Clinical Pharmacology & Biopharmaceutics
Open Access

Mometasone furoate (MF) (9,21-Dichloro-11b-hydroxy-16amethyl- 3,20-dioxopregna-1,4-dien-17-yl furan-2- carboxylate [1], (Figure 1) is a glucocorticoid or corticosteroid used topically to reduce inflammation of the skin or in the airways [2]. Tolnaftate (TF) (O-Naphthalen-2-yl methyl (3-methylphenyl) carbamothioate [1], (Figure 1) is a synthetic over-the-counter anti-fungal agent. It is used to treat jock itch, athlete's foot and ringworm [2].

Figure 1: The chemical structures of (a) MF and (b) TF.

A review of the literature showed that methods reported for the determination of (MF) alone or in combinations were spectrophotometry [3-5], HPLC [6-17], HPTLC [9,18-20], electrochemical method [21], and LC [22]. Several methods were reported for (TF) alone or in combinations as spectrophotometry [23], spectrofluorometry [24], HPLC [25] and HPTLC [26] spectrophotometry [27-29], spectrofluorometry [24,29-31], HPLC [32], and HPTLC [33].

Eriochrome black T is (sodium;3-hydroxy-4-{(1- hydroxynaphthalen-2-yl) diazenyl}-7-nitronaphthalene-1-sulfonate), (C20H12N3NaO7S) with MW of 461.38. It is a complexometric indicator. Eriochrome black T is also known as Mordant Black 11. It is used to detect the presence of rare earth metals [34]. Several drugs are determined spectrophotometrically through ion-pair complex formation with EBT as, (sulpiride, olanzapine, clozapine, and aripiprazole) [35], (bisoprolol fumarate, propranolol hydrochloride, and timolol maleate) [36], (gatifloxacin and cefotaxime sodium) [37], (haloperidol) [38], (diphenhydramine hydrochloride) [39], and (nifedipine) [40]. Methyl orange is (sodium; 4{4-(dimethyl amino) phenyl diazenyl} benzenesulf--onate), (C14H14N3NaO3S) with MW of 327.33 is a pH indicator. Methyl orange is also known as Acid Orange 52, Helianthin, and Orange III with Color Index 13025. Several drugs can be analyzed by forming ion-pair complex with MO as, (bisoprolol fumarate, propranolol hydrochloride, and timolol maleate) [36], (tropicamide) [41], (diphenhydramine hydrochloride) [39], (benzydamine HCl, levamisole HCl, and mebeverine HCl) [42], (terbinafine) [43], (oxiconazole) [44], and (enrofloxacin and pefloxacin) [45]. Chrome azurol S is (trisodium;5-{(E)-(3-carboxy-5-methyl-4- oxocyclohexa-2,5-dien-1-ylidene) -(2,6-dichloro-3-sulfonatophenyl) methyl}-3-methyl-2oxidobe--nzoate), (C23H11Cl2Na3O9S) with M.W. of 605.29 which is belongs to sulfonephthalein compounds. Chrome azurol S is also known as Color Index 43825. It is used for determination of cationic surfactant, copper, aluminum, beryllium, uranium, and other metals [46]. Various drugs can be also determined thought ion-pair formation with CRS as, (pipazethate HCl) [47], (iron and aluminium) [48], (vanadium (IV)) [49], (imipramine) [50], and (fluoxetine and fluvoxamine) [51].

The proposed methods are depending on the formation of ionpair complexes between the cited drugs, namely, MF and TF with eriochrome black T (EBT), methyl orange (MO) and Chrome azurol S (CRS) reagents in acidic buffers. Various factors altering these complexes formation are studied then Beer’s law is performed. These methods are rapid, simple and inexpensive matching other approaches.

Apparatus and software

• All the absorbance spectral measurements were made using Ultraviolet/Visible spectrophotometer (Spectronic Genesys® with WINPEC® application software) with1 cm quartz cell, Spectronic, (USA). HANNA model HI 98127-pH meter used for pH measurements.

• The centrifugation system: Laboratory Centrifuge, Sigma 2-16KL, Sigma 2-16KHL, with order number 10350, 10353.

• Sonicated water bath BranSonic 220, (Zurich, Switzerland).

• All calculations and statistics were carried out on computer using MATLAB® program version 7.9.

Chemicals and reagents

Standard mometasone furoate (MF) and tolnaftate (TF) were kindly donated by SIGMA Pharma Co., Quesna, Egypt. The purity was found to be 100.08% ± 0.39 [4] and 99.30% ± 0.55 [52] according to reported methods, respectively. The studied market dosage forms, Elcon® cream (SEDICO Co., 6 October city, Egypt), were assigned to contain 1 mg/g MF (batch No.1016335) and Tinea cure® cream (KAHIRA PHARM. & CHEM. IND. CO. – EGYPT), were assigned to contain 1% TF (batch No.1670292). All of the chemicals used were of analytical or pharmaceutical grade and used without further purification as methanol and chloroform (Analar grade).

