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RP–HPLC AND SPECTROPHOTOMETRY METHOD FOR FROVATRIPTAN

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RP–HPLC AND SPECTROPHOTOMETRY METHOD FOR  FROVATRIPTAN Powered By Docstoc
					                                         N. Usha Rani et al., IJSID 2011, 1 (1), 53-61

                                                                                                   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 AND SPECTROPHOTOMETRY METHOD FOR THE ANALYSIS OF FROVATRIPTAN IN
                                FOMULATIONS

                            N. Usha Rani1*, R. Sreenivasa Rao1, K .Saraswathi2, T.E.G. K. Murthy3
                    1Dept   of Chemistry, B.C.A.S, Bapatla, Guntur (D.T), A.P; 2 S.V University, Titrupathi, A.P
                                    3Bapatla college of Pharmacy, Bapatla, Guntur (D.T), A.P




                                                                                  ABSTRACT

   Received: 21.06.2011                                     A simple, rapid and precise reverse phase high

   Modified: 26.07.2011                            performance liquid chromatography method was developed
                                                   for the analysis of Frovatriptan. Chromatographic separation
   Published: 12.08.2011
                                                   of Frovatriptan was performed by using a kromosil C18, 250 x
   Keywords: Frovatriptan,
   accuracy, precision,                            4.6mm, 5 µm column, mobile phase comprising of 1%
   linearity, 230nm,
   colorimetry                                     Acetonitrile:Methanol:           80:20 (v/v) at a flow rate of
                                                   1.0ml/min and UV detection at 230nm. The linearity of
   *Corresponding Author
                                                   Frovatriptan is in the range of 10 ppm to 60 ppm. The limit of
                                                   detection for Frovatriptan was found to be 0.05ppm. The
                                                   proposed method was found to be accurate, precise and rapid
                                                   for the analysis of Frovatriptan. The colorometric methods
                                                   also developed and validated for analysis of Frovatriptan. The
                                                   following reagents are used for analysis. i.e MBTH at 570 nm,
   Address:                                        TPoo at 490nm, Brucine at520 nm, A.Red.S at 420 nm, 2,4 bi
   Name: N. Usha Rani
   Place: Guntur, AP                               pyrimidine at 590 nm, 1,10 Phenanthroline at 520 nm, FCF
   E-mail:

   nannapaneniusharani73@gmail.com                 at420 nm, K3FECN6 at 780nm,PNA at 560 nm, 1,10 WFBBL at
                                                   600 nm, Beer’s Law




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                                      N. Usha Rani et al., IJSID 2011, 1 (1), 53-61

                                                 INTRODUCTION
        Frovatriptan is a triptan drug developed by Vernalis for the treatment of migraine headaches and
for short term prevention of menstrual migraine. Frovatriptan inhibits excessive dilation of arteries that
supply blood to the head. Molecular formula is C14H17N3O I.U.P.A.C Name of Frovatriptan is (+)-(R)-3-
methylamino-6-carboxamido-1, 2, 3, 4-tetrahydrocarbazole. Molecular weight is 243.304 g/mol. Figure-1
represents the chemical structure of Frovatriptan.




                                      Figure-1: Structure of Frovatriptan

                                          MATERIALS AND METHODS
Chromatographic separation of Frovatriptan was performed by using a kromosil C18, 250 x 4.6mm, 5 µm
column, mobile phase comprising of 1% Acetonitrile:Methanol: 80:20 (v/v) at a flow rate of 1.0ml/min
and UV detection at 230nm. The linearity of Frovatriptan is in the range of 10 ppm to 60 ppm.
                                          RESULTS AND DISCUSSIONS
H.P.L.C Method development and validation: Methanol, Acetonitrile, Tri-ethyl amine, Methanol used
was analytical grade. Chromatographic separation was performed with PEAK high performance liquid
chromatography having LC-P7000 isocratic pump, equipped with PEAK LC-UV7000 variable wavelength
detector. Chromatograms and data were recorded by means of PEAK Chromatographic Software version
1.06.
Preparation of standard solution: 10mg of Frovatriptan was taken in a 100ml volumetric flask and
100ml of mobile phase was added to obtain 100 ppm of Frovatriptan standard solution. Chromatographic
conditions were tabulated in table-1. Figure-2 represents the standard solution.
                                     Table 1: Chromatographic Conditions
                      Mobile phase           Acetonitrile (80%), methanol (20%)
                      PH                     5.6
                      Analytical Column      Kromosil C18 column (250mm x 4.6mm) 5µ
                      UV detection           245 nm
                      Flow rate              1 ml/min
                      Injection Volume       20µl
                      Temperature            Ambient
                      Run time               10 min
                      Retention time         3.06 min.
                      Linearity Range        0.2- 1.0 ppm
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                                      N. Usha Rani et al., IJSID 2011, 1 (1), 53-61




