The document describes the development and validation of an RP-HPLC method for the estimation of Ketoconazole in pharmaceutical formulations. A Phenomenex Luna C18 column with a mobile phase of acetonitrile and phosphate buffer was used to separate Ketoconazole. The retention time of Ketoconazole was 4.768 minutes. The method was validated for accuracy, precision, linearity, robustness and specific parameters. The developed and validated method can be used for the routine analysis of Ketoconazole in formulations.
RP-HPLC Method Development and Validation of Ketoconazole in Bulk and Pharmaceutical Dosage Form
1. PROJECT GUIDE :
DR.ARJUN GOJE
ASSOCIATE PROFESSOR.
PROJECT MEMBERS
R.SWATHI [14AD1R0024]
S.SRAVYA [14AD1R0026]
U.SRAVANTHI [14AD1R0028]
1
2. INTRODUCTION
AIM AND OBJECTIVE
PLAN OF WORK
LITERATURE SURVEY
MATERIALS AND METHODS
DRUG PROFILE
METHOD DEVELOPMENT
METHOD VALIDATION
CONCLUSION
REFERENCES
2
3. High-performance liquid chromatography (HPLC) is a
technique in analytical chemistry used to separate, identify,
and quantify each component in a mixture.
When a mixture of components are introduced into the
column . various chemical and/or physical interactions take
place between the sample molecules and the particles of the
column packing.
3
4. Reverse Phase HPLC:
The stationary phase is nonpolar
(hydrophobic) in nature, while the mobile
phase is polar(hydrophilic) in nature
4
5. The main principle of separation is adsorption.
When a mixture of components are introduced in to a
HPLC column, They travel according to their relative
affinities towards the stationary phase.
The component which has more affinity towards the
stationary phase, travels slower.
The component which has less affinity towards the
stationary phase travels faster.
5
6. AIM AND OBJECTIVES
• Review of literature for Ketoconazole gave information
regarding its physical and chemical properties.
• Literature survey reveals that several
spectrophotometric methods were reported for
estimation of Ketoconazole.
• In view of the need for a suitable RP-HPLC method for
routine analysis of Ketoconazole in formulations,
attempts were made to develop simple, precise and
accurate analytical method.
• Validation is a necessary and important step in both
framing and documenting the capabilities of the
developed method.
6
7. Literature survey
Selection of
drug
Study of
physiochemical
properties of
drugs
Result and
discussion
Validation of
developed
method
Optimization of
analytical
method
Summary and
conclusion
PLAN OF WORK
7
8. S.NO YEAR AUTHOR DESCRIPTION
1. 2017 Verma Vikrant*, et al. •Stationary phase:Inertsil ODS-C18 column
(150mm,4.6mm,5μm) .
•Mobile phase: Buffer (pH-4) and Methanol in the mixture
of 30:70.
•Wavelength:274nm.
•. Injection volume :20μl.
•Flow rates: 1ml/min.
2. 2017 Olga Popovska, et al, •Stationary phase:LiChrospher®100 C-18 column (150
mm length x 4.6 mm i.d., 5 µm particle size)
•Mobile phase: Methanol and water (90:10 v/v)
•pH :8.9 with phosphate buffer.
•Flow rate: 1.0 ml/min.
3. 2012 Rakesh Kumar Jat, et
al.
•Stationary phase:Promosil C-18, (250 mm, 4.6 mm,
5µm).
•Mobile phase: Water : acetonitrile : buffer ph 6.8 (51:45:4
v/v) .
•Wavelength:238nm.
•Flow rate: 1.0 ml/min.
• Retention times: 2.713 min.
•Concentration range: 1-50µg/ml.
8
9. • All experiments will be carried out in the Analytical R
& D of Syncorp Clincare Technologies Pvt. Ltd.
Dilsuknagar, Hyderabad.
• Pure samples of Ketoconazole will be procured from
industries involved in bulk manufacture of this drug.
• Dosage formulation will be procured from local market.
• The methods will be developed and validated in
Analytical R & D of Syncorp Clincare Technologies
Pvt. Ltd. Dilsuknagar, Hyderabad.
• The methods will be first developed, then Validated as
per ICH guidelines, then the method will be applied to
the formulations.
9
10. DRUG PROFILE
• Name :
• Description :
• Structure :
• IUPAC Name :
• Chemical formula
Ketoconazole
Broad spectrum antifungal agent used for long periods at high doses,
especially in immunosuppressed patients.
: C26H28Cl2N4O4
• Molecular mass : 531.431g/mol
• Solubility : Ketoconazole is soluble in organic solvents such as methanol, ethanol.
• Category : Anti-Infective Agents, Antifungal Agents.
1-[4-(4-{[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-
ylmethyl)-1,3-dioxolan-4-yl]methoxy}phenyl)piperazin-1-
yl]ethan-1-one
10
12. • A suitable HPLC having
isocratic system equipped
with manual injector with UV
detector.
• LABINDIA T-60 UV – Vis
spectrophotometer.
• Analytical Balance, capable
of measuring the 0.01mg.
• Ultra Sonicator
• Vaccum filtration kit
• Usual laboratory glass ware
of class-A
12
13. Separations
Separation is based upon differential
migration between the stationary and
mobile phases.
Stationary Phase - the phase
which remains fixed in the
column. e.g. C18, C8.
Mobile Phase - carries the sample
through the stationary phase as it
moves through the column. e.g.
methanol, ACN,OPA.
