Characterization of currently marketed heparin products: composition analysis by 2D-NMR
Characterization of currently marketed heparin products: composition analysis by 2D-NMR
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DOI：
10.1039/c3ay40226f
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发表时间：
2013-01-01
影响因子：
3.1
通讯作者：
Al-Hakim, Ali
中科院分区：
文献类型：
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作者：
Keire, David A.;Buhse, Lucinda F.;Al-Hakim, Ali
每种类型的肝素在其核磁共振光谱中都有独特的信号模式，这是因为制造它们所用的特定过程化学反应的影响。因此，对于肝素钠，1D-H-1-核磁共振鉴定试验是美国药典(USP)和欧洲药典(EP)专著的一部分。以前的工作表明，二维(2D)核磁共振数据可以用于识别肝素类型，并用于计算肝素组成。因此，在这项研究中，我们应用二维H-1-C-13异核单量子相干(HSQC)光谱实验来表征2009年夏天在美国市场上上市的USP级样品中完整肝素钠或低分子肝素(LMWH)分子组成的正常变化。按美国药典USP肝素钠鉴别标准，对6家药厂的7个肝素钠有效成分(API)样品进行了2D法检测。此外，我们还检测了3家不同类型的低分子肝素原料药制造商的10份低分子肝素原料药样品和美国药典的依诺肝素鉴别标准。使用分配给特定肝素种类的HSQC交叉峰的积分体积，计算单糖或双糖的百分比组成。这些数据建立了供应美国市场的制造商之间2D分析中每种肝素类型的组成和正常变异性范围。从核磁共振测试获得的值与在同一样本集上对肝素消化进行质谱分析获得的值相似。通过改变共同添加的瞬变次数或松弛延迟，将采集时间从类似于3小时改变到类似于68小时，以测试该分析的稳健性；在这些变化中，获得的百分比组成值没有显着变化。我们的结论是，执行计算的方式最大限度地减少了由于光谱信噪比、肝素(1)J(CH)通过键耦合常数或弛豫时间的差异而引起的实验误差。
Each type of heparin has a unique pattern of signals in their NMR spectra because of the effects of the process specific chemical reactions used to manufacture them. Thus, for heparin sodium, 1D-H-1-NMR identification tests are part of the United States Pharmacopeia (USP) and European Pharmacopeia (EP) monographs. Previous work has shown that two-dimensional (2D) NMR data can be used to identify heparin types and be used to calculate heparin composition. Therefore, in this study, we applied a 2D H-1-C-13-heteronuclear single quantum coherence (HSQC) spectroscopy experiment to characterize the normal variation of intact heparin sodium or low molecular weight heparin (LMWH) molecular composition in USP grade sample lots which were available on the US market in the summer of 2009. We tested 7 heparin sodium active pharmaceutical ingredient (API) samples by the 2D method representing lots from 6 manufacturers and the USP heparin sodium identification standard. In addition, we tested 10 LMWH API samples from 3 manufactures of different types of LMWH and the USP enoxaparin identification standard. Using the integrated volumes of HSQC cross-peaks assigned to specific heparin species the monosaccharide or disaccharide percent compositions were calculated. These data establish the composition and normal range of variability for each of the heparin types in the 2D assay across manufacturers supplying the US market. The values obtained from the NMR test were similar to those obtained from mass spectrometric analysis of heparin digests on the same sample set. The robustness of the assay was tested by varying the acquisition time from similar to 3 to similar to 68 h by changing the number of transients co-added or the relaxation delay; across these changes the percent composition values obtained did not vary significantly. We conclude that the manner in which the calculations are performed minimizes experimental errors that arise from differences in spectral signal-to-noise, heparin (1)J(CH) through bond coupling constants or relaxation times.