Verification of Bound Aminoguanidine as the Marker Residue for the Banned Antibiotic, Nitrovin, in Pig Tissues.

Nitrovin (NTV) belongs to a class of antibiotics called nitrofurans, which are classified as nonallowed pharmacologically active substances that do not have a maximum residue limit listed in EU legislation. The objectives of this study were to confirm aminoguanidine (AGN) as a suitable marker residue to monitor NTV abuse and to investigate its persistence in porcine tissues. In this work, pigs were fed with NTV-medicated feed (50 mg/kg), and tissues (kidney, muscle, and liver) and plasma were collected on different withdrawal days. All samples were analyzed for bound AGN, total AGN, and the parent drug NTV itself. The highest concentrations of AGN residues were found in the liver, while the lowest were in muscle. Parent NTV was only detected in the kidney at low levels on day 0 of withdrawal. The findings are in support of using AGN as the marker residue for monitoring the illegal use of NTV in animal-derived products.

Development of a multi-residue high-throughput UHPLC-MS/MS method for routine monitoring of SARM compounds in equine and bovine blood.

Selective androgen receptor modulators (SARMs) are a group of anabolic enhancer drugs posing threats to the integrity of animal sports and the safety of animal-derived foods. The current research describes for the first time the development of a semi-quantitative assay for the monitoring of SARM family compounds in blood and establishes the relative stability of these analytes under various storage conditions prior to analysis. The presented screening method validation was performed in line with current EU legislation for the inspection of livestock and produce of animal origin, with detection capability (CCß) values determined at 0.5 ng/mL (Ly2452473), 1 ng/mL (AC-262536 and PF-06260414), 2 ng/mL (bicalutamide, GLPG0492, LGD-2226, ostarine, S-1, S-6, and S-23), and 5 ng/mL (andarine, BMS-564929, LGD-4033, RAD140, and S-9), respectively. The applicability of the developed assay was demonstrated through the analysis of blood samples from racehorses and cattle. The developed method presents a high-throughput cost-effective tool for the routine screening for a range of SARM compounds in sport and livestock animals.

Enhanced UHPLC-MS/MS screening of selective androgen receptor modulators following urine hydrolysis.

Selective androgen receptor modulators (SARMs) represent non-steroidal agents commonly abused in human and animal (i.e. equine, canine) sports, with potential for further misuse as growth promoting agents in livestock-based farming. As a direct response to the real and possible implications of illicit application in both sport as well as food production systems, this study incorporated enzymatic hydrolysis (ß-glucuronidase/arylsulfatase) into a previously established protocol while maintaining the minimal volume (200 µL) of urine sample required to detect SARMs encompassing various pharmacophores in urine from a range of species (i.e. equine, bovine, human, canine and rodent). The newly presented semi-quantitative UHPLC-MS/MS-based assay is shown to be fit-for-purpose, being rapid and offering high-throughput, with validation findings fulfilling criteria stipulated within relevant doping and food control legislation.•CCß values determined at 1 ng mL-1 for majority of analytes.•Deconjugation step included in the method led to significantly increased relative abundance of ostarine in analysed incurred urine samples demonstrating the requirement for hydrolysis to detect a total form of emerging SARMs.•Assay amenable for use within routine testing to ensure fair play in animal and human sports and that animal-derived food is free from contamination with SARM residues.

Investigation of stability of selective androgen receptor modulators in urine.

Selective androgen receptor modulators (SARMs) are a class of new emerging "designer" steroid compounds gaining popularity over more well established anabolic-androgenic steroids (AAS) amongst both non-professional and elite athletes. Moreover, due to their anabolic activity, SARM compounds may also potentially be abused in livestock animals to increase meat production. Consequently, SARM residues should be monitored as a part of routine testing employed within both anti-doping and drug residue laboratories. Since only a limited amount of information on SARM compound stability is currently available within the peer-reviewed literature, this study reports a practical approach to assess optimal storage conditions for 15 SARM compounds in solvent solutions (standard stock and intermediate mixed standard solutions) stored at -20°C for up to 1 year, as well as in a range of urine test matrices (bovine, equine, canine and human) under frozen (-20°C, -80°C) storage for up to 20 weeks and post freeze-thaw cycles. Moreover, SARM storage stability within solvent extracts was assessed at -20°C (0-4 weeks) and 4°C (0-2 weeks). Findings demonstrate that SARM analytes are stable in reference solutions when stored at -20°C, apart from PF-06260414 (stock solution) which should be stored at lower temperatures (e.g. -80°C). A limited degree of compound instability was observed for a number of SARM analytes in urine both when stored at -20°C, and after repeated freeze-thaw cycles. Moreover, SARM compounds within reconstituted urine solvent extracts were found to be effectively stable when stored for up to 4 weeks at -20°C and for 2 weeks at 4°C. The long-term stability testing data reported here will inform the more timely and effective development and validation of analytical methods for SARM residue detection and analysis, ensuring confidence in findings from monitoring of livestock animals and anti-doping processes.

