Lavinia G. Hinescu, Cristina M. Ranetti, Mihaela Ionescu, Constantin Mircioiu, Cristiana Cosmescu, Victor A. Voicu <br>Development and Validation of a Separation Method for an Analgesic Drug and its Main Metabolite from Plasma

Lavinia G. Hinescu, Cristina M. Ranetti, Mihaela Ionescu, Constantin Mircioiu, Cristiana Cosmescu, Victor A. Voicu
Development and Validation of a Separation Method for an Analgesic Drug and its Main Metabolite from Plasma

Abstract:

The objectives of this study were the development and validation of an extraction method for the simultaneous HPLC determination of Tramadol and its active metabolite o-desmethyltramadol (M1) in plasma in order to use it in bioequivalence studies. A reversed-phase mechanism with fluorescence detection and liquid/liquid extraction from basic plasma samples were chosen for the determination of Tramadol and M1. The bioequivalence study was made for 24 healthy volunteers. Fluconazol, Trimethoprim, Verapamil and Metoprolol were tested as internal standard (IS). It was chosen Metoprolol. During the validation it was studied the optimal extraction solvent (tert-butylmethylether or diisopropylether); the influence of the aqueous layer pH, ratio between phases and vortexing time to the extraction yield were also studied. The optimal extraction solvent was diisopropylether with 1:3 ratio between plasma and the organic phase; the organic layer was evaporated to dryness under N2, at 40°C. The recovery for M1 (95-101%) and Tramadol (70-84.81%) was satisfactory to allow the determination of the lowest plasma concentration of the drug. The method was linear between 10-600 ng/mL (r=0.9924, n=8) for Tramadol and 5-300ng/mL (r=0.9957, n=8) for metabolite. The lowest limit of quantification (LLOQ) was 5ng/mL for M1 and 10ng/mL for Tramadol. The extraction procedure was applied in a new, selective and sensitive HPLC method for quantitative determination of Tramadol and M1 in a bioequivalence study. Keywords: Tramadol separation, quantitative determination, liquid-liquid extraction, bioequivalence study

Issue: 2009, Volume 60, Issue 9

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