反相高效液相色谱法测定软骨中的II型胶原蛋白

Determination of type II collagen in cartilage by reversed-phase high performance liquid chromatography

建立了高效液相色谱法测定软骨中Ⅱ型胶原蛋白的方法。以ZORBAX300SB-C18为色谱分离柱,流动相:A为5%乙腈、0.05%TFA,B为80%乙腈(v/v),采用线形梯度洗脱;检测波长:220nm;流速:1mL/min,柱温:35℃;进样量:10μL。该方法对Ⅱ型胶原的检出限为0.02mg/mL,线性范围20～250μg/mL,样品测定的变异系数为0.51%,加标回收率为99.13%～100.06%。该方法具有快速准确、重现性好、所需样品用量少、易于自动化的优点,从而为Ⅱ型胶原提供了一种新的测定方法。

Reversed-phase high performance liquid chromatography（ RP-HPLC） method was demonstrated to determine the concentration of type II collagen in cartilage. Chromatography was performed with ZORBAX 300 SB-C18 and an injection volume of 10μL.The mobile phase consisted of two solvent： （ A） 5% acetonitrile and 0.05% trifluoroacetic（ TFA） and（ B） 80% acetonitrile（ v/v） .The separation was performed using the linear gradient of A-B（ v/v） . Flow rate was maintained 1mL/min. Absorbance was monitored at 220nm. The linear range was from 20μg /mL to 250μg /mL and the detection limit was 20μg /mL.The variation coefficient of determination was 0.51%. The recoveries of spiked samples were 99.13%～100.06%.The RP-HPLC has proved to be convenient,sensitive, accurate and reproducible on base of avoiding the use of a time-consuming pretreatment procedure.