Determination of steroid reference intervals in a pregnancy population

Steroids, including mineralocorticoids, glucocorticoids, and sex hormones play a critical role in pregnancy. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis offers an opportunity to conduct simultaneous multiplex steroid analysis within a given sample. This paper describes the LC-MS/MS steroid analysis method developed for assessing plasma specific reference ranges of 18 steroids from plasma samples (200 µL) of pregnant women participating in the PREDO study. Samples were prepared using supported liquid extraction and analyzed on an Acquity I-Class UPLC and a QTrap6500 + mass spectrometer. Mass spectrometry parameters were optimized for each steroid and chromatographic separation of 18 steroids was developed. Changes in steroid levels across pregnancy were assessed. Samples were collected after an overnight fast between 07:00 and 09:00. Data from 257 samples from 96 women with uncomplicated pregnancy (women with pre-pregnancy normal weight and no diabetes or hypertensive disorders before or during pregnancy, who delivered a live child at ≥ 37 weeks of gestation with appropriate for gestational age birth weight) were analyzed to calculate steroid reference ranges over three time points, between 11.6 and 14.3, 17.7-22.9, and 25.6-29.0 pregnancy weeks. Levels of progestogens, glucocorticoids, mineralocorticoids, estrogens, their precursors, and metabolites increased significantly across pregnancy. Androgen levels remained stable, except for a decrease in 5α-dihydrotestosterone. The LC-MS/MS method also showed validity in analyses of 917 samples of 328 women with complicated pregnancies. The method is suitable for the analysis of 18 steroids in plasma during pregnancy and the investigation of pregnancy complications, fetal growth, and development after birth.