Investigations into the elimination profiles and metabolite ratios of micro-dosed selective androgen receptor modulator LGD-4033 for doping control purposes.

LGD-4033 (ligandrol) is a selective androgen receptor modulator (SARM), which is prohibited in sports by the World Anti-Doping Agency (WADA) and led to 62 adverse analytical findings (AAFs) in 2019. But not only deliberate doping with LGD-4033 constitutes a problem. In the past years, some AAFs that concerned SARMs can be attributed to contaminated dietary supplements (DS). Thus, the urgency to develop methods to differentiate between inadvertent doping and abuse of SARMs to benefit from the performance-enhancing effect of the compound in sports is growing. To gain a better understanding of the metabolism and excretion patterns of LGD-4033, human micro-dose excretion studies at 1, 10, and 50 µg LGD-4033 were conducted. Collected urine samples were prepared for analysis using enzymatic hydrolysis followed by solid-phase extraction and analyzed via LC-HRMS/MS. Including isomers, a total of 15 phase I metabolites were detected in the urine samples. The LC-HRMS/MS method was validated for qualitative detection of LGD-4033, allowing for a limit of detection (LOD) of 8 pg/mL. The metabolite M1, representing the epimer of LGD-4033, was synthesized and the structure elucidated by NMR spectroscopy. As the M1/LGD-4033 ratio changes over time, the ratio and the approximate LGD-4033 concentration can contribute to estimating the time point of drug intake and dose of LGD-4033 in doping control urine samples, which is particularly relevant in anti-doping result management.

Urinary phenylethylamine metabolites as potential markers for sports drug testing purposes.

The misuse of 2-phenylethylamine (PEA) in sporting competitions is prohibited by the World Anti-Doping Agency. As it is endogenously produced, a method is required to differentiate between naturally elevated levels of PEA and the illicit administration of the drug. In 2015, a sulfo-conjugated metabolite [2-(2-hydroxyphenyl)acetamide sulfate (M1)] was identified, and pilot study data suggested that the ratio M1/PEA could be used as a marker indicating the oral application of PEA. Within this project, the required reference material of M1 was synthesized, single and multiple dose elimination studies were conducted and 369 native urine samples of athletes were analyzed as a reference population. While the oral administration of only 100 mg PEA did not affect urinary PEA concentrations, an increase in urinary concentrations of M1 was observed for all volunteers. However, urinary concentrations of both PEA and M1 showed relatively large inter-individual differences and establishing a cut-off-level for M1/PEA proved difficult. Consequently, a second metabolite, phenylacetylglutamine, was considered. Binary logistic regression demonstrated a significant (P < 0.05) correlation of the urinary M1 and phenylacetylglutamine concentrations with an oral administration of PEA, suggesting that assessing both analytes can assist doping control laboratories in identifying PEA misuse.

A toxicogenomics approach reveals characteristics supporting the honey bee (Apis mellifera L.) safety profile of the butenolide insecticide flupyradifurone.

Flupyradifurone, a novel butenolide insecticide, selectively targets insect nicotinic acetylcholine receptors (nAChRs), comparable to structurally different insecticidal chemotypes such as neonicotinoids and sulfoximines. However, flupyradifurone was shown in acute toxicity tests to be several orders of magnitude less toxic to western honey bee (Apis mellifera L.) than many other insecticides targeting insect nAChRs. The underlying reasons for this difference in toxicity remains unknown and were investigated here. Pharmacokinetic studies after contact application of [14C]flupyradifurone to honey bees revealed slow uptake, with internalized compound degraded into a few metabolites that are all practically non-toxic to honey bees in both oral and contact bioassays. Furthermore, receptor binding studies revealed a lack of high-affinity binding of these metabolites to honey bee nAChRs. Screening of a library of 27 heterologously expressed honey bee cytochrome P450 enzymes (P450s) identified three P450s involved in the detoxification of flupyradifurone: CYP6AQ1, CYP9Q2 and CYP9Q3. Transgenic Drosophila lines ectopically expressing CYP9Q2 and CYP9Q3 were significantly less susceptible to flupyradifurone when compared to control flies, confirming the importance of these P450s for flupyradifurone metabolism in honey bees. Biochemical assays using the fluorescent probe substrate 7-benzyloxymethoxy-4-(trifluoromethyl)-coumarin (BOMFC) indicated a weak, non-competitive inhibition of BOMFC metabolism by flupyradifurone. In contrast, the azole fungicides prochloraz and propiconazole were strong nanomolar inhibitors of these flupyradifurone metabolizing P450s, explaining their highly synergistic effects in combination with flupyradifurone as demonstrated in acute laboratory contact toxicity tests of adult bees. Interestingly, the azole fungicide prothioconazole is only slightly synergistic in combination with flupyradifurone - an observation supported by molecular P450 inhibition assays. Such molecular assays have value in the prediction of potential risks posed to bees by flupyradifurone mixture partners under applied conditions. Quantitative PCR confirmed the expression of the identified P450 genes in all honey bee life-stages, with highest expression levels observed in late larvae and adults, suggesting honey bees have the capacity to metabolize flupyradifurone across all life-stages. These findings provide a biochemical explanation for the low intrinsic toxicity of flupyradifurone to honey bees and offer a new, more holistic approach to support bee pollinator risk assessment by molecular means.

