Highlights
•
Ibutamoren is a growth hormone secretagogue, banned by WADA (class S2.2.4) •
A specific method has been developed for ibutamoren testing in hair using LC-MS/MS. •
The limit of detection of the method is 0.1 pg/mg. •
A single 10 mg dose of ibutamoren is detectable in hair. •
It is possible to discriminate ibutamoren doping use from an incidental contamination via a hair test.
Abstract
Background and aims
On numerous occasions during hearings at the Court of Arbitration for Sport (CAS), it has been advocated by the anti-doping authorities that the results of hair tests should be interpreted with utmost precautions. This was due to the lack of data for the interpretation of the results. In particular, the knowledge of the expected concentrations after a single exposure and after a doping regimen can be missing.
Material and method
A single 10 mg dose of ibutamoren, a growth hormone secretagogue, was administered to a male volunteer and his hair was collected 4 weeks after exposure. In addition, the hair specimen of a subject who took 60 capsules of 10 mg ibutamoren over 90 days was collected 4 weeks after drug discontinuation. After addition of bicalutamide-D4 used as internal standard and incubation in phosphate buffer pH = 9.5, 25 mg of hair samples were extracted and purified by liquid/liquid extraction.
Results
Linearity was verified from 0.5 to 250 pg/mg (R2 = 0.9961). The limit of detection was 0.1 pg/mg. After a single exposure to 10 mg, ibutamoren tested positive at 1.3 pg/mg (segment 0 to 1 cm). After consumption of 60 capsules at 10 mg (whole vial), the 0 to 4 cm hair segment of a user for enhancement of athletic performances tested positive at 224 pg/mg, highlighting for the first time the incorporation in hair of this molecule.
Conclusion
The interpretation of the findings of ibutamoren in human hair can be established in the light of these new data as the minimal detectable dose has been established, as well as the range of concentrations after a regimen of 60 days for performance enhancement.
Keywords
Ibutamoren
;
MK-677
;
Doping
;
Hair
;
Contamination
Introduction
On numerous occasions during hearings at the Court of Arbitration for Sport (CAS), it has been advocated by the anti-doping authorities that the results of hair tests should be interpreted with utmost precautions and in most cases, they suggested that the hair test were not conclusive. This was due to the lack of data for the interpretation of the results. In particular, the knowledge of the expected concentrations after a single exposure and after a doping regimen can be missing.
Although the minimal detectable dose in hair is not yet established for anabolic steroids, this parameter has been recently established for several other doping agents, including trimetazidine [1], letrozole [2], clomiphene [3], chlortalidone [4], ostarine [5], adrafinil [6] and octodrine [7].
After a negative hair test, the question of importance is to know if the analytical procedure is sensitive enough to identify traces of a specific drug [8]. This is particularly relevant when the urine specimen tests positive during an anti-doping control and the hair specimen test negative in a laboratory chosen for the defence of the athlete. For example, back in 2000, Segura et al [9] have published that a single administration of testosterone esters or nandrolone decanoate is not detectable in hair [9]. It has been accepted by the scientific community that a negative hair result cannot exclude the administration of a particular drug, or one of its precursors and the negative findings should not overrule a positive urine result. For example, this can be due to the limited hair incorporation of the drug. Nevertheless, the negative hair findings can, on occasion, cast doubt on the positive urine analysis, resulting in substantial and tedious debate about the interest of the hair test. The concept of minimal detectable dosage in hair is therefore recommended to document any negative findings [10].
Ibutamoren is one of the most popular drugs on the Internet and numerous e-vendors are proposing it to help building muscle mass. Little is known about this compound (Fig. 1). Ibutamoren, or MK-677 (its developmental code), was developed at Merck research Laboratories [11].
Ibutamoren is long-acting, potent, orally active, non-peptide and selective agonist of the growth hormone secretagogue receptor, also known as ghrelin receptor [12]. It first increases production of growth hormone releasing hormone (GHRH) which is a hormone that controls metabolism and growth. Secondly, it works by reducing somatostatin, a hormone that helps to reduce GH release. Thirdly, MK-677 works by promoting GHRH signals in the pituitary gland and lastly, it restrains somatostatin receptor signals. To increase growth hormone levels, MK-677 mimics the action of the natural ghrelin to stimulate the release of growth hormone from the brain [12].
