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RP-HPLC/ELSD METHOD DETERMINATION OF TOPIRAMATE IN PHARMACEUTICAL PRODUCTS

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RP-HPLC/ELSD METHOD DETERMINATION OF TOPIRAMATE IN PHARMACEUTICAL PRODUCTS Powered By Docstoc
					                                 Viswanath Reddy Pyreddy et al., IJSID 2011, 1 (2), 126-133



                                                                                                    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

  RP-HPLC/ELSD METHOD DETERMINATION OF TOPIRAMATE IN PHARMACEUTICAL PRODUCTS
   Viswanath Reddy Pyreddy1*, Useni Reddy Mallu1, Pingili Sunil Reddy2, K. Hussain Reddy1, Maheswara
                                          Reddy Musirike1
    1Department    of Chemistry, Sri Krishnadevaraya University, Anathapur-515003, A.P. India, 2 Analytical Research and
                        Development, IPDO, Dr. Reddys Laboratories Ltd, Bachupally, Hyderabad, India.


 Received: 02.08.2011

 Modified: 28.09.2011
                                                                           ABSTRACT
 Published: 27.10.2011
                                              Topiramate is chemically sulfamate-substituted monosaccharide
 Keywords: Topiramate,
 HPLC, ELS detector,                  group drug. It is used to prevent the seizures of epilepsy. The present
 pharmaceutical dosage                work describes RP-HPLC/ELSD method for estimation of topiramate in
 forms.
                                      bulk samples and formulations. Best peak shape was achieved with the

 *Corresponding Author
                                      mobile phase having composition of acetonitrile and buffer (1.0gm of
                                      ammonium acetate in 1000ml of HPLC water) in the ratio 35:65v/v,
                                      Xterra C18, 150 x4.6mm, 5µ column, 20µL injection volume, column oven
                                      temperature 40°C, 1.0mL per min flow rate and ELS detector conditions
                                      are 90°C drift tube temperature, 22psi gas pressure, 100 is the gain for
                                      sample and standard detection was used. The linearity response of the
                                      HPLC system was obtained over the range of 500-3000ppm with a
 Address:
 Name: Viswanath Reddy Pyreddy
                                      correlation coefficient of 0.983. The result of recovery analysis showed

 Place: Hyderabad, AP,   India        excellent recoveries ranging from 98.0% to 102.0%. Specificity study
 E-mail:
                                      indicated that no interference due to common excipients and diluent was
 drusenireddymallu@gmail.com
                                      observed with the developed method.


                                                    INTRODUCTION


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                                   Viswanath Reddy Pyreddy et al., IJSID 2011, 1 (2), 126-133

                                                      INTRODUCTION
        Topiramate   (1-15)   is a sulfamate-substituted monosaccharide. It is an oral drug that is used to prevent the
seizures of epilepsy. (It is an anti-epileptic or anti-seizure drug). Seizures may be classified as either partial if they
involve only a small portion of the brain or generalized if they involve major portion of the brain. Topiramate is
available in 25mg, 50mg and 100mg tablet dosage forms. Topiramate side effects are eye effects (sudden decrease
in vision with or without eye pain and redness; blockage of fluid in the eye causing increased pressure in the eye
(secondary angle closure glaucoma), fever, Tingling in arms and legs, loss of appetite, nausea, taste change,
diarrhea, weight loss, nervousness, and upper respiratory tract infection. Symptoms could include hyperventilation
(rapid, deep breathing), tiredness and loss of appetite, irregular heartbeat, or changes in the level of alertness. Call
your doctor immediately if you get these symptoms. Your doctor may want to do simple blood tests. Chronic,
untreated metabolic acidosis may increase the risk for kidney stones or bone disease. Topiramate has little or no
effect on the pharmacokinetics of other antiepileptic drugs, but it can increase the clearance of the estrogenic
component of oral contraceptives by up to 30%. Figure-1 represents the chemical structure of Topiramate.




                                        Figure-1: Chemical structure of Topiramate
        The literature survey reveals that no method was developed by using the evaporative light scattering
detector and all reported methods were chemical and prederivatized methods. The present study is to develop a
simple and accurate HPLC/ELSD method for the analysis of Topiramate in pharmaceutical products.
                                                MATERIALS AND METHODS
Chromatographic conditions: Complete chromatographic conditions were tabulated in table-1
                                          Table-1: Chromatographic conditions.
       Parameter                                                   Conditions
   Column                      Waters make X-Terra C18, 150 x 4.6mm, 5micron.
   Mobile phase                Buffer (1.0gm of ammonium acetate in 1000ml of HPLC water: Acetonitrile (65:35).
   ELSD conditions             Gain-100, Drift tube temperature-90°C, nebulizer 80% power level, Mode-Heating
                               and 22psi gas pressure
   Flow Rate                   1.0 ml per min
   Inj. volume                 20 μl.
   Column temparature          40°C
   Diluent                     Water
   Runtime                     10min
   Standard                    Prepared the 2000ppm solutions from high pure standards with water as diluent
   Test                        and samples also prepared to equal concentration.


