Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR CEP: 81531-980, Brazil.
Analyst. 2011 Jun 7;136(11):2330-8. doi: 10.1039/c0an01010c. Epub 2011 Apr 14.
Recently, oversulfated chondroitin sulfate (OSCS) was identified in contaminated heparin preparations, which were linked to several adverse clinical events and deaths. Orthogonal analytical techniques, namely nuclear magnetic resonance (NMR) and capillary electrophoresis (CE), have since been applied by several authors for the evaluation of heparin purity and safety. NMR identification and quantification of residual solvents and non-volatile low molecular contaminants with USP acceptance levels of toxicity was achieved 40-fold faster than the traditional GC-headspace technique, which takes ~120 min against ~3 min to obtain a (1)H NMR spectrum with a signal/noise ratio of at least 1000/1. The procedure allowed detection of Class 1 residual solvents at 2 ppm and quantification was possible above 10 ppm. 2D NMR techniques (edited-HSQC (1)H/(13)C) permitted visualization of otherwise masked EDTA signals at 3.68/59.7 ppm and 3.34/53.5 ppm, which may be overlapping mononuclear heparin signals, or those of ethanol and methanol. Detailed NMR and ESI-MS/MS studies revealed a hitherto unknown contaminant, tris(2-n-butoxyethyl) phosphate (TBEP), which has potential health risks.
最近，在受污染的肝素制剂中发现了过度硫酸化的软骨素硫酸盐（OSCS），这些制剂与一些不良临床事件和死亡有关。此后，一些作者应用正交分析技术，即核磁共振（NMR）和毛细管电泳（CE），来评估肝素的纯度和安全性。NMR 可以鉴定和定量残留溶剂和非挥发性低分子污染物，其毒性的 USP 接受水平比传统的 GC-顶空气相色谱技术快 40 倍，后者需要大约 120 分钟才能获得信噪比至少为 1000/1 的（1）H NMR 谱。该方法可以检测到 2 ppm 级别的 Class 1 残留溶剂，并且可以在 10 ppm 以上进行定量。二维 NMR 技术（编辑-HSQC (1)H/(13)C）可以可视化否则被掩蔽的 EDTA 信号，其化学位移在 3.68/59.7 ppm 和 3.34/53.5 ppm，这些信号可能是单核肝素信号的重叠，或者是乙醇和甲醇的信号。详细的 NMR 和 ESI-MS/MS 研究揭示了一种以前未知的污染物，三（2-正丁氧基乙基）磷酸酯（TBEP），它具有潜在的健康风险。