Standard solutions

Stock solutions: Pure MF and TF were prepared separately by dissolving 6 mg and 2 mg of drugs in a 100 mL volumetric flask, respectively. Working solutions were freshly prepared by appropriate dilution with methanol to obtain working standard solutions covering the range of 1-15 μg/mL for MF and TF.

Reagents: A6 × 10^–4 M EBT (sodium;3-hydroxy-4-[(1- hydroxynaphthalen-2-yl) diazenyl]-7-nitronaphthalene-1-sulfonate) and 6 × 10^–4 M MO (sodium;4-[[4-(dimethyl amino) phenyl]diazenyl] benzenesulfonate) and 2 × 10^–3 M CRS (trisodium;5-[(E)-(3-carboxy- 5-methyl-4-oxocyclohexa-2,5-dien-1-ylidene)-(2,6-dichloro-3- sulfonatophenyl)methyl]-3-methyl-2-oxidobenzoate) stock solutions were prepared by dissolving 27.68 mg, 19.64 mg and 121 mg of EBT, MO, and CRS, respectively in a 100 mL volumetric flask with methanol. Series of buffer solutions: Of acetate buffer (pH 3.5 and 5.5) and citrate buffer (pH 4.5) were prepared by standard methods.

General recommended procedure

Procedure for calibration curve: Aliquot volumes (3-15 μg/mL) of MF and (1-5 μg/mL) of TF (as shown in Table 1) were separately transferred into a series of separating funnels, and then 1 mL of 6 × 10^–4 M of EBT, 0.3 mL of 6 × 10^–4 M of MO, and 0.1 mL of 2 × 10^–3 M of CRS was added, and 1.0 mL of acetate buffer of pH 3.5 or 5.5 in the case of EBT and MO, respectively, and 1.0 mL of citrate buffer of pH 4.5 in the case of CRS, then the solutions were diluted to 10 mL with water. The ion-pairs complexes were extracted with 10 mL of chloroform by shaking for 2.0 min and then, the combined chloroform extracts were dried over anhydrous sodium sulphate. The absorbance of colored ion-pair complexes was measured within 5.0 min of extraction against the blank prepared in the same manner without addition of drugs.

Table 1: Analytical characteristics of the proposed methods using EBT, MO, and CRS reagents with MF and TF.

Procedure for cream- Elcon® cream: A two-gram portion of the cream was transferred to a 50 mL volumetric flask, taking care to avoid sticking cream to the walls of the volumetric flask. A 35 mL of methanol was added to the flask, and the cream was allowed to melt by warming at 60°C in a water bath with constant shaking. The solution was allowed to cool to room temperature. The volume was made up to the mark with methanol and mixed. The solution was centrifuged at 10000 rpm for 10 min, and the clear supernatant solution was obtained. A portion of the supernatant was diluted quantitatively with methanol to obtain a final concentration of 20 μg/mL.

TINEA CURE® cream: A one-gram portion of the cream was transferred to a 50 mL volumetric flask, then the procedure was completed as mentioned in section 4.4.2.

Absorption spectra

The absorption spectra of the ion-pair complexes were measured in the range 400-610 nm against blank (chloroform, dyes and buffers). MF was found to react with EBT, MO and CRS dye anions in acidic buffer donating an intense color with a maximum absorption at 510, 440, and 530 nm for EBT, MO and CRS, respectively, Figure 2. So, all the measurements are achieved at 510, 440, and 530 nm (EBT, MO, and CRS) for MF and at 550, 482, and 499 nm (EBT, MO, and CRS) for TF against blank. The influence of the dye concentrations, stability of formed complexes, extractive organic solvents, effect of pH, and effect of interferences were checked via monitoring experiment parameters. The optimum conditions were assured by changing one variable and watching its influence on the absorbance of the colored complex.

Figure 2: Absorption spectra of MF–dye complex extracted in chloroform: EBT (1), MO (2), and CRS (3).

Effect of dyes volume

The effect of dyes volume on the intensity of the color developed and the absorbance at the selected wavelength using constant drugs concentrations was checked using different volumes of EBT solution 0.03 % (w/v), MO solution 0.02 % (w/v) and CRS solution 0.12 % (w/v). The results indicated the maximum absorbance for EBT, MO, and CRS; for both MF and TF as shown in Figures 3 and 4. Thus 1, 0.3 and 0.1 mL of EBT, MO and CRS were used, respectively, for both MF and TF.

Figure 3: Effect of EBT (1), MO (2), and CRS (3) volumes (1.0, 0.3, and 0.1 mL, respectively) on the absorbance of MF (9 μg/mL) for all the studied complexes.

Figure 4: Effect of EBT (1), MO (2), and CRS (3) volumes (1.0, 0.3, and 0.1 mL, respectively) on the absorbance of TF (3 μg/mL) for all the studied complexes.

