An LC/MS/MS method for analyzing the steroid metabolome with high accuracy and from small serum samples[S]

Analysis of the global steroid metabolism in human can shed light on the etiologies of steroid-related diseases. However, existing methods require large amounts of serum, and lack accuracy evaluation. Here, we developed an LC-MS/MS method for simultaneous quantification of 12 steroid hormones, including testosterone, pregnenolone, progesterone, androstenedione, corticosterone, 11-deoxycortisol, cortisol, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, estriol, and estradiol. Steroids and the spiked internal standards in 100 μL serum were extracted by protein precipitation and liquid-liquid extraction. The organic phase was dried by evaporation and isonicotinoyl chloride was added for  steroid derivatization, followed by evaporation under nitrogen and redissolving in 50% methanol. Chromatographic separation was performed on a reverse-phase PFP column, and analytes were detected on a triple quadrupole mass spectrometer with ESI. The lower limits of quantification ranged from 0.005 ng/mL for estradiol to 1 ng/mL for cortisol. Apparent recoveries of steroids at high-, medium- and low- concentrations in quality-control samples were between 86.4% and 115.0%. There were limited biases (-10.7%-10.5%) between the measured values and the authentic values, indicating that the method has excellent reliability. An analysis of the steroid metabolome in pregnant women highlighted the applicability of the method in clinical serum samples. We conclude that the LC-MS/MS method reported here enables steroid metabolome analysis with high accuracy and reduced serum consumption, indicating that it may be useful tool for both clinical and scientific laboratory research.