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Chapter 2

Assay

Method Validation

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2.1 Experimental Details and Chromatographic condition for Assay:

2.1.1 Instrument:

Chromatography was performed using HPLC of Waters 2695 Alliance separation module

system, Waters 996 with PDA detector and column oven. Chromatograms and data were

recorded by means of Empower software version

2.1.2 Chemical and Reagents:

United States Pharmacopeia reference standard of Acyclovir ,Valacyclovir, Acyclovir

Related compound A and Valacyclovir related compound C were supplied by Cipla Ltd ,

Mumbai .Samples containing Acyclovir and Valacyclovir (Label claim : Each tablet

contain 200mg of Acyclovir and 500mg of Valacyclovir) were procured from Local

market. Acetonitrile of HPLC grade, Ammonium acetate was procured from Merck. Milli-

Q water was used. GF/C filter paper was obtained from company name Whatmann. All

preparation of standard and sample solution dilutions were prepared in class A volumetric

flasks.

2.2 Method development for Assay and Related Substances:

2.2.1 Optimisation of mobile phase:

The main target of the chromatographic method is to detect and quantify the acyclovir and

Valacyclovir by utilizing same chromatographic setup in single run eluted at reasonable

time and proper separation of all the components. Optimization of conditions for simple,

accurate and reproducible analysis involves analyzing system suitability solution on

varying stationary phase, strength of aqueous phase, pH, different proportion water with

acetonitrile, change in flow rate and at different column oven temperature.

Initially Reverse phase chromatography with mobile phase containing buffers such as

Potassium dihydrogen orthophosphate, Ammonium dihydrogen phosphate with organic

modifier acetonitrile was tested in isocratic mode by varying the composition. The

retention time of Acyclovir and Valacyclovir was found closely eluted which was not

suitable due to the possible placebo interference .Also different concentrations of

acetonitrile and different pHs did not make significant change in the elution pattern of

Acyclovir and Valacyclovir

Chemical structure of Acyclovir, Acyclovir related impurity A, Valacyclovir and

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Valacyclovir Related comp. c are shown in (Page 20 to 24). Different mobile phase and

stationary phases were employed to develop a suitable LC method for the quantitative

determination of Acyclovir and Valacyclovir in their respective formulations. A number of

columns containing various packing materials of ODS supplied by different manufacturers

and different mobile phase composition were tried to get good peak shapes and selectivity

for the impurities present in Acyclovir and Valacyclovir. Refer Table 3.

Table-3: Method development Trials

Sr. no. Mobile phase Stationary Phase Result

1) Phosphate buffer, acetonitrile and methanol (50:25:25 v/v/v)

waters symmetry C18 (150 x 4.6mm, 5.0µm) .

Acyclovir Impurity-A and Acyclovir co-eluted

2) Phosphate buffer, acetonitrile and methanol (50:30:20 v/v/v)

waters symmetry C18 (150 x 4.6mm, 5.0µm) .

Acyclovir Impurity-A and Acyclovir co-eluted

3) 0.1% Phosphate buffer in water: Acetonitrile and Methanol(80:10:10 v/v/v)

Phenomenex luna C18 (250 x 4.6mm, 5.0µm)

Peak shape of analytes and impurities was not symmetrical


Inertsil ODS (250 x 4.6mm, 5.0µm)

Analyte and impurities co-eluted



Peak shape not symmetrical and Analyte and impurities co-eluted

6) 0.1% Ammonium Acetate, Acetonitrile and methanol (75:15:10 v/v/v/)



7) 0.1% Ammonium Acetate, Acetonitrile and methanol (75:15:10 v/v/v/)

Inertsil Cyno (250 x 4.6mm, 5.0µm)


8) 0.1% Ammonium Acetate and Acetonitrile (75:25v/v/)








Analyte and impurities are well separated and peak shape are symmetrical

The separation was achieved using isocratic program of Buffer (A Buffer used was of

0.1% Ammonium acetate in water): Acetonitrile. The method was optimized based on the

peak shapes and resolution of Acyclovir (fig. 7), Acyclovir Imp-A (fig. 9), Valacyclovir

(fig. 8) and Valacyclovir related comp. c (fig. 10) and for resolution chromatogram refer

fig. 5.

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2.2.2 Selection of Chromatographic Column:

The polarity of both the active compounds are so strong that they do not retain on C18

column and C8 column even if the mobile phase has very low organic solvent , whereas

Acyclovir impurity A and Valacyclovir related compound C shows good retention on Cyno

column. But, investigation of column selectivity of the method showed improvement in the

peak profile, Newer HPLC columns with highly purified silica supports, which are less

acidic, minimize the potential problem of surface acidic, while separating basic

compounds. So, Inertsil Cyno, a porous, spherical packing material column was utilized as

separation unit .Separation of all related substances was achieved by optimizing isocratic

program and column temperature.

The high column temperature in the procedure evidenced the impurities and actives were

stable throughout the column separation process. Wavelength was selected by scanning

over the wide range of wavelength 200nm to 400nm. Both the components show

reasonably good response at 254 nm which is in line with Pharmacopeial wavelength for

individual drugs. This is shown in Figure 1. A typical chromatogram showing the

separation of the impurities spiked at specification level was given in Figure 2.

