Page 1 of 54

Edited by: James Sasanya

Date Issued: 18/09/09

Test Method;
Anthelmintics

1.

Identification
Determination of anthelmintic residues in beef by LC-MS/MS

2.

Scope
Method 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 tested

As outlined in Scope.

4.

Parameters/quantities 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), aminoflubendazole (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), triclabendazolesulphone (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 technical performance requirements.

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 MgSO4 and 1 g NaCl (Biotage).

5.2

Tube B: Centrifuge tubes, 50 ml, polypropylene, containing 1.5 g

MgSO4 and 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, Sigma laboratory centrifuge (Vienna, Austria).

5.7

Vortex mixer (Labinco, Breda, The Netherlands).

_________________________________________________________________

5.8

TurboVap LV 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 C18 Atlantis analytical column (100

mm x 2.1 mm x 3 m, Waters, UK; guard cartridge, 2.1x10mm) and
detected 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 LCMS/MS system is controlled by Masslynx software, and the results are
processed by TargetLynx software and microsoft excel.

6.

Reference standards and reference materials required

No reference materials are used in this test method.

7.

Safety measures to be observed

No 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, MgSO4 and 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.

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
3

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 in
the 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 a concentration 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
4

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 in
the 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 a concentration 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 a concentration 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
5

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.
9.2.12.14

Hydroxy-Flubendazole (FLU-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 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 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.

9.2.12.16

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 5
ml MeOH (9.2.2) in a 10 ml volumetric flask for 5 min, and make up
6

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 of
Analysis] 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 the
flask 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 of
Analysis] 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 turning
the flask upside down. This gives a concentration 1 mg/ml.
7

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 the
flask 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 of
Analysis] 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 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.

9.2.12.31

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.
8

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 turning
the 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.

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 well
by 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 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.

9.2.12.42

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 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.

9.2.12.44

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
10

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 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
1mg/ml.

9.2.12.50.0

Preparation of standard curves and standards for

infusion.

9.2.12.50.1

Preparation 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 concentration
of 10 g/ml.

9.2.12.50.2 Preparation of 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.

11

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 flask
and 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 0.05
g/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

9.2.13.1

Preparation 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 ml
volumetric flask, and bring to the 10 ml mark with methanol (9.2.2).
12

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-SO2D3 (9.2.12.40), FBZ-SO-D3 (9.2.12.41), FBZ-D3 (9.2.13.42), ABZSO2-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 concentration of the IS (e.g. 2 to 4 g/ml so
that a higher final concentration of 20 to 40 g/kg is obtained) may
ease data processing, especially 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.

13

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 MgSO4 and 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 samples
analyzed.
14

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 variation
in 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 9.4.19.4.5.3. 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 9.4.19.4.4.5. 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 the
11 internal standards is spiked to obtain a final concentration of 8 g/kg.
15

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:
Solvents
A%
B%

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

75:25, v/v (Methanol:Acetonitrile); 5
mM amonium formate
Continuous
130.0 l
1.0
345
Atlantis T3 2.1 x 100 mm, 3m
Atlantis T3 2.1 x 10 mm
Sentry 2.1 mm
0.25 ml/min
0.50
10 l
20l
45C
22 min

Degasser
Stroke volume
Min pressure (Bar)
Max pressure (Bar)
Column
Guard cartridge
Guard holder kit
Flow
Flow ramp
Injection volume
Injection loop
Column temperature
Total run time
Autosampler
Sample temperature
Sample temperature limit
Draw speed
Needle depth (mm)
Purge loop volumes

22oC
2oC
Normal
1.00
0.2

16

Table 1: The table below shows the Water Alliance 2695 HPLC pump
gradient timetable for the inlet method to be used.
Time
0.00
2.00
10.10
50.0
12.0050.0
15.000.25
16.10 6
16.20
0.50
20.00
22.00

