Validation of a Rapid Lateral Flow Test for the Simultaneous Determination of B-Lactam Drugs and Flunixin in Raw Milk

® Validation of a Rapid Lateral Flow Test for the Simultaneous Determination of b-Lactam Drugs and Flunixin in Raw Milk DAVID DOUGLAS,1 KATIE BANASZEWSKI,2 RIMA JUSKELIS,2 FADWA AL-TAHER,2 YANG CHEN,2 JACK CAPPOZZO,2 LINDSAY MCROBBIE,1 AND ROBERT S. SALTER1* 1Charm Sciences, Inc., 659 Andover Street, Lawrence, Massachusetts 01843; and 2Institute for Food Safety and Health–National Center for Food Safety and Technology at the Illinois Institute of Technology, 6502 South Archer Road, Bedford Park, Illinois 60501, USA 1270 Journal of Food Protection, Vol. 75, No. 7, 2012, Pages 1270–1277 doi:10.4315/0362-028X.JFP-11-570 Copyright G, International Association for Food Protection Validation of a Rapid Lateral Flow Test for the Simultaneous Determination of b-Lactam Drugs and Flunixin in Raw Milk DAVID DOUGLAS,1 KATIE BANASZEWSKI,2 RIMA JUSKELIS,2 FADWA AL-TAHER,2 YANG CHEN,2 JACK CAPPOZZO,2 LINDSAY MCROBBIE,1 AND ROBERT S. SALTER1* 1Charm Sciences, Inc., 659 Andover Street, Lawrence, Massachusetts 01843; and 2Institute for Food Safety and Health–National Center for Food Safety and Technology at the Illinois Institute of Technology, 6502 South Archer Road, Bedford Park, Illinois 60501, USA MS 11-570: Received 30 December 2011/Accepted 17 March 2012 ABSTRACT b-Lactam antibiotics are the most commonly used drugs on dairy farms. b-Lactam residues in milk are kept out of the human milk supply with good agricultural practices and mandatory truck screening performed by the dairy industry under Appendix N of the Pasteurized Milk Ordinance. Flunixin, a nonsteroidal and anti-inflammatory drug, appears in dairy cattle tissue residues with a frequency similar to the occurrence of penicillin G. This creates concern that flunixin residues could be in milk and would go undetected under current milk screening programs. A single test that combines mandatory b-lactam screening with voluntary flunixin screening is an economical approach for monitoring and controlling for potential flunixin or 5-hydroxyflunixin, the primary flunixin metabolite marker in milk. The objective of this study was to validate a b-lactam and flunixin rapid lateral flow test (LFT) and compare the results obtained with a liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS/ MS) method for the simultaneous determination of flunixin and 5-hydroxyflunixin in raw milk with a limit of detection of ,1 ppb, equivalent to 1 ng/ml. Using the LFT, three combined manufactured lots of test strips detected penicillin G at 2.0 ppb, ampicillin at 6.8 ppb, amoxicillin at 5.9 ppb, cephapirin at 13.4 ppb, ceftiofur (total metabolites) at 63 ppb, and 5-hydroxyflunixin at 1.9 ppb at least 90% of the time with 95% confidence. The LFT also detected incurred flunixin milk samples that were analyzed with the LC-MS/MS and diluted to tolerance in raw milk. The detection levels for the LFT are lower than the U.S. safe levels or tolerances and qualify the test to be used in compliance with U.S. milk screening programs. b-Lactam drugs are the most commonly used antibiot- used in combination with antibiotic treatment. Its presence ics in dairy cattle health management in the United States has been detected in the tissues of dairy cattle with a (8). Proper drug management includes disposal of milk for frequency comparable to that of penicillin G according to prescribed withhold times to prevent drug residues from the national tissue monitoring program conducted by the passing into the human food chain. It is a public health U.S. Department of Agriculture Food Safety Inspection requirement that all milk tankers prior to unloading into Service (16). Since the drug is detected in dairy cattle, there dairies be tested for b-lactam drugs using methods is concern that the milk from flunixin-treated cows may be recommended to the National Conference of Interstate Milk contaminated with the drug and that the drug would not be Shipments and evaluated by the U.S. Food and Drug detected under the current evaluated screening methods Administration (FDA) as dictated in the Pasteurized Milk recommended for milk tankers. Thus, there is a need for Ordinance Appendix N program (19). Tests are evaluated rapid, sensitive, and reliable procedures for the detection of for specificity, sensitivity, and ability to detect incurred drug residues in milk. levels at drug tolerances or safe levels expressed as parts per A method for determination of flunixin in milk by billion, which is equivalent to nanograms per milliliter. liquid chromatography (LC) and confirmation by gas Numerous b-lactam drug-screening tests and other FDA- chromatography–mass spectrometry (MS) has been reported evaluated and National Conference of Interstate Milk (11). Analysis of samples from cattle treated by intravenous Shipments–approved antibiotic screening tests for tetracy- administration of a normal dose of 2.2 mg/kg of body clines, sulfa drugs, and chloramphenicol are listed in FDA weight of [14C]flunixin for 3 consecutive days indicated that memorandum M-A-85 (20). 