Pharmaceutical Institute, Christian-Albrechts-University, Gutenbergstrasse 76, 24118, Kiel, Germany.
Anal Bioanal Chem. 2011 Jan;399(2):681-90. doi: 10.1007/s00216-010-4252-0. Epub 2010 Oct 16.
There are several methods for sensitive detection of oversulfated chondroitin sulfate (OSCS) in heparin. Although contamination with OSCS is unlikely to be repeated, use of other compounds to counterfeit heparin must be considered. We have previously developed a two-step fluorescence microplate assay (two-step FI assay) for detection of OSCS. First, the heparin sample is incubated with heparinase I, then its increasing effect on the fluorescence intensity (FI) of the sensor molecule Polymer-H is measured (PolyH assay). The high sensitivity of the assay is shown to be based on heparinase I inhibition by OSCS. The objective of this study was to evaluate another assay option - indirect quantification of OSCS after heparinase I incubation by means of the anti-Factor Xa (aXa) activity of the remaining undegraded heparin (two-step aXa assay). We also examined, whether other heparin mimetics (HepM), direct Factor Xa inhibitors (DXI), and protein impurities are detectable by use of these assays. Heparin was spiked with different amounts of HepM including OSCS, pentosan polysulfate, dextran sulfate, curdlan sulfate, the natural contaminant dermatan sulfate, the DXI rivaroxaban, and BSA as a protein. These samples were compared with pure heparin in the two-step FI assay, the two-step aXa assay, and in the PolyH assay and the aXa assay without heparinase I incubation. Both two-step assays sensitively measured contamination with all the HepM (LOD ≤ 0.5%, LOQ ≤ 0.7%). The two-step aXa assay also detected rivaroxaban (LOD 0.3%, LOQ 0.4%), whereas the two-step FI assay was shown to be suited to determination of protein impurities (LOD 0.11%, LOQ 0.13%). Use of two different heparinase I inactivation procedures enabled clear differentiation between protein, HepM, and both contaminants. Finally, with the aXa assay the heparin potency can be determined in the same assay run, whereas the FI increase in the PolyH assay was shown to be useful for identification. In conclusion, both the two-step FI assay and the two-step aXa assay are sensitive, rapid, and simple tests for the detection of counterfeit heparin. Comprehensive information about heparin quality can be obtained by their combined use and the parallel measurement of non-incubated heparin samples.
有几种方法可用于敏感检测硫酸乙酰肝素（OSCS）在肝素中的存在。尽管 OSCS 的污染不太可能重复，但必须考虑使用其他化合物来伪造肝素。我们之前开发了一种两步荧光微孔板检测法（两步 FI 检测法）用于检测 OSCS。首先，将肝素样品与肝素酶 I 孵育，然后测量传感器分子 Polymer-H 的荧光强度（FI）的增加（PolyH 检测法）。该检测法的高灵敏度基于 OSCS 对肝素酶 I 的抑制作用。本研究的目的是评估另一种检测方案 - 通过剩余未降解肝素的抗 Xa 因子（aXa）活性间接定量肝素酶 I 孵育后的 OSCS（两步 aXa 检测法）。我们还研究了其他肝素类似物（HepM）、直接 Xa 因子抑制剂（DXI）和蛋白杂质是否可以通过这些检测法检测到。将不同量的 HepM（包括 OSCS、戊聚糖多硫酸酯、硫酸葡聚糖、卡拉胶硫酸酯、天然污染物硫酸皮肤素、DXI 利伐沙班和 BSA 作为蛋白）加入到肝素中。将这些样品与纯肝素在两步 FI 检测法、两步 aXa 检测法和 PolyH 检测法和没有肝素酶 I 孵育的 aXa 检测法中进行比较。两种两步检测法均能灵敏地测量所有 HepM 的污染（LOD≤0.5%，LOQ≤0.7%）。两步 aXa 检测法还能检测到利伐沙班（LOD 0.3%，LOQ 0.4%），而两步 FI 检测法则适用于确定蛋白杂质（LOD 0.11%，LOQ 0.13%）。使用两种不同的肝素酶 I 失活程序，能够清楚地区分蛋白、HepM 和两种污染物。最后，通过 aXa 检测法可以在同一检测运行中确定肝素的效价，而 PolyH 检测法中的 FI 增加则可用于鉴定。总之，两步 FI 检测法和两步 aXa 检测法都是检测假冒肝素的灵敏、快速和简单的方法。通过联合使用这些方法并平行测量未孵育的肝素样品，可以获得有关肝素质量的综合信息。
