Long-term Metabolite Research Articles

Differential short-term and long-term metabolic and cytokine responses to infection of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the SFTS virus (SFTSV), which has a wide geographic distribution. The primary clinical manifestations of SFTS are fever and thrombocytopenia, with multiorgan failure being the leading cause of death. While most patients recover with treatment, little is known about the potential long-term metabolic effects of SFTSV infection. This study aimed to shed light on dysregulated metabolic pathways and cytokine responses following SFTSV infection, which pose significant risks to the short-term and long-term health of affected individuals. Fourteen laboratory-confirmed clinical SFTS cases and thirty-eight healthy controls including 18 SFTSV IgG-positive and 20 IgG-negative individuals were recruited from Taizhou city of Zhejiang province, Eastern China. Inclusion criteria of healthy controls included residing in the study area for at least one year, absence of fever or other symptoms in the past two weeks, and no history of SFTS diagnosis. Ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS) was used to obtain the relative abundance of plasma metabolites. Short-term metabolites refer to transient alterations present only during SFTSV infection, while long-term metabolites persistently deviate from normal levels even after recovery from SFTSV infection. Additionally, the concentrations of 12 cytokines were quantified through fluorescence intensity measurements. Differential metabolites were screened using orthogonal projections to latent structures discriminant analysis (OPLS-DA) and the Wilcoxon rank test. Metabolic pathway analysis was performed using MetaboAnalyst. Between-group differences of metabolites and cytokines were examined using the Wilcoxon rank test. Correlation matrices between identified metabolites and cytokines were analyzed using Spearman's method. We screened 122 long-term metabolites and 108 short-term metabolites by analytical comparisons and analyzed their correlations with 12 cytokines. Glycerophospholipid metabolism (GPL) was identified as a significant short-term metabolic pathway suggesting that the activation of GPL might be linked to the self-replication of SFTSV, whereas pentose phosphate pathway and alanine, aspartate, and glutamate metabolism were indicated as significant long-term metabolic pathways playing a role in combating long-standing oxidative stress in the patients. Furthermore, our study suggests a new perspective that α-ketoglutarate could serve as a dietary supplement to protect recovering SFTS patients.

Soil and unsaturated zone as a long-term source for pesticide metabolites in groundwater

Pesticide metabolites are frequently detected in groundwater, often exceeding the concentrations of their parent pesticides. Ceasing the application of certain pesticides has often not led to the expected decrease in metabolite concentrations in groundwater, which is potentially caused by residues in soil. Whereas pesticide residues in soils are well-documented, there are only few studies about metabolite residues. We investigated if the soil/unsaturated zone can act as a long-term source for metabolites in groundwater by combining soil analysis, groundwater analysis and numerical modelling. The field study focused on the herbicide chloridazon (CLZ) and its frequently detected metabolites desphenyl-chloridazon (DPC) and methyl-desphenyl-chloridazon (MDPC) while in the model additional pesticides and metabolites were considered. In soil samples from an agricultural area, where the last CLZ application was 5 to 10 years ago, we observed 10 times (DPC: 0.22 – 7.4 µg kg−1) and 6 times (MDPC: 0.12 – 3.1 µg kg−1) higher metabolite concentrations compared to CLZ (< 0.050 – 1.0 µg kg−1). Calculations suggested that the majority of the metabolites (DPC: 63 - 96%, MDPC: 74 – 97%) were sorbed despite their lower sorption tendency. The metabolite retention was in particular related to the organic carbon content. The calculated pore water concentrations were highest in the deepest part of the soil profile (75 – 100 cm) with median concentrations of 3.6 and 1.7 µg L−1 for DPC and MDPC, respectively. The groundwater concentrations of DPC and MDPC were 3 to 3.5 times higher in monitoring wells downgradient from the agricultural zone than upgradient of it. This increase highlights the potential of soil and unsaturated zone as a long-term metabolite source after the application stop of pesticides, consistent with the calculated elevated pore water concentrations. Numerical flow and transport model simulations suggested that this input from soil and unsaturated zone can cause elevated metabolite concentrations (> 0.1 µg L−1) in groundwater over more than one decade. The study highlights that soil and unsaturated zone can act as a long-term source of pesticide metabolites even if they have much higher mobility than the parent compound.