Determination of Cotton as a Larval Feeding Source for Lepidopteran Moths Using a Derivative from Cotton Metabolites as a Marker by LC-MS/MS Method.

Determination of the feeding history of polyphagous insect pests, such as noctuid moths (Lepidoptera: Noctuidae), is a critical element in developing population and resistance management strategies for such pests. To identify reliable markers for larval host plant determination and to develop simple extraction and detection methods, a metabolomics approach was implemented after acid hydrolysis of adult moth samples. We identified a derivative from cotton metabolites as a marker in adult moths that were fed cotton tissues as a larval diet, and we propose that the marker is tricycloheliocide H4 based on NMR and mass fragmentation analysis. Using this derivative from cotton metabolites as a marker, a targeted LC-MS/MS method reliably identified cotton as a larval diet in extracts of three noctuid moth species: Helicoverpa zea (cotton bollworm), Chloridea (Heliothis) virescens (tobacco budworm) and Chrysodeixis includens (soybean looper). We are using similar approaches to identify markers for other host plants including soybean.

Pharmacokinetics of Three Neonicotinoid Insecticides upon Contact Exposure in the Western Honey Bee, Apis mellifera.

Neonicotinoid insecticides differ in their acute contact toxicity to honey bees. We investigated the uptake, metabolic fate, and excretion of imidacloprid and two much less toxic chemotypes, thiacloprid and acetamiprid, upon contact exposure in honey bees because ADME data for this mode of entry are lacking. Pharmacokinetic parameters were analyzed by tracking a 14C-label and by HPLC coupled to ESI-MS. Imidacloprid penetrates the honey bee cuticle much faster and more readily compared to thiacloprid and acetamiprid, thus revealing a pharmacokinetic component, i.e., faster penetration and higher steady-state internal body concentrations, contributing to its higher acute contact toxicity.

Mass spectrometric characterization of the selective androgen receptor modulator (SARM) YK-11 for doping control purposes.

RATIONALE: Selective androgen receptor modulators (SARMs) represent an emerging class of therapeutics targeting inter alia conditions referred to as cachexia and sarcopenia. Due to their anabolic properties, the use of SARMs is prohibited in sports as regulated by the World Anti-Doping Agency (WADA), and doping control laboratories test for these anabolic agents in blood and urine. In order to accomplish and maintain comprehensive test methods, the characterization of new drug candidates is critical for efficient sports drug testing. Hence, in the present study the mass spectrometric properties of the SARM YK-11 were investigated. METHODS: YK-11 was synthesized according to literature data and three different stable-isotope-labeled analogs were prepared to support the mass spectrometric studies. Using high-resolution/high-accuracy mass spectrometry following electrospray ionization as well as electron ionization, the dissociation pathways of YK-11 were investigated, and characteristic features of its (product ion) mass spectra were elucidated. These studies were flanked by density functional theory (DFT) computation providing information on proton affinities of selected functional groups of the analyte. RESULTS AND CONCLUSIONS: The steroidal SARM YK-11 was found to readily protonate under ESI conditions followed by substantial in-source dissociation processes eliminating methanol, acetic acid methyl ester, and/or ketene. DFT computation yielded energetically favored structures of the protonated species resulting from the aforementioned elimination processes particularly following protonation of the steroidal D-ring substituent. Underlying dissociation pathways were suggested, supported by stable-isotope labeling of the analyte, and diagnostic product ions for the steroidal nucleus and the D-ring substituent were identified. Further, trimethylsilylated YK-11 and its deuterated analogs were subjected to electron ionization high-resolution/high-accuracy mass spectrometry, complementing the dataset characterizing this new SARM. The obtained fragment ions resulted primarily from A/B- and C/D-ring structures of the steroidal nucleus, thus supporting future studies e.g. concerning metabolic pathways of the substance. Copyright © 2017 John Wiley & Sons, Ltd.

