Spectrofluorimetric and TLC-densitometric methods for a stability indicating assay of valacyclovir hydrochloride in the presence of its degradation product

Article abstract of DOI:10.1039/c4ra06677d

Two stability-indicating methods were developed and validated for determination of valacyclovir HCl (VAC) in the presence of its degradation product, acyclovir. The first was a TLC-densitometric method, in which chloroform:::methanol:::ammonia (50:::14:::2 v/v/v) was used as a mobile phase. Silica gel 60 F₂₅₄ was used as a stationary phase and the chromatogram was scanned at 253 nm. Using this chromatographic system, VAC can be readily separated from its degradation product and gives a compact spot at an R_F value of (0.55 ± 0.03). The peak area concentration plot is rectilinear over the range 20-300 ng per band. The second method represents the first attempt for spectrofluorimetric determination of VAC. The method was based on the reaction between VAC and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in an alkaline medium (pH 8.5) to form a highly fluorescent product that was measured at 500 nm after excitation at 465 nm. The fluorescence intensity concentration plot is rectilinear over the range 1-10 μg ml^-1. The proposed methods were successfully applied for the determination of VAC in its commercial tablets with average percentage recovery of 100.13 ± 0.33 and 98.50 ± 1.75 for TLC-densitometric and spectrofluorimetric methods, respectively, without interference from common excipients. The results of the proposed methods were statistically analyzed and found to be in accordance with those given by a reported method. In addition the proposed methods were extended to a stability study of VAC, where the drug was exposed to acidic, alkaline, oxidative and photolytic degradation according to ICH guidelines. This journal is

Full text of DOI:10.1039/c4ra06677d

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Spectrofluorimetric and TLC-densitometric
methods for a stability indicating assay of
valacyclovir hydrochloride in the presence of its
degradation product
Cite this: RSC Adv., 2014, 4, 42308
*
Sayed M. Derayea, Islam M. Mostafa and Mahmoud A. Omar
Two stability-indicating methods were developed and validated for determination of valacyclovir HCl (VAC)
in the presence of its degradation product, acyclovir. The first was a TLC-densitometric method, in which
chloroform : methanol : ammonia (50 : 14 : 2 v/v/v) was used as a mobile phase. Silica gel 60 F254 was
used as a stationary phase and the chromatogram was scanned at 253 nm. Using this chromatographic
system, VAC can be readily separated from its degradation product and gives a compact spot at an RF
value of (0.55 ꢀ 0.03). The peak area concentration plot is rectilinear over the range 20–300 ng per band.
The second method represents the first attempt for spectrofluorimetric determination of VAC. The
method was based on the reaction between VAC and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl)
in an alkaline medium (pH 8.5) to form a highly fluorescent product that was measured at 500 nm after
excitation at 465 nm. The fluorescence intensity concentration plot is rectilinear over the range 1–10 mg
mlꢁ1. The proposed methods were successfully applied for the determination of VAC in its commercial
tablets with average percentage recovery of 100.13 ꢀ 0.33 and 98.50 ꢀ 1.75 for TLC-densitometric and
spectrofluorimetric methods, respectively, without interference from common excipients. The results of
the proposed methods were statistically analyzed and found to be in accordance with those given by a
reported method. In addition the proposed methods were extended to a stability study of VAC, where the
drug was exposed to acidic, alkaline, oxidative and photolytic degradation according to ICH guidelines.
Received 5th July 2014
Accepted 28th August 2014
DOI: 10.1039/c4ra06677d
www.rsc.org/advances
technique for analysis of drugs due to its low operating cost, high
sample-throughput, and need for minimum sample clean-up. The
1. Introduction
major advantage of TLC-densitometric lies in several samples can
be run simultaneously using a small quantity of mobile phase
thus lowering analysis time and cost per analysis, Unlike HPLC,
wherein substantial amounts of mobile phase and time are
required for analysis of multiple samples.25 TLC-densitometric
method has the advantages of being sensitive, cheap and one of
the simplest chromatographic technique that does not need large
precautions as in HPLC technique which should be taken to
prevent several problems in HPLC such as plugging of the column
and variation of retention time. Both proposed methods of anal-
ysis of the cited drug are able to determine the drug in pure form
and pharmaceutical tablets without interference from tablet
excipients. Furthermore, both methods were developed to be
stability-indicating methods that can be used for determination of
VAC in presence of its degradation products.
