A Simple Sample Preparation with HPLC–UV Method for Estimation of Amlodipine from Plasma: Application to Bioequivalence Study

A simple, rapid and selective method was developed for the estimation of amlodipine from human plasma. The method involves a simple protein precipitation techniques using nifedipine as internal standard. Chromatographic separation was carried out on a reverse phase C18 column using mixture of 50 mM potassium di hydrogen ortho phosphate (pH 7.5) and acetonitrile (60:40, v/v) at a flow rate of 1.0 mL/min with UV detection at 239 nm. The retention time of amlodipine and internal standard were 4.12 and 8.31min, respectively. The method was validated and found to be linear in the range of 0.5-50.0 ng/mL. An open, randomized, two-treatment, two period, single dose crossover, bioequivalence study in 24 fasting, healthy, male, volunteers was conducted. After dosing, serial blood samples were collected for the period of 168.0 h. Various pharmacokinetic parameters including AUC0–t, AUC0– , Cmax, Tmax, T1/2, and elimination rate constant (Kel) were determined from plasma concentration of both formulations of test (Amlodipine 5 mg tablets) and reference (Amlodipine 5 mg tablets). Log transformed values were compared by analysis of variance (ANOVA) followed by classical 90% confidence interval for Cmax, AUC0–t and AUC0– and was found to be within the range. These results indicated that the analytical method was linear, precise and accurate. Test and reference formulation were found to be bioequivalent. INTRODUCTION Amlodipine besylate is chemically described as 3-ethyl-5methyl(±)-2-[(2-aminoethoxy)methyl]-4-(2-chloro phenyl)1,4-dihydro-6-methyl-3,5-pyridine dicarboxylate mono benzene sulphonate. It is a dihydropyridine calcium channel blocker; activity resides mainly in the (-) isomer, that inhibits the transmembrane influx of calcium ions into vascular smooth muscle and cardiac muscle. The contractile processes of cardiac muscle and vascular smooth muscle are dependent upon the movement of extracellular calcium ions into these cells through specific ion channels. Only few bioanalytical methods were reported for the determination of amlodipine in biological samples [1-7]. There were no simple, rapid and reproducible methods so far reported for the estimation of amlodipine in plasma. The objective of the present investigation was to develop a new, rapid and sensitive RP-HPLC method for the estimation of amlodipine in human plasma using perchloric acid as a precipitating agent with C18 column and this method was applied to a bioequivalence study of amlodipine 5.0 mg tablets using human volunteers. The outcome of a study depends upon the reliability, reproducibility and sensitivity of the analytical methodology employed. Therefore, the bioanalytical method was validated in accordance with USFDA guidelines prior to the initiation of the study. EXPERIMENTAL Chemicals and Reagents Working standard of amlodipine besylate with 99.96% purity (German Remedies Ltd., Mumbai, India), nifedipine *Address correspondence to this author at the Department of Pharmaceutical Analysis, J.S.S. College of Pharmacy, Tamilnadu, India; Tel: +91-4232443393; Fax: +91-423-2442937; E-mail: [email protected] with 99.56% purity (Cadila Health Care Ltd., Ahmedabad, India), HPLC grade acetonitrile, methanol (Qualigens Fine Chemicals, Mumbai, India), potassium dihydrogen ortho phosphate, ortho phosphoric acid, and perchloric acid (S.D. Fine Chem. Ltd., Mumbai, India) were used. In house mill Q water was used throughout the study. Fresh frozen human plasma used in the method development was obtained from the Vijay Hospital, Ooty, India and was stored at 20 C until required. Instrumentation and Chromatography The HPLC system consisted of a HPLC 2010 A HT (Shimadzu Ltd., Japan), Auto injector port with 100 L loop (Rheodyne, USA) and UV detector (Shimadzu Ltd., Japan). The wavelength of the detector was set at 239 nm. Detector output was quantified on CLASS VP (Version 6.01) chromatography software. Separation was carried out on a Princeton SHER C18, 4.6 m 250 mm, Japan, using mixture of 50 mM potassium di Hydrogen ortho phosphate (pH 7.5) and acetonitrile (60:40, v/v) as a mobile phase, at a flow rate of 1 mL/min. Total analysis time was 10 min. All analysis was performed at room temperature. Preparation of Calibration Standard Stock solutions of amlodipine and nifedipine (I.S) (1 mg/ml) were dissolved in water and stored at 4 C. The stock solution of amlodipine was further diluted with water to give series of standard solutions. Calibration standard of amlodipine (0.50,1.0,2.5,5.0,10.0,15.0,25.0 and 50.0 ng/mL) were used by spiking appropriate amount of the standard solution in blank plasma. Quality Control Standards Lowest quality