Development and validation of RP-HPLC method for the determination of valacyclovir hydrochloride and its related substances in tablet formulation

Abstract

A chiral high performance liquid chromatographic method was developed and validated for the separation of Valacyclovir drug substance and its related substances V1 (guanine), V2 (acyclovir) and V3 unknown impurity). The Valacyclovir and its impurities were resolved on 150 × 4.0 mm (i.d.), stainless steel, packed with 5μm Daicel Chiral Phase Crownpack CR (+) column at 15°C using 0.1% aqueous Phosphoric acid (85%): Methanol (90:10 V/V) as a mobile phase. A PDA detector set at 254 nm was used for detection. The linearity for related substances was obtained within concentrations ranging from 0.3 to 6μg/mL. The correlation coefficient of Valacyclovir was 0.9997. Relative standard deviations of peak areas from six measurements were always less than 2%. TThe proposed method was found to be suitable and accurate for the quantitative determination of Valacyclovir in bulk drug substance and tablet formulation. Validation parameters showed that the method is specific, accurate, precise and reproducible. The method can be used for routine quality control and stability analysis of Valacyclovir drug substance.

Full text

Int. J. Pharm. Sci. Rev. Res., 25(1), Mar – Apr 2014; Article No. 08, Pages: 53-58 ISSN 0976 – 044X
International Journal of Pharmaceutical Sciences Review and Research
Available online at
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Girija B. Bhavar*1, Sanjay S. Pekamwar2, Kiran B. Aher1, Sanjay R. Chaudhari1
1Amrutvahini College of Pharmacy, Amrutnagar, Sangamner, Maharashtra, India.
2School of Pharmacy, Swami Ramanand Teerth Marathwada University, Nanded, Maharashtra, India.
*Corresponding author’s E-mail: girijabhavar@gmail.com
Accepted on: 13-12-2013; Finalized on: 28-02-2014.
ABSTRACT
A chiral high performance liquid chromatographic method was developed and validated for the separation of Valacyclovir drug
substance and its related substances V1 (guanine), V2 (acyclovir) and V3 unknown impurity). The Valacyclovir and its impurities were
resolved on 150 x 4.0 mm (i.d.), stainless steel, packed with 5µm Daicel Chiral Phase Crownpack CR (+) column at 15°C using 0.1%
aqueous Phosphoric acid (85%): Methanol (90:10 V/V) as a mobile phase. A PDA detector set at 254 nm was used for detection. The
linearity for related substances was obtained within concentrations ranging from 0.3 to 6µg/mL. The correlation coefficient of
Valacyclovir was 0.9997. Relative standard deviations of peak areas from six measurements were always less than 2%. TThe
proposed method was found to be suitable and accurate for the quantitative determination of Valacyclovir in bulk drug substance
and tablet formulation. Validation parameters showed that the method is specific, accurate, precise and reproducible. The method
can be used for routine quality control and stability analysis of Valacyclovir drug substance.
Keywords: HPLC, Related substances, Valacyclovir, Validation.
INTRODUCTION
alaciclovir hydrochloride (VAL) [(S)-2-[(2-amino-6-
oxo-6,9-dihydro-3H-purin-9-yl)methoxy]ethyl-2-
amino-3-methylbutanoate] (Figure 1) is a
hydrochloride salt of L-Valyl ester of acyclovir.1-3 It is an
oral antiviral drug used to treat infections with herpes
zoster (shingles), herpes simplex genitalis (genital herpes),
and herpes labialis (cold sores). It inhibits the replication
of viral DNA. It is a prodrug intended to increase the
bioavailability of acyclovir by increasing lipophilicity.
Valacyclovir is converted by esterase to active drug
acyclovir via hepatic first pass metabolism.3
Literature survey revealed that few Spectrophotometric
methods4-11, HPLC methods12-18, and LC-MS methods for
biological fluids19-23 are reported in the literature for the
determination of VAL in Bulk, pharmaceutical
formulations and serum samples.
The aim of the present work was to develop a simple and
economic liquid chromatographic method that would be
suitable for determination of VAL and its impurities in
bulk and dosage form. The proposed method is found to
be simple, accurate, reproducible and suitable for routine
determination of VAL from its pharmaceutical dosage
form.
Figure 1: Structure of Valacyclovir Hydrochloride
MATERIALS AND METHODS
Chemicals and reagents
Valacyclovir Hydrochloride (VAL) reference standard,
Valacyclovir bulk drug and related substances were
procured from GlaxoSmithkline Nashik (India). Methanol
used was of HPLC grade (Qualigens, Mumbai).
Valcivir tablets 500mg (Cipla Pharmaceutical Ltd., Goa,
India) were used for the assay.
Equipments
HPLC system : Water’s HPLC system
Column : 150 x 4.0mm (i.d.), Stainless steel,
packed with 5µm Daicel Chiral Phase Crownpack CR (+)
Balance : Mettler Toledo 205
Ultrasonicator : ENERTECH Electronics Pvt. Ltd.
Preparation of solutions
Standard Stock solution
Standard stock solution containing 1000 µg/mL of the VAL
was prepared in 0.1N hydrochloric acid using VAL
reference standard.
Diluent
A 0.1% v/v aqueous phosphoric acid (85%) was used as a
diluent.
Stock solutions of related substances
The separate stock solutions of related substances V1
(guanine), V2 (acyclovir) and V3 of concentrations
100µg/mL were prepared by dissolving 10mg of each
Development and Validation of RP-HPLC Method for the Determination of Valacyclovir
Hydrochloride and its Related Substances in Tablet Formulation
V
Research
Article

