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A NOVEL RP-HPLC METHOD FOR THE QUANTIFICATION OF ESOMEPRAZOLE IN FORMULATIONS

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A NOVEL RP-HPLC METHOD FOR THE QUANTIFICATION OF ESOMEPRAZOLE IN FORMULATIONS Powered By Docstoc
					                                       AVN. Gupta et al., IJSID 2011, 1 (2), 165-171



                                                                                               ISSN:2249-5347
                                                                                                         IJSID
                         International Journal of Science Innovations and Discoveries               An International peer
                                                                                               Review Journal for Science


Research Article                                                     Available online through www.ijsidonline.info
       A NOVEL RP-HPLC METHOD FOR THE QUANTIFICATION OF ESOMEPRAZOLE IN
                                FORMULATIONS

   A.V.N Guptha, A. Babu, D. Masatan, G. Srilatha, P. Sambaiah, CH. Koteswara Rao, P. Parvathi*
              Dept of P.G Chemistry, S.S.N College, Narasaraopeta, Andrapradesh, India


 Received: 03.08.2011

 Modified: 14.10.2011

 Published: 27.10.2011
                                                                     ABSTRACT
                                             A simple accurate RP-HPLC method was developed and
                                      validated for rapid assay of Esomeprazole in formulation. Isocratic
 *Corresponding Author
                                      pump elution at a flow rate of 0.8ml/min was employed on
                                      symmetry Chromosil C18, 250x4.6mm, 5µm specification at 27. c
                                      temperature. The mobile phase consisted of Methanol: ACN: THF
                                      60:30:10 (V/V). The UV detection wavelength was 272 nm and
                                      20µl sample was injected. The retention time for Esomeprazole
                                      was 6.05 min. The percentage RSD for precision and accuracy of
 Address:                             the method was found to be less than 2%. The method was

 Name: P. Parvathi                    validated as per the ICH guidelines.
                                      Key Words: Esomeprazole, RP-HPLC, UV detection, recovery,
 Place: Guntur, AP, India
                                      precise, 272 nm
 Email:

  parvathi.pothuri@gmail.com


                                                   INTRODUCTION




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                                     AVN. Gupta et al., IJSID 2011, 1 (2), 165-171

                                                 INTRODUCTION
       Esomeprazole is a proton pump inhibitor which is used in the treatment of dyspepsia, peptic ulcer
disease (PUD), gastroesophageal reflux disease (GORD/GERD) and Zollinger-Ellison syndrome.
Esomeprazole reduces acid secretion through inhibition of ATPase in gastric parietal cells. By inhibiting
the functioning of this enzyme, the drug prevents formation of gastric acid. Common side effects include
headache, diarrhoea, nausea, gas, decreased appetite, constipation, dry mouth, and abdominal pain. More
severe side effects are severe allergic reactions, chest pain, dark urine, fast heartbeat, fever, paresthesia,
persistent sore throat, severe stomach pain, unusual bruising or bleeding, unusual tiredness, and
yellowing of the eyes or skin. Esomeprazole is a competitive inhibitor of the enzymes CYP2C19 and
CYP2C9, and may therefore interact with drugs that depend on them for metabolism, such as diazepam
and warfarin. The drug is rapidly cleared from the body, largely by urinary excretion of
pharmacologically-inactive       metabolites       such       as     5-hydroxymethylesomeprazole           and    5-
carboxyesomeprazole[4]




                                Figure.1: Chemical structure of Esomeprazole
                                                 EXPERIMENTAL
Materials
       Working standard of Esomeprazole was obtained from well reputed research laboratories. HPLC
grade water, methanol, Acetonitrile ,Tetrahydrofuran was purchased from E. Merck (Mumbai, India).
Apparatus
       A Series HPLC system PEAK LC7000 isocratic HPLC with PEAK 7000 delivery system. Rheodyne
manual sample injector with switch (77251),Analytical column Chromosil C18. 250×4.6mm, Electronic
balance-DENVER (SI234), A manual Rheodyne injector with a 20 μl loop was used for the injection of
sample., PEAK LC software were used. UV 2301 SPECOPHOTOMETER was used to determine the
wavelength of maximum absorbance




