METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF AMLODIPINE AND INDAPAMIDE BY DIFFERENT SPECTROPHOTOMETRIC AND RP-HPLC METHODS IN BULK DRUG AND PHARMACEUTICAL FORMULATION

ABOUT AUTHORS:
Madhuri tadiparthi
Chalapathi institute of pharmaceutical sciences,
guntur, a.p, india.
tadiparthimadhuri@gmail.com

ABSTRACT:
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 (λ_maxof AMLO) and 279 nm (λ_maxof 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 (λ_maxof 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 (λ_maxof AMLO) and 600 nm (λ_maxof 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.

DRUG PROFILE

2.1. Amlodipine ^[27]

Drug name: Amlodipine besylate

Therapeutic category: Anti-hypertensive

Chemical structure:

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 (P^Ka =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 ^[28]

Drug name: Indapamide

Therapeutic category: Thiazide like diuretic

Chemical structure:

Description : White crystalline powder

Solubility: Slightly soluble in water and freely soluble in methanol Molecular

formula: C₁₆H₁₆ClN₃O₃S

Molecular weight : 365.835

Chemical name: 4-chloro-N-(2-methyl-2,3-dihydroindol-1-yl)-3-sulfamoyl-
benzamide

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.

Pharmacokinetics:
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.

LITERATURE REVIEW

3.1. Amlodipine
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. ^[29]

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 (λ_max 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.^[30]

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^-1for 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.^[31]

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.^[32]

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.^[33]

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 (λ_Ex 360 nm) and 378 nm (λ_Ex 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.^[34]

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.^[35]

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.^[36]

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%.^[37]

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.^[38]

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CH.M.M.Prasada Rao etal.,developed asimple, rapid and selective HPLC method for quantization of Amlodipine Besylate and Metoprolol succinate from bulk drug and pharmaceutical formulations using a mobile phase consisting mixture of 0.02 M phosphate buffer solution and Acetonitrile as 80:20 at the flow rate of 1 mL/min. An Inertsil ODS-CV column was used as stationary phase. The retention time of Amlodipine Besylate and Metoprolol succinate were 3.92 and 10.43 respectively. Linearity was observed in the concentration range of 2.5 to 15 μg/ml for Amlodipine Besylate and 25 to 150 μg/ml for Metoprolol succinate, with good linearity response greater than 0.999. Percent recoveries obtained for Amlodipine Besylate and Metoprolol succinate were 100.03 and 100.48% respectively. The proposed method is precise, accurate, selective and rapid for the simultaneous determination of Levofloxacin hemihydrate and Orinadozole.^[39]

S. D. Kayal etal.,developed a simple, accurate and precise method for the simultaneous determination of amlodipine besylate and telmisartan in bulk drug and pharmaceutical dosage by RP-HPLC method. Separation was performed on a 5μm Prontosil C18 column (250 × 4.6mm ID) with methanol: potassium dihydrogen phosphate buffer at pH 4.5 (75:25v/v) , flow rate of 1.4 ml/ min and UV detection wavelength 240 nm. The calibration of the method was performad by concentration range of 2-20μg/ml for amlodipine and 16-160 μg/ml for telmisartan. The validation of proposed method was carried out for accuracy , precision, ruggedness,specificityfor both amlodipine and telmisartan the method can be used for routine quality analysis of tittled drug in tablet formulation.^[40]

Pratap Y. Pawar etal.,developedTwo simple, rapid, precise and accurate spectrophotometric methods for determination ofAmlodipine Besylate (AMB) and Telmisartan (TEL) by simultaneous equation method and first order derivative method in combined dosage form. The simultaneous equation method is based on measurement of absorbance at 367 nm and 292 nm as two wavelengths selected for quantification of AMB and TEL. The second method is first order derivative based on the measurement of absorbance at 270nm and 295nm as two wavelength selected as for quantification of AMB and TEL. Both methods obeyed Beer’s law in the concentration range of 20-100 μg/ml for AMB and 5-30 μg/ml for TEL. The proposed methods were validated and can be used for analysis of combined dosage tablet formulation containing AMB and TEL.^[41]

K.S.Raut, etal.,developeda simple, accurate and reproducible spectrophotometric method for the simultaneous estimation of amlodipine besylate (AML) and telmisartan (TEL) in combined tablet dosage forms. The tablet is determined by the multi-wavelength technique, at the wavelengths of 360 nm and 298 nm over the concentration ranges of 15-75 mcg ml-1 and 1-10 mcg ml-1 with mean recovery more than 98% for both drug amlodipine besilate and telmisartan respectively. The results of the analysis were validated statistically and recovery studies were carried out as per ICH guidelines. Thus the proposed method can be successfully applied for simultaneous determination of amlodipine besilate and telmisartan in routine analysis work.^[42]

Pournima s. Patil,etal.,developed a RP?HPLC method for simultaneous estimation of Amlodipine Besylate (AML) and Olmesartan Medoxomil (OLM) from tablet. Best resolution of two drugs was achieved with the mobile phase having composition of acetonitrile and water in the ratio 60:40. The linearity response of the HPLC system for both OLM and AML was obtained over the range of 5?35 μg ml?1. Optimum retention time with greater resolution of the two drugs and internal standard eluting within six minutes was achieved with a flow rate of 1 ml min?1. After recording the spectra of the two drugs and internal standard, 248 nm was selected as suitable wavelength for estimation. The result of analysis showed excellent recoveries for both the drugs ranging from 99.75 % to 100.62 % for OLM and 98.91 % to 102.05 % for AML. Which suggested the good accuracy of the method. The results of analysis of tablet indicated that no interference due to common tablet excipients was observed with the developed method.^[43]

S. B. Wankhede etal.,developed Two UV Spectrophotometric and one reverse phase high performance liquid chromatography methods have for the simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form. First UV spectrophotometric method was a determination using the simultaneous equation method at 237.5 nm and 255.5 nm over the concentration range 10-50 μg/ml and 10-50 μg/ml, for amlodipine besilate and olmesartan medoxomil with accuracy 100.09%, and 100.22% respectively. Second UV spectrophotometric method was a determination using the area under curve method at 242.5-232.5 nm and 260.5-250.5 nm over the concentration range of 10-50 μg/ml and 10-50 μg/ml, for amlodipine besilate and olmesartan medoxomil with accuracy 100.10%, and 100.48%, respectively. In reverse phase high performance liquid chromatography analysis carried out using 0.05M potassuim dihydrogen phosphate buffer:acetonitrile (50:50 v/v) as the mobile phase and Kromasil C18 (4.6 mm i.d.×250 mm) column as the stationery phase with detection wavelength of 238 nm. Flow rate was 1.0 ml/min. Retention time for amlodipine besilate and olmesartan medoxomil were 3.69 and 5.36 min, respectively. Linearity was obtained in the concentration range of 4-20 μg/ml and 10-50 μg/ml for amlodipine besilate and olmesartan medoxomil, respectively. Proposed methods can be used for the estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form.^[44]

Pratap Y. Pawar etal.,developedTwo simple, accurate, precise, reproducible, requiring no prior separation and economical procedures for simultaneous estimation of amlodipine besylate and benazepril HCl in capsule dosage form. First method is simultaneous equation method; in this method 366 nm and 238 nm were selected to measure the absorbance of drugs at both wavelengths. The second method is Q?value analysis based on measurement of absorbance at 213 nm (as an iso?absorptive point) and 238 nm (λmax of benazepril HCl). At selected wavelength both drugs show linearity in a concentration range of 10-50 μg/ml. Both methods are validated as per ICH guideline. The proposed methods are recommended for routine analysis since it is rapid, simple, accurate, and sensitive and specific as it does not required heating and organic solvent for extraction.^[45]

RB Kakde etal.,developed a Simple spectrophotometric method for simultaneous estimation of amlodipine besylate and bisoprololfumarate in combined dosage form. The method employed simultaneous equation method for analysis using 10%methanol as a solvent. The two wavelengths 222 nm and 365 nm were selected for estimation of bisoprolol fumarate andamlodipine besylate respectively. Linearity was observed in the concentration range of 5-100 μg/ml for both the drugsamlodipine besylate and bisoprolol fumarate. The recovery studies ascertained the accuracy of the proposed method andthe results were validated as per ICH guidelines. The method can be employed for estimation of pharmaceuticalformulations with no interference from any other excipients and diluents.^[46]

V.C.Chandnani etal.,developedTwo simple spectrophotometric methods for simultaneous determination ofAmlodipine besylate and Nebivolol hydrochloride in tablet formulation. The first method is Absorbance correctionmethod based on determination of Amlodipine besylate at 365 nm using its absorptivity value and Nebivololhydrochloride at 280 nm after deduction of absorbance due to Amlodipine besylate. The second method is based onAbsorbance ratio in which wavelengths selected were 269 nm, an isoabsorptive point and 280 nm as λmax of Nebivololhydrochloride. The methods were validated in terms of accuracy, precision, ruggedness and specificity. The methods canbe routinely adopted for quality control of these drugs in tablet.^[47]

Ay?egül gölcüa etal.,developed a coloured ion-pair complex formation reaction among amlodipine and acid-dye bromophenol blue at pH 3.2 for the colorimetric determination of the drug. The complex formed was extracted into chloroform and the maximum absorbance of the solution was measured at 414 nm against blank. The calibration curve calculated obeys Beer's law over the concentration range of 6-30 μg/ml and the regression equation was A=0.055C-0.018 (r=0.9997). The recovery of the drug from a commercial tablet was 100.7 % of the label claim with a relative standard deviation of 1.24 %. The results were compared with those of the spectrophotometric method currently used by the manufacturer of the tablets and no significant difference was found.^[48]

Sushant K Shrivastava etal., developed and validated a simple and rapid isocratic reversed-phase high-performance liquid chromatographic method (RP-HPLC) for the simultaneous estimation of Amlodipin and Telmisartan in combined dosage form. The chromatographic separation was achieved by using mobile phase acetonitrile and 0.05M sodium dihydrogen phosphate buffer (60:40) adjusted to p^H6.0, a C-18 column, perfect target ODS3 (150 mm?4.6 mm i.d., 5 µm). The mobile phase was pumped at a flow rate of 0.8 ml/min and the eluents were monitered at 254 nm. Retention times were 4.0 min and 8.2 min for Amlodipine and Telmisartan respectively.^[49]

3.2. Indapamide
Nadia f. Youssef etal.,developed three sensitive spectrophotometric,spectrofluorimetric, and densitometric methods for the determination of indapamide. The first and second methods are based on the oxidative coupling reaction of indapamide with 3-methyl-2-benzothiazolinone hydrazone HCl (MBTH) in the presence of cerium(IV) ammonium sulfate in an acidic medium. The absorbance of the reaction product is measured at the _max, 601 nm. With the same reaction, indapamide is determined by its quenching effect on the fluorescence of excess cerous ions at the emission _max, 350 nm, and the excitation at _max, 300 nm. The reaction conditions were optimized, and Beer’s law was obeyed for indapamide at 1.2–9.6 _g/mL with mean recoveries of 99.92 _ 0.83 and 99.97 _ 1.11%, respectively. The third method, a stability-indicating densitometric assay, was developed for the determination of indapamide, using toluene–ethyl acetate– glacial acetic acid (69 + 30 + 1, v/v/v) as the developing system and scanning at the _max , 242 nm, in the presence of the degradation product and related substance; for the indapamide concentration range of 0.6–6 _g/spot, the mean recovery was 99.73 _ 0.71%. The proposed methods were successfully applied to the determination of indapamide in bulk powder and commercial tablets, and the results of the analysis agreed statistically with those obtained with the official method.^[50]

