INTERNATIONAL JOURNAL OF PHARMACEUTICAL RESEARCH … 1424.pdf · The sample of Miconazole was...
Transcript of INTERNATIONAL JOURNAL OF PHARMACEUTICAL RESEARCH … 1424.pdf · The sample of Miconazole was...
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METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION
OF MICONAZOLE AND ORNIDAZOLE IN PHARMACEUTICAL DOSAGE FORM BY
RP-HPLC
ASEEM VERMA 1, PROF. SUSHIL AGARWAL2 1. Student, M.Pharma (Quality Assurance) 4th Semester, Student of ,Mahatma Gandhi College of Pharmaceutical Sciences, Jaipur (Rajasthan)
2. M Pharma. Mahatma Gandhi College of Pharmaceutical Sciences, Jaipur (Rajasthan).
Accepted Date: 10/01/2018; Published Date: 27/02/2018
Abstract: The liposomes are concentric bilayered vesicle in which an aqueous volume is entirely enclosed by the membranous lipid bilayer mainly composed of the natural or synthetic phospholipids. The liposomes are one of unique drug delivery system which can be use in controlling & targeting drug delivery system. The liposomes are generally classified based upon the structure, method of preparation, composition and application, conventional liposome, and specialty liposome. The liposomes can be filled with drugs & used to deliver the drugs for cancer & other diseases. This review is focused on the various aspects of liposomes like classification, methods of preparation, characterization and applications of liposomes.
Keywords: Liposomes, Phospholipids, Targeting drug delivery system, Bilayered
INTERNATIONAL JOURNAL OF
PHARMACEUTICAL RESEARCH AND BIO-SCIENCE
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Corresponding Author: MR. ASEEM VERMA
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Aseem Verma, IJPRBS, 2018; Volume 7(1): 56-86
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INTRODUCTION
Indian sub-continent, being Tropical region has high temperature and high humidity conditions,
which are favourable for the growth of fungal and other infections. Presently, the treatment is
the Azole group/antibiotics. With continuous efforts, the combination of Azole have become a
preferred choice for the better response and therapeutics efficacy. To develop separate
methods of quantification for two drugs requires more efforts for the chemist and less output
in terms of number of samples analysed. In lieu of above, efforts are now being put in
developing a common analytical method for multicomponent formulation analysis.
Thus, despite of various analytical methods developed, still no work has been done in
developing Simultaneous method of Quantification of these drugs. As there is no Stability
indicating HPLC method reported for these drugs in combined dosage forms.
Therefore it was wisely thought worthwhile to develop Stability indicating RP-HPLC method for
simultaneous estimation of Micronazole and Ornidazole in the combined Pharmaceutical
dosage form. Moreover, there are two methods develop for the simultaneous estimation of
Ornidazole and Miconazole by HPLC, one is for cream formulation and the other has been
developed for Tablet formulation. Moreover the run time for Chromatogram is higher, thus the
method is superior, accurate and more reliable than the developed one.
Looking from the QA point of view, the method can be successfully adopted for routine quality
analysis of Ornidazole and Miconazole in combined tablet dosage from without any
interferences from common excipients and impurities. Also method can be transferred to utilize
for routine Laboratory analysis and Assay of Ornidazole and Miconazole in their tablet dosage
form.
DRUG PROFILE9-10
1.4.1. MICONAZOLE9
INTRODUCTION
Name Miconazole
Official in IP-2010, BP-2009, USP30-NF25
Description Miconazole is an imidazole antifungal agent that is used topically and by intravenous infusion.
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Structure
Chemical Formula
C18H14Cl4N2O
Mol. Weight 416.13
IUPAC Name 1-[2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxy]ethyl]-1-Himidazole
Categories Anti infective
Solubility Very slightly soluble in Water, freely soluble in Methanol, soluble in Ethanol (96 per cent)
PHARMACOLOGY
Classes Benzoids
Mechanism of action Miconazole interacts with 14αdemethylase, a cytochrome P450enzyme necessary to convert lanosterol to ergosterol. As ergosterol is an essential component membrane, inhibition of its synthesis results in increased cellular permeability causing leakage of cellular contents.
PROPERTIES
State Solid.