                                          Figure-2: Standard solution

Linearity:
        In order to check the linearity for the developed method, solutions of five different
concentrations ranging from 0.2ppm-1.0ppm were prepared. The chromatograms were recorded and the
peak areas were given in table-1. A linear relationship between areas versus concentrations was
observed in about linearity range. This range was selected as linear range for analytical method
development of Frovatriptan. Linearity graph was shown in figure-3.




                                   Figure-3: Linearity Graph
Precision (Repeatability): 1ppm standard solution was prepared to calculate the precision for the
developed method. The prepared solution was injected into injector at same concentrations and same
chromatographic conditions. The chromatograms were recorded. R.S.D for the values calculated is 0.14.
Results reveal that the developed method was precise.
Limit of Quantification (LOQ) and Limit of Detection (LOD): The LOQ and LOD were established at a
signal to noise ratio. The LOD of Frovatriptan is 0.02ppm. The LOQ of Frovatriptan is 0.06.
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                                     N. Usha Rani et al., IJSID 2011, 1 (1), 53-61

Recover studies: Method recovery study was evaluated with standard spiking to placebo with different
concentration levels. The results were satisfactory and results were tabulated in table-2.
                                           Table-2: Accuracy results
     % of Recovery Amount of drug added                       Amount drug found                % of recovery
          50 %                    0.2                                   0.195                        97.5
          100%                    0.4                                   0.402                       100.5
          150%                    0.6                                   0.597                        99.5
       The Reverse Phase High Performance Liquid Chromatography method was developed a stability
indicating assay method. Pure drugs chromatogram was run in different mobile phases containing
methanol, acetonitrile, THF, and different buffers like potassium dihydrogen phosphate, sodium
dihydrogen phosphate,Ortho phosphoric acid in different volumes ratios. Different columns like C8, C18,
phenyl, cyano with different dimensions were used. Then retention time and tailing factor were
calculated. Finally acetonitrile: methanol: 80:20 v/v and Kromosil C18 analytical column was selected
which gave a sharp and symmetrical peak with 1.68 tailing. Calibration graph was found to be linear at
range 0.2 ppm to 1.0 ppm. Five different concentrations of Frovatriptan in range given above were
prepared and 20µl of each concentration injected in HPLC. The slope (m) and intercept (c) obtained were
found to be 199194.2 and -0.005.The correlation of coefficient (r2) obtained was found to be 0.999. It was
observed that the concentration range showed a good relationship. The limit of detection for
Frovatriptan was found to be 0.02pm and the limit of quantification was found to be 0.06ppm. It proves
the sensitivity of method. The low values of standard deviation and coefficient of variation at each level of
the recovery experiment indicate high precision of the method.
Spectrophotometric Methods Development and validation:
Instrumentation:
Spectral and absorbance measurements are made with Genesys 10 UV split beam Spectrophotometer
procured from Thermo Scientific Company marketed by Merck.
Standard and Sample solution of Frovatriptan
10 mg of Frova (bulk dosage form), 10 mg of API was dissolved in 100 ml of was brought to 100 ml with
methanol to give a concentration of 100 ppm.
1. WFBBL Method:
WFB BL Solution: Prepared by dissolving Weigh accurately 200 mg of Wool Fast Blue and is dissolving in
100 mL of distilled water.



