Anthelmintic SOP.pdf

8/9/2019 Anthelmintic SOP.pdf

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Page 1 of 54 Date Issued: 18/09/09 Test Method;

Edited by: James Sasanya Anthelmintics

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1. IdentificationDetermination of anthelmintic residues in beef by LC-MS/MS

2. ScopeMethod is suitable for the screening/confirmatory analysis according to EU

criteria [1] of residues of metabolites of anthelmintic and flukicide drugs in beef.

3. Description of items to be testedAs outlined in Scope.

4. Parameters/quanti ties and ranges to be determined

Limits/units

Albendazole (2.5g/kg), albendazole sulphoxide (10g/kg), albendazole

sulphone (10g), albendazole amino sulphone (10g/kg), closantel (1g/kg),

eprinomectin (5g/kg), morantel (2.5g/kg), oxyclozanide (5g/kg), clorsulon(10g/kg), bithionol (5g/kg), abamectin (5g/kg), emamectin (1g/kg),

doramectin (5g/kg), ivermectin (5g/kg), cambendazole (2.5g/kg),

fenbendazole (1g/kg), fenbendazole-sulphone (2.5g/kg), oxfendazole

(fenbendazole-sulphoxide) (2.5g/kg), flubendazole (1g/kg), amino-

flubendazole (2.5g/kg), hydroxy-flubendazole (2.5g/kg), mebendazole

(1g/kg), amino-mebendazole (5g/kg), hydroxy-mebendazole (1g/kg),

oxibendazole (1g/kg), thiabendazole (5g/kg), 5-hydroxy-thiabendazole

(10g/kg), coumaphos (5g/kg), coumaphos-oxon (2.5g/kg), haloxon

(5g/kg), nitroxynil (5g/kg), triclabendazole (5g/kg), triclabendazole-

sulphone (5g/kg), triclabendazole-sulphoxide (5g/kg), levamisole

(2.5g/kg), niclozamide (2.5g/kg), rafoxanide (1g/kg), moxidectin (20

g/kg).

5.0 Apparatus and equipment, including techni cal per formance requi rements.Usual laboratory apparatus not otherwise specified, and the following

items:

5.1 Tube A: Centrifuge tubes with screw caps, 50 ml polypropylene,

containing 4 g MgSO4and 1 g NaCl (Biotage).

5.2 Tube B: Centrifuge tubes, 50 ml, polypropylene, containing 1.5 gMgSO4and 0.5 g C18(Biotage).

5.3 Centrifuge tubes with screw caps polypropylene 50 ml, 120 mm 36

mm (Sarstedt or equivalent).

5.4 25 ml glass test tubes.

5.5 Glass dispenser for acetonitrile.

5.6 AK15, Sigmalaboratory centrifuge (Vienna, Austria).

5.7 Vortex mixer (Labinco, Breda, The Netherlands).


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5.8 TurboVapLV evaporator (Zymark, Runcorn, UK) set at 50C under a

stream of nitrogen.

5.9 Whatman 0.2 m PTFE, 13 mm diameter syringe filters (Whatman,

Florham Park, NJ, USA).

5.10 Millipore HVLP 0.45 m membrane filters (Durapore, Carrigtwohill,

Ireland).

5.11.1 Blendor (New Hartford, Connecticut, USA).

5.11.2 Homogeniser set at level 3 (Ultraturax, Staufen, Germany).

5.11.3 Ultrasonicator (VWR, Model USC 300T, Made in Malaysia)

5.12 LC-MS/MS system, consisting of C18Atlantis analytical column (100

mm x 2.1 mm x 3 m, Waters, UK; guard cartridge, 2.1x10mm) anddetected using a Triple Quadrupole Micromass micro Mass

Spectrometer (MS, Waters, UK) with electrospray ionization (ESI)

interface. The inlet method involves use of a Waters Alliance HPLC

2695 series (Waters, UK). The pumps are operated at a flow rate of

0.25 ml/min while the column temperature is set at 45oC (5). The LC-

MS/MS system is controlled by Masslynx software, and the results are

processed by TargetLynx software and microsoft excel.

6. Reference standards and reference materi als requi redNo reference materials are used in this test method.

7. Safety measures to be observedNo particular safety measures apply other than routine laboratory

safety procedures.

8. Description of procedure

9.1 Principle

The sample is prepared for analysis using a modified QuEChERS method.The sample is extracted by shaking in acetonitrile, MgSO4and NaCl before

being cleaned up by dispersive solid phase extraction (SPE), using C18

and MgSO4. The extract is concentrated, filtered and transferred to a

HPLC vial. The anthelmintic residues are determined by liquid

chromatography (LC) on a reverse phase C18 Atlantis analytical column

coupled to a mass spectrometer. Anthelmintic residues are quantified using

internal standards added to the sample before extraction.


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9.2.0 Reagents

All reagents shall be of analytical quality. The water used shall be

nanopurified water or water of equivalent purity. Materials mentioned

here have been found to be suitable. Expiry dates for prepared

solutions shall be 12 months unless otherwise indicated.

9.2.1 Water (Millipore, 18.2 Mcm).

9.2.2 Methanol (HPLC grade, LiChrosol, Germany).

9.2.3 Acetonitrile (HPLC grade, LiChrosol, Germany).

9.2.4 Isopropyl alcohol (IPA, Merck, Darmstadt, Germany).

9.2.5 Dimethylsulphoxide (DMSO, Analar Grade, Sigma Chemical, St.

Louis, MO, USA).

9.2.6 Ammonium formate (Alfa Aesar, Karlsruhe, Germany)

9.2.7 LC mobile phase A: Water (9.2.1): acetonitrile (9.2.3), 90:10 (v/v).

Filter (5.10) and degas in an ultrasonic bath (5.11.3) for 15 min.

Prepare daily.

9.2.8 LC Mobile phase B: 5mM ammonium formate (9.2.6) in methanol

(9.2.2): acetonitrile (9.2.3), 75:25 (v/v). Dissolve 0.3153 g ammonium

formate in 1 L of 75:25 methanol: acetonitrile. Sonicate for

approximately 5 min to ensure the ammonium formate is fully

dissolved. Filter the mobile phase using 0.45 m filter paper (5.10) and

degas in an ultrasonic bath (5.11.3) for 15 min. Prepare daily.

9.2.9 Wash Solution one: water (9.2.1): methanol (9.2.2), 80:20 (v/v).

Combine the appropriate volumes of water and methanol. Degas in an

ultrasonic bath (5.11.3) for 15 min. Prepare as required.

9.2.10 Wash solution two: water (9.2.1): methanol (9.2.2): IPA (9.2.4), 10:80:10

(v/v/v). Combine the appropriate volumes of water, methanol and IPA.