Valacyclovir hydrochloride (VAC) is L-valine 2-[(2-amino-1,6-dihy-
dro-6-oxo-9H-purin-9yl)methoxy]ethyl ester hydrochloride (Fig. 1).
Aer its oral administration, VAC is rapidly converted into
acyclovir which has demonstrated antiviral activity, against herpes
simplex virus type I (HSV-1), type 2 (HSV-II) and Varicella zoster
virus.1 The oral bioavailability of acyclovir is higher aer admin-
istration of VAC relative to acyclovir itself.2,3 The mechanism of
action of acyclovir involves the highly selective inhibition of virus
DNA replication, via enhanced uptake in virus-infected cells and
phosphorylation by viral thymidine kinase.4,5 Few analytical
methods have been reported for the determination of VAC. These
methods include: spectrophotometric,6–8 chromatographic,9–22
capillary electrophoretic23 and electrochemical methods.24 To the
present time there is no spectrouorometric method reported for
analysis of VAC. Accordingly we here present the rst attempt for
spectrouorimetric method in addition to TLC-densitometry for
the determination of VAC in pure form and pharmaceutical
2. Experimental
tablets. TLC-densitometric method is becoming
a routine
2.1. Apparatus
Camag TLC scanner 3 S/N 130319 in the absorbance mode
utilizing deuterium lamp as a source of radiation with Wincats
Anal. Chem. Dept., Faculty of Pharmacy, Minia University, Minia, Egypt. E-mail:
momar71g@yahoo.com
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2.4. Preparation of sample solutions
An accurately weighed amount of nely powdered twenty tablets
equivalent to 25 mg of VAC was transferred into a 50 ml volu-
metric ask. The powder was dissolved in 25 ml methanol,
sonicated for 15 min, diluted to the mark with methanol and
mixed well. The resulting solution was centrifuged at 3000 rpm
for 5 min. Two ml of this solution was diluted to 10 ml with
methanol.
2.5. General analytical procedure
2.5.1. For TLC-densitometric method. TLC-densitometric
method was performed in aluminum plates (10 ꢂ 20 cm) coated
with 0.2 mm layers of silica gel 60 F254. Standard or sample
solutions were applied as 6.0 mm at bands at the concentration
range of 20–300 ng per band to the plate at 10 mm from the
bottom and 10 mm from the side edges of the plate. The band
was dried with the aid of an online nitrogen gas. Linear
ascending development with chloroform : methanol : ammonia
(50 : 14 : 2 v/v/v) as a mobile phase was performed in a Camag
twin-trough glass chamber previously saturated with mobile
phase for 15 min at room temperature. Aer development, the
plates were dried in a current of hot air using a hair-dryer.
Densitometric scanning in reectance mode at 253 nm was
performed at 20 mm sꢁ1 scanning speed and the slit dimension
was kept at 6.0 ꢂ 0.30 mm. The TLC chromatograms were
manipulated by WINCATS soware.
Fig. 1 Chemical structure of valacyclovir HCl and acyclovir.
soware. Samples were applied with Camag micro-syringe
100 ml, using a Camag Linomat 5 autosampler, (Camag, Mut-
tenz, Switzerland).
A Perkin Elmer LS 45 luminescence spectrometer (United
Kingdom) equipped with 150 watt xenon arc lamp and 1 cm
quartz cell. Slit width for both monochromators were set at
10 nm. The spectrometer is connected to a PC loaded with the
FL Winlab™ soware, Spectronic™ Genesys™ 2 PC. Ultravi-
olet/visible spectrophotometer (Milton Roy Co, USA) with
matched 1 cm quartz cell and was connected to PC loaded with
Winspec™ application soware.
