1. Syed N Alvi, Ph.D Clinical Studies & Empirical Ethics Department King Faisal Specialist Hospital & Research Centre Riyadh 11211, Kingdom of Saudi Arabia Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

2. Determination of Amlodipine in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry and its Application in Pharmacokinetic & Bioequivalence Study Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

3. The objective of the study: o Develop and validate the method to determine amlodipine levels in small volumes of human plasma by LC-MS/MS. o Assess the stability under various clinical laboratory conditions. o Application to asses bioequivalence study. Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

4. Amlodipine Chemically : Derivative of Dihydropyridine Molecular Formula: C 20 H 25 ClN 2 O 5 (FW: 408.8) Class of Drug: Calcium channel blockers Uses: In treatment of hypertension and angina pectoris. Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

5. Pharmacokinetic Study Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain  Single Dosage: 10 mg  C max: 6-12 ng/ml  Time Max: 8-10 hrs  Half life: 35-45 hrs  Bioavailability : 60-65%

6. Study Design  Single Dose : Amlodipine (10 mg Tablet)  Formulation : Four (Three generic and reference)  Randomized, 4Treatment and 4 Group Crossover Study  Participants: 68 (4 Groups, 4 periods)  No. of sample: 18 x 4 = 72  Study was conducted after the approval of Ethical Committee, KFSHRC Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

7. References: 1.HPLC-UV: IL FARMACO 60 (2005) 789-792 2.HPLC-UV: The Open Chemical and Biomedical Method Journal 1 (2008) 22-27. 3. HPLC-CE: J. Chromatography B. Biomedical Sciences and Applications 672:2 (1995) 310-313. 4. HPLC-FL: Pre-column Derivatization with NBD-Cl (Nitrobenzofuran) J. of Pharmaceutical & Biomedical Analysis 36:1 (2004) 163-168. 5. LCMS-MS: Anal. Bioanal Chem 382 (2005) 1049-1054. 6. LCMS/MS: Pharmacology & Pharmacy 4 (2013) 191-200. Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

8. Material/Reagents & Equipment  Amlodipine (Ref. Standard), Tizanidine (Int. Standard)  Acetonitrile (HPLC-Grade), Formic acid (AR-Grade) (Both from Fisher Scientific, NJ, USA).  HPLC grade water prepared by reverse osmosis and further purified by passing through a Synergy Water Purification System (Millipore, Bedford, MA, USA)  Instrument: MS/MS Micromass, Triple quadruple, HPLC, Alliance 2695, (Waters Associates Inc. Milford, MA, USA) Methodology: Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

9. Standards and Quality Control Preparations Stock Solutions: -Amlodipine & Tizanidine (1 µg/ml, methanol) Working Solutions: Amlodipine : 20 ng/ml in drug free human plasma Tizanidine : 30 ng/ml in methanol Calibration Curve (Nine concentrations) Range: 0.2 ng/ml – 20 ng/ml plasma Quality Control Samples 1: LLQ (0.2 ng/ml), 2: 3xLLQ (0.6 ng/ml) 3: 0.5 HLQ (10 ng/ml) 4: 0.9 HLQ (18 ng/ml) Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

10. Analytical Conditions Liquid Chromatograph: - Column: Atlantis dC18 (2.1 x 100 mm, 3 μm) - Guard Column: Symmetry C18 (2.1 x 10 mm, 5 μm) - Mobile Phase: Acetonitrile and 1.0 mM Formic acid (80: 20, V/V) - Sample Temperature: 4°C - Flow rate: 0.3 ml/min. Mass Spectrometer: Spray: Electrospray ionization (positive) Voltages: Capillary 4.0 kV, Cone 30 V Temperature: Source 105°C, Desolvation 350°C Cone Gas Flow: 600 L/hr. Collision Energy ; 25 eV Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

11. Product Ion Spectra of Amlodipine and IS Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain Amlodipine (409.8 > 238.4) Tizanidine, IS (254.3 > 43.9)

12.  Human plasma (0.5 ml) + Tizanidine, (Internal standard) (100 µl of 30 ng/ml, methanol )  Add 3.0 ml- Tert. Butylmethylether+DCM (3:1,v/v)  Vertex, then centrifuge @ 6000 rpm for 10 minutes at 30°C  Separate organic layer and evaporate solvent at 40 °C  Reconstitute 100 µL (MeOH+Water, 1:1), inject volume 10.0 µL  Run Time: 3 Minutes Sample Preparation Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

13. Representative MRM Chromatogram of blank human plasma Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain Amlodipine Tizanidine (IS)

14. Representative MRM Chromatogram of plasma spiked with IS Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain Amlodipine Tizanidine (IS)