Simultaneous quantification of TB-500 and its metabolites in in-vitro experiments and rats by UHPLC-Q-Exactive orbitrap MS/MS and their screening by wound healing activities in-vitro

BackgroundTB-500 (Ac-LKKTETQ), derived from the active site of thymosin β4 (Tβ4), has various biological functions in its unacetylated form, LKKTETQ. These functions include actin binding, dermal wound healing, angiogenesis, and skin repair. The biological effects of TB-500, however, have not been documented. And the analysis of TB-500 and its metabolites have been neither simultaneously quantified nor structurally identified using synthesized authentic standards. MethodsThis study was aimed to investigating simultaneous analytical methods of TB-500 and its metabolites in in-vitro and urine samples by using UHPLC-Q-Exactive orbitrap MS, and to comparing the biological activity of its metabolites with the parent TB-500. The metabolism of TB-500 was investigated in human serum, various in-vitro enzyme systems, and urine samples from rats treated with TB-500, and their biological activities measured by cytotoxicity and wound healing experiments were also evaluated in fibroblasts. ResultsThe simultaneous analytical method for TB-500 and its metabolites was developed and validated. The study found that Ac-LK was the primary metabolite with the highest concentration in rats at 0–6 h intervals. Also, the metabolite Ac-LKK was a long-term metabolite of TB-500 detected up to 72 hr. No cytotoxicity of the parent and its metabolites was found. Ac-LKKTE only showed a significant wound healing activity compared to the control. ConclusionThe study provides a valuable tool for quantifying TB-500 and its metabolites, contributing to the understanding of metabolism and potential therapeutic applications. Our results also suggest that the previously reported wound-healing activity of TB-500 in literature may be due to its metabolite Ac-LKKTE rather than the parent form.

Targeted Metabolic Investigation of Ligandrol and Analytical Methods Validation for Its Main Long-term Metabolite.

Prompted by the need for related analytical reference material in the frame of the fight against doping in sports, synthetic efforts towards the main long-term bishydroxylated metabolite (LGD-LTM1) of the nonsteroidal selective androgen receptor modulator (SARM) ligandrol have produced related derivatives that were exploited for a targeted metabolite analysis of urine samples obtained in the course of previous excretion studies of this SARM. Further clarifying ligandrol's metabolic profile, the availability of synthetic reference material permitted the structural elucidation of a previously reported pyrrolidinone-type metabolite and revealed its potential analytical utility as an additional long-term marker. Moreover, synthetic reference material enabled the comparison and validation of liquid chromatography coupled with mass spectrometry (LC-MS)-based and gas chromatography coupled with mass spectrometry (GC-MS)-based detection and identification methods focusing on the LGD-LTM1 marker.

Detection time comparison of non-hydrolysed sulphated metabolites of metenolone, mesterolone and 17α-methyltestosterone analysed by four different mass spectrometric techniques.

The frequent detection of anabolic androgenic steroids (AAS) indicates their popularity among rule-breaking athletes. The so called long-term metabolites play a crucial role in their detection, and non-hydrolysed sulphated metabolites have gained renewed interest, as research has demonstrated their extended detection time compared to the more conventional markers (e.g., for metenolone and mesterolone). Their potential has been investigated using liquid and gas chromatography-mass spectrometry (LC- and GC-MS). However, due to their complementary nature, chances are that the most promising metabolite on one technique does not necessarily exhibit the same behaviour on the other and vice versa. Therefore, a comparison was carried out where as a trial model, metenolone, mesterolone and 17α-methyltestosterone were selected and the most likely long-term sulphated metabolites identified on four mass spectrometric instruments. Additionally, using a modified sample preparation procedure, comparison between conventional and non-hydrolysed sulphated metabolites between different GC-MS instruments was also included. When focusing on each individual marker, no cases were observed where a single metabolite provided a superior detection time on all instruments. Furthermore, for each AAS, there were incidences where a metabolite provided the best detection time on one instrument but could only be detected for a shorter period or not at all on other instruments. This demonstrates that metabolite detection windows and hence their added-value as target substance are unique and dependent on the analytical technique and not only on their pharmacokinetic behaviour. Consequently, in each case, a metabolite versus instrument evaluation is needed to maximise the probabilities of detecting doping offences.

