NOVEL RP-HPLC METHOD DEVELOPMENT AND VALIDATION OF LOSARTAN POTASSIUM AND AMLODIPINE DRUGS IN PURE AND PHARMACEUTICAL DOSAGE FORMS

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ABOUT AUTHORS:
1Kumari Jyothsna*, 2N.Chandana
1,2Department of Pharmaceutical Analysis and Quality Assurance,
Nimra College of Pharmacy, Jupudi, Vijayawada, A.P, India
jyothsna.s.k24@gmail.com

ABSTRACT
A fast, robust and accurate RP-HPLC method was developed and validated for simultaneous determination of Losartan potassium and Amlodipine in tablets. The mobile phase was mixture of aqueous Tri ethyl amine with pH 2.0 and Acetonitrile(70:30), effluent flow rate monitored at 1.0 ml/min. the stationary phase was C18 column, 3µm(4.6×250mm). The solutions of standard and the sample were prepared in methanol. The retention times was found to be 2.916min and 5.214min for Losartan potassium and Amlodipine respectively at 246nm. Calibration graphs constructed at their wavelengths of determination were linear in the concentration range of 50-150µg/ml. The percentage assay for Losartan potassium and Amlodipine were found to be 101% and 100%respectively. The method was validated and it was found to be accurate, precise, linear and reproducible as per ICH guidelines.

REFERENCE ID: PHARMATUTOR-ART-2003

1. INTRODUCTION
Losartan potassium is a [2-butyl-4-chloro-1-({4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl}methyl)-1H-imidazol-5-yl]methanol monopotassium salt. It competitively inhibits the binding of angiotensin II to AT1 in many tissues including vascular smooth muscle and the adrenal glands. 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. Losartan potassium is 1,000 times more selective for AT1 than AT2. Inhibition of aldosterone secretion may increase sodium and water excretion while decreasing potassium excretion. It is effective for reducing blood pressure and may be used to treat left ventricular hypertrophy and diabetic nephropathy.

Amlodipine (3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-1,4-     dihydropyridine-3,5-dicarboxylate) decreases arterial smooth muscle contractility and subsequent vasoconstriction by inhibiting the influx of calcium ions through L-type calcium channels. Calcium ions entering the cell through these channels bind to calmodulin. Calcium-bound calmodulin then binds to and activates myosin light chain kinase (MLCK). Activated MLCK catalyzes the phosphorylation of the regulatory light chain subunit of myosin, Inhibition of the initial influx of calcium decreases the contractile activity of arterial smooth muscle cells and results in vasodilation. The vasodilatory effects of amlodipine result in an overall decrease in blood pressure. Another possible mechanism is that amlodipine inhibits vascular smooth muscle carbonic anhydrase I activity causing cellular pH increases which may be involved in regulating intracelluar calcium influx through calcium channels.

Literature survey revealed that very few methods have been reported for the analysis of Losartan potassium and Amlodipine combinational dosage forms which include UV spectroscopy, High performance Liquid Chromatography, HPTLC methods. The present study illustrate development and validation of simple,  selective, accurate, economical precise RP-HPLC method for the determination of Losartan potassium and Amlodipine in bulk and Pharmaceutical dosage forms as per ICH guidelines.

The aim of this study is to develop rapid, economical HPLC method for the analysis of Losartan potassium and Amlodipine in combined dosage form using most commonly employed column(C18) and simple mobile phase preparation.

In the present work a successful attempt had been made to develop a method for the simultaneous estimation of Losartan potassium and Amlodipine pharmaceutical dosage form and validate it. The method would help in estimation of drugs in single run which reduces the time of analysis and does not require separate method for each drug. Thus, the paper reports an economical, simple and accurate RP-HPLC method for the above said pharmaceutical dosage forms.           

2. MATERIALS AND METHODS

Equipments used:Quantitative HPLC was performed on a high performance liquid chromatography -Waters e2695Alliance HPLC system connected with PDA Detector 2998 and Empower2 Software. The drug analysis data were acquired and processed using Empower2 software running under Windows XP on a Pentium PC and Agilent Zorbax C18, (3μ, 250 x 4.6mm). In addition an analytical balance (DENVER 0.1mg sensitivity), digital pH meter (Eutech pH 510), a sonicator (Unichrome associates UCA 701) were used.

Standards and chemicals used:Pharmaceutical gradeLosartan potassium and Amlodipinewere kindly supplied as a gift sample by Dr.Reddy’s Laboratory, Hyderabad, India. Acetonitrile and methanol was of HPLC grade, Purchased from E. Merck, Darmstadt, Germany. Triethylamine was analytical reagent grade supplied by Fischer Scientific Chemicals. Water HPLC grade was obtained from a Milli-QRO water purification system.

Losartan potassium and Amlodipine Tablets available in the market as Losar-A (Unichem pharmaceuticals, Himachal pradesh, India.)  in composition of Losartan potassium (50mg) Amlodipine (5mg).

