Salvianolic acid A (SalA) is one of the main active constituents in Salvia miltiorrhiza (Danshen). Although the pharmacokinetics of SalA in rats after intravenous (i.v.) administration of Danshen injection has been reported, the information relevant to the metabolites of SalA in vivo is absent so far. In this study, by means of liquid chromatography with time-of-flight mass spectrometry (LC/TOFMS) and liquid chromatography with ion trap mass spectrometry (LC/MS(n)) techniques, the unknown metabolites of SalA in rat plasma after i.v. administration of the purified SalA at the dose of 20 mg/kg body weight were identified. A liquid-liquid extraction method was established to separate the metabolites from the plasma and the chromatographic separations were performed on a Xterra MS C(18) column (100 mm x 4.6 mm i.d., 3.5 microm) with acetonitrile/methanol/water/formic acid (20.5:19.5:64: 0.05, v/v/v/v) as the mobile phase at a constant flow rate of 0.2 mL/min. Based on the data obtained from the LC/TOFMS determination (the total ion chromatograms, MS spectra and extracted ion chromatograms), in combination with the characteristic fragment ions acquired from the LC/MS(n) determination, five metabolites were identified as SalA-monoglucuronide, monomethyl-SalA-monoglucuronide, mono-methyl-SalA, dimethyl-SalA and dimethyl-SalA-monoglucuronide, and the possible chemical structures were deduced. The results indicated that SalA might mainly undergo two metabolic pathways in vivo in rats, which were methylation and glucuronidation. The present studies have laid a solid foundation for the metabolic mechanism of SalA in vivo.
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