Agilent QQQ Data

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LC/MS Food SafetyApplications

J uly, 2006

Agilent TechnologiesAgilent Technologies

6000 Series LC/MS6000 Series LC/MS

for Food Safetyfor Food Safety



Group/LC/MS Food SafetyApplications

J uly, 2006

Agilent LC/MS SolutionsAgilent LC/MS Solutions

• Single provider of:

– Single Quad

– Triple Quad– Ion Trap

– TOF

– Q-TOF

• Major solutions for Food

– High performance/low cost Agilent 6410 Triple Quad LC/MS

– High performance 6210 Agilent TOF LC/MS




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Nitrofuran MetabolitesNitrofuran Metabolites -- Agilent 6410 LC/MS TripleAgilent 6410 LC/MS Triple

QuadrupoleQuadrupole




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The four drugs furazoidone, furaltadone, nitrofurazone

and introfurantoin are veterinary drugs that belong to

family of nitrofuran antibiotics

Widely used for the treatment of gastrointestinal

infections in cattle, pigs and poultry

The European Union (EU) banned the use of nitrofuran

antibiotics in food-producing animals by listing them in

AnnexⅣ of Council Regulation 2377/90

The EU set a Minimum Required Performance Limit(MRPL) of 1 ug/kg that laboratories should at least detect

and confirm

BackgroundBackground




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EU RegulationsEU Regulations-- Minimum Required PerformanceMinimum Required Performance

Limits (MRPL)Limits (MRPL)

COMMISSION DECISIONof 12 August 2002

implementing Council Directive 96/23/EC concerning the performance of analytical

methods and

the interpretation of results

(2002/657/EC)




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2002/657/EC2002/657/EC

•Set’s criteria for screening (< 5 % β error)

•Set’s criteria for confirmation

–For LC/MS

•MS Points for confirmation

•MS ratio of at least two ions•Retention time tolerance

•Does not set level for nitrofurans




J uly, 2006

EU RegulationsEU Regulations --NitrofuransNitrofurans

COMMISSION DECISION

of 13 March 2003

amending Decision 2002/657/EC as regards the setting of minimum required performance limits

(MRPLs) for certain residues in food of animal origin

(2003/181/EC)

Nitrofuran metabolites:

— furazolidone

— furaltadone— nitrofurantoin

— nitrofurazone’

Poultry meat

Aquaculture products 1 µg/kg for all




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Structures of Structures of NitrofuransNitrofurans andand MetaboliteMetabolitess

O

NN NH

O

O

N

O

O

H2NN NH

O

O

N

O O

NN

NH

O

O

Nitrofurantoin AHD NPAHD

O

NNH NH2

O

N

O

O

H2NNH NH2

O

Nitrofuranzone SEM NPSEM

NO O

NNH

O

NH2




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O

N NO

O

N

O

O

H2N N O

O

NO O

N N

OO

Furazolidone AOZ NPAOZ

O

NN

ON

O

O

O

N

O

Furaltadone AMOZ NPAMOZ

H2NN

O

O

N

O

N

O O

NN

OO

N

O

Structures of Structures of NitrofuransNitrofurans andandmetabolitemetabolitess




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HPLCHPLC ParametersParameters◆ HPLC system : Agilent 1100 series

◆ Column : C18, 2.1×150, 5 µm

◆ Injection Volumn : 50 µL◆ Flow rate : 0.3 mL/min

◆ TempTemp : 40: 40℃

◆ Mobile phase : A-0.1% Formic acid; B-Acetonitrile

◆ Gradient

109017.01109022

9551795512.0160401250501010900BATime




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◆ MS system : Agilent 6410 LC/MS/MS◆ Ion source : ESI

◆ Polarity : Positive

◆ Nebulizer gas : Nitrogen

◆ Ion spray voltage : 4000V

◆ Source temperature : 350℃

◆ Resolution : Q1 (unit) Q3 (unit)

◆ Scan mode : Multiple Reaction Monitoring (MRM)

◆ Conditions of MRM

MS ParametersMS Parameters




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Mass spectra of SC at different collision energiesMass spectra of SC at different collision energies

4x10

0

0.2

0.4

0.6

0.8

11.2

1.4

1.6

1.8

Abundance vs. Mass-t o-Charge (m/z)100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

