High Pressure Liquid Chromatographic Analysis of Barbiturates in the Picomole Range by Fluorometry of Their DANS-Derivatives

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Abstract
The sensitive determination of barbiturates is of special interest in the therapy of epilepsy and in barbiturate intoxications. The highly fluorescent DANS-derivatives of barbiturates have been obtained by refluxing ethyl acetate solutions with DANS-CI and solid potassium carbonate. A micro refluxer was used in case of blood samples. After direct injection of the reaction mixture the DANS-derivatives were separated by a reverse phase high pressure liquid chromatographic system and determined in the picomole range by fluorometry. The detection limit was 0.3–0.5 ng ml eluate. The determination of therapeutic levels (i.e. ∼1 ppm) of barbiturates in 20 µl blood samples is possible with this method in principle.