| Objective(s):To establish an ultra-high performance liquid chromatography tandem mass spec-trometry(UPLC-MS/MS)method for rapid qualitative and quantitative determination of 10 ganoderma triterpenoids;the single factor and orthogonal test were used to optimize the extraction method,and the optimum extraction conditions were selected by comparing the extraction conditions comprehensively;the established method was used for qualitative and quantitative detection of triterpenoids in 154 samples of Ganoderma from Yunnan Province;the lid and stalk of Ganoderma were detected to compare the total contents of triterpenoids between them.Methods:An ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer(AB Sciex 6500)was used to search for parent ions and product ions of 10 ganoderma triterpenoids under ESI positive and negative mode.The optimal mass spectrum conditions such as DP and CE were explored.After the optimal mass spectrum conditions were obtained,the chromatographic conditions such as column,mobile phase,elution gradient and flow rate were optimized.Through single factor and orthogonal tests,methanol concentration,solid-liquid ratio,ultrasonic time,ultrasonic temperature and extraction times were used as indexes to optimize the sample extraction method,and the total content of 10 ganoderma triterpenoids was used as evaluation index to obtain the optimal extraction conditions combination.The established UPLC-MS/MS method was used for qualitative and quantitative determination of 10 triterpenoids in Ganoderma.Correlation analysis and principal component analysis of 10 triterpenoids in Ganoderma were performed by SPSS 20.0 and the results were evaluated.Results:1.An UPLC-MS/MS method was developed for the qualitative and quantitative determination of 10 ganoderma triterpenoids.2.The linear range of ganoderic acid C2 and ganoderenic acid D was 0.5-50μg/L,and the linear range of ganoderic acid I,N,B,H,C6,F,lucidenic acid B and ganoderenic acid C was 1-100 μg/L.The linear relationship was good(r was0.9970-0.9993),and the average recoveries ranged from 82.1% to 103%.The precision was 0.81%-8.5%.The detection limits of ganoderic acid I,B,H,F,lucidenic acid B and ganoderenic acid D were 0.1 μg/L,and the limits of quantification were0.33 μg/L.The detection limit and quantitation limit of ganoderenic acid C were 0.07μg/L and 0.23 μg/L respectively.The detection limit of ganoderic acid C2 was 0.025μg/L and the limit of quantification was 0.083 μg/L.The detection limit of C6 was 0.2μg/L and the limit of quantification was 0.67 μg/L.The detection limit of ganoderic acid N was 0.05 μg/L,and the limit of quantification was 0.17 μg/L.The method was sensitive and could meet the detection needs.3.Through single factor and orthogonal test,the optimal extraction conditions were as follows: Filter the Ganoderma through a 10-mesh sieve after crushed,Weigh0.5 g Ganoderma sample,extract with 100% methanol,the ratio of solid to liquid was1:30,ultrasonic was carried out at 25 ℃ for 85 min(power 300 W,frequency45 k Hz),and the supernatant was centrifuged through 0.22 μm nylon filter membrane.4.The detection rate of 10 triterpenoids in Ganoderma were over 94.2% except for ganoderic acid B(71.4%).The content of ganoderic acid C2 ranged from 0.2 to1018.7 mg/kg,ganoderic acid N ranged from 0.05 to 349.0 mg/kg,ganoderic acid I ranged from 0.1 to 416.3 mg/kg,ganoderic acid H ranged from 0.8 to 1750.4 mg/kg and lucidenic acid B ranged from 0.04 to 124.35 mg/kg,ganoderenic acid C was0.13-208.13 mg/kg,ganoderma acid C6 was 3.3-1080.0mg/kg,ganoderma acid F was0.8-1467.1 mg/kg,ganoderenic acid D was 0.25-343.49 mg/kg and ganoderma acid B was 8.9-1258.0 mg/kg,the top 5 levels were ganoderic acid H,ganoderic acid F,ganoderic acid B,ganoderic acid C2 and ganoderic acid C6.The total content of 10 substances ranged from 2.2 to 6293.5 mg/kg.According to morphological classification,this batch of Ganoderma was divided into 8 kinds: G.lingzhi,G.sinense,G.tsugae,G.flexipes,G.ahmadii,G.tropicum,G.weberianum and G.orbiforme.5.After correlation analysis,ganoderic acid B and ganoderic acid N,ganoderic acid B and lucidenic acid B,ganoderic acid B and ganoderenic acid D were not significantly correlated(P>0.05),the other substances were positively correlated(P<0.05);By the principal component analysis method,it was found that the three principal components extrated have already contained 78.67% of the original index information,which could represent the original index to evaluate the efficacy components of Ganoderma.By calculating the comprehensive score,only 10 ganoderma triterpenoids were used as evaluation indexes,and it was known that the content of functional components of G.ahmadii,G.lingzhi and G.tsugae were higher.6.The total content of triterpenoids in Ganoderma cap ranged from 106.2 to3340.9 mg/kg,while the corresponding content in stalk of Ganoderma ranged from22.2 to 1202.1 mg/kg,The overall distribution of triterpenoids was different between the two(P< 0.05),and content of Ganoderma cap was much higher than that of the stalk.Conclusion(s):1.The established UPLC-MS/MS method for the qualitative and quantitative detection of 10 ganoderma triterpenoids in was rapid,accurate,sensitive and reliable.The extraction efficiency of triterpenoids from Ganoderma was high and the recovery rate was good when the samples were extracted by the optimal extraction conditions optimized by experiments of single factor and orthogonal test.2.The detection rate of 10 triterpenoids in Ganoderma was high,and the difference of content was great.Principal component analysis showed that extracting three principal components could represent 10 ganoderma triterpenoids to evaluate functional components of Ganoderma,which found that Ganoderma with higher functional components were G.ahmadii,G.lingzhi and G.tsugae.The total content of triterpenoids in Ganoderma cap was higher than that in stalk.3.Through the detection and analysis of 154 pieces of Ganoderma in Yunnan province,it provides the basis for the study on the effective components of Ganoderma in Yunnan province.Establishing the ganoderma triterpenoids mass spectral library,which can provide a method for the content detection of triterpenoids in Gano-derma in the future,as well as provides a basis for the development of local characteristic industries in Yunnan Province. |
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