Investigation of in vitro generated metabolites of LGD-4033, a selective androgen receptor modulator, in homogenized camel liver for anti-doping applications.

RATIONALE: The use of selective androgen receptor modulators (SARM) in sports is prohibited by the World Anti-Doping Agency (WADA) due to their potential as performance-enhancing drugs, offering an unfair advantage. LGD-4033 is a SARM known for its similarities to anabolic steroids and can be easily purchased online, leading to increased availability and misuse. Adverse analytical findings have revealed the presence of SARMs in dietary supplements. Although LGD-4033 misuse has been reported in human sports over the years, concerns also arise regarding its illicit use in animal sports, including camel racing. Although various studies have investigated the metabolism of LGD-4033 in humans, horse, and other species, there is limited research specifically dedicated to racing camels. METHODS: This study focuses on the in vitro metabolism of LGD-4033 in homogenized camel liver using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) to identify and characterize the metabolites. RESULTS: The findings indicated the presence of 12 phase I metabolites and 1 phase II metabolite. Hydroxylation was responsible for the formation of the main phase I metabolites that were identified. A glucuronic acid conjugate of the parent drug was observed in this study, but no sulfonic acid conjugate was found. The possible chemical structures of these metabolites, along with their fragmentation patterns, were identified using MS. CONCLUSIONS: These findings provide valuable insights into the metabolism of LGD-4033 in camels and aid in the development of effective doping control methods for the detection of SARMs in camel racing.

An extensive screening method for the identification and quantitation of ecdysteroids in equine urine and plasma using liquid chromatography coupled with mass spectrometry.

RATIONALE: Recently, there has been a report suggesting that ecdysteroids can enhance sports performance, making them relevant substances in doping control. Hence, it is imperative to examine the analytical characteristics of ecdysteroids in biological samples to identify their misuse in competitive sports. METHODS: To assess the doping of ecdysteroids such as ecdysone, ecdysterone, ponasterone A, turkesterone, and ajugasterone C, a fast and sensitive extraction and detection method was developed, optimized, and validated using equine urine and plasma. Different extraction techniques, namely, solid-phase extraction, liquid-liquid extraction, and dilute-and-inject, were explored to detect ecdysteroids from equine urine and plasma. RESULTS: The most suitable method of detection was solid-phase extraction using ABS Elut-NEXUS, while liquid-liquid extraction and dilute-and-inject methods encountered difficulties due to the high polarity of ecdysteroids and the presence of significant matrix interferences. Mass spectrometric parameters are optimized on both the Q Exactive high-resolution mass spectrometer and the TSQ Altis triple quadrupole mass spectrometer. However, the study indicated that the triple quadrupole mass spectrometer exhibited improved limit of detection when analyzing samples. To achieve optimal separation of the analytes under investigation from the matrix interferences, various liquid chromatography columns were compared. The Selectra PFPP LC column with a mobile phase consisting of 0.2% formic acid in water (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.5 mL/min demonstrated superior performance. CONCLUSIONS: The findings of this study will significantly contribute to the accurate identification of ecdysteroids, facilitating the investigation of their illicit use in horse racing.

Electrospray ionization mass spectrometry adduct formation by mobile phase additives: A case study using nitrile functional groups containing selective androgen receptor modulators.

RATIONALE: The formation of mass adducts is common during electrospray ionization mass spectrometry (ESI-MS). However, the mechanism that leads to adduct formation is poorly understood and difficult to control. Multiplication of mass adducts at once will adversely impact the sensitivity of mass analysis and cause misinterpretation of the level of detection. Prior studies on selective androgen receptor modulators (SARMs) revealed an immense mass adduct formation in both positive and negative ESI modes. METHODS: In this study, additives in the mobile phases are investigated as a potential means of controlling mass adduct formation in various SARMs. RESULTS: The first evidence of chloride adduct formation when SARMs are detected via ESI-MS has been reported in this research. A series of mobile phase combinations were tested to achieve the optimal condition for HPLC-MS. A comparison was also made between adduct formation on various grades of water used for preparing the mobile phase. A validation study using equine urine and plasma was also conducted to assess the suitability of the developed method. CONCLUSION: The results of this study will allow for a more accurate identification of SARMs, which will make it easier to investigate their illicit use in horse racing.

Metabolic study of selective androgen receptor modulator LY2452473 in thoroughbred horses for doping control.

RATIONALE: Since 2010, there has been an increasing number of adverse analytical findings related to selective androgen receptor modulators (SARMs) in competitive sports. It emphasizes the importance of comprehensive doping control analytical procedures that are capable of detecting SARM misuse. METHODS: In this study, it is described how LY2452473, a SARM, was metabolized in thoroughbred horses after a single-dose oral administration and in vitro with equine liver microsome preparations. An investigation of the metabolism of LY2452473 in horses' urine, plasma, and hair matrices was carried out during the study. The plausible structures of the detected metabolites were postulated using high-performance liquid chromatography-high resolution mass spectrometry. RESULTS: Under the experimental conditions 15 metabolites (12 phase I and three conjugates of phase I) were detected (M1-M15). The major phase I metabolites identified were formed by hydroxylation. Side-chain dissociated and methylated metabolites were also detected. In phase II, the glucuronic acid and sulfonic acid conjugates of hydroxy LY2452473 were detected as the major metabolites. In vitro analysis has confirmed the presence of all metabolites found in vivo except for the methylated analogs M11 and M12. A peak concentration of LY2452473 (0.5 pg/mg) in proximal hair segments was achieved 4 weeks after administration, according to hair analysis. CONCLUSIONS: Data obtained will aid in identifying LY2452473 and related substances faster. Furthermore, the results will assist in checking for the illegal use of these substances in competitive sports.

