INTRODUCTION:

Clotrimazole (CLO) is a broad spectrum antimycotic drug, it is work by inhibiting the fungal cytochrome P450 3A enzyme, lanosine 14α-demethylase, which is responsible for converting lanosterol to ergosterol, the main sterol in the fungal cell membrane. Chemically it is 1-[(2-Chlorophenyl)(diphenyl)methyl]-1H-imidazole^[1-2].

Figure 1: Structure of Clotrimazole^[3]

Some methods have been described for identification and quantification of active substance CLO such as UV^[4-5], HPLC^[6-15] and HPTLC methods^[16-18]. It is important to detect the impurities not only in the active ingredient but in the final formulation.

Impurity analysis is not only important for maintaining consistency and product quality. Patient safety and reliability are equally important. One method is available for determination of Clotrimazole and its impurities with the use of internal standard. Here we develop a method which gives the reliable and accurate results without the use of internal standard.

MATERIAL AND METHOD:

Selection of Wavelength:

Appropriate dilution of standard stock solution of Clotrimazole and 2-Chlorophenyl diphenyl methanol (Imp-A) were prepared. Both solutions were scanned separately in order to get absorbance. Both the solutions were scanned between 200 – 400 nm using UV-Visible spectrophotometer. Suitable wavelength was selected from the overlay spectra of above solutions. The overlay spectrum for selection of wavelength is given below.

Figure 2: Overlay Spectrum of CLT and Imp-A

Preparation of Standard Stock Solution:

Accurately 10 mg of Clotrimazole and 10 mg of 2-Chlorophenyl diphenyl methanol were weighed separately and transferred into two different 100 mL volumetric flask. Each drug was dissolved in few mL of methanol. The volume was made up to the mark with methanol to give final solutions containing 100 μg/mL of Clotrimazole and 100 μg/mL of 2-Chlorophenyl diphenyl methanol respectively. The solution was subjected to ultrasonication for 20 min and then filtered through 0.45 μm, 47 mm membrane filter paper.

Preparation of Standard Solution for Binary Mixtures of Clotrimazole and 2-Chlorophenyl diphenyl methanol:

Transfer 5 mL solution of Clotrimazole from standard stock solution (100 μg/mL) and 0.5 mL solution of 2-Chlorophenyl diphenyl methanol from standard stock solution (100 μg/mL) in to a 10 mL volumetric flask. Few ml of methanol was added. The solution was subjected to ultrasonication for 20 min and then filtered through 0.45 μm 47 mm membrane filter paper. The volume was made up to the mark with methanol to obtain a binary mixture containing 50 μg/mL of Clotrimazole and 5 μg/mL of 2-Chlorophenyl diphenyl methanol.

Selection of Mobile Phase:

The pure drug of Clotrimazole and 2-Chlorophenyl diphenyl methanol were injected into the HPLC system and run in different solvent systems. Different mobile phases like Methanol, Methanol: Water, Methanol: Acetonitrile, Methanol: Acetonitrile: Triethyl amine, Acetonitrile: Water, Water: Acetonitrile: Phosphate buffer, with varying mobile phase ratio were tried in order to find the best conditions for the separation of Clotrimazole and 2-Chlorophenyl diphenyl methanol. From the various chromatograms obtained with different mobile phase, Methanol: Acetonitrile in a ratio of 95:5 is selected for the separation of Clotrimazole and 2-Chlorophenyl diphenyl methanol.

Preparation of Mobile Phase:

Here, HPLC grade acetonitrile and methanol was filtered through 0.45 µm, 47 mm membrane filter paper and then ultrasonicated for 20 minutes on ultrasonicator. Mobile phase was prepared by mixing 95 mL of methanol and 5 mL of acetonitrile.

Chromatographic Separation:

Mixed standard of Clotrimazole and 2-Chlorophenyl diphenyl methanol was injected in column with 20 µL of micro-syringe. The chromatogram was run for appropriate minutes with mobile phase of methanol: acetonitrile (95:5 % v/v) which was previously degassed. The flow rate was set to 1 mL/min. and detection was carried out at 225 nm. After complete separation of two drugs, data related to peak area, height, retention time, resolution etc. were recorded using software.

