Determination of bromazepam in plasma by quantitative thin-layer chromatography

Summary

A method for determination of bromazepam (I) in plasma on thin-layer plates is described, which is highly specific and sensitive. After extraction from plasma, bromazepam is separated from interfering constituents by thin-layer chromatography. The quantitation of the spots can be performed in two different ways. Either it is possible to measure directly the UV reflectance on the plate, or bromazepam is hydrolyzed to 2-amino-5-bromobenzoyl-pyridine (ABBP, II) on the thin-layer plate, diazotized and coupled with N-(1-naphthyl) ethylenediamine to form an azo-dye, which can be evaluated densitometrically. The recovery from plasma was found to be above 90%, the sensitivity of both methods was about 5 ng of bromazepam per cm³ of plasma using 0.5 cm³. The relative standard deviation of both procedures was ±6%. In a series of plasma samples the concentration of bromazepam was determined by the thin-layer procedures described and by GLC with an electron capture detector. The different methods gave similar results, but the thin-layer chromatographic methods are easier to perform.