A specific radioimmunoassay for melatonin in biological tissue and fluids and its validation by gas chromatography-mass spectrometry.

A specific practical radioimmunoassay suited to determinations of melatonin in both tissues and body fluids is described. The rabbit antibody employed was raised to an antigen formed by condensation between N-acetylserotonin and the Mannich adduct of bovine serum albumin and formaldehyde. Substitution was shown by protonmagnetic resonance spectroscopy to occur exclusively at die 4 position of the indole nucleus. The antibody reacted with a variety of N-acetylated indoles, and absolute specificity was dependent upon the extraction procedure and column (Lipidex 5000) chromatography. In addition to the usual reliability criteria, the validity of the assay was checked by gas chromatography- mass spectrometry using [2H3]melatonin as an internal standard, the preparation of which is described. The occurrence of melatonin in the plasma of man, sheep, rat and chicken was confirmed, and its presence in the plasma of the pig (22–76 pg/ml), donkey (24–128 pg/ml), cow (20–320 pg/ml), camel (29–221 pg/ml) and a scinci...