Int. J. Pharm. Sci. Rev. Res., 25(1), Mar – Apr 2014; Article No. 08, Pages: 53-58 ISSN 0976 – 044X
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related substance in diluent separately and diluting to
100mL with the same solvent.
Resolution Check Solution
The solution containing 50µg/mL of VAL reference
standard and 0.8µg/mL of related substance V3 was
prepared in diluent. The resolution between peaks due to
V3 and VAL is not less than 1.5.
Chromatographic Conditions
Analysis was carried out on 150 x 4.0mm (i.d.), stainless
steel, packed with 5µm Daicel Chiral Phase Crownpack CR
(+) column using 0.1% v/v aqueous Phosphoric acid (85%):
Methanol (90:10 V/V) as mobile phase at a flow rate of
0.8mL per minute. The column temperature was set at
15°C. The volume injected was 10µl and detection was
carried out at 254 nm using a PDA detector.
Calibration curve
From the standard stock solution of VAL aliquots, (0.25,
0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mL) were transferred to
series of 10 mL volumetric flasks and volume was made
up to the mark with diluents to give solutions of
concentrations in the range of 25-500 g/mL. The
chromatograms and peak areas of these solutions were
measured at 254 nm and a calibration curve was
constructed, by plotting the area against the
corresponding drug concentration.
Related Substances by HPLC
A solution containing 100µg/mL of VAL and 0. 8µg/mL of
Guanine (V1), acyclovir (V2) and V3 was prepared in
diluent and injected in HPLC system. The retention time
for each substance was recorded. Figure 2 shows the
resolution of VAL and related substances.
Figure 2: Typical chromatogram of a) mobile phase (Blank) and b) Standard VAL and related substances V1, V2, V3
Validation of the Method25
Linearity
The linearity of peak areas versus different
concentrations was evaluated for VAL over the range of
25-500 g/mL and for all the related substances over the
range of 0.3µg/mL to 6µg/mL. The correlation
coefficient ( r2) for VAL and each related substances was
calculated.
Specificity
The analyte should have no interference from other
extraneous components and be well resolved from them.
To determine the specificity of the method, the mixture
of reference standard VAL and the related substances was
injected and chromatogram was recorded. The sample
solution (pharmaceutical dosage form) was then injected
and the chromatogram was obtained. The sample
chromatogram was compared with the standard
chromatogram.

