Method Development and Method Validation for the estimation of Valganciclovir in Tablet Formulation by RP- HPLC Method Presented by RAVITEJAPENTYALA DEPARTMENT OF PHARMACEUTICAL ANALYSIS
Jan 21, 2015
Method Development and Method Validation for the estimation of
Valganciclovir in Tablet Formulation by RP- HPLC Method
Presented by
RAVITEJAPENTYALA
DEPARTMENT OF PHARMACEUTICAL ANALYSIS
INTRODUCTION
Analytical Chemistry
Analytical chemistry may be defined as the science and art of
determining the composition of materials in terms of the elements of
compound contained. By means of analytical techniques both qualitative and
quantitative analysis can be done.
Qualitative methods :-
Quantitative analysis:-
Classification of analytical methods:-
Spectral methods
Chromatographic methods
Electrochemical methods
Miscellaneous methods
Hyphenated methods
CHROMATOGRAPHY
Chromatography is a technique used for the separation, purification and
identification of the compounds of mixtures by their continuous distribution,
between two phases. One is stationary phase and the other is mobile phase.
Principles of Chromatographic Separation:
Adsorption chromatography: a solid stationary phase and a liquid or gaseous
mobile phase.
Partition chromatography: a liquid stationary phase and a liquid or gaseous
mobile phase.
Size exclusion chromatography: an inert gel which acts as a molecular sieve,
and liquid mobile phase.
Ion exchange chromatography: a solid polymeric stationary phase containing
replaceable ions.
Mode of chromatographic operations:
There are three modes of chromatographic operation they are as follows:
1. Elution techniques
Isocratic method
Gradient method
2. Frontal techniques
3. Displacement techniques
Types of chromatography techniques:
Planar chromatography
Column chromatography
TYPES OF LIQUID CHROMATOGRAPHY:-
Basic operation of Liquid chromatography
1. Feed Injection:
2. Separation in the column:
3. Elution from the column:
4. Detection:
High performance liquid chromatography – [HPLC]
Different types of HPLC Techniques
1. Normal phase chromatography.
2. Reverse phase chromatography.
3. Size exclusion chromatography.
4. Ion exchange chromatography.
INSTRUMENTATION OF HPLC
Mobile phase
Pumps
Injector port
Stationary phase
Detector
ANALYTICAL METHOD DEVELOPMENT
Selecting an accurate assay procedure for each ingredient present in
pharmaceutical dosage forms, either individually or complex dosage formulation
containing several therapeutically and chemically compatible drugs with very
similar chemical nature is a monumental undertaking.
STEPS TO BE FOLLOWED IN METHOD DEVELOPMENT
1. Standard Analyte Characterization
2. Method Requirements
3. Literature Search and prior Methodology
4. Choosing of Method
5. Instrumental Setup and Initial Studies
6. Optimization
7. Documentation of analytical figures
8. Evaluation of Method Development with actual Sample
9. Determination of Percent Recovery of Actual Sample and Demonstration of
Quantitative Sample Analysis
VALIDATION OF ANALYTICAL METHOD DEVELOPMENT
Analytical method validation is the process of demonstrating that analytical
procedures are suitable for their intended use and provide accurate test results that
evaluate a product against its defind specification and quality attributes.
VALIDATION OF ANALYTICAL PROCEDURES
Different Types of Validation characteristics:
A. Precision
B. Accuracy
C. Specificity and Selectivity
D. Linearity and Range
E. Forced degradation Studies
F. Limit of Detection (LOD)
G. Limit of Quantification (LOQ)
H. Robustness
I. Ruggedness.
J. System Suitability
VALIDATION OF ANALYTICAL PROCEDURES
Different Types of Validation characteristics:
A. Precision
B. Accuracy
C. Specificity and Selectivity
D. Linearity and Range
E. Forced degradation Studies
F. Limit of Detection (LOD)
G. Limit of Quantification (LOQ)
H. Robustness
I. Ruggedness.
J. System Suitability
REVIEW OF LITERATURE
Baburao et al., reported a simple sensitive and economical UV spectrophotometric method
for the determination of valganciclovir in bulk and tablet dosage form. Valganciclovir shows
maximum absorbance at 254nm in methanol. Beer’s law was obeyed within the concentration
range of 5-30 mcg/ml with the correlation coefficient of 0.9999. The standard plot was clearly
showed a straight line passing through the origin. The method was extended to pharmaceutical
formulations.