Jain DS, subbaiah G etal.,developed a highly precise and sensitive method for the estimation of indapamide in human whole blood using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method developed is validated in human whole-blood matrix, with a sensitivity of 0.5 ng/ml as lower limit of quantification. The procedure for the extraction of indapamide and glimepiride as internal standard (IS) involves haemolysis and deprotienation of whole blood using ZnSO(4) followed by liquid-liquid extraction using ethyl acetate. The sample extracts after drying were reconstituted and analysed by LC-MS/MS, equipped with turbo ion spray (TIS) source, operating in the positive ion and selective reaction monitoring (SRM) acquisition mode to quantify indapamide in human whole blood. The mean recovery for indapamide was 82.40 and 93.23% for IS. The total run time was 2.5 min to monitor both indapamide and the IS. The response of the LC-MS/MS method for indapamide was linear over the range of 0.5-80.0 ng/ml with correlation coefficient, r>or=0.9991. The coefficient of variance (% CV) at 0.5 ng/ml was 4.02% and the accuracy was well within the accepted limit of +/-20% at 0.5 ng/ml and +/-15% at all other concentrations in the linear range. This method is fully validated for the accuracy, precision and stability studies.^[51]

Zhao L, Gu S etal.,aimed to compare two methods which were based on liquid chromatography with ultraviolet detection (LC-UV) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively, to determine indapamide (CAS 26807-65-8) and to apply them to bioequivalence studies. The universal parameters, including selectivity, linearity, precision, and quantification limit, served as gold standard for the comparison of the two methods. As a result, the two methods were both very consistent and reliable. Furthermore, the LC-MS/MS method required only one-fifth the blood volume needed by the other method and was approximately 25 times more sensitive than the other method. The total run time of the LC-MS/MS method was 3.5 min per sample as opposed to 11 min for the other method. Forty healthy male Chinese volunteers were selected as subjects. One half were orally administrered 2.5 mg indapamide immediate release tablets while the other half were orally administered 1.5 mg indapamide sus-tained release coated tablets. The collected blood samples were determined with the two methods described above. The pharmacokinetic parameters were determined using a noncompartmental method. For the bioequivalence studies, the pharmacokinetic parameters acquired here were in line with the literature and parameters met the criteria set by the State Food and Drug Administration of China (SFDA) for bioequivalence study, indicating that generic drugs are bioequivalent to branded drugs. The present study suggests that the two methods based on LC-UV and LC-MS/MS were suitable for bioavailability studies of indapamide with different pharmaceutical formulations.^[52]

Albu F, Georgi?? C. etal.,developed a method in which Indapamide and internal standard (5-chloro-2-methoxy-N-[2-(4-sulphamoylphenyl)ethyl]benzamide) were isolated from plasma by a single step liquid-liquid extraction in t-butyl methyl ether. The chromatographic separation was achieved on a reversed-phase C(18) monolithic column with a mobile phase consisting in a methanol/aqueous 0.1% formic acid mixture and a flow rate of 0.8 ml/min, in isocratic conditions, within 11 min. Target compounds were transferred in an ion trap analyzer via an atmospheric pressure electrospray interface (AP-ESI). The mass analyzer was used in a selected reaction monitoring (SRM) mode, in order to enhance on detection selectivity. Whole method produces quantitation limit for indapamide of 1 ng/ml. Method was successfully applied to assess bioequivalence of two sustained release marketed pharmaceutical formulations of indapamide 1.5 mg coated tablets, carried-out in a single/multiple doses, randomized design.^[53]

Ioan Tomutaa,etal.,described the development and application of NIR-chemometric methods for active content assay and pharmaceutical characterization (granulometric analysis and flowability assessment) of indapamide powder blends for tabletting. Indapamide powder blends were prepared and their NIR spectra were recorded in reflectance mode. Partial least-squares (PLS) regression followed by leave-one-out cross-validation was used to develop calibration models for predicting the indapamide content and pharmaceutical properties. The method for indapamide assay was validated in terms of trueness, precision, accuracy. The near infrared based property predictions were compared with the reference method results and no significant differences were found between the reference and predicted characteristics. The developed NIR-chemometric methods can be useful tools for prediction of active content, granulometric properties and parameters related to flowability of pharmaceutical powders.^[54]

Tarkase Kailash N, etal.,developed two new, precise and simple UV spectrophotometric methods for estimation of Indapamide from bulk andtablet formulation in phosphate buffer 7.4. The drug obeyed the Beer’s law with correlation coefficient 0.996 and0.998 respectively for Method I and Method II. It showed absorption maxima at 240 nm and 223 nm respectively formethod I and Method II; in phosphate buffer 7.4. The linearity was observed between 5 –40 μg/ml. The results ofanalysis were validated by recovery studies, accuracy, precision, LOD, LOQ and ruggedness. The method wasfound to be simple, accurate, precise, economical and robust.^[55]

Jyoti B. Pai etal.,developeda simple, precise, specific and accurate RP-HPLC method for the determination of Indapamide in bulk and pharmaceutical dosage forms. Chromatography was performed on a supelco RP C-18 Column (25cm x 4.6 mm i.d.,particle size 5 μm) with o-phosphoric acid (0.05%) buffer of pH 3.0 and Acetonitrile in the ratio of 60:40 (v/v) as a mobile phase at a flow rate of 1 ml/ min. Detection was performed at 240 nm. The retention time of Indapamide was found to be 6.76±0.0145 min. By adoption of this procedure Indapamide is eluted completely. Linear calibration plots were obtained between 10-100μg/ml. The method of analysis was used for quantification in pharmaceutical preparations with a coefficient of variation <2%. Results of analysis were validated statistically and by recovery studies. The method was validated according to the ICH guidelines with respect to specificity, linearity, accuracy, precision and robustness.^[56]

Nevin Erk, etal.,developed a new sensitive, simple, rapid and precise reversed-phase high performance liquid chromatographic (HPLC) and two spectrophotometric methods have been developed for resolving binary mixture of perindopril and indapamide in the pharmaceutical dosage forms. The first method is based on HPLC on a reversed-phase column using a mobile phase of phosphate buffer pH 2.4 and acetonitrile (7:3 v/v) was used. Linearity range for perindopril and indapamide was 5.0–70.0 and 8.0–35.0 _g ml−1. In the second method, the first derivative spectrophotometry with a zero-crossing technique of measurement is used for the simultaneous quantitative determination of perindopril and indapamide in binary mixtures without previous separation step. Linear calibration graphs of first derivative values at 225.7 and 255.4 nm for peridopril and indapamide, respectively. The third method is based on ratio derivative spectrophotometry, the amplitudes in the first derivative of the ratio spectra at 226.5 and at 255.3 nm were selected to determine perindopril and indapamide in the binary mixture. All the proposed methods showed good linearity, precision and reproducibility.^[57]

Rajesh Tiwari, etal.,developed aisocratic reversed-phased HPLC method for simultaneous determination of Indapamide and Perindopril. In the first stage of method development, pH value of the water phase, percentage of methanol, temperature of the column and flow rate of the mobile phase were investigated using fractional factorial design. This work is concerned with application of simple, accurate, precise and highly selective reverse phase high performance liquid chromatographic (RP-HPLC) method for simultaneous estimation of Indapamide and Perindopril in combined dosage form. Chromatographic separation was achieved isocratically at 25°C ± 0.5°C on phase Inertsil ODS C8 [250 mm x 4.6mm] column with a mobile phase composed of Phosphate buffer with pH 2.5 and methanol in the ratio of 40:60 at flow rate of 0.5 ml/min. Detection is carried out using a UV-PDA detector at 215nm. The retention time of Perindopril and Indapamide at 5.08 min and 6.91 min respectively. The correlation coefficients for all components are close to 1. The developed method was validated according to ICH guidelines and values of accuracy, precision and other statistical analysis were found to be in good accordance with the prescribed values.^[58]

P.S. Jain, etal.,developed a dissolution method with high performance liquid chromatography (HPLC) analysis was validated for perindopril erbumine and indapamide in combination tablet formulation. The method was validated to meet requirements for a global regulatory filing and this validation included specificity, linearity, accuracy, precision, range, robustness and solution stability studies. The dissolution method, which uses a USP apparatus 1 with basket rotating at 100 rpm, 1000 ml of phosphate buffer, pH 6.8, as the dissolution medium, and reversed-phased HPLC was carried out at 50 °C on a 4.6 mm×250 mm×5 μm cyano column that contained USP packing L1 with acetonitrile:buffer 40:60 (v/v), pH 2.8, as mobile phase. UV detector was set at 225 nm. The method was found to be selective, linear, accurate and precise in the specified ranges. Intra-day and inter-day variability for method was <2% RSD. This method was successfully used for quantification of perindopril erbumine and indapamide combination tablet formulations.^[59]

Juddy Joseph, etal.,developed a simple, specific, accurate and reproducible isocratic reversed phase high performance liquid chromatographic (RP-HPLC) method which is subsequently validated using ICH recommendations for the simultaneous estimation of Perindopril Erbumine (PE) and Indapamide (ID) in combined tablet dosage form. The determination was carried for a runtime of 20min at 40?C on Inertsil ODS-3V column having 250mm x 4.6mm i.d. with 5μm particle size and potassium dihydrogen phosphate buffer adjusted to pH 3.0 using ortho phosphoric acid and acetonitrile (60:40 v/v) was used as eluent at a constant flow rate of 1.0ml/min with UV detection wavelength of 215nm. The retention time of PE and ID was about 11.9 and 4.9min with correlation coefficient of 0.9992 and 0.9990 respectively. The linearity was established at 8-24μg/ml for PE and 2.5-7.5μg/ml for ID and the mean recovery for both drugs were found to be 100.3% at a load volume of 50μl.^[60]

Mrinalini C Damle, etal.,developed a new sensitive, simple, rapid and precise high performance thin layer chromatographic (HPTLC) method has been developed for simultaneous determination of both the drugs in Pharmaceutical dosage form. The method was based on the separation of two drugs on plates precoated with silica gel 60 F254. The mobile phase used was Dichloromethane : Methanol : Glacial acetic acid in the ratio of 9.5:0.5:0.1 v/v/v. Both the drugs showed considerable absorbance at 215 nm. Linearity was obtained in the concentration range of 1-5 μg/band and 100-500 ng/band for perindopril and indapamide respectively. The method has been successfully applied to tablets and was validated according to ICH Harmonized Tripartite guidelines.^[61]

Vipul Prajapati, etal.,developed a simple and accurate methods to determine Indapamide, in pure powder form, and validated using liquid chromatography (LC). The LC separation was achieved on a Inertsil ODS 3V, 5μm, 150 x 4.6 mm,5μ in the isocratic mode using Mixture of 7 volume of acetoinitrile, 20 volume of Tetrahydrofuran and 73 volumes of a 1.5g/l solution of Triethylamine adjusted pH 2.8 with ortho-phosphoric acid at a flow rate of 1.4 ml/min. the methods were performed at 305nm; In LC method, quantification was achieved with PDA detection over the concentration range of 80 to 120 μg/ml. The methods were validated, and the results were compared statistically. They were found to be simple, accurate, precise, and specific. The methods were successfully applied for the determination of Indapamide in pure powder form without any interference from common excipients.^[62]

Patel Amit R, etal.,madean approach of forced degradation study was successfully applied for the development of a stability indicating assay method for simultaneous determination of Telmisartan and Indapamide in a formulation in the presence of its degradation products. The method showed adequate separation of Telmisartan and Indapamide from their associated main impurities and degradation products. Separation was achieved on an Amazon C18, 5micron, 150 x 4.6 mm the mobile phase (Buffer: Acetonitrile: Methanol) (45+25+30) KH₂PO₄& Triethaylamine P^H 3.0 with ortho phosphoric acid buffer flow rate of 1 mL/min and UV detection at 285 nm. Comprehensive stress testing of Telmisartan and Indapamide Rt= 4.7 min, 10.7 min was according to the International Conference on Harmonization (ICH) guideline Q1A (R2). The method was validated in terms of linearity, precision, accuracy, Specificity, robustness, and solution stability. The linearity of the proposed method was investigated in the range of 6-22.5 microg/mL (r2 = 0.999) for Telmisartan and 11.2-42 microg/mL (r² = 0.9997) for Indapamide.^[63]

