Establishment and validation of a sensitive LC-MS/MS method for quantification of urinary estrogens in women with gestational diabetes mellitus

Gestational diabetes mellitus (GDM) is not only a threat to the health of pregnant women, but also has profound effects on the health of offspring. Studies have shown that the imbalance of estrogen metabolism is associated with an increased risk of GDM. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established and validated for simultaneous quantification of thirteen estrogens in the urine of GDM women, including estrone (E1), estradiol (E2), estriol (E3), and their hydroxylated and methylated metabolites. The method was achieved on a Waters CORTECS C18 column (2.1 mm × 150 mm, 1.6 µm) within 8.5 min. The linear range of thirteen estrogens in urine was 2–1000 pg·mL−1. Both intra- and inter-day precision for each analyte were less than 15%, with accuracies ranging from 8.3% to 7.3%. The extraction recoveries rate were between 86% and 111%, and stability verification results met the requirements for determination of biological samples. The results suggested that the concentrations of estrogens in all urine samples range from 0.08 to 134.06 (pg·mg−1 creatinine). The mean levels of E1, E2 and most estrogen metabolites in the urine of GDM women were higher than those in healthy pregnant women. Notably, the mean level of 2-hydroxyestrone (2-OHE1) in GDM women was 13.2-fold lower than that in healthy pregnant women. The types of estrogens with the highest mean levels in the urine of GDM and healthy pregnant women were obviously different, which are 2-methoxyestrone (2MeOE1) and E3, respectively. Our results demonstrated that this specific and sensitive method is suitable for quantifying estrogens in human urine and could provide support for further research on estrogen-related pathological mechanisms in GDM and other diseases. •Development and validation of an UPLC-MS/MS method for quantification of estrogens.•Simultaneous separation and quantification of 13 endogenous estrogens in human urine.•Determination of urinary estrogens in GDM patients.•Changes in urinary E1, E2, 16α-OHE1 and 2OHE1 are relatively pronounced with GDM.