A HPLC-UV method for the determination of puerarin in rat plasma after intravenous administration of PEGylated puerarin conjugate.

A sensitive and reproducible HPLC method for quantitative determination of puerarin (PUE) in rat plasma was developed and validated using 4-hydroxybenzaldehyde as an internal standard. The separation of PUE was performed on a CAPCELL PAK C18 column by gradient elution with 0.2% aqueous phosphoric acid and acetonitrile as the mobile phase. The method was validated and found to be linear in the range of 80-12,000ng/mL. The limit of quantification was 80ng/mL based on 100μL of plasma. The variations for intra- and inter-day precision were less than 8.3%, and the accuracy values were between 98% and 105.2%. The extraction recoveries were more than 85%. The method was successfully applied in the comparative study of pharmacokinetics of PEGylated puerarin (PEG-PUE) versus PUE in rats. Compared with PUE, PEG-PUE showed a 5.2-fold increase in half-life of PUE and a 4.7-fold increase in mean residence time. In addition, this method was also successfully applied to determine the low plasma concentration of PUE regenerated from PEG-PUE in vitro.

Liu X ，Zhi H ，Du F ，Ye Z ，Wang N ，Qin W ，Li J ... -  《-》

A validated stability-indicating HPLC method for the determination of PEGylated puerarin in aqueous solutions.

The aim of this study was to develop a validated specific stability-indicating HPLC method for the quantitative determination of PEGylated puerarin (PEG-PUE) in aqueous solutions. The method was validated by subjecting PEG-PUE to forced degradation under stress conditions of acid, alkali, water hydrolysis, and oxidation. Both PEG-PUE and puerarin (PUE) were simultaneously determined and separated on CAPCELL PAK C18 column by gradient elution with 0.2% aqueous phosphoric acid and acetonitrile as the mobile phase. The flow rate was 1.0 mL min(-1) and detection wavelength was set at 250 nm. Both calibration curves showed good linear regression (r> or =0.9998) within test ranges. The LOD and LOQ of PEG-PUE were determined to be 3 and 9 microg mL(-1) respectively. Degradation of PEG-PUE followed pseudo-first-order kinetics with t(1/2) of 59 min at pH 9.0 and 17.79 h at pH 7.4. However, at pH 5.0 and 2.0, there was no significant degradation of PEG-PUE over time. In conclusion, the method was observed to have the necessary specificity, precision, and accuracy, and to be suitable for quantity monitoring the degradation process of PEG-PUE during stability studies. The degradation studies may give insight into useful information for formulation development of PEG-PUE.

Liu X ，Yu B ，Wang N ，Zhang B ，Du F ，He C ，Ye Z ... -  《-》

Simultaneous determination of acetylpuerarin and puerarin in rat plasma by liquid chromatography-tandem mass spectrometry: Application to a pharmacokinetic study following intravenous and oral administration.

A rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous determination of acetylpuerarin (AP) and its major metabolite puerarin (PUE) in rat plasma using genistein as the internal standard (IS). Plasma samples were pretreated by protein precipitation with a mixture of methanol and acetonitrile. Chromatographic separation was performed on a CAPCELL PAK C18 MGШ column with a mixture of 0.1% formic acid in water and methanol (35:65, v/v) as the mobile phase. The analytes were detected using a tandem mass spectrometer in the positive ionization and multiple-reaction monitoring mode. The ion transition of m/z 669.4→627.3, 417.5→297.6 and 271.3→153.0 was utilized to quantify AP, PUE and the IS, respectively. The calibration curves showed good linearity over the plasma concentration range of 1-2000ng/mL for AP and 2.5-5000ng/mL for PUE. The intra- and inter-day precisions (RSD %) for each analyte were less than 6.91%, and the accuracies ranged from -2.17% to 2.93%. The validated LC-MS/MS method was further successfully applied to a pharmacokinetic study of AP and PUE in rats following intravenous and oral administration.

Sun D ，Xue A ，Wu J ，Zhang B ，Yu J ，Li Q ，Sun C ... -  《-》

Quantitative determination of puerarin in dog plasma by HPLC and study on the relative bioavailability of sustained release tablets.

Ren F ，Jing Q ，Shen Y ，Ma H ，Cui J ... -  《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》

HPLC determination of safflor yellow A and three active isoflavones from TCM Naodesheng in rat plasma and tissues and its application to pharmacokinetic studies.

A high-performance liquid chromatographic method was developed for the simultaneous determination and pharmacokinetic studies of safflor yellow A, puerarin, 3'-methoxyl-puerarin, and puerarinapioside in the plasma and tissues of rats that had been administered with the traditional Chinese medicine (TCM) preparation Naodesheng via the caudal vein. Samples taken from rats were subjected to protein precipitation with acetone. Separation of these four compounds was accomplished on a Kromisil C18 stationary phase using a mobile phase of acetonitrile-0.1% phosphoric acid-tetrahydrofuran (8:92:2, v/v/v) at a flow-rate of 1.0 mL/min. The detection wavelength was set at 250 nm. The calibration curves of the four components were linear in the given concentration ranges. The intra- and inter-day precisions in plasma and tissues were less than 15% and the extraction recoveries were higher than 60%. The lower limits of quantitation of four components were low enough to determine the four components. These four components all exhibited kinetics that fitted a two-compartment model in rats. The elimination half-life was 1.19 h for safflor yellow A, 2.69 h for puerarin, 2.94 h for 3'-methoxyl-puerarin, and 0.87 h for puerarinapioside, respectively. Following administration of a single injection of Naodesheng, the concentration (C) of the four components in the tissues showed C(kidney) > C(lung), C(liver) > C(spleen), C(stomach), C(heart), approximately. The method is a reliable tool for performing studies of safflor yellow A and three puerarin isoflavones in different biological material.

Yu Z ，Gao X ，Zhao Y ，Bi K ... -  《BIOMEDICAL CHROMATOGRAPHY》