高效液相色谱法快速测定鱼类鲜度指标K值及方法学优化

Rapid determination of K value of fish freshness index by high performance liquid chromatography and optimization of methodology

对行业标准SC/T3048-2014测定鱼类鲜度K值(三磷酸腺苷,二磷酸腺苷,腺苷酸,肌苷酸,肌苷和次黄嘌呤)的分析方法进行优化。优化后的条件:以高氯酸溶液为提取剂,样品经高速均质结合超声波辅助提取后,用氢氧化钠溶液调节pH至6.0~6.4,一级水定容。采用C_(4)柱(4.6mm×250mm,5μm)分离,以磷酸二氢钾溶液(0.048mol/L,pH=5.7)~甲醇/磷酸二氢钾溶液(9/1,v/v)为流动相进行梯度洗脱,检测波长254nm,流速1.0mL/min,柱温30℃,外标法定量。优化结果表明:6种化合物的分析时间由行业标准方法的38min减少至11min。该方法的加标回收率(77.2%~112%),相对标准偏差(4.69%~7.81%),检出限(0.648~2.15mg/kg)和定量限(2.16~7.18mg/kg)均优于行业标准方法。结果表明该方法操作简便,分析快速,干扰较小,灵敏度高和重现性好,具有一定的实用价值。

A method for determination of K value(adenosine triphosphate,adenosine diphosphate,adenosine 5′-monophosphate,inosine 5′-monophosphate,inosine and hypoxanthine)of fish freshness in the industry standard SC/T 3048-2014 was optimized.The optimized conditions were as follows:Perchloric acid solution was the extractant.The samples were treated with high-speed homogenization and ultrasonic assisted extraction.The pH was adjusted to 6.0~6.4 with sodium hydroxide solution then diluted to scale with gradeⅠwater.The separation was performed on a C_(4) column(4.6mm×250mm,5μm)using potassium dihydrogen phosphate solution(0.048mol/L,pH=5.7)~methanol/potassium dihydrogen phosphate solution(9/1,v/v)as mobile phase by gradient elution.The detection was carried out at a wavelength of 254 nm and the flow rate was 1.0 mL/min.The column temperature was 30℃and the quantification was performed by external standard method.The optimized results showed that the analysis time of 6 compounds was decreased from 38 min of the industry standard method to 11 min.These parameters including spiked recoveries(77.2%~112%),relative standard deviations(4.69%~7.81%),limits of detection(0.648~2.15mg/kg)and quantitation(2.16~7.18mg/kg),were better than the ones under industry standard method.The method is found to be convenient,rapid,with less interference and high accuracy,and has certain potential for practical applications.