Development and Validation of Stability Indicating high performance liquid chromatographic Method for Indapamide

Rucha A Patel^1*, Meghna P Patel ¹, Hasumati Raj¹, Nehal Shah²

¹Department of Quality Assurance, Shree Dhanvantry Pharmacy College, Kim, Surat, Gujarat, India

²Dharmaj Degree of Pharmacy, Dharmaj, Anand, Gujarat, India

*Corresponding Author E-mail: ruchajigar6114@gmail.com

ABSTRACT:

An approach of forced degradation study was successfully applied for the development of a stability-indicating high performance liquid chromatographic method for Indapamide in the presence of its degradation products. In the present study a simple, accurate and precise reverse phase liquid chromatographic method has been developed of Olmesartan Indapamide in bulk. Developed Method was achieved on symmetry C18 (150 mm × 4.6 mm, 5 μ) column using a Acetonitrile: 0.02 M Na2HPO4(45:55 v/v) mobile phase and pH 7 adjusted with ortho phosphoric acid. Isocratic elution mode at a flow rate of 1.0 ml/min at Room temperature with a load of 20μl Injection volume. The detection was carried out at 240 nm. The linearity of the proposed method was investigated in the range of 10-50 microg/mL (r2 = 0.998) for Indapamide. The retention time of Indapamide was found to be around 4.79 min and 7.59 min respectively. The drug substances were subjected to stress conditions of acid hydrolysis, base hydrolysis, Oxidative, photolytic and thermal. The developed RP-HPLC method was validated with respect to linearity, accuracy, precision, robustness, LOD and LOQ.

KEY WORDS: Indapamide, Hypertension, stability study.

INTRODUCTION:

Indapamide (IND) is used in cardiac disease condition like Hypertension. Indapamide is an orally administered diuretic and anti‑hypertensive drug.[1] Its molecule contains both a polar sulfamoyl chlorobenzamide moiety and a lipid soluble methyl‑indoline moiety[2] Indapamide[Figure 1] is chemically 3-(aminosulfonyl)-4-chloro-N-(2, 3-dihydro-2-methyl-1H-indol-1-yl) benzamide It differs chemically from thiazide in a way that it does not possess the thiazide ring system and contains only one sulfonamide group. It is used for hypertension and also for oedema, including that associated with heart failure[3]. Currently most commonly prescribed medicines for hypertension are Angiotensin receptor blockers and diuretics.

Figure1. Chemical Structure of Indapamide

The clinical and pharmaceutical analysis of this drug requires effective analytical procedures for quality control and pharmacodynamics and pharmacokinetic studies as well as stability study Several HPLC assay method of Indapamide bulk and in tablet dosage form are available[4,5,6,7]. Indapamide is official in IP, BP, EP, JP[8,9,10,11] The method was validated as per International Conference on Harmonization (ICH) guidelines[12]

INSTRUMENTS:

· Chromatographic analysis was carried out on Operation-semi automatic, Pump-single pump Model- SPD 10 A-LC 10 AT Company-shimadzu, Japan Software-Winchrome software

· Semi micro analytical balance (Sartorius CD2250, Germany) was used for weighing purpose.

· HPLC water was obtained using Arium^®611VF(Sartorius).

· Magnetic stirrer (1 MLH, Remi) was used for mixing purpose.

· pH tutor (313927, Eutech Instruments) was used for pH measurement.

· Sonications of solutions were done using Ultrasonic cleaner (D 120/1H, Trans-O-Sonic).

· Column used was Inert Phenomenex C₁₈ (250mm×4.6mm i.d.) 5μm

· Nylon membrane filters (0.22 µm, 47 mm D)

· All volumetric glass wares used were calibrated.

EXPERIMENTAL WORK AND CONDITION:

Buffer preparation

Accurately weighed 2.83 gm Na₂HPO₄ was dissolved in to 1000 ml water, than pH was adjusted to 7 with ortho‑phosphoric acid.

A) Preparation of standard stock solution (1000 μg/mL):

10 mg of bulk drug was weighed accurately and transferred into a clean, dry 10 mL volumetric flask, dissolved in 1ml of Acetonitrile and volume was adjusted to 10 mL with mobile phase and further 0.1 ml dilute up to 10 ml with mobile phase to get a concentration of 10 μg/mL.

