[论文解读] Quantification of heparin in complex matrices (including urine) using a mix-and-read fluorescence assay

Ulrich Warttinger, Roland Krämer|arXiv (Cornell University)|Nov 8, 2016

Proteoglycans and glycosaminoglycans research参考文献 18被引用 5

一句话总结

本研究提出了一种简单、即混即测的荧光分析法，利用Heparin Red检测复杂基质（如尿液）中的肝素，具有高特异性和高灵敏度。该方法可直接、准确地检测低至微克每毫升级别的肝素，且不受常见杂质或基质成分的干扰，适用于质量控制和药代动力学研究。

ABSTRACT

Heparin is an important anticoagulant drug, about one billion doses are produced annually. It is a polydisperse sulfated polysaccharide, and the inherent heterogeneity makes the analysis of heparin difficult. The global crisis resulting from adulterated heparin in 2008 has drawn renewed attention to the challenges that are associated with the quality control and characterization of this complex biological medicine from natural sources. The present study addresses the need for simple and user-friendly analytical methods for the fast and accurate quantification of heparin in complex matrices. Direct quantification of heparin in the low microgram per mL range was accomplished using a specific commercially available assay based on the fluorescent molecular probe Heparin Red, simply by mixing the heparin containing sample and a reagent solution in a 96-well microplate followed by fluorescence readout. A screening of typical impurities in raw heparin (selected other glycosaminoglycans, residual nucleic acids and proteins), related to the extraction from animal tissues, as well as of components of the urine matrix (inorganic salts, amino acids, trace proteins) revealed that these compounds even in large excess have no or very little effect on the accuracy of heparin determination. Heparin spike detection in urine, a biological multicomponent matrix, also showed good accuracy. We envision applications of this mix-and-read assay in the process and quality control in heparin manufacturing, but also in pharmacokinetic studies as a convenient tool for measuring of the urinary excretion of heparins.

研究动机与目标

• 开发一种快速、用户友好的方法，用于定量分析复杂生物基质（如尿液）中的肝素。
• 解决由于肝素本身具有多分散性、异质性以及历史污染问题所带来的分析挑战。
• 评估该方法对原始肝素中常见杂质及尿液组分的特异性和稳健性。
• 实现该方法在制药工业质量控制和肝素尿排泄药代动力学研究中的实际应用。

提出的方法

• 采用市售的Heparin Red探针进行即混即测荧光分析，该探针能特异性结合肝素，并在结合后荧光信号显著增强。
• 样品与Heparin Red试剂在96孔微孔板中混合，实现高通量分析。
• 混合后直接测量荧光强度，定量基于已知肝素浓度标准曲线。
• 通过一系列潜在基质成分（包括糖胺聚糖、核酸、蛋白质、无机盐、氨基酸及痕量蛋白）测试干扰情况。
• 向尿液样品中添加肝素，以评估在生物相关条件下的回收率和准确性。
• 在复杂基质中对方法的线性范围、检测限和精密度进行了验证。

实验结果

研究问题

• RQ1 一种简单、即混即测的荧光分析法是否能在复杂基质（如尿液）中实现肝素的准确定量？
• RQ2 原始肝素中的常见杂质或尿液组分在多大程度上干扰基于荧光的肝素检测？
• RQ3 该方法是否足够灵敏和特异，能够检测生物样品中低至微克每毫升浓度的肝素？
• RQ4 该方法是否可可靠地应用于制药工业质量控制及涉及肝素尿排泄的药代动力学研究？

主要发现

• 该方法实现了对低至微克每毫升范围肝素的直接定量，具有高灵敏度和良好的重复性。
• 即使在大量过量存在下，所选糖胺聚糖、核酸、蛋白质、无机盐、氨基酸及痕量蛋白均未观察到显著干扰。
• 在尿液基质中添加肝素的回收率准确，证明该方法在复杂生物环境中的稳健性。
• 即混即测的格式实现了高通量、低成本且用户友好的分析，无需样品预处理。
• 该方法在工业质量控制及肝素排泄药代动力学研究中具有广阔应用前景。