Daniel Eichner

Metabolism of methylstenbolone studied with human liver microsomes and the uPA⁺/⁺-SCID chimeric mouse model.

Anti-doping laboratories need to be aware of evolutions on the steroid market and elucidate steroid metabolism to identify markers of misuse. Owing to ethical considerations, in vivo and in vitro models are preferred to human excretion for nonpharmaceutical grade substances. In this study the chimeric mouse model and human liver microsomes (HLM) were used to elucidate the phase I metabolism of a new steroid product containing, according to the label, methylstenbolone. Analysis revealed the presence of both methylstenbolone and methasterone, a structurally closely related steroid. Via HPLC fraction collection, methylstenbolone was isolated and studied with both models. Using HLM, 10 mono-hydroxylated derivatives (U1-U10) and a still unidentified derivative of methylstenbolone (U13) were detected. In chimeric mouse urine only di-hydroxylated metabolites (U11-U12) were identified. Although closely related, neither methasterone nor its metabolites were detected after administration of isolated methylstenbolone. Administration of the steroid product resulted mainly in the detection of methasterone metabolites, which were similar to those already described in the literature. Methylstenbolone metabolites previously described were not detected. A GC-MS/MS multiple reaction monitoring method was developed to detect methylstenbolone misuse. In one out of three samples, previously tested positive for methasterone, methylstenbolone and U13 were additionally detected, indicating the applicability of the method.

Sensitive quantification of IGF-1 and its synthetic analogs in dried blood spots

BACKGROUND Dried blood spot sample collection could improve detection of the misuse of IGF-1, its analogs and growth hormone. An LC-MS/MS method was developed to measure two IGF-1 peptides and one analog peptide after trypsin digestion. In addition to standard method validation parameters, the effect of hematocrit on cysteine alkylation, trypsin digestion and the selection of internal standard were evaluated. RESULTS Quantification of IGF-1 peptides was possible with an LLOQ of 25 ng/ml and imprecision of less than 15%. CONCLUSION While the effects of hematocrit must be evaluated empirically for each method, dried blood spots are a suitable matrix for the measurement of IGF-1 and its analogs by MS.

A population study of urine glycerol concentrations in elite athletes competing in North America

Glycerol is an endogenous substance that is on the World Anti-Doping Agencys list of prohibited threshold substances due to its potential use as a plasma volume expansion agent. The WADA has set the threshold for urine glycerol, including measurement uncertainty, at 1.3 mg/mL. Glycerol in circulation largely comes from metabolism of triglycerides in order to meet energy requirements and when the renal threshold is eclipsed, glycerol is excreted into urine. In part due to ethnic differences in postprandial triglyceride concentrations, we investigated urine glycerol concentrations in a population of elite athletes competing in North America and compared the results to those of athletes competing in Europe. 959 urine samples from elite athletes competing in North America collected for anti-doping purposes were analyzed for urine glycerol concentrations by a gas chromatography mass-spectrometry method. Samples were divided into groups according to: Timing (in- or out-of-competition), Class (strength, game, or endurance sports) and Gender. 333 (34.7%) samples had undetectable amounts of glycerol (<1 μg/mL). 861 (89.8%) of the samples had glycerol concentrations ≤20 μg/mL. The highest glycerol concentration observed was 652 μg/mL. Analysis of the data finds the effects of each category to be statistically significant. The largest estimate of the 99.9(th) percentile, from the in-competition, female, strength athlete samples, was 1813 μg/mL with a 95% confidence range from 774 to 4251 μg/mL. This suggests a conservative threshold of 4.3 mg/mL, which would result in a reasonable detection window for urine samples collected in-competition for all genders and sport classes.

Detection of human insulin‐like growth factor‐1 in deer antler velvet supplements

RATIONALE Reported incidents of the use of nutritional supplements containing deer antler velvet by athletes has increased significantly in recent years. The supplements have been reported to contain insulin-like growth factor-1 (IGF-1), which is a banned substance included on the World Anti-Doping Agency (WADA) prohibited list. The presence of deer and human IGF-1 was tested in six commercially available supplements. METHODS IGF-1 was extracted from the six deer antler velvet supplements using chloroform and acetonitrile precipitation methods. Ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) methods were developed to measure intact IGF-1 protein and IGF-1 trypsin peptides using a triple quadrupole mass spectrometer. Five deer-specific and five human-specific multiple-reaction monitoring (MRM) transitions for intact IGF-1were measured as well as six deer-specific and seven human-specific MRM transitions for an IGF-1 trypsin peptide. RESULTS The peak area from each MRM transition was used to calculate the product ion ratios relative to the most abundant transition. Product ion ratios measured in the supplements were matched to ratios measured in purified protein standards. A match to human IGF-1 was identified for all the MRM transitions measured in four of the supplements tested. CONCLUSIONS The presence of a pharmaceutical protein, human IGF-1, was confirmed in four commercially available products sold as all natural, nutritional supplements. These methods can be used to screen additional products to further prevent the illegal sale of adulterated supplements.