Deepmala Manore, etal.,developed aSimple, fast and precise simultaneous spectrophotometric methods for simultaneous estimation of two-component drug mixture of Telmisartan and Indapamide in combined capsule dosage form. The proposed first method is based on the formation and solving of simultaneous equations using 294.0 and 242.0 nm as two analytical wavelengths. The second method is absorbance ratio method, which uses 254.0 and 294.0 nm as two analytical wavelengths. Third method is based on multicomponent mode method. Developed method was applied to laboratory mixture and its marketed formulation. These methods were statistically validated and recovery studies confirmed the accuracy of the proposed method.^[64]

Neha Manish Munot, etal.,developeda simple, precise, accurate and reproducible Reverse Phase High Performance Liquid Chromatographic method for simultaneous estimation of Telmisartan and Indapamide. The method was carried out on a Hypersil Gold column [250×4.6mm;5μ] with a mobile phase consisting of Acetonitrile:0.1M Potassium dihydrogen phosphatebuffer [60:40v/v] at a flow rate 1.0ml/min. Detection was carried out at 237nm. The retention time of telmisartan and indapamide was 7.84, 3.38 min respectively. The developed method was validated for accuracy, precision, linearity, limit of detection, limit of quantification and solution stability as per ICH guidelines Q2 [R1]. The proposed method can be used for simultaneous estimation of these drugs in bulk drugs and formulations.^[65]

Venkateswarlu Ponneri, etal.,developed a simple, sensitive and precise high performance liquid chromatographic method for the analysis of atenolol and indapamide with UV detection at 231 nm, has been developed and used for the determination of these compounds in pharmaceutical dosage forms, in human blood and in human milk. The compounds were well separated on a Hypersil BDS C18 reversed?phase column with mobile phase consisting of, pH = 3.5, 0.01 M potassium dihydrogen orthophosphate buffer?acetonitrile (60:40; v:v) at a flow rate of 1.0 mL/min. The method showed good linearity in the range of 5?30 μg/mL for atenolol and 0.25?1.50 μg/mL for indapamide. Both the drugs were eluted within 5 minutes and give sharp peak with high theoretical plate count and low tailing factor. The reaction time for atenolol and indapamide was found to be 2.29 and 3.83 min, respectively. The validation was carried according to International Conference on Harmonisation (ICH) guidelines. In linearity curve correlation coefficients for atenolol and indapamide were found to be 0.9995 and 0.9991, respectively. The percent recovery was 99.81?100.02 for atenolol and indapamide indicating accuracy and reliability of method. So the method can be used for estimation of these drugs in tablet dosage form, human blood and milk.^[66]

G.Tulja Rani, etal.,developed a simple, fast, precise, selective and accurate RP-HPLC method and validated for the simultaneous determination of atenolol and indapamide from bulk and formulations. Chromatographic separation was achieved isocratically on a Waters C18 column (250×4.6 mm, 5 μ particle size) using a mobile phase, methanol and water (adjusted to pH 2.7 with 1% orthophosphoric acid) in the ratio of 80:20. The flow rate was 1 mL/min and effluent was detected at 230 nm. The retention time of atenolol and indapamide were 1.766 min and 3.407 min. respectively. Linearity was observed in the concentration range of 12.5-150 μg/mL for atenolol and 0.625-7.5 μg/mL for indapamide. Percent recoveries obtained for both the drugs were 99.74-100.06% and 98.65-99.98%, respectively. The method was validated according to the ICH guidelines with respect to specificity, linearity, accuracy, precision and robustness. The method developed can be used for the routine analysis of atenolol and indapamide from their combined dosage form.^[67]

P.V.Pawar, etal.,developedan UV spectrophotometric method using simultaneous equation for the simultaneous determination of Atenolol and Indapamide in a binary mixture. In the Proposed method, the signals were measured at 225.0 nm and 240.0 nm corresponding to absorbance maxima of Atenolol and Indapamide in methanol respectively. Linearity range was observed in the concentration range of 6-30 μg/ml for Atenolol and 0-10 μg/ml for Indapamide. Concentration of each drug was obtained by using the absorptivity values calculated for both drugs at two wavelengths, 225.0 nm and 240.0 nm and solving the simultaneous equation. Developed method was applied to laboratory mixture and its Pharmaceutical formulation. The method was validated statistically and recovery study was performed to confirm the accuracy of the method. The method was found to be rapid, simple, accurate and precise.^[68]

Naveen Kadian, etal.,developed a simple, sensitive, accurate and effective Reverse Phase High-Performance liquid chromatographic (RP- HPLC) method for the determination of Atenolol and Indapamide simultaneously in the tablets. The analysis was resolved by using a mobile phase methanol: water (60:40) with 0.1%v/v of Ammonium Hydroxide, at a flow rate of 1ml/min on an isocratic HPLC system consisting of SPD-20 A/20AV UV-VIS detector using C18 column at a wavelength of 260 nm. The retention time was found to be 7.5 and 8.9 min respectively. Linearity was established for Atenolol and Indapamide in the range of 4.5-500 µg/ml and 0.225-25µg/ml respectively.^[69]

David S.M. Ribeiro, etal.,developed The oxidative coupling reaction between aromatic amines and 3-methylbenzothiazolin-2-one hydrazone (MBTH) in the presence of cerium(IV) with quantitative analytical purposes. In the present work, the oxidative coupling reaction of indapamide (an oral antihypertensive diuretic drug) with MBTH in the presence of cerium(IV) was evaluated using distinct reaction approaches and was implemented in an automated multipumping flow system. The developed methodology was applied in the spectrophotometric control of the drug in pharmaceutical formulations. This work revealed the formation of products with different reaction kinetics, chemical stabilities and absorbance spectra, depending on the sequence of addition of the reagents. By exploiting a specific sequence in the addition of reagents, the proposed automatic system allowed the rapid quantification of indapamide in pharmaceutical formulations, with a determination rate of about 25 h⁻¹, and a relative deviation under 2.1% when comparing with the reference procedure. Detection limit was approximately 1 mg L⁻¹.^[70]

Singhvi and Goyal developed two simple, accurate, fast and economical methods for the quantitative estimation of aceclofenac and indapamide from their respective tablet formulation using Folin-Ciocalteu reagent. Aceclofenac forms a blue colored chromogen with the reagent, which shows absorbance maxima at 642.6 nm and linearity in the concentration range of 80-160 µg/ml of drug. Indapamide forms a green colored chromogen with the reagent, which shows absorbance maxima at 783.2 nm and linearity in the concentration range of 2-12 µg/ml of drug. The results of analysis for both the methods were validated statistically and by recovery studies.^[71]

AIM AND OBJECTIVE
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 drug marketed by Servier, generally used in the treatment of hypertension, as well as decompensated cardiac failure. Amlodipine and Indapamide combination is available as marketed formulation and prescribed by physician for the treatment of hypertension. The brands available in the market are NATRILAM (SERDIA) and AMLODAC-C tablets.

Literature survey reveals that different spectroscopic and chromatographic methods were developed and validated for Amlodipine and Indapamide individually and in combination with other drugs.

To the best of my knowledge no methods were reported for simultaneous estimation of Amlodipine and Indapamide in bulk drug and pharmaceutical formulation, previous to my work.

Thus, efforts were made to develop simple, accurate and precise methods for simultaneous estimation of Amlodipine and Indapamide by different spectroscopic and RP-HPLC method in bulk drug and pharmaceutical formulation.

The main aim of the present study was to develop UV, Colorimetry, RP-HPLC methods and validate as per ICH guidelines.

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MATERIALS AND INSTRUMENTS

5.1. INSTRUMENTS
*Shimadzu corporation Japan-electronic balance type AX200, 1 mg sensitivity.
*LABINDIA- UV 3092 UV/VIS spectrophotometer.
*Bruker –10048657, Alpha-T FTIR.
*Shimadzu- Model LC20AT, Spin chrome soft ware HPLC.
*Oscar ultrasonic’s – ultra probe sonicator.
*Vacuum filtration unit.
*Potentiometer-Titrasys 352.
*Colorimeter- ELICO SL 27- Spectrophotometer.

5.2. CHEMICALS AND REAGENTS
*HPLC grade water.
*HPLC grade Methanol.
*Acetonitrile.
*Triethylamine buffer of PH 3.0.
*MBTH reagent.
*Distilled water.

METHODOLOGY
6.1. METHOD DEVELOPMENT BY UV SPECTROSCOPY

6.1.1. SIMULTANEOUS EQUATION METHOD

6.1.1.1. Preparation of standard solutions

Preparation of standard solution of Amlodipine: Standard AMLO (10 mg) was dissolved in 10 ml of methanol. Further dilutions were prepared in distilled water.

Preparation of standard solution of Indapamide: Standard INDA (10 mg) was dissolved in 10 ml of methanol. Further dilutions were prepared in distilled water.

6.1.1.2. Preparation of sample solution: 20 tablets were powdered and an amount equivalent to 100 mg AMLO was weighed and dissolved in 25 ml methanol and the volume was made up to 100 ml with distilled water. Solutions were filtered using whatmann filter paper grade 1. Appropriate dilutions were prepared in distilled water taking suitable aliquots of the clear filtrates and subjected to analysis by the proposed method.

6.1.1.3. Procedure:
Diluted standard solution of AMLO (40 μg/ml) (Fig No.6) and INDA (15 μg/ml) (Fig No.7) were scanned over the range of 260-400 nm to select the λ_max. Wavelength (λ_max) of 365 nm and 279 nm was selected for analysis of these drugs by simultaneous equation method. The overlay UV spectrum of AMLO and INDA was given in (Fig No.5). The linearity was determined and found to be in the range of 10-50 μg/ml and 5-25 μg/ml of AMLO and INDA respectively. For quantification of AMLO and INDA simultaneously, absorptivity coefficient of both drugs at each wavelength were determined from different dilutions (5 independent determinations) of various standard solutions of AMLO and INDA drugs within Beer’s law concentration range limits. By using absorptivity coefficient, a set of two simultaneous equations 1 and 2 were obtained for determination of concentration of AMLO and INDA

C_x = (A₂ay₁-A₁ay₂)/ (ax₂ay₁-ax₁ay₂) -------------- 1

C_Y = (A₁ax₂-A₂ax₁)/ (ax₂ay₁-ax₁ay₂) -------------- 2

C_X = concentration of AMLO

C_Y = concentration of INDA

A₁=absorbance of samples at 365 nm.

A₂= absorbance of samples at 279 nm.

ax₁ is the absorptivity of AMLO at 365nm.

ax₂ is the absorptivity of AMLO at 279 nm.

ay₁ is the absorptivity of INDA at 365 nm.

ay₂ is the absorptivity of INDA at 279nm.

6.1.1.4. Method validation

6.1.1.4.1. Linearity
Each concentrations of working solution of AMLO and INDA were observed under UV system. The absorbances were determined for each concentration of the AMLO and INDA independently. The calibration curves were obtained with linear response of AMLO (Table No.4) and INDA (Table No.5) in the concentration ranges of 10-50 μg/ml, and 5- 25 μg/ml respectively. The regression equations of calibration curves were y=0.013x and y=0.003x for AMLO at 365 and 279 nm and y=0.024x and y=0.004x for INDA at 279 and 365 nm by simultaneous equation method respectively (Fig No.8 and Fig No.9).

6.1.1.4.2. Precision
The precision of the method was established by carrying out the determination of concentration of AMLO and INDA by repeatability (Intra-day) and intermediate precision (Inter-day precision). The intra-day precision was performed on the same day on same sample at particular time interval (Table No.6).The inter-day precision was performed on the three consecutive days on same sample solution (Table No.7 to Table No.9). The results were obtained and %RSD was calculated in order to establish the precision of the method.