Stability indicating property (forced degradation)

A. Acid Induced Degradation: Accurately weighed 10mg bulk drug was dissolved in 1 ml Acetonitrile and add 10 ml 0.1 N HCl in it.Then this solution is kept At 60˚C and pipette out 0.1 ml and dilute with mobile phase up to 10 ml and chromatogram was recorded.Samples were taken at 0min, 10min, 30min, 1 Hr, 2 Hr and 3Hr Blank solution was also injected without API.

B. Base Induced Degradation:

Accurately weighed 10mg bulk drug was dissolved in 1 ml Acetonitrile and add 10 ml 0.1 N NaOH in it. Then this solution is kept At 60˚C and pipette out 0.1 ml and dilute with mobile phase up to 10 ml and chromatogram was recorded. Samples were taken at 0min, 10min, 30min, 1 Hr, 2 Hr and 3Hr .Blank solution was also injected without API.

C. Hydrogen Peroxide Induced Degradation:

Accurately weighed 10mg bulk drug was dissolved in 1 ml Acetonitrile and add 10 ml 3% H₂O₂ in in it. Then this solution is kept At 60˚C and pipette out 0.1 ml and dilute with mobile phase up to 10 ml and chromatogram was recorded. Samples were taken at 0min, 10min, 30min, 1 Hr, 2 Hr and 3Hr .Blank solution was also injected without API.

D. Thermal degradation:

Accurately weighed 10mg bulk drug was kept in a hot air oven for 3 h at a temperature of 60°C, then made up with mobile phase. For further dilution, 1 mg sample was added to a 10 ml volumetric flask individually, and for tablet degradation, Average weight of 5 tablet powder was kept in hot air oven in same condition.10mg powder added to a 10 ml flask and made up with mobile phase and further dilute 0.1 ml in 10 ml mobile phase

E. Photochemical Degradation:

10 mg of drug was exposed to UV light for 3 hr, then made up with mobile phase up to 10ml. For further dilution, 1 ml of sample was added to a 10 ml volumetric flask individually, for tablet degradation Average weight of 5 tablet powder was exposed to UV light for 3 hr.10mg powder added to a 10 ml flask and made up with mobile phase and further dilute 0.1 ml in 10 ml mobile phase

Figure 2. Standard Indapamide (10 μg/mL)

Acid Degradation – 3 Hr at 60˚C

Figure- 3 Indapamide -Acid degradation of 3 Hr at 60˚C

Table- 1 Indapamide -Acid degradation of 3 Hr at 60˚C

	Retention Time (min.)	Area	% Degradation
IMP-B	1.12	4729
IMP-A	5.43	5620	29.73%
INDAPAMIDE	7.61	170800

[A]ACID DEGRADATION OF INDAPAMIDE

Accurately weighed 10mg bulk drug was dissolved in 1 ml Acetonitrile and add 10 ml 0.1 N HCl in it. Then this solution is kept AT 60˚C and pipette out 0.1 ml and dilute with mobile phase up to 10 ml and chromatogram was recorded. Samples were taken after 10min, 30min, 1 Hr, 2 Hr and 3Hr .Blank solution was also injected without API. (Figure-3)

[B ]BASE DEGRADATION OF INDAPAMIDE

Accurately weighed 10mg bulk drug was dissolved in 1 ml Acetonitrile and add 10 ml 0.1 N NaOH in it.Then this solution is kept at 60˚C and pipette out 0.1 ml and dilute with mobile phase up to 10 ml and chromatogram was recorded. Samples were taken after 10min, 30min, 1 Hr, 2 Hr and 3Hr .Blank solution was also injected without API. (Figure-4)

Table-2 Indapamide - Base Degradation of 2 Hr at 60˚C

		Retention Time (min.)	Area	% Degradation
IMP-C		3.14	75331	17.72%
IMP-D		3.45	31923
IMP-F		5.14	1433
INDAPAMIDE		7.28	249826
	IMP-E	9.05	1220

Base Degradation - 3 Hr at 60˚C

Figure- 4 Indapamide - Base Degradation of 3 Hr at 60˚C

[C]OXIDATION DEGRADATION OF INDAPAMIDE

Accurately weighed 10mg bulk drug was dissolved in 1 ml Acetonitrile and add 10 ml 3% H₂O₂ in it. Then this solution is kept At 60˚C and pipette out 0.1 ml and dilute with mobile phase up to 10 ml and chromatogram was recorded. Samples were taken after 10min, 30min, 1 Hr, 2 Hr and 3Hr .Blank solution was also injected without API. (Figure-5)

