1
Chapter 2
Assay
Method Validation
2
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
3
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
4) 0.1% Phosphate buffer in water: Acetonitrile and Methanol(80:10:10 v/v/v)
Inertsil ODS (250 x 4.6mm, 5.0µm)
Analyte and impurities co-eluted
5) 0.1% Phosphate buffer in water: Acetonitrile and Methanol(70:15:15 v/v/v)
Inertsil ODS (250 x 4.6mm, 5.0µm)
Peak shape not symmetrical and Analyte and impurities co-eluted
6) 0.1% Ammonium Acetate, Acetonitrile and methanol (75:15:10 v/v/v/)
Inertsil ODS (250 x 4.6mm, 5.0µm)
Analyte and impurities co-eluted
7) 0.1% Ammonium Acetate, Acetonitrile and methanol (75:15:10 v/v/v/)
Inertsil Cyno (250 x 4.6mm, 5.0µm)
Analyte and impurities co-eluted
8) 0.1% Ammonium Acetate and Acetonitrile (75:25v/v/)
Inertsil Cyno (250 x 4.6mm, 5.0µm)
Analyte and impurities co-eluted
9) 0.1% Ammonium Acetate and Acetonitrile (85:15v/v/)
Inertsil Cyno (250 x 4.6mm, 5.0µm)
Analyte and impurities co-eluted
10) 0.1% Ammonium Acetate and Acetonitrile (95:5v/v/)
Inertsil Cyno (250 x 4.6mm, 5.0µm)
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.
4
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
5
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.
10
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
11
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.
Acyclovir Imp A N.A. N.A. N.A. N.A. N.A. 2.77
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
12
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
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 NaOH
solution was added to the flask. Mixed and kept in boiling water
bath for 2 hour, then cooled, neutralized with 1M HCl solution
using pH paper and further Sample preparation same as in
Experimental details specificity chapter.
3.
Oxidative
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 10% H2O2
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solution 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.
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
Experimental details specificity chapter.
6. Reduction 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. 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
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. 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
17
Figure 18: Degradation study of Reduction Sample (1 hr) Chromatogram
Figure 19: Degradation study of Hydrolysis Chromatogram
18
Figure 20: Peak Purity graph of Resolution Solution chromatogram
19
Figure 21: Peak Purity graph of Control sample chromatogram
Figure 22: Peak Purity graph of Elevated sample chromatogram
20
Figure 23: Peak Purity graph of PH lux hr sample chromatogram
Figure 24: Peak Purity graph of watt hr sample chromatogram
21
Figure 25: Peak Purity graph of Acidic sample chromatogram
Figure 26: Peak Purity graph of Basic sample chromatogram
22
Figure 27: Peak Purity graph of Oxidation sample chromatogram
Figure 28: Peak Purity graph of Reduction sample chromatogram
23
Figure 29: Peak Purity graph of Hydrolysis sample chromatogram
24
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.
Preparation of 60% solution:
Pipetted out 6ml above standard stock solution into 100 ml volumetric flask and diluted to 100ml
with diluent. Then the solution was injected 2 times separately. The solution is injected and
recorded the chromatograms and peak areas.
Preparation of 80% solution:
Pipetted out 8ml above standard stock solution into 100 ml volumetric flask and diluted to 100ml
with diluent. Then the solution was injected 2 times separately. The solution is injected and
recorded the chromatograms and peak areas.
Preparation of 100% solution:
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
Preparation of 120% solution:
Pipetted out 12ml 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
25
Preparation of 140% solution:
Pipetted out 14ml 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
Preparation of 150% solution:
Pipetted out 15ml 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.
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
26
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:
Preparation of 50% 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.
Preparation of 100% solution:
Weighed 65 mg Acyclovir Placebo, 200 mg of Valacyclovir placebo and added 20 mg of
Acyclovir WS and 50 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 100% solutions 3 times separately. The solution
is injected and recorded the chromatograms and peak areas.
29
Preparation of 150% solution:
Weighed 65 mg Acyclovir Placebo, 200 mg of Valacyclovir placebo and added 30 mg of
Acyclovir WS and 75 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 150% solutions 3 times separately. The solution
is injected and recorded the chromatograms and peak areas.
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.
Acyclovir Imp A N.A. N.A. N.A. N.A. N.A. 3.47
Valacyclovir 0.2 1.20 6071 1.20 1.00 4.51
Valacyclovir rel. comp. C N.A. N.A. N.A. N.A. N.A. 1.74
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:
30
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.
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
32
retention time of Acyclovir and Valacyclovir were determined. Results for resolution system
suitability are presented in Table 16.
Table 16: Result of System suitability- Precision (Assay):
Analyte % RSD
T.P. T.F. Sim. Fac Resolution RT Area
Acyclovir 0.1 0.97 13870 1.34 0.99 N.A.