6.1.1.4.3. Accuracy
The accuracy of method was determined by carrying out recovery studies at three different levels (80, 100 and 120) on the basis of the label claim. At each level, three determinations were performed simultaneously and % recovery and % RSD was calculated to prove the accuracy of the method. Data of accuracy study is given in table (Table No.10 and Table No.11).

6.1.1.4.4. Limit of detection and Limit of quantification
The limit of detection and the limit of quantification of the drugwere derived by calculating the signal-to-noise ratio (S/N,i.e.,3.3 for LOD and 10 for LOQ) using the following equations designated by international conference on harmonization (ICH) guidelines.

LOD=3.3×σ/s

LOQ=10×σ/s

Where σ = the standard deviation of the response and
S = slope of the calibration curve

The results of LOD and LOQ are tabulated in (Table No.12).

6.1.1.5. Analysis of commercial formulation
The tablet sample solution was subjected to analysis by simultaneous equation method. Absorbances of sample solutions were recorded at 365 nm (λ_max of AMLO) and 279 nm (λ_max of INDA) and concentration of two drugs in the sample were determined by using equations 1 and 2. The values of % recovery from formulation areshown in the (Table No.13).

6.1.2. FIRST ORDER DERIVATIVE SPECTROSCOPIC METHOD

6.1.2.1. Preparation of standard solutions
Preparation of standard solution of Amlodipine: Standard AMLO (10 mg) was dissolved in 10 ml of methanol. Further dilutions were prepared in distilled water.

Preparation of standard solution of Indapamide: Standard INDA (10 mg) was dissolved in 10 ml of methanol. Further dilutions were prepared in distilled water.

6.1.2.2. Preparation of sample solution
20 tablets were powdered and an amount equivalent to 100 mg AMLO was weighed and dissolved in 25 ml methanol and the volume was made up to 100 ml with distilled water. Solutions were filtered using whatmann filter paper grade 1. Appropriate dilutions were prepared in distilled water taking suitable aliquots of the clear filtrates and subjected to analysis by the proposed method.

6.1.2.3. Procedure
The solutions of standard AMLO and INDA were prepared in the range of 10-50 μg/ml and 5-25 μg/ml respectively. The absorption spectra of the solutions of AMLO and INDA were recorded in the range of 260 nm to 400 nm and were stored in the memory of the instrument and transformed to first derivative. From the overlain spectra of both drugs (Fig No.10),wavelengths were selected. At 276 nm, INDA is having zero crossing point (Fig No.12) and AMLO can be determined. At 337 nm, AMLO is having zero crossing point (Fig No.11) and INDA can be determined. The amplitudes at 276 nm were plotted against respective concentrations of AMLO and the amplitudes at 337 nm were plotted against the respective concentrations of INDA for the preparation of calibration graph.

6.1.2.4. Method validation

6.1.2.4.1. Linearity
Appropriate volume of aliquot from AMLO and INDA standard stock solution was transferred to volumetric flask of 10 ml capacity. The volume was adjusted to the mark with distilled water to give solutions containing 10-50 µg/ml AMLO (Table No.14) and 5-25 µg/ml INDA (Table No.15). All D1 Spectra were recorded using above spectrophotometric condition. D1 absorbance at 276 nm and 337 nm were recorded for AMLO and INDA, respectively. Calibration curves were constructed by plotting absorbance versus concentrations for both drugs. Straight line equations were obtained from these calibration curves (Fig No.13 and Fig No.14).

6.1.2.4.2. Precision
The repeatability was evaluated by assaying 6 times of sample solution prepared for assay determination. The intra-day and inter-day precision study of AMLO and INDA was carried out by estimating median concentration of AMLO (30 µg/ml) and INDA (15 µg/ml), 3 times on the same day (Table No.16) and on 3 different days (first, second, third) (Table No.17 to Table No.19) and the results were reported in terms of %RSD.

6.1.2.4.3. Accuracy
Accuracy was assessed by determination of the recovery of the method by addition of standard drug to the pre-quantified sample preparation at 3 different concentration levels 80, 100 and 120 %, taking into consideration percentage purity of added bulk drug samples. Each concentration was analyzed 3 times and average recoveries were measured. % Recovery and % RSD was calculated to prove the accuracy of the method. Data of accuracy study is given in (Table No.20 and Table No.21).

6.1.2.4.4. Limit of detection and Limit of quantification
The limit of detection and the limit of quantification of the drug was derived by calculating the signal-to-noise ratio (S/N,i.e.,3.3 for LOD and 10 for LOQ) using the following equations designated by international conference on harmonization (ICH) guidelines.

LOD=3.3×σ/s

LOQ=10×σ/s

Where σ = the standard deviation of the response and

S = slope of the calibration curve

The results of LOD and LOQ are tabulated in (Table No.22)

6.1.2.5. Analysis of commercial formulation
The tablet sample solution was subjected to analysis by first order derivative spectroscopic method. Absorbances of sample solutions were recorded at 276 nm (zero crossing point of INDA) and 337 nm (zero crossing point of AMLO). The solution of required dilution was analysed by the proposed method. The values of % recovery from formulation areshown in (Table No.23).

6.1.3. Q ABSORPTION RATIO METHOD

6.1.3.1. Preparation of standard solutions
Preparation of standard solution of Amlodipine: Standard AMLO (10 mg) was dissolved in 10 ml of methanol. Further dilutions were prepared in distilled water.

Preparation of standard solution of Indapamide: Standard INDA (10 mg) was dissolved in 10 ml of methanol. Further dilutions were prepared in distilled water.

6.1.3.2. Preparation of sample solution
20 tablets were powdered and an amount equivalent to 100 mg AMLO was weighed and dissolved in 25 ml methanol and the volume was made up to the 100 ml with distilled water. Solutions were filtered using whatmann filter paper grade 1. Appropriate dilutions were prepared in methanol taking suitable aliquots of the clear filtrates and subjected to analysis by the proposed method.

6.1.3.3. Procedure
From the overlain spectra of AMLO and INDA, 365 nm was taken as λ_max for AMLO and 312 nm as iso-absorptive point at which the two drugs have same absorbance (Fig No.15). Series of different concentrations in range of 10-50 μg/ml for AMLOand 5-25 μg/ml for INDA were prepared from the working standard solutions. The calibration curves were plotted at 365 and 312 nm. The absorptivity (A1%, 1 cm) of both the drugs at both the wavelengths was determined. The concentration of sample was measured by this equation,

Where, A₁& A₂ are the absorbance of the mixture at 312 nm & 365nm respectively; aX₁ and aY₁are absorptivities of AMLO and INDA respectively at312nm; aX₂ and aY₂are absorptivities of AMLO and INDA respectively at365 nm; Q_M=A₂/A₁, Q_X= aX₂/ aX₁and

Q_Y= aY₂/ aY₁.

6.1.3.4. Method validation

6.1.3.4.1. Linearity
The Q absorption ratio method shows good linearity in the concentration range of 10-50 µg/ml and 5-25 µg/ml for AMLO (Table No.24) and INDA (Table No.25) respectively. The calibration graphs of AMLO and INDA were given in (Fig No.16 and Fig No.17).

6.1.3.4.2. Precision
The repeatability was evaluated by assaying 6 times of sample solution prepared for assay determination. The intra-day and inter-day precision study of AMLO and INDA was carried out by estimating median concentration of AMLO (30 µg/ml) and INDA (15 µg/ml), 3 times on the same day (Table No.26) and on 3 different days (first, second, third) (Table No.27 to Table No.29) and the results are reported in terms of %RSD.

6.1.3.4.3. Accuracy
Accuracy was assessed by determination of the recovery of the method by addition of standard drug to the pre-quantified sample preparation at 3 different concentration levels 80, 100 and 120 %, taking into consideration percentage purity of added bulk drug samples. Each concentration was analyzed 3 times and average recoveries were measured. % Recovery and % RSD was calculated to prove the accuracy of the method. Data of accuracy study is given in (Table No.30 and Table No.31).

6.1.3.4.4. Limit of detection and Limit of quantification:
The limit of detection and the limit of quantification of the drug was derived by calculating the signal-to-noise ratio (S/N,i.e.,3.3 for LOD and 10 for LOQ) using the following equations designated by international conference on harmonization (ICH) guidelines.

LOD=3.3×σ/s

LOQ=10×σ/s

Where σ = the standard deviation of the response and
S = slope of the calibration curve

The results of LOD and LOQ were given in (Table No.32).

6.1.3.5. Analysis of commercial formulations
The tablet sample solution was also subjected to analysis by q absorption ratio method. Absorbances of sample solutions were recorded at 312 nm (isobestic point) and 365 nm (λ_max of AMLO). The solution of required dilution was analysed by the proposed method. The values of % recovery from formulation areshown in (Table No.33).

6.1.4. DUAL WAVELENGTH METHOD

6.1.4.1. Preparation of standard solutions
Preparation of standard solution of Amlodipine: Standard AMLO (10 mg) was dissolved in 10 ml of methanol. Further dilutions were prepared in distilled water.

Preparation of standard solution of Indapamide: Standard INDA (10 mg) was dissolved in 10 ml of methanol. Further dilutions were prepared in distilled water.

6.1.4.2. Preparation of sample solution
20 tablets were powdered and an amount equivalent to 100 mg AMLO was weighed and dissolved in 25 ml methanol and the volume was made up to the 100 ml with distilled water. Solutions were filtered using whatmann filter paper grade 1. Appropriate dilutions were prepared in distilled water taking suitable aliquots of the clear filtrates and subjected to analysis by the proposed method.

6.1.4.3. Procedure
By appropriate dilutions from the working standard solutions of 100 μg/ml of AMLO and 100 μg/ml of INDA, the solutions of AMLO (40 μg/ml) and INDA (15 μg/ml) were prepared respectively and scanned over the range of 260-400 nm and the overlain spectra were observed for the development of suitable method for analysis. The overlain spectra of AMLO and INDA are shown in figure. From the overlay spectra 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 (Fig No.18). For calibration curve, from the working standard solutions, appropriate dilutions in the range of 10-50 μg/ml and 5-25 μg/ml for AMLO and INDA respectively were prepared and analyzed.

6.1.4.4. Method validation

6.1.4.4.1. Linearity
The calibration curves of AMLO and INDA were linear in the range of 10-50 µg/ml and 5-25 µg/ml respectively (Table No.34 and Table No.35). The calibration curves and regression equations were given in (Fig No.19 and Fig No.20).

6.1.4.4.2. Precision
The repeatability was evaluated by assaying 6 times of sample solution prepared for assay determination. The intra-day and inter-day precision study of AMLO and INDA was carried out by estimating median concentration of AMLO (30 µg/ml) and INDA (15 µg/ml), 3 times on the same day (Table No.36) and on 3 different days (first, second, third) (Table No.37 to Table No.39) and the results are reported in terms of %RSD.

6.1.4.4.3. Accuracy
Accuracy was assessed by determination of the recovery of the method by addition of standard drug to the pre-quantified sample preparation at 3 different concentration levels 80, 100 and 120 %, taking into consideration percentage purity of added bulk drug samples. Each concentration was analyzed 3 times and average recoveries were measured. % Recovery and % RSD was calculated to prove the accuracy of the method. Data of accuracy study is given in (Table No.40 and Table No.41).

6.1.4.4.4. Limit of detection and Limit of quantification
The limit of detection and the limit of quantification of the drug was derived by calculating the signal-to-noise ratio (S/N,i.e.,3.3 for LOD and 10 for LOQ) using the following equations designated by international conference on harmonization (ICH) guidelines.

LOD=3.3×σ/s

LOQ=10×σ/s

Where σ = the standard deviation of the response and
S = slope of the calibration curve

The results of LOD and LOQ were given in (Table No.42).

6.1.4.5. Analysis of commercial formulation
The tablet sample solution was subjected to analysis by dual wavelength method. Absorbances of sample solutions were recorded at 270 and 288nm for AMLO where INDA as same absorbance at these wavelengths, and 350 and 378nm where AMLO as same absorbance at these wavelengths. The solution of required dilution was analysed by the proposed method. The % recoveries of formulation are given in (Table No.43).

