INTRODUCTION:

Itraconazole in particular is active against Aspergillus. It is also licensed for use in blastomycosis, sporotrichosis, histoplasmosis and onchomycosis. Itraconazole is over 99% protein-bound and has virtually no penetration into cerebrospinal fluid. Chemically it is (2R,4S)-rel-1-(butan-2-yl)-4-{4-[4-(4-{[(2R,4S)-2-(2,4 dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-4-yl] methoxy} phenyl) piperazin-1-yl] phenyl}-4,5-dihydro-1H-1,2,4-triazol-5-one¹as shown in figure1. It is not official in any pharmacopoeias. Literature survey reveals that several methods like LCMS/MS^1-4, HPLC^5-7 and UV-Visible Spectrophotometric⁸ methods have been reported for its estimation in pharmaceutical dosage forms. The present work reports simple, rapid, precise and sensitive RP-HPLC method for routine analysis of Itraconazole in bulk and Pharmaceutical dosage forms.

Figure:1 Structure of Itraconazole

MATERIALS AND METHODS:

Chemicals and Solvents:

Itraconazole was obtained as a gift sample from MSN Laboratories, Hyderabad. Acetonitrile used of HPLC grade by MERCK. All other chemicals used throughout the experiment were of analytical grade unless specified.

Chromatographic Conditions:

Chromatographic separation was carried on Zorbax C18 (150× 4.6 mm, 5µm) column and the instrument used was Agilent 1100 series having a software of EZ chrome with a PDA detection at 262 nm. The injection volume was 10 µl with a flow rate of 1.0 ml/min with a run time of 7 minutes.

Preparation of Mobile Phase:

The mobile phase composition consisting of a binary mixture of acetonitrile and water in the ratio of 75:25 % v/v which was filtered through 0.45µ membrane filter paper before use.

Preparation of Standard Stock Solution:

Transfer accurately weighed quantity of 100 mg of itraconazole reference standard into a 100 ml volumetric flask. Added about 70 ml of methanol and sonicated for about 5 min. and made the volume up to the mark with methanol and filtered through 0.45 µ Millipore pvdf filter.

Preparation of Working Standard Solution:

1.0 ml of the above standard stock solution was pipette out and transferred into a 10 ml volumetric flask and then volume was made up to the mark with methanol to obtain the working concentration of 100 µg/ml. An aliquot was further diluted in a 10 ml volumetric flask with methanol to obtain final concentration of 16 µg/ml.

Assay in Capsules:

20 capsules were weighed and average weight of each capsule was calculated. An accurately weighed and transferred 561 mg of powder equivalent to 100 mg of itraconazole into a 100 ml volumetric flask and added 70 ml of methanol and sonicated for 5 min., and then volume was made up to the mark with methanol and filtered through 0.45 µ Millipore pvdf filter. Then 1.0 ml of above filtered solution was pipette out and transferred into a 10 ml volumetric flask and the volume was made up to the mark with methanol to obtain the final concentration of 100 µg/ml and an aliquot of the above solution was further diluted in a 10 ml volumetric flask with methanol to obtain the final concentration of 16 µg/ml for the assay procedure. Then 10 mL of both standard and sample solutions were injected in duplicate into the chromatographic system, chromatograms were recorded and peak areas were measured.

Method Validation:

Validation of the proposed RP-HPLC method was carried out as per ICH guidelines by means of following parameters.

System Suitability:

System suitability test was carried out by injecting 10 mL of the working standard solution five times into the chromatographic system and chromatograms were recorded. The system suitability parameters were evaluated.

Linearity:

Linearity standard solutions of different concentrations ranging from 4-40 µg/ml of itraconazole were prepared from the working standard solution (100 µg/ml) by diluting aliquots from 0.4 to 4.0 ml in 10 ml volumetric flasks for linearity studies. Then 10 µl of each solution was injected in to the HPLC system under the optimized chromatographic conditions and the peak area responses were recorded. Standard calibration curves were plotted between peak areas versus concentrations and regression parameters were calculated from the graph. All the measurements were carried out three times for each concentration.

Precision:

Method precision and system precision were performed by injecting five times of concentration of 16 µg/ml of both sample and standard solutions in to the chromatographic system respectively. Peak areas and % RSD were calculated from the chromatograms.

Accuracy:

Accuracy was determined by adding the known amount of standard drug to the pre analyzed concentrations of assay samples by standard addition method. The recovery studies were carried by injecting into the chromatographic system in triplicate of three different levels of concentrations at 50 %, 100 % and 150 % by spiking standard drug solution to the sample. Chromatograms were recorded and amount found and % mean recoveries were calculated from the peak area responses obtained.

