INTRODUCTION:

Itraconazole is a powerful antifungal triazole agent prescribed to patients with fungal infections that are used to treat mycoses. The product can be administered orally or intravenously.

Figure 1: Chemical structure of Itraconazole

Itraconazole (ITZ) is used orally in capsule-shaped treatment of dermatophyte infections, superficial fungal infections and systemic fungal infections. In pharmaceutical formulations, few methods have been published for quality control and stability testing of Itraconazole, as the drug is the product of spectro fluorimetry used in the raw material and dosage forms for Itraconazole assay. RP-HPLC method is used for Itraconazole determination in human plasma. In this method, the chromatographic separation was performed using the fluorescence detector on an octadecylsilane column. It does have the downside to be time-consuming though. Both of these studies have also emphasized the need for a rapid and efficient quality-control review of the pharmaceutical formulations containing Itraconazole. Because these methods are costly, we have attempted to develop a more reliable, quick, and economical spectrophotometric method for analyzing Itraconazole in bulk and dosage forms with greater precision, accuracy, and sensitiveness^1,2.

MATERIALS AND METHODS:

Materials:

The Itraconazole was obtained as a gift sample from FDC limited, Mumbai. The other reagents used were analytical grade reagents, and used without further purification.

Preparation of standard stock solution:

Accurately weighed quantity of 100mg Itraconazole reference standard has been transferred to 100ml volumetric flask and dissolved and diluted to the methanol mark to give 1000μg/ml strength to a stock solution. 100μg/ml normal working solution was prepared by diluting 1 ml of stock solution to 10ml of methanol^3-7.

Preparation of solutions:

Table No. 1: Dilution table for calibration curve

Sr. No	Concentration in (ppm)	Stock solution in (ml)	Make up volume with methanol (ml)
1	2	0.2	9.8
2	4	0.4	9.6
3	6	0.6	9.4
4	8	0.8	9.2
5	10	1.0	9.0
6	12	1.2	8.8
7	14	1.4	8.6

Determination of Absorption maxima:

For the selection of analytical wavelength, µg/ml solution of Itraconazole was scanned in the spectrum mode from 200nm to 400nm separately.

Validation:

Linearity:

The linearity of the analytical method is determined using concentrations taken from 2 to 14μg/ml. From the regular stock solution of Itraconazole (100μg/ml), pipettate aliquots of 0.2 to 1.4ml of solution transferred to the series of 10ml volumetric flask and final volume made up to the Methanol mark as a diluent to form solutions of 2 to 14μg/ml Itraconazole. Such solutions were then screened as blank against diluent within the range of 200-400nm, and the calibration curve was then plotted as absorbance vs concentration to check the linear relationship between absorbance and Itraconazole concentration^8-10.

Acceptance Criteria:

The Coefficient of correlation does not more than 0.999.

Precision:

The system's precision is determined by assaying three determinations at 100% test concentrations (6, 10 and 14 ppm) and calculating relative standard deviation (% RSD). Precision study results were reported in percentage relative standard deviation¹¹.

Acceptance Criteria:

The relative standard deviation does not exceed 2%.

Accuracy:

An analytical method's accuracy is calculated by applying the method to the analyzed samples, to which defined analyte quantities have been applied. The accuracy is determined based on the test results, as the percentage of the analyte recovered through the assay. By calculating the percentage recovery of Itraconazole at three different points, 80%, 100%, and 120%, by standard addition procedure, the accuracy of the method developed was achieved¹².

Procedure for preparation of sample solution:

It was conducted at three stages, 80%, 100%, and 120% of the nominal concentration.

80% Accuracy solution (8 ppm):

It was prepared by diluting 0.8ml of the stock solution with methanol up to 10ml.

100% Accuracy solution (10 ppm):

This was prepared by diluting 1.0ml of the stock solution with methanol up to 10ml.

120% Accuracy solution (12 ppm):

It was prepared with methanol by diluting 1.2ml of the stock solution up to 10ml.

The sample was prepared and analyzed in triplicate using the Ultra Violet spectrophotometer wavelength: 262nm ¹³.

Acceptance Criteria:

Mean recovery will be between 98% - 102%.

Limit of Detection:

It is the lowest volume of analyte in a sample which can be detected but not generally quantitated under the specified experimental conditions. The detection limit can be calculated using the following equation as set out in the ICH guidelines^14-15.

LOD = 3.3 × σ / S

Where,

σ =Standard deviation of the response and

S = Slope of the corresponding calibration curve

Limit of Quantification:

It is the lowest analyte concentration in a sample which can be measured with reasonable precision and accuracy under defined experimental conditions. The quantification limit can be determined according to ICH guidelines using the following equation^14-15.

