single rp-hplc method for the estimation of losartan ... - Ijsidonline.info

L. Bhaskar Reddy et al., IJSID 2011, 1 (1), 33-40ISSN:2249-5347IJSIDInternational Journal of Science Innovations and DiscoveriesAn International peerReview Journal for ScienceResearch ArticleAvailable online through www.ijsidonline.infoSINGLE RP-HPLC METHOD FOR THE ESTIMATION OF LOSARTAN POTASSIUM, ENALAPRILMALEATE AND HYDROCHLOROTHIAZIDE IN PHARMACEUCTIAL FORMULATIONSL. Bhaskar Reddy 1* , P. Raveendra Reddy 1 , L. Maheswara Reddy 1 and Viswanath Reddy Pyreddy 11Dept of Chemistry, Sri Krishnadevaraya University, Anantapur, AP, India-515055.ABSTRACTReceived: 15.06.2011Modified: 15.07.2011Published: 12.08.2011Keywords: Losartan Potassium,Enalapril Maleate,Hydrochlorothiazide,RP-HPLC and formulations.*Corresponding AuthorA simple, specific, precise and accurate RP-HPLC method hasbeen developed for simultaneous estimation of LosartanPotassium, Enalapril Maleate and Hydrochlorothiazide informulations. The separation was achieved on a 5-micron C18column (250 X 4.6 mm) using mobile phase consisting of amixture of Methanol: Acetonitrile: Water: 0.1% orthophosphoric acid (35:5:5:5% v/v/v/v), adjusted the pH to 3.0with diluted triethylamine. The flow rate was maintained at 1.0ml/min. The detection of the constituents was done using UVdetector at 230nm. The retention time of Enalapril maleate,hydrochlorothiazide and Losartan Potassium were about 1.8,2.7 and 4.6 min respectively. Recovery study values ofLosartan, enalapril maleate and hydrochlorothiazide is100.0%, 100.0% and 99.91% respectively, relative standardAddress:Name: L. Bhaskar ReddyPlace: HyderabadE-mail: bhaskarreddy1997@gmail.comdeviation for precision is less than 2% for the assay and Linearresponse obtained for three actives concentration range 50-100 μg/ml and correlation coefficient of 0.999 shows that themethod INTRODUCTIONis precise, accurate, linear, rugged and robust.International Journal of Science Innovations and Discoveries VOL1, Issue 1, July-August 201133

L. Bhaskar Reddy et al., IJSID 2011, 1 (1), 33-40INTRODUCTIONLosartan Potassium (1-3) , 2-butyl-4-chloro-1-[[2’-(1H-tetrazol-5-yl) [1, 1’-biphenyl]-4-yl] methyl]-1Himidazole- 5-methanol mono Potassium salt. It is the first member of a new class of nonpeptideangiotensin II receptor antagonist. It is used to treat hypertension by suppressing the effects ofangiotensin II at its receptors, thereby blocking the renin-angiotensin system. Losartan has beendemonstrated to be superior to previous peptide receptor antagonists and angiotensin convertingenzyme inhibitors because of its enhanced specificity, selectivity, and tolerability. Losartan potassium ismarketed alone or combined with hydrochlorothiazide.Hydrochlorothiazide (HCTZ) (4, 5) is a thiazide class diuretic drug. This reduces the volume of theblood, decreasing blood return to the heart. Hydrochlorothiazide is often used in the treatment ofhypertension, congestive heart failure, symptomatic edema and the prevention of kidney stones. Therecommended dose of hydrochlorothiazide for treating high blood pressure is hydrochlorothiazide 25 mgto 50 mg per day.Enalapril maleate (6-8) is used to treat high blood pressure (hypertension), it works by relaxingblood vessels, causing them to widen. Lowering high blood pressure helps prevent strokes, heart attacksand kidney problems. Enalapril maleate is supplied as 2.5 mg, 5 mg, 10 mg, and 20 mg tablets for oraladministration. Side effects are light-headed, fainting and urinating more or less than usual, fever, chills,body aches, flu symptoms, pale skin, easy bruising or bleeding. Figure-1 represents the chemicalstructure of three active ingredients.Losartan Potassium Hydrochlorothiazide Enalapril MaleateFigure-1: Chemical structure of Three active ingredientsThe literature reports many analytical methods were reported for the quantization of Losartanand hydrochlorothiazide in tablets using HPLC and methods for enalapril maleate andhydrochlorothiazide formulations by HPLC and methods for enalapril maleate and Losartan by HPLC. TheInternational Journal of Science Innovations and Discoveries VOL1, Issue 1, July-August 201134

