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.
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 |
Fig:
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.
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Received on 25.10.2013 Accepted on 30.11.2013
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