control standards (LQC), median quality control standards (MQC) & highest quality control standards HPLC–UV Method for Estimation of Amlodipine from Plasma The Open Chemical and Biomedical Methods Journal, 2008, Volume 1 23 (HQC) were used by spiking drug free plasma with amlodipine to give solutions containing 1.0, 10.0 and 50.0 ng/mL, respectively. They were stored at 20°C till analysis. Sample Preparation The sample was prepared by adding 1ml of plasma containing amlodipine (calibration standard) into 100 L of internal standard (10 g/mL) and followed by adding 100 L of 10% perchloric acid, which was vortexed followed by centrifugation at 4000 RFC for 15 min at 4 C. The supernatant solution was separated and injected. The sample chromatogram was presented in Fig. (1). Validation The validation parameter [8] like selectivity of the method was checked for interference from plasma. The standard curve consisting of five points ranging from 0.5 to 50.0 ng/mL was developed. Quality control samples of LQC (1.0 ng/mL), MQC (10.0 ng/mL) and HQC (50.0 ng/mL) were used to determine the intra and inter-day precision and accuracy of the assay. Peak area ratios of amlodipine to internal standard were fit to linear equation (y = 7e-05x+0.0006) and drug concentration in control samples along with the same day standard curve samples were calculated using this equation. For all the curves the correlation coefficients (r 2 ) were never lower than 0.9996. Clinical Design The study protocol was approved by the Institution ethics committee. Twenty four healthy male Indian subjects with mean age group 20-30 years and average weight 65.8 ± 6.1 kg were included in the study. Subjects were excluded from the study if one or more of following criteria were present at time of medical screening: allergic to amlodipine, history or clinical data of renal or liver disease, positive test for hepatitis B, HIV, history of alcohol, drug addiction or donated blood within 72 days prior to study. Test and reference formulations of amlodipine 5.0 mg tablet were administered with 240 ml of water. The study was conducted as 24 2 single dose, randomized, open, and complete crossover design. Volunteers were fasted overnight before and 4 h after drug administration. Blood sample (5 ml) were collected at 0.0, 0.5,1.0,2.0,4.0,6.0,8.0,10.0, 12.0, 18.0, 24.0, 36.0, 48.0, 72.0 and 168.0 h of post drug administration through an indwelling cannula into heparinized glass vials. After drug administration standard breakfast and lunch were provided at 4 and 6 h post dose. The blood samples were immediately centrifuged, plasma was separated and stored at 20°C until anaFig. (1). Sample Chromatogram of amlodipine and internal standard. Minutes 0 2 4 6 8 10 12 14 V ol ts 0.0 0.1 0.2 0.3 0.4 0.5 0.6 A M LO D IP IN E 4 .1 25 IN TE R N A L S TA N D A R D 8 .3 17 24 The Open Chemical and Biomedical Methods Journal, 2008, Volume 1 Meyyanathan et al. lyzed. After a washout period of 7 days, the study was repeated in the same manner to complete the crossover design. The plasma samples obtained at various time intervals were analzsed by the HPLC method developed. Pharmacokinetic Analysis The plasma concentration profile obtained was fed into PK solution, computer software on Microsoft Excel ® , to determine the pharmacokinetic parameters. The maximum amlodipine concentration Cmax and the corresponding peak time Tmax were determined by the inspection of the individual drug plasma concentration–time profiles. The elimination rate constant Kel was obtained from the least-square fitted terminal log-linear portion of the plasma concentration–time profile. The elimination half-life T1/2 was calculated as 0.693/Kel. The area under the curve to the last measurable concentration (AUC0–t) was calculated by the linear trapezoidal rule. The area under the curve extrapolated to infinity (AUC0– ) was calculated asAUC0–t +Ct/Kel where Ct is the last measurable concentration. Statistical Analysis For the purpose of bioequivalence analysis AUC0–t, AUC0– and Cmax were considered as primary variables. Bioequivalence of two formulations was assessed by means of an analysis of variance (ANOVA) for crossover design and calculating 90% confidence interval of the ratio of test/reference using log transformed data. The formulation was considered bioequivalent when the difference between two compared parameters was found statistically [9, 10] insignificant (p > 0.05) and confidence interval for these parameters fell within 80-125%. RESULTS AND DISCUSSION Selectivity Selectivity of the method described was investigated by screening six different batches of human blank plasma. UnFig. (2). Blank plasma chromatogram. Minutes 0 2 4 6 8 10 12 V ol ts 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16