Beyer T.,University of Würzburg |
Matz M.,TU Braunschweig |
Brinz D.,University of Würzburg |
Radler O.,Ratiopharm Group |
And 5 more authors.
European Journal of Pharmaceutical Sciences | Year: 2010
In 2008, some 900 cases of adverse events associated with the use of heparin were reported to the Food and Drug Administration of USA and the Federal Institute of Drugs and Medical Devices in Germany. 238 patients died from heparin in the USA. In March 2008, oversulfated chondroitin sulfate (OSCS) was identified to be responsible for these cases. NMR spectroscopic evaluation of heparin samples revealed OSCS, dermatan sulfate (DS), chondroitin sulfate A and C as well as various residual solvents to be present in heparin batches, which could not be identified by means of conventional methods described in various pharmacopoeias at that time. In order to evaluate the situation on the German market, 145 representative samples were collected in 2008 and analyzed by means of 1H NMR spectroscopy, water determination, optical rotation and sheep plasma clotting assay. 66 samples were found to contain pure heparin, 51 samples heparin plus DS, 5 samples heparin plus OSCS, and 23 samples heparin, DS and OSCS, each in varying amounts. In 94 out of 145 batches especially ethanol was found in strongly varying amounts up to about 9.5%. Traces of acetone and formic acid were found with concentrations up to 0.04%, as well as sodium acetate and methanol up to 0.5%. Additionally, in many batches the content of water was found to be relatively high. Whereas the optical rotation was able to identify samples with a high contamination of OCSC, all samples tested fulfilled the requirements of the anticoagulation potency assay of the European Pharmacopoeia 6.0. The presented analysis of a representative set of heparin samples proves the suitability of 1H NMR spectroscopy for the quality control of heparin of both glycosaminoglycans and residual solvents. © 2010 Elsevier B.V.
Alban S.,University of Kiel |
Luhn S.,University of Kiel |
Schiemann S.,University of Kiel |
Beyer T.,University of Würzburg |
And 7 more authors.
Analytical and Bioanalytical Chemistry | Year: 2011
The widespread occurrence of heparin contaminated with oversulfated chrondroitin sulfate (OSCS) in 2008 initiated a comprehensive revision process of the Pharmacopoeial heparin monographs and stimulated research in analytical techniques for the quality control of heparin. Here, a set of 177 heparin samples from the market in 2008 as well as pure heparin sodium spiked with defined amounts of OSCS and DS were used to evaluate established and novel methods for the quality control of heparin. Besides 1H nuclear magnetic resonance spectroscopy (NMR), the assessment included two further spectroscopic methods, i.e., attenuated total reflection-infrared spectroscopy (ATR-IR) and Raman spectroscopy, three coagulation assays, i.e., activated partial thromboplastin time (aPTT) performed with both sheep and human plasma and the prothrombin time (PT), and finally two novel purity assays, each consisting of an incubation step with heparinase I followed by either a fluorescence measurement (Inc-PolyH-assay) or by a chromogenic aXa-assay (Inc-aXa-assay). NMR was shown to allow not only sensitive detection, but also quantification of OSCS by using the peak-height method and a response factor determined by calibration. Chemometric evaluation of the NMR, ATR-IR, and Raman spectra by statistical classification techniques turned out to be best with NMR spectra concerning the detection of OSCS. The validity of the aPTT, the current EP assay, could be considerably improved by replacing the sheep plasma by human plasma. In this way, most of the contaminated heparin samples did not meet the novel potency limit of 180 IU/mg. However, also more than 50% of the uncontaminated samples had <180 IU/MG. In contrast to the aPTT, the PT specifically detects OSCS and other heparin mimetics (LOD 3%). About ten times more sensitive are both the Inc-PolyH-assay and the Inc-aXa-assay, two rapid and simple quantification assays for heparin mimetics. The determined OSCS contents of the heparin samples excellently correlated with those calculated from the NMR spectra. In conclusion, NMR proved to be the current spectroscopic method of choice. The two two-step-assays represent options to supplement NMR, especially as tests for the initial screening, since they detect any heparin mimetic without requiring special expertise for interpretation of the results. © 2010 Springer-Verlag.