The Validation of the Wheat Gluten ELISA Kit.

Background: It is important to analyze the presence of wheat/gluten in food to avoid wheat allergy or celiac disease. Objective: The Wheat/Gluten ELISA kit was developed to measure total wheat protein or gluten content in wheat, barley, and rye cereals as raw materials, and processed foods. Validation as to whether this kit is suitable for quantifying total wheat protein/gluten was carried out. Methods: The Wheat/Gluten ELISA kit was designed as a sandwich ELISA based on antigliadin polyclonal antibody. Selectivity, interference study, matrix study including incurred food, robustness, stability, and lot-to-lot consistency studies were conducted for the Wheat/Gluten ELISA kit. Incurred matrix studies were also conducted in an independent laboratory. Results: The analysis of 38 different substances revealed no cross-reactivity above the LOQ except for oats. Recoveries of the spiked samples were mostly in the range of 75-140%, including an independent laboratory result. The LOD of the ELISA was found to be 0.02-0.16 mg/kg. Robustness testing proved that extraction time and incubation time of first reaction and enzyme reaction had no significant influence on quantified value. The stability at 2-8°C was found to exceed 12 months. Good lot-to-lot consistency was observed. Conclusions: The Wheat/Gluten ELISA kit showed good analytical performance in the quantitative analysis of total wheat protein/gluten in the identified food products using the AOAC Performance Tested Method(s)SM program. Highlights: The Wheat/Gluten ELISA kit was validated and showed good analytical performance in the quantitative analysis of total wheat protein/gluten in food.

Validation of the BetaStar® Advanced for Beta-lactams Test Kit for the Screening of Bulk Tank and Tanker Truck Milks for the Presence of Beta-lactam Drug Residues.

A validation study was conducted for an immunochromatographic method (BetaStar® Advanced for Beta-lactams) for the detection of beta-lactam residues in raw, commingled bovine milk. The assay detected amoxicillin, ampicillin, cloxacillin, penicillin, cephapirin, and ceftiofur below the U.S. Food and Drug Administration tolerance levels but above the maximum sensitivity thresholds established by the National Conference on Interstate Milk Shipments. The results of internal and independent laboratory dose-response studies employing spiked samples were in agreement. The test detected all six drugs at the approximate 90/95% sensitivity levels in milk from cows treated with each drug. Selectivity of the assay was 100%, as no false-positive results were obtained in testing 1148 control milk samples. Testing the estimated 90/95% sensitivity level for amoxicillin (8.5 ppb), ampicillin (6.9 ppb), cloxacillin (8.9 ppb), penicillin (4.2 ppb), and cephapirin (17.6 ppb), and at 100 ppb for each antibiotic, resulted in 94-100% positive tests for each of the beta-lactam drugs. The results of ruggedness experiments established the operating parameter tolerances for the assay. Cross-reactivity testing established that the assay detects other certain beta-lactam drugs, but it does not cross-react with any of 30 drugs belonging to seven different drug classes. Abnormally high bacterial or somatic cell counts in raw milk produced no assay interference.

Validation of the BetaStar® Advanced for Tetracyclines Test Kit for the Screening of Bulk Tank and Tanker Truck Milks for the Presence of Tetracycline Drug Residues.

A validation study was conducted for an immunochromatographic method (BetaStar® Advanced for Tetracyclines) for detection of tetracycline antibiotic residues in raw, commingled bovine milk. The assay was demonstrated to detect tetracycline, chlortetracycline, and oxytetracycline at levels below the FDA tolerance levels but above the maximum sensitivity thresholds established by the National Conference on Interstate Milk Shipments. Results of internal and independent laboratory dose-response studies employing spiked samples were in agreement. All three drugs at the approximate 90/95% sensitivity levels were detected in milk collected from cows that had been treated with the specific drug. Selectivity of the assay was 100%, as no false-positive results were obtained in testing 881 control milk samples. Testing the estimated 90/95 sensitivity level for tetracycline (213 ppb), chlortetracycline (272 ppb), and oxytetracycline (180 ppb) and at 1000 ppb for each antibiotic resulted in 100% positive tests for each tetracycline. Results of ruggedness experiments established the operating parameter tolerances for the test. Results of cross-reactivity testing established that the assay detects certain other tetracycline drugs but does not cross-react with any of 32 drugs belonging to seven different drug classes. Abnormally high bacterial or somatic cell counts (SCC) in raw milk produced no assay interference.

