METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF AMLODIPINE AND INDAPAMIDE BY DIFFERENT SPECTROPHOTOMETRIC AND RP-HPLC METHODS IN BULK DRUG AND PHARMACEUTICAL FORMULATION
Chalapathi institute of pharmaceutical sciences,
guntur, a.p, india.
Amlodipine (as besylate, mesylate or maleate) is a long-acting calcium channel blocker (dihydropyridine (DHP) class) used as an anti-hypertensive and in the treatment of angina. Indapamide is a thiazide diuretic used in the treatment of hypertension, as well as decompensated cardiac failure. Six new, simple, accurate and precise methods have been developed and validated according to ICH guidelines for the simultaneous estimation of Amlodipine and Indapamide in their combined dosage form (four UV-Spectrophotometric, one colorimetric and one RP-HPLC methods).
First method is based on simultaneous estimation using two wavelengths, 365 nm (λmaxof AMLO) and 279 nm (λmaxof INDA) by simultaneous equation method. The second method involves the use of first order derivative technique, here 293 nm, the zero crossing point of AMLO, 279 nm, the zero crossing point of INDA were used for the estimation. The third method is based on Q-absorption Ratio method using two wavelengths 365 nm (λmaxof AMLO) and 312 nm (Isoabsorptive point). In the dual wavelength method two wave lengths 270 nm and 288 nm were selected as λ1 and λ2 for the estimation of AMLO, INDA shows the same absorbance at these wavelengths. Similarly, wavelengths 350 nm and 378 nm were selected as λ1 and λ2 for the estimation of INDA, AMLO shows the same absorbance at these wavelengths.Colorimetry: The method is based on use of MBTH reagent for simultaneous estimation of AMLO and INDA using two wavelengths, 626 nm (λmaxof AMLO) and 600 nm (λmaxof INDA).
REFERENCE ID: PHARMATUTOR-ART-1747
All the five methods obeys Beer’s linearity in the concentration range of 10-50 µg/ml for AMLO and 5-25 µg/ml for INDA respectively.
RP-HPLC Method: The system was used withC18 column and the detection was made at 250 nm in the UV region. Mobile phase consisted of Acetonitrile: Methanol: Triethylamine buffer 25:35:40(v/v) at a flow rate of 1.0 ml/min with linearity of 5-25 µg/ml for AMLO and 2-10 µg/ml for INDA, the Rt values were 3.4 and 4.5 min for AMLO and INDA respectively.
All the methods were validated and all the parameters were found to be within the acceptance criteria.
2.1. Amlodipine 
Drug name: Amlodipine besylate
Therapeutic category: Anti-hypertensive
Description: White crystalline powder Solubility: Slightly soluble in water and freely soluble in methanol
Molecular formula: C20H25ClN2O5.C6H6O3S
Molecular weight : 567.1
Chemical name: (RS)-3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4-(2- chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate benzene sulfonate Melting point : 2030C
Half life : 30-50hrs
PKa : 8.6
Mode of action: Amlodipine is a dihydropyridine calcium antagonist (calcium ion antagonist or slow-channel blocker) that inhibits the transmembrane influx of calcium ions into vascular smooth muscle and cardiac muscle. Experimental data suggest that Amlodipine binds to both dihydropyridine and non-dihydropyridine binding sites. 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. Amlodipine inhibits calcium ion influx across cell membranes selectively, with a greater effect on vascular smooth muscle cells than on cardiac muscle cells. Negative inotropic effects can be detected in vitro but such effects have not been seen in intact animals at therapeutic doses. Serum calcium concentration is not affected by Amlodipine. Within the physiologic pH range, Amlodipine is an ionized compound (PKa =8.6), and its kinetic interaction with the calcium channel receptor is characterized by a gradual rate of association and dissociation with the receptor binding site, resulting in a gradual onset of effect.
