Monitoring the Chemical Hydroxylation of Complex Phenolic Compounds
thesisposted on 23.05.2021, 17:25 by Wagday Mohammed Samrgandi
It might be industrially or bio-medically important to confirm and monitor the hydroxylation of phenolic amine substrates via mass spectrometry. Phenolic amines may be assayed by colorimetric reactions, liquid chromatography (LC) or thin layer chromatography (TLC), spectrophotometry (UV VIS) or other methods that may not confirm the product molecule with reasonable specificity. Phenolic amine compounds may easily enter the gas phase by electrospray ionization (ESI) and the compounds parent and subsequent fragment ions examined by tandem mass spectrometry (MS/MS). Thus a number of phenolic amine or other reaction products might be monitored and confirmed by liquid chromatography with electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS). L-tyrosine was reacted with dihydroxyfumaric acid (DHFA) at 0 °C in the presence of bubbling O2 in 400 mL flask respectively or ≥ 100 μL volume in a 96 well plate in an oxygen atmosphere resulting in the product L-DOPA (L-3,4 dihydroxyphenylalanine). The production of L-DOPA was examined with nitrite-molybdate in 0.5 M HCl followed the addition of 1 M of NaOH to form a red color quantified by absorbance at 510 nm. Thin layer chromatography with staining for amines by ninhydrin was used to detect the production of LDOPA. LC-ESI-MS/MS confirmed the molecular identity of the L-DOPA product with a parent ion predominately observed at an m/z value of 198 [M+1H] and the major fragment ions at 181m/z and 151m/z. Monitoring the 181 m/z fragment ion permitted the quantification of L-DOPA over time to ≤1 pM in the reaction vessel with respect to external standards. The hydroxylation of tyrosine was observed to require O2 and DHFA and produced a strong yield at pH 2 but was not dependent on Horseradish peroxidase (HRP) and proceeded in the presence of EDTA. The hydroxylation reaction of tyrosine was depending on DHFA, oxygen and acid (DOA).We conclude that DOA hydroxylation by LC-ESI-MS/MS may be directly applicable to monitoring the industrial modification of a wide class of phenolic amines.