;

Figure 1: Spectrum Plot of Acyclovir

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Figure 2: Spectrum Plot of Valacyclovir

2.3 Method Validation(Assay) :

2.3.1 Reagent and Reference Standards:

Acyclovir : working standard, B. No: WS/C/A73/01.

Acyclovir impurity A: EPCRS, B. No: 6.0 ID: OOKE20.

Valacyclovir: USP Reference Standard, Lot No. F0C210

Valacyclovir Related compound C: EP Reference Standard, Lot No. n01a,

Zovirax 200 mg Tablets:B. No.: P105

Manufactured date: 10/2010, Expiry date: 11/2013

Valtrax Tablets, (Each tablet contain 500mg ) B. No.: EVD001A

Manufactured date: 09/2010, Expiry date: 02/2012

Water (Milli Q).

Ammonium Acetate (GR Grade) - Merck

Acetonitrile (HPLC Grade). - Merck

2.3.2 Instruments and Equipments:

HPLC: Waters

Autosampler : Waters 2695

Detector : Waters 996 (Photodiode Array Detector)

Pump : Waters 2695

Software : Empower 2

Version no : 6.20.00.00

Balance: Mettler Toledo.

Filter s: Whatmann -GF/C filters – Cat no 1822-090 (0.45µm)

Whatmann- PVDF filter membrane GD/X (Poly- vinyl difluoride) – Cat no 6872-

2504 (0.45 µm)

Whatmann filter paper Ashless 41 –Cat no 1441090 (20µm)

Photostability chamber: company name: Thermolab, India

2.3.3 Chromatographic conditions:

Separation was achieved on HPLC column Inertsil cyno column, (25 cm × 4.6mm) having

particle size 5µm, with flow rate as 0.8 ml/min and column oven temperature as 50o C. The

buffer was prepared by dissolving 1.00g of Ammonium acetate in 1000ml water, filtered

through 0.45µm membrane filter and degassed in ultrasonic bath prior to use as mobile

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phase A. Acetonitrile is the organic solvent used as mobile phase B in isocratic mode. The

injection volume amounted to 5µl. The analysis was carried out under isocratic condition

as mobile phase A: Mobile phase B is (95:5). Detection was monitored at the wavelength

of 254nm .Diluent used was a mixture of acetonitrile and water in the ratio of (50:50) in

the preparation of analytical solutions.

The analytical method was analysed for the following parameters Specificity, linearity and

Range, accuracy, precision, robustness, system suitability, limit of detection , limit of

quantitation and reproducibility according to the International Conference on

Harmonization (ICH) guidelines[131][132][133] taking into consideration the specified limits

for Acyclovir and Valacyclovir Tablets.

2.4 Specificity and System Suitability:

2.4.1 Specificity:

The methos validation parameter specificity is determined by comparing chromatograms

of diluent, actives, the related impurities and the placebo (containing all the ingredients of

the formulation except the analytes) of the tablets as per the procedure applied to sample

solution.

2.4.2 Preparation of Resolution solution for Assay:

Weigh accurately about 200 mg Acyclovir, 3 mg Acyclovir impurity A, 500 mg

Valacyclovir and 15 mg of Valacyclovir related comp C, added 70 ml of diluent. Sonicated

for 5 minutes and make up to 100 ml with diluent. Further diluted 10 ml of this solution to

100 ml with diluent. Filter through 0.45 µm filter paper using syringe. The solution is

injected and recorded the chromatograms and peak areas.

2.4.3 Preparation of Standard solution:

Weigh accurately about 20mg Acyclovir and 50mg of Valacyclovir, added 70 ml of

diluent. Sonicated for 5 minutes and make up to 100 ml with diluent. Filter through 0.45

µm filter paper using syringe. The standard solution prepared twice and injected for

checking the similarity factor. Then the standard solution A was injected 5 times

separately. The solution is injected and recorded the chromatograms and peak areas.

2.4.4 Preparation of Sample solution:

Twenty tablets were weighed and ground to homogenous powder using a mortar and

pestle. An accurately weighed portion of the powder, equivalent to 200 mg of Acyclovir

and 500mg of Valacyclovir ( 1 Tablets of Zovirax and Valtrax each ) was transferred into a

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100 ml volumetric flask containing 70 ml of diluent and disperses with the aid of

ultrasound for 10 minutes with intermittent swirling. The flask was further shaken with the

means of mechanical shaker for 15 minutes and allowed to reach the ambient room

temperature. The volume was made up to 100 ml with diluent and mixed. Further diluted

10 ml of this solution to 100 ml with diluent. Filter through 0.45 µm filter paper using

syringe. The solution is injected and recorded the chromatograms and peak areas.

2.4.5 Placebo Preparation:

An accurately weighed portion of the placebo, equivalent to 200 mg of acyclovir and 500

mg of valacyclovir was transferred into a 100 ml volumetric flask containing 70 ml of

diluent and disperses with the aid of ultrasound for 10 minutes with intermittent swirling.

The flask was further shaken with the means of mechanical shaker for 15 minutes and

allowed to reach the ambient room temperature. The volume was made up to 100 ml with

diluent and mixed. Further diluted 10 ml of this solution to 100 ml with diluent. Filtered

the solution through GF/C.

