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Development and Validation of a Separation Method for an analgesic drug and its main metabolite from plasma

Domenii publicaţii > Chimie + Tipuri publicaţii > Articol în revistã ştiinţificã

Autori: Lavinia G. Hinescu, C. M. Ranetti, M. Ionescu, C. Mircioiu, C. Cosmescu, V. A. Voicu

Editorial: Revista de Chimie, ISSN 0034-7752, vol. 60(9), p.961-966, 2009.


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.

Cuvinte cheie: Tramadol separation, quantitative determination, liquid-liquid extraction, bioequivalence study