Investigation of in vitro generated metabolites of GLPG0492 using equine liver microsomes for doping control.

An effective alternative to testosterone therapy is selective androgen receptor modulators, a class of compounds that has a tissue-specific effect on muscle and bone. These drugs, which enhance performance, pose a severe abuse risk in competitive sports. GLPG0492 is one of the selective androgen receptor modulators discovered in recent decades. This compound has a unique tissue-specific action for muscle and bone against steroid receptors and acts as a partial agonist for androgen receptors. This study examined GLPG0492 and its metabolites in vitro using equine liver microsomes. Liquid chromatography-high-resolution mass spectrometry was utilized to determine the probable structures of detected metabolites. This study identified 39 metabolites of GLPG0492 (21 phase I and 18 phase II). The hydroxylation of GLPG0492 results in monohydroxylated and dihydroxylated metabolites. Additionally, the study detected dissociated side chains (3-methyl and 4-(hydroxymethyl)) and corresponding hydroxylated metabolites. A series of glucuronic acid- and sulfonic acid-conjugated analogs of GLPG0492 were detected during phase II of the study. The findings might help in the detection of GLPG0492 and the elucidation of its illegal use in equestrian sports.

In-depth metabolic study of nonsteroidal selective androgen receptor modulator GSK2881078 in thoroughbred horses and horse liver microsomes for doping control.

Nonsteroidal selective androgen receptor modulators (SARMs) are a novel class of compounds that have not yet been clinically approved; however, they appear to have a better anabolic/androgenic ratio than steroids and cause slighter side effects. Sports drug testing laboratories are required to maintain continuously updated doping control analytical methods in light of the widespread misuse of SARMs in elite and amateur sports. This paper describes the metabolic conversion of SARM GSK2881078 in thoroughbred horses following oral administration and in vitro with equine liver microsomes. A liquid chromatography-high-resolution mass spectrometry method was used to postulate the plausible structures of the detected metabolites. A total of five (M1-M5) in vivo metabolites and six (M1-M6) in vitro metabolites were detected under experimental conditions. Phase I metabolites mainly result from hydroxylation. Methoxylated and side-chain dissociated metabolites were also detected. Neither sulfonic acid nor glucuronic acid conjugated metabolites were observed in this study. Data reported here could aid in the detection of nonsteroidal SARM GSK2881078 and reveal its illicit use in competitive sports.

Is 9ß-dehydrohalogenation of betamethasone and dexamethasone hindering the detection of banned co-eluting meprednisone? A reverse-phase chiral liquid chromatography high-resolution mass spectrometry approach.

Mass spectral analysis of dexamethasone and betamethasone reveal intense signals at m/z 373.19994 (using a Thermo Q Exactive high-resolution mass spectrometer coupled with Dionex UltiMate 3000 UHPLC + operated in the positive ion mode), matching the signal of meprednisone, the 11-oxo version of methylprednisolone, along with its parent signal; possibly due to dehydrohalogenation of these drugs at MS. The parent mass of meprednisone is exactly same as that of dehydrohalogenated mass of dexamethasone and betamethasone; and are co-eluting, displaying same mass spectra. Specifically when they are administered together, identifying meprednisone (a drug for which there is zero tolerance in some regions of the world), is a great challenge with currently available techniques because it could be easily mistaken for dexamethasone or betamethasone, drugs allowed at certain threshold limits for therapeutic considerations. False negative results could be obtained in conventional reverse-phase chromatography and are liable to be abused; hence, establishing "zero tolerance" limits for these compounds often proves ineffective. In this paper, present an effective and reliable analytical method for simultaneously separating and identifying dexamethasone, betamethasone and meprednisone in equine urine and plasma using chiral liquid chromatography-electrospray ionization-mass spectrometry. From the various columns screened, the Lux i-Cellulose-5 chiral column produced high-quality results with extremely good separation. During this study, it is quite evident that dehydrohalogenation occurs only in the mass ionization source; the compounds are very stable in-vivo/in-vitro and do not break down either on-column or during sample preparation.

Mass spectrometric method for distinguishing isomers of dexamethasone via fragment mass ratio: An HRMS approach.

The major challenge in identifying dexamethasone, betamethasone, and paramethasone from a mixture of these corticosteroids is difficulty in achieving an efficient separation. In this study, we aimed to develop an efficient technique to identify these co-eluting isomers based on the mass spectral patterns of them and their corresponding phase II metabolites after electrospray ionization. Fragmentation pathways in tandem mass spectrometry revealed acceptable specificity within the groups of conjugates. The method was validated using individual isomers and mixtures at various compositions. The effects of concentration and collision energies on fragmentation patterns were also studied extensively. Matrix-fortified equine urine and plasma samples were also included so that matrix effects and interferences on fragmentation ratios could be elucidated. Preliminary results using biological samples demonstrated the suitability of this analytical strategy for direct measurement from their fragmentation patterns. Possible fragmentation pathways for each isomer were proposed.