Comprehensive Comparisons between 1-Phenyl-3-methyl-5-pyrazolones, 1-(4-Methoxyphenyl)-3-methyl-5-pyrazolones and 1-(2-Naphthyl)-3-methyl-5-pyrazolones as Labeling Reagents Used in LC-DAD-ESI-MS-MS Analysis of Neutral Aldoses and Uronic Acids

Comprehensive comparisons between three 1-aryl-3-methyl-5-pyrazolone (AMP) labeling reagents were carried out for the analysis of reductive monosaccharides using reversed phase high performance liquid chromatography diode array detection coupled to electrospray ionization mass spectrometry. AMP derivatives included 1-phenyl-3-methyl-5-pyrazolone (PMP), 1-(4-methoxyphenyl)-3-methyl-5-pyrazolone (PMPMP) and 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP). The separation of AMP-monosaccharides was found to be pH-dependent under reversed phase conditions and acceptable separations were obtained at pH < 4.5. The elution orders of AMP-aldoses were rationalized by geometric factors involved in the presence of hydroxyl groups at C2 and C3 positions of the saccharide moiety. When PMP or PMPMP were used as labeling agents Glucose and galactose were completely separated, while arabinose and xylose were observed to co-elute. The use of NMP revealed that arabinose and xylose could be separated while glucose and galactose were co-eluted. MS-MS data of AMP-monosaccharides gave characteristic fragment ions resulting from cleavage between the C2–C3 bond (m/z 373 for PMP derivatives, m/z 433 for PMPMP derivatives, and m/z 473 for NMP derivatives). Bond breakage between C1–C2 (m/z 359 for PMP derivatives, m/z 419 for PMPMP derivatives, and m/z 459 for NMP derivatives) of the saccharide moiety was also found to be characteristic for AMP-saccharides. Application to the analysis of hydrolyzed rape pollen polysaccharides revealed the presence of four unexpected monosaccharides, namely ribose, erythrose, threose and glyceraldehyde.