CAS NO. 22916-47-8
Melting point 159-163°C
Experimental properties
Property Value
Log P 5.86
PKa 6.77 (Basic)
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1.4.2. ORNIDAZOLE10
INTRODUCTION
Name Ornidazole
Official in IP 2010
Description Ornidazole is a drug that cures some protozoan infections.It has been investigated for use in Crohn's disease after bowel resection
Structure
Chemical Formula
C7H10ClN3O3
Mol.Wt 219.625 g/mol
IUPAC 1-chloro-3-(2-methyl-5-nitro-1H-imidazol-1-yl)propan-2-ol
Categories Anti amoebiasis
Solubility Soluble in chloroform and in methanol.=, Practically insoluble in water
PHARMACOLOGY
Classes Tissue amoebecides
Mechanism of action
It causes the inhibition of DNA synthesis resulting in to loss of helical structure.
PROPERTIES
State Solid.
CAS NO. 16773-42-5
Melting point 77-78°C
Experimental Properties
Property Value
Log P 1.28
pKa 2.4
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AIM: Literature review reveals that numbers of individual analytical methods available for
estimation of Miconazole and Ornidazole in their individual dosage forms. But no method has
been reported for simultaneous estimation of Miconazole and Ornidazole in combined
pharmaceutical dosage form by RP-HPLC. So it is develop reverse phase high performance liquid
chromatographic method for simultaneous estimation of Miconazole and Ornidazole in
Combined Dosage Form. So Aim of present work is to develop simple, accurate, precise, rapid,
specific, sensitive and selective Reverse Phase HPLC method for simultaneous estimation of
Miconazole and Ornidazole.
3.2 OBJECTIVE
1) To develop HPLC method for simultaneous estimation of, Miconazole and Ornidazole in
pharmaceutical dosage form.
2) Applying the newly developed, validated analytical method for the estimation of Miconazole
and Ornidazole in formulations.
The Premilinary Analysis:
MICONAZOLE:
1) Description
The sample of Miconazole was observed for its color and texture.
2) Melting point
The sample of Miconazole was taken in capillary and place into the melting point apparatus.
Observed the melting point and compared with the reference.
3) Identification Test
Potassium Bromide IR disc was prepared using 1mg of Miconazole on Hydraulic Pellet Press.
This disc was scanned in the region of 4000–400cm-1 in FTIR and obtained IR spectrum was
compared with the reference spectrum of Miconazole.
4) Solubility
The sample of Miconazole was taken in test tubes and observed for solubility in various
solvents like water, methanol, 0.1 N Hcl and 0.1 N NaOH.
ORNIDAZOLE:
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1) Description
The sample of Ornidazole was observed for its and texture.
2) Melting point
The sample of Ornidazole was taken in capillary and place into the melting point apparatus.
Observed the melting point and compared with the reference.
3) Identification Test
Potassium Bromide IR disc was prepared using 1mg of Ornidazole on Hydraulic Pellet Press. This
disc was scanned in the region 0f 4000–400cm-1 in FTIR and obtained IR spectrum was
compared with the reference spectrum of Ornidazole.
4) Solubility
The sample of Ornidazole was taken in test tubes and observed for solubility in various solvents
like water, methanol, 0.1 N Hcl and 0.1 N NaOH.
>Development and Validation for Simultaneous Estimation of Miconazole and Ornidazole By
RP-HPLC.
Apparatus and Instruments:
Model: Shimadzu HPLC System
Pump:-LC-20 AT
Column: C18 (250 mm × 4.6 mm i.d.,5µm)
Injector: 20μL fixed loop.
Detector: UV Detector
Software: Spinchrom
Analytical balance: Electronic Balance (Shimadzu AUX220)
REAGENTS AND MATERIALS:
Miconazole was procured from Yash Pharma
Ornidazole was procured from Yash Pharma
Water
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Methanol
Acetonitrile
Hydrogen phosphate
Preparation of standard solutions:
(A) Ornidazole standard stock solution: (500 μg/mL)
A 50 mg of Ornidazole was weighed and transferred to a 100 mL volumetric flask. Volume was
made up to the mark with methanol.
(B) Miconazole standard stock solution: (100 μg/mL)
A 10 mg of Miconazole was weighed and transferred to a 100 mL volumetric flask. Volume was
made up to the mark with methanol.