Detection of DHCMT long-term metabolite glucuronides with LC-MSMS as an alternative approach to conventional GC-MSMS analysis

Dehydrochloromethyltestosterone (DHCMT) is one of the most detected illicit used anabolic–androgenic steroids in professional sports. Therefore, a fast and accurate analysis of this substance is of great importance for a constructive fight against doping abuse. The conventional method for the analysis of this drug, GC-MSMS, is very sensitive and selective but also very time- and resource-consuming. With the presented work, a new approach for simple detection with LC-HRMSMS without any sample preparation is introduced. The method is based on the direct analysis of two newly described phase-II metabolites of the DHCMT long-term metabolite 4-chloro-18-nor-17β-hydroxymethyl-17α-methyl-5β-androst-13-en-3α-ol (M3). LC-HRMSMS, GC-MSMS, fractionation and derivatization experiments are combined to identify and characterize for the first time two different glucuronide-acid conjugates of this metabolite in positive human urine samples. In addition, a third glucuronide metabolite was identified, however without isomeric structure determination. The detection of these metabolites is particularly interesting for confirmation analyses, as the method is rapid and requires little sample material.

Ишемическая болезнь сердца с пограничными стенозами коронарных артерий. Клинические особенности. Соотношение паспортного и биологического возраста

Introduction. Coronary artery disease (CAD) remains the main cause of disability and mortality among working-age people. A significant part of stable angina patients are patients with intermediate (40–70%) coronary artery stenosis. Aim. To study cardiovascular risk factors and make a comparative assessment of the clinical angiographic picture and vascular stiffness in stable angina patients with intermediate coronary artery stenosis, depending on the chronological age / biological age ratio, as well as to determine the level of long-term metabolites of nitric oxide (NO) – nitrites (NO2) . Materials and methods. 40 stable angina class I–III patients (36 men) aged 33–68 years with intermediate coronary artery stenosis were examined. Depending on the values of the chronological age / biological age coefficient (K), the patients were divided into two groups. The 1st group included patients with a biological age exceeding the passport age (K &lt; 1.0 [0.53; 0.98]), the 2nd group included patients with a biological age equal or inferior to the chronological age (K ≥ 1.0 [1.1; 1.5], p &lt; 0.01 ). Results. Stable angina patients with intermediate coronary artery stenosis represent a rather challenging group of people in terms of prognosis. Patients with increased biological age are characterized by an unfavorable course of the disease and a less favorable cardiometabolic profile compared to patients whose biological age corresponded to the chronological one. At the same time, the level of long-term metabolites of nitric oxide (NO) – nitrites (NO2) in plasma did not depend on the biological age of patients. Conclusion. The study of the biological age and level of long-term metabolites of nitric oxide (NO) – nitrites (NO2)in stable CAD patients with intermediate coronary artery stenosis is of great interest and requires further research.

Structure revision and chemical synthesis of ligandrol's main bishydroxylated long-term metabolic marker.

Although the main bishydroxylated long-term metabolite of the WADA-banned anabolic agent ligandrol (LGD-4033) is an important metabolic marker, it is not readily available in sufficient quantities to facilitate the development and validation of related analytical protocols or sensors. A chemically more robust structure was postulated as an alternative to the one previously established. The NMR spectra of the synthesized material and its LC-HRMS comparison with a relevant metabolic sample support the proposed structural revision.

Changes in metabolomics profiles over ten years and subsequent risk of developing type 2 diabetes: Results from the Nurses' Health Study.

