The mission of the U.S. Department of Agriculture

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1 ALI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 1, DRUGS, COSMETICS, FORENSIC SCIENCES Simultaneous Determination of Eprinomectin, Moxidectin, Abamectin, Doramectin, and Ivermectin in Beef Liver by LC with Fluorescence Detection ALI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 1, 2000 M. SHER ALI, TUNG SUN, GINA E. MCLEROY, and EVAN T. PHILLIPPO U.S. Department of Agriculture, Food Safety and Inspection Service, Office of Public Health and Science, PO Box 6085, Athens, GA Eprinomectin, moxidectin, abamectin, doramectin, and ivermectin are drugs used to control parasitic infections in both meat-producing and nonmeat-producing animals. A number of analytical methods are available to analyze these anthelmintic drugs individually. A multiresidue screening method was developed for these drugs; however, the initial attempt to derivatize eprinomectin following the method published by Merck scientists was unsuccessful because the eprinomectin derivatization reaction was temperature- and time-dependent. The optimum time and temperature for the completion of eprinomectin derivatization were 90 min and 65 C, respectively, without appreciable effect on the remaining 4 drugs. Beef liver samples were fortified with 0, 25, 50, and 100 ppb mixed standards of eprinomectin, moxidectin, abamectin, doramectin, and ivermectin. Each set of 4 levels of recoveries was repeated 10 times with all 5 compounds. The average of 10 recoveries of 5 compounds at all 4 levels of fortification was > 70%; the coefficient of variation was < 20%. The mission of the U.S. Department of Agriculture (USDA) Technical Services Laboratory is to provide analytical data for regulating the meat, poultry, and egg product industries to ensure that these products moving in interstate commerce or exported to other countries are safe, wholesome, and accurately labeled. We have analyzed beef liver tissues in our laboratory for ivermectin residue since 1987 under the National Residue Plan for Food Safety and Inspection Service (FSIS). Ivermectin is a potent anthelmintic drug used in food animals to control parasitic infections. A number of similar antiparasitic drugs such as abamectin, moxidectin, doramectin, and eprinomectin, available in the United States and in the world agri-chemical markets, have been approved for use in meat-producing and nonmeat-producing animals worldwide. In the United States, Received April 15, Accepted by JM August 17, ivermectin, doramectin, eprinomectin, and moxidectin have been approved for use in meat-producing animals by the U.S. Food and Drug Administration (FDA), and abamectin has been approved as a pesticide by the U.S. Environmental Protection Agency (EPA). Each of these drugs consists of one major ( 80%) and one minor homologue (5 20%). The major homologue is used as a marker to calculate total residues in edible tissues. The minimum residue limits (MRLs) or tolerances of these drugs vary from species to species and from target tissue to target tissue. Tolerances for these drugs are summarized in Table 1. Of the 5 anthelmintic drugs, only ivermectin has been monitored in meat-producing animals in the United States under the FSIS National Residue Plan. However, each of these compounds has several liquid chromatographic (LC) methods of determination: ivermectin (9 11), doramectin (12 13), eprinomectin (14 16), abamectin (17 19), and moxidectin (20 21). A multiresidue method was needed in which all 5 antiparasitic drugs could be analyzed by one method, to reduce analysis time and solvent use and monitor compliance. As shown in Table 1, tolerance levels of anthelmintic drugs established by the FDA and the Joint Pesticide Residues Committee of the Food and Agriculture Organization/World Health Organization (FAO/WHO) of the United Nations vary from compound to compound and tissue to tissue. Therefore, the intended multiresidue LC method had to be designed to cover each compound at levels lower than or closer to its known tolerance level, and to obtain peak heights comparable to those of other anthelmintic drugs in the group in a liquid chromatogram. In this LC method with fluorescence detection, the lowest limits of reliable measurements of all 5 anthelmintic drugs in liver were 10 ppb, with limits of detection of 1 2 ppb. However, at these levels none of the drugs can be confirmed by mass spectrometry (MS). LC/MS confirmation requires ppb of each drug in liver. In this multiresidue method, 4 levels of fortification (0, 25, 50, and 100 ppb) were chosen for beef liver. Each set of 4 recovery levels was repeated 10 times for all 5 compounds, resulting in 200 recoveries.

