ReviewLC-MS/MS determination and pharmacokinetic study of five flavone components after solvent extraction/acid hydrolysis in rat plasma after oral administration of Verbena officinalis L. extract

Ethnopharmacological relevanceTraditional Chinese medicine (TCM) has been used in clinical practice for several thousand years. TCM has played an indispensable role in the prevention and treatment of diseases, especially the complicated and chronic ones. Pharmacokinetic study on active constituents in herbal preparations is a good way for us to explain and predict a variety of events related to the efficacy and toxicity of TCM.Aim of the studyA selective and sensitive HPLC-MS/MS method was first developed and validated for the determination of luteolin, kaempferol, apigenin, quercetol, and isorhamnetin in rat plasma.Materials and methodsThe LC system consisted of an Agilent Technologies Series 1200 system (Agilent, USA) equipped with an automatic degasser, a quaternary pump, and an autosampler. Chromatographic separations were performed on a Waters SunFire™ C18 column (150 mm × 4.6 mm, 5 μm), and the column temperature was maintained at 25 °C and the sample injection volume was 20 μL. The current LC-MS/MS assay was validated for linearity, intra-day and inter-day precisions, accuracy, extraction recovery and stability.ResultsThe validated method was successfully applied to monitoring the concentrations and pharmacokinetic studies of five flavone compounds in rat plasma after a single oral administration of Verbena officinalis L. extract with a dosage of 8.0 mL/kg. The time to reach the maximum plasma concentration (Tmax1) was 0.48 ± 2.14 h for luteolin, 0.25 ± 0.13 h for kaempferol, 0.97 ± 1.08 h for apigenin, 1.04 ± 4.25 h for quercetol and 0.25 ± 0.16 h for isorhamnetin, and the maximum plasma concentration (Tmax2) was 3.97 ± 1.48 h, 4.05 ± 0.46 h, 4.33 ± 0.58 h, 2.99 ± 0.48 h and 4.02 ± 0.34 h. The elimination half-time (t1/2) of luteolin, kaempferol, apigenin, quercetol and isorhamnetin was 4.02 ± 0.81, 7.65 ± 0.71, 3.30 ± 0.83, 4.55 ± 0.49 and 5.56 ± 1.32 h, respectively.ConclusionsThis paper described a simple, sensitive and validated LC-MS/MS method for simultaneous determination of luteolin, kaempferol, apigenin, quercetol and isorhamnetin in rat plasma after oral administration of V. officinalis L. extract, and investigated on their pharmacokinetic studies as well, with a short run time of 5 min.

Graphical abstractA selective and sensitive HPLC-MS/MS method was first developed and validated for the determination of luteolin, kaempferol, apigenin, quercetol, and isorhamnetin after solvent extraction/acid hydrolysis in rat plasma. Blood samples were collected via the fossa orbitalis vein at time intervals after oral administration and the concentrations of the five ingredients in plasma were analyzed by HPLC-MS/MS. Sulfamethoxazole (SMZ) was used as an internal standard, and plasma samples were pretreated by a single-step protein precipitation with methanol-hydrochloric acid (25%) (4:1, v/v). Chromatographic separation was achieved on a Waters SunFire™ C18 column (150 mm × 4.6 mm, 5 μm) at 25 °C with a mobile phase consisting of acetonitrile and 0.1% aqueous formic acid at a flow rate of 0.8 mL/min. A tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) under negative ionization mode with an electrospray ionization (ESI) source. Calibration curves of luteolin, kaempferol, apigenin, quercetol, and isorhamnetin were generated over the range 0.005-2.50, 0.005-2.50, 0.020-10.0, 0.025-12.5 and 0.020-10.0 μg/mL, respectively. The lower limits of the quantitation of these analytes were less than 25 ng/mL. The mean extraction recoveries for all compounds were between 81.5 and 108.1%. The intra- and inter-day precisions were less than 9.1% and 10.8%, and the accuracy was within ±8.3% for all the analytes.Download high-res image (89KB)Download full-size image