Development of validated RP-HPLC method for the estimation of Itraconazole in pure and pharmaceutical dosage form

 

B. Thangabalan, M. Salomi*, N. Sunitha, S. Manohar Babu

Department of Pharmaceutical Analysis, SIMS College of Pharmacy, Mangaldas Nagar, Guntur-522 002 (A.P.)

*Corresponding Author E-mail salomi.bpharm@gmail.com

 

 

ABSTRACT:

A simple, fast and precise RP–HPLC method was developed for the quantification of Itraconazole in pure and pharmaceutical dosage form. The quantification was carried out using Dionex C18 4.6 X 250mm, 5μm enhanced polar selectivity column and mobile phase comprised of methanol and pH 7.5 potassium dihydrogen phosphate in the ratio of 40:60 and degassed under ultrasonication. The flow rate was 1.5ml/min and the effluent was monitored at 306nm. The retention time of Itraconazole was found to be 5.2 min. The method was validated in terms of linearity, precision, accuracy, specificity, robustness, limit of detection and limit of quantitation in accordance with ICH guidelines. Linearity of Itraconazole was in the range of 200-600 μg/mL. The percentage recoveries of Itraconazole were 99.33% to 99.66% from the capsule formulation. The proposed method is suitable for determination of Itraconazole in pharmaceutical dosage form.

 

KEYWORDS: Chromatography, Itraconazole, Method validation.

 


 

INTRODUCTION:

Itraconazole is chemically 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- 4yl]methoxy}phenyl)piperazin-1-yl]phenyl}-4,5-dihydro-1H-1,2,4-triazol-5-one. (Figure:1), is an imidazole/Triazole1,2 type antifungal agent. Itraconazole is a highly selective inhibitor of fungal cytochrome P-450 sterol C-14 α-demethylation via the inhibition of the enzyme cytochrome P-450 14α-demethylase.

 

Figure 1: Chemical structure of Itraconazole

 

Several HPLC3, and LC/MS-MS4-6 methods have been reported for the analysis of Memantine in plasma that suffer from either undesirably long chromatographic run times and requirement for gradient analysis or use of an internal standard one spectrophotometric method7 have also been reported. One RP-HPLC method8 with have also been reported which suffer from long chromatographic run time and also high retention time.

 

The objective of this study was to develop reverse phase high performance liquid chromatography method for the estimation of Itraconazole in pure and capsule dosage form without any derivatization and having short retention time. This method was found to be linear, precise, accurate, sensitive, specific, and robust, and therefore suitable for routine analysis.

 

MATERIALS AND METHOD:

Chemicals and Reagents:

Itraconazole was obtained as a gift from Hetero Drugs Ltd, Hyderabad.  HPLC grade double distilled water and analytical grade potassium dihydrogen phosphate, methanol obtained from Qualigens Fine Chemicals Ltd, Mumbai. HPLC grade water was used to prepare all solutions.


Text Box: 5.278
 


Figure 2: Standard chromatogram for Itraconazole

 

 


HPLC Instrumentation and Chromatographic conditions:

The analytical separations were carried out on a Water HPLC system equipped with Photo Diode Array detector. The output of signal was monitored and integrated using Empower2 software. The analytical column was Dionex C18 (250 × 4.6mm, 5µ). Mobile phase consisted of pH 7.5 potassium Dihydrogen Ortho Phosphate, methanol in the ratio of 50:40. Mobile phase was mixed, filtered through 0.45µmembrane filter and degassed under ultrasonication. The methanol was used as diluent. The flow rate was 1.5 ml/min and runtime was 7 minutes. The column was maintained at 450C temperature. UV detection was measured at 306 nm and the volume of sample injected was 10 μl.

 

Preparation of standard stock solution:

Transfer accurately weighed quantity of 100 mg of Itraconazole working standard into 100ml volumetric flask. Add about 50 ml of methanol and sonicate for about 30min and make the volume up to the mark with methanol and filter through 0.45 µ Millipore PVDF filter.

 

Preparation of Working standard solution:

4 mL of standard stock solution was pipetted out and transferred into a 10 mL volumetric flask. Volume was made up to the mark with methanol. The standard chromatogram for Itraconazole (100μg/ml) was shown in figure 2.