Degas in an ultrasonic bath (5.11.3) for 15 min. Prepare as required.

9.2.11 Seal wash/purge solution: methanol (9.2.2): IPA (9.2.4), 80:20 (v/v).

Combine the appropriate volumes of methanol and IPA. Degas in an

ultrasonic bath (5.11.3) for 15 min. Prepare as required.

9.2.12.0 Anthelmintic standard stock solutions

9.2.12.1 Albendazole (ABZ)

Dissolve 0.01 (x 100/F)g [where F is the content (%) of the standard in

the particular batch of material as shown in the Certificate of Analysis]

of ABZ (Sigma Aldrich Ireland, Tallaght, Dublin 24) by sonicating in 5

ml DMSO (9.2.5) in a 10 ml volumetric flask for 5 min, and make up


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to the 10 ml mark. Mix well by repeatedly turning the flask upside

down. This gives a concentration of 1 mg/ml.

9.2.12.2 Albendazole-2-amino-sulphone (ABZ-NH2-SO2)

Dissolve 0.01 (x 100/F)g [where F is the content (%) of the standard inthe particular batch of material as shown in the Certificate of Analysis]

of ABZ-NH2-SO2 (Dr. Ehrenstorfer GmbH, Augsburg, Germany) by

sonicating in 5 ml DMSO (9.2.5) in a 10 ml volumetric flask for 5 min,

and make up to the 10 ml mark. Mix well by repeatedly turning the

flask upside down. This gives a concentration of 1 mg/ml.

9.2.12.3 Albendazole-sulphoxide (ABZ-SO)



of ABZ-SO (Witega, Berlin, Germany) by sonicating in 5 ml DMSO(9.2.5) in a 10 ml volumetric flask for 5 min, and make up to the 10

ml mark. Mix well by repeatedly turning the flask upside down. This

gives a concentration of 1 mg/ml.

9.2.12.4 Albendazole-sulphone (ABZ-SO2)



of ABZ-SO2 (Witega, Berlin, Germany) by sonicating in 5 ml DMSO

(9.2.5) in a 10 ml volumetric flask for 5 min, and make up to the 10


gives aconcentration of 1 mg/ml.

9.2.12.5 Thiabendazole (TBZ)



of TBZ (Sigma Aldrich, St. Louis, MO, USA) by sonicating in 5 ml

MeOH (9.2.2) in a 10 ml volumetric flask for 5 min, and make up to the

10 ml mark. Mix well by repeatedly turning the flask upside down. This

gives a concentration of 1 mg/ml.

9.2.12.6 5-Hydroxy-Thiabendazole (5-OH-TBZ)Dissolve 0.01 (x 100/F)g [where F is the content (%) of the standard in


of 5-OH-TBZ (Dr. Ehrenstorfer GmbH, Augsburg, Germany) by

sonicating in 5 ml DMSO (9.2.5) in a 10 ml volumetric flask for 5

min, and make up to the 10 ml mark. Mix well by repeatedly turning

the flask upside down. This gives a concentration of 1 mg/ml.

9.2.12.7 Fenbendazole (FBZ)


the particular batch of material as shown in the Certificate of Analysis]of FBZ (Dr. Ehrenstorfer GmbH, Augsburg, Germany) by sonicating in


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5 ml DMSO (9.2.5) in a 10 ml volumetric flask for 5 min, and make up



9.2.12.8 Oxfendazole (OXF) (synonym: Fenbendazole-sulphoxide)


of OXF (Dr. Ehrenstorfer GmbH, Augsburg, Germany) by sonicating

in 5 ml DMSO (9.2.5) in a 10 ml volumetric flask for 5 min, and make

up to the 10 ml mark. Mix well by repeatedly turning the flask upside


9.2.12.9 Fenbendazole-sulphone (FBZ-SO2)



of FBZ-SO2 (Witega, Berlin, Germany) by sonicating in 5 ml DMSO(9.2.5) in a 10 ml volumetric flask for 5 min, and make up to the 10


gives aconcentration of 1 mg/ml.

9.2.12.10 Mebendazole (MBZ)



of MBZ (Janssen Animal Health, Beerse, Belgium) by sonicating in 5

ml DMSO (9.2.5) in a 10 ml volumetric flask for 5 min, and make up



9.2.12.11 Hydroxy-Mebendazole (MBZ-OH)



of MBZ-OH (Janssen Animal Health, Beerse, Belgium) by sonicating



down. This gives aconcentration of 1 mg/ml.

9.2.12.12 Amino-Mebendazole (MBZ-NH2)Dissolve 0.01 (x 100/F)g [where F is the content (%) of the standard in


of MBZ-NH2(Janssen Animal Health, Beerse, Belgium) by sonicating




9.2.12.13 Flubendazole (FLU)


the particular batch of material as shown in the Certificate of Analysis]of FLU (Dr. Ehrenstorfer GmbH, Augsburg, Germany) by sonicating in


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5 ml DMSO (9.2.5) in a 10 ml volumetric flask for 5 min, and make



9.2.12.14 Hydroxy-Flubendazole (FLU-OH)


of FLU-OH (Janssen Animal Health, Beerse, Belgium) by sonicating in




9.2.12.15 Amino-Flubendazole (FLU-NH2)



of FLU-NH2 (Janssen Animal Health, Beerse, Belgium) by sonicatingin 5 ml DMSO (9.2.5) in a 10 ml volumetric flask for 5 min, and make



9.2.12.16 Oxibendazole (OXI)


the particular batch of material as shown in the Certificate of

Analysis] of OXI (Dr. Ehrenstorfer GmbH, Augsburg, Germany) by




9.2.12.17 Triclabendazole (TCB)



Analysis] of TCB (Sigma-Aldrich, St. Louis, MO, USA) by sonicating

in 5 ml MeOH (9.2.2) in a 10 ml volumetric flask for 5 min, and make



9.2.12.18 Triclabendazole-sulphone (TCB-SO2)Dissolve 0.01 (x 100/F)g [where F is the content (%) of the standard in


Analysis] of TCB-SO2 (Witega, Berlin, Germany) by sonicating in 5

ml MeOH (9.2.2) in a 10 ml volumetric flask for 5 min, and make up



9.2.12.19 Triclabendazole-sulphoxide (TCB-SO)



Analysis] of TCB-SO (Witega, Berlin, Germany) by sonicating in 5ml MeOH (9.2.2) in a 10 ml volumetric flask for 5 min, and make up


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9.2.12.20 Cambendazole (CAM)


the particular batch of material as shown in the Certificate ofAnalysis] of CAM (Dr. Ehrenstorfer GmbH, Augsburg, Germany)

by sonicating in 5 ml MeOH (9.2.2) in a 10 ml volumetric flask for 5



9.2.12.21 Eprinomectin (EPRI)

Dissolve 0.01 (x 100/F)g [where F is the content (%) of Eprinomectin

B1a in the particular batch of material as shown in the Certificate of

Analysis] of EPRI (Dr. Ehrenstorfer GmbH, Augsburg, Germany) by

sonicating in 5 ml MeCN (9.2.3) in a 10 ml volumetric flask for 5 min,

and make up to the 10 ml mark. Mix well by repeatedly turning theflask upside down. This gives a concentration of 1 mg/ml.