Calculation of Acyclovir: A W1 100 100 P 100 = ------ x ------- x ------- x ------------x --------- x ------------x Avg. wt B 100 W2 10 100 L.A. Where, A = Area of Acyclovir in sample solution B = Average peak area of peak in replicate injections of standard solution (A). W1 = Weigh of Acyclovir WS in Standard solution (A) in mg. W2 = Weigh of sample in gm. P = Purity of working standard L.A. = label amount of Acyclovir in Tablet Avg. wt = Average weight of Acyclovir tablets Calculation of Valacyclovir: A W1 100 100 P 100 = ------ x ------- x ------- x ------------x --------- x ------------x Avg. wt B 100 W2 10 100 L.A. Where, A = Area of Valacyclovir in sample solution B = Average peak area of peak in replicate injections of standard solution (A). W1 = Weigh of Valacyclovir WS in Standard solution (A) in mg. W2 = Weigh of sample in gm. P = Purity of working standard L.A. = label amount of Valacyclovir in Tablet Avg. wt = Average weight of Acyclovir tablets System suitability was performed by injecting resolution solution and determining

resolution between closely eluting peak of Acyclovir, Acyclovir impurity A, Valacyclovir

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and Valacyclovir related compound C. The parameters such as tailing factor, theoretical

plates were checked in resolution solution. The standard solution for Acyclovir and

Valacyclovir was prepared twice and injected. The parameters such as similarity factor,

and % RSD for peak area responses and retention time of Acyclovir and Valacyclovir were

determined. Results for resolution system suitability are presented in Table 4.

Table 4: Result of System suitability-Specificity(Assay):

Analyte % RSD

T.P. T.F. Sim.

Fac Resolution

RT Area

Acyclovir 0.2 1.16 14795 1.27 1.02 N.A.

Acyclovir Imp A N.A. N.A. N.A. N.A. N.A. 3.79

Valacyclovir 0.1 1.22 7368 1.18 1.01 4.61

Valacyclovir rel. comp.

C N.A. N.A. N.A. N.A. N.A. 1.71

The acceptance criterion for similarity factor was ≥0.98 ≤1.02, tailing factor for the peaks

due to Acyclovir and Valacyclovir was NMT 2.0, theoretical plates was not less than 2000

and % RSD for retention time and peak area response of Acyclovir and Valacyclovir

Standard solution of first replicate solution was not more than 1.0% and 2.0% respectively.

The acceptance criteria were met.

Diluent (Figure 3), Placebo Preparation (Figure 4), Resolution solution and standard

Preparation (Figure 5), were injected into the chromatograph. No peak was detected at the

retention time of Acyclovir and Valacyclovir and their respective impurities hence proving

the specificity of the method. Table 5 indicate the relative retention times (RRT) of all the

impurities.

Table 5: Identification Study of assay

Sr.

No Components

Retention

Time (mins)

Relative retention

time (RRT)

Placebo peak area

response

1 Acyclovir 4.467 1.00 Not detected

2 Acyclovir impurity A 5.249 1.17* Not detected

3 Valacyclovir 6.648 1.00 Not detected

4 Valacyclovir rel. comp. C 7.231 1.09** Not detected

*: calculated with respect to Acyclovir

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**: calculated with respect to Valacyclovir There were no peaks due to the Mobile phase, Diluent and Placebo at the retention time of

Acyclovir, Valacyclovir and their respective impurities. This shows that method is specific

by HPLC determination.

Figure 3: Chromatogram of diluent

Figure 4: Chromatogram of placebo

Figure 5 : Resolution chromatogram of Acyclovir, Valacyclovir and its related Impurities.

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Figure 6: standard solution chromatogram

Figure 7: Acyclovir ID Solution chromatogram

Figure 8: Valacyclovir ID Solution chromatogram

Figure 9: Acyclovir imp A ID Solution chromatogram

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Figure 10: Valacyclovir Rel. Comp. C ID Solution chromatogram

2.5 Forced Degradation Studies:

System suitability solution preparation, Standard solution preparation, Placebo preparation

and sample solution for control sample as per specificity chapter.

System suitability was performed by injecting resolution solution and determining

resolution between closely eluting peak of Acyclovir, Acyclovir impurity A, Valacyclovir

and Valacyclovir related compound C. The parameters such as tailing factor, theoretical

plates were checked in resolution solution. The standard solution for Acyclovir and

Valacyclovir was prepared twice and injected. The parameters such as similarity factor,

and % RSD for peak area responses and retention time of Acyclovir and Valacyclovir were

determined. Results for resolution system suitability are presented in Table 6.

Table 6: Result of System suitability-FD (Assay):

Analyte % RSD

T.P. T.F. Sim.

Fac Resolution

RT Area

Acyclovir 0.1 0.50 7153 1.31 1.01 N.A.


Valacyclovir 0.3 1.84 5307 1.00 1.00 4.12

Valacyclovir rel. comp. C N.A. N.A. N.A. N.A. N.A. 1.66

The acceptance criterion for similarity factor was ≥0.98 ≤1.02, tailing factor for the peaks

due to Acyclovir and Valacyclovir was NMT 2.0, theoretical plates was not less than 2000

and % RSD for retention time and peak area response of Acyclovir and Valacyclovir

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Standard solution of first replicate solution was not more than 1.0% and 2.0% respectively.

The acceptance criteria were met.