2 32 ALI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 1, 2000 METHOD The apparatus and reagents used in the method may be substituted with equivalent apparatus and reagents. Apparatus (a) Blender. Waring laboratory blender (Waring Products Div., New Hartford, CT) with L capacity glass blender jars for liver sample preparation. (b) N-Evap. Model 112 (Organomation Assoc., Inc., Berlin, MA). (c) Centrifuge. Sorvall Model T-6000B (DuPont Co., Newton, CT). (d) Mechanical shaker. Eberbach Model 610 equipped with shaker box Model 6040 (Thomas Scientific, Swedsboro, NJ). (e) Vortex mixer. Fisher Scientific, Norcross, GA. (f) Extraction columns. Prep-Sep-R (empty) Cat. No. P449R (Fisher Scientific). (g) Silane-treated glass wool. Cat. No (Supelco, Inc., Bellefonte, PA). (h) Polypropylene centrifuge tubes. 50 ml, Cat. No (Becton Dickinson Labware, Lincoln Park, NJ). (i) Screw cap glass centrifuge tubes. 50 ml, Cat. No B (Fisher Scientific). (j) EDP plus micropipets. 100, 250, and 1000 ml capacities (Rainin Instrument Co., Inc., Emeryville, CA). (k) Borosilicate disposable culture tube. Cat. No (Fisher Scientific). (l) LC inserts. Cat. No , 250 ml micro sample tube with stopper (Scienceware, Bel-Art Products, Pequannock, NJ). (m) LC apparatus. Waters Model 510 HPLC pump, 712 WISP autosampler, 474 scanning fluorescence detector, 600E Table 1. Tolerances of ivermectin, doramectin, eprinomectin, moxidectin, and abamectin Compound Species Tissue/Product Tolerance, ppb References Ivermectin Cattle Liver and muscle 50 a 1 Swine Liver 75 Kidney and fat 125 Sheep Liver, kidney, and fat 125 Muscle 25 Reindeer Liver 50 Muscle 25 Kidney 75 Doramectin Cattle Liver 100 a 2&3 Muscle 10 Kidney 39 Fat 150 Eprinomectin Cattle Liver 4.8 a,b 4&5 Milk 12 Moxidectin Cattle, sheep, deer Liver 100 c 6 Muscle 20 Kidney 50 Fat 500 Cattle Liver 200 a 7 Muscle 50 Abamectin Cattle Liver and fat 100 d 8 Kidney 50 Meat and meat by-products 20 e 10 Fat 150 a b c d e Established by FDA. In ppm. Established by FAO of the United Nations. Established by FAO/WHO of the United Nations. Established by EPA.

3 ALI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 1, Table 2. Derivatization reaction kinetics of 50 ppb mixed standards of anthelmintic drugs at 65 C Compound Run No. Peak heights, mm Initial 15 min 30 min 45 min 60 min 75 min 90 min 105 min Eprinomectin a Avg Moxidectin a Avg Avamectin a Avg Doramectin a Avg Ivermectin a Avg a Run No. 2 did not have a sample for 105 min.

