Chinese Academy of Inspection and Quarantine, Beijing 100176, China.
Chinese Academy of Inspection and Quarantine, Beijing 100176, China.
Carbohydr Polym. 2023 Jul 1;311:120488. doi: 10.1016/j.carbpol.2022.120488. Epub 2022 Dec 23.
Low molecular weight (LWM) hyaluronic acid (HA) and chondroitin sulfate (CS) have a wide range of applications. To determine their molecular weight (MW), we developed a gel permeation chromatography (GPC) method, which is calibrated based on serrated peaks in the chromatograms. MW calibrants were obtained from the enzymolysis of HA and CS using hyaluronidase. The identical structure of calibrants and samples ensured the soundness of the method. The highest confidence MWs were up to 14,454 and 14,605 for HA and CS, respectively, and the standard curves showed very high correlation coefficients. Thanks to the changeless relationship between MW and its contribution to the GPC integral, the second calibration curves could be derived via one GPC column, also embodied correlation coefficients of >0.9999. The discrepancies of MW values were minuscule, and the measurement of a sample could be conducted in <30 min. The accuracy of the method was verified using LWM heparins, and the measured Mw values showed a 1.2 %-2.0 % error relative to pharmacopeia results. The MW results obtained for LWM-HA and LWM-CS samples were also consistent with the results obtained by multiangle laser light scattering. The method was also verified be able to measure the very low MWs.
低分子量（LWM）透明质酸（HA）和硫酸软骨素（CS）具有广泛的应用。为了确定它们的分子量（MW），我们开发了一种凝胶渗透色谱（GPC）方法，该方法基于色谱图中的锯齿峰进行校准。MW 校准标准品是通过透明质酸酶和软骨素酶对 HA 和 CS 的酶解获得的。校准标准品和样品的相同结构确保了该方法的可靠性。HA 和 CS 的最高置信度 MW 分别高达 14454 和 14605，标准曲线显示出非常高的相关系数。由于 MW 与其在 GPC 积分中的贡献之间的不变关系，第二校准曲线可以通过一个 GPC 柱导出，也体现了>0.9999 的相关系数。MW 值的差异非常小，一个样品的测量可以在<30 分钟内完成。该方法的准确性通过低分子量肝素进行了验证，测量的 Mw 值与药典结果相比存在 1.2%-2.0%的误差。LWM-HA 和 LWM-CS 样品的 MW 结果也与多角度激光光散射的结果一致。该方法还能够测量非常低的 MW。
