一测多评法同时测定木瓜中5种五环三萜类成分的含量

Simultaneous determination of 5 kinds of pentacyclic triterpenoids in Chaenomeles speciosa by quantitative analysis of multi-components by single-marker

目的建立同时测定木瓜中3-O-乙酰坡模醇酸等5种五环三萜类成分含量的方法,并分析不同产地、不同炮制工艺木瓜的含量差异。方法以高效液相色谱法为检测手段,色谱柱为COSMOSIL 5 C18-MS-Ⅱ,流动相为乙腈-0.005 mol/L磷酸氢二铵溶液(用磷酸调pH至6.5)(70︰30,V/V),流速为1.0 mL/min,柱温为30℃,检测波长为210 nm,进样量为20μL。以齐墩果酸为内参物,结合一测多评法,对3-O-乙酰坡模醇酸等4种五环三萜类成分含量进行测定,并与外标法进行比较;同时,对不同产地、不同炮制加工工艺木瓜中齐墩果酸与熊果酸的总含量和5种成分的总含量进行比较。结果3-O-乙酰坡模醇酸、白桦脂酸、齐墩果酸、熊果酸、3-O-乙酰熊果酸检测质量浓度的线性范围分别为4.06~81.2、2.12~42.4、9.62~192.3、7.77~155.4、4.21~84.1μg/mL(R^(2)>0.999);精密度、重复性、稳定性(24 h)试验的RSD均小于3%;平均加样回收率为98.29%~101.38%(RSD<3%,n=6)。一测多评法测得3-O-乙酰坡模醇酸、白桦脂酸、熊果酸、3-O-乙酰熊果酸的含量分别为0.023%~0.060%、0.044%~0.528%、0.101%~0.368%、0.067%~0.221%,与外标法所测结果的相对偏差均小于8%。同一产地鲜切工艺加工木瓜中齐墩果酸与熊果酸的总含量和5种成分的总含量均高于传统工艺加工木瓜,且重庆黔江产木瓜中5种成分的总含量显著高于其他产地(P<0.05)。结论建立了同时测定木瓜中3-O-乙酰坡模醇酸、白桦脂酸、齐墩果酸、熊果酸、3-O-乙酰熊果酸含量的一测多评法,该方法简便、快速、准确。重庆黔江产木瓜中5种成分的总含量较高,且同一产地鲜切工艺加工木瓜总含量高于传统工艺加工木瓜。

OBJECTIVE To establish a method for simultaneous determination of 5 kinds of pentacyclic triterpenoids as 3-Oacetyl-pomolic acid in Chaenomeles speciosa,and to analyze the difference in the contents of C.speciosa from different producing areas by different processing technologies.METHODS HPLC method was adopted.The determination was performed on COSMOSIL 5 C18-MS-Ⅱ column with mobile phase consisted of acetonitrile-0.005 mol/L ammonium dihydrogen phosphate solution(p H value adjusted to 6.5 with phosphoric acid)(70∶30,V/V)at the flow rate of 1.0 mL/min.The column temperature was set at30℃.The detection wavelength was set at 210 nm,and sample size was 20μL.The contents of the other four pentacyclic triterpenoids were calculated according to quantitative analysis of multi-components by single-marker(QAMS)using oleanolic acid as internal reference.The results were compared with those determined by external standard method.The total content of oleanolic acid and ursolic acid,the total content of 5 components in C.speciosa from different producing areas and different processing technologies were compared.RESULTS The linear range of 3-O-acetyl-pomolic acid,betulinic acid,oleanolic acid,ursolic acid and 3-O-acetyl ursolic acid were 4.06-81.2,2.12-42.4,9.62-192.3,7.77-155.4,4.21-84.1μg/mL,respectively(R^(2)>0.999).RSDs of precision,reproducibility and stability(24 h)tests were all lower than 3%.The average recoveries were 98.29%-101.38%(RSDs<3%,n=6).The mass fraction of 3-O-acetyl-pomolic acid,betulinic acid,ursolic acid and 3-O-acetyl ursolic acid measured by QAMS were 0.023%-0.060%,0.044%-0.528%,0.101%-0.368%,0.067%-0.221%,respectively;the deviations from the results measured by external standard method were all within 8%.The total content of oleanolic acid and ursolic acid,the total content of 5 components in C.speciosa processed by fresh-cut technology from the same producing area were higher than those in C.speciosa processed by traditional technology,and the total content of 5 components in C.speciosa from Chongqing Qianjiang were significantly higher than those from other producing areas(P<0.05).CONCLUSIONS QAMS method is established for the simultaneous determination of 3-O-acetyl-pomolic acid,betulinic acid,oleanolic acid,ursolic acid and 3-O-acetyl ursolic acid in C.speciosa.Established method is simple,rapid and accurate.The total content of 5 components in C.speciosa produced in Chongqing Qianjiang is higher,and the total content of C.speciosa processed by fresh-cut technology from the same origin is higher than C.speciosa processed by traditional technology.