Development of Validated Q-absorbance Ratio, Derivative Spectrophotometric and Stability indicating RP-HPLC methods for the simultaneous estimation of Losartan Potassium & Hydrochlorothiazide in Pharmaceutical Dosage Form and comparison of the results by ANOVA

 

V.V.S. Ashritha, N. Usharani*, Soudamini Alekhacharan, , B. Alekhya, R. Venkatesh

Maharajah’s College of Pharmacy, Phool Baugh, Vizianagaram, Andhra Pradesh. India.

 

ABSTRACT:

Hypertension is estimated to cause 7.5 million deaths, leading to 12.8 % of the total of all deaths worldwide. In the recent years, the combination of Losartan Potassium and Hydrochlorothiazide has attained significant popularity in reducing the risk of heart diseases particularly in the patients with hypertension and ventricular hypertrophy. The literature survey revealed that there were several chromatographic and spectroscopic methods for the simultaneous estimation of the selected drugs but it was noted that there were no reported comparative studies of the developed methods. Hence the authors attempted to develop new validated Q- absorbance ratio, Derivative spectrophotometric and Stability indicating RP-HPLC methods for the simultaneous estimation of Losartan Potassium and Hydrochlorothiazide in pharmaceutical dosage form. The authors also propose to compare the results obtained by ANOVA. The two Spectrophotometric methods were carried out using an Agilent Cary 60 UV-Visible double beam Spectrophotometer. In Q-absorbance ratio method, samples were measured at two wavelengths, 230 nm (isoabsorptive point) and 225.5 nm (λ_max of Hydrochlorothiazide). In Derivative spectroscopy, analysis was performed by derivatizing the zero order spectrum to the corresponding second order spectrum. Stability indicating RP-HPLC method was performed using Eclipse XDB C₁₈ column where the samples were analyzed using a mixture of Acetonitrile and Water in the ratio of 70:30 % v/v as mobile phase at a flow rate of 1 ml/min. The drugs were monitored at 230 nm. The retention times for Losartan Potassium and Hydrochlorothiazide were found to be 1.010 minutes and 1.387 minutes respectively. The proposed methods were validated as per ICH guidelines. The results obtained from the three developed methods were then compared by ANOVA which indicated that the proposed methods can successfully be used for the analysis of Losartan Potassium and Hydrochlorothiazide in bulk or in combined dosage forms.

 

 

 

 

INTRODUCTION:

Losartan Potassium is an Angiotensin-receptor blocker used for the treatment of hypertension¹. Chemically it is Potassium; (2-butyl-4-chloro-1-{[2`-(1H-tetrazol-5-yl) biphenyl-4-yl] methanol. The structure of Losartan Potassium is shown in Figure 1.

 

 

Hydrochlorothiazide belongs to thiazide class of diuretics which blocks Na/Cl co-transporter in distal convoluted tubule. Chemically it is 6-chloro-3, 4-dihydro-2H-1, 2, 4-benzothiadiazine-7-sulfonamide 1, 1-dioxide².  The structure of Hydrochlorothiazide is shown in Figure 2.

Figure.1 Chemical Structure of Losartan Potassium

 

Figure.2 Chemical Structure of Hydrochlorothiazide

 

The literature survey revealed that though there are several chromatographic (HPLC, HPTLC) and spectrophotometric methods (first order derivative spectroscopy) existing for the simultaneous estimation of the selected drugs, statistical comparison studies on the results of the developed methods were not found to be reported. Hence the authors attempted not only to develop new validated Q- absorbance ratio, Derivative spectroscopic and stability indicating RP-HPLC methods for the simultaneous estimation of Losartan Potassium and Hydrochlorothiazide in pharmaceutical dosage form but also to compare the results obtained using ANOVA.

 

Q-Absorbance ratio method:

Q-Absorbance ratio method has been used in the present study where absorbance for a drug is measured at two wavelengths; i) isoabsorptive point of the two drugs and ii) at the absorption maximum of any one of the two drugs^3,4. The concentration of each drug was calculated using the equations,

 

 

Where, X and Y are the two selected drugs;

 

C_X and C_Y are the concentrations of drugs X and Y respectively.

 

Q_m represents the ratio of the absorbances

Q_x, Q_y represents the ratio of the absorptivity’s for the drugs X, Y at their respective is absorptive points.