Stability of the Ion-pair complexes

The stability of the complex after extraction with the chloroform was studied. It was found that the absorbance remained constant up to 30 min.

Choice of organic solvent

Various organic solvents as chloroform, dichloromethane, and ether were checked as extractive solvents for the proposed methods. Chloroform was favored to other solvents because it obtained highest absorbance. A one extraction was satisfactory to have a quantitative recovery of the complexes. Shaking time of 0.5-5 min afforded constant absorbance and the 2.0 min was the optimum shaking time.

Effect of pH

The effect of pH on the formed complexes of MF and TF with the three dyes has been checked using various types of buffers of various media. The best buffer related with the maximum color intensity and maximum absorbance is sodium acetate buffer of pH 3.5 in case of the EBT and pH 5.5 in case of MO and sodium citrate buffer of pH 4.5 in case of CRS. The volume of the used buffers was detected by applying the same procedure and changing the volume orderly (0.5-3.0 mL). The highest absorbance was achieved by using 1.0 mL of buffer solutions.

Composition of the Ion-pair complexes

The formed ion-pair complexes between MF and TF and the three dyes; (EBT, MO, and CRS) were determined by Job’s method [53]. A series of solutions were arranged with total volume of the drugs and reagent (5.0 mL) and complete the procedures as described under general procedures. Plot the absorbance of each reading against mole fraction of drug. The plot attained to the maximum value at a mole fraction of 0.5 in case of MF and the three reagents, which determined that a 1:1 (drug: dye) ion-pair are formed through the electrostatic attraction between dyes anions and positive protons of drugs in all cases, Figure 5. In case of TF with EBT and MO the maximum value was found also at a mole fraction of 0.5 where the ratio is (1:1) except in (TF:CRS) case where the ratio is (2:1), Figure 5. The suggested mechanism for the reaction product of TF-CRS, TF-EBT and TF-MO ion-pair complex formation for example, is given in Figures 6-8.

Figure 5: Job’s plots of equimolar solutions of MF with EBT (a), MO (b), and CRS (c) and Job’s plots of equimolar solutions of TF with EBT (1), MO (2), and CRS (3).

Figure 6: Suggested mechanism of TF–CRS ion-pair complex formation.

Figure 7: Suggested mechanism of TF–EBT ion-pair complex formation.

Figure 8: Suggested mechanism of TF–MO ion-pair complex formation.

The calibration graphs were constructed for the considered drugs Figures 9 and 10, with the optimum conditions detailed before. The concentration range, molar absorptivity, correlation coefficient, the limit of detection (LOD) and limit of quantitation (LOQ) for each drug are listed in Table 1. A linear relationship was constructed between the absorbance and the concentration of the investigated drugs within the range 1-15 μg/mL.

Figure 9: Calibration graphs MF–dye complex extracted in chloroform: EBT (1), MO (2), and CRS (3).

Figure 10: Calibration graphs TF–dye complex extracted in chloroform: EBT (1), MO (2), and CRS (3).

Regression analysis of Beer’s law plotted at λmax reveals a good correlation (r2=0.9988-0.9999). The graphs displayed a negligible intercept, which was determined from the regression equations, A=a+bC (where A is the absorbance of 1.0 cm layer, b is the slope, a is the intercept and C is the concentration of the studied solution in μg/ mL [54].

The high molar absorptivities of the formed complexes indicated high sensitivity of the methods (2.17 × 10⁴ 7.44 × 10⁴). The TF–EBT method was found to be the most sensitive of all methods with high molar absorptivity value.

Assay precision and accuracy

The inter-day and intra-day precision (intermediate precision) of the proposed method were determined by the analysis of different concentrations of studied drugs, within the linearity range, by three replicate analyses on a single day and three consecutive days, for the intra-day and inter-day precisions, respectively. The results expressed as percentage recoveries and RSD is illustrated in Table 2.

Table 2: Evaluation of intra-day and inter-day precision for the determination of MF and TF by the proposed methods using EBT, MO, and CRS reagents.

To study the accuracy of the proposed method, repeated analysis (three times) of different concentrations of MF and TF with the three reagents within the linearity range were performed.

Selectivity

To confirm the absence of interference of excipients in the analysis of the standard drugs by the proposed methods, standard addition technique was applied. The attained results exhibit the accuracy of the proposed methods for the detection of drugs in creams without any interference of the excipients as shown in Table 3.

Table 3: Application of standard addition technique for the determination of MF and TF in pharmaceutical formulations using EBT, MO, and CRS reagents.

Analysis of pharmaceutical formulations

The dosage forms were analyzed by the proposed and reported methods in three replicate determinations. The results of the proposed methods were compared with well-established reported methods by means of t-test and F-test at 95% confidence level. The results are listed in Table 4. It was found that, there was no significant difference between results obtained by the proposed and reported methods, indicating good accuracy and precision.

Table 4: Statistical comparison between the results obtained by the proposed method and the reported methods for the determination of MF and TF in pure powder form.