中国临床药学杂志

目的 建立同时测定3种碱性氨基酸（精氨酸、赖氨酸和组氨酸）含量的HPLC法，并测定该3种碱性氨基酸是否具有肝素中和作用。方法 采用异硫氰酸苯酯（PITC）柱前衍生法。色谱条件：Diamonsil C18色谱柱（150 mm×4.6 mm,5μm），柱温30℃，以乙腈-乙酸钠溶液（冰醋酸调p H至6.3）为流动相进行梯度洗脱，流速1.0 m L·min-1，检测波长254 nm，进样量20μL。将氨基酸混合液与肝素进行体外中和实验，测定混合前后氨基酸浓度的变化。SD大鼠注射肝素钠注射液后随机分为3组，空白对照组、实验组和阳性对照组大鼠分别注射生理盐水、氨基酸混合液或鱼精蛋白注射液，不同时间取血测定活化部分凝血活酶时间（APTT）。结果 3种氨基酸在5～100μg·mL-1范围呈线性，稳定性、精密度、回收率结果良好。体外肝素中和试验前后氨基酸浓度无显著变化。体内凝血试验显示，实验组与空白对照组APTT相近（P> 0.05），阳性对照组与空白对照组或实验组相比APTT明显延长（P <0.05）。结论 柱前衍生HPLC方法可以同时准确测定精氨酸、赖氨酸和组氨酸的含量，体内体外研究显示3种碱性氨基酸不具备肝素中和作用。

AIM To establish a high performance liquid chromatography(HPLC) method for the determination of concentrations of three alkaline amino acid(arginine, lysine and histidine) simultaneously and determine the neutralization of three alkaline amino acids after admixed with heparin. METHODS Precolumn derivatization was used and phenylisothioeyanate(PITC) was selected as derivatization reagent. Analysis was performed on a Diamonsil C18column(150 mm×4.6 mm, 5 μm) with the mixture of acetonitrile and sodium acetate solution(pH was adjusted to 6.3 with glacial acetic acid) as mobile phase in gradient elution, and the flow rate was 1.0 mL·min-1, pre-column derevatization was operated before analysis. The detection wavelength was 254 nm. The column temperature was 30℃ and the sample size was 20 μL. In vitro neutralization of mixed amino acid solution and heparin was tested through the determination of amino acid mixture concentration changes. SD rats were divided into 3 groups randomly after heparin injection, each group(blank, experimental and positive control) was administrated with saline solution, amino acid mixture and protamine injection respectively. Blood samples of different time were drawn for activated partial thromboplastin time(APTT).RESULTS Three kinds of free amino acid showed good linear response within the range of 5-100 μg·mL-1 with good stability, precision and recovery. No obvious differences among the concentrations of amino acids were observed from in vitro neutralization test. Meanwhile, in vivo coagulation test indicated similar APTT between blank and experimental group(P > 0.05), while positive control group showed longer APTT compared with them(P < 0.05). CONCLUSION The precolumn derivatization HPLC method can accurately determine the concentrations of arginine, lysine and histidine simultaneously, and the tests in vivo and in vitro demonstrate that three alkaline amino acids are unavailable for heparin neutralization.