Terbium sensitized luminescence for the determination of fexofenadine in pharmaceutical formulations

2.1 Apparatus

Luminescence measurements were performed using Perkin Elmer LS-55 luminescence spectrometer (Perkin Elmer, Beaconsfield UK) equipped with a xenon discharge flash lamp. The operation and data processing were controlled by the fluorescence Data Manager Software. The excitation and emission wavelengths were set at 220 nm and 550 nm respectively. The instrument was operated in a phosphorescence mode with a gate time of 0.04 ms and a delay time of 0.2 ms used throughout the measurements. Absorption spectra were recorded on a Varian CARY 50 Conc UV visible spectrophotometer (Connecticut, USA) with 1 cm matched quarts cell.

2.2 Reagents

HPLC grade methanol, ethanol, 1-propanol, acetone and acetonitrile were purchased from Sigma–Aldrich (St Louis, Mo, USA). Terbium (III) chloride hexahydrate and triethylamine were purchased from Kanto Chemical CO. INC (Tokyo, Japan). Zinc nitrate was obtained from (BDH Chemicals Ltd pool England). FEX standard was obtained from the Quality Control Laboratory, Ministry of Health, Muscat, Sultanate of Oman.

Pharmaceutical formulations: Telfadin 180 mg (Muscat, Sultanate of Oman), Telfast 120 mg (France) and Fexodine 120 mg (Tabuk, Saudi Arabia) were purchased from local retail outlets.

2.3 Preparation of standard solutions

1.14 × 10⁻³ M of FEX stock standard solution was prepared in methanol. Working standard solutions were freshly prepared by appropriate dilutions of the stock with methanol.

Terbium (III) chloride hexahydrate stock solution (1.31 × 10⁻³ M) was prepared by dissolving 24.5 mg of Tb³⁺ in 50 mL methanol. Working standard solutions were freshly prepared by appropriate dilution of the stock solution with methanol. 2.00 × 10⁻³ M stock solution of triethylamine was prepared by dissolving 0.01 g of the reagent in 50 ml methanol.

Stock solutions (2.00 × 10⁻³ M) of various co-luminescent agents such as zinc nitrate, gadolinium, samarium, lanthanum, and europium were prepared in methanol.

2.4 Preparation of drug samples

Three commercial products containing known amounts of FEX were analyzed. Five tablets of each product were separately weighed, and then the tablets were powdered and mixed. A portion of the powder known to contain an equivalent amount of one tablet was accurately weighed and sonicated with methanol for 30 min. It was then filtered into a volumetric flask and completed to the mark with methanol to make a stock solution. Working solutions of 50 ppm in methanol were prepared. Appropriate aliquots from the working solution were taken for the determination of FEX in samples.

3.1 Spectral characteristics

The absorption spectrum of FEX and Tb–FEX complex in methanol was found to exhibit two bands in the ultraviolet region at 202 nm and 219 nm. A slight enhancement in the absorption was observed in the presence of the complex when compared to the ligand alone.

On the other hand, the fluorescence spectrum of 1.0 × 10⁻⁵ M of FEX in methanol revealed a notable fluorescence signal in the low UV region with an emission wavelength at 298 nm when excited at 252 nm. The emission characteristic at low UV region will not be analytically useful particularly for the analysis of the drug in biological solution. This is due to the possible interference of short-life emitting species from the matrix.

In order to improve the analytical characteristics for the determination of FEX in pharmaceutical formulation, the system of (Tb–FEX) in methanol was investigated. FEX was used to sensitize the luminescence of terbium ion. Terbium exhibited a weak luminescence in methanol. However, when FEX was added to the Tb³⁺ solution, the intensity of Tb³⁺ peaks was enhanced about six times compared to that of Tb³⁺ alone as shown in Fig. 2. The signal of Tb–FEX resulted in the well-structured emission bands of Tb³⁺ while the excitation spectra of Tb³⁺ revealed the presence of a band at 216 nm. Upon addition of FEX to Tb³⁺ solution, a broad band slightly red shifted to 220 nm was observed (figure not shown).