Acyclovir Imp A N.A. N.A. N.A. N.A. N.A. 3.74
Valacyclovir 0.2 0.74 6171 1.22 0.98 4.73
Valacyclovir rel. comp. C N.A. N.A. N.A. N.A. N.A. 1.77
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.
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 18.
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.
Acyclovir Imp A N.A. N.A. N.A. N.A. N.A. 3.75
34
Valacyclovir 0.2 0.74 6121 1.21 0.99 4.79
Valacyclovir rel. comp. C N.A. N.A. N.A. N.A. N.A. 1.78
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.
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:
System suitability solution, Standard solution, sample solution and Placebo solution preparation
is as per specificity chapter.
2.10.1 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.
System suitability was performed by injecting resolution s
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 standar
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 20
35
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 sample solution is also
2.10 Intermediate Precision or Ruggedness:
System suitability solution, Standard solution, sample solution and Placebo solution preparation
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
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
Table 20.
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 %
le solution is also
System suitability solution, Standard solution, sample solution and Placebo solution preparation
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
olution 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
d 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
36
Table 20: Result of System suitability- Intermediate Precision (Assay):
Analyte % RSD
T.P. T.F. Sim. Fac Resolution RT Area
Acyclovir 0.1 1.22 13747 1.37 0.99 N.A.
Acyclovir Imp A N.A. N.A. N.A. N.A. N.A. 3.66
Valacyclovir 0.1 1.87 7013 1.20 0.99 4.75
Valacyclovir rel. comp. C N.A. N.A. N.A. N.A. N.A. 1.77
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.
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)
System suitability solution, Standard solution, sample solution and Placebo solution preparation
is as per specificity chapter.
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.
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 22.
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.
Acyclovir Imp A N.A. N.A. N.A. N.A. N.A. 3.75
Valacyclovir 0.2 0.06 6871 1.33 0.99 4.79
Valacyclovir rel. comp. C N.A. N.A. N.A. N.A. N.A. 1.78
38
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.
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
acceptance criterion of NMT 2.0% .The results were presented in Table 24.
Table 24: Robustness-I (Flow rate changed by –0.1ml) cumulative %RSD of Acyclovir and
Valacyclovir
Sample No. Assay of Acyclovir Assay of 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:
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.
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 25.
Table 25: System suitability table for robustness-II (Assay)
Analyte % RSD
T.P. T.F. Sim. Fac Resolution RT Area
Acyclovir 0.3 0.97 11944 1.30 1.02 N.A.
Acyclovir Imp A N.A. N.A. N.A. N.A. N.A. 3.47
Valacyclovir 0.6 0.64 6071 1.20 1.02 4.51
Valacyclovir rel. comp. C N.A. N.A. N.A. N.A. N.A. 1.74
40
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.
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 26.
Table 26: Robustness-II (Flow rate changed by +0.1ml) Assay of Acyclovir and
Valacyclovir
Sample No. Assay of Acyclovir Assay of 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
acceptance criterion of NMT 2.0% .The results were presented in Table 27.
Table 27: Robustness-I (Flow rate changed by +0.1ml) cumulative %RSD of Acyclovir and
Valacyclovir
Sample No. Assay of Acyclovir Assay of 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)
(Resolution standard solution, Standard solution, Sample solution (three preparations and
placebo solution prepared as robustness-I chapter)
2.11.6 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..
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 28.
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.
Acyclovir Imp A N.A. N.A. N.A. N.A. N.A. 3.74
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
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.
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 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
Sample No. Assay of Acyclovir Assay of 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
acceptance criterion of NMT 2.0% .The results were presented in Table 30.
Table 30: Robustness-III (Column oven Temp. changed by +2) cumulative %RSD of
Acyclovir and Valacyclovir
Sample No. Assay of Acyclovir Assay of Valacyclovir
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)
(Resolution standard solution, Standard solution, Sample solution (three preparations and
placebo solution prepared as robustness-I chapter)
2.11.8 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.
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 31.
44
Table 31: System suitability table for robustness-IV (Assay)
Analyte % RSD
T.P. T.F. Sim. Fac Resolution RT Area
Acyclovir 0.1 1.84 12730 1.12 1.02 N.A.
Acyclovir Imp A N.A. N.A. N.A. N.A. N.A. 3.47
Valacyclovir 0.5 1.50 6927 0.97 1.02 4.84
Valacyclovir rel. comp. C N.A. N.A. N.A. N.A. N.A. 1.90
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.
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 32.
Table 32: Robustness-IV (Column oven Temp. changed by -2) cumulative %RSD of
Acyclovir and Valacyclovir
Sample No. Assay of Acyclovir Assay of Valacyclovir
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
acceptance criterion of NMT 2.0% .The results were presented in Table 33.
Table 33: Robustness-IV (Column oven Temp. changed by -2) cumulative %RSD of
Acyclovir and Valacyclovir
Sample No. Assay of Acyclovir Assay of 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
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