HILIC-MS/MS技术同时检测蛹虫草中虫草素、虫草酸、腺苷和肌苷含量的方法学
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摘要
蛹虫草是我国具有极高药用价值的滋补中药材。对蛹虫草成分的含量进行准确的定性定量分析,可为相关产品质量控制、筛选高活性蛹虫草及进一步研究蛹虫草的功效提供技术手段。建立一种灵敏稳定的亲水作用色谱法-串联质谱(HILIC-MS/MS)方法,同时对蛹虫草中虫草素、虫草酸(D-甘露糖醇)、腺苷和肌苷的含量进行测定。研究发现其线性范围分别为0.02～5μg/mL(R~2=0.995 3)、0.2～50μg/mL(R~2=0.999 4)、0.002～0.5μg/mL(R~2=0.997 0)和0.002～0.5μg/mL(R~2=0.995 0)。采用HILIC-MS/MS技术,测定结果精密度小于10.0%,准确度在8.0%左右,具有较好的特异性、灵敏度和重现性。
Cordyceps militaris is a nourishing Chinese herbal medicine with high medicinal value.Accurate qualitative and quantitative analysis of the content of Cordyceps militaris can provide technical means for quality control of related products,screening of highly active Cordyceps militaris and further research on the efficacy.A sensitive and stable hydrophilic interaction chromatography-tandem mass spectrometry(HILIC-MS/MS) method was established to determine the content of cordycepin,cordycepic acid(D-mannitol),adenosine and inosine in Cordyceps militaris simultaneously.The test found that their linear ranges are 0.02～5 μg/mL(R~2=0.995 3),0.2～50 μg/mL(R~2=0.999 4),0.002～0.5 μg/mL(R~2=0.997 0) and 0.002～0.5 μg/mL(R~2=0.995 0).The precision of the measurement results was less than 10.0%,the accuracy was around 8.0%.And it has good specificity,sensitivity and reproducibility.
Cordyceps militaris is a nourishing Chinese herbal medicine with high medicinal value.Accurate qualitative and quantitative analysis of the content of Cordyceps militaris can provide technical means for quality control of related products,screening of highly active Cordyceps militaris and further research on the efficacy.A sensitive and stable hydrophilic interaction chromatography-tandem mass spectrometry(HILIC-MS/MS) method was established to determine the content of cordycepin,cordycepic acid(D-mannitol),adenosine and inosine in Cordyceps militaris simultaneously.The test found that their linear ranges are 0.02～5 μg/mL(R~2=0.995 3),0.2～50 μg/mL(R~2=0.999 4),0.002～0.5 μg/mL(R~2=0.997 0) and 0.002～0.5 μg/mL(R~2=0.995 0).The precision of the measurement results was less than 10.0%,the accuracy was around 8.0%.And it has good specificity,sensitivity and reproducibility.
引文
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[15] INOUE K,MIYAZAKY Y,UNNO K,et al.Stable isotope dilution HILIC-MS/MS method for accurate quantification of glutamic acid,glutamine,pyroglutamic acid,GABA and theanine in mouse brain tissues[J].Biomed Chromatogr,2016,30(1):55-61.
[2] YANG Q,YIN Y,Yu G,et al.A novel protein with anti-metastasis activity on 4T1 carcinoma from medicinal fungus Cordyceps militaris[J].Int J Biol Macromol,2015,80:385-391.
[3] CHEN X,WU G,HUANG Z.Structural analysis and antioxidant activities of polysaccharides from cultured Cordyceps militaris[J].Int J Biol Macromol,2013,58:18-22.
[4] NALLATHAMBY N,GUAN SEM L,VIDYADARAN S,et al.Ergosterol of Cordyceps militaris attenuates LPS induced inflammation in BV2 microglia cells[J].Nat Prod Commun,2015,10(6):885-886.
[5] LIU J Y,FENG C P,LI X,et al.Immunomodulatory and antioxidative activity of Cordyceps militaris polysaccharides in mice[J].Int J Biol Macromol,2016,86:594-598.
[6] YU S H,DUBEY N K,LI W S,et al.Cordyceps militaris treatment preserves renal function in type 2 diabetic nephropathy mice[J].PLoS ONE,2016,11(11):166342.
[7] XIAO Y,SUN M,ZHANG Q,et al.Effects of Cordyceps militaris (L.) Fr.fermentation on the nutritional,physicochemical,functional properties and angiotensin I converting enzyme inhibitory activity of red bean (Phaseolus angularis[Willd.]W.F.Wight.) flour[J].J Food Sci Technol,2018,55(4):1244-1255.
[8] CHA J Y,AHN H Y,CHO Y S,et al.Protective effect of cordycepin-enriched Cordyceps militaris on alcoholic hepatotoxicity in sprague-dawley rats[J].Food Chem Toxicol,2013,60:52-57.
[9] YONG T,CHRN S,XIE Y,et al.Cordycepin,a characteristic bioactive constituent in Cordyceps militaris,ameliorates hyperuricemia through URAT1 in hyperuricemic mice[J].Front Microbiol,2018,9:58.
[10] RUKACHAISIRIKUL V,PRAMJIT S,PAKAWATCHAI C,et al.10-membered macrolides from the insect pathogenic fungus Cordyceps militaris BCC 2816[J].J Nat Prod,2004,67(11):1953-1955.
[11] CHAN J S,BARSEGHUAN G S,ASATIANI M D,et al.Chemical composition and medicinal value of fruiting bodies and submerged cultured mycelia of caterpillar medicinal fungus Cordyceps militaris CBS-132098 (ascomycetes)[J].Int J Med Mushrooms,2015,17(7):649-659.
[12] ZHOU X,CAI G,HE Y,et al.Separation of cordycepin from Cordyceps militaris fermentation supernatant using preparative HPLC and evaluation of its antibacterial activity as an NAD+-dependent DNA ligase inhibitor[J].Exp Ther Med,2016,12(3):1812-1816.
[13] LEE H H,KANG N,PARK I,et al.Characterization of newly bred Cordyceps militaris strains for higher production of cordycepin through HPLC and URP-PCR analysis[J].J Microbiol Biotechnol,2017,27(7):1223-1232.
[14] YANG F Q,LI D Q,FENG K,et al.Determination of nucleotides,nucleosides and their transformation products in cordyceps by ion-pairing reversed-phase liquid chromatography-mass spectrometry[J].J Chromatogr A,2010,1217(34):5501-5510.
[15] INOUE K,MIYAZAKY Y,UNNO K,et al.Stable isotope dilution HILIC-MS/MS method for accurate quantification of glutamic acid,glutamine,pyroglutamic acid,GABA and theanine in mouse brain tissues[J].Biomed Chromatogr,2016,30(1):55-61.