A salt-free isocratic high-pressure liquid chromatographic method for the quantitation of benzodiazepines in serum

A simple, rapid high-pressure liquid chromatography (HPLC) method was developed for detecting and quantifying benzodiazepines in serum. Seven major benzodiazepines were extracted from spiked serum samples using solid-phase extraction with prazepam as the internal standard. The eluted drugs were then resolved isocratically by HPLC within 11 min using a reversed-phase C8 column with a mobile phase consisting of acetonitrile, methanol, water, and perchloric acid. All drugs gave responses that varied linearly with concentration over the ranges studied. Within-day imprecision (CV) varied from 3.9 to 14.9%, day-to-day CV from 4.8 to 17.0%, absolute recoveries from 67% to 114%, and detection limits from 10 to 110 ng/mL. Tricyclic antidepressants did not interfere, and clinical results were in good agreement with those obtained by a gas chromatographic method. The advantage of this method is that it uses a salt-free isocratic mobile phase that can be easily manipulated to effect difficult benzodiazepine separations.