4 34 ALI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 1, 2000 Figure 1. Chromatograms of derivatization reaction kinetics of 50 ppb mixed standards: (a) 20 L portion mixed standard analyzed immediately after adding and mixing derivatizing reagents; (b) after 15 min at 65 C; (c) after 30 min at 65 C; (d) after 45 min at 65 C; (e) after 60 min at 65 C; (f) after 75 min at 65 C; (g) after 90 min at 65 C; and (h) after 105 min at 65 C. Fluorescence detection excitation and emission at 340 and 418 nm, respectively. system controller, and 740 data module (Waters Associates, Milford, MA). (n) LC column. Zorbax ODS mm id C 18 column (DuPont Co., Wilmington, DE). (o) Guard column. Brownlee Labs Spheri-5-RP-18, mm, 5 µm guard column (Applied Biosystems, Inc., Foster City, CA). (p) Column heater. Bio-Rad (Hercules, CA) LC column heater. Reagents (a) Acetonitrile (ACN), methanol, and water. LC grade. (b) Acetonitrile, 99.8%, anhydrous. Cat. No. 27,100-4 (Aldrich Chemical Co., Inc., Milwaukee, WI). (c) Alumina. Neutral Type WN-3, activity grade 1 (Sigma Chemical Co., St. Louis, MO). Store at 135 C until used. Prepare deactivated alumina for column chromatography by adding 6 ml distilled water to 44 g alumina. Mix on mechanical shaker for 45 min. Store alumina at room temperature in tightly closed container. Make fresh weekly. (d) Alumina column. Weigh 2.0 g deactivated alumina into empty Prep-Sep column. Tap gently to settle alumina. (e) C 18 bulk packing material. Waters Bondapak 125 A, mm, Cat. No. WAT (Waters Associates, Inc.). (f) C 18 SPE cartridges. Place small silanated glass wool plug into neck of 5.75 in. disposable transfer pipet. Add 0.1 g C 18 bulk packing material into disposable pipet. Tap gently to settle. (g) 1-Methylimidazole. Redistilled, 99+ %; Cat. No. 33,609-2 (Aldrich Chemical Co.). (h) Trifluoroacetic anhydride (TFAA). 99+ %, Cat. No. 10,623-2 (Aldrich Chemical Co.). (i) Derivatizing reagents. Prepare 2 ml 1-methylimidazole ACN (1 + 1, v/v) in dry 5 ml test tube, and 2 ml TFAA ACN (1 + 1, v/v) in dry 5 ml test tube. Stopper or cover with aluminum foil. These reagents are stable for 1 day. Prepare fresh daily. (j) LC mobile phase. Methanol water (97 + 3, v/v). (k) Standards. Eprinomectin standard Cat. No. L-653, C012, ivermectin standard Cat. No. L-640, P004, and abamectin standard Cat. No. L-676, A003 (Merck & Co., Inc., Rahway, NJ); doramectin standard (Pfizer, Lee-Summit, MO); moxidectin standard Cat. No (American Cyanamid Co., Princeton, NJ). Preparation of Standard Solutions (a) Mixed stock standard solution (125 g/ml). Dissolve appropriate amount each of eprinomectin, moxidectin, abamectin, doramectin, and ivermectin in ACN to make concentration of 125 µg/ml.

5 ALI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 1, (b) Working standard solution (0.5 g/ml). Dilute 1 ml stock solution to 250 ml with anhydrous ACN in 250 ml volumetric flask. This working solution is used as fortification solution for repeatability studies and to generate standard curves. Store mixed stock standard and working standard solutions at 20 C. Under this condition, stock standard is stable for 1 year and working standard is stable for 3 months. LC Operating Parameters Mobile phase flow rate, 1.8 ml/min; column temperature, 30 C; injection volume, 20 µl; detector settings: excitation wavelength, 365 nm and emission wavelength, 465 nm; detector gain, 100; attenuation, 8; and run time, 16 min. Studies of Derivatization Reaction Kinetics A 50 ppb level mixed standard solution in 2.5 ml anhydrous ACN was derivatized by adding 200 µl 1-methylimidazole ACN and 200 ml TFAA ACN, and was immediately transferred into eight 250 µl LC inserts. (Anhydrous ACN was used in this step to decrease moisture content which would affect the derivatization reaction.) One insert was analyzed immediately and the remaining inserts (completely full) were capped tightly, placed in a 10 ml beaker, covered with aluminum foil, and incubated at 65ºC. At every 15 min interval, an insert was removed from the incubator and analyzed by LC. The last insert was analyzed after 105 min at 65ºC. This experiment was repeated 6 times to verify that completion of the