dwell Polarity Points+ve -ve +ve ion Switch -ve ion Switch Total 3 sec

1 0.050 0.020 0.070 432 0.100 0.040 0.140 213 0.150 0.060 0.210 144 0.200 0.080 0.280 115 0.250 0.100 0.350 91 1 0.050 0.020 0.100 0.050 0.100 0.320 92 1 0.100 0.040 0.100 0.050 0.100 0.390 83 1 0.150 0.060 0.100 0.050 0.100 0.460 74 1 0.200 0.080 0.100 0.050 0.100 0.530 65 1 0.250 0.100 0.100 0.050 0.100 0.600 5

Dwell Delay Polarity Dwell Polarity Points+ve -ve +ve ion Switch -ve ion Switch Total 3 sec

1 0.005 0.005 0.010 3002 0.010 0.010 0.020 1503 0.015 0.015 0.030 1004 0.020 0.020 0.040 755 0.025 0.025 0.050 601 1 0.005 0.005 0.020 0.005 0.020 0.055 552 1 0.010 0.010 0.020 0.005 0.020 0.065 463 1 0.015 0.015 0.020 0.005 0.020 0.075 404 1 0.020 0.020 0.020 0.005 0.020 0.085 355 1 0.025 0.025 0.020 0.005 0.020 0.095 32

Compounds per run

Compounds per run

Quattro Instrument

MRM Dwell = 50ms

Delay = 20ms

Polarity switch = 100ms

TQD

MRM Dwell = 5ms

Delay = 5ms

Polarity switch = 20ms

©2007 Waters Corporation 18

Waters TQ DetectorWaters TQ DetectorKey FeaturesKey Features

Tandem quadrupole mass spectrometer

Small footprint :—Only 13” wide

—25% smaller box than Quattro micro

Z-Spray ion sources—ESI and ESCi are standard

—IonSabre APCI, APPI options

—Integrated infusion fluidics

—Tool free routine maintenance

T-Wave collision cell for Hi-Speed UPLC-MS/MS

Scan rate up to 10,000 Da per second

Polarity switching in 20 ms

©2007 Waters Corporation 19

TQ DetectorTQ DetectorMassLynx ViewMassLynx View

Short-cut button to TQ console page

IntelliStart warns the analyst when the TQ Detector is not ready

©2007 Waters Corporation 20

Tandem Quadrupole MS/MS Tandem Quadrupole MS/MS AdvantagesAdvantages

High Selectivity —reduce or eliminate matrix interferences

High Sensitivity—trace levels - low reporting limits

Quantitative Accuracy—reproducibility, stability and dynamic range

—accurate quantitation of targets at low levels in matrix

Robustness—complex sample matrices, reduced sample clean-up

©2007 Waters Corporation 21

Waters Waters IntelliStartIntelliStartTMTM SystemSystem

IntelliStart is designed to automatically monitor Instrument ‘health’ to ensure it is ready for use.

Where possible, the system may also take corrective action to rectify any failed system checks

IntelliStart comprises:— Automated system checking software

— Diagnostic electronics

— Integrated fluidics device

— 2 built-in vial locations for set-up/user solutions

©2007 Waters Corporation 22

Sample PreparationSample Preparation

Extraction of the baby food, orange and potato samples— Weigh 10g homogenised sample into a centrifuge tube

— Add 10ml acetonitrile with 1% acetic acid, 4g anhydrous MgSO4

and 1.66g sodium acetate and shake immediately

— Centrifuge at 4300g for 5min

— Transfer 1ml aliquot of acetonitrile layer to a micro-centrifuge vial

— Add 150mg anhydrous MgSO4 + 50mg PSA and vortex for 30s

— Centrifuge at 5000g for 1min

— Transfer 100μl into a LC vial and add 900μl water

— Submit for LC/MS/MS analysis

©2007 Waters Corporation 23

Acquity UPLC BEH C18 Column, 2.1 x 50mm, 1.7μm at 40°CFlow rate = 0.6ml/min

Mobile phase— Solvent A, Water

— Solvent B, Methanol

Injection volume = 20μl

UPLC ConditionsUPLC Conditions

100% B4min

100% B5min

90% A0minTotal run time

7min

©2007 Waters Corporation 24

MS ConditionsMS Conditions

MS System: TQD Detector

Electrospray — Positive and negative switching in 0.02s

— Capillary voltage = 2.5 kV

Cone Voltage — 35 V

Desolvation gas— Nitrogen at 400 °C with a flow rate of 800l/hr

Cone gas— Nitrogen with a flow rate of 50l/hr

Source temperature— 140 °C

Collision gas— Argon at a cell pressure of 4.0 x 10-3 mBar

©2007 Waters Corporation 25

MRM ConditionsMRM Conditions

10163>106

8150.025

163>880.85Methomyl

7223>76

12230.015

223>860.72Aldicarb sulfone

7223>166

10170.015

223>1060.68Butoxycarboxim

10207>132

14160.015

207>890.64Aldicarb sulfoxide

36218>79

17250.015

218>1050.61Pymetrozine

6207>132

12170.015

207>750.62Butocarboxim sulfoxide

15214>155

12200.015

214>1830.60Omethoate

18184>125

8160.015

184>1430.53Acephate

13142>125

14220.015

142>940.43Methamidophos

Collision Energy (eV)Cone Voltage (V)Dwell time (s)MRM TransitionsRTPesticide

©2007 Waters Corporation 26

MRM ConditionsMRM Conditions

18199>111

8170.020

199>1281.41Cymoxanil

15223>56

20270.020

223>1261.32Acetamiprid

24242>168

13220.020

242>1851.26Methiocarb sulfoxide

10230>199

20170.020

230>1251.27Dimethoate

32202>131

25400.020

202>1751.18Thiabendazole

20256>175

16220.020

256>2091.15Imidacloprid

30192>132

18250.025

192>1601.01Carbendazim

17263>121

17260.025

263>1690.90Demeton-S-methyl sulfone

28247>109

14200.025

247>1690.86Oxydemeton-methyl

Collision Energy (eV)Cone Voltage (V)Dwell time (s)MRM TransitionsRTPesticide

©2007 Waters Corporation 27

MRM ConditionsMRM Conditions

24293>115

30180.020

293>972.29Phorate sulfone

32233>107

24440.030

233>1512.30Lenacil (-ve)

20277>143

32180.020

277>972.25Phorate sulfoxide

16355>108

16150.020

355>882.19Thiodicarb

28202>127

10180.020

202>1452.08Carbaryl

7208>89

770.020

208>1161.64Aldicarb

10213>156

15240.020

213>751.61Butocarboxim

37253>90

20280.020

253>1261.49Thiacloprid

37258>107

20220.020

258>1221.40Methiocarb sulfone

Collision Energy (eV)Cone Voltage (V)Dwell time (s)MRM TransitionsRTPesticide

©2007 Waters Corporation 28

MRM ConditionsMRM Conditions

16296>99

10140.015

296>702.85Triadimenol

8321>203

18150.015

321>1192.84Iprovalicarb

15294>197

21220.015

294>692.77Triadimefon

35247>126

28450.030

247>1802.65Fludioxonil (-ve)

30404>329

15220.020

404>3722.60Azoxystrobin

19226>121

10160.020

226>1692.63Methiocarb

15249>182

16280.020

249>1602.56Linuron

20297>69

20300.020

297>1592.52Imazalil

8318>261

8140.020

318>1602.46Azinphos-methyl

Collision Energy (eV)Cone Voltage (V)Dwell time (s)MRM TransitionsRTPesticide

©2007 Waters Corporation 29

MRM ConditionsMRM Conditions

41336>159

24250.015

336>1873.11Zoxamide

10347>238

28190.015

347>1373.06Tolylfluanid

32298>100

20320.015

298>1443.04Spiroxamine

12302>116

20210.015

302>883.00Fenoxycarb

9309>289

11200.100

309>1562.96Diflubenzuron (-ve)

25226>108

33450.015

226>932.95Cyprodinil

10364>194

20170.015

364>1522.88Flufenacet

35302>55

25350.015

302>972.86Fenhexamid

10333>224

24220.015

333>1232.86Dichlofluanid

Collision Energy (eV)Cone Voltage (V)Dwell time (s)MRM TransitionsRTPesticide

©2007 Waters Corporation 30

MRM ConditionsMRM Conditions

22487>329

16270.020

487>1563.62Flufenoxuron (-ve)

13482>462

14340.020

482>1563.58Flucycloxuron (-ve)

22509>326

40220.020

509>1753.49Lufenuron (-ve)

17463>398

21260.020

463>4163.50Fluazinam

15379>339

25180.020

379>1963.47Teflubenzuron (-ve)

39459>175

22220.020

459>2763.31Hexaflumuron (-ve)

32261>97

12110.015

261>753.15Phorate

Collision Energy (eV)Cone Voltage (V)Dwell time (s)MRM TransitionsRTPesticide

©2007 Waters Corporation 31

ACQUITY UPLCACQUITY UPLC--TQD TQD Total Ion Chromatogram (TIC)Total Ion Chromatogram (TIC)

0.1mg/kg matrix-matched standard in cereal-based baby food.

4min gradient for 52 pesticides.

©2007 Waters Corporation 32

ACQUITY UPLCACQUITY UPLC--TQD TQD Improved ResolutionImproved Resolution

Aldicarb sulfoxide

Butocarboxim sulfoxide

HPLCRs = 0.9

UPLCRs = 1.3

©2007 Waters Corporation 33

ACQUITY TQD ACQUITY TQD Polarity SwitchingPolarity Switching

Chromatography achieved by two co-eluting compounds undertaking polarity switching.

Rapid polarity switching enables positive and negative analytes to be analysed in a single run.

Lenacil, -ve

Phorate sulfone, +ve

©2007 Waters Corporation 34

Calibration curve /Polarity SwitchingCalibration curve /Polarity Switching0.010.01µµg/ml in Baby Foodg/ml in Baby Food

r2 = 0.9952

r2 = 0.9988

Compound name: LenacilCorrelation coefficient: r = 0.999385, r^2 = 0.998771Calibration curve: 6195.2 * x + -1.79023Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

µg/ml0.000 0.100 0.200 0.300 0.400 0.500

Res

pon

se

0

1000

2000

3000

Compound name: Phorate sulfoneCorrelation coefficient: r = 0.997579, r^2 = 0.995165Calibration curve: 192494 * x + 58.2215Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

µg/ml0.000 0.100 0.200 0.300 0.400 0.500

Res

pon

se

0

20000

40000

60000

80000

©2007 Waters Corporation 35

Calibration curveCalibration curve

Lenacil 0.0005 to 2.5 µg/mL in fruit-based baby food

Compound name: LenacilCorrelation coefficient: r = 0.999623, r^2 = 0.999246Calibration curve: 6193.91 * x + -1.82154Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

µg/ml0.00 0.50 1.00 1.50 2.00 2.50

Res

pon

se

0

10000

µg/mlRes

idual

-10.0

0.0

r2 = 0.9952

©2007 Waters Corporation 36

Robustness ACQUITY TQDRobustness ACQUITY TQD

Acquity TQD over 100 injections of three compounds in fruit-based baby food

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

0 20 40 60 80 100Injection Number

Peak A

rea /

Con

cen

trati

on

Azoxystrobin, 4.41% RSDCarbendazim, 4.25% RSDAcetamiprid, 5.71% RSD

©2007 Waters Corporation 37

ACQUITY TQDACQUITY TQDAcquisition speedAcquisition speed

Even at fast acquisition speeds with multiple compounds, ACQUITY TQD can maintain maximum data quality

5 2010 50 100 200 500 ms

Short cycle time enables UPLC compatible detection speeds

while maintaining sensitivity

©2007 Waters Corporation 38

% Recovery% Recovery

96 (2)Demeton-S-methyl sulfone

91 (5)Oxydemeton-methyl

100 (3)Methomyl

103 (8)Aldicarb sulfone

101 (6)Butoxycarboxim

93 (6)Aldicarb sulfoxide

76 (7)Pymetrozine

90 (5)Butocarboxim sulfoxide

88 (8)Omethoate

89 (6)Acephate

79 (4)Methamidophos

Recovery at 0.01mg/kg from cereal-based baby food (n = 5)

Mean % Recovery and % RSD (in parenthesis)

104 (7)Aldicarb

106 (6)Butocarboxim

101 (3)Thiacloprid

100 (6)Methiocarb sulfone

93 (11)Cymoxanil

97 (4)Acetamiprid

95 (4)Methiocarb sulfoxide

103 (3)Dimethoate

87 (4)Thiabendazole

100 (9)Imidacloprid

97 (4)Carbendazim

©2007 Waters Corporation 39

% Recovery% Recovery

73 (14)Dichlofluanid

93 (6)Triadimenol

100 (7)Iprovalicarb

104 (9)Triadimefon

107 (5)Fludioxonil

104 (5)Azoxystrobin

103 (5)Methiocarb

105 (6)Imazalil

104 (3)Linuron

113 (19)Azinphos-methyl

106 (5)Phorate sulfone

94 (3)Lenacil

102 (6)Phorate sulfoxide

99 (3)Thiodicarb

101 (5)Carbaryl

114 (3)Flufenoxuron

105 (9)Flucycloxuron

109 (5)Lufenuron

99 (4)Fluazinam

107 (6)Teflubenzuron

124 (9)Hexaflumuron

96 (6)Phorate

100 (8)Zoxamide

85 (13)Tolylfluanid

98 (7)Spiroxamine

102 (7)Fenoxycarb

100 (11)Diflubenzuron

95 (6)Cyprodinil

99 (8)Flufenacet

93 (8)Fenhexamid

©2007 Waters Corporation 40

Application to Real SamplesApplication to Real Samples

• 3 potato samples with suspected incurred residues

• Potato matrix-matched standards used for calibration

• TargetLynx application manager used for automatic quantification and confirmation

• 2 MRM transitions per residue were acquired with the confirmation criteria dependent on their relative abundance

— >0.5 ± 20% >0.2 < 0.5 ± 25%

— >0.1 < 0.2 ± 30% < 0.1 ± 50%

Reporting Level = 0.005mg/kg

©2007 Waters Corporation 41

AldicarbAldicarb SulfoxideSulfoxide0.019mg/kg in Potato0.019mg/kg in Potato

©2007 Waters Corporation 42

AldicarbAldicarb sulfone (sulfone (AldoxycarbAldoxycarb))0.016mg/kg in Potato0.016mg/kg in Potato

©2007 Waters Corporation 43

SummarySummary

A fast and simple UPLC-MS/MS method involving polarity switching has been developed for the determination of 52 pesticides.Method gave satisfactory recoveries for the majority of pesticides in potatoes, oranges and cereal-based baby foods.

The Acquity UPLC method showed significant advantages including the increase in the speed of the separation without the loss of resolution, as shown by the separation of the structural isomers butocarboxim sulfoxide and aldicarbsulfoxide.

The Tandem quadrupole detector (TQD) was capable of very fast polarity switching, allowing the determination of positive and negative compounds in a single run.

©2007 Waters Corporation 44

AIM

This applications note will assess the suitability of the Acquity TQD

tandem quadrupole mass spectrometer for pesticide residue analysis in

baby food. Polarity switching, differing dwell times and ion ratio

robustness will also be assessed.

INTRODUCTION

The European Union residue monitoring programme 2005-2007

establishes the need to cover fifty-five active ingredients in various foods,

including baby foods1,2. Twenty of these pesticides are suitable for multi-

residue LC-MS analysis, of which, only one has a negative polarity in

electrospray mode, normally requiring two injections (one in each polarity

ion mode). Consequently compounds with negative polarity are often

excluded from monitoring programmes. Ideally, these should be

determined in a single analysis with polarity switching.

Furthermore, chemists analysing pesticide residues are under increasing

pressure to broaden the range of pesticides determined in a single

analysis, to improve limits of detection, precision and quantitation, to

increase confidence in the validity of residue data, to provide faster

methods and to reduce usage of hazardous solvents whilst maintaining or

reducing costs. In order to meet these demanding requirements the

scope, sensitivity, efficiency and speed of multi-residue methods of

analysis must be improved.