Int. J. Pharm. Sci. Rev. Res., 25(1), Mar – Apr 2014; Article No. 08, Pages: 53-58 ISSN 0976 – 044X
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Figure 3: Linearity plot of standard VAL
Precision
Precision of the method was studied in terms of
repeatability and intermediate precision.
Repeatability
Repeatability was performed by analyzing six separate
VAL solutions of concentration 50 µg/mL that were
prepared by spiking the related substances to give 1
µg/mL of each of V1, V2 and V3. The %R.S.D for each
related substance was evaluated.
Intermediate precision
The intermediate precision of the method for VAL and
related substances was determined on three separate
sample solutions prepared by spiking the related
substances by two different analysts on two different
days. The mean values of results for each day and for
each analyst were compared.
Accuracy (Recovery studies)
To ensure the accuracy of method, recovery studies were
performed by standard addition method at 80 %, 100 %
and 120 % levels of drug concentrations, to the pre-
analyzed samples and they were re-analyzed.
Accuracy of the method for all the related substances was
determined by analyzing VAL sample solutions spiked
with all the related substances at three different
concentration levels of 1%, 2% and 4% of sample
concentration each in triplicate.
Application of the method for estimation of VAL in bulk
drug
The bulk drug VAL was dissolved in 0.1N hydrochloric acid
and diluted to give the solution containing 50 µg/mL of
the VAL. The solution was injected and peak area was
recorded.
Assay of VAL in tablets
Twenty tablets were accurately weighed, their average
weight was determined and they were finely powdered.
The powder equivalent to 50 mg of VAL was transferred
to 100 mL volumetric flask and 50mL of 0.1N hydrochloric
acid was added. The solution was sonicated for 10
minutes and volume was made up to the mark with 0.1N
hydrochloric acid. The solution was filtered through
0.45µm filter and 1 mL of filtrate was diluted to 10 mL
with diluent to give the solution containing 50µg/mL of
the VAL. The solution was injected and peak area was
recorded.
System Suitability Parameters
System suitability test was performed to verify that the
resolution and reproducibility of the chromatographic
systems were adequate for the analysis. Five injections of
the standard were injected for this purpose. The
retention time, areas, theoretical plates, peak asymmetry
and resolution were calculated for standard solutions.
RESULTS AND DISCUSSION
A new HPLC method was developed for the
determination of Valacyclovir related substances in
Valacyclovir drug substance using 0.1%aqueous
Phosphoric acid (85%): Methanol (90:10 V/V) as a mobile
phase. Three related substances were detected by this
method. The developed method was validated as per the
ICH guidelines and was applied to estimate the drug from
tablet formulations. The details of findings are as below.
Related Substances by HPLC
Three related substances were detected and well
resolved by the method. The retention data for VAL and
related substances is indicated in Table 1.
Table 1: Typical Retention Data
Component Retention time
(minutes) Relative retention
time (RRT)
Guanine (V1) 2.2 0.49
Acyclovir(V2) 2.8 0.57
V3 3.9 0.80
VAL 4.9 1.0
Linearity
The linearity for VAL was obtained in the concentration
range of 25-500 g/mL and for the related substances in
the concentration range of 0.3 - 6 g/mL. Linearity data
for VAL and Related substances is summarized in Table 2.
Table 2: Linearity data for VAL and Related substances
Parameter VAL V1 V2 V3
Linearity range
(µg/mL) 25-500 0.3-6 0.3-6 0.5-6
Slope 26078 20113 20027 12363
Y-intercept 46889 1556 813 867
Correlation
coefficient (r2) 0.9997 0.9999 1 0.9999
Specificity
Specificity of the method was demonstrated by no
interference of related substances with the VAL peak. All
the related substances were separated with good

