LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY OF CONJUGATES AND OXIDATIVE METABOLITES OF INDOLE-3-ACETIC-ACID

Indole-3-acetic acid and some of its hydroxylated derivatives and amide and ester-linked conjugates were analysed by high-performance liquid chromatography/mass spectrometry (HPLC/MS). The effluent from a capillary reversed-phase HPLC column was introduced into the ion source of a double-focusing mass spectrometer via a frit interface, and ions were generated by fast atom bombardment with 1% glycerol in the mobile phase acting as a matrix. Full-scan positive ion mass spectra were obtained with 1-2 ng of non-polar and 100 ng of polar indoles. The spectra of both amide and ester-linked indole-3-acetic acid conjugates contained a dominant molecular ion, glycerol adducts of the molecular ion and an m/z 130 quinolinium ion. The spectra of oxidized indole-3-acetic acid derivatives, such as oxindole-3-acetic acid and 5-hydroxy-indole-3-acetic acid, were characterized by the presence of a strong response at m/z 146, representing an oxygenated quinolinium ion, and the absence of an ion at m/z 130. Further oxidation products of oxindole-3-acetic acid, such as 7-hydroxy-oxindole-3-acetic acid, yielded spectra containing an intense m/z 162 representing a hydroxy-2-oxoquinolonium ion. The HPLC/MS system was used to identify indole-3-acetylalanine in a purified extract from elongating shoots of Picea abies. The structure elucidation was based on the positive ion fast atom bombardment spectrum, daughter ion spectra and an accurate mass determination by high-resolution mass spectrometry.