Determination of Tramadol in Human Plasma by HPLC with Fluorescence Detection

Tramadol is a centrally acting analgesic, atypical opioid, and although it is generally considered as a medicinal drug with a low potential for dependence, there is growing evidence of tramadol abuse in some countries. The ultraviolet detection is not suitable for analysis of tramadol in plasma, due to the lack of sensitivity and selectivity. However, it was shown that tramadol has a weak fluorescence, and the latest techniques for determination of tramadol in plasma include liquid chromatographic methods with fluorescence detection (FL). The objective of the paper was to develop a HPLC-FL method applicable for quantification of tramadol in human plasma.The separation was achieved by reverse phase HPLC method, using as stationary phase C18 – Kromasil^® column and a mobile phase consisted of acetonitrile: 0.1% formic acid (20:80). The fluorescence detection has been applied with λ_ex/_em = 280/310 nm. A solid phase extraction procedure using C18 cartridge was carried out. The linearity of the method has been demonstrated in the range of both therapeutic and toxic plasma tramadol levels (concentrations of 0.100 – 1 μg/mL). The selectivity, precision, and accuracy of the method have been demonstrated. The limit of detection (LOD = 0.010 μg/mL) and the limit of quantification (LOQ = 0.100 μg/mL) have been established.The proposed method can be used to assess tramadol levels in human plasma in pharmacokinetic studies, as well as in overdose cases. The utility of the method for the quantification of therapeutic levels of tramadol has been shown on the plasma samples from the patients with tramadol treatment as analgesic (doses ranging from 100 mg to 400 mg/day).The developed method is rapid, using simple experimental conditions and an accurate and short extraction procedure.