A%
50.0
50.0
0.0
0.0
0.0
0.0
50.0
50.0
50.0

B%
50.0
50.0
100.0
100.0
100.0
100.0
50.0
50.0
50.0

Flow
(ml/min)
Curve
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25

Curve
1
6
6
6
6
6
6
6
6

Table 2. The table below shows Quattro Micro Mass tune parameters used to
determine suitable MS/MS conditions for subsequent analysis involving an LC
inlet method.
Parameter
Capillary voltage
Extractor
RF Lens
LM 1/HM 1 Resolution
Ion Energy 1
Entrance
Exit
LM 2/HM 2 Resolution
Ion Energy 2
Source T (C)
Multiplier (V)
Desolvation T (C)/Gas
flow
Cone Gas Flow
Cone Gas
Syringe pump flow

Positive ESI
3.29 kV
5V
0.1 V
15.0/15.0
0.5
-1
2
13.5/13.5
0.5
150
650
400/650L/hr

Negative ESI
2.55 kV
3V
0.5 V
14/13.5
0.2
-1
2
13.2/13.0
1.0
150
650
400/650L/hr

50
Argon, p = 3.17e-3 bar
30 l/min

50
Argon, p = 3.17e-3 bar
30 l/min

17

Table 3: MS/MS fragmentation conditions for the standards (9.2.12.1 9.2.12.51).

Component

Parent ion
(m/z)

Daughter
ion (m/z)

Dwell
time
(s)

Cone
Energy
(V)

Collision
Energy
(eV)

Polarity

RT
(min)

LEVAMISOLE

205.00

178.00
122.9.

0.100
0.100

35
35

27
25

POS

0.0-5.0

LEVAMISOLE-D5

210.10
ALT

183.10
95.80

0.085
0.085

33
33

20
34

POS

0.0-5.0

5-OH-TBZ

218.00

191.00
147.00

0.100
0.100

45
45

24
32

POS

0.0-4.0

Used
ABZ-NH2-SO2

274.00

238.20
131.00

0.100
0.100

15
15

11
33

NEG

0.0-5.0

ABZ-NH2-SO2-D2

240.00
ALT

131.0
147.00

0.1
0.1

30
30

24
21

NEG

0.0-5.0

TBZ

202.00

131.00
175.00

0.010
0.010

43
43

30
24

POS

0.0-5.5

TBZ-NH-D6

208.20

180.00
136.00

0.085
0.085

48
48

24
29

POS

0.0-5.0

MOR

221.00

111.00
164.00

0.01
0.1

15
15

25
28

POS

0.0-5.0

NITROX

289.00

127.00
162.00

0.010
0.100

36
36

23
20

NEG

0.0-4.5

CLORS

380.00

344.00
342.00

0.500
0.500

25
25

12
11

NEG

0.0-4.5

MBZ-NH2

238.00

105.00
133.00

0.015
0.100

50
50

24
44

POS

0.0-5.0

FLU-NH2

256.00

23
23
35
35
27

35
36
34
19
21

0.0-5.5

282.00

0.100
0.100
0.08
0.08
0.010

POS

ABZ-SO

95.00
123.00
158.90
208.00
222.00

POS

0.0-4.7

ALT

18

158.9
194.00

0.080
0.080

15
15

30
18

POS

0.0-4.7

266.00
159.00
See MBZ OH 224.00

0.010
0.010
0.010

37
37
37

20
35
26

POS

0.0-5.0

0.100
0.100
0.100

30
30
30

22
32
40

POS

0.0-5.0

See ABZ SO2

266.00
160.00
219.83

ABZ-SO2-D3

301.00
ALT

159.20
266.10

0.030
0.030

31
31

38
20

POS

0.0-5.0

OXF (FBZ-SO)

