Quantitative and qualitative determination of estrogen sulfates in human urine by liquid chromatography/tandem mass spectrometry using 96-well technology

A sensitive and robust method to determine five estrogen sulfates in human urine has been developed employing high-throughput solid-phase extraction with 96-well technology, and HPLC coupled with negative turbo ion spray tandem mass spectrometry in the selected reaction monitoring mode. The five estrogen sulfates determined include three major endogenous estrogen sulfates in the human, estrone 3-sulfate (E-1-3S), estriol 3-sulfate (E-3-3S), and 17 beta-estradiol 3-sulfate (E-2-3S), and two biochemical synthetic estrogen sulfates, 17 beta-estradiol 17-sulfate (E-2-17S) and 17 beta-estradiol 3,17-disulfate (E-2-3,17S), For E-2-3,17S, E-3-3S, and E-2-17S, external standard calibration was used for quantitation, and for the remaining two compounds, internal standard calibration using a stable isotopic labeled internal standard was employed. A total of 96 samples may be prepared with 96-well C18 extraction disk plate techniques performed by a robot within 25 min including the time for evaporation of solvent. The lower level of quantitation (LOQ) for these estrogen sulfates in human urine was determined at 0.2 ng/mL based on 100-mu L aliquots of human urine using the optimum tuning parameters for each individual selected precursor ion/product ion transition. The assay was validated with a linear concentration range of 0.2-200 ng/mL, and the interassay accuracy, intraassay precision, and interassay precision do not exceed 8.6%, 12%, and 12%, respectively, by analysis of quality control samples at five concentration levels including the LOQ of 0.2 ng/mL, from four 96-well plates. The target endogenous test articles were qualitatively determined by comparing the full-scan LC/MS/MS mass spectra and retention time in test samples and reference standards. The LOQ is significantly improved compared to previous reports for the targeted compounds using LC/MS/MS, The described simple and automated sample preparation procedure recovered 91% of the target compounds. A total of 192 samples can be analyzed within 1 day(22 h), The method can measure the endogenous estrogen sulfates in urine from both gravid and nongravid subjects.