Development of a solid phase extraction method for the simultaneous determination of steroid hormones in H295R cell line using liquid chromatography-tandem mass spectrometry

Abstract

The H295R in vitro cell line produces the majority of the steroidogenesis, for which reason it is commonly used as a screening tool for endocrine disrupting chemicals. Simultaneous determination of the precursor cholesterol and key steroid hormones could give a broad insight into the mechanistic disruption of the steroidogenesis. Steroid hormones have primarily been extracted from H295R incubation medium by means of liquid-liquid extraction (LLE) and the obtained recoveries and matrix effects have typically not been stated or assessed. In the present study a solid-phase extraction (SPE) method was developed and validated for the simultaneous extraction of cholesterol and five key steroid hormones pregnenolone, 17-hydroxyprogesterone, testosterone, cortisol and aldosterone from H295R incubation medium, and finally detected by LC-MS/MS. Cholesterol was recovered at a level of 55.7%, while steroid hormone recoveries ranged from 98.2 to 109.4%. Matrix effects varied between -0.6% and 62.8%. Intra-day precision was deemed acceptable, but the inter-day precision for pregnenolone and aldosterone exceeded the precision limit of 15% RSD. Although LLE has been the most frequently used extraction method in H295R studies, however, our investigation has shown that SPE may relatively easily extract and recover steroid hormones, potentially replacing LLE.

Keywords: 17-hydroxyprogesterone; 17OHProg; ALD; Chol; Chol IS; Cholesterol; F; F IS; GC; H295R; H295R incubation medium; HPLC; IS; LC; LC–MS/MS; LLE; LOD(instr); LOQ(instr); MS/MS; OECD; Preg; RSD; SPE; Solid phase extraction; Steroid hormones; Steroidogenesis; T; T IS; aldosterone; cholesterol; cortisol; d(3)-testosterone; d(4)-cortisol; d(6)-cholesterol; gas chromatography; high performance liquid chromatography; human adrenocortical carcinoma cell line; instrumental limit of detection; instrumental limit of quantification; internal standard; liquid chromatography; liquid–liquid extraction; organization for economic cooperation and development; pregnenolone; relative standard deviation; solid phase extraction; tandem mass spectrometry; testosterone.