DEVELOPMENT AND VALIDATION OF REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHYMETHOD FOR SIMULTANIOUS ESTIMATION OF AMLODIPINE BESYLATE AND IRBESARTAN IN SYNTHETIC MIXTURE

DEVELOPMENT AND VALIDATION OF REVERSE PHASE HIGH

PERFORMANCE LIQUID CHROMATOGRAPHYMETHOD FOR

SIMULTANIOUS ESTIMATION OF AMLODIPINE BESYLATE AND

IRBESARTAN IN SYNTHETIC MIXTURE.

PATEL SEJAL K., DARJI MAUSAM S.

Department of Pharmaceutical Quality Assurance,

Shree S. K. Patel College of Pharmaceutical Education & Research,

Ganpat University, Kherva – 382711, Mehsana, Gujarat, India.

Date Received:

30TH Jan 2014

Date of Accepted:

6th Feb 2014

Date Published:

11th Feb 2014

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Abstract:

This research manuscript describes simple, sensitive, accurate, precise and repeatable reverse phase high performance liquid chromatography method for the simultaneous determination of Amlodipine besylate(AMLO) and Irbesartan(IRBE) in synthetic mixture. The sample was analyzed by reverse phase C18 column (Phenomenex C18, 250 mm × 4.6 mm, 5µ) as

stationary phase; acetonitrile : methanol : water, pH 3.0 (25 : 20 : 55 , v/v/v) as a mobile phase at a flow rate of 1.0 ml/min. Quantification was achieved with Photo Diode Array detector at 238 nm. The retention time for Amlodipine besylate was found to be 3.6 min and for Irbesartan was found to be 6.3min. The linearity was obtained in the concentration range of 0.3-1.1 µg/ml and 1-6µg/ml for AMLO and IRBE respectively. The method was successfully applied to synthetic mixture because no chromatographic interferences from formulation excipients were found. The method retained its accuracy and precision when the standard addition technique was applied.

Keywords:

Introduction

Amlodipine is a long-acting 1,4-dihydropyridine calcium channel blocker.[1]Chemically it is 3-Ethyl-5-methyl(±)-2-[(2-aminoethoxy)methyl]-4-(2-chloro phenyl)-1,4-dihydro-6-methyl 3,5-pyridine dicarboxylate, mono benzene sulphonate.[2] IRBE is chemically 2-butyl-3-[p-(o -1H-tetrazol-5-ylphenyl)benzyl]-l,3 diazaspiro[4,4]non-l -en-4-one.[3]IRBE is official in US Pharmacopoea (USP)[4]. AMLO is official in Indian Pharmacopoeia (IP), British Pharmacopoeia (BP), US Pharmacopoeia (USP), Japanese Pharmacopoeia (JP) and European Pharmacopoeia (EP). AMLO is estimated by potentiometric titration as per JP.[4] The analytical

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synthetic mixture.[22] The present developed method is simple,, precise and accurate for simultaneous determination of both drugs in their synthetic mixture as per International Conference on Harmonization (ICH) guidelines.

MATERIALS & METHODS

Apparatus

The chromatography was performed on a Shimadzu

(Japan) RP-HPLC instrument (LC-2010CHT) equipped

with Photo Diode Array(PDA) detector and LC-solution software, Phenomenex (Torrance, CA) C18 column (250

mm × 4.6 mm id, 5µm particle size) was used as stationary phase. Sartorius CP224S analytical balance (Gottingen, Germany), an ultrasonic cleaner (Frontline FS 4, Mumbai, India), Digital pH meter (LI 712 pH analyzer, Elico Ltd., Ahmedabad) were used in the study. Reagents and materials

Amlodipine besylate bulk powder was kindly supplied as a gift samples from Astron research limited, Ahmedabad, Gujarat, India and Irbesartan from the medwin Pharmaceuticals Ltd. The synthetic mixture containing 100mg IRBE and 10mg AMLO was prepared in the laboratory using pharmaceutical excipients. Acetonitrile, Methanol, triple distilled water (S. D. Fine Chemicals Ltd., Mumbai, India) used were of HPLC grade. Potassium dihydrogen ortho-phosphate and Ortho-phosphoric acid (S.D Fine Chemicals Ltd., Mumbai, India) used were of AR grade. Nylon 0.45 µm – 47 mm membrane filter (Gelman Laboratory, Mumbai, India) and Whatman filter paper no. 41. (Whatman International Ltd., England) were used in the study.

Preparation of Acidic Water

Acidic Water pH 3 was prepared by accurately adding 0.7ml of Triethyl Amine in 100 ml HPLC-grade water and the pH adjusted to 3.0 by diluted ortho-phosphoric acid.