Quantitative Identification of Biogenic Nonextractable Pesticide Residues in Soil by (14)C-Analysis.

Quantification of nonextractable residues (NER) of pesticides in soil is feasible by use of radioactively labeled compounds, but structural information on these long-term stabilized residues is usually lacking. Microorganisms incorporate parts of the radiolabeled ((14)C-) carbon from contaminants into microbial biomass, which after cell death enters soil organic matter, thus forming biogenic nonextractable residues (bioNER). The formation of bioNER is not yet determinable in environmental fate studies due to a lack of methodology. This paper focuses on the development of a feasible analytical method to quantify proteinaceous carbon, since proteins make up the largest mass portion of bacterial cells. The test substance (14)C-bromoxynil after 56 days forms more than 70% of NER in soil. For further characterization of NER the amino acids were extracted, purified, and separated by two-dimensional thin-layer chromatography (TLC). Visualization of the (14)C-amino acids was performed by bioimaging, unambiguous identification by GC-MS and LC-MS/MS. Our analysis revealed that after 56 days of incubation about 14.5% of the (14)C-label of bromoxynil was incorporated in amino acids. Extrapolating this content based on the amount of proteins in the biomass (55%), in total about 26% of the NER is accounted for by bioNER and thus is not environmentally relevant.

Identification of selected in vitro generated phase-I metabolites of the steroidal selective androgen receptor modulator MK-0773 for doping control purposes.

Research into developing anabolic agents for various therapeutic purposes has been pursued for decades. As the clinical utility of anabolic-androgenic steroids has been found to be limited because of their lack of tissue selectivity and associated off-target effects, alternative drug entities have been designed and are commonly referred to as selective androgen receptor modulators (SARMs). While most of these SARMs are of nonsteroidal structure, the drug candidate MK-0773 comprises a 4-aza-steroidal nucleus. Besides the intended therapeutic use, SARMs have been found to be illicitly distributed and misused as doping agents in sport, necessitating frequently updated doping control analytical assays. As steroidal compounds reportedly undergo considerable metabolic transformations, the phase-I metabolism of MK-0773 was simulated using human liver microsomal (HLM) preparations and electrochemical conversion. Subsequently, major metabolic products were identified and characterized employing liquid chromatography-high-resolution/high- accuracy tandem mass spectrometry with electrospray (ESI) and atmospheric pressure chemical ionization (APCI) as well as nuclear magnetic resonance (NMR) spectroscopy. MK-0773 produced numerous phase-I metabolites under the chosen in vitro incubation reactions, mostly resulting from mono- and bisoxygenation of the steroid. HLM yielded at least 10 monooxygenated species, while electrochemistry-based experiments resulted predominantly in three monohydroxylated metabolites. Elemental composition data and product ion mass spectra were generated for these analytes, ESI/APCI measurements corroborated the formation of at least two N-oxygenated metabolites, and NMR data obtained from electrochemistry-derived products supported structures suggested for three monohydroxylated compounds. Hereby, the hydroxylation of the A-ring located N- bound methyl group was found to be of particular intensity. In the absence of controlled elimination studies, the produced information enables the implementation of new target analytes into routine doping controls and expands the focus of anti-doping efforts concerning this new anabolic agent.