The introduction of the Acquity TQD detector (Figure 1) allows scientists

to perform analysis of pesticides whilst harnessing all the benefits that

EXPERIMENTAL

The sample extraction method has been previously reported3,. Extracts of

blank baby food matrix in acetonitrile were provided along with mixtures

of the compounds in acetonitrile by the Central Science Laboratory (CSL),

York, UK. Extracts for injection were prepared by spiking the compounds

into the baby food matrix. The supernatant was analysed on the Acquity

TQD tandem quadrupole mass spectrometer following dilution with water

(1:9) v/v.

UPLC Method

LC System: Waters ACQUITY UPLC System

Column: ACQUITY UPLC BEH C18 Column

2.1 x 50 mm, 1.7 µm

Column Temp: 40 ˚CFlow Rate: 600 µL/min.

Mobile Phase A: Water

Mobile Phase B: Methanol

Gradient: Time 0 min 90% A

Time 4 min 100% B

Time 5 min 100% B

Total run time 7 min

Injection volume 20 µL

APPLICATION OF ACQUITY TQD FOR THE ANALYSIS OF PESTICIDE RESIDUES IN BABY FOODJames Morphet and Peter Hancock, Waters Corporation, Manchester, M22 5PP, UK.

Figure 1. Waters Acquity UPLC coupled to a TQD mass spectrometer.

©2007 Waters Corporation 45

AcknowledgementsAcknowledgements

Waters Corporation, Manchester, UK— Peter Hancock

— Anthoinetta Gledhill

— James Morphet

Central Science Laboratory, York, UK— Richard J. Fussell

— Cristiana Leandro

University of York, York, UK— Cristiana Leandro

— Brendan J. Keely

©2007 Waters Corporation

The application of UPLC-Travelling Wave Tandem Mass Spectrometry for multiresidue

steroid analysis- But now what do we do with all the data

generated.

©2007 Waters Corporation 47

Lays down rules for identificationvalidationinterpretation

MRL and banned compounds

Veterinary Drug Residue Analysis

©2007 Waters Corporation 48

Identification Points (IPs)Identification Points (IPs)

“Score” for different detection stepsMinimum requirementMRL compounds: 3 IPsBanned compounds: 4 IPs

Chromatographic Separation

HPLC - 2.5 % toleranceGC - 0.5 % tolerance

©2007 Waters Corporation 49

With single quad mass spectrometry, each ion and its ratio may be attributed 1 “identification point” (IP).

Since 4 IPs are required for confirmation of group A substances 4 fragment ions are required, with ratios matching that of a standard solution.

With Tandem MS each transition ion is worth 1.5 IPsand 1 IP may be assumed for the parent. Therefore, confirmation may be achieved by monitoring 2 transition ions with appropriate ratios.

Ion Ratios and Identification PointsIon Ratios and Identification Points

©2007 Waters Corporation 50

MS Technique IPs earned per ion

Low resolution (LR) MS 1.0LR-MSn Precursor ion 1.0LR-MSn Transition products 1.5High resolution (HR) MS 2.0HR-MSn Precursor ion 2.0HR-MSn Transition products 2.5

Relationship between mass fragment Relationship between mass fragment classes & classes & IPsIPs: Selected Ion Monitoring: Selected Ion Monitoring

©2007 Waters Corporation 51

Relative Intensity (% of base peak)

EI- GC-MS (relative)

CI GC-MS, GC-MSn, LC-MS, LC-MSn (relative)

> 50 ± 10 ± 20> 20 - 50 ± 15 ± 25> 10 - 20 ± 20 ± 30≤ 10 ± 50 ± 50

Relative ion intensities in samples must correspond to calibration solutions or spiked samples within above tolerances.

Ion Ratio Tolerances Dependent on Ion Ratio Tolerances Dependent on Relative IntensitiesRelative Intensities

©2007 Waters Corporation 52

Prohibits the use of:

hormones (e.g. zeranol)ß-Agonists (e.g. clenbuterol)Thyreostats (e.g. Thiouracil)

• for the purposes of growth promotion• in food-producing animals

©2007 Waters Corporation 53

ACQUITY UPLC / Quattro Premier*ACQUITY UPLC / Quattro Premier*

*Following slides are courtesy from Paul YoungChemical Surveillance Department,

Veterinary Sciences Division, Agri-Food and Biosciences Institute (AFBI)

paul.young@afbini.gov.uk

©2007 Waters Corporation 54

Quattro Premier XEQuattro Premier XE

High Sensitivity.

No loss of sensitivity even at extremely short dwell times.

Travelling wave technology to minimise cross-talk.

Acquity UPLCAcquity UPLC

Increased Resolution

Reduced Run-Time

©2007 Waters Corporation 55

Acquity UPLC : RepeatabilityAcquity UPLC : Repeatability

16 repeat injections

©2007 Waters Corporation 56

Quattro Premier XE : Time SectoringQuattro Premier XE : Time Sectoring

Short Dwell Time

Narrow Retention windows

The goal:

31 Steroids

Minimum 2 transitions

+

14 DeuteratedISTDs

©2007 Waters Corporation 57

Acquity UPLC - Travelling Wave MS with Fast Polarity Switching

Acquity UPLC - Travelling Wave MS with Fast Polarity Switching

107 Transitions in 5 minutes

10 -ve ion21 +ve ion

This method is achievable on Quattro Premier XE as a result of the ability for extremely rapid polarity changes

With a large number of drugs to be screened the goal is to increase the scope of each analytical technique

©2007 Waters Corporation 58

Acquity UPLC : Separation of 31 SteroidsAcquity UPLC : Separation of 31 Steroids

50 X 2.1 mm UPLC Column

31 analytes eluted in 5 minutes, system re-equilibrated and ready to inject again after 6 minutes

©2007 Waters Corporation 59

Quattro Premier UPLC MethodQuattro Premier UPLC Method

31 Analytes + 14 Deuterated ISTD

50 X 2.1 mm HPLC Column

107 Transition ions monitored

UPLC run time 5 minutes

1 minute for re-equilibration

Total run time 6 minutes

©2007 Waters Corporation 60

UPLC Method Creates 3 Major ProblemsUPLC Method Creates 3 Major Problems

1. Large number of calculations required to address EU identification criteria

2. Volume of paperwork created and associated archiving problems

3. Extraction and recording of quality assurance data

©2007 Waters Corporation 61

Quattro Premier UPLC Method - CalculationsQuattro Premier UPLC Method - Calculations

31 Analytes + 14 Deuterated ISTD

107 Transition ions monitored

Each Injection

31 ISTD Corrected Concentration Calculations

31 ISTD Corrected Relative RT Comparisons

62 Ion Ratio Calculations and Comparisons

40 Sample Batch = almost 5000 Calculations

©2007 Waters Corporation 62

Targetlynx Checks Identification CriteriaTargetlynx Checks Identification Criteria

©2007 Waters Corporation 63

Masslynx Sample TableMasslynx Sample Table

©2007 Waters Corporation 64

Targetlynx Checks Identification CriteriaTargetlynx Checks Identification Criteria

©2007 Waters Corporation 65

Targetlynx ReportTargetlynx Report

©2007 Waters Corporation 66

Typical Report SizeTypical Report Size

Typical Report

Summary Pages 31 analytes + 14 ISTD 45 Pages

Calibration Pages 45 (3 per page) 15 Pages

Chromatograms 10 Pages per injection 400 Pages

Total 460 Pages

Sample extraction involves automated dual stage SPE

Capacity = 80 SPE Cartridges

Therefore analytical run = 40 injections (6 MMStd levels, 6 QA + 28 Unknowns)

6 minutes between injections - analytical run-time = 4 hours

5 Runs per week ~ 120,000 Pages / Year

©2007 Waters Corporation 67

Scientific Data Management System (SDMS)

Scientific Data Management System (SDMS)

Captures print data on way to printer

Stores in a readable form in an Oracle Database

Allows indexing of “TAG” fields extracted at print capture

Allows subsequent review, searching and data extraction of stored reports

©2007 Waters Corporation 68

SDMS Print CaptureSDMS Print Capture

Print

SDMS will capture ANY print data either instead of, or in addition to a hardcopy printer output

©2007 Waters Corporation 69

SDMS can search stored reports for all occurrences of text items

SDMS can search stored reports for all occurrences of text items

SDMS can search entire database for any occurrence of a text item anywhere in any report

©2007 Waters Corporation 70

Stored reports can be retrieved and reviewedStored reports can be retrieved and reviewed

©2007 Waters Corporation 71

Reports can be captured from any instrumentReports can be captured from any instrument

Biacore Biosensor

Optical Biosensor

Rapid Screening Technique

©2007 Waters Corporation 72

Select Report and Run Extraction TemplateSelect Report and Run Extraction Template

©2007 Waters Corporation 73

VBA Code

login to SDMS

Selects report and runs template

Outputs data to XML file

Parses XML file

Transfers data to Excel

SDMS Operation transparent to usersSDMS Operation transparent to users

©2007 Waters Corporation 74

SDMS Invisibly creates link between almost any instrument and Excel (or LIMS)

SDMS Invisibly creates link between almost any instrument and Excel (or LIMS)

©2007 Waters Corporation 75

•EU Legislation imposes strict criteria for the identification of veterinary drug residues

•UPLC/Travelling wave MS allows for significant increases in throughput whilst simultaneously meeting identification criteria

•Targetlynx can automate the calculations and comparisons required to ensure demonstration of compliance with legislative requirements

• SDMS provides solutions for storage, indexing and retrieval of most electronic data and for integration of most scientific instruments with either office software or a LIMS database

ConclusionsConclusions

©2007 Waters Corporation 76

AcknowledgmentAcknowledgment

Paul YoungChemical Surveillance Department, Veterinary Sciences Division, Agri-Food and Biosciences Institute (AFBI)

paul.young@afbini.gov.uk

©2007 Waters Corporation

Application of Biomarkers in the Food Application of Biomarkers in the Food IndustryIndustry

©2007 Waters Corporation 78

OverviewOverview

Identification of marker ions using metabonomics approach: statistical analysis and database searching

Comparison of different teas

Analysis of rice from various geographies

©2007 Waters Corporation

Introduction to Introduction to MetabonomicsMetabonomics

©2007 Waters Corporation 80

MetabonomicsMetabonomicsDefinitionDefinition

“Quantitative measurement of timetime--relatedrelatedmultiparametricmultiparametric metabolic responsesresponses of

multicellular systems to pathophysiological stimuli or genetic modification”.

--Professor Jeremy Nicholson, Imperial College, London, UKProfessor Jeremy Nicholson, Imperial College, London, UK

©2007 Waters Corporation 81

The Metabonomics EndeavorThe Metabonomics Endeavor

Focus on endogenous compounds rather than xenobiotics

Identification and quantification of small molecules in a biological system

Down-regulation and Up-regulation of these compounds are indicative of biological insults due to disease, toxicity, genetic modification or environmental factors

Knowledge of these endogenous compounds may be used for diagnosis and screening

©2007 Waters Corporation 82

Evolution of MetabonomicsEvolution of Metabonomics

1982

1983

1984

1989

1999

2000

2001

2002

2004

Van De GreefVan De Greef: publication of MS for urine profilingSadler, Buckingham and Sadler, Buckingham and

Nicholson:Nicholson: First publication on 1H-NMR of blood and plasma Nicholson, et al.: Nicholson, et al.: Multi-component

analysis of spectra data from rat urine

Nicholson and Wilson: NMR spectroscopy of biofluids

Nicholson: Nicholson: Definition of MetabonomicsHaselden, et al.:Haselden, et al.: First

independent Pharma publication of Metabonomics Nicholson, Lindon, and Holmes:Nicholson, Lindon, and Holmes:

Publication in Nature on MetabonomicsHolmes and Antti Holmes and Antti Explanation

of statistics in Metabonomics

Increasing # of publicationsIncreasing # of publications

©2007 Waters Corporation 83

Challenges of MetabonomicsChallenges of MetabonomicsSample Complexity and Data HandlingSample Complexity and Data Handling

Each sample has..,Each sample has..,a wide range of compound classes

a wide variation in metabolite concentrations

a large number of potential components

Each group of samples hasEach group of samples has……,,many sample analyses are required for statistical relevance

a complex raw dataset that needs to be processed

differences between sample groups which need to be highlighted

©2007 Waters Corporation 84

The Metabonomics ApproachThe Metabonomics Approach

Data AcquisitionData Acquisition

Data ReductionData Reduction

Statistical AnalysisStatistical Analysis

Interpretation of ResultsInterpretation of Results

©2007 Waters Corporation 85

Data ReductionData Reduction

Align like variables

Normalize

Extract variables

Exact MassChromatographic Peaks

Control 1

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 16.00Time8

100

%

H030918JGa047 1: TOF MS ES+ TIC

3.68e4

Zucker PM M1

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 16.00Time9

100

%

H030918JGa019 1: TOF MS ES+ TIC

3.95e4

Sample Group 1 Sample Group 2

Populate Markers Table

©2007 Waters Corporation 86

Multivariate Data ArrayMultivariate Data ArraySa

mpl

es

Retention time_m/z pairs

….

©2007 Waters Corporation 87

Principal Components AnalysisPrincipal Components Analysis

• Complex data is reduced to principal components and data is shown graphically

• Similar groups cluster in same regions

• Components responsible for clustering are identified

• Other statistical tools exist, including tools such as PLS-DA

©2007 Waters Corporation 88

From Data Table to Variable SpaceFrom Data Table to Variable Space

One object inOne object in3D variable 3D variable

spacespace

The whole table yields a swarm of points in

variable space

var. 3

var. 1

var. 2

©2007 Waters Corporation 89

CenteringCentering

var. 1

var. 2

var. 3

var. 1

var. 2

var. 3

Move center of point swarm to the variable origin

Mean

©2007 Waters Corporation 90

The First Principal ComponentThe First Principal Component

var. 1

var. 2

var. 3

(i)

ti1

PCPC1 1 (t(t11pp’’11))

The first principal component (PC1)is set to describe the largest variation in thedata, which is the same as the direction in which the points spread most in the variable space

The Score value (ti1) for the point i is the distance from the projection of the point on the 1st component to the origin.

PC1 hence is the first latent variable in a newcoordinate system that describes the variation in the data.

©2007 Waters Corporation 91

The Second Principal ComponentThe Second Principal Component

The second principal component(PC2) is set to describe the second largest variation in the data, and is perpendicular (orthogonal) to the 1st component

The Score value (ti2) for the point i is the distance from the projection of the point on the 2nd component to the origin

var. 1

var. 2

var. 3

PCPC11

PCPC22

(i)

ti1 ti2

©2007 Waters Corporation 92

Scores and Loadings PlotsScores and Loadings Plots

Two PC’s make up a plane (window) in theK-dimensional variable space

If the points are projected down on the plane, it can be lifted out and be viewed as a 2D-plot describing the object’s relationships, a so called scores plot (t1/t2). In this plot similarities/dissimilarities between objects (samples) can be seen

The corresponding loadings plot (p1/p2)describes the variables relationships and is also a means for interpreting the scores plotby telling which variables are responsible for similarity/dissimilarities between objects

The perpendicular distance from the object to the projection on the plane is the residual (E) or the variation not described by the two PCs

x1

x2

x3

PCPC11

PCPC22

Plane

observation observation ii

projection

©2007 Waters Corporation 93

Understanding PCA for MetabonomicsUnderstanding PCA for MetabonomicsScores and Loadings PlotsScores and Loadings Plots

Principal Component 1

Prin

cipa

l Com

pone

nt 2

Scores Plot

Principal Component 1

Prin

cipa

l Com

pone

nt 2

Loadings Plot

Group 1

Group 2

Group 3

xxxx xx

xx x x

xx

xxx

xx

xx

x

x

xx

x

x

x

x

xxxx xx

xx x x

xx

xxx

xx

xx

x x xxx

xxxx xx

xx x

xx

xxx

xx

x

xx

xx

x

xx

Marker 6

Marker 5

Marker 1

Marker 2

Marker 3

Marker 4

©2007 Waters Corporation 94

Data HandlingData HandlingMarkerLynx Application Manager for MassLynxMarkerLynx Application Manager for MassLynx™™

Sample List

Marker Table

Trend Line

PCA

©2007 Waters Corporation 95

Areas of ApplicationAreas of Application

Biomedical Research

Biomarker discovery— Pharmaceutical Industry

Food Safety— Product release

— Raw material acceptance

Industrial Process— Process optimization

Agrochemicals— GMO monitoring

©2007 Waters Corporation 96

Challenges in Metabolic ProfilingChallenges in Metabolic ProfilingHow to Get From Raw Data to ResultsHow to Get From Raw Data to Results

Reliable peak detectionAccurate peak quantitationPeak alignment processing from many injectionsStatistical analysis of resulting data for clustering infoSearching of databases for potential biomarkersConformation of structureLink to metabolic pathways

©2007 Waters Corporation 97

Waters Metabonomics SolutionWaters Metabonomics SolutionLC/MS and GC/MSLC/MS and GC/MS

ACQUITYUPLC™

SensitivityThroughputResolution

LCT Premier™

Exact MassPos/Neg

ESCi

MarkerLynx™ db

DatabaseStatistical AnalysisDynamic Review

Q-Tof Premier™

MS/MSExact Mass

High Resolution

GCT Premier™

Fast ScanningExact Mass

EI Library Search

Screening Discovery InformaticsUPLC +

©2007 Waters Corporation 98

MarkerLynxMarkerLynx™™ dbdbAttributesAttributes

Simple to use interactive browser for database searching— Exact mass searching

Customizable database

Built on Oracle Database Technology

Structural storage

Search results returned to MarkerLynx db for reporting

©2007 Waters Corporation 99

Analysis of TeaAnalysis of Tea

Millions of people consume tea everyday.