9.2.12.22 Moxidectin (MOXI)



Analysis] of MOXI (Dr. Ehrenstorfer GmbH, Augsburg, Germany) by

sonicating in 5 ml MeCN (9.2.3) in a 10 ml volumetric flask for 5



9.2.12.23 Emamectin benzoate (EMA)

Dissolve 0.01 (x 100/F)g [where F is the content (%) of Emamectin


Analysis] of EMA (Sigma Aldrich St. Louis, MO, USA) by

sonicating, in 5 ml MeCN (9.2.3) in a 10 ml volumetric flask for 5



9.2.12.24 Doramectin (DORA)

Dissolve 0.01 (x 100/F)g [where F is the content (%) of the standard

in the particular batch of material as shown in the Certificate ofAnalysis] of DORA (Dr. Ehrenstorfer GmbH, Augsburg, Germany)

by sonicating in 5 ml MeCN (9.2.3) in a 10 ml volumetric flask for 5



9.2.12.25 Abamectin (ABA)

Dissolve 0.01 (x 100/F)g [where F is the content (%) of Abamectin


Analysis] of ABA (Dr. Ehrenstorfer GmbH, Augsburg, Germany) by

sonicating in 5 ml MeCN (9.2.3) in a 10 ml volumetric flask for 5

min, and make up to the 10 ml mark. Mix well by repeatedly turningthe flask upside down. This gives a concentration 1 mg/ml.


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9.2.12.26 Ivermectin (IVER)

Dissolve 0.01 (x 100/F)g [where F is the content (%) of Ivermectin


Analysis] of IVER (Dr. Ehrenstorfer GmbH, Augsburg, Germany) by

sonicating in 5 ml MeCN (9.2.3) in a 10 ml volumetric flask for 5 min,

and make up to the 10 ml mark. Mix well by repeatedly turning theflask upside down. This gives a concentration of 1 mg/ml

9.2.12.27 Haloxon (HALOX)



Analysis] of HALOX (Sigma Aldrich, Milwaukee, USA) by sonicating




9.2.12.28 Closantel (CLOS)



Analysis] of CLOS (Sigma Aldrich, St. Louis, MO, USA) by sonicating




9.2.12.29 Levamisole-HCl (LEVA)


the particular batch of material as shown in the Certificate ofAnalysis] of LEVA (Dr. Ehrenstorfer GmbH, Augsburg,

Germany) by sonicating in 5 ml MeOH (9.2.2) in a 10 ml volumetric

flask for 5 min, and make up to the 10 ml mark. Mix well by

repeatedly turning the flask upside down. This gives a concentration of

1 mg/ml.

9.2.12.30 Rafoxanide (RAFOX)



Analysis] of RAFOX (Sigma Aldrich Ireland, Tallaght, Dublin 24)

by sonicating in 5 ml MeOH (9.2.2) in a 10 ml volumetric flask for 5min, and make up to the 10 ml mark. Mix well by repeatedly turning


9.2.12.31 Oxyclozanide (OXYCLOZ)



Analysis] of OXYCLOZ (Sigma Aldrich/Riedel-de Haen, Seelze,

Germany) by sonicating in 5 ml MeOH (9.2.2) in a 10 ml volumetric

flask for 5 min, and make up to the 10 ml mark. Mix well by


1 mg/ml.


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9.2.12.32 Niclozamide (NICLOZ)



Analysis] of NICLOS (Sigma Aldrich Ireland, Tallaght, Dublin 24)

by sonicating in 5 ml MeOH (9.2.2) in a 10 ml volumetric flask for 5

min, and make up to the 10 ml mark. Mix well by repeatedly turningthe flask upside down. This gives a concentration of 1 mg/ml.

9.2.12.33 Nitroxynil (NITROX)

Dissolve 0.01 (x 100/F)g [where F is the content (%) of the standard

in the particular batch of material as shown in the Certificate of

Analysis] of NITROX (Sigma Aldrich Ireland, Tallaght, Dublin 24) by

sonicating in 5 ml MeOH (9.2.2) in a 10 ml volumetric flask for 5



9.2.12.34 Clorsulon (CLOR)



Analysis] of CLOR (Sigma Aldrich St. Louis, MO, USA) by sonicating




9.2.12.35 Bithionol (BITH)



Analysis] of BITH (Dr. Ehrenstorfer GmbH, Augsburg, Germany) by

sonicating in 5 ml MeOH (9.2.2) in a 10 ml volumetric flask for 5



9.2.12.36 Morantel tartrate monohydrate (MOR)



Analysis] of MOR (Dr. Ehrenstorfer GmbH, Augsburg, Germany) by

sonicating in 5 ml MeOH (9.2.2) in a 10 ml volumetric flask for 5 min,and make up to the 10 ml mark. Mix well by repeatedly turning the


9.2.12.37 Coumaphos (COUMA)



Analysis] of COUM (Sigma Aldrich/Riedel-de Haen, Seelze,

Germany) by sonicating in 5 ml of MeOH (9.2.2) in a 10 ml

volumetric flask for 5 min, and make up to the 10 ml mark. Mix well

by repeatedly turning the flask upside down. This gives a concentration

of 1 mg/ml.


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9.2.12.38 Coumaphos-Oxon (COUMA-O)



Analysis] of COUMA-O (Sigma Aldrich/Riedel-de Haen, Seelze,

Germany) by sonicating in 5 ml of MeOH (9.2.2) in a 10 ml

volumetric flask for 5 min, and make up to the 10 ml mark. Mix wellby repeatedly turning the flask upside down. This gives a concentration

of 1 mg/ml.

9.2.12.39 Selamectin (SELA)

Dissolve 0.01 g of SELA (Pfizer, Kent, UK) by sonicating in 5 ml

MeCN (9.2.3) in a 10 ml volumetric flask for 5 min, and make up to

the 10 ml mark. Mix well by repeatedly turning the flask upside down.

This gives a concentration of 1 mg/ml.