2.5.2. For spectrouorimetric method. One milliliter of the
working standard or sample solution in the concentration range
of 10–100 mg mlꢁ1 was transferred into 10 ml test tubes, then 1.0
ml of borate buffer solution (pH 8.5) and 1.2 ml of NBD-Cl
reagent (0.1% w/v) were added. The reaction was allowed to
proceed at 70 ꢃC in a water bath for 20 min. Aer cooling the test
tubes in ice bath, 0.2 ml of conc. hydrochloric acid was added at
the wall of the test tube and mixed well. Finally the contents of
the test tube were quantitatively transferred into 10 ml cali-
brated asks and completed to the mark with methanol. The
uorescence intensity of the resulting solutions was measured
at 500 nm (lex, 465 nm). A blank experiment was performed
similarly omitting the drug.
MLW type thermostatically controlled water bath (Memmert
GmbH, Schwabach, Germany) was used for heating purposes.
2.2. Material and chemicals
VAC was obtained as a gi from (Hikma Pharma, Cairo, Egypt).
Valtovir® tablets (Hikma Pharma, Cairo, EGYPT) labeled to
contain 1110 mg VAC per tablet was purchased from local
market. Acyclovir was obtained as a gi from GlaxoSmithKline
(Cairo, Egypt). 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole NBD-Cl
(Sigma Chemical Co., St. Louis, USA) was freshly prepared as
0.1% w/v in methanol. Chloroform, hydrochloric acid,
ammonia solution, hydrogen peroxide (30% v/v), sodium
hydroxide, boric acid and methanol were purchased from El-
Nasr Chemical Co. (Cairo, Egypt). All chemicals and reagents
used were of analytical grade.
2.6. Procedures for stability indicating assay
2.6.1. Alkali induced degradation. Into 10 ml test tube,
1 ml of VAC solution (1 mg mlꢁ1) was transferred and 1 ml of
0.05 M NaOH was added. The solution was le at room
temperature for 2 hours. Aer the specied time, the solution
was neutralized to pH 7 using 1 ml of 0.05 M HCl solution and
completed to the volume with methanol.
Aluminum plate precoated with silica gel 60 F254, (20 ꢂ
10 cm) with 250 mm thickness (Merck, Darmstadt, Germany).
2.3. Standard solution preparation
An accurately weighed amount (10 mg) of VAC was quantita-
2.6.2. Acid induced degradation. Into 10 ml volumetric
tively transferred into a 100 ml calibrated ask, dissolved in ask, 5 ml of VAC solution (1 mg mlꢁ1) was transferred and
20 ml methanol, and then completed to volume with the same reuxed with 5 ml of 1 M HCl for 2 hours. Aer the specied
solvent to obtain a stock solution of 0.1 mg mlꢁ1. The stock time, the solution was neutralized to pH 7 using 1 M NaOH
solution was further diluted with methanol to obtain working solution. The solution was transferred quantitatively into 50 ml
solutions in the concentration range of 5–75 mg mlꢁ1 and volumetric ask and completed to the volume with methanol.
10–100 mg mlꢁ1 for TLC-densitometric and spectrouorimetric
methods, respectively.
Oxidative induced degradation with hydrogen peroxide was
carried out by transferring 1.0 ml of VAC solution working
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solution (1 mg mlꢁ1) into 10 ml volumetric ask then 1 ml of
hydrogen peroxide (30.0% v/v) was added. The solution was kept
at room temperature for a period of 2 hours. The solution was
heated in boiling water bath for 10 minutes to completely
remove the excess of hydrogen peroxide. The volume was
completed to 10 ml with methanol.
The photochemical stability of the drug was studied by
exposing the stock solution (0.1 mg mlꢁ1) to direct articial day
light for ve days.
For TLC-densitometric method 6 ml of the above solution
was diluted to 10 ml with methanol in volumetric ask then 4 ml
was applied to TLC plate and the plate was developed and
analyzed as described in “general analytical procedures”. For
spectrouorimetric, 1.0 ml of the above solution was used in the
“general analytical procedures”.