B.dagontopa et al., reported a rapid, sensitive, and specific reverse phase high
performance liquid chromatography with diode array detection procedure for the simultaneous
determination of abacavir, efavirenz and valganciclovir in spiked human serum is described.
Separation was performed on a 5µm waters spherisorb column (256×4.6 mm ID) with
acetonitrile: methanol: KH2PO4 (at PH 5.0) (40:20:40 v/v/v) isocratic elution at a flow rate of
1.0 ml min-1. Calibration curves were constructed in the range of 50-30,000 ng mL-1 for
abacavir and efavirenz, and 10-30,000 ng mL-1 for serum samples. The limit of detection and
limit of quantification concentration of the HPLC method were 3.80 and 12.68 ng mL-1 for
abacavir, 2.61 and 8.69 ng mL-1 for efavirenz, 1.30 and 4.32 ng ml-1 for valganciclovir. The
method for the simultaneous determination of these three compounds in human serum.
DRUG PROFILEVALGANCICLOVIR
Structural formula
Common Name : Valganciclovir
Chemical Name IUPAC : 2-[(2-amino-6,9-dihydro-3H-purin-9-yl)methoxy]-3-
hydroxy propyl(2s)-2-amino-3-methyl butanoate.
Empirical formula : C14H22N6O5.HCL
Molecular weight : 390.83
Nature : White crystalline powder
Solubility : Soluble in methanol, slightly soluble in water
Melting point : 1800C
Purity : 98.0% to 102.0%
Category : Anti viral, Anti-Herpes virus
INSTRUMENTS AND REAGENTS
Instruments:
S.No Name of the Instrument
Make Model
1. HPLC Water Alliance-2695 PDA Waters-2996
2. Electronic balance
Shimadzu AY 220
3. Digital PH meter Digisun
Electronics 7007
4.
Centrifuge Thermolab R8C
5.
PhotoStability Chamber
Thermolab 943/03/0607
Reagents & Chemicals:-
S.No Name
GradeManufacture
r/ Supplier
1. Valganciclovior
Working standard -
Aurobindo
labs
2. Acetonitrile HPLC Merck
3.
Potassium di hydrogen ortho
phosphate
HPLC
Merck
4.
Methanol HPLC Merck
5. Water
- -
6. Milli Q Water
HPLC
-
OBJECTIVE AND PLAN OF THE WORK
The literature survey indicates that are some methods reported for the
determination of valganciclovir in human plasma and in combination with other
drugs like penciclovir, ganciclovir, aciclovir by LC-MS, ESI-MS/MS,
Fluorescence, HPLC and UV with longer quantitation time.
The aim of present work is to develop and validate simple RPHPLC method
by isocratic mode for the quantification of valganciclovir in bulk and it’s
formulation.
METHOD DEVELOPMENT AND OPTIMIZATION
1. Selection of wave length:
S. No. Wavelength Absorbance
1. 234 0.549
2. 240 1.014
3. 246 1.469
4. 252 1.821
5. 254 2.047
6. 264 1.936
Optimization of chromatographic parameters:
a. selection of mode of separation.
b. Selection and standardization of mobile phase and column
Standard solution of valganciclovir:-
FIXED CHROMATOGRAPHIC CONDITION
Instrument: waters 2695 separations module, UV- 2998 PDA detector
Column : Symmetry C8 (4.6×150, 15µm)
Wavelength: 254 nm
Temperature: Ambient temperature (250C)
Flow rate: 0.6ml/min
Injection: 20µl
Mobile phase: Acetonitrile: Phosphate buffer (pH4) (45:55)
Retention time: Valganciclovir – 3.68
Standard preparation :
QUANTITATIVE DETERMINATION OF THE DRUG BY USING THE
DEVELOPED METHOD
Sample: Valganciclovir
Label claim: 450mg
Sample solution of valganciclovir:-
Amount of drug present in the tablet:
Sample area Standard dilution
------------------ x --------------------- x Average weight of tablet
Standard area Sample dilution
Amount present
Percentage content = ----------------------- x 100
Label claim
Blank
Standard preparation
Sample solution
Quantitative Estimation
Acceptance criteria: 98.0- 102%w/v.
