Determination of nanogram amounts of aromatic compounds by spectrophotometry on thin-layer chromatograms

A highly sensitive method is described for the simultaneous determination of codeine and chlorpheniramine in plasma. Thin-layer chromatography is used for the separation of the drugs. The spots are then rendered visible by nitration of the substances on the thin-layer plate. Codeine can be quantified by direct measurement of the resulting fluorescence. After reduction, the aromatic amines are diazotized and coupled with N-(1-naphthyl)ethylenediamine on the thin-layer plate. For codeine the fluorimetric measurement is more reliable than the colorimetric determination. The sensitivity limits are 8 ng/ml of plasma for codeine phosphate and 1-2 ng/ml of plasma for chlorpheniramine maleate. This procedure is also applicable to other aromatic compounds which can be nitrated by the described method. The method has been applied to compare a determination of the plasma levels of codeine and chlorpheniramine after administration in normal capsules and retard tablets.