Development and validation of a HPLC-MS/MS method the determination of genistein and equol in serum, urine and follicular fluid

Abstract

Soy isoflavones exert estrogen-like synergistic or antagonistic effects by binding to estrogen receptors, and potentially impact the function of female reproductive system, but their distribution profile in human remains little clarified. To determination of genistein (GEN) and equol (EQ) in human urine, serum and follicular fluid (FF), an analytical method based on high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed and validated. The enrichment and clean-up are performed on a solid-phase extraction (SPE) column; the elution is a gradient one, with the mobile phase (A) of 0.1 % (v/v) formic acid aqueous solution and the mobile phase (B) of 0.1 % (v/v) formic acid in acetonitrile; the column temperature is 40 °C. Mass spectrometry is performed using negative ion mode electrospray ionization (ESI -) in multiple reaction monitoring (MRM) mode. The method was validated over the linear ranges of 7.8–1000.0 ng/mL and 39.1–5000.0 ng/mL, for serum and urine, with correlation coefficients (r) of 0.9948–0.9984. The precision, accuracy and stability meet the U.S. Food and Drug Administration guidance. This method has been used to detect genistein (GEN) and equol (EQ) in serum, follicular fluid, and urine, to report equol in follicular fluid for the first time, and to study the correlation between genistein and equol in three body fluids. The study showed that the average concentration of EQ in follicular fluid was 18.5 ng/mL and there was a significant positive Spearman's correlation between concentrations of GEN in serum and FF (r = 0.44, p ≤ 0.05).