315.82

284.1
158.98
191.1
159.00
194.00

0.100
0.100
0.100
0.080
0.080

40
40
40
16
16

18
33
20
30
18

POS

0.0-5.0

POS

0.0-5.0

ABZ-SO-D3

285.00
ALT

ABZ-SO2

298.10

MBZ-OH

297.90

Used
FBZ SO-D3

319.10
ALT

FBZ-SO2

332.00

300.00
159

0.100
0.100

35
35

18
36

POS

0.0-5.0

FBZ-SO2-D3

335.04
ALT

299.9
158.8

0.080
0.080

19
19

19
32

POS

0.0-5.0

OXI

250.00

176.00
218.00

0.080
0.080

36
36

26
18

POS

2.0-9.0

ABZ

266.10

191.00
234.00

0.015
0.100

32
32

32
20

POS

4.00-7.50

ABZ-D3

269.12

191.10
233.85

0.080
0.080

35
35

19
19

POS

4.00-7.50

MBZ

296.00

264.00
105.00

0.100
0.015

35
35

20
31

POS

1.0-5.8

CAM

303.00

217.00
261.00

0.080
0.080

30
30

27
18

POS

2.0-9.0

FLU

314.00

123.00
282.00

0.010
0.100

35
35

35
20

POS

2.0-7.0

19

FLU-OH

316.00

97.00
125.00

0.100
0.100

40
40

40
33

POS

1.0-5.8

FBZ

300.00

159.00
268.00

0.010
0.100

35
35

33
23

POS

6.00-11.00

FBZ-D3

303.1
ALT

158.70
268

0.080
0.080

28
28

18
33

POS

6.00-11.00

TCB

359.00
Used

197.00
344.00

0.100
0.100

46
46

35
23

NEG

8.00-14.50

TCB-D3

362.00

197.00
344.00

0.080
0.080

20
20

37
23

NEG

8.00-14.50

Used
NICLOZ

325.00

171.00
289.00

0.100
0.100

35
35

25
18

NEG

8.00-14.50

BITH

355.00

161.00
194.00

0.010
0.100

32
32

22
23

NEG

8.00-14.50

TCB-SO

375.00

359.70
181.00

0.100
0.100

32
32

20
41

NEG

8.00-14.50

TCB-SO2

330.00

29
29
35
35

25
38
27
21

7.00-12.00

400.00

0.100
0.100
0.100
0.100

NEG

OXYCLOZ

184.00
118.00
176.00
202.00

NEG

7.00-16.00

RAFOX

624.00

127.00
345.00

0.100
0.100

52
52

46
35

NEG

10.00-15.00

CLOS

661.00

127.00
345.00

0.100
0.100

55
55

45
39

NEG

10.00-15.00

MOXI

662.30

240.10
337.00
498.25
528.36

0.100
0.100
0.015
0.015

44
44
17
17

35
32
11
10

POS

9.50-16.00

144.80
648.30
158.00
126.00

0.100
0.080
0.100
0.100

36
60
45
45

30
33
34
37

POS
POS
POS

ALT. 640.09

SELA
EMA

770.50
ALT. 792.10
887.00

20

POS

11.00-17.00
11.00-16.00

ABA

895.00

751.50
327.30

0.015
0.015

58
58

18
29

POS

12.60-17.00

IVER

897.00

153.10
329.1
183.20

0.015
0.015
0.015

55
55
55

55
55
55

POS

12.60-17.00

ALT
EPRI

936.00

490.40
352.20

0.100
0.100

25
25

53
54

POS

12.60-17.00

DORA

921.00

777.20
183.20
449.4

0.100
0.100
0.100

40
40
40

29
40
28

POS

12.60-17.00

ALT
COUMA

363.10

227.00
307.10

0.100
0.100

32
32

25
16

POS

9.50-16.00

COUMA-O

347.00

291.00
211.00

0.080
0.080

30
30

19
29

POS

2.0-9.0

HAL

415.00

211.05
273.05

0.100
0.100

38
38

30
23

POS

4-11

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.

