Kinetic Spectrophotometric Determination of Antifungal Drugs in Pharmaceuticals

 

B.S. Virupaxappa¹*, R.M. Kulkarni², K.H. Shivaprasad¹ and Latha M.S.³

¹Department of Chemistry, VSK University, Bellary, Karnataka, (INDIA)

²Department of Chemistry, Gogte Institute of Technology, Udyambag, Belgaum, Karnataka, (INDIA)

³Department of Chemistry, GM Institute of Technology, Davangere, Karnataka, (INDIA)

ABSTRACT:

Simple and sensitive kinetic spectrophotometric methods have been developed and validated for the determination of Antifungal drugs, Flucanazole and Ketoconazole in their pharmaceutical dosage forms. The method is based on the oxidation of Flucanazole and Ketoconazole with alkaline and acidic potassium permanganate respectively. In alkaline medium permanganate (Mn⁺⁷) oxidizes Flucanazole and undergoes one electron reduction to give a green colored manganate ions (Mn⁺⁶) which has wavelength maximum at 610 nm, and unreacted permanganate at 526 nm. In acidic medium the course of the reaction was conveniently followed by measuring the absorbance of permanganate at 526 nm, as the permanganate undergoes five electrons reduction to give divalent manganese ions (Mn⁺²) . The calibration graphs are linear in the concentration ranges from 30.8–308 μgmL−¹ and 20.6 – 206 μgmL−¹ using the Fixed time methods and Rate constant methods respectively. The proposed method has a great value in its application to the analysis of Flucanazole and Ketoconazole in quality control laboratories.

 

 

 

INTRODUCTION:

Antifungal drugs are widely used and commercially available in different pharmaceutical dosage forms¹ two of the investigated drugs namely flucanazole and ketoconazole possess imidazole ring to which almost all chemical and physical properties are attributed². The studied drugs possess either no significant absorption or relatively low absorption in the UV range. Reported methods for their analysis are mostly titrimetry³, or based on ion pair complex reaction^{4, 5} or derivative spectrophotometry^6,7 chromatographic method^8,9,10 and electrochemical method¹¹. All pharmaceutical methods for their determinations are either chromatographic^{12, 13} that are expensive or non aqueous titrimetry¹⁴, Gas Chromatography (GC) ^{15, 16} and High performance liquid chromatography (HPLC) ^{17, 18, 19} methods or the determination of Flucanazole in biological fields, HPLC for eye drops²⁰ and creams²¹ which are less sensitive methods.

               

Flucanazole is chemically 2-(2, 4-difluorophenyle-1, 3-bis (1H-1, 2, 4-triazole-1-y-1, 2, 4-triazole-1-y1)-2-propanol, a synthetic triazole derivative antifungal agent that has been shown to be effective against a wide range of systematic and superfacial fungal infections, following both oral and intravenous administrations²².

 

Ketoconazole, Cis-1-acetetyl-4-[4-2(2, 4-dichlorophenyl)-2-(1H-imidazole-1, 3- dioxolan-4-yl) methoxy piperazine, is widely used as a typical antifungal drugs in the treatment of tinea infections²³. Ketoconazole is a potent inhibitor of cytochrome p-450 dependent steroid hydroxylation in the adrenals²⁴. It is used to treat a wide variety of superficial and systematic mycoses^{25, 26}. However, some of these methods need expensive equipment and or are time consuming.  

 

Information on kinetic estimation of antifungal drugs in pharmaceutical dosage forms is scarce. This investigation was undertaken with the intent of developing simple and accurate kinetic spectrophotometric methods for antifungal drugs. The chemical structure of Flucanazole and ketoconazole are shown in (Fig1).

 

1. Flucanazole

 

2.Ketoconazole

 

Fig 1. Structures of studied antifungal drugs.

 

EXPERIMENTAL:

Apparatus

A Peltier Accessory (Temperature controlled) Varian Cary 50 model UV-Vis spectrophotometer equipped with 10mm quartz cell was used for all spectral measurements. Systronics pH meter were used for the accurate pH determinations.

 

Materials and reagents

All the materials were of analytical reagent grade, and the solutions were prepared with double distilled water, samples of Flucanazole and ketoconazole were generously supplied by their respective manufactures and were used without further purification. The Flucanazole and Ketoconazole were procured as a gift sample from (Abbot Chemicals, Goa) and (Dr. Reddy lab, Hyderabad) respectively. Potassium permanganate (Merck, Germany) 1 x 10^-3 M solution was prepared by dissolving 0.0395g KMnO₄ in 100 ml of double distilled water, followed by boiling and filtration through sintered glass. Potassium permanganate solution should be freshly prepared and its molarity was checked titrimetrically. Sodium hydroxide (Merck, Germany), 2M NaOH was prepared by dissolving 8g of NaOH in 100ml of double distilled water. 2 M perchloric acid was prepared by dissolving 17.5 ml of HClO₄ in 100 ml of double distilled water. 2M NaClO₄: was prepared by dissolving equal proportions of 2M NaOH and 2M HClO₄.

