A high-performance liquid chromatography–diode array detector method (HPLC-DAD) was developed and
validated for the determination of tramadol in human saliva. Samples of saliva were prepared utilizing liquid–liquid
extraction with hexane-ethyl acetate (4:1, v/v). Propranolol was used as an internal standard (IS). The linear range
and average recovery of tramadol were 0.25–4.00 μg/mL and 94.70%, respectively. The intra- and inter-assay
precisions at low, intermediate and high concentrations were within 1.40–9.79 % and 0.77–3.98%, respectively.
Intra- and inter-assay accuracies were within 98.50–107.13% and 100.44–103.89%, respectively. The proposed
method was successfully applied to determine tramadol in saliva concentrations in three healthy volunteers for 24
hours after administration of 100 mg oral doses of tramadol.