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The optimized method for HPLC determination of tramadol and its metabolite O-desmethyl tramadol in human plasma using sotalol as internal standard has been developed and validated by a new approach. The determination by fluorescence detection was performed on re-eluted solution, obtained after liquid-liquid extraction with ethyl acetate of the three analytes from plasma. The chromatographic separation of tramadol under a gradient elution was achieved at a temperature of 15 degrees C with a RP-18 column, guarded by a C18 precolumn. The mobile phase was a mixed aqueous solution containing ortho-phosphoric acid, triethylamine, acetonitrile and methanol in a complex gradient mode. The quantitative determination of tramadol was performed at different successive pairs of excitation/emission wavelengths (200/300 nm, 200/295 nm, 212/305 nm) with lower limits of quantification: LLOQ=4.078 ng/ml for tramadol, respectively LLOQ=3.271 ng/ml for O-desmethyl tramadol. For the LLOQ limits, were calculated the values of the coefficient of variation and difference between mean and the nominal concentration. For tramadol analyte they were CV%=5.147% and bias%=-7.273% in the intra-days and CV%=4.894% and bias%=0.836% in the between-days assay, respectively for the metabolite O-desmethyl tramadol they were CV%=11.517% and bias%=0.337% in the intra-days and CV%=6.41% and bias%=3.259% in the between-days assay. In addition, the stabilities of the analytes were verified in different conditions. Both, tramadol and its metabolite proved to be stable in plasma for four weeks, frozen at -20 degrees C, but also for 48 h at 15 degrees C in the re-eluted solution after liquid-liquid extraction.

Indigene Stachys species have a high content in interesting compounds as iridoids, but also high quantities of flavonoids and phenol-carboxylic acids. These compounds present vasorelaxing effect on smooth vascular muscle interfering with metabolic degradation of nitric oxide pathways (inhibition of peroxynitrites formation) and by inhibition of phosphodiesterase. This work shows flavonoids spectrum of 3 Stachys species using HPLC methods. We identified quercetol, rutoside, miricetin and phenol-carboxylic acids (chlorogenic, caffeic and ferulic acids).