Int. J. Pharm. Sci. Rev. Res., 25(1), Mar – Apr 2014; Article No. 08, Pages: 53-58 ISSN 0976 – 044X
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Available online at
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56
resolution by the mobile phase developed for the
method. Specificity of peak of VAL from tablet was
determined by comparing with the peak of reference
standard. No interference of excipients with the VAL peak
was observed.
Precision
Precision of the method was studied in terms of
repeatability and intermediate precision. Precision was
expressed in terms of % R.S.D. All values for precision
were within recommended limits.
Repeatability
Analysis of six separate solutions of VAL with
concentration 50 µg/mL spiked with the related
substances to give 1 µg/mL of each of V1, V2 and V3
showed the repeatability of the method. Satisfactory
agreement of VAL retention times and peak area counts
was observed as given in Table 3.
Table 3: Repeatability of VAL and Related substances
Concentration found (µg/mL)
Mean ± SD (n=6) % RSD
VAL 48.12 ± 0.036 0.07
V1 1.02 ± 0.003 0.284
V2 1.02 ± 0.006 0.626
V3 0.999 ± 0.006 0.588
n=6: Mean of six determinations
Intermediate precision
Comparisons of the mean values for each day and for
each analyst as summarized in Table 4 indicated that the
method is precise and reproducible.
Table 4: Results of intermediate precision
Component
Concentration found (µg/mL) Mean ± %RSD, n=3
Analyst 1 Analyst 2
Day 1 Day 2 Day 1 Day 2
VAL 49.98 ± 0.14 49.98 ± 0.19 50.02 ± 0.09 50.00 ± 0.07
V1 1.02 ± 0.461 1.03 ± 0.386 1.02 ± 0.536 1.03 ± 0.414
V2 1.02 ± 0.480 1.02 ± 0.688 1.02 ± 0.660 1.02 ± 0.506
V3 0.99 ± 0.770 1.00 ± 1.070 1.00 ± 0.542 1.00 ± 0.780
n=3: Mean of three determinations
Accuracy
The results of recovery studies showed the accuracy of
the method. The recoveries for VAL and related
substances were ranged between 100.11 – 101.15 % and
98.43 – 101.77 %, respectively. Results obtained for VAL
and related substances are given in Table 5 and Table 6
respectively.
Table 5: Recovery of VAL
Level Amount added
(µg/mL) Amount found
(µg/mL) %Recovery*
(Mean ± SD)
80% 40 40.14 100.34 ± 0.39
100% 50 50.05 100.11 ± 0.14
120% 60 60.69 101.15 ± 0.07
*-Mean of three determinations
Table 6: Recovery of Related substances
Amount added
(µg/mL)
V1 V2 V3
Amount found
(µg/mL) %Recovery ± SD
* Amount found
(µg/mL) %Recovery ±
SD* Amount found
(µg/mL) %Recovery ±
SD*
0.5 0.502 100.31 ± 1.0 0.499 99.79 ± 0.40 0.492 98.43 ± 0.89
1 1.018 101.77 ± 0.12 1.015 101.52 ± 0.50 0.986 98.65 ± 0.17
2 2.02 100.85 ± 0.16 2.013 100.67 ± 0.13 2.012 100.59 ± 0.41
* - Mean of three determinations
Assay of VAL in Bulk drug
The solution of bulk drug containing 50 µg/mL of the VAL
was analyzed. The results of assay are as given in Table 7.
Table 7: Results of assay in Bulk drug
Concentration taken ( g/mL) 50
Concentration found (g/mL), mean  SD 50.01 0.03
% RSD (n=6) 0.05
Assay of VAL in tablets
The concentration of tablet solution was determined
using linear regression equation (using slope and Y-
intercept) and amount of drug in tablet was determined.
The results of assay in tablets are summarized in Table 8.
System Suitability Parameters
System suitability parameters were tested for the
chromatographic conditions and results are as shown in
Table 9.

Int. J. Pharm. Sci. Rev. Res., 25(1), Mar – Apr 2014; Article No. 08, Pages: 53-58 ISSN 0976 – 044X
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Table 8: Results of assay in tablets
Labeled claim (mg) 500
Amount found (mg) 499.62
% labeled claim 100.61
% RSD (n=6) 1.05
Table 9: System Suitability Parameters
Parameter Average % RSD
Retention time 4.91 0.23
Peak Area 1351591 0.05
HETP 7691
Tailing Factor 1.20
Resolution 1.4
CONCLUSION
The results of the validation tests indicate that the
method is simple, accurate, precise and specific. This
method is suitable for the routine quality control of the
tablet dosage form.
Acknowledgements: The authors are very thankful to
GlaxoSmithKline Pharmaceutical Ltd., Nashik, India for
providing samples for this research work and
management of Amrutvahini Sheti and Shikshan Vikas
Sanstha and Amrutvahini College of Pharmacy,
Sangamner, M.S., India for providing the facilities for the
research work.
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Source of Support: Nil, Conflict of Interest: None.