HPLC法快速测水产品中游离芳香族氨基酸含量的研究

Rapid analysis of free aromatic amino acids in aquatic product by high performance liquid chromatography

建立了水产品中3种游离芳香族氨基酸含量的HPLC测定方法。试验确定了流动相的最佳pH、流速及浓度,并评价了方法的准确度、精密度和检测限。采用5%高氯酸溶液提取,用KOH溶液中和后进行HPLC分析。采用WatersAtlantis T3 C18(4.6×250 mm,5μm)色谱柱分离,以15%甲醇-85%磷酸盐溶液为流动相,用磷酸调pH为6.0;色谱条件流速:0.9 mL/min;柱温:27℃;检测波长:256.7 nm;12 min内完成测定。结果表明:Tyr、Phe、Trp分别在6～500μg/mL、30～1 000μg/mL和3～250μg/mL范围内有良好的线性关系,其线性相关系数(R2)在0.998 6～0.999 5之间。三者的检测限分别为2μg/mL、10μg/mL、1μg/mL,加标回收率在88.0%～105.2%之间。方法操作简便且定性可靠、定量准确。

To establish a HPLC method for the determination of free aromatic amino acids in aquatic product. Optimal conditions such as pH, flow rate and concentration of mobile phase were confirmed. Accuracy, precision and limit of quantitation were surveyed and evaluated. The method is based on an extraction of amino acids from aquatic product with a solution of 5% perchloric acid followed by neutralization by the addition of KOH just prior to HPLC analysis. HPLC system consisted of a Waters Atlantis T3 C18 （4.6×250 mm,5μm） column. The mobile phase consisted of 85% 0.05 mol/L sodium phosphate buffer and 15% methanol, adjusted to pH 6.0 with phosphoric acid. The flow rate was 0.9 mL/min, the column temperature was 27℃, and the detection wavelength was 256.7 nm. The three amino acids were separated in 12 min. The standard curves of Tyr, Phe, Trp were linear within a range of 10 - 500, 50 - 1 000 and 5 - 250μg/mL, and the correlation coefficient deviations range from 0. 999 86 to 0. 995. The limits of detection were 2, 10 and 1 μg /mL. The average recovery of spiked sample deviations ranges from 88.0% to 105.2%, In conclusion, this method proved to be simple, rapid, reliable and with high accuracy and precision. It has practical value for detecting the three amino acids in aquatic product.