Effects of molecular oxygen on multiphoton-excited photochemical analysis of hydroxyindoles

We have examined the effects of dissolved molecular oxygen on multiphoton-e xcited (MPE) photochemical derivatization of serotonin (5HT) and related ce llular metabolites in various buffer systems and find that oxygen has a pro found effect on the formation efficiency of visible-emitting photoproducts. Previously, end-column MPE photoderivatization provided low mass detection limits for capillary electrophoretic analysis of hydroxyindoles, but relie d on the use of Good's buffers to generate high-sensitivity visible signal. In the present studies, visible emission from 5HT photoderivatized in diff erent buffers varied by 20-fold under ambient oxygen levels but less than 2 -fold in the absence of oxygen; oxygen did not significantly alter the phot oproduct excited-state lifetime (similar to 0.8 ns), These results support a model in which oxygen interferes with formation of visible-emitting photo products by quenching a reaction intermediate, an effect that can be suppre ssed by buffer molecules. Deoxygenation of capillary electrophoresis separa tion buffers improves mass detection limits for 5-hydroxyindoles fractionat ed in 600-nm channels by approximately 2-fold to less than or equal to 30 0 00 molecules and provides new flexibility in identifying separation conditi ons for resolving 5HT from molecules with similar electrophoretic mobilitie s, such as the catecholamine neurotransmitters.