Development and Validation of (HPLC) Method for Simultaneous Determination of Atomoxetine HCl & Fluoxetine HCl in their Pharmaceutical Dosage Forms of (HPLC)

pre-menstrual dysphoric disorder [4]. Literature ABSTRACT The present work described the development of a high-performance liquid chromatographic (HPLC) method for determination atomoxetine & fluoxetine in their pharmaceutical dosage forms Strattera . The analysis was achieved on Thermo Hypersil BDS C18 column (250mm x 4.6mm i.d, 5  m particle size) using mixture of 375ml of distilled water containing 0.1ml tetra-n-butylammonium hydroxide + 0.4ml triethylamine (adjust pH to 3.5 with phosphoric acid) & 625ml Acetonitrile (375: 625, v/v) as mobile phase at flow rate of 1ml/min and UV detection at 220nm. The developed method was linear over the concentration range of 1-16  g/ml (r = 0.99999) with a limit of detection 0.028 and 0.065  g/ml for atomoxetine & fluoxetine respectively and a limit of quantitation 0.085 and 0.198  g/ml for atomoxetine & fluoxetine respectively. The developed HPLC method was validated with respect to specificity, linearity, accuracy, precision, limit of detection and limit of quantitation. The statistical analysis proved that the developed method for quantification of atomoxetine & fluoxetine as bulk drug and from pharmaceutical preparation is reproducible and selective. The proposed HPLC method can be used for the quality control of formulated products containing atomoxetine


Materials and Reagents
Acetonitrile was of HPLC grade, phosphoric acid, tetra-nbutylammonium hydroxide and triethylamine were analytical grades. a) Authentic: atomoxetine HCl, kindly supplied by Lilly.

HPLC Conditions
The HPLC separation and quantitation were achieved on:

Standard Solution
Stock solution of atomoxetine (1mg/ml) was prepared by dissolving a weight of atomoxetine HCL which is equivalent to 100 mg of atomoxetine base in 100ml of mobile phase. Stock solution of fluoxetine (1mg/ml) was prepared by dissolving a weight of fluoxetine HCl which is equivalent to 100 mg of fluoxetine base in 100ml of mobile phase.

Preparation of Calibration Curve
The working solution was prepared by further dilution of the stock standard solution with the mobile phase to reach the concentration range of 1-16µg/ml for both atomoxetine & fluoxetine.
Triplicate 20µl injections were made for each concentration and chromatographed under the specified chromatographic conditions described previously. The peak area values were plotted against corresponding concentrations, linear relationship was obtained.

Pharmaceutical formulation preparation
The content of ten capsules of both Strattera® & Prozac®

System Suitability
The system suitability parameters including capacity factor (k′), selectivity (α), resolution (R s ), tailing factor (T) and theoretical plate (N) listed in Table 1. All parameters were satisfactory with good specificity for the stability assessment of atomoxetine & fluoxetine.

Application to Pharmaceutical Formulation
The proposed method was successfully applied to determine atomoxetine and fluoxetine in their dosage forms (Strattera® and Prozac®) capsules. Five replicates determination were made.
Satisfactory results were obtained for atomoxetine and fluoxetine in good agreement with label claims ( Table 2). The results obtained were compared statistically by Student′s t -test (for accuracy) and variance ratio F -test (for precision) with the reported methods [32,33]. The results in Table 3 showed that the t and F values were smaller than the tabulated values indicating that there was no significant difference between the proposed and reported methods.

Results and Discussion
After the trials of various mobile phase systems such as 0.1%

Precision
To prove the validity and applicability of the proposed method and reproducibility of the results mentioned, three concentrations of the cited drugs were carried out. Table 5 show the values of Intraday and Inter-day relative standard deviation (RSD %) for different concentrations.

Range
The calibration range was established through consideration of the practical range necessary, according to the drug concentration present in the pharmaceutical product, to give accurate, precise and linear results. The calibration range of the proposed method is given in Table 4.
where C is the concentration of compound in g /ml and Y is the peak area.

Limit of Detection and Quantitation
According to ICH recommendation

Accuracy
This study was performed by adding known amounts of the studied drugs compound to known concentration of the commercial pharmaceutical capsules (standard addition method). The resulting mixtures were analyzed and the results obtained were compared with the expected results (Table 6) suggested the good accuracy of the proposed methods.

Robustness
Robustness is the measure of capacity of analytical methods to remain unaffected by small but deliberating variations of the operation parameters. Variation of the pH of (aqueous triethylamine and tetra-n-butylammonium hydroxide) of the mobile phase by ± 0.2, organic solvent strength of the mobile phase by ± 2 % and detector wavelength by ± 2nm did not have significant effect on chromatographic resolution of the HPLC method.

Solution stability
Solution of studied drug in mobile phase exhibit no absorbance or chromatographic change for 2weeks when kept at room temperature, and for 3 months when stored in the refrigerator at

Conclusion
The proposed HPLC method provide simple, accurate, and reproducible quantitative analysis for determination of atomoxetine and fluoxetine in bulk and in pharmaceutical dosage forms without any interference from the excipients. The method was completely validated. Owing to its sensitivity, simplicity and short analysis time, it is suitable for routine analysis in quality control laboratories to assay the two drugs.