HPLC法同时测定不同来源半夏及其伪品中9种核苷类成分的含量

Simultaneous determination of nine nucleosides in the rhizome of Pinellia ternata from different origins and its counterfeit by HPLC

目的:建立反相高效液相色谱法测定半夏及其主要伪品掌叶半夏、水半夏与天南星中尿嘧啶、胞苷、次黄嘌呤、尿苷、腺嘌呤、次黄嘌呤核苷、鸟苷、胸苷和腺苷含量。方法:采用Diamonsil C_(18)色谱柱(150 mm×4.6 mm,5μm),以水-甲醇为流动相,梯度洗脱,流速0.8 m L·min–1,检测波长262 nm,柱温25℃,进样量50μL。结果:尿嘧啶、胞苷、次黄嘌呤、尿苷、腺嘌呤、次黄嘌呤核苷、鸟苷、胸苷和腺苷质量浓度分别在0.12~5.80μg·mL^(–1)(r=1.000 0)、0.17~8.40μg·mL^(–1)(r=0.999 9)、0.17~8.48μg·mL^(–1)(r=0.999 9)、0.16~8.08μg·mL^(–1)(r=1.000 0)、0.16~8.16μg·mL^(–1)(r=0.999 9)、0.15~7.68μg·mL^(–1)(r=0.999 8)、0.16~7.84μg·mL^(–1)(r=0.999 9)、0.17~8.72μg·mL^(–1)(r=1.000 0)、0.16~8.24μg·mL^(–1)(r=0.999 8)范围内线性关系良好;平均加样回收率(n=6)分别为102.31%(RSD=1.08%)、101.67%(RSD=0.33%)、101.77%(RSD=1.37%)、99.24%(RSD=0.85%)、99.98%(RSD=0.73%)、96.73%(RSD=4.05%)、102.11%(RSD=0.47%)、103.56%(RSD=1.97%)、97.01%(RSD=1.62%);不同来源与产地的半夏与其伪品(掌叶半夏、水半夏、天南星)的各核苷含量差别较大,其中胸苷在天南星中未检出。半夏中9种核苷总量在626.7~1 063μg·g–1之间,掌叶半夏在638.6~1 132μg·g–1之间,水半夏核苷总量为107 6μg·g–1,天南星为756.2μg·g–1。结论:本法初步建立了测定半夏及其伪品中9种核苷类成分含量的方法,可用于半夏药材的质量控制。

Objective：To establish an HPLC method for determination of nine nucleosides（uracil,cytidine,hypoxanthine,uridine,adenine,inosine,guanosine,thymidine and adenosine）in Pinelliae Rhizoma and itscounterfeit（Pinelliae Pedatisectae Rhizoma,Typhonii Flagelliformis Rhizoma,Arisaemae Rhizoma）.Methods：The chromatographic separation was achieved on a Diamonsil C18 column（150 mm×4.6 mm,5 μm）with water（A）and methanol（B）as mobile phase in gradient elution program.The flow rate was 0.8 m L·min–1,the column temperature was maintained at 25 ℃,the injection volume was 50 μL,and the detection wavelength was set at 262 nm.Results：The linear ranges of uracil,cytidine,hypoxanthine,uridine,adenine,inosine,guanosine,thymidine and adenosine were 0.12-5.80 μg·mL^–1（r=1.000 0）,0.17-8.40 μg·mL^–1（r=0.999 9）,0.17-8.48 μg·mL^–1（r=0.999 9）,0.16-8.08 μg·mL^–1（r=1.000 0）,0.16-8.16 μg·mL^–1（r= 0.999 9）,0.15-7.68 μg·mL^–1（r=0.999 8）,0.16-7.84 μg·mL^–1（r=0.999 9）,0.17-8.72 μg·mL^–1（r=1.000 0）,0.16–8.24 μg·mL^–1（r=0.999 8）,respectively.The average recoveries（n=6）were 102.31%（RSD=1.08%）,101.67%（RSD=0.33%）,101.77%（RSD=1.37%）,99.24%（RSD=0.85%）,99.98%（RSD=0.73%）,96.73%（RSD=4.05%）,102.11%（RSD=0.47%）,103.56%（RSD=1.97%）,97.01%（RSD=1.62%）,respectively.The contents of uracil,cytidine,hypoxanthine,uridine,adenine,inosine,guanosine,thymidineand adenosine in Pinelliae Rhizoma and its counterfeit（Pinelliae Pedatisectae Rhizoma,Typhonii Flagelliformis Rhizoma,Arisaemae Rhizoma）from different sources were significantly different.Thymidine was not detected in samples of Rhizoma Arisaemae.The total content ranges of nine nucleosides in Pinelliae Rhizoma and Pinelliae Pedatisectae Rhizoma were 626.7-1 063 μg·g–1,638.6-1 132 μg·g–1,respectively.The total contents of nine nucleosides in Typhonii Flagelliformis Rhizoma and Arisaemae Rhizoma were 107 6 μg·g–1 and 756.2 μg·g–1,respectively.Conclusion：The established method can be used for the determination of nine nucleosides in Pinelliae Rhizoma and its counterfeit,which can be applied for quality control of Pinelliae Rhizoma.