Simultaneous quantification of Kirenol and ent-16ß,17-dihydroxy-kauran-19-oic acid from Herba Siegesbeckiae in rat plasma by liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic studies.

A rapid and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method was developed for the simultaneous determination of two active diterpenoids: Kirenol and ent-16ß,17-dihydroxy-kauran-19-oic acid (DHKA) from Herba Siegesbeckiae in rat plasma using osthole as an internal standard (IS). Plasma sample pretreatment involved a one-step liquid-liquid extraction with ethyl acetate. Chromatographic separation was performed on a Waters Symmetry C18 column (2.1mm×100mm, 3.5µm) with isocratic elution using methanol-5mmol/L aqueous ammonium acetate (80:20, v/v) as the mobile phase at a flow rate of 0.2mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode under positive and negative electrospray ionization. The calibration curves were linear over the range of 50.0-25,000ng/mL for Kirenol, and 25.0-12,500ng/mL for DHKA. The extraction recoveries of the two analytes and the IS were all over 85%. The intra- and inter-day precision (relative standard deviation) values were less than 16.8% and the accuracy (relative error) ranged from -10.7 to 10.6% at four quality control levels. The validated method was successfully applied to a comparative pharmacokinetic study of the two diterpenoids in rat plasma after intragastric administration of Kirenol, DHKA and Herba Siegesbeckiae extract. The results showed that there were obvious differences between the pharmacokinetic behaviors after oral administration of Herba Siegesbeckiae extract compared with each of the substances alone.

Simultaneous determination of seven major diterpenoids in Siegesbeckia pubescens Makino by high-performance liquid chromatography coupled with evaporative light scattering detection.

A novel HPLC method with evaporative light scattering detection was developed for the simultaneous quantification of seven major diterpenoids of two types, including ent-pimarane type: Kirenol, Hythiemoside B, Darutigenol, and ent-kaurane type: ent-16ß,17,18-trihydroxy-kauran-19-oic acid, ent-17,18-dihydroxy-kauran-19-oic acid, ent-16ß,17-dihydroxy-kauran-19-oic acid, 16α-hydro-ent-kauran-17,19-dioic acid in the aerial parts of Siegesbeckia pubescens Makino, an important traditional Chinese medicinal herb. Chromatographic separation was achieved on a Waters Symmetry Shield(TM) RP18 column (250 mm× 4.6 mm id, 5 µm) with a gradient mobile phase (A: 0.3% v/v aqueous formic acid and B: acetonitrile) at a flow rate of 1.0 mL/min. The drift tube temperature of evaporative light scattering detection was set at 103°C, and nitrogen flow rate was 3.0 L/min. The method was validated for accuracy, precision, LOD, and LOQ. All calibration curves showed a good linear relationship (r > 0.999) in test range. Precision was evaluated by intra- and interday tests that showed RSDs were less than 3.5%. Accuracy validation showed that the recovery was between 96.5 and 102.0% with RSDs below 2.8%. The validated method was successfully applied to determine the contents of seven diterpenoids in the different parts of Siegesbeckia pubescens Makino from two sources and to determine the contents of ent-pimarane, ent-kaurane, and total diterpenoids.