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Although filtration is one of the most common steps in sample preparation for chemical analysis, filter membrane materials can leach contaminants and/or retain some analytes in the filtered solutions. In multiclass, multiresidue analysis of veterinary drugs, it is challenging to find one type of filter membrane that does not retain at least some of the analytes before injection in ultrahigh‐performance liquid chromatography‐tandem mass spectrometry (UHPLC‐MS/MS). In this study, different filter membranes were tested for use in UHPLC‐MS/MS analysis of 183 diverse drugs in bovine muscle, kidney, and liver tissues. Membranes evaluated consisted of polytetrafluoroethylene (PTFE), polyvinylidene difluoride (PVDF), polyethersulfone, nylon, and regenerated cellulose. Drug classes represented among the analytes included β‐agonists, β‐lactams, anthelmintics, macrolides, tetracyclines, sulfonamides, tranquilizers, (fluoro)quinolones, anti‐inflammatories, nitroimidazoles, coccidiostats, phenicols, and others. Although the presence of a matrix helped reduce the binding of analytes on surface active sites, all of the filter types partially retained at least some of the drugs in the final extracts. In testing by flow‐injection analysis, all of the membrane filters were also observed to leach interfering components. Ultimately, filtration was avoided altogether in the final sample preparation approach known as the quick, easy, cheap, effective, rugged, safe, efficient, and robust (QuEChERSER) mega‐method, and ultracentrifugation was chosen as an alternative. [ABSTRACT FROM AUTHOR]

Analyte stability is more commonly a confounding factor in analytical chemistry than many analysts recognize. In this study, we assessed the stability of 31 common veterinary drugs in water and final extracts of bovine (milk and kidney/liver) and chicken (muscle and egg) matrices. Two different sample preparation methods were evaluated for one-month storage of the final extracts at typical room, refrigerator, and freezer temperatures. Liquid chromatography – mass spectrometry (LC-MS) by triple quadrupole and high-resolution techniques was used for analysis of the extracts spiked at different relevant concentrations for general regulatory purposes (10–1000 ng/g sample equivalent). Comparison of results between two labs demonstrated that stable drugs (≤20% loss) at all tested conditions consisted of danofloxacin, enrofloxacin, florfenicol, flubendazole, hydroxy-flubendazole, flumequine, flunixin, 5-hydroxy-flunixin, lincomycin, and meloxicam. The tested drugs found to be the most unstable (>20% loss at room temperature within a matter of days) consisted of the β-lactams (ampicillin, cefalexin, cloxacillin, and penicillin G). Curiously, the following antibiotics (mostly macrolides) were apparently more stable in sample extracts than water: emamectin, erythromycin, ivermectin, lasalocid, monensin, tilmicosin, tulathromycin, and tylosin. Those and the other drug analytes (ciprofloxacin, doxycycline, florfenicol amine, 2-amino-flubendazole, oxytetracycline, sulfadiazine, sulfadimethoxine, sulfamethazine, and trimethoprim) were mostly stable for a month in refrigerated extracts, especially at higher concentrations, but not in all cases. In practice, freezer storage of extract solutions was found to be acceptable for at least a month, with a few exceptions. [ABSTRACT FROM AUTHOR]

In 2018, AOAC International issued Standard Method Performance Requirements (SPMR) 2018.010 - Screening and Identification Method for Regulated Veterinary Drug Residues in Food. In response, we compared 4 different multiresidue methods of sample preparation using the same analytical method entailing ultrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). Tilapia was chosen for testing, and the analytes and monitoring levels were from SPMR 2018.010. The methods consist of efficient procedures with published validation results from the US Department of Agriculture (USDA), Food and Drug Administration (FDA), and Canadian Food Inspection Agency (CFIA), and an enhanced-matrix removal (EMR)-Lipid protocol from China. Each method was used to prepare 102 final extracts of tilapia spiked or not at different levels with the 78 targeted analytes plus metabolites. The same FDA/USDA rules of mass spectral identification were employed in all analyses to assess rates of false positives and negatives. Quantitative accuracy of the methods was also compared in terms of recoveries and reproducibility of spiked tilapia, incurred catfish, and spiked and certified reference material of bovine muscle. Each method yielded generally acceptable results for the targeted veterinary drugs, but the USDA "extract & inject" method was the fastest, simplest, and cheapest to achieve equally or more acceptable results for the widest scope of analytes for the tested food matrices. [ABSTRACT FROM AUTHOR]

The way to maximize scope of analysis, sample throughput, and laboratory efficiency in the monitoring of veterinary drug residues in food animals is to determine as many analytes as possible as fast as possible in as few methods as possible. Capital and overhead expenses are also reduced by using fewer instruments in the overall monitoring scheme. Traditionally, the highly polar aminoglycoside antibiotics require different chromatographic conditions from other classes of drugs, but in this work, we demonstrate that an ion-pairing reagent (sodium 1-heptanesulfonate) added to the combined final extracts from two sample preparation methods attains good separation of 174 targeted drugs, including 9 aminoglycosides, in the same 10.5-min ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The full method was validated in bovine kidney, liver, and muscle tissues according to US regulatory protocols, and 137-146 (79-84%) of the drugs gave between 70 and 120% average recoveries with ≤ 25% RSDs in the different types of tissues spiked at 0.5, 1, and 2 times the regulatory levels of interest (10-1000 ng/g depending on the drug). This method increases sample throughput and the possible number of drugs monitored in the US National Residue Program, and requires only one UHPLC-MS/MS method and instrument for analysis rather than two by the previous scheme. [ABSTRACT FROM AUTHOR]

