Simultaneous determination of multiple bioactive components of Bu-zhong-yi-qi-tang in rat tissues by LC-MS/MS: Application to a tissue distribution study

A liquid chromatography coupled with electrospray ionization mass spectrometry method was developed and validated for simultaneous determination of seven bioactive constituents including astragaloside IV, calycosin, glycyrrhizic acid, enoxolone, saikosaponin D, ferulic acid and hesperiden in rats' various tissues using diclofenac as the internal standard (IS). Biological samples were pretreated by protein precipitation with acetonitrile. The chromatographic separation was carried out on a C18 column with a gradient mobile phase consisting of acetonitrile and water (containing 0.1% formic acid and 4 mM ammonium acetate). All analytes and IS were quantitated through electrospray ionization in negative ion multiple reaction monitoring mode. The mass transitions were as follows: m/z 829.7 → 783.3 for astragaloside IV, m/z 283.3 → 267.7 for calycosin, m/z 821.6 → 350.0 for glycyrrhizic acid, m/z 469.9 → 425.2 for enoxolone, m/z 825.7 → 779.6 for saikosaponin D, m/z 192.5 → 133.9 for ferulic acid, m/z 609.1 → 301.0 for hesperiden and m/z 293.6 → 249.9 for the IS, respectively. The lower limits of quantification for the seven analytes in different rat tissues were 0.2-20 ng/mL. Bu-zhong-yi-qi-tang (Hochuekkito in Japan, Bojungikki-tang in Korea) is one of the most frequently prescribed traditional herbal formulas used in Korea, Japan, and China to treat gastrointestinal diseases, cancer and chronic fatigue syndrome. The validated method was successfully applied to a tissue distribution study of the seven components in rat tissue after oral administration of Bu-zhong-yi-qi-tang concentrated granule. The results of the tissue distribution study showed that the high concentration of seven components were mainly in the gastrointestinal tract.