Acyclovir drug substance ,Valacyclovir drug substance, Zovirax and Valtrax tablets,

(200/500) mg, and Placebo for Acyclovir and Valacyclovir tablets were individually

subjected to acidic, basic, oxidative, thermal and photolytic degradation to obtain

degradants impurities in at least one of the forced degradation conditions.

The forced degradation Sample Preparation were prepared as described in Table 7 and

analyzed as the developed method

Table 7: Degradation Experiment sample preparation procedure (Assay):

Sr. No Degradation Sample Preparations

1. Acid

degradation

An accurately weighed portion of the powdered tablets,

equivalent to 200 mg of Acyclovir and 500 mg of valacyclovir

was transferred into a 100 ml volumetric flask containing 70 ml

of diluent, kept for sonication for 10 minutes with intermittent

swirling, then further shaking for 15 minutes.5 ml of 1M HCl

solution was added to the flask. Mixed and kept in boiling water

bath for 2 hour, then cooled, neutralized with 1M NaOH solution

using pH paper and further Sample preparation same as in

Experimental details specificity chapter.

2. Alkali

degradation





swirling, then further shaking for 15 minutes.5 ml of 1M NaOH


bath for 2 hour, then cooled, neutralized with 1M HCl solution

using pH paper and further Sample preparation same as in


3.

Oxidative

degradation





swirling, then further shaking for 15 minutes. 5 ml of 10% H2O2

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bath for 2 hour, then cooled and further Sample preparation same

as in Experimental details specificity chapter.

4. Thermal

degradation

Acyclovir and Valacyclovir placebos and tablets of Acyclovir

and Valacyclovir were exposed at 80°C for 48 hrs. Further

Sample preparation same as in Experimental details specificity.

5. Photolytic

degradation

Acyclovir and Valacyclovir placebos and tablets of Acyclovir

and Valacyclovir were exposed in photostability chamber for

stipulated period ( exposure to light intensity of 1.2 million lux

and 200 watts/hr). further Sample preparation same as in


6. Reduction Degradation





swirling, then further shaking for 15 minutes. 10% aq. Sodium

Bisulphite was added to the flask. Mixed and kept in boiling

water bath for 2 hour, then cooled and further Sample

preparation same as in Experimental details specificity chapter.

7. Hydrolysis

Degradation





swirling, then further shaking for 15 minutes. 10 ml purified

water was added to the flask. Mixed and kept in boiling water

bath for 2 hour, then cooled and further Sample preparation same

as in Experimental details specificity chapter.

A Control Sample Preparation of Zovirax and Valtrax tablets, (200/500) mg, and Placebo

for Acyclovir and Valacyclovir tablets, was prepared as per the method and analyzed along

with the Forced degradation samples utilizing a photodiode array detector. The

observations are presented in Table 8.

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Table 8: Recovery of Acyclovir and Valacyclovir (Assay)

Condition % Recovery of Acyclovir % Recovery of Valacyclovir

Control 99.16 94.99

EV 100.97 96.66

Lux 100.66 96.51

Watt 102.04 97.87

Acidic 1Hr 90.77 104.35

Basic 1Hr 0.1M 90.22 105.98

Oxidation 2Hr 100.97 96.65

Reduction 1Hr 96.78 108.77

Hydrolysis 2Hr 98.88 106.11

The Purity Angle value is required to be less than Purity threshold value in order to

demonstrate the absence of co elution and specificity of the method. The chromatograms

acquired were processed for Peak Purity of the Acyclovir and Valacyclovir peak in the

range of 210-400 nm to demonstrate the specificity. The observations are presented in

Table 9.

Table 9: Peak Purity of Acyclovir and Valacyclovir (Assay):

Condition Acyclovir Valacyclovir

PA PT PA PT

Control 0.147 0.238 0.040 0.231

EV 0.212 0.253 0.054 0.256

Lux 0.166 0.241 0.055 0.235

Watt 0.200 0.248 0.052 0.251

Acidic 1Hr 0.215 0.254 0.045 0.242

Basic 1Hr 0.1M 0.125 0.218 0.048 0.247

Oxidation 2Hr 0.202 0.263 0.058 0.266

Reduction 1Hr 0.129 0.219 0.075 0.241

Hydrolysis 2Hr 0.149 0.241 0.041 0.245

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2.5.1 Observations

Based upon the observations, Zovirax and Valtrax tablets, 200/500 mg, are comparatively

stable in Oxidation, Hydrolysis and Photolytic degradation conditions. The peak purity is

confirmed from the peak angle and peak threshold value for Acyclovir and Valacyclovir

peak in degradation samples indicates absence of co elution (Peak angle should be less

than peak threshold). Thus, illustrating the stability indicating nature of the method for the

determination of Assay in Zovirax and Valtrax tablets, also specific and selective.

