The determination of 5-hydroxytryptamine, related indolealkylamines and 5-hydroxyindoleacetic acid in the bovine eye by gas chromatography—negative ion chemical ionization mass spectrometry

Abstract

Methods were developed for the analysis of 5-hydroxytryptamine, related indolealkylamines (tryptamine, melatonin, 5-methoxytryptamine, N-acetyl-5-hydroxytryptamine and 6-hydroxymelatonin) and 5-hydroxyindole-3-acetic acid (5HIAA) in bovine retina, aqueous and vitreous humours. 5-Hydroxytryptamine and related indolealkylamines were extracted and derivatized to form their corresponding pentafluoropropionyl spirocyclic derivatives. 5HIAA was extracted and derivatized to the corresponding pentafluoropropionamide-trifluoroethyl derivative. Identification and quantitation by gas chromatography—negative ion chemical ionization mass spectrometry was made with reference to deuteriated internal standards. 5-Hydroxytryptamine was present in all ( n = 34) retinal samples analysed (20.53 ± 1.64 ng) while N-acetyl-5-hydroxytryptamine was detected in half of the samples of retina (0.06 ± 0.02 ng). Melatonin (0.15 ± 0.06 ng) and tryptamine (0.78 ± 0.34 ng) were detected in only a small number of retinas. 5-Methoxytryptamine was not present in retina. 5-Hydroxytryptamine was also present in aqueous (0.76 ± 0.17 ng ml −1) and vitreous (0.35 ± 0.05 ng ml −1) humours from bovine eye. Tryptamine, melatonin, 5-methoxytryptamine and N-acetyl-5-hydroxytryptamine were not detected in bovine aqueous and vitreous humours. 5HIAA was found in both bovine aqueous (2.03 ± 0.38 ng ml −1) and vitreous (0.65 ± 0.06 ng ml −1) humours, but its consistent determination in retina was obviated by interference from spurious peaks.