Pharmacokinetics: The metabolism and excretion of Amlodipine have been studied in healthy volunteers following oral administration of 14C-labelled drug. Amlodipine is well absorbed by the oral route with a mean oral bioavailability of approximately 60%. Renal elimination is the major route of excretion with about 60% of an administered dose recovered in urine, largely as inactive pyridine metabolites. The major metabolite identified was 2-([4-(2-chlorophenyl)-3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl- 2-pyridyl] methoxy) acetic acid and this represented 33% of urinary radioactivity. Amlodipine concentrations in plasma declined with a mean half-life of 33 h, while elimination of total drug-related material from plasma was slower.
Adverse effects: Adverse side effects of the use of Amlodipine may be
* Very often: peripheral edema in 8.3% of users, fatigue in 4.5% of users
* Often: dizziness; palpitations; muscle-, stomach- or headache; dyspepsia; nausea- in 1 in 100 users
* Sometimes: blood disorders,developmentof breasts in men (gynecomastia),impotence, depression, insomnia, tachycardia, gingival enlargement - in 1 in 1,000 users,
* Rarely: erratic behavior, hepatitis, jaundice - in 1 in 10,000 users
* Very rarely: hyperglycemia, tremor, Stevens–Johnson syndrome in 1 in 100,000 users
2.2. Indapamide 
Drug name: Indapamide
Therapeutic category: Thiazide like diuretic
Description : White crystalline powder
Solubility: Slightly soluble in water and freely soluble in methanol Molecular
Molecular weight : 365.835
Chemical name: 4-chloro-N-(2-methyl-2,3-dihydroindol-1-yl)-3-sulfamoyl-
Melting point : 1610C
Half life : 14hrs
PKa : 8.8
Mode of action: Indapamide enhances excretion of sodium, chloride and water by interfering with the transport of sodium ions across the renal tubular epithelium.Indapamide blocks the slow component of delayed rectifier potassium current (IKs) without altering the rapid component (IKr) or the inward rectifier current. Specifically it blocks or antagonizes the action the proteins KCNQ1 and KCNE1. Indapamide is also thought to stimulate the synthesis of the vasodilatory hypotensive prostaglandin PGE2.
Absorption: Rapidly and completely absorbed from the GI tract (oral). Peak plasma levels are achieved within 2-2.5 hr.
Distribution: Widely distributed, preferentially and reversibly bound to erythrocytes.
Metabolism: Extensively metabolised in the liver.
Excretion: Via urine (60-70% as metabolites, 5-7% as unchanged), via faeces (16-23% remaining dose); 14 hr (elimination half-life).
Adverse effects: Commonly reported adverse events are hypokalemia (low potassium levels), fatigue, orthostatic hypotension (blood pressure decrease on standing up) and allergic manifestations.
J. Bagyalakshmi etal.,developed a RP-HPLC method for the estimation of s(-) amlodipine in tablet dosage form by fixing the parameters as Phenomenex C8 ODS column (150 x 4.6 mm), 5m particle size with mobile phase 20 mM sodium dihydrogen phosphate buffer: acetonitrile (65: 35% v/v) adjusted to pH 8 was used. Mobile phase flow rate was maintained at 1.2ml/min and detected at 239nm. The retention time was 4.20± 0.02 minutes. 
Nafisur Rahman etal., developed Three new spectrophotometric methods for the determination of amlodipine besylate. The first two methods, i.e. A and B, are based on the oxidation of the drug with Fe(III) and the estimation of Fe(II) produced after chelation with either 1,10-phenanthroline or 2,2′-bipyridyl at 500 and 515 nm, respectively. The Beer's law was obeyed in the concentration ranges of 2–10 and 4–14 μg ml–1 with molar absorptivity of 2.9 × 104 and 2.7 × 104 l mol–1 cm–1 for methods A and B, respectively. The third procedure depends on the interaction of amlodipine besylate with ammonium heptamolybdate tetrahydrate, which resulted in the formation of molybdenum blue (λmax 825 nm). The linear dynamic range and the molar absorptivity values were found to be 15–59 μg ml–1 and 1.8 × 104 l mol–1 cm–1, respectively. The results of the proposed procedures were validated statistically and compared with those obtained by the reference method. The proposed methods were applied successfully to the determination of amlodipine besylate in commercial tablets.
