INTRODUCTION:

Norfluoxetine (N-FLX), is chemically 3-phenyl-3-[4-(trifluoromethyl)phenoxy],propan-1-amine which is obtained after Fluoxetine is extensively metabolized by cytochrome P450 (CYP) isoenzymes in the liver. Norfluoxetine has similar potency and selectivity with regard to the serotonin reuptake inhibiting effect of parent drug^. Fluoxetine is well absorbed after oral administration and disappears from plasma with half time of 1-3 days. and its metabolite N-FLX has a plasma half-time of 7-15 days. After administration of FLX, approximately 65% of the administered dose of this drug is recovered in urine and about 15% in faces. The therapeutic dosage for fluoxetine is 20 mg/day which is metabolized in the liver to N-FLX and other unidentified metabolites. The literature survey reveals that FLX and N-FLX have been simultaneously estimated by TLC by using derivatization reagent ¹, spectrophotometry², HPLC ^3-5,GC ^6,7 , LC-MS ^7,8 . The aim of this work was to develop simple, fast, precise and accurate colorimetric method for the estimation of N-FLX in urine.

EXPERIMENTAL:

The instrument used for the present study was PC based Jasco V-530 UV-Visible double beam Spectrophotometer with 1 cm matched pair quartz cell and spectral bandwidth of 2 nm.

Preparation of standard stock solutions:

Standard stock solution of N-FLX was prepared by dissolving 10 mg of N-FLX in 40 ml of water and then final volume was made up to 100 ml with the same to get stock solution containing 100 µg/ml of N-FLX.

Preparation of reagents:

Metanil yellow (0.02% w/v):

20 mg of metanil yellow was dissolved in 100 ml of pH2.4 buffer and it was freshly prepared each time.

Buffer solution:

Accurately weighed quantity of 4.086 gm potassium hydrogen phthalate was dissolved in 100 ml double distilled water to obtain 0.2M potassium hydrogen phthalate solution. To 25 ml of this solution, 21.1 ml of 0.2M hydrochloric acid was added and diluted to 100 ml with double distilled water to obtain pH 2.4 buffer.

Procedure for plotting calibration curve:

Appropriate aliquots of working standard solution (02-1.2ml of 100 μg/ml) of N-FLX were mixed with 2ml of dye solution (0.02% w/v) in 125 ml separating funnel and the drug-dye complex was extracted with 10 ml chloroform by equilibrating the two phase for 2 minutes. The absorbance of chloroform layer was measured at 405nm against the reagent blank in the concentration range of 2-12 μg/ml and calibration curve of N-FLX was plotted.

Fig.1. Overlain spectra of complex of N-FLX with metanil yellow.

Fig. 2. Overlain spectra of N-FLX, N-FLX– Dye complex and metanil yellow reagent.

Assay of Norfluoxetine from urine:

An amount equivalent to 10 mg of N-FLX was weighed and dissolved in 10 ml of urine. The solution was filtered through whatmann filter paper no. 41 and then final volume of the solution was made up to 100 ml with glass distilled water to get a stock solution containing 100 µg/ml of N-FLX. After appropriate dilution to 6µg/ml and addition of reagents as per procedure described for calibration curve absorbance was measured at 405 nm and the concentration of each analyte was determined with the equation generated. The statistical data obtained after replicate of nine determinations is shown in Table 2.

METHOD VALIDATION:

The proposed method was validated according to ICHQ2B guidelines for validation of analytical procedures. The reagent was not found to react with the soluble matters of the body fluids like plasma and urine. Metanil yellow can be used for the spectrophotometric determination of Fluoxetine. The intensity of absorption is more in case of FLX–Dye complex in comparison to that of N-FLX– Dye complex and also the λmax shifts from 405 nm to 410 nm Fig. 4. The results of analysis are shown in Table 3 and 4.

Table 1: Optical characteristics:

Parameters	Values for AB
λ_max	405 nm
Beer’s law limit (μg ml^-1)	2-12 µg/ml
Correlation coefficient	0.998
*Regression equation (Y)**
Slope (B)	0.0524
Intercept (A)	-0.0236

Y= A + B*C, where C is the concentration in µg ml^-1 and Y is absorbance unit.

Table 2: Results of analysis of laboratory sample from urine:

Analyte

% Concentration Estimated*

(Mean ± S.D.)

% R.S.D

N-FLX

101.17 ± 2.00

1.20

*Average of nine determinations.

Table 3: Results of specificity:

The reagent was not found to react with the soluble matters of the body fluids like plasma and urine.

Analyte (1 ml)	Absorbance at 405 nm	% R.S.D
Plasma	-0.00672*	0.45
Urine	0.00031*	0.78
N-FLX	0.40375*	0.69

* Average of six determinations.

