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					                                                                                          ISSN 2229 – 6867
                                IJPI’s Journal of Analytical Chemistry

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Optimization and Validation of Quantitative Spectrophotometric Methods for Determination of
                Formoterol Bulk Drug and its Pharmaceutical Dosage Forms

                   Mohd. Gousuddin*, S.Appala Raju, Sultanuddin, Shobha Manjunath


                 Department of Pharmaceutical Analysis, H.K.E. Society’s College of Pharmacy,
                                 Sedam Road, Gulbarga-585 105 (K.S.) India



   Corresponding Author: Mohd. Gousuddin                  Email address: gousuddin.pharma@rediffmail.com




                                               ABSTRACT:

          Two simple, sensitive, selective, accurate, precise and economical methods (method A and B)
   has been developed for the estimation of Formetrol in bulk drug and its pharmaceutical formulations.
   Method A is based on formation of stable blue colored chromogen due to reduction of Folin-
   ciocalteau reagent in presence of 20% sodium hydroxide by Formetrol and exhibiting absorption
   maximum at 734 nm and obeying beers law in the concentration range of 5 – 25 µg/ml. Method B is
   based on formation of blood red colored chromogen with ferric chloride and 1, 10 phenanthroline
   which exhibit absorption maximum at 509 nm and obeyed Beer’s law in the concentration range of 2
   – 10 µg/ml. The results of analysis for both the methods have been validated statistically and by
   recovery studies. The proposed methods are economical and sensitive for estimation of Formetrol in
   bulk drug and its rotacaps dosage form.

              Key words: Formetrol, Folin- ciocalteau, 1, 10 phenanthroline, Ferric chloride
Vol 1:4 (2011)                                                                 IJPI’s Journal of Analytical Chemistry


1. INTRODUCTION

       Formoterol1-2 is a long-acting beta2-agonist used in the management of asthma and/or chronic obstructive
pulmonary disease (COPD). Inhaled formoterol works like other beta2-agonists, causing bronchodilatation by relaxing
the smooth muscle in the airway so as to treat the exacerbation of asthma.
       N-[2-hydroxy-5-[1-hydroxy-2-[1-(4-methoxyphenyl)           propan-2-ylamino] ethyl]  phenyl]     formamide
phenylethylamine derivative with one phenolic hydroxyl and one secondary amino group.

2. OBJECTIVE

        The aim of this study Formoterol fumarate is latest anti asthmatic drug. It is available in rotacap dosage form. It
is official in Indian pharmacopoeia.3 and British pharmacopoeia4. The literature survey reveals that one
spectrophotometric5, few HPLC Chromatographic6 analytical methods have been reported for determination of
formoterol in rotacaps.In present investigation we have developed a simple isocratic RP-HPLC method for quantitative
estimation of Formoterol fumarate in bulk drug and pharmaceutical formulations with high accuracy and precision.
         Hence in the present work spectrophotometric methods has been developed for the estimation of Formetrol
using FC in presence of sodium hydroxide in method A and 1, 10 phenanthroline in presence of FeCl 3 in method B. The
above methods are simple, sensitive, accurate and precise and can be used for the routine quality control of this drug in
bulk as well as in pharmaceutical formulation
                                                  Structure: Formoterol

                                              O

                                              CH
                                         NH
                              HO                                                   OCH3
                                                              CH3

                                                         N
                                                         H
                                                  OH


3. MATERIALS AND METHODS

3.1 Instruments and reagents:
         A Shimadzu model 1601 double beam UV/Visible spectrophotometer with spectral width of 2 nm, wavelength
accuracy of 0.5 nm and a pair of 10 mm matched quartz cells was used to measure absorbance of the
resulting solution.
         A Sartorius CP224S analytical balance, an ultra-sonic cleaner (Frontline FS4), Formoterol pure powder
(sun pharma Mumbai India ), Folin-Ciocalteu’s (FC) reagent (diluted to 1:4 with distilled water), 20% sodium
hydroxide solution in double distilled water, 0.2 % 1, 10 phenanthroline solution 0.4% Ferric Chloride in double
distilled water were used. All the chemicals and reagents used were of analytical reagent grade.
The marketed formulation in rotocaps forms (Cipla lab goa)
3.2 Preparation of standard drug solutions:
        Accurately weighed 100 mg of Formoterol (bulk drug or its formulation) was dissolved in 40 ml of distilled
methanol and volume was made up to 100 ml with distilled methanol (i.e. 1000 µg/mL). Further dilution was made
with distilled methanol to get the concentration of 100µg/mL.