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                                     N. Usha Rani et al., IJSID 2011, 1 (1), 53-61

Buffer solution (pH 1.5) : Prepared by mixing 289 mL of glycine solution ( 37.52 g glycine and 29.24 g of
NaCl were dissolved in 500 mL distilled water) with 711 mL of 0.1 M HCl and the pH of solution was
adjusted to 1.5.
Procedure: Into a series of 125 mL separating funnels containing aliquots of standard drug solution [0.5
–2.5 mL 20 µg/mL, 0.5 - 2.5 mL, 80 µg/mL, 6.0 Ml] of buffer solution [pH 1.5 (M1a) or 0.1 M HCl] and 2.0
mL of dye solution WFB BL is added. The total volume of aqueous phase in each separating funnel was
adjusted to 15mL with distilled water and 10 mL of CHCl3 was added. The contents were shaken for 2
min. The two phases were allowed to separate and the absorbance of the separated organic layer was
measured at 590 nm against a similar reagent blank after 10 min. The amount of drug was deduced from
the calibration curve.
2. TP ooo Method:
TP ooo solution: Weigh accurately 200 mg of tropaeolin ooo and is dissolved in 100 mL of distilled water.
HCl solution: dissolve 8.6 mL of concentrated hydrochloric acid in 1000 mL of distilled water and
standardized.
Chloroform: AR grade chloroform was used.
Procedure: Into a series of 125 mL separating funnels containing aliquots of standard drug solution [0.5
–2.5 mL 20 µg/mL , 0.5 - 2.5 mL, 80 µg/mL ], 6.0 mL of buffer solution [ pH 1.5 or 0.1 M HCl (M1b) ] and
2.0 mL of dye solution TPooo is added. The total volume of aqueous phase in each separating funnel was
adjusted to 15mL with distilled water and 10 mL of CHCl3 was added. The contents were shaken for 2
min. The two phases were allowed to separate and the absorbance of the separated organic layer was
measured at 480 nm against a similar reagent blank after 10 min. The amount of drug was deduced from
the calibration curve.
3. PNA Method:
PNA solution: Dissolve 100 mg of PNA in 100 ml of 0.2 M HCl.
NaNO2 solution: Dissolve 100 mg of NaNO2 in 100 mL of distill water.
NaOH solution: Weigh accurately 4g of NaOH, dissolved in few ml of distill water and made up to the
mark in a in 100 ml volumetric flask with distill water.
Procedure: Into a series of 10 ml graduated test tubes 1.0 ml of PNA solution and 1.0 ml of NaNO2
solution were successively added and allowed to stand for 2 min. Later, aliquots of the standard drug
delivered into the test tubes. Then 1.5 ml of NaOH solution was added and the volume in each tube was
made up to 10 ml distill water. The absorbance was measured at 480 nm against a reagent blank.
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                                       N. Usha Rani et al., IJSID 2011, 1 (1), 53-61

