高效液相色谱波长切换法结合双标线性校正法用于茯神木指纹图谱的定性研究

Qualitative study on fingerprint of Pini Radix in Poria by HPLC wavelength switching method combined with double standard linear correction

目的:建立茯神木高效液相色谱波长切换法指纹图谱,考察双标线性校正法对色谱峰定性的可行性。方法:采用高效液相色谱-二极管阵列检测器(HPLC-DAD)波长切换法建立指纹图谱,使用C18柱,流动相为乙腈(A)-0.1%磷酸(B),梯度洗脱(0~6 min,60%A;6~30 min,60%A→80%A;30~34 min,80%A→90%A;34~40 min,90%A),流速1.0 mL·min^(-1),检测波长242 nm(0~28 min)、210 nm(28~30.5 min)、242 nm(30.5~40 min),柱温30℃,进样量5μL。以相似度分析进行质量评价,并使用DRS Origin软件进行双标线性校正,以16α-羟基松苓新酸、松苓新酸为双标化合物,预测其余待测峰的保留时间,并在另1根C18色谱柱上进行验证,将预测结果与相对保留时间法进行比较,以辅助色谱峰的定性分析。结果:10批茯神木指纹图谱相似度≥0.972,表明不同批次间茯神木的成分比例稳定,整体相似度很高。双标线性校正法对保留时间的预测准确率(86%)更高,且有更高的色谱柱适用性(11根色谱柱)。结论:双标线性校正法在多波长条件下同样具有优势,可辅助色谱峰的定性。该方法可行、准确、经济。

Objective:To establish HPLC wavelength switching fingerprint of Pini Radix in Poria and investigate the feasibility of dual standard linear correction method for qualitative analysis of chromatographic peaks.Methods:The fingerprints were established by high performance liquid chromatography-diode array detector(HPLC-DAD)wavelength switching method,using a C_(18)column,the mobile phase was acetonitrile(A)-0.1%phosphoric acid(B)with gradient elution(0-6 min,60%A;6-30 min,60%A→80%A;30-34 min,80%A→90%A;34-40 min,90%A)at a flow rate of 1.0 mL min^(-1),the detection wavelengths were 242 nm(0-28 min),210 nm(28-30.5 min)and 242 nm(30.5-40 min),the column temperature 30℃,and the injection volumn was 5μL.Quality evaluation using similarity analysis,and double standard linearity correction was performed using DRS Origin software,with 16α-hydroxydehydrotrametenolic acid and 3β-hydroxy-5α-lanosta-7,9(11),24-trien-21-oic acid are double standard compounds.The retention time of the remaining peaks to be measured was predicted and verified on another C_(18)chromatographic column.The predicted results were compared with the relative retention time method to assist the qualitative analysis of chromatographic peaks.Results:The fingerprint similarity of 10 batches of Pini Radix in Poria were above or equal to 0.972,indicating that the proportion of components of Pini Radix in Poria among different batches was stable and the overall similarity was high.The dual-standard linear calibration method had higher retention time prediction accuracy(86%)and higher column adaptability(11 columns).Conclusion:The double standard linear correction method also has advantages under the condition of dual wavelength.It can better assist the qualitative determination of chromatographic peaks.The method is feasible,accurate,economical and practical.