method development and validation for simultaneous...

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Indo American Journal of Pharmaceutical Research, 2013 ISSN NO: 2231-6876

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INDO AMERICAN

JOURNAL OF

PHARMACEUTICAL

RESEARCH

Method Development and Validation for Simultaneous Estimation of

Enalapril Maleate and Losartan Potassium in Bulk and Pharmaceutical

Dosage Form

BHAUMIK C PATEL*

Department of Quality Assurance, Gujarat Technological University, Shivam Pharmaceutical Studies and Research Center,

Valasan, Anand , Gujarat- 388326, India.

Corresponding author

Bhaumik C Patel

Department of Quality Assurance, Gujarat Technological University,

Shivam Pharmaceutical Studies and Research Center,

Valasan, Anand , Gujarat- 388326, India.

Copy right © 2013 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical

Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly

cited.

ARTICLE INFO ABSTRACT

Article history Received 11/05/2013

Available online

29/05/2013

Keywords Enalapril Maleate,

Losartan Potassium,

Phosphate Buffer,

Acetonitrile,

RP-HPLC,

Validation, Stability

Study.

The objective of present work was to develop and validate a simple, linear, precise,

accurate and stability indicating RP‐HPLC method for quantitative determination of

Enalapril Maleate and Losartan Potassium in tablet formulation. This developed

HPLC method, is cost effective as it does not involve use of expensive solvents and

clean up. This method is very Simple and Robust as both peaks are well separated

from its excipient peaks and with total runtime of 10 min, makes the developed

method suitable for routine quality control analysis work. Moreover proposed

method is more accurate, more precise, more stable and robust developed method. In

RP-HPLC, analysis is carried out using Buffer-Acetonitrile(60:40 v/v) pH4.5

adjusted With o-Phosphoric Acid as a mobile phase and Hyperchrom phase C-18

BDS Hypersil column (250mm × 4.6 mm id 5µm) as stationary phase at 235nm and

1 ml/min flow rate. The retention time of Enalapril Maleate and Losartan Potassium

was found to be 3.150 and 5.420 minutes respectively. Linearity was obtained in the

concentration range of 5-15 μg/ml and 25-75 μg/ml with % recoveries were found to

be 98.47% – 100.68% and 98.49% –100.61% for Enalapril Maleate and Losartan

Potassium respectively. LOD were found to be 0.120μg/ml and 0.606μg/ml at

235 nm for Enalapril Maleate and Losartan Potassium respectively. Limit of

Quantification and Limit of Detection for Enalapril Maleate was found to be 0.365

µg/ml and 0.120 and for Losartan Potassium 1.839 µg/ml and 0.606 respectively.

Stability method shows that in stress conditions i.e. acidic, basic, oxidation, thermal

and photolytic, comparison of % degradation of tablet dosage form of drug and its

Active Pharmaceutical Ingredient( API) is satisfactory and less. As per ICH

guidelines HPLC method for Enalapril Maleate and Losartan Potassium was

developed and validated.

Please cite this article in press as Bhaumik C Patel et al. Method Development and Validation for Simultaneous Estimation of

Enalapril Maleate and Losartan Potassium in Bulk and Pharmaceutical Dosage Form . Indo American Journal of Pharm

Research.2013:3(5).

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Vol 3, Issue 5, 2013. Bhaumik C Patel et al. ISSN NO: 2231-6876

INTRODUCTION :

Enalapril Maleate

((2S)-1-[(2S)-2-{[(2S)-1-ethoxy-1-oxo-4-phenylbutan-2-yl]amino}propanoyl]pyrrolidine-2-carboxylic acid) is it’s

chemical IUPAC name. It is Antihypertensive agent and Angiotensin-Converting Enzyme Inhibitor. Mechanism of

action of Enalapril Maleate is in following way: ACE inhibitors bind to and inhibit the activity of both functionally

active domains, N and C, which arise from tandem gene duplication Enalaprilat, the principle active metabolite of

enalapril, competes with ATI (Angiotensin 1) for binding to ACE and inhibits and enzymatic proteolysis of ATI to

ATII. Decreasing ATII (Angiotensin 2) levels in the body decreases blood pressure by inhibiting the pressor effects of