Preparation of standard stock solutions

An accurately weighed quantity of AMLO(10 mg) and IRBE(10mg) was transferred to a separate 100 ml volumetric flask and dissolved and diluted to the mark with methanol to obtain standard solution each having concentration of 100 µg/ml.

Chromatographic Condition

Stationary phase: C18 column (150 mm x 4.6 mm id., 5

µm).

Mobile phase: Acetonitrile: Methanol: Water pH 3.0 (25: 20: 55, v/v/v)

Flow rate: 1.0 ml/min

Injection volume: 20 µL Temperature: 40 °C

Detection: At 238 nm using PDA detector.

Preparation of calibration curve

Accurately measured standard stock solutions of IRBE (0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 ml) were transferred to a series of 10 ml corning volumetric flasks, and the volume was made up to the mark with methanol. and same the conc.of AMLO 0.3-1.1µl/ml was prepared.An aliquot (20 µl) of each solution was injected under the operating chromatographic condition as described above and responses were recorded. Calibration curves were constructed by plotting the peak areas versus the concentration, and the regression equations were calculated. Each response was average of three determinations.

Preparation of sample solution

The synthetic mixture containing 100mg IRBEand AMLO 10mg and other excipients was prepared in the laboratory. A quantity of powder equivalent to 100mg of IRBE and 10mg AMLO was transferred to a 100 ml volumetric flask. Methanol (50 ml) was added and sonicated for 15 min. The flask was allowed to stand for 5 min at room temperature and the volume was adjusted up to the mark with methanol. The solution was then filtered through Whatman filter paper no. 41. The solution was suitably diluted with mobile phase to get a final concentration of 5 µg/ml of IRBE and 0.5 µg/ml sof

AMLO. An aliquot (20 µl) of sample solution was

injected under the operating chromatographic condition as described above and responses were recorded. The analysis procedure was repeated three times with synthetic mixture.

Method Validation

The method was validated in compliance with ICH guidelines[6].

Accuracy (recovery study)

To study the accuracy of the proposed method, recovery studies were carried out by standard addition method at three different levels (50%, 100%, 150%). A known amount of drug was added to preanalyzed sample powder and percentage recoveries were calculated.

Method precision

Precision of the method was determined by performing inter day variation and intraday variation(%RSD).Intra- day precision (%RSD) was assessed by analyzing standard drug solutions within the calibration range, three times on the same day. Inter-day precision (%RSD) was assessed by analyzing drug solutions with

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in the calibration range on three different days over a period of 7days.

Limit of detection and Limit of quantification

The limit of detection (LOD) and the limit of quantification (LOQ) 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[6].

LOD = 3.3 × σ/S LOQ = 10 × σ/S

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

Robustness

The robustness was studied by analyzing the same samples of AMLO and IRBE by deliberate variations in the method parameters. The change in the responses of AMLO and IRBE were noted. Robustness of the method was studied by changing the extraction time of AMLO and IRBE from synthetic mixture by ± 2 min, composition of mobile phase by ± 2 % of organic solvent, flow rate by ± 2 ml/min and column oven temperature by± 2 oC. The parameters used in system suitability test were asymmetry of the chromatographic peak, tailing factor and theoretical plates, as RSD of peak area for replicate injections.

To optimize the RP-HPLC parameters, several mobile phase compositions were tried. A satisfactory separation and good peak symmetry for AMLO and IRBE were obtained with a mobile phase comprising of acetonitrile: methanol: Water, pH 3.0 (25: 20: 55, v/v/v) at a flow rate of 1.0 ml/min to get better reproducibility and repeatability. Quantification was achieved with PDA detection at 238 nm based on peak area. The peak with clear baseline was obtained (Figure 1). The retention time for AMLO was found to be 3.6 min and for IRBE 6.3min,(Figure 1). Linear correlation was obtained between peak area versus concentrations of AMLO and IRBE in the concentration ranges of 0.3-1.1 µg/ml and 1-6µg/ml respectively (Table 1) (Figure 2 & 3). The method was found to be specific as no significant changes in the responses of AMLO and IRBE was observed after 24 hr. The mean recoveries obtained were 99.89 ± 0.79 % for AMLO and 98.79±0.69 (Table 1 and 2), which indicates accuracy of the proposed method. The % RSD value for bothwere found to be <2 %, which indicates that the proposed method is repeatable. The low % RSD values of interday and intraday variations for both, reveal that the proposed method is precise. LOD value for AMLO and IRBE was found to be 0.1637

µg/ml and 0.1463µg/ml LOQ value for AMLO and IRBE

was found to be 0.4963 µg/ml and 0.365 µg/ml (Table 1). These data show that the proposed method is sensitive.