Injector
Detector
Column
Solvents
Mixer
Pumps
High Performance Liquid Chromatograph
Waste
13
35. 1. Accuracy
2. Precision
a) Repeatability
b) Intermediate precision
• Intra-day(or)intra-assay
• Inter-day(or)inter-assay
c)Reproducibility
3.Linearity
4.Range
5.Robustness
6.Ruggedness
7.Limit of detection(LOD)
8.Limit of quantification(LOQ)
9.System suitability parameter
10.Assay of marketed formulation
METHOD VALIDATION PARAMETERS
36
36. To determine the accuracy of the proposed method, recovery studies were carried out by adding
different amounts (80%, 100%, and 120%) of pure drug of Ketoconazole were taken and added
to the pre-analyzed formulation of concentration 10g/ml. From that percentage recovery values
were calculated
Sample ID
Concentration (g/ml)
Peak Area
% Recovery of
Pure drug
Statistical AnalysisAmount
Added
Amount
Found
S1 : 80 % 8 8.157 595625 101.962 Mean= 101.387%
S.D. = 0.516599
% R.S.D.= 0.509532
S2 : 80 % 8 8.099 591457 101.237
S3 : 80 % 8 8.077 589875
100.962
Chromatogram for accuracy-1 replicate-1
Chromatogram for accuracy-1 replicate-2
Chromatogram for accuracy-1 replicate-3
Accuracy
37
37. Sample ID
Concentration (g/ml)
Peak Area
% Recovery of
Pure drug
Statistical AnalysisAmount
Added
Amount
Found
S4 : 100 % 10 10.077 734587 100.77 Mean= 100.43%
S.D. = 0.833727
% R.S.D.= 0.830157
S5 : 100 % 10 9.948 725268 99.48
S6 : 100 % 10 10.104 736524
101.04
Chromatogram for accuracy-2 replicate-1
Chromatogram for accuracy-2 replicate-2
Chromatogram for accuracy-2 replicate-3
38
38. Sample ID
Concentration (g/ml)
Peak Area
% Recovery of
Pure drug
Statistical AnalysisAmount
Added
Amount
Found
S7 : 120 % 12 11.989 872949 99.908
Mean= 100.6997%
S.D. = 0.841254
% R.S.D.= 0.835409
S8 : 120 % 12 12.190 887456 101.583
S9 : 120 % 12 12.073 878975
100.608
Chromatogram for accuracy-3 replicate-1 Chromatogram for accuracy-3 replicate-2
Chromatogram for accuracy-3 replicate-3 39
39. Precision:
Repeatability
Repeatability Results of Precision
HPLC Injections Retention Time
(Minutes)
Peak Area
(AUC)
Replicates of Ketoconazole
Replicate – 1 4.765 725542
Replicate – 2 4.768 726334
Replicate – 3 4.773 727283
Replicate – 4 4.768 724365
Replicate – 5 4.768 728387
Replicate – 6 4.767 725342
Average 4.768167 726208.8
Standard Deviation 0.002639 1449.807
% RSD 0.055356 0.19964
Chromatogram for Repeatability- 1 Chromatogram for Repeatability-2
40
40. Chromatogram for Repeatability-3 Chromatogram for Repeatability-4
Chromatogram for Repeatability-5 Chromatogram for Repeatability-6
41
42. Chromatogram for linearity-1 Chromatogram for linearity-2
Chromatogram for linearity-3
Chromatogram for linearity-4
Chromatogram for linearity-5
43
43. Robustness
Change in parameter % RSD
Flow (1.1 ml/min) 0.08
Flow (0.9 ml/min) 0.52
Temperature (270C) 0.12
Temperature (230C) 0.17
Wavelength of Detection (244 nm) 0.68
Wavelength of detection (240 nm) 0.73
44
44. • HPLC methods play a critical role in analysis
of pharmaceutical product
• Validation of HPLC should demonstrate that
the method is suitable for its intended us
• Data for acceptance,release,stability will only
be trustworthy if the methods used are reliable 45
45. • Drug profile- www.drug bank.com.
• www. Wikepedia.com.
• Verma Vikrant *, Singh Umesh Kumar, Ketoconazole hplc method
development and validation: a novel approach, international
research journal of pharmacy, 2017, 8 (8), Pages-74-81.
• Rakesh Kumar Jat1*, S Sharma2, R.C. Chhipa1, Rambir Singh1 and
Imran Alam, Development and validation of reverse-phase hplc
method for estimation of ketoconazole in bulk drug, Pharmacophore
2012, Vol. 3 (2), 123-129.
• Olga Popovska*, Zoran Kavrakovski, Vesna Rafajlovska, A RP-
HPLC Method for the Determination of Ketoconazole in
Pharmaceutical Dosage Forms, Current Pharmaceutical Analysis,
Volume 13 , Issue 6 , 2017. Page: [505 - 511].
46
![PROJECT GUIDE :
DR.ARJUN GOJE
ASSOCIATE PROFESSOR.
PROJECT MEMBERS
R.SWATHI [14AD1R0024]
S.SRAVYA [14AD1R0026]
U.SRAVANTHI [14AD1R0028]
1](https://image.slidesharecdn.com/sravya14ad26-180422062345/85/RP-HPLC-Method-Development-and-Validation-of-Ketoconazole-in-Bulk-and-Pharmaceutical-Dosage-Form-1-320.jpg)