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

The sensitivity of HPLC was determined from LOD and LOQ which is calculated from the calibration curve using the following equations:

LOD = 3.3 σ/S

LOQ = 10 σ/S

Where σ = Standard deviation of y intercept of regression line

S = Slope of the calibration curve

Robustness:

Robustness is the measure of the ability of an analytical method to remain unaffected by small but deliberate variations in method parameters, such as temperature change (± 5 °C), mobile phase organic composition (± 10 %), flow rate (± 0.2 ml/min), wavelength (± 2 nm) and temperature (± 5 ^oC). The sample solution containing concentration of 16 µg/ml of itraconazole was injected under the varied conditions and change in the peak area responses was recorded.

Ruggedness:

As a part of ruggedness, the change from analyst to analyst, column to column and instrument to instrument was performed.

Specificity:

According to ICH guidelines, specificity is defined as the ability to assess unequivocally the analyte in the presence of components that may be expected to be present, such as impurities, degradation products and matrix components. In the case of assay, demonstration of specificity requires that it can be shown that the procedure is unaffected by the presence of impurities or excipients.

RESULTS AND DISCUSSIONS:

The proposed RP-HPLC method for the estimation of itraconazole in capsule dosage form was carried out using Zorbax C18 column (150× 4.6 mm, 5µm) with a mobile phase composition of acetonitrile and water in the ratio of 75:25 %v/v at a flow rate of 1.0 ml/min and the injection volume at 10 µl with 7 minutes runtime. The detection was carried out using PDA detector at 262 nm and proposed method was validated as per ICH guidelines and results were reported.

System Suitability Studies:

The column efficiency, peak asymmetry and % RSD were calculated using standard drug solution of itraconazole and the values obtained demonstrated the suitability of the system for analysis of itraconazole in capsule dosage form. The results are reported in table 1.

Table 1: System suitability parameters of itraconazole

Parameter	Results
Parameter	Standard	Sample
Retention time (min)	3.753	3.733
Theoretical plates	8609	5287
USP Tailing factor	1.13	1.21
Peak area	1001731	1097997
% RSD	0.52	0.46

Linearity:

The calibration curve was found to be linear over the concentration range of 4 - 40 µg/ml (as shown in Figure 2). The correlation coefficient (R²) was found to be 0.9993. The calibration data are reported in table 2.

Figure: 2 Calibration curve of itraconazole

Precision:

The proposed HPLC method was found to be highly precise as the % RSD values of six replicate injections of the standard and sample solutions of itraconazole were found to be below 2.0 % which is under the limit as per recommendations of ICH guidelines. The low % RSD values indicate the proposed method was very precise. The results are reported in table 3.

Assay of Marketed Formulation:

Capsule dosage form was analyzed and the results of assay showed that the amount of itraconazole was in good agreement with the label claim of formulation as indicated by % assay which was found to be 99.92 % for itraconazole. The retention time of standard and sample was found to be 3.753 min and 3.733 min respectively as shown in figure 3 and figure 4. All the results were found to be within the limits and therefore the proposed method was found to be free from interferences from excipients.

Table 2: Data of Linearity studies of itraconazole

S.No.	Concentration (µg/ml)	Peak area	USP Plate count	Retention time (min)	Statistical Data
1.	4	251422	5637	3.740	Slope=61262 Intercept= -3908 R²=0.9993
2.	8	514636	5730	3.733
3.	12	701531	5561	3.733
4.	16	998514	5399	3.733
5.	20	1164803	5618	3.747
6.	24	1455970	5287	3.720
7.	28	1735241	5445	3.733
8.	32	1958463	5828	3.720
9.	36	2204877	5726	3.727
10.	40	2456123	5938	3.753

Figure: 3 Chromatogram of standard peak of itraconazole

Figure: 4 Chromatogram of sample peak of itraconazole

Limit of detection (LOD) and Limit of Quantification (LOQ):

LOD and LOQ values of itraconazole were determined from their respective calibration curves. LOD and LOQ were found to be 0.624 µg/ml and 2.26 µg/ml respectively. The results are shown in table 3 and figure 5 and figure 6.

Figure: 5 Chromatogram of LOD solution

Table 3: Validation Parameters of the Proposed RP-HPLC Method

Parameter	Results
Beer’s law limit (µg/ml)	4 - 40
Regression equation	y =61262 x -3908
Correlation coefficient (r²)	0.9993
LOD (µg/ml)	0.624
LOQ (µg/ml)	0.54
Repeatability (% RSD) *	0.23
Intra - day precision (% RSD) *	0.34
Inter - day precision (% RSD) *	0.45
% Assay	99.92

Figure: 6 Chromatogram of LOQ solution

Accuracy:

The mean % recovery of itraconazole was found to be 99.83 - 100.12. Hence the proposed RP-HPLC method was said to be accurate. The results are shown in table 4.

Table 4: Recovery Studies of itraconazole

Name of the drug	Level	Average Peak area	Mean % Recovery ± SD*	% RSD*
Itraconazole	50 %	1464490	99.83 ± 0.55	0.56
	100 %	1960227	100.12 ± 0.25	0.24
	150 %	2424980	99.96 ± 0.38	039

*Mean of three determinations.