RESULTS AND DISCUSSION:

The results obtained by this method are precise and reproducible for the Clotrimazole and its Impurity. Reproducibility of the method was done on six samples of Clotrimazole and its Impurity. The robustness of the method was confirmed by varying the concentration of organic phase and change in flow rate. With the increase in flow rate retention time and tailing factor decreases and with decrease in flow rate retention time and tailing factor increases. The best result is obtained with the flow rate of 1 ml/min. The results obtained with the variation in the various parameters are within the acceptable limit, so the method was found to be robust in the conditions specified (table 15). The system suitability parameters were calculated to confirm the specificity of the developed method and shown in table 1. The high percentage of recovery and low standard deviation data were satisfactory and confirms the accuracy, precision and reliability of the method. Further this method eliminates the use of internal standard as it is used by published method. This two estimated within 3 min, hence the present method is cost effective and faster, can be used to the routine analysis of drug with its impurity.

Figure 3: Chromatogram for Clotrimazole

Figure 4: Chromatogram for 2-Chlorophenyl diphenyl methanol

Figure 5: Chromatogram for Clotrimazole and 2-Chlorophenyl diphenyl methanol

Table 1: Validation and system suitability parameters

Parameters	Clotrimazole	CLT Imp A
Retention Time (min.)	2.14	2.81
Resolution	1.72
Linearity (µg/mL)	10-80	2-12
Correlation Coefficient (r²)	0.999	0.998
Slope	1699	990.6
Y – intercept	61323	9864
LOD (µg/mL)	0.6383	1.9344
LOQ (µg/mL)	0.5853	1.9511
Specificity (RT ± SD) n=6	2.789 ± 0.0233	2.326 ± 0.0421
Repeatability (% RSD) n=6	0.5166	0.5519
Intraday Precision (% RSD) n=6	0.9056	0.5038
Interday Precision (% RSD) n=6	0.9597	0.9450
Accuracy (%)	99.00-101.00	99.00-101.00

Table 2: Calibration table of Clotrimazole

Sr. No.	Concentration of Clotrimazole (µg/mL)	Area Under Curve (AUC)
1	10	79653.8
2	20	96526.3
3	30	110861.5
4	40	127185.0
5	50	145973.1
6	60	163203.4
7	70	180331.7
8	80	198589.5

*n=6 times

Figure 6: Standard calibration curve of Clotrimazole

Table 3: Calibration table of 2-Chlorphenyl diphenyl methanol

Sr. No.	Concentration of 2-Chlorophenyl diphenyl methanol (µg/mL)	Area Under Curve (AUC)
1	2	11988.4
2	4	13769.9
3	6	15538.2
4	8	17964.0
5	10	19753.5
6	12	21782.1

Figure 7: Standard calibration curve of 2-Chlorophenyl diphenyl methanol

Table 8: Statistical Validation of Synthetic Mixture

Drug	Mean Drug Found in Mixture (%)*	Standard Deviation*	*Relative Standard Deviation (%)**	Standard Error*
CLT	100.01	0.1722	0.1720	0.070
TNZ	99.93	0.1862	0.1863	0.070

*n=6 times

Table 9: Recovery data for Clotrimazole from synthetic mixture

Conc. Level (%)	Amount present in mixture (mg/300 mg)	Amount Spiked (mg)	Total Amount Present (mg)	Amount Present in Prepared Solution (µg/mL)	Amount Found in Prepared Solution (µg/mL)	Recovery (%)
80	50	40	90	50	49.9	99.8
80	50	40	90	50	50.02	100.02
80	50	40	90	50	49.95	99.9
100	50	50	100	50	50.01	100.02
100	50	50	100	50	49.98	99.96
100	50	50	100	50	50.09	100.18
120	50	60	110	50	49.95	99.81
120	50	60	110	50	50.25	100.5
120	50	60	110	50	49.90	99.8

Table 10: Recovery data for 2-Chlorophenyl diphenyl methanol from synthetic mixture

Conc. Level (%)	Amount Present in mixture (mg /300 mg)	Amount Spiked (mg)	Total Amount Present (mg)	Amount Present in Prepared Solution (µg/mL)	Amount found in Prepared Solution (µg/mL)	Recovery (%)
80	5	4	9	5	4.98	99.75
80	5	4	9	5	4.96	99.25
80	5	4	9	5	5.01	100.25
100	5	5	10	5	4.95	99.06
100	5	5	10	5	5.02	100.4
100	5	5	10	5	4.95	99.04
120	5	6	11	5	4.99	99.66
120	5	6	11	5	5.00	100.16
120	5	6	11	5	5.01	100.33

