High-temperature liquid chromatography-isotope ratio mass spectrometry methodology for carbon isotope ratio determination of anabolic steroids in urine

Background: Gas Chromatography Isotope Ratio Mass Spectrometry (GC-C-IRMS) has long been used in routine laboratories to determine the delta 13 C values of anabolic steroids in urine, differentiating between, e.g., endogenous and synthetic testosterone (T) in sports doping control. Until now, liquid chromatography (LC-IRMS) has not been used. The LC-IRMS setup doesn't allow organic solvents or modifiers in the mobile phase for delta 13 C determinations. Mid-to non-polar analytes such as steroids can be analysed in water heated to High Temperatures (HT, up to 200 degrees C) because at 200 degrees C has a similar polarity as 80/20 methanol/water at ambient temperature. In this work, we developed a method for steroids in urine, extending the application of the LC-IRMS to non-polar analytes in complex matrices. Result: An HT-LC-IRMS method capable of determining the delta 13 C values of four steroids (i.e., testosterone (T), 5 alpha-androstane-3 alpha,17 beta-diol beta- diol (alpha alpha beta), beta ), 5 beta-androstane-3 alpha,17 beta- beta- androstane-3 alpha,17 beta- diol (beta alpha beta) beta alpha beta ) and pregnanetriol (PT)) in urine was developed and validated. Accuracy ranged from 0.23 %o (alpha alpha beta beta and beta alpha beta ) to 0.49 %o (T), and the detection limit was set at 10 ng mL-1-1 (T, alpha alpha beta+beta alpha beta). beta + beta alpha beta ). The validation data and a comparison of authentic urine samples analysed with HT-LC-IRMS and GC-C-IRMS indicated a comparable performance between HT-LC-IRMS and GC-C-IRMS. Significance: HT-LC-IRMS can be used to determine delta 13 C values of anabolic steroids, extending the applicability of both HT-LC and LC-IRMS to non-polar substances determined in a complex matrix in routine laboratory practice.