Biphenyl based stationary phases for improved selectivity in complex steroid assays

The measurement of steroid hormones and their corticoid precursors is an important aspect in endocrinology since these analytes are biomarkers for several endocrine disorders. Over the last few years, HPLC-MS/MS has become the method of choice to analyze these compounds. There are already several methods using stationary phases modified with C18 groups. However, since these columns sometimes do not enable sufficient separation of some isobaric steroids, we investigated the potential of a different RP modification using biphenyl groups for the separation of challenging isobars such as corticosterone, 11- and 21-deoxycortisol. The aim of our work was the development of an isotope dilution UHPLC-MS/MS assay for clinical research that combines simple and effective sample preparation with a powerful MS method quantifying a broad steroid panel (aldosterone, corticosterone, cortisol, cortisone, 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17-OH-progesterone, progesterone, and testosterone) in human serum. After a manual protein precipitation step using zinc trifluoroacetate (ZnTFA) in methanol, the supernatants were directly injected into the UHPLC-MS system. Chromatographic baseline separation of all isobaric compounds (corticosterone↔11-deoxycortisol↔21-deoxycortisol, 17-OH-progesterone↔11-deoxycorticosterone, and aldosterone↔cortisone) was achieved using a Kinetex Biphenyl column (150×2.1mm, 1.7μm) with a mobile phase consisting of 0.2mM ammonium fluoride in water and methanol. The total run time was 10min. For detection we used a Xevo TQ-S mass spectrometer operating in the ESI positive and negative modes. The method was validated according to the EMA guideline for bioanalytical method validation. The results for accuracy (within-run: 92.3%-115%, between-run: 92.4 %-113%) and imprecision (within-run: 0.80%-9.05%, between-run: 1.98 %-15.2%) were satisfying. The recovery ranged from 95% to 111%. The matrix effect was between 93% and 112% and an excellent linearity with R²>0.99 for all analytes was achieved. It was demonstrated that biphenyl based columns are a powerful tool for comprehensive, MS based steroid assays including various isobaric substances. Additionally, we could evince that ZnTFA is a convenient precipitation agent suitable for steroid analysis.