High-performance liquid chromatographic analysis on radial compression column of the neurotoxic tri-o-cresyl phosphate and metabolites.

A method utilizing high-performance liquid chromatography (HPLC) has been developed for the analysis of tri-o-cresyl phosphate (TOCP) and its possible metabolites, o-cresyl dihydrogen phosphate, di-o-cresyl hydrogen phosphate, o-hydroxybenzyl alcohol, o-cresol, saligenin cyclic-o-tolyl phosphate [2-(o-cresyl)-4H-1:3:2:benzodioxaphosphoran-2-one], salicylic acid, salicylaldehyde, hydroxymethyl TOCP (di-o-cresyl o-hydroxymethylphenyl phosphate), and dihydroxymethyl TOCP (o-cresyl di-o-hydroxymethylphenyl phosphate). TOCP and its possible metabolites were analyzed on a reverse-phase C18 cartridge fitted into RCM-100 radial-compression separation system. Compounds were separated using a linear gradient of 25 to 80% acetonitrile in 2% aqueous acetic acid at a flow rate of 1.3 ml/min in a period of 22 min. Quantification was achieved by monitoring the ultraviolet absorbance of the column eluates at 254 nm and measuring peak areas. Retention times and peak areas were highly reproducible for all compounds analyzed. The relationship between peak area and amount injected was linear over a 100-fold range for all compounds analyzed. The minimum detectable level was 3 ng for salicylaldehyde, 25 ng for o-hydroxybenzyl alcohol and salicylic acid, and 50 ng for the remaining compounds. A mixture of TOCP and its possible metabolites was added to samples of cat liver, kidney, and plasma and then extracted and analyzed. High recovery and reproducibility for most compounds was observed in tissues analyzed.