 

Preparation of sample solutions:

Twenty capsules were weighed average weight of capsule was determined. A portion of the powder equivalent to about 100mg of Itraconazole was weighed accurately and transferred into100mL volumetric flask and added 50 ml of methanol and sonicated for 30 min, and made volume upto the mark with methanol and filter through 0.45 µ Millipore PVDF filter. 4 mL of above was pipetted out and transferred into a 10 mL volumetric flask. Volume was made up to the mark with methanol and used for analysis.

 

RESULTS AND DISCUSSION:

HPLC method development and optimization:

To optimize the chromatographic conditions, different columns, mobile phases, flow rates etc., were tested.  Methanol and pH 7.5 potassium dihydrogen ortho phosphate in the ratio of 40:50 was preferred as mobile phase because it resulted in a greater response to Itraconazole after several preliminary investigatory runs compared with the different mobile phase combinations. The effect of the flow rate was studied in the range 0.9 to 1.5 ml/min and 1.5 ml/min was preferred to be effective. Under these conditions, the analyte peak obtained was well-defined and free from tailing. The retention time (RT) was found to be 5.278 min. The optimized chromatographic parameters were listed in table 1.

 

Table 1: Optimized chromatographic parameters

Optimized Chromatographic parameters

Elution                   Isocratic

Mobile phase          methanol and pH 7.5 KH2PO4  (40:50)

Column                  Dionex C18column

Flow rate                1.5ml/min

Detection                306nm

Injection volume    10μl

Temperature           450C

Retention time        5.278 min

Run time                 7 min

Concentration         200-600μg/ml

 

Validation of the method:

When method development and optimization are complete, it is necessary to accomplish method validation. The validation studies include linear range (correlation coefficient), method precision (RSD, %), method accuracy (% recovery and RSD, %), sensitivity studies (LOD and LOQ), and robustness.

 

System suitability studies: 

System-suitability tests are an integral part of method development and are used to ensure adequate performance of the chromatographic system. Retention time (RT), number of theoretical plates (N), tailing factor (T), and peak asymmetry (AS), resolution (RS) were evaluated. The system suitability test was performed using five replicate injections of standards before analysis of samples. The system suitability method acceptance criteria set in each validation run were: capacity factor > 2.0, tailing factor ≤ 2.0 and theoretical plates > 2000. In all cases, the relative standard deviation (R.S.D) for the analytic peak area for two consecutive injections was < 2.0%. System suitability parameters were shown in table 2.

 

Table 2: System suitability parameters

Parameters

Values

Retention time

5.278 min

Theoretical plates

8609.000

Tailing factor

1.138

 

Linearity:

The linearity of the method was evaluated by preparing six series of standard solutions of Itraconazole in the range of

200 - 600 µg/ml in methanol and injecting the solutions into the HPLC system. Excellent correlation between Itraconazole peak area and concentration was observed with R2 = 0.999 (Figure.3). The regression equation was found to be Y = y = 17820x + 10162. Statistical data are presented in table 3 and the calibration curve was shown in figure 3.

 

Table 3: Linearity results for Itraconazole

S.No

Concentration (μg/ml)

Area  AU (n=6)

1

200

3564715

2

300

5346123

3

400

7179256

4

500

8907315

5

600

10694359

 

Figure 3: Calibration curve of Itraconazole

 

Precision:

System precision: (Repeatability)

To study precision, five replicate standard solutions of Itraconazole (400µg/ml) were prepared and analyzed using the proposed method. The percent relative standard deviation (% RSD) for peak responses was calculated. Results of system precision studies were shown in table 4.

 

Table 4: Results of system precision for Itraconazole

S.No

Rt (min)

Peak Area (AU)

1

5.278

7139718

2

5.270

7141270

3

5.264

7180014

4

5.271

7189738

5

5.268

7199631

MEAN

5.2702

7170074

S.D

0.005119

27885

%RSD

0.0971

0.4

 

Method precision: (Reproducibility)

The intraday and inter-day precision of the proposed method was determined by analyzing the corresponding responses 5 times on the same day and on different days for concentration of sample solutions of 400µg/ml.  The result was reported in terms of relative standard deviation (% RSD). Results of method precision studies were shown in table 5.