Fig. 3 Effect of the pH on the condensation reaction of VAC (4 mg mlꢁ1
and NBD-Cl.
)
3. Results and discussion
3.1. Method optimization for the TLC-densitometric
measurements
method for determination of VAC. The reaction product was
colored yellow and exhibited highest uorescence intensity at
lem 500 nm aer excitation at lex 465 nm, (Fig. 2). The experi-
mental parameters affecting the development and stability of
the reaction product were investigated and optimized. Each
parameter was changed individually while the others were kept
constant. These parameters include; pH, volume of the buffer,
concentration of NBD-Cl, reaction time, temperature and
diluting solvent.
3.2.1. Effect of pH. The effect of the solution pH on the
reaction product formation was studied in the pH range of 7–10.
The uorescence intensity of the reaction product was highly
dependent on pH. The highest intensity was obtained in the pH
range of (8.3–8.8). Higher or lower solution pH resulted in a
decrease on the uorescence intensity. Therefore the optimum
pH was 8.5 using borate buffer solutions (Fig. 3).
TLC procedure was optimized with a view to develop a stability-
indicating assay method. The pure forms of drug and its
degradation product, acyclovir were spotted on TLC plates and
run in different solvent systems. Initially, chloroform–methanol
in varying ratios was tried. Chloroform–methanol (5 : 1.4 v/v) as
a mobile phase gave good resolution with RF value of 0.56 ꢀ 0.03
for VAC and 0.3 ꢀ 0.03 for acyclovir unfortunately with tailing
peaks. Addition of 0.2 ml ammonia to the above mobile phase
gave compact spots for both drug and acyclovir. The nal
mobile phase consisted of chloroform : methanol : ammonia
(5 : 1.4 : 0.2 v/v/v) which gave a sharp and symmetrical peaks. To
obtain a well-dened spots the chamber was saturated with the
mobile phase for 15 min at room temperature prior to the
analysis.
3.2.2. Effect of the volume of the buffer. Several volumes
(0–3 ml) of borate buffer of pH 8.5 were used for general
assay procedure. The maximum uorescence intensity was
obtained when the buffer volume was in the range of 0.8–1.5 ml.
Lower or higher volume showed marked decrease in the
uorescence intensity, Therefore the selected volume of borate
buffer was 1 ml.
3.2. Method optimization for the spectrouorimetric
measurements
VAC contains a primary amino group that could react with NBD-
Cl through a condensation reaction. This reaction was investi-
gated in this study to develop the rst spectrouorimetric
3.2.3. Effect of the volume of NBD-Cl solution. Different
volumes of NBD-Cl (0.1% w/v) were used in performing the
general analytical procedure. Increasing NBD-Cl volume
produced a gradual increase in the uorescence intensity until
reaching a steady state at 1.0–1.5 ml of NBD-Cl. Further increase
in the volume resulted in slight decrease in the uorescence
intensity. So, 1.2 ml NBD-Cl was chosen for subsequent works
(Fig. 4).
3.2.4. Effect of heating time and temperature. The general
analytical procedure was carried at different temperature or for
different heating period. It was found that either increasing the
temperature of the reaction mixture or heating for longer time
produced a corresponding increase in the uorescence intensity
of the reaction product. However, it was noticed that heating at
a relatively low temperature (70 ꢃC) for a longer period (20 min)
was better than heating at a higher temperature for a shorter
Fig. 2 Excitation and emission spectra of the reaction between NBD-
Cl and VAC (4 mg mlꢁ1).
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Fig. 4 Effect of the volume of NBD-Cl reagent on the fluorescence
intensity of the reaction with VAC (4 mg mlꢁ1).
Fig. 5 The stoichiometry of the reaction between NBD-Cl and VAC.
period of time, regarding the reproducibility of the uorescence
intensities.