9.2.12.40 Fenbendazole-sulphone-D3 (FBZ-SO2-D3)Dissolve 0.01 g of FBZ-SO2-D3 (Witega, Berlin, Germany) by


min, and make up to the 10 ml Mark. Mix well by repeatedly

turning the flask upside down. This gives a concentration of 1 mg/ml.

9.2.12.41 Fenbendazole-sulphoxide-D3 (FBZ-SO-D3)

Dissolve 0.01 g of FBZ-SO-D3 (Witega, Berlin, Germany) by

sonicating in 5 ml DMSO (9.2.5) in a 10 ml volumetric fl ask for 5

min, and make up to the 10 ml mark. Mix well by repeatedly


9.2.12.42 Fenbendazole-D3 (FBZ-D3)

Dissolve 0.01 g of FBZ-SO2-D3 (Witega, Berlin, Germany) by


min, and make up to the 10 ml mark. Mix well by repeatedly


9.2.12.43 Albendazole-sulphone-D3 (ABZ-SO2-D3)

Dissolve 0.01 g of ABZ-SO2-D3 (Witega, Berlin, Germany) by

sonicating in 5 ml DMSO (9.2.5) in a 10 ml volumet ric flask for

5 min, and make up to the 10 ml mark. Mix well by repeatedly


9.2.12.44 Albendazole-sulphoxide-D3 (ABZ-SO-D3)

Dissolve 0.01 g of ABZ-SO-D3 (Witega, Berlin, Germany) by

sonicating in 5 ml DMSO (9.2.5) in a 10 ml volumetric flask for

5 min, and make up to the 10 ml mark. Mix well by repeatedly

turning the flask upside down. This gives a concentration of 1

mg/ml.

9.2.12.45 Triclabendazole-D3 (TCB-D3)

Dissolve 0.005 g of TCB-D3 (Witega, Berlin, Germany) by sonicating

in 3 ml MeOH (9.2.2) in a 5 ml volumetric flask for 5 min, and


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make up to the 5 ml mark. Mix well by repeatedly turning the flask

upside down. This gives a concentration of 1mg/ml.

9.2.12.46 Albendazole-2-amino-sulphone-D2 (ABZ-NH2-SO2-D2)

Dissolve 0.002 g of ABZ-NH2-SO2-D2 (Quechem, Belfast, UK) by




9.2.12.47 Albendazole-D3 (ABZ-D3)

Dissolve 0.01 g of ABZ-D3 (Witega, Berlin, Germany) by sonicating in




9.2.12.48 Thiabendazole NH-D6 (TBZ-D6)Transfer 1.1 ml of 100 ng/l of TBZ-NH-D6 (Dr. Ehrenstorfer GmbH,

Augsburg, Germany) supplied in acetone into a 10 ml volumetric flask,

and make up to the 10 ml mark with methanol (9.2.2). Mix well by


11 g/ml.

9.2.12.49 Levamisole-D5 (Leva-D5)

Dissolve 0.01 g of Leva-D5 (Dr. Ehrenstorfer GmbH, Augsburg,Germany) by sonicating in 5 ml MeOH (9.2.2) in a 10 ml volumetricflask for 5 min, and make up to the 10 ml mark. Mix well by

repeatedly turning the flask upside down. This gives a concentration of1mg/ml.

9.2.12.50.0 Preparation of standard curves and standards for

infusion .

9.2.12.50.1 Preparat ion of 10 g/ml individual standard.

Transfer 0.1 ml of the (9.2.12.1 to 9.2.12.38) standard into a 10 ml

volumetric flask, and bring to the 10 ml mark with methanol. Mix well

by repeatedly turning the flask upside down. This gives a concentrationof 10 g/ml.

9.2.1 2.50.2 Preparat ion o f 20 g/ml intermediate standards, mixed intermidiate

mixed standard containing the 38 anthelmintics.

Transfer 0.5 ml of the 1 mg/ml stock solutions of each anthelmintic

standard (9.2.12.1 to 9.2.12.38) into a 25 ml volumetric flask, and

make up to the 25 ml mark. Mix well by repeatedly turning the flask

upside down. This gives a concentration of 20 g/ml.


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9.2.12.50.3 Working standards (10, 5, 4, 3, 2, 1, 0.5, 0.25, 0.2, 0.1, and 0.05 g/ml)

These standards are prepared from the intermediated mixed standards

by transferring:

a.

5 ml of the 20 g/ml mixed standard into a 10 ml volumetric flaskand bringing to the mark with methanol (9.2.2) to obtain a 10 g/ml

mixed standard;

b. 5 ml of the 10 g/ml mixed standard into a 10 ml volumetric flask

and bringing to the mark with methanol (9.2.2) to obtain a 5 g/ml

mixed standard;

c. 8 ml of the 5 g/ml mixed standard into a 10 ml volumetric flask


mixed standard;

d. 7.5 ml of the 4 g/ml mixed standard into a 10 ml volumetric flask


mixed standard;e.

6.7 ml of the 3 g/ml mixed standard into a 10 ml volumetric flask


mixed standard;

f. 5 ml of the 2 g/ml mixed standard into a 10 ml volumetric flask


mixed standard;

g. 5 ml of the 1 g/ml mixed standard into a 10 ml volumetric flask

and bringing to the mark with methanol (9.2.2) to obtain a 0.5

g/ml mixed standard;

h.

5 ml of the 0.5 g/ml mixed standard into a 10 ml volumetric flask


g/ml mixed standard;

i. 0.5 ml of the 10 g/ml each standard into a 25 ml volumetric flask


g/ml mixed standard; This was then used to prepare 0.1 and 0.05

g/ml mixed standards by transferring:

j. 5 ml of the 0.2 g/ml mixed standard into a 10 ml volumetric flask


g/ml mixed standard; and then

k. 5 ml of the 0.1 g/ml mixed standard into a 10 ml volumetric flask

and bringing to the mark with methanol (9.2.2) to obtain a 0.05g/ml mixed standard.

Note: Contents in the volumetric flasks above are mixed well by

repeatedly turning them upside down.

9.2.13 Intermediate and working internal standards

9.2.1 3.1 Preparat ion of 10 g/ml individual internal standards.

Transfer 0.1 ml of the (9.2.12.39 to 9.2.12.49) standard into a 10 mlvolumetric flask, and bring to the 10 ml mark with methanol (9.2.2).


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Mix well by repeatedly turning the flask upside down. This gives a

concentration of 10g/ml.

9.2.13 . Internal standard mix (0.8 g/ml).

