Picomole analyses of tryptophan by derivatization to 9-hydroxymethyl-beta-carboline

A new method specific for the determination of subpicomole quantities of tryptophan has been developed by elaboration of the Pictet-Spengler reaction. It permitted reproducible quantitation of tryptophan in less than 1 microliter of plasma ultrafiltrate or 1 mg of brain tissue. Samples deproteinized by trichloroacetic acid were boiled for 15 min with formaldehyde and potassium ferricyanide at controlled acidity, where tryptophan was converted to a single new product identified as 9-hydroxymethyl-beta-carboline. It was quantitated by either direct spectrofluorometry or a reversed-phase HPLC system developed for beta-carbolines. Under our conditions, peptides containing N-terminal tryptophan such as Trp-Leu and delta sleep-inducing peptide gave N-(9-hydroxymethyl-beta-carboline-3-carbonyl) peptides which retained all amino acid residues except tryptophan.