3.2.5. Effect of diluting solvent. In order to select the most
appropriate solvent for dilution, different solvents were tested
including; methanol, ethanol, acetonitrile, and acetone. Meth-
anol was found to be a best diluting solvent as it gave highest
uorescence intensity, and therefore was selected as diluting
solvent. Unfortunately the high reading obtained with meth-
anol was accompanied with the presence of high uorescence
background. This was attributed to the hydrolysis of NBD-Cl to
the corresponding hydroxy derivative, namely, 4-hydroxy-7-
nitrobenzo-2-oxa-1,3-diazole (NBD-OH).26 The uorescence of
NBD-OH could be quenched by decreasing the pH of the reac-
tion medium to less than one.27 Therefore acidication of the
reaction mixture prior to dilution and measurement of the
relative uorescence intensity was necessary to effectively
decrease the background uorescence. In the same time, the
uorescence of the reaction product was not affected by acidi-
cation, thus the sensitivity was ultimately enhanced. The
suitable amount of hydrochloric acid required for acidication
was found to be 0.2 ml of the concentrated acid.
the proton of amino group acidic therefore no condensation
reaction could occur with NBD-Cl.
3.4. Method validation
The proposed methods have been validated according to ICH
guidelines.29 The investigated parameters include: linearity and
range, sensitivity, precision, accuracy, recovery and robustness.
The International Conference on Harmonization (ICH)
guideline entitled ‘stability testing of new drug substances
and products’ requires the stress testing to be carried out to
elucidate the inherent stability characteristics of the active
substance.30 An ideal stability-indicating method is one that
quanties the drug and resolves its degradation products.
3.4.1. Linearity and range. The proposed methods showed
a good linear correlation in concentration range of 20–300 ng
per band (r2 ¼ 0.9992) for densitometric method and 1–10 mg
mlꢁ1 (r2 ¼ 0.9991) for spectrouorimetric method. The high
value of correlation coefficient assured the validity of the
proposed methods (Table 1).
3.3. Stoichiometry and mechanism of the reaction
Job's method of continuous variation28 was employed for
investigating the stoichiometry of the reaction. Equimolar
solutions of both the drug and NBD-Cl reagent (1 ꢂ 10ꢁ3 M)
were prepared. Then portions of mixture of master solutions of
the drug and reagent were made up comprising different
complementary
proportions
(0.2 : 2.8,
0.4 : 2.6,
0.5 : 2.5.2.6 : 0.4, 2.8 : 0.2). Results suggested that VAC reacts
with NBD-Cl in the ratio 1 : 1 (Fig. 5). Based on this ratio the
suggested mechanism of the condensation reaction with NBD-
Cl was presented in (Fig. 6).
It should be noted that, the degradation product, acyclovir
could not undergo the condensation reaction with NBD-Cl and
this can be inferred from the absence of any uorescence
property due to the reaction. This may be attributed to
Fig. 6 The suggested mechanism of the condensation reaction of
tautomerism of the amino group in the guanine base making VAC with NBD-Cl.
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Table 1 Regression equation and validation parameters for the proposed TLC-densitometric and spectrofluorimetric methods for determi-
nation of VAC
Parameters
TLC-densitometric method
Spectrouorimetric method
Linearity range
20–300 (ng per band)
0.9996
0.9992
1–10 (mg mlꢁ1
0.9996
0.9991
)
Correlation coefficient (r)
Determination coefficient (r2)
Intercepta ꢀ SD
249.75 ꢀ 24.31
104.03 ꢀ 1.67
Condence limit of interceptb
Slopeb ꢀ SD
192.25
99.74
12.48 ꢀ 0.14
12.16
6.42 (ng per band)
19.47 (ng per band)
22.2 ꢀ 0.29
21.45
Condence limit of slopeb
LOD
0.25 (mg mlꢁ1
0.75 (mg mlꢁ1
)
)
LOQ
a
Number of determination is 9 for TLC-densitometric method and 7 for spectrouorimetric method. b At 95% condence limit.