A₁ and a_x1 are the absorbance and absorptivity of drug X at isoabsorptive point

 

Derivative spectrophotometric method:

The proposed Derivative spectrophotometry is based on zero-crossing technique which eliminates the spectral interferences. The spectral characteristics of Losartan Potassium and Hydrochlorothiazide revealed that the signal of second order derivative spectrum was relevant for quantification⁵

 

Stability indicating RP-HPLC method:

Stability indicating RP-HPLC method for the simultaneous estimation of Losartan Potassium and Hydrochlorothiazide was carried out by deliberately subjecting the sample solutions to certain conditions like acid hydrolysis, base hydrolysis, photolytic and thermal degradation as per ICH guidelines^6,7. The method was carried out using Eclipse XDB plus C₁₈ column (4.6 × 150 mm, 5μm particle size). After several trials, mobile phase consisting a mixture of Acetonitrile and Water in the ratio of 70:30 % v/v at a flow rate of 1ml/min was found to be suitable for the separation of principle peaks from that of the degradants at 230 nm.

 

Analysis of Variance (ANOVA):

The developed methods were subjected to the statistical application namely ANOVA to determine whether there is any significant difference among the results of the proposed methods. In this process, F-test was applied to the obtained results in order to check and specify the best out of the three proposed methods⁸.

 

MATERIALS AND METHODS:

Instrument and materials:

UV spectrophotometric methods were carried out using an Agilent Cary 60 UV-Visible double beam Spectrophotometer with 1 cm matched quartz cells. For Stability indicating RP- HPLC method development, Agilent prominence 1220 infinity LC equipped with Eclipse XDB plus C₁₈ column, dual-channel gradient pump and variable wavelength detector (190-600nm) was used. Losartan Potassium and Hydrochlorothiazide standards were procured from Yarrow chem products, Mumbai. Commercially available brand (REPACE H) containing 50 mg of Losartan Potassium and 12.5 mg of Hydrochlorothiazide was purchased from local market Vizianagaram, AP, India. HPLC grade Acetonitrile and water were purchased from Fisher scientific, Mumbai.

 

Preparation of standard solutions:

Standard solutions of Losartan Potassium (100 µg/ml) and Hydrochlorothiazide (100 µg/ml) were prepared by dissolving 10 mg of each drug in the solvent (water and methanol in the ratio of 90:10 v/v) separately in 100 ml volumetric flasks. The volumes were then made upto the mark with the same solvent. From the above solutions 30 µg/ml of Losartan Potassium and Hydrochlorothiazide were prepared separately and scanned from 200-400 nm where Losartan Potassium showed λ_max at 230 nm and Hydrochlorothiazide showed λ_max at 225.5 nm. In the overlay spectra isoabsorptive point was found to be at 230 nm (Figure 3).

 

Figure. 3 Overlay Spectra of Losartan Potassium and Hydrochlorothiazide

Q- Absorbance ratio method:

In Q- Absorbance ratio method, the absorbance of Losartan Potassium solutions (10-50 µg/ml) and Hydrochlorothiazide solutions (2.5-12.5 µg/ml) were measured at 230 nm (isoabsorptive point) and at 225.5 nm (λ_max of Hydrochlorothiazide). The amount of the selected drugs in the tablet dosage form were estimated from the absorbance and absorptivity values calculated using the corresponding formulae as mentioned before in introduction^9,10,11.

 

Derivative spectrophotometric method:

In derivative spectrophotometric method, the corresponding zero order spectrum was converted to second order derivative spectra (Figure. 4). One particular wavelength was selected for each drug at which the absorbance of the other was found to be zero. From the second order overlain derivative spectra, the zero crossing points obtained for Losartan Potassium and Hydrochlorothiazide were found to be 212.4 nm, 227.8 nm, 232.3 nm and 221.2 nm, 223.8 nm, 229.7 nm respectively. The working wavelengths were selected as 232.3 nm for Losartan Potassium where Hydrochlorothiazide exhibited zero absorbance and 229.7 nm for Hydrochlorothiazide where Losartan Potassium exhibited zero absorbance¹². The regression equations for the second order derivative spectra were obtained as y = 0.5286 x + 0.0013 for Losartan Potassium and y = 0.0722 x + 0.008 for Hydrochlorothiazide.

