高效液相法测定人凝血因子Ⅷ中甘氨酸含量

目的: 建立HPLC法测定人凝血因子Ⅷ中甘氨酸含量. 方法: 采用氨基键合柱进行分离,流动相为乙腈-磷酸盐缓冲液(50: 50),流速1.0 mL·min^-1,紫外检测波长为205 nm,柱温40℃,进样量20 μL;用面积外标法计算含量. 结果: 在本实验条件下,甘氨酸与精氨酸的分离度符合要求;甘氨酸质量浓度在1.02~5.04 mg·mL^-1范围内线性关系良好;3批样品平均含量分别为8.70、8.76、8.80 mg·mL^-1,RSD分别为0.57%、0.32%和0.50%. 结论: 该方法经方法学验证,可用于人凝血因子Ⅷ中甘氨酸含量的测定.

Objective: To investigate an HPLC method to determine glycine content in human coagulation factor Ⅷ. Methods: The amino bonded column was applied to separate glycine;the mobile phase consisted of acetonitrile-phosphate buffer(50: 50)at a flow rate of 1.0 mL·min^-1;the detection wavelength was 205 nm,the column temperature was 40℃,and the injection volume was 20 μL.The content of glycine in human coagulation factor Ⅷ was calculated by area external standard method. Results: In this experiment,the results showed that the separation of glycine and arginine met the requirement,and the linearity was good at the range of 1.02 mg·mL^-1 to 5.04 mg·mL^-1.The average contents in three batches of samples were 8.70 mg·mL^-1,8.76 mg·mL^-1,and 8.80 mg·mL^-1;RSD were 0.57%,0.32% and 0.50%. Conclusion: The results of methodology validation show that the HPLC method can be used to determine the content of glycine in human coagulation factor Ⅷ.

[1] ChP 2010.VolⅢ(中国药典2010年版.三部)[S].2010:236
[2] CAO HJ(曹海军),ZHANG XJ(张学俊),YANG XF(杨显福),et al.Comparison of several additives about human coagulation factor Ⅷ(人凝血因子Ⅷ几种冻干保护剂的比较)[J].Chin J Blood Transfus(中国输血杂志),2009,22(7):558
[3] ZHANG T(张彤),WU HY(武晗燕),ZHANG W(张伟),et al.Content determination of glycine in the blood products by HPLC with post-column derivatization(柱后衍生HPLC法测定血液制品中甘氨酸的含量)[J].China Pharm(中国药房),2013,24(25):2378
[4] WU YQ(吴艳亲).HPLC determination of amino acids in human prothrombin complex(HPLC 法测定人凝血酶原复合物中氨基酸含量)[J].Chin J Pharm Anal(药物分析杂志),2012,32(11):1929
[5] WU XM(吴小曼),XU Y(徐滟),SHI BJ(石蓓佳).Determination of glycine and methionine in compound glycyrrhizin tablets by HPLC(HPLC法同时测定复方甘草酸苷片中甘氨酸和蛋氨酸的含量)[J].Chin J Biochem Pharm(中国生化药杂志),2010,31(6):395
[6] CUI SM(崔素梅),GAO HQ(高红琴).Determination of glycine in human coagulation factor Ⅷ by pre-column derivation(柱前衍生法测定人凝血因子Ⅷ中甘氨酸含量)[J].J Shanxi Med Univ(山西医科大学学报),2012,43(7):505
[7] NI WF(倪维芳),JIN O(金瓯),ZHANG JS(张劲松).HPLC determination of amino acids in compound amino acid capsules(HPLC法测定复合氨基酸胶囊中氨基酸的含量)[J].Chin J Pharm Anal(药物分析杂志),2004,24(4):403
[8] DING YS(丁永胜),MOU SF(牟世芬).Development of analytical methods for amino acids and their applications(氨基酸的分析方法及其应用进展)[J].Chin J Chromatogr(色谱),2004,22(5):210
[9] PAN W(潘葳),GUO GH(郭根和),SU DS(苏德森),et al.The comparison of methods of determinating amino acids by RP-HPLC and IEC(反相高效液相色谱法与离子交换色谱法测定氨基酸比对)[J].Mod Sci Instrum(现代科学仪器),2004(3):25
[10] XING J(邢健),LI QL(李巧玲),GENG TH(耿涛华),et al.Research development for amino acid analysis and determination(氨基酸分析方法的研究进展)[J].China Food Addit(中国食品添加剂),2012(5):187