HPLC-一測多評法同時測定壯藥羅漢茶中6種黃酮類成分及多元統計分析

燕霞 朱雪妍 何頌華 張慧 羅軼 黃清泉

中圖分類號 R284 文獻標志碼 A 文章編號 1001-0408（2021）20-2485-07

DOI 10.6039/j.issn.1001-0408.2021.20.09

摘 要 目的：建立同時測定羅漢茶中新落新婦苷、落新婦苷、新異落新婦苷、異落新婦苷、槲皮苷和黃杞苷含量的方法，并進行多元統計分析。方法：采用高效液相色譜-一測多評法。色譜柱為Phenomenex? SuperLu C18，流動相為乙腈-0.1%甲酸溶液（19 ∶ 81，V/V），檢測波長分別為254 nm（新落新婦苷、落新婦苷、新異落新婦苷、異落新婦苷、黃杞苷）、291 nm（槲皮苷），流速為1.0 mL/min，柱溫為30 ℃，進樣量為10 μL。以落新婦苷為內參物，計算其他5種成分的相對校正因子，再根據相對校正因子計算各成分的含量，并與外標法進行比較;采用SPSS 22.0軟件進行聚類分析和主成分分析。結果：新落新婦苷、落新婦苷、新異落新婦苷、異落新婦苷、槲皮苷和黃杞苷檢測進樣量的線性范圍分別為0.007～0.311、0.871～18.184、0.002～0.119、0.052～1.251、0.105～2.202、0.020～2.319 μg（r>0.999）;精密度、重復性、穩定性（24 h）試驗的RSD均小于3%;平均加樣回收率分別為97.32%、94.89%、97.15%、96.90%、97.52%、97.53%（RSD為1.09%～2.60%，n=6）。新落新婦苷、新異落新婦苷、異落新婦苷、槲皮苷、黃杞苷相對于落新婦苷的平均相對校正因子分別為1.252 6、1.198 3、0.958 6、0.807 1、1.138 1。一測多評法測得新落新婦苷、新異落新婦苷、異落新婦苷、槲皮苷、黃杞苷等5種成分的含量分別為0.394 2～2.067 2、0.139 1～0.804 7、2.864 8～8.554 8、4.581 2～11.371 1、1.028 9～13.401 5 mg/g;外標法測得新落新婦苷、落新婦苷、新異落新婦苷、異落新婦苷、槲皮苷和黃杞苷等6種成分的含量分別為0.367 2～1.925 3、46.361 1～126.342 1、0.138 1～0.798 8、2.966 2～8.857 8、4.642 5～11.523 3、0.970 6～12.641 9 mg/g;兩種方法含量測定結果的相對誤差均不高于3.55%。聚類分析結果顯示，9批羅漢茶樣品可聚為兩類，S8聚為一類、其余聚為一類。主成分分析結果顯示，前2個主成分的累計方差貢獻率為84.745%，分類結果與前者一致。結論：所建高效液相色譜-一測多評法準確、可行，重復性良好，可用于羅漢茶中6種黃酮類成分的同時測定，并可為其質量控制提供參考。

關鍵詞 羅漢茶;高效液相色譜法;一測多評法;黃酮;含量;聚類分析;主成分分析

Simultaneous Determination of 6 Flavonoids in Zhuang Medicine Engelhardia roxburghiana by HPLC-QAMS and Multivariate Statistical Analysis

YAN Xia，ZHU Xueyan，HE Songhua，ZHANG Hui，LUO Yi，HUANG Qingquan（Guangxi Zhuang Autonomous Region Institute for Food and Drug Control/NMPA Key Laboratory for Quality Monitoring and Evaluation of Traditional Chinese Medicine， Nanning 530021， China）

ABSTRACT? ?OBJECTIVE： To establish a method for simultaneous determination of neoastilbin，astilbin，neoisoastilbin，isoastilbin，quercitrin and engeletin in Engelhardia roxburghiana， and conduct multivariate statistical analysis. METHODS： HPLC-QAMS method was adopted. The determination was performed on Phenomenex SuperLu C18 column with mobile phase consisted of acetonitrile-0.1% formic acid （19 ∶ 81， V/V） at the flow rate of 1.0 mL/min. The detection wavelengths were set at 254 nm （neoastilbin， astilbin， neoisoastilbin， isoastilbin， engeletin） and 291 nm （quercitrin）. The column temperature was 30 ℃， and sample size was 10 μL. Using astilbin as internal substance， and the relative correction factors of other 5 factors were calculated. The contents of each component were calculated according to relative correction factor， and were compared with the results of external standard method. SPSS 22.0 software was used for cluster analysis and principal component analysis. RESULTS： The linear range of neoastilbin，astilbin，neoisoastilbin，isoastilbin，quercitrin and engeletin were 0.007-0.311， 0.871-18.184， 0.002-0.119， 0.052-1.251， 0.105-2.202， 0.020-2.319 μg （r>0.999）， respectively. RSDs of precision， reproducibility and stability （24 h） tests were all lower than 3%. The average recoveries were 97.32%， 94.89%， 97.15%， 96.90%， 97.52% and 97.53% （RSDs were 1.09%-2.60%， n=6）， respectively. The relative correction factors of neoastilbin，neoisoastilbin，isoastilbin，quercitrin and engeletin were 1.252 6，1.198 3，0.958 6，0.807 1 and 1.138 1， respectively. The contents of neoastilbin， neoisoastilbin，isoastilbin，quercitrin and engeletin measured by QAMS were 0.394 2-2.067 2，0.139 1-0.804 7，2.864 8-8.554 8，4.581 2- 11.371 1，1.028 9-13.401 5 mg/g; the contents of neoastilbin，astilbin，neoisoastilbin，isoastilbin，quercitrin and engeletin were 0.367 2-1.925 3， 46.361 1-126.342 1， 0.138 1-0.798 8，2.966 2-8.857 8，4.642 5-11.523 3，0.970 6-12.641 9 mg/g，respectively. Relative errors of two methods was lower than or equal to 3.55%. The results of cluster analysis showed that 9 batches of samples could be clustered into two categories; S8 sample was one category and others were one category. The results of principal component analysis showed that accumulative contribution rate of former 2 principle components was 84.745%， and the results of sample classification were consistent with those of cluster analysis. CONCLUSIONS： The established HPLC-QAMS method is accurate，feasible and repeatable，and can be used for simultaneous determination of 6 flavonoids in E. roxburghiana， and it can provide reference for quality control.