 

Pharmaceutical formulations

The following available commercial preparations were analyzed. Fluzole and Flunova tablets (150 mg each). Forcan and Fungicon capsules (150  mg each), containing Flucanazole drug and Nizaral , Finazole, Fungicide, Ketafung tablets (200 mg each) and Ketovate cream (15 mg), containing Ketoconazole drug  are used for the determination.

 

Preparations of standard solution.

A Working standard solution of 0.01 M Flucanazole was prepared by dissolving  0.306 g in 100 ml of 0.1 N NaOH and Ketoconazole was prepared by dissolving 0.531 g in 10% Acetic acid.

 

Kinetic procedure for antifungal drugs determination

All kinetic measurements were performed under pseudo first order conditions where Flucanazole and Ketoconazole used were at least 10 fold excess over permanganate at a constant ionic strength of 0.4 mol dm^-3.The reaction was initiated by mixing previously thermostatted solutions of KMnO₄ and antifungal drugs Flucanazole and ketoconazole, which also contained the required quantities of HClO₄ and NaClO₄ to maintain the required acidity and ionic strength respectively. The temperature maintained at 25 ±0.1 ^oC. The course of the reaction was followed by monitoring the decrease in the absorbance of KMnO₄ at 526 nm for both Flucanazole and Ketoconazole in alkaline and acidic medium respectively and also increase in the absorbance of manganate ion (Mn⁺⁶) at 610nm was monitored for Flucanazole oxidation in alkaline medium_.

 

Determination of studied drugs in pharmaceutical formulations

Twenty tablets were weighed and finely powdered. Quantities of the mixed powder equivalent to 100 mg of Antifungal drugs were transferred into a 100 ml calibrated flask. Dissolved in about 30 ml of acetone and the mixture were shaken for 5 min. The mixture was filtered using Whatman No. 42 filter paper and the filtrate was evaporated to dryness on a water bath.

 

For Flucanazole: The residue was washed thoroughly several times with water before dissolving it in 0.1 N NaOH. The solution was then transferred into a 50 ml volumetric flask, made up to the mark with .1 N NaOH and 10 % Acetic acid and suitable aliquot was then subjected to analysis using the procedure described under method 2.5 after diluting to 0.01 M   solution.

 

For Ketaconazole: The residue was washed thoroughly several times with water before dissolving it in 10 % Acetic acid. The solution was then transferred into a 50 ml volumetric flask, made up to the mark with 10 % Acetic acid and suitable aliquot was then subjected to analysis.

 

The cream is dissolved in 10 % Acetic acid and then the procedure was continued as described under tablets after diluting to 0.01 M Solution.

 

RESULT AND DISCUSSIONS:

Potassium permanganate as strong oxidizing agent has been used in oxidimetric analytical method for determination of many compounds^{27, 28, 29, 30}. During the course of the reaction, the valence of manganese changes from heptavalent manganese ion to the green color (Mn VI) in alkaline medium, while in neutral and acid medium, the permanganate is reduced to color less (Mn II). The behavior of permanganate was the basis for its use in its development of spectrophotometric method. The absorption spectrum of aqueous potassium permanganate solution in alkaline medium exhibited an absorption band at 526 nm. The additions of any of the studied drugs to this solution produce a new characteristic band at 610 nm. This band is due to the formation of manganate ion, which resulted from the oxidation of antifungal drugs by potassium permanganate in alkaline medium. The intensity of the color increases with time; therefore a kinetically based method was developed for the determination of antifungal drugs in their pharmaceutical dosage formulations. The different variables that affect the formation of manganate ion were studied and optimized. Calibration graph of various kinetic procedures are given below.

 

Kinetic determination of Antifungal drugs Flucanazole and ketoconazole in Pharmaceutical Formulations

The initial rate, rate constant, fixed concentration method and Fixed time methods were used for determining Flucanazole and Ketoconazole, and the best method was choosen based on applicability, the slope of the calibration graph, the intercept and the Correlation coefficient (r).

 

Initial Rate method

In this method, graphs of the rate (at the beginning of the reaction) versus the drug concentration were not easy to obtain because the reaction was fast. Thus, the tangents to the curves at zero time were not easy to draw.