Place 0.8 ml of the 10 g/m1 solutions of Sela (9.2.12.39), FBZ-SO2-

D3 (9.2.12.40), FBZ-SO-D3 (9.2.12.41), FBZ-D3 (9.2.13.42), ABZ-SO2-D3 (9.2.12.43), ABZ-SO-D3 (9.2.12.44), TCB-D3 (9.2.12.45),

ABZ-NH2-SO2-D3 (9.2.12.46); ABZ-D3 (9.2.12.47), Leva-D5

(9.2.1.49), 0.727 ml of TBZ NH-D6 (9.2.12.48) into a 10 ml

volumetric flask. Make up to the 10 ml mark with methanol (9.2.2).

Mix well by repeatedly turning the flask upside down. This gives a

concentration of 0.8 g/ml.

Note: Using a higher concentr ation of the I S (e.g. 2 to 4 g/ml sothat a higher f inal concentr ation of 20 to 40 g/kg is obtained) may

ease data processing, especial ly the integration step.

Store all standard solutions at -20oC.

9.3.0 Sample

9.3.1 Samples for method development and validation are purchased from

local grocery stores and expected to contain no residues of the drugs

under study. Nevertheless, the samples are analyzed to check for or

confirm absence of these residues.

9.4.0 Procedure

9.4.1 Preparation of sample

Mince the meat samples using the Blendor (5.11.1) (or an equivalent

tool) to ensure sample homogeneity prior to taking the analytical sample.

9.4.2.0 Extraction

9.4.2.1 Weigh 10 ( 0.01)g of sample into a 50 ml polypropylene tube (5.3).

9.4.2.2 Spike all samples with internal standard (9.2.12.39 to 9.2.12.51)

(except blank samples designated for preparing matrix matched

standards and internal standards) and spike the recovery samples as

described in section 9.4.6.

9.4.2.3 Leave sampled to stand for 15 min at room temperature.

9.4.2.4 Add 10.0 ml of acetonitrile (9.2.3) to the sample.

9.4.2.5 Homogenise using an ultraturax (5.11.2) for 30 s.


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9.4.2.6 Add the contents of Tube A (5.1) to the sample in sarstedt tubes.

Dispense 2 ml of acetonitrile (9.2.3) to Tube A (5.1), shake, and

transfer to the sample.

9.4.2.7 Shake vigorously by hand for 1 min.

9.4.2.8 Centrifuge (5.7) at 2602 g for 12 min, at 4C.

9.4.3.0 Clean up

9.4.3.1 Pour the supernatant into Tube B (5.2) containing 1.5 g MgSO4and 0.5

g C18.

9.4.3.3 Vortex sample for 30 s (5.7).

9.4.3.4 Centrifuge (5.6) at 2602 for 10 min.

9.4.4.0 Concentration

9.4.4.1 Add 6 ml of the supernatant to two sets of 25 ml glass tubes containing

0.5 ml and 0.3 ml of DMSO (5.4) and mix for 30 s.

9.4.4.2 Place samples on the Turbovap (5.8) at 50oC and evaporate off the

acetonitrile layer until the 0.5 ml mark for the recovery samples.

9.4.4.3 Evaporate standard matrix matched samples until 0.3 ml of the solvent

remains. To this test tube add 0.1 ml of the standard mixture and 0.1 ml

of the internal standard mixture.

9.4.4.4 Filter the resulting extract through a 0.2 m filter (5.9) directly into

HPLC vials. Samples are stored at 20oC if they will not be analyzed

immediately.

9.4.5.0 Determination

9.4.5.1 Determine acceptable performance of the LC-MS/MS system by

injecting matrix matched samples containing all the 38 anthelmintics

and internal standards (9.2.12) at least in duplicate and measuring the

system suitability parameters including retention time shifts and

response. Record measured values in a designated form and compare

with the ranges of acceptable values specified. Run system suitability

test in between batches or every 8 to 12 samples. Repeat injection of

system suitability at the end of each sequence to assess performance.

Note: Using the matrix matched samples as system suitability saves

time instead of injecting separate system suitability samples. Also

analytes in matrix provide a representative profile of the samplesanalyzed.


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9.4.5.2 Analyse the sample extracts on the LC-MS/MS system (5.12) together

with appropriate standards. Inject 10 l of sample extracts and

standards. A set of standard should be injected at the beginning and

end of each run and evenly dispersed throughout the run to generate a

standard curves and monitor response and retention time. The variationin retention time for standard over the chromatographic run should not

be greater than 2% of the medium time.

9.4.5.3 A peak determined in a sample shall be deemed to be the analyte if it

meets all the criteria set out in section on expression of results. The

sample may be reanalysed for further confirmation.

9.4.6 Control and recovery samples

9.4.6.1 Negative, matrix matched and recovery samples are described as

below.

9.4.6.2 Negative controls : Two 10 g aliquots of a control sample

(beef muscle without anthelmintic and flukicide residues) are

prepared and analyzed as mentioned in the procedure in 9.4.1-

9.4.5.3. Where a measurable response is obtained following analysis,

this is subtracted from the response for the fortified samples when

calculating recovery.

9.4.6.3 Recovery internal standards: Two blank samples are spiked with 0.1

ml of 0.8 g/ml of the 11 internal standards to obtain a final concentration

of 8 g/kg. The samples are then prepared as in section 9.4.1-9.4.5.3.

9.4.6.4 Recovery samples: These are negative samples, fortified with

standard and internal standard mixtures. From a proven blank beef muscle, 6

test portions of 10.0 ( 0.01) g are weighed into 50 ml centrifuge tubes

(5.3). These blank samples are then fortified with 0.1 ml of the

appropriate standard and 0.1 ml of internal standard, and left to

equilibrate for 15 min, before preparation using the procedure in 9.4.1-

9.4.4.5. For example, the final concentration of the analytes in the recovery

samples would be 40, 20, 10, 5, 2.5, and 1 g/kg after adding 0.1 ml of 4, 2,1, 0.5, 0.25 and 0.1 g/ml mixed standards, respectively.

9.4.6.5 Matrix matched standards and internal standards: Eight proven blank

muscles (10.0 0.01 g) are weighed into 50 ml centrifuge tubes (5.3) and

prepared as the blank and recovery samples. These blank samples that

are then fortified with 0.1 ml of standard and 0.1 ml of internal

standard after step 9.4.4.3 following evaporation of a mixture

containing 6 ml of the supernatant and 0.3 ml of DMSO. After step

9.4.4.3, 0.1 ml of the following standards in solvent: 4, 3, 2, 1, 0.5, 0.25, 0.1

and 0.05 g/ml are added to obtain 40, 30, 20, 10, 5, 2.5, 1 and 0.5 g/kg

matrix matched standards. Also, 0.1 ml of 0.8 g/ml of the mixture of the11 internal standards is spiked to obtain a final concentration of 8 g/kg.