Table 1 Regression analysis data and summary of validation parameter for theproposed RP-HPLC method

Parameters AMLO IRBE

Concentration range (µg/ml) 0.3-1.1 µg/ml 1-6 µg/ml

Slope 42443 36548

Intercept 4065 6745

Correlation Coeficient 0.9997 0.9986

LODa (µg/ml) 0.1637 0.1463

LOQb (µg/ml) 0.4963 0.3653

Accuracy (nc = 3) 99.8±0.78 98.75±0.63

Repeatability (% RSDd, n = 6) 0.78 0.47

Precision (%RSD)

Interday(n=3)

Intraday(n=3)

0.95-1.92

0.48-0.97

1.04-1.47

0.38-0.80

a = Limit of detection b = Limit of quantification

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Table 3 System suitability test parameters for the proposed RP-HPLC method

Parameters AMLO± CV, %

(n = 6)

IRBE ± CV, %

(n = 6)

Retentiontime (min) 3.655± 0.12235 6.303± 0.0859

Tailing factor 1.223±1.195 1.13133± 1.2817

Theoretical plates 2280 ± 1.915 3623.5 ± 1.880

Resolution 5.2943 ± 1.170

Table 4 Analysis of synthetic mixture of LSD by proposed RP-HPLC method (n = 3)

Sample

No AmontTaken AmontFound

%

Assay

AMLO

mg

IRBE

mg

AMLO

Mg

IRBE

Mg

AMLO

mg

IRBE

mg

1 10 100 9.51 99.79 95.1 99.79

2 10 100 10.3 99.96 103 99.96

3 10 100 9.87 100.6 98.7 100.6

Figure 1. Chromatogram of AMLO+IRBE (5µg/ml) at 238 nm

DRUG LEVEL AMOUNT OF SAMPLE

TAKEN (mg/ml) AMOUNT OF STANDARDSPIKED (%)

MEAN

% RECOVERY±SD

AMLO I 1 _{50 99.20 ± 0.98}

II 1 _{100 99.48 ± 0.81}

III 1 _{150 100.33± 0.38}

IRBE I 10 _{50 100.46 ± 0.37}

II 10 _{100 100.61 ± 0.30}

Figure 2(above). Linearity curve of Amlodipine besylate(0.3

1.1µg/ml)

Figure 3Linearity curve of Irbesartan(1-6

CONCLUSION

A simple, sensitive, repeatable and specific RP method has been developed for the estimation of Amlodipine besylate and Irbesartan using a PDA detector. The method was validated for accuracy, precision, linearity, specificity, LOD &LOQ and robustness. In this proposed method the linearity is observed in the concentration range of 0.3-1.1

AMLO and 1-6 µg/mlfor IRBE with co correlation, (R2) = 0.9997 and (R2)=0.9986.

the analysis of synthetic mixture by the proposed method is highly reproducible and reliable and it is in good agreement with the label claim of the drug. The method can be used for the routine analysis of the AMLO and IRBE in pharmaceutical dosage form without any interference of excipients.

ACKNOWLEDGEMENT

The authors are thankful to Astron research limited Ahmedabad, Gujarat, Indiaand Medwin Pharmaceuticals Ltd,Ahmedabad,Gujarat,India for providing gift sample of Amlodipine besylate and Irbesartan for research. The authors are highly thankful to Shree S. K. Patel College of Pharmaceutical Education & Research, Ganpat University, Kherva, Mehsana, Gujarat, India for providing all the facilities to carry out the work.

y = 22275x + 14199 R² = 0.999

0 100000 200000 300000 400000 500000

0 0.5 1

Amlodipine

besylate(0.3-6µg/ml)

A simple, sensitive, repeatable and specific RP-HPLC method has been developed for the estimation of Amlodipine besylate and Irbesartan using a PDA detector. The method was validated for accuracy, precision, linearity, specificity, LOD &LOQ and robustness. In this proposed method the linearity is 1.1 µg/ml for for IRBE with co-efficient of The result of the analysis of synthetic mixture by the proposed method is highly reproducible and reliable and it is in good agreement with the label claim of the drug. The method sis of the AMLO and IRBE in pharmaceutical dosage form without any

The authors are thankful to Astron research limited, and Medwin Pharmaceuticals ng gift sample of Amlodipine besylate and Irbesartan for research. The Shree S. K. Patel College of Pharmaceutical Education & Research, Ganpat University, Kherva, Mehsana, Gujarat, India for providing all the

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