derivatization reaction of eprinomectin at 65 C requires 90 min. Identification Under the described LC conditions, the retention times (min) of the 5 anthelmintic drugs were: eprinomectin, 3.609; moxidectin, 5.366; abamectin, 6.712; doramectin, 8.390; and ivermectin, Total analysis time was ca 16 min. Sample Extraction, Derivatization, and Determination Figure 2. (A D) Liquid chromatograms of reagent blank and mixed standards of eprinomectin, moxidectin, abamectin, dormectin, and ivermectin at 25, 50, and 100 ppb levels used in the method. (A D ) Liquid chromatograms of blank beef liver and recoveries of eprinomectin. For the recovery study, fortify a 2.5 g portion of blended defrosted liver sample in four 50 ml polypropylene centrifuge tubes with 0, 125, 250, and 500 µl 0.5 µg/ml mixed standards, equivalent to 0, 25, 50, and 100 ppb, respectively, of eprinomectin, abamectin, moxidectin, doramectin, and ivermectin. Add 8 ml ACN and mix on Vortex mixer for 30 s, and then centrifuge for 3 min at 1500 rpm. Pour ACN extract through deactivated alumina column. Collect eluate in 50 ml glass centrifuge tube. Re-extract tissue with additional 8 ml ACN, centrifuge, and decant through same alumina column, combining eluates. Evaporate combined extracts of each sample under gentle stream of nitrogen at ca 65 C. Reconstitute dried sample in 0.5 ml anhydrous ACN to decrease moisture content in subsequent reaction mixture in derivatizing step. Load each sample extract in 0.5 ml anhydrous ACN onto pre-wet C 18 SPE cartridge. (Pre-wet C 18 SPE cartridge with 1.0 ml anhydrous ACN. Make sure the cartridge does not dry up until elution of sample extract is complete. Drying up the cartridge at this step will result in low recovery.) Collect eluate in small 5 ml test tube (borosilicate disposable culture tube) at gravity flow. Rinse each sample tube with 2 ml anhydrous ACN and add to SPE cartridge. Collect eluate in same culture tube for total volume of 2.5 ml. For each set of regulatory samples, extract a blank and a 50 ppb fortified sample as a recovery to be used to correct positive sample results. Derivatize the sample by adding 200 µl 1-methylimidazole ACN (1 + 1) reagent and 200 µl TFAA ACN (1 + 1) reagent to each test tube containing 2.5 ml sample extract, and mix. Completely fill LC inserts with sample extracts, cap tightly, place in small 10 ml beaker, cover with aluminum foil, and incubate in oven at 65 C for 90 min. Using LC parameters described, samples are analyzed immediately after completion of reaction. (Note: Derivatized samples decompose on exposure to light and after standing.) Four levels of mixed standard (0, 25, 50, and 100 ppb) are similarly reconstituted to 2.5 ml each with anhydrous ACN,

6 36 ALI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 1, 2000 Table 3. Overall performance of 5 anthelmintics at 0, 25, 50, and 100 ppb fortifications in beef liver Compound Concn, ppb No. of recoveries Mean recovery, % SD CV, % Avg. recovery, ppb Eprinomectin Moxidectin Abamectin Doramectin Ivermectin derivatized, and analyzed by LC to generate a standard curve for each analyte. A set of recoveries consisting of 4 blank beef liver samples was fortified with 0, 25, 50, and 100 ppb mixed standards of 5 anthelmintic drugs. Ten sets of recoveries of each fortification were analyzed on 10 different days. The samples and standards were quantitated by peak height measurements. Peak heights were plotted vs concentration of standards (in ppb), to construct a 4-point linear curve for each compound. The standard curve should be linear with a correlation coefficient (r) of Results and Discussion Ivermectin is a potent anthelmintic drug used in food animals to control parasitic infections. The method for determining ivermectin residue in liver tissue by LC with fluorescence detection was provided by the manufacturer (Merck Sharpe & Dohme Research Laboratories, Rahway, NJ) as part of a New Animal Drug Application (NADA; 9) method which was later published by Merck scientists (10). This method was modified several times in our laboratory (22 24); the extraction part of the method was adapted to automation (25) for analysis of ivermectin under the National