 

Figure.4 Second Order Derivative Spectra of Losartan Potassium and Hydrochlorothiazide

 

Stability indicating RP-HPLC method:

Losartan Potassium (40 mg) and Hydrochlorothiazide (10 mg) were accurately weighed, transferred into a 50 ml volumetric flask and dissolved in the mobile phase consisting a mixture of Acetonitrile and Water in the ratio of 70:30 v/v. The solution was then made upto the mark with the same mobile phase. When suitable aliquots of mixed standard solutions of the proposed drugs were spiked in the HPLC system at a flow rate of 1 ml/min and at the detection wavelength of 230 nm, the typical chromatogram obtained showed the retention times of Losartan Potassium and Hydrochlorothiazide at 1.010 minutes and 1.387 minutes respectively (Figure 5). The calibration curves showed good linearity within the range of 10-60 µg/ml and 2.5-15 µg/ml for Losartan Potassium and Hydrochlorothiazide respectively^13,14,15.

 

Figure. 5 Chromatogram of Losartan Potassium and Hydrochlorothiazide

 

Forced degradation studies were undertaken to assess the quality of the selected drugs with time under the influence of a variety of factors such as acid hydrolysis, base hydrolysis, photolytic and thermal degradation¹⁶. 

 

Acid and Base degradation:

Twenty tablets were taken and made into fine powder. A quantity of powder equivalent to 50 mg was taken and dissolved in little quantity of mobile phase in a 50 ml volumetric flask. The volume was then made upto mark with mobile phase. Twenty five ml of the resulting solution was subjected to acid hydrolysis by adding 1 N Hydrochloric acid and the remaining 25 ml solution was subjected to alkaline hydrolysis by adding 1 N NaOH. The solutions were then refluxed for 6 hrs at 60 ⁰C and cooled to room temperature. The solutions resulting from acid hydrolysis and base hydrolysis were neutralized with 1 N NaOH and 1 N Hydrochloric acid respectively. The solutions were filtered through a 0.45 µ membrane filter. Then suitable aliquots prepared from the respective filtrates were injected into the column and the chromatograms for acid and base degradation of Losartan Potassium and Hydrochlorothiazide were recorded (Figure. 6- 7).

 

Figure.6 Chromatogram showing Acid degradation of Losartan Potassium and Hydrochlorothiazide

 

Figure.7 Chromatogram showing Base degradation of Losartan Potassium and Hydrochlorothiazide

 

Photolytic and Thermal degradation:

To conduct photolytic degradation studies twenty tablets were taken, made into fine powder and exposed to sunlight for 8 days. For thermal degradation studies another set of twenty tablets were taken, made into fine powder and exposed to dry heat at 80 ⁰C for 2 days. Twenty five mg of the resulting powder from each of photolytic and thermally degraded samples were taken and dissolved in 15 ml of mobile phase in a 25 ml volumetric flask separately. The solutions were shaken well, volumes were made upto the mark with mobile phase and filtered through a 0.45 µ membrane filter. From the resulting solutions, suitable aliquots were prepared and injected into the column. Typical chromatograms for photolytic and thermal degradation of Losartan Potassium and Hydrochlorothiazide were recorded (Figure. 8- 9).

 

Figure.8 Chromatogram showing Photolytic degradation of Losartan Potassium and Hydrochlorothiazide

 

Figure.9 Chromatogram showing Thermal degradation of Losartan Potassium and Hydrochlorothiazide

 

Assay of marketed formulations:

Twenty tablets were taken weighed and made into fine powder. An amount equivalent to Labeled claim was taken in a volumetric flask, 20 ml of solvent was added to it and sonicated for 15 mins. Then the solution was filtered and made upto the mark with the solvent. From this solution, suitable aliquots were prepared and their absorbances were taken at 230 nm for both the developed UV spectroscopic methods.

 

For HPLC method, a quantity of powder equivalent to labeled claim was weighed and dissolved in 50 ml of mobile phase in a 50 ml volumetric flask. The volume was made up to give a concentration of 1000 µg/ml and filtered through 0.45 µ nylon membrane filter paper.

 

From the above filtrate, different dilutions of Losatan potassium and Hydrochlorothiazide were taken in 10 ml volumetric flasks and made upto the mark with mobile phase¹⁷.  20 µL of each of these solutions were injected and the amount of Losatan potassium and Hydrochlorothiazide present in each tablet formulation was calculated by comparing the peak area of the tablet solution with that of standard. The chromatogram of marketed solution was recorded (Figure 10) and the results were tabulated (Table 1).

