INTRODUCTION:

Fluoxetine is chemically known as N-methyl-3-[(α,α,α- Trifluro-P-tolyl) oxy] propylamine. (Figure 1). It acts as a selective serotonin reuptake inhibitor and used as Anti -depressant. It has basically toluene derivative¹. The estimation of fluoxetine hydrochloride by Gas chromatographic-mass spectrometric method², high performance liquid chromatography [HPLC]³, high performance thin layer chromatography [HPTLC]⁴, Micellar electrokinetic capillary chromatography⁵is reported in literature.

The objective of this work is to report a simple, accurate, cost effective and reproducible UV method for estimation of fluoxetine hydrochloride.

MATERIALS AND METHOD:

Instrument and materials:

Instrument used was Jasco UV V-630 Spectrophotometer. Fluoxetine Hydrochloride pure drug was procured from Wockhardt Pharma. Ltd. Aurangabad as gift sample and was used without further purification. All chemicals and reagents used were of analytical grade.

Method:

Selection of Solvent

A main criterion for solvent selection was solubility. Literature surveys have revealed that UV Spectrophotometric method for determination of Fluoxetine has been done by K. Sujatha et al by using distilled water as a solvent⁶. The solubility of Fluoxetine in methanol is more as compared to that of water hence methanol was selected as a solvent.

Preparation of standard stock solution

Standard drug solution of fluoxetine hydrochloride was prepared by dissolving 25mg fluoxetine hydrochloride in 10ml methanol and transferred it to 25ml volumetric flask and volume was made upto mark with methanol to obtain stock solution of 1000μg/ml concentration.

Preparation of working stock solution

5.0 mL of the above stock solution was diluted to 100.0 mL in 100 mL volumetric flask with distilled water. Then a solution having concentration about 20.0 µg was prepared by diluting 2.0 mL of the working stock solution to 10.0 mL with distilled water and this solution was scanned from 200-400 nm.

Spectra stability (24 hrs)

Stability of this UV spectrum was determined over a period of 24 hrs, which shows negligible change in its shape and λ_max^.

Figure 1- Chemical structure of Fluoxetine Hydrochloride

Selection of Detection wavelength:

A solution having concentration about 20.0 µg/mL was prepared by diluting 2.0 mL of the working stock solution to 10.0 mL with distilled water and this solution was scanned from 200-400 nm. The λ_max value was found to be 226.0 nm. (Figure 2)

Preparation of calibration curve:

Calibration curve was prepared in methanol at λmax 226 nm using Jasco UV-visible spectrophotometer. For this stock solution of 1000 µg/ml was prepared. Serial dilution of 5, 10, 20, 30, 40 and 50µg/ml were prepared and absorbance was taken at λ max 226 nm. The calibration curve was plotted (n=5) (Figure 3).

Figure 2 :Overlain spectra of 24 hrs stability of fluoxetine hydrochloride

Application of proposed method for determination in capsules:

Contents of twenty capsules were weighed. The amount of capsule powder equivalent to 25 mg of fluoxetine hydrochloride was weighed accurately and transfered to 10 ml methanol and kept for 15 min with frequent shaking and volume was made up to 25ml mark with methanol. The solution was then filtered through whatmann filter paper # 41. This filtrate was diluted suitably with solvent to get the solution of 1000µg/mL concentration. This solution was used as stock solution. The working solution of drug (20μg/ml) was prepared from standard stock solution in water. The absorbance of working solution was measured against blank and amount of fluoxetine was calculated from the calibration curve. The readings were taken in triplicate. The drug content of the preparation was calculated using standard calibration curve.

RESULTS AND DISCUSSION:

Validation:

Precision:

Assay of method precision (intra-day precision) was evaluated by carrying out three independent assays of fluoxetine hydrochloride. The intermediate precision (inter-day precision) of the method was also evaluated using two different analysts, systems and different days in the same laboratory. The relative standard deviation (RSD) and assay values obtained by two analysts were 0.1257, 99 .89and 0.3006, 100.39 respectively (Table no.2).

Table no. 1–Validation parameters

Sr. no.	Parameter	Result
1	Absorption maxima (nm)	226
2	Linearity Range(µg/mL)	5-50
3	Standard regression equation	y =0.027x+ 0.030
4	Correlation Coefficient (r2 )	0.999
5	Accuracy (% recovery ±SD)	100.01± 0.445
6	Precision	99.89% (intra-day), 100.39% (inter-day)
7	Specificity	Method is specific
8	Robustness	Method is robust

Table No.2- Determination of Precision

Sample number	Assay of fluoxetine as % of labeled amount
	Analyst-I (Intra-day precision)	Analyst-II (Inter-day precision)
1	99.81	100.83
2	99.93	100.54
3	99.93	99.99
4	99.69	100.52
5	100.03	100.15
6	99.99	100.32
Mean	99.89	100.39
SD	0.1256	0.3018
% RSD	0.1257	0.3006

Accuracy (Recovery Test):

Accuracy of the method was studied by recovery experiments. The recovery experiments were performed by adding known amounts to capsule powder. The recovery was performed at five levels, 80, 90, 100, 110 and 120% of fluoxetine hydrochloride standard concentration. The recovery samples were prepared in afore mentioned procedure. Three samples were prepared for each recovery level. The solutions were then analyzed, and the percentage recoveries were calculated from the calibration curve. The recovery values for fluoxetine hydrochloride ranged from 100.01± 0.445 (Table no.3).

Specificity:

Specificity studies were performed by weighing seven samples of capsule powder and transferred to 25.0 mL volumetric flasks. All these samples were stored for 24 hrs under following different conditions.