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Note: Adding both standards and internal standards to one test tube containing 0.3 ml of

DMSO excludes the need to prepare two separate sets of matrix matched standards and

internal standards. The final volume (0.5 ml) of the matrix matched sample is the same as

that of the recovery samples or any experimental samples to be analyzed.

A higher concentration of the internal standard mixture (2 to 4 g/ml) is recommended in

order to obtain a final concentration of 20 to 40 g/kg internal standard in spiked samples

(should 0.1 ml be added). This reduces the time/effort required for to integrate

chromatograms hence (possibly) reduced data processing time.

9.5 LC-MS/MS Analysis

LC conditions:

SolventsA% 90:10, v/v (Water:Acetonitrile)

B% 75:25, v/v (Methanol:Acetonitrile); 5

mM amonium formate

Degasser Continuous

Stroke volume 130.0 l

Min pressure (Bar) 1.0

Max pressure (Bar) 345

Column AtlantisT3 2.1 x 100 mm, 3m

Guard cartridge AtlantisT3 2.1 x 10 mm

Guard holder kit Sentry2.1 mm

Flow 0.25 ml/min

Flow ramp 0.50

Injection volume 10 l

Injection loop 20l

Column temperature 45C

Total run time 22 min

Autosampler

Sample temperature 22oC

Sample temperature limit 2oC

Draw speed Normal

Needle depth (mm) 1.00Purge loop volumes 0.2


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Table 1: The table below shows the Water Alliance 2695 HPLC pump

gradient timetable for the inlet method to be used.

Time A% B% Flow

(ml/min)

Curve

0.00 50.0 50.0 0.25 12.00 50.0 50.0 0.25 610.10 0.0 100.0 0.25 612.00 0.0 100.0 0.25 615.00 0.0 100.0 0.25 616.10 0.0 100.0 0.25 616.20 50.0 50.0 0.25 620.00 50.0 50.0 0.25 622.00 50.0 50.0 0.25 6

Table 2. The table below shows Quattro Micro Mass tune parameters used todetermine suitable MS/MS conditions for subsequent analysis involving an LC

inlet method.

Parameter Positive ESI Negative ESI

Capillary voltage 3.29 kV 2.55 kV

Extractor 5 V 3 V

RF Lens 0.1 V 0.5 V

LM 1/HM 1 Resolution 15.0/15.0 14/13.5

Ion Energy 1 0.5 0.2

Entrance -1 -1

Exit 2 2

LM 2/HM 2 Resolution 13.5/13.5 13.2/13.0

Ion Energy 2 0.5 1.0

Source T (C) 150 150

Multiplier (V) 650 650

Desolvation T (C)/Gas

flow

400/650L/hr 400/650L/hr

Cone Gas Flow 50 50

Cone Gas Argon, p = 3.17e-3 bar Argon, p = 3.17e-3 bar

Syringe pump flow 30 l/min 30 l/min


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Table 3: MS/MS fragmentation conditions for the standards (9.2.12.19.2.12.51).

Component Parent ion(m/z)

Daughterion (m/z)

Dwelltime(s)

ConeEnergy

(V)

CollisionEnergy

(eV)

Polarity RT(min)

LEVAMISOLE 205.00 178.00 0.100 35 27 POS 0.0-5.0

122.9. 0.100 35 25

LEVAMISOLE-D5 210.10 183.10 0.085 33 20 POS 0.0-5.0

ALT 95.80 0.085 33 34

5-OH-TBZ 218.00 191.00 0.100 45 24 POS 0.0-4.0

Used 147.00 0.100 45 32

ABZ-NH2-SO2 274.00 238.20 0.100 15 11 NEG 0.0-5.0

131.00 0.100 15 33

ABZ-NH2-SO2-D2 240.00 131.0 0.1 30 24 NEG 0.0-5.0

ALT 147.00 0.1 30 21

TBZ 202.00 131.00 0.010 43 30 POS 0.0-5.5

175.00 0.010 43 24

TBZ-NH-D6 208.20 180.00 0.085 48 24 POS 0.0-5.0

136.00 0.085 48 29

MOR 221.00 111.00 0.01 15 25 POS 0.0-5.0

164.00 0.1 15 28

NITROX 289.00 127.00 0.010 36 23 NEG 0.0-4.5

162.00 0.100 36 20

CLORS 380.00 344.00 0.500 25 12 NEG 0.0-4.5

342.00 0.500 25 11

MBZ-NH2 238.00 105.00 0.015 50 24 POS 0.0-5.0

133.00 0.100 50 44

FLU-NH2 256.00 95.00 0.100 23 35 POS 0.0-5.5

123.00 0.100 23 36

ABZ-SO 282.00 158.90 0.08 35 34 POS 0.0-4.7

208.00 0.08 35 19ALT 222.00 0.010 27 21


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ABZ-SO-D3 285.00 158.9 0.080 15 30 POS 0.0-4.7

ALT 194.00 0.080 15 18

ABZ-SO2 298.10 266.00 0.010 37 20 POS 0.0-5.0159.00 0.010 37 35

See MBZ OH 224.00 0.010 37 26

MBZ-OH 297.90 266.00 0.100 30 22 POS 0.0-5.0

160.00 0.100 30 32

See ABZ SO2 219.83 0.100 30 40

ABZ-SO2-D3 301.00 159.20 0.030 31 38 POS 0.0-5.0

ALT 266.10 0.030 31 20

OXF (FBZ-SO) 315.82 284.1 0.100 40 18 POS 0.0-5.0

Used 158.98 0.100 40 33

191.1 0.100 40 20

FBZ SO-D3 319.10 159.00 0.080 16 30 POS 0.0-5.0

ALT 194.00 0.080 16 18

FBZ-SO2 332.00 300.00 0.100 35 18 POS 0.0-5.0

159 0.100 35 36

FBZ-SO2-D3 335.04 299.9 0.080 19 19 POS 0.0-5.0

ALT 158.8 0.080 19 32

OXI 250.00 176.00 0.080 36 26 POS 2.0-9.0

218.00 0.080 36 18

ABZ 266.10 191.00 0.015 32 32 POS 4.00-7.50

234.00 0.100 32 20

ABZ-D3 269.12 191.10 0.080 35 19 POS 4.00-7.50

233.85 0.080 35 19

MBZ 296.00 264.00 0.100 35 20 POS 1.0-5.8

105.00 0.015 35 31

CAM 303.00 217.00 0.080 30 27 POS 2.0-9.0

261.00 0.080 30 18

FLU 314.00 123.00 0.010 35 35 POS 2.0-7.0

282.00 0.100 35 20


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FLU-OH 316.00 97.00 0.100 40 40 POS 1.0-5.8

