Syed N Alvi Ph D Clinical Studies Empirical

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

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

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

Amlodipine Chemically : Derivative of Dihydropyridine Molecular Formula: C 20 H 25 Cl. N 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

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

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

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

Methodology: 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) Analyitica 2015 (1 -3 Sept. 2015) Valencia, Spain

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: 3 x. LLQ (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

Analytical Conditions Liquid Chromatograph: - Column: Atlantis d. C 18 (2. 1 x 100 mm, 3 μm) - Guard Column: Symmetry C 18 (2. 1 x 10 mm, 5 μm) - Mobile Phase: Acetonitrile and 1. 0 m. M 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 k. V, Cone 30 V Temperature: Source 105°C, Desolvation 350°C Cone Gas Flow: 600 L/hr. Collision Energy ; 25 e. V Analyitica 2015 (1 -3 Sept. 2015) Valencia, Spain

Product Ion Spectra of Amlodipine and IS Amlodipine (409. 8 > 238. 4) Tizanidine, IS (254. 3 > 43. 9) Analyitica 2015 (1 -3 Sept. 2015) Valencia, Spain

Sample Preparation § § § 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 (Me. OH+Water, 1: 1), inject volume 10. 0 µL Run Time: 3 Minutes Analyitica 2015 (1 -3 Sept. 2015) Valencia, Spain

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

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

MRM Chromatograms of plasma spiked with Amlodipine and IS (Conc. 0. 6 ng/ml) (Conc. 10 ng/ml) (Conc. 18 ng/ml) Analyitica 2015 (1 -3 Sept. 2015) Valencia, Spain

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

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

Extraction Recovery Human Plasma Amlodipine (ng/ml) Mobile Phase SD Mean Height (n=5) SD Recovery (%) 133 17 216 40 62 0. 6 390 44 573 37 68 10 6857 785 8632 506 79 18 9758 990 12873 792 76 IS (30) 17874 1283 18896 1154 95 Mean Height (n=5) 0. 2 Analyitica 2015 (1 -3 Sept. 2015) Valencia, Spain Mean (%) 71 95

Matrix Effect Amlodipine (ng/ml) Post-Extracted Analyte Aqueous standard SD Mean Height (n=4) SD Suppression (%) 205 13 224 15 -8. 6 7582 236 7996 462 -5. 2 Mean Height (n=4) 0. 2 10 Analyitica 2015 (1 -3 Sept. 2015) Valencia, Spain

Linearity Nominal Conc. (ng/ml) AMO-PH IS-PH Ratio Measured Conc. (ng/ml) 0. 2 169 24603 0. 0069 0. 178 89 0. 5 723 41073 0. 0180 0. 469 94 1. 0 1222 36240 0. 0337 0. 883 88 2. 0 3495 56361 0. 0620 1. 625 81 4. 0 11710 74541 0. 1571 4. 119 103 8. 0 23876 73044 0. 3269 8. 573 107 12. 25783 54052 0. 4770 12. 512 104 16 35142 55192 0. 6367 16. 702 104 20 40142 55192 0. 7273 19. 078 95 Analyitica 2015 (1 -3 Sept. 2015) Valencia, Spain Acc. (%)

Representative Standard Calibration Curve Amlodipine 0, 9 Peak Height Ratio 0, 8 0, 7 0, 6 0, 5 0, 4 Y 0, 3 Predicted Y 0, 2 0, 1 0, 0 0 10 20 Concentration (ng/ml) 30 Analyitica 2015 (1 -3 Sept. 2015) Valencia, Spain

Precision & Accuracy INTRA-DAY (n=10) Nominal Measured (ng/ml) INTER-DAY (n=20) SD CV (%) Bias (%) Measured (ng/ml) SD CV (%) Bias (%) 0. 22 0. 03 14. 2 9. 8 0. 21 0. 03 12. 7 5. 3 0. 64 0. 09 13. 7 5. 9 0. 66 0. 08 11. 61 9. 2 10 10. 54 1. 52 14. 4 5. 4 10. 07 1. 37 13. 6 0. 7 18 20. 47 2. 14 10. 5 13. 7 20. 01 2. 35 11. 7 11. 2 Analyitica 2015 (1 -3 Sept. 2015) Valencia, Spain

Stability: Processed & unprocessed samples Storage Condition Nominal (ng/ml) Measured (ng/ml SD Base line/None 0. 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 0. 6 18 0. 55 16. 93 0. 02 1. 40 91 94 FT: Cycle-1 0. 6 18 0. 56 17. 55 0. 08 0. 78 94 97 FT: Cycle-2 0. 6 18 0. 57 17. 42 0. 12 2. 67 95 96 FT: Cycle-3 0. 6 18 0. 54 15. 94 0. 06 2. 02 89. 88 68 wks (-40ºC) Analyitica 2015 (1 -3 Sept. 2015) Valencia, Spain Stability (%)

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

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

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

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

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

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

Acknowledgements Ø Ø Ø 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 -BIO 96). Dr. Muhammad M Hammami, MD. Ph. D, Chairman, Department Clinical Studies & Empirical Ethics, KFSHRC Staff members of Clinical & Bio-analysis Laboratories CSEED, KFSHRC Analyitica 2015 (1 -3 Sept. 2015) Valencia, Spain

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