柱前衍生高效液相色谱法测定注射用骨肽中15种氨基酸含量

目的建立测定注射用骨肽中15种氨基酸含量的柱前衍生高效液相色谱法。方法色谱柱为ZorbaxEclipseAAA柱(150 mm×4.6 mm,5μm),衍生剂为邻苯二甲醛(自动衍生化进样程序),流动相A为40 mmol/L磷酸二氢钠溶液(pH 7.8),流动相B为乙腈-甲醇-水(45∶45∶10,V/V/V),梯度洗脱,流速为2.0 mL/min,检测波长为338 nm,柱温为40℃。结果门冬氨酸、谷氨酸、丝氨酸、组氨酸、甘氨酸、苏氨酸、精氨酸、丙氨酸、酪氨酸、缬氨酸、甲硫氨酸、苯丙氨酸、异亮氨酸、亮氨酸、赖氨酸的质量浓度均在0.01~1.59 mg/mL范围内与峰面积线性关系良好(r≥0.9976);检测限为0.0083 nmol/μL,定量限为0.025 nmol/μL,稳定性和重复性试验结果的RSD均小于2.0%(n=6);平均加样回收率为92.63%~108.28%,RSD为0.87%~2.66%(n=9);3批样品中氨基酸总含量分别为1.83%,1.75%,1.73%。结论该方法操作简便,准确度高,稳定性和重复性均较好,可用于测定注射用骨肽中15种氨基酸的含量。

Content Determination of 15 Amino Acids in Ossotide for Injection by Precolumn Derivatization HPLC

Objective To establish a precolumn derivation high-performance liquid chromatography(HPLC)method for the content determination of 15 amino acids in Ossotide for Injection.Methods The chromatographic column was Zorbax Eclipse AAA column(150 mm×4.6 mm,5μm),the derivatization agent was O-phthaladehyde(OPA)with automatic injection procedure,the mobile phase A was 40 mmol/L sodium dihydrogen phosphate solution(pH 7.8),the mobile phase B was acetonitrile-methanol-water(45∶45∶10,V/V/V)with gradient elution,the flow rate was 2.0 mL/min,the detection wavelength was 338 nm,and the column temperature was 40℃.Results The mass concentrations of aspartic acid,glutamic acid,serine,histidine,glycine,threonine,arginine,alanine,tyrosine,valine,methionine,phenylalanine,isoleucine,leucine and lysine showed a good linear relationship with the peak area in the range of 0.01-1.59 mg/mL(r≥0.9976).The limit of detection(LOD)was 0.0083 nmol/μL,and the limit of quantitation(LOQ)was 0.025 nmol/μL.The RSDs of stability and repeatability tests were less than 2.0%(n=6).The average recoveries of 15 amino acids were in the range of 92.63%-108.28%with RSDs of 0.87%-2.66%(n=9).The total contents of amino acids in three batches of samples were 1.83%,1.75%and 1.73%,respectively.Conclusion The method is simple,accurate,stable and reproducible,which can be used for the content determination of 15 amino acids in Ossotide for Injection.