ATII. Enalapril also causes an increase in plasma renin activity likely due to a loss of feedback inhibition mediated by

ATII on the release of renin. [1,2]

Figure-1 Structure of Enalapril Maleate

Losartan Potassium

[2-butyl-4-chloro-1-({4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl}methyl)-1H-imidazol-5-yl]methanol is it’s

chemical IUPAC name. It is Antihypertensive Agent, Angiotensin II Receptor Antagonist, Angiotensin II Type

1 Receptor Blocker, Anti-Arrhythmia Agent. Mechanism of action of Losartan Potassium is in following way:

Losartan competitively inhibits the binding of angiotensin II to AT1 (Angiotensin 1) in many tissues including

vascular smooth muscle and the adrenal glands. Losartan is metabolized to its active metabolite, E-3174, which

is more potent than losartan and acts as a non-competitive AT1 antagonist. Inhibition of angiotensin II binding to

AT1 inhibits its AT1-mediated vasoconstrictive and aldosterone-secreting effects and results in decreased

vascular resistance and blood pressure. :[3,4]

Figure-2 Structure of Losartan Potassium

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In literature survey few methods like UV, HPTLC, etcetera are reported on simultaneous determination of these

two drugs either alone or their combined dosage form along with other drugs. Simultaneous Estimation of

Enalapril Maleate, Hydrochlorthiazide, Aspirin and Atorvastatin In Pure and Its Stimulated Dosage Form

Using Isocratic RP-HPLC [5]. HPTLC Determination of Enalapril Maleate and Hydrochlorthiazide In

Pharmaceutical Dosage Form[6].Simultaneous UV Spectrophotometric Estimation of Enalapril Maleate and

Hydrochlorthiazide In Tablets[7]. Development And Validation Of RP-HPLC Method For Simultaneous

Estimation of Enalapril Maleate and Amlodipine Besylate In Combined Dosage Form[8]. UV-Visible

Spectroscopic Estimation and Validation of Enalapril Maleate In Bulk and Pharmaceutical Dosage Forms[9].

Difference Spectrophotometric Estimation of Enalapril Maleate From Tablet Dosage Form[10]. Development

and Validation of A Reversed Phase HPLC Method For Simultaneous Estimation of Enalapril

Maleate,Hydrochlorthiazide And Paracetamol In Pure and Its Pharmaceutical Dosage Form[11]. HPLC-UV

Method For the Determination of Enalapril In Bulk, Pharmaceutical Formulations and Serum[12]. Development

and Validation of UV Spectrophotometric Method For Determination Of Enalapril Maleate[13]. Determination

of Felodipine and Enalapril In Binary Mixture Using Second Derivative Spectrophotometric, First Derivative of

the Ratio Spectra UV Methods and High Performance Liquid Chromatographic Method[14]. Simultaneous UV

Spectrophotometric Methods For Estimation of Losartan Potassium and Amlodipine Besylate inTablet Dosage

Form[15]. RP-HPLC Method For the Determination of Losartan Potassium and Perindopril Erbumine In

Combined Tablet dosage Form[16]. Quantitative Estimation of Losartan Potassium In Pharmaceutical Dosage

Form By UV Spectrophotometry[17]. Development and Validation of RP-HPLC Method For Development and

Estimation Of Hydrochlorthiazide and Losartan Potassium In Tablet Dosage Form[18]. Simultaneous Estimation

of Losartan, Atenolol, Hydrochlorthiazide and Valsartan In Pharmaceutical Dosage Form[19]. Evaluation of

Losartan Potassium In Capsules By UV Spectrophotometry[20]. Analytical Method Development and

Validation of Losartan Potassium And Amlodipine Besylate In Tablet Dosage Form By RP-HPLC[21].

Simultaneous UV Spectrophotometric Method For Estimation of Losartan Potassium and Amlodipine Besylate

In Tablet Dosage Form[22]. RP-HPLC Method For Simultaneous Determination of Losartan,

Irbesertan,Hydrochlorthiazide and Chlorthalidone In Comdined Drug Product[23]. RP-HPLC Method For

Simultaneous Estimation of Losartan Atrovaststin In Tablet Formulations[24]. Method Development and

Validation of Losartan Potassium By RP-HPLC[25]. Single RP-HPLC Method For the Estimation of

Losartan Potassium, Enalapril Maleate and Hydrochlorthiazide In Tablet Frormulation[26].