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Figure 2 Luminescence emission spectra of: (1) Tb³⁺ in MeOH system, (2) Tb³⁺ in presence FEX in MeOH. [Tb³⁺] = 2.3 × 10⁻⁶ M, [FEX] = 1.0 × 10⁻⁶ M, λ_ex = 220 nm, λ_em = 550 nm.

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The enhancement of the luminescence of Tb³⁺ ions upon addition of FEX is probably due to complexation of the ions by the drug with the ensuing efficient transfer of energy from FEX to the chelated Tb³⁺, which can overcome the low extinction coefficient of the ions. For efficient energy transfer to occur, the sensitizing moiety must absorb light in the ultraviolet region, which is the case for FEX. The close agreement between the absorption spectra of the ligand at 219 nm and the excitation spectra at 220 nm support that the excitation of the antenna may be the only photophysical pathway leading to the luminescence enhancement of the complex. Furthermore, a complete overlap between the absorption spectrum of FEX and the excitation spectrum of Tb–FEX complex is observed. The emission spectrum of Tb–FEX complex when excited by the π–π^∗ absorption band of the complex at 220 nm revealed the well-known bands of terbium luminescence. Among these transitions, the ⁵D₄–⁷F₅ around 545 nm is the strongest followed by the one at 490 nm. Therefore, the peak height at 550 nm was used to measure the luminescence intensity of the Tb–FEX complex. Moreover a large stokes shift of 330 nm was observed, indicating that there is no overlap between the Tb³⁺ emission band and the antenna chromophore absorption bands.

The luminescence intensity of Tb³⁺ at 550 nm is affected by the coordination environment. Hence the effect of solvent, concentration of Tb³⁺ and FEX, Et₃N and Zn²⁺ and effect of different co-luminescence reagents were investigated in order to maximize the luminescence intensity.

3.2 Effect of solvent

The influence of the solvents on the luminescence intensity of FEX–Tb³⁺ complex was investigated carefully and the results are shown in Fig. 3. It can be seen that the enhancing effect of organic solvents in the Tb–FEX system is arranged in the order CH₃OH > CH₃CN > C₂H₅OH > C₃H₇OH > (CH₃)₂CO > H₂O. The dielectric constants (ε) of these solvents are methanol 32.6, acetonitrile 37.5, ethanol 24.5, propanol 20.3, acetone 20.4 and water 78.8. It is clear that there is an agreement between dielectric constant (ε) of the solvents that contain oxygen atoms and the luminescence in the system apart from methanol and water. It has been reported, that the difference in intensity of the emission line between the four alcoholic solutions indicates the different coordination behavior of alcohols with terbium metal with methanol exhibiting the strongest coordination ability (AbdEl-Hay et al., 2012). Similar results were previously reported for terbium and europium complexes, where maximum sensitization was observed in the presence of methanol (Al-Kindy et al., 2008; Dang et al., 2007; Qin et al., 2003). The emission intensity decreases with increase in the alkyl chain in alcohols (Ansari et al., 2007). The low sensitization in water may be due to the O–H oscillators of water molecules that are known to quench the excited state of the lanthanide ions by interaction with high-energy vibrations associated with O–H. Solvent molecules can usually replace coordinated water and avoid high frequency O–H bond vibration—thus greatly decreasing energy loss resulting through the coordination of water molecules, and increasing the fluorescence intensity of the emitting lanthanide ion. This suggests that the chemical environment plays an important role in determining the fluorescence intensity of the complexes; hence, the choice of methanol was ideal in studying the luminescence of the Tb–FEX complex.

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Figure 3 Effect of type of solvent on terbium sensitized luminescence emission spectra. [FEX] = 1.03 × 10⁻⁵ M, [Tb³⁺] = 2.89 × 10⁻⁵ M, λ_ex = 220 nm, λ_em = 550 nm (1) methanol, (2) acetonitrile, (3) ethanol, (4) propanol, (5) acetone, and (6) water.