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                                 Viswanath Reddy Pyreddy et al., IJSID 2011, 1 (2), 126-133

Apparatus: The instrument used for the present study was Waters make Alliance HPLC instrument (2690module)
and connected with 2247 module ELS detector and the data controlled by Empower-2 software, Waters make X-
terra C18 column -5 (4.6 mm x 150 mm) was used for this study.
Reagents and materials: Pure standard of Topiramate was used for this study. Acetonitrile and HPLC grade water
were purchased from Merck India Ltd. Double distilled water was used throughout the experiment.
System suitability
1.   %RSD of five replicate standard solutions area is not more than 5.0%
2.   Tailing factor of Topiramate peak in standard solution is not more than 2.0
                                                RESULTS AND DISCUSSIONS
Method development:
        Based on sample solubility, stability and suitability various mobile phases and compositions were tried to
get a sharp peak. The following mobile phases were tried:
        1. Acetonitrile: Methanol [20:80 % v/v].
        2. Acetonitrile: Methanol [60:40 % v/v].
        3. Acetonitrile: Methanol: Water [30:30:50 % v/v/v].
        4. Water: Acetonitrile [30:70 % v/v].
        5. Ammonium acetate buffer: Acetonitrile [60:40 % v/v].
        Each mobile phase was filtered through 0.22μ membrane filter and degassed by sonication for 20 minutes.
Finally, ammonium acetate buffer and acetonitrile in 65:35% v/v proportions was selected as mobile phase. The
suitable ELS detector conditions were selected.
        The resulting chromatograms were recorded and the chromatographic parameters such as selectivity and
asymmetric factor. Optimisation in HPLC is the process of finding a set of conditions that adequately analyze the
quantification of the analyte with acceptable accuracy, precision, sensitivity, specificity, cost, ease, and speed of
analysis.

                  14.00

                  12.00

                  10.00

                   8.00

                   6.00
            LSU




                   4.00

                   2.00

                   0.00

                  -2.00

                  -4.00

                     0.00     1.00       2.00       3.00       4.00        5.00       6.00      7.00      8.00
                                                              Minutes
                                                 Figure-2: Diluent chromatogram
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                                    Viswanath Reddy Pyreddy et al., IJSID 2011, 1 (2), 126-133

       System suitability parameters were analyzed on freshly prepared standard solution. Both the solutions
(std/spl) were injected into the chromatographic system under the optimized chromatographic conditions.
Parameters were validated according to ICH Q2B guidelines for validation of analytical procedures in order to
determine the linearity, sensitivity, precision and accuracy. Diluent, standard solution chromatograms were
represented in figure-2 and 3. Figure-4 represents the five replicate standard solution injections. Table-2
represents the system suitability results.

                       400.00




                                                                    Topiramate - 4.081
                       300.00
                 LSU




                       200.00



                       100.00



                         0.00


                           0.00          1.00    2.00     3.00    4.00                   5.00   6.00      7.00      8.00
                                                                 Minutes
                                                Figure-3: Standard chromatogram

                                                Table-2: System suitability results
                                                                          Parameter
                                  Std.
                                                        Std Area       Retention time                  Tailing factor
                                 Inj-1                  2263314              4.08                           1.08
                                 Inj-2                  2232173              4.07                           1.08
                                 Inj-3                  2273069              4.08                           1.05
                                 Inj-4                  2262082              4.08                           1.06
                                 Inj-5                  2412006              4.08                           1.08
                                Average                 2288529              4.078                          1.07
                                 %RSD                  3.089542           0.109665                       1.321695


       Figure-5 represents the overlaid chromatogram of diluent and standard solution and figure-6 represents
the overlaid chromatogram of placebo and test solution. This indicates the developed method have no interference
with diluent and placebo.