Preparation of mobile phase: Transfer water into 1000ml of beaker dissolve and diluted volume with water. Then adjust its pH to 2.0  with Trithylamine. The Water adjusted pH to 2 with Triethylamine: acetonitrile (70:30 v/v) and filtered through 0.45µ membrane filter and degassed by sonication.

Preparation of calibration standards:10mg Losartan potassium and 10mg Amlodipine was taken into a 50, 10 ml of  Volumetric flask and add 10ml ofDiluent and sonicated for 10 minutes and made up with Diluent.It was further diluted to get stock solution of Losartan potassium and Amlodipine (To get 0.2 ppm and 0.02 ppm solution Respectively). This is taken as a 100% concentration. Working standard solutions of Losartan potassium and Amlodipine was prepared with mobile phase. To a series of 10 ml volumetric flasks, standard solutions of Losartan potassium and Amlodipine in the concentration range of 0.1-0.3µg/ml and 0.01-0.03µg/ml were transferred respectively.

System suitability: System suitability is an integral part of chromatographic system. At first the HPLC system was stabilized for 40 min.One blank followed by six replicate analysis of solution containing 100% target concentration of Losartan potassiumand Amlodipinewere injected to check the system suitability. To ascertain the system suitability for the proposed method, a number of parameters such as theoretical plates, retention time were taken and results along with optimized chromatographic conditions were presented in Table 1.

Recommended procedure:

Calibration curves for Losartan potassium and Amlodipine:Replicate analysis of solution containing 0.1-0.3µg/ml and 0.01-0.03µg/ml of Losartan potassiumand Amlodipinesample solutions  respectively were injected into HPLC according to the procedure in a sequence and chromatograms were recorded. Calibration curves were constructed by plotting by taking concentrations on X-axis and ratio of peak areas of standards on Y-axis and regression equation were computed for both drugs and represented in Table 2.

Analysis of  marketed formulation: The content of Ten tablets were weighed accurately. Their average weights were determined. Powder of tablets equivalent to two tablets weight(600.04mg) were weighed and taken in a 50 ml volumetric flask, dissolved in diluents, shaken and sonicated  for about 20 minutes then filtered through 0.45µ membrane filter. The filtered solution was further diluted (5 to 50ml) in the diluent to make the final concentration of working sample equivalent to 100% of target concentration.The prepared sample and standard solutions were injected into HPLC system according to the procedure. from the peak areas of Losartan potassium and Amlodipinethe amount of the drugs in the sample were computed. The contents were calculated as an average of six determinations and experimental results were presented in Table 3. The representive standarad and sample chromatograms were shown in fig. 3and fig.4.

Validation study of Losartan potassium and Amlodipine: An integral part of analytical method development is validation. Method validation is the process to confirm that the analytical procedure employed for a specific test is suitable for its intended use. The newly developed RP-HPLC method was validated as per International Conference on Harmonization (ICH) guidelines for parameters like specificity, system suitability, accuracy, linearity,  precision (repeatability), limit of detection(LOD),  limit of Quantification(LOQ)  and robustness.

Specificity: The effect of wide range of excipients and other additives usually present in the formulation of Losartan potassium and Amlodipine in the determination under optimum conditions were investigated. The specificity of the RP-HPLC method was established by injecting the mobile phase and placebo solution in triplicate and recording the chromatograms. The common excipients such as lactose anhydrous, microcrystalline cellulose and magnesium state have been added to the sample solution injected and tested.

Precision: precision study of sample (Losartan potassium and Amlodipine) was carried out by estimating corresponding responses 6 times on the same day for the 100% target concentration. The percent relative standard deviation (%RSD) is calculated which is within the acceptable criteria of not more than 2.0.

Linearity: The linearity graphs for the proposed assay methods were obtained over the concentration range of 0.1-0.3µg/ml and 0.01-0.03µg/ml (50-150%) Losartan potassiumand Amlodipinerespectively. Method of least square analysis is carried out for getting the slope, intercept and correlation coefficient, and the results were presented in Table 2. A calibration curve was plotted between concentration and area response and statistical analysis of  the calibration curves were shown in fig 6,7.

Accuracy (Recovery studies): The accuracy of the method is determined by calculating recovery of Losartan potassiumand Amlodipineby the method of addition. Known amount of Losartan potassium and Amlodipineat 50%, 100%, 150% is added to a pre quantified sample solution. The recovery studies were carried out in the tablet in triplicate each in the presence of placebo. The mean percentage recovery of Losartan potassium and Amlodipineat each level is not less than 99% and not more than 101%.

Robustness: The robustness is evaluated by the analysis of Losartan potassiumand Amlodipineunder different experimental conditions such as making small changes in flow rate (±0.2 ml/min), column temperature (±5).

LOD and LOQ: Limit of detection is the lowest concentration in a sample that can be detected but not necessarily quantified. Under the stated experimental conditions. The limit of quantification is the lowest concentration of analyte in asample that can be determined with acceptable precision and accuracy. Limit of detection and limit of quantification were calculated using following formula LOD=3.3(SD)/S and LOQ=10(SD)/S, where SD= standard deviation of response (peak area) and S= average of the slope of the calibration curve.

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