Endogenous steroid hormones with similar structure, poor content and high efficacy are difficult and vital to be quantitatively detected. In this study, a validated method was established for the simultaneous quantification of thirteen steroids in human serum, and applied to the study of type 2 diabetes mellitus (T2DM). An ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) was developed for the simultaneous determination of thirteen steroid hormones in human serum, including androstenedione, corticosterone (B), cortisol (F), cortisone, 18-hydroxycortisol (18OHF), 11-deoxycorticosterone, 11-deoxycortisol, pregnenolone, progesterone, 17-hydroxyprogesterone, testosterone, androstanolone and estradiol. Under the optimum conditions, method was achieved with a BEH Shield RP18 column within 18 min. The lower limits of quantitation for steroids were 0.08-7.81 ng/mL. The intra- and inter-day precision for all the analytes were less than 15 %, and the accuracy ranged from -14.19 % to 12.89 % at three quality control levels. The proposed method, indicating high steady and sensitivity, was successfully applied to the quantification of thirteen steroids levels in serum from patients with T2DM and healthy individuals. The serum concentrations of 18OHF and F were significantly increased in the patients compared with the healthy individuals, while B was significantly decreased. The fold change was 1.98, 1.25 and 0.79 respectively. The ratio of 18OHF to B (18OHF/B) exhibited a 2.51-fold increase in T2DM patients and presented a more significant change. 18OHF/B was identified as a prospective serum marker, which deserves further attention.
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