(C) Preparation of standard solution of binary mixtures of Ornidazole (50 μg/mL) and
Miconazole (10 μg/mL)
Take 1 mL from the Ornidazole stock solution and 1mL from Miconazole stock solution and
transferred to 10 mL volumetric flask and volume made up to the mark by mobile phase which
was used in particular trials.
Selection of wavelength:
Standard solution of Ornidazole (5 μg/mL) and Standard solution of Miconazole (10 μg/mL)
were scanned between 200-400 nm using UV-visible spectrophotometer.
Both solutions were scanned between 200 - 400 nm.
Wavelength was selected from the overlay spectra of above solutions.
Chromatographic separation:
Standard solutions of 25-75 μg/ml of Ornidazole and 5-15 μg/ml of Miconazole were injected in
column with 20 μl micro-syringes. The chromatogram was run for appropriate minutes with
mobile phase Buffer(pH 5): Methanol (50:50)
The detection was carried out at wavelength 240 nm. The chromatogram was stopped after
separation achieved completely. Data related to peak like area, height, retention time,
resolution etc were recorded using software.
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System suitability test:
It is an integral part of chromatographic method. These tests are used to verify that the
resolution and reproducibility of the system are adequate for the analysis to be
performed. System suitability tests are based on the concept that the equipment,
electronics, analytical operations and samples constitute an integral system that can be
evaluated as a whole. System suitability testing provides assurance that the method will
provide accurate and precise data for its intended use.
Chromatographic conditions:
Column: C18 (250 mm × 4.6 mm i.d.,5µm)
Mobile Phase: Buffer (pH 5):Methanol(50:50)
Flow Rate : 1.0 ml/min
Detection Wavelength: 240 nm
Runtime: 10 min
Injection volume : 20.0 μl
Validation of Developed RP-HPLC method
Linearity
The linearity for Miconazole and Ornidazole were assessed by analysis of combined
standard solution in range of 5-15 μg/ml and 25-75 μg/ml respectively,
5,7.5,10,12.5,15 ml solutions were pipette out from the Stock solution of Ornidazole(500
μg/ml) and Miconazole(100 μg/ml) and transfer to 100 ml volumetric flask and make up with
mobile phase to obtain 5,7.5,10,12.5,15 μg/ml and 25,37.5,50,62.5 and 75 μg/ml for
Miconazole and Ornidazole respectively.In term of slope, intercept and correlation co-efficient
value. The graph of peak area obtained verses respective concentration was plotted.
Precision: Results should be expressed as Relative standard deviation (RSD) or coefficient
of variance.
A. Repeatability
Standard solution containing Ornidazole (50µg/ml) and Miconazole (10 µg/ml) was injected six
times and areas of peaks were measured and % R.S.D. was calculated.
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B. Intra-day precision
Standard solution containing (25, 50, 75 µg/ml) of Ornidazole and (5, 10, 15 µg/ml) of
Miconazole were analyzed three times on the same day and % R.S.D was calculated.
C. Inter-day precision
Standard solution containing (25, 50, 75 µg/ml) of Ornidazole and (5, 10, 15 µg/ml) of
Miconazole were analyzed three times on the different day and % R.S.D was calculated.
Accuracy
For Miconazole
5 µg/ml drug solution was taken in three different flask label A, B and C. Spiked 80% , 100%,
120% of standard solution in it and diluted up to 10ml. The area of each solution peak was
measured at 240 nm. The amount of Miconazole was calculated at each level and % recoveries
were computed.
For Ornidazole
25 µg/ml drug solution was taken in three different flask label A, B and C. Spiked 80% , 100%,
120% of standard solution in it and diluted up to 10ml. The area of each solution peak was
measured at 240 nm. The amount of Ornidazole was calculated at each level and % recoveries
were computed.
LOD and LOQ
The LOD was estimated from the set of 3 calibration curves used to determination
method linearity. The LOD may be calculated as,
LOD = 3.3 × (SD/Slope)
Where,
SD= Standard deviation of Y-intercepts of 3 calibration curves.
Slope = Mean slope of the 3 calibration curves.
The LOQ was estimated from the set of 3 calibration curves used to determine method
linearity. The LOQ may be calculated as,
LOQ = 10 × (SD/Slope)
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Where,
SD = Standard deviation of Y-intercepts of 3 calibration curves.
Slope = Mean slope of the 3 calibration curves.
Robustness:
Following parameters were changed one by one and their effect was observed on system
suitability for standard preparation.