125.00 0.100 40 33

FBZ 300.00 159.00 0.010 35 33 POS 6.00-11.00268.00 0.100 35 23

FBZ-D3 303.1 158.70 0.080 28 18 POS 6.00-11.00

ALT 268 0.080 28 33

TCB 359.00 197.00 0.100 46 35 NEG 8.00-14.50

Used 344.00 0.100 46 23

TCB-D3 362.00 197.00 0.080 20 37 NEG 8.00-14.50

Used 344.00 0.080 20 23

NICLOZ 325.00 171.00 0.100 35 25 NEG 8.00-14.50

289.00 0.100 35 18

BITH 355.00 161.00 0.010 32 22 NEG 8.00-14.50

194.00 0.100 32 23

TCB-SO 375.00 359.70 0.100 32 20 NEG 8.00-14.50

181.00 0.100 32 41

TCB-SO2 330.00 184.00 0.100 29 25 NEG 7.00-12.00

118.00 0.100 29 38

OXYCLOZ 400.00 176.00 0.100 35 27 NEG 7.00-16.00

202.00 0.100 35 21

RAFOX 624.00 127.00 0.100 52 46 NEG 10.00-15.00

345.00 0.100 52 35

CLOS 661.00 127.00 0.100 55 45 NEG 10.00-15.00

345.00 0.100 55 39

MOXI 662.30 240.10 0.100 44 35 POS 9.50-16.00

337.00 0.100 44 32

ALT. 640.09 498.25 0.015 17 11 POS

528.36 0.015 17 10

SELA 770.50 144.80 0.100 36 30 POS 11.00-17.00

ALT.792.10 648.30 0.080 60 33 POS

EMA 887.00 158.00 0.100 45 34 POS 11.00-16.00126.00 0.100 45 37


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ABA 895.00 751.50 0.015 58 18 POS 12.60-17.00

327.30 0.015 58 29

IVER 897.00 153.10 0.015 55 55 POS 12.60-17.00329.1 0.015 55 55

ALT 183.20 0.015 55 55

EPRI 936.00 490.40 0.100 25 53 POS 12.60-17.00

352.20 0.100 25 54

DORA 921.00 777.20 0.100 40 29 POS 12.60-17.00

183.20 0.100 40 40

ALT

449.4

0.100

40

28

COUMA 363.10 227.00 0.100 32 25 POS 9.50-16.00

307.10 0.100 32 16

COUMA-O 347.00 291.00 0.080 30 19 POS 2.0-9.0

211.00 0.080 30 29

HAL 415.00 211.05 0.100 38 30 POS 4-11

273.05 0.100 38 23

Note: some of these details may vary slightly with laboratory; Alt = alternative ions

and its parameters; POS = positive mode; NEG = Negative mode. Unless otherwise

indicated (as Used), ions in the first raw of each compound are used for

quantification.


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Table 4. The table below shows 38 anthelmintics and flukicides standard compounds

used in this study. The corresponding internal standards (11) used for quantification

are also shown against each compound.

Standard (S) Internal standard (IS)ABA SELA

ABZ ABZ-D3

ABZ- SO2 ABZ-SO2-D3

ABZ-SO ABZ-SOD3

ABZ-NH2-SO2 ABZ-NH2-SO2-D2

FLU-NH2 FBZ-D3

MBZ-NH2 FBZ-D3

BITH TCB-D3

CAM FBZ-D3

CLORS NONECLOS TCB-D3

COUMA LEVA-D5

COUMA-O LEVA-D5

DORA SELA

EMA SELA

EPRI SELA

FBZ FBZ-D3

FBZ-SO2 FBZ-SO2-D3

FLU FBZ-D3

HALOX LEVA-D5

FLU-OH FBZ-D3MBZ-OH FBZ-D3

IVER SELA

LEVA LEVA-D5

MBZ FBZ-D3

MOR TBZ-NH D6

MOXI SELA

NICLOZ TCB-D3

NITROX TCB-D3

FBZ-SO FBZ SO-D3

OXI FBZ-D3OXYCLOZ TCB-D3

RAFOX TCB-D3

TCB TCB-D3

TCB-SO TCB-D3

TCB-SO2 TCB-D3

TBZ TBZ-NH-D6

5-OH-TBZ TBZ-NH D6


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Table 5: MRM parameters

MRM Inter-scan delay Inter-channel delay

1 0.1 0.020

2 0.1 0.020

3 0.1 0.020

4 0.1 0.020

5 0.1 0.020

6 0.1 0.020

7 0.1 0.020

8 0.1 0.020

9 0.1 0.020

10 0.1 0.020

11 0.1 0.020

12 0.1 0.020

13 0.1 0.02014 0.1 0.020

15 0.1 0.020

16 0.1 0.020

17 0.1 0.020

18 0.1 0.020

19 0.1 0.020

20 0.1 0.020

21 0.1 0.020

22 0.1 0.020

23 0.1 0.020

24 0.1 0.020

25 0.1 0.020

26 0.1 0.020

27 0.1 0.020

28 0.1 0.020

29 0.1 0.020

30 0.1 0.020

10.0 Expression of Results

Calculate the ion ratio (Eq. I) and the relative deviation of the ion ratio (Eq. II)

for each analyte compared to the matrix matched samples.

Calculate the relative retention time (Eq. IV) and the deviation of the relative

retention time (Eq. V) for each analyte compared to the matrix matched

samples. See Table 4 for list of internal standards.

The identity of the analyte is confirmed when the EU criteria for ion ratio and

retention time similarity to matrix matched samples are met [1]. For deviation

in ion ratio the value must be less than 10-20 % and for deviation in retention

time the value should be less than 2.5 %.


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Calculate the response factor for each analyte the amount of the response

factor (RF) (Eq. III).

Recovery is calculated by dividing the residue concentrations determined in

the recovery controls (minus concentration in blank) by the fortification

concentrations. This is expressed as a percentage.

The linearity of the positive control curve shall be assessed from the

correlation coefficient for the positive controls used to make up the curve. An

acceptable limit for the correlation coefficient is normally 0.98.

Equation I: Calculation of the ion ratio (R)

R = (Alowest intensity ion/Ahigest intensity ion) *100

R= ion ratio (%)

Alowest intensity ion= peak area of the product ion of lowest intensityAhighest intensity ion= peak area of the product ion of highest intensity

Equation II:Calculation of the relative deviation of the ion ratio (R).

(R) = (Rsample_Rmean/Rmean)*100%

R = relative deviation of the ion ratio of the analyte in the sample compared to the

average ion ratio of the same analyte in the positive control samples (fortified

samples).