The purpose of this research is to develop validated HPLC method for simultaneous quantitation of Enalapril

Maleate and Losartan Potassium in bulk drug and in dosage form. The present work describes the

development of a validated RP-HPLC method which can quantify these components simultaneously from a

combined dosage form which is fast, simple, precise, stable and reliable method for routine analytical needs .

The present RP-HPLC method was validated and Stability Study was carried out following the ICH

guidelines[27-29]. Stability Study is also carried out to assure that method is stable.

MATERIALS & METHODS:

Materials:

Pure Enalapril Maleate and Losartan Potassium were obtained as gift samples from Cadila

Pharmaceuticals Limited respectively.The combined dose Tablet formulation containing Enalapril Maleate

and Losartan Potassium was purchased from local pharmacy as a Brand Name: (Losapril-(Microlabs Ltd.)

(Content: Enalapril Maleate-10mg, Losartan Potassium-50mg, Excipients-q.s).

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The water for RP-HPLC (Merck Ltd., Ahmedabad, India). The Methanol for RP-HPLC (Merck Ltd.,

Ahmedabad, India). The Potassium Dihydrogen Phosphate (Merck Ltd., Ahmedabad, India). The

Orthophosphoric acid (AR Grade) (Merck Ltd., Ahmedabad, India). TheAcetonitrile (HPLC Grade) (Merck

Ltd.,Ahmedabad,India).

Instrumentation:

A HPLC instrument (Shimadzu LC-20 AT) equipped with SPD-20A detector, Rheodyne injector with 20 μl

loop was used for the analysis. Separation was achieved on BDS Hyersil C-18 (250 mm × 4.6 mm id, 5 μm

particle size), analyzed with Spinchrom software. Analytical balance with 0.1 mg accuracy, model, AX200,

Manufacture by Shimadzu. Chromatographic Software: spincrom. PH Meter: Model: CL110 ( Chemiline

Digital PH meter) . Ultra Bath Sonicator: 1-SLSOH (Janki Impex PVT. LTD).

Preparation of standard stock solution :

Enalapril Maleate standard stock solution (100 g/ml):

Enalapril was accurately weighed 10 mg and transferred to a 100 ml volumetric flask and dissolved in 10 ml

of mobile phase. The flask was then sonicated for 10 min.Volume was made up to the mark with mobile

phase to obtain a solution containing 100 μg/ml Enalapril. From this solution 1 ml was transfered to 10 ml

volumetric flask. The volume was adjusted up to the mark with the mobile phase to obtain a final

concentration of 10μg/ml Enalapril.

Losartan Potassium standard stock solution(500 g/ml):

Losartan was accurately weighed 50 mg and transferred to a 100 ml volumetric flask and dissolved in 10 ml

mobile phase. The flask was then sonicated for 10 min.Volume was made up to the mark with mobile phase

to obtain a solution containing 500 μg/ml Losartan. From this solution 1 ml was transfer to 10 ml volumetric

flask. The volume was adjusted up to the mark with the mobile phase to obtain a final concentration of

50μg/ml Losartan.

Calibration curve for Enalapril Maleate and Losartan Potassium:

Appropriate volume of aliquot from standard Enalapril Maleate and Losartan Potassium stock solution was

transferred to same volumetric flask of 10 ml capacity. The volume was adjusted to the mark with mobile

phase to give a solution containing 5, 7.5, 10, 12.5 and 15μg/ml Enalapril Maleate (50% to 150%) and 25,

37.5, 50, 62.5 and 75μg/ml Losartan Potassium (50% to 150%). The mixed standard solution was

chromatographed for 10 minutes using mobile phase at a flow rate of 1ml/min.The calibration curve were

plotted for peak area vs. concentration for both the drugs.

Figure 3: Overlain UV spectra of Enalapril Maleate and Losartan Potassium in mobile phase.

