Determination of Hyaluronic Acid in a Chitosan-Based Formulation by RP C18 and HILIC LC–ESI-MS: an Evaluation of Matrix Effect

Abstract

Two simple and fast C18 and HILIC liquid chromatography–electrospray mass spectrometry methods for the determination of hyaluronic acid (HA) in a mucoadhesive chitosan-based formulation were developed and validated. The performances of both methods were compared in terms of validation parameters and matrix effect. A simple sample preparation method based on sulphuric acid-based degradation was optimized for the detection of HA fragments (i.e. m/z 380 2-mer, m/z 759 4-mer, m/z 1,138 8-mer and m/z 1,518 16-mer). By operating under selected ion-monitoring mode, excellent selectivity towards chitosan fragments was obtained. For validation, good linearity, detection limits (<4 μg mL⁻¹) and precision (RSD % < 16 %) were generally obtained on matrix with both columns. However, HILIC column exhibited improved performances in terms of HA fragment separation and selectivity. By analyzing on the C18 column the chitosan-based formulation and sample extracts from pig mucosa treated with the formulation, matrix effects exhibited a dependence of signal suppression degree (ranging from 37 to 83 %) as a function of the HA fragment dimension. The HILIC column afforded instead a significantly reduced suppression degree (ranging from 1 to 16 %) and a better separation. These findings demonstrated the improved performances of the HILIC column with respect to conventional C18 mechanism for the analysis of HA fragments in complex matrices.