医用透明质酸钠凝胶的绝对分子量及其分布的测定方法研究

目的:建立医用透明质酸钠凝胶的绝对分子量及其分布的测定方法。方法: 采用多角度激光光散射仪与体积排阻色谱法联用(MALLS-GPC)测定医用透明质酸钠凝胶的绝对分子量及其分布。根据透明质酸钠特有的绝对折射率增量(dn/dc)和样品示差信号强度求得透明质酸钠含量。通过数据分析得到医用透明质酸钠凝胶的结构特性。结果: 本方法测得聚乙二醇(PEG)标准品重均分子量(M_w)与标示值的相对偏差为 4.7%,医用透明质酸钠凝胶样品分散度接近于1,分子量大于100万,所得的均方根半径(RMS radius)约为140 nm。本方法测得的透明质酸钠含量和糖醛酸法所得结果相差不大。结论: 该方法准确性及精密度较好,在实验过程中可同时得到均方根旋转半径、聚集态及样品中的透明质酸钠含量等信息,可用于医用透明质酸钠凝胶的质量控制和应用研究。

Objective: To establish a new method for the determination of the absolute molecular weight and molecular weight distribution in medical sodium hyaluronate gel. Methods: A multi angle light scatterer and a gel permeation chromatograph(MALLS-GPC)was used to determine the molecular weight and the distribution of medical sodium hyaluronate gel.The contents of sodium hyaluronate were calculated by the specific refractive index increment(dn/dc)of sodium hyaluronate and signal strength of sample.The structural properties of medical sodium hyaluronate gel were obtained through statistical analysis. Results: The relative deviation of the measured value and the nominal value(by measuring Polyethylene Glycol standard)was 4.7%.The dispersity of the samples were about 1,the weight-average molecular weight(M_w)of the samples were greater than 1 million,and their RMS radius were about 140 nm.The sodium hyaluronate contents measured by MALLS-GPC method are similar to the uronic acid content method. Conclusion: The accuracy and precision obtained by this method were proved to be reliable.Data such as the gel's RMS radius,conformational status,and the sodium hyaluronate content in samples and etc can be obtained simultaneously.The MALLS-GPC method can be used for the quality control and application study of the medical sodium hyaluronate gel.

1 Surendrakumar K,Martyn G,Hodgers EC,et al.Sustained release of insulin from sodium hyaluronate based dry powder formulations after pulmonary delivery to Beagle dogs.J Control Rel,2003,91(3):385
2 Laurent TC,Laurent UB,Fraser JR.The structure and function of hyaluronan:An overview.Immunol Cell Biol,1996,74(2):A1
3 Arshinoff SA.Dispersive and cohesive viscoelastic materials in phacoemulsification revisited.Ophthalmic Pract,1998,16(1):24
4 Goa KL,Benfield P.Hyaluronic acid:A review of its pharmacology and use as a surgical aid in ophthalmology,and its therapeutic potential in joint disease and wound-healing.Drugs,1994,47(3):536
5 Laurent TC.The Chemistry Biology and Medical Applications of Hyaluronan and Its Derivatives.London:Portland Press,1998.213
6 Morra M.Engineering of biomaterials surfaces by hyaluronan.Biomacromolecules,2005,6(3):1205
7 LI Guo-feng(李国凤),ZHANG Kai-jun(张凯军),CUI Xiang-zhen(崔向珍),et al.The sodium hyaluronate molecular weight comparison for different diameter with viscometer test(不同内径乌式粘度计检测透明质酸钠分子量的比较).Shandong Pharm Ind(山东医药工业),2003,22(6):44
8 EP 5.0.2004.2434
9 DONG Ke-yun(董科云),LUAN Yi-hong(栾贻宏),GUO Xue-ping(郭学平).Determination of the Content of Sodium Hyaluronic Acid by GPC(凝胶色谱法测定透明质酸钠的含量).Shandong Province Medicine in 2006 Biochemical and Biotech Drugs Proceedings(山东省药学会2006年生化与生物技术药物学术研讨会论文集),2006.137
10 CHEN Zhong-you(陈重酉).Gel permeation chromatography analysis precision and accuracy(凝胶渗透色谱分析的精度和准确性).Chin J Chromatogr(色谱),1989,7(3):138 TE
11 Almond A,Hardingham TE.Hyaluronan:current macomolecular and micromocular views..http://www.glycoforum.gr.jp/science/hyaluronan/HA31/HA31E_20111024.pdf
12 Wen J,Arakawa T,Philo JS.Size-exclusion chromatography with on-line-light-scattering,absorbance,and refractive index detectors for studying proteins and their interactions.Anal Biochem,1996,240(2):155
13 American Polymer Standards Corporation.Light Scattering dn/dc Values.http://www.ampolymer.com/dndn.html
14 Jason W,Danielle L.Charaterization of hyaluronic acid with on-line differential viscometry,multiangle light scattering,and differential refractometry.LC GC North Am,2005,23(3):302