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.

Dietary supplementation of branched-chain amino acids increases muscle net amino acid fluxes through elevating their substrate availability and intramuscular catabolism in young pigs.

Branched-chain amino acids (BCAA) have been clearly demonstrated to have anabolic effects on muscle protein synthesis. However, little is known about their roles in the regulation of net AA fluxes across skeletal muscle in vivo. This study was aimed to investigate the effect and related mechanisms of dietary supplementation of BCAA on muscle net amino acid (AA) fluxes using the hindlimb flux model. In all fourteen 4-week-old barrows were fed reduced-protein diets with or without supplemental BCAA for 28 d. Pigs were implanted with carotid arterial, femoral arterial and venous catheters, and fed once hourly with intraarterial infusion of p-amino hippurate. Arterial and venous plasma and muscle samples were obtained for the measurement of AA, branched-chain α-keto acids (BCKA) and 3-methylhistidine (3-MH). Metabolomes of venous plasma were determined by HPLC-quadrupole time-of-flight-MS. BCAA-supplemented group showed elevated muscle net fluxes of total essential AA, non-essential AA and AA. As for individual AA, muscle net fluxes of each BCAA and their metabolites (alanine, glutamate and glutamine), along with those of histidine, methionine and several functional non-essential AA (glycine, proline and serine), were increased by BCAA supplementation. The elevated muscle net AA fluxes were associated with the increase in arterial and intramuscular concentrations of BCAA and venous metabolites including BCKA and free fatty acids, and were also related to the decrease in the intramuscular concentration of 3-MH. Correlation analysis indicated that muscle net AA fluxes are highly and positively correlated with arterial BCAA concentrations and muscle net BCKA production. In conclusion, supplementing BCAA to reduced-protein diet increases the arterial concentrations and intramuscular catabolism of BCAA, both of which would contribute to an increase of muscle net AA fluxes in young pigs.

Detection, quantification and confirmation of anabolic steroids in equine plasma by liquid chromatography and tandem mass spectrometry.

Anabolic androgenic steroids are related to the male sex hormones and are abused in equine sports. In an effort to deter the abuse of anabolic steroids, a sensitive LC-MS/MS method was developed for detection, quantification and confirmation of eight major anabolic steroids (testosterone, normethandrolone, nandrolone, boldenone, methandrostenolone, tetrahydrogestrinone (THG), trenbolone, and stanozolol) in equine plasma. Formation of solvent adduct ions of the analytes was observed under electrospray ionization (ESI) conditions, and desolvation of the solvent adduct ions by source collision-induced decomposition (CID) increased the abundance of the [M+H]+ ions as well as the multiple-reaction monitoring (MRM) signals. ESI (+) and APCI (+) were compared with respect to sensitivity for the analytes and the former provided better sensitivity. The matrix effect on ion suppression or enhancement was evaluated, and was negligible. Confirmation of the analytes was performed using criteria of three ion transitions and LC retention time of each analyte. The limit of detection (LOD) and quantification (LOQ) was 25 pg/mL. The limit of confirmation (LOC) was 25 pg/mL for boldenone; 50 pg/mL for normethandrolone, nandrolone, and methandrostenolone; and 100 pg/mL for testosterone, THG, trenbolone, and stanozolol. The analytes were evaluated for stability and found to be stable in plasma for 24h at room temperature, 13 days at 4 degrees C, and 34 days at -20 and -70 degrees C. The method was successfully applied to analyses of equine plasma samples for pharmacokinetics study. This method is sensitive and useful for detection, quantification and confirmation of these anabolic steroids in equine plasma.

Quantitation of anabolic hormones and their metabolites in bovine serum and urine by liquid chromatography-tandem mass spectrometry.

A specific and sensitive method based on tandem mass spectrometry with on-line high-performance liquid chromatography using atmospheric pressure chemical ionisation (LC-APCI-MS-MS) for the quantitation of anabolic hormone residues (17beta-19-nortestosterone, 17beta-testosterone and progesterone) and their major metabolites (17alpha-19-nortestosterone and 17alpha-testosterone) in bovine serum and urine is reported. [2H2]17Beta-testosterone was used as internal standard. The analytes were extracted from urine (following enzymatic hydrolysis) and serum samples by liquid-liquid extraction and purified by C18 solid-phase extraction. Ionisation was performed in a heated nebulizer interface operating in the positive ion mode, where only the protonated molecule, [M+H]+, was generated for each analyte. This served as precursor ion for collision-induced dissociation and two diagnostic product ions for each analyte were identified for the unambiguous hormone confirmation by selected reaction monitoring LC-MS-MS. The overall inter-day precision (relative standard deviation) ranged from 6.37 to 2.10% and from 6.25 to 2.01%, for the bovine serum and urine samples, respectively, while the inter-day accuracy (relative error) ranged from -5.90 to -3.18% and from -6.40 to -2.97%, for the bovine serum and urine samples, respectively. The limit of quantitation of the method was 0.1 ng/ml for all the hormones in bovine serum and urine. On account of its high sensitivity and specificity the method has been successfully used to confirm illegal hormone administration for regulatory purposes.