柱前衍生-气相色谱-质谱联用法同时测定茯苓中的可溶性糖与三萜酸

Simultaneous determination of soluble sugars and triterpene acids in Poria cocos by gas chromatography-mass spectrometry with pre-column derivatization

[目的]通过建立柱前衍生-气相色谱-质谱(GC-MS)法实现茯苓(Poria cocos)各成分的快速高效分离检测,为茯苓有效成分的分析提供新方法并为茯苓原料品质判断提供参考.[方法]采用超声辅助提取法,以反油酸为内标物,甲氧胺(MOX)和N-甲基-N-(三甲基硅烷)三氟乙酰胺[含1%(质量分数)三甲基氯硅烷](MSTFA+1%TMCS)为肟化-硅烷化衍生试剂,采用GC-MS同时检测茯苓中的果糖、葡萄糖、海藻糖、阿拉伯糖醇、甘露糖醇、山梨糖醇、麦芽糖醇、茯苓酸、茯苓新酸A和茯苓新酸B.[结果]优化后的衍生条件为:200μL茯苓的甲醇提取液氮气吹干后,加入50μL MOX,于40℃反应2 h,然后加入250μL(MSTFA+1%TMCS),于40℃下反应30 min.衍生后的目标化合物在40 min内完成分离分析,线性关系良好,相关系数均在0.9935以上,该方法的定量限为0.008~1.000μg/mL,检测限为0.003~0.300μg/mL.茯苓样品加标回收率为82.0%~122.4%,相对标准偏差为1.05%~4.43%.[结论]本方法样品用量少,结果稳定且灵敏度高,可用于检测茯苓样品中可溶性糖和微量的三萜酸类化合物,同时也为检测其他样品中的三萜酸类化合物提供更多的选择.

[Objective]Poria cocos has long been used in China as a medicinal and food mushroom due to its sedative,tranquilizing,and diuretic properties.Previous research has confirmed that soluble sugars and triterpene acids are its major nutritional and functional components,and the contents are substantially affected by factors such as the planting area,harvesting period,and processing methods.It is vital to comparatively identify and quantify soluble sugars and triterpene acids rapidly and accurately for the further development of fermented mycelia.[Methods]P.cocos was extracted using ultrasonic extraction with methanol,followed by derivatization with methoxyamine(MOX)and N-methyl-N-(trimethylsilyl)trifluoroacetamide(MSTFA)containing 1%(mass fraction)trimethylchlorosilane(TMCS).The effects of various variables(e.g.,volume ratio of menthanol,extraction time,volume of MSTFA,volume of MOX,methoximation time,and silylation time)on the extraction and derivation processes were investigated through single factor experiments.The derivatives were analyzed using gas chromatography-mass spectrometry(GC-MS),qualitatively in full scan mode with a scan range of m/z 40800,and quantitatively in selected ion scan mode.Derivatives were identified based on their retention time and electron impact(EI)-MS library match using commercial National Institute of Standards and Technology(NIST)library and standards.[Results]Methanol was selected as the extraction solvent,due to the highest extraction yields of pachymic acid(PA),poricoic acid A(PAA),and poricoic acid B(PAB),as well as shorter nitrogen blow-dry time.The extraction yields of derivatives increased sharply with increasing extraction time from 30 min to 60 min,then plateaued.Considering extraction efficiency,the extraction time was determined to be 60 min.For the volume of MOX,the yields of reducing sugars derivatives increased and reached the peak at 50μL.The efficiency of further silylation derivations would be reduced by the presence of pyridine in the methoximation reagent.For reducing sugars,methoximation time is the main factor inhibiting the ring formation.The yields of derivative by-products and incomplete derivative products decreased significantly with an increase in methoximation time,and the yield of reducing sugars derivatives remained constant when the time exceeded 120 min.There was a notable improvement in the peak areas of derivatives with increasing volume of MSTFA with 1%TMCS from 50μL to 200μL.Because excessive derivative reagents would further dilute the sample,the yields of derivatives decreased at the volume of 250μL.The yields of derivatives increased with silylation time from 15 min to 60 min,then decreased when time exceeded 60 min.According to the yield of derivatives,the optimal conditions for derivation were established as:50μL MOX(20 mg/mL),a methoximation time of 120 min,200μL MSTFA with 1%TMCS,and a silylation time of 60 min.Under the optimum GC-MS conditions,baseline separation of the soluble sugar derivatives and the triterpene acid derivatives was obtained within 40 min.There were two peaks of trimethylsilyl(TMS)oxime derivatives of reducing sugar such as glucose(Glu).For sugar alcohols and triterpene acids,there were no keto or aldehyde groups,and no methoximation occurred.Based on the chemical structures and EI-MS,the triterpene acids derivatives were estimated as biTMS-PA,triTMS-PAA,and triTMS-PAB for PA,PAA,and PAB,respectively.Using elaidic acid as an internal standard,the linear range of analytes was 2200μg/mL and correlation coefficients were above 0.9935.The limits of detection ranged from 0.003μg/mL to 0.300μg/mL and the limits of quantitation were from 0.008μg/mL to 1.000μg/mL.The recovery was between 82.0%and 122.4%and the relative standard deviation ranged from 1.05%to 4.43%.[Conclusions]In this study,a new GC-MS method with derivatization was developed and validated for the simultaneous determination of arabitol(Ara),fructose,Glu,mannitol(Man),sorbitol,trehalose,maltitol,PA,PAA,and PAB in P.cocos.This method,noted for its small sample consumption,high sensitivity,excellent selectivity,and good repeatability,has been applied to analyze the chemical profile of P.cocos from three districts in Sanming,Fujian Province.The highest contents of Glu,Ara,and PA were observed in the monosaccharides,sugar alcohols,and triterpene acids of fermented mycelia,respectively,and varied across different regions.There were other similar peaks of triterpene acids observed in the sample spectra,and the specific substances should be further confirmed through structural analysis of the standard and MS.This established method holds powerful potential for trace analysis of triterpene acids from plant samples.