S.No Brand
Name
content Label
Claim(mg)
Peakarea Amount
Present(m
g)
Percentag
e
Content(%
)
1. Valcyte Valganciclovir 450mg 1648428 440mg 98.6%
SPECIFICITY
The specificity of an analytical method is its ability to measure accurately
and specifically the analytes in the presence of compounds that may be expected
to be present in the sample matrix.
Blank
Valganciclovir Standard:
Placebo
Valganciclovir standard + placebo
Specificity for valganciclovir
S.No Sample Area obtained Percentage content
of Drug
1. Placebo 0
0
2. Standard 1648919 98.6%
3.Standard+Placebo 1631852 99.5%
Linearity-20µg/ml
Linearity-30 µg/ml
LINERARITY AND RANGE
The linearity of an analytical method is its ability to elicit test solution that are
directly (or by a well defined mathematical transformation) proportional to the concentration of
analyte in samples within a given range.
Linearity-40 µg/ml
Linearity-50 µg/ml
Linearity-60 µg/ml
STANDARD LINEARITY OF VAGANCICLOVIR
Linearity Results
S.NoLinearity
LevelConcentration Area
1 I 20µg/ml 8180702 II 30µg/ml 12219563 III 40µg/ml 1656338
4 IV 50µg/ml 2065429
5 V 60 µg/ml 2479515
Correlation Coefficient 0.999
ANALYTICAL PERFORMANCE PARAMETERS
S.No Drug Linearity &
range
(µg/ml)
Correlation
Coefficient
Slope
1 Valganciclovir 20-60 0.999 1656338
ACCURACY :-The accuracy of an analytical method is the closeness of that results
obtained by that method to the true value. Accuracy may often be expressed as
percent recovery by the assay of known added amount of analyte.
Standard preparation
Valganciclovir 50% solution-
Valganciclovir 100% solution
Valganciclovir 150% solution-
Recovery study of Valganciclovir
S.No Sample - idStandard
Added
Standard
Area
Standard
Found
Percentag
e
Recovery
(%)
1. 50%
5.02 831427 4.98 99.2%
5.07 839156 5.01 98.9%
5.05 834192 4.99 98.8%
2. 100%
10.1 1666403 9.96 98.6%
10 1645021 9.84 98.4%
10.03 1665379 9.96 99.3
3. 150%
15 2488675 14.88 99.2%
15.3 2538991 15.18 99.2%
15.1 2501759 14.96 99.1%
PRECISION
Precision of an analytical method is the degree of agreement
among individual test results when the procedure is applied
repeatedly to multiple sampling of a homogenous sample precision
of analytical method is usually expressed as the standard deviation
and relative standard deviation.
A. System Precision
B. Method precision
System precision
System precision data
Injection Area
Injection-1 1642147
Injection-2 1676409
Injection-3 1676588
Injection-4 1670567
Injection-5 1676215
Injection-6 1672426
Average 1669058
Standard Deviation 13415
%RSD 0.81
Method Presicion
Method precision of Valganciclovir
S.No Area Obtained Amount present
(m.g)
Percentage
Content(%)
1. 1665599 9.96 99.6
2. 1657621 9.91 99.1
3. 1644289 9.85 98.2
4. 1660606 9.93 99.3
5. 1661522 9.93 99.3
6. 1653829 9.89 98.9
MEAN 99.06
STANDARD DEVIATION 0.4844
RELATIVE STANDARD DEVITATION.
0.488
Limit of Detection: (LOD)
It is the lowest amount of analyte in a sample that can be detected but not
necessarily quantities as an exact value under the stated, experimental
conclusions. The detection limit is usually expressed as the concentration of
analyte.
The Standard deviation of the response and the slope
3.3 X Standard deviation (σ)
LOD=
S S= slope of the calibration curve of the analyte.
3.3 X 658793
=
1656338
= 1.31
Limit of Quantitation: (LOQ)
The quantitation limit of an analytical procedure is the lowest amount of
analyte in a sample which can be quantitatively determined with suitable precision
and accuracy.
