DTDPA-PITC联合柱前衍生化HPLC法同时测定复方氨基酸注射液中18种氨基酸的含量

Simultaneous Determination of 18 Kinds of Amino Acids in Compound Amino Acid Injection by DTDPA-PITC Combined with Pre-column Derivatization HPLC

目的：建立同时测定含有半耽氨酸或胱氨酸的复方氨基酸注射液中18种氨基酸含量的方法。方法：采用高效液相色谱法，以二硫代二丙酸（DTDPA）和异硫氰酸苯酯（PITC）联合作为柱前衍生化试剂。色谱柱为Kromasil C18m流动相[A为0.1mol／L醋酸盐缓冲液（pH5．38）-乙腈（93：7，V/V）、B为乙腈-水（80：20，V/V），采用梯度洗脱]，检测波长为254nm，流速为1．0ml／min，进样量为3μl，柱温为35℃。结果：18种氨基酸在各自测定浓度的20％～180％的范围内与各自峰面积积分值呈良好的线性关系（r≥0．9990）；精密度、稳定性、重复性试验的RSD≤1．91％；平均回收率在95．7％～99．7％之间，RSD≤1．99％（n=9）。结论：该方法准确、高效，实现了同时对复方氨基酸注射液中半胱氨酸以及其他17种氨基酸的准确定量。

OBJECTIVE： To develop a method for simultaneous determination of 18 kinds of amino acids in Compound amino acid injection including cysteine or cystine. METHODS： HPLC method was adopted. The samples were derived with 3,3＇ -dithiodipropionic acid （DTDPA） and phenylisothiocyanate （PITC）. The determination was performed on Kromasil C18 column with mobile phase A consisted of 0.1 mol/L acetate buffer （pH 5.38） -acetonitrile （93： 7, VIV） and mobile phase B consisted of acetonitrile-water （80： 20, VIV） at the flow rate of 1.0 mllmin （gradient elution） and the detection wavelength of 254 nm. The column temperature was 35 ℃, and the injection volume was 3 ul, RESULTS： The linear range of 18 kinds of amino acids was 20%-180% （r= 0.999 0）; RSDs of precision, stability and reproducible were 〈 1.91 %; average recoveries were 95.7 %-99.7 % （RSD≤ 1.99 %, n= 9）. CONCLUSIONS： The method can accurately and efficiently determine cysteine and other 17 kinds of amino acids in simultaneously.