A new spectrophotometric method for the determination of fluvoxamine maleate in pure form and in pharmaceutical formulation

Article history: Received on: 17/12/2014 Revised on: 17/01/2015 Accepted on: 04/02/2015 Available online: 27/02/2015 A simple, accurate and highly sensitive spectrophotometric methods are proposed for the rapid and accurate determination of fluvoxamine maleate (FXA) using bromocressol green (BCG), methyl orange (MO) and bromothymol blue (BTB). The developed methods involve formation of stable yellow colored chloroform extractable ion-associate complexes of the amino derivative (basic nitrogen) of the FXA with three sulphonphthalein acid dyes, namely; BCG, MO and BTB, in potassium hydrogen phthalate buffer pH 3.3, 3.6 and 3.4 respectively. The ion-associates exhibit absorption maxima at 420, 420 and 410 nm for BCG, MO and BTB, respectively. FXA can be determined up to 2.0–16, 2.0–15 and 2.0–20 μgmL for BCG, MO and BTB, respectively. The effect of optimum conditions via pH on the ion pair formation, reagent concentration, time and temperature, and solvent was studied. The composition of the ion pairs was found 1:1 by Job’s method. The low relative standard deviation values indicate good precision and high recovery values. These methods have been successfully applied for the assay of FXA in pure form and in pharmaceutical formulations and the results are in good agreement with those obtained by the official method.

No official (pharmacopoeia) method has been found for the assay of FXA in its formulations.However, many studies have been reported for the determination of FXA in pharmaceuticals including visible spectroscopy (Kishore et al., 2010, Jat et al., 2010), HPLC (Tadashi OHKUBO et al., 2003, Ulu et al., . 2007), fluorimetriy (EI-Enany, 2007), capillary gas chromatography (Berzas Nervado et al., 2000), capillary electrophoresis (Nevado et al., 2000), gas Chromatography-Mass Spectrometry (Maurer et al., 2000), solid-phase microextraction gas chromatography-mass spectrometry (Petinal et al., 2005).No extractive spectrophotometric methods for analysis of FXA in pure form and in pharmaceutical formulation.The present study has shown the development of an accurate, reproducible, fast and adequately sensitive extractive spectrophotometric method based on an ion-pair complex formed between FXA and an anionic dye, BCG, MO and BTB (Nafisur et al., 2000, Nafisur et al., 2004, Gowda et al., 2001, Sevgi et al 2007, Faten et al., 2006).An important advantage of the extractive spectrophotometric method is that it can be applied to the determination of individual compounds in a multi-component mixture.Unlike gas chromatographic (GC) and HPLC procedures, the instrument is relatively simple and affordable.The sensitivity (in terms of molar absorptivity) and precision (in terms of relative standard deviation, RSD) of the methods are suitable for the determination of the drug in pure and mixed dosage forms.
The reagents utilized in the proposed method are relatively cheap and readily available, and the procedure does not involve any critical reaction condition or tedious sample preparation.These advantages, coupled with reasonable accuracy and precision, render the methods suitable for routine quality control.The proposed method was applied to the determination of FXA in pharmaceutical formulations.
No interference was observed in the assay of FXA from common excipients in levels found in pharmaceutical formulations.The proposed method was validated by statistical data (Barary et al., 1991, Abdelmageed et al., 1993, Botello et al., 1995, Sastry et al., 1995).

Apparatus
A Shimadzu UV-160A UV-Vis spectrophotometer with 1 cm glass cells was used.UV-Vis spectra were automatically obtained by Shimadzu UV-160A system software.

Chemicals
FXA and its tablets were procured from Jazz Pharmaceuticals, Inc. (US).Methyl orange (MO), bromothymol blue (BTB) and bromocresol green (BCG) were purchased from Merck (Darmstadt, Germany).Analytical grade chloroform was used for extraction.Solvents and other chemicals were of analytical grade (Merck, Darmstadt, Germany).

Standard solution
Standard solution 100 µg mL -1 of FXA was prepared by dissolving 10 mg of pure drug (pharmaceutical grade) in the least amount of distilled water and made up to 100 mL in measuring flask with distilled water.The solution remained stable for 1 month when kept refrigerated.
Potassium hydrogen Phthalate-HCl buffer (pH = 3.0-3.7)was prepared by dissolving 1.020 g of potassium hydrogen phthalate in water and completed to 50 mL with distilled water and adjusting pH by addition of 0.1M HCl solution.Freshly prepared solutions were always employed.

