Agilent QQQ Data

Agilent Technologies 6000 Series LC/MS for Food Safety
LC/MS Food Safety Applications July, 2006
Agilent 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
Group/LC/MS Food Safety Applications July, 2006
Nitrofuran Metabolites - Agilent 6410 LC/MS Triple Quadrupole
Background
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 Annex 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
EU Regulations- Minimum Required Performance Limits (MRPL)
COMMISSION DECISION of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results (2002/657/EC)
2002/657/EC
Sets criteria for screening (< 5 % error) Sets 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
EU Regulations -Nitrofurans
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 Poultry meat furaltadone 1 g/kg for all Aquaculture products nitrofurantoin nitrofurazone
Structures of Nitrofurans and Metabolites

O O N O O N N O Nitrofurantoin NH N O AHD O O N NH O Nitrofuranzone NH2 H2N NH O SEM NPSEM NH2 NPAHD O N NH O NH2 O NH O O O N N
NH O
H2N
O N O
Structures of Nitrofurans and metabolites

O N O O O N N O Furazolidone O O N O O N O N N O H2N O O N N O O O H2N N O AOZ O N NPAOZ O O N N N O O N O O N N O
Furaltadone
AMOZ
NPAMOZ
HPLC Parameters

HPLC system Column Injection Volumn Flow rate Mobile phase Gradient
: Agilent 1100 series : C18, 2.1150, 5 m : 50 L : 0.3 mL/min : 40 : A-0.1% Formic acid; B-Acetonitrile
Time 0 10 12 12.01 17 17.01 22 A 90 50 40 5 5 90 90 B 10 50 60 95 95 10 10
Temp
MS Parameters

MS system Ion source Polarity Nebulizer gas Ion spray voltage Resolution Scan mode Conditions of MRM
: Agilent 6410 LC/MS/MS : ESI : Positive : Nitrogen : 4000V : Q1 (unit) Q3 (unit) : Multiple Reaction Monitoring (MRM)
Source temperature : 350
Mass spectra of SC at different collision energies

+ Product Ion (9.738 min) (209.2 -> **) nitrofuran_MS2_100_Frag_5CE_3.d x10 4 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 107.9 119.4 147.9 134.2 166.1 192.1 209.0
CE 5V
Abundance vs. M ass-t o-Charge (m /z)
x10 4 1 0.8 0.6 0.4 0.2 0
+ Product Ion (9.721 min) (209.2 -> **) nitrofuran_MS2_100_Frag_10CE_4.d 192.2
CE 10V
134.2
149.1
166.2
103.8
119.0
209.1
100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
MRM Conditions of nitrofuran metabolites
Time 0
Compound AMOZ
Precursor 335.1 335.1
Product 291.4 262.4 192.3 166.3 134.2 104.2 134.3 104.3
Dwell (ms) 150 150 150 150 150 150 150 150
Fragmentor (V) 92 92 92 92 92 92 92 92
Collision Engergy (V) 5 5 5 5 5 5 5 5
SC
209.1 209.1
9.95
AH
249.1 249.1
10.8
AOZ
236.0 236.0
High Outlier Low Outlier
Quant Ion Integration
Rel Ion Ratio
Ion Ratios- Instant Graphic Review

335.1 -> 291.1 Relative Abundance (%) x10 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 -0.1 2.4 2.6 2.8 3 3.2 3.4 3.6
2
, 262.1 Ratio=24.0
Relative Peak Abundance Quant Ion
}
Acquisition Time (min)
20 % Tolerance
Relative Peak A... Qualifier (outside limits-colored)
Calibration curve of AMOZ
R2=0.99985
Linear range: 10 ppt ~ 10ppb

MRM EIC of Nitrofuran Metabolites (100 ppt)

+ MRM MRM (249.1 -> 134.3) Quan_Qualify_100ppt_2.d Smooth (1) x10 2 1 7 6.5 6 5.5 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 1 2 3 4 5 6 7 8 12 23
AOZ
34
AMOZ
SC AH
Abundance vs. Acqui si t i on Ti m e (m i n)
10
11
12
13
14
15
16
17
EIC at Transitions m/z 291 and m/z 262