CHROMATOGRAMS

Figure 11: Degradation study of Resolution Chromatogram

Figure 12: Degradation study of Elevated Sample Chromatogram

Figure 13: Degradation study of PH lux hr Sample Chromatogram

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Figure 14: Degradation study of Watt hr Sample Chromatogram

Figure 15: Degradation study of Acidic Sample (1 hr) Chromatogram

Figure 16: Degradation study of Basic Sample (1 hr/0.1M) Chromatogram

Figure 17: Degradation study of Oxidation Sample Chromatogram

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Figure 18: Degradation study of Reduction Sample (1 hr) Chromatogram

Figure 19: Degradation study of Hydrolysis Chromatogram

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Figure 20: Peak Purity graph of Resolution Solution chromatogram

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Figure 21: Peak Purity graph of Control sample chromatogram

Figure 22: Peak Purity graph of Elevated sample chromatogram

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Figure 23: Peak Purity graph of PH lux hr sample chromatogram

Figure 24: Peak Purity graph of watt hr sample chromatogram

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Figure 25: Peak Purity graph of Acidic sample chromatogram

Figure 26: Peak Purity graph of Basic sample chromatogram

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Figure 27: Peak Purity graph of Oxidation sample chromatogram

Figure 28: Peak Purity graph of Reduction sample chromatogram

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Figure 29: Peak Purity graph of Hydrolysis sample chromatogram

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2.6 Linearity and Range:

The concentration of serial standard solution is 50, 60, 80, 100, 120, 140 & 150% of assay

concentration (working level 200 ppm for Acyclovir and 500 ppm for Valacyclovir) was

prepared and injected.

2.6.1 Linearity solution preparation:

Linearity Stock solution preparation:

Weighed 200 mg of Acyclovir WS and 500 mg of Valacyclovir WS into 100 ml of volumetric

flask and added 75 ml diluent. Shook well to dissolve and dilute to volume with diluent.

Preparation of 50% solution:

Pipetted out 5ml above standard stock solution into 100 ml volumetric flask and diluted to 100ml

with diluent. Then the solution was injected 6 times separately. The solution is injected and

recorded the chromatograms and peak areas.










Pipetted out 10ml above standard stock solution into 100 ml volumetric flask and diluted to

100ml with diluent. Then the solution was injected 2 times separately. The chromatograms are

recorded and measure the peak response





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100ml with diluent. Then the solution was injected 6 times separately. The solution is injected

and recorded the chromatograms and peak areas.

2.6.2 Procedure:

First level (low conc. 50% solution) and Last level (high conc. 150% solution) is injected

separately 6 times and other levels (exception of low & high conc.) are injected separately 2

times (5 µl).

The chromatogram is to be recorded and measured the peak response.

Calculation:

% Y-Intercept:

Y-intercept % Y-Intercept = ------------------------------------------------------ X 100 Mean area of working level conc.

Response Factor:

Mean Response of each Linearity Response Factor = --------------------------------------------------------------- Concentration of Respective linearity level A series of Standard Solutions of Acyclovir and Valacyclovir were prepared over the range of 50

% to 150% of the specified limit for Assay for both the actives it is standard concentration. The

results obtained are presented in Table 10 and Table 11.

Table 10: Linearity and Range study for Acyclovir (104.0– 312.0 µµµµg/ml)

Sr.

No. Concentration Level

Concentration

( µµµµg/ml ) Mean Peak Area Counts

1 50% 104.00 1286820

2 60% 124.80 1579153

3 80% 166.40 2162204

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4 100% 208.00 2668691

5 120% 249.60 3214760

6 140% 291.20 3700867

7 150% 312.00 3966572

Correlation Coefficient 1.000

Table 11: Linearity and Range study for Valacyclovir (260.5– 781.5 µµµµg/ml)

Sr. No. Concentration Level Concentration

( µµµµg/ml ) Mean Peak Area Counts

1 50% 260.50 1401062

2 60% 312.60 1723123

3 80% 416.80 2343240

4 100% 521.00 2894386

5 120% 625.20 3492264

6 140% 729.40 4030802

7 150% 781.50 4322194

Correlation Coefficient 1.000

Five Solutions containing concentrations of Acyclovir and Valacyclovir in the range of 50% to

150% of the working level (i.e. 104 µg/ml to 312 µg/ml of Acyclovir and 260.5 µg/ml to

781.5µg/ml of Valacyclovir) were injected into the chromatograph. The peak area responses

were found to be linear with respect to the concentration. The regression coefficient (r2) for

Acyclovir and Valacyclovir was determined as 1.000 and 1.000 respectively, the % Y-intercept

for Acyclovir and Valacyclovir was -0.43% and -0.59% respectively, the Response factor for

Acyclovir and Valacyclovir was 1.6% and 1.4% respectively.

The criteria for regression coefficient was not less than 0.999, % Y-intercept was not more than

± 2.0% The % RSD of responses factor of Acyclovir and Valacyclovir peak was not more than

3.00%. The regression coefficient, the %Y-intercept, %RSD of peak area response, %RSD of

retention time and % RSD of responses factor were within the acceptance range.

2.6.3 The Linearity co-efficient of Acyclovir and Valacyclovir:

27

The linearity curve is plotted between y-axis in mean response of injection replicate (peak area

of Acyclovir and Valacyclovir) and x-axis in respective serial concentration (in ppm). The Linear

co-efficient (R2) and the % y-intercept are to be determined.

Figure 30: Linearity curve of Acyclovir:

Figure 31: Linearity curve of Valacyclovir:

y = 52,426.64x + 1,026.86

R² = 1.00

0

20000

40000

60000

80000

100000

0% 20% 40% 60% 80% 100% 120% 140% 160%

A

r

e

a

Concentration

Linearity of Acyclovir

y = 40,345.2836x + 180.3108

R² = 1.00

0

10000

20000

30000

40000

50000

60000

70000

0% 50% 100% 150% 200%

A

r

e

a

Concentration

Linearity of Valacyclovir

28

2.6.4 Range:

The % of RSD for peak area and RT of Acyclovir and valacyclovir from lower concentration

(Lower level) and higher concentration (Higher level).