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                              Viswanath Reddy Pyreddy et al., IJSID 2011, 1 (2), 126-133




                                                                 Topiramate - 4.081
        400.00
  LSU




        200.00


          0.00

        400.00




                                                                 4.082
  LSU




        200.00


          0.00

        400.00




                                                                 4.082
  LSU




        200.00


          0.00

        400.00
                                                                 4.083
  LSU




        200.00


          0.00

        400.00
                                                                  4.089
  LSU




        200.00


          0.00
               0.00       1.00         2.00       3.00         4.00                      5.00                6.00    7.00          8.00
                                                             Minutes
                                       Figure-4: System suitability solution
                                                                                        Topiramate - 4.081




        2.00


        1.50


        1.00
LSU




        0.50


        0.00


      -0.50

                       1.00             2.00             3.00                         4.00                    5.00          6.00
                                                           Minutes


                      Figure-5: Overlaid chromatogram of diluent and standard solution
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                                 Viswanath Reddy Pyreddy et al., IJSID 2011, 1 (2), 126-133




                                                                                                      Topiramate - 4.097
                 0.80

                 0.60

                 0.40
           LSU




                 0.20

                 0.00

                 -0.20

                         1.00    1.50       2.00        2.50         3.00           3.50           4.00                           4.50
                                                                Minutes
                         Figure-6: Overlaid chromatogram of Placebo and Test solution
Precision: In case of intra-day precision, %RSD was found to be in the range of 1.2-1.4. In case of inter-day
precision, %RSD was found to be in the range of 0.91-1.20. Reproducibility results were tabulated in table-3.
                                           Table-3: Reproducibility Results.
                                                        Sample preparations                                                              Average
           Parameter
                                  Prep-1       Prep-2      Prep-3       Prep-4             Prep-5              Prep-6                     (%)
            Precision              101.1        98.9           99.6         99.2           100.5                     101.4               100.11
     Intermediate Precision        101.5        100.8      100.38           100.1           99.6                           99.4          100.29
Recovery study: Accuracy of the method was confirmed by recovery study from marketed sample at three level of
standard addition. % Recovery for topiramate was found to be 98.5-102.5. results were tabulated in table-4.
                                               Table-4: Accuracy Results.
                                               Spike level                                                                   %
                         25%       50%         75%         100%         125%               150%               Recovery
                         101.1     100.1        99.6        99.2            101.5          100.8                           100.38
Linearity: The linearity of Topiramate was found to be in the range of 500-3000ppm with correlation coefficient
0.983. Linearity chromatograms were represented in figure-7 and results were shown in Table-5.




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                                         Viswanath Reddy Pyreddy et al., IJSID 2011, 1 (2), 126-133

                1000.00




                                                                            T opiram ate - 4.097
                 800.00


                 600.00
         L SU




                 400.00


                 200.00


                   0.00

                          3.50   3.60   3.70     3.80   3.90   4.00         4.10                     4.20    4.30         4.40   4.50   4.60   4.70   4.80   4.90
                                                                                                   Minutes
                                     Figure-7: Linearity solutions
                                      Table-5: Linearity Results.
                        Linearity Level Concentration (ppm) Peak area
                                1                   500              156183
                                2                  1000              819275
                                3                  1500             1680972
                                4                  2000             2732638
                                5                  2500             3955438
                                6                  3000             5867372
                                          Co-relation Coefficient 0.983261
Robustness and Robustness: The robustness was checked by small but deliberate change in two parameters like
mobile phase flow rate0.9ml/min to 1.1 ml/min, mobile phase composition ±10% organic modifier. Overall %RSD
was found to be 0.40. The robustness data are shown in table for two parameters like mobile phase flow rate,
mobile phase composition. Robustness data is shown in Table 6.

                                                         Table-6: Robustness Results.
                                                               System suitability Results
                     Parameter (Variation)                     USP           %RSD for                                                            Limit
                                                              Tailing    5replicate std.inj.
                                               0.9ml/min              1.0                                           0.6
                      Flow rate                1.0ml/min              0.9                                           0.8                    Tailing factor:
                                               1.1ml/min              1.0                                           0.9                   Not more than 2.0
                                               90% ratio              1.1                                           1.0                        %RSD:
                 Organic                                                                                                                 Not more than 2.0%
                                               100% ratio             1.0                                           0.7
         variation(acetonitrile)
                                               110% ratio             0.8                                           0.9


                                                                      CONCLUSION
       A specific, accurate, precise and robust isocratic HPLC-ELSD method has been developed for the
determination of Topiramate in pharmaceutical products. The recovery studies revealed excellent
accuracy and high precision of the method. Therefore the proposed method could be applied for routine
analysis in quality control laboratories.
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                               Viswanath Reddy Pyreddy et al., IJSID 2011, 1 (2), 126-133

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Description: RP-HPLC/ELSD METHOD DETERMINATION OF TOPIRAMATE IN PHARMACEUTICAL PRODUCTS