LOQ = 10 × σ / S

Where,

σ = Standard deviation of the response and

S = Slope of the corresponding calibration curve

RESULTS AND DISCUSSION:

Determination of Absorption maxima:

The maximum wavelength (λ max) was found to be 262 nm from the Itraconazole spectra, which was chosen for the study.

Figure 2: UV Spectra of Itraconazole

Method development and optimization:

The present paper describes the application of the absorption ratio method to the estimation of Itraconazole in bulk and solid dosage form. The method was validated for linearity, accuracy, precision, LOD, and LOQ.

Validation:

Linearity:

The calibration curve was taken in the range of 2-14 µg/ml for Itraconazole at, λ max 262nm. The Itraconazole was found to be linear within the concentration range of 2-14µg/ml with a regression coefficient of 0.9994 by the absorbance ratio method. There was an excellent correlation between absorbance and concentration.

Table 2: Calibration data of Itraconazole API by UV spectroscopy:

Concentration in PPM	Absorbance Mean ± SD (n=3)	% SD
2	0.104 ± 0.007	0.67
4	0.207 ± 0.002	0.17
6	0.302 ± 0.006	0.60
8	0.390 ± 0.008	0.80
10	0.487 ± 0.010	0.97
12	0.572 ± 0.006	0.55
14	0.678 ± 0.018	1.76

Figure 3: Calibration curve of Itraconazole in methanol by UV spectroscopy

Precision:

The intraday and interday precision of an analytical method were determined by assaying 3 test concentrations (6, 10 and 14ppm). % Relative Standard Deviation (% RSD) were calculated statistically. It was found to be less than 2%.

Table No. 3: Results of Intraday precision

Concentration in PPM	Absorbance Mean ± SD (n=3)	% SD
6	0.302 ± 0.006	0.60
10	0.487 ± 0.010	0.97
14	0.678 ± 0.018	1.76

Table No. 4: Results of Interday precision

Concentration in PPM	Absorbance Mean ± SD (n=3)	% SD
6	0.309 ± 0.012	1.17
10	0.483 ± 0.016	1.63
14	0.674 ± 0.006	0.64

Accuracy:

The accuracy was evaluated by Itraconazole's standard addition method of three replicate determinations of three different solutions containing 8, 10, and 12μg/ml (i.e. 80%, 100%, and 120% of nominal concentration). The overall recovery percentage was 99.40% for three different concentrations.

Table No. 5: Calculation for accuracy

Sample Identity	Conc. (PPM)	Abs.	Mean	Amount	% recovered	Average % recovered
Sample Identity	Conc. (PPM)	Abs.	Abs.	Recovered In ppm	% recovered	Average % recovered
Accuracy 80% set-1	8	0.385	0.389	7.87	98.38	99.35
	8	0.389		7.96	99.44
	8	0.392		8.02	100.24
Accuracy 100% set-1	10	0.472	0.483	9.72	97.18	99.58
	10	0.487		10.04	100.36
	10	0.491		10.12	101.21
Accuracy 120% set-1	12	0.576	0.575	11.93	99.38	99.26
	12	0.568		11.76	97.97
	12	0.582		12.05	100.44

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

Based on the absorbance ratio, the overlain spectra of both drugs showed that the peaks are well-resolved, thereby satisfying the requirement for optimum precision. The detection limit was 0.175μg/ml, and the quantification limit was 0.531μg/ml. For correct determination of Itraconazole, the criteria being the concentration should lie beyond the range 0.1–2.0.

Table 6: Summary of validation parameters of UV spectroscopy

Sr. No	Parameters		Results
1	λ _max		262 nm
2	Regression line equation		y = 0.0471x + 0.0143
3	Correlation coefficient (R2)		0.9994
4	Precision (% RSD)	Intraday	0.60-1.76
		Interday	0.64-1.17
5	Accuracy (%Recovery)		99.26-99.58
6	LOD		0.175 µg/ml
7	LOQ		0.531 µg/ml

CONCLUSION:

Fast, economical, rapid, and non-toxic UV spectrophotometric methods were developed for Itraconazole assessment. The method is also validated according to ICH guidelines so that the existing UV spectrophotometric device can also be used in bulk and solid dosage form for routine quality control analysis of Itraconazole and is found to be linear, accurate and precise.

ACKNOWLEDGEMENT:

Authors thankful to FDC limited, Mumbai (India) for providing gift sample of Itraconazole. Authors also thankful to Ashokrao Mane College of Pharmacy, PethVadgaon for providing facilities to carry out research work.

CONFLICT OF INTEREST:

The authors declare that they have no conflict of interest.

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Received on 15.08.2020 Revised on 17.09.2020

Asian J. Pharm. Res. 2021; 11(1):13-16.

DOI: 10.5958/2231-5691.2021.00004.6