RATIONALE Monitoring of veterinary drug residues in foods is often conducted using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Results have high economic stakes for producers, but the ions monitored are usually selected due to signal intensities without structural interpretation. In this study, the ion transitions were characterized by high-resolution mass spectrometry. METHODS The 62 veterinary drugs from the LC/MS/MS method consisted of sulfonamides, β-lactams, phenicols, macrolides, tetracyclines, fluoroquinolones, non-steroidal anti-inflammatory drugs (NSAIDs), and corticosteroids. They were individually infused into a quadrupole time-of-flight (Q-TOF) mass spectrometer using electrospray ionization (ESI) operated in positive mode. The MS and collision-induced dissociation (CID) MS/MS spectra for each analyte were obtained for structural elucidation. The Q-TOF instrument was calibrated to obtain a mass accuracy error <5 ppm for the MS and MS/MS spectra. RESULTS The use of high-resolution ESI-Q-TOF-MS for the generation of the MS/MS product ions allowed for the determination of chemical formulae for the analytes, some of which led to new findings. Assigned structures were based on rational interpretation of the most stable possible products with comparison with the scientific literature. In difficult cases, isotopically labeled drugs or hydrogen/deuterium (H/D) exchange experiments were used to help confirm the structures of the product ions. CONCLUSIONS The use of ESI-Q-TOF-MS in this study has allowed structure elucidation of 186 MS/MS product ions previously selected for the LC/MS/MS analysis of 62 veterinary drugs. This serves to reduce the chances of false positives and negatives in the monitoring program, and provides justification and defense in regulatory enforcement actions. Published in 2014. This article is a US Government work and is in the public domain in the USA. [ABSTRACT FROM AUTHOR]

Abstract: In this study, optimization, extension, and validation of a streamlined, qualitative and quantitative multiclass, multiresidue method was conducted to monitor >100 veterinary drug residues in meat using ultrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). Optimization centered on extensive ruggedness evaluation of the method. Various clean-up sorbents were tested and the amount of co-extractives were weighed, matrix effects were measured using post-column infusion of representative analytes, the effect of extract dilution before injecting was studied, and analyte recoveries and reproducibilities were determined. In order to extend our previous method, more drug analytes were added that possessed a wider range of chemical properties, and a re-appraisal of different types of C18 in dispersive solid-phase extraction clean-up and mobile phases in UHPLC–MS/MS was done. Ultimately, end-capped C18 and post-column infusion of ammonium formate as an ionization enhancer for the late-eluting anthelmintics were found to give improved qualitative results for greater analytical scope. A multi-day, multi-analyst validation demonstrated that the final method is suitable for screening of 113 analytes, identifying 98 and quantifying (recoveries between 70–120% and RSD<25%) 87 out of the 127 tested drugs at or below US regulatory tolerance levels in bovine muscle. [Copyright &y& Elsevier]

Abstract: Rapid screening tests can be used as part of an efficient program designed to monitor veterinary drug residues in cattle. In this work, three rapid tests designed to screen samples for the presence of antibiotic residues, the Fast Antimicrobial Screen Test (FAST), Premi® and Kidney Inhibition Swab (KIS™) tests, were compared using beef kidney juice and serum samples. In order to provide a realistic assessment, potentially incurred samples of beef kidney juice and serum were obtained from 235 carcasses which had been retained by inspectors in a processing plant for further testing. In addition, liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis was conducted on these samples to identify what antibiotics were present, if any, and their levels. The comparison of the three rapid screening test results with those from LC–MS/MS analysis allowed for a more complete comparison of the relative sensitivity of these analytical methods, as well as valuable information on false positive and negative response rates. [Copyright &y& Elsevier]

Abstract: A liquid chromatographic-tandem mass spectrometric (LC–MS/MS) multi-residue method for the simultaneous quantification and identification of 38 residues of the most widely used anthelmintic veterinary drugs (including benzimidazoles, macrocyclic lactones, and flukicides) in milk and liver has been developed and validated. For sample preparation, we used a simple modification of the QuEChERS method, which was initially developed for pesticide residue analysis. The method involved extracting sample (10g) with acetonitrile (10mL), followed by phase separation from water (salting out) with MgSO4:NaCl (4:1, w/w). After centrifugation, an aliquot of the extract (1mL) was purified by dispersive solid-phase extraction with MgSO4 (150mg) and C18 (50mg), prior to LC–MS/MS analysis. Two injections of the same extract were required with the LC–MS/MS instrument to cover the 30 electrospray positive and 8 electrospray negative analytes. The limit of quantitation of the method was 5μgkg−1 for 37 analytes (and 10μgkg−1 for dichlorvos). The method was successfully validated according to the 2002/657/EC guidelines. Recovery of analytes was typically in the 70–120% range, with repeatabilities and reproducibilities typically <15% in milk and <20% in liver. [Copyright &y& Elsevier]