There are many varieties of teas that are enjoyed around the world.

Can we distinguish between these tea varieties?

Can we identify any of the analytes represented by m/z and RT pairs that distinguish the teas?

©2007 Waters Corporation 100

Instrumental Requirements Instrumental Requirements produce usable Datasetsproduce usable Datasets

The m/z values should have mass accuracy of < 5 ppm to make analyte identification easy.

Very high chromatographic resolution is required to reduce ion suppression in the mass spectrometer.

Retention times should be very reproducible sample-to-sample so that the multivariate data matrix can be easily assembled.

N

N

OO

©2007 Waters Corporation 101

Chromatographic ReproducibilityChromatographic ReproducibilityACQUITY UPLC of a Urine MetaboliteACQUITY UPLC of a Urine Metabolite

Time2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00

%

0

100

2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00

%

0

100

2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00

%

0

100

2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00

%

0

100

2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00

%

0

100

2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00

%

0

100

2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00

%

0

100

2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00

%

0

1000401305_PR_ESI+_034 1: TOF MS ES+

205.0988.85e3

2.58

0401305_PR_ESI+_033 1: TOF MS ES+ 205.0989.75e3

2.58

0401305_PR_ESI+_032 1: TOF MS ES+ 205.0988.86e3

2.58

0401305_PR_ESI+_031 1: TOF MS ES+ 205.0989.21e3

2.58

0401305_PR_ESI+_030 1: TOF MS ES+ 205.0988.41e3

2.58

0401305_PR_ESI+_029 1: TOF MS ES+ 205.0989.07e3

2.58

0401305_PR_ESI+_028 1: TOF MS ES+ 205.0989.11e3

2.58

0401305_PR_ESI+_027 1: TOF MS ES+ 205.0988.83e3

2.58

N

N

OO

XIC Raw Data

Average RT = 2.58 min

Standard Deviation = 0

Eight Replicate Injections

Time2.00 4.00 6.00 8.00

%

0

1000401305_PR_ESI+_028 1: TOF MS ES+

BPI1.03e5

5.39

0.77

0.68

0.51

2.58

1.82

0.94 2.24

4.373.62

3.50 3.80

5.15

5.11

4.56

6.986.36

5.55

6.04

7.51

9.739.44

©2007 Waters Corporation 102

Types of Tea AnalyzedTypes of Tea Analyzed

Caffeinated— Japanese Green Tea— Traditional English Tea— Earl Grey— Lotus Green Tea— Chai Tea

Decaffeinated— Tension Tamer Tea— Cranberry, Raspberry, and Elderflower Tea— Lemon and Ginger Tea— Black Current, Ginseng, and Vanilla— Chamomile Tea

©2007 Waters Corporation 103

Separation of English Tea UPLC and HPLCUPLC and HPLC

Caffeine is the largest detected peak in the caffeinated teasCaffeine is the largest detected peak in the caffeinated teas

English Tea

Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00

%

0

100

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00

%

0

100051105_PR_ESI+_003_ACQ 1: TOF MS ES+

TIC2.53e5

5.71

0.41

0.672.29

1.01

1.56 4.753.38

13.178.348.126.46 9.01 15.9813.94 16.83

051105_PR_ESI+_005_Alliance 1: TOF MS ES+ TIC

2.32e58.51

0.69

0.04

0.93

1.26

7.442.692.194.28 7.60

17.2216.738.799.86 11.8610.5211.19 12.16 13.87

17.6719.55 20.55

FWHM = 0.14 minutesPc = 214

FWHM = 0.09 minutesPc = 33456 % increase

UPLC

HPLC

*

*

©2007 Waters Corporation 104

Statistical Analysis Statistical Analysis UPLC/LCTUPLC/LCT--P DataP Data

Samples5 10 15 20 25 30 35 40 45 50 55 60

%

0

100

3.557e+003

Caffeinated•Japanese Green Tea•Traditional English

Tea•Earl Grey•Lotus Green Tea•Chai Tea

Loadings: Component 1 - Component 2

Component 10.00 0.25 0.50 0.75

Com

pone

nt 2

-0.200

0.000

0.200

0.400

0.600

1.12581.15

0.42144.10

0.36118.09

0.28166.99

1.88611.16

2.04498.26

0.47209.030.28

144.000.28138.990.28162.01

1.96597.18

0.28146.03

1.25127.042.02

257.080.35295.120.46

260.120.46

278.130.40

116.072.57

611.200.33

104.111.10276.15

1.96595.17

0.65438.981.10

294.16

0.38138.06

0.35277.110.52262.13

0.41287.20

0.34266.13

0.66215.02

0.28190.000.28151.01

0.65437.97

0.51130.08

1.53177.06

2.08495.11

1.58639.12

0.35248.12

0.37235.17

1.32163.04

1.33355.100.35259.10

2.10463.10

1.21261.131.10324.11

1.10162.06

1.96177.06

0.48223.052.78551.172.00

479.081.57

388.251.21

224.010.41

365.110.53

280.141.55379.10

1.61449.11

0.47191.02

1.74597.14

1.18163.04

0.46150.091.71432.28

1.96195.07

0.34133.060.38203.06

0.35258.12

1.15769.140.37160.10

1.86476.31

0.56182.99

2.31463.09

1.59354.23

2.64303.09

1.89153.06

0.30185.00

2.66455.23

2.37579.17

0.45236.150.30

218.990.30

250.941.14

449.110.31

336.852.41

581.180.29

234.972.00

520.331.13

310.131.96

379.100.66

129.02

2.52463.15

1.57393.21

2.49224.01

2.08449.11

1.12328.14

0.50433.94

2.86261.11

1.71437.24

2.02419.13

1.61224.01

0.31122.93

2.43282.10

2.57633.18

1.86481.26

2.28224.01

1.16265.15

3.13224.01

2.60163.04

0.39278.09

0.28148.98

1.87194.08

2.46273.08

2.02298.11

3.52224.01

1.94801.15

2.58509.13

0.57170.08

1.25146.06

1.57371.23

2.46433.11

1.11166.09

2.87519.11

2.24625.17

1.87544.22

0.50169.040.38309.13

1.98365.16

2.62188.07

2.56710.11

0.53224.01

0.31320.87

1.97692.12

1.50613.14

1.10465.10

1.40274.15

2.86301.11

1.86383.13

1.34377.08

1.16262.19

0.32175.12

2.01525.29

1.25753.152.47609.18

1.11787.15

1.28263.01

2.91224.01

0.60314.16

1.37107.05

1.12427.21

1.69352.21

0.34208.98

2.51261.11

2.61496.06

2.82419.13

1.56767.15

1.73465.10

1.36181.03

0.65127.04

2.98340.14

2.05237.19

1.87181.08

1.86461.07

2.55449.15

1.80234.15

1.71415.26

2.34499.23

2.63147.04

2.32517.13

2.82163.04

0.55214.01

1.75303.051.16

327.072.40

346.062.06

336.222.75

275.102.59

706.141.27

157.091.68

207.141.86

455.091.88

224.01

2.00650.09

2.64324.08

0.54181.01

1.25206.12

0.40603.16

1.23168.07

2.10252.16

2.06565.15

0.42605.07

1.87134.06

3.04169.98

1.34495.05

3.62182.99

0.48113.03

2.67463.09

2.17551.10

0.50973.33

1.14615.15

1.28266.99

0.25182.99

1.75232.10

2.80282.15

0.30131.13

0.44829.29

1.17771.16

1.17355.10

1.99352.21

2.76253.17

1.75484.25

2.63481.170.43

469.970.43

131.010.52

326.992.51

556.221.31

305.16

1.88633.14

1.19321.06

1.23599.15

0.43353.08

2.62741.22

2.65565.12

1.83565.15

0.45467.12

0.40112.05

1.33496.05

1.33559.11

2.39107.05

2.86279.13

2.43266.13

1.30418.11

2.36317.07

1.25305.07

0.48145.02

2.06513.07

2.82613.09

2.85189.06

0.51335.06

2.49473.11

0.40620.24

0.71121.07

1.15105.07

0.51372.14

1.50231.12

0.29137.03

1.17109.03

0.69136.08

1.33147.05

2.16717.14

1.21583.14

1.23442.05

1.76826.12

0.34296.14

1.16267.14

0.30158.98

1.27131.05

2.41457.11

2.46487.21

1.36597.18

0.48157.10

1.98487.081.62

319.052.65

717.140.37

460.170.52

229.160.39

162.112.22

401.160.87

611.141.43

426.050.41

139.041.21

595.140.87

593.13

1.99465.100.29

128.021.15

611.142.70

869.161.21

289.070.39

204.091.59

165.062.36

238.151.34

439.161.94

433.11

1.59147.05

1.99303.051.20

188.071.59

339.110.43

157.071.59

206.081.26

447.061.49

917.181.15

222.100.36

184.081.76

275.091.26

287.062.41

427.101.51

333.060.35

446.162.27

287.060.35

148.061.09

171.031.87

535.152.29

449.111.24

333.062.02

757.221.18

763.151.75

773.221.10

345.082.16

595.171.39

138.071.39

236.12

1.39195.090.51

175.111.13

181.070.71

337.160.51

158.09

1.98443.101.21

307.081.49

459.101.45

291.09

Scores: Component 1 - Component 2

Component 1-0.100 0.000 0.100

Com

pone

nt 2

-0.100

0.000

0.100

0.200

0.300

Decaffeinated

Caffeinated

CaffieneDecaffeinated•Tension Tamer•Cranberry •Lemon and Ginger•Black Current, •Chamomile

©2007 Waters Corporation 105

MarkerLynx AnalysisMarkerLynx Analysiswithout Caffeinewithout Caffeine

Loadings: Component 1 - Component 2

Component 1-0.200 0.000 0.200

Com

pone

nt 2

-0.200

0.000

0.200

0.400

1.12581.15

0.36118.09

0.28166.99

0.42144.10

1.88611.16

2.04498.260.47

209.030.28144.00 0.28

138.99

0.28162.01

1.96597.18

1.25127.042.02

257.080.38

138.060.35

295.120.46

260.120.46

278.132.57

611.200.40

116.071.10

276.150.28

146.03

0.65438.980.33

104.11

0.35277.110.41287.20

1.10294.16

0.34266.13

0.52262.13

0.37235.17

0.66215.02

1.33355.100.28190.00

0.35258.120.48

223.051.32

163.040.35259.10

1.53177.06

1.96595.17

2.08495.11

0.47191.021.74

597.14

0.35248.12

0.46150.090.65437.97

1.21261.13

1.15769.141.18

163.042.00479.08

0.51130.08

0.34133.06

1.96177.06

1.59354.232.78551.171.10162.06

0.37160.100.53

280.141.10324.11

2.10463.10

0.38203.06

0.31336.852.64

303.091.89

153.062.31

463.091.57

388.251.96

195.070.28

151.01

1.55379.101.58

639.12

2.41581.182.52

463.151.86

476.311.71

432.280.30

185.002.66

455.23

0.29234.97

0.45236.15

0.30218.990.30

250.941.14

449.112.86

261.112.37

579.171.13

310.130.66

129.021.21

224.011.61

449.111.96

379.10

0.50433.940.56182.99

0.39278.09

0.28148.981.87

194.082.43

282.102.00

520.332.49

224.011.16

265.152.02

419.132.57

633.181.12

182.99

2.43159.07

0.41365.11

0.66393.98

0.46322.97

1.12328.14

1.57393.21

0.29120.98

2.16293.17

2.24625.171.87

544.222.02

298.111.86

481.262.31

229.14

1.10465.102.46

273.083.13

224.011.71

437.242.33

302.14

1.50613.14

1.25753.15

0.50169.04

1.40274.15

2.86301.11

2.28224.01

1.34377.08

1.16262.19

1.28263.01

3.35224.01

2.58509.13

1.57371.23

2.10224.01

2.16564.36

0.31122.93

1.14217.11

1.11787.15

0.60314.16

1.12427.21

2.99299.112.08449.11

2.56710.11

1.33147.051.88

633.141.75

303.051.36

181.031.69

352.211.61

224.012.87

519.112.46

433.111.56

625.171.95

435.132.60

163.043.52

224.01

0.34208.98

2.82419.13

2.51261.11

1.28347.01

1.23168.07

2.43241.07

2.06336.22

1.73465.10

2.98340.14

0.65127.04

2.75275.10

2.32517.13

1.96289.07

1.16327.072.40

346.062.55

449.150.40

458.190.43

117.00

0.42605.07

1.87134.06

2.59706.14

2.16569.31

2.62188.07

0.50973.33

0.40603.16

1.86383.13

1.25146.06

1.14615.15

2.05237.19

1.75232.10

2.17551.10

1.28266.99

2.00650.09

0.44829.29

1.17771.16

1.75484.25

1.19321.06

0.71121.07

0.51335.06

2.49473.11

1.86676.26

1.63333.16

2.85189.06

0.40620.24

0.34280.15

2.06513.07

2.98575.23

1.47169.98

0.48159.97

2.50451.12

1.15105.07

1.41303.05

0.52375.06

1.09152.06

1.50231.12

2.83255.07

1.65757.22

1.25305.070.41

765.212.86

243.102.57

393.191.47

486.141.48

207.072.83

257.082.03

305.071.88

711.083.13

314.142.51

309.101.18

367.152.68

501.142.70

621.120.31

290.851.05

146.971.02

167.002.92

611.141.93

369.161.95

412.222.27

849.081.57

275.181.78

337.090.45

363.011.40

349.091.93

417.170.42

353.161.54

425.150.29

128.021.60

627.150.29

137.031.82

608.070.88

127.041.11

166.09

1.98487.08

1.76826.12

1.23442.050.87

611.140.87

593.131.66

411.191.27

131.050.28

123.022.70

869.161.82

261.170.41

139.040.48

157.102.41

457.111.55

373.190.37

460.171.16

267.140.39

204.091.59

165.061.26

287.061.62

319.051.45

180.071.43

426.051.68

469.142.18

617.151.21

289.071.21

595.141.59

339.112.46

487.210.48

348.070.52

229.162.22

401.161.42

261.171.59

206.081.52

467.081.34

439.160.70

301.140.43

157.072.36

238.150.70

319.150.29

174.031.26

447.060.40

246.060.35130.05

2.09599.20

1.27365.12

1.50594.05

2.92565.13

1.40441.17

1.76275.09

1.51333.061.94

433.110.88

171.031.34

915.161.86

579.170.34

351.070.39

292.111.11120.08

1.99465.101.10

345.081.20188.07

1.15611.14

1.49917.180.40

194.091.10

212.062.01

741.220.39

310.121.83

473.110.36

184.081.15

222.101.36

747.151.99

123.052.41

427.100.40

193.071.62

731.161.14

298.101.70

595.16

0.35446.16

0.35148.06

2.27287.06

1.09171.03

1.24333.06

1.98578.06

0.51129.10

1.87535.15

1.59147.051.99

303.051.20

139.041.49

289.071.98

273.082.29

449.111.12

130.091.88

757.221.49

139.041.18

763.152.02

757.221.62

565.161.36

579.15

1.75773.222.16

595.171.39

236.121.39

138.07

1.21307.081.49

459.101.45291.09

0.51175.111.13

181.070.71337.16

0.51158.09

1.98443.10

Samples5 10 15 20 25 30 35 40 45 50 55 60

%

0

100

5.521e+003

Caffeinated and Caffeinated and decaffeinated teas still decaffeinated teas still separate in scores and separate in scores and

trends plots.trends plots.

