Complexation and oxidation of Flutamide with Fe³⁺ and 1,10-phenanthroline: Few analytical applications

1 Introduction

Flutamide (FLD), chemically known as 2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]-propanamide (Fig. 1) is widely used as a antineoplastic (hormonal) and antiandrogen drug (Budavari et al., 1989; Brogden and Chrisp, 1991; McLeod, 1993; APFH, 2004). It is a powerful nonsteroidal androgen antagonist, commonly used to treat prostate cancer, is believed to block androgen receptor sites. Flutamide and its primary hydroxyl metabolites decrease metabolism of C-19 steroids by the cytochrome P-450 system at the target cells in the secondary sex organ (Osol and Hoover, 1996). It has been recently included in the European Pharmacopoeia which involves a liquid chromatographic method for the determination of the drug (EP, 2005).

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Fig. 1 Chemical structure of FLD.

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Several methods have been reported for the assay and the determination of FLD in pure form, in biological and pharmaceutical preparation methods, including spectrophotometric (Nagaraja et al., 2002; Reddy et al., 2003; Rangappa et al., 2000; Hemavathi et al., 2012), spectrofluorometric (Smith et al., 2008), Voltammetric (Reddy et al., 2011; Hammam et al., 2004) and HPLC (Filip et al., 2007; Niopas and Daftsios, 2001; Smith et al., 2009; Salgado et al., 2005).

The spectrophotometric method includes the formation of yellow color with hydrochloric acid with λ_max of 380 nm which suffers from various drawbacks Zarapkar et al., 1996). The recently reported three spectrophotometric methods for the determination of FLD using promethazine hydrochloride or resorcinol or (NEDA) N-(1-naphthyl) ethylenediamine dihydrochloride (Rangappa et al., 2000) have been reported. Two newly spectrophotometric methods published make use of p-dimethylamino cinnamaldehyde, NEDA, chromotropic acid and resorcinol (Reddy et al., 2001).

We have recently reported a detailed study of the interactions of copper(II) with flutamide in the presence of anionic surfactants by resonance rayleigh scattering, second-order scattering and frequency doubling scattering (Mohd et al., 2011).

Although highly expensive, sophisticated and time consuming techniques are available, we are reporting in this paper, a simple, precise and accurate spectrophotometric method for rapid determination of FLD. The method is based on the complexation and oxidation of the drug by Fe³⁺ ion in the presence and absence of phenanthroline. Under both the conditions the reactions were monitored spectrophotometrically by measuring the increase in absorbance at 510 nm.

2 Experimental

3 Results and discussion

The methanolic solutions of FLD and FLD–Fe³⁺ complex were found to absorbance at maximum wavelength at 210, 230, 270 and 510 nm, respectively (Figs. 2 and 3). FLD–Fe³⁺ was allowed to react with 1.10-phenanthroline resulting in the formation of red colored product and obtain the maximum absorbance at 510 nm (Fig. 3).

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Fig. 2 Absorbtion spectra of flutamide.

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Fig. 3 Absorption spectra of (A) mixture condition for FLD = 0.8 μg mL⁻¹, Fe³⁺ = 2.2 × 10⁻³ mol L⁻¹,pH = 3.5 at T = 25 °C (B) FLD = 1.0 μg mL⁻¹, 1,10-phenanthroline = 1.30 × 10⁻³ mol L⁻¹, Fe³⁺ = 2.2 × 10⁻³ mol L⁻¹ and pH = 5.0 at 50 °C.

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The FLD reacted with Fe³⁺ at pH 3.5 at room temperature, forming a colored complex according to the equation given below. However, in acetate buffer of pH 5 at 50 °C, Fe³⁺ is reduced by FLD in Eq. (2) and subsequently forms a colored complex with 1,10-phenanthroline which is called ferroin in Eq. (3) (Marczenko, 1986). The mechanism of ferroin is shown in Scheme 1.

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Scheme 1 A detailed mechanistic scheme for the complexation and oxidation of FLD with Fe3⁺ and 1,10-phenanthroline.