Characterization of a non-approved selective androgen receptor modulator drug candidate sold via the Internet and identification of in vitro generated phase-I metabolites for human sports drug testing.

RATIONALE: Potentially performance-enhancing agents, particularly anabolic agents, are advertised and distributed by Internet-based suppliers to a substantial extent. Among these anabolic agents, a substance referred to as LGD-4033 has been made available, comprising the core structure of a class of selective androgen receptor modulators (SARMs). METHODS: In order to provide comprehensive analytical data for doping controls, the substance was obtained and characterized by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography/electrospray ionization high resolution/high accuracy tandem mass spectrometry (LC/ESI-HRMS). Following the identification of 4-(2-(2,2,2-trifluoro-1-hydroxyethyl)pyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile, the substance was subjected to in vitro metabolism studies employing human liver microsomes and Cunninghamella elegans (C. elegans) preparations as well as electrochemical metabolism simulations. RESULTS: By means of LC/ESI-HRMS, five main phase-I metabolites were identified as products of liver microsomal preparations including three monohydroxylated and two bishydroxylated species. The two most abundant metabolites (one mono- and one bishydroxylated product) were structurally confirmed by LC/ESI-HRMS and NMR. Comparing the metabolic conversion of 4-(2-(2,2,2-trifluoro-1-hydroxyethyl)pyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile observed in human liver microsomes with C. elegans and electrochemically derived metabolites, one monohydroxylated product was found to be predominantly formed in all three methodologies. CONCLUSIONS: The implementation of the intact SARM-like compound and its presumed urinary phase-I metabolites into routine doping controls is suggested to expand and complement existing sports drug testing methods.

Investigations into Annona fruit consumption as a potential source of dietary higenamine intake in the context of sports drug testing.

Higenamine is prohibited in sports as a ß2 -agonist by the World Anti-Doping Agency. As a key component of a great variety of plants, including the Annonaceae family, one aim of this research project was to evaluate whether the ingestion of Annona fruit could lead to higenamine adverse analytical findings. Single-dose administration studies including three Annona species (i.e., Annona muricata, Annona cherimola, and Annona squamosa) were conducted, leading to higenamine findings below the established minimum reporting level (MRL) of 10 ng/mL in urine. In consideration of cmax values (7.8 ng/mL) observed for higenamine up to 24 h, a multidose administration study was also conducted, indicating cumulative effects, which can increase the risk of exceeding the applicable MRL doping after Annona fruit ingestion. In this study, however, the MRL was not exceeded at any time point. Further, the major urinary excretion of higenamine in its sulfo-conjugated form was corroborated, its stability in urine was assessed, and in the absence of reference material, higenamine sulfo-conjugates were synthesized and comprehensively characterized, suggesting the predominant presence of higenamine 7-sulfate. In addition, the option to include complementary biomarkers of diet-related higenamine intake into routine doping controls was investigated. A characteristic urinary pattern attributed to isococlaurine, reticuline, and a yet not fully characterized bismethylated higenamine glucuronide was observed after Annona ingestion but not after supplement use, providing a promising dataset of urinary biomarkers, which supports the discrimination between different sources of urinary higenamine detected in sports drug testing programs.

Comparison of the in vitro assays to investigate the hepatic metabolism of seven pesticides in Cyprinus carpio and Oncorhynchus mykiss.

Liver S9 fractions from common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) were incubated with seven pesticides (fenamidone, fenoxaprop-p-ethyl, penflufen, spirotetramat, tebuconazole, tembotrione and trifloxystrobin) and the metabolic pathways of the applied chemicals were determined by HPLC-high-resolution mass spectrometry. Five of the seven pesticides (fenamidone, penflufen, spirotetramat, trifloxystrobin and fenoxaprop-p-ethyl) revealed a higher metabolic capacity of rainbow trout liver fractions compared to carp liver fractions. The other two pesticides (tebuconazole and tembotrione) showed a similar and marginal biotransformation for liver S9 fractions of both species. Furthermore, four compounds (penflufen, spirotetramat, tembotrione and tebuconazole) were incubated with cryo-preserved hepatocytes of rainbow trout showing additional conjugated metabolites compared to liver S9 fractions. The incubations were performed with concentrations of 1 and 10 µM for experiments with liver S9 fractions and 5 µM with hepatocytes for up to 120 (liver S9 fractions) or 240 min (hepatocytes). A set of positive controls was used to confirm the metabolic capability of the in vitro systems. The comparison of the in vitro results from hepatocyte assays of penflufen and tebuconazole with the data from corresponding in vivo studies performed according to OECD (Organisation for Economic Co-operation and Development) guideline 305 exhibited a similar metabolic behavior for these pesticides and emphasizes the reliability of the in vitro assays. Besides investigation of the metabolism of plant protection products for research purposes, inter-species comparison by in vitro assays and the use of PBTK modelling approaches will allow improved environmental and dietary risk assessments.