SummaryBackgroundMetabolomics profiles were consistently associated with type 2 diabetes (T2D) risk, but evidence on long-term metabolite changes and T2D incidence is lacking. We examined the associations of 10-year plasma metabolite changes with subsequent T2D risk.MethodsWe conducted a nested T2D case-control study (n=244 cases, n=244 matched controls) within the Nurses' Health Study. Repeated metabolomics profiling (170 targeted metabolites) was conducted in participant blood specimens from 1989/1990 and 2000/2001, and T2D occurred between 2002 and 2008. We related 10-year metabolite changes (Δ-values) to subsequent T2D risk using conditional logistic models, adjusting for baseline metabolite levels and baseline levels and concurrent changes of BMI, diet quality, physical activity, and smoking status.FindingsThe 10-year changes of thirty-one metabolites were associated with subsequent T2D risk (false discovery rate-adjusted p-values [FDR]<0.05). The top three high T2D risk-associated 10-year changes were (odds ratio [OR] per standard deviation [SD], 95%CI): Δisoleucine (2.72, 1.97-3.79), Δleucine (2.53, 1.86-3.47), and Δvaline (1.93, 1.52-2.44); other high-risk-associated metabolite changes included alanine, tri-/diacylglycerol-fragments, short-chain acylcarnitines, phosphatidylethanolamines, some vitamins, and bile acids (ORs per SD between 1.31and 1.82). The top three low T2D risk-associated 10-year metabolite changes were (OR per SD, 95% CI): ΔN-acetylaspartic acid (0.54, 0.42-0.70), ΔC20:0 lysophosphatidylethanolamine (0.68, 0.56-0.82), and ΔC16:1 sphingomyelin (0.68, 0.56-0.83); 10-year changes of other sphingomyelins, plasmalogens, glutamine, and glycine were also associated with lower subsequent T2D risk (ORs per SD between 0.66 and 0.78).InterpretationRepeated metabolomics profiles reflecting the long-term deterioration of amino acid and lipid metabolism are associated with subsequent risk of T2D.

HepG2 as promising cell-based model for biosynthesis of long-term metabolites: Exemplified for metandienone.

In order to detect the abuse of substances in sports, the knowledge of their metabolism is of undisputable importance. As in vivo administration of compounds faces ethical problems and might even not be applicable for nonapproved compounds, cell-based models might be a versatile tool for biotransformation studies. We coincubated HepG2 cells with metandienone and D3 -epitestosterone for 14 days. Phase I and II metabolites were analyzed by high-performance liquid chromatography (HPLC)-tandem mass spectrometry and confirmed by gas chromatography-mass spectrometry (GC-MS). The metandienone metabolites formed by HepG2 cells were comparable with those renally excreted by humans. HepG2 cells also generated the two long-term metabolites 17β-hydroxymethyl-17α-methyl-18-nor-androst-1,4,13-trien-3-one and 17α-hydroxymethyl-17β-methyl-18-nor-androst-1,4,13-trien-3-one used in doping analyses, though in an inverse ratio compared with that observed in human urine. In conclusion, we showed that HepG2 cells are suitable as model for the investigation of biotransformation of androgens, especially for the anabolic androgenic steroid metandienone. They further proved to cover phase I and II metabolic pathways, which combined with a prolonged incubation time with metandienone resulted in the generation of its respective long-term metabolites known from in vivo metabolism. Moreover, we showed the usability of D3 -epitestosterone as internal standard for the incubation. The method used herein appears to be suitable and advantageous compared with other models for the investigation of doping-relevant compounds, probably enabling the discovery of candidate metabolites for doping analyses.