+ProductIon(9.738min)(209.2->**)nitrofuran_MS2_100_Frag_5CE_3.d

209.0166.1 192.1

134.2

107.9119.4 147.9

4x10

0

0.2

0.4

0.6

0.8

1

Abundance vs. Mass-t o-Charge (m/z)100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

+ProductIon(9.721min)(209.2->**)nitrofuran_MS2_100_Frag_10CE_4.d

192.2

149.1134.2 166.2

209.1

103.8119.0

CE 10VCE 10V

CE 5VCE 5V




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MRM Conditions of nitrofuran metabolitesMRM Conditions of nitrofuran metabolites

559292150150166.3166.3209.1209.1

559292150150134.2134.2249.1249.1AHAH9.959.95

559292150150104.2104.2249.1249.1

559292150150134.3134.3236.0236.0AOZAOZ10.810.8

559292150150104.3104.3236.0236.0

559292150150192.3192.3209.1209.1SCSC88

559292150150262.4262.4335.1335.1

559292150150291.4291.4335.1335.1AMOZAMOZ00

Collision

Engergy (V)

Fragmentor

(V)

Dwell

(ms)ProductPrecursor CompoundTime




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Low Outlier

High Outlier

Quant Ion Integration

Rel Ion Ratio




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Ion RatiosIon Ratios-- Instant Graphic ReviewInstant Graphic Review

R e l a t i v e A b u n d a n c e ( % )

AcquisitionTime(min)

2x10

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

2.4 2.6 2.8 3 3.2 3.4 3.6

335.1->291.1 ,262.1

Ratio=24.0

Relative Peak AbundanceQuant Ion

Relative Peak A... Qualifier

(outside limits-colored)

}20 %

Tolerance




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Calibration curve of AMOZCalibration curve of AMOZ

Linear range: 10 ppt ~ 10ppb

RR22=0.99985=0.99985




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MRM EIC of MRM EIC of Nitrofuran MetabolitesNitrofuran Metabolites (100(100 pptppt))

2x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

Abundance vs. Acqui si t i on Ti me (mi n)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

+MRMMRM(249.1->134.3)Quan_Qualify_100ppt_2.dSmooth(1)

1 1 2 2 3 3 4

AMOZAMOZ

SCSC

AHAH

AOZAOZ




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2x10

0.5

1

1.5

2

2.5

3

3.5

Abundance vs. Acqui si t i on Ti me (mi n)0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8


4.5131 1

1x10

4.8

55.2

5.4

5.6

5.8

6

6.2

6.4

6.6


4.481

4.592

1.997

1 1

AMOZ 100AMOZ 100

pptppt

331.3331.3→291.4 (Quant Ion)291.4 (Quant Ion)

AMOZ 100AMOZ 100 pptppt

335.1335.1→262.4 (Qualify Ion)262.4 (Qualify Ion)

EIC at TransitionsEIC at Transitions m/zm/z 229191 andand m/zm/z 262262




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2x10

1

2

3

4

5

6

Abundance vs. Acqui si t i on Ti me (mi n)11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5 20 20.5 21 21.5 2


11.194

12.323 13.15911.702

4 4

2x10

0.5

0.6

0.7

0.8

0.9

1

1.1


11.197

11.767

4 4

EIC Transitions atEIC Transitions at m/zm/z 113434 andand m/zm/z 110404

AOZ 100AOZ 100 pptppt


AOZ 100AOZ 100 pptppt





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2x10

0.5

0.6

0.7

0.8

0.9

Abundance vs. Acqui si t i on Ti me (mi n)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 2

+MRMMRM(335.1->291.4)10ppt_2.dSmooth(1) Noise(RMS)=8.54;SNR(4.481min)=6.5

4.481

3.175

7.999

1 1 2 2 3 3 4 4

1x10

5

5.5

6

6.5

7

7.5

+MRMMRM(209.1->192.3)10ppt_2.dSmooth(1) Noise(RMS)=14.69;SNR(9.838min)=4.0

9.838

9.480

1 1 2 2 3 3 4 4

MRM EIC SpeMRM EIC Specctrum of AMOZ and SC (10trum of AMOZ and SC (10 pptppt))

S/N=6S/N=6

S/N=4S/N=4




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2x1 0

0. 6

0. 8

1

1. 2

1. 4

1. 6

1. 8

2

2. 2

2. 4

2. 6

2. 8

3

3. 2

Ab u n d a n c e v s . Ac q u i s i t i o n T i me ( mi n )1 2 3 4 5 6 7 8 9 1 0 11 1 2 1 3 1 4 1 5 16 1 7 1 8 1 9 20 21 2 2

