Liquid chromatographic assay of dityrosine in human cerebrospinal fluid

A micro-scale method for separation and measurement of dityrosine in human cerebrospinal fluid (CSF) is described utilizing liquid-liquid extraction and ion-paired, reversed-phase high-performance liquid chromatography with fluorimetric detection. A mobile phase containing 1-heptanesulfonic acid linearly increased in methanol from 0 to 100% over 30 min allows the resolution of dityrosine from other fluorescent compounds with excitation at 285 nm and emission at 410 nm. As little as 0.15 ml CSF sample can be utilized with a detection limit of 60 pg dityrosine on the column. This method facilitates the use of CSF dityrosine as a measure of free radical mediated protein damage in the central nervous system.