Specific and Sensitive High Performance Liquid Chromatographic Determination of Alprazolam or Triazolam

A specific and highly sensitive liquid chromatographic procedure has been developed for the rapid determination of intact alprazolam or triazolam in dog serum, using these structurally similar triazolobenzodiazepines as mutual internal standards. The procedure consists of (1) extracting one ml of alkali buffered serum with toluene, (2) evaporating an aliquot of the toluene to dryness, and (3) quantitating the redissolved residue by HPLC using ultraviolet detection (221 nm). Samples were chromatographed on a microparticulate reverse phase column using a mobile phase composed of acetonitrile: isopropanol: water (94:5:1) and a flow rate of 0.75 ml/min. Metabolites of alprazolam and triazolam did not interfere in the assay. The lower limit of detection was approximately 1 ng/ml of serum extracted. The utility of the analytical methodology for the determination of alprazolam or triazolam in pharmacokinetic studies in the dog was demonstrated.