In vitro metabolism of tebuconazole, flurtamone, fenhexamid, metalaxyl-M and spirodiclofen in Cannabis sativa L. (hemp) callus cultures.

BACKGROUND: Cannabis sativa L. (hemp) is a medicinal plant producing various cannabinoids. Its consumption is legalized for medical use due to the alleged positive health effects of these cannabinoids. To satisfy the demand, C. sativa plants are propagated in contained growth chambers. During indoor propagation, pesticides usually are used to ensure efficient production. However, pesticide registration and safe application in C. sativa has not been investigated in detail. RESULTS: With this study the metabolic degradation of pesticides in recently established C. sativa callus cultures was examined. Tebuconazole, metalaxyl-M fenhexamid, flurtamone and spirodiclofen were applied at 10 µm for 21 days. Results were compared with metabolism data obtained from Brassica napus L., Glycine max (L.) Merr., Zea mays L. and Tritium aestivum L. callus cultures as well as in metabolism guideline studies. The successfully established C. sativa callus cultures were able to degrade pesticides by oxidation, demethylation, and cleavage of ester bonds in phase I, as well as glycosylation and conjugation with malonic acid in phase II and III. Initial metabolites were detected after Day (D)7 and were traced at D21. CONCLUSION: The resulting pathways demonstrate the same main degradation strategies as crop plants. Because metabolites could be the main residue, the exposure of consumers to these residues will be of high importance. We present here an in vitro assay for a first estimation of pesticide metabolism in C. sativa. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Evaluation of Callus Cultures to Elucidate the Metabolism of Tebuconazole, Flurtamone, Fenhexamid, and Metalaxyl-M in Brassica napus L., Glycine max (L.) Merr., Zea mays L., and Triticum aestivum L.

Plant cell cultures can be used to identify the metabolic degradation of pesticides in crops. Therefore, Brassica napus L., Glycine max (L.) Merr., Zea mays L. and Triticum aestivum L. were used to elucidate the metabolic degradation of the following pesticides: tebuconazole, flurtamone, fenhexamid, and metalaxyl-M. Callus cultures were treated with 10 µM of the named pesticides by passive diffusion out of the nutrition agar while young plants were hydroponically exposed to it. After 14 days, the comparison of in planta and in vitro experiments showed that the metabolic degradation is well described by in vitro callus cultures. The intracellular uptake of all pesticides and a broad spectrum of exemplarily hydroxylated and conjugated metabolites were detectable. Overall, the comparability of the nature of residues out of both experiments with the regulatory guideline metabolism studies could be demonstrated. Therefore, we recommend it as a potential screening tool to elucidate the metabolism of pesticides in crops.

Safening activity and metabolism of the safener cyprosulfamide in maize and wheat.

