Development of an LC-MS method for simultaneous quantitation of amentoflavone and biapigenin, the minor and major biflavones from Hypericum perforatum L., in human plasma and its application to real blood.

Biflavones of Hypericum perforatum L. are bioactive compounds used in the treatment of inflammation and depression. Determination of amentoflavone and biapigenin from blood is challenging owing to their similar structures and low concentrations. To develop a rapid, sensitive and accurate method based on liquid-phase extraction followed by high-performance liquid chromatography and electrospray ionisation mass spectrometry (HPLC-ESI-MS) for quantification of biflavones in human plasma. After extraction from blood, the analytes were subjected to HPLC with an XTerra® MS C(18) column and a binary mobile phase consisting of 2% formic acid in water and acetonitrile under isocratic elution conditions, with ESI-MS detection in the negative ion mode and multiple reaction monitoring (MRM). Both calibration curves showed good linearity within the concentration range 1-500  ng/mL. Limits of detection (S/N = 3) were 0.1  ng for pure substances and the limits of quantitation (S/N = 5) were 1.0  ng/mL from analyte-spiked serum. The grand mean recovery was 90% from several subsamples of each biflavone. The imprecision (RSD) of peak areas was between 5% (intraday) and 10% (interday) for high concentrations (250  ng/mL) and between 10% (intraday) and 15% (interday) for low concentrations (1  ng/mL). Inaccuracy of the mean was less than 20% at the lower limit of quantitation. The developed and validated method for determination of biflavones from human plasma was effectively applied to pharmacokinetic studies of 13 probands and preliminary results indicate biphasic concentration-time curves.