Concurrent separation of catecholamines, dihydroxyphenylglycol, vasoactive intestinal peptide, and neuropeptide Y in superfusate and tissue extract

A method is described for separation and quantification of 3,4-dihydroxyphenylglycol (DO-PEG), norepinephrine (NE), dopamine (DA), vasoactive intestinal peptide (VIP), and neuropeptide Y (NPY) from single samples of tissue homogenate and from superfusate from in vitro dog blood vessel preparations using cartridges containing 0.4 g of octadecylsilane (Sep-Pak C-18). Samples were passed through the cartridge at pH 7.4. A step-gradient system was used to first selectively desorb the catechols (DOPEG, NE, DA) with a moderately polar eluent; subsequently VIP and NPY were eluted with 2.5 ml of a mixture of 1% trifluoroacetic acid, 80% acetonitrile. Five Sep-Pak catechol eluents were tested. Catechols were quantified by HPLC with electrochemical detection and peptides by radioimmunoassay. An HPLC solvent system is described which is particularly useful for chromatography of the more hydrophilic catechols DOPEG, 3,4-dihydroxymandelic acid, and 3,4-dihydroxyphenylalanine concurrently with catecholamines. For superfusion studies, sample cleanup time was reduced to about 4 min per sample by attachment of the cartridges directly to the bottom of the superfusion chamber. Superfusate was subsequently pulled through the cartridges immediately after they were passed over the tissue. Batches of 12 high-speed tissue supernates were processed through the method in about 30 min. The method was used to analyze DOPEG, NE, DA, VIP, and NPY in various rat and dog tissues. The values obtained were similar to values obtained previously by other methods. Because the catechols and peptides are separated from a single sample, the method has several advantages over those described previously; e.g., it is rapid, simple, and more sensitive.