Table-12: % of RSD for Retention Time and peak area: Name of the Standard 50 % level 150 % level

Retention time Peak area Retention time Peak area

Acyclovir 0.2% 0.93% 0.3% 0.97%

Valacyclovir 0.2% 1.62% 0.2% 0.99%

Limit NMT 1.0% NMT 2.0% NMT 1.0% NMT 2.0%

2.7 Accuracy and Recovery:

Working level concentration of standard solution that is 50,100 & 150 % of assay concentration

prepared and injected. System suitability solution, Standard solution and Placebo preparation is

as per specificity chapter.

2.7.1 Preparation of Accuracy solution:


Weighed 65 mg Acyclovir Placebo, 200 mg of Valacyclovir placebo and added 10 mg of

Acyclovir WS and 25 mg of Valacyclovir WS Standard into 100 ml volumetric flask. Added

75ml of diluent Sonicated for 15 minutes and make up to 100ml with diluent. Filter through 0.45

µm filter paper using syringe. Prepare and inject 50% solutions 3 times separately. The solution

is injected and recorded the chromatograms and peak areas.







29







System suitability was performed by injecting resolution solution and determining resolution

between closely eluting peak of Acyclovir, Acyclovir impurity A, Valacyclovir and Valacyclovir

related compound C. The parameters such as tailing factor, theoretical plates were checked in

resolution solution. The standard solution for Acyclovir and Valacyclovir was prepared twice

and injected. The parameters such as similarity factor, and % RSD for peak area responses and

retention time of Acyclovir and Valacyclovir were determined. Results for resolution system

suitability are presented in Table 13.

Table 13: Result of System suitability- Accuracy(Assay):

Analyte % RSD

T.P. T.F. Sim. Fac Resolution RT Area

Acyclovir 0.2 1.21 11944 1.30 1.00 N.A.


Valacyclovir 0.2 1.20 6071 1.20 1.00 4.51


The acceptance criterion for similarity factor was ≥0.98 ≤1.02, tailing factor for the peaks due to

Acyclovir and Valacyclovir was NMT 2.0, theoretical plates was not less than 2000 and % RSD

for retention time and peak area response of Acyclovir and Valacyclovir Standard solution of

first replicate solution was not more than 1.0% and 2.0% respectively. The acceptance criteria

were met.

2.7.2 Procedure:

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The working Standard Solution is injected separately 5 times and sample solutions (50%

solution, 100% solution & 150% solution) is also injected separately 3 times (about 5µl)

respectively.

The % Recovery of Acyclovir and Valacyclovir is determined by the following Expression. Sample area Std wt Purity Amount Found = × × 1000 × _______ Std Area Std dilution 100 Std wt Purity Amount Added = × × 1000 Std dilution 100 The % Recovery is determined by the following Expression. Amount Found % Recovery = × 100 Amount Added The accuracy study of the analytes was determined for the following levels, 50%, 100% and

150% of the specified limit. Both the Analytes showed the recovery between 98% to 102 %

concluding the accuracy of the method. The results for both the analytes are presented in Table

14 and Table 15 which have met the acceptance criteria.

Table 14: Accuracy and recovery of Acyclovir:

Added (µg) Recovered (µg) %Recovery Mean Recovery

100.5

99.5 99.00

98.65 98.94 98.44

98.99 98.50

208.0

208.19 100.09

100.77 210.39 101.15

210.24 101.08

305.0

302.65 99.23

99.48 301.62 98.89

305.95 100.31

31

Table 15: Accuracy and recovery of Valacyclovir

Added (µg) Recovered (µg) %Recovery Mean Recovery

242.5

241.17 99.45

99.22 240.78 99.29

239.87 98.92

501.0

506.46 100.69

101.47 512.53 101.90

512.19 101.83

752.0

744.64 99.02

99.09 740.28 98.44

750.65 99.82

At each respective level, the mean recovery for each analyte, from the tablet solution, is within

the limit of 98 % - 102 %. Thus, illustrating the HPLC analytical method for the determination of

Assay of Acyclovir and Valacyclovir is accurate.

2.8 Precision:

System suitability solution, Standard solution, sample solution and Placebo solution preparation

is as per specificity chapter.

2.8.1 Procedure:

The working standard solution is injected separately 5 times and six assay sample preparations

are injected separately 1 times respectively (5 µl). The solution is injected and recorded the

chromatogram and peak areas.






32



Table 16: Result of System suitability- Precision (Assay):

Analyte % RSD


Acyclovir 0.1 0.97 13870 1.34 0.99 N.A.


Valacyclovir 0.2 0.74 6171 1.22 0.98 4.73


The acceptance criterion for similarity factor, tailing factor for the peaks due to Acyclovir and

Valacyclovir , theoretical plates and % RSD for retention time and peak area response of

Acyclovir and Valacyclovir are as specificity chapter.

