ANALYTICAL VALIDATION OF CLOPIDOGREL IN HUMAN PLASMA THROUGH ULTRAHIGHPERFORMANCE LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

Objective: This study developed a sensitive, selective, and valid method for analyzing clopidogrel in human plasma using liquid chromatographytandemmass spectrometry (LC-MS/MS).Methods: The chromatography separation was performed on the waters ACQUITY UPLC Class BEH C18 1.7 μm (2.1 × 100 mm) column, consisting of0.1% formic acid in water and 0.1% formic acid in acetonitrile (30-70) as the mobile phase, and an isocratic elution with a flow rate of 0.2 mL/minutes.The mass detection was performed using a Waters Xevo TQD equipped with positive electrospray ionization in the multiple reaction monitoringmodes. The clopidogrel was detected at m/z 322.086>212.097 and irbesartan as an internal standard at m/z 429.233>207.131. The sample wasprepared with protein precipitation method using acetonitrile, vortex mixed for 10 minutes, and centrifuged at 13,000 rpm for 20 minutes.Results: This method showed a linear result at the concentration range of 0.2-10 ng/ml with r≥0.9997.Conclusions: The developed method provides sensitivity, linearity, precision, and accuracy and is suitable for analysis of clopidogrel in human plasmausing LC-MS/MS

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

PEMBENTUKAN AKRILAMIDA DALAM MAKANAN DAN ANALISISNYA

Acrylamide is a chemical substance which derived from acrylonitrile, is the material used in polyacrylamide production. Recent research has found acrylamide is contained in some food, especially food is rich in carbohydrate and treated in high temperature (more than 120°C). Due to its nature, acrylamide is classified as a hazardous material to be contained in human’s food. The International Agency for Research on Cancer (IARC) has classified acrylamide into group 2A (probably carcinogenic for humans). Many methods that used to analyse the acrylamide in some foods with sophisticated equipment, and in department of pharmacy FMIPA-UI there were also develop the method with simple extraction and conventional HPLC. Keywords : acrylamide, HPLC, carcinogenic

METHOD DEVELOPMENT AND VALIDATION OF LERCANIDIPINE IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY TANDEM-MASS SPECTROMETRY

Objective: To obtain an optimum and validated method for analyzing lercanidipine in plasma using Ultra Performance Liquid Chromatography of Tandem Mass Spectrometry (UPLC-MS/MS).Methods: The separation was carried out using 1.7μm (2.1 x 100 mm) Waters AcquityTM UPLC C18 column, a mobile phase of the 0.1% formic acid-methanol mixture (20:80 v/v) with isocratic elution, 30 °C column temperature, 0.2 ml/min flow rate and amlodipine as an internal standard. Mass detection was performed with a positive XBL TQD type Electrospray Ionization (ESI) in Multiple Reaction Monitoring modes. Lercanidipine was detected at m/z value of 612.11>280.27 and amlodipine was detected at m/z value 409.1>238.15. The optimum sample preparation method was a liquid-liquid extraction using 5 ml of n-hexane-ethyl acetate (50:50 v/v), vortex mixed for 3 min, centrifuged at 4000 rpm for 20 min, evaporated with nitrogen at 50 °C for 30 min, and the residue was reconstituted with 100 μl of mobile phase.Results: The method was linear in the range of 0.025-10 ng/ml with r ≥ 0.9986. Accuracy and precision within-run and between-run met the requirements with %diff and %CV, not exceeding ± 15% and not more than ± 20% for Lower Limit of Quantification (LLOQ) concentration.Conclusion: It was concluded that the developed method met the requirements of selectivity, carry over, stability, the integrity of dilution, and matrix effects under the Guideline on Bioanalytical Method Validation by the European Medicines Agency in 2011.Â

Analysis of Derivate Compound Between Sodium Alendronat with Chloride by High Performance Liquid Chromatography

Sodium alendronate is one of biphosphonate group drugs for the treatment of osteoporosis,. The aim of this research was to obtain the optimum condition for forming derivative of sodium alendronate with dansyl chloride. By adding 270 μL dansyl chloride to 0.1 M sodium carbonate buffer at pH 10.0 mixing with thermomixer at 50.oC for 50 minutes, resulted in a stable derivative within 30 minutes. The compound was analysed by high performance liquid chromatography method using C18 column acetonitrilemethanol-buffer (25 mM KH2PO4 and 25 mM citric acid) (20:15:65;v/v) as mobile phase at a flow rate of 1.0 mL/minute; (detected at wavelength of excitation 320 nm and emission 495 nm). The retention time of the derivative was 19.758 minutes, the calibration’s curve was linear at concentration range of 0.2-1 μg/mL with coefficient of correlation (r) 0.9995 and limit of quantitation 0.114 μg/mL.Key word : HPLC, sodium alendronat, dansyl chloride, fluorescenc