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Optimized Chromatographic conditions:

Stationary phase : BDS Hyersil C-18 (250 mm × 4.6 mm id, 5 μm particle size): Mobile phase :- Buffer

:Acetonitrile(60:40 v/v) pH4.5 adjusted with o-phosphoric acid; Detection wavelength: 235 nm; Injection

volume : 10 µl, Flow rate : 1ml/min.

1st peak= Enalapril Maleate, 2

nd peak= Losartan Potassium

Figure 4: Chromatogram of mixed standard solution containing 10 µg/ml Enalapril Maleate and 50 µg/ml Losartan

Potassium using mobile phase buffer: Acetonitrile (60:40, v/v) pH 4.5 adjusted with o-phosphoric acid.

Table1 System Suitability Parameters:-

System Suitability

Parameters

Proposed Method Range Inference

ENALAPRIL LOSARTAN

Retention times (RT)

(min)

2.980 ± 0.04 5.027± 0.05 - -

Theoretical plates

(N)

6550±35.69 7142± 31.72

> 2000

Criteria met

Resolution (RS) 10.626 ± 1.11 >2 Criteria met

Tailing factor (AS) 1.421± 0.01 1.438± 0.03 < 2 Criteria met

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STABILITY STUDY :

Forced Degradation Study of Enalapril Maleate and Losartan Potassium by RP-HPLC

In order to establish whether the analytical method for the assay was stability-indicating, pure active

pharmaceutical ingredient (API) and tablet of Enalapril and Losartan was subjected to various stress

conditions to conduct forced degradation studies. Stress studies were carried out under the conditions of

acid/base hydrolysis, oxidation, thermal as mentioned in ICH Q1A (R2) [27,28].

Preparation of standard solutions:

Preparation of Enalapril Maleate stock solution(100μg/ml)

Accurately weighed Enalapril(10 mg) was transferred into 100 ml volumetric flask and dissolved in mobile

phase and diluted up to the mark to give a stock solution having strength 1 mg/ml (100μg/ml).

Preparation of Losartan Potassium stock solution(500μg/ml)

Accurately weighed Losartan (50 mg) was transferred into 100 ml volumetric flask and dissolved in mobile

and diluted up to the to give a stock solution having strength 1 mg/ml (500μg/ml).

Working standard solution of Enalapril Maleate (10μg/ml)

From this Enalapril stock solution 1 ml was transfered to 10 ml volumetric flask. The volume was adjusted

up to the mark with the mobile phase to obtain a final concentration of 10μg/ml Enalapril.

Working standard solution of Losartan Potassium (50μg/ml)

From this Losartan stock solution 1 ml was transfer to 10 ml volumetric flask. The volume was adjusted up

to the mark with the mobile phase to obtain a final concentration of 50μg/ml Losartan.

Preparation of 0.1N sodium hydroxide

Accurately weighed sodium hydroxide (400 mg) was transferred into 100 ml volumetric flask and dissolved

in water and diluted up to the mark with distilled water.

Preparation of 0.1N hydrochloric acid

Accurately 0.85 ml concentrated HCl was transferred in to 100 ml volumetric flask and diluted up to the

mark with distilled water.

Procedure for Preparation of Samples for Force Degradation Study:

Degradation study of Enalapril Maleate in 0.1N NaOH at temperature 300C 2

Accurately weighed Enalapril (10 mg) was transferred into 10 ml volumetric flask and dissolved in 10 ml of

freshly prepared 0.1N NaOH. The flask was kept at room temperature and at interval of half an hour, 1 ml of

sample was withdrawn and transferred to 10 ml volumetric flask, makeup volume up to mark with mobile

phase, and injected to system with stated chromatographic conditions and analyzed. Similar procedure is

applied for both API and tablet.