Robustness:

The developed method is robust with deliberate changes with variation of organic mobile phase composition, flow rate, temperature and wavelength as % RSD shows below 2.0 % with meeting system suitability parameters as per ICH guidelines. The results are given in table 5.

CONCLUSION:

The developed and validated RP-HPLC method was found to be rapid, accurate, precise and reliable which is useful for routine quantification of itraconazole in quality control analysis and in pharmaceutical dosage form. The system suitability parameters indicate good sensitivity, more ruggedness and robustness of the method. Therefore the proposed method has proven to be simple, selective and specific which meeting the ICH guidelines for analytical method validation.

ACKNOWLEDGMENTS:

The author is grateful to Bapatla College of Pharmacy for providing the necessary facilities during the course of study and also gratified to MSN laboratories Ltd., Hyderabad for providing the gift sample of itraconazole.

REFERENCES:

1. Cox S, Orosz S, Brunette J and Frazier D. Micro assay determination of Itraconazole and hydroxyl itraconazole in plasma and tissue biopsies. Journal of Chromatography B. 702; 1997: 175-180.

2. Vijaya Bharathi D , Kishore Kumar Hothvidya Sagar P.V, Sanagapati Sirish Kumar, Pandu Ranga Reddy, Naidu A, Mullangi Ramesh. Development and validation of a highly sensitive and robust LCMS/MS with electro spray ionization method for simultaneous estimation of Itraconazole in Human plasma: Application to a bioequivalence study. Journal of chromatography. B. 868(2); 2008: 70-76.

3. Constantinos Kousoulos, Georgia Tsatsou, Constantinos Apostolou, Yannis Dotsikas and Yannis L. Loukas. Development of a high throughput method for the determination of Itraconazole and its hydroxyl metabolite in human plasma, employing automated liquid-liquid extraction based on 96-well format plates and LC/MS/MS. Analytical and Bio analytical chemistry. 384(1); 2006: 199-207.

4. Sarath Chandiran, K.N. Jayaveera and Raghunadha Reddy. S. Development and validation of High-Throughput Liquid Chromatography-Tandem Mass Spectroscopic Method for Quantification of Itraconazole and its metabolite in Human plasma.Der Pharmacia Lettre. 3(2); 2011: 316-328.

5. Stefanie Redmann and Bruce G. Charles. A rapid HPLC method with flurometric detection for determination of plasma Itraconazole and hydroxyl-itraconazole concentrations in cystic fibrosis children with allergic bronchopulmonary aspergillosis. Biomedical Chromatography. 20(4); 2006: 343-348.

6. Ming Yao, Laishun Chen, Nuggehally R Srinivas, quantification of Itraconazole in rat heparinized plasma by LCMS. Journal of Chromatography B: Biomedical sciences and Applications. 752 (1, 5); 2001: 9-16.

7. Srvatsan V,. Dasgupta A.K, Prashant Kale, Rama Raju Datla, Devangi Soni, Mahendra Patel, Rakesh Patel and Chandrakant Mavadhiya. Simultaneous determination of Itraconazole and hydroxyitraconazole in human plasma by high performance liquid chromatography. Journal of Chromatography. 1031(1-2); 2004: 307-313.

8. Shalin K. Parikh, Ankit D. Patel,. J.B. Dave, C.N. Patel and D.J. Sen. Development and validation of UV Spectrophotometric method for estimation of itraconazole in bulk and pharmaceutical form. International Journal of Drug Development and Research. 3(2); 2011: 324-328.

Received on 02.10.2014 Modified on 25.10.2014

Asian J. Research Chem 8(4): April 2015; Page 236-240

DOI: 10.5958/0974-4150.2015.00041.3

S. No.	Parameter	Change Level	Retention time (min)	USP plate count	USP tailing	% RSD
1.	Flow rate (± 0.2 ml/min)	0.8	4.707	8286	0.96	0.32
1.	Flow rate (± 0.2 ml/min)	1.2	2.983	6112	0.97	0.63
2.	Mobile organic phase composition (± 5 % v/v)	70: 30	3.467	5954	1.1	0.43
2.	Mobile organic phase composition (± 5 % v/v)	80: 20	2.507	5782	0.94	0.57
3.	Wavelength (± 5 nm)	257	3.760	7140	1.03	0.52
3.	Wavelength (± 5 nm)	267	3.773	7202	1.15	0.44

Table 3: Validation Parameters of the Proposed RP-HPLC Method

Parameter

Results

Beer’s law limit (µg/ml)

4 - 40

Regression equation

y =61262 x -3908

Correlation coefficient (r2)

0.9993

LOD (µg/ml)

0.624

LOQ (µg/ml)

0.54

Repeatability (% RSD) *

0.23

Intra - day precision (% RSD) *

0.34

Inter - day precision (% RSD) *

0.45

% Assay

99.92

Correlation coefficient (r²)