L. Bhaskar Reddy et al., IJSID 2011, 1 (1), 33-40purpose of this study was to develop a single RP-HPLC method for the estimation of Losartan Potassium,enalapril maleate and hydrochlorothiazide simultaneously.MATERIALS AND METHODSMaterials: All the chemicals and solvents used were of AR. The pure drugs Losartan Potassium, enalaprilmaleate and hydrochlorothiazide were used as standards. All market samples were procured and usedfor this study.Standard Solution: Prepared the standard solution to get the known concentration of all activeingredients 70ppm with diluent.Chromatographic conditions:The following Chromatographic conditions were established for separation of active samples fromall its degradation products and maintained throughout the method.Column:Intersil C18, 250 mm x 4.6 mm, 5 μMobile phase Methanol: ACN: Water: 0.1% ortho-phosphoric acid (35:5:5:5% v/v/v/v), with pHto 3.0 with triethylamine.Detection: wavelength 230nmSample size: 20μL.Temperature: Room temp.Run time: 8min.Flow rate: 1.0ml per minAnalysis of Marketed Formulation: All market samples were prepared to get known concentration ofall active ingredients 70ppm with diluent.RESULTS AND DISCUSSIONMethod development:Method development trials were performed with a C18 column and a mobile phase containingacetonitrile and water in 60:40 proportions and tried with changing the proportion of organic solventswere studied. Acetonitrile, methanol, phosphate buffer and water in various ratios were tested to get anappropriate mobile phase composition. The mixtures of acetonitrile, methanol and water at various ratioswere examined, which resulted in very close retention times of the peaks. Best resolution was achievedwith the mobile phase having composition of Methanol: ACN: Water: 0.1% ortho-phosphoric acid(35:5:5:5% v/v/v/v) with pH to 3.0 with triethylamine. Figure-2 represents the individual injections ofthree actives.International Journal of Science Innovations and Discoveries VOL1, Issue 1, July-August 201135

L. Bhaskar Reddy et al., IJSID 2011, 1 (1), 33-40Figure-2: Chromatogram of three active ingredients.Validation of Method:System Suitability: Having optimized the efficiency of a chromatographic separation the quality of thechromatography was monitored by applying the following system suitability tests: Resolution, tailingfactor and theoretical plates. The system suitability method acceptance criteria set in each validation runwere: Resolution >2.0, tailing factor ≤2.0 and theoretical plates >2000. In all cases, the relative standarddeviation (R.S.D) for the analyte peak area for two consecutive injections was < 2.0%. A chromatogramobtained from reference substance solution is presented in Figure-3.Figure-3: Chromatogram of standard solution.Specificity: The excipients in the tablets used in this study contained the following inactive ingredients:microcrystalline cellulose, lactose, corn starch, magnesium stearate, hydroxypropyl cellulose,International Journal of Science Innovations and Discoveries VOL1, Issue 1, July-August 201136

L. Bhaskar Reddy et al., IJSID 2011, 1 (1), 33-40hydroxypropyl methylcellulose, titanium dioxide. The capsules tested contained lactose, corn starch,magnesium stearate and colloidal silicon dioxide as excipients. Injections of placebo tablet solutionsshowed no interference with the main peak.Range of linearity: Standard curves were constructed daily, for three consecutive days, using sixdifferent concentrations in a range 50 to 100 μg/ml for all actives. This concentration range correspondsto levels of 50-150% w/w of the nominal analytical concentration. The linearity of peak area responsesversus concentrations was demonstrated by linear least square regression analysis. The linearity ofcorrelation coefficient of Losaratin Potassium is 0.999, Enalapril maleate is 0.999 andHydrochlorothiazide is 0.999. Results were tabulated in table-1, overlaid chromatograms wererepresented in figure-4. Linearity graph plotted between area and concentration and represented ingraph-1.LinearitylevelTable-1: Linearity resultsLinearity ResultsLosartan Potassium Hydrochlorothiazide Enalapril MaleateConc.Area Conc. Area Conc. Area(ppm)(ppm)(ppm)1 50 239629 50 199926 50 1271212 60 285466 60 234067 60 1476083 70 334490 70 265294 70 1693714 80 380586 80 304913 80 1896665 90 424743 90 338487 90 2115316 100 481017 100 378888 100 235212Correlationcoefficient0.9995 0.9993 0.9997Figure-4: Overlaid linearity chromatogramsInternational Journal of Science Innovations and Discoveries VOL1, Issue 1, July-August 201137

L. Bhaskar Reddy et al., IJSID 2011, 1 (1), 33-40Graph-1: Linearity GraphPrecision: Precision of the analytical method is expressed as the series of the measurement. It wasascertained by replicate estimation of the drug by the proposed methods. Method precision wasdemonstrated by the assay of six samples, prepared as described above, on different systems, analystsand columns. Results were calculated and tabulated in table-2.International Journal of Science Innovations and Discoveries VOL1, Issue 1, July-August 201138

