METHOD DEVELOPMENT AND VALIDATION OF STABILITY INDICATING METHOD FOR ANALYSIS OF LOSARTAN POTASSIUM IN BULK DRUG AND DOSAGE FORM FORMULATION

METHOD DEVELOPMENT AND VALIDATION OF STABILITY INDICATING METHOD FOR ANALYSIS OF LOSARTAN POTASSIUM IN BULK DRUG AND DOSAGE FORM FORMULATION

Elsadig H. Rudwan ^*1, Amna B. W. E Mohammed Hussien ² and Ahmed E. M. Saeed ³

Amipharma Laboratories Ltd ¹, Khartoum, Sudan.

College of Animal Production science and Technology ², Sudan University of Science and Technology, Sudan.

Department of Chemistry ³, College of Science, Sudan University of Science and Technology, Sudan

ABSTRACT: Losartan potassium in tablet dosage form, separation achieved with C 18 column (Kromasil 100-5C18250mm x4.6mm E82860   i.d., 5µm particle), ambient temperature with isocratic mode with mobile phase containing 40:60 Acetonitrile:  Buffer consist of 10.5mM of disodium hydrogen phosphate and 10mM potassium dihydrogen phosphate, pH 3.5 adjusted by 10 % Orthophosphoric acid and at flow rate 1.0ml/minute the eluent was monitored at 250nm. The selected chromatographic conditions were found to be effectively separate Losartan potassium 9.98min. The proposed method was found to be linear over the range of 6.4-9.6µg/ml. The drug was subjected to oxidation, acid, alkaline, photolysis and heat stress conditions for degradation. The method was validated for specificity, linearity, accuracy, robustness and solution stability

Losartan potassium, reversed phase HPLC, Experimental design, stress condition, validation

INTRODUCTION: According to an FDA guidance document, a stability-indicating method is “a validated quantitative analytical procedure that can detect the changes with time in the pertinent properties of the drug substance and drug product. A stability-indicating method accurately measures the active ingredients, without interference from degradation products, process impurities, excipients, or other potential impurities ⁷.  Information on the stability of the drug substance is an integral part of the systematic approach to stability evaluation.

Stress testing (or forced degradation studies) is a critical component of the drug development process. The ICH guideline indicates that stress testing is designed to help “determine the intrinsic stability of the molecule by establishing degradation pathways in order to identify the likely degradation products and to validate the stability indicating power of the analytical procedures used. Stress testing also is becoming increasingly important in testing new molecules. Methods developed by stress testing and the stability information gained from those methods can have a significant effect on the actual compound selected for development

Literature review revealed that several methods have been reported for analysis of the drug in pharmaceutical formulation alone or with other drugs in combination. USP described HPLC method for assay and impurities of Losartan potassium, Valsartan and their impurities B, C (RP-HPLC), Valsartan impurity a (normal phase HPLC) and Irbesartan (ion pair HPLC), ion pair HPLC for Telmisartan.

BP described a potentiometric titration for assay of Losartan potassium. For impurities BP used a gradient RP-HPLC for Losartan potassium, some methods have been published for simultaneous determination of studied ARA-II-drugs including ⁸.

Losartan potassium is chemically:2-butyl-4-chloro-1-[[2’-(1H-tetrazol-5-yl) [1, 1’-biphenyl]-4-yl] methyl]-1H-imidazole-5-methanol monopotassium salt. Losartan is a phenyl tetrazole substituted imidazole compound which acts as a selective, angiotensin II receptor type I antagonist and is employed in the management of essential hypertension ^{2, 3}.

Losartan potassium is white to off white crystalline powder, it is freely soluble in water, soluble in alcohols, and slightly soluble in common organic solvents, such as acetonitrile and methyl ethyl ketone.

FIG.1: LOSARTAN POTASSIUM

The aim of this work, we report the development and validation of a stability indicating LC method for determination Losartan potassium in pharmaceutical dosage form. It separates drug components from degradation products under ICH suggested stress conditions (acid base hydrolysis, oxidation, photolysis and thermal stress) ⁴.

The target in developing the stability indicating method is to achieve the resolution between the losartan potassium and its degradation products. Our goal of chromatographic method development was to achieve peak tailing factor <2, retention time between 3 to 10min and capacity factor K >3.

Method validation study include system suitability, linearity, precision, accuracy, specificity, robustness, limit of detection, limit of quantification and stability of  sample, reagents, instruments.

