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Abstract
High-pressure liquid chromatography was used to separate the fluorescent adduct formed from the reaction of histamine with o-phthalaldehyde (OPT) from other biogenic amines in tissue, cerebrospinal fluid (CSF), sweat and urine samples. Using off-column derivatization and isocratic elution techniques fluorescent OPT adducts can be detected in the low picogram range. Perchloric acid extracts of tissue samples from Aplysia california and urine specimens collected from healthy adult males, including internal standard, were derivatized with OPT buffer, pH 9.5 and extracted with ethylacetate to increase sensitivity and stabilization of the fluorescent adduct prior to chromatography. Sweat and CSF samples were reacted with OPT buffer and aliquots of this mixture injected directly onto the chromatographic column (μBondapak CN) with methanol/0.08 mol/liter acetic acid (52/48 by volume) as the mobile phase. Assay of pooled urine containing added histamine (1 μg/ml) gave a with-in run coefficient of variation of 2.5%. The use of o-phthalaldehyde as an off-column HPLC derivatization agent for fluorometric determination of low-levels of biogenic amines is rapid, sensitive and easily adapted to routine use in a clinical or neurobiological laboratory.