Thin-layer chromatographic method for the quantitative analysis of nalidixic acid in human plasma

A sensitive and highly selective thin-layer chromatographic method for determining plasma levels of nalidixic acid is presented. Plasma (50 microliter) was acidified with 50 microliter M orthophosphoric acid and extracted with 100 microliter toluene. A 40-microliter aliquot of the extract was spotted onto the thin-layer plate with the aid of a Desaga Autospotter and, after irrigation, the nalidixic acid on the plate was converted into a fluorescent compound by exposing the plates to hydrogen chloride gas for 10 min and then to strong ultraviolet radiation from a mercury lamp for 10 min. The fluorescence was measured quantitatively using a spectrofluorimeter equipped with a thin-layer chromatogram scanning attachment.