SIMULTANEOUS DETERMINATION OF FIVE MAJOR CONSTITUENTS IN THE EFFECTIVE FRACTIONS OF HUANGQICHIFENG MEDICINAL BROTH, BY UPLC-MS/MS

An UPLC method coupled with tandem quadrupole mass spectrometry (MS/MS) was established for the simultaneous quantitative analysis of five major constituents in the effective fractions of Huangqichifeng Medicinal Broth (EFHQCF), namely paeoniflorin (PA), prim-O-glucosylcimifugin (PR), ononin (ON), calycosin (CA), and formononetin (FO). The analytes were analyzed on an ACQUITY BEH C-18 VanGuard precolumn and an ACQUITY UPLC BEH C-18 column using gradient elution. The mobile phase A was ammonium acetate (10mM) in water containing 0.2% acetic acid (v/v); mobile phase B was ammonium acetate (10mM) in methanol containing 0.2% acetic acid (v/v). The solvent flow rate was 0.25mL/min. The retention times of PA, PR, ON, CA, and FO were 9.9min, 18.2min, 26.6min, 30.6min, and 35.7min, respectively. The investigated compounds were detected by MS/MS using a negative ion multiple reaction monitoring mode for the simultaneous quantitative detection. The limits of detection ranged from 0.001ug/mL to 0.043 mu g/mL, and the limits of quantitation varied from 0.003 mu g/mL to 0.148 mu g/mL. Moreover, the linearities (r (2)>0.999) and recoveries (from 96.34% to 104.75%) were acceptable. Therefore, the present study suggested that this UPLC-MS/MS method should be reliable and could be used to control the quality of EFHQCF.