+ MRM MR M ( 2 3 6 .0 -> 1 34 . 3 ) N F r e a l s a m p l e B l a n k _ 1 . d Sm o o t h ( 1 )

1 1 2 2 3 3 4 4

2x1 0

0. 6

0. 8

1

1. 2

1. 4

1. 6

1. 8

2

2. 2

2. 4

2. 6

2. 8

3

3. 2

3. 4

3. 6

3. 8

Ab u n d a n c e v s . Ac q u i s i t i o n T i me ( mi n )1 2 3 4 5 6 7 8 9 1 0 11 1 2 1 3 14 15 16 17 1 8 1 9 20 2 1 2

+ M R M M RM (2 4 9 .1 - > 13 4 .2 ) N F _ M RM s p i k e 1 00 p p t _ 2.d S m o o t h ( 1)

1 0 . 0 9 3

1 1 2 2 3 3 4 4

Spike Nitrofuran Metabolites in TilapiaSpike Nitrofuran Metabolites in Tilapia (100(100 pptppt))

BlankBlank

SpikeSpike AMOZ AMOZ

SC SC

AOZ AOZ

AH AH




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The Reproducibility of Nitrofuran MetabolitesThe Reproducibility of Nitrofuran Metabolites

1.051.050.10.1AMOZAMOZ

2.012.010.10.1SCSC

1.581.580.10.1AOZAOZ

8.078.070.10.1AHAH

RSDRSD

(%)(%)

ConcentrationConcentration

((ng/mng/mLL))CompoundsCompounds

a Each value was the average of 8 replicates (n=8).




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Malachite Green and MetaboliteMalachite Green and Metabolite

Agilent 6410 Triple QuadrupoleAgilent 6410 Triple Quadrupole




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MalachiteMalachite GGreen (MG)reen (MG)

Among other veterinary drugs, theAmong other veterinary drugs, the triphenyltriphenyl--methanemethanedye malachite green (MG) is a popular substance, anddye malachite green (MG) is a popular substance, and

prevents fungal and parasitic infections in aquaculture,prevents fungal and parasitic infections in aquaculture,includingincluding saprolegniasissaprolegniasis, white spot disease, and, white spot disease, andciliates.ciliates.

MG is prevalently reduced intoMG is prevalently reduced into leucoleuco--malachite greenmalachite green(LMG) and deposited in fatty tissue of the fish.(LMG) and deposited in fatty tissue of the fish.

C

N

N

Cl

Malachite Green

C

N

N

H

Leucomalachite Green

reduction

(Green) (Colorless)




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EU Regulatory Limit for Malachite Green and itsEU Regulatory Limit for Malachite Green and its

MetaboliteMetabolite




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HPLC equipped with a postHPLC equipped with a post--column unit for oxidation of LMG andcolumn unit for oxidation of LMG and

with either an absorbance or MS detector for detection in troutwith either an absorbance or MS detector for detection in trout

(eggs, fry and muscle), catfish (muscle and plasma) and in trout(eggs, fry and muscle), catfish (muscle and plasma) and in trout

muscle. The reactorsmuscle. The reactors (5 cm(5 cm××4mm)4mm)used in these studies were filledused in these studies were filled

withwith 2525%% PbOPbO22

Other method LCOther method LC--APCIAPCI--MS in catfish and trout without the use of MS in catfish and trout without the use of

a reactor for the conversion of LMG. This study aimed at thea reactor for the conversion of LMG. This study aimed at the

development and validation of a procedure for sample processingdevelopment and validation of a procedure for sample processing

and of an HPLC analysis method for determination of residues of and of an HPLC analysis method for determination of residues of

MGMG

The use of LCThe use of LC – –ESIESI--MSMS – –MS analysis was explored for MS analysis was explored for

confirmation of detected residues of this drug in such matrices.confirmation of detected residues of this drug in such matrices.