1. Flow rate of mobile phase was changed (± 0.2 ml/min) 0.8 ml/min and 1.2 ml/min.
2. pH of Mobile phase was changed ( ± 0.2 ) 5.2 and 4.8.
3. Ratio of Mobile phase was changed (±2) Buffer: Methanol (48:52) and Buffer: Methanol
(52:48)
Analysis of formulation
Take Tablet Powder equivalent to 50 mg of Ornidazole and 10 mg of Miconazole was
transferred to a 100 ml volumetric flask, add 60 ml of Mobile phase and shake well till 15
minutes and made up volume up to the mark with mobile phase. The solution was filtered
through Whatman filter paper no. 42 and first few drops of filtrate were discarded. 1 ml of this
solution was diluted to 10 ml with mobile phase. The solution was injected 20 µl. The areas of
resulting peak were measured at 240 nm.
Identification of Drugs
Determination of Solubility
Drug Solubility
Miconazole Slightly Soluble in Water & Freely Soluble in Methanol
Ornidazole Insoluble in water in Soluble Methanol
Determination of Melting Point:
Drug Melting Point (Observed) Melting Point (Observed)
Miconazole 158-162°C 158-163°C
Ornidazole 76-78°C 76-78°C
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Identification by IR
Fig: IR Spectra of Sample Ornidazole
Functional group Frequency
NO2 (Aromatic) 1500
C-N 1200
C=C 1250
C-Cl 750
Fig. : IR Spectra of sample Miconazole
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Functional group Frequency
C-O 1050
C-N 1300
C=C 1690
C-Cl 730
Development of RP-HPLC Method
1 Selection of wavelength
The sensitivity of HPLC method that uses UV detection depends upon proper selection of
detection wavelength. An ideal wavelength is the one that gives good response for the drugs
that are to be detected. In the present study drug solutions of Miconazole (10 ppm) and
Ornidazole (50 ppm) were prepared in Methanol. These drug solutions were than scanned in
UV region of 200-400 nm and overlay spectrums were recorded.
Figure: UV Spectra of Miconazole (10 ppm) and Ornidazole (50 ppm) in Methanol
(Wavelength Taken 240 nm)
2 Selection of Mobile Phase
Trail contains various mobile phase which are considered of Methanol, Water and Acetonitrile
in different proportions and different volumes at different flow rate were tried. On the basis of
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various trails the mixture of Buffer (pH 5.0): Methanol (40:60), at 1.0 mL/min flow rate, proved
to be better than the other mixture in terms of peak shape, theoretical plate and asymmetry.
Trials are summarizes in following table.
TABLE: List of Mobile Phase trials
Sr. Mobile Phase Remark
1 Water : Methanol (20:80) Two Peaks Observed
2 Water : Methanol (20:80) First peak was of Miconazole, Confirmed by injecting Miconazole (10ppm) individual
3 Water : Methanol (20:80) Second peak was of Ornidazole, Confirmed by injecting Ornidazole (50ppm) individual
4 Water : Methanol (30:70) Both Peak are merged and Peak shape of peak of Ornidazole is Irregular
5 Phosphate Buffer (pH 6.0): Methanol (50:50)
Peak Shape Became sharp but peak of Miconazole observed at Solvent peak time
6 Phosphate Buffer (pH 6.0): Methanol (60:40)
Retention time of Second peak increased but peak of Miconazole still observed at Solvent peak time
7 Phosphate Buffer (pH 6.0): Methanol (70:30)
Again Retention time of Peak of Ornidazole Increased by increasing the proportion of Buffer but Peak of Miconazole is as it is.