Oxidation Degradation – 3 Hr at 60˚C

Figure- 5 Indapamide-Oxidation Degradation – 3 Hr at 60˚C

Table-3 Indapamide - OxidationDegradation Of 3 Hr At 60˚C

Impurity	Retention Time (min.)	Area	% Degradation
IMP-H	2.2	4673
3% H2O2	2.44	92553
IMP-G.	2.85	113381	50.50%
IMP-I	3.79	1202
INDAPAMIDE	6.39	118889

DEGRADATION SUMMARY

Table 4 Summary of Overall Degradation Study

Sr. No.		CONDITION	% DEGRADATION at 60 ^˚C
			INDAPAMIDE
1		Acid hydrolysis	29.73%
2	Base hydrolysis		17.72%
3	Oxidation		50.50%
4	Thermal		3.87%
5		Photolytic	3.42%

CONCLUTION:

The study shows that the developed HPLC Method is fast, precise, specific, accurate and stability indicating. The stability-indicating method resolved the drug peak and also the peaks of degradation products formed under variety of conditions. After exposure of indapamide to stress condition like acid, base hydrolysis; oxidation, Indapamide IMP-A, IMP-B, IMP-C, IMP-D, IMP-E, IMP-F, IMP-G, IMP-H, IMP-I and IMP-J were observed Therefore this method can be employed for monitoring the stability of and Indapamide drug substance commercially.

Substantiation of developed Stability indicating method for estimation of Indapamide were good at both its specificity and recovery; the degradation product does not interfere in the estimation of Drug(Olmesartan medoxomil and Indapamide) the results of Stability indicating method validation fulfills the FDA guideline.

These developed methods provide selective quantification of drug without any interference from blank and placebo and are highly sensitive, reproducible, reliable, rapid and specific. Analysis by these methods were found to be simple, accurate, reproducible, precise, and in good agreement with labeled claim of the drug.

Validation of developed rp-hplc method ^[13]

The RP-HPLC method has been developed on symmetry C₁₈ (150 mm × 4.6 mm, 5 μ) column using a Acetonitrile: 0.02 M Na₂HPO₄(45:55 v/v) mobile phase and pH 7 adjusted with ortho phosphoric acid. Isocratic elution mode at a flow rate of 1.0 ml/min at Room temperature with a load of 20μl Injection volume. The detection was carried out at 240 nm. with retention time of Indapamide at \ 7.52 min . As the HPLC method has been developed, the validation using various parameters was performed to ensure that the performance characteristic of the method meets the requirements for the intended analytical applications. Validation was carried out with respect to various parameters, as required under ICH guideline Q2 (R1). The developed method validated with respect to parameters such as linearity, precision, accuracy, LOD, LOQ robustness, and solution stability.

[A] LINEARITY

Purpose

The linearity of the analytical method is its ability to elicit test results which are directly proportional to the concentration of the analyte in the sample.

Methodology

The portions 1 mL, 2 mL, 3 mL, 4 mL and 5 mL of 100 µg/mL of standard stock solution of Indapamide (Sec: 5.3.1.3.1) were transferred separately to a series of 10 mL of volumetric flasks and volume was adjusted to 10 mL with Mobile phase to obtain the concentrations of 10 μg/mL, 20 μg/mL, 30 μg/Ml, 40 μg/mL and 50 μg/mL respectively Calibration curve was constructed by plotting peak areas v/s concentrations of Indapamide. The results are presented in Table 7 figure 6.

Acceptance Criteria

The regression coefficient (r²) should be ideally 1.0 or value as lose as possible to 1.0

[B] ACCURACY (% RECOVERY)

Purpose

The accuracy of an analytical method is the closeness of test results obtained by that method to the true value. The accuracy of the method was determined by calculating recovery of Indapamide by the standard addition method.

Methodology

Accuracy expresses the closeness of agreement between the values which are accepted either as a conventional true value or an accepted reference value and the value found practically recovery was performed by preparing concentration 10 μg/mL of Indapamide sample solution. Three samples were prepared for each recovery level of 80%, 100% and 120% spiking of standard solution. The solutions were then analyzed, and the percentage recoveries were calculated from the calibration curve. The accuracy was calculated as the percentage of the drug recovered from the formulation matrix.