6.2.METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF AMLO AND INDA BY COLORIMETRY USING MBTH REAGENT

Reaction:

Step-I: formation of oxidation form of MBTH as intermediate

Step-2: formation of coloured complex

6.2.1. Preparation of standard solutions

Preparation of standard solution of Amlodipine: Standard AMLO (10 mg) was dissolved in 10 ml of methanol. Further dilutions were prepared in distilled water.

Preparation of standard solution of Indapamide: Standard INDA (10 mg) was dissolved in 10 ml of methanol. Further dilutions were prepared in distilled water.

6.2.2. Preparation of reagents

6.2.2.1. Preparation Of 0.5%MBTH Reagent: Accurately weigh 0.5 gms of MBTH reagent and dissolve in 100ml of distilled water.

6.2.2.2. Preparation of 1% Fecl₃reagent: Accurately weigh 1 gm of ferric chloride reagent and dissolve in 100 ml of distilled water. Filter if necessary and keep it in a dark place.

6.2.3. Preparation of sample solution
20 tablets were powdered and an amount equivalent to 100 mg AMLO was weighed and dissolved in 25 ml methanol and the volume was made up to 100ml with distilled water. Solutions were filtered using whatmann filter paper grade 1. Appropriate dilutions were prepared in distilled water taking suitable aliquots of the clear filtrates and subjected to analysis by the proposed method.

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6.2.4. Procedure
Diluted standard solution of AMLO (50μg/ml) and INDA (25μg/ml) were scanned over the range of 400-800 nm to select the λ_max. Wavelength (λ_max) of 626 nm and 600 nm was selected for analysis of these drugs by simultaneous equation method.

To the drug solution add 1.0 ml of 1% Fecl₃ reagent and 1 ml of 0.5% MBTH reagent. Keep aside for 2 min and make up the volume to 10 ml with distilled water and measure the absorbance of resulting green coloured solution against the reagent blank prepared in the same manner but without the drug solution.

The overlay spectrum of AMLO and INDA was given in (Fig No.21) The linearity was determined and found to be in the range of 10-50 μg/ml and 5-25 μg/ml of AMLO and INDA respectively. For quantification of AMLO and INDA simultaneously, absorptivity coefficient of both drugs at each wavelength were determined from different dilution (5 independent determinations) of various standard solutions of AMLO and INDA drugs within Beer’s law concentration range limits. By using absorptivity coefficient, a set of two simultaneous equations 1 and 2 were obtained for determination of concentration of AMLO and INDA

C_x = (A₂ay₁-A₁ay₂)/ (ax₂ay₁-ax₁ay₂) ----------------- 1

C_Y = (A₁ax₂-A₂ax₁)/ (ax₂ay₁-ax₁ay₂) ----------------- 2

C_X = concentration of AMLO

C_Y = concentration of INDA

A₅=absorbance of samples at 626 nm.

A₂= absorbance of samples at 600 nm.

ax₁ is the absorptivity of AMLO at 626 nm.

ax₂ is the absorptivity of AMLO at 600 nm.

ay₁ is the absorptivity of INDA at 626 nm.

ay₂ is the absorptivity of INDA at 600 nm.

6.2.5. Method validation

6.2.5.1. Linearity
Each concentrations of working solution of AMLO and INDA were observed under UV system. The absorbencies were determined for each concentration of the AMLO and INDA independently. The calibration curves were obtained with linear response of AMLO (Table No.44) and INDA (Table No.45) in the concentration ranges of 10-50 μg/ml, and 5-25 μg/ml respectively. The regression equations of calibration curves were y=0.018x and y=0.004x for AMLO at 626 and 600 nm and y=0.034x and y=0.003x for INDA at 600 and 626 nm by simultaneous equation method respectively (Fig No.22 and Fig No.23).

6.2.5.2. Precision
The precision of the method was established by carrying out the determination of concentration of AMLO and INDA by repeatability (Intra-day) and intermediate precision (Inter-day precision). The intra-day precision was performed on the same day on same sample at particular time interval (Table No.46). The inter-day precision was performed on the three consecutive days on same sample solution (Table No.47 to Table No.49). The results were obtained and %RSD was calculated in order to establish the precision of the method.

6.2.5.3. Accuracy
The accuracy of methods was determined by carrying out recovery studies at three different levels (80,100 and 120) on the basis of the label claim. At each level, three determinations were performed simultaneously and % Recovery and % RSD was calculated to prove the accuracy of the methods. Data of accuracy study is given in (Table No.50 and Table No.51).

6.2.5.4. Limit of detection and Limit of quantification
The limit of detection and the limit of quantification of the drug were derived by calculating the signal-to-noise ratio (S/N,i.e.,3.3 for LOD and 10 for LOQ) using the following equations designated by international conference on harmonization (ICH) guidelines.

LOD=3.3×σ/s

LOQ=10×σ/s

Where σ = the standard deviation of the response and

S = slope of the calibration curve

The results of LOD and LOQ were given in (Table No.52).

6.2.6. Analysis of commercial formulations
The tablet sample solution was subjected to analysis by simultaneous equation method. Absorbances of sample solutions were recorded at 626 nm (λ_maxof AMLO) and 600 nm (λ_maxof INDA) and concentration of two drugs in the sample were determined by using equations 1 and 2. The % recovery of formulation was given in (Table No.53).

6.3. METHOD DEVELOPMENT FOR SIMULTANEOUS ESTIMATION OF AMLO AND INDA BY RP-HPLC

6.3.1. Trail 1

6.3.1.1. Chromatographic conditions

Mobile phase: Methanol + HPLC grade water (50+50)

Flow rate: 1ml/min

Column: C18 column

Detection wavelength: 250nm

Injection volume: 20µl

The chromatograms of this trail are given in (Fig No.24 and Fig No.25).

Result: No peak was eluted for Indapamide.

6.3.2. Trail 2

6.3.2.1. Chromatographic conditions

Mobile phase: Methanol + HPLC grade ACN (50+50)

Flow rate: 1ml/min

Column: C18 column

Detection wavelength: 250nm

Injection volume: 20µl

The chromatograms of this trail are given in (Fig No.26 to Fig No.28).

Result: No resolution between the two peaks.

6.3.3. Trail 3

6.3.3.1. Chromatographic conditions

Mobile phase: Acetonitrile: methanol: triethylamine buffer 25:35:40(v/v)

Flow rate : 1ml/min

Column : C18 column

Detection wavelength: 237nm

Injection volume : 20µl

The chromatograms of this trail are given in (Fig No.29 to Fig No.31).

Result: Broad peak of Indapamide was observed.

6.3.4. Optimized method

6.3.4.1. HPLC Instrument details

Make: Shimadzu

Model: LC-20AT

Detector: SPD-20A PROMENENCE UV/VIS DETECTOR

Column make: EQUISIL C-18 ODS column

Particle Size: 5 m

Column length: 250 x 4.6mm i.d

Injector Type: MANUAL Rheodyne type injector.

Injection Volume: 20 ml

Detection wavelength: 250 nm

Run time: 10 min

Mobile Phase Used: Acetonitrile: Methanol: Triethylamine buffer 25:35:40(v/v)

6.3.4.2. Reagents
All chemicals and reagents used were of HPLC grade. Pure standards of Amlodipine and Indapamide employed in the study were obtained as gift sample from MICRO LABS, Bangalore. The other reagents used were Methanol and Acetonitrile from Qualigens ltd. Mumbai, India, Triethylamine from Hi-media, Mumbai, India, and HPLC grade water from Merck chemicals, Mumbai, India.

6.3.4.3. Preparation of mobile phase
The mobile phase used was Acetonitrile, Methanol and freshly prepared Triethylamine buffer solution (P^H3.0⁾ in the ratio of 25:35:40(v/v) and the mobile phase was filtered through 0.45 mmembrane filter and sonicated before use. Sample solution was prepared by dissolving the drugs in mobile phase by sonication for 30 minutes. The mobile phase was delivered isocratically at a flow rate of 1 ml/min. All solutions were filtered through a 0.45 mmembrane filter before use.Equisil C-18 ODS column 250 x 4.6 mm i.d with 5 mparticle size and the column was maintained at ambient temperature. The injection volume was 20 ml and the total run time was 10 min. The detection was carried out at 250 nm.

6.3.4.4. Preparation of buffer (P^H-3.0)
Buffer solution was prepared by taking accurately a quantity of 0.7 ml of Triethylamine dissolved in 100 ml HPLC grade water. The P^H of the solution was adjusted to 3.0 with Ortho- phosphoric acid and degassed for about 30 min in a ultra bath sonicator.

6.3.4.5. Preparation of Mobile phase
The mobile phase was prepared by mixing Acetonitrile, Methanol, Trietylamine buffer in the ratio of 25:35:40(v/v) sonicated for about 15 min and filtered.

6.3.4.6. Preparation of standard
About 10 mg of each drug AMLO and INDA was accurately weighed and dissolved in methanol in a 10ml standard flask and make up to the volume.

6.3.4.7. Preparation of working standard
From the above standard solution 1ml is taken and diluted to 10 ml to give a concentration of 100 mg/ml of each standard solution of AMLO and INDA.

6.3.4.8. Validation of the Method

6.3.4.8.1. Linearity
Linearity was assessed by performing single measurement at several analyte concentration varying quantities of stock standard solution diluted with the mobile phase to give a concentration of 5, 10, 15, 20 and 25µg/ml of AMLO (Table No.54) and 2, 4, 6, 8 and 10 µg/ml of INDA (Table No.55). Injection was made at intervals of 10.0 min. Linearity of AMLO was found to be exist between 5-25 µg/ml (Fig No.34 to Fig No.38) and for INDA 2-10 µg/ml (Fig No.40 to Fig No.44). The chromatograms were recorded and linearity graph was plotted by using peak area of drug against respective concentrations to obtain the linearity range. The calibration curves and regression equations for both the drugs were given in (Fig No.32 and Fig No.39). The chromatogram of formulation was given in (Fig No.46).

6.3.4.8.2. Precision
The intra-day and inter-day precision studies were carried out by estimating the corresponding responses 3 times on the same day (Table No.56) and on 3 different days (Table No.57 to Table No.59) for middle concentrations of AMLO (15 µg/ml) and INDA (6 µg/ml), and the results were reported in terms of relative standard deviation.

6.3.4.8.3. Accuracy
The accuracy of the method was determined by calculating recoveries of AMLO and INDA by method of standard additions. Known amount of AMLO and INDA were added to a pre quantified sample solution (containing AMLO and INDA in 5:1.5 μg/ ml proportion, respectively), and the amount of AMLO and INDA were estimated by measuring the peak areas and by fitting these values to the straight-line equation of calibration curve. The accuracy data of AMLO and INDA was given in (Table No.60 and Table No.61).

6.3.4.8.4. Limit of detection and Limit of quantification
The limit of detection (LOD) is defined as the lowest concentration of an analyte that can reliably be differentiated from background levels. Limit of quantification (LOQ) of an individual analytical procedure is the lowest amount of analyte that can be quantitatively determined with suitable precision and accuracy.

LOD and LOQ were calculated using the following equation as per ICH guidelines.

LOD = 3 .3 × σ / S;

L OQ = 1 0 × σ / S;

Where σ is the standard deviation of y-intercepts of regression lines and S is the slope of the calibration curve.

The results of LOD and LOQ were given in (Table No.62).

6.3.4.9. Analysis of marketed formulation
A total of 20 tablets were accurately weighed and powdered in a mortar. An amount equivalent to one tablet (Containing 5 mg of AMLO and 1.5 mg of INDA) was transferred to 25 ml volumetric flask, 10 ml of methanol was added and content of the flask were ultrasonicated for 10 min. The solution was filtered through whatmann filter paper No. 41 and filter paper was washed with methanol twice and volume was then made up to the mark with methanol. The sample solution thus prepared was diluted with methanol to get the solution containing AMLO and INDA in 5:1.5 μg/ ml proportion, respectively. The solution was injected at above chromatographic conditions and peak areas were measured. The quantification was carried out by keeping these values to the straight line equation of calibration curve. The % recovery of formulation was given in (Table No.63).