 

Figure.10 Chromatogram of Marketed formulation

 

Table 1: Assay data of the marketed formulations

Formulation		Amount found (mg)		% Recovery
REPACE H	Losartan potassium (50 mg)	Q- absorbance ratio method	49.70	99.40
		Derivative spectrophotometric method	49.99	99.98
		RP-HPLC method	49.98	99.96
	Hydrochlorothiazide (12.5 mg)	Q- absorbance ratio method	12.45	99.60
		Derivative spectrophotometric method	12.49	99.92
		RP-HPLC method	12.48	99.89

 

RESULTS AND DISCUSSION:

The developed methods were validated using different validation parameters like linearity, accuracy, precision, LOD and LOQ as per International Council for Harmonization^{18, 19}.

 

Linearity:

The linearity was obtained within the range of 10-50 µg/ml for Losartan Potassium and 2.5-12.5 µg/ml for Hydrochlorothiazide using a mixture of water and methanol (90:10 v/v) as solvent in Q-absorbance ratio and Derivative spectrophotometric methods. In RP-HPLC method, Losartan Potassium and Hydrochlorothiazide showed linearity within the range of 10-60 µg/ml and 2.5-15 µg/ml respectively. The results for Linearity of the developed methods were tabulated (Table 2).

 

Accuracy:

To assess the accuracy of the proposed methods, recovery studies were carried out by standard addition method. A known amount of drug was added to preanalyzed tablet powder at 80%, 100% and 120% of label claim and the percentage recoveries obtained (Table 3)

 

Precision:

Method and system precision were calculated in terms of intraday and interday studies by analyzing responses in triplicate on same day and on consecutive days²⁰.  The results were tabulated as shown in Table 4- 5.

 

 

Table 2: Linearity of the proposed methods

Statistical parameters	Losartan potassium			Hydrochlorothiazide
	Q-absorbance ratio method	Derivative spectrophotometric method	RP-HPLC method	Q-absorbance ratio method	Derivative spectrophotometric method	RP-HPLC method
Linearity (μg/ml)	10-50	10-50	10-60	2.5-12.5	2.5-12.5	2.5-15
Correlation coefficient (R2)	0.999	0.997	0.999	0.999	0.998	0.999
Regression equation y=mx+c	y = 0.045x + 0.183	y = 0.5286x + 0.0013	y = 54710x + 124647	y = 0.3987x + 0.1887	y = 0.2886x + 0.008	y = 59004x + 126868
Slope (m)	0.045	0.5286	54710	0.3987	0.2886	59004
Intercept(c)	0.183	0.0013	124647	0.1887	0.008	126868

Table 3: Accuracy of the proposed methods

Drug	Methods	Amount of sample taken µg/ml	Amount of Standard added µg/ml	Amount recovered mean (n=3)	% Recovery	SD* (n=3)	% RSD
Losartan potassium	Q-absorbance ratio method	30	24 30 36	54.84 60.13 66.36	101.55 100.2 100.5	0.114 0.146 0.095	0.462 0.484 0.262
	Derivative spectrophotometric method	30	24 30 36	53.99 59.99 88.01	99.98 99.98 100.01	0.022 0.015 0.020	0.068 0.037 0.041
	RP-HPLC method	30	24 30 36	107.98 119.99 132.0	99.98 99.99 100.00	0.031 0.022 0.026	0.064 0.036 0.036
Hydrochlorothiazide	Q-absorbance ratio method	10	8 10 12	8.66 9.74 12.05	101.22 99.56 100.08	0.017 0.038 0.030	0.068 0.059 0.045
	Derivative spectrophotometric method	10	8 10 12	7.99 9.99 12.00	99.88 99.90 100.00	0.025 0.017 0.030	0.313 0.170 0.250
	RP-HPLC method	10	8 10 12	7.00 9.98 12.01	100.00 99.93 100.03	0.017 0.022 0.020	0.140 0.146 0.111

 

 

Table 4: Method precision of the proposed methods

Parameter			Losartan potassium	Hydrochlorothiazide
Precision (%RSD)	Q-absorbance ratio method	Intra day	0.198 0.102 0.074	0.110 0.126 0.083
		Inter day	0.064 0.048 0.032	0.033 0.033 0.033
	Derivative spectrophotometric method	Intra day	0.086 0.066 0.099	0.050 0.086 0.086
		Inter day	0.050 0.066 0.049	0.086 0.073 0.050
	RP-HPLC method	Intra day	0.039 0.087 0.043	0.698 1.044 1.411
		Inter day	0.062 0.476 0.243	0.633 1.427 0.835

