A HIGH PERFORMANCE LIQUID CHOMATOGRAPHIC ASSAY FOR FORMOTEROL IN BULK DRUG AND ITS PHARMACEUTICAL DOSAGE FORMS

A simple, rapid and reproducible high performance reverse phase liquid chromatographic method has been developed for quantitative estimation of Formetrol in rotacap using a C-8 column and UV detection at 254nm. The isocratic elution was used to quantify the analyte. The samples were chromatographed on C-8 column and the mobile phase was ammonium acetate: Acetonitrile (80:20, w/v) was pumped at 1 mL/min. The method was linear between 30-180µg mL -1 , statistically validated for its linearity, precission and accuracy. The intra-and - inter day variation was found to be less than 1% showing high precission of the assay method. It was found that the excipients in the commercial rotocaps did not interfere with the method.


Introduction
Formoterol 1-2 is a long-acting beta2agonist 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. 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  was used. A mobile phase contains ammonium acetate and Acetonitrile a (80:20, w/v) pumped at a flow rate 1.0mL/min through a ACE C-8 column (4.6x150mm, 5µm). The injection volume 10µL and peaks were detected at 254nm. The total run time for assay was 8min. assay was done at ambient temperature. The HPLC system was equipped with the software Empower-2.

Standard solution:
The standard Formoterol fumarate solution was prepared by accurately weighing 30mg of which Formoterol was transferred to a 100ml volumetric flask, add about 70ml of diluents (ammonium acetate: Acetonitrile,80:20 w/v) and sonicate to dissolve it completely for 20min and make up to the mark with the diluents. From this stock take 1ml and make up to the mark with diluents to get final concentration of 120µg mL -1 .A typical chromatogram was shown in

Sample preparation:
For quantitative determination of Formoterol in rotacap, 100 rotacap were weighed to be obtaining the average weight remove the capsules shells and transfer the content of test substance 10ml volumetric flask; add about 1mL dilute hydrochloric acid and sonicate to dissolve it completely for 20min and make up to the mark with the diluent. Mix well and filter through 0.45µm filter paper the sample solution was injected five times at 10 µg mL -1 . A typical chromatogram was shown in Fig 4.into the columns at a flow rate 1.0 mL/min, the peak area of the sample drug were calculated.

Assay:
Formoterol in rotacap, 100 rotacap were weighed to be obtaining the average weight remove the capsules shells and transfer the content of test substance 10ml volumetric flask; add about 1mL dilute hydrochloric acid and sonicate to dissolve it completely for 20min and make up to the mark with the diluent. Mix well and filter through 0.45µm filter paper. This solution (10 µL) was injected 5 times in to the column. The mean values of peak areas of five such determinations were calculated and the content in the rotacaps was quantified using regression equation obtained above. The same procedure was followed for the estimation of Formoterol in other commercially available rotacaps dosage forms.

Results and discussion
Formoterol can be analyzed by using proposed HPLC method both as pure drug and pharmaceutical dosage forms. The aim of the study was to develop a chromatographic procedure for the analysis of Formoterol in pure drug and pharmaceutical dosage forms. Ammonium acetate and Acetonitrile was chosen as the organic solvents because of Formoterol solubility and reversed phase chromatography was used to perform the assay. The column used in this research (150mm x 4.6mm) showed a high resolution separation. Moreover the mobile phase was modified. The influence of different organic solvents in the mobile phase was studied aiming to establish the best experimental conditions for the analysis of Formoterol.
The UV absorption spectrum of Formoterol was found at 254nm. The retention time for Formoterol was 4.66min for a run time of 8min. Each of the samples was injected 5 times and same retention times were observed in all cases. The system was validated for system suitability, precision, accuracy, linearity, ruggedness and robustness.
Linearity was obtained in the concentration range of 30-180µg mL -1 . A total 10µL volume of each sample was injected into the column. The peak areas were shown in Table 2. The standard calibration curve of Formoterol was constructed by plotting the peak area versus the respective drug concentration shown in Fig 4. The correlation coefficient was 0.9993 indicating excellent linearity. The regression equation for Formoterol was calculated by the equation: y =5125+9136 C, where C and y are concentration and area respectively.
The precision was determined for intra-and inter day on three different days. Precision was assessed by performing replicate analyses of quality control samples against calibration standard. The results obtained for Inter and Intra day variations were shown in Table 2 Recovery studies were carried out at three different levels 50%, 100%, 150%. The results were shown in Table 3. About 98.7% -100.1% Formoterol could be recovered from the preanalysed sample indicating the high accuracy of the proposed HPLC method. No interfering peaks were found in the chromatograms due to rotocaps excipients. Formoterol was stable during all the procedure.
The robustness of the method was determined by carrying out the analysis under conditions during which the flow rate, temperature and different columns at intra-and inter day variation, there are insignificant variations were observed in those above analyses shown in Table 4. The drug content in the rotocaps was quantified using the proposed method. The mean content of Formoterol in commercial rotocaps dosage form is shown in Table 5.

Conclusion
The proposed reverse phase HPLC method was found to be simple, precise, highly accurate, specific and less time consuming for determination of Formetrol in pure drug and rotacaps Method validation yields good results and presented good precision and accuracy. It was also found that the excipients in the commercial tablet dosage form did not interfere with the assay. Therefore, the proposed HPLC method is precise and accurate, and can be used for quantitative estimation in of Formetrol rotocaps.