 

Rate constant method

Pseudo-first order rate constants were calculated for Flucanazole and Ketoconazole Concentrations in the range from 30.6 – 275 µg/ml and 53.2 – 274.4 µg/ml and are presented in (Table 1 and 2) respectively. A plot of K_obs versus [FLU, KC] is drawn, which was used as a calibration graph (Fig 2). The range of FLU and KC concentrations giving the most acceptable calibration graph with the above equation (30.6 – 275 µg/ml and 53.2 – 532.4 µg/ml).

 

Fig 2. Calibration graph of Rate constant method for Flucanazole and Ketacanazole @ 610 and 526 nm

 

 

Table 1 .Various Kinetic methods for the determination of Flucanazole @ 610nm and 526nm in alkaline medium.

[FLU]x 103/mol dm-3	Rate constant method K obs x 103 / S-1		Fixed-concentration method(Abs = 0-15)		Fixed-time method (t = 120 s) Abs.
	610 nm	526 nm	610 nm	526 nm	610 nm	526 nm
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0	1.63 1.75 1.81 1.92 2.01 2.15 2.23 2.34 - -	1.92 1.85 1.76 1.65 1.54 1.47 1.39 1.32 -	300 250 200 140 100 060 - - -         -	180 160 150 130 110 090 -         - -        -	1.8 2.2 2.5 2.8 3.2 3.7 4.1 4.3 4.6         4.8	3.8 3.6 3.3 3.2 3.0 2.8 2.6 2.4 2.2 1.9

  a. Experimental and calculated.

 

Table 2. Various Kinetic methods for the determination of Ketoconazole @ 526 in acidic medium.

[KC] x 103/ Method moldm-3	Rate constant method K obs x 103 / S-1	Fixed-concentration  method (Abs = 0-15)	Fixed-time method (t = 120 s) Abs.
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0	1.1 1.21 1.34 1.42 1.48 1.53 1.64	240   210   190       160          140	6.7     6.4     6.1                                         5.9           5.5           5.2           4.9           4.6           4.3           4.1

a. Experimental and calculated

               

Fixed Concentration method

A preselected value of the absorbance was fixed and time was measured for different Drugs concentrations (Table1and2).The time versus the initial Concentration of Antifungal drugs was plotted, which could be used as a calibration graph (Fig 3).

 

v

 

 

Fig3. Calibration graph of Fixed Concentration method for Flucanazole and Ketacanazole @ 610 and 526 nm 

Fixed-time method

 

A pre-selected time was fixed and the absorbance was measured for different concentrations of antifungal drugs (Table 1and 2). A plot of the absorbance versus the initial concentration of antifungal drugs was drawn, which was linear and could be used as a calibration graph (Fig 4).

 

The range of the drug concentrations giving the most acceptable calibration graph with the above was   30.6- 275 µg/ml).

 

The best correlation coefficient was obtained for the fixed time method, and the value of the slope was also high. Even though the range was limited compared to the rate-constant method, the fixed-time method was found to be more applicable.

 

Fig 4. Calibration graph of Fixed time method for Flucanazole and Ketacanazole @ 610 and 526 nm 

 

Statistical analysis of the results in comparison with the official method.

The performance of the proposed method was judged by calculating the student t-test and variance ratio F-test. At the 95% confidence level, the calculated t- test and F-values do not exceed the theoretical values, indicating that there is no significant difference between the proposed method and the official method.

 

From an analytical point of view, it is concluded that the described procedure allows for the determination of antifungal drugs in pure and pharmaceutical dosage forms. Unlike the spectrofluorometer, as well as gas chromatographic and HPLC procedures, the instrument is simple and inexpensive. Its importance lies in the chemical reaction upon which the procedure is based, rather than sophistication of the instrument. This aspect of the kinetic method of determination is of major interest in analytical pharmacy, since it offers a distinct possibility for the assay of a particular component in complex dosage formulations.

 

Validation of the proposed method

Concentration range is established by confirming that the analytical kinetic procedures provide a suitable degree of precision, accuracy and linearity when applied to the sample containing the amount of analyte within or at the specified range of the analytical procedure. In this work concentrations ranging from 30.6- 306 µg/ml and 53.2 – 532 µg/ml were studied for the investigated drugs in the fixed time method (at preselected fixed time for 100 secs) and concentration ranging from 30.6 – 275 µg/ml and 53.2 – 274.4 µg/ml were studied for the investigated drugs in the rate constant method. The whole sets of experiments were carried out through this range to ensure the validation of the proposed procedure. Linear calibration graphs were obtained for all the studied drugs by plotting the logarithm of rate constant method of the reaction versus Absorbance of molar concentration of analyte in the sample within the specific range.