The Analysis of Phenylbutazone and Its Active Metabolite, Oxyphenbutazone, in Equine Tissues (Muscle, Kidney, and Liver), Urine, and Serum by LC-MS/MS.

This study reports the use of two validated LC with tandem MS (MS/MS) methods to study the residue depletion profile of phenylbutazone (PBZ) and its metabolite oxyphenbutazone (OXPBZ) from equine serum, urine, and muscle, kidney, and liver tissues. One LC-MS/MS method, with an LOQ of 1.0 ng/mL for PBZ and 2.0 ng/mL for OXPBZ, was used for the analysis of the two drugs in the biological fluids (equine urine and serum); the other LC-MS/MS method, with an LOQ of 0.5 ng/g for PBZ and OXPBZ, was used for the analysis of the drugs in the equine tissue samples. PBZ was administered intravenously to two horses dosed with 8.8 mg/kg PBZ once daily for 4 days and sacrificed humanely at a slaughter plant 7 days after the last drug administration. Urine, serum, and kidney, liver, and muscle tissues were collected from the two horses and shipped on ice to the laboratory and stored at -20°C until analysis. The concentrations of PBZ and OXPBZ residues in the biological fluid and tissue samples collected at slaughter were measured with the two validated LC-MS/MS methods using deuterated internal standards. The results demonstrate that the validated methods are fit for studying the depletion kinetics of PBZ residues in equine tissues and biological fluids.

A multi-residue method for 17 anticoccidial drugs and ractopamine in animal tissues by liquid chromatography-tandem mass spectrometry and time-of-flight mass spectrometry.

A new and sensitive multi-residue liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QToF-MS) method was developed and validated for the determination and confirmation of residues of 17 anticoccidials, plus free ractopamine in poultry muscle and liver, and bovine muscle, liver, and kidney tissues. The 17 anticoccidials are lasalocid, halofuginone, narasin, monensin, semduramicin, ethopabate, robenidine, buquinolate, toltrazuril as its sulfone metabolite, maduramicin, salinomycin, diclazuril, amprolium, decoquinate, dinitolmide, clopidol, and the nicarbazin metabolite DNC (N,N1-bis(4-nitrophenyl)urea). The analytes were extracted and cleaned up within a 3-hour period by simply extracting the analytes into a solvent mixture with salts followed by centrifugation, dilution, and filtration. The validated method was used in a pilot study for the analysis of 173 samples that included quail liver, bovine kidney, liver, muscle, and horse muscle. The predominant residues found in this study were monensin, ractopamine, and lasalocid. The results of this pilot study showed that this new method is applicable to real samples, and is fit for use in a regulatory testing programme. © 2016 Her Majesty the Queen in Right of Canada. Drug Testing and Analysis. © 2016 John Wiley & Sons, Ltd.

Analysis of phenylbutazone residues in horse tissues with and without enzyme-hydrolysis by LC-MS/MS.

Phenylbutazone (PBZ) is permitted to be used for the treatment of musculoskeletal pain and inflammation in race horses but it is not approved for use in horses destined for human consumption. In a recent study initiated in our laboratory to study the disposition of PBZ and its oxyphenbutazone (OXPBZ) metabolite in equine tissues, we compared the effect of an additional enzymatic hydrolysis step with ß-glucuronidase on the results of the analysis for PBZ without enzymatic hydrolysis. Incurred tissue samples obtained from a female horse dosed with PBZ at 8.8 mg/kg for 3 days and sacrificed 6 days following the last administration were used for this study. Liver, kidney, and muscle tissues were collected, extracted, cleaned up on a silica-based solid-phase extraction (SPE) preceded by a weak-anion exchange SPE and analyzed with our in-house validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for PBZ and OXPBZ. Addition of the hydrolysis step resulted in a significant increase in recovery of both PBZ and OXPBZ residues. © 2016 Her Majesty the Queen in Right of Canada. Drug Testing and Analysis © 2016 John Wiley & Sons, Ltd.

