Indapamid Articol Original

7/25/2019 Indapamid Articol Original

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International Journal of PharmTech ResearchCODEN (USA): IJPRIF ISSN : 0974-4304

Vol.3, No.3, pp 1482-1487, July-Sept 2011

Development and Validation of RP-HPLCMethod for Quantitative estimation of

Indapamide in Bulk and Pharmaceuticaldosage forms

Jyoti B. Pai*, A. Sathish Kumar Shetty**, Gopinath B., Gangaprasad chenna.

Department of Pharmaceutical Analysis, National College of Pharmacy,Balaraj Urs Road, Shimoga (District), Karnataka, India 577 201.

Corres. author: [email protected]**[email protected]*

Cell: 09449100007,Fax: 08182 273796.

Abstract : A simple, precise, specific and accurate RP-HPLC method has been developed for the determination ofIndapamide 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 m) 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 ofIndapamide was found to be 6.760.0145 min. By adoption of this procedure Indapamide is eluted completely. Linearcalibration plots were obtained between 10-100g/ml. The method of analysis was used for quantification in

pharmaceutical preparations with a coefficient of variation


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A. Sathish Kumar Shettyet al/Int.J. PharmTech Res.2011,3(3) 1483

N

CH3

NH

Cl

O

S

O O

NH2

Figure 1: Chemical Structure of Indapamide

EXPERIMENTAL

MATERIALS AND METHODS

A gradient HPLC (Shimadzu, class VP-Series)equipped with Supelco Reverse Phase C-18 Column(25cm x 4.6 mm i.d.,particle size 5 m) was used, LC-

10 AT VP pump, UV/VIS detector SPD-10A VP withN-2000 CHROMTECK (Shimadzu) software, o-

Phosphoric acid AR grade, Double distilled water andAcetonitrile (ACN) HPLC grade. The optimizedchromatographic conditions are summarized in Table

I.

PREPARATION OF MOBILE PHASE200ml of HPLC grade ACN was mixed with

300ml of 0.05% of orthophosphoric acid which wasprepared in double distilled water and its pH wasadjusted to 3.0 using KOH. Then it was ultrasonicated

for 20 minutes and then filtered through 0.4 mmembrane filter paper

PREPARATION OF STANDARD STOCK

SOLUTION OF INDAPAMIDE

25 mg of standard Indapamide was weighedaccurately and transferred to 25 ml volumetric flaskand dissolved in 10 ml of mobile phase and then

volume was made up to the mark with mobile phase toget 1000 g/ml of standard stock solution A of the

drug. These stock solutions were filtered through0.4m membrane filter paper.

PREPARATION OF MARKETED

FORMULATIONS

Twenty tablets of Indapamide were weighedand the average weight was determined. The tablets

were then crushed to fine powder and powderequivalent to 100 mg was weighed and transferred to100 ml volumetric flask and dissolved in sufficientquantity of mobile phase. The contents wereultrasonicated for 20 minutes and the final volume was

made up to the mark with mobile phase to get 1000

g/ml of standard stock solution B of the drug. Thenthe above prepared solution was filtered through0.4m membrane filter paper.

CHROMATOGRAPHIC CONDITION

The mobile phase containing ACN and o-Phosphoric acid (0.05%) in the ratio of (40:60) wasselected as the optimum composition of mobile phase,because it was found that this solvent system eluted the

drug with good resolution. The flow rate was set to 1.0ml/min and UV detection was carried out at 240 nm.The mobile phase and samples were degassed by

ultrasonication for 20 min and filtered through 0.4mmembrane filter paper. All determinations were

performed at constant column temperature (250C).

ANALYSIS AND PREPARATION OF

CALIBRATION CURVE FOR INDAPAMIDEAppropriate aliquots were pipetted out from

the standard stock solution A (1000 g/ml) in to aseries of 10 ml volumetric flasks. The volume wasmade up to the mark with the mobile phase to get a setof solutions having the concentration range, rangingfrom 20-100 g/ml of Indapamide. 20 l of each

solution were injected into the HPLC system and theirchromatograms were recorded under the same

chromatographic conditions as described above. TheIndapamide was eluted at 6.76 min as shown in Figure:2, the calibration curve was constructed by plottingaverage peak area versus concentration and waspresented in Figure: 3. The method was extended for

determination of Indapamide in pharmaceutical dosageform containing 2.5 mg. The linearity range was found

to be 10-100 g/ml.

ANALYSIS OF INDAPAMIDE IN

FORMULATIONS

From this stock solution B, various dilutions

of the sample solution were prepared and analyzed. A20 l volume of each sample solution was injected in

to the sample injector of HPLC system and theirchromatograms were recorded under the samechromatographic conditions as described above. Thearea of each peak was determined at 240 nm and theamount of drug present in the sample was determined.The proposed methods were validated as per the ICHguidelines.