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4 Effect of pH

The effect of pH on the reaction of FLD with Fe³⁺ was studied in the range of 2–7. In the first procedure only FLD forms complex with Fe³⁺. The absorbance of FLD with pH increases and attains a maximum at pH 3.5. Since at higher pH absorption decreases an optimum pH of 3.5 was chosen for the first set. Fig. 4 shows the effect of pH on the oxidation of FLD by Fe³⁺ in the presence of 1,10-phenanthroline under the second set of conditions. Since in this case the absorbance attains a maximum at pH 5 the subsequent experiments were done at this pH.

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Fig. 4 Effect of pH on the complex formation reaction and oxidation of FLD (A) FLD = 0.8 μg mL⁻¹, Fe³⁺ = 2.2 × 10⁻³ mol L⁻¹, at 25 °C (B) FLD = 1.0 μg mL⁻¹, 1,10-phenanthroline = 1.30 × 10⁻³ mol L⁻¹ and Fe³⁺= 2.2 × 10⁻³ mol L⁻¹ at 50 °C.

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5 Effect of the concentration of Fe³⁺

The effect of Fe³⁺ concentration on the complex formation in Fig. 5 of FLD was studied in the range of 6.0 × 10⁻⁵ to 5.8 × 10⁻³mol L⁻¹. The absorbance of FLD increased with increasing Fe³⁺ concentration up to 2.2 × 10⁻³ mol L⁻¹ and remained constant at higher concentration. A concentration of 2.2 × 10⁻³ mol L⁻¹ Fe³⁺ ion was therefore chosen for the first set of conditions. Fig. 3 also shows the effect of concentration of Fe³⁺ on the oxidation of FLD under the second set of condition in the range of 6.4 × 10⁻⁵ to 5.8 × 10⁻³ mol L⁻¹. The absorbance of FLD increases with increasing Fe³⁺ concentration and attains a maximum at 3.8 × 10⁻⁴ mol L⁻¹. The concentration 3.8 × 10⁻⁴ mol L⁻¹ of Fe³⁺ was therefore, selected for the second set of conditions.

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Fig. 5 Effect of Fe³⁺ ion concentration on the complex formation and oxidation of FLD (A) FLD = 0.8 μg mL^–1, pH = 3.5 M, at 25 °C (B) FLD = 1.0 μg mL^–1, 1,10-phenanthroline = 1.30 × 10⁻³ mol L⁻¹ and pH = 5.0 at 50 °C.

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6 Effect of the concentration of 1,10-phenanthroline

The effect of 1,10-phenanthroline concentration on the absorbance of the solution under the second set of condition was studied in the range of 6.0 × 10⁻⁵–2.40 × 10⁻³ mol L⁻¹ shown in Fig. 6. The absorbance of the solution increases with increasing 1,10-phenanthroline concentration with a maximum at 1.30 × 10⁻³ mol L⁻¹. It was therefore, selected as optimum concentration.

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Fig. 6 Effect of 1,10-phenanthroline concentration on the absorbance of the ferroin produced from the oxidation of (A) FLD (B) FLD Fe³⁺ ion. Condition FLD = 1.0 μg mL^–1, Fe³⁺ = 2.2 × 10⁻³ mol L⁻¹ and pH = 5.0 at 50 °C.

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7 Effect of temperature

In order to examine the effect of temperature, the reaction of FLD with Fe³⁺ was studied between 25 and 50 °C. The variation in temperature had no effect on the absorbance and hence experiment was done at 25 °C for the first set. Since in the second set of conditions the absorbance increases rapidly up to 50 °C and becomes slow at higher temperature, a temperature of 50 °C was chosen for the reaction.

7.1

7.1 Kinetic studies

It is clear from the plots in Figs. 7 and 8 that the rate is proportional to the concentration of FLD and obeys the equation:

(4)

$$\text{Rate}=K^{\prime }{[\text{FLD}]}^n$$ where K′ is the pseudo first-order rate constant of the reaction and n is the order of the reaction. The rate may be estimated by the variable-time method measured as ΔA/Δt, where A is the absorbance and t is the time in seconds (Weisberger et al., 1953). Taking logarithms of rates and concentration as shown in Table 1, Eq. (4) is transformed into:

(5)

$$\text{log}(\text{rate})=\text{log}\mathrm{\Delta }A/\mathrm{\Delta }t=\text{log}{K}^{\prime }+n\text{log}[\text{FLD}]$$

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Fig. 7 Absorbance vs. time graphs for the reaction of FLD Oxidation with Fe³⁺ ion in the presence of 1,10-phenanthroline by method B.