 

 

 

 

 

Table 5: System precision of the proposed RP-HPLC method

S. No	Peak area for Losartan potassium	Peak area for Hydrochlorothiazide
1	230642	706612
2	231174	711634
3	230187	712218
4	230016	709981
5	231681	716413
6	230114	712996
Mean	230635	711642
%RSD	0.458	0.723

 

Table 6: Sensitivity of the proposed methods

Parameters	Methods	Losartan potassium	Hydrochlorothiazide
LOD (μg/ml)	Q-absorbance ratio method	0.594	0.3
	Derivative spectrophotometric method	0.093	1.188
	RP-HPLC method	0.015	0.909
LOQ (μg/ml)	Q-absorbance ratio method	1.8
	Derivative spectrophotometric method	0.283	3.601
	RP-HPLC method	0.047	0.024

 

 

 

Ruggedness:

Ruggedness of the proposed methods was determined by analyzing the standard and sample solutions by two different analysts to check the reproducibility²¹. The results were summarized as shown in Table 7.

 

Robustness:

Robustness for the developed Q- absorbance ratio and Derivative spectrophotometric methods was assessed by slight variation in the wavelength (± 5 nm). In RP-HPLC method robustness was determined by slight variation in wavelength (± 5 nm) and flow rate (± 0.1 ml/min). The results of robustness studies were shown in Table.8

 

Specificity:

The peak purity of Losartan Potassium and Hydrochlorothiazide was tested by using statistical parameter like correlation at the peak start (s), peak apex (m) and peak end (e) positions.  Correlation between these spectra confirmed the purity of Losartan Potassium peak (correlation r (s,m)= 0.9988, r (m,e)= 0.9996) and Hydrochlorothiazide peak (correlation r (s,m)= 0.9989, r (m,e)= 0.9997) respectively as shown in Figure 17- 18.

 

 

Table 7: Ruggedness for proposed methods

S. No	Parameters	Methods	Amount of Losartan potassium recovered	Amount of Hydrochlorothiazide recovered
1	Analyst-01	Q-absorbance ratio method	99.8% w/w	99.9 %w/w
		Derivative spectrophotometric method	99.9% w/w	99.8 %w/w
		RP-HPLC method	0.037 % RSD	0.042 % RSD
2	Analyst-02	Q-absorbance ratio method	99.7% w/w	99.5% w/w
		Derivative spectrophotometric method	99.6% w/w	99.9% w/w
		RP-HPLC method	0.041 % RSD	0.062 % RSD

 

Table 8: Robustness for proposed methods

Parameters	Losartan potassium		Hydrochlorothiazide
Q-absorbance ratio method (%RSD)
Wavelength (225 nm)	0.069		0.031
Wavelength (230 nm)	0.056		0.027
Wavelength (235 nm)	0.066		0.032
Derivative spectrophotometric method (%RSD)
Wavelength (225 nm)	0.043		0.039
Wavelength (230 nm)	0.059		0.044
Wavelength (235 nm)	0.072		0.056
RP-HPLC method
	Retention time (mins)	Tailing factor	Retention time (mins)	Tailing factor
Flow rate (0.9 ml/min)	1.077	1.14	1.393	1.12
Flow rate (1 ml/min)	1.010	1.25	1.387	1.00
Flow rate (1.1 ml/min)	0.973	0.92	1.267	1.10
Wavelength (235 nm)	1.077	1.16	1.387	0.983
Wavelength (230 nm)	1.010	1.25	1.387	1.00
Wavelength (225 nm)	1.070	0.97	1.390	1.13

 

 

Figure. 17 Peak purity spectra of Losartan potassium

 

Figure. 18 Peak purity spectra of Hydrochlorothiazide System suitability

Six replicates of mixed standard solutions were injected and the parameters like theoretical plate number (N), tailing factor (K) and resolution (Rs) were calculated to check the system suitability²².  The results were tabulated as shown in Table 9.

