To establish an HPLC analysis method for the determination of diazepam，nordiazepam and oxazepam in hair samples.

The determination of drug content and related substances was performed by high performance liquid chromatography (HPLC). Ultrasonic method combined with liquid-liquid extraction method was used as the pretreatment method. The chromatographic column was Athena C₁₈-WP column (250 mm×4.6 mm，5 μm)；the mobile phase was the mixture of methanol：0.02 mol·L^-1 sodium dihydrogen phosphate (adjusted to pH 3.4 with phosphoric acid)：acetonitrile (30:43:27，v/v/v) in an isometric elution；the column temperature was 50 ℃；the flow rate was 0.7 mL·min^-1；the running time was 30 min；the injection volume was 20 μL. The detection wavelength was 254 nm. The internal standard was clonazepam.

The linearity of all the analytes were good in the concentration range of 0.1 to 25 ng·mg^-1（r>0.995）. The limit of quantitative was 0.1 ng·mg^-1 and the limit of detection was 0.05 ng·mg^-1. The intra-batch accuracies were 88.2%-103.9% (n=6) and the inter-batch accuracies were 88.5%-106.2% with imprecisions ≤9.19%. The recoveries were stable for three anlytes，which met the requirement of methodology. The present method was applied to the authetic hair samples，in which the concentrations of diazepam，norazepam and oxazepam were (0.307±0.016) ng·mg^-1，(0.244±0.012) ng·mg^-1 and (0.478±0.053) ng·mg^-1 respectively. Besides，C_norazepam/C_diazepam was 0.795，which was in consistent with the literature reported.

The established HPLC method is fast and easy to operate，with good reproducibility，applicability，and reliability.