HarmitaDepartemen Farmasi FMIPA-UI VALIDASI METODA ANALISISValidasi metoda analisis adalah suatu tindakan penilaian terhadap parameter tertentu, berdasarkan percobaan laboratorium, untuk membuktikan bahwa parameter tersebut memenuhi persyaratan untuk penggunaannya. PARAMETER PENAMPILAN ANALISISBeberapa parameter analisis yang harus dipertimbangkan dalam validasi metode analisis diuraikan dan didefinisikan sebagaimana cara penentuannya. Kecermatan (accuracy)Definisi

This research aimed to quantitatively bioanalyze 6-mercaptopurine (6-MP), 6-methylmercaptopurine (6-MMP), and 6-thioguanosine-5′-monophosphate (6-TGMP) in dried blood spots (DBS) prepared from a small volume of acute lymphoblastic leukemia (ALL) patients. Analytes on the DBS card were extracted using 90% methanol with 5-fluorouracil (5-FU) as an internal standard. Analytical separation was performed on a Waters Acquity® UPLC BEH AMIDA column of 1.7 μm (2.1 × 100 mm) with a mobile phase mixture of 0.2% formic acid in water and 0.1% formic acid in acetonitrile-methanol, with gradient elution and a flow rate of 0.2 mL/min. Mass detection of 6-MP, 6-MMP, 6-TGMP, and 5-FU showed m/z values of 153.09 > 119.09, 167.17 > 126.03, 380.16 > 168.00, and 129.09 > 42.05, respectively. This DBS method had a run time of 5 min and yielded a linear calibration curve over a range of 25.5–1020 ng/mL for 6-MP, 6-MMP, and 6-TGMP. Analyte analysis in 22 of 24 ALL patients showed that the measured value of 6-TGMP as an active metabolite was in the range of 29–429 pmol/8 × 108 erythrocytes. Five of 22 patients had concentrations in a therapeutic range, which indicates that the treatment is effective, while 17 of 24 patients had concentrations below the therapeutic range, which indicates that a treatment dose adjustment is needed. The measured value of 6-MMP, an inactive metabolite, was in the range of 28–499 pmol/8 × 108 erythrocytes, which includes concentrations below the hepatotoxic range. The method employed here can thus be effectively utilized to support therapeutic drug monitoring.

Objective: The aim of this study was to develop transferosome vesicles for the transdermal drug delivery of lynestrenol.Methods: The lynestrenol transferosome vesicle was made by encapsulating the drug in a variation of phosphatidylcholine and Tween 80 by the thinlayerhydration method. The resulting transferosome vesicles were modified with a time variation of 30, 60, 90, and 120 min, and sonication variationswere paused and not paused. Particle size evaluation, polydispersity (PDI), and entrapment efficiency (%EE) were carried out on the variation ofsonication time.Results: The evaluation results showed that sonication without pauses showed better %EE and particle size than sonication with pauses andincreasing concentration of Tween 80 (edge activator). The %EE increased, and particle size decreased with increasing sonication time; PDI of vesicleswas heterogeneous with increasing sonication time. The %EE in formulas F1 and F2 after 120 min was 73.06% and 76.06% (paused) and 80.40% and82.97% (without paused). The particle size of formula F1 and F2 after 120 min 575.4 nm and 471.6 nm (paused) and 524.1 nm and 434.7 nm (withoutpaused). The PDI formulas of F1 and F2 after 120 min were 0.69 and 0.763 (paused) and 0.84 and 0.59 (without paused).Conclusion: Based on the results of the transferosome vesicle characteristics, it was shown that the optimal vesicle composition for packaginglynestrenol was vesicles that were composed of phosphatidylcholine and Tween 80 without pauses and could potentially be used as a transdermaldrug delivery system.

Objective: To analyze and validate 6-mercaptopurine (6-MP) and 6-methylmercaptopurine (6-MMP) in dried blood spots (DBS) using liquid chromatography-tandem mass spectrometry (LC/MS-MS).Methods: Bio-sampling dried blood spot with DBS-CAMAG ® paper diameter of 8 mm and extracted with acetonitrile-methanol (1:3) containing internal standard 5-fluorouracil (5-FU). Separation was performed with C 18 column Acquity ® 1.7 μm (2.1 mm × 100 mm), with a mobile phase mixture of 0.1% formic acid in water 0.1% formic acid in acetonitrile with gradient elution and flow rate 0.2 ml/min. Mass detection was Waters Xevo TQD with positive electrospray ionization (ESI) for 6-MP, 6-MMP and negative ESI for 5-FU in multiple reaction monitoring modes. The ions of 6-MP was detected at m/z 153.09->119.09, 6-MMP at m/z 167.17->126.03, and 5-FU at m/z 129.15->42.05. Results:This method fulfill the requirements of selectivity, linearity, lower limit of quantification, accuracy, precision, carry-over, matrix effects, and stability which refers to the european medicines agency (EMEA) guidelines. The linearity of 0.99 was 26-1000 ng/mL for 6-MP and 6-MMP, respectively. The validated method was applied to two childhood ALL maintenance phase. Retrieved 6-MP levels of 10.37 pmol/8×10 8 erythrocytes, respectively. The levels of 6-MMP gained 16.59 pmol/8×10 8 erythrocytes, respectively. Conclusion:The developed LC/MS-MS method is valid to analysis 6-MP and 6-MMP in DBS simultaneous in vitro according to EMEA guidelines. The method was successfully applied to authentic capillary blood samples from two childhood patients with ALL in the maintenance phase.

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