Purification of testosterone and its metabolites in urine using two-dimensional high-performance liquid chromatography for C/C ratios analysis by gas chromatography/combustion/isotope ratio mass spectrometry.

Two-dimensional high-performance liquid chromatography (2D-HPLC) is employed as a sample purification method for the isolation and enrichment of testosterone and its main metabolites in urine samples, thereby facilitating subsequent determination of C/C ratios (δC) using Gas Chromatography/Combustion/Isotope Ratio Mass Spectrometry (GC/C/IRMS). The orthogonality of the HPLC columns was optimized by leveraging the distinct differences in polarity and steric hindrance effects between the stationary phases, enabling effective separation of endogenous interferents from testosterone in urine. Urine samples purified with 2D-HPLC exhibit significantly reduced matrix interferences in the fractions of testosterone (T), 5α-androstane-3α,17β-diol (5α-diol), and 5β-androstane-3α,17β-diol (5β-diol), thereby enhancing the accuracy and reliability of GC/C/IRMS analysis. The method achieves limit of quantification as low as 2 ng/mL for testosterone, and 3 ng/mL for both 5β-diol and 5α-diol. Furthermore, the simple instrument configuration enables direct transfer of 1D-purified fractions to 2D-HPLC for further isolation and enrichment, providing a flexible and efficient workflow for method development, particularly in handling complex biological matrices.