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Page 1 of 54 Date Issued: 18/09/09 Test Method;
Edited by: James Sasanya Anthelmintics
_________________________________________________________________
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)
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-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)
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-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
ml mark. Mix well by repeatedly turning the flask upside down. This
gives aconcentration of 1 mg/ml.
9.2.12.5 Thiabendazole (TBZ)
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 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
the particular batch of material as shown in the Certificate of Analysis]
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)
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 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
to the 10 ml mark. Mix well by repeatedly turning the flask upside
down. This gives a concentration of 1 mg/ml.
9.2.12.8 Oxfendazole (OXF) (synonym: Fenbendazole-sulphoxide)
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 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
down. This gives a concentration of 1 mg/ml.
9.2.12.9 Fenbendazole-sulphone (FBZ-SO2)
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 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
ml mark. Mix well by repeatedly turning the flask upside down. This
gives aconcentration of 1 mg/ml.
9.2.12.10 Mebendazole (MBZ)
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 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
to the 10 ml mark. Mix well by repeatedly turning the flask upside
down. This gives a concentration of 1 mg/ml.
9.2.12.11 Hydroxy-Mebendazole (MBZ-OH)
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 MBZ-OH (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 to the 10 ml mark. Mix well by repeatedly turning the flask upside
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
the particular batch of material as shown in the Certificate of Analysis]
of MBZ-NH2(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 to the 10 ml mark. Mix well by repeatedly turning the flask upside
down. This gives a concentration of 1 mg/ml.
9.2.12.13 Flubendazole (FLU)
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 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
up to the 10 ml mark. Mix well by repeatedly turning the flask upside
down. This gives aconcentration of 1 mg/ml.
9.2.12.14 Hydroxy-Flubendazole (FLU-OH)
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 FLU-OH (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 to the 10 ml mark. Mix well by repeatedly turning the flask upside
down. This gives a concentration of 1 mg/ml.
9.2.12.15 Amino-Flubendazole (FLU-NH2)
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 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
up to the 10 ml mark. Mix well by repeatedly turning the flask upside
down. This gives aconcentration of 1 mg/ml.
9.2.12.16 Oxibendazole (OXI)
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 OXI (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.
9.2.12.17 Triclabendazole (TCB)
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 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
up to the 10 ml mark. Mix well by repeatedly turning the flask upside
down. This gives a concentration of 1 mg/ml.
9.2.12.18 Triclabendazole-sulphone (TCB-SO2)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 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
to the 10 ml mark. Mix well by repeatedly turning the flask upside
down. This gives a concentration of 1 mg/ml.
9.2.12.19 Triclabendazole-sulphoxide (TCB-SO)
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 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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to the 10 ml mark. Mix well by repeatedly turning the flask upside
down. This gives a concentration of 1 mg/ml.
9.2.12.20 Cambendazole (CAM)
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 CAM (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.
9.2.12.21 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)
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 MOXI (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
the flask upside down. This gives a concentration of 1 mg/ml.
9.2.12.23 Emamectin benzoate (EMA)
Dissolve 0.01 (x 100/F)g [where F is the content (%) of Emamectin
B1a in the particular batch of material as shown in the Certificate of
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
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.
9.2.12.24 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
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.
9.2.12.25 Abamectin (ABA)
Dissolve 0.01 (x 100/F)g [where F is the content (%) of Abamectin
B1a in the particular batch of material as shown in the Certificate of
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
B1a in the particular batch of material as shown in the Certificate of
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)
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 HALOX (Sigma Aldrich, Milwaukee, 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.
9.2.12.28 Closantel (CLOS)
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 CLOS (Sigma Aldrich, St. Louis, MO, USA) 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.
9.2.12.29 Levamisole-HCl (LEVA)
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 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)
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 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
the flask upside down. This gives a concentration of 1 mg/ml.
9.2.12.31 Oxyclozanide (OXYCLOZ)
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 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
repeatedly turning the flask upside down. This gives a concentration of
1 mg/ml.
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9.2.12.32 Niclozamide (NICLOZ)
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 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
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.
9.2.12.34 Clorsulon (CLOR)
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 CLOR (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.
9.2.12.35 Bithionol (BITH)
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 BITH (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.
9.2.12.36 Morantel tartrate monohydrate (MOR)
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 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
flask upside down. This gives a concentration of 1 mg/ml.
9.2.12.37 Coumaphos (COUMA)
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 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)
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 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
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.
9.2.12.41 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
turning the flask upside down. This gives a concentration of 1 mg/ml.
9.2.12.42 Fenbendazole-D3 (FBZ-D3)
Dissolve 0.01 g of FBZ-SO2-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.
9.2.12.43 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
turning the flask upside down. This gives a concentration of 1 mg/ml.
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
sonicating in 1 ml DMSO (9.2.5) in a 2 ml volumetric flask for 5 min,
and make up to the 2 ml mark. Mix well by repeatedly turning the
flask upside down. This gives a concentration of 1 mg/ml.
9.2.12.47 Albendazole-D3 (ABZ-D3)
Dissolve 0.01 g of ABZ-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.
9.2.12.48 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
repeatedly turning the flask upside down. This gives a concentration of
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
and bringing to the mark with methanol (9.2.2) to obtain a 4 g/ml
mixed standard;
d. 7.5 ml of the 4 g/ml mixed standard into a 10 ml volumetric flask
and bringing to the mark with methanol (9.2.2) to obtain a 3 g/ml
mixed standard;e.
6.7 ml of the 3 g/ml mixed standard into a 10 ml volumetric flask
and bringing to the mark with methanol (9.2.2) to obtain a 2 g/ml
mixed standard;
f. 5 ml of the 2 g/ml mixed standard into a 10 ml volumetric flask
and bringing to the mark with methanol (9.2.2) to obtain a 1 g/ml
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
and bringing to the mark with methanol (9.2.2) to obtain a 0.25
g/ml mixed standard;
i. 0.5 ml of the 10 g/ml each standard into a 25 ml volumetric flask
and bringing to the mark with methanol (9.2.2) to obtain a 0.2
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
and bringing to the mark with methanol (9.2.2) to obtain a 0.1
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.