_{Accuracy was calculated using the following equation:}

_{% Recovery = [(Spiked Area – Unspiked Area)]}

_{_______________________________________________________x 100}

_{Standard Area}

Acceptance Criteria

The % recovery at each level should be between 98.0%-102.0%l

[C] PRECISION

Purpose

The precision of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found.

Methodology

Intraday precision

The portions of 1 mL, 2 mL and 3 mL of 100 µg/mL of standard stock solution of Indapamide was transferred to a separate 10 mL of volumetric flasks and volume adjusted up to 10 mL with Mobile phase to obtain a concentrations of 10 μg/mL, 20 μg/mL and 30 μg/mL for Indapamide.20 μl of each of these standard solutions of Indapamide were injected under the operating chromatographic conditions into system three times on the same day and chromatograms were recorded

Interday Precision:

The portions of 1 mL, 2 mL and 3 mL of 100 µg/mL of standard stock solution of Indapamide was transferred to a separate 10 mL of volumetric flasks and volume adjusted up to 10 mL with Mobile phase to obtain a concentrations of 10 μg/mL, 20 μg/mL and 30 μg/mL for Indapamide.20 μl of each of these standard solutions of Indapamide were injected under the operating chromatographic conditions into system three times on the three consecutive days and chromatograms were recorded. The results are presented in terms of % relative standard deviation (% RSD) in Table 8 and Table 9.

Acceptance Criteria

The % RSD of the absorbance for μl of each of these standard solutions of obtained should be NMT 1.0%.

[D ] Limit of detection and limit of quantification

Purpose

LOD and the LOQ of the drug were calculated using the following equations as per _{International Conference on Harmonization (ICH) guidelines.}

LOD = 3.3 × σ/S

LOQ = 10 × σ/S

_Where,

σ = Standard deviation of the response

S = Slope of calibration curve.

The results of LOD and LOQ are presented in Table 11

[E] ROBUSTNESS

Robustness was measured by changing the pH of mobile phase, Ratio of Mobile phase and flow rate. The of mobile phase was set ±2 ºC, The , Ratio of Mobile phase was set ±5ml and Flow rate was set ±2 mL/Min. solution of both the drugs was injected three times.The results are presented in Table no. 12 ,Table 13 and Table 14

RESULTS AND DISCUSSION:

SYSTEM SUITABILITY

It includes various parameters like theoretical plates, tailing factor, system precision and capacity factor. Various system suitability parameters for the developed method are shown in Table 6.

Table 6: System suitability parameters for HPLC Method

Sr. No.	System suitability parameter	Values obtained	Acceptance limit according to FDA guidelines
1	Theoretical Plates (N)	73829	>2000
2	Retention time	7.59 min	-
3	Tailing Factor (T)	1.45	≤2

[A] LINEARITY

The linearity of the response of indapamide was found to be 10-50 μg/mL. The calibration graphs were obtained by plotting the peak area versus the concentration. Regression coefficient was found to be 0.998 for indapamide.The linearity of the analytical method is its ability to elicit test results which are directly proportional to the concentration of the analyte in the sample.

Figure 6: Calibration of Indapamide

Table 7: Peak area of Indapamide

Sr.no	Concentration(μg/ml)	Peak Area±SD(n=6)
1	10	482722±5586
2	20	865436±221
3	30	1297231±3580
4	40	1787321±1101
5	50	2219382±105

[B] PRECISION:

Method precision (intra-day precision) was evaluated by carrying out three independent measurements of standard drug solution at three times on the same day .The results obtained for Mean % Relative Standard Deviation (RSD) values obtained for three concentrations of Indapamide (10 μg/mL, 20 μg/mL and 30 μg/mL) at intraday precision was 0.28% and at interday precision was 0.64% for Indapamide .All the data are within the acceptance criteria of 1% (Table-8, 9).

