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.

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.

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.

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.

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.

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.