Division of Pharmaceutical Analysis, Food and Drug Administration, CDER, St. Louis, Missouri 63101, USA.
Anal Chem. 2010 Dec 1;82(23):9865-70. doi: 10.1021/ac102301j. Epub 2010 Nov 11.
Here we report results from the analyses by enzymatic digestion and reversed-phase ion-pairing liquid chromatography mass spectrometry (RPIP-LC-MS) of active pharmaceutical ingredient (API) unfractionated heparins (UFHs) from six different manufacturers and one USP standard sample. We employed a reverse phase ion-pairing chromatography method using a C(18) column and hexylamine as the ion-pairing reagent with acetonitrile gradient elution to separate disaccharides generated from the digestion of the heparins by lyase I and III (E.C. 4.2.2.7 and 4.2.2.8) before introduction into an ion-trap mass spectrometer by an electrospray ionization (ESI) interface. Extracted ion chromatograms (EICs) were used to determine the relative abundance of the disaccharides by mass spectrometry. Eight disaccharides were observed and a similar composition profile was observed from digests of 20 UFH samples. The compositional profile determined from these experiments provides a measure of the norm and range of variation in "good" heparin to which future preparations can be compared. Furthermore, the profile obtained in the RPIP-LC-MS assay is sensitive to the presence of the contaminant, oversulfated chondroitin sulfate A (OSCS), in heparin.
在这里，我们报告了通过酶消化和反相离子对液相色谱-质谱联用（RPIP-LC-MS）分析来自六个不同制造商和一个 USP 标准样品的未分级肝素（UFH）的活性药物成分（API）的结果。我们采用了反相离子对色谱法，使用 C（18）柱和己胺作为离子对试剂，采用乙腈梯度洗脱，在将肝素通过裂解酶 I 和 III（E.C. 4.2.2.7 和 4.2.2.8）消化生成的二糖引入离子阱质谱仪之前，在电喷雾电离（ESI）接口之前。提取离子色谱图（EIC）用于通过质谱法确定二糖的相对丰度。观察到八种二糖，并且从 20 个 UFH 样品的消化物中观察到相似的组成谱。这些实验确定的组成谱提供了“良好”肝素的标准和变化范围的衡量标准，未来的制剂可以与之进行比较。此外，RPIP-LC-MS 测定中获得的谱图对肝素中存在的污染物，过度硫酸化软骨素 A（OSCS）敏感。