4. Potassium ferricyanide Method:
Potassium ferricyanide solution: Dissolve 100 mg of pot. Ferricyanide in 100 mL of Double distills water.
Fe(III) solution ( Wilson Labs, 0.054 %, 3.32 X10-3 M) : Prepared by dissolving 54 mg of anhydrous ferric
chloride in 100 mL of distill water.
HCl solution (1N): Solution was prepared by diluting 86 ml of conc. HCl to 1000 mL with distill water and
standardized.
Procedure: Into a series of 10ml of calibrated tubes, aliquots of standard drug were transferred and 1
mL of Fe(III) solution was added. The tubes were stopper immediately and shaken well for 5 min. Then
0.5 ml of Pot. Ferricyanide solution was added into each tube and was closed with lids immediately. After
5 min, 1 mL of 1N HCl was added and the final volume was made upto 10 mL with distill water. The
absorbance of the solution in each tube was measured immediately at 740 nm against a similar reagent
blank.
5. MBTH Method:
MBTH solution: Solution was Prepared by dissolving 200 mg of MBTH in 100 mL of distill water.
Fe(III) solution: Solution is Prepared by dissolving 250 mg o Fe(III) in 100 mL of distill water.
NaOH solution: Solution was prepared by dissolving 400 mg of NaOH to 100 mL of distill water and
standardized.
Procedure: 1.0 mL of standard chloroformic BUD solution containing were transferred into a series of 25
ml calibrated tubes and gently evaporated on a boiling water-bath to dryness. Then 1 mL of water, 0.5 ml
of 0.5% MBTH and 0.5 ml of 0.1 N NaOH was added to each tube. The contents were heated for 10 min in
a water bath at 100 0C and cooled for 5 min in a water bath at 15 0C. Then 0.5 mL of 1N HCl and 2 ml of
Fe(III) solutions were added successively and kept side for 1 hr. The absorbance was measured at 620
nm against a reagent blank prepared in a similar way.
5. Brucine Method:
Brucine 0.2%: Weigh accurately 200 mg of Brucine and is dissolved in 100 ml distill water.
NaIO4: Analytical grade is used.
H2SO4 (2.3M): Dissolve 6.38 ml of 18 M H2SO4 in 100 ml distill water.
Procedure: Into a series of 10ml of calibrated tubes, aliquots of standard drug were transferred and add
3 mL of Brucine, 15 mL of NaIO4, 2 mL of H2SO4 is added and total volume made upto 10 ml with distill
water then heat for 15 mts. Cool, Readjust volume to 10 ml, measure absorbance at 510 – 520 nm, Stable
for 40 mts.
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                                      N. Usha Rani et al., IJSID 2011, 1 (1), 53-61

6. Picric acid Method:
Weigh accurately 300 g of monosodium phosphate dihydrate and 9 grams of of NaOH and are dissolved
in 750 ml of distill water.
Picric acid: Analytical grade 0.1% picric acid is used.
Procedure: Into a 25 ml of separating funnel, introduced 10 ml of sample solution in chloroform, 2 mL of
buffer, and 1 ml of reagent, shake for 1 min, allow the phase to separate, color organic layer is separated
and absorbance is measured at 390 nm.
7. F.C.F METHOD
F.C. reagent (2N) supplied by S.d. Fine chem. India, Ltd., was used by diluting 50 mL to 100 mL with
distilled water. Sodium hydroxide solution (4%) was prepared by dissolving 4g of sodium hydroxide in
100 mL of distilled water. 300 µg/mL Stock reference solution was freshly prepared from pure sample of
Frovatriptan by dissolving 0.03 g in 100 mL of distilled water. General procedure In to 10 mL measuring
flasks, different aliquots of working standard solution were transferred to provide final concentration
range 30.0 – 150.0 µg mL-1. To each flask, 1.5 mL of sodium hydroxide and 1.5mL of F-C were
successively added and kept a side for 5 min. The solutions were made up to volume with distilled water.
The absorbance of each solution was measured at 760 nm against the reagent blank. The calibration
graph was then prepared by plotting the absorbance versus concentration of the drug. The concentration
of the unknown was read from the calibration graph or computed from the regression equation.
8. 1, 10 phenanthroline Method
 To the working concentrated drug solution, 1 ml 1,10 Phenanthroline reagent added and diluted up to
50 ml. Absorbance measured at 520 nm wavelength. Fixed wavelength linearity, precision, recovery
stability studies are carried out at this fixed wave length.
9. 2, 2-Bi pyrimidine
To the working concentrated drug solution, 2 ml 2, 2 Bi pyrimidine reagents added and diluted up to 25
ml. Absorbance measured at 590 nm wavelength. Fixed wavelength linearity, precision, recovery stability
studies are carried out at this fixed wave length.
10. Alizarin Red S
   To the working concentrated drug solution, 0.5 ml Alizarin Red .S reagents added and diluted up to
100 ml. Absorbance measured at 420 nm wavelength. Fixed wavelength linearity, precision, recovery
stability studies are carried out at this fixed wave length.


