柱后衍生HPLC法测定血液制品中甘氨酸的含量

Content Determination of Glycine in the Blood Products by HPLC with Post-column Derivatization

目的:建立测定血液制品中甘氨酸含量的方法。方法:采用高效液相色谱法。以邻苯二甲醛(OPA)为柱后衍生剂,测定4种血液制品中的氨基酸与衍生剂衍生后所得衍生产物的含量,并与2010年版《中国药典》标准方法(柱前衍生法)比较。色谱柱为ThermoC18,流动相为柠檬酸钠缓冲液(pH4.0),流速为1.0ml/min,柱温为60℃,检测波长为336nm,内标(正缬氨酸)法定量。结果:甘氨酸检测质量浓度线性范围为2～200mg/L(r=0.9999),4种血液制品中甘氨酸的平均回收率分别为99.6%、98.5%、100.2%、98.6%,RSD分别为2.5%、0.3%、1.6%、2.2%,定量限为20ng。该法与标准方法测定含量结果基本一致。结论:该方法灵敏度高,结果准确可靠。

OBJECTIVE： To establish the method for the content determination of glycine in the blood products. METHODS： Using O-phthaldialdehyde （OPA） as post-column derivating agent, the derivant content of derivating agent and amino acid of 4 blood products were determined by HPLC. It was compared with the pre-column derivatization stated in Chinese Pharmacopeia （2010 edition）. The determination was performed on Thermo C18 column with mobile phase composed of sodium citrate buffer （pH=4.0） at the flow rate of 1.0 ml/min. The column temperature was 60 ℃ and detection wavelength was 336 nm. The quantitative method was carried out by internal standard （norvaline）. RESULTS： The linear range of glycine was 2-200 mg/L （r=0.999 9）, and the recovery rates of 4 blood products were 99.6% （RSD=2.5%）, 98.5% （RSD=0.3%）, 100.2% （RSD=1.6%）, 98.6% （RSD=2.2%）. The limit of quantitation was 20 ng. The method was found to be basically in line with standard method. CONCLUSIONS： The method is sensitive, accurate and reliable.