PITC柱前衍生-高效液相色谱法测大米肽中的16种氨基酸含量  

Determination of 16 Amino Acids in Rice Peptides by PITC Pre-column Derivatization High Performance Liquid Chromatography

作　　者：黄莹 黄晶 杨晓 陈曦 胡武瑶 HUANG Ying;HUANG Jing;YANG Xiao;CHEN Xi;HU Wuyao(Anti-aging Nutrition&Health Research and Development Center of China Health Promotion Foundation,Wuhan 430000,China)

机构地区：[1]中国健康促进基金会抗衰老营养与健康研发中心,湖北武汉430000

出　　处：《食品工业科技》2023年第24期279-285,共7页Science and Technology of Food Industry

摘　　要：为了建立一种以高效液相色谱检测大米肽中氨基酸含量的方法,以异硫氰酸苯酯(Phenyl isothiocyanate,PITC)为柱前衍生剂,实现大米肽中16种氨基酸(天冬氨酸(Asp)、谷氨酸(Glu)、丝氨酸(Ser)、甘氨酸(Gly)、组氨酸(His)、精氨酸(Arg)、苏氨酸(Thr)、丙氨酸(Ala)、脯氨酸(Pro)、酪氨酸(Tyr)、缬氨酸(Val)、蛋氨酸(Met)、异亮氨酸(Ile)、亮氨酸(Leu)、苯丙氨酸(Phe)、赖氨酸(Lys))能同时、快速、有效地分析检测。采用Athena AAA色谱柱为分析柱,以甲醇:乙腈:水=20:60:20和50 mmol/L乙酸钠(pH6.5)为流动相,进行梯度洗脱,流速为1.0 m L/min,柱温35℃,紫外检测器波长为254 nm。结果表明,16种氨基酸在0.125~2.5μmol/m L范围内具有良好的线性关系,14种氨基酸线性关系的决定系数R2≥0.9999,另外2种氨基酸的决定系数R^(2)分别为0.9998和0.9994,检出限为0.003%~0.018%,定量限为0.010%~0.059%,加标回收率在82.21%~103.59%,RSD均小于5%。该方法使得大米肽中16种氨基酸很好地分离,其中含量最高的氨基酸是谷氨酸(Glu),含量范围为13.11%±0.39%~21.38%±0.48%,含量最低的氨基酸是组氨酸(His)或蛋氨酸(Met),含量范围为1.59%±0.05%~2.27%±0.03%或1.26%±0.05%~2.32%±0.06%,其准确度和精密度佳,灵敏度和回收率高,适用于大米肽的氨基酸含量分析。To establish a methodology for detecting the amino acid content in rice peptides,the research employed the precolumn derivative phenyl isothiocyanate(PITC)for realizing simultaneous,rapid,and effective analysis of 16 amino acids,including aspartate(Asp),glutamic acid(Glu),serine(Ser),glycine(Gly),histidine(His),arginine(Arg),threonine(Thr),alanine(Ala),proline(Pro),tyrosine(Tyr),valine(Val),methionine(Met),isoleucine(Ile),leucine(Leu),phenylalanine(Phe),and lysine(Lys).The analysis was carried out using an Athena AAA chromatographic column as the analysis column,with a flow rate of 1.0 mL/min,column temperature of 35℃,and UV detector wavelength of 254 nm.A gradient elution was performed with a mobile phase consisting of methanol:acetonitrile:water=20:60:20 and 50 mmol/L sodium acetate(pH6.5).The results revealed a strong linear relationship among the 16 amino acids within the range of 0.125 to 2.5μmol/mL,with a coefficient of determination R^(2)≥0.9999 for 14 amino acids.The remaining 2 amino acids exhibited coefficient of determination R2 were 0.9998 and 0.9994,respectively.The detection limit ranged from 0.003% to 0.018%,while the quantitative limit ranged from 0.010% to 0.059%.The spiked recovery rate ranged from 82.21% to 103.59%,and the relative standard deviation(RSD)was less than 5% for all measurements.This method effectively separates the 16 amino acids in rice peptides,with glutamic acid(Glu)having the highest content ranging from 13.11%±0.39% to 21.38%±0.48%.The amino acids with the lowest content are histidine(His)or methionine(Met),ranging from 1.59%±0.05% to 2.27%±0.03% or 1.26%±0.05% to 2.32%±0.06%,respectively.The experimental method employed in this study demonstrates high accuracy,precision,sensitivity and recovery rate,thereby,indicating its suitability for analyzing the amino acid content in rice peptides.

关 键 词：大米肽 氨基酸 异硫氰酸苯酯 柱前衍生 高效液相色谱 

分 类 号：TS207.3[轻工技术与工程—食品科学]