15. MRM Chromatograms of plasma spiked with Amlodipine and IS Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain (Conc. 0.6 ng/ml) (Conc. 10 ng/ml) (Conc. 18 ng/ml)

16. Method Validation ----------------------------------------------- Parameters Acceptable limits ----------------------------------------------- Specificity : Blank plasma (6) Commonly used drugs Extraction Recovery : Consistent Matrix Effect : Confirm Linearity : Det. Response/Analyte Conc : (6-8, Zero and Blank) Accuracy & Precision : ±15% (3 levels) :±20% for LLQ Stability : Confirm -------------------------------------------------- Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

17. Specificity  Six different batches of human plasma screened  Eight commonly used medications: Acetaminophen, Ibuprofen, Aspirin, Omeprazole, Nicotinic acid, Ascorbic acid, Ranitidine and Caffeine. Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

18. Extraction Recovery Amlodipine (ng/ml) Human PlasmaMobile Phase Recovery (%) Mean (%) Mean Height (n=5) SD Mean Height (n=5) SD 0.2133172164062 71 0.6390445733768 106857785863250679 1897589901287379276 IS (30)17874128318896115495 Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

19. Matrix Effect Amlodipine (ng/ml) Post-Extracted Analyte Aqueous standard Suppression (%) Mean Height (n=4) SD Mean Height (n=4) SD 0.22051322415-8.6 1075822367996462-5.2 Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

20. Linearity Nominal Conc. (ng/ml) AMO-PH IS-PHRatio Measured Conc. (ng/ml) Acc. (%) 0.2169246030.00690.17889 0.5723410730.01800.46994 1.01222362400.03370.88388 2.03495563610.06201.62581 4.011710745410.15714.119103 8.023876730440.32698.573107 12.25783540520.477012.512104 1635142551920.636716.702104 2040142551920.727319.07895 Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

21. Representative Standard Calibration Curve Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

22. Precision & Accuracy INTRA-DAY (n=10)INTER-DAY (n=20) Nominal (ng/ml) Measured (ng/ml) SDCV (%) Bias (%) Measured (ng/ml) SDCV (%) Bias (%) 0.20.220.0314.29.80.210.0312.75.3 0.60.640.0913.75.90.660.0811.619.2 1010.541.5214.45.410.071.3713.60.7 1820.472.1410.513.720.012.3511.711.2 Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

23. Stability: Processed & unprocessed samples Storage Condition Nominal (ng/ml) Measured (ng/ml SDStability (%) Base line/None0.6 18 0.60 18.09 0.05 1.21 Processed 24 h. (RT) 0.6 18 0.57 16.87 0.16 2.35 95 93 Processed 48 h. (4ºC) 0.6 18 0.55 18.44 0.09 2.71 92 102 Unprocessed 24 h. (RT) 0.6 18 0.56 18.44 0.04 1.17 93 102 68 wks (-40ºC)0.6 18 0.55 16.93 0.02 1.40 91 94 FT: Cycle-10.6 18 0.56 17.55 0.08 0.78 94 97 FT: Cycle-20.6 18 0.57 17.42 0.12 2.67 95 96 FT: Cycle-30.6 18 0.54 15.94 0.06 2.02 89. 88 Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

24. Ruggedness & Robustness Ruggedness: Mobile Phase: Altering strength of formic acid (±5%) Proportion of formic acid and acetonitrile (±2%) Robustness: Analyst Split Analysis Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

25. Method Application  No. of Samples collected: 72 within 24 hrs.  Processed: According to method  Analyzed: LC-MS/MS Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

26. Representative MRM chromatograms of plasma sample obtained from healthy volunteer before and 2 & 6 hrs. after oral a single 10 mg Amlodipine dose Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain Time: Zero hr. Time : 2 hrs. Time: 6hrs.

27. Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain Results of Volunteer’s Sample Analysis

28. Measured levels  Samples collected from a health volunteer before and after ingestion of a single oral dose of 10 mg amlodipine analyzed according method.  Measured concentration: Range 0.2 – 6.45 ng/ml Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

29. Conclusions A simple, precise, and accurate assay for the measurement of amlodipine in human plasma was developed and fully validated. The assay was successfully applied to monitor stability of amlodipine under various condition routinely encountered by the laboratory. The assay was applied to determine the level of amlodipine in 0.5 ml plasma sample obtained from a healthy volunteer. Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

30. Acknowledgements  This work was funded by a grant from the King Abdul- Aziz City for Science and Technology, under National Comprehensive Plan for Science and Technology, Riyadh, Saudi Arabia, (Biotech:10-BIO96).  Dr. Muhammad M Hammami, MD. PhD, Chairman, Department Clinical Studies & Empirical Ethics, KFSHRC  Staff members of Clinical & Bio-analysis Laboratories CSEED, KFSHRC Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

31. THANKS FOR YOUR ATTENTION Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain