Online RPLC x HILIC – MS for the analysis of enzymatically-digested cross-linked hyaluronic acid hydrogels
Résumé
Hyaluronic acid is a glycosaminoglycan formed by a repetition of disaccharide units composed of a glucuronic acid and a N-acétyl-glucosamine. This naturally occurring polysaccharide in the body is widely used in some medical and aesthetic applications. The body fastly metabolizes hyaluronic acid. For this reason, it is necessary to cross-link it to improve the resistance to degradation and the mechanical properties of the resulting hydrogel. To better understand the interconnections of the polysaccharide chains with the crosslinker in the hydrogel, methods to characterize their structure are needed. For this purpose, a method of two-dimensional liquid chromatography coupled with mass spectrometry (LCxLC - MS) was developed. During hydrogel depolymerization, a complex mixture of highly polar and chemically related compounds is formed, including anionic derivatives of hyaluronic acid and zwitterionic fragments. The HILIC mode was chosen in order to separate compounds resulting from enzymatic digestion according to the polysaccharide chain length. The RP and cation exchange mixed mode were selected to separate the compounds according to the number of cross-linker molecules linked to the polysaccharide. Due to their high polarity, digested products are poorly soluble in acetonitrile, making it difficult to inject the sample directly into HILIC. In addition, the use of HILIC just prior to the detection improves the mass detection. Accordingly, the RP mode was used as the first dimension and the HILIC mode as the second dimension. After optimization of the different two-dimensional parameters, the method was applied for the comparison of hydrogels presenting different degrees of cross-linking.
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