Degradation study of Losartan Potassium in 0.1N NaOH at temperature 30 ± 2oC

Accurately weighed Losartan (50 mg) was transferred into 100 ml volumetric flask and dissolved in 100 ml

of freshly prepared 0.1N NaOH. The flask was kept at room temperature and at interval of half an hour, 1 ml

of sample was withdrawn and transferred to 10 ml volumetric flask, makeup volume up to mark with mobile

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Degradation study of Enalapril Maleate in 0.1N HCl at 600C

Accurately weighed Enalapril (10 mg) was transferred into 10 ml volumetric flask and dissolved in 10 ml of

freshly prepared 0.1N HCl. The flask was kept at room temperature and at interval of half an hour, 1 ml of


phase, and injected to system with stated chromatographic conditions and analyzed Similar procedure is


Degradation study of Losartan Potassium in 0.1N HCl at 600C

Accurately weighed Losartan (50 mg) was transferred into 100 ml volumetric flask and dissolved in 10 ml of

freshly prepared 0.1N HCl. The flask was kept at room temperature and at interval of half an hour, 1 ml of




Peroxide degradation

It was carried out as per the procedure described for the acid degradation using 10 % v/v H2O2 instead of

0.1N HCl. For both Enalapril Maleate and Losartan Potassium.

Thermal degradation

Powder of API of both the Enalapril and Losartan and tablet powder were heated at 100°C for 1hr, and from

this final concentration of 10 µg/ml Enalapril and 50 µg/ml of Losartan and chromatographed to check the

specificity.All the solutions were passed through whatman filter 0.45µ before injection.For each degradation,

blank solutions were also prepared without taking API or tablet powder as per the same procedure described

above. All the blank solutions were passed through whattman filter 0.45µ before injection. Each blank was

injected separately and chromatoghraphed.

Photolytic degradation

Solid drug was spread in 1 mm thickness uniform layer on a Petridish and exposed it with the light of energy

1.2 million lux/hours in photo stability chamber for 4 hours.Add 30 ml of diluent to it, and sonicate the

solution for about 10 mins with occasional shaking. Make the volume up to 100 ml with diluent and

mix.Filter the solution and after suitable dilution record the chromatogram.Similar procedure is applied for

both API and tablet.

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Figure 5,6,7 : Chromatogram of acid degradation spectra of :

Enalapril

Losartan

Tablet dosage form

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Figure 8,9,10: Chromatogram of alkali degradation spectra of :

Enalapril

Losartan

Tablet dosage form

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Figure 11,12,13 : Chromatogram of Peroxide gradation spectra of API of :

Enalapril

Losartan

Tablet dosage form

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Figure 14,15,16 : Chromatogram of Thermal gradation spectra of :

Enalapril

Losartan

Tablet dosage form

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Figure 17,18,19 : Chromatogram of Photolytic gradation spectra of :

Enalapril

Losartan

Tablet dosage form

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Validation of the method:

Validation of the optimized HPLC method was carried out with respect to the following parameters.

Linearity and Range:

Linearity was observed in the range of 5-15 μg/ml for enalapril maleate and 25-75 μg/ml for losartan

potassium respectively . The regression coefficient is near indicating good correlation between peak area and

concentration. Calibration graph for the two drugs are taken.

Precision:-

The precision of an analytical method is the degree of agreement among individual test results when the

method is applied repeatedly to multiple samplings of homogenous samples. It provides an indication of

random error results and was expressed as %RSD. The %RSD value for Intraday precision and Interday

precision were calculated respectively revealing method to be precise.

Repeatability:

Standard mixture solutions of Enalapril(5,7.5,10,12.5,15 µg/ml) and Losartan (25,37.5,50,62.5,75 µg/ml)

were prepared and chromatograms were recorded. Area was measured of the same concentration solution six

times and %RSD was calculated.

Accuracy (%Recovery) :-

Accuracy is the closeness of the test results obtained by the method to the true value. To study the accuracy

placebo powdered mixture was prepared using common excipients and analysis of the same was carried

out. Recovery studies were carried out by addition of standard drug to the sample at 3 different concentration

levels taking into consideration percentage purity of added bulk drug samples.

Robustness:-

The sample solutions were prepared and then analyzed with change in the typical analytical conditions like

change in flow rate, change in mobile phase ratio, change in pH..

Reproducibility:-

The areas were measured by another analyst and the values obtained were evaluated using t-test to verify

their reproducibility.