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                                      Table 3A: Spectrophotometric conditions
                 TEST                  MBTH              FCF          K3Fe(CN)6            PNA          WFBBL
         Linearity range              5-40 ppm        1-10 ppm         20-40 ppm         10-20 ppm      50 -75 ppm
         Precision R.S.D                 0.21             1.6              0.32             0.047           0.42
         Slope                        0.022261         0.02659          0.01765            0.0227        0.009629
         Intercept                      0.007         -0.00124            -0.01           -0.03224       -0.09395
         Correlation coefficient        0.9993          0.999            0.9998             0.999          0.999
         Stability period              180 min         165 min          190 min           145 min        150 min
         Accuracy                      96.65%          99.44%           98.99%             99.79%         99.24%
         Wave length                   570 nm          420 nm           780 nm             560 nm         600 nm
                                      Table 3 B: Spectrophotometric conditions
       TEST                        Brucine    A.Red.S,       2,4bi-pyrimidine 1,10Phenanthroline                 TPOOO
          Linearity (ppm)            5 -40     0.3-100                2-12                      1-32               2-14
          Precision R.S.D             0.44       0.24                0.769                     0.829               0.45
               Slope               0.02276     0.0123               0.0765                    0.02284             0.0582
             Intercept             0.007578   0.003236             0.002321                   -0.0084            0.00583
       Correlation coefficient      0.9994     0.9994               0.9997                     0.999               0.995
          Stability period         135 min    150 min              170 min                    140 min            160 min
             Accuracy              99.83 %     99.76%               99.61%                    99.50%             101.16%
            Wave length             520 nm     420 nm               590 nm                    520 nm              490 nm
        From Table 2A, 2B maximum linearity range shows A.R.S method i.e 0.3-100 ppm. Low precision
R.S.D shows M.B.T.H method. Maximum stability period shows K3Fe (CN)6 method i.e. 190 min.
Maximum accuracy showing method is Brucine method, 99.83 %. But all methods are validated as per
I.C.H guidelines.
                                           CONCLUSION
        The above developed methods were applicable for regular analysis of the dosage forms.

                                                      REFERENCES
1.   "Frova". Vernalis. Archived from the original on 2007-09-27. Retrieved 2007-11-28.
2.   "Patient Information Sheet -- Frovatriptan succinate (marketed as Frova)". Food and Drug Administration.
     07/2006. Archived from the original on 2007-09-29. Retrieved 2007-11-28.
3. Chiral separation of Frovatriptan isomers by HPLC using amylose based chiralstationary phaseMuzaffar Khan,
     Balaji Viswanathan, D. Sreenivas Rao and Rajasekhar Reddy,Journal of Chromatography B Volume 846, Issues
     1-2, 1 February 2007, Pages 119-123
4. Cafer Saka ,Review of Analytical Methods for Identification and Determination of Triptans,Critical Reviews in
     Analytical Chemistry,Volume 39, Issue 1, 2009, Pages 32 – 42
5. RecentAdvancesintheImpurityProfilingofDrugs, Bartos,Dorottya; Gorog,Sando, Current                       Pharmaceutical
     Analysis, Volume 4, Number 4, November 2008 , pp. 215-230(16)



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6. Paterson, John R, Baxter, Gwendoline Dreyer, Jacob S, Halket, John M, Flynn, Robert, Lawrence and R James,
   Salicylic acid sans aspirin in animals and man: persistence in fasting and biosynthesis from benzoic acid,
   Journal of Agricultural and Food Chemistry, 2008, 56 (24), 11648–11652.
7. Delaney JA et al., Drug drug interactions between antithrombotic medications and the risk of gastrointestinal
   bleeding, Canadian Medical American Journal, 2007, 177 (4): 347–51.
8. http://antoine.frostburg.edu/chem/senese/101/acidbase/faq/buffered-aspirin.shtml.
9. UK Prospective Diabetes Study Group, Efficacy of atenolol and captopril in reducing risk of macrovascular and
   microvascular complications in type 2 diabetes: UKPDS 39, British Medical Journal, 1998, 317 (7160), 713-720.




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Description: RP–HPLC AND SPECTROPHOTOMETRY METHOD FOR FROVATRIPTAN