Direct injection HPLC method for the determination of selected benzodiazepines in plasma using a Hisep column

A direct plasma injection HPLC method has been developed for the determination of selected benzodiazepines (nitrazepam, clobazam, oxazepam, lorazepam). The method uses an analytical hydrophobic shielded phase (Hisep) column equipped with a Hisep guard column, are easy to perform and requires 20 ul of a filtered plasma sample. The chromatographic run time is less than 15 min using a mobile phase of 15:85 v/v acetonitrile-0.18 M ammonium acetate pH 2.5. The method is good for 175 injections before replacement of the guard column. The method was linear in the range 0.5-18 ug ml(-1) (r>0.99, n=6) for the analytes with R.S.D. less than 10.82%. Interday and intraday variability were found to be less than 14%. The limits of detection and quantitation were 0.16 (s/n>3) and 0.5 ug ml(-1) (s/n>10), respectively, for each of the four benzodiazepines.