反相离子对高效液相色谱法测定黄柏及其颗粒剂中小檗碱及巴马汀的含量

Reversed-phase ion pair high performance liquid chromatography in determination of berberine and palmatine in Phellodendron chinense Schneid.and its granules

目的:探讨应用反相离子对高效液相色谱法测定黄柏药材(Phellodendron chinense Schneid.)及其颗粒剂中小檗碱及巴马汀的含量.方法:采用反相离子对高效液相色谱法并进行方法学考察,色谱条件:Lichrospher C18色谱柱(4.6 mm×250 mm,5 μm);流动相为乙腈∶25 mmol/L磷酸二氢钠∶25 mmol/L十二烷基硫酸钠(2∶1∶1);流速1.0 ml/min;检测波长345 nm;柱温25℃.黄柏药材及其颗粒剂以盐酸甲醇溶液(1∶100)提取.结果:小檗碱和巴马汀色谱峰的理论塔板数分别为14 906和14 847,分离度分别为2.33和2.86,拖尾因子分别为1.09和1.06,符合定量分析的要求.回归方程分别为Y=698 278X-3 846, r=1.000和Y=536 632X-7 738, r=0.999 9,线性范围分别在40～500 ng和20～250 ng之间.方法学考察结果表明,小檗碱和巴马汀低、中、高3个进样量的日内精密度和日间精密度的RSD均小于2.5%和1.5%,48 h内稳定性试验的RSD分别为0.66%和0.70%,重现性试验的RSD分别为0.11%和0.12%(n=5),最低检测限分别为2.0 ng和1.0 ng,加样回收率结果分别为100.4%,RSD＝0.12%(n=3)和99.80%,RSD＝0.22%(n=3).测定3批黄柏药材和5批黄柏颗粒剂中小檗碱及巴马汀的含量.结论:该方法简便可行,结果可靠,能够用于黄柏药材及其颗粒剂中小檗碱及巴马汀含量测定的研究.

Objective:To determine berberine and palmatine in Phellodendron chinense Schneid.and its granules.Methods: The reversed-phase ion pair high performance liquid chromatography(RP-HPLC) was used and the validation of the method was tested.The chromatography condition was with Lichrospher C18 column(4.6 mm×250 mm,5 μm), mobile phase was ACN∶25 mmol/L NaH 2PO 4∶25 mmol/L SDS(2∶1∶1),flow speed was 1.0 ml/min,detection wavelength was 345 nm,and temperature of column was 25℃,Phellodendron chinense Schneid.and its granules were extracted with methanol solution of hydrochloric acid.Results: The theoretical plate number of berberine and palmatine were 14 906 and 14 847,the resolution were 2.33 and 2.86,the tailing factor were 1.09 and 1.06,respectively; all the parameters were suitable for determination.The calibration curves were linear in the range of 40-500 ng,Y=698 278X-3 846,r=1.000(berberine) and 20-250 ng, Y= 536 632X- 7 738, r=0.999 9,r=0.999 4(palmatine).The intra-day and inter-day precision(RSD) at low,middle and high injection amount was all less than 2.5%(berberine) and 1.5%(palmatine).The stability(RSD) was 0.66%(berberine) and 0.70% (palmatine) in 48 h.The recurrence(RSD,n=5) was 0.11%(berberine) and 0.12%(palmatine).The limits of detection was 2.0 ng(berberine) and 1.0 ng(palmatine).The recoveries were 100.4% (RSD=0.12%,n=3) for berberine and 99.80%(RSD=0.22%,n=3) for palmatine.The contents of berberine and palmatine in 3 batch of Phellodendron chinense Schneid.and 5 batch of its granules were determined.Conclusion: Our method can be used for determination of berberine and palmatine in Phellodendron chinense Schneid.and its granules, which is simple and reliable.