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                                     AVN. Gupta et al., IJSID 2011, 1 (2), 165-171

Determination of wavelength of maximum absorbance
       The standard solutions of Esomeprazole were scanned in U.V range against mobile phase as a
blank. Esomeprazole showed maximum absorbance at 272 nm. So the wavelength selected for the
determination of Esomeprazole was 272 nm.
Chromatographic equipment and conditions
       The development and validation of the assay was performed on A Series 200 HPLC system PEAK
LC7000 isocratic HPLC with PEAK 7000 delivery system. Rheodyne manual sample injector with switch
(77251),Analytical column Chromosil 100-5 C18. 250×4.6mm, , manual injector rheodyne valve) with
20μL fixed loop, PEAK LC software were used.
    The mobile phase consisted of a Methanol, Acetonitrile Tetrahydrofuran 60:30:10 (v/v). Injections
were carried out using a 20 μl loop at room temperature (20 + 2 °C) and the flow rate was 0.8 ml/min.
Detection was performed at 272 nm with 10 min runtime.
Standard and sample solutions
       A 10 mg amount of Esomeprazole reference substance was accurately weighed, dissolved in10ml
mobile phase in a 10 ml volumetric flask to obtain 1000 ppm concentrated solution. From standard
solution by the serial dilution we prepared required concentrations of 100ppm.3ml of above sample was
taken and diluted to 10ml using mobile phase. A composite of 20 tablets was prepared by grinding them
to a fine, uniform size powder. 10 mg of Esomeprazole was accurately weighed and quantitatively
transferred into a 100 ml volumetric flask. Approximately 26 ml mobile phase were added and the
solution was sonicated for 15 min. The flask was filled to volume with mobile phase, and mixed. After
filtration, an amount of the solution was diluted with mobile phase to a concentration of 30 μg/ml.
Method validation
       Method validation was performed following ICH specifications for specificity, range of linearity,
accuracy, precision and robustness
                                          RESULTS AND DISCUSSION
System Suitability
       Having optimized the efficiency of a chromatographic separation the quality of                          the
chromatography was monitored by applying the following system suitability tests: capacity factor, tailing
factor and theoretical plates. The system suitability method acceptance criteria set in each validation run
were: capacity factor >2.0, tailing factor ≤2.0 and theoretical plates >2000 13. In all cases, the relative
standard deviation (R.S.D) for the analytic peak area for two consecutive injections was < 2.0%. A



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                                    AVN. Gupta et al., IJSID 2011, 1 (2), 165-171

chromatogram obtained from reference substance solution is presented. System suitability parameters
were shown in Table.1. Standard chromatogram was given in Figure.2
                          Table.1 System suitability parameters
                   Mobile phase                    Methanol,Acetonitrile:Tetrahydrofuran
                                                   60:30:10 (v/v)
                   Pump mode                       Isocratic
                   pH                              5.8
                   Diluents                        Mobile phase
                   Column                          Zodiac C18 column (250 X 4.6 mm, 5μ)
                   Column Temp                     Ambient
                   Wavelength                      272nm
                   Injection Volume                20 μl
                   Flow rate                       0.8 ml/min
                   Run time                        10 minutes
                   Retention Time                  6.05 minutes