 MATERIAL AND METHODS:

Instrument and Chromatographic equipment conditions:

Chromatographic separation was performed on SYKAM HPLC isocratic system equipped with a pump S1121 delivery system, variable- wavelength programmable UV-visible detector S3200, Auto sampler S5200 sample iniector, and software chromostar version 6.35. Samples (20µL) were injected using 20 µL loops; compound separated on column Kromasil 250x4.6mm100-5C1825cmx4.6mm E82860i.d., 5µm particle .The mobile phase was 40:60 ACN:  Buffer consist of 10.5mM of disodium hydrogen phosphate and 10mM potassium dihydrogen phosphate, pH 3.5 adjusted by 10 % Orthophosphoric acid and at flow rate 1.0ml/minute theeluent was monitored at 250nm_.

 Reagents and Chemicals:

HPLC grade of potassium dihydrogen phosphate, disodium hydrogen phosphate, orthophosphoric acid, acetonitrile 99.99%, methanol, Sodium hydroxide, hydrogen peroxide and losartan potassium working standard 99.85% from GPT Company India. The commercial formulation of losartan potassium from local market.

 Preparation of mobile phase and stock solution:

Mobile phase: The mobile phase containing 40:60 Acetonitrile:  Buffer consist of 10.5mM of disodium hydrogen phosphate and 10mM potassium dihydrogen phosphate, pH 3.5 adjusted by 10 % Orthophosphoric acid. The mobile phase   sonicated for 10min and filtrate through 0.45µm membrane filter which is used as a mobile phase.

 Stock solution (1000µg/ml) -stock solutions were prepared by weighing 100mg of losartan potassium, the weighed  drug  was transferred into  100ml  volumetric  flask  and  adjust the  volume to  the  mark with  methanol , sonicate for  10 min and  filtrate.

Working standard solutions (  8µg/ml)-  take 0.2ml  solution  from  stock solution and  transfer into 25 ml volumetric  flask,   the volume  was completed   to  the mark with  diluent.

The  HPLC  analysis   was  performed on  reversed  phase  high performance  liquid  chromatography system with  isocratic elution mode using  a mobile  phase  of acetonitrile: buffer of 10.5mM of  disodium hydrogen phosphate and 10mM potassium  dihydrogen phosphate( 40:60) on Kromasil 250x4.6mm100-5 C1825cmx4.6mm E82860i.d., 5µm particle ,  with 1.0ml/min flow rate   at 250nm using  UV detector.

Preparation of degradation samples:

The effect of acid and base on instability of losartan potassium:

Accurately weight 54.63mg of losartan potassium and transferred into 100 volumetric flask, to it 10ml of water was added and sonicateted for 15 minutes with intermittent shaking. To it 5 ml of 1M of HCl was added and 5ml of 1M NaOH were added separately. The sample was heated on boiling water bath for 45 minutes, cool to room temperature and diluted to volume with diluent, mixed well. The acidic degradation and the alkaline forced degradation was performed in dark in order to exclude the possible degradation effect of light. This solution was filtered through 0.45um filter, 5ml of  the  filtrate  was transferred to 25 ml volumetric flask, diluted to  volume  with diluent(9:1)  acetonitrile and  water, mixed  well  and  injected into  the  HPLC system.

 Effect of UV radiation on losartan potassium:

Accurately weight 54.63mg of losartan potassium and transferred into 100 volumetric flask, to it 10ml of water was added and sonicateted for 15 minutes with intermittent shaking. Kept in UV 254nm (UV radiation for 16hours), completed to the mark with diluent. This solution was filtered through 0.45um filter, 5ml of  the  filtrate  was transferred to 25 ml volumetric flask, diluted to  volume  with diluent(9:1)  acetonitrile and  water, mixed  well  and  injected into  the  HPLC system.

 Effect of hydrogen peroxide on losartan potassium:

Accurately weight 54.63mg of losartan potassium and transferred into 100 volumetric flask, to it 10ml of water was added and sonicateted for 15 minutes with intermittent shaking. To it 5 ml of 3% H₂O₂ was added. The sample was heated on boiling water bath for 45 minutes, cool to room temperature and diluted to volume with diluent, mixed well. This solution was filtered through 0.45um filter, 5ml of  the  filtrate  was transferred to 25 ml volumetric flask, diluted to  volume  with diluent(9:1)  acetonitrile and  water, mixed  well  and  injected into  the  HPLC system. 

