基于一测多评法的半夏碱基和核苷含量测定

Content determination of Pinellia ternate bases and nucleosides based on quantitative analysis of multi-components by single marker

目的:采用高效液相色谱法(HPLC)建立半夏中2种碱基(尿嘧啶、次黄嘌呤)和3种核苷(尿苷、鸟苷、胸苷)的定量检测方法,以验证该方法在半夏质量控制中应用的可行性和技术适应性。方法:超声提取半夏粉末,采用HPLC法检测尿嘧啶、次黄嘌呤、尿苷、鸟苷、胸苷的含量。使用Agilent ZORBAX SB-C色谱柱(4.6 mm×250 mm,5μm),以水-甲醇为流动相,流速0.8 ml/min,检测波长260 nm,柱温30℃,进样量10μl,梯度洗脱(0~7 min,2%~2%甲醇;7.1~13 min,3%~3%甲醇;13~25 min,3%~20%甲醇;25~40 min,20%~98%甲醇)。以鸟苷为内参物,建立尿嘧啶、次黄嘌呤、尿苷、胸苷相对于鸟苷的相对校正因子,并进行含量计算,实现一测多评。同时采用外标法测定10批半夏药材中5种成分的含量,比较计算值与实测值的差异,验证一测多评的准确性。结果:此种方法建立的相对校正因子在不同仪器中的耐用性良好。计算值与实测值之间无显著性差异(RSD<1.3%)。结论:本研究建立的半夏中5种成分同时测定的一测多评法,简单易行,成本较低,可为后续物质基础及作用机制研究提供借鉴。

Objective:To establish a high-performance liquid chromatography(HPLC)method for quantification of two types of bases(uracil and hypoxanthine)and three types of nucleosides(uridine,guanosine,and thymidine)in Pinellia ternate,and to validate the feasibility and technical adaptability of this method in the quality control of Pinellia ternate.Methods:Ultrasound extraction was used to obtain Pinellia ternate powder,and HPLC was used to measure the content of uracil,hypoxanthine,uridine,guanosine,and thymidine.HPLC was performed on an Agilent ZORBAX SB-Ccolumn(4.6 mm×250 mm,5μm)with a mobile phase of water-methanol for gradient elution(0~7 minutes:2%~2% methanol;7.1~13 minutes:3%~3%methanol;13~25 minutes:3%~20%methanol;25~40 minutes:20%~98% methanol)at a flow rate of 0.8 ml/min,a detection wavelength of 260 nm,a column temperature of 30℃,and a sample size of 10μl.Guanosine was used as the internal reference to establish the relative correction factors of uracil,hypoxanthine,uridine,and thymidine to guanosine,and the content was calculated to realize the quantitative analysis of multi-components by single marker.In addition,the external standard method was used to measure the content of five components in Pinellia ternate,and the calculated values were compared with the measured values to validate the accuracy of quantitative analysis of multi-components by single marker.Results:The relative correction factors established by this method had good durability in different instruments,and there was no significant difference between the calculated values and the measured values,with a relative standard deviation of<1.3%.Conclusion:This study establishes the quantitative analysis of multi-components by single marker for simultaneous determination of five components in Pinellia ternate,which is simple and convenient with low costs and provides a reference for subsequent studies on material basis and mechanism of action.