Mohd. Gousuddin et al                                                                                             Page 2
Vol 1:4 (2011)                                                               IJPI’s Journal of Analytical Chemistry


3.3 Preparation of calibration curves:
Method A- FC Method
Fresh aliquots of Formoterol ranging from 0.5 - 2.5 mL (1 ml-100µg/mL) was transferred into a series of 10 mL
volumetric flasks to provide final concentration range of 5 - 25 μg/mL. To each flask 1ml of aqueous sodium
hydroxide (1N) solution and 0.5 ml of Folin cio-calteu reagent was added. The solution in each tube were made upto
the mark with distilled methnol The absorbance of blue colored chromogen was measured at 734 nm against the
reagent blank. The amount of Formoterol present in the sample solution was computed from its calibration curve.
Method B- 1, 10 phenanthroline
Fresh aliquots of Formoterol ranging from 0.2 - 1 mL (1 ml-100 µg/mL) was transferred into a series of 10 mL
volumetric flasks to provide final concentration range of 2 - 10 µg/mL. To each flask 0.5 ml of aqueous Ferric chloride
(0.4 %) solution and 1 ml of alcoholic 1, 10 phenanthroline (0.2%) was added and heated at 40 0C for 10 min. The
solutions were cooled to room temperature and made upto mark with distilled water. The absorbance of blood red
colored chromogen was measured at 509nm against the reagent blank. The amount of Formoterol present in the sample
solution was computed from its calibration curve.
3.4 Analysis of pharmaceutical preparations:
        A total 20 rotacaps (each rotacaps contains 12 mcg drug is available) each from different batches was dissolved
in a mixture of 1 ml of sodium hydroxide and 4 ml of distilled methanol and filterd through through Whatman filter
paper No.41.
        The amount of drugs was determined by referring to the calibration curve. The analysis procedure was
repeated five times with pharmaceutical formulations and the results of analysis of pharmaceutical formulations are
reported in Table 1.
3.5 Recovery studies:
        To study the accuracy and reproducibility of the proposed method, recovery studies were carried out by adding
known amount of the drugs to the preanalyzed formulations and reanalyzing the mixture by proposed method. Results
of recovery studies are reported in Table 1.
                   Table 1: Assay and Recovery of Formoterol in pharmaceutical dosage form

                                         Amount found by proposed         Reference         Recovery of proposed
   Pharmaceutical        Labelled
                                                  methods                   method              methods (%)
    dosage form          Amount
                                             A                B          (UV method)             A              B
          B1               12 µg         11.89±0.01      11.85±0.01        11.98±0.1        98.79±0.11        98.89
          B2               12 µg         11.88±0.05      11.94±0.08        11.96±0.2        98.88±0.11        98.92
B1, B2 are rotacaps from same manufacturers, average of 5 determinations (12 µg of Formoterol was added and
recovered)

4. RESULT AND DISCUSSION

        As per Lewis acid-base theory, nitrogen-containing group is basic in nature having unshared pair of
electrons so it can act as a reducing agent and can reduce tungstate and/or molybdate, which are present in
Folin Ciocalteu’s (FC) reagent in alkaline medium The above mentioned drug contains nitrogen in their structure
and therefore reduces the FC reagent in alkaline condition forming blue colored chromogen molybdenum blue.
Formoterol reacts positively with FC reagent and producing blue colored chromogen. Therefore the proposed work