8 Phosphate Buffer (pH 5.0): Methanol (70:30)
Now retention time of Peak of Miconazole increased,
9 Phosphate Buffer (pH 5.0): Methanol (60:40)
Run time decreased
10 Phosphate Buffer (pH 5.0): Methanol
(50:50)
Again run time decreased
11 Phosphate Buffer (pH 5.0): Methanol
(40:60)
First peak observed at solvent peak time, so
above mobile phase is final
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Figure: HPLC Chromatogram of Miconazole (10ppm) and Ornidazole (50ppm) in Water:
Methanol (20:80)
Figure 6.5: HPLC Chromatogram of Miconazole (10ppm) in Water : Methanol (50:50)
Figure : HPLC Chromatogram of Ornidazole (50ppm) in Water : Methanol (50:50)
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Figure: HPLC Chromatogram of Miconazole (10ppm) and Ornidazole (50ppm) in Water :
Methanol (30:70)
Figure: HPLC Chromatogram of Miconazole (10ppm) and Ornidazole (50ppm) in Buffer (pH
6.0): Methanol (50:50)
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Figure : HPLC Chromatogram of Miconazole (10ppm) and Ornidazole (50ppm) in Buffer (pH
6.0): Acetonitrile (60:40)
Figure: HPLC Chromatogram of Miconazole (10ppm) and Ornidazole (50ppm) in Buffer (pH
6.0): Methanol (70:30)
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Figure: HPLC Chromatogram of Miconazole (10ppm) and Ornidazole (50ppm) in Buffer (pH
6.0):Methanol (70:30)
Figure: HPLC Chromatogram of Miconazole (10ppm) and Ornidazole (50ppm) in Buffer (pH
5.0):Methanol (60:40)
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Figure: HPLC Chromatogram of Miconazole (10ppm) and Ornidazole (50ppm) in Buffer (pH
5.0):Methanol (50:50) (Final)
Figure: HPLC Chromatogram of Miconazole (10ppm) and Ornidazole (50ppm) in Buffer (pH
5.0):Methanol (40:60)
Mobile Phase was selected based on the review of literature. Various mobile phases were tried.
Trial contains various mobile phases which consisted of Methanol, Water, Triethylamine in
different proportions with various pH and different volumes at flow rate 1 ml/min were tried.
On the basis of various trials the mixture of Buffer (pH 5.0):Methanol (50:50)
Parameters Miconazole Ornidazole
Retention Time 3.320 4.707
Theoretical Plates 7539 7262
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Asymmetry 1.350 1.400
Resolution 7.418
Optimization of flow rate
1ml/min flow rate, proved to be better than the other in terms of resolution, peak shape and
shorter retention time.
Table: RP-HPLC optimized chromatographic conditions
Parameters Chromatographic Condition
Mode of elution Isocratic
Mobile Phase Buffer (pH 5.0):Methanol (50:50)
Column C18 (25cm x 0.46 cm) Hypersil BDS
Flow rate 1ml/min
Runtime 6 min
Injection volume 20 µL
Detection wavelength 240 nm
Validation of RP-HPLC method:
Specificity:
Fig. :- Chromatogram of Miconazole and Ornidazole std
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Fig. :- Chromatogram of Miconazole and Ornidazole sample
Fig. :- Chromatogram of Miconazole and Ornidazole Blank
The Chromatograms of Miconazole and Ornidazole standards and Miconazole and Ornidazole
sample show no interference with the Chromatogram of Miconazole and Ornidazole Blank, so
the Developed method is Specific.
Linearity:
The regression line equation for Ornidazole and Miconazole are as following:
For Ornidazoley = 98.439x - 0.8074 and For Miconazole: y = 84.221x + 0.6068
Table: Linearity data for Ornidazole
Sr.No Concentration(µg/ml) Area
1 25 2517.111
2 37.5 3636.106
3 50 4923.711
4 62.5 6082.409
5 75 7446.404
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Sr.No Concentration(µg/ml) Area
1 5 430.105
2 7.5 621.961
3 10 847.982
4 12.5 1040.391
5 15 1273.655
Table: Linearity data for Miconazole.
Fig.: Overlay chromatogram of different concentrations of mixtures of Ornidazole and
Miconazole
Fig. : Calibration Curve of Miconazole (5-15μg/ml).
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Fig. : Calibration Curve of Ornidazole (25-75 μg/ml)
Precision I. Repeatability
Ornidazole
Sr No. Conc (μg/ml) Area Mean ± S.D (n=6) % R.S.D
1.
50
4956.633 4962.454±26.202
0.528
4951.608
4986.296
4961.373
4996.102
4922.712
Table: Repeatability data for Ornidazole.
Miconazole
Sr No. Conc (μg/ml) Area Mean ± S.D (n=6) % R.S.D
1.
10
847.956 849.973 ±3.179
0.374
847.155
853.119
848.838
854.802
847.970
Table : repeatability data for Miconazole.
