Quantification of hyaluronic acid fragments in pharmaceutical formulations using LC-ESI-MS

Three different hyaluronic acid fragment preparations (HAF) derived from hyaluronic acid (HA) by hyaluronate lyase digestion have been investigated. The amount of these fragment mixtures in pharmaceutical formulations was determined by liquid chromatography-electrospray tandem mass spectrometry (LC/MS/MS). HAF analysis was performed in less than 8 min using a Nucleosil 100-7 C2 column. Based on the assumption that the mass distribution is kept constant, which is confirmed by the calibration results, quantification can be carried out relating to the most intense fragments. For that purpose, the ratios of the peak areas of product ions of m/z=378 (tetramer, hexamer, octamer) to the peak area of m/z=83 ([2xmaltose-H(+)], internal standard) were calculated. Calibration was done for each HAF and good linearity from 5 to 80 microg/ml has been shown. To evaluate the molecular weight distribution of the fragment preparations used in this approach MALDI-TOF, mass spectra have been collected.