An UPLC-MS/MS method for the simultaneous quantification of 6 steroids from the androgen "backdoor pathway" in human...

Hyperandrogenism poses significant diagnostic challenges in women's healthcare, requiring precise identification of elevated androgen sources and types for accurate diagnosis and optimal treatment. Key steroid metabolites of the androgen backdoor pathway, which are critical for differentiating androgen sources, remain underutilized due to constraints in measurement methodologies. We developed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneous quantifying six backdoor pathway steroids in human plasma.

Plasma samples (500 μL) underwent liquid-liquid extraction with ethyl acetate/hexane (8:2, v/v). Steroids were separated on a C18 column using methanol-water gradient elution with 0.05 % (v/v) formic acid and analyzed by positive electrospray ionization in multiple reaction monitoring (MRM) mode. Total runtime was 6.2 min. The method was validated following the CLSI C62-A guidelines.

Lower limits of quantification for six steroids ranged from 0.0138 to 0.0538 nmol/L. Linearity was excellent (R² ≥ 0.996). Intra- and inter-day coefficients of variation were < 8 %, and recoveries ranged from 85.8 % to 110.5 %. Preliminary application to healthy controls and patients with congenital adrenal hyperplasia (CAH), including hyperandrogenic cases [21-hydroxylase deficiency (21-OHD) and 11β-hydroxylase deficiency (11β-OHD)] and hypoandrogenic cases [17α-hydroxylase deficiency (17α-OHD)], demonstrated the method's utility in identifying the type and source of androgen dysregulation.

This sensitive, rapid method enables simultaneous quantification of six key steroids in the androgen backdoor pathway, thereby providing enhanced metabolic profiling for patients with hyperandrogenism and improving diagnostic capabilities for androgen disorders.