Rapid monitoring of benzodiazepines in clinical samples by using on-line column switching HPLC

We have determined routinely administered benzodiazepines and some of their metabolites in clinical samples by using on-line column switching for sample clean-up and reversed-phase high-pressure liquid chromatography (HPLC). The plasma sample is directly injected onto a cyanopropyl (CN) pre-column, washed with buffer, and eluted onto an analytical column (C18, octadecylsilane). Analytical recovery from drug-supplemented plasma samples was greater than 85% for all the benzodiazepines tested. The response of the analytical column varied linearly with drug concentrations over the range 0.01-1 mg/L in plasma, with CVs ranging from 0.3% to 1.8% within-day and 5.5% to 15.7% between days. The detection limit (signal-to-noise ratio greater than 3) was 0.01 mg/L (1 ng on column) for all of the benzodiazepines and their metabolites. The advantages of the technique include the extreme cleanliness of the traces, no requirement for off-line sample preparation, and ease of automation.