Maria Kristina Parr

Analytical strategies for the detection of non-labelled anabolic androgenic steroids in nutritional supplements

Recent studies showed that non-hormonal supplements such as vitamins, minerals and amino acids can contain anabolic androgenic steroids not declared on the labels of the products. These undeclared substances (often prohormones of testosterone or 19-nortestosterone) can cause health risks to consumers and might lead to positive results in sports doping control, especially for the nandrolone metabolite norandrosterone. The analysis of nutritional supplements for anabolic steroids has proven to be rather difficult due to the different matrices in the various products. To conduct a broad-based analysis, a few robust methods capable of analysing various matrices are needed. To obtain a sensitive gas chromatography-mass spectrometry (GC-MS) analysis, a method including extraction and purification of the analytes followed by GC-MS analysis of the trimethylsilyl (TMS) derivatives of the steroids was developed. The limit of detection was improved by the addition of a mixture of 1-N,N-diisopropylamino-n-alkanes (DIPAs) to the final extract. In pure creatine monohydrate powder the limits of detection were demonstrated to be 0.1 ng μg −1 for dehydroepiandrosterone (DHEA) and estr-4-ene-3β,17β-diol, 0.7 ng g−1 for 5α-androstane-3β,17β-diol and androsta-1,4-diene-3,17-dione, 1 ng g−1 for estr-5-ene-3β,17β-diol, estr-4-ene-3,17-dione, 19-nortestosterone, androst-4-ene-3, 17-dione and testosterone, and 2 ng g−1 for androst-4-ene-3β,17β-diol and androst-5-ene-3β,17β-diol. The recovery (determined at 200 ng g−1) ranged from 32% for 19-nortestosterone to 92% for androst-5-ene-3β,17β-diol. During the investigation of different nutritional supplements, several analytical difficulties occurred. Aspects of homogenization, extraction, separation, derivatization and GC-MS measurement as well as strategies for the solution of problems arising were optimized. For quantitative measurements of the steroids in nutritional supplements, deuterated internal standards of the specific steroids or standard addition are necessary to compensate for matrix effects.

Detection and characterization of a new metabolite of 17α-methyltestosterone.

The misuse of the anabolic steroid methyltestosterone is currently routinely monitored in doping control laboratories by gas chromatography-mass spectrometry (GC-MS) of two of its metabolites: 17α-methyl-5β-androstane-3α,17β-diol and 17α-methyl-5α-androstane-3α,17β-diol. Because of the absence of any easy ionizable moiety, these metabolites are poorly detectable using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI). In this study, the metabolism of methyltestosterone has been reinvestigated by the use of a precursor ion scan method in LC-ESI-MS/MS. Two metabolites have been detected using this method. Both compounds have been confirmed in postadministration urine samples of an urokinase plasminogen activator-severe combined immunodeficiency (uPA-SCID) mouse with humanized liver and were characterized by LC-MS/MS and GC-MS using both quadrupole and time of flight analyzers. From the detailed study of the fragmentation, these metabolites were proposed to be epimethyltestosterone and a dehydrogenated compound. Epimethyltestosterone has previously been described as a minor metabolite, whereas the occurrence of the oxidized metabolite has not been reported. Comparison with the synthesized reference revealed that the structure of the dehydrogenated metabolite is 6-ene-epimethyltestosterone. A selected reaction monitoring method including three transitions for each metabolite has been developed and applied to samples from an excretion study and to samples declared positive after GC-MS analysis. 6-Ene-epimethyltestosterone was found in all samples, showing its applicability in the detection of methyltestosterone misuse.

CYP21-catalyzed production of the long-term urinary metandienone metabolite 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one: a contribution to the fight against doping

Abstract Anabolic-androgenic steroids are some of the most frequently misused drugs in human sports. Recently, a previously unknown urinary metabolite of metandienone, 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one (20OH-NorMD), was discovered via LC-MS/MS and GC-MS. This metabolite was reported to be detected in urine samples up to 19 days after administration of metandienone. However, so far it was not possible to obtain purified reference material of this metabolite and to confirm its structure via NMR. Eleven recombinant strains of the fission yeast Schizosaccharomyces pombe that express different human hepatic or steroidogenic cytochrome P450 enzymes were screened for production of this metabolite in a whole-cell biotransformation reaction. 17,17-Dimethyl-18-norandrosta-1,4,13-trien-3-one, chemically derived from metandienone, was used as substrate for the bioconversion, because it could be converted to the final product in a single hydroxylation step. The obtained results demonstrate that CYP21 and to a lesser extent also CYP3A4 expressing strains can catalyze this steroid hydroxylation. Subsequent 5 l-scale fermentation resulted in the production and purification of 10 mg of metabolite and its unequivocal structure determination via NMR. The synthesis of this urinary metandienone metabolite via S. pombe-based whole-cell biotransformation now allows its use as a reference substance in doping control assays.