                                Figure.2: Standard solution chromatogram
Range of linearity
      Standard curves were constructed daily, for three consecutive days, using six standard
concentrations in a range of 10, 20, 30, 40, 50,60μg/ml. for Esomeprazole. The linearity of peak area
responses versus concentrations was demonstrated by linear least square regression analysis. The linear
regression equation was y = 42598 + 6031x (r= 0.997). Linearity values can shown in Table: 2
Precision
      To study precision, six replicate standard solutions of Esomeprazole(30 ppm) were prepared and
analyzed using the proposed method. The percent relative standard deviation (% RSD) for peak
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                                               AVN. Gupta et al., IJSID 2011, 1 (2), 165-171

responses was calculated and it was found to be 0.89which is well within the acceptance criteria of not
more than 2.0%. Results of system precision studies are shown in Table.
                                                      Table.2: Linearity results
                                              Concentration of Esomeprazole                    peak area
                   Level                                 In ppm
                  Level - 1                                 10                                   65149
                  Level - 2                                 20                                  154690
                  Level - 3                                 30                                  254613
                  Level - 4                                 40                                  327894
                  Level - 5                                 50                                  399456
                  Level - 6                                 60                                   49675
                                                          Slope                                  8328
               Range:10ppm                              Intercept                                7214
                 to 60ppm                         Correlation coefficient                       0.9988


                                600000

                                500000

                                400000
                    PEAK AREA




                                300000

                                200000

                                100000

                                     0
                                          0        10        20        30      40        50      60        70
                                -100000
                                                                  SAMPLE CONCENTRATION


                                                        Graph.1: Linearity graph
Precision Results for Esomeprazole:
                                    Table.3: Precision Results
                    Conc. (in ppm)       Injection No.   Intraday  Interday
                                               1          254932    254112
                                               2          254614    254327
                                               3          254032    254918
                           30
                                               4          254516    254716
                                               5          254739    254510
                                               6          254419    254403
Limit of Detection and Limit of Quantification:
       To determine the Limit of Detection (LOD) sample was dissolved by using Mobile phase and
injected until peak was disappeared. After 0.02 ppm dilution Peak was not clearly observed, based on
which 0.02 ppm is considered as Limit of Detection and Limit of Quantification is 0.06 ppm.
         International Journal of Science Innovations and Discoveries Vol 1, Issue 2, September-October 2011

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                                       AVN. Gupta et al., IJSID 2011, 1 (2), 165-171

                                                Table.4: LOD and LOQ results
                                     Parameter                       Measured Value
                                Limit of Quantification                    0.06 ppm
                                  Limit of Detection                       0.02 ppm
Robustness
          Typical variations in liquid chromatography conditions were used to evaluate the robustness of
the assay method. In this study, the chromatographic parameters monitored were retention time, area,
capacity factor, tailing factor and theoretical plates. The robustness acceptance criteria set in the
validation were the same established on system suitability test describe above.
                                                 Table.5: Robustness results
 S.NO        PARAMETER                          CONDITION                        AREA           % OF CHANGE
      1         Standard                    Standard conditions                  254613                 100%
      2       Mobile phase            Methanol 80%,ACN 10%,THF 10%.              254562                99.97%
      3      Mobile phase pH                        5.6                          254179                99.82%
      4       Wavelength                          270 nm                         254833                 100%
Recovery
          Recover test was performed at 3 different concentrations i.e. 20ppm,40ppm,60ppm. Results are
given in table.6
                                                   Table.6: Recovery results
          Recovery                  Conc of sample                   Recovery                 % of recovery
            50%                         20PPM                           19.85                      99.25
            100%                        40PPM                           40.02                      100.05
            150%                        60PPM                           59.95                      99.91




                                            Figure.3: Formulation sample
                                                 Table.7: Assay results
S.NO Formulation Dosage                Sample conc        Drug estimated         % of Drug Estimated in Tablet
  1          RACIPER        40 mg          40 ppm              39.95ppm                         99.8

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                                           AVN. Gupta et al., IJSID 2011, 1 (2), 165-171

                                                         CONCLUSION
         The proposed method for the assay of Esomeprazole in formulations is very simple and rapid. It
should be emphasized it is isocratic. The method was validated for specificity, linearity, precision,
accuracy and robustness.From our validated reports we are concluded that this method may use for
analysis of Esomeprazole in formulations.
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