 

Precision

It was checked at three concentration levels. Eight replicate measurements were recorded at each concentration level. The calculated relative standard deviation were all below 2.2% indicating excellent precision of the proposed procedures at both level of repeatability and intermediate precision. ³¹

Limit of detection (LOD)    LOD was calculated based on standard deviation of response and the slope of calibration curve^{32, 33}. The limit f detection was expressed as ³¹,      

Where, σ is the standard deviation of intercept s is the slope of calibration curve. The results were summarized in (Tables 3 and 4) indicating good sensitivity of the proposed method. According to USP XXV guidelines³², the calculated LOD values should be further validated by laboratory experiments. In our work, good results were obtained where the calculated by LOD equations were actually detected in these experiments.

 

Table 3. Analytical parameters of Kinetic procedures for the determination of Flucanazole drug with Alkaline KMno4

 

Table 4. Analytical parameters of Kinetic procedures for the determination of Ketoconazole drug with Acidic KMno4.

Drug/ Methods	Linear range(µg/ml)	Intercept	Correlation coefficient(R2)	LOD	LOQ	Sand ell’s sensitivity
Ketoconazole A.Rate constant method	53.2 – 372.4	0.0010	0.99492	0.1761	0.5336	0.0003585
B.Fixed time method	53.2 - 532	0.5313	0.99504	0.0004	0.0011	0.0000073

 

Table 5. Analysis of Flucanazole in pharmaceutical formulations (Found values^a ± SD% and comparison with the official method.

Drug	Labeled	Found (X ± SD)
		Proposed method	Reference method
Tablets 1.Flunova   2.Fluzova   Capsules   3.Forcan   4.Fungicon	150 mg   150 mg       150 mg   150 mg	148 ± 0.78 t = 0.043, F = 1.267 149 ± 0.48 t = 1.786, F = 2.571     150 ± 0.16  t = 0.945, F = 3.680 147 ± 0.88 t = 0.615, F = 2.915	152.2 ± 0.22 mg   150.2 ± 0.32 mg       152.2 ± 0.22 mg   151.2 ± 0.12 mg

 

Table 6. Analysis of Ketoconazole in pharmaceutical formulations (Found values^a ± SD% and comparison with the official method).

Drug	Labeled	Found (X ± SD)
		Proposed method	Reference method
Tablets:   Nizaral   Finazole   Fungicide   Ketafung   Ointment: Ketovate   Shampoo:	200mg   200mg   200mg   200mg     2 %   2 %	208 ± 2.0 mg t = 0.52, F = 2.39 208 ± 2.0 mg t = 0.32, F = 4.39 208 ± 2.0 mg t = 0.62, F = 3.39 208 ± 2.0 mg t = 0.72, F = 4.39   2.10 ± 0.15 % t = 0.29, F = 16 1.38 ± 0.03 %	207.5 ± 1.97 mg   207.2 ±  2.01 mg   208.2 ±  1.87 mg   206.8 ±  1.78 mg     2.02 ± 0.17 %   -

 

 

Limit of Quantification (LOQ)

LOQ was calculated based on standard deviation of intercept and slope of calibration curve. In this method, the limit of quantization is expressed as³¹

                        LOQ= 10 σ S

The results were summarized in (Table 3 and 4) indicating the good sensitivity of the proposed method. According to USP XXV guidelines, the calculated LOQ values should be further validated by laboratory experiments. In our work, good results were obtained by LOQ equations were actually quantities in these experiments.

 

Application to pharmaceutical Dosage forms.

The fixed time and rate constant methods for the investigated drugs have been tested on commercial pharmaceutical dosage forms. The concentration of investigated drugs was computed from its responding regression equations. The results of proposed methods (Fixed time, fixed absorbance and rate constant methods) were statistically compared with those of reported methods in respect to accuracy and precision. The obtained mean recovery values were recorded in (Table 5 and 6), which ensures that there is no interference of other additives present in the studied formulations.

 

In the t- and F- tests, no significant differences were found between the calculated (Table 5 and 6) and theoretical values of both the proposed and the reported methods at 95% confidence level. This indicates good precision and accuracy in the analysis of investigated Antifungal drugs in dosage forms.  

 

CONCLUSION:

The Fixed time and rate constant methods can be easily applied for determination of investigated antifungal drugs in pure and dosage forms that do not require elaborate treatment and tedious extraction of chromophore produced. The proposed methods (Fixed time and rate constant method) are sensitive enough to enable determination of lower amounts of drug; these advantages encourage the application of proposed method in routine quality control of investigated antifungal in industrial laboratories. Finally our methods provides advantage of improving selectivity, avoiding interference of colored and or are turbidity background of samples because it measures the increase in absorbance with time against blank treated similarly.

 

ACKNOWLEDGEMENT:

The author thanks to Mr. Girish Bolakatte, Lecturer Bapuji Pharmaceutical College Davangere for his support to carry out this work.

 

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Received on 03.05.2012        Modified on 02.06.2012

Accepted on 09.06.2012        © AJRC All right reserved