Techniques for Detection of Clinical Used Heparins

Heparins and sulfated polysaccharides have been recognized as effective clinical anticoagulants for several decades. Heparins exhibit heterogeneity depending on the sources. Meanwhile, the adverse effect in the clinical uses and the adulteration of oversulfated chondroitin sulfate (OSCS) in heparins develop additional attention to analyze the purity of heparins. This review starts with the description of the classification, anticoagulant mechanism, clinical application of heparins and focuses on the existing methods of heparin analysis and detection including traditional detection methods, as well as new methods using fluorescence or gold nanomaterials as probes. The in-depth understanding of these techniques for the analysis of heparins will lay a foundation for the further development of novel methods for the detection of heparins.

Preparation and Application of Nanosensor in Safeguarding Heparin Supply Chain

Heparin has been in clinical use as an anticoagulant for the last eight decades and used worldwide in more than 100 million medical procedures every year. This lifesaving drug is predominantly obtained from ~700 million pig intestines or bovine organs through millions of small and medium-sized slaughterhouses. However, the preparations from animal sources have raised many safety concerns, including the contamination of heparin with potential pathogens, proteins, and other impurities. In fact, contaminated heparin preparations caused 149 deaths in several countries, including the United States, Germany, and Japan in 2008, highlighting the need for implementing sensitive and simple analytical techniques to monitor and safeguard the heparin supply chain. The contaminant responsible for the adverse effects in 2008 was identified as oversulfated chondroitin sulfate (OSCS). We have developed a very sensitive, facile method of detecting OSCS in heparin lots using a nanosensor, a gold nanoparticle–heparin dye conjugate. The sensor is an excellent substrate for heparitinase enzyme, which cleaves the heparin polymer into smaller disaccharide fragments, and therefore facilitates recovery of fluorescence from the dye upon heparitinase treatment. However, the presence of OSCS results in diminished fluorescence recovery from the nanosensor upon heparitinase treatment, because OSCS inhibits the enzyme. The newly designed nanosensor can detect as low as 1 × 10–9% (w/w) OSCS, making it the most sensitive tool available to date for the detection of trace amounts of OSCS in pharmaceutical heparins. In this report, we describe a simple methodology for the preparation of nanosensor and its application in the detection of OSCS contaminants.

Crude Heparin Preparations Unveil the Presence of Structurally Diverse Oversulfated Contaminants

Nowadays, pharmaceutical heparin is purified from porcine and bovine intestinal mucosa. In the past decade there has been an ongoing concern about the safety of heparin, since in 2008, adverse effects associated with the presence of an oversulfated chondroitin sulfate (OSCS) were observed in preparations of pharmaceutical porcine heparin, which led to the death of patients, causing a global public health crisis. However, it has not been clarified whether OSCS has been added to the purified heparin preparation, or whether it has already been introduced during the production of the raw heparin. Using a combination of different analytical methods, we investigate both crude and final heparin products and we are able to demonstrate that the sulfated contaminants are intentionally introduced in the initial steps of heparin preparation. Furthermore, the results show that the oversulfated compounds are not structurally homogeneous. In addition, we show that these contaminants are able to bind to cells in using well known heparin binding sites. Together, the data highlights the importance of heparin quality control even at the initial stages of its production.

A “turn on” fluorescent probe for heparin and its oversulfated chondroitin sulfate contaminant

The conjugation of tetraphenylethene and a heparin binding peptide afforded a “turn on” fluorescent probe for reliable determination of heparin and its contaminant oversulfated chondroitin sulfate.