高效液相色谱法同时测定微生物酶法产L-丙氨酰-L-谷氨酰胺反应体系中5种物质及其样品处理方法对色谱峰的影响分析  

Simultaneous Determination of 5 Substances in the Microbial Enzymatic Reaction System of L-Alanyl-L-glutamine by High Performance Liquid Chromatography and the Influence of Sample Preparation on Chromatographic Peaks

作　　者：魏照辉[1] 张震宇[1] 孙付保[1] 

机构地区：[1]江南大学生物工程学院工业生物技术教育部重点实验室糖化学与生物技术教育部重点实验室,江苏无锡214122

出　　处：《分析测试学报》2016年第10期1317-1322,共6页Journal of Instrumental Analysis

基　　金：国家自然科学基金(30970058;21176106);江苏省自然科学基金(BK2012554)

摘　　要：建立了同时测定微生物酶法产L-丙氨酰-L-谷氨酰胺反应体系中5种物质(L-丙氨酰-L-谷氨酰胺、L-谷氨酰胺、L-丙氨酸甲酯、L-谷氨酸、L-丙氨酰-L-丙氨酸)的高效液相色谱分析方法。考察了样品处理中存在的发酵液、磷酸、三氯乙酸对色谱峰的影响,建立了一种对色谱峰无干扰的样品处理方法。选用邻苯二甲醛(OPA)为衍生剂,以在线衍生的方式实现了标准品或样品的柱前自动衍生。选用C18色谱柱进行分离,柱温40℃,以12.5 mmol/L磷酸盐缓冲液(88%,pH 7.4)与12%乙腈为流动相进行等度洗脱,流速为1 m L/min,在λex=338 nm,λ_(em)=450 nm下进行荧光检测,外标法定量。结果显示,该色谱条件下5种物质的测定线性范围广,相关系数均大于0.999 4。低、中、高3组加标水平下的回收率为93.5%~104.9%,相对标准偏差(RSD)小于7%。1日内间隔2 h进行6次进样,5种物质保留时间的RSD均小于0.12%,峰面积RSD均小于1.2%。该方法操作简单,分离度与灵敏度高,定量精确,重复性好。A method of high performance liquid chromatography （ HPLC ） with fluorescence detector （FLD） was developed for the simultaneous determination of 5 substances in the microbial enzymatic reaction system of L-alanyl-L-glutamine, including L-alanyl-L-glutamine, L-glutamine, L-alanine methyl ester, L-glutamic acid and L-alanyl-L-alanine. Standard substances and samples were derivat- ed with phthaldialdehyde （OPA） by online automated precolumn derivatization. The analytes were separated on a C^8 column using acetonitrile （ 12% ） and 12. 5 mmol/L potassium phosphate buffer （88% , pH 7.4 ） as mobile phase by isocratic elution and analyzed quantitatively by the external standard method at an excitation wavelength of 338 nm and an emission wavelength of 450 nm. The flow rate was set at 1 mL/min and the column temperature was 40 ~C. The calibration curves for five standard substances were all linear in a broad range with correlation coefficients more than 0. 999 4. The average recoveries of five substances at three spiked levels ranged from 93.5% to 104.9% with relative standard deviations（RSDs） less than 7%. Injecting six times every two hours one day, the RSD values of retention time for five substances were all less than 0. 12% , and the RSD values of peak area of five substances were all less than 1.2%. The method was simple, sensitive, accurate and repeatable with a high resolution.

关 键 词：高效液相色谱法 柱前自动化衍生 L-丙氨酰-L-谷氨酰胺反应体系 样品处理方法 

分 类 号：O657.72[理学—分析化学]