The Standard deviation of the response and the slope
10 X Standard deviation (σ)
LOQ =
S
S= slope of the calibration curve of the analyte.
10 X 658793
=
1656338
= 3.97
RUGGEDNESS
Ruggedness as the degree of reproducibility of test result obtained by the
analysis of the same of the samples under verity of normal test conditions, such as
different labs, different analysis, and different lots of reagents, different elapsed
assay times, different assay temperature, different days etc.
RUGGEDNESS OF THE METHOD
S.No Column
code
Instrument
Code
Analyst Result
Obtained
(%)
1. C-01 W-29 I 100.1%
2. C-02 W-30 II 99.6%
3. C-03 W-31 III 99.8%
Parameter
Result observed
Acceptance Criteria
%Content 99.83 98 – 102%
Robustness floe rate: 0.4ml/min
Robustness flow rate: 0.6ml/min
ROBUSTNESS
It is a measure of ability to remain unaffected by small but deliberate
variations in method parameters and provides an indication of its reliability during
normal usage.
Robustness flow rate: 0.8ml/min
S.NoFlow Rate
(ml/min)
Sampl
e
area
System Suitability Results
USP Plate Count USP Tailing
1 0.416326
312301.20 1.21
2 0.616453
472649.91 1.26
3 0.816218
402125.70 1.11
* Results for actual flow (0.6ml/min) have been considered from Assay standard.
ROBUSTNESS MOBILE PHASE (44:56)
ROBUSTNESS MOBILE PHASE (45:55):
ROBUSTNESS MOBILE PHASE (46:54)
S.No
Change in Organic
Composition in the
Mobile Phase
Sample
area
System Suitability Results
USP Plate Count USP Tailing
1 44:56 1637097 2142.56 1.08
2 45:55 1645347 2649.91 1.26
3 46:54 1623206 2179.15 1.20
Results for actual Mobile phase composition (45:55 Acetonitrile: Buffer) have been considered.
SYSTEM SUITABILTY PARAMETERS
System suitability testing is an integral part of many analytical procedures. The tests are based on the equipment, electronics, analytical operation and sample to be analyzed constitute an integral system that can be evaluated as such. System suitability test parameters to be established for a particular procedure depend on the procedure type being validated.
System Suitability Parameters:
Parameters Results
Tailing factor
1.28
%RSD
0.89
Number of Theoretical
plates
2588.81
RESULTS & DISCUSSION
Method development:
The developed method has an advantage of determination of valganciclovir in
RP-HPLC. The following tables give the results of the method development,
quantitation and validation parameters.
FIXED CHROMATOGRAPHIC CONDITION
Instrument: waters 2695 separations module, UV- 2998 PDA detector
Column : Symmetry C8 (4.6×150, 15µm)
Wavelength: 254 nm
Temperature: Ambient temperature (250C)
Flow rate: 0.6ml/min
Injection: 20µl
Mobile phase: Acetonitrile: Phosphate buffer (pH4) (45:55)
Retention time: Valganciclovir – 3.71
S. No. Content Label claim
(mg)
Peak area Amount
present (mg)
Percentage purity
1. Valganciclovir 450mg 1648428 440mg 98.6%
Quantitative Estimation
.
Acceptance criteria: 98– 102% w/v
S.No ParametersHPLCresult Acceptance Criteria
1. SPECIFICITY 99.5% No interference of excipients
2. LINEARITY 0.999 0.99
3. LOD 1.31µg/ml -4. LOQ 3.97µg/ml -5. ACCURACY 98.9% 98 – 102%
6.PRECISION
System Precision 0.81% 2% (RSD)
Method Precision 0.488% 2% (RSD)
7.RUGGEDNESS 99.83% 98-102%
0.815% 2% (RSD)
8.
ROBUSTNESSa)Change in flow rate
(+0.2 ml/min)(-0.2 ml/min)
100.1%99.3%
98 – 102%
b)Change in mobilephase
(44:56)(46:54)
99.1%98%
98– 102%
9.System suitability
a)Theoritical Platesb)Taling factor
c)RSD%
2588.811.160.89
NLT 2000 NMT 2
NMT 2.0%
CONCLUSION