Analytical Approaches for Determination of Analytical Approaches for Determination of

Residues of MGResidues of MG




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Tissue sample (5g)

Add 1mL 0.25 g/mL Hydroxylamine hydrochloride

Add 2mL 1mol/L p-toluene sulfonic acid

Add 2ml 0.1 mol/L NH4Ac buffer, pH4.5

Add 40ml acetonitrile

homogenized for 2 min

PPtSupernatant(acetonitrile phase)

Add 20ml acetonitrile

filter

Supernatant

35ml water, 30ml dichloromethane

Collect dichloromethane phase, add 20ml dichloromethane in upper layer

LC/MS/MS

Concentration

The analysis of residues of MG and LMG in TilapiaThe analysis of residues of MG and LMG in Tilapia




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HPLCHPLC ParametersParameters

◆ HPLC system : Agilent 1100 series

◆ Column : C18, 2.1×150, 5 µm

◆ Injection Volumn : 10 µL

◆ Flow rate : 0.3 mL/min

◆ Mobile phase : A-10mmol NH4Ac (pH=4.5); B-Acetonitrile◆ Gradient

30701330708.0195589552

5050130700BATime




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◆ MS system : Agilent 6410 LC/MS/MS◆ Ion source : ESI

◆ Polarity : Positive

◆ Nebulizer gas : Nitrogen

◆ Ion spray voltage : 4000V

◆ Source temperature : 350℃

◆ Resolution : Q1 (unit) Q3 (unit)

◆ Scan mode : Multiple Reaction Monitoring (MRM)◆ Conditions of MRM

MS ParametersMS Parameters




J uly, 2006

TIC of MG and LMG withTIC of MG and LMG with

DifferentDifferent Fragmentor Fragmentor VoltagesVoltages

7x10

1

2

3

4

5

Abundance vs. Acquisition Time (min)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 1

+EIC(329.4,331.4m/z)ScanoptimizingFRG70_3.d

1 1

7x10

1

2

3

4

5


1 1

7x10

1

2

3

4

5

6


1 1

70V

90V

110V




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Mass spectra of MG at different collisionMass spectra of MG at different collision

energiesenergies5x10

0

0.5

1

1.5

2

2.5

3

3.5

4

Abundance vs. Mass-to-Charge (m/z)50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 35

+ProductIon(5.549min)(329.3->**)optimizingMS2_FRG100_CE20_2.d

329.3

313.5208.3 284.3236.9193.1

268.4

5x10

0

0.2

0.4

0.6

0.81

1.2

1.4

1.6

1.8

2


329.3

313.4

208.2

285.3251.4237.2 270.3192.8164.6 298.9217.4134.3

5x10

0

0.2

0.4

0.6

0.8

1

1.2


313.3

208.2

329.4284.2165.3 241.4

270.3192.1 251.4221.0134.1 298.9

91.5117.9 202.0179.8

CE 20VCE 20V

CE 30VCE 30V

CE 40VCE 40V

C

N

N

[M+H]+ m/z 329

-CH4 -dimethylaniline

m/z 313 m/z 208




J uly, 2006

MRM Conditions of MG and LMGMRM Conditions of MG and LMG

30301001004040239.2239.2331.3331.3

30301001004040316.3316.3331.3331.3LeucomalachLeucomalach

iteite greengreen77

40401001004040208.2208.2329.3329.3

40401001004040313.3313.3329.3329.3MalachiteMalachite

greengreen00

Collision

Engergy (V)

Fragmentor

(V)

Dwell

(ms)ProductPrecursor CompoundTime




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Calibration curve of malachite greenCalibration curve of malachite green


RR22=0.9995=0.9995




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Calibration curve of Calibration curve of LeucomalachiteLeucomalachite greengreen


RR22=0.9994=0.9994




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3x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

Abundance vs. Acqui si t i on Ti me (mi n)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5

+MRMMRM(329.3,331.3->**)malachitegreen_200606127.d

8.409

5.440

8.118 9.660

1 1 2

MRM EIC of MG and LMG (100MRM EIC of MG and LMG (100 pptppt))

MGMG

LMGLMG




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EIC spectra of ions atEIC spectra of ions at m/zm/z 313 and313 and m/zm/z 208208

2x10

0

0.5

1

1.5

2

2.5

3

3.5


+MRM(329.3->313.3)malachitegreen_200606127.d

5.4481 1 2

2x10

00.2

0.4

0.6

0.8

1

1.2

1.4

1.6

+MRMMRM(329.3->208.2)malachitegreen_200606127.d

5.4361 1 2

Malachite Green 100Malachite Green 100 pptppt


Malachite Green 100Malachite Green 100 pptppt

329.3329.3→208.2 (208.2 (QualQual Ion)Ion)