+ MRM MRM (335.1 -> 291.4) Quan_Qualify_100ppt_2.d Smooth (1) x10 2 1 3.5 3 2.5 2 1.5 1 0.5 + MRM MRM (335.1 -> 262.4) Quan_Qualify_100ppt_2.d Smooth (1) x10 1 1 6.6 6.4 6.2 6 5.8 5.6 5.4 5.2 5 4.8 4.481 1
AMOZ 100 ppt 331.3291.4 (Quant Ion)
4.513
AMOZ 100 ppt 335.1262.4 (Qualify Ion) 1.997
4.592
0.2 0.4 0.6 0.8
1.2 1.4 1.6 1.8
2.2 2.4 2.6 2.8
3.2 3.4 3.6 3.8
4.2 4.4 4.6 4.8
5.2 5.4 5.6 5.8
6.2 6.4 6.6 6.8
7.2 7.4 7.6 7.8
EIC Transitions at m/z 134 and m/z 104

+ MRM MRM (236.0 -> 134.3) Quan_Qualify_100ppt_2.d Smooth (1) x10 2 4 6 5 4 3 2 1 11.702 12.323 13.159 11.194
AOZ 100 ppt 236.0134.3 (Quant Ion)
+ MRM MRM (236.0 -> 104.3) Quan_Qualify_100ppt_2.d Smooth (1) x10 2 4 1.1 1 0.9 0.8 0.7 0.6 0.5 11 11.5 11.767 12 12.5 13 13.5 14 14.5 15 11.197
AOZ 100 ppt 236.0104.3 (Qualify Ion)
15.5
16
16.5
17
17.5
18
18.5
19
19.5
20
20.5
21
21.5
22
MRM EIC Spectrum of AMOZ and SC (10 ppt)

+ MRM MRM (335.1 -> 291.4) 10ppt_2.d Smooth (1) Noise (RMS) = 8.54; SNR (4.481 min) = 6.5 x10 2 1 0.9 0.8 0.7 0.6 0.5 + MRM MRM (209.1 -> 192.3) 10ppt_2.d Smooth (1) Noise (RMS) = 14.69; SNR (9.838 min) = 4.0 x10 1 1 7.5 7 6.5 6 5.5 5 1 2 3 4 5 6 7 8 9.480 12 9.838 23 34 3.175 7.999 4.481 12 23 34
S/N=6
S/N=4
10
11
12
13
14
15
16
17
18
19
20
21
22
Spike Nitrofuran Metabolites in Tilapia (100 ppt)

+ M R M M R M ( 2 3 6 .0 -> 1 3 4 .3 ) N F r e a ls a m p le B la n k _ 1 .d S m o o th ( 1 ) x1 0 2 3 .2 3 2 .8 2 .6 2 .4 2 .2 2 1 .8 1 .6 1 .4 1 .2 1 0 .8 0 .6 1 2 3 4 5 6 7 8 1 1 2 2 3 3 4 4
Blank
A b u n d a n c e v s . A c q u i s i t i o n T i me ( mi n )
10
11
12
13
14
15
16
17
18
19
20
21
22
+ M R M M R M (2 4 9 .1 - > 1 3 4 .2 ) N F _ M R M s p ik e 1 0 0 p p t_ 2 .d S m o o th (1 ) x1 0 2 3 .8 3 .6 3 .4 3 .2 3 2 .8 2 .6 2 .4 2 .2 2 1 .8 1 .6 1 .4 1 .2 1 0 .8 0 .6 1 2 3 4 5 6 7 8 1 0 .0 9 3 1 1 2 2 3
3 4
AOZ
AMOZ SC AH
10 11 12 13 14 15
Spike
16
17
18
19
20
21
22
The Reproducibility of Nitrofuran Metabolites
Concentration Compounds AMOZ SC AH AOZ

a
RSD (%) 1.05 2.01 8.07 1.58
(ng/mL) 0.1 0.1 0.1 0.1
Each value was the average of 8 replicates (n=8).
Malachite Green and Metabolite Agilent 6410 Triple Quadrupole
Malachite Green (MG)