Precision of the method was studied for repeatability and intermediate precision. Samples of

zovirax and valtrax tablets showed assay value within the limit, hence the precision of the

method was done and % RSD of by analyzing sample of zovirax and valtrax tablets six times

separately. The RSD for recovered Acyclovir and Valacyclovir were well within the limit of

2.00% confirming the precision of the method. Table 17 represents the method precision results.

Table 17: Method Precision Results of Acyclovir and Valacyclovir:

Sr. No. % Recovery of Acyclovir % Recovery of Valacyclovir

Sample-1 98.62 100.06

Sample-2 98.11 98.08

Sample-3 99.41 99.36

Sample-4 99.28 99.31

Sample-5 98.29 97.97

Sample-6 97.95 97.89

33

Mean 98.61 98.78

SD 0.61 0.92

%RSD 0.62 0.93

The relative standard deviation of six sample preparation of acyclovir and valacyclovir each

from the tablet solution was found to be within the acceptance criteria of not more than 2.00%.

Thus, illustrating the HPLC analytical method for the determination of Assay of zovirax and

valtrax tablets is precise.

2.9 Solution Stability:

Stability of solutions in Room temperature from 0th hr ,2nd hr,4th hr,6th hr,8th hr, 12th hr, 16th hr,

20th hr and then up to 24th hrs. System suitability solution, Standard solution, sample solution and

Placebo solution preparation is as per specificity chapter.

2.9.1 Procedure:

Mobile phase and fresh standard was prepared for system suitability. The working standard

solution (freshly prepared) and the assay solution (freshly prepared) is injected separately 5 and 1

replicates respectively and this solution was kept in room temperature from 0hrs to 24th hr.








Table 18: Result of System suitability- Solution stability (Assay):

Analyte % RSD

T.P. T.F. Sim.

Fac Resolution

RT Area

Acyclovir 0.1 1.34 13689 1.37 1.00 N.A.


34

Valacyclovir 0.2 0.74 6121 1.21 0.99 4.79


The acceptance criterion for similarity factor , tailing factor for the peaks due to Acyclovir and



The stability of sample solutions were determined at time periods representative for storage. The

stability of sample solution was confirmed on the sample by comparing the values of 0th sample

at different time interval to its initial value. The % relative difference should not be more than

2.00. The data for sample solution stability in Table 19.

Table 19: Sample solution stability of Acyclovir and Valacyclovir:

Condition % Assay of Acyclovir % Assay of Valacyclovir

Sample-0th HR 101.33 99.64

Sample-2nd HR 102.05 97.57

Sample-4th HR 102.88 98.23

Sample-8th HR 99.62 98.64

Sample-12th HR 100.30 100.82

Sample-16th HR 101.66 99.95

Sample-20th HR 99.62 98.64

Sample-24th HR 100.42 98.76

Mean 100.98 99.03

SD 1.19 1.04

%RSD 1.17 1.05

Figure 32: Standard solution stability Curve

The standard solution was found to be stable till 24 hrs since the % RSD of the peak area

response of Acyclovir and valacyclovir is not more than 2.0 %. The absolute difference of %

assay in sample solution was not more than 2.0 %. Indicating that the samp

stable for 24 hrs.

2.10 Intermediate Precision or Ruggedness:



2.10.1 Procedure:

The working standard solution is injected separately 5 times and six assay sample preparations


chromatogram and peak areas.

System suitability was performed by injecting resolution s



resolution solution. The standar



suitability are presented in Table 20

35



assay in sample solution was not more than 2.0 %. Indicating that the sample solution is also

2.10 Intermediate Precision or Ruggedness:


solution is injected separately 5 times and six assay sample preparations








Table 20.



le solution is also


solution is injected separately 5 times and six assay sample preparations


olution and determining resolution



d solution for Acyclovir and Valacyclovir was prepared twice



36

Table 20: Result of System suitability- Intermediate Precision (Assay):

Analyte % RSD


Acyclovir 0.1 1.22 13747 1.37 0.99 N.A.


Valacyclovir 0.1 1.87 7013 1.20 0.99 4.75






were met.

It was determined on six separate sample solutions prepared from same batch by a different

analyst using different mobile phase, diluents preparation and same instrument on a different day

with different lot of same brand column. The overall RSD was evaluated and were within the

acceptance criterion of NMT 2.0% .The results were presented in Table 21.

Table 21: Intermediate Precision Study

Instrument Waters – Empower Waters – Empower

Analyst A B

% Acyclovir % Valacyclovir

Sr. No. (A) (B) (A) (B)

1. 98.62 98.26 100.06 97.27

2. 98.11 97.77 98.08 96.79

3. 99.41 96.90 99.36 95.94

4. 99.28 97.61 99.31 96.72

5 98.29 98.09 97.97 97.09

6 97.95 96.81 97.89 95.49

Mean 98.09 97.66

37

SD 0.79 1.40

%RSD 0.81 1.43

2.11 Robustness:

In all the deliberate varied chromatographic conditions carried out (mobile phase flow rate and

column temperature in the variants),

2.11.1 Robustness-I: (Flow rate changed by –0.1ml)



2.11. 2 Procedure:

The working standard solution is injected separately 5 times and three assay sample preparations

are injected separately 1 times ( 5 µl). The solution is injected and recorded the chromatogram

and peak areas.








Table 22: System suitability table for robustness-I (Assay)

Analyte % RSD

T.P. T.F. Sim.

