柱前衍生高效液相色谱法测定缩宫素的氨基酸组成

目的: 建立一种测定缩宫素氨基酸组成的柱前衍生化-HPLC法。 方法: 缩宫素经酸水解产生的游离氨基酸用异硫氰酸苯酯衍生后,以0.1 mol·L^-1醋酸钠溶液-乙腈 (97∶ 3,pH 5.20)和乙腈-水(4∶ 1)为二元流动相,流速为1.0 mL·min^-1,经RP-C₁₈色谱柱梯度洗脱,于254 nm处测定。通过外标法计算样品的氨基酸组成。 结果: 氨基酸浓度在0.12~0.28 μmol·mL^-1范围时与峰面积呈良好的线性关系(r均大于0.999);定量限介于4.73~31.7 ng;回收率范围为90.4%~101.8%,RSD范围为0.25%~3.2%。 结论: 本实验建立的分析方法分离效果好、灵敏、可靠,成功用于缩宫素的氨基酸组成测定研究。

Objective: An HPLC method with precolumn derivation has been established to determine amino acid composition of oxytocin. Methods: The oxytocin was hydrochloric acid hydrolyzed and free amino acids derived with phenylisothiocyanate (PITC).The derivates were gradient eluted by reverse-phase high performance liquid chromatography and determined at the wavelength of 254 nm.The mobile phase A was 0.1 mol·L^-1-sodium acetate buffer solution-acetonitrile(93∶ 7,pH 5.20) and B was acetonitrile-water(8∶ 20).The flow rate was 1.0 mL min^-1.The content of amino acids in oxytocin was calculated by HPLC-external standard method. Results: The calibration curves were linear over the concentration range of 0.12-0.28 μmol·mL^-1 with all correlation coefficients above 0.999.The limit of quantification of 8 amino acids were between 4.73 ng and 31.7 ng.The recoveries were between 90.4% and 101.8% with RSD between 0.25% and 3.2%. Conclusion: The proposed HPLC method,with the advantagement of high efficiency,sensitive and reliable,had been successfully applied in quantitative analysis of amino acid composition of oxytocin.