基于UPLC-Q/TOF-MS技术的没药及其炮制品中倍半萜类成分分析及HPLC含量测定研究

Chemical Constituents of Myrrh and Its Processed Product Based on UPLC-Q/TOF-MS Technology and Establishment of HPLC Method for Determination of Four Components

目的基于超高效液相色谱-串联四极杆飞行时间质谱(UPLC-Q/TOF-MS)技术结合多元统计学分析没药及其炮制品的倍半萜类成分,并基于HPLC技术建立同时测定没药不同炮制品中4种倍半萜类成分含量的方法。方法UPLC-Q/TOF-MS法采用Waters acquity BEH C_(18)(2.1 mm×100 mm,1.7μm)液相色谱柱,柱温40℃,流动相为乙腈(A)-体积分数0.1%甲酸水溶液(B),梯度洗脱,流速0.3 mL·min^(-1);电喷雾离子源,正、负离子模式下采集质谱数据,质量扫描范围m/z 50~1200。使用SIMCA-P 14.0进行主成分分析(PCA)及正交偏最小二乘法-判别分析(OPLS-DA),筛选得到差异性(VIP>1)的倍半萜类成分。HPLC使用YMC-Pack ODS-A色谱柱(4.6 mm×250 mm,5μm),流动相为乙腈(A)-体积分数0.1%磷酸水(B),梯度洗脱,流速1.0 mL·min^(-1),检测波长分别210和240 nm;柱温30℃,同时测定没药中2-甲氧基-5-乙酰基-呋喃吉玛烷-1(10)-烯-6-酮(FSA)、[1(10)E,2R,4R]-2-甲氧基-8,12-环氧吉玛-1(10),7,11-三烯-6-酮(MCS134)、9-甲氧基没药酮和没药酮等4种倍半萜类成分的含量。结果基于UPLC-Q/TOF-MS技术在正、负离子模式下检测出78个化合物;通过PCA法和OPLS-DA法可以快速、有效区分没药和醋没药,筛选出二者的差异性成分21种。采用HPLC进行4种倍半萜类成分的含量测定,各成分在各自线性范围内呈良好线性关系(r>0.9990),精密度、稳定性等方法学考察均符合要求。结论基于UPLC-Q/TOF-MS技术结合多元统计学方法可以快速、准确区分没药不同炮制品;建立的HPLC测定没药及其炮制品中4种倍半萜类成分的含量方法稳定、可靠,可为其炮制及质量控制提供参考。

OBJECTIVE To investigate the primary chemical constituents of myrrh using UPLC-Q/TOF-MS combined with multivariate statistical method for comparing the different chemical constituents of two different types of myrrh products,and develop an HPLC method for simultaneous determination of four chemical constituents in two different types of myrrh products.METHODS Nontargeted analysis was carried out by UPLC-Q/TOF-MS method using an Waters acquity BEH C_(18)(2.1 mm×100 mm,1.7μm)column,temperature was maitained at 40℃,gradient elution was performed at a flow rate of 0.3 mL·min^(-1) using acetonitrile(A)-0.1%formic acid aqueous solution(B)as the mobile phase;electrospray ionization(ESI)source was used to acquire mass spectrometry data in positive and negative ion modes with the scanning range of m/z 50-1200.The PCA and OPLS-DA methods were performed with SIMCA-P14.0 to discover distinct metabolites(VIP>1).HPLC method was used for quantitative analysis of the four chemical components of Myrrh,i.e.,(1(10)E,2R*,5R*)-2-methoxy-5-acetoxyfuranogermacr-1(10)-en-6-one,(1(10)E,2R,4R)-2-methoxy-8,12-epoxygemacra-1(10),7,11-trien-6-one,9-methoxymyrrhone,and myrrhone,which was performed on a YMCPack ODS-A column(4.6 mm×250 mm,5μm),with a gradient elution of acetonitrile(A)-0.1%aqueous phosphoric acid(B)as the mobile phase at a flow rate of 1. 0 mL·min - 1 . The column temperature was maintained at 30 ℃,and the detection wavelengthswere set at 210 and 240 nm. RESULTS Based on UPLC-Q/TOF-MS technology,78 chemical components were identified in myrrh,and 21 components with significant differences in content were screened and identified in positive and negative ion modes. For quantitativeanalysis,a technique for concurrently identifying four chemical components in myrrh using HPLC was established. Within the investigatedlinear ranges,satisfactory linearities were attained with good coefficients ( r > 0. 999 0) ,and the methodological analysis revealedthat the method had good reproducibility,precision and stability. CONCLUSION An accurate and effective qualitative analytical techniquefor identifying the chemical components of myrrh is provided by UPLC-Q/TOF-MS. The developed HPLC method for the contentdetermination of four chemical components is stable and reliable,which can be used for the processing and quality control of myrrh.