Fac Resolution

RT Area

Acyclovir 0.1 0.05 13665 1.37 0.98 N.A.


Valacyclovir 0.2 0.06 6871 1.33 0.99 4.79


38





were met.

The resolution had no significant changes, when the parameters were modified .Method

robustness shows that the minor change of the operational parameters does not affect the

chromatographic separation.

It was determined on three separate sample solutions prepared from same batch. The % RSD was

evaluated and was within the acceptance criterion of NMT 2.0% .The results were presented in

Table 23.

Table 23: Robustness-I (Flow rate changed by –0.1ml) Assay of Acyclovir and Valacyclovir

Sample No. Assay of Acyclovir Assay of Valacyclovir

1 100.06 100.64

2 99.07 99.61

3 99.37 99.94

Mean 99.50 100.07

SD 0.51 0.53

%RSD 0.51 0.53

The cumulative % RSD was evaluated with the method precision samples and was within the


Table 24: Robustness-I (Flow rate changed by –0.1ml) cumulative %RSD of Acyclovir and

Valacyclovir


1 98.62 100.06

2 98.11 98.08

3 99.41 99.36

4 99.28 99.31

5 98.29 97.97

39

6 97.95 97.89

7 100.06 100.64

8 99.07 99.61

9 99.37 99.94

Mean 98.91 99.21

SD 0.71 1.00

%RSD 0.71 1.01

2.11.3 Robustness -II:(Flow rate changed by +0.1ml)

(Resolution standard solution, Standard solution, Sample solution (three preparations and

placebo solution prepared as robustness-I chapter)

2.11.4 Procedure:


are injected separately 1 times (5 µl). The solution is injected and recorded the chromatogram

and peak areas.








Table 25: System suitability table for robustness-II (Assay)

Analyte % RSD


Acyclovir 0.3 0.97 11944 1.30 1.02 N.A.


Valacyclovir 0.6 0.64 6071 1.20 1.02 4.51


40









Table 26.

Table 26: Robustness-II (Flow rate changed by +0.1ml) Assay of Acyclovir and

Valacyclovir


1 100.58 99.87

2 100.36 100.34

3 99.56 101.05

Mean 100.17 100.42

SD 0.54 0.59

%RSD 0.54 0.59

The cumulative % RSD was evaluated with the method precision samples and were within the


Table 27: Robustness-I (Flow rate changed by +0.1ml) cumulative %RSD of Acyclovir and

Valacyclovir


1 98.62 100.06

2 98.11 98.08

3 99.41 99.36

4 99.28 99.31

5 98.29 97.97

41

6 97.95 97.89

7 100.58 99.87

8 100.36 100.34

9 99.56 101.05

Mean 99.13 99.33

SD 0.96 1.13

%RSD 0.96 1.14

2.11.5 Robustness -III: (Column oven Temp. changed by +2)



2.11.6 Procedure:



and peak areas..








Table 28: System suitability table for robustness-III (Assay)

Analyte % RSD

T.P. T.F. Sim.

Fac Resolution

RT Area

Acyclovir 0.1 1.53 13675 1.39 1.02 N.A.


Valacyclovir 0.5 0.76 6116 1.20 1.02 4.73

42

Valacyclovir rel. comp.

C N.A. N.A. N.A. N.A. N.A. 1.77








evaluated and were within the acceptance criterion of NMT 2.0% .The results were presented in

Table 29.

Table 29: Robustness-III (Column oven Temp. changed by +2) Assay of Acyclovir and

Valacyclovir


1 96.41 97.89

2 97.51 99.28

3 96.73 99.98

Mean 96.88 99.05

SD 0.56 1.06

%RSD 0.58 1.07

The cumulative % RSD was evaluated with the method precision samples and were within the


Table 30: Robustness-III (Column oven Temp. changed by +2) cumulative %RSD of



43

1 98.62 100.06

2 98.11 98.08

3 99.41 99.36

4 99.28 99.31

5 98.29 97.97

6 97.95 97.89

7 96.41 97.89

8 97.51 99.28

9 96.73 99.98

Mean 98.03 98.87

SD 1.03 0.91

%RSD 1.05 0.92

2.11.7 Robustness -IV :( Column oven Temp. changed by -2)



2.11.8 Procedure:



and peak areas.








44

Table 31: System suitability table for robustness-IV (Assay)

Analyte % RSD


Acyclovir 0.1 1.84 12730 1.12 1.02 N.A.


Valacyclovir 0.5 1.50 6927 0.97 1.02 4.84










Table 32.

Table 32: Robustness-IV (Column oven Temp. changed by -2) cumulative %RSD of



1 98.76 98.27

2 99.39 100.03

3 100.24 100.10

Mean 99.46 99.47

SD 0.74 1.04

%RSD 0.75 1.04

45

The cumulative % RSD was evaluated with the method precision samples and was within the


Table 33: Robustness-IV (Column oven Temp. changed by -2) cumulative %RSD of



1 98.62 100.06

2 98.11 98.08

3 99.41 99.36

4 99.28 99.31

5 98.29 97.97

6 97.95 97.89

7 98.76 98.27

8 99.39 100.03

9 100.24 100.10

Mean 98.89 99.01

SD 0.74 0.95

%RSD 0.75 0.96

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