Detection of LongR3 -IGF-I, Des(1-3)-IGF-I, and R3 -IGF-I using immunopurification and high resolution mass spectrometry for antidoping purposes.

Insulin-like growth factor-I (IGF-I) and its analogs LongR3 -IGF-I, Des(1-3)-IGF-I, and R3 -IGF-I are prohibited substances in sport. Although they were never approved for use in humans, they are readily available as black market products for bodybuilding and can be used to enhance physical performance. This study's aims were to validate a fast and sensitive detection method for IGF-I analogs and to evaluate their detectability after intramuscular administration in rats. The sample preparation consisted of an immunopurification on MSIA™ microcolumns using a polyclonal anti-human-IGF-I antibody. The target substances were then directly analyzed by nano-liquid chromatography coupled with high-resolution mass spectrometry. Abundant signs of lower quality, oxidized peptide forms were found in black market products, justifying the need to monitor at least both the native and mono-oxidized forms. The analytical performance of this method (linearity, carry over, detection limits, precision, specificity, recovery, and matrix effect) was studied by spiking the analogs into human serum. Following a single intramuscular administration (100 µg/kg) in rats, detection was evaluated up to 36 h after injection. While unchanged Des(1-3)-IGF-I and R3 -IGF-I were detected until 24 h after administration, LongR3 -IGF-I disappeared rapidly after 4 h. Des(1)-LongR3 -IGF-I, a new N-terminal Long-R3 -IGF-I degradation product, was detected in addition to Des(1-10)-LongR3 -IGF-I and Des(1-11)-LongR3- IGF-I: the latter was detected up to 16 h. The same products were found after in vitro incubation of the analogs in human whole blood, suggesting that observations in rats may be extrapolated to humans and that the validated method may be applicable to antidoping testing.

Use of capillary dried blood for quantification of intact IGF-I by LC-HRMS for antidoping analysis.

Background: IGF-I is used as a biomarker to detect Growth Hormone doping in athletes' blood samples. Objective: Our aim was to develop and validate a fast, high-throughput and accurate quantification of intact IGF-I from volumetric absorptive microsampling (VAMS) dried blood using LC coupled to high resolution mass spectrometry (LC-HRMS). Methodology & results: IGF-I was extracted from the VAMS, released from its binding proteins, concentrated using microelution SPE and analyzed by LC-HRMS. The method was successfully validated in accordance with the World Anti-Doping Agency's requirements. Subsequently, IGF-I measurements from capillary dried blood and serum were compared. Conclusion: The combination of VAMS, microelution SPE and LC-HRMS is a promising strategy applicable to IGF-I quantification in athletes' samples.

Mass spectrometric characterization of the hypoxia-inducible factor (HIF) stabilizer drug candidate BAY 85-3934 (molidustat) and its glucuronidated metabolite BAY-348, and their implementation into routine doping controls.

The development of new therapeutics potentially exhibiting performance-enhancing properties implicates the risk of their misuse by athletes in amateur and elite sports. Such drugs necessitate preventive anti-doping research for consideration in sports drug testing programmes. Hypoxia-inducible factor (HIF) stabilizers represent an emerging class of therapeutics that allows for increasing erythropoiesis in patients. BAY 85-3934 is a novel HIF stabilizer, which is currently undergoing phase-2 clinical trials. Consequently, the comprehensive characterization of BAY 85-3934 and human urinary metabolites as well as the implementation of these analytes into routine doping controls is of great importance. The mass spectrometric behaviour of the HIF stabilizer drug candidate BAY 85-3934 and a glucuronidated metabolite (BAY-348) were characterized by electrospray ionization-(tandem) mass spectrometry (ESI-MS(/MS)) and multiple-stage mass spectrometry (MSn ). Subsequently, two different laboratories established different analytical approaches (one each) enabling urine sample analyses by employing either direct urine injection or solid-phase extraction. The methods were cross-validated for the metabolite BAY-348 that is expected to represent an appropriate target analyte for human urine analysis. Two test methods allowing for the detection of BAY-348 in human urine were applied and cross-validated concerning the validation parameters specificity, linearity, lower limit of detection (LLOD; 1-5 ng/mL), ion suppression/enhancement (up to 78%), intra- and inter-day precision (3-21%), recovery (29-48%), and carryover. By means of ten spiked test urine samples sent blinded to one of the participating laboratories, the fitness-for-purpose of both assays was provided as all specimens were correctly identified applying both testing methods. As no post-administration study samples were available, analyses of authentic urine specimens remain desirable. Copyright © 2016 John Wiley & Sons, Ltd.

Glucocorticoid administration in athletes: Performance, metabolism and detection.

It is generally acknowledged in the sporting world that glucocorticoid (GC) use enhances physical performance. This pharmacological class is therefore banned by the World Anti-Doping Agency (WADA) in in-competition samples after systemic but not local (defined as any route other than oral, intravenous, intramuscular or rectal) administration, which thus allows athletes to use GCs for therapeutic purposes. According to the 2016 WADA list, the urine reporting level for all GCs is set at 30ng/ml to distinguish between the authorized and banned routes of administration. The actual data on the ergogenic effects of GC intake are nevertheless fairly recent, with the first study showing improved physical performance with systemic GC administration dating back only to 2007. Moreover, the studies over the last decade coupling ergogenic and metabolic investigations in humans during and after GC intake have shown discrepant results. Similarly, urine discrimination between banned and authorized GC use remains complex, but it seems likely to be improved thanks to new analytical studies and the inclusion of the authorized GC uses (local routes of administration and out-of-competition samples) in the WADA monitoring program. In this review, we first summarize the current knowledge on the ergogenic and metabolic GC effects in humans during various types of exercise. We then present the antidoping legislation and methods of analysis currently used to detect GC abuse and conclude with some practical considerations and perspectives.