Turbulent-flow Chromatography Coupled On-line to Fast High-performance Liquid Chromatography and Mass Spectrometry for Simultaneous Determination of Verticine, Verticinone and Isoverticine in Rat Plasma

A method based on the on-line turbulent-flow chromatography and fast high-performance liquid chromatography/mass spectrometry (TFC-LC/MS) was developed for sensitive and high throughput pharmacokinetic study of traditional Chinese medicines (TCMs). In this method, an on-line extraction column (Waters Oasis HLB) and a fast HPLC column with sub-2 microm particle size (Agilent Zorbax StableBond-C(18), 4.6 mm x 50 mm, 1.8 microm) in a column-switching set-up were utilized. HLB is a reversed-phase extraction column with hydrophilic-lipophilic balanced copolymer (2.1 mm x 20 mm, 25 microm particle size), which will exhibit some turbulent-flow properties at a high-flow rate. The method combines the speed and robustness of turbulent-flow extraction and the sensitivity and separation efficiency of fast HPLC-MS to analyze multiple and trace constituents of TCMs in plasma matrix. This method was successfully applied for pharmacokinetic study of verticine, verticinone and isoverticine, the chemical markers of Fritillaria thunbergii, after oral administration of total steroidal alkaloids extract of F. thunbergii to rats. Each plasma sample was analyzed within 7 min. The method demonstrated good linearity (R>0.999) ranged from 0.505 to 96.0 ng/mL with satisfactory accuracy and precision, and the lower limit of quantifications of verticine, verticinone and isoverticine were estimated to be 0.120, 0.595 and 0.505 ng/mL, respectively. These results indicate that the proposed method is fast, sensitive, and feasible for pharmacokinetic study of TCMs.