1. Normal

2. At 50⁰C after addition of 1.0 mL of 0.1N NaOH (alkali)

Table No.3- Determination of Accuracy by percentage recovery method

Ingredient	Tablet amount (µg/ml)	Level of addition (%)	Amount added (mg)	Drug found (µg/ml)	% Recovery
Fluoxetine hydrochloride*	17.5	80	2.5	2.5008	100.03
	17.5	90	5	5.0070	100.14
	17.5	100	7.5	7.5502	100.66
	17.5	110	10	9.97	99.69
	17.5	120	12.5	12.4374	99.51
Mean SD %RSD					100.01 0.445 0.444

* Fluoxetine hydrochloride having brand name Fludac-20

Table no.4 –Determination of specificity

Sr. no.	Treatment	Wt.of capsule powder eq.to bulk drug	% Labeled claim	SD	%RSD
1	Normal	23.99	99.93	0.2685	0.2687
2	Acid	24.0	99.60	1.041	1.2763
3	Alkali	24.0	99.32	1.373	1.800
4	Peroxide	24.4	96.17	1.401	1.456
5	Heat	24.2	99.03	0.950	0.9593
6	UV	24.1	99.99	0.0964	0.096
7	75% RH	24.0	100.83	0.4572	0.4534

Table no.5 – Determination of ruggedness

Sr. no.	Analyst	% Labeled claim*	Days	% Labeled claim*	Instruments	% Labeled claim*
1	I	99.80	I	100.46	I	99.93
2	II	100.32	II	99.93	II	100.19
3	III	99.99	III	100.14	_	_
Mean SD % RSD		100.03 0.2631 0.2630	Mean SD % RSD	100.17 0.2668 0.2663	Mean SD % RSD	100.06 0.1838 0.1836

*Indicates mean of three determinations

3. At 50⁰C after addition of 1.0 mL of 0.1N HCL (Acid)

4. At 50⁰C after addition of 1.0mL of 3% H₂O₂ (Oxide)

5. At 60⁰C (Heat)

6. In UV chamber

7. At 75% RH at room temperature

The solutions of samples were made and analysed. The % labelled claim were calculated. The results shown in Table no.4

Figure 3 – Calibration curve

Linearity:

The linearity of the response of the drug was found to be between 5-50µg/ml concentrations. The calibration graphs were obtained by plotting the absorbance versus the concentration data and were treated by linear regression analysis (Table no.1). The equation of the calibration curve for fluoxetine hydrochloride obtained y = 0.027+0.030, the calibration curve was found to be linear in the aforementioned concentrations. The correlation coefficient (r²) of determination was 0.999.

Table no. 6 –Determination of robustness

Sr. no.	Wavelength	%Labelled claim*	pH	%Labelled claim*
1	224	101.11	3.5	100.03
2	226	99.99	4.0	100.32
3	228	101.33	4.5	100.52
Mean SD % RSD		100.81 0.7186 0.7128	Mean SD % RSD	100.29 0.2463 0.2455

*Indicates mean of three determinations

Ruggedness:

The ruggedness studies were performed by analyzing the capsule powder samples using proposed method by different analysts, different instruments and on different days. Results are as shown in Table no.5

Robustness:

Repeatability is based on the results of the method operating over short time interval under same conditions. The low RSD values of intra-day precision (Table no.2), recovery (Table no.3), showed high repeatability. Making deliberate small changes in pHof solvent and wavelength used tested the robustness of method (Table no.6).

CONCLUSION:

The proposed UV method is simple, accurate, precise, specific and highly sensitive; developed and validated for the determination of fluoxetine hydrochloride in bulk and in dosage form. The method is economical, rapid and do not require any sophisticated apparatus in contrast to chromatographic methods. Hence, the proposed method can be successfully used for routine quality control analysis of drug in marketed preparations.

AKNOWLEDGEMENT:

Authors are very much thankful to the Chairman, Mrs. Fatma Rafiq Zakaria, Maulana Azad Educational Trust, for providing necessary facilities for the project work. We are thankful to Wockhardt Pharma. Ltd. Aurangabad for providing the gift sample of Fluoxetine Hydrochloride.

REFERENCES:

1. The Merck Index,13^th edition, Merck Research Laboratories, Merck and Co., Inc., Whitehouse Station, NJ. 2001: pp.741

2. Addison RS, Franklin ME, Hooper WD. Sensitive high-Throughput Gas chromatographic-Mass Spectrometric assay for fluoxetine and norfluoxetine in human plasma and its application to pharmacokinetic studies. J Chromatogr B: Biomed and Appli. 1998;716(1-2):153-160

3. Alvarez J, Bothua D, Collignon I et al. Determination of fluoxetine and its metabolite norfluoxetine in serum and brain areas using high performance liquid chromatography with ultra violet detection. J Chromatogr B. 1998;707:175-180

4. Jagadeeswaran M, Mahibalan S, Gopal N. Estimation of fluoxetine in capsule dosage form by high performance thin layer chromatographic method. Inter J Pharmacy and Pharma Sci. 2009;1(2):71-73

5. Labat L, Deveaux M. Seperation of new antidepressants and their metabolites by micellar electrokinetic capillary chromatography. J Chromatogr B:Analytical Tech in the Biomed and Life Sci. 2002;773(1):17-23

6. Sujatha K, Chitra K, Ashok Kumar et al. Spectrophotometric determination of fluoxetine hydrochloride. Ind J Pharma Sci. 2004;8(6):457-458

Received on 25.12.2009 Modified on 19.01.2010

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 545-548