Automated precolumn derivatization device to determine neurotransmitter and other amino acids by reversed-phase high-performance liquid chromatography.

A device to derivatize amino acids with o-phthaldialdehyde, which is directly connected to high-performance liquid chromatographic equipment is described. Its principle is that a sample (10-500 microliters) is mixed with a reagent (containing o-phthaldialdehyde, 2-mercaptoethanol and sodium hydrogen carbonate buffer), using a peristaltic pump. This mixture is pumped into a loop of a pneumatically controlled injection valve at atmospheric pressure. When the derivatization is complete the valve switches, so that the sample is applied to a column and the amino acid derivatives are separated with a gradient of methanol-phosphate buffers. The reproducibility is such that brain perfusates or tissue extracts can be analyzed for the amino acid transmitter content and no internal standard is necessary. The major advantages of the present device are that it produces thorough mixing of reagent and sample, so that a high and constant degree of derivatization occurs (thus producing high sensitivity; less than 0.1 pmol can be detected) and its low cost.