General analytical procedure for the analysis of bulk drug
Aliquots of (0.2-1.6 mL of BCG; 0.2-1.5 mL of MO; 0.2-2.0mL of BTB) the working drug solutions (100µg/mL) were transferred to 10 mL measuring flasks and added 3.0 mL potassium hydrogen phthalate buffers of pH 3.3, 3.6 and 3.4 using BCG, MO and BTB, respectively, then add 1.0 mL of BCG, MO and BTB.The mixture was extracted twice with 5.0 mL chloroform by shaking for 2.0 min and then allowed to stand for clear separation of the two phases and the chloroform layer was passed through anhydrous sodium sulphate.The absorbance of the yellow colored complexes was measured at 420, 420 and 410 nm for BCG, MO and BTB, respectively, against corresponding reagent blank similarly prepared.All measurements were made at room temperature (25±2°C).The procedures were repeated for other analyte aliquots and calibration plots were drawn to calculate the amount of drug in unknown analyte samples.

General analytical procedure for the analysis of tablets
The average tablet weight was calculated from the contents of 20 tablets that had been finely powdered and weighed.A portion of this powder, equivalent to 50 mg of FXA, was accurately weighed.The samples were shaken with 25mL of distilled water.The mixtures were then introduced into an ultrasonic bath for 25 min and diluted with distilled water in a 50 mL calibrated flask and filtered.An aliquot of 10 mL of the filtrate was diluted to 100 mL to prepare of working sample solutions (100µg mL -1 ).An appropriate volume of 100 µg mL -1 sample solution (0.2-1.6 mL of BCG; 0.2-1.5 mL of MO; 0.2-2.0mL of BTB) was added and mixed.This solution was analyzed as in the general analytical procedure for the bulk drug.The amount of FXA per tablet was calculated using the calibration curve method.The proposed method was applied to the determination of FXA in tablets.

Absorption spectra
The nitrogenous drugs are present in positively charged protonated forms and anionic dyes of sulphonpthalein group present mainly in anionic form at pH≥3.So when treated with an acid dye at pH 3.3, 3.6 and 3.4 of potassium hydrogen phthalate buffer using BCP, MO and BTB, respectively, a yellow ion-pair complex which is extracted with chloroform is formed.The absorption spectra of the ion-pair complexes, which were formed between FXA and each of BCG, MO and BTB were measured in the range 400-500 nm against the blank solution and shown in Figure 2. The ion-pair complexes show maximum absorbance at 420, 420 and 410 nm for BCG, MO and BTB, respectively.The optimum reaction conditions for determination of the ion-pair complexes were established.Then linearity, accuracy, precision, sensitivity, and stability of proposed methods were described and these developed methods applied to pharmaceutical preparations as tablets and obtained results evaluated statistically.

Optimum reaction conditions for complex formation
The reaction conditions of the method were carefully studied to achieve complete reaction, highest sensitivity and maximum absorbance.Reaction conditions for the formation of the ion-pair complex were optimized by studying preliminary experiments involving pH of buffer, type of organic solvent, volume of the dye and shaking time for the extraction of ion-pair complexes.

Effect of time and temperature
The optimum reaction time was investigated from 0.5 to 4.0 min by following the color development at ambient temperature (25±2 °C).Complete color intensity was attained after 2.0 min of mixing for all complexes (Figure 3).Raising the temperature up to 30 °C has no effect on the absorbance of the formed complexes, whereas above 30 °C, the absorbance starts to decay.The absorbance remains stable for at least 24 h.

Effects of pH on the ion-pair formation
The effect of pH was studied by extracting the colored complexes in the presence of various buffers such as KCl-HCl (pH = 1.5-5.0),NaOAc-HCl (pH = 1.5-5.0)and potassium hydrogen phthalate-HCl (pH = 1.5-5.0).
It was noticed that the maximum color intensity and highest absorbance value were observed in potassium hydrogen phthalate-HCl buffer of pH 3.3, 3.6 and 3.4 for BCG, MO and BTB in addition to the stability of the color without affecting the absorbance at pH 3.3, 3.6 and 3.4 for BCG, MO and BTB methods, respectively (Figure 4).Further, 3.0 mL potassium hydrogen phthalate buffers gave maximum absorbance and reproducible results.

Effect of dye Concentration
The effect of the concentration of the dye on the intensity of the color developed at the selected wavelength and constant drug concentration (10µg mL -1 ) was tested using different volumes of MO, BCG and BTB (0.5 -2.0 mL).It was observed that 1.0 mL of 0.2% (w/v) dye was necessary for maximum color development of the ion-pair complex.Above this volume, the absorbance remained constant (Figure 5).

Effect solvents
The effect of several organic solvents viz., chloroform, carbon tetrachloride, ethyl acetate, xylene, diethyl ether, butyl acetate, toluene, dichloromethane and chlorobenzene were tried for effective extraction of the colored species from aqueous phase.Chloroform was found to be the most suitable solvent for extraction of colored complex for all reagents, yielding maximum absorbance intensity and considerably lower extraction ability for the reagent blank and was also observed that only double extraction was adequate to achieve a quantitative recovery of the complex and the shortest time to reach the equilibrium between both phases.

