Development and validation of a sensitive liquid chromatography–tandem mass spectrometry assay to simultaneously measure androgens and estrogens in serum without derivatization

BackgroundImmunoassays are widely used to quantify steroid hormones in biological samples. However, they lack specificity, especially at low levels. This study aimed to develop a sensitive LC–MS/MS method to measure serum androgens and estrogens without derivatization within a single run.MethodsA stable-isotope dilution LC–MS/MS method was established using atmospheric pressure photoionization to quantify testosterone (T), dihydrotestosterone (DHT), estradiol (E2) and estrone (E1) from serum. Sample preparation involved liquid–liquid extraction (LLE) with hexane:ethyl acetate (3:2) containing deuterated internal standards. Accuracy was assessed by spiked recovery of serum pools, and imprecision by quality controls.ResultsUsing 200 μL serum, limits of quantification were 0.3 pg (1.5 pg/mL) E1, 0.5 pg (2.5 pg/mL) E2, 2 pg (10 pg/mL) T and 10 pg (50 pg/mL) DHT. Accuracy (93–110%) and precision (median 4%, all <15%) were determined to be well within acceptable limits for bioanalytical method validation. An analysis time of less than 10 min allowed up to 150 samples (600 analytes) to be processed per day.ConclusionsThe method is sufficiently sensitive and precise to accurately quantify serum T levels in females and E2 in males, and is readily adapted to tissue and non-human samples.