Residue Plan for FSIS. A number of similar antiparasitic drugs such as abamectin, moxidectin, doramectin, and eprinomectin are available in the United States and in the world agri-chemical markets. All 5 anthelmintic drugs, eprinomectin, moxidectin, abamectin, doramectin, and ivermectin, are included in the current study because of their structural similarities and their similar antiparasitic properties. A reversed-phase LC column was used to separate the compounds. The most significant aspect of the present study was the discovery of the time and temperature sensitivity of the eprinomectin derivatization reaction. The LC analytical method for eprinomectin developed by Merck scientists (15) used on-line derivatization by adding 30% TFAA in ACN to residues dissolved in 30% 1-methylimidazole in ACN with reversed-phase fluorescence detection. Because we did not have the on-line derivatization equipment, we were unable to analyze eprinomectin by the published method (15). However, following an alternative suggestion that derivatization can also be performed manually (15), we were not able to obtain reproducible chromatograms of eprinomectin even by using extreme precautions (e.g., performing derivatization reaction in inert atmosphere with anhydrous reagents and under yellow light). The reaction kinetics of the derivatization step of all 5 anthelmintic drugs were studied by using a 50 ppb mixed standard. The eprinomectin derivatization reaction proceeded very slowly at room temperature, whereas derivatization of moxidectin, abamectin, doramectin, and ivermectin was nearly instantaneous. The derivatization reaction of eprinomectin peaked at about 7 h at room temperature,

7 ALI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 1, whereas the peak heights of moxidectin, abamectin, doramectin, and ivermectin diminished considerably on standing. However, the derivatization reaction of eprinomectin was sped up by heating the reaction mixture. Heating the reaction mixture increased overall peak heights by 10 15% for the remaining 4 compounds, which meant that an optimum temperature is needed to complete derivatization of these compounds. The reaction kinetic experiment was repeated 6 times to ascertain the validity of this observation (Table 2). Chromatograms of a typical reaction kinetic study are shown in Figure 1. The optimum time required for completion of derivatization reaction of eprinomectin at 65 C was about 90 min. Peak heights of the remaining 4 anthelmintics were relatively unchanged at 65 C for 90 min. The derivatization reaction of subsequent recoveries was performed under these conditions because they produced the least variability in peak heights for all compounds. Immediately after adding derivatizing reagents to standards and recoveries, each resulting solution is transferred into LC insert and capped to prevent loss by evaporation. They are then placed in a small beaker, covered with aluminum foil, and incubated at 65 C for about 90 min, to complete the reaction. The samples in LC inserts are analyzed with the LC conditions discussed earlier. Derivatized solutions should be analyzed soon after completion of reaction. Because derivatized samples decompose on exposure to light and after standing, they should be protected from exposure. Figure 2 (A D) shows chromatograms of reagent blank and mixed standards of all 5 anthelmintic drugs at the 4 levels of interest used in the method. Figure 2 (A D ) shows chromatograms of recoveries of these drugs in beef liver at the 4 levels of interest. Recovery data were based on analysis of bovine liver samples fortified with each of the 5 anthelmintic drugs at 0, 25, 50, and 100 ppb. The extraction and analysis were performed on 10 different days at each of the fortification levels of all 5 anthelmintics, resulting in a total of 50 recoveries at each level. Overall performance summary for the 5 anthelmintics at each of the 4 fortification levels in beef liver is shown in Table 3. The combined average recoveries of all 5 anthelmintic drugs in bovine liver tissue at all 3 levels of fortification was > 70%, with a coefficient of variation (CV) of < 20%. The general FSIS guidelines for a