BACKGROUND: Safeners extend the application of existing herbicides by selectively enhancing tolerance in large-grained cereal crops. While their activity is linked to enhanced herbicide metabolism, their exact mode of action and reasons for their crop specificity have yet to be determined. In this study, we investigated the selectivity of the recently developed sulfonamide safener cyprosulfamide (CSA) in maize (Zea mays L.) and wheat (Triticum aestivum), focusing on its uptake, distribution and metabolism in the two species. RESULTS: CSA protected maize, but not wheat, from injury by thiencarbazone-methyl (TCM). This correlated with the selective enhanced detoxification of the herbicide in maize. CSA underwent more rapid metabolism in maize than in wheat, with the formation of a specific hydroxylated metabolite correlating with safening. Studies with the nsf1 mutant sweetcorn line showed that the hydroxylation of CSA was partly mediated by the cytochrome P450 CYP81A9. However, primary metabolites of CSA were chemically synthesised and tested for their ability to safen TCM in maize but when tested were inactive as safeners. CONCLUSION: The results of this study suggest that the protection against TCM injury by CSA is linked to enhanced herbicide metabolism. This selective activity is due to the specific recognition of parent CSA in maize but not in wheat. Subsequent rapid oxidative metabolism of CSA led to its inactivation, demonstrating that cytochrome P450s regulate the activity of safeners as well as herbicides. © 2020 Society of Chemical Industry.

Metabolism and spatial distribution of metalaxyl in tomato plants grown under hydroponic conditions.

Knowledge about translocation of plant protection products (PPP's) in plants is important to understand the uptake via the root system. Here we report the combination of analysis of tissue extracts by LC-HRMSn, autoradiography of 14C-labeled compounds and MALDI-MSI, which combine qualitative and quantitative information of chemical composition and the spatial distribution of PPP's and their metabolites in situ. Therefore, the uptake of the phenylamide fungicide metalaxyl was studied in tomato plants (Solanum lycopersicum) using a hydroponic system. The plants have been cultivated in perlite until the two-leaf stage and were transferred into the hydroponic test system afterwards. The radioactive labeled fungicide was readily taken up by the roots during the normal water consumption and radioactivity was translocated uniformly to the aboveground part of the tomato plants, while only small proportion of the applied radioactivity were observed in the roots. The distribution of metalaxyl after the plant uptake experiment in the primary roots where analyzed by a transversal tissue section in the zone of maturation. Metalaxyl is mainly localized in root xylem and in cortex located at the epidermis. With LC-HRMSn and radiochemical analyses of stem and leaf, no parent compound was detectable. Four polar metabolites were the main identified components of the residue and could be visualized by MALDI-imaging mass spectrometry. With these results we could show, that the fungicide metalaxyl is taken up by the plant via the roots during the regular water consumption and transported to xylem.

Studies on the in vivo metabolism of methylstenbolone and detection of novel long term metabolites for doping control analysis.

The anabolic-androgenic steroid methylstenbolone (MSTEN; 2α,17α-dimethyl-17ß-hydroxy-5α-androst-1-en-3-one) is available as a so-called designer steroid or nutritional supplement. It is occasionally detected in doping control samples, predominantly tested and confirmed as the glucuronic acid conjugate of methylstenbolone. The absence of other meaningful metabolites reported as target analytes for sports drug testing purposes can be explained by the advertised metabolic stability of methylstenbolone. In 2013, a first investigation into the human metabolism of methylstenbolone was published, and two hydroxylated metabolites were identified as potential targets for initial testing procedures in doping controls. These metabolites were not observed in recent doping control samples that yielded adverse analytical findings for methylstenbolone, and in the light of additional data originating from a recent publication on the in vivo metabolism of methylstenbolone in the horse, revisiting the metabolic reactions in humans appeared warranted. Therefore, deuterated methylstenbolone together with hydrogen isotope ratio mass spectrometry (IRMS) in combination with high accuracy/high resolution mass spectrometry were employed. After oral administration of a single dose of 10 mg of doubly labeled methylstenbolone, urine samples were collected for 29 days. Up to 40 different deuterated methylstenbolone metabolites were detected in post-administration samples, predominantly as glucuronic acid conjugates, and all were investigated regarding their potential to prolong the detection window for doping controls. Besides methylstenbolone excreted glucuronidated, three additional metabolites were still detectable at the end of the study on day 29. The most promising candidates for inclusion into routine sports drug testing methods (2α,17α-dimethyl-5α-androst-1-ene-3ß,17ß-diol and 2α,17α-dimethyl-5α-androst-1-ene-3α,17ß-diol) were synthesized and characterized by NMR.

Studies on the in vivo metabolism of the SARM YK11: Identification and characterization of metabolites potentially useful for doping controls.