Effect of hydrogen peroxide on losartan potassium:

Accurately weight 54.63mg of losartan potassium and transferred into 100 volumetric flask, to it 10ml of water was added and sonicateted for 15 minutes with intermittent shaking. To it 5 ml of 3% H₂O₂ was added. The sample was heated on boiling water bath for 45 minutes, cool to room temperature and diluted to volume with diluent, mixed well. This solution was filtered through 0.45um filter, 5ml of  the  filtrate  was transferred to 25 ml volumetric flask, diluted to  volume  with diluent(9:1)  acetonitrile and  water, mixed  well  and  injected into  the  HPLC system.

 RESULTS AND DISCISSION:

Optimization of Chromatographic Condition:

The Chromatographic method development for the determination of Losartan potassium was optimized by applying factorial design experiment.

 Effect of column type:

To have good separation of degradation products, we used a stationary phase. This column was found optimum hence; it become the column of work study. The bests electivity was observed on a C18 based stationary phase. The chromatographic separation was achieved using RP C18 (Kromasil250x4.6mm100-5C1825cmx4.6mm E82860i.d., 5µm particle).

Effect of mobile phase Mode:

Change the composition of mobile phase optimized chromatographic method by applying factorial design approach. Opitimization experiments were conducted through a process of screening and optimization. The purpose of screening design is  to identify the  factors that  have significant effects on the  selected chromatographic responses, and for  this purpose a factorial  design   2³ was   used to  develop a stability  indicating   method  it  was necessary to  maintain  proportion of the mobile phase Table 1, 2: (isocratic  mode  for  determination  losartan  potassium peak was  studied. Isocratic elution was firstly using different Acetonitrile   ration with   phosphate buffer pH 3.5.

By Appling   statistical software (SPSS 22, Minitab 17). Themodel was fit  R=90.30 if  we consider    P –value 0.05 and  standard  deviation about 1.055, acetonitrile has p-value 0.0066  is  highly  significant, but  buffer  composition  no  significant with  respect  to  flow  rate is  mild  affected  the  elution  of  peak.

The obtained adjusted R² were within acceptable limit of R²≥0.80, indicating that the experimental data were a good fit to the equation ⁴.

Y = ß₀+β₁ X₁ +β₂X₂+β₃X₃+ β₄X₁X₂+ β₅X₂X₃+ β₆X₁X₃+ β₇X₁X₂X₃

Where, Y is the level of the measured response, ß0 is the intercept, β₁ to β₇ are the regression coefficients, X_1, X₂ and X₃ stand for the main effects, X₁ X₂, X₂ X₃ and X₁ X₃ are the two- way  interaction between  the main effects and  X₁ X₂ X₃ is  the three-way interact between the main effect.

As a result the optimized composition of the mobile phase Acetonitrile: buffer pH 3.5 40: 60v/v   was selected.

TABLE 1: FACTORS LEVEL

TABLE 2: CHROMATOGRAPHIC CONDITION EMPLOYED AS FACTORIAL DESIGN   2³

Effect of wavelength:

The wavelength was important for detection of the components for HPLC as a detector. Losartan potassium   have wavelength 250nm, this wavelength was selected as optimum wavelength.

 Effect of Flow rate:

The  flow  rate of  mobile  phase was very important  to  achieved  the   good    resolution   between drug  and  its  degradation products. 1.0ml/min was efficient to resolve all peaks within appropriate time about 20 min.

 Method validation: ^{5, 6}

Method validation is  the  process  which  to  confirm that  the  analytical procedure employed for  specific test is  suitable for  its  intended  use.

 System suitability:

System suitability parameters   were measured to confirm or verify the system performance. System precision was determined on six replicate injection of standard preparation. All important characteristics were measured, including peak resolution, capacity  factor, tailing factor and  theoretical  plate.

 Specificity:

Specificity is ability to assess unequivocally the analyte in the presence of compounds which may be present. The describe method  was  validated with respect  to specificity, linearity, system suitability, accuracy, robustness, limit  of  detection (LOD) and  limit  of  quantitation(LOQ).

The excipients in the tablets contained the following in active ingredients: Microcrystalline cellulose, Povidone K 30, Maze starch, Magnesium stearate, Lactose and Talc powder as excipients, chromatograms of placebo solution showed no interference with the main peak of losartan potassium Table 9 and Fig. 6.