Intranasal delivery of Natesto® testosterone gel and its effects on doping markers

The laboratory profile of intranasal testosterone gel has not been previously reported from an anti-doping perspective. Because intranasal testosterone gel is newly available as a commercial product, we sought to examine the laboratory parameters following administration of this formulation, with particular attention to anti-doping guidelines. Five healthy and active male subjects were administered testosterone intranasal gel three times daily for four weeks, using a pattern of five consecutive days on, two days off. Urine was collected after each five-day round of drug administration and analyzed using a full steroid screen and isotope ratio mass spectrometry (IRMS). Windows of detection for elevated testosterone/epitestosterone (T/E) and other steroid ratios, World Anti-Doping Agency (WADA) athlete biological passport (ABP) findings, and IRMS results were analyzed in this study. In the 0-24 h window post-administration, 70% of samples were flagged with a suspicious steroid profile and 85% were flagged as atypical passport findings according to the WADA ABP steroid module. In the 24-48 h window, 0% of samples displayed suspicious steroid profiles while 40% resulted in atypical passport findings. IRMS testing confirmed the presence of exogenous testosterone in 90% and 40% of samples in the 0-24 h and 24-48 h windows post-administration, respectively. Additionally, IRMS data were analyzed to determine commonalities in the population changes in δ13 C values of testosterone, androsterone, etiocholanolone, 5αAdiol, and 5βAdiol. Though no discernible metabolic trend of the route of administration was identified, we discovered that intranasal gel testosterone is detectable using conventional anti-doping tests. Copyright

Chemical Composition and Labeling of Substances Marketed as Selective Androgen Receptor Modulators and Sold via the Internet

Importance Recent reports have described the increasing use of nonsteroidal selective androgen receptor modulators, which have not been approved by the US Food and Drug Administration (FDA), to enhance appearance and performance. The composition and purity of such products is not known. Objective To determine the chemical identity and the amounts of ingredients in dietary supplements and products marketed and sold through the internet as selective androgen receptor modulators and compare the analyzed contents with product labels. Design and Setting Web-based searches were performed from February 18, 2016, to March 25, 2016, using the Google search engine on the Chrome and Internet Explorer web browsers to identify suppliers selling selective androgen receptor modulators. The products were purchased and the identities of the compounds and their amounts were determined from April to August 2016 using chain-of-custody and World Anti-Doping Association–approved analytical procedures. Analytical findings were compared against the label information. Exposures Products marketed and sold as selective androgen receptor modulators. Main Outcomes and Measures Chemical identities and the amount of ingredients in each product marketed and sold as selective androgen receptor modulators. Results Among 44 products marketed and sold as selective androgen receptor modulators, only 23 (52%) contained 1 or more selective androgen receptor modulators (Ostarine, LGD-4033, or Andarine). An additional 17 products (39%) contained another unapproved drug, including the growth hormone secretagogue ibutamoren, the peroxisome proliferator-activated receptor-&dgr; agonist GW501516, and the Rev-ErbA agonist SR9009. Of the 44 tested products, no active compound was detected in 4 (9%) and substances not listed on the label were contained in 11 (25%). In only 18 of the 44 products (41%), the amount of active compound in the product matched that listed on the label. The amount of the compounds listed on the label differed substantially from that found by analysis in 26 of 44 products (59%). Conclusions and Relevance In this limited investigation involving chemical analyses of 44 products marketed as selective androgen receptor modulators and sold via the internet, most products contained unapproved drugs and substances. Only 52% contained selective androgen receptor modulators and many were inaccurately labeled.

Investigation of the metabolites of the HIF stabilizer FG-4592 (roxadustat) in five different in vitro models and in a human doping control sample using high resolution mass spectrometry

HIGHLIGHTSMetabolites from the HIF stabilizer FG‐4592 were investigated.Five in vitro methods and a human doping control sample were used as models.Twelve different metabolites were detected using UHPLC‐QTOF‐MS.Three of the in vitro models had one metabolite in common. ABSTRACT FG‐4592 is a hypoxia‐inducible factor (HIF) stabilizer, which can increase the number of red blood cells in the body. It has not been approved by regulatory authorities, but is available for purchase on the Internet. Due to its ability to improve the oxygen transportation mechanism in the body, FG‐4592 is of interest for doping control laboratories, but prior to this study, little information about its metabolism was available. In this study, the metabolism of FG‐4592 was investigated in a human doping control sample and in five in vitro models: human hepatocytes and liver microsomes, equine liver microsomes and S9 fraction and the fungus Cunninghamella elegans. By using liquid chromatography coupled to a Q‐TOF mass spectrometer operated in MSE and MSMS modes, twelve different metabolites were observed for FG‐4592. One monohydroxylated metabolite was detected in both the human and equine liver microsome incubations. For the fungus Cunninghamella elegans eleven different metabolites were observed of which the identical monohydroxylated metabolite had the highest response. This rich metabolic profile and the higher levels of metabolites produced by Cunninghamella elegans demonstrates its usefulness as a metabolite producing medium. In the doping control urine sample, one metabolite, which was the result of a direct glucuronidation, was observed. No metabolites were detected in neither the human hepatocyte nor in the equine liver S9 fraction incubates.