RESULTS AND DISCUSSION

7.1. METHOD DEVELOPMENT BY UV SPECTROSCOPY

7.1.1. SIMULTANEOUS EQUATION METHOD

7.1.1.1. Absorption spectra

Fig No.5: overlain spectrum of AMLO and INDA

Fig No.6: UV- Spectrum of Amlodipine (λ_max-365 nm)

Fig No.7: UV- Spectrum of Indapamide (λ_max-279 nm)

7.1.1.2. Method validation

7.1.1.2.1. Linearity

Table No.4: Linearity of AMLO at 365 and 279 nm

S.NO	Concentrations (µg/ml)	AMLO
S.NO	Concentrations (µg/ml)	365 nm	279 nm
1	10	0.133	0.025
2	20	0.259	0.043
3	30	0.398	0.062
4	40	0.514	0.085
5	50	0.667	0.101
6	Equation(y= m x)	Y=0.013 x	Y=0.002 x
7	Slope	0.013	0.002
8	Correlation coefficient	0.999	0.995
9	Molar absorptivity
10	Sandell’s Sensitivity

Fig No.8: Calibration graphs of AMLO at 365 and 279 nm

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Table No.5: Linearity of INDA at 365 and 279 nm

S.NO	Concentrations (µg/ml)	INDA
S.NO	Concentrations (µg/ml)	365 nm	279 nm
1	5	0.019	0.130
2	10	0.038	0.243
3	15	0.064	0.369
4	20	0.085	0.483
5	25	0.098	0.622
6	Equation(y=m x)	y=0.004x	y=0.024x
7	Slope	0.004	0.024
8	Correlation coefficient	0.993	0.999
9	Molar absorptivity
10	Sandell’s Sensitivity

Fig No.9: Calibration graphs of INDA at 365 and 279 nm

7.1.1.2.2. Precision

Table No.6: Intra-day precision

S.NO	AMLO		INDA
S.NO	365 nm	279 nm	365 nm	279 nm
1	0.398	0.062	0.064	0.372
2	0.396	0.064	0.062	0.369
3	0.401	0.062	0.063	0.368
4	0.411	0.064	0.061	0.370
5	0.395	0.063	0.062	0.365
6	0.406	0.065	0.063	0.375
Mean	0.4011	0.0633	0.0625	0.3698
SD	0.00624	0.00121	0.00104	0.00343
%RSD	1.5560	1.9122	1.678	0.9275

Table No.7: Inter-day precision (DAY-1)

S.NO	AMLO		INDA
S.NO	365 nm	279 nm	365 nm	279 nm
1	0.398	0.0627	0.0642	0.3682
2	0.396	0.0623	0.0645	0.3680
3	0.401	0.0624	0.0639	0.3670
4	0.411	0.0620	0.0635	0.3679
5	0.395	0.0622	0.0644	0.3675
6	0.406	0.0598	0.0638	0.3685
Mean	0.4011	0.0619	0.06405	0.3678
SD	0.00624	0.001055	0.000383	0.000532
%RSD	1.556	1.7035	0.5986	0.14461

Table No.8: Inter-day precision (DAY-2)

S.NO	AMLO		INDA
S.NO	365 nm	279 nm	365 nm	279 nm
1	0.402	0.0622	0.0640	0.3679
2	0.398	0.0620	0.0639	0.3675
3	0.387	0.0624	0.0635	0.3672
4	0.389	0.0599	0.0634	0.3669
5	0.390	0.0623	0.0632	0 .3665
6	0.384	0.0621	0.0642	0.3662
Mean	0.391667	0.061817	0.0637	0.36703
SD	0.00689	0.00095	0.00039	0 .000631
%RSD	1.75904	1.53609	0.612044	0.1720

Table No.9: Inter-day precision (DAY-3)

S.NO	AMLO		INDA
S.NO	365 nm	279 nm	279 nm	365 nm
1	0.405	0.0599	0.0635	0.3680
2	0.395	0.0597	0.0633	0.3679
3	0.399	0.0595	0.0631	0.3675
4	0.403	0.0600	0.0629	0.3672
5	0.389	0.0601	0.0628	0.3683
6	0.385	0.0605	0.0626	0.3682
Mean	0.396	0.05995	0.063033	0.36785
SD	0.007874	0.000345	0.000333	0.000423
%RSD	1.9883	0.575419	0.527762	0.115015

7.1.1.2.3. Accuracy

Table No.10: Accuracy of AMLO

Level of % recovery	Amount of drug spiked(µg/ml)	Drug recovered	%Recovery	Mean	SD	%RSD
80	16	16.1	100.6	100.366	0.4932	0.4914
		16.12	100.7
		15.98	99.8
100	20	20.10	100.5	99.9666	0.8386	0.83892
		20.08	100.4
		19.80	99.0
120	24	24.20	100.8	100.23	0.6658	0.6642
		24.1	100.4
		23.89	99.5

Table No.11: Accuracy of INDA

Level of % recovery	Amount of drug spiked(µg/ml)	Drug recovered	%Recovery	Mean	SD	%RSD
80	8	8.13	101.6	99.93	1.755	1.757
		8.01	100.1
		7.85	98.1
100	10	10.12	101.2	100.733	1.553	1.542
		10.20	102.0
		9.90	99.0
120	12	12.21	101.7	99.56	1.858	1.866
		11.80	98.3
		11.85	98.7

7.1.1.2.4. Limit of detection and Limit of quantification

Table No.12: LOD and LOQ of AMLO and INDA

S.NO	Results observed	AMLO			INDA
S.NO	Results observed	365 nm	279 nm		365 nm	279 nm
1	SD	0.00624		0.00121	0.00104	0.00343
2	LOD(µg/ml)	1.584		1.9	0.858	0.462
3	LOQ(µg/ml)	4.8		6.0	2.6	1.4

7.1.1.2.5. Results of analysis of commercial formulation

Table No.13:

Tablet	Label claimed(mg)		Conc.found (mg)		% Recovery
Amlodac-c Tablets	AMLO	INDA	AMLO	INDA	AMLO	INDA
Amlodac-c Tablets	5.0	1.5	5.03	1.49	100.6	99.3

7.1.2. FIRST ORDER DERIVATIVE SPECTROSCOPIC METHOD

7.1.2.1. Absorption spectra

Fig No.10: overlain spectra of AMLO and INDA

Fig No.11: First order derivative spectra of AMLO

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Fig No.12: First order derivative spectra of INDA

7.1.2.2. Method validation

7.1.2.2.1. Linearity

Table No.14:Linearity of AMLOat 279 nm (zero crossing point of INDA)

S.NO	Concentrations (µg/ml)	AMLO
S.NO	Concentrations (µg/ml)	279 nm
1	10	-0.013
2	20	-0.024
3	30	-0.035
4	40	-0.045
5	50	-0.054
6	Equation(y=mx)	y=-0.001x
7	Slope	-0.001
8	Correlation coefficient	0.994
9	Molar absorptivity
10	Sandell’s Sensitivity

Fig No.13: Calibration graph of AMLO at 279 nm

Table No.15: Linearity OF INDA at 293 nm (zero crossing point of AMLO)

S.NO	Concentrations (µg/ml)	INDA
S.NO	Concentrations (µg/ml)	293 nm
1	5	-0.011
2	10	-0.023
3	15	-0.035
4	20	-0.047
5	25	-0.059
6	Equation(y=mx)	y=-0.002
7	Slope	-0.002
8	Correlation coefficient	0.999
9	Molar absorptivity
10	Sandell’s Sensitivity

Fig No.14: Calibration graph of INDA at 293 nm

7.1.2.2.2. Precision

Table No.16: Intra-day precision

S.NO	AMLO 279 nm	INDA 293 nm
1	-0.034	-0.035
2	-0.036	-0.035
3	-0.035	-0.034
4	-0.035	-0.034
5	-0.035	-0.035
6	-0.035	-0.034
Mean	-0.035	-0.0345
SD	0.000632	0.000548
%RSD	-1.80702	-1.5876

Table No.17: Inter-day precision (DAY-1)

S.NO	AMLO 279 nm	INDA 293 nm
1	-0.034	-0.035
2	-0.034	-0.036
3	-0.034	-0.035
4	-0.035	-0.034
5	-0.035	-0.035
6	-0.035	-0.035
Mean	0.0345	-0.035
SD	0.000548	0.000632
%RSD	-1.5876	-1.80702

Table No.18: (Inter-day precision (DAY-2)

S.NO	AMLO 279 nm	INDA 293 nm
1	-0.033	-0.033
2	-0.034	-0.033
3	-0.033	-0.033
4	-0.033	-0.032
5	-0.034	-0.032
6	-0.034	-0.033
Mean	-0.0335	-0.03267
SD	0.000548	0.000576
%RSD	-1.63499	-1.58081

Table No.19: Inter-day precision (DAY-3)

S.NO	AMLO 279nm	INDA 293nm
1	-0.032	-0.032
2	-0.032	-0.032
3	-0.033	-0.033
4	-0.033	-0.033
5	-0.032	-0.033
6	-0.032	-0.033
Mean	-0.03233	-0.03267
SD	0.000516	0.000516
%RSD	-1.59711	-1.5808

7.1.2.2.3. Accuracy

Table No.20: Accuracy of AMLO

Level of % recovery	Amount of drug spiked(µg/ml)	Drug recovered	%Recovery	Mean	SD	%RSD
80	16	16.20	101.2	100.5	0.964	0.959
		15.91	99.4
		16.15	100.9
100	20	20.12	100.6	100.1	0.585	0.584
		19.90	99.5
		20.08	100.4
120	24	24.20	100.8	100.2	0.665	0.664
		24.10	100.4
		23.90	99.5

Table No.21: Accuracy of INDA

Level of % recovery	Amount of drug spiked(µg/ml)	Drug recovered	%Recovery	Mean	SD	%RSD
80	8	8.05	100.6	100.2	1.096	1.094
		7.92	99.0
		8.09	101.1
100	10	10.10	101.0	100.1	1.040	1.039
		10.05	100.5
		9.90	99.0
120	12	12.10	100.8	100.4	1.331	1.325
		11.89	99.0
		12.20	101.6

Table No.22: Limit of detection and Limit of quantification

S.NO	Results observed	AMLO	INDA
S.NO	Results observed	279 nm	293 nm
1	SD	0.000632	0.000548
2	LOD(µg/ml)	2.08	0.904
3	LOQ(µg/ml)	6.32	2.74

Table No.23: Analysis of commercial formulation

Tablet	Label claimed(mg)		Conc.found (mg)		% Recovery
Amlodac-c Tablets	AMLO	INDA	AMLO	INDA	AMLO	INDA
Amlodac-c Tablets	5.0	1.5	5.03	1.48	100.6	98.6

7.1.3. Q ABSORPTION RATIO METHOD

7.1.3.1. Absorption spectrum

Fig No.15: overlain spectrum of AMLO and INDA showing iso- absorptive point at 312nm

7.1.3.2. Method validation

7.1.3.2.1. Linearity

Table No.24: Linearity of AMLO at 312 nm

S.NO	Concentrations (µg/ml)	AMLO
		312 nm
1	10	0.018
2	20	0.041
3	30	0.063
4	40	0.086
5	50	0.115
6	Equation(y=mx)	y=0.002x
7	Slope	0.002
8	Correlation coefficient	0.993
9	Molar absorptivity
10	Sandell’s Sensitivity

Fig No.16: Calibration graph of AMLO at 312 nm

Table No.25: Linearity of INDA at 312 nm

S.NO	Concentrations (µg/ml)	INDA
		312 nm
1	5	0.030
2	10	0.047
3	15	0.069
4	20	0.091
5	25	0.117
6	Equation(y=mx)	Y=0.004x
7	Slope	0.004
8	Correlation coefficient	0.994
9	Molar absorptivity
10	Sandell’s Sensitivity