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II. Intraday precision
Ornidazole
SR. NO. Conc. (µg/ml)
Area Mean ± S.D. (n=3)
% R.S.D
1 25 2482.292 ± 3.734 0.150
2 50 5000.660 ± 28.846 0.577
3 75 7438.857± 30.997 0.417
Table: Intraday precision data for estimation of Ornidazole
Miconazole
SR. NO. Conc. (µg/ml)
Area Mean ± S.D. (n=3)
% R.S.D
1 5 424.703 ± 1.082 0.255
2 10 856.778 ± 3.470 0.405
3 15 1272.391 ± 5.272 0.414
Table: Intraday precision data for estimation of Miconazole
III. Interday precision
Ornidazole
SR. NO. Conc. (µg/ml)
Area Mean ± S.D. (n=3)
% R.S.D
1 25 2460.137 ± 31.438 1.277
2 50 4955.970± 61.494 1.241
3 75 7470.511± 37.323 0.500
Table: Interday precision data for estimation of Ornidazole.
Miconazole
SR. NO. Conc. (µg/ml)
Area Mean ± S.D. (n=3)
% R.S.D
1 5 421.478 ± 3.597 1.049
2 10 849.944± 7.245 0.852
3 15 1278.500± 5.597 0.438
Table: Interday precision data for estimation of Miconazole.
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Accuracy:
SR. NO.
Conc. Level (%)
Sample amount (μg/ml)
Amount Added (μg/ml)
Amount recovered (μg/ml)
% Recovery
% Mean Recovery ± S.D
1 80 % 25 20 20.136 100.681 99.679 ± 1.726
2 25 20 19.537 97.686
3 25 20 20.134 100.670
4 100 % 25 25 24.970 99.880 100.539 ± 0.977
` 25 25 25.019 100.074
6 25 25 25.415 101.662
7 120 % 25 30 29.891 99.638 99.589 ± 0.799
8 25 30 29.630 98.766
9 25 30 30.109 100.362
Table: Recovery data for Ornidazole
SR. NO.
Conc. Level (%)
Sample Amount
Amount Added
Amount recovered (μg/ml)
% Recovery
% Mean Recovery ± S.D
1 80 % 5 4 4.038 100.945 100.572 ± 0.648
2 5 4 3.993 99.824
3 5 4 4.038 100.947
4 100 % 5 5 5.006 100.112 100.326 ± 0.218
5 5 5 5.016 100.318
6 5 5 5.027 100.547
7 120 % 5 6 5.991 99.848 99.986 ± 0.545 8 5 6 5.971 99.524
9 5 6 6.035 100.587
Table: Recovery data for Miconazole
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LOD and LOQ:
Calibration curve was repeated for five times and the standard deviation (SD) of the intercepts
was calculated. Then LOD and LOQ were calculated as follows:
LOD = 3.3 * SD/slope of calibration curve
LOQ = 10 * SD/slope of calibration curve
Where, SD = Standard deviation of intercepts
Limit of Detection:
Ornidazole Miconazole
LOD = 3.3 x (SD / Slope) = 3.3 x (71.061/98.43) = 2.382µg/ml
LOD = 3.3 x (SD / Slope) = 3.3 x (12.469/84.22) = 0.489 µg/ml
Table: Limit of Detection data for Miconazole and Ornidazole.
Limit of Quantitation:
Ornidazole Miconazole
LOQ = 10 x (SD / Slope) = 10 x (71.061/98.43) = 7.219 µg/ml
LOQ = 10 x ( SD / Slope ) = 10 x (12.469/84.22) = 1.481 µg/ml
Table: Limit of Quantitation data for Miconazole and Ornidazole.
Robustness:
Table: Robustness data for Ornidazole.
SR NO. Area at Flow rate (- 0.2 ml/min)
Area at Flow rate (+ 0.2 ml/min)
Area at pH (-0.2)
Area at pH (+0.2)
Area at Mobile phase(-2)
Area at Mobile phase(+2)
1 5136.090 3333.630 5076.642 5001.410 5031.404 4912.148
2 5115.490 3282.563 5042.385 5001.071 5021.296 4909.360
3 5115.459 3374.725 5081.618 4991.104 5021.230 4966.243
% R.S.D 0.232 1.386 0.422 0.117 0.117 0.651
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SR NO. Area at Flow rate (- 0.2 ml/min)
Area at Flow rate (+ 0.2 ml/min)
Area at pH (- 0.2)
Area at pH (+ 0.2)
Area at Mobile phase(-2)
Area at Mobile phase(+2)
1 878.606 2282.685 868.529 855.633 860.790 840.382
2 875.087 2354.415 868.557 859.950 848.874 843.762
3 872.713 2368.847 869.428 853.941 859.081 849.686
% R.S.D 0.339 1.976 0.059 0.362 0.753 0.558
Table: Robustness data for Miconazole.
