Extraction

Sample preparation commences with liquid-liquid extraction (LLE). A slightly modified method of steroid extraction was used²⁸. Briefly, 50 µL of plasma was mixed with the mixture of IS. Plasma steroids were extracted to 2 mL of tert-butyl methyl ether (MTBE). Samples were gently vortexed for 10 min and were centrifuged at 1000 g for 10 min. The supernatant was collected and the solvent was evaporated under a stream of nitrogen gas. During the extraction, the steroids were protected against oxidation by the addition of 0.05 mg/mL BHT to the extraction solvent (MTBE). MTBE, diethyl ether, dichloromethane and a mixture of hexane and diethyl ether are the most common solvents used for LLE⁷. MTBE was found to be the best for a satisfactory extraction yield after testing a few solvents and solvent mixtures. Optimisation of the extraction procedure was controlled by thin layer chromatography as described in Ubhayasekera et al.³⁰.