syed n alvi, ph.d clinical studies & empirical ethics department king faisal specialist hospital...

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


The objective of the study:

oDevelop and validate the method to determine amlodipine levels in small volumes of human plasma by LC-MS/MS.oAssess the stability under various clinical laboratory conditions.oApplication to asses bioequivalence study.


Amlodipine

Chemically : Derivative of DihydropyridineMolecular Formula: C20H25ClN2O5 (FW: 408.8) Class of Drug: Calcium channel blockersUses: In treatment of hypertension and angina pectoris.


Pharmacokinetic Study


Single Dosage: 10 mg C max: 6-12 ng/ml Time Max: 8-10 hrs Half life: 35-45 hrs Bioavailability : 60-65%

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


References:1. HPLC-UV: IL FARMACO 60 (2005) 789-7922. 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.


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:


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)


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


Product Ion Spectra of Amlodipine and IS


Amlodipine(409.8 > 238.4)

Tizanidine, IS (254.3 > 43.9)

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


Representative MRM Chromatogram of blank human plasma


Amlodipine

Tizanidine (IS)

Representative MRM Chromatogram of plasma spiked with IS


Amlodipine

Tizanidine (IS)

MRM Chromatograms of plasma spiked with Amlodipine and IS


(Conc. 0.6 ng/ml)

(Conc. 10 ng/ml)

(Conc. 18 ng/ml)

Method Validation-----------------------------------------------Parameters Acceptable limits-----------------------------------------------Specificity : Blank plasma (6) Commonly used drugsExtraction Recovery : ConsistentMatrix Effect : ConfirmLinearity : Det. Response/Analyte Conc : (6-8, Zero and Blank)Accuracy & Precision : ±15% (3 levels) :±20% for LLQStability : Confirm--------------------------------------------------


Specificity Six different batches of human plasma

screened Eight commonly used medications: Acetaminophen, Ibuprofen, Aspirin, Omeprazole, Nicotinic acid, Ascorbic acid, Ranitidine and Caffeine.


Extraction Recovery

Amlodipine

(ng/ml)

Human Plasma

Mobile Phase

Recovery

(%)

Mean (%) Mean

Height (n=5)

SDMean

Height(n=5)

SD

0.2 133 17 216 40 62

710.6 390 44 573 37 6810 6857 785 8632 506 7918 9758 990 12873 792 76

IS (30) 17874 1283 18896 1154 95 95Analyitica 2015 (1-3 Sept. 2015) Valencia, Spain

Matrix Effect

Amlodipine(ng/ml)

Post-ExtractedAnalyte

Aqueous standard

Suppression

(%)Mean

Height (n=4)

SDMean

Height(n=4)

SD

0.2 205 13 224 15 -8.610 7582 236 7996 462 -5.2


Linearity Nominal Conc. (ng/ml)

AMO-PH IS-PH Ratio Measured Conc. (ng/ml)

Acc. (%)

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


Representative Standard Calibration Curve


Regression StatisticsMultiple R 0.9976R Square 0.9952Adjusted R Square 0.9946Standard Error 0.0208Observations 9

Precision & Accuracy INTRA-DAY (n=10) INTER-DAY (n=20)

Nominal (ng/ml)

Measured(ng/ml)

SD CV (%)

Bias (%)

Measured(ng/ml)

SD CV (%)

Bias (%)

0.2 0.22 0.03 14.2 9.8 0.21 0.03 12.7 5.3

0.6 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


Stability: Processed & unprocessed samples Storage Condition

Nominal (ng/ml)

Measured (ng/ml

SD Stability (%)

Base line/None 0.618

0.6018.09

0.051.21

Processed24 h. (RT)

0.618

0.5716.87

0.162.35

9593

Processed48 h. (4ºC)

0.618

0.5518.44

0.092.71

92102

Unprocessed24 h. (RT)

0.618

0.5618.44

0.041.17

93102

68 wks (-40ºC) 0.618

0.5516.93

0.021.40

9194

FT: Cycle-1 0.618

0.5617.55

0.080.78

9497

FT: Cycle-2 0.618

0.5717.42

0.122.67

9596

FT: Cycle-3 0.618

0.5415.94

0.062.02

89.88


Ruggedness & Robustness Ruggedness: Mobile Phase:

•Altering strength of formic acid (±5%)•Proportion of formic acid and acetonitrile (±2%)

Robustness: Analyst •Split Analysis


Method Application No. of Samples collected: 72

within 24 hrs. Processed: According to method Analyzed: LC-MS/MS


Representative MRM chromatograms of plasma sample obtained from healthy volunteer before and 2 & 6 hrs. after oral a single 10 mg Amlodipine dose


Time: Zero hr.

Time : 2 hrs.

Time: 6hrs.


Time (h) Conc. (nglml)0 0.0001 0.7702 2.8394 2.0685 3.6486 4.7017 3.2968 4.5449 3.622

10 3.48512 6.48424 2.88848 2.65372 2.72096 1.150

144 0.713192 0.364240 0.364

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

0 50 100 150 200 250

Amlo-36

Time (hours)

Conc

entr

ation

(ng

/ml

)

Results of Volunteer’s Sample Analysis

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


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.


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


THANKS FOR YOUR ATTENTION


syed n alvi, ph.d clinical studies & empirical ethics department king faisal specialist hospital...

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