  Heparin is an important anticoagulant drug, about one billion doses are
produced annually. It is a polydisperse sulfated polysaccharide, and the
inherent heterogeneity makes the analysis of heparin difficult. The global
crisis resulting from adulterated heparin in 2008 has drawn renewed attention
to the challenges that are associated with the quality control and
characterization of this complex biological medicine from natural sources. The
present study addresses the need for simple and user-friendly analytical
methods for the fast and accurate quantification of heparin in complex
matrices. Direct quantification of heparin in the low microgram per mL range
was accomplished using a specific commercially available assay based on the
fluorescent molecular probe Heparin Red, simply by mixing the heparin
containing sample and a reagent solution in a 96-well microplate followed by
fluorescence readout. A screening of typical impurities in raw heparin
(selected other glycosaminoglycans, residual nucleic acids and proteins),
related to the extraction from animal tissues, as well as of components of the
urine matrix (inorganic salts, amino acids, trace proteins) revealed that these
compounds even in large excess have no or very little effect on the accuracy of
heparin determination. Heparin spike detection in urine, a biological
multicomponent matrix, also showed good accuracy. We envision applications of
this mix-and-read assay in the process and quality control in heparin
manufacturing, but also in pharmacokinetic studies as a convenient tool for
measuring of the urinary excretion of heparins.
