Standardized LC–MS/MS based steroid hormone profile-analysis

In order to overcome many limitations of immunoassays, high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) has the potential to find its place in the clinical laboratory medicine for quantification of steroid hormones. A prerequisite for the application of a new analytical procedure in clinical diagnostics is standardization to minimize analytical intra- and interlaboratory variability and inaccuracy. We evaluate a newly standardized HPLC–MS/MS assay in kit-format, developed for routine determination of 16 steroid hormones in human serum samples. Fifteen metabolites can be measured quantitatively, which include aldosterone, androstenedione, androsterone, corticosterone, cortisol, cortisone, 11-deoxycortisol, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), 17β-estradiol (E2), estrone (E1), etiocholanolone, 17α-hydroxyprogesterone (17OHP), progesterone, and testosterone. 11-Deoxycorticosterone is the only compound rated as semi-quantitative in this kit. The sample preparation is performed by solid phase extraction (SPE) on a 96-well plate. The standardized assay has been validated for human serum in terms of lower and upper limit of quantification (LLOQ 0.01–32 ng/mL, ULOQ 5–8000 ng/mL), linear correlation coefficient of calibration (R2 > 0.9966), intra- and inter-day precision (intra-day 1.1–8.8%, inter-day 5.2–14.8% and 8.2–18.6% for 11-deoxycorticosterone), accuracy (intra-day 88.3–115.5% and 109.3–128.2% for 11-deoxycorticosterone, inter-day 91.4–117.2% and 102.3–137.1% for 11-deoxycorticosterone), analytical total error (3.6–17.8%), proficiency test accuracy (85.4–113.4%), recovery (68–99%), and metabolite stability (freeze/thaw stability 95.5–108.1%, short term stability 86.9–107.2%). Inter-assay comparison with a routine reference HPLC–MS/MS assay and seven immunoassays demonstrates the outstanding high performance of this HPLC–MS/MS based kit by improvements in accuracy for progesterone, androstenedione, and 17OHP. Finally, results of two metyrapone tests demonstrate the potential of the standardized HPLC–MS/MS assay for the analysis of a comprehensive steroid hormone profile in clinical diagnostics.

► Standardized LC–MS/MS in kit-format analyzing 16 steroid hormones in human serum. ► Assay showed excellent validation data fulfilling the FDA criteria. ► Inter-assay comparison with immunoassay with improvements in accuracy. ► Steroid profiles of metyrapone test samples were analyzed.