Analytical methods for the detection of clenbuterol

Clenbuterol is therapeutically used for the treatment of pulmonary diseases such as bronchial asthma or for tocolytic reasons. In cattle feeding as well as in sports it is illicitly misused due to its anabolic properties to promote muscle growth. Sample preparation procedures and analytical techniques used for the detection of clenbuterol are manifold and vary with the objectives of the investigation. Methods for its detection in biological specimens, drug preparations, the environment, food and feed products are reported. They are mainly based on immunochemical, chromatographic and mass spectrometric techniques, or on capillary electrophoresis. Sample preparation primarily includes liquid-liquid extraction and solid-phase extraction. Depending on the aim of the method clenbuterol can be determined in single- or multi-analyte methods. In biological and environmental samples concentrations are generally low due to the potency of the drug. Thus, highly sensitive procedures are required for expedient analyses.

Estrogen receptor beta is involved in skeletal muscle hypertrophy induced by the phytoecdysteroid ecdysterone

SCOPE The phytoectysteroid ecdysterone (Ecdy) was reported to stimulate protein synthesis and enhance physical performance. The aim of this study was to investigate underlying molecular mechanisms particularly the role of ER beta (ERβ). RESULTS In male rats, Ecdy treatment increased muscle fiber size, serum IGF-1 increased, and corticosteron and 17β-estradiol (E2) decreased. In differentiated C2C12 myoblastoma cells, treatment with Ecdy, dihydrotestosterone, IGF-1 but also E2 results in hypertrophy. Hypertrophy induced by E2 and Ecdy could be antagonized with an antiestrogen but not by an antiandrogen. In HEK293 cells transfected with ER alpha (ERα) or ERβ, Ecdy treatment transactivated a reporter gene. To elucidate the role of ERβ in Ecdy-mediated muscle hypertrophy, C2C12 myotubes were treated with ERα (ALPHA) and ERβ (BETA) selective ligands. Ecdy and BETA treatment but not ALPHA induced hypertrophy. The effect of Ecdy, E2, and BETA could be antagonized by an ERβ-selective antagonist (ANTIBETA). In summary, our results indicate that ERβ is involved in the mediation of the anabolic activity of the Ecdy. CONCLUSION These findings provide new therapeutic perspectives for the treatment of muscle injuries, sarcopenia, and cachectic disease, but also imply that such a substance could be abused for doping purposes.

Unexpected contribution of cytochrome P450 enzymes CYP11B2 and CYP21, as well as CYP3A4 in xenobiotic androgen elimination – Insights from metandienone metabolism

The metabolism of a variety of anabolic steroids frequently misused for doping purposes has been investigated in the last years. This research mainly focused on main and long-term metabolites suitable for detection, but detailed clearance mechanisms have rarely been elucidated. Recent studies on metandienone focused on the identification of 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one (20βOH-NorMD) as long-term metabolite, however, the metabolic pathway of its generation remained unclear. Metandienone and its Wagner-Meerwein rearrangement product 17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one (NorMD) were hydroxylated by different human cytochrome P450 enzymes (CYPs). Some of their hydroxylation products were chemically synthesized and characterized by mass spectrometry to allow for their trace detection in urine samples. Following oral administration of metandienone or NorMD in one human volunteer each the post administration urines were checked for the presence of those hydroxylated metabolites using GC-MS/MS analysis. The human mitochondrial steroid hydroxylating enzymes CYP11B1 and CYP11B2 were capable to metabolize metandienone leading to the formation of 11β-hydroxymetandienone and 18-hydroxymetandienone. Following Wagner-Meerwein rearrangement, the resulting products could be assigned to 20βOH-NorMD and 11βOH-NorMD. The contribution of CYP11B1 and CYP11B2 in human metabolism of metandienone was confirmed by analysis of post-administration samples of metandienone and NorMD. Combined with the results from a previous study, enzymatic pathways were identified that involve CYP21 and CYP3A4 in the hydroxylation of NorMD, while CYP21, CYP3A4 and CYP11B2 take part in 20βOH-NorMD generation from MD. The current study represents a valuable contribution to the elucidation of clearance mechanisms of anabolic steroids and also indicates that mainly non-liver CYPs seem to be involved in these processes.

