基于响应面分析的微波水解-亲水作用色谱法测定肉中羟脯氨酸含量  

Determination of L-hydroxyproline in meat by microwave hydrolysis-hydrophilic interaction chromatography based on response surface analysis

作　　者：朱帅 张水锋 盛华栋 黄润韬 孙琦琦 郑仕剑 王佳萍 ZHU Shuai;ZHANG Shuifeng;SHENG Huadong;HUANG Runtao;SUN Qiqi;ZHENG Shijian;WANG Jiaping(Zhejiang Fangyuan Test Group Co.Ltd.,Food Safety Key Lab of Zhejiang Province,Hangzhou 310018,China)

机构地区：[1]浙江方圆检测集团股份有限公司,浙江省市场监管生物安全重点实验室,杭州310018

出　　处：《分析试验室》2024年第10期1443-1451,共9页Chinese Journal of Analysis Laboratory

基　　金：国家市场监督管理总局技术保障专项(2020YJ014)资助。

摘　　要：采用微波水解-磺酸甜菜碱型两性离子亲水相互作用色谱建立了肉中羟脯氨酸的测定方法。基于响应面优化微波水解条件,通过单因素实验确定微波水解盐酸浓度为6 mol/L。以微波水解温度、水解时间、液料比为自变量,羟脯氨酸提取量为响应变量,进行Box-Behnken实验设计,运用响应面分析法建立多元回归模型。结果发现,各因素对羟脯氨酸提取量的影响顺序为:液料比>微波水解时间>微波水解温度。优化后的提取条件为:微波水解时间60 min,微波水解温度130℃,液料比12.5 mL/g。水解液经异硫氰酸苯酯(PITC)衍生化后,以乙酸钠溶液和乙腈为流动相,由两性离子-亲水作用色谱柱(ZIC-HILIC)分离,紫外检测器测定。羟脯氨酸在1~500 mg/L范围内线性关系良好,定量限为10 mg/kg,回收率在82.0%~107.5%之间,相对标准偏差在1.5%~6.2%之间。该方法适用于肉中羟脯氨酸的高通量检测。A method was developed for the determination of hydroxyproline in meat by microwave hydrolysis and betaine sulfonate zionophilic interaction chromatography.The concentration of hydrochloric acid hydrolyzed by microwave was determined to be 6 mol/L by single factor test based on response surface optimization.The BoxBehnken experiment was designed with microwave hydrolysis temperature,time and liquid-solid ratio as independent variables,and hydroxyproline extraction amount as response variable.A multiple regression model was established by response surface analysis method,and it was found that the order of the influence of each factor on the extraction amount of hydroxyproline was liquid-solid ratio>microwave hydrolysis time>microwave hydrolysis temperature.The optimized extraction conditions were as follows:microwave hydrolysis time of 60 min,microwave hydrolysis temperature of 130℃,and liquid-solid ratio of 12.5 mL/g.After derivatization with phenyl isothiocyanate(PITC),the hydrolysate was separated on a ZIC-HILIC column with sodium acetate solution and acetonitrile as mobile phase,and determined by UV detector.The linear relationship of hydroxyproline was good in the range of 1-500 mg/L,the limit of quantitation was 10 mg/kg,the recoveries ranged from 82.0%to 107.5%,and the relative standard deviations ranged from 1.5%to 6.2%.The method is suitable for high throughput detection of hydroxyproline in meat.

关 键 词：响应面 微波水解 羟脯氨酸 亲水作用色谱法 

分 类 号：TS207.3[轻工技术与工程—食品科学]