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3.3 Stoichiometry of the Tb–FEX complex

Since the sensitization results from complex formation between FEX and Tb³⁺, it is necessary to optimize the ratio of the concentration of the metal to the ligand that will result in maximum complexation and hence maximum intensity of the emission line of terbium ion. The optimum concentration of terbium was determined by measuring the luminescence intensity of various molar ratios of Tb³⁺:FEX. The luminescence intensity increased with an increased concentration of FEX and reached a maximum value at a ratio of 1:3 after which the intensity remained constant (Fig. 4) indicating the formation of Tb (FEX)₃ complex.

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Figure 4 Influence of [FEX]: [Tb³⁺] ratio on terbium sensitized luminescence. [Tb³⁺] = 1 × 10⁻⁵ M, λ_ex = 220 nm. λ_em = 550 nm.

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The stoichiometry of the complex was corroborated using Job’s plot of continuous variation. Equal concentration of Tb³⁺ and FEX (2.0 × 10⁻⁵ M) was used, and the molar ratio of Tb³⁺:FEX was varied. The luminescence intensity increased with an increase in the mole fraction of Tb³⁺:FEX and reached a maximum at a mole fraction of 0.3 (Fig. 5), indicating a stoichiometry of Tb³⁺:FEX of 1:3.

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Figure 5 Continuous variation method of the Tb (FEX) complex in MeOH. [Tb³⁺] = [FEX] = 2.0 × 10⁻⁵ M, λ_em = 550 nm, λ_ex = 220 nm.

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3.4 Luminescence enhancement of Tb–FEX complex by zinc and Et₃N

One of the strategies to improve the luminescence of lanthanide system is the addition of co-luminescence agent. Non-fluorescing lanthanide ions such as La³⁺, Gd³⁺ and Lu³⁺ have been used. In the presence of these ions, the fluorescence of Tb³⁺ and Eu³⁺ complexes is enhanced (Yang et al., 1994). In this case, the intermolecular energy flow is believed to occur between the non-fluorescing donor complexes of Ln³⁺, Gd³⁺ and Sm³⁺ to the fluorescing acceptor complexes of Tb³⁺, Eu³⁺ or Dy³⁺.

Initially the effect of addition of co-luminescence agents such as Eu³⁺, La³⁺, Gd³⁺, Sm³⁺ and Zn²⁺ with the concentration of 5.00 × 10⁻⁶ M was investigated. Addition of Zn²⁺ gave the highest luminescence intensity in this system. However, other co-luminescence quenched the fluorescence of Tb³⁺ as shown in Fig. 6.

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Figure 6 The effect of co-luminescence agents on the luminescence spectra of Tb³⁺–FEX system. [Tb³⁺] = 2.25 × 10⁻⁶ M, [FEX] = 7.5 × 10⁻⁷ M, [co-luminescence] = 5.00 × 10⁻⁶ M.

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Most of the work on co-luminescence effect was reported in aqueous system in the presence of micelles and synergistic agents such as TOPO or 1, 10 phenanthroline. Recently, (Dang et al., 2007) a new co-luminescence system was reported in methanol, the system used β-diketonate as ligand. The system of Tb-N-(2-pyridinyl) ketoacetamide was greatly enhanced in the presence of zinc nitrate and triethylamine (Et₃N).

In this study, the luminescence properties of Tb–FEX complex were enhanced in the presence of Zn²⁺ and Et₃N (Fig. 7). It is clear from the figure that Tb³⁺ exhibited a weak signal in methanol. When adding Et₃N and FEX an enhancement of the terbium ion signal is observed. However, a huge enhancement in the signal is observed when Zn²⁺ was added to Tb³⁺ in the presence of FEX and Et₃N.

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Figure 7 The emission spectra of Tb–FEX–Et₃N–Zn (1), Tb–FEX–Et₃N, (2), Tb–FEX, (3) Tb³⁺ alone, (4) system in methanol. [Tb³⁺] = 2.25 × 10⁻⁶ M, [FEX] = 7.50 × 10⁻⁷ M, [Et₃N] = 5.00 × 10⁻⁶ M and [Zn²⁺] = 5.00 × 10⁻⁶ M.