Among other veterinary drugs, the triphenyl-methane dye malachite green (MG) is a popular substance, and prevents fungal and parasitic infections in aquaculture, including saprolegniasis, white spot disease, and ciliates. MG is prevalently reduced into leuco-malachite green (LMG) and deposited in fatty tissue of the fish.
H C N reduction C N
N Cl Malachite Green (Green)
Leucomalachite Green (Colorless)

EU Regulatory Limit for Malachite Green and its Metabolite
Analytical Approaches for Determination of Residues of MG

HPLC equipped with a post-column unit for oxidation of LMG and with either an absorbance or MS detector for detection in trout (eggs, fry and muscle), catfish (muscle and plasma) and in trout muscle. The reactors (5 cm4mm)used in these studies were filled with 25% PbO2 Other method LC-APCI-MS in catfish and trout without the use of a reactor for the conversion of LMG. This study aimed at the development and validation of a procedure for sample processing and of an HPLC analysis method for determination of residues of MG The use of LCESI-MSMS analysis was explored for confirmation of detected residues of this drug in such matrices.
The analysis of residues of MG and LMG in Tilapia

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
Supernatant
(acetonitrile phase)
PPt
Add 20ml acetonitrile filter
Supernatant
35ml water, 30ml dichloromethane
Collect dichloromethane phase, add 20ml dichloromethane in upper layer Concentration LC/MS/MS
HPLC Parameters

HPLC system Column Injection Volumn Flow rate Mobile phase Gradient
: Agilent 1100 series : C18, 2.1150, 5 m : 10 L : 0.3 mL/min : A-10mmol NH4Ac (pH=4.5); B-Acetonitrile
Time 0 1 2 8 8.01 13 A 70 50 5 5 70 70 B 30 50 95 95 30 30
MS Parameters

MS system Ion source Polarity Nebulizer gas Ion spray voltage Resolution Scan mode Conditions of MRM
: Agilent 6410 LC/MS/MS : ESI : Positive : Nitrogen : 4000V : Q1 (unit) Q3 (unit) : Multiple Reaction Monitoring (MRM)
Source temperature : 350
TIC of MG and LMG with Different Fragmentor Voltages

+ EIC(329.4, 331.4 m/z) Scan optimizing FRG70_3.d
x10 7 5 4 3 2 1
70V
x10 7 5 4 3 2 1
90V
x10 7 6 5 4 3 2 1
110V
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 12
Abundance vs. Acquisition Time (min)
Mass spectra of MG at different collision energies

+ Product Ion (5.549 min) (329.3 -> **) optimizing MS2_FRG100_CE20_2.d x10 5 4 3.5 3 2.5 329.3
CE 20V
2 1.5 1 0.5 0 193.1 + Product Ion (5.549 min) (329.3 -> **) optimizing MS2_FRG100_CE30_3.d 329.3 208.3 236.9 268.4 284.3 313.5
N
x10 5
[M+H]+ m/z 329 -CH4 m/z 313
2 1.8
-dimethylaniline1.6
1.4
CE 30V
313.4 208.2 164.6 134.3 + Product Ion (5.549 min) (329.3 -> **) optimizing MS2_FRG100_CE40_4.d 313.3 192.8 217.4 237.2 251.4 270.3 285.3 298.9
m/z 208
1.2 1 0.8 0.6 0.4 0.2 0 x10 5 1.2 1 0.8 0.6 0.4 165.3 0.2 0 50 60 70 80 91.5 90 100 110 120 130 140 150 160 170 117.9 134.1 179.8 180 192.1 190 202.0 200 210 221.0 220 230 240 241.4 251.4 250 260 270.3 208.2
CE 40V
329.4 298.9 280 290 300 310 320 330 340 35
284.2
270
MRM Conditions of MG and LMG