Controlled administration of dehydrochloromethyltestosterone in humans: Urinary excretion and long-term detection of metabolites for anti-doping purpose

Dehydrochloromethyltestosterone (DHCMT) is an anabolic-androgenic steroid that was developed by Jenapharm in the 1960s and was marketed as Oral Turinabol®. It is prohibited in sports at all times; nevertheless, there are several findings by anti-doping laboratories every year. New long-term metabolites have been proposed in 2011/12, which resulted in adverse analytical findings in retests of the Olympic games of 2008 and 2012. However, no controlled administration trial monitoring these long-term metabolites was reported until now. In this study, DHCMT (5 mg, p.o.) was administered to five healthy male volunteers and their urine samples were collected for a total of 60 days. The unconjugated and the glucuronidated fraction were analyzed separately by gas chromatography coupled to tandem mass spectrometry. The formation of the described long-term metabolites was verified, and their excretion monitored in detail.Due to interindividual differences there were several varieties in the excretion profiles among the volunteers. The metabolite M3, which has a fully reduced A-ring and modified D-ring structure, was identified by comparison with reference material as 4α-chloro-17β-hydroxymethyl-17α-methyl-18-nor-5α-androstan-13-en-3α-ol. It was found to be suitable as long-term marker for the intake of DHCMT in four of the volunteers. In one of the volunteers, it was detectable for 45 days after single oral dose administration. However, in two of the volunteers M5 (already published as long-term metabolite in the 1990s) showed longer detection windows. In one volunteer M3 was undetectable but another metabolite, M2, was found as the longest detectable metabolite.The last sample clearly identified as positive was collected between 9.9 and 44.9 days. Furthermore, the metabolite epiM4 (partially reduced A-ring and a modified D-ring structure which is epimerized in position 17 compared to M3) was identified in the urine of all volunteers with the help of chemically synthesized reference as 4-chloro-17α-hydroxymethyl-17β-methyl-18-nor-androsta-4,13-dien-3β-ol. It may serve as additional confirmatory metabolite.It is highly recommended to screen for all known metabolites in both fractions, glucuronidated and unconjugated, to improve identification of cheating athletes. This study also offers some deeper insights into the metabolism of DHCMT and of 17α-methyl steroids in general.

New Insights into the Metabolism of Methyltestosterone and Metandienone: Detection of Novel A-Ring Reduced Metabolites.

Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a 17α-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. Following the previously reported detection of long-term metabolites with a 17ξ-hydroxymethyl-17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone (“oral turinabol”), in this study we investigated the formation of similar metabolites of metandienone and 17α-methyltestosterone with a rearranged D-ring and a fully reduced A-ring. Using a semi-targeted approach including the synthesis of reference compounds, two diastereomeric substances, viz. 17α-hydroxymethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol and its 5α-analog, were identified following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5β-metabolite was detected. Additionally, 3α,5β-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides the classical metabolites included in the screening procedures. Besides their applicability for anti-doping analysis, the results provide new insights into the metabolism of 17α-methyl steroids with respect to the order of reductions in the A-ring, the participation of different enzymes, and alterations to the D-ring.

Synthesis of human long-term metabolites of dehydrochloromethyltestosterone and oxymesterone

We herein report the synthesis of the long-term metabolites “M4” (IUPAC: 4-chloro-17-hydroxymethyl-17-methyl-18-norandrosta-4,13-dien-3-ol) of dehydrochloromethyl-testosterone (DHCMT, Oral Turinabol) and “Oxy M9” (4-hydroxy-17β-hydroxymethyl-17α-methyl-18-norandrosta-4,13-dien-3-one) of oxymesterone (Oranabol). Both compounds were derived from a common synthetic route starting from dehydroepiandrosterone acetate. Four different stereoisomers were evaluated for metabolite M4. The previously assigned structure could be corrected regarding the C-3 and C-17 stereocenters.

Searching for new long-term urinary metabolites of metenolone and drostanolone using gas chromatography-mass spectrometry with a focus on non-hydrolysed sulfates.