INTRADAY:

Table 8: Intraday result of of Indapamide

Conc. (µg/ml)	Peak Area ± SD (n=3)	%RSD	Mean % RSD
10	485271±670	0.13%	0.28%
20	866080±544	0.06%
30	1295498±3205	0.23%

INTERDAY

Table 9: Interday result of of Indapamide

Conc. (µg/ml)	Peak Area ± SD (n=3)	%RSD	Mean % RSD
10	508178±2913	0.57%	0.64%
20	876320±114	0.01%
30	130313±1284	0.75%

[C] ACCURACY (RECOVERY STUDY)

Accuracy expresses the closeness of agreement between the values which are accepted either as a conventional true value or an accepted reference value and the value found practically recovery was performed by preparing concentration 10 μg/mL of Indapamide sample solution. Three samples were prepared for each recovery level of 80%, 100% and 120% spiking of standard solution. The solutions were then analyzed, and the percentage recoveries were calculated from the calibration curve. The accuracy was calculated as the percentage of the drug recovered from the formulation matrix. (Table-10)

_{% Recovery = [(Spiked Area – Unspiked Area)]}

_{_______________________________________________________x 100}

_{Standard Area}

[D] LOD and LOQ

The peak area of ten solutions containing 50 µg/mL were measured at 240 nm and calculated according to equation of LOD [3.3 x MSD/ slope] and LOQ [10 x MSD/ slope]. (Table-11)

Table 10: Accuracy of Indapamide

Conc (μg/ml )	%Spiking	Total Conc	Peak Area	Recovered Amt.	%Recovery	Mean % Recovery
10	-	10	442680	10.06	100.62%	100.35%
10	80%	18	792100	18.02	100.11%
10	100%	20	873055	19.86	99.32%
10	120%	22	967900	22.02	100.09%

Table 11: LOD and LOQ of Indapamide

Sr No.	Parameter	INDA
1.	S.D of the Y-intercepts of 6 calibration curve	11885
2.	Mean slope of the 6 calibration curves.	43950
3.	LOD = 3.3 × (SD/Slope) (μg/ml)	0.89 μg/ml
4.	LOQ = 10 × (SD/Slope) (μg/ml)	2.70 μg/ml

[E] ROBUSTNESS

The %RSD for Olmesartan medoxomil and indapamide for Robustness was measured by changing the pH of mobile phase, Ratio of Mobile phase and flow rate. The of mobile phase was set ±2 ºC, The , Ratio of Mobile phase was set ±5ml and Flow rate was set ±2 mL/Min. (Table-12)

FLOW RATE CHANGE

Table 12: Flow rate in rubustness of Indapamide

Sr. No.	Flow rate (ml/min)	Conc. (µg/ml)	Peak area ± SD ( n=3 )	%RSD	Mean % RSD
1	0.8	10	482882±881	0.18	0.47%
2	1.2	10	475063±3613	0.76

pH CHANGE

Table 13: pH change in rubustness of Indapamide

Sr. No.	Wavelength (ml/min)	Conc. (µg/ml)	Peak area ± SD ( n=3 )	%RSD	Mean % RSD
1	6.8	10	489929±3391	0.69%	0.705%
2	7.2	10	505025±3643	0.72%

RATIO OF MOBILE PHASE CHANGE

Table 14: Ratio of mobile phase change in rubustness of Indapamide

Sr. No.	Ratio (µg/ml)		Peak area ± SD ( n=3 )	%RSD	Mean % RSD
	Acetonitrile	Buffer	INDA	INDA	INDA
1	50	50	439522±1297	0.29%	0.30%
2	40	60	615715±722	0.11%

[F] SOLUTION STABILITY:

The standard and sample solutions were found stable up to 24 hr at room temperature. After 3,6,24 hr the solutions were analyzed and results related to solution stability were summarized in table-15.

Table 15:

Times(Hr)	Area	%RSD
3	242536	0.09 %
6	242563
24	242942

SUMMARY OF THE OBTAINED RESULTS

Table 16: Summary of validation of Indapamide

Sr No.	Parameters	INDAPAMIDE
1.	Wavelength (nm)	240 nm
2.	Linearity range (μg/ml)	10-50 µg/ml
3.	Standard Regression equation	Y=43950x + 11885
4.	Correlation coefficient (R^2)	0.998
5.	Precision (%RSD) Intraday Interday	0.28 0.64
6.	% Recovery (Accuracy, n = 3)	100.35%
7.	LOD (μg/ml)	0.89%
8.	LOQ (μg/ml)	2.70%
9.	Robustness Flow rate change pH change Ratio change	0.47% 0.70% 0.30%

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Received on 08.04.2015 Accepted on 22.04.2015

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Tech. 2015; Vol. 5: Issue 3, July- Sept. Pg 158-164

DOI: 10.5958/2231-5713.2015.00023.9