L. Bhaskar Reddy et al., IJSID 2011, 1 (1), 33-40Table-2: Precision ResultsResults (% of assay)S. No. Losartan Hydrochlorothiazid Enalapril MaleatePotassiumePrecisionInt.Precision Precision Int.PrecisionPrecision Int.Precision1 99.17 99.4 99.8 101.2 99.9 99.62 98.9 100.2 101.5 99.5 98.7 98.93 100.1 99.8 100.4 98.7 99.5 98.44 99.8 99.9 99.9 99.3 99.2 99.35 99.5 101.1 98.9 99.4 98.6 99.76 99.1 99.7 99.7 98.9 99.4 100.1Average 99.42 100.01 100.03 99.5 99.21 99.33%RSD 0.46 0.59 0.87 0.89 0.50 0.61Accuracy: Accuracy of each of the proposed method was ascertained on the basis of recovery studiesperformed by the addition of standard to placebo solution and recovery results found to be satisfactory.Results were tabulated in talbe-3Table-3: Recovery ResultsSample No. Spiked Level Recovery % of recovery1 50 99.682 50 98.999.483 50 99.871 100 100.102 100 99.8999.963 100 99.91 150 99.72 150 98.999.463 150 99.8Ruggedness: The stability study of Standard preparation and test preparation on bench top wasconducted at Initial, after 1 day and 2 days. The test preparation and standard preparation wereestimated against freshly prepared standard every time. The difference in percentage assay of testpreparation from initial up to 2days results were found to be within the accepted criteria. The similarityfactor for standard preparation from initial to 2 days should be in the range of 0.98 to 1.02 and was foundto be within the limits. Standard solution was prepared as per test method and system suitabilityparameters were evaluated by using the same lot of Mobile phase each time. The system suitability wasfound to be within the Limits.Robustness: A study was conducted to determine the effect of slight changes in the chromatographicconditions such as flow rate, mobile phase composition, pH of the mobile phase and filter validation. TheInternational Journal of Science Innovations and Discoveries VOL1, Issue 1, July-August 201139

L. Bhaskar Reddy et al., IJSID 2011, 1 (1), 33-40result was found to be within the limits. Based on the above results the method was proven to pass theparameters described as per Q2 guidelines of ICH and can be used for analysis.CONCLUSIONThe developed method is the first report for simultaneous estimation of Losartan potassium,Enalapril maleate and Hydrochlorothiazide. The developed HPLC method was found to be more accurate,precise with good repeatability. The recovery study result reveals that the method has accuracy and highprecision. Therefore the proposed method has applicable for regular analysis.REFERENCESS1. Guo ZX and Qiu MC, Losartan down regulates the expression of transforming growth factor beta typeI and type II receptors in kidney of diabetic rat], Zhonghua Nei Ke Za Zhi, 2003, 42(6), 403-408.2. Habashi JP, Judge DP, Holm TM, Cohn RD, Loeys BL, Cooper TK, et al. Losartan, an AT1 antagonist,prevents aortic aneurysm in a mouse model of Marfan syndrome, and preserves muscle tissuearchitecture in DMD mouse models. Science 2006, 312(5770), 117-121.3. University of Michigan Health System, Breast Cancer Gene Can Be Blocked By Blood Pressure Drug,Science Daily, 2009.4. Beermann B, Groschinsky-Grind M and A Rosén, Absorption, metabolism and excretion ofhydrochlorothiazide, Clinical Pharmacology Therapy, 1976, 19 (5), 531–537.5. Dvorak MM, De Joussineau C and DH Carter, Thiazide diuretics directly induce osteoblastdifferentiation and mineralized nodule formation by interacting with a sodium chloride cotransporterin bone, Journal of the American Society of Nephrology, 2007, 18 (9), 2509–2516.6. Tocco DJ et al., The physiological disposition and metabolism of enalapril maleate in laboratoryanimals, Drug metabolism and Disposition, 1982, 10 (1), 15-19.7. Ulm EH, Enalapril maleate, a potent, nonsulfhydryl angiotensin-converting anzyme inhibitor:absorption, disposition and metabolism in man, Drug metabolism Review, 1983, 14 (1), 99-110.8. Merlin ME et al., Enalapril maleate affects 2-oxoglutartate metabolism in mitochondria form the ratkidney cortex, Cell Biochemical Function, 1994, 12 (1), 21-28.International Journal of Science Innovations and Discoveries VOL1, Issue 1, July-August 201140