The role of validated analytical methods in JECFA drug assessments and evaluation for recommending MRLs.

The Joint Food and Agriculture Organization and World Health Organization (FAO/WHO) Expert Committee on Food Additives (JECFA) is one of three Codex committees tasked with applying risk analysis and relying on independent scientific advice provided by expert bodies organized by FAO/WHO when developing standards. While not officially part of the Codex Alimentarius Commission structure, JECFA provides independent scientific advice to the Commission and its specialist committees such as the Codex Committee on Residues of Veterinary Drugs in Foods (CCRVDF) in setting maximum residue limits (MRLs) for veterinary drugs. Codex methods of analysis (Types I, II, III, and IV) are defined in the Codex Procedural Manual as are criteria to be used for selecting methods of analysis. However, if a method is to be used under a single laboratory condition to support regulatory work, it must be validated according to an internationally recognized protocol and the use of the method must be embedded in a quality assurance system in compliance with ISO/IEC 17025:2005. This paper examines the attributes of the methods used to generate residue depletion data for drug registration and/or licensing and for supporting regulatory enforcement initiatives that experts consider to be useful and appropriate in their assessment of methods of analysis. Copyright © 2016 Her Majesty the Queen in Right of Canada. Drug Testing and Analysis © 2016 John Wiley & Sons, Ltd.

A single laboratory-validated LC-MS method for the analysis of tulathromycin residues in bison and deer sera and selected tissues of white-tailed deer.

The performance characteristics of a newly developed liquid chromatography-mass spectrometry (LC-MS) method were validated and demonstrated to be fit for purpose in a pharmacokinetic and tissue depletion study of white-tailed deer and bison. Tulathromycin was extracted from bison and deer sera with acetonitrile or trifluoroacetic acid and K2 HPO4 (pH 6.8) buffer solution and cleaned up on a conditioned Bond-Elut cartridge. Tulathromycin, retained on the cartridge; it was eluted with methanol containing 2% formic acid, dried, re-constituted in methanol/1% formic acid, and analyzed by LC-MS. The limit of quantification (LOQ) of the method was 0.6 ng/mL in serum and 0.6 ng/g in tissue with RSDs ≤ 10% and accurate over the linear calibration range of 0.8-100 ng/mL for bison serum, 0.6-50 ng/mL for deer serum, 100-2500 ng/g for deer muscle tissue, and 500-5000 ng/g for deer lung tissue, all with coefficients of determination, r(2) ≥0.99. The validated method was used to quantify the concentration of tulathromycin residues in serum of bison and deer and selected tissue (lung and muscle tissue) samples obtained from 10 healthy, white-tailed deer that were administered the therapeutic dose approved for cattle (i.e., a single 2.5 mg/kg subcutaneous injection of tulathromycin in the neck). The deer were included in a tulathromycin drug depletion study. © 2016 Her Majesty the Queen in Right of Canada. Drug Testing and Analysis © 2016 John Wiley & Sons, Ltd.

Review: Application of High Resolution Mass Spectrometry to Monitor Veterinary Drug Residues in Aquacultured Products.

High resolution MS (HRMS) instruments provide accurate mass measurements. With HRMS, virtually an unlimited number of compounds can be analyzed simultaneously because full-scan data are collected, rather than preselected ion transitions corresponding to specific compounds. This enables the development of methods that can monitor for a wide scope of residues and contaminants in aquacultured fish and shellfish including antibiotics, metabolites, and emerging contaminants. Applications of HRMS to the analysis of veterinary drug residues in aquacultured products are summarized in this review including methods for screening, quantifying, and identifying drug residues in these matrixes. The use of targeted, semi-targeted, and nontargeted analysis of HRMS data and the implications to the global aquaculture industry are also reviewed.

