HPLC-UV 法测定聚乙二醇葛根素缀合物静脉注射后大鼠血浆中的葛根素。

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

机构信息

Department of Chinese Medicinal Prescription, China Pharmaceutical University, Nanjing 2111198, China.

出版信息

J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Dec 1;878(31):3297-302. doi: 10.1016/j.jchromb.2010.10.011. Epub 2010 Oct 21.

DOI:10.1016/j.jchromb.2010.10.011

PMID:21056017

Abstract

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.

摘要

建立并验证了一种灵敏、重现的 HPLC 法，用于定量测定大鼠血浆中的葛根素（PUE），以内标 4-羟基苯甲醛（4-OHB）进行定量。采用梯度洗脱，以 0.2%磷酸水溶液和乙腈为流动相，在 CAPCELL PAK C18 柱上进行分离。该方法线性范围为 80-12000ng/mL，定量下限为 80ng/mL（取 100μL 血浆）。日内和日间精密度的变异系数均小于 8.3%，准确度在 98%-105.2%之间。提取回收率均大于 85%。该方法成功应用于 PEG 化葛根素（PEG-PUE）与 PUE 在大鼠体内药代动力学的比较研究。与 PUE 相比，PEG-PUE 使 PUE 的半衰期延长了 5.2 倍，平均驻留时间延长了 4.7 倍。此外，该方法还成功应用于测定体外 PEG-PUE 中 PUE 的低血浆浓度。