Development of an LC-MS method for determination of three active constituents of Shuang-huang-lian injection in rat plasma and its application to the drug interaction study of Shuang-huang-lian freeze-dried powder combined with levofloxacin injection.

A sensitive and specific high performance liquid chromatography coupled with mass spectrometric (LC-MS) method was developed and validated for the simultaneous determination of three main active constituents of Shuang-huang-lian injection with and without the combination use of levofloxacin injection in rat plasma. After addition of the internal standard rutin, plasma samples were protein precipitated with acetonitrile, the chromatographic separation was achieved on a Kromasil C₁₈ column (250 mm × 4.6 mm, 5 μm), using a gradient mobile phase system of acetonitrile-water containing 0.05% formic acid. The analytes were detected without interference in the selected ion monitoring (SIM) mode with positive electrospray ionization. The linear range was 0.04-20 μg/mL for chlorogenic acid, 0.8-400 μg/mL for baicalin and 0.01-5.0 μg/mL for phillyrin, respectively. The accuracy (relative error, R.E.%) were between -2.7 and 3.4%, while the intra-day and inter-day precisions were less than 9.2 and 9.6% for the three analytes, respectively. This method was successfully applied to the drug interaction study of Shuang-huang-lian freeze-dried powder combined with levofloxacin injection after intravenous administration to rats. The results indicated that there were obvious differences in the pharmacokinetic behaviors after combination compared with only administration of Shuang-huang-lian injection.