Scores: Component 1 - Component 2

Component 1-0.100 0.000 0.100

Com

pone

nt 2

-0.100

0.000

0.100

0.200

0.300Decaffeinated

Caffeinated

©2007 Waters Corporation 106

Separating Tea VarietiesSeparating Tea Varietiesthey really are different!they really are different!

Loadings: Component 1 - Component 2

Component 1-0.100 0.000 0.100 0.200 0.300

Com

pone

nt 2

-0.100

0.000

0.100

0.200

0.300

1.39195.09

1.24333.06

1.36163.04

0.28151.01

2.93565.14

1.18763.15

0.28166.99

1.60147.051.12

130.09

0.28146.030.34

266.13

1.33355.10

0.87268.100.38138.061.18

127.042.63

147.040.51

661.271.71

186.13

0.28174.03

0.46347.10

2.56611.19

0.65438.980.34248.12

1.51333.06

1.34915.16

2.01741.22

1.17109.03

0.33104.11

1.39236.12

2.63188.07

0.46260.12

1.90132.08

0.40136.060.88

171.03

1.12328.14

0.37203.06

1.13181.07

0.39162.11

0.51130.07

1.16168.07

1.15311.07

0.46278.13

1.22196.100.48

305.140.40

116.07

1.36597.18

0.65215.02

0.34351.07

2.31499.122.01

257.081.21

224.01

0.45702.131.47

865.192.31

517.13

0.40193.07

0.31122.93

0.51175.112.28

449.11

1.11166.09

1.12310.13

2.56517.13

0.31206.89

1.18293.15

0.37259.10

1.10276.14

1.99465.10

1.99303.05

2.26287.062.18

595.17

0.87611.14

0.87593.13

1.50594.05

1.14222.10

1.21261.13

1.28143.04

2.56163.042.31

163.040.87

136.062.73

224.010.28

162.011.14

178.13

1.08152.06

0.65437.970.46

342.14

2.79551.18

2.35334.182.34

303.05

2.75577.15

0.56182.99

2.70869.16 0.88

127.040.29

218.99

2.65717.140.28

128.02

1.86476.31

0.41126.060.31

274.88

0.31181.012.75

563.18

2.01525.29 2.39

179.07

0.35295.12

0.54218.01

0.06159.97

3.11224.01

1.11120.081.26287.06

0.44325.12

2.64325.090.47352.08

1.75451.20

1.76275.09

1.27365.12

1.24581.13

2.01520.33

1.21583.142.16

717.14

2.16569.31 1.10

294.16

2.56547.350.06

182.99

1.39138.07

2.36197.12

0.31290.851.86481.26

0.65129.02

1.71204.14

2.03401.22

0.65393.99

2.04603.132.47

565.132.94

564.121.24

761.13

1.28211.01

1.70437.24

2.52224.01

2.16564.36

0.30160.05

0.28123.02

1.56393.21

1.75446.24

1.97487.081.59

165.061.43

735.150.31

320.872.18

435.092.36

238.15

2.38477.27

1.46937.17

1.35181.031.93312.17

2.39332.17

2.74253.17

0.52262.13

0.50425.04

2.34302.14

1.61224.01

1.07346.06

0.06177.99

0.39204.092.27

471.091.12144.081.16

609.121.30785.15

0.06173.99

0.31185.01

0.06205.98

2.29919.191.45

408.180.29242.011.15217.11

1.24778.16

2.27755.14

0.31542.74

2.51429.08

1.10199.11

0.42215.061.52

913.142.17

551.101.63333.16

1.29214.00

2.81581.10

0.71121.07

1.76885.15

1.70177.06

2.66565.130.42111.05

3.00182.992.63118.05

1.14136.061.24

112.082.28423.03

2.30608.39

1.71691.210.23190.990.30

120.991.82

174.09

1.05177.98

1.85459.28

2.14336.20

0.70307.03

0.25159.97

0.88153.01

1.70531.172.82169.98

1.46289.13

1.83428.21

2.64243.282.02

298.11

0.77271.002.18

224.01

1.26406.15

1.47307.08

2.02419.13

0.78113.04

1.27131.05

1.27163.082.93427.10

1.16627.13

2.17361.15

2.06403.23

1.41140.04

1.52930.17

2.87739.152.70341.150.78359.02

2.34439.022.56

499.121.28157.09

0.69136.081.77

139.042.55

535.111.14801.15

3.98204.99

1.75371.07

1.41356.19

2.09318.18

2.65716.142.85211.01

3.13314.14

1.42931.16

0.45100.08

0.32458.782.82181.02

2.31229.14

2.47564.13

1.65643.15

2.13853.13

2.49887.26

2.82553.10

1.84540.19

1.75333.061.63169.98

1.41182.99

2.09581.18

0.55214.01

1.25169.98

1.65330.03

1.93641.172.34

581.18

0.41139.040.69

165.061.10

233.152.41

228.00

2.41562.07

2.91224.02

1.69439.09

2.04602.13

1.35475.09

0.71123.04

1.45613.17

0.45114.09

1.55553.15

2.90781.16

2.25565.141.82565.15

1.59339.11

0.51402.122.42

256.081.29

613.171.92

609.122.88

397.170.86

633.120.86

305.130.24

162.991.98

471.114.55

177.983.94

190.99

2.75919.25

2.70932.16

1.53163.04

0.66218.99

1.86169.98

1.60211.01

1.66455.02

2.18617.15

2.52503.191.59

206.081.44190.05

1.96311.07

0.70200.99

3.28143.06

2.86219.114.14173.99

2.39543.18

2.52520.192.77

887.262.04

209.160.34

296.142.51

565.064.46

173.992.82

222.022.77

731.161.74

347.084.33

195.090.56

139.992.05

207.141.94

559.041.97

272.070.47

991.343.86

177.981.17

181.02

0.51158.081.88

633.140.69

182.081.93591.21

0.47293.18

3.12100.08

3.84218.99

2.28512.12

3.01242.03

1.76199.03

1.46194.04

2.29502.02

3.25323.94

3.28194.04

2.96232.01

1.44715.14

1.86435.131.96

441.082.55

331.163.56

167.002.93

155.123.29

332.892.08

503.192.02

811.140.45

220.091.97

427.060.72

190.013.17

282.912.78

100.070.45

829.294.51

182.992.09

222.021.15

363.052.12

177.174.10

177.983.55

146.983.32

100.080.25

114.09

1.98578.06

0.29181.99

0.23153.01

1.15138.07

2.95139.041.04

173.993.10

139.994.55

153.002.73

889.242.02479.08

0.43117.00

2.34219.11

2.39267.16

2.74155.12

3.56114.09

3.34192.06

1.48144.98

2.64189.11

3.54336.93

3.26433.83

2.64278.99

3.58139.99

4.70171.98

2.51428.07

2.47245.14

2.45236.13

3.45211.01

0.24169.99

2.44657.36

3.10186.98

3.02273.17

3.20155.12

1.87211.01

2.91313.07

3.08118.08

3.34196.02

1.95439.02

2.22355.18

2.09621.18

0.06201.00

3.64201.00

2.88429.12

3.15275.97

2.64627.24

3.07288.96

3.29242.03

3.56282.91

4.48146.96

4.46121.97

2.07452.06

3.47169.98

3.23146.98

2.36179.11

1.50426.052.05

763.202.54

493.131.99

219.103.08

192.062.59

257.142.29

543.042.41

799.233.09

168.993.08

141.104.55

190.992.68

476.124.28

200.994.51

187.973.73

139.992.29

455.123.05

219.112.68

745.420.43

127.042.67

366.183.13

208.043.02

587.202.29

155.122.76

636.392.10

181.022.76

143.063.53

100.082.77

784.493.10

344.923.06

323.942.33

504.302.45

433.12

0.95261.09

0.23224.01

0.72348.07

2.70183.00

3.32130.53

1.74228.00

3.53154.99

3.25114.09

2.26114.09

1.06204.99

3.06194.04

2.51441.25

1.30360.15

2.68740.46

2.69411.20

2.82275.14

2.91439.02

3.35227.99

2.53208.04

2.64579.22

1.34480.06

2.37383.07

2.17228.00

2.10652.11

2.21207.11

3.07169.98

3.24118.09

0.43124.042.41

257.080.54

169.982.30

273.172.56

303.060.27

213.012.53

543.191.18

349.142.73

369.043.05

165.992.25

625.173.12

246.002.70

621.122.06

728.262.85

587.192.28

373.180.25

171.982.07

731.160.70

390.072.32

181.022.82

609.230.86

236.980.96

200.990.87

259.131.42

257.140.86

249.994.54

167.002.71

395.213.39

182.993.12

215.013.44

143.060.39

278.100.43

527.161.71

181.023.13

167.990.30165.03

2.92749.25

0.30336.85

2.79841.24

2.49554.17

3.53167.99

3.34214.00

2.83825.24

0.94181.99

1.07139.99

3.03196.02

2.54470.18

2.28439.23

2.83244.14

2.76453.12

0.23139.99

2.34783.23

1.01330.06

0.77307.15

0.76119.05

2.54429.15

1.55899.162.71433.12

0.87132.10

2.36260.13

0.30158.98

2.71493.14

1.43344.23

2.29799.23

0.47124.08

2.05447.10

0.97231.14

1.43349.19

0.93195.98

2.43560.19

2.07374.14

2.05429.15

2.48397.22

2.82873.24

2.54413.18

2.58292.23

0.78159.97

2.13211.01

0.25196.98

2.19695.092.92

611.142.44

652.411.63

587.14

1.81147.051.69447.18

0.56201.00

1.83649.10

2.70373.22

0.43157.07

2.41139.04

1.05146.97

0.44206.11

2.55355.17

1.41381.04

0.65227.02

2.16130.072.28

199.030.52

719.250.45

349.141.17

261.081.07

313.062.18

694.081.48

332.121.53

654.132.71

411.110.25

229.001.24

448.180.45

363.011.69

427.172.68

483.131.45

717.21

0.25173.99

1.43617.12

1.93369.16

1.30123.05

1.38377.07

1.43327.20

1.99287.06

2.24277.07

2.16291.101.92531.17

2.07745.14

1.17455.122.87

532.291.15

469.132.45

371.212.11

513.07

1.04190.991.71

415.262.16

245.102.46

595.112.19

601.171.70

453.170.32

251.041.82

570.161.18

132.081.29

618.151.49

500.122.48

428.151.29

265.150.34

277.152.19

690.112.07

379.101.69

449.110.70

359.152.06

535.151.41

349.091.15

376.142.04

426.591.18

367.150.72

190.991.80

337.091.68

434.58

0.70301.14

0.45428.04

1.35427.12

1.06218.99

0.42738.16

1.41311.13

0.70167.060.25

200.992.09

214.000.65

287.981.95

305.07

1.88611.16

0.35148.06

1.44595.17

2.41427.10

0.31236.09

2.42471.23

1.16368.101.17

493.18

2.55545.200.89

304.150.45

495.022.47

177.172.31

449.182.22

385.190.88

355.07

1.34439.161.16

220.121.41

194.080.42

381.080.45

396.041.42

425.201.15

323.081.06

241.16

0.47111.09

2.04570.16

0.28137.03

1.81715.16

0.51129.101.79

617.120.88

307.080.90

314.09

1.69261.17

2.16601.12

1.45273.08

0.43261.04

2.10399.141.19764.16

2.09427.10

0.52337.16

1.39426.050.43783.22

2.55783.23

1.18159.09

1.53212.062.22

442.191.33

339.112.55

453.121.44

295.080.47

217.121.33

147.041.68

514.10

0.37460.171.45123.05

1.26465.07

0.48157.102.09163.04

0.70319.15

0.41365.11

1.86434.20

1.56373.19

0.46322.97

1.33395.13

2.09599.20

2.41592.08

1.45607.13

1.76303.05

1.25652.11

1.52420.192.46

518.14

1.87535.15

1.23442.05

1.82563.21

0.33304.161.92275.191.65319.05

1.18146.062.22

331.160.42

353.161.59

480.061.28

419.101.27

321.051.28

362.091.52

789.211.54

418.151.52

275.190.29138.99

0.35130.05

1.10212.06

1.09171.031.22169.05

1.28867.21

2.03779.20

1.65481.10

1.99402.16

1.69209.15

1.09229.16

2.02810.131.97

885.19

1.01268.11

0.45460.21

1.34323.08

1.23305.07

1.50867.21

1.69389.22

0.44146.09

1.14298.100.40

246.06

0.39292.11

0.52229.16

1.75561.19

1.16267.141.95417.171.76

595.16

1.94433.11

1.25303.021.76

795.191.86

439.161.20

205.101.45

180.07

2.41457.11

1.76826.12

1.69411.201.52

425.140.38310.12

1.82289.07

0.35446.161.52

467.081.49

289.07

2.22401.160.36

258.112.46

482.261.82

608.070.32

175.121.82

261.171.87

579.17

0.71337.16

1.88757.22

1.60627.15

0.35184.08

1.62731.16

0.48348.07

1.36747.151.40441.171.42261.17

0.45236.15

0.40194.09

1.21595.14

1.69469.13

1.10345.082.02

757.221.26

447.061.98

273.08

2.46487.211.99123.05

1.45291.09

1.35579.15

1.62565.16

1.21289.07

1.13611.141.49

917.181.20

139.041.83

473.111.75

773.22 1.98443.10

1.21307.081.49

459.10

Samples2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

%

0

100

3.564e+003

Separation amongst Separation amongst caffeinated tea caffeinated tea

varieties.varieties.m/z = 195.08

Scores: Component 1 - Component 2

Component 1-0.200 0.000 0.200

Com

pone

nt 2

-0.200

-0.100

0.000

0.100

0.200

0.300 Traditional English

Earl Grey Lotus Green

Japanese GreenChai

©2007 Waters Corporation 107

Separating Tea VarietiesSeparating Tea Varietiescaffeine signal removedcaffeine signal removed

Separation amongst Separation amongst caffeinated tea caffeinated tea

varieties, caffeine varieties, caffeine excluded.excluded.