 

Table 9: System suitability of the proposed RP-HPLC method

Drugs	Retention time (min)	No. of theoretical plates	Tailing factor	Resolution
Losartan potassium	1.010	2260	1.25	3.73
Hydrochlorothiazide	1.387	2174	1

 

Stability studies:

Stability studies revealed that the principle peaks of Losartan Potassium and Hydrochlorothiazide were well separated. The chromatograms obtained after acid hydrolysis showed a decrease in the percent peak area from 99.43% to 96.46% for Losartan Potassium and from 99.64% to 98.25% for Hydrochlorothiazide. There was also a decrease in the peak resolution from 3.73 to 2.86. After alkaline hydrolysis, the chromatograms showed a decrease in the percent peak area from 99.43% to 95.36% for Losartan Potassium and from 99.64% to 96.26% for Hydrochlorothiazide. The photolytic degradation results of the proposed drugs showed that there was a decrease in peak resolution from 3.73 to 1.565. From thermal degradation studies it was found that there was decrease in the percent peak area from 99.43% to 96.17% for Losartan Potassium and from 99.64% to 97.27% for Hydrochlorothiazide. The net degradation results (Table 10) along with the additional peaks obtained for the degradation products were shown in Figure 6 - 9.

 

ANOVA:

The % recoveries obtained for the selected drugs by the three developed methods were used to calculate TSS (Total Sum of Squares), BSS (Between Sum of Squares) and WSS (Within Sum of Squares) as per the principle of ANOVA. The calculated F value (0.058) for (2, 3) degree of freedom at 5% level of significance was found to be less than the tabulated F value (9.55). Hence the null hypothesis can be accepted indicating that there was no significant difference among the results obtained from the three proposed methods as shown in Table 11.

 

Table 10: Degradation study result for Losartan potassium and Hydrochlorothiazide by RP-HPLC method

S. No	Degradation study	Drugs	Peak area (mV*min)	Degradation assay (%)	Standard assay (%)	% Net degradation
1	Acid hydrolysis	Losartan potassium	1971238	96.46	99.4	2.94
		Hydrochlorothiazide	633228	98.25	99.6	1.35
2	Base hydrolysis	Losartan potassium	1948843	95.36	99.4	4.04
		Hydrochlorothiazide	620346	96.26	99.6	3.34
3	Photolytic degradation	Losartan potassium	2014860	98.59	99.4	0.81
		Hydrochlorothiazide	634968	98.52	99.6	1.08
4	Thermal degradation	Losartan potassium	1965420	96.17	99.4	3.23
		Hydrochlorothiazide	626867	97.27	99.6	2.33

Table 11: Result of ANOVA for the proposed methods

Source	Degree of freedom	Sum of squares (SS)	Mean of sum of squares (MSS)	F = Mean of BSS/ Mean of WSS	F- value
Between sum of squares (BSS)	2	0.01	0.005	0.05/ 0.086	0.058
Within sum of squares (WSS)	3	0.26	0.086
Total sum of squares (TSS)	5	Tabulated F- value within (2,3) degree of freedom at 5% level of significance was 9.55 The calculated F-value is less than the tabulated value. (0.058˂ 9.55)

 

CONCLUSION:

A new Q-absorbance ratio, Derivative spectrophotometric and Stability indicating RP-HPLC methods for the simultaneous estimation of Losartan Potassium and Hydrochlorothiazide were developed. The optimized methods were validated for various parameters as per ICH guidelines. The developed methods when applied to analyze the marketed formulations have given the results within the acceptance criteria. The stability studies showed the principle peaks of the selected drugs well separated from those of the corresponding degradation products. The results obtained from the three developed methods were then analyzed by ANOVA where it was found that there was no significant difference among the results. Hence it can be concluded that any one of the developed methods can be used in the routine analysis of Losartan Potassium and Hydrochlorothiazide in combined tablet dosage forms depending upon the availability of instruments and materials.

 

ACKNOWLEDGEMENT:

The authors are thankful to Yarrow Chem Products, Fisher scientific and SD-fine chemicals, Mumbai for providing all the reference materials and chemicals required for the investigation. The authors acknowledge the laboratory facilities provided by Maharajah’s College of Pharmacy, Vizianagaram, Andhra Pradesh, India for this study.

 

CONFLICT OF INTEREST:

There is no conflict of interest.

 

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20.   Ashutosh Kumar S, Manidipa Debnath and Seshagiri Rao JVLN. Development and validation of stability indicating RP-HPLC method for simultaneous estimation of Losartan Potassium, Ramipril and Hydrochlorothiazide in bulk as well as in pharmaceutical formulation. Indo American Journal of Pharmaceutical Research. 2013; 3(6): 5423-5445.

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Received on 31.01.2019          Modified on 16.03.2019

Accepted on 18.04.2019        © RJPT All right reserved