A.P. Argekar etal.,developed a new simple, precise, rapid and selective high-performance thin-layer chromatographic (HPTLC) method for the simultaneous determination of atenolol (ATL) and amlodipine (AMLO) in tablets, using methylene chloride:methanol:ammonia solution (25% NH3) (8.8:1.3:0.1; v:v) as the mobile phase and Merck HPTLC plates (0.2mm thickness) precoated with 60F254 silica gel on aluminium sheet as the stationary phase. Detection was carried out densitometrically using a UV detector at 230 nm. The retention factors of ATL and AMLO were 0.33 and 0.75, respectively. Calibration curves were linear in the range 10–500 mg ml-1 for both. Assays of ATL and AMLO were 49.87 mg per tablet (relative standard deviation (R.S.D.), 1.3%) and 4.90 mg per tablet (R.S.D., 1.38%) for brand I, and 49.27 mg per tablet (R.S.D., 1.12%) and 4.98 mg per tablet (R.S.D., 1.42%) for brand II, respectively. The percentage recoveries for ATL and AMLO for brands I and II were 99.06 and 99.30%, and 99.27 and 99.15%, respectively.
Permender rathee etal.,developed a new UV-Spectrophotometric method for the simultaneous assay of Amlodipine Besylate and Atenolol in bulk drug and in tablet dosage forms using aqueous medium as the solvent. The method is based on simultaneous equation or Vierodt’s method. The λmax values for Amlodipine Besylate and Atenolol in the solvent medium were found to be 238.4 nm and 273.4 nm respectively. The systems obey Beer’s law in the range of 4.0 to 32.0 mg/ml and 20.0 to 200.0 mg/ml with correlation coefficient of 0.9984 and 0.9996 for Amlodipine Besylate and Atenolol respectively. Repeatability, Interday and intraday precision were found to be 0.562, 0.474, 0.456 and 0.238, 1.31, 0.337 respectively. No interference was observed from common tablet adjuvants. t –test and F-test have been applied for the recovery studies of the method. The method was successfully applied to the assay of Amlodipine Besylate and Atenolol in tablet formulations.
Bhusari Vidhya k. etal.,developed a HPLC method for simultaneous determination of Amlodipine besylate, Atenolol and Aspirin in formulation. This method is based on HPLC separation of the three drugs on the Thermo Hypersil BDS–C18 (250 mm × 4.6mm, 5.0 μ) from Germany with isocratic conditions and simple mobile phase containing methanol: 10 mM phosphate buffer with pH 7.0 adjusted with ortho phosphoric acid (70: 30) at flow rate of 1 mL/min using UV detection at 235 nm with Rt of 2.58 min for Amlodipine besylate, Rt of 3.40 min for Atenolol and 4.23 min for Aspirin. This method has been applied to formulation without interference of excipients of formulation. The linear regression analysis data for the calibration plots showed a good linear relationship over the concentration range of 2-12 μg/mL for Amlodipine besylate, Atenolol and 4-24 μg/mL for Aspirin, respectively. The mean values of the correlation coefficient, slope and intercept were 0.9993 ± 0.63, 2134.1 ± 0.54 and 1676 ± 0.89 for Amlodipine besylate, 0.9994 ± 0.91, 21326 ± 1.02 and 42960 ± 0.74 for Atenolol and 0.9993 ± 1.02, 15182 ± 0.48 and 64910 ± 0.64 for Aspirin, respectively. The method was validated for precision, robustness and recovery. The limit of detection (LOD) and limit of quantitation (LOQ) was 0.5 μg/mL and 1 μg/mL for Amlodipine besylate and Atenolol and 1 μg/mL and 2 μg/mL for Aspirin, respectively. Statistical analysis showed that the method is repeatable and selective for the estimation of Amlodipine besylate, Atenolol and Aspirin.
