Safety And Quality Control Of Chitosan For Hemostatsis Usage

Chitin and chitosan are not only biocompatible and outstanding in hemostasis effect, but alsobiodegradable. Therefor they have been widely studied as hemostatic materials. However, present qualitycontrol methods are inadequate to ensure the safety and efficiency of chitosan. The adverse effect ofchitosan and other absorbable material were also reported recently. Researching on the safety andeffectiveness related quality factors of chitosan, and establishing the standard of chitosan for hemostasisusage is of great importance to ensure the quality of chitosan for hemostasis usage, and will provide a solidtheoretical basis for the research and application of chitosan as a hemostatic material.Protein residue could possibly cause allergy reaction and therefore affect the safety of chitosan forhemostasis usage. Meanwhile alkai extraction and other deproteinization methods were inefficient becausethe deproteinization was hetergenous reaction. The deproteinization could also induce the degradation ofchitsoan. Deproteinization of chitosan with immobilized enzyme was studied. Enzyme was immobilizedusing sol-gel method with tetramethoxysilane （TMOS） and3-aminopropyltriethoxysilane （APTES） assilanes for immobilization. The immobilization condition was studied by estimating the immobilizationefficiency, activity and protein selectivity. The optimum condition was immobilizing pepsin withTMOS:APTES （90:10）, with enzyme activity125.3%±14.3%to free enzyme and substrate selectivityincreased2.7times. Chitosan deproteinization with chitosan solution incubated with immobilized pepsin atpH4.5,45oC for160min. After the deproteinization with immobilized pepsin,53.8%-80.4%protein inchitosan was removed and the residue protein content lowered to0.12%⁰.34%, which was significantlysuperior to traditional NaOH deproteinization.As chitosan influenced the determination of protein residue in chitosan, in present workfluorenylmethyloxycarbonyl chloride （FMOC-Cl） derivation and HPLC method was studied to minimizethe influence of chitosan. The derivation of amino acids in large amount of glucosamine, chitosanhydrolysis product, and the chromatographic behavior of the derivation products were optimized and thedetermination of protein residue in chitosan was studied and established. The established method was goodin calibration （r>0.99）, precision （RSD1.6%-8.5%）, sensentive (LOQ1.9-10.9ng·mL^-1) and accurate（recovery84.4-98.6）. The method was also proved superior to Bradford method, LC-MS/MS method andelemental analysis.Virus in chitosan was another safety related factor of chitosan but few related study reported. Theinactivation of virus in chitosan with γ irradition or NaOH-ethanol incubation was evaluated and studied,with Porcine parvovirus （PPV） and Bovine viral diarrhea virus （BVDV） as indicators. Irradation ofchitosan wiht γ ray for5kGy, after incubation with8mol·L^-1NaOH consisting10%ethanol at35oC for1hour was proved effective in inactivation of virus in chitosan(ΔlgTCID₅₀>4.0) and only inducedmarginal degradation of chitosan for8.4%.Because the browning degradation of chitosan in acid hydrolysis, the recovery of chitoan inquantification was about90%in most reports, which was inadequate for quantification of chitosan inhemostasis application. The quantification of chitosan was studied by optimizing and evaluating thebrowning degradation of glucosamine and chitosan in hydrolysis with hydrochloric acid and phosphoricacid. Minimum brwoning degradation was proved with hydrochloric acid and phosphoric acid （4.5:1.5inmolar ratio） hydrolysis. Chitosan was quantified with hydrochloric acid and phosphoric acid （4.5:1.5inmolar ratio） hydrolysis and FMOC-Cl derivation HPLC determination. The method was linear at20-300μg·mL-（1r>0.99）and with good precision. The accuracy （recovery97.7%） was significantly superiorto the traditional HCl hydrolysis （recovery85.1%） and can be used for the quality control of chitosan forhemostatic usage.As lack proper standard of chitosan for hemostasis usage, the main quality related factors of chitosanwere evaluated, and the standard of chitosan was then proposed to ensure the quality control of chitosan forhemostasis usage, with the consult of current chitosan standard and the general requirements of implantmaterials.The present work studied on the deproteinization and purification of chitosan, which was significantlysuperior to NaOH deproteinization. Studied and established a sensitive and accurate pre-column derivationHPLC method for the determination of trace protein residue in chitosan. Studied on the HCl-H3PO4hydrolysis coupled pre-column derivation HPLC method for the quantification of chitosan, this improvedthe recovery of the method. These were meaninful to the total quality control of chitosan.