Rsample= ion ratio of the analyte in the sample (Eq.1)

Rmean= average ion ratio of the analyte in the positive control samples (%) (Eq.1)

Equation III: Calculation of the response factor (RF) and analyte present in the

sample

Response factor (peak areacomponent)/peak areaTrue/matrix matched IS) Vs

concentration, R2and equation Y c generated

Y = (peak area unknown/peak area Recovered IS) X= Conc. (amount) of analyte in sample. X is multiplied by 2 to obtain

the true concentration of the analyte in the original 10 g of the sample

extracted. Although 12 ml of the extraction solvent was used (10 ml for

extraction and 2 ml for washing the extraction salts, hence 12 ml of

acetonitrile used for extraction), only 6 ml of supernatant was

concentrated down to 0.5 ml of DMSO.

Recovery = (XConc. in blank/Conc. of ES added)%

Only peak area of the quantification ion is used.

Data is first processed using target-lynx and exported to Microsoft excel for further

processing such as to calculate means, standard and relative standard deviations etc.


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Equation IV:Calculation of the relative retention time (RRT)

RRT = (RTcomponent/RTIS)

RRT= relative retention time

RRTcomponent= retention time of analyteRTIS= retention time of the IS

Equation V:Calculation of the deviation of the relative retention (RRT).

(RRT) = (RRTsample_ RRTmean/RRTmean)*100

RRT = relative retention time of the analyte in the sample compared to the RRT of

the same analyte in the matrix matched standards.

RRTsample= relative retention time of the analyte in the sample

RRTmean = average relative retention time of the analyte in the matrix matchedstandards.

11.0 Criteria and/or requirements for approval/rejection of results

Acceptable recovery of analyte from fortified samples (typically 60-120%)

deem the assay to be satisfactory. With an internal standard added to each

sample, recovery values outside this range 60-120% are acceptable as the

internal standard is used to correct for recovery and gives an absolute value for

the amount of analyte present.

12.0 Notes on procedure

12.1 When adding the sample to the Tube A, it is critical that the tube is shaken

straight away to avoid clumping of the salts.

13.0 Data to be recorded and method of analysis and presentation

Test report: The test report should show the result obtained (this result is already

corrected for recovery, based on internal standard recovery correction). The test

report should mention any operating conditions not specified in this method.

14.0 Procedure for estimating uncertainty

Where required, derive an estimate for uncertainty of measurement based on

published literature.

15.0 References

1.

Commission Decision (2002/657/EC) of 12 August 2002 implementing

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

methods and interpretation of results, O. J. Europ. Comm.L 221, 8-36.


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16.0 Specimen chromatograms:

Figure 1. ABZ (40 ppb in beef matrix)

Figure 2. ABZ SO (40 ppb in beef matrix)


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Figure 3. ABZ SO2(40 ppb in beef matrix)

Figure 4. ABZ NH2SO2 (40 ppb in beef matrix)


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Figure 5. CAM (40 ppb in beef matrix)

Figure 6. FBZ (40 ppb in beef matrix)


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Figure 7. FBZ SO2(40 ppb in beef matrix)

Figure 8. OXF (40 ppb in beef matrix)


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Figure 9. FLU (40 ppb in beef matrix)

Figure 10. FLU NH2(40 ppb in beef matrix)


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Figure 11. FLU OH (40 ppb in beef matrix)

Figure 12. MBZ (40 ppb in beef matrix)


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Figure 13. MBZ NH2(40 ppb in beef matrix)

Figure 14. MBZ OH (40 ppb in beef matrix)


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Figure 15. OXI (40 ppb in beef matrix)

Figure 16. TCB (40 ppb in beef matrix)


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Figure 17. TCB SO (40 ppb in beef matrix)

Figure 18. TCB SO2(40 ppb in beef matrix)


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Figure 21. LEVA (40 ppb in beef matrix)

Figure 22. BITH (40 ppb in beef matrix)


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Figure 23. CLORS (40 ppb in beef matrix)

Figure 24. CLOS (40 ppb in beef matrix)


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Figure 25. COUMA (40 ppb in beef matrix)

Figure 26. COUMA-O (40 ppb in beef matrix)


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Figure 27. MOR (40 ppb in beef matrix)

Figure 28. NICLOZ (40 ppb in beef matrix)


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Figure 29. NITROX (40 ppb in beef matrix)

Figure 30. OXYCLOZ (40 ppb in beef matrix)


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Figure 31. RAFOX (40 ppb in beef matrix)

Figure 32. ABA (40 ppb in beef matrix)


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Figure 33. DORA (40 ppb in beef matrix)

Figure 34. EMA (40 ppb in beef matrix)


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Figure 35. EPRI (40 ppb in beef matrix)

Figure 36. IVER (40 ppb in beef matrix)


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Figure 37. MOXI (50 ppb in beef matrix)

Figure 38. SELA (8 ppb in beef matrix)


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Figure 39. ABZ-SO-D3 (8 ppb in beef matrix)

Figure 40. ABZ-SO2-D3 (8 ppb in beef matrix)


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Figure 41. ABZ-NH2-SO2-D2 (8 ppb in beef matrix)

Figure 42. FBZ-D3 (8 ppb in beef matrix)


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Figure 43. LEVA-D5 (8 ppb in beef matrix)

Figure 44. FBZ-SO-D3 (8 ppb in beef matrix)


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Figure 45. FBZ-SO2-D3 (8 ppb in beef matrix)

Figure 46. TBZ NH-D6 (8 ppb in beef matrix)


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Figure 49. Haloxon (40 ppb in beef matrix)

17.0. Proposed washing plan (pump head, column and probe capillary tube).

17.1. Seal wash (9.2.11): Autosampler purge is done between sample injection using

a mixture of methanol (9.2.2) and IPA (9.2.4) (80:20, v/v). This is

automatically set up in the inlet method with the stroke length/volume set at

130 l and the degasser set in continuous mode (9.5).

17.2. Wash step one: After sample analysis, each solvent in lines A and B arereplaced with wash solution one (9.2.9) water:methanol (80:20, v/v). Wet

priming is done for 3 min. The injector is also purged for 3 min. The new

solvents are then pumped through lines A and B at 50:50 percent delivery

washing both column and probe capillary for 60 min. The flow rate is 0.5

ml/min while the column temperature is maintained at 45C. To avoid

washing of salts (from the original mobile phase) into the ion source, the

source is closed and the MS turned off after cooling. The wash solution then

drips onto a pack of paper towel placed under the probe tip (under close

observation). Alternatively, the probe may be removed so that the wash

solution is collected outside the source closure.

17.3. Wash step two: Stop the pumps and replace the weak wash solution with wash

solution two (9.2.10). Perform wet priming, and run both pumps as in 17.2 for

30 min (may be longer if deemed necessary). Store column in recommended

solvent after wash step two.

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