PHARMACOKINETIC PROFILE AND INCURRED ESOMEPRAZOLE SAMPLE STABILITY IN PLASMA USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY - PHOTODIODE ARRAY

Objective: Esomeprazole (ESO) is one of the proton-pump inhibitors and is used to treat gastroesophageal reflux. It is sensitive to low pH, heat,moisture, and oxidation, which often means that ESO in clinical samples is degraded at the time of storage, affecting analysis results. This study aimedto analyze the in vivo stability of ESO in subjects’ plasma samples by testing the incurred sample stability (ISS) of ESO in plasma following 7, 14, and28 days of storage at two concentrations close to Cmax and one concentration in the elimination phase.Methods: Samples were analyzed using high-performance liquid chromatography with a C18 column with detection at 300 nm using a photodiodearray detector. Lansoprazole was used as an internal standard.Results: The ESO pharmacokinetics profile in the plasma samples yielded the values of Cmax 704.57–1425.85 ng/mL; tmax is 2.25 h; and AUC0-t is2444 ng.h/mL. ISS testing of plasma samples values were 6.50%, 5.73%, and 4.57% on first Cmax concentration; 3.55%, 4.84%, and 3.68% on 2nd Cmaxconcentration; and 4.04%, 4.80%, and 4.98% on elimination phase concentration.Conclusion: ISS testing results of plasma samples from six healthy subjects who were administered doses of 40 mg of ESO stored for 28 days showedthat it fulfilled the acceptance criteria (<20%) of the 2011 EMEA Bioanalytical Guidelines with a %diff value in all incurred samples of 6.5%

THE EFFECT OF ANTICOAGULANT TYPES ON THE IN VITRO ANALYSIS OF CLOPIDOGREL IN HUMAN PLASMA USING LIQUID CHROMATOGRAPHY TANDEM-MASS SPECTROMETRY

Objective: The aim of this study was to optimize and validate a plasma clopidogrel analysis method using liquid chromatography tandem-massspectrometry.Methods: Plasma samples were analyzed using a BEH C18 column (1.7 μm; 100 mm×2.1 mm), the mobile phase was 0.1% formic acid in acetonitrile(30:70, v/v). The flow rate was 0.2 mL/min, with a column temperature set to 35°C, an injection volume of 5 μL, an analysis time of 4 min, andirbesartan as the internal standard. Aliquots were obtained by liquid-liquid extraction using ammonium acetate and diethyl ether. The stability andpeak area ratio of the respective plasma area responses were evaluated using ANOVA.Results: No significant differences (p>0.05) were observed between anticoagulants regarding analyte stability. However, the peak area ratioshowed significant differences (p<0.05) between the anticoagulants. The accuracy and precision of the analysis with citrate, heparin, andethylenediaminetetraacetic acid (EDTA) plasma met the quality requirements, and a linear calibration curve was created with concentrations rangingfrom 0.02 to 5.0 ng/mL.Conclusion: The results showed that improved analysis of clopidogrel was achieved using citrate or heparin plasma compared with EDTA plasma

SIMULTANEOUS ANALYTICAL METHOD DEVELOPMENT OF 6-MERCAPTO¬PURINE AND 6-METHYLMERCAPTOPURINE IN PLASMA BY HIGH PERFOR¬MANCE LIQUID CHROMATOGRAPHY-PHOTODIODE ARRAY

Objective: 6-Mercaptopurin is antineoplastic drug that is included in antimetabolite group and is used in acute lymphoblastic leukemia medication. 6-Mercaptopurin is inactive pro-drug that will be metabolized into metabolites. One of its metabolites is 6-methylmercaptopurine. This study is aimed to optimize the analytical conditions and perform validation for the analysis of 6-mercaptopurine and 6-methylmercaptopurine in plasma. Method: Separation was performed using Waters 2996 HPLC, C18 SunfireTM column (5μm, 250 x 4.6 mm) with the mobile phase containing water-methanol-acetonitrile with gradient elution, and detected at 303 nm. 5-Fluorouracil was used as internal standard. Plasma extraction was done by liquid-liquid extraction using dichloromethane. Result: The method was linear at concentration range of 2.0 – 200.0 ng/mL with r > 0.9991 for 6-mercaptopurine and 20 – 2000 ng/mL with r > 0.9993 for 6-methylmercaptopurine. Accuracy and precision within-run and between-run fulfill the acceptance criteria with % RE and relative standard deviation (% RSD) ≤ 20% (LLOQ) and ≤ 15% (QC samples). 6-Mercaptopurine and 6-methylmercaptopurine was stable in plasma at least for 21 days when stored at -20ºC. Conclusion: The bio-analytical method was sensitive, selective and all the parameters fulfilled the acceptance criteria of the EMA Bio-analytical Method Validation Guideline, 2011