Simultaneous determination of fourteen main active components in Gumiganghwal-tang tablet by using a newly developed UPLC-ESI-MS/MS method.

The purpose of this study was to develop a method for simultaneous analysis of fourteen major active components in Gumiganghwal-tang tablet widely prescribed for cold related diseases using UPLC-ESI-MS/MS. Twelve of these 14 components were separated using 0.1% formic acid and acetonitrile as a mobile phase by gradient elution at a flow rate of 0.3 mL/min equipped with a KINETEX C column (2.1 × 50 mm, 1.7 μm). The remaining two components were separated using 10 mM aqueous ammonium formate containing 0.01% formic acid and acetonitrile as a mobile phase by gradient elution at a flow rate of 0.2 mL/min equipped with an Inertsil C-3 column (2.1 × 100 mm, 2.0 μm). Quantitation of this analysis was performed on a triple quadrupole mass spectrometer using electrospray ionization technique operating in multiple reaction monitoring mode. Full validation of the analysis method was carried out, including its linearity, selectivity, sensitivity, precision, accuracy, recovery, and stability. Chromatograms showed high resolution, sensitivity, and selectivity without interference by impurities. Calibration curves of all 14 components ranged from 0.5 to 1000 ng/mL, displaying excellent linearity (correlation coefficients >0.99). The relative standard deviations (RSD) of intra- and inter-day were <11.75%. Recoveries were within the range 95.41-103.24% (RSD value of 1.62-9.09%). These results demonstrate that the developed method is simple, rapid, reliable, specific, accurate, and sensitive for the quantification of bioactive components of Gumiganghwal-tang. The developed method was successfully applied to the analysis of Gumiganghwal-tang tablet. The developed UPLC-ESI-MS/MS method could be useful not only for quality control, but also for effectiveness and safety evaluation of Gumiganghwal-tang tablet.