Sulfated metabolites have been shown to have potential as long-term markers of anabolic-androgenic steroid (AAS) abuse. In 2019, the compatibility of gas chromatography-mass spectrometry (GC-MS) with non-hydrolysed sulfated steroids was demonstrated, and this approach allowed the incorporation of these compounds in a broad GC-MS initial testing procedure at a later stage. However, research is needed to identify which are beneficial. In this study, a search for new long-term metabolites of two popular AAS, metenolone and drostanolone, was undertaken through two excretion studies each. The excretion samples were analysed using GC-chemical ionization-triple quadrupole MS (GC-CI-MS/MS) after the application of three separate sample preparation methodologies (i.e. hydrolysis with Escherichia coli-derived β-glucuronidase, Helix pomatia-derived β-glucuronidase/arylsulfatase and non-hydrolysed sulfated steroids). For metenolone, a non-hydrolysed sulfated metabolite, 1β-methyl-5α-androstan-17-one-3ζ-sulfate, was documented for the first time to provide the longest detection time of up to 17 days. This metabolite increased the detection time by nearly a factor of 2 in comparison with the currently monitored markers for metenolone in a routine doping control initial testing procedure. In the second excretion study, it prolonged the detection window by 25%. In the case of drostanolone, the non-hydrolysed sulfated metabolite with the longest detection time was the sulfated analogue of the main drostanolone metabolite (3α-hydroxy-2α-methyl-5α-androstan-17-one) with a detection time of up to 24 days. However, the currently monitored main drostanolone metabolite in routine doping control, after hydrolysis of the glucuronide with E.coli, remained superior in detection time (i.e. up to 29 days).

A photochemical approach to 18-nor-17β-hydroxymethyl-17α-methylandrost-13-ene steroids

A photochemical approach to 18-nor-17β-hydroxymethyl-17α-methylandrost-13-ene unit of the long-term metabolites of 17-methylated androgenic anabolic steroids (AAS) is reported. It is based on a visible light-promoted radical decarboxylative alkynylation of steroidal redox-active ester. The developed method was used in synthesis of the long-term metabolite of AAS oxymesterone.

Differentiation of soluble aqueous humor metabolites in primary open angle glaucoma and controls

We report an analysis of the aqueous humor (AH) metabolome of primary open angle glaucoma (POAG) in comparison to normal controls. The AH samples were obtained from human donors [control (n = 35), POAG (n = 23)]. The AH samples were subjected to one-dimensional 1H nuclear magnetic resonance (NMR) analyses on a Bruker Avance 600 MHz instrument with a 1.7 mM NMR probe. The same samples were then subjected to isotopic ratio outlier analysis (IROA) using a Q Exactive orbitrap mass spectrometer after chromatography on an Accela 600 HPLC. Clusterfinder Build 3.1.10 was used for identification and quantification based on long-term metabolite matrix standards. In total, 278 metabolites were identified in control samples and 273 in POAG AH. The metabolites identified were fed into previously reported proteome and genome information and the OmicsNet interaction network generator to construct a protein-metabolite interactions network with an embedded protein-protein network. Significant differences in metabolite composition in POAG compared to controls were identified indicating potential protein/gene pathways associated with these metabolites. These results will expand our previous understanding of the impeded AH metabolite composition, provide new insight into the regulation of AH outflow, and likely aid in future AH and trabecular meshwork multi-omics network analyses.

Synthesis of a human long-term oxymetholone metabolite

A long-term metabolite of the doping agent oxymetholone (OXM-M2, 17β-hydroxymethyl-2,17α-methyl-18-norandrost-13-en-3-one) which has been identified by GC-MS/MS was synthesized from commercially available materials. Two efficient synthetic routes to access both C-17 epimers of tentative metabolites were developed. The identity and molecular configuration of the in vivo metabolite: 17β-hydroxymethyl-2α,17α-methyl-18-norandrost-13-en-3-one was confirmed by single crystal X-ray diffraction.