Analysis of marketed formulation by developed method
Sample Stock Solution (Miconazole 100 μg/mL, and Ornidazole 500 μg/mL):
Take Tablet Powder equivalent to 10 mg of Miconazole, and 50 mg of Ornidazole was
transferred to a 100 ml volumetric flask, Add 60 ml Mobile phase and Shake for 15 min and
make up volume with Mobile phase. The solution was filtered through Whatman filter paper
no. 42.
Working Sample Preparation (Miconazole 10 μg/mL, and Ornidazole 50 μg/mL):
Take 1 mL from standard stock solution and transferred to 10 ml volumetric flask and made up
volume up to the mark with the mobile phase
Inject above Solution 20 μl for Assay Analysis.
Tablet Candifem
Label claim Miconazole (100mg) Ornidazole (500mg)
Assay (% of label claim*) Mean ± S. D.
97.402±0.885 97.214±1.180
Table: Analysis on marketed formulation
The assay results were comparable to labelled value of each drug in Combined dosage form.
These results indicate that the developed method is accurate, precise, simple and rapid. It can
be used in the routine quality control of dosage form in industries
SUMMARY AND CONCLUSION
RP-HPLC method was developed for simultaneous estimation Miconazole and Ornidazole. In
RP-HPLC method, good resolution and separation of two drugs was achieved. Buffer (pH
5.0):Methanol (50:50) was used as mobile phase. Retention time of Miconazole and
Ornidazole were found to be 3.320 min and 4.707 min respectively with a flow rate of 1
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ml/min. The proposed method was accurate and precise. Therefore proposed method can
be used for routine analysis of Miconazole and Ornidazole in Combined Dosage form.
Validation parameters like Linearity, Accuracy, Precision, Robustness, System suitability,
Specificity were tested.
Observation of all these parameters leads to the point that developed RP-HPLC
method is linear, accurate, precise, specific and robust.
It can be successfully adopted for routine quality control analysis of Miconazole And
Ornidazole in combined Tablet dosage form without any interference from common
excipients and impurity.
This method can now transfer to utilize for routine laboratory analysis and assay of
Miconazole And Ornidazole In Their Tablet Dosage Form.
CONCLUSION:
RP-HPLC method was developed for simultaneous estimation Miconazole and Ornidazole. In
RP-HPLC method, good resolution and separation of two drugs was achieved. Buffer (pH
5.0):Methanol (50:50) was used as mobile phase. Retention time of Miconazole and
Ornidazole were found to be 3.320 min and 4.707 min respectively with a flow rate of 1
ml/min. The proposed method was accurate and precise. Therefore proposed method can
be used for routine analysis of Miconazole and Ornidazole in Combined Dosage form.
Validation parameters like Linearity, Accuracy, Precision, Robustness, System suitability,
Specificity were tested.
Observation of all these parameters leads to the point that developed RP-HPLC
method is linear, accurate, precise, specific and robust.
It can be successfully adopted for routine quality control analysis of Miconazole And
Ornidazole in combined Tablet dosage form without any interference from common
excipients and impurity.
This method can now transfer to utilize for routine laboratory analysis and assay of
Miconazole And Ornidazole In Their Tablet Dosage Form
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COMPARISON WITH THE EXISTING METHOD:
Developed Method Existing Method
Mobile Phase: Phosphate Buffer (pH 5.0): Methanol 50:50
Mobile Phase: Acetonitrile: Methanol 80:20
Column: C18 (250 mm × 4.6 mm i.d.,5µm) Column:C18 (250 mm × 4.6 mm i.d.,5µm)
Detection Wavelength: 240nm Detection Wavelength: 219nm
Flow rate: 1ml/min Flow rate: 1ml/min
Injection Volume: 20 μl Injection Volume: 20 μl
Run time: 6min Run time: 10min
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