Production and certification of BOTS-1: bovine muscle-certified reference material for incurred veterinary drug residues.

A freeze-dried bovine muscle-certified reference material (CRM), known as BOTS-1 (DOI: https://doi.org/10.4224/crm.2018.bots-1 ), containing incurred residues of commonly used veterinary drugs was produced and certified for the mass fraction of eight veterinary drug residues. Value assignment was carried out using liquid chromatography tandem mass spectrometry (LC-MS/MS) methods in conjunction with isotope dilution and standard addition approaches involving stable isotope internal standards. Data from the National Research Council of Canada (NRC), Canadian Food Inspection Agency (CFIA), United States Department of Agriculture (USDA), and the Federal Office of Consumer Protection and Food Safety in Germany (BVL) were used for value assignment. Results for two drug residues were also obtained through an international inter-laboratory comparison CCQM-K141/P178 organized under the auspices of the International Bureau of Weights and Measures (BIPM). Quantitative NMR (1H-qNMR) was used to characterize primary standards of all veterinary drugs certified. The certified mass fractions of the veterinary drug residues were 490 ± 100 µg/kg for chlorpromazine, 44 ± 4.4 µg/kg for ciprofloxacin, 3.3 ± 1.4 µg/kg for clenbuterol, 9.5 ± 0.8 µg/kg for dexamethasone, 57 ± 4.8 µg/kg for enrofloxacin, 3.0 ± 0.4 µg/kg for meloxicam, 12.4 ± 1.2 µg/kg for ractopamine, and 2290 ± 120 µg/kg for sulfadiazine with expanded uncertainties quoted (95% confidence) which include the effects due to between-bottle inhomogeneity, instability during long-term storage and transportation, and characterization.

Simplified Liquid Chromatography-Mass Spectrometry Methods for Gestagen Analysis in Animal Fat and Liver.

Gestagens, a class of veterinary drugs also called progestogens, are synthetic hormones used to increase feed efficiency and rate of gain in heifers. The Canadian Food Inspection Agency analyzes progestogens melengestrol acetate (MGA), megestrol acetate, and chlormadinone acetate using liquid chromatography-mass spectrometry (LC-MS). Our conventional gestagen method for kidney fat has many time-consuming steps, including solid-phase extraction. A sample preparation procedure having fewer clean-up steps was developed for routine diagnostic analysis of kidney fat and provided similar results faster, and at lower cost. A confirmatory liver method for gestagens, developed using salt-assisted extraction, employed minimal clean-up steps that resulted in high chemical background at the desired lower limit of quantification (LLOQ). Differential ion mobility spectrometry, specifically high-field asymmetric waveform ion mobility spectrometry (FAIMS), was used to filter chemical background in the gas phase. The effect of the ionization probe position on FAIMS parameters, including sensitivity, is described. With LC-FAIMS-MS, chemical background for each gestagen was virtually eliminated, resulting in a quantitative liver method having the desired 0.6 ng/g LLOQ and estimated limits of detection (LODs) up to 140 times lower than LC-MS. Incurred MGA samples, analyzed using kidney fat and liver methods from the same animal, show levels within the quantitative ranges of both methods.

Improved Thyreostatic Drug Detection in Animal Tissues Using Liquid Chromatography-High-Field Asymmetric Waveform Ion Mobility Spectrometry-Mass Spectrometry.

Thyreostatic drugs (thyreostats) interfere with thyroid function and have been used illegally in animals slaughtered for food. Thyreostat use leads to poorer quality meat, and the drug residues can cause adverse effects in humans. These drugs, with the exception of thiouracil, do not occur naturally and require sensitive methodologies for their detection in animal tissues. Because thyreostats are low-molecular-weight polar analytes, liquid chromatography-mass spectrometry (LC-MS) is typically used for detection and, in particular, triple quadrupole mass spectrometry with selective reaction monitoring (i.e., LC-SRM). However, LC-SRM thyreostat methods suffer from chemical background noise and endogenous interferences arising from the complex tissue matrix. An improved high-field asymmetric waveform ion mobility spectrometry interface (FAIMS Pro), which separates ions based on differential ion mobility, was combined with LC-SRM to minimize these interferences. Using the same samples and conditions, LC-FAIMS-SRM showed improvements in the signal-to-noise ratio (S/N) of up to 50 times compared with our validated LC-SRM method. In addition, wider linear ranges, including substantial improvements in the lower limit of quantification (approximately an order of magnitude for tapazole and methylthiouracil), were observed with LC-FAIMS-SRM.

The Establishment of Tandem Mass Spectrometric Fingerprints of Phytosterols and Tocopherols and the Development of Targeted Profiling Strategies in Vegetable Oils.