Human Metabolism of The Anabolic Steroid Methasterone: Detection and Kinetic Excretion of New Phase I Urinary Metabolites and Investigation of Phase II Metabolism by GC-MS and UPLC-MS/MS

Methasterone is a designer anabolic steroid that is prohibited for athletes and is monitored by anti-doping laboratories. In this work, our objective is to discover new human phase I metabolites, define their excretion kinetics for 30 days and analyze their phase II metabolism (sulfate, cysteine and N-acetylcysteine conjugates). Urine samples from four volunteers were analyzed by chromatographic techniques. Through gas chromatography coupled to mass spectrometry analysis it was possible to detect methasterone and its nine phase I metabolites in the urine samples after glucuronide enzymatic hydrolysis, from which one were previously unreported. These nine compounds were not excreted in free form. The new proposed metabolite is 17β-hydroxy-2α,17α-dimethyl-5β-androstan-3-one, obtained from the epimerization at C5. The 3α-hydroxy metabolite, currently monitored by anti-doping laboratories, was the most abundant and was detected for the longest time. Furthermore, four other long-term metabolites were identified. By ultra-performance liquid chromatography coupled to tandem mass spectrometry, only the drug and a known metabolite were detected after glucuronide hydrolysis, and phase II metabolites were not found. Thus, our results contribute to elucidating methasterone metabolism, including long-term metabolites besides of the 3α-hydroxy in routine doping analysis, which is very important due to variation in human metabolism.

Isotopically labeled boldenone as a better marker of derivatization efficiency for improved quality control in anti-doping analysis.

At present, anti-doping laboratories use androsterone, a major urinary steroid metabolite, to evaluate completeness of the derivatization step. This is typically done by calculating the ratio of mono-trimethylsilyl (TMS) androsterone to the total mono- and di-TMS androsterone. Certain samples may show an elevated percentage of mono-TMS androsterone indicating a failed derivatization step. In such cases, the laboratory would have to repeat the analysis or perform other remedial actions to ensure that completeness of derivatization is achieved. We have noticed that a poorly derivatized positive control sample spiked with various target analytes has a disproportionally low abundance of the di-TMS derivatives of boldenone and 18-nor-17β-hydroxymethyl-17α-methylandrosta-1,4,13-trien-3-one (methandienone long-term metabolite). A follow-up investigation confirmed that 1,4-diene-3-one steroids are more likely to fail during the trimethylsilylation step. To better control derivatization efficiency, 13 C3 -boldenone (13C-BLD) was incorporated into our routine procedure as an additional internal standard. Analysis of a large number of urine samples has shown that derivatization of 13C-BLD could be grossly incomplete even in cases when mono-TMS androsterone is well below 1%. In other words, one or both of boldenone and the long-term metabolite of methandienone could remain undetected unless the laboratory has the means to recognize samples where derivatization of 1,4-diene-3-one steroids failed.

Human in vivo metabolism study of LGD-4033.

Selective androgen receptor modulators (SARMs) are an emerging class of therapeutics targeted to cachexia, sarcopenia, and hypogonadism treatment. LGD-4033 is a SARM which has been included on the Prohibited List annually released by the World Anti-Doping Agency (WADA). The aim of the present work was the investigation of the metabolism of LGD-4033 in a human excretion study after administration of an LGD-4033 supplement, the determination of the metabolites' excretion profiles with special interest in the determination of its long-term metabolites, and the comparison of the excretion time of the phase I and phase II metabolites. The results were also compared to those derived from previous LGD-4033 studies concerning both in vitro and in vivo experiments. Supplement containing LGD-4033 was administered to one human male volunteer and urine samples were collected up to almost 21days. Analysis of the hydrolyzed (with β-glucuronidase) as well as of the non-hydrolyzed samples was performed using liquid chromatography-high resolution mass spectrometry (LC-HRMS) in negative ionization mode and revealed that, in both cases, the two isomers of the dihydroxylated metabolite (M5) were preferred target metabolites. The gluco-conjugated parent LGD-4033 and its gluco-conjugated metabolites M1 and M2 can be also considered as useful target analytes in non-hydrolyzed samples. The study also presents two trihydroxylated metabolites (M6) identified for the first time in human urine; one of them was recently reported in an LGD-4033 metabolism study in horse urine and plasma.