Sample Preparation for Headspace GC Analysis of Residual Solvents in Hyaluronic Acid Derivative Fiber

Abstract

Hyaluronic acid (HA) is a linear polysaccharide built from repeating disaccharides of alternating D-glucuronic acid and N-acetyl-D-glucosamine. HA can be isolated from biological sources such as vitreous humor, human umbilical cord and rooster and hen comb. HA has biocompatibility and its average molecular weight is usually several million. HA is very viscous in aqueous solution because of its molecular conformation. Due to its viscoelastic properties, HA is used in the medical applications as ophthalmology, dermatology, osteoarthritis, urology and wound healing. HA derivative has an improved rheological property without losing natural biocompatibility of HA. Because HA derivative is water-insoluble or slowly dispersible in human body, it lasts much longer than HA in human body. Therefore, HA derivatives have been widely developed as post-operative adhesion-preventing films or gels, materials for wrinkle treatment, materials for soft tissue augmentation, materials for the arthritis treatment, vehicles for drug delivery, etc. In this study, a HA derivative fiber is obtained from amide reaction of chitosan and HA using N(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) hydrochloride and N-hydroxysuccinimide (NHS). HA derivative fiber is turned into HA derivative gel in water. Headspace gas chromatography (HS-GC) method has been used for the determination of residual solvents in pharmaceutical compounds. Direct injection of analytes evaporated through equilibration between liquid (or solid) phase and gas phase to GC system minimized the contamination of GC system and the deterioration of GC column. In addition, the automation of equilibrium and injection procedure reduced analysis time and improved reproducibility in injection procedure. In this study, HA derivative fiber was hydrolyzed by hyaluronidase (HAse) in buffer solution for the minimization of variation in the viscosity of HA derivative fiber in water resulting in the higher extraction of residual solvents from the gel matrix. HAse is an enzyme that randomly hydrolyzes the linkage between the N-acetyl-D-glucosamine and the D-glucuronate residues in HA. The hydrolysis rate is dependent on pH and concentration of HAse. In this study viscosimetric assay was applied because it was a simple and rapid method for the measurement of activity of HAse. The aim of this study is to develop efficient sample preparation method for HS-GC analysis of residual solvents in HA derivative fiber. Compared to direct extraction of residual solvents from HA derivative fiber, the extraction through the hydrolysis of HA derivative fiber by HAse gave more complete and higher reproducible quantification of residual solvent. To validate HS-GC analysis method of residual solvents, specificity, limits of detection and quantification, linearity, accuracy and precision are investigated in the study.