高效液相色谱法检测食品中17种游离氨基酸

Determination of 17 free amino acids in food by high performance liquid chromatography

中文关键词:  高效液相色谱法  异硫氰酸苯酯  前处理  游离氨基酸  衍生

英文关键词:high performance liquid chromatography  phenylisothiocyanate  pretreatment  free amino acid  derivation

中文摘要:

      目的 使用异硫氰酸苯酯衍生-高效液相色谱法对食品中17种游离氨基酸进行检测。方法 对17种游离氨基酸及其苯胺硫甲酰基衍生物稳定性的分析, 研究游离氨基酸的最佳衍生条件。同时, 对盐沉淀法和C18 固相萃取(solid phase extraction, SPE)小柱法2种样品除杂方法的加标回收率进行测定。结果 胱氨酸加热干燥5 h后, 回收率为68.46%±4.01%, 热稳定性差; 其它16种氨基酸加热1~5 h后回收率均在90%~110%之间, 热稳定性较好。胱氨酸的苯胺硫甲酰基衍生物, 6 h后回收率仅为11.04%±1.65%, 稳定性差; 其它氨基酸的苯胺硫甲酰基衍生物24 h后回收率均大于90%, 比较稳定。另外, 前处理中使用C18 SPE除杂时, 样品中游离氨基酸的加标回收率均大于70%, 而使用水杨酸和三氯乙酸除蛋白后苯胺硫甲酰基衍生物降解严重。 结论 除胱氨酸外, 其余16种氨基酸的热稳定性均较好, 且生成的苯胺硫甲酰基衍生物比较稳定, 可以通过异硫氰酸苯酯衍生进行定量分析。相比于水杨酸和三氯乙酸沉淀法, C18 SPE柱是游离氨基酸除杂方法的首选, 而无机盐影响异硫氰酸苯酯与氨基酸衍生, 因此此方法不适用于含盐样品的衍生。

英文摘要:

      Objective To determine 17 kinds of free amino acids in food by high performance liquid chromatography with derivatization of phenylisothiocyanate. Methods The thermal stability of 17 amino acids and their aniline thioformyl derivatives were analyzed, and the optimal derivative conditions of free amino acids were obtained. At the same time, The standard recovery rates of salt precipitation method and C18 solid phase extraction(SPE) column method for impurity removal were compared. Results After heating and drying for 5 h, the recovery of cystine was 68.46%±4.01%, with poor thermal stability; after heating for 1-5 h, the recovery rate of the other 16 amino acids were 90%-110%, with good thermal stability. The standard recovery rate of derivative cystine was only 11.04%±1.65% after 6 h, with poor thermal stability. The derivatives of other amino acids of aniline were stable and the standard recovery rate was more than 90% after 24 h. In addition, when C18 SPE was used to remove impurities in the pretreatment, the standard recoveries of the free amino acids in the samples were all more than 70%, and the aniline thioformyl derivatives were seriously degraded after the protein removal by salicylic acid and trichloroacetic acid. Conclusion Except cystine, the other 16 amino acids are thermally stable, and the aniline thioformyl derivatives of amino acid are relatively stable, which can be quantitatively analyzed by benzene isothiocyanate derivatives. Compared with salicylic acid and trichloroacetic acid precipitation, C18 SPE column is the first choice for free amino acid impurity removal, while inorganic salt affects the derivation of phenylisothiocyanate and amino acid, therefore, this method is not suitable for the derivation of salt samples.