A steroidal compound was recently detected in a seized black market product and was identified as (17α,20E)-17,20-[(1-methoxyethylidene) bis (oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11). This compound is described to possess selective androgen receptor modulator- and myostatin inhibitor-like properties. As YK11 is an experimental drug candidate and a non-approved substance for humans, scientific data on its metabolism is scarce. Due to its steroidal backbone and the arguably labile orthoester-derived moiety positioned at the D-ring, substantial metabolic conversion in vivo was anticipated. To unambiguously detect urinary metabolites of YK11, an elimination study with six-fold deuterated YK11 was conducted. Post-administration specimens were analyzed using hydrogen isotope ratio mass spectrometry coupled to single quadrupole mass spectrometry to identify metabolites alongside basic mass spectrometric data. Further characterization of those metabolites relevant to sports drug testing was accomplished using gas chromatography-high resolution-high accuracy mass spectrometry. Fourteen deuterated urinary metabolites were detected comprising unconjugated, glucuronidated, and sulfoconjugated metabolites. As expected, no intact YK11 was observed in the elimination study urine samples. While the unconjugated metabolites disappeared within 24 hours post-administration, both glucuronidated and sulfated metabolites were traceable for more than 48 hours. The chemical structures of the two most promising glucuronidated metabolites (5ß-19-nor-pregnane-3α,17ß,20-triol and 5ß-19-nor-pregnane-3α,17ß-diol-20-one) were verified by in-house synthesis of both metabolites and confirmed by nuclear magnetic resonance analysis. In order to elucidate their potential in sports drug testing, both were successfully implemented into the currently applied analytical method for the detection of anabolic agents.

Screening for adiponectin receptor agonists and their metabolites in urine and dried blood spots.

AdipoRon and 112254 represent two known synthetic adiponectin receptor (adipoR) agonists. Although AdipoRon is the more potent compound, both have physiological properties that are similar to adiponectin - an adipokine with antidiabetic and antiatherogenic effects. Several transcriptional regulators are activated by adipoR-agonists leading to increased mitochondrial DNA content in vitro, an effect that can be abused by athletes for performance enhancing purposes. In the context of preventive anti-doping research, detection of AdipoRon and 112254 in routine doping control specimens would be valuable. Here, we describe our process for incorporating AdipoRon and 112254 into routine doping control methods involving urine and dried blood spot (DBS) analysis. Method validation including evaluation of specificity, limit of detection, identification capability, carryover, matrix interference, recovery, interday and intraday precision and linearity to standards provided by WADA. For identification in human urine, a liquid chromatography-tandem mass spectrometry-based testing approach was implemented for both adipoR agonists and two respective phase-I metabolites. Recovery of 85-104%, satisfactory limits of detection (i.e., 0.5-1 ng/mL), and imprecision values over three days at three concentration levels of <19% demonstrated the assay's fitness-for-purpose. For identification from DBS a liquid chromatography-high-resolution/high-accuracy tandem mass spectrometry with online solid-phase extraction was implemented for AdipoRon and 112254. Here also, acceptable recoveries (i.e., 22-33%), limits of detection of 5-10 ng/mL, and imprecision values over three days at three concentration levels of <23%, were demonstrated. Hence, two methods for doping control screening from urine and DBS were established and shown to be fit-for-purpose for routine use.

Complementing the characterization of in vivo generated N-glucuronic acid conjugates of stanozolol by collision cross section computation and analysis.