Food safety evaluation for the use of albendazole in fish: residual depletion profile and withdrawal period estimation.

Due to the lack of drugs regulated for aquaculture, we have evaluated the use of albendazole (ABZ) - a potential drug to be regulated for fish - under food safety perspectives assessing the depletion profile of ABZ and its main metabolites (albendazole sulphoxide - ABZSO, albendazole sulphone - ABZSO2 and albendazole amino sulphone - ABZ-2-NH2SO2) in fish fillets (muscle and skin) after single dose oral administration of 10 mg ABZ kg-1 body weight. For the drug administration, a suitable procedure for ABZ incorporation into fish feed was employed, obtaining good homogeneity of ABZ concentration among feed pellets (CV<4.1%) and low drug leaching when medicated feed remained in the water for up to 60 min (<2.7%). After medication, fish were euthanised at 8, 12, 24, 48, 72, 96 and 120 h and fillets collected. Depletion studies in various fish species (patinga and tilapia) were conducted simultaneously, under water temperature at 30.4 ± 0.3 °C and pH 6.8 ± 0.1. The highest concentrations for the sum of residues (ABZ, ABZSO, ABZSO2 and ABZ-2-NH2SO2) in fish fillet were 1210 ng g-1 in patinga and 637 ng g-1 in tilapia. Under the employed rearing conditions, the obtained results did not indicate a requirement for a minimum withdrawal period to be proposed for tilapia considering the maximum residue limit of 100 µg g-1, since the determined residual concentration was

Monitoring of selective androgen receptor modulators in bovine muscle tissue by ultra-high performance liquid chromatography-tandem mass spectrometry.

Selective androgen receptor modulators (SARMs) are non-steroidal compounds widely reported as drugs of abuse in human and animal sports, with potential for misuse as growth promoters in animal-based food production. In this study, a first analytical methodology to simultaneous screen for a panel of emerging SARMs in bovine muscle was developed, validated (CCß values from 0.5-5 ng g-1), and applied to detect 15 structurally diverse compounds from nine SARM families. Muscle samples (200 mg) were homogenised in extraction solvent (MeCN:H2O, 4:1, v/v) before clean-up (end-capped C18 dSPE), defatting (n-hexane pre-saturated with MeCN partitioning) and concentration prior to UHPLC-MS/MS analysis. In the absence of incurred bovine muscle, method applicability was demonstrated by the analysis of rodent muscle tissue. The developed screening assay serves as a rapid, simple and cost-effective tool for surveillance monitoring of SARM abuse in livestock production systems as a pre-emptive measure ensuring food safety.

Development and validation of a semi-quantitative ultra-high performance liquid chromatography-tandem mass spectrometry method for screening of selective androgen receptor modulators in urine.

A semi-quantitative method was developed to monitor the misuse of 15 SARM compounds belonging to nine different families, in urine matrices from a range of species (equine, canine, human, bovine and murine). SARM residues were extracted from urine (200 µL) with tert-butyl methyl ether (TBME) without further clean-up and analysed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). A 12 min gradient separation was carried out on a Luna Omega Polar C18 column, employing water and methanol, both containing 0.1% acetic acid (v/v), as mobile phases. The mass spectrometer was operated both in positive and negative electrospray ionisation modes (ESI±), with acquisition in selected reaction monitoring (SRM) mode. Validation was performed according to the EU Commission Decision 2002/657/EC criteria and European Union Reference Laboratories for Residues (EU-RLs) guidelines with CCß values determined at 1 ng mL-1, excluding andarine (2 ng mL-1) and BMS-564929 (5 ng mL-1), in all species. This rapid, simple and cost effective assay was employed for screening of bovine, equine, canine and human urine to determine the potential level of SARMs abuse in stock farming, competition animals as well as amateur and elite athletes, ensuring consumer safety and fair play in animal and human performance sports.

Determination of the persistence of dimetridazole, metronidazole and ronidazole residues in black tiger shrimp (Penaeus monodon) tissue and their stability during cooking.

