The UHPLC-MS/MS determination of ostarine in food supplements for athletes

Abstract

Ostarine, also called enobosarm or MK-2866, is one of the Selective Androgen Receptor Modulators (SARMs). A number of preclinical data suggest the beneficial use of these substances to increase muscle mass growth and bone density. Unlike anabolic steroids, they have suppressed androgenic side effects and are therefore thought to be useful in the treatment of health conditions associated with insufficient muscle growth. For the same reason, substances from the SARMs group are abused in sports and bodybuilding. They have been added on the World Anti-Doping Agency (WADA) list in 2008 and their use in sports is prohibited. In many countries, products containing these substances are available through a number of e-shops offering supplements for athletes. The positive effects on muscle growth and body fat loss and the absence of side effects are emphasized. Because SARMs are banned in sports and are not approved for any therapeutic use, they are marketed as “Research Only”. However, the products are sill quite popular among athletes. As with anabolic steroids, there are a variety of dosing regimens for both beginners and advanced athletes, which end up recommending a combination of three to four SARMs over several weeks. Although more extensive clinical data are lacking, case studies are beginning to appear describing liver damage or heart attack in people taking ostarine or other SARMs products. However, due to the lack of clinical data, no positive or negative effect on the organism is objectively confirmed. For these reasons, it is very appropriate to have an analytical method available that allows the rapid and correct determination of the ingredients in preparations that have ostarine declared in the list of ingredients. Equally important is the possibility of identifying this substance in preparations that should not contain any active substance and appear as purely natural products. For the ostarine determination a simple and fast UHPLC-MS/MS method was developed. For extraction of ostarine the content of the studied capsule was weighted and transferred to a volumetric flask, which was then made up with methanol to 100 mL. The mixture was shaken for one hour. After filtration 10 μL of extract was transferred to a new flask and made up to 5 mL with 10% methanol with 0,1% acetic acid and 10 mM ammonium acetate. This solution was used for UHPLC-MS/MS analysis. An UHPLC system Infinity 1290 and chromatographic column Eclipse Plus C18 (50 × 2.1 mm; 1.8 μm) (both Agilent Technologies, USA) were used for the separation. 0.1% acetic acid and 10 mM ammonium acetate in water and methanol were chosen as mobile phases for gradient elution. For the MS/MS detection 6460 Triple Quadrupole mass spectrometer (Agilent Technologies, USA) with negative ESI ionization were used. For dynamic MRM mode two transitions of m/z were chosen for the detection: 388.1 → 117.9 and 269.0. Limit of detection was 0.2 ng/mL and limit of quantification 0.5 ng/mL. The method was used for quantitation of ostarine in dietary supplements for athletes. The content of ostarine in one capsule ranged from 10 to 15 mg and did not differ from the quantity indicated on the label. The described method is suitable for the determination of ostarine and can be used in toxicological and forensic practice.