Loadings: Component 1 - Component 2

Component 1-0.100 0.000 0.100 0.200 0.300

Com

pone

nt 2

-0.150

-0.100

-0.050

-0.000

0.050

0.100

0.150

1.13181.07

2.93565.14

0.28151.01

1.18763.15

1.36163.04

0.28146.03

0.41144.10

1.33355.10

0.51158.080.51

175.112.28449.11

1.99465.101.51

333.06

1.60147.051.12

130.09

0.34266.130.28

166.99

0.87268.10

2.01741.221.34915.16

0.28174.03

1.18127.04

0.65438.98

0.38138.06

0.51661.27

1.71186.13

2.63147.04

1.11120.082.18

595.171.99

303.05

0.46347.10

2.56611.19

1.11166.09

0.39162.11

0.40136.06

0.34248.12

0.88171.03

1.17109.03

0.51130.07

1.36597.18

0.46260.12

1.90132.082.63

188.07

0.34351.070.33

104.111.39

236.121.12

328.140.37

203.06

2.26287.06

0.40193.07

1.15311.07

0.46278.13

1.27365.12

1.26287.062.70

869.16

0.87593.13

1.16168.07

1.14222.10

0.31206.89

0.65215.02

0.40116.07

0.48305.14

0.31122.931.21

224.01

1.22196.10

2.01257.08

0.87611.14

0.45702.131.47

865.192.31

499.122.31

517.13

2.65717.14

1.24581.13

0.65437.97

1.12310.13

1.18293.15

2.56517.131.50

594.050.37

259.10

1.21583.14

2.16717.14

0.29218.99

2.73224.01

2.56163.04

2.34303.05

0.87136.06

2.31163.04

2.47565.13

1.21261.131.14

178.131.28

143.04

0.88127.04

0.46342.14

1.08152.06

1.10276.14

2.79551.18

2.35334.18

2.75577.15

2.04603.13

1.24761.13

0.31290.85

0.28128.02

2.01525.29

1.86476.31

0.28162.01

0.41126.06

1.97487.08

2.94564.12

0.31181.01

1.39138.07

0.65393.99

0.28123.02

2.64325.09

2.75563.18

3.11224.010.31274.88

0.54218.01

0.35295.122.39

179.07

1.59165.06

0.31320.871.86

481.26

2.38477.27 2.16

569.310.65

129.02 0.06159.97

0.56182.99

0.47352.081.75

451.200.44

325.12

1.46937.17

2.01520.33

1.43735.15 1.71

204.142.56

547.35

1.50887.17

1.76275.091.16

609.12

2.18435.09

2.03401.22

0.06182.99

2.29919.19

1.70437.24

2.36197.12

2.52224.01

2.16564.36

0.39204.092.27

471.091.12

144.080.31

542.742.27

755.14

2.75575.14

1.24778.16

0.71121.071.35

181.031.56393.21

1.28211.011.75

446.242.73

595.17

1.27131.05

2.81581.10

1.76885.151.45

408.18

0.33190.11

2.64324.08

1.63193.05

1.52913.14

1.30785.150.30

160.051.10

245.150.53224.02

0.69136.081.27

163.082.51

429.082.83

419.14

2.17551.10

2.55535.111.14

136.061.70

177.062.74

253.171.28

199.031.35

496.051.57

388.25

2.93706.09

0.29242.01

1.57371.232.39

332.172.36

238.151.93

312.172.34302.14

0.50425.04

2.93701.10

1.17599.14

1.53733.14

2.66565.13

2.47401.09

0.52262.13

1.07346.061.93317.12

1.77139.040.42215.06

0.06177.99

1.71691.212.28423.03

0.06173.99

0.31185.01

0.06205.981.70432.28

0.33380.10

0.69165.061.10233.151.26

406.15

1.63333.16

0.47150.09

1.10294.16

1.24112.08

1.16627.13

1.70531.170.42111.05

1.10199.11

1.61224.01

2.93427.10

1.15182.99

3.00182.99

2.79334.181.15

217.11

0.23190.99

1.14801.15

2.87739.151.75

378.25

1.52930.17

1.23160.11

2.65716.141.47

307.08

2.56449.16

1.42931.16

2.93305.04

1.29214.00

1.88611.16

2.41427.101.88

633.140.41139.041.59

206.081.59

339.112.49887.26

0.45100.08

0.32458.781.05177.98

2.41562.07

2.82553.10

1.84540.19

2.63118.05

2.83431.14

2.47564.13

1.65643.15

2.11238.09 2.16

356.19

2.82169.980.50509.15

1.85459.280.77130.053.43

224.01

2.91224.02

2.13853.132.70341.15

1.75333.06

2.14336.20

1.82174.09

1.41182.992.85211.010.25182.99

0.70307.03

0.88153.01

1.69439.09

0.30120.99

2.70868.15

1.46289.13

1.83428.21

2.81717.13

0.25159.97

2.04602.13

2.02298.11

0.51402.12

2.31613.34 2.64

243.282.30

608.39

0.43256.09

2.09581.18

0.77271.00

2.27849.08

1.83729.14

0.78113.04

2.02419.13

2.09477.27

2.06403.23

1.41140.04

1.17745.13 2.82

181.02

2.39341.14

1.06159.97

2.34439.02

0.78359.02

2.56499.12

0.69182.08

2.18617.15

1.45613.17

0.71123.04

1.29625.12

2.58696.44

1.75371.07

2.17361.151.41

356.19

3.07211.01

3.98204.99

2.09318.18

2.52503.19

2.68629.15

2.80716.06

2.66371.14

0.49174.08

1.66190.093.13

314.141.34

495.053.02

378.17

2.52169.982.01

333.06

0.86710.06

2.67564.13

1.35475.093.15222.02

1.63169.98

1.69583.14

1.29613.17

2.42256.08

0.89434.99

2.29951.12

1.55553.15

1.50540.30

2.90781.16

2.88903.252.31229.14

1.74179.04

1.67207.14

2.41362.25

2.53579.17

1.46199.03

2.41228.00

0.55214.01

1.65330.03

2.78219.07

1.93641.17

0.43157.07

2.41257.08

1.44715.14

2.52520.19

1.44190.05

0.45114.09

1.92609.12

2.77279.18

2.25565.143.09214.00

2.34581.18

0.86305.13

2.89561.072.71287.06

2.71549.16

2.18224.01

1.96746.07

2.77887.262.88

397.170.86

633.122.70

932.160.86

139.042.72488.27

2.66336.20

1.41433.14

2.85199.032.88194.04

1.53163.043.59227.99

1.65735.16

2.70896.11

1.53563.13

2.86219.11

2.75919.250.25177.98

3.40199.03

2.39158.16

1.68409.18

1.53717.07

2.64857.092.24310.15

2.50199.03

0.55197.092.11

341.142.56

181.022.33

169.982.82

257.08

1.84883.172.39

543.180.28

272.962.83222.15

1.84316.17

2.82362.17

1.86169.98

1.98471.11

1.28157.09

2.58741.20

2.12280.120.66

218.99

2.44419.10

2.82716.13

3.09455.16

3.17257.08

0.77247.99

1.81165.05

2.64853.12

1.60211.01

2.48190.08

1.46635.16

1.63215.13

2.65439.23

1.96311.07

3.28143.06

1.86435.131.93

591.212.54

287.122.11

239.092.10

143.064.14

173.992.88

579.213.10

181.02

2.48701.10

2.27401.09

2.10867.182.47257.05

2.21535.132.35405.18

2.34607.11

3.31344.92

2.59401.16

0.70200.99

2.99341.143.66182.99

2.91584.113.32

139.99

2.54493.13

1.96441.08

2.77140.00

3.07114.09

2.24335.221.31305.16

0.70233.12

4.46173.99

2.82222.02

3.51192.07

2.77731.16

2.82579.11

4.93232.97

4.51200.99

4.33195.09

3.54194.04

1.77182.99

3.87173.99

1.90889.24

3.30236.00

3.39208.04

3.18304.89

0.71177.98

2.34219.11

4.48142.98

3.25186.98

3.20151.05

2.59344.92

4.94205.98

2.51565.06

2.99358.15

0.56139.99

3.39222.02

3.85182.99

3.67176.01

2.55331.162.08

503.191.74

347.083.32

282.912.09

222.023.12

100.083.00

535.831.94

559.043.86

177.982.04

209.160.56

190.033.84

218.992.78

100.072.28

512.124.29

218.982.93

155.121.95

439.021.87

211.012.91

313.072.74

155.123.56

167.003.11

199.031.97

272.073.34

317.921.71

273.10

2.79221.19

3.30211.01

2.55222.02

3.55146.98

0.43152.06

1.13171.11

3.17282.91

3.08130.53

3.01242.03

2.05207.14

3.56114.09

2.96232.01

3.25323.94

2.26222.02

3.29332.89

0.24169.99

0.47991.34

4.10177.98

4.70171.98

3.54181.01

4.51182.99

2.39267.16

1.17181.02

0.45220.09

0.47293.18

3.32100.08

3.28194.04

1.97427.06

1.81147.05

2.54429.152.36179.11

2.05763.201.46194.04

1.99219.10

2.22355.18

1.15363.05

3.54336.93

4.55153.00

3.10139.99

0.72190.01

0.45829.29

2.02811.14

3.34192.06

1.15138.07

2.12177.17

2.95139.04

2.73889.24

3.26433.83

4.57139.99

2.07452.06

2.44657.36

0.29181.99

0.23153.01

2.64189.11

3.20155.12

2.47245.14

2.45236.13

1.48144.98

3.08118.08

2.64278.99

2.51428.072.02479.08

2.29543.04

3.15275.97

1.76199.03

3.02273.17

3.10186.98

3.07288.96

3.34196.02

1.04173.991.49600.04

2.51441.25

0.43117.00

3.56282.91

3.58139.99

3.29242.03

3.45211.01

2.29502.02

0.43127.04

4.48146.96

1.07205.98

2.67366.18

3.08192.06

4.46121.97

1.55899.160.34

296.140.06

201.002.77

784.493.10

344.922.29

455.123.64

201.00

0.95261.09

3.13208.04

3.07169.98

2.29155.12

2.09621.18

4.55190.99

4.51187.97

2.64627.24

3.09168.99

4.28200.99

3.02587.20

3.73139.99

2.76636.39

2.41799.232.53

543.19

1.74228.00

3.06323.94

2.33504.300.25114.09

0.23224.01

2.68745.42

2.10181.02

2.68740.463.25

114.092.76

143.063.47

169.983.53

154.992.59

257.140.72348.07

3.32130.53

3.53100.08

0.35148.06

1.44595.171.34439.161.81715.160.44

206.112.55

355.172.41

139.040.87

132.102.56

303.062.17

228.002.70

183.003.06

194.042.82

275.143.35

227.992.64

579.223.24

118.091.50

426.051.30

360.152.91

439.022.37

383.072.70

621.122.26

114.092.53

208.042.32

181.020.87

259.132.06

728.262.28

373.181.18

349.142.07

731.163.05

165.992.21

207.110.86

236.982.73

369.043.12

246.00

2.30273.17

1.71181.02

2.85587.19

2.10652.11

2.25625.17

3.13167.99

0.86249.99

0.43527.16

2.82609.23

0.27213.01

4.54167.00

0.25171.98

3.44143.06

0.96200.99

0.54169.983.12215.01

2.29799.23

3.34214.00

0.94181.99

2.79841.24

2.76453.12

0.39278.10

1.07139.99

1.47219.15

0.30336.85

1.30913.15

3.00143.06

3.39182.99

2.92749.25

3.03196.02

2.54470.18

2.06328.92

0.29190.00

1.16220.122.16

291.100.43

124.042.13211.010.70390.07

0.78159.97

3.53167.99

2.49554.17

1.01330.06

2.34783.23

0.97231.14

0.77307.15

2.28439.231.06204.99

2.07374.14

2.05447.10

0.47124.08

0.76119.05

1.41381.04

2.36260.13

2.92611.142.71

493.142.54

413.182.05

429.150.93

195.982.05

419.102.19

695.090.45

349.141.69

447.182.82

873.240.68

173.990.25

196.982.58

292.23

1.04190.990.34

277.150.25

173.991.92

531.170.65

227.021.61

503.082.16

245.102.46

595.111.71

415.262.07

379.102.40

273.08

1.98578.06

2.19690.111.41

349.091.69

449.110.72

190.992.06

535.151.29

265.152.48

428.151.80337.09

0.70359.15

0.51129.10

2.10399.140.31236.09

1.41311.13

0.70167.06

2.55545.201.68434.58

2.10182.99

0.45396.04

0.89304.15

1.35427.12

0.45428.04

2.04426.59

1.15376.14

1.79617.12

0.88307.080.65287.98

1.06218.99

0.28137.03

2.09214.00

0.42381.08

0.25200.99

1.18367.15

1.41194.08

2.16601.122.42

471.23

1.95305.07

0.43261.040.47

111.092.04

570.161.16368.10

0.45495.02

0.88355.072.22

385.19

1.17493.18

2.47177.170.42

738.16

1.56373.19

0.37460.17

1.42425.201.69

261.17 1.15323.08

2.31449.18

1.12510.16

1.06241.16

2.09599.200.48157.100.43783.22

1.18159.09

1.45273.08

1.40534.09

0.90314.09

2.55783.23

2.09427.10

1.19764.16

0.70301.14

1.53212.06

1.39426.052.55453.12

1.33147.04

2.22442.19

0.52337.16

1.44295.08

1.33339.11

1.14298.10

0.35130.05

1.10212.061.09171.030.33304.161.45123.051.12458.11

1.14355.10

1.87535.15

0.47217.12

0.51123.06

1.45607.13

1.68514.102.09

163.04 1.87224.01

2.41592.08

0.40246.06

2.46518.14

1.25652.11

1.26465.07

1.16267.14

1.75561.19

1.99402.16

1.18146.06

1.23442.05

1.82563.21

1.86434.200.46

322.971.33

395.131.20

188.071.52

420.191.65

319.050.41365.11

1.22169.05

0.70319.15

1.01268.11

0.45460.21

1.97885.192.22331.16

1.92275.19

0.42353.16

1.28867.211.28419.10

1.34323.08

1.54418.15

1.27321.05

1.28362.09

2.03779.20

1.59480.06

1.23305.07

1.50867.21

1.52275.19

1.52789.210.29138.99

2.02810.131.09229.16

1.65481.10

1.69209.15

0.52229.16

1.94433.11 1.69

389.22

0.44146.09

1.76595.16

1.20205.10

1.25303.021.95

417.17

0.39292.11

2.41457.111.86

439.161.76795.19

0.35446.161.52

467.081.45180.071.76826.12

0.35184.081.36

747.151.62

731.16

0.36258.11

1.82289.07

1.52425.14 2.22

401.16

1.88757.222.46

482.261.82

608.071.69

411.201.49

139.04

0.32175.12

0.38310.12

1.60627.15

1.49289.07

1.82261.17

1.87579.170.71

337.16

1.10345.080.45

236.15

1.40441.17 0.48

348.071.21

595.141.69

469.131.26

447.06

1.42261.172.46

487.21

0.40194.09

1.45291.091.35

579.15

1.13611.142.02

757.221.99

123.05 1.98273.08

1.62565.161.21

289.071.49

917.181.20

139.041.83473.11

1.75773.22

1.98443.10

1.21307.08

1.49459.10

Samples2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

%

0

100

7.498e+002

m/z = 307.08

Scores: Component 1 - Component 2

Component 1-0.200 0.000 0.200

Com

pone

nt 2

-0.200

-0.100

0.000

0.100

0.200

0.300 Traditional English

Earl Grey

Lotus Green

Japanese Green

Chai

©2007 Waters Corporation 108

Comparison of Analysis TimeComparison of Analysis TimeUPLC/LCTUPLC/LCT--Premier DataPremier Data

3, 10 and 30 minute 3, 10 and 30 minute analysis timeanalysis time

min2.0 4.0 6.0 8.0 10.0

%

0

100

3.102e+0052.95

0.36

2.520.50

2.400.97

4.173.41 4.31 5.208.397.99

7.45

min5.0 10.0 15.0 20.0 25.0 30.0

%

0

100

1.472e+0050.52

9.63

0.73

6.771.01

6.491.35

9.89

14.0110.3224.8915.01

min1.00 2.00 3.00 4.00

%

0

100

1.955e+0051.39

0.27 1.10

0.38

0.511.50 1.98

2.35 2.56

©2007 Waters Corporation 109

Effect of Run TimeEffect of Run Timem/z 443.09m/z 443.09

Samples5 10 15 20 25 30 35 40 45 50 55 60

%

0

100

4.245e+002

From 30 minute Separation.

No loss of information.

Samples5 10 15 20 25 30 35 40 45 50 55 60

%

0

100

4.734e+002

From 3 minute separation.

From 10 minute separation.

