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Background: Simultaneous assessment of steroid hormone concentrations in amniotic fluid is of importance for elucidating long-term consequences of intrauterine processes, and of broad scientific and clinical relevance. The objective of the study was to develop sensitive and specific analytical ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) methods for determination of steroid hormone concentrations in human amniotic fluid, and to provide proof-of-concept of their applicability. Methods: Methods were validated according to linearity, limit-of-detection, limit-of-quantification, recovery, intra- and inter-assay precision, and applied to 275 amniotic fluid samples. Results: Limits-of-quantification (S/N = 10:1) were 0.05 ng/mL for cortisol, dehydroepiandrosterone sulfate (DHEAS), estradiol, estriol, and testosterone, and 0.01 ng/mL and 1.0 ng/mL for cortisone and dehydroepiandrosterone (DHEA), respectively. Good inter- and intra-assay precision were observed. Cortisol, cortisone, DHEAS, estradiol and estriol concentration were quantified in all samples. By lack of sensitivity of the analytical method, DHEA was quantified in 11 samples only. Testosterone was quantified in 119 of 275 samples analyzed. 116 of the quantified testosterone samples were from male offspring (out of 138 male offspring). Conclusion: These specific and sensitive methods offer a simple and non-invasive way to measure cortisol, cortisone, DHEAS, estradiol, estriol, and testosterone concentrations in human amniotic fluid.

Quantifying steroid hormones in amniotic fluid by ultra-performance liquid chromatography and tandem mass spectrometry