Physicochemical characterization of biopharmaceuticals

Biopharmaceuticals are gaining interest in therapy due to their high target selectivity. Most of the recently approved biopharmaceuticals represent drugs that are produced by biotechnological processes involving recombinant DNA. Thus, this review article mainly focusses on protein therapeutics. However analogous considerations also apply for other large molecule therapeutics. As early approved blockbuster biopharmaceuticals run out of patent protection shortly, a growing interest in biosimilar production results in the need of proper analytical characterization and comparison of inventor and biosimilar. In contrast to small molecule drugs small variations in the production process may strongly impact the final biological. Thus, quality assurance of biopharmaceuticals results in much higher analytical effort compared to small molecules. This review gives an overview on analytical methods for characterization of protein biologicals. Classical methods such as gel electrophoresis and liquid chromatography are summarized and complemented with state-of-the-art mass spectrometric investigations. Full molecule investigations of native or denaturized proteins as well as methods including digestion (middle-down and bottom-up) are discussed. Furthermore, literature on glycoprotein analysis using glycopeptide, released glycan and monosaccharide analysis is reviewed.

Ecdysteroids: A novel class of anabolic agents?

Increasing numbers of dietary supplements with ecdysteroids are marketed as “natural anabolic agents”. Results of recent studies suggested that their anabolic effect is mediated by estrogen receptor (ER) binding. Within this study the anabolic potency of ecdysterone was compared to well characterized anabolic substances. Effects on the fiber sizes of the soleus muscle in rats as well the diameter of C2C12 derived myotubes were used as biological readouts. Ecdysterone exhibited a strong hypertrophic effect on the fiber size of rat soleus muscle that was found even stronger compared to the test compounds metandienone (dianabol), estradienedione (trenbolox), and SARM S 1, all administered in the same dose (5 mg/kg body weight, for 21 days). In C2C12 myotubes ecdysterone (1 µM) induced a significant increase of the diameter comparable to dihydrotestosterone (1 µM) and IGF 1 (1.3 nM). Molecular docking experiments supported the ERβ mediated action of ecdysterone. To clarify its status in sports, ecdysterone should be considered to be included in the class “S1.2 Other Anabolic Agents” of the list of prohibited substances of the World Anti-Doping Agency.

INGESTION OF DESIGNER SUPPLEMENTS PRODUCED POSITIVE DOPING CASES UNEXPECTED BY THE ATHLETES

Since the end of the last century, the sport community has been grappling with contamination and faking of dietary or nutritional supplements. Publication of the results of many studies on this problem has resulted in educational and informative actions on risk connected with consumption of unchecked products. As compared to the past, nowadays athletes have become incomparably more aware of the potential risk of a positive result in anti-doping tests as a consequence of consumption of contaminated dietary supplements. However, this problem is still topical, which is confirmed by regular reports, presented e.g. during the Cologne Workshops on Dope Analysis held annually in Germany. This paper presents a few doping cases caused by the use of supplements with doping substances by athletes, noted by two WADA accredited laboratories (Cologne and Warsaw). Still more education on the health and doping risks of dietary supplement products seems to be necessary for the protection of both athletes and the general public.

SFC-MS/MS as an orthogonal technique for improved screening of polar analytes in anti-doping control

AbstractHPLC is considered the method of choice for the separation of various classes of drugs. However, some analytes are still challenging as HPLC shows limited resolution capabilities for highly polar analytes as they interact insufficiently on conventional reversed-phase (RP) columns. Especially in combination with mass spectrometric detection, limitations apply for alterations of stationary phases. Some highly polar sympathomimetic drugs and their metabolites showed almost no retention on different RP columns. Their retention remains poor even on phenylhexyl phases that show different selectivity due to π–π interactions. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to HPLC may help to overcome these issues. Selected polar drugs and metabolites were analyzed utilizing SFC separation. All compounds showed sharp peaks and good retention even for the very polar analytes, such as sulfoconjugates. Retention times and elution orders in SFC are different to both RP and HILIC separations as a result of the orthogonality. Short cycle times could be realized. As temperature and pressure strongly influence the polarity of supercritical fluids, precise regulation of temperature and backpressure is required for the stability of the retention times. As CO2 is the main constituent of the mobile phase in SFC, solvent consumption and solvent waste are considerably reduced. Graphical AbstractSFC-MS/MS vs. LC-MS/MS