Characterization of Hydrogel Drug Delivery System Stability Using Column-Switching Multimodal Liquid Chromatography–Mass Spectrometry

Drug delivery systems (DDSs) have demonstrated effectiveness in enhancing bioavailability, controlled drug release, and targeted drug delivery and minimizing toxicity. However, DDS components can vary greatly, requiring numerous analytical methods for an exhaustive characterization protocol. Herein, we evaluated the stability of a hyaluronic acid-based hydrogel DDS under forced alkaline and enzymatic <i>in vitro</i> degradation. To consolidate the analyses and maximize data collection from a single injection, a column-switching multimodal liquid chromatograph (LC) was configured with two sets of binary pumps and a two-position, six-port valve, which enabled a multipath separation. Restricted access medium (RAM) was used to trap small, relatively hydrophobic analytes while directing larger species to a size-exclusion (SEC) column for size determination. Captured molecules were subsequently back eluted toward a reversed phase (RP) column with mass spectrometric detection. The optimized multimodal LC method separated an array of analytes relevant to DDS. Representative analytes for small drug molecules, process impurities, bioconjugation linkers, stealth polymers, and small proteins were observed in the RP channel. The active therapeutic, polymeric carrier, and its associated degradation products were observed in the SEC channel. Hydrogel degradation kinetics were monitored with the instrumental setup, revealing distinct degradation profiles during enzymatic digestion and alkaline hydrolysis of the gel; the active therapeutic was present at all time-points. Samples were spiked with acetaminophen for system suitability, which was observed at all time-points, demonstrating efficient trapping throughout the extent of the study. From this, the multimodal configuration proved to be a viable means to simultaneously analyze vastly different analytes comprising a DDS.