The power of hyphenated chromatography-Time of flight mass spectrometry for unequivocal identification of spirostanes in bodybuilding dietary supplements.

A previously unidentified purported botanical ingredient was found in dietary supplements marketed for anabolic benefits. In an attempt to assess the 'naturalness' of a group of steroid-like compounds called laxogenins, a UHPLC-QToF method was developed. Several dietary supplements claim to contain 5α-hydroxy laxogenin, which is a derivative of a naturally occurring spirostane-type steroid, laxogenin. Although laxogenin has been isolated from the rhizomes of Smilax sieboldii, 5α-hydroxy laxogenin has not been isolated or reported from any natural source. These derivatives of laxogenins have untested anabolic properties. Due to the low UV absorbance of the spirostanes, a mass spectrometric method in positive ion mode was developed for unambiguous identification of laxogenin and closely related compounds. To show the utility of the developed method, twelve dietary supplements labeled to contain 5α-hydroxy laxogenin or laxogenin as 5α-hydroxy laxogenin were analyzed as a proof-of-concept. Five supplements did not contain any 5α-hydroxy laxogenin, whereas in the remaining seven samples, spirostane-type contaminants were identified along with the labeled 5α-hydroxy laxogenin. The identity of some of these contaminants was established based on reference standards along with mass fragmentation patterns. One of the unlabeled contaminants was identified as the phytosteroid saponin, diosgenin, a common starting precursor of several steroidal drugs. Several synthetic derivatives of diosgenin were identified in the eight products. These findings indicate that the labeled 5α-hydroxy laxogenin along with other spirostanes found in supplements are synthetic and signify a lack of quality controls. Additionally, an unlabeled, anabolic androgenic steroid, arimistane, an aromatase inhibitor, was also identified in one product. Laxogenin, was not detected in any of the samples analyzed during this investigation.