Stoichiometric Ratio
Job's Method (Inczedy, 1976) of Continuous Variation of equimolar solutions was employed.A 1.0 x 10 −3 M standard solution of FXA and 1.0 x 10 −3 solution of BCG, MO and BTB were used.A series of solutions was prepared in which the total volume of drug and reagent was kept at 10 mL.The absorbance was measured at 420, 420 and 410 for BCG, MO and BTB, respectively.The molar ratio of the reagent (drug: dye) in the ionpair complex was determined by the Method of Continuous Variation (Job's Method) and the results indicated that 1: 1 (drug: dye) was the optimum ratio for the reaction (Figure 6).

Linearity and range
Beer's law range, molar absorptivity, Sandell's sensitivity, regression equation and correlation coefficient determined for each method are given in Table 1.A linear relationship was found between the absorbance at λ max and the concentration of the drug in the range 2.0-16 μg mL −1 for BCG, 2.0-15 μg mL −1 for MO and 2.0-20 μg mL −1 for BTB method in the final measured volume of 10 ml.Regression analysis of the Beer's law plots at λ max reveals a good correlation.The graphs show negligible intercept and are described by the regression equation, A = mC + b (where A is the absorbance of 1 cm layer, m is the slope, b is the intercept and C is the concentration of the measured solution in μg ml −1 ) obtained by the least-squares method (Miller et al., 1993).The high molar absorptivities of the resulting colored complexes indicate the high sensitivity of the methods.Calibration curve for three proposed methods are shown in Figure 7.

Validation of the methods
Samples of pure FXA were prepared and tested at four levels of drug using the proposed procedures.The complete set of validation assays was performed for drug, determined by the proposed methods.The results obtained for pure drugs are given in Table 2.The precision and accuracy of the methods were tested by analyzing six replicates of the drug.The standard deviation, relative standard deviation, recovery and 95% confidence limits of different amounts tested were determined from the calibration curve, as recorded in Table 2.The accuracy of the method is indicated by the excellent recovery (100.00-100.16%),(99.77-100.33%)and (99.83-100.66%)for MO, BCG andBTB methods, respectively (ICH, 1996, Shabir, 2003).

Tablets analysis
The proposed methods were applied to the determination of FXA in commercial tablets.The accuracy of the proposed methods is evaluated by applying standard addition technique, in which variable amounts of the drug were added to the previously analyzed portion of pharmaceutical preparations and the results are tabulated in Table 3. Six replicates determinations were made.Satisfactory results were obtained for drug and were in a good agreement with the label claims (Table 3).The results were reproducible with low R.S.D. values.The average percent recoveries obtained were quantitative (99.50-100.0%for MO, 100.33-101.0%for BCG and 100.50-101.5% for BTB), indicating good accuracy of the methods.The results of analysis of the commercial tablets and the recovery study of drug suggested that there is no interference from any excipients (such as starch, lactose, titanium dioxide, and magnesium stearate), which are present in tablets.

CONCLUSIONS
The three proposed methods (MO, BCG and BTB) can be used for determination of FXA in tablets.The three methods are rapid, simple and have great sensitivity and accuracy.Proposed methods make use of simple reagents, which an ordinary analytical laboratory can afford.Methods are sufficiently sensitive to permit determination even down to 2.0 μg mL −1 .The three proposed methods are suitable for routine determination of FXA in its formulations.The commonly used additives such as starch, lactose, titanium dioxide, and magnesium stearate do not interfere with the assay procedures.

ACKNOWLEDGEMENT
We are very much grateful to Principal, Ratnam Institute of Pharmacy, Andhra Pradesh for providing necessary facilities to conduct the research work.
Barary MH, Wahbi AM.Spectrophotometric determination of chlorpheniramine maleate and chlorphenoxamine hydrochloride each in

Fig. 3 :
Fig. 3: Effect of shaking time on the ion-pair complexes.

Fig. 4 :
Fig. 4: Effect of pH of potassium hydrogen phthalate buffer solution on the absorbance of FXA (10µg/mL).

a
Mean of six determination.bConfidence limit at 95% confidence level and five degrees of freedom.

Table 1 :
Statistical data of the regression equations for determination of BTB, MO, BCG.

Table 3 :
Evaluation of accuracy and precision of FXA tablets by standard addition method (n= 6).number of determination, R.S.D.%, percentage relative standard deviation; Er %, percentage relative error.Confidence limit at 95% confidence level and five degrees of freedom.
a Mean of six determination.b