Carbon Isotope Ratio Analysis of Steroids by High-Temperature Liquid Chromatography-Isotope Ratio Mass Spectrometry

Generally, compound-specific isotope analysis of steroids is carried out by gas chromatography combined with isotope ratio mass spectrometry. Thus, a derivatization of the steroids prior to the measurement is compulsory, and a correction of the isotopic data is often necessary. To overcome this limitation, we present a new approach of high-temperature liquid chromatography coupled with photodiode array detection and isotope ratio mass spectrometry (HT-LC/PDA/IRMS) for the carbon isotope ratio analysis of unconjugated steroids. A steroid mixture containing 19-norandrosterone, testosterone, epitestosterone, androsterone, and 5 beta-pregnane-3 alpha,17 alpha,20 alpha-triol was fully separated on a C-4 column under high-temperature elution with water as the sole eluent. The accuracy for isotope analysis (+/- 0.5 parts per thousand) was around 20 mu g g(-1) for testosterone, epitestosterone (79 ng steroid absolute on column), and 30 mu g g(-1) for 19-norandrosterone, androsterone, and 5 beta-pregnane-3 alpha,17 alpha,20 alpha-triol (119 ng steroid absolute on column). The applicability of the method was tested by measuring a pharmaceutical gel containing testosterone. With this work, the scope of LC/IRMS applications has been extended to nonpolar compounds.