Mohd. Gousuddin et al                                                                                          Page 3
Vol 1:4 (2011)                                                                IJPI’s Journal of Analytical Chemistry


is based on the similar reaction. principle. In the present work, the quantitative reaction of the drug with FC
reagent is proposed The reaction is based on the reduction of phosphomolybdotungstic acid, the FC reagent by
formoterol in presence of 20% sodium hydroxide .It was found that 3 ml FC reagent with 1.5 ml 20% sodium
hydroxide solution for formoterol was sufficient for the development of maximum colour intensity. Colored
chromogen was found to be stable for more than 3 h at room temperature for both the drugs. The linearity was
found in the concentration range of 5 to 25 µg/ml (r =0.9999) .
        Formoterol Fumarate by oxidation followed by complex formation reaction. Using 1,10 phenanthroline
formation of orange red coloured complex when Formoterol is treated with 1,10 phenanthroline in presence Fe 2+
resulted from oxidation of drug with Fe3+. The orange red coloured chromogen exhibited absorption maximum at
509 nm and obeyed Beer’s law in the concentration range of 2-10 mcg /ml The coloured chrmogen was stable for more
than 2 hour. (r=0.9999) The optical characteristics such as Beers law limit, sandell’s sensitivity, molar extinction
coefficient, percent relative standard deviation (calculation from eight measurements containing ¾ th of the amount of
the upper Beers law limit) were calculated. Regression Characteristics like slope, intercept, correlation coefficient and
percentage range of errors (0.05 and 0.01 confidence limits), LOD, LOQ, Error’s in bulk sample and standard error of
estimation were calculated.
        Commercial formulation of Formoterol was successfully analyzed by proposed spectrophotometric methods
and results are calculated. To evaluate validity and reproducibility of the methods, fixed amounts of drug were added to
the preanalyzed formulation. These results of percentage recovery are calculated. There is no interference of additive
and exicipients in proposed analytical methods. The proposed spectrophotometric methods for the estimation of
Formoterol are simple, sensitive, accurate and precise and can be used for the routine quality control of this drug in
bulk as well as in pharmaceutical formulations.
                                   Table 2: Optical Characteristics and Precision

                                    Parameters                          Method-A         Method-B
                                      max (nm)                             734              509
                           Beer’s law limits (g/ml) (c)                   5-25              2-10
                                         Color                             Blue          orange red
                         Molar absorptivity (lit/mol-1 cm-1)            1.699 x 104      3.997 x 104
                        Limit of Detection (LOD/ mcgml-1)                  0.140           0.05989
                      Limit of Quantification (LOQ/ mcgml-1)               0.426           0.18148
                    Sandell’s sensitivity (g/ml/0.001 abs units)         0.0035            0.0024
                             Regression equation (Y*)
                                       Slope (b)                          0.0447           0.1097
                                     Intercept (a)                     9.333 x 10-4     -5.999 x 10-4
                         Standard error of estimation (Se)              0.0006745        0.0002672
                             Correlation coefficient (r)                  0.9999           0.9999
                                        % RSD                              0.283            0.284
                         Confidence limits with 0.05 level               0.00159          0.00632
                         Confidence limits with 0.01 level               0.00236          0.00935
                           0% Error in bulk Samples***                      0.31             0.12
                 *Y=bC+a, where C is the concentration of Formoterol in g/ml and Y is the absorbance at the
                  respective maximum absorbency,
                 **Average for eight determination, ***Average for three determination



Mohd. Gousuddin et al                                                                                            Page 4
Vol 1:4 (2011)                                                          IJPI’s Journal of Analytical Chemistry


5. REFERENCES

   (1) O’Neil MJ Editor, the Merck Index: An Encyclopedia of Chemicals, Drugs and Biologicals, 14 th Edn. Merck
       & Co., INC; 2006; 4244.

   (2) Sweetman SC Editor, Martindale: The Complete Drug Reference, Pharmaceutical Press, London, 35th Edn.
       2007; 1007.

   (3) Indian Pharmacopoeia, Ministry of Health and Family Welfare, Government of India, New Delhi, 2007.1145-
       1146.

   (4) British pharmacopoeia (2008) London, Medicines and Health care Products regulatory agency (MHRA). vol1.
       965.-966.
   (5) Pai, PNS Sandeep, SK; Sekhar ABP; Indian J. Pharma Sciences 2003; 65(6), 649.

   (6) Ramana Rao G, Kanjilal G, Mohan KR: Analyst; 1978; 103, 993.




Mohd. Gousuddin et al                                                                                   Page 5

				
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