Samples5 10 15 20 25 30 35 40 45 50 55 60

%

0

100

6.109e+002

O

O

O

OH

OH

OH

OH

OHOH

OH

Catechin Gallate

©2007 Waters Corporation 110

Negative ion AnalysisNegative ion Analysisseparation of decaffeinated and separation of decaffeinated and caffeinatedcaffeinated

Loadings: Component 1 - Component 2

Component 1-0.100 0.000 0.100 0.200

Com

pone

nt 2

-0.100

-0.050

0.000

0.050

0.100

0.150

0.38195.06

0.38165.05

0.41439.15

1.98441.10

1.42289.081.10

343.100.52

173.111.50

457.090.88

169.04

1.60337.10

1.34353.030.41

191.011.73

367.111.98

883.181.21

611.140.35357.16 2.17

593.19

1.42289.14

1.10191.09 1.26

633.211.59337.15

1.18411.060.89

125.051.74771.30

0.35132.06

0.41383.191.59173.05

1.19761.301.82471.151.36577.27

1.50457.17

2.03755.26

1.39581.29

1.49503.20

1.64479.01

2.24447.102.41

425.111.98883.25

0.36357.22

1.17305.17

1.42913.39

0.41383.25

1.10343.17

1.37577.131.42

289.011.27

387.152.09597.21

1.49747.34

1.41579.161.99905.25

1.50747.24

1.10687.231.88

609.140.42

533.070.44

431.161.49

579.051.50

747.031.19

483.081.59

675.121.20

305.002.54

567.231.21

593.16

1.16331.11

1.99905.19

1.56395.18

1.64729.07

1.92431.08

0.89168.98

0.41533.251.63

563.051.40

325.081.71

593.131.50

502.991.88

577.100.51

481.092.70

867.281.57

785.10

1.35337.02

1.15410.961.53

635.002.29

896.271.10

191.02

0.39135.05

2.71867.241.52501.07

2.05739.33

1.75807.34

1.75583.17

2.87555.38

0.35458.08

1.19491.11

1.59337.21

1.30579.070.35

444.081.69817.28

0.35146.021.38

577.061.70

177.041.27

443.191.35

912.932.00

906.141.99

791.122.70

715.150.43

431.37

1.41463.15

1.81337.07

2.71867.17

2.02801.43

1.44595.10

2.13761.20

1.28758.93

1.26632.920.88

609.062.50

426.992.18

593.011.72

771.131.94

927.14

1.68593.071.48

593.050.44

431.021.34

299.080.42

781.361.55

395.130.42

729.131.86

713.110.46

168.97

0.28273.022.63761.24

1.86529.03

1.82745.09

1.40325.02

1.19323.14

0.58173.03

1.96463.022.77419.29

2.46261.09

2.45687.14

3.70146.98

1.16218.151.41

473.131.53

455.080.83

290.111.26

341.041.00

146.972.46

541.33

0.41377.02

0.31280.98

1.21631.10

0.84132.900.78

243.082.08

307.912.34

405.191.98

692.132.81

617.262.70

479.23

1.67433.16

2.28419.16

0.85134.89

2.74451.31

0.31242.98

2.68639.42

0.53450.92

1.35251.13

2.24307.90

1.48406.18

2.64209.101.35

707.092.85

473.252.33

614.211.20

375.070.50

343.07

2.73479.37

1.41363.16

2.01499.23

2.24714.22

1.39137.02

0.19337.32

1.11164.12

1.08282.08

2.12461.02

2.72367.23

0.56146.93

2.03431.33

1.63515.00

2.82616.19

2.85559.21

2.87515.26

1.73207.02

1.52401.12

0.52346.91

2.28134.89

2.69134.04

0.45317.121.45175.03

2.51153.05

2.48385.26

1.46484.06

2.46576.19

1.82371.08

2.41535.31

1.33190.87

0.72597.96

1.68595.31

2.71352.88

1.1599.931.09

282.191.81

371.282.53

744.381.60

433.08

1.89397.131.44293.21

0.94369.10

2.79717.43

0.77227.98

1.28176.99

1.92493.37

0.75598.08

0.90317.03

1.55201.13

2.7999.942.3199.93

1.28443.29

2.08377.22

1.45429.18

2.05485.13

2.27561.07

1.30183.031.33739.41

1.16218.041.71339.18

2.81483.19

1.14783.071.46393.31

0.64389.03

2.12319.13

2.81387.32

1.89307.88

2.27509.33

1.54387.120.42

719.280.52

216.981.91

711.281.56

201.180.54

594.182.10

694.23

2.32614.11

1.25593.062.40334.02

1.93711.57

2.33625.17

2.12469.13

2.27157.89

1.74694.13

2.59651.39

1.93329.07

1.62623.931.68

711.122.66

599.32

1.77361.16

1.30467.93

1.11335.05

2.12469.08

1.72235.88

1.85657.23

2.32843.44

2.81183.12

2.04303.11

1.70493.10

2.64779.27

1.36639.07

1.37451.08

1.41509.03

1.38715.11

1.96173.04

1.5799.931.80

466.082.43317.18

1.89247.08

1.41323.13

1.65755.36

1.26465.04

2.74157.88

2.45443.23

2.23649.42

2.75317.17

1.61447.02

2.39501.38

1.38669.08

2.47389.26

1.52583.12

1.24655.06

1.26445.06

1.52733.33

2.30261.17

0.85261.95

1.94428.12

2.11293.10

2.81616.09

1.26445.12

1.63624.11

0.83235.92

1.74351.13

1.17203.06

0.29289.01

0.43601.04

1.69146.921.75

325.070.56

116.952.81

539.162.12345.28

1.84377.12

2.62519.23

1.23180.07

2.84361.15

2.66421.28

2.56371.182.01648.23

0.30242.88

1.10737.42

2.65421.22

1.18152.971.19359.19

1.93391.28

2.05623.38

0.77273.00

2.08461.33

1.48473.31

2.12345.13

2.53715.38

2.62810.21

2.82617.171.93711.49

1.10527.19

2.75595.18

2.67636.24

1.41279.06

0.53241.92

1.84147.01

2.85439.14

0.44763.63

2.94352.92

1.68353.13

1.97694.10

2.70417.31

1.68549.30

1.71309.13

0.93483.22

2.71609.12

2.17581.221.45393.18

2.24649.26

2.04835.38

2.57727.38

0.27601.36

1.75595.050.38134.93

2.57609.191.58

447.091.68

385.102.28

723.422.58

520.282.27449.33

2.11671.24

1.38395.19

2.14641.22

1.46153.00

1.83225.14

1.23452.99

2.66655.17

2.32561.28

2.34383.14

1.57500.13

1.40561.14

2.09507.16

2.45352.91

1.98493.30

2.55190.96

0.31296.77

1.22515.06

2.88473.18

1.08142.08

2.53375.201.48502.17

1.90593.062.87

473.122.81

361.222.28

723.311.11

497.231.14

767.13

0.51499.23

0.90127.05

1.88723.27

1.68385.132.06

563.322.95

197.142.09

923.31

1.88385.170.33312.37

1.93329.23

1.23371.99

2.93389.22

2.42340.08

0.47600.99

0.42781.08

2.87517.08

1.33451.941.25351.07

1.74335.05

0.33329.87

1.35352.98

2.76451.25

0.31349.00

1.10282.02

1.41339.03

1.33421.02

2.40477.37

2.42535.17

2.24661.28

1.56623.38

0.64628.972.66537.23

2.83515.18

2.54715.31

0.44197.92

2.46523.26

1.92343.21

2.81539.21

0.85175.02

0.51247.921.48391.240.68263.11

1.92537.35

2.70235.99

1.32116.89

0.94483.11

2.36543.18

2.07235.93

2.44261.220.52703.31

1.61176.99

1.69463.02

1.55423.08

1.11142.03

2.82616.130.51119.27

1.14185.07

0.63127.03

0.78159.07

1.18383.14

1.41339.16

2.82391.261.54623.34

1.13368.16

1.10737.36

1.47293.08

1.25707.18

1.80131.082.24649.32

2.30615.41

1.48611.15

2.20449.27

2.69199.160.53401.07

2.43340.03

1.87395.171.47593.32

1.76319.10

1.52623.28

2.85439.19

1.72217.08

0.33113.07

2.04516.13

1.11164.06

0.42781.291.30369.02

1.62525.02

1.28339.021.33

351.021.56

423.031.61

399.041.45

451.161.39

235.882.58

461.371.45

451.260.52

346.852.86

345.220.29

264.932.35

383.191.10

265.081.89

247.150.70

235.032.41

503.322.00585.20

1.70493.18

2.24649.37

2.31515.33

2.61739.39

1.45553.04

1.94593.33

1.16218.09

2.11593.43

1.52313.05

2.09539.08

1.30161.021.64415.21

1.79367.04

2.26711.42

2.26711.30

2.09987.39

2.58553.27

0.42719.07

1.11497.09

2.47579.43

1.29285.05

0.33301.15

0.54351.06

0.29265.02

1.28339.12

2.65519.18

2.83475.25 0.28

441.47

0.31242.931.22345.08

2.54415.31

2.64332.06

0.90199.02

1.58281.14

2.71243.19

2.25615.27

1.87719.46

0.50189.10

2.35543.110.28418.83

1.63467.28

1.17251.15

1.12597.14

1.42369.08

0.51597.21

2.00146.93

1.97593.390.48432.01

2.74607.37

0.38391.11

2.09638.15

0.51432.07

2.57655.22

0.42729.27

1.62399.17

1.52408.140.43781.18

2.42579.26

0.33285.08

0.48189.24

2.50146.96

1.56453.970.52

216.931.13

515.111.45153.04

1.10595.13

1.36190.992.03

373.221.97

631.27 1.12613.31

2.05349.181.79

367.261.33421.181.74

351.24

0.28288.89

2.88235.961.13613.14

2.63719.33

0.34225.00

2.10461.08

1.88719.34

2.80517.27

2.16191.06

1.29369.13

0.28402.76

2.02549.39

1.40385.071.44293.16

2.22177.06

2.16475.292.73379.302.62609.251.10595.23

0.44925.41

0.51615.18

2.36503.39

2.42447.16

0.43341.14

1.13363.070.40539.24

1.56711.38

0.31130.98

1.25311.09

1.80357.14

1.84235.95

1.96631.22

2.75481.27

0.34292.98

0.41438.89

0.48127.06

0.27419.071.09595.19

2.56711.452.66

493.231.67431.210.38208.92

2.00477.11

1.55387.22

2.57515.18

2.32515.17

0.47194.94

1.31353.081.68

431.130.45

132.992.75

373.301.17

361.211.96

595.38

0.41387.04

1.55401.072.51477.17

2.47235.99

2.57445.24

2.05417.28

1.97593.27

0.29174.20

0.30147.02

0.85117.04

1.21453.12

2.65493.28

1.49385.14

0.51615.23

0.65405.14

0.30280.91

1.56454.112.23146.96

1.94593.17

2.00595.31

0.51247.860.38

391.181.41

367.11

2.72479.30

1.98397.18

2.82391.201.29235.97

2.99285.14

0.35321.15

1.28355.06

0.75499.34

1.64467.20

1.10523.13

0.43267.07

2.32515.26

1.10160.05

2.65563.16

0.33104.06

0.31113.021.66371.19

1.82305.070.47379.01

2.61405.20

1.82305.18

2.30461.13

2.82146.96

2.00391.14

0.94129.02

2.40477.30

1.17314.970.41

438.99

2.52116.95

1.25341.16

1.23345.18

0.37181.10

2.45431.22

2.14369.13

0.38208.991.46235.96

1.34335.13

1.12326.20

1.18353.09

2.63719.42

1.79146.97

1.57236.10

2.57655.27

1.10292.11

2.66477.32

2.81463.302.21235.97

1.15597.24

0.43404.152.50607.400.41387.34

1.75248.14

1.22705.35

2.55507.250.90217.08

2.74595.37

1.96641.21

0.35293.03

1.27351.16

0.50167.03

1.52407.982.58

553.190.37179.072.41

201.161.13

329.16

1.20597.312.05417.21

0.38164.93

0.42533.18

2.79461.37

1.29285.14

0.27288.831.40147.07

0.39209.12

1.63525.191.37563.17

2.32515.21

1.22179.06

1.12389.03

0.41729.32

0.75499.16

1.40281.08 1.37

335.06

2.07493.08

2.62461.17

0.52188.87

2.33477.23

1.54313.12

0.28402.860.44369.060.47188.93

0.28299.08

0.28334.092.41625.34

0.65190.981.87359.22

2.78417.24

0.43267.12

2.61477.26

0.42386.93

0.65405.03

2.24116.95

2.51607.30

1.35531.22

1.11515.24

0.45206.87

0.28146.96 0.43

763.20

1.16315.03

2.87269.06

2.54415.24

0.28272.92

0.40190.96

2.79116.95 1.32

707.25

2.62503.29

1.82116.94

0.28458.97

2.54717.371.10265.14

0.41190.91

2.56515.26

0.90217.03

2.45579.31

0.65436.06

2.38623.34

1.87405.23

2.74191.051.55401.181.93537.18

0.34225.140.42781.23

1.13613.230.44369.15

2.58507.18

1.62371.11

0.28175.031.41

367.200.43

207.07

2.13323.09 1.74

367.17

1.96355.17

0.44197.83

0.44195.83

1.55355.040.40191.07

2.63359.14

0.46115.01

0.42377.150.77

128.050.34

145.110.34

225.060.27

418.881.10292.202.02

116.940.28

419.02

1.12579.28

1.94391.19

2.56609.37

2.74595.32

2.05469.26

0.27402.92

1.31109.05 0.35

293.20

0.28304.91

2.03549.17

1.97355.20

0.44317.071.55

355.090.28

272.971.39

146.96 1.96641.26

0.38164.98

1.29313.12

1.42116.94

0.41601.142.35

477.17

0.39194.98

1.87359.16

0.39149.00

2.31461.19

2.34337.132.69

193.072.34

337.181.87

405.18

2.45431.160.31112.971.97593.222.71

147.092.54717.30

2.56655.40

0.40223.06

1.17315.08

0.42719.23

0.44160.862.82

517.171.96

401.221.97

161.060.48

219.06

0.49379.13

0.34145.06

1.82305.12

0.65435.97

0.51541.20

2.09539.21

1.98173.10 0.28

305.01

0.34114.05

1.99595.19

2.81517.22

2.63359.18

1.19153.04

0.35293.10

2.42195.10

2.79461.31

1.68431.29

2.76461.26

1.96711.41

1.96193.09

1.56711.32

2.00595.26

2.05303.17

1.12579.21

0.27288.93

1.37209.09

2.62711.30

0.75499.26

0.33131.09

0.28174.95

0.66111.02

2.60609.30

2.04417.16

1.73595.25

0.46133.040.32131.030.30

158.95

1.36191.06

1.95401.172.12

323.140.42

387.130.41

439.091.47

179.05

2.00549.24

1.37165.09

2.07493.20

1.96711.33

0.38209.06

0.43207.02

2.64187.12

0.41377.09

1.16315.15

0.48127.01

1.55355.15

1.31353.15

0.65191.04

min0.50 1.00 1.50 2.00 2.50 3.00 3.50

%

0

100

1.322e+0050.42

0.38

1.97

1.881.32

1.10

0.650.77

1.591.82

2.622.54

2.10 2.742.83

Scores: Component 1 - Component 2

Component 1-0.100 0.000 0.100 0.200

Com

pone

nt 2

-0.200

-0.100

0.000

0.100

0.200

CaffeinatedCaffeinated DecaffeinatedDecaffeinated

3 minute UPLC separation3 minute UPLC separation

©2007 Waters Corporation 111

Fresh vs. Refrigerated Tea Fresh vs. Refrigerated Tea without Caffeinewithout Caffeine

