Advancement in steroid hormone analysis by LC-MS/MS in clinical routine diagnostics - A three year recap from serum cortisol to dried blood 17α-hydroxyprogesterone.

Steroid analysis by LC-MS/MS in daily clinical routine diagnostics requires high-throughput conditions including fast chromatographic separation. Hereby, signal interferences may occur due to limited specificity in complex biologic matrices. During the last three years of routine steroid analysis in our laboratory and roughly 50,000 measurements, about 1% was affected by interferences, mainly serum cortisol (>90%) and dried blood 17α-hydroxyprogesterone (17-OHP). To overcome specificity problems, enhanced chromatography, ionization polarity switching, and detection via two-stage fragmentation (MS3) using a quadrupole linear ion trap were investigated in our study. Signal interferences of serum cortisol were eliminated by applying a protocol for automated method switching without changing the basic high-throughput LC-MS/MS setup. This approach includes negative ionization and extended chromatography from 4 to 6.6 min using the fourfold column length. From 9 samples affected by cortisol interference using the high-throughput method, 8 could be reliably analyzed applying the method switching protocol. Moreover, the applicability of the high-throughput method as second tier analysis in congenital adrenal hyperplasia (CAH) diagnostics from dried blood was verified with 100% diagnostic specificity. In addition, the combination of fast LC and MS3 detection enables specific quantitation of 17-OHP from dried blood spots on a screening time scale. This approach may be an alternative to the newborn screening for CAH by immunoassay due to its higher specificity, reducing the number of false positive results by 90%. In this work we recap experiences from three years of clinical routine steroid analysis via LC-MS/MS and present a unique analytical setup that enables both high-throughput and enhanced resolution analysis of steroid hormones in serum and dried blood.