Identification of potentially counterfeit ingredient in 4-Chlorodehydromethyl testosterone tablets by LC-ESI-MS

Abstract

A reversed-phase high performance liquid chromatography-electrospray ionization mass spectrometry method has been developed to identify methyl testosterone from 4-chlorodehydromethyl testosterone in potentially counterfeit steroid products. Counterfeit tablets are suspected to contain not only the declared active pharmaceutical ingredient 4-chlorodehydromethyl testosterone but also the cheaper active ingredient methyl testosterone that is not shown in label. Chromatographic separation was achieved on a Phenomenex Gemini RP C18 column (5 μm, 150 × 4.6 mm i.d.). Elution was isocratic using a mobile phase consisting of methanol and water (60:40, v/v) at a flow rate of 1.0 mL/min. The detection was performed by UV-VIS absorption at wavelength of 254 nm. Excellent linearity between peak area versus standard concentration of methyl testosterone and 4-chlorodehydromethyl testosterone was both obtained in the concentration range of 0.01-0.8 mg/mL. The average recoveries of methyl testosterone and 4-chlorodehydromethyl testosterone in 4-chlorodehydromethyl testosterone tablet were 99.8 % and 100.8 %, respectively, with the relative standard deviations of 1.0 % and 1.4 %. Moreover, the ESI mass spectra for these two steroids were presented with the interpretation of fragmentation pattern. Methyl testosterone can be distinguished from 4-chlorodehydromethyl testosterone by its characteristic fragment ions m/z 284.0 and 270.0 in ESI-MS. The method provides a tool for the control of substandard or counterfeit commercial preparations of steroids.