Assay of Marketed Formulation:-

Applicability of the proposed method was tested by analyzing the commercially available tablet formulation (

Losapril- Microlabs. Ltd. Enalapril maleate-10mg, Losartan potassium-50mg). Prepare mobile phase as per

method and run for 45 minutes. Standard stock solution is prepared in following way: Dissolve 10mg of

Enalapril in 100ml of solvent and dissolve 50mg of Losartan in 100ml of solvent to form 100 & 500 ppm

respectively. From this prepared solution take 1ml of Enalapril and 1ml of Losartan and dilute it upto 10ml to

form 10 & 50ppm respectively. Similarly prepare sample preparation in this way by using sample of both

drugs instead of standard ingredient. Inject mix standard preparation. Prepare stock three times and inject

them single. Calculate %Assay of Enalapril maleate and Losartan potassium by comparing area in standard

and sample preparation.

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RESULTS AND DISCUSSION:

STABILITY STUDY :

Table 2 : Results of force degradation study of Enalapril Maleate and Losartan Potassium:

Stress condition/

duration/ state

% Degradation of API % Degradation of Tablet

Dosage form

Enalapril Losartan Enalapril Losartan

Acidic /0.1N HCl/60°C/

30 min/ Solution

16.580 25.973 16.712 26.225

Alkaline / 0.1N NaOH /

Room temperature /30 min/

Solution

15.784 19.110 15.784 19.386

Oxidative /10% H2O2 /

60°C / 30 min/Solution 19.847 10.074 19.909 10.340

Thermal/100°C/1 hr/Solid 20.114 16.905 20.267 17.058

Photolytic/sunlight/4 hr/Solid 12.506 10.159 12.826 10.627

VALIDATION OF THE DEVELOPED HPLC METHOD:

Linearity and Range:

Linearity was observed in the range of 5-15 μg/ml for enalapril maleate and 25-75 μg/ml for losartan

potassium respectively . The regression coefficient is near indicating good correlation between peak area and

concentration. Calibration graph for the two drugs and Regression parameters are mentioned.

ENALAPRIL MALEATE:

Table 3: Linearity data for LOSARTAN POTASSIUM

Conc. (µg/ml) Area*±SD %RSD

5 537.075±9.42 1.75

7.5 789.365±12.68 1.60

10 1039.909±13.02 1.25

12.5 1292.256±13.64 1.05

15 1539.416±16.83 1.09

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Fig 20: Calibration curve for ENALAPRIL MALEATE

LOSARTAN POTASSIUM:

Table 4: Linearity data for LOSARTAN POTASSIUM

Conc.(µg/ml) Area*±SD %RSD

25 2965.837±24.48 0.82

37.5 4448.952±41.97 0.94

50 5800.082±49.33 0.85

62.5 7231.769±60.75 0.84

75 8637.881±71.02 0.82

* mean of three determination, SD- standard deviation, RSD- relative standard deviation

Fig 21: Calibration curve for LOSARTAN POTASSIUM

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Table 5: Statistical data for ENALAPRIL and LOSARTAN by RP- HPLC method

Parameters ENALAPRIL LOSARTAN

Linearity(µg/ml) 5-15 25-75

Regression equation Y = 100.7X + 31.84 Y=112.6X + 178.3

Slope 100.7 112.6

Intercept 31.84 178.3

Correlation Coefficient (R²) 0.999 0.999

SD 3.685 20.709

LOD 0.120 0.606

LOQ 0.365 1.839

Precision:-

The precision of an analytical method is the degree of agreement among individual test results when the

method is applied repeatedly to multiple samplings of homogenous samples. It provides an indication of

random error results and was expressed as %RSD. The %RSD value for Intraday precision were in the range

of 0.95-1.30 and 1.02-1.75 for ENALAPRIL and LOSARTAN respectively, for Interday precision the range

obtained was 1.34-1.65 and 0.90-1.26 for ENALAPRIL and LOSARTAN respectively revealing method to

be precise.

Table 6: Intraday and Interday precision for Enalapril.

CONCENTRATION

(μg/ml)

INTRADAY

(Area*±SD) n=3

%RSD INTERDAY

(Area*±SD)

%RSD

5 (50%) 507.08 ± 4.85 0.95 547.41 ± 9.08 1.65

10(100%) 1036.53 ± 12.44 1.20 1053.91 ± 14.17 1.34

15(150%) 1552.12 ± 20.20 1.30 1574.69 ± 22.84 1.45

Table 7: Intraday and Interday precision for Losartan.