A multi-residue method for the determination of seven polypeptide drug residues in chicken muscle tissues by LC-MS/MS.

A new multi-residue method for the determination of seven polypeptides, namely, polymixin B1, polymixin B2, polymixin E1 (colistin A), polymixin E2 (colistin B), enduracidin A (enramycin A), enduracidin B (enramycin B), and bacitracin A, in food of animal origin was developed and validated for chicken muscle tissue. Chicken muscle tissue was extracted with acidified methanol (1 % TFA). After homogenization, shaking, and centrifugation, the acidified methanol extract was decanted. A second extraction was performed with methanol (1 % TFA) and formic acid (1 %) 25:75, v/v. The pooled extract was cleaned up and concentrated on a solid-phase extraction cartridge. The retained analytes were eluted with methanol/acetonitrile. The extract was evaporated to dryness, reconstituted in mobile phase, filtered, and quantified by LC-MS/MS under ESI conditions. The method has a LOQ of 50.0 µg/kg for polymixin E2 (colistin B), 39.0 µg/kg for polymixin E1 (colistin A), 74.0 µg/kg for polymixin B1, 71.0 µg/kg for polymixin B2, 66.0 µg/kg for enduracidin A, 50.0 µg/kg for enduracidin B, and 30.0 µg/kg for bacitracin A in chicken muscle tissues. This is the first sensitive, suitable, multi-residue method reported for the seven polypeptide drug residues in chicken muscle tissue.

Analysis of antithyroid drug residues in food animals.

In the late 1970s, concerns were raised regarding why antithyroid drugs were being administered to food animals to promote growth despite the fact that they had been implicated as being carcinogenic and teratogenic; the growth promotion process produced an inferior quality meat with increased water retention in the animal's gastrointestinal tract. An increased incidence of aplasia cutis (a characteristic scalp defect) in consumers in Spain was linked to an increased consumption of antithyroid-contaminated meat. Therefore, to protect human health, the EU banned the use of antithyroid drugs in food animal production in 1981. This article reviews the impact of this regulatory decision on the regulatory analysis of these compounds in foods of animal origin. It discusses the physiology of the thyroid gland, the chemistry of antithyroid drugs and critically evaluates the suitability of the analytical methods that have been developed and validated to support enforcement of the regulation.

Validation of a new screening, determinative, and confirmatory multi-residue method for nitroimidazoles and their hydroxy metabolites in turkey muscle tissue by liquid chromatography-tandem mass spectrometry.

A new and sensitive multi-residue method (MRM) with detection by LC-MS/MS was developed and validated for the screening, determination, and confirmation of residues of 7 nitroimidazoles and 3 of their metabolites in turkey muscle tissues at concentrations ≥ 0.05 ng/g. The compounds were extracted into a solvent with an alkali salt. Sample clean-up and concentration was then done by solid-phase extraction (SPE) and the compounds were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The characteristic parameters including repeatability, selectivity, ruggedness, stability, level of quantification, and level of confirmation for the new method were determined. Method validation was achieved by independent verification of the parameters measured during method characterization. The seven nitroimidazoles included are metronidazole (MTZ), ronidazole (RNZ), dimetridazole (DMZ), tinidazole (TNZ), ornidazole (ONZ), ipronidazole (IPR), and carnidazole (CNZ). It was discovered during the single laboratory validation of the method that five of the seven nitroimidazoles (i.e. metronidazole, dimetridazole, tinidazole, ornidazole and ipronidazole) and the 3 metabolites (1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole (MTZ-OH), 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI, the common metabolite of ronidazole and dimetridazole), and 1-methyl-2-(2'-hydroxyisopropyl)-5-nitroimidazole (IPR-OH) included in this study could be detected, confirmed, and quantified accurately whereas RNZ and CNZ could only be detected and confirmed but not accurately quantified.

Analytical determination of virginiamycin drug residues in edible porcine tissues by LC-MS with confirmation by LC-MS/MS.