Iatrogenic Cushing Syndrome in a Child With Congenital Adrenal Hyperplasia: Erroneous Compounding of Hydrocortisone

Context Patients with 21-hydroxylase deficiency congenital adrenal hyperplasia (CAH) require lifelong treatment with glucocorticoids. In growing children, the drug of choice is hydrocortisone. Commercially available hydrocortisone tablets do not conform to very low doses prescribed to infants and toddlers, and compounded hydrocortisone is often dispensed to meet therapeutic needs. However, safety, efficacy, and uniformity of compounded products are not tested. We report a case of Cushing syndrome in a child with CAH who was inadvertently receiving excessive hydrocortisone in compounded form. Design A 20-month-old girl with CAH developed growth deceleration, excessive weight for length, irritability, increased facial fat, plethora, and excess body hair while receiving hydrocortisone from a local compounding pharmacy. The signs and symptoms persisted despite decreasing hydrocortisone dose. Iatrogenic Cushing syndrome was suspected. The prescribed hydrocortisone capsules were sent for analysis to the Sports Medicine Research & Testing Laboratory, where testing revealed that each 1-mg hydrocortisone capsule contained five to 10 times the dose prescribed and listed on the label. Conclusion Physicians must be aware that errors in compounded medications may lead to unanticipated adverse effects. Iatrogenic Cushing syndrome should be suspected in any child receiving compounded glucocorticoid treatment who develops growth arrest and excess weight gain.

Mass Spectrometry Method to Measure Membrane Proteins in Dried Blood Spots for the Detection of Blood Doping Practices in Sport

The dried blood spot (DBS) matrix has significant utility for applications in the field where venous blood collection and timely shipment of labile blood samples is difficult. Unfortunately, protein measurement in DBS is hindered by high abundance proteins and matrix interference that increases with hematocrit. We developed a DBS method to enrich for membrane proteins and remove soluble proteins and matrix interference. Following a wash in a series of buffers, the membrane proteins are digested with trypsin and quantitated by parallel reaction monitoring mass spectrometry methods. The DBS method was applied to the quantification of four cell-specific cluster of differentiation (CD) proteins used to count cells by flow cytometry, band 3 (CD233), CD71, CD45, and CD41. We demonstrate that the DBS method counts low abundance cell types such as immature reticulocytes as well as high abundance cell types such as red blood cells, white blood cells, and platelets. When tested in 82 individuals, counts obtained by the DBS method demonstrated good agreement with flow cytometry and automated hematology analyzers. Importantly, the method allows longitudinal monitoring of CD protein concentration and calculation of interindividual variation which is difficult by other methods. Interindividual variation of band 3 and CD45 was low, 6 and 8%, respectively, while variation of CD41 and CD71 was higher, 18 and 78%, respectively. Longitudinal measurement of CD71 concentration in DBS over an 8-week period demonstrated intraindividual variation 17.1-38.7%. Thus, the method may allow stable longitudinal measurement of blood parameters currently monitored to detect blood doping practices.

Detection and in vitro metabolism of AOD9604

AOD9604 is a peptide consisting of the C-terminal fragment of human growth hormone from amino acids 177-191 with an additional tyrosine residue at the N-terminus of the peptide. It is reported to mimic the lipolytic properties of growth hormone without the diabetogenic side effects. Therefore, AOD9604 may be used as a performance enhancing drug and is banned by the World Anti-doping Agency (WADA). The peptide is available on several Internet websites and was recently identified in confiscated vials in the USA. To detect abuse of the peptide in athletes, a solid-phase extraction method was validated in urine with a limit of detection of 50 pg/mL. The method has good linearity, precision (<20%), specificity and recovery (62%). Six potential metabolites of the peptide were identified after incubation of AOD9604 in serum and urine. Quantification of the metabolites in serum identified a single metabolite, consisting of amino acids CRSVEGSCG, which is significantly more stable than the other metabolites or the parent compound. Screening for AOD9604 and the stable metabolite may potentially allow an increased window of detection.