Dwell Fragmentor Collision (ms) (V) Engergy (V)
Time
Compound
Precursor
Product
Malachite green Leucomalach ite green
329.3 329.3
313.3 208.2 316.3 239.2
40 40 40 40
100 100 100 100
40 40 30 30
331.3 331.3
Calibration curve of malachite green
R2=0.9995

Calibration curve of Leucomalachite green
R2=0.9994

MRM EIC of MG and LMG (100 ppt)

+ MRM MRM (329.3, 331.3 -> **) malachite green_200606127.d x10 3 1 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 8.118 9.660 8.5 9 9.5 10 10.5 11 11.5 12 8.409
LMG
MG
5.440
5.5
6.5
7.5
EIC spectra of ions at m/z 313 and m/z 208

+ MRM (329.3 -> 313.3) malachite green_200606127.d x10 2 1 3.5 3 2.5 2 1.5 1 0.5 0 + MRM MRM (329.3 -> 208.2) malachite green_200606127.d x10 2 1 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 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 5.436 12 5.448 12
Malachite Green 100 ppt 329.3313.3 (Quant Ion)
Malachite Green 100 ppt 329.3208.2 (Qual Ion)
EIC spectra of ions at m/z 239 and m/z 316

+ MRM MRM (331.3 -> 239.2) malachite green_200606127.d x10 3 1 1 0.8 0.6 0.4 0.2 0 + MRM MRM (331.3 -> 316.3) malachite green_200606127.d x10 1 1 6 5 4 3 2 1 0 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 12 8.395 8.118 9.660 12 8.409
Leucomalachite Green 100 ppt 331.3239.2 (Quant Ion)
Leucomalachite Green 100 ppt 331.3316.3 (Qualify Ion)

MRM EIC Spectrum of MG and LMG (10 ppt)

+ MRM MRM (331.3 -> 239.2) MG_10ppt_MRM_a_2.d x10 2 1 1.3 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 8.725 5 5.5 6 6.5 7 7.5 8 8.5 9 9.452 9.5 10 10.5 11 11.5 12 8.394
LMG
MG
Spike MG and LMG in Tilapia (1 ppb)

+ M R M M R M ( 3 3 1 .3 -> 2 3 9 .2 ) S p ik e _ B la n k _ 1 .d 8 7 .5 7 6 .5 6 5 .5 5 4 .5 4 3 .5 3 2 .5 2 1 .5 1 0 .5 1 1 .5 2 2 .5 3 3 .5 4 4 .5 1 1 2 2
Blank
5 .5
6 .5
7 .5
8 .5
9 .5
10
1 0 .5
11
1 1 .5
12
+ M R M M R M (3 2 9 .3 - > 3 1 3 .3 ) S p ik e 1 p p b _ 1 .d x1 0
3
LMG Spike
2
1 2
3 .6 3 .4 3 .2 3 2 .8 2 .6 2 .4 2 .2 2 1 .8 1 .6 1 .4 1 .2 1 0 .8 0 .6 0 .4 0 .2 0 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
MG
10
1 0 .5
11
1 1 .5
Spike MG and LMG in Tilapia (100 ppt)

+ M R M M R M ( 3 3 1 .3 -> 2 3 9 .2 ) S p ik e _ B la n k _ 1 .d 8 7 .5 7 6 .5 6 5 .5 5 4 .5 4 3 .5 3 2 .5 2 1 .5 1 0 .5 1 1 .5 2 2 .5 3 3 .5 4 4 .5 1 1 2 2
Blank
5 .5
6 .5
7 .5
8 .5
9 .5
10
1 0 .5
11
1 1 .5
12
+ M R M M R M (3 3 1 .3 - > 2 3 9 .2 ) S p ik e 1 0 0 p p t_ 1 .d x1 0 2 1 .7 1 .6 1 .5 1 .4 1 .3 1 .2 1 .1 1 0 .9 0 .8 0 .7 0 .6 0 .5 0 .4 0 .3 0 .2 0 .1 0 0 .5 1 1 .5 2 2 .5 3 3 .5 4 4 .5 5 1
MG
LMG
2
1 2
Spike
5 .5
6 .5
7 .5
8 .5
9 .5
10
1 0 .5
11
1 1 .5
12
The Reproducibility of MG and LMG
Compounds
Concentration (ng/mL)
RSD (%) 3.52 2.25
MG (m/z 329.3313.3) LMG (m/z 331.3239.2)
0.1 0.1
Each value was the average of 8 replicates (n=8).


