柱前衍生化-HPLC法测定市售3种胶类药材中L-羟脯氨酸和胶原蛋白的含量

Content Determination of L-Hyp and Collagen in 3 kinds of Gelatinous Chinese Medicines by Pre-column Derivation-HPLC

目的:建立测定胶类药材中L-羟脯氨酸(L-Hyp)和胶原蛋白含量的方法,并比较对照药材与市售药材中两种成分的含量。方法:采用柱前衍生化法进行前处理,并采用高效液相色谱法测定样品中L-Hyp的含量:色谱柱为Kromasil C18,流动相为[乙腈-0.1 mol/L醋酸钠缓冲液(p H 6.5,7∶93,V/V)]-[乙腈-水(4∶1,V/V)](梯度洗脱),流速为1.0 m L/min,检测波长为254 nm,柱温为43℃,进样量为20μL;再通过折算系数计算样品中胶原蛋白的含量。结果:L-Hyp检测质量浓度线性范围为2.5~40μg/m L(r=0.999 9);定量限为0.20μg/m L,检测限为0.05μg/m L;精密度、稳定性、重复性试验的RSD<4.0%;加样回收率为96.03%~102.07%(RSD=2.20%,n=9)。28批市售药材中L-Hyp和胶原蛋白的含量有一定差异,其中13批市售阿胶药材与阿胶对照药材中两种成分的含量较为接近,而5批龟甲胶和7批鹿角胶药材中两种成分的含量高于其对照药材。结论:该方法准确、可靠,适用于测定胶类药材中L-Hyp和胶原蛋白的含量。

OBJECTIVE： To establish the method for the content determination of L-Hyp and collagen in gelatinous medicinal material, and to compare the contents of two components in reference medicinal material and commercially available medicinal ma- terial. METHODS： Pre-column derivatization was adopted for pretreatment. The content of L-Hyp was determined by HPLC. The determination was performed on Kromasil Cta column with mobile phase consisted of acetonitrile-0.1 mol/L sodium acetate buffer （pH 6.5,7 ： 93, V/V）-acetonitrile-water （4 ： 1, V/V） （gradient elution） at the flow rate of 1.0 mL/min. The detection wavelength was set at 254 nm, and column temperature was set at 43 22. The sample size was 20 μL. The content of collagen was calculated by us- ing convert coefficient. RESULTS： The linear range of L-Hyp were 2.5-40 μg/mL（r=0.999 9）. LOQ was 0.20 μg/mL, and LOD was 0.05 μg/mL. RSDs of precision, stability and reproducibility tests were all lower than 4.0%. The recoveries were 96.03%-102.07% （RSD=2.20% ,n=9）. There was difference in the contents of L-Hyp and collagen among 28 batches of commer- cially available medicinal material. The contents of two components in 13 batches of commercially available Colla Corii Asini were relative close to reference medicinal material; 5 batches of Colla Carapacis et Plastri Testudinis and 7 batches of Cervi Comus Colla were much higher than those of reference medicinal material. CONCLUSIONS： The method is accurate, reliable and suitable for the content determination of L-Hyp and collagen in gelatinous medicinal material.