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3x10

0

0.2

0.4

0.6

0.8

1



8.409

8.118 9.660

1 1 2

1x10

0

1

2

3

4

5

6


8.3951 1 2

EIC spectra of ions atEIC spectra of ions at m/zm/z 239 and239 and m/zm/z 316316

LeucomalachiteLeucomalachite Green 100Green 100pptppt


LeucomalachiteLeucomalachite Green 100Green 100

pptppt





J uly, 2006

2x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3


+MRMMRM(331.3->239.2)MG_10ppt_MRM_a_2.d

8.394

9.4528.725

1 1 2

MRM EIC SpeMRM EIC Specctrum of MG and LMG (10trum of MG and LMG (10 pptppt))

MGMG

LMGLMG




J uly, 2006

1

1 .5

2

2 .5

3

3 .5

4

4 .5

5

5 .5

6

6 .5

7

7 .5

8

Ab u n d a n c e v s . Ac q u i s i t i o n T i me ( mi n )0 .5 1 1 .5 2 2 .5 3 3 .5 4 4 .5 5 5 .5 6 6 .5 7 7 .5 8 8 .5 9 9 .5 1 0 1 0 .5 1 1 1 1 .5 1

+ M R M M RM (3 3 1 .3 - > 23 9 .2 ) S p i k e _ B l a n k _ 1 .d

1 1 2 2

3x1 0

0

0 .2

0 .4

0 .6

0 .8

1

1 .2

1 .4

1 .6

1 .8

2

2 .2

2 .4

2 .6

2 .8

3

3 .2

3 .4

3 .6

Ab u n d a n c e v s . Ac q u i s i t i o n T i me ( mi n )0 .5 1 1 .5 2 2 .5 3 3 .5 4 4 .5 5 5 .5 6 6 .5 7 7 .5 8 8 .5 9 9 .5 1 0 1 0 .5 1 1 1 1 .5

+ M R M M RM (3 2 9 .3 - > 31 3 .3 ) S p i k e 1 p p b _ 1 . d

1 1 2 2

Spike MG and LMGSpike MG and LMG

in Tilapiain Tilapia

(1 ppb)(1 ppb)

BlankBlank

SpikeSpikeMGMG

LMGLMG




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1

1 .5

2

2 .5

3

3 .5

4

4 .5

5

5 .5

6

6 .5

7

7 .5

8

Ab u n d a n c e v s . Ac q u i s i t i o n T i me ( mi n )0 .5 1 1 .5 2 2 .5 3 3 .5 4 4 .5 5 5 .5 6 6 .5 7 7 .5 8 8 .5 9 9 .5 1 0 1 0 .5 1 1 1 1 .5 1

+ M R M M RM (3 3 1 .3 - > 23 9 .2 ) S p i k e _ B l a n k _ 1 .d

1 1 2 2

2x1 0

0

0 .1

0 .2

0 .3

0 .4

0 .5

0 .6

0 .7

0 .8

0 .9

1

1 .1

1 .2

1 .3

1 .4

1 .5

1 .6

1 .7

Ab u n d a n c e v s . Ac q u i s i t i o n T i me ( mi n )0 .5 1 1 .5 2 2 .5 3 3 .5 4 4 .5 5 5 .5 6 6 .5 7 7 .5 8 8 .5 9 9 .5 1 0 1 0 .5 1 1 11 .5 1

+ MRM MRM (3 3 1 . 3 -> 23 9 . 2 ) S p i k e 1 0 0 p p t _ 1. d

1 1 2 2

Spike MG and LMGSpike MG and LMG

in Tilapiain Tilapia

(100(100

pptppt

))

BlankBlank

SpikeSpike

MGMG LMGLMG




J uly, 2006

The Reproducibility of MG and LMGThe Reproducibility of MG and LMG

2.252.250.10.1LMG (LMG (m/zm/z 331331.3.3→239239.2.2))

3.3.52520.10.1MG (MG (m/zm/z 329329.3.3→313.3313.3))

RSDRSD

(%)(%)

ConcentrationConcentration

((ng/mng/mLL))

CompoundsCompounds

a Each value was the average of 8 replicates (n=8).

Documents

Agilent QQQ Data