CONCENTRATION

(μg/ml)

INTRADAY

(Area*±SD) n=3

%RSD INTERDAY

(Area*±SD)

%RSD

25(50%) 2826.46±35.03 1.23 3044.97 ± 27.52 0.90

50(100%) 5789.02 ± 59.09 1.02 5900.23 ± 68.38 1.15

75(150%) 8630.80 ± 151.54 1.75 8794.52 ± 111.34 1.26

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Table 8: Precision %RSD Range.

Precision (%RSD) ENALAPRIL LOSARTAN %RSD Limit

Intraday 0.95 – 1.30 1.02 - 1.75 NMT 2.0

Interday 1.34 – 1.65 0.90 - 1.26

Limit Of Detection (L.O.D.)

The limit of detection for Enalapril Maleate and Losartan Potassium was found to be 0.120μg /ml and

0.606μg/ml respectively.

Limit Of Quantification (L.O.Q.)

The limit of quantification for Enalapril Maleate and Losartan Potassium was found to be 0.365μg /ml and

1.839μg/ml respectively.

Repeatability:

Standard mixture solutions of Enalapril (5,7.5 ,10,12.5,15 µg/ml) and Losartan (25,37.5,50,62.5,75

µg/ml) were prepared and chromatograms were recorded. Area was measured of the same concentration

solution six times and %RSD was calculated.

Table 9 : Repeatability data for Enalapril Maleate

Conc.(μg/ml) 5 7.5 10 12.5 15

Area

531.432 791.507 1040.649 1289.363 1541.254

548.596 788.449 1019.379 1274.498 1537.491

544.648 807.158 1054.799 1311.246 1566.643

525.733 771.466 1038.988 1298.498 1528.749

534.966 788.248 1045.731 1287.674 1522.943

539.452 783.98 1047.781 1244.56 1557.234

Mean 537.470 788.468 1041.221 1284.307 1542.385

S.D. 8.48 11.55 12.08 22.97 16.72

%RSD 1.57 1.46 1.16 1.78 1.08

SD= standard deviation, RSD= relative standard deviation

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Table 10: Repeatability data for Losartan Potassium

Conc. (μg/ml) 25 37.5 50 62.5 75

Area

2971.694 4455.965 5819.257 7209.514 8618.15

2954.489 4389.468 5760.482 7165.499 8626.479

2973.892 4479.851 5736.878 7277.954 8693.559

2997.468 4493.459 5854.349 7311.458 8535.456

2931.642 4426.017 5829.445 7194.418 8715.761

2989.124 4467.234 5632.557 7278.347 8589.349

Mean 2969.718 4451.98 5772.16 7239.531 8629.773

S.D. 23.87 38.27 81.39 57.57 66.55

%RSD 0.80 0.85 1.41 0.79 0.77

SD= standard deviation, RSD= relative standard deviation

Accuracy (%Recovery) :-

Recovery studies were carried out by addition of standard drug to the sample at 3 different concentration

levels taking into consideration percentage purity of added bulk drug samples.Good recoveries in the range

of 98.47% – 100.68%, for Enalapril and 98.49% –100.61% for Losartan respectively by the standard addition

method.

Table 11 : Accuracy (Recovery study for Enalapril Maleate)

Sample amt

(µg/ml)

Amt added

(µg/ml) STD

Amt recovered

(µg/ml) STD

%Reco-

very

Avg SD %RSD

80% 10 8 8.04 100.60 99.64 1.08 1.08

80% 10 8 7.98 99.84

80% 10 8 7.87 98.47

100% 10 10 10.06 100.68 99.32 1.19 1.19

100% 10 10 9.87 98.78

100% 10 10 9.84 98.49

120% 10 12 11.98 99.91 99.61 0.67 0.67

120% 10 12 11.86 98.84

120% 10 12 12.01 100.09

STD= standard sample, SD= standard deviation, RSD= relative standard deviation

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Table 12 : Accuracy (Recovery study for Losartan Potassium)

Sample

amt (µg/ml)

Amt added

µg/ml STD

Amt recovered

(µg/ml) STD

%Recovery Avg SD %RSD

80% 50 40 39.91 99.77 99.85 0.72 0.72

80% 50 40 40.24 100.61

80% 50 40 39.66 99.16

100% 50 50 49.79 99.58 99.38 0.81 0.81

100% 50 50 50.04 100.08

100% 50 50 49.24 98.49

120% 50 60 59.62 99.38 99.55 0.84 0.85

120% 50 60 60.28 100.48

120% 50 60 59.28 98.81

Robustness:-

The sample solutions were prepared and then analyzed with change in the typical analytical conditions like

change in flow rate, change in mobile phase ratio, change in pH..

