Analysis of Acidic Metabolites of Biogenic Amines in Bovine Retina and Vitreous and Aqueous Humour by Gas Chromatography‐Negative Ion Chemical Ionisation Mass Spectrometry

Abstract

Acidic metabolites of a number of biogenic amines have been identified and quantified by reaction with either acetic or propionic anhydride in the aqueous phase followed by extraction into ethyl acetate, esterification of carboxyl groups with ditrifluoromethylbenzyl bromide (DTFMBzBr), and then conversion of the remaining free hydroxyl groups to acetates. Subsequent analysis of these derivatives revealed that most (greater than 60%) of the ion current was carried by the ion resulting from the loss of DTFMBz from the molecular ion. This made the method highly specific and practical--limits of detection were established at approximately 200 pg with a potential limit of detection below the picogram level. This method establishes unequivocally that the metabolites of tyramine, dopamine, and adrenaline/noradrenaline (4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, and dihydroxymandelic acid, respectively) are present in bovine retina and in vitreous and aqueous humour. In addition, high concentrations of the dopamine metabolite homovanillic acid were found in retina and vitreous, but not in aqueous humour. p-Hydroxymandelic acid, the acidic metabolite of p-octopamine/p-synephrine, was identified in vitreous and in aqueous humour.

K+ and Cl‐ transport mechanisms in bovine pigment epithelium that could modulate subretinal space volume and composition.

“…Metabolites of adrenaline and dopamine have also been isolated in the bovine retina (Midgley, Watson, MacFarlane, MacFarlane & McGhee, 1990). It has been speculated that light-induced release of adrenaline or other catecholamines could provide a paracrine signal to the RPE in the bovine retina.…”

Determination of Acidic Metabolites of Biogenic Amines in Human Aqueous Humour by Gas Chromatography‐Negative Ion Chemical Ionisation Mass Spectrometry

“…Internal standards that were deuterated in the ring and side chain were prepared as described previously (Midgley et al, 1990).…”

“…Samples of aqueous humour were diluted with 1 ml of phosphate buffer ( 1 M, pH 7.2); the appropriate deuterated internal standards (10 ng of each) were added. Extraction and derivatisation were then carried out as described previously (Midgley et al, 1990), yielding the acids as their acetyl/propyl/ditrifluoromethylbenzyl (DTFMBz) derivatives. DTFMBz/trimethylsilyl derivatives of several samples were also prepared by direct extraction of the acids from the acidified aqueous or vitreous humour followed by formation of a DTFMBz derivative as described previously (Midgley et al, 1990) and then reaction with N,O-bis(trimethy1-sily1)acetamide to form trimethylsilyl derivatives of the free hydroxyl groups.…”

“…GC-NICIMS was camed out using a Hewlett-Packard model 5988A gas chromatograph-mass spectrometer interfaced with an HP RTEd/VM data system. The instrument conditions previously (Midgley et al, 1990).…”

“…In previous studies we have determined the concentration of biogenic amines and their metabolites in bovine retina and vitreous and aqueous humour (Macfarlane et al, 1989;Midgley et al, 1990). We have also reported preliminary results on the determination of levels of acidic metabolites of biogenic amines in human aqueous humour (Watson et al, 1989).…”