The depletion of three banned nitroimidazole drugs - dimetridazole (DMZ), metronidazole (MNZ) and ronidazole (RNZ) - was investigated in black tiger shrimp (Penaeus monodon) following in-water medication. The highest concentrations of residues were measured immediately after the 24-h immersion (d0). At this time, MNZ and MNZ-OH residues were measured in shrimp tissue samples at concentrations ranging from 361 to 4189 and from 0.28 to 6.6 µg kg(-1), respectively. DMZ and its metabolites HMMNI ranged in concentration between 31,509 and 37,780 and between 15.0 and 31.9 µg kg(-1), respectively. RNZ and HMMNI concentrations ranged from 14,530 to 24,206 and from 25.0 to 55 µg kg(-1), respectively. MNZ, DMZ and RNZ were the more persistent marker residues and can be detected for at least 8 days post-treatment. MNZ-OH was only detectable on d0 following treatment with MNZ. HMMNI residues were only detectable up to d1 (0.97-3.2 µg kg(-1)) or d2 (1.2-4.5 µg kg(-1)) following DMZ and RNZ treatment, respectively. The parent drugs MNZ, DMZ and RNZ were still measureable on d8 at 0.12-1.0, 40.5-55 and 8.8-18.7 µg kg(-1), respectively. The study also investigated the stability of nitroimidazole residues under various cooking procedures (frying, grilling, boiling, and boiling followed by microwaving). The experiments were carried out in shrimp muscle tissue containing both high and low concentrations of these residues. Different cooking procedures showed the impact on nitroimidazole residue concentration in shrimp tissue. Residue concentration depleted significantly, but partially, by boiling and/or microwaving, but the compounds were largely resistant to conventional grilling or frying. Cooking cannot therefore be considered as a safeguard against harmful nitroimidazole residues in shrimp.

Determination of nitroimidazole residues in aquaculture tissue using ultra high performance liquid chromatography coupled to tandem mass spectrometry.

An UHPLC-MS/MS method was developed for the quantitative confirmatory analysis of residues of nitroimidazole drugs (dimetridazole, ipronidazole, metronidazole, ornidazole and ronidazole) and their corresponding hydroxy metabolites (HMMNI, ipronidazile-OH and metronidazole-OH) in aquaculture tissue. Samples were extracted by shaking in acetonitrile, water, MgSO4 and NaCl before being defatted with n-hexane pre-saturated with acetonitrile and concentrated under nitrogen. Nitroimidazole residues were determined by UHPLC-MS/MS operating in positive electrospray ionisation mode using a reversed phase BEH C18 column. The method was validated according to the EU Commission Decision 2002/657/EC guidelines. The following performance studies were carried out: specificity, selectivity, linearity, within laboratory repeatability (WLr)/reproducibility (WLR), accuracy, precision, decision limit (CCα), detection capability (CCß), absolute recovery and stability. The analytical range of the method is 0.1-20 µg kg(-1). Accuracy and precision of the method, under within-laboratory reproducibility conditions, ranged from 83 to 105% and 2.3 to 14.0%, respectively. CCα were 0.07-1.0 µg kg(-1) depending on analyte and matrix. A total of 50 samples can be analysed in a single day using the assay. The method has been extensively evaluated through application to real test samples.

Determination of 20 coccidiostats in egg and avian muscle tissue using ultra high performance liquid chromatography-tandem mass spectrometry.

A quantitative, comprehensive multiresidue method which includes 20 coccidiostat residues has been developed. The method described uses a simple one-step liquid extraction with acetonitrile to isolate analytes from both the polyether ionophore and chemical classes of coccidiostats. Subsequent to a further concentration step, samples were analysed via UHPLC-MS/MS. The method was validated according to the Commission Decision 2002/657/EEC in egg and avian muscle. The method permitted quantitative confirmation for 13 compounds below target concentrations, and screening for a further 7 compounds. Within-laboratory repeatability gave accuracy values in the range of 68-129%, while reproducibility ranged between 75 and 123%. Calibration ranges were typically 1-50 µg kg⁻¹, although higher ranges were used for dinitrocarbanilide, imidocarb and toltrazuril residues. A regression coefficient (R²) value of greater than 0.98 was obtained for all analytes. Precision results ranged from 2.3 to 19.7% CV for egg and from 2.6 to 23.6% CV in muscle. CCα was in the range from 1.13 µg kg⁻¹ (clopidol) to 179 µg kg⁻¹ (lasalocid) in egg. In muscle, CCα ranged from 2.25 µg kg⁻¹ (aprinocid) to 4579 µg kg⁻¹ (dinitrocarbanilide). CCß was from 1.29 µg kg⁻¹ (clopidol) to 209 µg kg⁻¹ (lasalocid) in egg, and 2.58 µg kg⁻¹ (arprinocid) to 6060 µg kg⁻¹ (dinitrocarbanilide) in muscle. Limits of quantification were 1 µg kg⁻¹ for all compounds, except imidocarb and dinitrocarbanilide (10 µg kg⁻¹), and toltrazuril and metabolites (50 µg kg⁻¹).