Table 13: Robustness for Enalapril Maleate

Changed Condition Area*±SD %RSD

Flow rate +0.2(1.2ml/min) 1006.467±8.71 0.86

Flow rate -0.2(0.8ml/min) 1111.956±7.86 0.70

Mobile Phase Ratio

(58:42)

1055.793±11.79 1.11

Mobile Phase Ratio

(62:38)

1058.284±10.66 1.01

pH +0.2 (4.7) 1057.792±9.77 0.92

pH -0.2 (4.3) 1059.799±12.37 1.16

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Table 14: Robustness for Losartan Potassium

Condition Area*±SD %RSD

Flow rate +0.2(1.2ml/min) 5628.032±48.67 0.86

Flow rate -0.2(0.8ml/min) 6219.929±43.52 0.69

Mobile Phase Ratio

(58:42)

5904.69±65.68 1.11

Mobile Phase Ratio

(62:38)

5918.763±59.38 1.01

pH +0.2 (4.7) 5916.193±54.16 0.91

pH -0.2 (4.3) 5927.139±69.54 1.17

Reproducibility:-

The areas were measured by another analyst and the values obtained were evaluated using t-test to verify

their reproducibility.

Table 15: Reproducibility data for Enalapril Maleate (10 μg/ml)

Analyst 1

Mean ± S.D (n=3)

Analyst 2

Mean ± S.D (n=3)

Result of t-test* Inference

1004.753 ± 13.70

965.766 ± 8.98

0.015

Not significant

difference * At 95% confidence interval, (t-Tabulated = 2.45)

Table 16: Reproducibility data for Losartan Potassium (50 μg/ml)

Analyst 1

Mean ± S.D (n=3)

Analyst 2

Mean ± S.D (n=3)

Result of t- test*

test*

Inference

5497.997 ± 83.66

5434.186 ± 81.14

0.131

Not significant

difference * At 95% confidence interval, (t-Tabulated = 2.45)

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ASSAY OF MARKETED FORMULATION:-

Table 17 : Assay Result of Enalapril maleate

STD= standard sample

Table 18 : Assay Result of Losartan potassium

Area of Std 3390.884

Sr.no Area of samples %Assay

1 2644.341 98.48

2 2664.807 99.24

3 2654.739 98.87

Avg assay 98.87

SD 0.381

%RSD of assay 0.385

STD= standard sample

CONCLUSION:

HPLC method was developed and validated as per ICH guidelines. Stability study was also carried out as per

ICH guidelines. The procedure has been evaluated for the linearity, accuracy, precision and robustness in

order to ascertain the suitability of the analytical method. The method was also applied to marketed samples.

Statistical analysis proves that the method is suitable for the analysis of Enalapril Maleate and Losartan

Potassium as bulk drug and in pharmaceutical formulation without any interference from the excipients.

Future work on this method may be extended to study the degradation kinetics (Stability studies), HPTLC

of Enalapril Maleate and Losartan Potassium as well as its estimation in plasma and other biological fluids.

Area of Std 728.239

Sr.no Area of Samples %Assay

1 1223.126 98.43

2 1245.097 100.20

3 1238.467 99.66

Avg assay 99.43

SD 0.906

%RSD of assay 0.912

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ACKNOWLEDGEMENT:

The author would like to thank, Cadila Pharmaceuticals Limited. Dholka (Gujarat, India) for providing a gift

sample of standard Enalapril Maleate and Losartan Potassium respectively. The authors would like to thank,

Dr. H.N.Kakrani, Principal, Shivam institute of pharmaceutical studies and research center, Gujarat,

India for providing necessary facilities to carry out the work.

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