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Abstract
This paper describes the development and validation of an analytical method for the quantitative determination of genistein and daidzein in human plasma by liquid chromatography with negative electrospray ionization tandem mass spectrometric detection (LC/ESI−‐MS‐MS). In this work, 4‐hydroxybenzophenone was used as an internal standard (I.S.). Genistein and daidzein, together with the I.S., were first extracted from human plasma by a Speed Scan ABN (3 mL) cartridge, then separated by a Nova‐Pak® C8 column (5 µm, 2 × 150 mm) with isocratic elution (45% acetonitrile + 55% 0.5 mM ammonium formate at pH 4.0), and detected by ESI−‐MS‐MS with multiple‐reaction‐monitoring (MRM) mode. The retention times of genistein, daidzein, and the I.S. were 3.4, 2.6, and 4.4 min, respectively. Quantitation using MRM mode was carried out at m/z 269 > 133 for genistein, m/z 253 > 208 for daidzein, and m/z 197 > 92 for the I.S. The average recoveries for genistein, daidzein, and the I.S. from human plasma were 78.8, 77.5, and 85.5%. The linear calibration ranges were 8.50–1000 ng/mL for both genistein and daidzein with correlation coefficients r 2 > 0.998. The intra‐assay precision (%CV, n = 5) and the inter‐assay precision (%CV, n = 3) were <5 and 8% at three concentration levels (10.0, 50.0, 250 ng/mL). The limit of detection and the limit of quantification for both genistein and daidzein were 2.50 and 8.50 ng/mL with a 20‐µL sample injection. This method has a total instrument analysis time of 6 min per sample. It may be useful for the measurement of genistein and daidzein in human plasma for clinical studies of these compounds.