Development and validation of a deep eutectic solvent-assisted liquid-liquid extraction method for simultaneous quantification of six steroid hormones in serum by liquid chromatography-tandem mass spectrometry

•DES-assisted LLE method was first used to the pretreatment of serum to determine steroid hormones.•A tailor-made DES was synthesized and characterized for this assay.•Optimum parameters were obtained by Box-Behnken design and validated.•The process extended the application of DES in biological samples.•DES-assisted LLE method was distinguished a more simple and low-cost procedure compared with SPE method. Steroid hormones have been reported to be associated with endocrine system diseases. This paper proposes a novel procedure of deep eutectic solvent (DES)-assisted liquid–liquid extraction (LLE) to extract six steroid hormones (including cortisone, cortisol, androstenedione, testosterone, 17-hydroxyprogesterone, and progesterone) from serum coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of five types of L-proline, choline chloride, and citric acid-based DESs were tailored; the DES from L-proline and ethylene glycol at a molar ratio of 1:4 with 20 % acetonitrile was selected as the best-fit assisted solvent for the six steroid hormones compared with other DESs. The parameters for extraction by selected DES were optimized using Box-Behnken design (BBD), and the optimal extraction conditions are 200 µL of acetonitrile, 100 µL of the sample, and 80 µL of DES. Under optimum conditions, the method has good linear calibration ranges (between 0.07 ng mL−1 and 600 ng mL−1), correlation coefficients of determination (r2>0.99), and low limits of quantification (between 0.02 and 0.60 ng mL−1). The extraction recoveries were in the range of 81.84–114.43 %, and the intra-day and inter-day relative standard deviations (RSDs) were less than 10 %.In general, the DES-LC-MS/MS method is a simple and environmentally-friendly method, which can be complementary to the presently available methods for determining steroid hormones in serum.