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Fig. 8 Absorbance vs. time graphs for the reaction of FLD with Fe³⁺ ion by the method A.

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Table 1 Logarithms of the rates for different concentrations of FLD applying methods A and B.

Log ΔA/Δt	Log [FLD], (mol L–1)
Reaction with Fe3+ (Method A)
−3.942	−4.854
−3.690	−4.553
−3.491	−4.376
−3.360	−4.244
Oxidation with Fe3+ ion in the presence of 1,10-phenanthroline (Method B)
−4.066	−3.824
−3.750	−3.523
−3.552	−3.251
−3.500	−3.222

Regression of log (rate) versus log (FLD) gave the regression equations: $$\text{log}(\text{rate})=-0.9874+1.9920\text{log}\text{C}(r=0.9996)\text{Method A}$$ $$\text{log}(\text{rate})=9.48\times 10-3+0.848\text{log}\text{C}(r=0.9998)\text{Method B}$$

Hence K′ = 1.023S⁻¹ or 1.054S⁻¹, applying Methods A or B, respectively, and the reactions can be approximated to first order (n ≈ 1) with respect to FLD concentration under the optimized experimental conditions. The concentration of FLD was determined using an excess of Fe³⁺ and acetate buffer of pH 3.5, applying Method A.

Under second procedure excess of 1,10-phenanthroline, Fe³⁺ and acetate buffer of pH 5 with respect to the initial concentration of FLD was taken.

However, the rate will be directly proportional to drug concentration in a pseudo-first rate equation as follows:

(6)

$$\text{Rate}=K^{\prime }[\text{drug}]$$

where K’ is the pseudo-order rate constant. Several experiments were then carried out to obtain drug concentration from the rate data according to Eq. (6). The rate constant, fixed-concentration and fixed time methods (Yatsimirskii, (1966), Laitinen and Harris, (1975)) were tried and the most suitable analytical method was selected taking into account the applicability, the sensitivity, the correlation coefficient (r) and the intercept.

8 TLC of FLD and its reaction products

The FLD drug, Fe³⁺ and its reaction products were analyzed by thin layer chromatography. The spots test of FLD, reaction products such as red color ternary complex of methods A and B were with Rf values of 0.65, 0.45 and 0.60, detected respectively.

9 Determination of FLD in urine

Authors have already carried out a detailed investigation on the FLD determination in urine by the resonance rayleigh scattering (RRS) method in their previous study (Mohd et al., 2011) as shown in Table 7. The results portrayed that the RRS method has good repeatability. The % RSD and % recovery are found to lie between 0.44 and 0.89 and 99.9 and 100.3 respectively. Therefore, the method can be applied for the determination of FLD in urine sample by future researchers.

10 Application to pharmaceutical dosage forms

The optimized methods have been applied for the determination of FLD in commercial pharmaceutical dosage forms. The concentration of FLD determination was computed from the regression method. The obtained results of proposed methods were statistically compared with those of reported literatures (Saleh et al., 2003; Khan et al., 2015). The mean recovery values were 99.51 and 99.83 respectively, which ensures that there is no interference in the FLD of other active compounds. The calculated and theoretical values of both the proposed and the reported methods at 95% confidence level. This indicates good precision and accuracy for the determination of FLD in pharmaceutical dosage forms.

11 Conclusion

The determination of FLD by the proposed method is based on its complexation and oxidation by Fe³⁺. Two sets of conditions were established. In one set of conditions only FLD forms complex with Fe³⁺ while in the other set FLD is oxidized by Fe³⁺ in the presence of 1,10-phenanthroline. In both the sets, the reactions can be monitored spectrophotometrically by measuring the increase in absorbance at 510 nm. The proposed method is sensitive enough to enable determination of low amount of drug. These advantages encourage the application of the proposed method in routine analysis of FLD in industrial laboratories.