Trace analysis of diazepam in serum using microbore high-performance liquid chromatography and on-line...

The feasibility of determining trace analytes in human serum using on-line preconcentration and microbore high-performance liquid chromatography has been demonstrated. The serum is subjected to ultracentrifugation and injected onto a hydrophobic pre-column using water as the mobile phase. This traps the components of interest which are then backflushed onto a microbore analytical column using a stronger mobile phase. The column-switching apparatus was evaluated using highly dilute aqueous paraben solutions and sample enrichment factors as high as 1500 were obtained. The procedure was then applied to diazepam in serum. Recovery was linear and quantitative over the range from at least 4 to 1000 ng/ml. The method was specific against caffeine and the three major metabolites of diazepam: oxazepam, temazepam, and nordiazepam. The effects of varying pre-column dimensions, pre-column loading time, and sample volume were evaluated.