Fig No.17: Calibration graph of INDA at 312 nm

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7.1.3.2.2. Precision

Table No.26: Intra-day precision

S.NO	AMLO 312 nm	INDA 312 nm
1	0.063	0.069
2	0.063	0.068
3	0.064	0.070
4	0.064	0.068
5	0.062	0.069
6	0.062	0.069
Mean	0.063	0.068833
SD	0.000894	0.000753
%RSD	1.4197	1.0936

Table No.27:Inter-day precision (DAY-1)

S.NO	AMLO 312 nm	INDA 312 nm
1	0.062	0.068
2	0.062	0.068
3	0.063	0.067
4	0.064	0.068
5	0.061	0.070
6	0.062	0.069
Mean	0.06233	0.06833
SD	0.001033	0.001033
%RSD	1.65689	1.51140

Table No.28:Inter-day precision (DAY-2)

S.NO	AMLO 312 nm	INDA 312 nm
1	0.063	0.069
2	0.060	0.069
3	0.062	0.067
4	0.062	0.068
5	0.062	0.069
6	0.062	0.067
Mean	0.061833	0.06816
SD	0.000983	0.000983
%RSD	1.590068	1.442336

Table No.29:Inter-day precision (DAY-3)

S.NO	AMLO 312 nm	INDA 312 nm
1	0.062	0.068
2	0.061	0.067
3	0.061	0.065
4	0.061	0.066
5	0.063	0.065
6	0.062	0.065
Mean	0.061667	0.066
SD	0.000816	0.001265
%RSD	1.32404	1.916532

7.1.3.2.3. Accuracy

Table No.30: Accuracy of AMLO

Level of % recovery	Amount of drug spiked(µg/ml)	Drug recovered	%Recovery	Mean	SD	%RSD
80	16	15.95	99.6	99.9	0.360	0.360
		16.06	100.3
		15.98	99.8
100	20	19.98	99.9	99.7	0.513	0.514
		20.04	100.2
		19.85	99.2
120	24	24.01	100.0	100.0	0.152	0.152
		23.98	99.9
		24.05	100.2

Table No.31: Accuracy for INDA

Level of % recovery	Amount of drug spiked(µg/ml)	Drug recovered	%Recovery	Mean	SD	%RSD
80	8	8.03	100.3	100.4	0.763	0.760
		7.99	99.8
		8.11	101.3
100	10	10.15	101.5	100.7	1.628	1.616
		9.89	98.9
		10.19	101.9
120	12	12.12	101.0	100.6	0.907	0.901`
		11.96	99.6
		12.16	101.3

Table No.32: Limit of detection and Limit of quantification

S.NO	Results observed	AMLO	INDA
S.NO	Results observed	312 nm	312 nm
1	SD	0.000894	0.000753
2	LOD(µg/ml)	1.475	0.621
3	LOQ(µg/ml)	4.47	1.882

Table No.33: Analysis of commercial formulation

Tablet	Label claimed(mg)		Conc.found (mg)		% Recovery
Amlodac-c Tablets	AMLO	INDA	AMLO	INDA	AMLO	INDA
Amlodac-c Tablets	5.0	1.5	4.97	1.52	99.4	101.3

7.1.4. DUAL WAVELENGTH METHOD

7.1.4.1. Absorption spectrum

Fig No.18: Spectrum showing dual wavelength

7.1.4.2. Method validation

7.1.4.2.1. Linearity

Table No.34: Linearity of AMLO (difference of absorbences at 270nm and 288nm)

S.NO	Concentrations (µg/ml)	AMLO
1	10	0.042
2	20	0.084
3	30	0.128
4	40	0.166
5	50	0.211
6	Equation(y=mx)	Y=0.004
7	Slope	0.004
8	Correlation coefficient	0.999

Fig No.19: Calibration graph of AMLO at 288 and 270 nm

Table No.35: Linearity of INDA (difference of absorbences at 350 nm and 378 nm)

S.NO	Concentrations (µg/ml)	INDA
1	5	0.006
2	10	0.011
3	15	0.017
4	20	0.022
5	25	0.027
6	Equation(y=mx)	Y=0.001X
7	Slope	0.001
8	Correlation coefficient	0.998

Fig No.20: Calibration graph of INDA at 378 and 350 nm

7.1.4.2.2. Precision

Table No.36: Intra-day precision

S.NO	AMLO	INDA
1	0.128	0.0170
2	0.128	0.0172
3	0.127	0.0171
4	0.128	0.0172
5	0.128	0.0169
6	0.127	0.0172
Mean	0.12766	0.0171
SD	0.000516	0.000126
%RSD	0.4044	0.7397

Table No.37: Inter-day (DAY-1)

S.NO	AMLO	INDA
1	0.128	0.0170
2	0.128	0.0172
3	0.128	0.0169
4	0.127	0.0167
5	0.129	0.0170
6	0.127	0.0170
Mean	0.127833	0.016967
SD	0.000753	0.000163
%RSD	0.58887	0.9624

Table No.38: Inter-day (DAY-2)

S.NO	AMLO	INDA
1	0.127	0.0172
2	0.126	0.0170
3	0.123	0.0172
4	0.127	0.0169
5	0.127	0.0167
6	0.123	0.0169
Mean	0.1255	0.016983
SD	0.001975	0.000194
%RSD	1.5735	1.142762

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Table No.39: Inter-day (DAY-3)

S.NO	AMLO	INDA
1	0.126	0.0169
2	0.124	0.0167
3	0.126	0.0169
4	0.126	0.0168
5	0.124	0.0170
6	0.125	0.0169
Mean	0.1251	0.016867
SD	0.000983	0.000103
%RSD	0.7855	0.612329

7.1.4.2.3. Accuracy

Table No.40: Accuracy of AMLO

Level of % recovery	Amount of drug spiked(µg/ml)	Drug recovered	%Recovery	Mean	SD	%RSD
80	16	16.21	101.3	100.5	0.750	0.746
		16.10	100.6
		15.98	99.8
100	20	20.12	100.6	100.1	1.021	1.019
		19.80	99.0
		20.18	100.9
120	24	24.21	100.8	100.3	0.450	0.449
		24.10	100.4
		23.98	99.9

Table No.41: Accuracy of INDA

Level of % recovery	Amount of drug spiked(µg/ml)	Drug recovered	%Recovery	Mean	SD	%rsd
80	8	8.03	100.3	100.6	0.814	0.809
		8.13	101.6
		8.01	100.1
100	10	10.12	101.2	99.5	1.436	1.442
		9.85	98.5
		9.90	99.0
120	12	12.25	102.0	100.1	1.607	1.604
		11.88	99.0
		11.95	99.5

Table No.42:Limit of detection and Limit of quantification

S.NO	Results observed	AMLO	INDA
1	SD	0.000516	0.000126
2	LOD(µg/ml)	0.425	0.415
3	LOQ(µg/ml)	1.29	1.26

Table No.43: Analysis of commercial formulation

Tablet	Label claimed(mg)		Conc.found (mg)		% Recovery
Amlodac-c Tablets	AMLO	INDA	AMLO	INDA	AMLO	INDA
Amlodac-c Tablets	5.0	1.5	4.99	1.51	99.8	100.6

7.2. METHOD DEVELOPMENT BY COLORIMETRY

7.2.1. Absorption spectra

Fig No.21: Overlay spectrum of AMLO and INDA by using MBTH reagent

7.2.2. Method validation

7.2.2.1. Linearity

Table No.44: Linearity of AMLO at 626 and 600 nm

S.NO	Concentrations (µg/ml)	AMLO (626 nm)	AMLO (600 nm)
1	10	0.164	0.023
2	20	0.341	0.042
3	30	0.529	0.063
4	40	0.732	0.084
5	50	0.956	0.098
6	Equation(y=mx)	y=0.018x	y=0.002x
7	Slope	0.018	0.002
8	Correlation coefficient	0.995	0.995

Fig No.22: Calibration graphs of AMLO at 626 and 600 nm

Table No.45: Linearity of INDA at 626 and 600 nm

S.NO	Concentrations (µg/ml)	INDA (600 nm)	INDA (626 nm)
1	5	0.148	0.014
2	10	0.362	0.036
3	15	0.529	0.052
4	20	0.714	0.071
5	25	0.836	0.083
6	Equation(y=mx)	y=0.034x	y=0.003x
7	Slope	0.034	0.003
8	Correlation coefficient	0.995	0.994

Fig No.23: Calibration graphs of INDA at 626 and 600 nm

7.2.2.2. Precision

Table No.46: Intra-day precision

S.NO	AMLO		INDA
S.NO	626 nm	600 nm	626 nm	600 nm
1	0.529	0.063	0.052	0.530
2	0.529	0.062	0.052	0.0529
3	0.528	0.063	0.051	0.529
4	0.529	0.063	0.052	0.530
5	0.529	0.062	0.052	0.530
6	0.528	0.063	0.051	0.530
Mean	0.528667	0.062667	0.051667	0.529667
SD	0.000516	0.000516	0.000516	0.000516
%RSD	0.0976	0.824039	0.9948	0.097495

Table No.47: Inter-day precision (DAY-1)

S.NO	AMLO		INDA
S.NO	626 nm	600 nm	626 nm	600 nm
1	0.527	0.062	0.051	0.3682
2	0.527	0.062	0.051	0.3680
3	0.528	0.062	0.051	0.3670
4	0.528	0.063	0.052	0.3679
5	0.528	0.062	0.052	0.3675
6	0.527	0.063	0.051	0.3685
Mean	0.5275	0.062333	0.051333	0.3678
SD	0.000548	0.000516	0.000516	0.000532
%RSD	0.103834	0.828446	1.00597	0.14461

Table No.48: Inter-day precision (DAY-2)

S.NO	AMLO		INDA
S.NO	626 nm	600 nm	626 nm	600 nm
1	0.528	0.061	0.051	0.528
2	0.528	0.061	0.050	0.527
3	0.527	0.062	0.050	0.529
4	0.528	0.061	0.051	0.528
5	0.528	0.061	0.051	0.528
6	0.528	0.062	0.050	0.527
Mean	0.527833	0.061333	0.0505	0.527833
SD	0.000408	0.000516	0.000548	0.000753
%RSD	0.077344	0.841953	1.084599	0.142616

Table No.49: Inter-day precision (DAY-3)

S.NO	AMLO		INDA
S.NO	626 nm	600 nm	626 nm	600 nm
1	0.526	0.060	0.050	0.527
2	0.526	0.061	0.049	0.528
3	0.526	0.060	0.049	0.527
4	0.527	0.061	0.049	0.527
5	0.528	0.061	0.050	0.528
6	0.527	0.060	0.049	0.528
Mean	0.526667	0.0605	0.049333	0.5275
SD	0.000816	0.000548	0.000516	0.000548
%RSD	0.155031	0.905327	1.046752	0.103834

7.2.2.3. Accuracy

Table No.50: Accuracy of AMLO

Level of % recovery	Amount of drug spiked(µg/ml)	Drug recovered	%Recovery	Mean	SD	%RSD
80	16	16.05	100.3	100.1	0.513	0.512
		16.1	100.6
		15.95	99.6
100	20	20.10	100.5	99.9	0.838	0.838
		20.08	100.4
		19.80	99.0
120	24	24.05	100.2	100.1	0.360	0.360
		24.10	100.4
		23.95	99.7

Table No.51: Accuracy of INDA

Level of % recovery	Amount of drug spiked(µg/ml)	Drug recovered	%Recovery	Mean	SD	%RSD
80	8	8.13	101.6	99.9	1.755	1.757
		8.01	100.1
		7.85	98.1
100	10	10.12	101.2	100.7	1.553	1.542
		10.20	102.0
		9.90	99.0
120	12	12.21	101.7	99.5	1.858	1.866
		11.80	98.3
		11.85	98.7