 

Table 5: Results of Method precision for Memantine

S. No

Itraconazole           Standard Area = 7152073

Peak Area

% Labelled Claim

1

7126360

99.62

2

7148338

99.94

3

7173736

100.3

4

7137998

99.80

5

7163444

100.12

6

7140485

99.82

MEAN

7148393

99.83

S.D

17463

0.23

%RSD

0.2

0.244

 

Accuracy:

Accuracy of the method was confirmed by the standard addition method, which was carried out by performing recovery studies at 3 different concentrations 50%, 100% and 150% of these expected, in accordance with ICH guidelines, by replicate analysis (n=3).For a pre analyzed sample solution 100 µg/ml, 50%, 100%, 150% standard drug solution was added and percentage drug content was measured. The closeness of obtained value to the true value indicates that the proposed method is accurate. Recovery studies were shown in table 6.

 

Limit of Detection and Quantitation:

The LOD and LOQ were calculated based on the S/N ratio of the standard injection. The chromatograms for LOD and LOQ were shown in fig: 4 and 5.

 


 

Table 6: Results of recovery studies for Itraconazole

Name

Accuracy

Peak area

% Recovery

Average % Recovery

Itraconazole

50 %

50 %

50 %

3578856

3547046

3573014

100

99

99

 

99.33 + 0.05

100

100

100

7114998

7165041

7171581

99

100

100

 

99.66+ 0.05

150

150

150

10680557

10681963

10683103

99

99

100

 

99.33 + 0.05

 

Text Box: 5.270Fig: 4. Chromatogram for LOD

 

Fig: 5. Chromatogram for LOQ

 

 


Limit of detection and limit of quantification was calculated based on S/N ratio. The S/N ratio was found to be 645.374091.

Ø  LOD of itraconazole was found to be 1.8594 µg/ml

Ø  LOQ of itraconazole was found to be 6.197 µg/ml

 

Specificity

Specificity of an analytical method is its ability to measure the analyte accurately and specifically in the presence of component that may be expected to be present in the sample matrix. Chromatograms of standard and sample solutions were compared in order to provide an indication of specificity of the method.

 

Robustness:

The robustness study was performed to evaluate the influence of small but deliberate variation in the chromatographic condition. The Robustness was performed at different flow rates and at different column temperatures by using working standard solution of itraconazole. Robustness values for change in flow rate and change in temperature were given in Table: 7.

Table 7:  Robustness data of Itraconazole

Analyte

Itraconazole

Itraconazole

Flow

Flow 1(1.4mL/min)

Flow 2(1.6mL/min)

Retention time(min)

5.86

4.9

Tailing

1.140

1.169

Theoretical plates

7619

6084

Temperature

Temp 1(430C)

Temp 2(470C)

Retention time(min)

5.264

5.184

Tailing

1.189

1.123

Theoretical plates

6008

6419

 

Assay of pharmaceutical formulation:

The proposed validated method was successfully applied to determine Itraconazole in their capsule dosage form (Namenda). And the % Assay results were shown in table 8.

 

Table 8: Results of % assay

S.No

Amount Found (mg/ capsule)

% Assay

1

2

3

99.905

99.961

99.954

99.33

99.66

99.64

 

Table 9: Summary of validated parameters for proposed method

Parameter

Result

Linearity range

 200 – 600  µg/ml 

Regression equation

 y = 17820x + 10162

Slope

17820

Intercept   

10162

Correlation coefficient 

0.999

System precision (% RSD, n=5)

0.402

Method precision (% RSD, n=5)

0.2402

LOD (µg/ml)

1.8594

LOQ (µg/ml)

6.197

% Recovery (Accuracy, n =3)

99.33%

% Assay (% Assay, n=3)

99.50%

 

CONCLUSION:

A simple, rapid, accurate, and precise RP-HPLC method for the analysis of Itraconazole in pure and in capsule dosage forms had been developed and validated in accordance with ICH guidelines. The RP-HPLC method developed is cost-effective due to short retention time which enabled analysis of Itraconazole samples with a small amount of mobile phase. From the % RSD values of precision and recovery studies the method was found to be precise and accurate. The low detection and quantification limits achieved indicate the method is very sensitive. The robustness data gathered during method validation showed that the method is not susceptible to small changes in chromatographic conditions. The proposed RP-HPLC method developed by the author is suitable for routine analysis and quality assessment of Itraconazole in pharmaceutical products.

 

REFERENCES:

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Received on 25.10.2013          Accepted on 30.11.2013        

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Asian J. Pharm. Ana. 3(4): Oct. - Dec. 2013; Page  119-123