DEVELOPMENT AND VALIDATION OF ANALYTICAL METHOD OF 3, 4-METHYLENEDIOXY-N-ETHYLAMPHETAMINE IN DRIED BLOOD SPOT USING GAS CHROMATOGRAPHY-MASS SPEC-TROMETRY
Objective: This study aims to develop and validate the analytical method to determine 3,4-Methylenedioxy-N-ethylamphetamine (MDEA) in DBS using GC-MS.
Methods: This research used liquid-liquid micro-extraction for sample preparation and analysis was performed by GC-MS. In the method development, the optimized parameters were flow rate, column temperature, the spot of blood volume, % haematocrit, extraction and reconstitution of solvent volume, and sonication duration. Validation of the chosen method was performed based on EMEA bioanalytical guideline in 2011.
Results: The optimum chromatographic conditions were obtained using HP-5 MS capillary columns (30 m x 0.25 mm i.d; 0.25 μm ); helium with 99.9% purity as a mobile phase; flow rate of 1.0 ml/min; column temperature was 250 °C; MS detection using 4 fragments at m/z values of 72.00 and 44.00 for MDEA and 58.00 and 77.00 for ephedrine HCl as an internal standard. The DBS paper with the volume of blood spot 40 μl was then extracted using liquid-liquid micro-extraction with methanol 700 μl, sonication for 5 min, evaporated with nitrogen gas then reconstituted with 50 μl ethyl acetate. The validation results fulfilled the requirements based on the EMEA bioanalytical guideline in 2011.
Conclusion: It can be concluded that the optimum condition of the analytical method by using GC-MS was obtained and fulfilled validation criteria with a range concentration of 15-250 ng/ml.
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