21

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)
ABA
ABZ
ABZ- SO2
ABZ-SO
ABZ-NH2-SO2
FLU-NH2
MBZ-NH2
BITH
CAM
CLORS
CLOS
COUMA
COUMA-O
DORA
EMA
EPRI
FBZ
FBZ-SO2
FLU
HALOX
FLU-OH
MBZ-OH
IVER
LEVA
MBZ
MOR
MOXI
NICLOZ
NITROX
FBZ-SO
OXI
OXYCLOZ
RAFOX
TCB
TCB-SO
TCB-SO2
TBZ
5-OH-TBZ

Internal standard (IS)

SELA
ABZ-D3
ABZ-SO2-D3
ABZ-SOD3
ABZ-NH2-SO2-D2
FBZ-D3
FBZ-D3
TCB-D3
FBZ-D3
NONE
TCB-D3
LEVA-D5
LEVA-D5
SELA
SELA
SELA
FBZ-D3
FBZ-SO2-D3
FBZ-D3
LEVA-D5
FBZ-D3
FBZ-D3
SELA
LEVA-D5
FBZ-D3
TBZ-NH D6
SELA
TCB-D3
TCB-D3
FBZ SO-D3
FBZ-D3
TCB-D3
TCB-D3
TCB-D3
TCB-D3
TCB-D3
TBZ-NH-D6
TBZ-NH D6

22

Table 5: MRM parameters

MRM
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

10.0

Inter-scan delay
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1

Inter-channel delay
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020

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 %.
23

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 intensity
Ahighest 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, R2 and 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 = (X Conc. 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.

24

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

RRT = (RTcomponent/RTIS)
RRT = relative retention time
RRTcomponent = retention time of analyte
RTIS = 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 matched
standards.
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.

25

16.0

Specimen chromatograms:

Figure 1. ABZ (40 ppb in beef matrix)

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

26

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

Figure 4. ABZ NH2 SO2 (40 ppb in beef matrix)

27

Figure 5. CAM (40 ppb in beef matrix)

Figure 6. FBZ (40 ppb in beef matrix)

28

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

Figure 8. OXF (40 ppb in beef matrix)

29

Figure 9. FLU (40 ppb in beef matrix)

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

30

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

Figure 12. MBZ (40 ppb in beef matrix)

31

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

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

32

Figure 15. OXI (40 ppb in beef matrix)

Figure 16. TCB (40 ppb in beef matrix)

33

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

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

34

Figure 19. TBZ (40 ppb in beef matrix)

Figure 20. TBZ-5-OH (40 ppb in beef matrix)

35

Figure 21. LEVA (40 ppb in beef matrix)

Figure 22. BITH (40 ppb in beef matrix)

36

Figure 23. CLORS (40 ppb in beef matrix)

Figure 24. CLOS (40 ppb in beef matrix)

37

Figure 25. COUMA (40 ppb in beef matrix)

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

38

Figure 27. MOR (40 ppb in beef matrix)

Figure 28. NICLOZ (40 ppb in beef matrix)

39

Figure 29. NITROX (40 ppb in beef matrix)

Figure 30. OXYCLOZ (40 ppb in beef matrix)

40

Figure 31. RAFOX (40 ppb in beef matrix)

Figure 32. ABA (40 ppb in beef matrix)

41

Figure 33. DORA (40 ppb in beef matrix)

Figure 34. EMA (40 ppb in beef matrix)

42

Figure 35. EPRI (40 ppb in beef matrix)

Figure 36. IVER (40 ppb in beef matrix)

43

Figure 37. MOXI (50 ppb in beef matrix)

Figure 38. SELA (8 ppb in beef matrix)

44

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

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

45

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

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

46

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

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

47

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

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

48

Figure 47. TCB-D3 (8 ppb in beef matrix)

Figure 48. ABZ-D3 (8 ppb in beef matrix)

49

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 are
replaced 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.

50