Table 4: Method Validation Parameters:

Parameters	Norfluoxetine
Linearity range	2-12 μg/ml.
Correlation coefficient (r²)	0.998
Precision (n = 9) (%CV)	0.54
Specificity	No interference from any of the body fluid components.
Limit of detection (LOD)	0.362 μg/ml
Limit of quantitation (LOQ)	1.21 μg/ml

RESULT AND DISCUSSION:

The method employs metanil yellow reagent in presence pH 2.4 buffer to form yellow complex with N-FLX. The yellow colored drug-dye complex showed absorption maxima at 405 nm. On the basis of literature cited and the results it can be concluded that the coloured compound formed is an ion-association complex. The amino nitrogen of aliphatic chain of N-FLX is responsible for formation of the complex. The colour remained stable for more than 2 hours. The results showed that optimal pH for the quantitative reaction of metanil yellow reagent is 2.4. The concentration of N-FLX over a range of 2-12 µg/ml was found to obey Beer’s law in the stated range. The equation for straight line was found to be Abs = 0.0236 + 0.0524 * C and C = concentration of N-FLX. The correlation coefficient value for this curve was 0.998. The molar absorptivity and sandell’s sensitivity were found to be 2.2387 × 10⁴ lit mol^-1cm^-1 and 0.0154 µg/cm²/0.001 absorbance unit. After extracting the coloured components the absorbance reaches its maximum within 1 minute at room temperature and remains stable for 2 hours. Temperature of reaction, quantity, concentration and addition of reagents were optimized after several experimental trails. The optimum quantity and reagent composition for metanil yellow was found to be 2 ml and 0.02% w/v in pH 2.4 buffer. The proposed method is highly sensitive, specific and the reagent was not found to react with the soluble matters of the body fluids like plasma and urine.

Fig. 3. Calibration curve for FLX at 405 nm

Figure 4. Visible spectra of N-FLX– Dye complex and FLX– Dye complex

CONCLUSION:

The yellow colour complex formed under the above mentioned conditions and measured spectrophotometrically can be regarded as an ion-pair complex formation between the dye (metanil yellow) and the drug. The method developed is free from interference due to biological matrices and is accurate, precise and sensitive. Simplicity, ease of operation and short time required for analysis makes the method useful for routine analysis. The only limitation could be the lack of desired level of qualitative distinction between the colorimetric reaction for the metabolite and fluoxetine. The coloured compound formed leads to an over ten fold increase in the absorbtivity of the analyte and thus improves the sensitivity for detection and quantitation of N-FLX. Thus method described here is suitable for estimation of N-FLX from biological matrices and could be useful in a clinical laboratory for therapeutic drug monitoring, metabolic and bioequivalence studies.

AKNOWLEDGEMENT:

Authors are thankful to the Principal, Bharati Vidyapeeth College of Pharmacy, Kolhapur for providing the necessary facilities.

REFERENCES:

1. Oztunc A, Onal A, Erturk S .7, 7, 8, 8- Tetracynoquinodimethane as a new derivation reagent for high-performance liquid chromatography and thin-layer chromatography: rapid screening of plasma for some antidepressants. J. Chromatogr. B: Anal. Tech. Biomed. Life Sci 2002; 774: 149-155.

2. Staczewska B, Puzanowska H, Baranowska K. Investigation and analytical application of the reactions of eriochrome cyanine with fluvoxamine and Fluoxetine. J. Pharm. Biomed. Anal. 2000 ; 23: 477-481.

3. El-dawy M, Mabrouk M, El-Barbary F.Liquid chromatographic determination of Fluoxetine. J. Pharm. Biomed. Anal. 2002; 30: 561-571.

4. Lerena A, Dorado P, Berecz R, Gonzalez A, Norberto M, Rubia A, Caceres M. Determination of fluoxetine and norfluoxetine in human plasma by high-performance liquid chromatography with ultraviolet detection in psychiatric patients. J. Pharm. Biomed. Anal. 2003;783: 25-31.

5. Li K, Thompsan M, McGregor I . Rapid quantitation of fluoxetine and norfluoxetine in serum by micro-disc solid-phase extraction with high-performance liquid chromatography–ultraviolet absorbance detection. J. Chromato. B. 2004; 804: 319-326

6. Ulrich S. Direct stereoselective assay of ﬂuoxetine and norﬂuoxetine enantiomers in human plasma or serum by two-dimensional gas liquid chromatography with nitrogen–phosphorus selective detection. J. Chromatogr B. 2003;783: 481-490.

7. Addison R, Franklin M. Hooper W. Sensitive high-throughput gas chromatographic–mass spectrometric assay for ﬂuoxetine and norﬂuoxetine in human plasma and its application to pharmacokinetic studies. J Chromatogr.1998; 716: 53-160.

8. Nevado J, Salcedo A, Lierena M. Micellar electrokinetic capillary chromatography for the determination of ﬂuoxetine and its metabolite norﬂuoxetine in biological ﬂuids. J. Chromatogr. B. 2003;769: 261-268.

9. Silva M, Kelmann R, Foppa T, Cruz A, Bertol C, Sartori T, Granada A, Carmignan F, Murakami F. Thermoanalytical study of fluoxetine hydrochloride. J. Therm. Anal. Colori. 2007 ,87: 463-467.

Received on 11.02.2009 Modified on 09.04.2009

Asian J. Research Chem. 2(4):Oct.-Dec. 2009 page 390-392