摘要
目的 建立人凝血因子Ⅷ中氨基酸含量的柱前衍生HPLC检测方法。方法 采用6-氨基喹啉基-N-羟基琥珀酰亚氨基氨基甲酸酯(6-aminoquinoline-N-hydroxysuccinimide carbamate,AQC)在一定条件下与氨基酸形成稳定的衍生产物(柱前衍生),再通过高效液相色谱仪,采用氨基酸分析柱(150 mm×3.9 mm,4μm)检测,以不同流动相进行梯度洗脱[在《中国药典》三部(2015版)的基础上进行调整],柱温为37℃,流速为1.0 mL/min,检测波长为248 nm。同时验证方法的系统适应性、专属性、线性范围、精密性及准确度。结果 甘氨酸及盐酸赖氨酸均可在25 min内获得较好分离,与相应相邻色谱峰之间的分离度均>1.5,拖尾因子在0.95~1.40之间;各样品的氨基酸种类均能正确检出,混合样品与单纯样品出峰时间保持一致,且供试品溶液与对照品溶液主峰保留时间相对偏差均<2%;当甘氨酸浓度在12.5~62. 5μg/mL,盐酸赖氨酸浓度在4.0~20.0μg/mL时,与相应氨基酸与内标峰面积比呈良好的线性关系,相关系数(r)分别为0.999 9和0.999 8;甘氨酸及盐酸赖氨酸重复性的相对标准偏差(RSD)分别为0.4%和0.5%,中间精密度RSD分别为1.3%和1.0%;两种氨基酸的回收率均在95%~105%范围内,RSD均≤2%。结论 建立的方法具有良好的专属性、精密性及准确度,可用于测定人凝血因子Ⅷ中的氨基酸含量。
Objective To establish a method for determination of amino acid content in human coagulation factorⅧ(FⅧ)by HPLC with pre-column derivation.Methods A stable derivative(pre-column derivatization)was prepared with amino acids under a certain condition by 6-aminoquinoline-N-hydroxysuccinimide carbamate(AQC).HPLC was performed by using amino acid analysis column(150 mm×3.9 mm,4μm),and subjected to gradient elution with mobile phases A,B and C,which had been adjusted according to the requirements in Chinese Pharmacopoeia(VolumeⅢ,2015 edition),at a column temperature of 37℃,a flow rate of 1.0 mL/min and a detection wavelength of 248 nm.The developed method was verified for systemic suitability,specificity,linear range,precision and accuracy.Results Under the given chromatographic condition,glycine and lysine hydrochloride were separated well within 25 min,while the separation degree of the adjacent chromatographic peaks was more than 1.5,with a tailing factor of 0.95~1.40.The kinds of amino acids in various samples were identified correctly.The time of appearance of peak of mixed samples was consistent with that of simplex samples.The relative deviation between the retention time of main peaks of sample solution and control solution was less than 2%.The linear ranges of glycine and lysine hydrochloride were 12.5~62.5μg/mL and 4.0~20.0μg/mL,with correlation coefficients(r)of 0.9999 and 0.9998,respectively.The RSDs of reproducibility of glycine and lysine hydrochloride were 0.4%and 0.5%,while those of intermediate precision were 1.3%and 1.0%,respectively.The recoveries of the two kinds of amino acids were 95%~105%,with RSDs of not more than 2%.Conclusion The developed method was specific,precise and accurate,which might be used for determination of amino acid content in FⅧ.
作者
王月
喻剑虹
刘莹
罗晶
郑宵蓓
张雪
龚钦
WANG Yue;YU Jian-hong;LIU Ying;LUO Jing;ZHENG Xiao-bei;ZHANG Xue;GONG Qin(Sinopharm Wuhan Plasma-derived Biotherapies Co.,Ltd.,Wuhan 430030,Hubei Province,China)
出处
《中国生物制品学杂志》
CAS
CSCD
2019年第12期1402-1406,1410,共6页
Chinese Journal of Biologicals
关键词
人凝血因子Ⅷ
氨基酸含量
柱前衍生化
HPLC
甘氨酸
盐酸赖氨酸
Human coagulation factorⅧ(FⅧ)
Amino acid content
Pre-column derivatization
HPLC
Glycine
Lysine hydrochloride