5-hydroxyflunixin was the major residue in bovine milk. Flunixin (2-{[2-methyl-3-(trifluoromethyl) phenyl]amino}- Therefore, 5-hydroxyflunixin has been established as the 3-pyridinecarboxylic acid), is a nonsteroidal anti-inflammatory marker residue to be used for monitoring of flunixin drug administered intravenously to cattle and is sometimes residues in bovine milk (5). More recently, an LC–tandem MS (LC-MS/MS) method was developed for the determi- * Author for correspondence: Tel: 978-687-9200; Fax: 978-687-9216; nation and confirmation of 5-hydroxyflunixin at 1- and 2- E-mail: [email protected]. ppb levels, respectively, in unpasteurized bovine milk (1). J. Food Prot., Vol. 75, No. 7 b-LACTAM AND FLUNIXIN TEST FOR RAW MILK 1271 Screening tests are substantially less time consuming volume 300-ml pipet with 200- to 1,000-ml disposable pipet tips and less expensive than chromatographic techniques, such were supplied by Charm Sciences. as LC-MS/MS. Recently, a new 8-min immunological and receptor-based lateral flow test (LFT) has been developed Specificity and selectivity study for b-lactam drugs and flunixin in raw milk. Fresh raw commingled cow’s milk from for the detection of flunixin and five of the b-lactam drugs local dairy producer tanks or silos tested to be b-lactam free using approved for dairy cattle in the United States. The new the SL b-lactam test (13) and Charm II b-lactam quantitative assay lateral flow assay is similar in principle to the SL6 b-lactam (18) was used for the spiking experiments. The age of the milk from test evaluated and approved in 2003, which used an antibody cow milking was 5 days or less, and the milk was maintained at 4uC. for cloxacillin to achieve detection of a sixth b-lactam drug b-Lactam drugs were dissolved in buffers as described in the that was not detected by the b-lactam receptor binder (3). In United States Pharmacopeia and prepared on the day of testing (15). the flunixin lateral flow method, a flunixin antibody replaces 5-Hydroxyflunixin was dissolved in 50% methanol-water to yield the cloxacillin antibody, so that while detection of cloxacillin 100-mg/liter stock solutions. Standard stock solutions were then at U.S. tolerance is lost, the detection of flunixin and flunixin diluted in milk for a final concentration of 1 mg/liter to be used for metabolites is gained. Another commercially available the spiking tests. These stock solutions were made fresh daily. enzyme-linked immunosorbent assay method claims to detect Each drug’s specificity and sensitivity was evaluated on a flunixin in tissue and milk (10). This method takes more than separate day. Thirty test samples were prepared at concentrations 30 min and is unable to measure both the b-lactam drugs and that define the screening test dose response as determined by an flunixin at the same time. FDA statistician based on the manufacturer’s submitted data, as follows: 3.0, 4.0, 5.0, 6.0, 8.0, and 10.0 ppb (ng/ml) for A third-party laboratory evaluation and validation has amoxicillin; 4.0, 5.0, 6.0, 7.0, 8.0, and 10.0 ppb for ampicillin; been designed by the FDA’s Center for Veterinary Medicine 8.0, 12.0, 14.0, 16.0, 18.0, and 20.0 ppb for cephapirin; 1.0, 2.0, (FDA-CVM) for evaluating screening methods for flunixin 2.5, 2.75, 3.0, and 5.0 ng/ml for penicillin G; and 0.2, 0.4, 0.8, 1.2, in bulk commingled milk for sensitivity and selectivity, and 1.6, and 2.0 ppb for 5-hydroxyflunixin. Sixty samples of the raw it is similar to the b-lactam screening test protocol used to milk were prepared for a negative control (0 ng/ml) on the day of validate bulk tanks and tanker trucks as specified in the test. Samples were scrambled and blind coded and tested Appendix N of the Pasteurized Milk Ordinance (20–22). according to the manufacturer’s instructions (Fig. 1), and tests The objective of this study was to validate the new b-lactam conducted consistent with good laboratory practices (17). For these and flunixin combination test following the FDA-CVM experiments, there was a team of sample preparers and coders in protocol developed in association with the Institute for Food the test laboratory that then passed the samples to testers without Safety and Health (IFSH). Separate method validations were their knowledge of the sample, preparation, or order sequence. done for flunixin and the b-lactam drugs so that the Incurred residue studies for b-lactam drugs and flunixin screening method could be used to comply with mandated in raw milk.

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