A fast and robust hydrophilic interaction liquid chromatography tandem mass spectrometry method for determining methylpentose, hexose, hexosamine and hexonic acid in pneumococcal polysaccharide vaccine hydrolysates

The conventional UV/Vis spectroscopy methods recommended by the European Pharmacopoeia (EP) for determining hexosamine, hexonic acid and methylpentose in pneumococcal polysaccharide vaccine (PPSV) hydrolates are time-consuming due to derivatization process (typically, an analysis cycle is more than 4 h) and improvements of selectivity and precision of the methods are in demand. In this study, a new approach based on hydrophilic interaction liquid chromatography and triple quadrupole mass spectrometry (HILIC-MS/MS) was optimized to overcome the drawbacks of the EP methods for simultaneous determination of methylpentose, hexose, hexosamine and hexonic acid in PPSV hydrolysates. The chromatographic, MS and sample hydrolysis conditions were systematically investigated. A zwitterionic column, Click Cys, using a gradient elution with a mobile phase of 10 mM ammonium formate (pH 4.3) in acetonitrile from 72% to 21% in 6 min was applied for separating the targets, which exhibited low column bleeding, easy equilibration and long-term stability. The HILIC-MS/MS method showed a high sensitivity (LOD = 0.98 μg L−1 for hexonic acid), a good repeatability (RSD of peak area less than 1.669%), accuracy (92.9%-104.2%), recovery (97.6%-99.3%) and a wide linear range. The RSD of retention time obtained from more than 3000 injections in three months was less than 1.64%. The new method was compared with the EP method for determining hexosamine in 23 serotypes of PPSV hydrolysates. The results indicated that the new HILIC-MS/MS method was highly selective, accurate, stable and extremely fast due to without need of derivatization, as compared to the conventional EP methods.