Agilent QQQ Data

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Agilent Technologies 6000 Series LC/MS for Food Safety

LC/MS Food Safety Applications July, 2006

Agilent LC/MS Solutions

Group/LC/MS Food Safety Applications July, 2006

Nitrofuran Metabolites - Agilent 6410 LC/MS Triple Quadrupole

Group/LC/MS Food Safety Applications July, 2006

EU Regulations- Minimum Required Performance Limits (MRPL)

Group/LC/MS Food Safety Applications July, 2006

Does not set level for nitrofurans

Group/LC/MS Food Safety Applications July, 2006

Group/LC/MS Food Safety Applications July, 2006

Structures of Nitrofurans and Metabolites

Group/LC/MS Food Safety Applications July, 2006

Structures of Nitrofurans and metabolites

Group/LC/MS Food Safety Applications July, 2006

Source temperature : 350

Group/LC/MS Food Safety Applications July, 2006

Mass spectra of SC at different collision energies

Abundance vs. M ass-t o-Charge (m /z)

x10 4 1 0.8 0.6 0.4 0.2 0

+ Product Ion (9.721 min) (209.2 -> **) nitrofuran_MS2_100_Frag_10CE_4.d 192.2

Abundance vs. M ass-t o-Charge (m /z)

Group/LC/MS Food Safety Applications July, 2006

MRM Conditions of nitrofuran metabolites

Precursor 335.1 335.1

Product 291.4 262.4 192.3 166.3 134.2 104.2 134.3 104.3

Collision Engergy (V) 5 5 5 5 5 5 5 5

Group/LC/MS Food Safety Applications July, 2006

High Outlier Low Outlier

Quant Ion Integration

Rel Ion Ratio

Group/LC/MS Food Safety Applications July, 2006

Ion Ratios- Instant Graphic Review

Relative Peak Abundance Quant Ion

Relative Peak A... Qualifier (outside limits-colored)

Group/LC/MS Food Safety Applications July, 2006

Calibration curve of AMOZ

Linear range: 10 ppt ~ 10ppb

MRM EIC of Nitrofuran Metabolites (100 ppt)

Abundance vs. Acqui si t i on Ti m e (m i n)

Group/LC/MS Food Safety Applications July, 2006

EIC at Transitions m/z 291 and m/z 262

AMOZ 100 ppt 331.3291.4 (Quant Ion)

AMOZ 100 ppt 335.1262.4 (Qualify Ion) 1.997

0.2 0.4 0.6 0.8

1.2 1.4 1.6 1.8

2.2 2.4 2.6 2.8

3.2 3.4 3.6 3.8

Abundance vs. Acqui si t i on Ti m e (m i n)

4.2 4.4 4.6 4.8

5.2 5.4 5.6 5.8

6.2 6.4 6.6 6.8

7.2 7.4 7.6 7.8

Group/LC/MS Food Safety Applications July, 2006

EIC Transitions at m/z 134 and m/z 104

AOZ 100 ppt 236.0134.3 (Quant Ion)

AOZ 100 ppt 236.0104.3 (Qualify Ion)

Abundance vs. Acqui si t i on Ti m e (m i n)

Group/LC/MS Food Safety Applications July, 2006

MRM EIC Spectrum of AMOZ and SC (10 ppt)

Abundance vs. Acqui si t i on Ti m e (m i n)

Group/LC/MS Food Safety Applications July, 2006

Spike Nitrofuran Metabolites in Tilapia (100 ppt)