METHOD VALIDATION[13-16]

ACCURACY

The procedure for the preparation of solutionsfor Accuracy determination at 80%, 100% and 120%level were prepared in the same manner as explainedabove. The solutions were filtered through 0.4 m

membrane filter paper and then they were subjected toanalysis by RP-HPLC method under the samechromatographic conditions as described above. Ateach level, six determinations were performed. The


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results obtained were compared with expected resultsand were statistically validated.

PRECISION

Intraday and inter-day precision were carriedout for the various concentrations of the sample atdifferent time intervals in the same day and at same

time on different days. The concentration of thesample solution was determined as per the proceduregiven for the tablet formulation by determining peak

area at selected analytical wavelength 240 nm. Thevariation of the results within the same day wasanalyzed and statistically validated.

LINEARITY AND RANGEAppropriate aliquots were pipetted out from

the standard stock solution A in to a series of 10 ml

volumetric flasks. The volume was made up to themark with the mobile phase to get a set of solutions

having the concentration range, ranging from 20-100g/ml of the drug.The solutions were injected using a 20 l fixed loop into the chromatographic system at the flow rate of 1.0

ml / min and the effluents were monitored at 240 nm,chromatograms were recorded.

ROBUSTNESS

The evaluation of robustness showed thereliability of analysis with respect to deliberatevariations in method parameters. The various

concentrations were prepared and injected into sampleinjector of HPLC six times under different parameters

like deliberate variations in flow rate, detection (nm).

Figure 2: Chromatogram of Indapamide by RP-HPLC method.

Figure 3: Calibration curve of Indapamide at 240 nm by RPHPLC method.


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Table 1: Optimized Chromatographic conditions for the proposed method

Parameters Optimized condition

Linearity range (g/ml) 10-100

Detection wavelength (nm) 240 Temperature 250C

Retention Time (t) (min) 6.76

Run time (min) 15.0 Limit of Detection (g/ml) 0.173 Limit of Quantification (g/ml) 0.525

Table 2: System Suitability Test Parameters for the proposed method


Retention Time (t) (min) 6.76

Theoretical plates (N) 13432.91 Peak asymmetry 1.299

Table 3: Regression analysis of the Calibration curve for the proposed method


Linearity range (g/ml) 10-100 Regression equation (Y=mx +c) Slope (m) 24184

Intercept (c) 1983

Correlation coefficient (r2

) 0.9995 Relative standard deviation (%) 0.8727

Retention time 6.76

Table 4: Summary of Validation Parameters for the proposed method

Parameters Values

Limit of detection (g/ml) 0.173 Limit of quantitation (g/ml) 0.525 *

Accuracy ( % RSD)

80% 0.4025 100% 1.4773

120 % 0.2018 *

Precision ( % RSD)

Intra Day 0.2779 Inter Day 0.6979

*Robustness (% RSD)

Change in Flow rate 0.8 ml/min 0.1578 1.2 ml/min 0.3673 Change in Detection wavelength 239nm 0.3589

241nm 0.2780 *Mean of six determinations, RSD indicates relative Standard deviation


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Table 5: Statistical Data of Tablet Formulation by RP-HPLC method.

Brand name Label claim (mg) %Obtained S.D*

Lorvas 2.5 100.470.6782

*n=6

RESULTS AND DISCUSSION

In this method the conditions were optimizedto obtain elution of Indapamide. Mobile phase andflow rate selection was based on peak parameters(height, tailing factor, theoretical plates, capacity or

asymmetry), run time, resolution. The system with o-

Phosphoric acid (0.05%): ACN (60:40 v/v) with pH3.0 with system suitability parameters as shown inTable 1, 2.

The run time was set at 15 min and Rt forIndapamide was found 6.760.0145 min with standarddeviation less than 1% with a good linear relationship(r

2= 0.9995) was observed between the concentration

of Indapamide and the respective peak areas in the

range 10-100 g/ml. The regression of Indapamidewas found to be Y = 24184X + 19834, where Y is thepeak area and X is the concentration of Indapamide(Table 3).

The proposed RP-HPLC method was validatedfor intra and inter-day precision with %RSD, 0.2779and 0.6979 respectively. A known amount of the pure

drug solution (80, 100 and 120 %) was added to thepowder sample of the tablet formulation and subjected

for the recovery studies. High recovery was obtainedindicating that the proposed method is highly accurate.Robustness was determined by changing the

parameters like flow rate and detection using similaroperational and environmental conditions. (Table 4)

The proposed method was validated inaccordance with ICH parameters and applied for

analysis of the same in marketed formulations. (Table5)

CONCLUSIONThus, it can be concluded that the method

developed in the present investigation was simple,sensitive, accurate, robust, rapid and precise. Hence,

the above said method can be successfully applied forthe estimation of Indapamide in pharmaceutical dosageforms.

ACKNOWLEDGEMENT

We would like thank to Supra chemicals, NaviMumbai, India for providing reference sample ofIndapamide to facilitate this work.

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