On 19 June 2017, the USA Food and Drug Administration granted Orphan Drug Designation for ibutamoren mesylate (MK-0677) for the treatment of growth hormone deficiency, while on 20 June 2017, the European Commission did the same. However, the drug is not available therapeutically and is not approved for human use.
A daily dose of 5 mg of MK-677 results in a significant increase in growth hormone levels. The clinically tested approach of MK-677 is via oral administration. The dosage may range from 5 mg to 25 mg per day, depending on factors such as weight and age. It’s best to take the MK-677 during early morning or late evening, on an empty stomach. However, MK-677 may interfere with sleep that is why it’s not recommended to be taken during night time [13].
For many athletes, a 5 mg–10 mg capsule per day for 8 weeks seems to work for the bulking phase (the phase where the subject maximizes muscle growth and strength heavy training and elevated calories intake). During the cutting phase (the phase where the subject preserves muscle while losing fat by increased cardio and reduced calories intake), MK-677′s recommended dosage is 3 mg to 5 mg per day for 8 weeks. For the recomping phase (the phase where the subject simultaneously builds muscle and loses fat by consistent training and balanced diet), the recommended dosage is 5 mg to 8 mg per day for 8 weeks.
As MK-677 increases IGF-1 and growth hormone levels, which then leads to a reduction of body fat, increasing muscle strength and increasing muscle mass, the drug is listed under “S2.2.4 Growth hormone releasing factors” on the world anti-doping 2025 prohibited list. Ibutamoren is a substance prohibited at all times (in- and out-competition) [14]. The WADA testing figures [15] suggest that the prevalence of adverse analytical findings (AAFs) due to ibutamoren is low over the recent years (Table 1).
As ibutamoren can be present as contaminant in supplements [16,17] sometimes combined with selective androgen receptor modulators (SARMs) or even indicate MK-677 as a SARM, several athletes have challenged their anti-doping rules violation (ADRV) and requested additional investigations to confirm their claims. When it comes to contamination, the strategy of this laboratory is to document the findings with a hair test [18].
The detection of ibutamoren has been reported in equine hair [19]. After administration of 125 mg of MK-0677 on 3 consecutive days to a 4-year old horse, the drug was identified in the range not detected to 1.9 pg/mg in tail and mane hair specimens, collected at various time (2 to 358 days post-administration). However, there is no citation in PubMed, Web of Science, and ScienceDirect with the key words “human hair and ibutamoren”. Therefore, a dedicated method was developed, using liquid chromatography tandem mass spectrometry (LC-MS/MS).
Section snippets
Chemicals and reagents
Formic acid Normapur quality was purchased from VWR Prolabo (Fontenay-sous-Bois, France). Ammonium formate 99 % was purchased from Alfa Aesar (Schiltigheim, France). Acetonitrile of LC-MS grade, dichloromethane, hexane, and methanol were purchased from Merck (Molsheim, France). Diethyl ether was purchased from Honeywell Riedel-de Haën (Seelze, Germany). Water for the formate buffer (eluent A for chromatographic separation) was purified using a Millipore Milli-Q system (Molsheim, France).
Results and discussion
Table 3 summarises the validation parameters. Linearity (using 9 concentrations) was fixed with ibutamoren concentrations ranging from 0.5 to 250 pg/mg. The correlation coefficient was above 0.99 (R2 = 0.9961). The limit of quantitation of the method is therefore 0.5 pg/mg (first point of the calibration curve). With an aliquot of 25 mg of hair, the limit of detection was 0.1 pg/mg. Precisions were found acceptable for both levels (2 and 200 pg/mg), with calculated coefficients of variation
Conclusion
Although the number of AAFs involving ibutamoren seems limited, significant violation cases are still observed each year. In the recent period, some of these violations were challenged for possible contamination, particularly from contaminated supplements [23]. This paper is the first contribution to the identification of ibutamoren in human hair with reported concentrations obtained after a single use and after a doping regimen. The interpretation of a hair test result is facilitated when the
CRediT authorship contribution statement
Pascal Kintz: Writing – review & editing, Writing – original draft, Validation, Conceptualization. Laurie Gheddar: Methodology, Investigation, Formal analysis, Data curation.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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