Performance enhancement in the measurement of 5 endogenous steroids by LC-MS/MS combined with differential ion mobility spectrometry

Background: Challenges for steroid analysis by LC-MS/MS include low ionization efficiency, endogenous isobars with similar fragmentation patterns and chromatographic retention. Differential ion mobility spectrometry (DMS) provides an additional degree of separation prior to MS/MS detection, and shows promise in improving specificity of analysis. We developed a sensitive and specific method for measurement of corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, 17-hydroxyprogesterone and progesterone in human serum and plasma using an ABSciex 5500 mass spectrometer equipped with a differential ion mobility interface. Methods: 250 μL aliquots of serum were spiked with deuterated internal standards and extracted with MTBE. The samples were analyzed using positivemode electrospray LC-DMS-MS/MS. Themethod was validated and compared with immunoassays and LC-MS/MS methods of reference laboratories. Results: Inter and intra assay imprecision was b10%. Limits of quantification and detection in nmol/L were 0.18, 0.09 for corticosterone and 17-hydroxyprogesterone, 0.30, 0.16 for 11-deoxycortisol, 0.12, 0.06 for progesterone and 0.06, 0.03 for 11-deoxycorticosterone. Comparison for progesterone and 17-hydroxyprogesterone with immunoassay showed slopes of 0.97 and 1.0, intercepts of 0.16 and 0.10 and coefficients of determination (r2) of 0.92 and 0.97, respectively. Progesterone by immunoassay showed positive bias in samples measuring b3.18 nmol/L. Reference intervals for progesterone and 11-deoxycorticosterone in post-menopausal women were found to be b2.88 and b0.28 nmol/L respectively. Conclusions: We developed and validated an LC-DMS-MS/MS method for analysis of five endogenous steroids suitable for routine measurements in clinical diagnostic laboratories. Specificity gained with DMS allows reducing the complexity of sample preparation, decreasing LC run times and increasing speed of the analysis.