regulatory method are that it meets established criteria for concentration of analyte, expected recovery range, and % CV. If the concentration of an analyte varies from 1 to 400 ppb, the expected recovery range should be % (expected recovery is defined as ± 2 standard deviations of the statistical mean recovery), the repeatability CV should be 20%, and the reproducibility CV should be 30%. Data obtained for the method extension studies are well within the limits of acceptable criteria for a regulatory method (26). Conclusion The determinative part of the present multiresidue LC screening method is relatively simple and sensitive. It is reliable for routine, simultaneous determination of eprinomectin, moxidectin, abamectin, doramectin, and ivermectin in bovine liver tissue. This method could be applied for screening a variety of animal tissues and other products with complex matrixes for eprinomectin, moxidectin, abamectin, doramectin, and ivermectin. References (1) 51 FR (Federal Register/Vol. 51) 27021, July 29, 1986 (Tolerances for Residues of New Animal Drugs), as amended at 53 FR 27958, July 26, 1988; 54 FR 18281, Apr. 28, 1989; 59 FR 50830, Oct. 6, 1994; and 21 CFTR (Code of Federal Regulations) Part 556; Washington, DC (2) Committee for Veterinary Medicinal Products Working Party on the Safety of Residues (1994) Injection Site Residues, Brussels, Belgium, Commission of the European Communities (unpublished document III/5033/94; available on request from Directorate-General III/E/3, Pharmaceuticals, Commission of the European Communities, Brussels, Belgium) (3) General Principles for Evaluating the Safety of Compounds used in Food Producing Animals (rev. 1994) 1, Guidelines for Metabolism Studies and for Selection of Residues for Toxicological Testing, U.S. FDA, Rockville, MD (4) Federal Register: FR Doc Filed under section ; Washington, DC (5) Joint FAO/WHO Expert Committee on Food Additives (1996) Food and Nutrition Paper 41/8, Rome, Italy (6) 21 CFR (Code of Federal Regulations) , April 15, 1997, Washington, DC (7) Joint FAO/WHO Meeting on Pesticide Residues (1996) FAO Plant Production and Protection Paper 133, Rome, Italy (8) CFR (Code of Federal Regulations) , 1998; U.S. Government Printing Office, Washington, DC (9) Merck & Co., Inc. (1983) New Animal Drug Applications and , Ivermectins Application, Rahway, NJ, p. 40 (10) Tway, C.P., Wood, J.S., Jr, & Downing, G.V. (1981) J. Agric. Food Chem. 29, (11) Rupp, H.S., Turnipseed, S.B., Walker, C.C., Roybal, J.E., & Long, A.R. (1998) J. AOAC Int. 81, (12) Pfizer Drug Metabolism Department (1995) Animal Drug Application , Method Validation: A Determinative Procedure for the Quantitation of Doramectin in Cattle Liver, Groton, CT (13) Harrison, A.C., & Walker, D.K. (1998) J. Pharm. Biomed. Anal. 16, (14) Merck & Co., Inc. (1996) New Animal Drug Application , Method Validation: HPLC-Fluorescence Method for the Determination of Eprinomectin (MK-0397) Residue in Bovine Liver Tissue, Rahway, NJ (15) Payne, L.D., Mayo, V.R., Morneweck, A.A., Hicks, M.B., & Wehner, T.A. (1997) J. Agric. Food Chem. 45, (16) Ballard, J.M., Payne, L.D., Egan, R.S., Wehner, T.A., Rahn, G.S., & Tom, S. (1997) J. Agric. Food Chem. 45, (17) Jongen, M.J., Engel, R., & Leecheers, L.H. (1991) Am. Ind. 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8 38 ALI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 1, 2000 (19) Chamkasem, N., Papathakis, M.L., & Lee, S.M. (1993) J. AOAC Int. 76, (20) Khunachak, A., Dacunha, R.A.R., & Stout, S.J. (1993) J. AOAC Int. 76, (21) Alvinerie, M., Sutra, J.F., Capela, D., Galtier, P., Fernandezsuarez, A., Horne, E., & Okeefe, M. (1996) Analyst 121, (22) Reising, K.P. (1992) J. AOAC Int. 75, (23) Reising, K.P. (1998) J. AOAC Int. 81, (24) Reising, K.P., & Di Benedetto, N.M. (1998) J. AOAC Int. 81, (25) U.S. Department of Agriculture (1997) Analytical Chemistry Laboratory Guidebook, Residue Chemistry, Ivermectin LC, Automated Method Revised 2/20/97, USDA, FSIS, Quality Assurance Branch, Washington, DC (26) U.S. Department of Agriculture (1995) Chemistry Quality Assurance Manual, Food Safety and Inspection Service, Science and Technology Quality Systems Branch, Washington, DC