Determination of gentiopicroside and its excretion in human urine by LC/MS

Aim:To develop a solid-phase extraction and LC/MS method for the determination of gentiopicroside excreted in human urine and assessment of its elimination route.Methods:Human urine samples,collected from 12 healthy volunteers exposed to 80 mg,240 mg,and 400 mg of gentiopicroside by iv dosing in the randomized crossover design,added with caffeine as internal standard,handled by solid-phase extraction,then measured by LC-MS.Analyses were performed on Rescek C_8 column(150 mm×2.1 mm,5 μm)with a mobile phase of methanol-10 mmol/L NH_4OAC buffer-acetonitrile(50∶40∶10)at a flow rate of 0.2 mL/min.Mass spectrometer with the mode of multiple reaction monitoring(MRM) was applied in the detection.Results:It indicated good linearity in the range of 30～9000 ng/mL(r=0.998 0) for assay of gentiopicroside in human urine.The extraction recovery and overall recovery were 100.52%～103.83% and 91.10%～96.21%,respectively.The intra-day and inter-day deviations at lower,middle and high levels were estimated to be less than 10%.The percentages of urinary excretive amount of the parent compound in 24 h following the administration of three dose sizes were estimated to be(76.59±10.02)%,(71.95±12.12)%,and(79.76±8.54)%,respectively.The cumulative amount of the urinary excretion was shown to be directly proportional to the magnitude of dose.In addition,the maximal amount of the parent compound was excreted in urine in 0～5.5 h.Conclusion:This method proved to be sensitive,accurate,rapid,specific,and suitable for clinical pharmacokinetics.The results indicated that gentiopicroside,following iv dosing,is mainly eliminated via urinary route in the unchanged form.