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Many multinuclear heterometallic 3d–4f complexes were reported (Towatari et al., 2013; Lü et al., 2010; Yang et al., 2005; Bi et al., 2009). The formation of these complexes was evidenced by characterization by X-ray crystallography which revealed the existence of functional bridge in these 3d–4f complexes. The donation of OH groups was the main facilitator of the formation of such complexes. In the coordination compounds involving Zn²⁺ and lanthanide ions, it was reported that complexation of a pyridine group to Zn²⁺ ions in these complexes resulted in congestion around the coordination sphere of the 4f Ln³⁺ ions (Bi et al., 2009). This has led to a significant minimization of the luminescence quenching from solvents molecules around the lanthanide ions. On the other hand, the involvement of heavy atoms in the structure improves the antenna energy transfer significantly. We believe that a similar mechanism might be responsible for the enhancement of the luminescence of Tb³⁺–ZN Et₃N.

However, further work needs to be done to explain the actual mechanism of the energy transfer, including synthesizing the complex in a large scale and verify it is structured by X-ray crystallography.

Moreover, the influence of the amount of Et₃N on the fluorescence intensity was studied. The maximum intensity of luminescence was at the concentration of Et₃N equal to 5.0 × 10⁻⁶ M. Therefore, 5.0 × 10⁻⁶ M was employed as the final Et₃N concentration.

After optimizing the concentration of Et₃N, the effect of concentration of Zn²⁺ on the system was investigated. The luminescence intensity increased with an increase in the concentration of zinc and maximum intensity was observed at a concentration 5.0 × 10⁻⁶ M of zinc. On increasing the concentration of zinc further, a decrease in the luminescence intensity was observed. This could be due to the fact that a large excess of Zn ions could result in favoring the formation of mononuclear Zn-complexes at the expense of the heteronuclear metallic complexes. Hence, a 5.0 × 10⁻⁶ M of Zn²⁺solution was used for the subsequent work.

3.5 Effect of Tb³⁺ concentration

The effect of Tb³⁺ concentration on the luminescence intensity of the system was examined after optimizing all the other parameters. The luminescence intensity starts to increase with an increase in the concentration of Tb³⁺ until it reaches the maximum at 2.25 × 10⁻⁶ M after which a decrease in the luminescence intensity was observed. Hence, an optimum concentration of terbium for this study was taken to be a concentration of 2.25 × 10⁻⁶ M.

3.6 Analytical appraisal

A series of standard solutions of FEX (0–1000 ppb) were analyzed by the proposed method. The obtained calibration curves were evaluated using linear regression methods. The luminescence intensity I, versus FEX concentration was found to be linear over the range 10–800 ppb. The calibration equation was I = 974.6 C–0.566, with a correlation coefficient R² of 0.9999. The detection limit (S/N ratio = 3) was 1.00 ppb. The lower limit of quantitation (S/N = 10) was 4.00 ppb. The reproducibility of the method was obtained for standard solutions of FEX of 200 ppb and was found to be less than 2% (n = 7).

In the presence of Zn²⁺ and Et₃N, the luminescence intensity I, versus FEX concentration was also found to be linear over the range 10–800 ppb. The calibration equation was: I = 1994 C + 0.108, with a correlation coefficient R² of 0.999. Lower detection and quantitation limits of 0.3 ppb and 1.00 ppb were obtained respectively. The figures of merit of the proposed method compared favorably with most of the published methods for the determination of FEX (Table 1). In addition, the method offered an advantage over other luminescence methods, in that its emission is at a longer wavelength and free from interference from short-life emitting species present in these matrices.

3.7 Determination of FEX in pharmaceutical preparations

The proposed method was applied for the determination of the concentration of FEX in pharmaceutical formulations of different bands as shown in Table 2. The tablets are known to contain many excipients as fillers or binder. Three samples were prepared as described above and were analyzed by the proposed method. It is clear from the results that good recovery with no interference from the excipients was obtained indicating that the method exhibits good selectivity. For comparison, FEX in the pharmaceutical formulation was determined using a spectrofluorimetric method (Ibrahim et al., 2011). These determinations were carried out on the same batch of samples. The results obtained were compared statistically using Student t-test and the F-test (Table 3). Both methods produced comparable accuracy and precision, which indicates that there is no significant difference between the two methods.