Identification of tanshinones and their metabolites in rat bile after oral administration of TTE-50, a standardized extract of Salvia miltiorrhiza by HPLC-ESI-DAD-MSⁿ

TTE-50 is a standardized extract of Salvia miltiorrhiza which mainly consisted of tanshinones. A sensitive and specific method using liquid chromatography-diode array detection-electrospray ionization (ESI) ion trap mass spectrometry was established for the study of the constituents and metabolites of TTE-50 in rat bile sample after oral administration. The bile samples were extracted with ethyl acetate (EtOAc) of three-fold volume for three times. The chromatographic separation was carried out on a Zorbax Extend-C₁₈ column with a gradient elution program whereas acetonitrile-water was used as mobile phase. Mass spectra were acquired in positive ionization mode and data-dependant scan was used for the identification of the tanshinones and metabolites in the bile samples. Identification and structural elucidation of the tanshinones and their metabolites in bile samples were performed by comparing their retention-times and full scan MSⁿ spectra with those of reference compounds and data in the literatures. Sixteen tanshinones in TTE-50 along with seventeen phase I metabolites were identified simultaneously. The metabolic modification could take place in the C-4 side chain of tanshinone IIA, from methyl to primary alcohol, then to aldehyde group was proposed for the first time. The established method was valuable for the study of the metabolism of complex system such as herbal extracts or traditional Chinese medicine (TCM) formula.