Method Development and Method Validation for the estimation of Valganciclovir in Tablet Formulation by RP- HPLC Method project work details

1. Presented by
26103423
Under the supervision of
Mr. V. Sivanand, M.Pharm.,Ph.D., Asst Professor,
DEPARTMENT OF PHARMACEUTICAL ANALYSIS
Ultra college of pharmacy
The Tamilnadu Dr.M.G.R Meical University
Madurai-20

2. 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

3. 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.

4.  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

5. 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.

6. 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.

7. 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

8. 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

9. 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

10. 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.

11. DRUG PROFILE
VALGANCICLOVIR
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

12. INSTRUMENTS AND REAGENTS
Instruments:
S.No Name of the Make Model
Instrument
1. HPLC Water Alliance-2695
PDA Waters-2996
2. Electronic Shimadzu AY 220
balance
3. Digital PH meter Digisun 7007
Electronics
4. Centrifuge Thermolab R8C
5. PhotoStability Thermolab 943/03/0607
Chamber

13. Reagents & Chemicals:-
Name Grade Manufacturer/
S.No
Supplier
1. Valganciclovior Working - Aurobindo
standard labs
2. Acetonitrile HPLC Merck
3. Potassium di hydrogen Merck
ortho phosphate HPLC
4. Methanol HPLC Merck
5. Water - -
HPLC
6. Milli Q Water -

14. 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.

15. 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

16. 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

17. Standard preparation :

18. 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

19. Blank
Standard preparation

20. Sample solution
Quantitative Estimation
S.No Brand content Label Peakarea Amount Percentage
Name Claim(mg) Present(mg) Content(%)
1. Valcyte Valganciclovir 450mg 1648428 440mg 98.6%
Acceptance criteria: 98.0- 102%w/v.

21. 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

22. Placebo
Valganciclovir Standard:

23. 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%

24. 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-20µg/ml
Linearity-30 µg/ml

25. Linearity-40 µg/ml
Linearity-50 µg/ml

26. Linearity-60 µg/ml
STANDARD LINEARITY OF VAGANCICLOVIR

27. Linearity Results
Linearity
S.No Concentration Area
Level
1 I 20µg/ml 818070
2 II 30µg/ml 1221956
3 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 & Correlation Slope
range Coefficient
(µg/ml)
1 Valganciclovir 20-60 0.999 1656338

28. 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-

30. Valganciclovir 100% solution

31. Valganciclovir 150% solution-

32. Recovery study of Valganciclovir
Percentage
Standard Standard Standard
Recovery
S.No Sample - id Added Area Found
(%)
5.02 831427 4.98 99.2%
1. 50% 5.07 839156 5.01 98.9%
5.05 834192 4.99 98.8%
10.1 1666403 9.96 98.6%
2. 100% 10 1645021 9.84 98.4%
10.03 1665379 9.96 99.3
15 2488675 14.88 99.2%
15.3 2538991 15.18 99.2%
3. 150%
15.1 2501759 14.96 99.1%

33. 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

34. System precision

36. 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

37. Method Presicion

39. Method precision of Valganciclovir
S.No Area Obtained Amount present Percentage
(m.g) 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

40. 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

41. 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

42. 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.

45. RUGGEDNESS OF THE METHOD
S.No Column Instrument Analyst Result
code Code 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%

46. 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 floe rate: 0.4ml/min
Robustness flow rate: 0.6ml/min

47. Robustness flow rate: 0.8ml/min
System Suitability Results
Flow Rate Sample
S.No USP Plate Count USP Tailing
(ml/min) area
1 0.4 1632631 2301.20 1.21
2 0.6 1645347 2649.91 1.26
3 0.8 1621840 2125.70 1.11
* Results for actual flow (0.6ml/min) have been considered from Assay standard.

48. ROBUSTNESS MOBILE PHASE (44:56)
ROBUSTNESS MOBILE PHASE (45:55):

49. ROBUSTNESS MOBILE PHASE (46:54)
System Suitability Results
Change in Organic
Sample
S.No Composition in the
area USP Plate Count USP Tailing
Mobile Phase
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.

50. 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.

52. System Suitability Parameters:
Parameters Results
Tailing factor 1.28
%RSD 0.89
Number of Theoretical 2588.81
plates

53. RESULTS
&
DISCUSSION

54. 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

55. Quantitative Estimation
.
S. No. Content Label claim Peak area Amount Percentage purity
(mg) present (mg)
1. Valganciclovir 450mg 1648428 440mg 98.6%
Acceptance criteria: 98– 102% w/v

56. HPLC
S.No Parameters
result 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%
PRECISION
6. System Precision 0.81% 2% (RSD)
Method Precision 0.488% 2% (RSD)
7.
RUGGEDNESS 99.83% 98-102%
0.815% 2% (RSD)
8. ROBUSTNESS
a)Change in flow rate
98 – 102%
(+0.2 ml/min) 100.1%
(-0.2 ml/min) 99.3%
b)Change in mobile
phase
98– 102%
(44:56) 99.1%
(46:54) 98%
System suitability
9. a)Theoritical Plates 2588.81 NLT 2000
b)Taling factor 1.16 NMT 2
c)RSD% 0.89 NMT 2.0%

57. CONCLUSION