Scores: Component 1 - Component 2

Component 1-0.100 0.000 0.100 0.200

Com

pone

nt 2

-0.200

-0.100

0.000

0.100

Loadings: Component 1 - Component 2

Component 10.000 0.200 0.400 0.600

Com

pone

nt 2

-0.200

-0.100

0.000

0.100

0.200

0.300

1.14181.07

1.88611.160.51

158.080.51

175.112.18595.171.99

465.102.27

449.111.24

333.06

2.26287.06

2.93565.14

1.19763.15

0.27146.03

1.99303.05

1.11120.081.52

333.06

1.12130.09

0.41144.10

1.36163.04

1.60147.04

0.27151.01

2.01741.22

1.34915.16

0.40136.061.35

355.10

0.27166.99

1.75275.091.24

287.062.70

869.16

0.34266.130.38138.05

0.51661.27

2.64717.14

1.11166.09

2.56611.19

0.88611.14

0.88593.130.41193.07

1.89132.08

1.19127.04

1.24581.130.39

162.11

0.28174.03 0.27

162.012.63

147.04

1.36597.180.89171.03

1.23196.10

2.14717.141.14

222.100.28

128.02

1.12310.13

1.17109.03

0.51130.07 0.34

248.12

0.33104.11

1.45865.20

0.28123.01 1.12

328.14

0.40116.070.95

268.102.62

188.07

1.21583.14

1.18599.14

0.46260.12

1.17168.071.39236.12

1.10276.14

2.47565.13

1.39138.070.30206.89

0.36259.10

2.41427.101.27365.12

1.27131.052.04

603.130.30290.85

1.09152.06

1.23761.13

0.48305.14

2.00257.08

0.66182.08

1.17293.15

2.32499.12

1.71186.13

1.12144.081.27

163.080.66

136.080.42

139.04

0.46347.10

1.17967.24

1.60165.05

0.65438.98 2.57

517.132.32

517.131.54

186.13

0.65437.97 2.34

303.050.46

702.13

0.45442.19

0.31122.93

0.34351.07

2.56535.11

1.53733.14

2.74577.15

0.44278.13

1.43735.15

0.66165.05

1.13171.11

2.93564.13

1.46937.17

0.30320.87

1.15311.07

0.27138.99

1.53913.140.47

174.080.89

127.040.94136.06

0.28218.991.27143.04

2.35238.142.57163.04

1.10294.16

0.40204.09

1.09233.15

0.48191.10

1.14801.15

0.35295.12

0.37203.06

1.59339.11

1.45408.18

2.50429.08

0.31542.74

2.28755.152.35197.12

0.53262.13

1.21224.01

2.75563.18

2.32163.04

0.77130.05

0.28144.00

2.73224.01

2.40179.07

2.81581.09

1.44595.171.88633.141.76

139.042.27

471.091.60

206.081.71

177.061.24

112.081.86476.31

2.79551.18

2.30477.27

0.66121.06

1.35181.03

0.65215.020.44325.12

1.30785.15

1.96487.08

1.18103.05

1.60119.050.33190.11

2.52503.192.29

919.190.44117.00

0.35234.10

2.19551.10

1.76885.15

1.24778.16

1.71531.170.65393.98

2.18435.09

1.86481.26

1.49887.17

1.29186.09

2.01520.33

1.27172.08

2.48887.26

2.87739.15

1.74179.03

0.34277.13

0.31274.88

0.46342.14

3.11224.01

1.16609.121.57388.25

0.42215.060.31181.01

1.71691.212.52224.01

1.10245.15

2.66565.131.14178.13

2.01525.29

1.17160.11

1.14136.06

1.53930.171.28211.01

1.63193.05

0.56182.99

2.93427.100.41126.06

2.35334.18

1.56553.15

1.54433.12

1.18163.04

1.57371.23

2.75575.14

2.03401.21

2.71595.17

1.56393.21

1.83540.19

2.91224.02

2.04209.16

2.16564.36

1.75451.20

1.75446.24

0.54218.01

1.71432.28

2.16569.31

0.51402.12

1.54899.17

1.81147.05

1.29613.17

1.29625.12

2.47401.092.13334.18

2.57499.12

1.15278.09

0.45291.05

1.65643.152.56449.15

2.64325.09

2.82419.14

0.40160.10

2.17617.15

0.32458.78

0.65129.02

0.29160.05

0.44150.09

0.47352.08

0.34133.06

1.71437.24

2.41257.08

2.87397.17

1.26406.152.87336.20

0.33147.08

1.71439.09

0.34418.16

0.22190.99

1.79133.07

2.94701.10

1.78333.06

2.93706.09

1.32222.08

2.63324.08

1.78180.07

2.87903.26

0.29242.01

1.79174.09

1.42931.16

1.69583.14

2.73253.17

1.63534.17

2.37341.14

0.44114.09

1.16627.13

0.98174.08

2.83431.14

2.76334.18

2.81553.10

2.47564.13

0.42152.06

0.76348.07

1.33266.13

1.45613.17

1.83729.14

2.14238.09

1.93641.17

1.54204.14

2.81717.13

1.35475.09

2.09581.18

2.28423.03

2.32581.19

0.56214.01

0.30120.99

1.50169.98

1.28199.03

3.00332.17

2.58380.19

1.59451.12

2.87219.100.89

434.992.76

887.260.47

127.092.80

222.152.04

163.152.34

302.141.50

594.05

2.20761.14

2.02477.27

2.88561.07

3.11903.26

2.94195.14

1.50211.01

1.35496.05

1.10199.11

2.56465.14

0.45649.22

2.69868.15

1.07289.14

1.67207.14

2.89169.98

2.44419.10

2.69341.14

0.56280.14

1.07346.06

1.87535.15

0.41246.062.09

599.201.16

220.121.93

591.212.34

423.091.82

206.081.30

779.142.39

332.171.63

333.162.41

562.072.69

629.152.54

287.123.00

182.992.57

169.980.86

305.130.45

100.081.85

459.280.54

303.992.30

608.39

0.44206.11

2.41139.04

0.43124.04

1.28360.15

2.36799.23

2.22565.15

2.55177.09

2.81181.02

2.66371.14

1.92609.13

1.86169.98

1.81165.05

3.00378.17

1.92667.15

2.92305.04

0.43256.09

2.52520.19

1.00330.06

0.47173.09

2.29951.12

2.75919.25

2.11341.14

2.42256.08

3.60214.00

0.45131.01

2.82257.08

2.03219.10

2.01333.06

2.71287.06

1.33225.10

0.24177.98

2.63243.28

3.13314.14

1.44715.15

1.85435.13

0.63405.01

2.27849.08

0.86710.06

0.86139.04

2.58741.20

1.65729.14

2.06403.23

2.54331.16

1.46751.16

1.92377.09

2.69932.17

2.31169.98

2.52325.14

2.00401.16

2.67564.13

2.87318.18

2.05207.14

1.14229.12

2.48533.20

0.77344.13

1.91106.07

0.40458.19

2.10867.18

2.80716.06

0.85132.10

0.86742.23

2.80249.11

1.97889.25

2.58696.44

2.92298.18

2.93236.17

2.80747.13

0.36460.17

1.56373.190.42

174.042.16

291.102.54

493.132.41

107.050.86

633.122.31

493.102.13

177.161.07

182.991.97

441.082.12

280.120.72

204.992.06

374.151.21

493.162.11

311.130.55

197.090.77

307.152.30

273.171.71

181.022.87

401.161.07

214.012.92

743.181.66

360.110.96

261.091.94

743.202.09

222.021.95

439.022.92

889.241.96

194.040.06

201.002.54

429.150.44

261.042.82

873.252.77

784.490.24

169.992.75

100.071.58

563.170.54

279.140.26

214.012.44

425.093.38

155.124.76

171.983.10

344.922.82

609.232.64

627.242.76

636.392.71

493.142.49

441.251.78

774.212.92

749.252.28

373.182.33

219.102.07

763.210.53

719.240.24

173.990.43

527.162.05

429.152.23

881.161.66

659.211.30

435.111.93

369.160.32

236.082.55

355.172.44

595.111.92

531.172.19

695.090.65

227.020.70

167.062.07

379.102.42

731.160.50

160.111.44

327.202.16

245.091.42

617.111.85

883.171.41

381.042.16

130.071.15

114.090.42

127.041.61

503.081.24

633.111.79

337.091.38

377.072.10

182.990.45

495.021.15

323.082.26

513.071.16

368.102.41

273.081.50

500.120.46

275.990.07

205.000.06

177.98

1.44219.15

1.34439.16

1.81715.16

1.69447.17

1.93287.06

1.23448.18

1.75558.25

1.16469.13

1.16455.12

1.74275.19

0.47277.98

2.02479.08

2.63466.27

1.33339.112.18224.01

2.05535.14

0.45157.082.10399.14

1.76595.16

0.46396.04

0.88304.15

2.22442.18

1.06218.98

2.48428.15

0.88307.08

0.47111.09

1.95305.07

1.23169.98

0.47217.12

0.48190.99

0.30165.03

0.06182.99

2.10169.98

1.30913.15

2.24277.07

1.94412.22

0.54169.98

1.14355.10

2.09427.10

1.17367.15

1.93433.110.51

129.101.79

617.122.16

601.120.44

783.221.58

321.061.05

190.990.42

381.081.35

427.121.12

458.111.05

204.992.41

592.081.45

295.080.46

322.970.41738.16

2.46518.14

2.45177.16

0.51229.16

0.33304.15

2.55783.23

1.36463.17

2.55453.12

1.40194.08

1.33395.13

1.92275.19

1.53212.06

0.98314.09

1.43607.13

1.25652.11

1.12510.16

1.45273.08

2.09163.04

1.81563.21

1.07241.16

1.41311.12

0.33296.14

0.28137.03

1.74514.10

1.40534.09

1.34147.04

1.59480.06

1.98570.16

1.75561.19

2.22331.16

1.33323.08

1.28419.10

1.18159.09

1.48275.190.70301.14

1.42426.05

2.03810.13

1.19764.16

2.03779.20

1.95417.17

0.29190.00

1.44291.09

1.98443.101.14

298.100.35130.052.00402.16

1.26465.07

0.35184.081.50867.21

1.53420.19

1.23442.05

0.29158.98

1.87224.01

1.95139.04

1.45123.04

1.28867.21

1.76303.05

1.28321.05

1.77795.19

1.31362.09

0.42353.16

0.70319.15

1.53789.21

2.40457.11

1.86434.20

1.98578.06

1.09229.15

1.64319.05

1.86439.16

1.69389.22

1.53425.14

1.69411.20

0.35148.061.19205.10

1.18146.06

0.45460.20

1.26303.02

1.77826.12

1.82608.07

0.41365.11

1.65261.17

1.69209.15

1.22169.05

2.22401.16

1.83289.07

1.64481.10

1.10345.080.35

446.161.09

171.031.10

212.061.58418.151.82261.17

1.15267.13

1.96885.191.23305.07

1.52467.08

2.46482.260.32

175.120.41

146.090.51

337.16

0.49348.07

1.44180.07

1.62731.161.19188.071.41441.17

1.60627.15

1.36747.15

1.32261.16

0.39292.11

0.45236.15

1.87579.17

1.25447.061.69

469.132.46

487.210.40

194.091.87757.22

0.35258.111.98

123.040.39310.12

1.98273.081.49

139.041.49289.07

1.36579.15

1.62565.16

1.13611.141.21

595.141.21

289.071.49

917.18

1.21139.042.02

757.221.82473.11

0.71337.16

1.76773.22

1.21307.08

1.49459.10

©2007 Waters Corporation 112

Database SearchingDatabase Searchingdetecting detecting biomakersbiomakers

Identified Analytes

©2007 Waters Corporation 113

Potential BiomarkersPotential Biomarkersreviewing using the database browserreviewing using the database browser

Catechin Catechin GallateGallate

©2007 Waters Corporation 114

XIC of (XIC of (--) ) EpigallocatechinEpigallocatechinfrom 3 minute ESI+ datafrom 3 minute ESI+ data

Caffeinated TeasCamomile Bottom 6

Time1.00 2.00 3.00 4.00

%

0

100

1.00 2.00 3.00 4.00%

0

100

1.00 2.00 3.00 4.00

%

0

100

1.00 2.00 3.00 4.00

%

0

100

1.00 2.00 3.00 4.00

%

0

100041505_PR_ESI_pos_137 1: TOF MS ES+

307.082 0.05Da135

2.13

1.590.421.100.88 1.18 2.07 2.822.65

2.93

041505_PR_ESI_pos_105 1: TOF MS ES+ 307.082 0.05Da

1651.100.50

0.44

0.350.88

1.781.131.59

1.93

2.352.64 2.77

041505_PR_ESI_pos_089 1: TOF MS ES+ 307.082 0.05Da

77.01.10

0.510.36 0.86

1.222.41

1.782.28 2.43 2.86

3.04 3.36 4.644.47

4.24

041505_PR_ESI_pos_057 1: TOF MS ES+ 307.082 0.05Da

1410.50

0.430.40 0.87 1.10

1.592.272.19 2.37 2.88

041505_PR_ESI_pos_009 1: TOF MS ES+ 307.082 0.05Da

2081.81

0.87

0.790.50 1.09 1.582.12 2.86

2.44 2.923.07

Decaffeinated TeasLotus Green Tea Bottom 6

Time1.00 2.00 3.00 4.00

%

0

100

1.00 2.00 3.00 4.00

%

0

100

1.00 2.00 3.00 4.00

%

0

100

1.00 2.00 3.00 4.00

%

0

100

1.00 2.00 3.00 4.00

%

0

100041505_PR_ESI_pos_153 1: TOF MS ES+

307.082 0.05Da4.83e4

1.22

1.13

041505_PR_ESI_pos_121 1: TOF MS ES+ 307.082 0.05Da

4.61e31.22

1.13

0.88

041505_PR_ESI_pos_073 1: TOF MS ES+ 307.082 0.05Da

4.58e31.22

1.12

0.88 1.48

041505_PR_ESI_pos_041 1: TOF MS ES+ 307.082 0.05Da

3.71e31.21

1.12

0.46 0.87 1.47

041505_PR_ESI_pos_025 1: TOF MS ES+ 307.082 0.05Da

4.24e41.21

1.12

1.0 x

11.4 x

1.2 x

1.2 x

13 x

# 2

Lotus Green

Earl Grey

TraditionalEnglish

Chai

JapaneseGreen

Camomile

Black Current

Lemon andGinger

Cranberry

TensionTamer

©2007 Waters Corporation 115

Comparison of Important IonsComparison of Important Ionscaffeinated and decaffeinated teacaffeinated and decaffeinated tea

++++++++++++Lotus Green

Camomile

++++++++Earl Grey

Black Current

Lemon & Ginger

++++++++English

Cranberry

+++++++Chai

++++++++++++Japanese Green

Tension Tamer

Catechin GallateEpigallocatechinCaffeineTea

©2007 Waters Corporation

A Metabonomic Analysis A Metabonomic Analysis of Rice Using of Rice Using

Waters Metabonomic SystemWaters Metabonomic System

©2007 Waters Corporation 117

Sample Preparation MethodSample Preparation Method

3 grams of ground rice10 mL H2OHomogenize 30 mL EtOHWrist action shake 30 min.Let settle 5 min.Spin 500 mL @ 14,000 rcf 15 min.Speedvac until dryReconstitute 100 uL 15:85 MeOH:H20

©2007 Waters Corporation 118

ACQUITY UPLCACQUITY UPLC™™ / MS/ MSParametersParameters

UPLC/MS ConditionsUPLC/MS ConditionsACQUITY UPLC with LCT PREMIER™

Gradient elution over 10 minutes 0-95%B— A: Water (0.1% formic acid)— B: Acetonitrile (0.1% formic acid)

Column: ACQUITY UPLC BEH C18Flow Rate: 600uL/minColumn Temperature: 40CFunction type: TOF MSIonization Mode: Positive ion ESIResolution: W-Optics™, 12,000 FWHMScan duration (secs): 0.30Inter-scan delay (secs): 0.02Retention window (mins): 0.0 to 12.0

©2007 Waters Corporation 119

Typical ACQUITY UPLC Typical ACQUITY UPLC ChromatogramChromatogram (TIC) of Extracted Rice(TIC) of Extracted Rice

Korea Rice_ESI+_090205_1

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100Korea Rice_ESI+_090205_1 1: TOF MS ES+

TIC2.94e5

4.07

3.81

1.92

1.50

0.50

0.38

1.14

3.48

2.32

3.272.48

3.23

3.10

3.71

4.21

10.50

4.52

6.23

5.344.84

5.22 5.915.82

9.366.29

6.79

6.62 8.287.907.49

10.14

©2007 Waters Corporation 120

Results of MarkerLynxResults of MarkerLynx™™ db Analysis Showing db Analysis Showing Identification of Identification of NeopterinNeopterin as a Significant Markeras a Significant Marker

KORJPN CHN

AUS

©2007 Waters Corporation 121

Metabolic Pathway in Which Metabolic Pathway in Which NeopterinNeopterin is is involved involved (highlighted enzymes are in rice genome)(highlighted enzymes are in rice genome)

©2007 Waters Corporation 122

Results of MarkerLynx™ db Analysis With Thai Jasmine Rice and USA Instant Rice Included in The Analysis

THAI

USA KOR

JPN CHN

AUS

©2007 Waters Corporation 123

ConclusionsConclusions

UPLC combined with time of flight mass spectrometry is an ideal tool for profiling food and pharmaceutical batches