Phytosterols and tocopherols are essential for plant biochemistry, and they possess beneficial health effects for humans. Evaluating the tandem mass spectrometric (MS/MS) behavior of phytosterols and tocopherols is needed for the development of a qualitative and quantitative method for these biologically active plant metabolites. Herein, the MS/MS dissociation behavior of phytosterols and tocopherols is elucidated to establish generalized MS/MS fingerprints. MS/MS and multistage (MS3) analysis revealed common fragmentation behavior among the four tested phytosterols, namely ß-sitosterol, stigmasterol, campesterol, and brassicasterol. Similar analysis was conducted for the tocopherols (i.e., alpha (α), beta (ß), gamma (γ), and delta (δ)). As such, a universal MS/MS fragmentation pathway for each group was successfully established for the first time. Based on the generalized MS/MS fragmentation behavior of phytosterols, diagnostic product ions were chosen for the development of profiling methods for over 20 naturally occurring phytosterols. A precursor ion scan-triggered-enhanced product ion scan (PIS-EPI) method was established. Due to enhanced chromatographic peaks, multiple ion monitoring-triggered-enhanced product ion scan (MIM-EPI) was employed for confirmation. The screening approach was applied successfully to identify blinded samples obtained from standard mixtures as well as sesame and olive oils. The oil samples contain other phytosterols, and their successful identification indicates that, the generalized MS/MS fragmentation behavior is applicable to various structures of phytosterols. A similar approach was attempted for tocopherols and was only hindered by the low concentration of these bioactive metabolites present in the oil samples.

Development and validation of determinative and confirmatory LC-MS/MS methodologies for total florfenicol and tulathromycin residues in bovine, equine and porcine kidney, liver and muscle tissues.

Separate methods for the quantitation and confirmation of regulatory relevant residue concentrations of total florfenicol and tulathromycin residues in multiple tissue matrices were developed and validated. Total florfenicol residues, determined and expressed as florfenicol amine (FFA) equivalents, were quantified and confirmed over a concentration range of 100-4000ng/g, with an LOD of 33ng/g, while total tulathromyicn residues, determined as CP-60,300 and expressed as tulathromycin equivalents, were quantified and confirmed over a concentration range of 500-10,000ng/g, with an LOD of 300ng/g. A 2 or 1h acid digestion for the FFA and tulathromycin methods, respectively, followed by extraction, cleanup, and concentration using mixed-mode strong cation-exchange SPE cartridges was used. Quantitation and confirmation were accomplished using commercially available tri-deuterated FFA (FFA-D3) as internal standard and multiple reaction monitoring (MRM) of three transitions per target analyte. Mean recoveries and matrix effects were 60% and 25%; and 100% and 110%, respectively for the target analytes florfenicol amine and CP-60,300. Intra-lab method reproducibilities ranged from 7 to 11% RSD, which are within the AOAC recommended HORRATr guidelines for method reproducibilities estimated from single laboratory validation studies. Blind spikes showed that method bias was generally less than 15% for both methods within the calibration range. Both methods have been shown to meet requirements for use in national chemical residue monitoring programs.

Multiclass, multiresidue drug analysis, including aminoglycosides, in animal tissue using liquid chromatography coupled to tandem mass spectrometry.

A multiresidue, multiclass semiquantitative screening analysis of 39 drug residues covering 8 drug classes, including aminoglycosides in veal muscle, based on a single multiresidue extraction routine and using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), is presented. Sample preparation involves extraction of a 5 g diced tissue sample with 10 mL of acetonitrile/ water (86:14), incubated at 60 degrees C for 1 h, and then cooled for 10 min in ice. Formic acid is added to the suspension, then mixed, and centrifuged. The supernatant is retained, and the pellet is extracted with 10 mL of water for aminoglycosides and again centrifuged. Approximately 9.5 mL of each of the supernatants from both extracts is combined and diluted with water to 25 mL. The final solution is then defatted with 20 mL of hexane prior to analysis. Liquid chromatography for the aminoglycosides is carried out with ZIC-HILIC and for the remainder of the compounds with an Atlantis dC18 minicolumn. LC-ESI-MS/MS in positive and negative ionization modes (three injections total) is carried out, and two ion transitions per analyte are monitored. The method provides semiquantitative analysis to identify incurred positive drug classes in a rapid and cost-effective manner. Of particular interest is the detection of numerous compounds in the low nanograms per gram concentration range, which are not typically detected using receptor-based screening methods. All identified drugs were confirmed using internationally recognized regulatory methods, with no apparent false positives.

Ultratrace analysis of nine macrolides, including tulathromycin A (Draxxin), in edible animal tissues with minicolumn liquid chromatography tandem mass spectrometry.

The analysis of nine macrolides is presented, including tulathromycin A (Draxxin), in beef, poultry, and pork muscle with a simple multiresidue extraction and analysis method using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The sample preparation method involves extraction with acetonitrile and defatting with hexane followed by dilution of the extracts for analysis. Separation of the nine macrolides was performed using an Atlantis dC 18, 3 mum, 3.9 mm x 20 mm minicolumn (guard column). Detection was carried out with two multiple reaction monitoring experiments per macrolide. The method detection limits (MDLs) were based on three times standard deviation of eight repeat spikes at 3.0 ng/g of a mix of the nine macrolides in the various tissues. The MDLs and retention times for the macrolides were as follows: lincomycin, 0.19 ng/g (t R = 5.00 min); tulathromycin, 0.46 ng/g (t R = 5.63 min); spiramycin, 0.21 ng/g (t R = 6.06 min); pirlimycin, 0.10 ng/g (t R = 6.04 min); clindamycin, 0.16 ng/g (t R = 6.20 min); tilmicosin, 0.29 ng/g (t R = 6.38 min); erythromycin, 0.19 ng/g (t R = 6.62 min); tylosin, 0.10 ng/g (t R = 6.72 min); and josamycin, 0.09 ng/g (t R = 6.98 min). Precision at 25 ng/g (n = 4) ranged from 2.3 to 9.4% for the compounds from beef muscle. Of interest is the detection of incurred residues of tulathromycin A in edible calf tissue at 0.10-7 mug/g, which is presented here for the first time.