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Development and validation of separation method for an analgesic drug and its main metabolite from plasma

Domenii publicaţii > Ştiinţe medicale + Tipuri publicaţii > Articol în volumul unei conferinţe

Autori: Hinescu L. G., Ionescu M , Ranetti M.C., Ionica E

Editorial: 10th Congress of Balkan Military Medical Committee, Athens, Greece, 2006.


Tramadol hydrochloride is a centrally acting opioid analgesic agent marketed for the treatment of moderate to severe pain.
Objectives. The objectives were development and validation of a extraction method for the simultaneous HPLC determination of Tramadol and its active metabolite O-desmethyltramadol (M1) in plasma in order to use in bioequivalence studies.
Material and method. A reversed-phase mechanism with fluorescence detection and liquid/liquid extraction from basic plasma samples were chosen for determination of Tramadol and M1. The bioequivalence study was made for 24 healthy volunteers
Results. Fluconazol, Trimethoprim, Verapamil and Metoprolol were tested as internal standard (IS). Metoprolol was our choice. During the validation we studied the optimal extraction media between tert-butylmethylether and diisopropylether; the influence of the aqueous layer pH, ratio between phases and vortexing time to the extraction yield were also studied.
The optimal extraction media was: diisopropylether with 1:3 ratio between plasma and organic phase; the organic layer was evaporated to dryness under N2 at 40°C. The recovery for M1 (95-101%) and Tramadol (80-84.81%) is satisfactory to allow the determination of the lowest plasma concentration of drug. The method is linear between 10 and 600 ng/ml (r=0.9924, n=8) for tramadol and 5 to 300ng/ml (r=0.9957, n=8) for metabolite. The lower limit of quantification was 5ng/ml for M1 and 10ng/ml for Tramadol.
Conclusion. Validation of the method was in conformity with GLP specifications.
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: Validation, HPLC, separation, drug detection, pharmacokinestics