A UPLC-MS/MS method for simultaneous determination of danshensu, protocatechuic aldehyde, rosmarinic acid, and ligustrazine in rat plasma, and its application to pharmacokinetic studies of Shenxiong glucose injection in rats

A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of the four major active ingredients, danshensu, protocatechuic aldehyde, rosmarinic acid, and ligustrazine, in the traditional Chinese medicine Shenxiong glucose injection in rat plasma. Acidified and alkalized plasma samples were extracted using ethyl acetate, and separated on a Waters C18 column (2.1 mm × 50 mm, 1.7 μm) by using a gradient mobile phase system of acetonitrile-water containing 0.1% formic acid and luteoloside as an internal standard. Electrospray ionization in the positive-ion mode and multiple reaction monitoring were used to identify and quantitate the active components. All calibration curves showed good linearity (r > 0.994) over the concentration range, with a lower limit of quantification (LLOQ) between 0.02 and 0.21 μg/mL. The precision of the in vivo study was evaluated by intra- and inter-day assays, and the percentage of relative standard deviation was within 15%. Moreover, satisfactory extraction efficiency was obtained (between 83.94 and 117.81%) by liquid-liquid extraction. The validated method was successfully applied in a pharmacokinetic study in rats after intravenous administration of Shenxiong glucose injection. The results showed that the four bioactive ingredients in Shenxiong glucose injection have linear pharmacokinetic properties in rats after intravenous injection within the administered dose range and partially different ones compared to single ingredient.