Rasha A Shaalan etal.,developed a simple, sensitive, and reliable spectrofluorimetric method for the simultaneous determination of the two antihypertensive drugs; amlodipine besylate (AML) and valsartan (VAL) in their combined tablets. The method involved measurement of the native fluorescence at 455 nm (λEx 360 nm) and 378 nm (λEx 245 nm) for AML and VAL, respectively. Analytical performance of the proposed spectrofluorimetric procedure was statistically validated with respect to linearity, ranges, precision, accuracy, selectivity, robustness, detection, and quantification limits. Regression analysis showed good correlation between fluorescence intensity and concentration over the concentration ranges 0.2–3.6 and 0.008–0.080 µg mL−1 for AML and VAL, respectively. The limits of detection were 0.025 and 0.0012 µg mL−1 for AML and VAL, respectively. The proposed method was successfully applied for the assay of the two drugs in their combined pharmaceutical tablets with recoveries not less than 98.85%. No interference was observed from common pharmaceutical additives. The results were favourably compared with those obtained by a reference spectrophotometric method.
Nashwah Gadallah Mohamed etal.,developed a spectrophotometric method for simultaneous determination of amlodipine (Aml) and valsartan (Val) without previous separation. In this method amlodipine in methanolic solution was determined using zero order UV spectrophotometry by measuring its absorbency at 360.5 nm without any interference from valsartan. Valsartan spectrum in zero order is totally overlapped with that of amlodipine. First, second and third derivative could not resolve the overlapped peaks. The first derivative of the ratio spectra technique was applied for the measurement of valsartan. The ratio spectrum was obtained by dividing the absorption spectrum of the mixture by that of amlodipine, so that the concentration of valsartan could be determined from the first derivative of the ratio spectrum at 290 nm. Quantification limits of amlodipine and valsartan were 10–80 μg/ml and 20–180 μg/ml respectively. The method was successfully applied for the quantitative determination of both drugs in bulk powder and pharmaceutical formulation.
Mohammad Younus etal.,developed a simple, specific, accurate and precise Reverse Phase High Performance Liquid Chromatographic method for simultaneous estimation of Amlodipine Besylate (AB), Valsartan (VAT) and Hydrochlorothiazide (HTZ) in tablet dosage form on RP C-18 Column (Hypersil 250*4.6 mm) using Acetonitrile: Mixed Phosphate buffer (6.8 pH) (55:45) ( buffer was prepared by mixing the equal proportions of 0.01M Potassium dihydrogenphosphate and 0.001M Dipotassium hydrogenphosphate. pH was adjusted with Orthophosphoric acid) as mobile phase. The flow rate was 1.0 ml/min and effluent was monitored at 237nm. The retention time for VAT, HTZ and AB was found to be as 2.28, 2.99 and 4.57 respectively. Proposed method was validated for Precision, Accuracy, Linearity range, Robustness and Ruggedness.
Praveen S. Rajput etal.,developeda Simple and precise HPLC method for the simultaneous estimation of Ramipril and Amlodipine in pure drug and pharmaceutical dosage forms. The separation was carried out using C18 Column (250 × 4.6 mm i.d. 5 μm particle size), with mobile phase compressing of Acetonitrile, Sodium phosphate buffer and Methanol in the ratio of 50: 20:25 v/v/v, pH= 6.8 (pH adjusted with OPA). The flow rate was 0.8 ml/min and the detection was carried out using PDA detector at 210 nm. The retention times were 2.64 and 7.45 mins for Ramipril and Amlodipine respectively. Calibration curves were linear with correlation coefficient 0.998 and 0.996 over concentration range of 1 - 16 μg/ml for Ramipril and 0.2 – 3.2 μg/ml for Amlodipine respectively. Recovery was found in between 100.21% and 100.82% for Ramipril and Amlodipine respectively. Method was found to be reproducible with relative standard deviation (R.S.D) for intra and inter day precision less than 2%.
A. Lakshmana Rao etal.,developeda simple, reproducible and efficient reverse phase high performance liquid chromatographic method for simultaneous determination of lisinopril and amlodipine in tablets. A column having 150 × 4.6 mm i.d. in isocratic mode with mobile phase containing acetonitrile: phosphate buffer (60:40; adjusted to pH 3.0) was used. The flow rate was 0.5 ml/min and effluent was monitored at 215 nm. The retention time (min) and linearity range (μg/ml) for lisinopril and amlodipine were (4.111, 3.097) and (20-60, 10-30), respectively. The developed method was found to be accurate, precise and selective for simultaneous determination of lisinopril and amlodipine in tablets.
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