HPLC法同时测定九苷肽蛹虫草软胶囊中还原型及氧化型谷胱甘肽含量

目的：建立HPLC-SPD法同时测定九苷肽蛹虫草软胶囊中还原型和氧化型谷胱甘肽的含量。方法：以流动相为提取溶剂对样品进行超声提取；选用Atlantis T3（4.6 mm×250 mm，5 μm）色谱柱，以0.1%三氟乙酸-乙腈（95∶5）为流动相，流速1.0 mL·min^-1，紫外检测波长203 nm，柱温35℃。结果：样品中还原型和氧化型谷胱甘肽在8 min内得到很好的分离；质量浓度分别在50.4~504 μg·mL^-1（r=0.999 7）和6.2~62μg·mL^-1（r=0.999 4）线性良好；检测限分别为0.44和0.12 mg·g^-1；平均回收率（n=9）分别为97.5%和93.4%；3批样品中还原型和氧化型谷胱甘肽含量分别是70.2、70.5、70.1 mg·g^-1和1.3、1.4、1.3 mg·g^-1，其含氧化型谷胱甘肽均小于谷胱甘肽总量的2.0%。结论：本方法能够满足日常分析检测的需要，为含谷胱甘肽保健食品的质量控制提供科学依据。

Objective: To establish an HPLC-SPD method for the simultaneous determination of reduced and oxidized glutathione in Jiugantai Yongchongcao softgel.Methods: The samples were ultrasonically extracted with the mobile phase as extraction solvent;The chromatographic separation was achieved on an Atlantis T3 column (4.6 mm×250 mm,5 μm),using 0.1% trifluoroacetic acid-acetonitrile(95:5)as the mobile phase at a flow rate of 1.0 mL·min^-1,the detection wavelength was set at 203 nm,and the column temperature was 35℃.Results: Reduced and oxidized glutathione were well separated within 8 minutes; the calibration curves of reduced and oxidized glutathione showed good linearity in the range of 50.4-504 μg·mL^-1(r=0.999 7)and 6.2-62 μg·mL^-1 (r=0.999 4), respectively; the detection limits were 0.44 mg·g^-1 and 0.12 mg·g^-1, respectively; the average recovery(n=9)were 97.5% and 93.4%, respectively;The contents of reduced and oxidized glutathione in three batches were 70.2, 70.5, 70.1 mg·g^-1 and 1.3, 1.4, 1.3 mg·g^-1, which containing oxidized glutathione were less than 2.0% of the total.Conclusion: This method can meet the needs of daily analytical testing for the quality control of health foods containing glutathione.

[1] 金春英,崔京兰,崔胜云.氧化型谷胱甘肽对还原型谷胱甘肽清除自由基的协同作用[J].分析化学, 2009, 37(9):1349 JIN CY, CUI JL, CUI SY. Synergetic effect by oxidized glutathione for radical scavenging activities of reduced glutathione[J]. Chin J Chem,2009, 37(9):1349
[2] WU GY,FANG YZ,YANG S, et al. Glutathione metabolism and its implications for health[J]. J Nutr, 2004, 134(3):489
[3] BARFIELD M, SPOONER N, LAD R, et al. Application of dried blood spots combined with HPLC-MS/MS for the quantification of acetaminophen in toxicokinetic studies[J]. J Chromatogr B Anal Technol Biomed Life Sci,2008, 870(1):32
[4] PAULSEN CE, CARROLL KS. Orchestrating redox signaling networks through regulatory cysteine switches[J]. ACS Chem Biol, 2010, 5(1):47
[5] HANSENA JM, HARRISB C. Glutathione during embryonic development[J]. Biochim Biophys Acta,2015, 1850(8):1527
[6] FORMAN HJ, ZHANG H, RINNA A. Glutathione:overview of its protective roles, measurement, and biosynthesis[J]. Mol Aspects Med, 2009, 30(1-2):1
[7] KANĎÁR R, ŠTRÁMOVA X,DRÁBKOVÁ P, et al. Determination of total glutathione in dried blood spot samples using a highperformance liquid chromatography[J]. J Chromatogr Sci,2015,53(6):879.
[8] 方允中,杨胜,伍国耀.自由基、抗氧化剂、营养素与健康的关系[J].营养学报, 2003, 25(4):340 FANG YZ,YANG S,WU GY. Free radicals,antoxidants,and nutrients in relation to health[J]. Acta Nutr Sin,2003,25(4):340
[9] MUSGRAVE WB, YI H, KLINE D, et al. Probing the origins of glutathione biosynthesis through biochemical analysis of glutamate-cysteine ligase and glutathione synthetase from a model photosynthetic prokaryote[J]. Biochem J,2013, 450(1):63
[10] GAWLI MKRZYZANOWSKA W,,BOGUMILA GM,et al. Optimization of determination of reduced and oxidized glutathione in rat striatum by HPLC method with fluorescence detection and pre-column derivatization[J]. Acta Chromatogr,2014,24(2):335
[11] FRATERNALE A, BRUNDU S, MAGNANI M. Glutathione and glutathione derivatives in immunotherapy[J]. Biol Chem, 2017, 398(2):261
[12] 中国药典2015年版.二部[S]. 2015:512 ChP 2015. Vol Ⅱ[S]. 2015:512
[13] 颜燕,谢玮,郭婕,等.人工蛹虫草与谷胱甘肽组方对酒精性肝损伤保护作用的实验研究[J].毒理学杂志,2014, 28(6):471 YAN Y, XIE W, GUO J, et al. Experimental study of artificial Cordyceps and glutathione prescription for the protective effect of alcoholic liver injury[J]. J Toxicol,2014, 28(6):471
[14] RABAB MS, GHADA MH, RANDA AAS, et al. Quantitative determination of glutathione in presence of its degradant in a pharmaceutical preparation using HPLC-DAD and identification by LC-ESI-MS[J]. J Liq Chromatogr Relat Technol,2013,37(4):548
[15] 林志兴,吕永琴,谭天伟.高效液相色谱-蒸发光散射检测器定量分析谷胱甘肽含量[J].食品与发酵工业, 2007, 33(4):119 LIN ZX, L YQ, TAN TW. Quantification analysis of glutathione by HPLC with evaporative light scatter detector(ELSD)[J]. Food Ferment Ind,2007, 33(4):119
[16] 林宏琳. HPLC法同时测定核苷酸花粉胶囊中5种核苷酸含量[J]. 药物分析杂志,2015, 35(12):2121 LIN HL. Simultaneous determination of five kinds of nucleotids in nucleotide pollen capsules by HPLC[J]. Chin J Pharm Anal. 2015, 35(12):2121