Determination of bromazepam in plasma with an internal standard by gas-liquid chromatography

A gaschromatographic assay method is described for the determination of Ro 5-3350 (bromazepam) using Ro 5-4547 (methylbromazepam) as internal standard. After alkaline ether extraction the bromazepam and methylbromazepam obtained from sulfuric acid reextraction are hydrolyzed to ABBP and to MABBP, respectively. After neutralization, the bromo-pyridine-benzophenones are extracted with ether and dissolved in hexane after evaporation of the ether. Under the described gaschromatographic conditions it was found that MABBP and ABBP have retention times of 10.5 and 12.5 min, respectively. The limit of sensitivity is situated at 5 ng/ml of plasma. The specificity is satisfactory since the metabolite which might have interfered (hydroxy-3-bromazepam) appears only at very low concentrations in the blood. The linearity of the calibration curve was confirmed for plasma concentrations up to 100 ng/ml.