 Linearity and calibration Curve:-

The  linearity of  analytical procedure is  its ability( within a given range) to obtain  test  results which  are  directly proportional  to  the  concentration ( amount) of  analyte.

Five point's   calibration graphs were constructed covering the   range 6.4-9.6 µg/ml    (80-120%), for losartan potassium. Solution of linearity were injected in triplicate. The calibration graphs were obtained by plotting peak area response against concentration of the drug. The equation of the calibration curve of losartan potassium y =29.87x-5.772, the  graph  was  found  to  be  linear with   correlation coefficients 0.9998,  standard  error of  intercept 4.512 and  for  slope 0.558.

Precision (repeatability):

The  precision  of  analytical  procedure expresses the  closeness of  agreement(  degree of  scatter)  between a series of  measurements obtained  from  multiple  sampling of  the  same  homogenous sample under prescribed  conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility .Repeatability expresses the precision under the same operating   conditions over a short interval time.

 Accuracy:

The  accuracy  of  analytical  method  is extent to  which test  results  generated by  method and  true  value agree. Accuracy of method was studied by recovery experiments. The  accuracy of  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.

The recovery  was  performed at  three  levels  80%,100% and  120% of  the  label  claim of losartan potassium  50 mg, placebo equivalent to  one  tablet was  transferred into 100ml  volumetric  flask  and  the  amount of  losartan  potassium at  80,100 and  120%  were  added. Three levels   were analyzed and the percentage recoveries were calculated from the calibration curve. The  recovery value of  losartan   range  from %  to  %,the  average  recovery and %  RSD of  three  levels(nine determination) for  losartan was 99.61%(1.068)  RSD shown  in  parenthesis

 Limit of detection and Quantitation (LOD and LOQ):

The  detection limit of  an individual analytical procedure is  the  lowest amount of  analyte in  the sample which can  be detected but  not  necessary quantitated as  exact value. The quantitation limit of an individual analytical procedure is the lowest amount of analyte in   a sample which can be quantitatively determined with suitable precision and accuracy.

The LOD of losartan potassium was 0.176µg/ml and LOQ was 0534µg/ml.

 Robustness:

The robustness of  analytical procedure is a  measure of  its  capacity to  remain unaffected by  small but deliberate variations in method  parameters and  provides an indication of  its reliability during  normal usage. For the determination of method's robustness a number of chromatographic parameters (flow rate, buffer pH).

 TABLE 3: CALIBRATION CURVE OF LOSARTAN POTASSIUM

FIG. 4: CALIBRATION CURVE OF LOSARTAN POTASSIUM

TABLE 4: OPTIMIZED CHROMATOGRAPHIC CONDITIONS

 TABLE 5: REGRESSION CHARACTERISTIC AND SYSTEM SUITABILITY PARAMETERS OF PROPOSED METHOD

TABLE 6: ROBUSTNESS STUDY

TABLE 7: RESULTS OF THE RECOVERY TEST FOR THE LOSARTAN POTASSIUM

 TABLE 8: EFFECT OF SOME EXCIPIENTS ON REACTION KINETIC RATE OF LOSARTAN POTASSIUM

FIG.6: EXCIPIENT EFFECT ON LOSARTAN POTASSIUM

TABLE 9: STRESS TESTING OF DEGRADATION SAMPLE FOR LOSARTAN POTASSIUM

CONCLUSION: A new  HPLC analytical  method was  developed to  be  as routinely  applied to  estimation of  losartan  potassium in  pharmaceutical  dosage  form. In this work the stability of losartan potassium in present of dosage form was established through employment of ICH recommended stress condition. The developed procedure was evaluated for specificity, linearity, accuracy, precision and robustness to    assure the stability –indicating method. The simplicity of the method allows for application in laboratories that lack sophisticated an analytical instrument such as LC-MS/MS that are complicated, costly and time consuming than a simple HPLC-UV method .The method was found to be specific. Linear, precise, accurate, robust and stability –indicating. So the method is recommended for routine quality control analysis and stability sample analysis.

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   How to cite this article:

   Rudwan EH, Mohammed Hussien ABWE and Saeed AEM: Method Development and Validation of Stability Indicating Method for Analysis of Losartan Potassium in Bulk Drug and Dosage Form Formulation. Int J Pharm Sci Res 2016; 7(4): 1413-21.doi: 10.13040/IJPSR.0975-8232.7(4).1413-21.

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