Simultaneous determination of formononetin, biochanin A and their active metabolites in human breast milk, saliva and urine using salting-out assisted liquid-liquid extraction and ultra high performance liquid chromatography-electrospray ionization tandem mass spectrum

Isoflavonoid phytoestrogens, referred as “dietary estrogens” are widely distributed in the plant kingdom. Formononetin, biochanin A and their active metabolites daidzein and genistein are known to be the most potent among other isoflavonoid phytoestrogens. Thus there is a growing need to determine accurately their concentration in different biological fluids. In the present work, a sensitive analytical method was developed for the quantitative determination of these compounds in human breast milk, saliva and urine. The glycoside conjugates of these compounds were enzymatically hydrolysis prior to salting-out assisted liquid-liquid extraction. Quantitative analysis was done by ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The obtained results showed high correlation coefficients (r² > 0.998) for the linear range established for formononetine, biochanin A, daidzein and genistein. The limits of detection (LODs) and low limits of quantitation (LLOQs) were in the ranges of 0.05–1.0 ng/mL and 1.0–4.0 ng/mL for all analytes in human biological fluids, respectively. The average recoveries ranged from 83.29% to 115.24% for the analytes with relative standard deviation (n = 5) values from 1.84% to 9.75% in samples. Both intra-day and inter-day precisions and accuracy were found to be within 12.53% and ± 12.92% respectively. Under different conditions of stability, the concentrations for four isoflavonoid phytoestrogens deviated within ±12.87% of norminal values. The developed method was successfully validated and applied to human breast milk, saliva and urine. The average concentrations of daidzein and genistein found in breast milk, saliva and urine samples ranged from 0 to 104.2 µg/kg, 18.17 to 786.0 µg/kg, 0 to 10974 µg/kg, respectively. Their presence in breast milk samples shows exposure of breast-fed baby to isoflavones. It also allows for the rapid screening of human biological fluids when testing for formononetin, biochanin A, daidzein and genistein production status in human.