Detailed structural information on metabolites serving as target analytes in clinical, forensic, and sports drug testing programmes is of paramount importance to ensure unequivocal test results. In the present study, the utility of collision cross section (CCS) analysis by travelling wave ion mobility measurements to support drug metabolite characterization efforts was tested concerning recently identified glucuronic acid conjugates of the anabolic-androgenic steroid stanozolol. Employing travelling-wave ion mobility spectrometry/quadrupole-time-of-flight mass spectrometry, drift times of five synthetically derived and fully characterized steroid glucuronides were measured and subsequently correlated to respective CCSs as obtained in silico to form an analyte-tailored calibration curve. The CCSs were calculated by equilibrium structure minimization (density functional theory) using the programmes ORCA with the data set B3LYP/6-31G and MOBCAL utilizing the trajectory method (TM) with nitrogen as drift gas. Under identical experimental conditions, synthesized and/or urinary stanozolol-N and O-glucuronides were analyzed to provide complementary information on the location of glucuronidation. Finally, the obtained data were compared to CCS results generated by the system's internal algorithm based on a calibration employing a polyalanine analyte mixture. The CCSs ΩN2 calculated for the five steroid glucuronide calibrants were found between 180 and 208 Å(2) , thus largely covering the observed and computed CCSs for stanozolol-N1'-, stanozolol-N2'-, and stanozolol-O-glucuronide found at values between 195.1 and 212.4 Å(2) . The obtained data corroborated the earlier suggested N- and O-glucuronidation of stanozolol, and demonstrate the exploit of ion mobility and CCS computation in structure characterization of phase-II metabolic products; however, despite reproducibly measurable differences in ion mobility of stanozolol-N1'-, N2'-, and O-glucuronides, the discriminatory power of the chosen CCS computation algorithm was found to be not appropriate to allow for accurate assignments of the two N-conjugated structures. Using polyalanine-based calibrations, significantly different absolute values were obtained for all CCSs, but due to a constant offset of approximately 45 Å(2) an excellent correlation (R(2) = 0.9997) between both approaches was observed. This suggests a substantially accelerated protocol when patterns of computed and polyalanine-based experimental data can be used for structure elucidations instead of creating individual analyte-specific calibration curves.

A new ursane triterpenoic acid and other potential anti-inflammatory and anti-arthritic constituents from EtOAc extracts of Vitellaria paradoxa stem bark.

OBJECTIVE: Vitellaria paradoxa (shea tree) is used in traditional medicine for the treatment of various ailments, including, inflammation and fever. Therefore the present research investigates the anti-inflammatory and anti-rheumatic effects of V. paradoxa stem bark extracts in rats and the isolation and characterization of its active constituents. METHODS: The anti-inflammatory activity of ethyl acetate extract of V. Paradoxa (VPEE) was evaluated by use of the carrageenan-induced paw oedema model in rats. Moreover, rheumatoid arthritis (RA) was induced by injection of Freund's Completed Adjuvant (FCA) into the subplantar surface of the hind paw of the male Wistar rats. Paw volume was measured plethysmometrically. Joint swelling was measured using electronic vernier caliper. Hot plate test was used to assess the effect of VPEE on hyperalgesia while open field was used to assess the locomotors activity. The relative weight of spleen, liver and thymus was obtained as well as some haematological parameters. Tibiotarsal joint was extracted for histopathology under light microscope. Chemical analysis was carried out by high resolution mass spectrometry and one and two-dimensional NMR techniques. RESULTS: LC-MS analysis of the EtOAc extract revealed the presence of a new triterpenoid and several known compounds. The structure of the novel compound was elucidated by means of LC-MS and selected 1D and 2D-NMR experiments. The biological effects of ethyl acetate (VPEE), methanol (VPME) and water extracts (VPAE) of V. paradoxa were tested on carrageenan model of acute inflammation and FCA-induced rheumatoid arthritis animal model. In the carrageenan-induced inflammation, VPEE (150 mg/kg) significant (66.67%) inhibited the first (after 1h) and the second phase (4-6h) of edema formation. On the Complete Freund's adjuvant-induced rheumatoid arthritis, VPEE at the same dose showed a significantly protective effect. On days 19-28th of treatment, the maximum inflammatory percentage was between 9.60 and 8.91% for the VPEE compared to 30.91-24.29% for the controls. All the extracts significantly reduced the score of arthritis but the maximal reduction was obtained with the VPEE on day 24th of the experimentation. The altered haematological parameters in the arthritic rats were significantly recovered to near normal by the treatment with VPEE at the dose of 150 mg/kg. Further histological studies revealed the anti-arthritic activity by preventing cartilage destruction of the arthritic joints of adjuvant arthritic rats. The spleen hypertrophy induced by the FCA was also significantly inhibited. CONCLUSION: These findings provide pharmacological basis for the application of the VPEE in inflammatory disorders.