Table 11: Statistical Validation of Recovery Studies for Clotrimazole

Concentration Level (%)	*Mean Recovery (%)**	Standard Deviation*	*Relative Standard Deviation (%)**	Standard Error*
80	99.96	0.2082	0.2082	0.1202
100	100.05	0.1137	0.1131	0.0656
120	100.03	0.4013	0.4000	0.2317

*n=3

Table 12: Statistical validation of recovery studies for 2-Chlorophenyl diphenyl methanol

Concentration Level (%)	*Mean Recovery (%)**	Standard Deviation*	*Relative Standard Deviation (%)**	Standard Error*
80	99.75	0.5000	0.5012	0.2887
100	99.05	0.7795	0.7869	0.4500
120	100.05	0.3483	0.3481	0.2011

*n=3

Table 13: Robustness data for Clotrimazole

Change in Flow Rate
Flow Rate (mL/min.)	Amount of drug present (µg/mL)			Level		Retention Time (min.)		Resolution (min.)	Amount found (%)
0.9	50			-1		2.932		1.89	100.04
1	50			0		2.765		1.72	99.94
1.1	50			+1		2.571		1.61	99.67
Change in % of Acetonitrile in Mobile Phase
% of Acetonitrile in Mobile Phase		Amount of drug present (µg/mL)	Level		Retention Time (min.)		Resolution (min.)		Amount found (%)
4		50	-1		2.789		1.81		99.98
5		50	0		2.765		1.72		100.1
6		50	+1		2.741		1.64		99.99

Table 14: Robustness data for 2-Chlorophenyl Diphenyl Methanol

Change in Flow Rate
Flow Rate (mL/min.)	Amount of drug present (µg/mL)	Level	Retention Time (min.)	Resolution (min.)	Amount found (%)
0.9	5	-1	2.321	1.78	99.99
1	5	0	2.307	1.72	99.96
1.1	5	+1	2.214	1.59	100.03
Change in % of Acetonitrile in Mobile Phase
% of Acetonitrile in Mobile Phase	Amount of drug present (µg/mL)	Level	Retention Time (min.)	Resolution (min.)	Amount found (%)
4	5	-1	2.389	1.75	99.93
5	5	0	2.307	1.72	99.83
6	5	+1	2.265	1.68	99.89

Table 15: Statistical validation of robustness data

Drug	Change in Flow Rate		Change in % of Acetonitrile in Mobile Phase
Drug	Mean Retention Time (min.)*	Standard Deviation*	Mean Retention Time (min.)*	Standard Deviation*
CLT	2.756	0.1807	2.765	0.0240
IMP -A	2.280	0.0581	2.320	0.063

CONCLUSION:

The method includes development and validation of RP-HPLC method for simultaneous estimation of Clotrimazole and its impurity from bulk and synthetic powder mixture. The method was validated using various parameters like linearity, precision, accuracy, robustness etc. and the method was found to be specific, accurate, precise, repeatable and reproducible.

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Received on 22.03.2016 Modified on 24.05.2016

Research J. Pharm. and Tech. 2018; 11(3): 894-898.

DOI: 10.5958/0974-360X.2018.00165.8

Parameter	Mean*	Standard Deviation*	% Relative Standard Deviation*	Standard Error*
Slope	1699	5.514	0.3245	2.251
Intercept	61323	34.038	0.0555	13.896
Regression coefficient (r²)	0.999	0.0005	0.0500	0.0002

Parameter	Mean*	Standard Deviation*	% Relative Standard Deviation*	Standard Error*
Slope	990.6	2.685	0.2710	1.096
Intercept	9864	6.501	0.0659	2.654
Regression coefficient (r²)	0.998	0.0008	0.0801	0.0003

Sr No.	Amount Present (mg/ 300 mg)	Amount found (mg/300 mg)	(%) found
1	50	49.98	99.9
2	50	49.90	99.8
3	50	50.10	100.2
4	50	49.96	99.9
5	50	50.10	100.2
6	50	50.05	100.1

Sr No.	Amount Present (mg/300 mg)	Amount found (mg/300 mg)	(%) found
1	5	4.99	99.9
2	5	4.98	99.7
3	5	4.99	99.8
4	5	4.99	99.9
5	5	5.10	100.2
6	5	5.05	100.1