Caffeinated versus decaffeinated teas can be distinguished as can tea Varieties

Integrated database helps identify important marker ions

Pharmaceutical products from different sources can be differentiated

Potential markers can be identified using exact mass and MS/MS analysis

©2007 Waters Corporation

Environmental MonitoringEnvironmental Monitoring

©2007 Waters Corporation 125

Environmental applicationsEnvironmental applicationswww.waters.comwww.waters.com

Perfluorinated alkyl compounds — Screening and quantitative methods in relevant matrices— Collaborations with van Bavel (MTM, Orebro) and US-EPA NERL (Triangle Park)

Endocrine disrupting compounds — Focus on LC/MS but consider GC/MS if advantageous— Develop on-line SPE solution

Pharmaceuticals — Collaborations with Barcelo (CSIC) and US-EPA (Cincinnati)

Water disinfectant byproducts — Proof of principle work covering main legislated compounds by LC/MS and

GC/MS— Possible collaboration with US-EPA (Athens)

Brominated flame retardants (PBDEs, HXBCD etc) — Need to commercialise previous work for lower end labs— Address WEEEs and RoHS legislation— Possible collaborations with MOE (Toronto), MTM (Orebro)

Polychlorinated alkanes — Proof of principle work on GC/MS/MS

Pesticides and other established priority pollutants — Develop sensitive screening solutions— Demonstrate on-line SPE solution

Homeland defense applications— Collaboration with US-EPA ORD (Las Vegas)

©2007 Waters Corporation 126

SummarySummary

Analytical results black list priority pollutants in drinking and

surface water

— Introduction

— Instrumentation

— Method optimisation

Synthetic Pyrethroids analysis

Conclusions

©2007 Waters Corporation

Multi residue analysis of EU black list priority Multi residue analysis of EU black list priority pollutants in drinking and surface water using pollutants in drinking and surface water using

solid phase extraction and GC Tandem solid phase extraction and GC Tandem Quadrupole MSMSQuadrupole MSMS

©2007 Waters Corporation 128

EU directive 76/464/EECEU directive 76/464/EEC

• EU council directive 76/464/EEC lists a number of compound classes which have restricted levels in drinking and surface water

• Of these compounds groups >100 individual compounds are GC amenable– The GC amenable compounds cover a wide range of compound

groups, with differing chemical properties and polarities

• Typically, each group would have a dedicated analytical method

• A single injection confirmatory method could save laboratories a vast amount of time

©2007 Waters Corporation 129

Compounds analysedCompounds analysed

benzidines chloronitrotoluenes organochloro pesticides organophosphorus pesticidesbenzidine 2-chloro-6-nitrotoluene hexachlorobenzene dichlorvos3,3’-dichlorobenzidine 4-chloro-2-nitrotoluene α-hexachlorocyclohexane trichlorfonchloroanilines 4-chloro-3-nitrotoluene β-hexachlorocyclohexane mevinphos(Z)2-chloroaniline 2-chloro-3-nitrotoluene lindane mevinphos(E)3-chloroaniline chlorophenols δ-hexachlorocyclohexane omethoate4-chloroaniline 2-chlorophenol heptachlor demeton-S-methyl2,6-dichloroaniline 2,4-dichlorophenol aldrin dimethoate2,4-dichloroaniline 3-chlorophenol isodrin demethon-O2,5-dichloroaniline 4-chlorophenol o,o'-DDE disulfoton2,3-dichloroaniline 4-chloro-3-methylphenol α-chlordane parathion-methyl3,5-dichloroaniline 2,3,5-trichlorophenol α-endosulfan fenitrothion3,4-dichloroaniline 2,4,6-trichlorophenol o,p'-DDE malathion4-chloro-2-nitroaniline 2,4,5-trichlorophenol γ-chlordane fenthionchloronitrobenzenes 2,3,4-trichlorophenol dieldrin parathion-ethyl1-chloro-3-nitrobenzene 2,3,6-trichlorophenol p,p'-DDE triazophos1-chloro-4-nitrobenzene 2-amino-4-chlorophenol o,p'-DDD azinphos-methyl1-chloro-2-nitrobenzene 3,4,5-trichlorophenol endrin azinphos-ethyl3,5-dichloronitrobenzene pentachlorophenol β-endosulfan coumaphos2,5-dichloronitrobenezene chlorotoluidines p,p'-DDD phenylurea pesticides2,4-dichloronitrobenzene 2-chloro-4-toluidine o,p'-DDT monolinuron3,4-dichloronitrobenzene miscellaneous p,p'-DDT linuron2,3-dichloronitrobenzene biphenyl PCBs Semi volatile halogenated comopunds1-chloro-2,4-dinitrobenzene tributhyl phosphate PCB28 hexachlorobutadienePAHs trifluraline PCB52 1,2-dichloronaphthalenenaphthalene propanil PCB101 1,2,3,4-tetrachloronaphthalenePhenanthrene bentazone PCB77 triazinesanthracene pyrazon PCB118 simazinefluoranthene volatile amines PCB153 atrazinebenzo[b]fluoranthene cumene PCB138 benzo[k]fluoranthene PCB126benzo[a]pyrene PCB180indeno(1,2,3-cd)pyrene PCB169benzo[ghi]peryleneDibenz(a,h)anthracene

©2007 Waters Corporation 130

Choice of methodsChoice of methods

• Published1 methods use a combination of SIR screening and full scan confirmation in separate injections

• Tandem Quadrupole GC-EI/MSMS can offer a single injection for combined low level screening and confirmation

• Confirmatory method - Two MRM transitions per compound– Confirmation of target compounds at low levels

• Screening method - single MRM transition per compound– Increased duty cycle per compound

1. Anal. Chem.2000, 72,1430-1440; Broad Spectrum Analysis of 109 Priority Compounds Listed in the 76/464/CEE Council Directive Using Solid-Phase Extraction and GC/EI/MS; Sílvia Lacorte, Ingrid Guiffard,Daniel Fraisse,DamiàBarceló

©2007 Waters Corporation 131

Choice of methodsChoice of methods

• Extraction/clean-up needs to be as ‘generic‘ as possible– Wide range of compound groups and polarities, all requiring good

recoveries

– How many compounds can be included in one extraction/clean-up process?

• GC separation must be optimised based upon the detection technique and compounds of interest– Lower detection selectivity requires greater chromatographic

separation

– compounds having common fragmentation?

– Acquisition time window switching

©2007 Waters Corporation 132

Waters Quattro Micro GC Tandem Quadrupole mass spectrometer operated in EI+ mode— Acquisition performed in MRM mode

Agilent 6890 GC fitted with cool on column and Split/Splitless injectors

4mm ID double taper deactivated liner for splitless injections

CTC Combi PAL autosampler

InstrumentationInstrumentation

©2007 Waters Corporation 133

Quattro Micro GC Quattro Micro GC

Quattro micro GC

Tandem Quadrupole GC/MS/MS

©2007 Waters Corporation 134

Full scan analysis— Records all ions within a specified mass range, need to select specific

mass chromatograms for quantification if there is high background from sample matrix

— Can suffer interference, lower sensitivity

SIR analysis— Records specific ions for each target compound

— More selective when compared with full scan, greater sensitivity

MRM analysis— Records compound specific transitions for each target analyte

— High selectivity, High sensitivity

Quattro Micro GC Quattro Micro GC Why MSMS?Why MSMS?

©2007 Waters Corporation 135

Quattro Micro GC Quattro Micro GC Selectivity & SensitivitySelectivity & Sensitivity

MS1Collision

Cell MS2

StaticCIDStatic

Multiple Reaction Monitoring (MRM)minimises matrix interference

high sensitivityaccurate and reproducible quantitation

©2007 Waters Corporation 136

Priority PollutantsPriority PollutantsGCGC--MSMS optimisationMSMS optimisation

• Two columns evaluated– 30m 250 μm 0.25 μm DB17-ms

– 30m 250 μm 0.25 μm VF5-ms

• Two injectors evaluated– Split/splitless operated in pulsed splitless mode

– Cool on column (COC) operated in track oven mode, 2m 0.53mm I.D. Retention gap fitted

• Injection technique and columns evaluated for maximum separation and response– close eluting compounds such as 3 & 4 Chlorophenol, E & Z

mevinphos, trichlorophenols, o,p‘-DDT & p,p‘-DDD

©2007 Waters Corporation 137

GCGC--MSMS optimisationMSMS optimisationDB17DB17--ms + COC injectionms + COC injection

• Reconstructed total ion chromatogram from MRM acquisition– 40ºC/1min, 3ºC/min to 160ºC, 7ºC/min to 240ºC,15ºC/min to

305ºC, hold 15mins. 1ml/min He flow

©2007 Waters Corporation 138

GCGC--MSMS optimisationMSMS optimisationDB17ms separationDB17ms separation

• Mevinphos (E & Z) separation

• o,p‘-DDT and p,p‘-DDD separation

©2007 Waters Corporation 139

GCGC--MSMS optimisationMSMS optimisationVF5VF5--ms with pulsed splitless injectionms with pulsed splitless injection

• Reconstructed total ion chromatogram from MRM acquisition– 40ºC/0.8min, 6ºC/min to 160ºC, 8ºC/min to 310ºC, hold 2mins.

0.9ml/min He flow

– Injector 250ºC, purge time 0.8mins,110kPa pulse for 0.8mins

©2007 Waters Corporation 140

GCGC--MSMS optimisationMSMS optimisationVF5ms separationVF5ms separation

• Mevinphos (E & Z) separation

• o,p‘-DDT and p,p‘-DDD separation

©2007 Waters Corporation 141

Method sensitivityMethod sensitivity

• Summary of method LODs, based upon S/N of 3:1, using confirmatory transition– Confirmatory LODs (two transitions per compound) and

screening LODs (one transition per compound)

– Method LOD based upon average (n=5) LOD from 0.5µg/L spiked water extracts (DCM elution)

0.32977Method LOD,Confirmatory

0.11294Method LOD,Screen

0.016100Instrument LOD, Screen

10

No Compounds >0.1 μg/L

0.03

Avg LOD μg/L

96Instrument LOD, Confirmatory

No Compounds <0.1μg/L

©2007 Waters Corporation 142

Synthetic Synthetic PyrethroidsPyrethroids in the in the EnvironmentEnvironment

In the environment, they have limited persistence compared to many other pesticide classesThey are extremely toxic to fish and aquatic organisms

An estimated 200 million litres of sheep dip is disposed of each year from around 50,000 dipping facilities in the UK—Discharged directly into water courses —Via a soakaway that allows the dip to soak into the ground —Pumped out and spread onto surrounding farmland

©2007 Waters Corporation 143

Electron ImpactElectron ImpactPermethrinPermethrin MSMS

©2007 Waters Corporation 144

Electron ImpactElectron ImpactPermethrinPermethrin MS/MSMS/MS

Quantification Transition

Confirmation Transition

Ar = 3.0 x 10-3mBar

©2007 Waters Corporation 145

Electron ImpactElectron ImpactMS MethodMS Method

MRM functions arranged into 5 time windows—More flexible use of dwell times—Improved signal to noise (S/N) ratios for some analytes—Overlapping allows for small changes in retention time

©2007 Waters Corporation 146

Total Ion Chromatogram (TIC)Total Ion Chromatogram (TIC)

Bi fe

nthr

in

Cy f

lut h

rin

λ-C

y hal

othr

in

De l

tam

eth r

in

Pe r

me t

hri n

Cy p

erm

ethr

in

Fenv

a ler

a te

©2007 Waters Corporation 147

Electron Impact SensitivityElectron Impact Sensitivity

0.4ng/L0.4pg/μL9:11pg/μLFenvalerate I

15:1

6:1

10:1

6:1

15:1

6:1

50:1

150:1

S/N

0.5ng/L0.5pg/μL1pg/μLCyfluthrin

0.5ng/L0.5pg/μL1pg/μLcis-Permethrin

1pg/μL

1pg/μL

1pg/μL

1pg/μL

1pg/μL

1pg/μL

Standard Concentratio

n

0.4ng/L0.4pg/μLCypermethrin

0.2ng/L0.2pg/μLtrans-Permethrin

0.06ng/L0.06pg/μLλ-Cyhalothrin

0.5ng/L0.5pg/μLFenvalerate II

Method LOD

Instrumental LOD

Synthetic Pyrethroid

0.2ng/L0.2pg/μLDeltamethrin

0.02ng/L0.02pg/μLBifenthrin

• LOD based on S/N ≥ 3:1 of the confirmation MRM transition• Method LOD assumes 10μL is injected• EU Regulations, Permethrin = 10ng/L, Cyfluthrin = 1ng/L

©2007 Waters Corporation 148

Electron ImpactElectron ImpactWastewater ExtractWastewater Extract

Permethrin, ~ 0.03μg/L

% Difference = 1.6%Confirmation Criteria

± 10%

Expected Ratio = 0.62Measured Ratio = 0.63

©2007 Waters Corporation 149

Pyrethroids fragment around the ester oxygen single bond

Compounds with a positive electron affinity ionise well in negative chemical ionisation mode, e.g. halogenated

The selectivity of negative chemical ionisation is greater than electron impact for the synthetic pyrethroids

This gain in selectivity should improve the LOD achievable

Negative Chemical IonisationNegative Chemical Ionisation

OO

O

CH3 CH3

Cl

Cl

Negative

Positive

©2007 Waters Corporation 150

Pyrethroid SensitivityPyrethroid SensitivityEI+ versus CIEI+ versus CI--

7 PyrethroidsMean = 20x

Fenvalerate, 10pg on columnEI+

CI-

©2007 Waters Corporation 151

Negative Chemical IonisationNegative Chemical IonisationBifenthrinBifenthrin MSMS

Methane = 1.6 x 10-4mBar

©2007 Waters Corporation 152

Negative Chemical IonisationNegative Chemical IonisationBifenthrinBifenthrin MS/MSMS/MS

Quantification Transition

Confirmation Transition

Ar = 3.0 x 10-3mBar

©2007 Waters Corporation 153

Negative Chemical IonisationNegative Chemical IonisationWastewater ExtractWastewater Extract

Fenvalerate, ~ 0.04μg/L

% Difference = 0%Confirmation Criteria

± 15%

Expected Ratio = 0.31Measured Ratio = 0.31

©2007 Waters Corporation 154

Negative Chemical Ionisation Negative Chemical Ionisation Wastewater ExtractWastewater Extract

Deltamethrin, ~ 0.007μg/L

% Difference = 2.0%Confirmation Criteria

± 10%

Expected Ratio = 0.99Measured Ratio = 0.97

©2007 Waters Corporation 155

Negative Chemical Negative Chemical Ionisation SensitivityIonisation Sensitivity

0.001ng/L0.001pg/μL430:10.1pg/μLFenvalerate I

25:1

180:1

8:1

6:1

8:1

5:1

650:1

1200:1

S/N

0.05ng/L0.05pg/μL0.1pg/μLCyfluthrin

0.1ng/L0.1pg/μL0.1pg/μLcis-Permethrin

0.1pg/μL

0.1pg/μL

0.1pg/μL

0.1pg/μL

0.1pg/μL

0.1pg/μL

Standard Concentr

0.05ng/L0.05pg/μLCypermethrin

0.05ng/L0.05pg/μLtrans-Permethrin

0.001ng/L0.001pg/μLλ-Cyhalothrin

0.002ng/L0.002pg/μLFenvalerate II

MethodLOD

Instrumental LOD

Synthetic Pyrethroid

0.02ng/L0.015pg/μLDeltamethrin

0.0005ng/L0.0005pg/μLBifenthrin

• LOD based on S/N ≥ 3:1 of the confirmation MRM transition• Method LOD assumes 10μL is injected• EU Regulations, Permethrin = 10ng/L, Cyfluthrin = 1ng/L

©2007 Waters Corporation 156

SummarySummary

Quattro micro GC provides:—High selectivity to reduce matrix interferences

—High sensitivity to reach the levels required by legislation

—Accurate quantification of targets at low levels in matrix

—Quantitative and confirmatory data in a single injection

PTV in solvent vent mode provides:—Ability to inject larger volumes onto the GC column

Negative chemical ionisation provides:—Greater sensitivity and selectivity for the synthetic

pyrethroids