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Table No.52: Limit of detection and Limit of quantification

S.NO	Results observed	AMLO			INDA
S.NO	Results observed	626 nm	600 nm		626 nm	600 nm
1	SD	0.000516		0.000516	0.000516	0.000516
2	LOD(µg/ml)	0.0946		0.851	0.567	0.050
3	LOQ(µg/ml)	0.28		2.58	1.72	0.151

Table No.53:Analysis of commercial formulation

Tablet	Label claimed(mg)		Conc.found (mg)		% Recovery
Amlodac-c Tablets	AMLO	INDA	AMLO	INDA	AMLO	INDA
Amlodac-c Tablets	5.0	1.5	5.03	1.49	100.6	99.3

7.3. METHOD DEVELOPMENT BY HPLC

7.3.1. Trail 1

Fig No.24: AMLO 100 µg/ml

Fig No.25: INDA 100 µg/ml

7.3.2. Trail 2

Fig No.26: AMLO 100 µg/ml

Fig No.27: INDA 100 µg/ml

Fig No.28: Mixture

7.3.3. Trail 3

Fig No.29: AMLO 100 µg/ml

Fig No.30: INDA 100 µg/ml

Fig No.31: Mixture

7.3.4. Optimized method

7.3.4.1. Validation parameters

7.3.4.1.1. Linearity

Table No.54: Linearity of AMLO

Concentration (µg/ml)	Retention Time	Area	Height	efficiency [th.pl]	Eff/l [t.p./m]	Asym metry	Capacity factor	Tailing factor
5 µg/ml	3.420	358575	12513	3673	73467	1.775	5.071	1.073
10 µg/ml	3.430	691556	14268	4039	80773	1.724	5.231	1.061
15 µg/ml	3.433	1081521	14953	4285	85697	1.776	5.744	1.030
20 µg/ml	3.430	1451468	15724	4789	95771	1.716	5.111	1.023
25 µg/ml	3.460	1842135	16125	5481	109624	1.785	5.984	1.050

Fig No.32: Calibration graph of AMLO

Fig No.33: Chromatogram Of Blank

Fig No.34: Chromatogram of AMLO 5 µg/ml

Fig No.35: Chromatogram of AMLO 10 µg/ml

Fig No.36: Chromatogram of AMLO 15 µg/ml

Fig No.37: Chromatogram of AMLO 20 µg/ml

Fig No.38: Chromatogram of AMLO 25 µg/ml

Table No.55: Linearity of INDA

Concentration (µg/ml)	Retention Time	Area	Height	efficiency [th.pl]	Eff/l [t.p./m]	Asym metry	Capacity factor	Tailing factor
2 µg/ml	4.460	289124	10315	5622	108991	1.234	12.558	1.044
4 µg/ml	4.467	591568	11985	5918	112448	1.353	12.459	1.015
6 µg/ml	4.510	890541	12498	6033	118354	1.368	12.657	1.070
8 µg/ml	4.526	1205846	12956	6367	120661	1.215	13.636	1.016
10 µg/ml	4.460	1510214	13742	6199	127332	1.169	13.420	1.025

Fig No.39: Calibration graph of INDA

Fig No.40: Chromatogram of INDA 2 µg/ml

Fig No.41: Chromatogram of INDA 4 µg/ml

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Fig No.42: Chromatogram of INDA 6 µg/ml

Fig No.43: Chromatogram of INDA 8 µg/ml

Fig No.44: Chromatogram of INDA 10 µg/ml

Fig No.45: Chromatogram of standard mixture

Fig No.46: Chromatogram of formulation

7.3.4.1.2. Precision

Table No.56: Intra-Day

S.NO	AMLO	INDA
1	1081521	890541
2	1081256	890265
3	1079852	890215
4	1082413	891426
5	1086315	891754
6	1079514	890475
Mean	1081812	890779.3
SD	2455.786	648.1644
%RSD	0.227007	0.072764

Table No.57: Inter-day (day 1)

S.NO	AMLO	INDA
1	1078259	890126
2	1083695	890421
3	1084237	890825
4	1081595	890618
5	1073572	890481
6	1075285	890523
Mean	1079441	890499
SD	4446.402	230.8896
%RSD	0.411917	0.025928

Table No.58: Inter-day (day 2)

S.NO	AMLO	INDA
1	1079651	889159
2	1075491	889754
3	1077426	885693
4	1079945	890624
5	1078264	890489
6	1072248	889025
Mean	1077171	889124
SD	2903.91	1805.154
%RSD	0.269587	0.203026

Table No.59: Inter-day (day 3)

S.NO	AMLO	INDA
1	1073841	889028
2	1076195	887375
3	1078428	885243
4	1079619	881415
5	1078357	886948
6	1075729	889245
Mean	1077028	886542.3
SD	2143.725	2909.249
%RSD	0.199041	0.328157

7.3.4.1.3. Accuracy

Table No.60: Accuracy of AMLO

Level of % recovery	Amount of drug spiked(µg/ml)	Drug recovered	%Recovery	Mean	SD	%RSD
80	8	8.13	101.6	99.9	1.755	1.757
		8.01	100.1
		7.85	98.1
100	10	10.12	101.2	100.7	1.553	1.542
		10.20	102.0
		9.90	99.0
120	12	12.21	101.7	99.5	1.858	1.866
		11.80	98.3
		11.85	98.7

Table No.61: Accuracy of INDA

Level of % recovery	Amount of drug spiked(µg/ml)	Drug recovered	%Recovery	Mean	SD	%RSD
80	3.2	3.18	99.3	99.7	0.513	0.514
		3.21	100.3
		3.19	99.6
100	4	4.05	101.2	99.8	1.278	1.279
		3.98	99.5
		3.95	98.7
120	4.8	4.79	99..7	99.6	0.750	0.753
		4.82	100.4
		4.75	98.9

Table No.62: Limit of detection and limit of quantification

S.NO	Results observed	AMLO	INDA
1	SD	2455.786	648.1644
2	LOD(µg/ml)	0.44	0.0712
3	LOQ(µg/ml)	1.35	0.215

Table No.63: Analysis of Commercial Formulation

Tablet	Label claimed(mg)		Conc.found (mg)		% Recovery
AMLODAC-C Tablets	AMLO	INDA	AMLO	INDA	AMLO	INDA
AMLODAC-C Tablets	5.0	1.5	5.02	1.51	100.4	100.6

SUMMARY AND CONCLUSION

8.1. Method development by UV spectroscopy
8.1.1. Estimation of AMLO and INDA by Simultaneous equation method
The proposed method was validated as per the ICH guidelines. Linearity was determined at different concentration, AMLO and INDA shows linearity in the concentration range of 10-50 µg/ml and 5-25 µg/ml with correlation coefficient of 0.999 at 365 and 279 nm respectively. Limit of detection (LOD) and Limit of quantitation (LOQ) were determined by standard deviation of response and slope of calibration curve. LOD and LOQ were found to be 1.58, 4.80 and 1.90, 6.00 for AMLO at 365 and 279 nm respectively. LOD and LOQ were found to be 0.85, 2.60 and 0.46, 1.40 for INDA at 365 and 279 nm respectively. There was no interference from the common excipients present in tablets. The recovery of drug was determined at 80, 100 and 120 % levels. The percent recovery of AMLO was found to be 100.27±0.31 and 99.97±1.16 for INDA indicating that method has required accuracy. Precision was performed under different conditions different days, and intraday. The results show the % RSD values < 2.0% signifies the precision of the method. The proposed method for simultaneous estimation of AMLO and INDA in combined dosage form was found to be simple, accurate and rapid.

8.1.2. Estimation of AMLO and INDA by First order derivative spectroscopic method
Based on the results, obtained from the analysis of described method, it can be concluded that the method has linear response in the range of 10-50 μg/ml and 5-25 μg/ml for AMLO and INDA, respectively with co-efficient of correlation, (r²)=0.994 and (r²) = 0.999 for AMLO and INDA, respectively. The result of the analysis of pharmaceutical formulation by the proposed method is highly reproducible and reliable and it is in good agreement with the label claim of the drug. The recovery experiment was performed by the standard addition method. The results of recovery studies indicate that the proposed method is accurate. Limit of detection (LOD) and Limit of quantitation (LOQ) were determined by standard deviation of response and slope of calibration curve. LOD and LOQ were found to be 2.08 and 6.32 for AMLO at 279nm and 0.90 and 2.74 for INDA at 293 nm. The additives usually present in the pharmaceutical formulation of the assayed sample did not interfere with determination of AMLO and INDA. The method can be used for the routine analysis of the AMLO and INDA in combined dosage form without any interference of excipients.

8.1.3. Estimation of AMLO and INDA Q absorption ratio method
In absorbance ratio method (Q-analysis), the primary requirement for developing a method for analysis is that the entire spectra should follow the Beer’s law at all the wavelength, which was fulfilled in case of both these drugs. The two wavelengths were used for the analysis of the drugs were 312 nm (iso-absorptive point) and 365 nm (λ-max of AMLO) at which the calibration curves were prepared for both the drugs. The overlain UV absorption spectra of AMLO (365 nm) and INDA (279 nm) showing iso-absorptive point (312 nm) in methanol is shown in Figure 11. The validation parameters were studied at all the wavelengths for the proposed method. Accuracy was determined by calculating the recovery and the mean was determined. The results of recovery show that the method has required accuracy. Precision was calculated as repeatability for both the drugs and the results of SD, % RSD were found to be within the limit. Hence, the method can be employed for the routine analysis of these two drugs in combined dosage form.

8.1.4. Estimation of AMLO and INDA by Dual wavelength method
The proposed method was found to be simple, sensitive, rapid, accurate, precise and economic for the routine simultaneous estimation of two drugs. The linearity range for AMLO and INDA were found to be 10-50 µg/ml and 5-25 µg/ml respectively.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.Precision was calculated as repeatability (% RSD) and intra and inter day variation (% RSD) for both the drugs. Accuracy was determined by calculating the recovery and the mean was determined. The LOD and LOQ were found to be 0.42 and 1.29 µg/ml respectively for AMLO and 0.41 and 1.26 µg/ml respectively for INDA indicates sensitivity of the proposed method. The method was successfully used to determine the amounts of AMLO and INDA present in tablets. The results obtained are in good agreement with the corresponding labeled amount. By observing the validation parameters, the method was found to be sensitive, accurate and precise. Hence the method can be employed for the routine analysis of these drugs in combinations.

8.2. Estimation of AMLO and INDA by Colorimetry using MBTH Reagent
The colorimetric method demonstrated was applicable to the estimation of AMLO and INDA in pure as well as in existing dosage forms. To prove the reliability of the method, validation as per ICH guidelines have been carried out to a possible extent. The results demonstrated that simultaneous estimation by LABINDIA-UV 3092 UV/VIS Spectrophotometer could be useful technique for determination of AMLO and INDA whenthey are given in same dosage form. The wavelengths selected were 600 and 626 nm for AMLO and INDA respectively. LOD and LOQ were found to be 0.09, 0.28 for AMLO at 626 nm and 0.05, 0.15 for INDA at 600 nm.

8.3. Estimation of AMLO and INDA by RP-HPLC
An isocratic reversed-phase liquid chromatographic assay method was developed for the quantitative determination of Amlodipine Besylate (AMLO) and Indapamide (INDA) in combined dosage form. A C-18 column with a mobile phase containing acetonitrile, methanol and triethylamine buffer (25:35:40v/v) total pH-adjusted to 3 using o-phosphoric acid was used. The flow rate was 1.0 ml/min and effluents were monitored at 250 nm. The retention times of amlodipine besylate and Indapamide were 3.4 min and 4.5 min, respectively. The proposed method was validated with respect to linearity, accuracy, precision, and robustness. The method was successfully applied to the estimation of Amlodipine Besylate and Indapamide in combined tablet dosage forms.

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