A liquid chromatographic-mass spectrometric (LC-MS) method was developed and validated for the determination and confirmation of virginiamycin (VMY) M1 residues in porcine liver, kidney, and muscle tissues at concentrations > or =2 ng/g. Porcine liver, kidney, or muscle tissue is homogenized with methanol-acetonitrile. After centrifugation, the supernatant is diluted with phosphate buffer and cleaned up on a C18 solid-phase extraction cartridge. VMY in the eluate is partitioned into chloroform and the aqueous upper layer is removed by aspiration. After evaporating the chloroform in the residual mixture to dryness, the dried extract is reconstituted in mobile phase and VMY is quantified by LC-MS. Any samples eliciting quantifiable levels of VMY M1 (i.e., at concentrations > or =2 ng/g) are subjected to confirmatory analysis by LC-MSIMS. VMY S1, a minor component of the VMY complex, is monitored but not quantified or confirmed.

A determinative and confirmatory method for residues of the metabolites of carbadox and olaquindox in porcine tissues.

Carbadox (CBX) and olaquindox (OLQ) are used in swine feed for growth promotion, to improve feed efficiency, increase the rate of weight gain, control swine dysentery and bacterial enteritis in young swine. In 1991, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) recommended maximum residue limits (MRLs) of 30 and 5mugkg(-1) in liver and muscle tissues of pigs, respectively, based on the concentration of, and expressed as, quinoxaline-2-carboxylic acid (QCA) as marker residue. In 1998, the European Commission (EC) banned the use of CBX and OLQ in food animal production together with four other feed additives, following reports that CBX and desoxycarbadox (DCBX) are suspect carcinogens and mutagens. In 2001, the sale of CBX was halted in Canada. In 2003, JECFA recommended the withdrawal of the previously recommended acceptable daily intake (ADI) and MRLs and concluded that QCA was not a suitable marker residue for CBX, based on new sponsor studies reporting that DCBX, the suspect carcinogen, persisted in animal tissues much longer than had previously been thought. This paper presents a very sensitive LC-MS/MS method that was developed by CFIA scientists for the simultaneous determination and confirmation of DCBX residues at concentrations >/=0.050 ngkg(-1) and QCA and mQCA residues at concentrations >/=0.50 ngkg(-1)in bovine muscle, pork liver and muscle tissues.

Evaluation of high-field asymmetric waveform ion mobility spectrometry mass spectrometry for the analysis of the mycotoxin zearalenone.

A high-field asymmetric waveform ion mobility spectrometry (FAIMS)-based method for the determination of the mycotoxin zearalenone (ZON) and its metabolites alpha-zearalenol (alpha-ZOL), beta-zearalenol (beta-ZOL), and beta-zearalanol (beta-ZAL), in a cornmeal (maize) matrix is described. Detection limits achieved using the FAIMS device coupled with electrospray ionization (ESI) and mass spectrometric (MS) detection are 0.4 ng mL(-1) for ZON and 3 ng mL(-1) for alpha-ZOL+beta-ZOL, and beta-ZAL. This represents a significant improvement when compared to detection limits determined using ESI-MS or ESI-tandem mass spectrometry (MSMS) analytical methods. The developed flow-injection (FIA)-ESI-FAIMS-MS method was applied to reference materials ERM-BC-716 and ERM-BC-717 certified for ZON and excellent agreement with the certified values was observed.

Bacterial isolates from equine infections in western Canada (1998-2003).

All bacterial samples of equine origin submitted to the diagnostic laboratory at the Western College of Veterinary Medicine from January 1998 to December 2003 from either "in-clinic" or Field Service cases were accessed (1323 submissions). The most common bacterial isolates from specific presenting signs were identified, along with their in vitro antimicrobial susceptibility patterns. The most common site from which significant bacterial isolates were recovered was the respiratory tract, followed by wounds. Streptococcus zooepidemicus was the most common isolate from most infections, followed by Escherichia coli. Antimicrobial resistance was not common in the isolates and acquired antimicrobial resistance to multiple drugs was rare. The results are compared with previous published studies from other institutions and used to suggest appropriate antimicrobial treatments for equine infections in western Canada.