King J Timothy, Desai Umesh R
Institute for Structural Biology and Drug Discovery, and Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23298, USA.
Anal Biochem. 2008 Sep 15;380(2):229-34. doi: 10.1016/j.ab.2008.05.046. Epub 2008 Jun 4.
Clinically used low molecular weight heparins (LMWH) are anticoagulants of choice and are phenomenally complex mixtures of millions of distinct natural and unnatural polymeric sequences. The FDA recommends that each LMWH be considered as an independent drug with its own activity profile, placing significant importance on the biophysical characterization of each intact LMWH. We report a robust protocol for fingerprinting these pharmaceutical agents. Capillary electrophoresis of three LMWHs, enoxaparin, tinzaparin, and a Sigma preparation, under reverse polarity conditions in the presence of selected linear alkyl polyamines gives an electrophoretic pattern that is characteristic of the nature of the starting material. The buffers that best provided optimal resolution without compromising sensitivity and speed of analysis were 50 mM sodium phosphate, pH 2.3, and 100 mM ammonium formate, pH 3.5. Resolution was strongly dependent on the structure of polyamine with pentaethylenehexamine being most effective for enoxaparin and Sigma LMWH. In contrast, tinzaparin could be best resolved with tetraethylenepentamine. Cyclic polyamines were ineffective. Resolution was also dependent on the concentration of resolving agents and displayed a narrow window that provides optimal resolution. These features suggest a strong structural origin of the fingerprint pattern. Overall, the simple protocol will find special use in assessing LMWH quality and batch-to-batch variability.
临床使用的低分子量肝素（LMWH）是首选的抗凝剂，是由数百万种不同的天然和非天然聚合物序列组成的极其复杂的混合物。美国食品药品监督管理局（FDA）建议将每种低分子量肝素视为具有自身活性特征的独立药物，高度重视每种完整低分子量肝素的生物物理特性。我们报告了一种用于这些药物指纹识别的可靠方案。在选定的线性烷基多胺存在下，在反相条件下对三种低分子量肝素（依诺肝素、替扎肝素和Sigma制剂）进行毛细管电泳，得到的电泳图谱是起始材料性质的特征。在不影响分析灵敏度和速度的情况下，能提供最佳分离度的缓冲液是50 mM磷酸钠，pH 2.3和100 mM甲酸铵，pH 3.5。分离度强烈依赖于多胺的结构，五乙烯六胺对依诺肝素和Sigma低分子量肝素最有效。相比之下，替扎肝素用四乙烯五胺能得到最佳分离效果。环状多胺无效。分离度还取决于分离剂的浓度，并且显示出一个提供最佳分离度的狭窄窗口。这些特征表明指纹图谱有很强的结构起源。总体而言，这个简单的方案将特别用于评估低分子量肝素的质量和批次间的变异性。
