Method Development and Method Validation for the estimation of

Valganciclovir in Tablet Formulation by RP- HPLC Method

Presented by 26103423

Under the supervision of Mr. V. Sivanand, M.Pharm.,Ph.D., Asst Professor,

DEPARTMENT OF PHARMACEUTICAL ANALYSISUltra college of pharmacy

The Tamilnadu Dr.M.G.R Meical UniversityMadurai-20

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 PROCEDURESDifferent 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 : ValganciclovirChemical 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 : 390.83Nature : White crystalline powderSolubility : Soluble in methanol, slightly soluble in water Melting point : 1800CPurity : 98.0% to 102.0%Category : Anti viral, Anti-Herpes virus

INSTRUMENTS AND REAGENTSInstruments:

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 Grade

Manufacturer/

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 OPTIMIZATION1. 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 BrandName

content LabelClaim(mg)

Peakarea AmountPresent(m

g)

Percentage

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.No Linearity Level Concentration Area

1 I 20µg/ml 8180702 II 30µg/ml 12219563 III 40µg/ml 16563384 IV 50µg/ml 20654295 V 60 µg/ml 2479515

Correlation Coefficient 0.999ANALYTICAL PERFORMANCE PARAMETERS

S.No Drug Linearity &range

(µg/ml)

CorrelationCoefficient

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

AddedStandard

AreaStandard

Found

Percentage

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 Columncode

InstrumentCode

Analyst ResultObtained

(%)

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)

Sample

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 Parameters HPLCresult Acceptance Criteria

1. SPECIFICITY 99.5% No interference of excipients

2. LINEARITY 0.999 0.993. 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