Table 2 Standard addition method for the recovery studies
TLC-densitometric method
Concentration (ng per band)
Spectrouorimetric method
Concentration (mg mlꢁ1
)
Taken
% Added
% Recoverya ꢀ SD
Taken
% Added
% Recoverya ꢀ SD
80
80
80
80
0
99.56 ꢀ 0.46
99.70 ꢀ 0.21
99.85 ꢀ 0.11
99.90 ꢀ 0.12
2
2
2
2
0
99.74 ꢀ 1.29
99.10 ꢀ 0.99
98.55 ꢀ 0.69
99.35 ꢀ 0.45
50%
100%
150%
50%
100%
150%
a
The value is the average of six determinations.
deliberate variation in method parameters and provides an
indication of its reliability in routine analysis.
3.4.2. Accuracy. Standard addition method was applied to
examine of the accuracy of the proposed methods. Known
amounts of pure drug were added to a previously analyzed
sample solution. The mixtures were re-analyzed by the proposed
procedures in six replicates and the percentage recovery was
calculated. The calculated high percentage average recovery and
low relative standard deviation indicate the suitable accuracy of
the method (Table 2).
3.4.3. Precision. The interday and intraday precisions31
were examined by analyzing six replicates of VAC solutions at
three concentration levels (80, 120 and 300 ng per band for TLC-
densitometric and 3, 4 and 6 mg mlꢁ1 for spectrouorimetric
method) in seven successive days. The low value of RSD (less
than 1.28%) indicates fairly the good, repeatability and repro-
ducibility of the proposed method (Table 3).
Robustness of TLC-densitometric method was performed by
introducing small changes in the mobile phase composition,
saturation time and wavelength, then examining their effects on
the obtained results. Mobile phases having different composi-
tion of chloroform : methanol : ammonia (4.9 : 1.3 : 0.1 and
5.1 : 1.5 : 0.3, v/v/v) and different wavelengths (248 and 258 nm).
Robustness for spectrouorimetric method was performed by
Table 3 The intra- and inter-day precision for the determination of
VAC by the proposed methods
Intra-day precision
Inter-day precision
3.4.4. Sensitivity. Sensitivity of the proposed method was
evaluated by calculating limit of detection (LOD) and limit of
quantication (LOQ) using the formula; “x ¼ ns/S”, where x is
LOD or LOQ, n is a numerical value equal to 3.3 or 10 for LOD or
LOQ, respectively and s is the standard deviation of intercept
and S is the slope of calibration graph.30–32 The calculated LOD
and LOQ values for TLC-densitometric were 6.4 and 19.5 ng per
band, respectively and for spectrouorimetric method were 0.25
and 0.75 mg mlꢁ1 respectively. The low values of these param-
eters indicate the high sensitivity of the proposed method
(Table 1).
Amounta % Recoveryb ꢀ % RSD Amounta % Recoveryb ꢀ % RSD
TLC-densitometric method
80
120
300
99.89 ꢀ 0.33
98.60 ꢀ 0.52
99.45 ꢀ 0.49
0.08
0.12
0.30
98.41 ꢀ 0.29
99.73 ꢀ 0.26
99.55 ꢀ 0.39
Spectrouorimetric method
3
4
6
99.07 ꢀ 1.28
101.2 ꢀ 0.26
100.8 ꢀ 1.11
3
4
6
99.86 ꢀ 1.22
99.32 ꢀ 0.45
98.65 ꢀ 0.93
a
The amount is ng per band for TLC-method and mg mlꢁ1 for the
b
3.4.5. Robustness. The robustness of an analytical proce-
dure refers to its capability to remain unaffected by small and
spectroourimetric method.
The value is the average of six
determinations.
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Fig. 7 TLC chromatograms of VAC degradation. (a) Alkali induced degradation. (b) Acid induced degradation. (c) Photolytic degradation. (d)
Oxidative degradation.
making small changes in general analytical procedure, such as appearance of an additional peak at RF value of 0.3 ꢀ 0.03
NBD-Cl volume (ꢀ0.2 ml), solution pH (ꢀ0.2 unit), and volume (Fig. 7a and b). The new peak appears before the parent drug
of the buffer (ꢀ0.5 ml). The obtained results from small varia- peak which gives an indication that the degradation product is
tion in any of these parameters did not signicantly affect the more hydrophilic and contains polar group. The degradant was
results of both methods. Therefore the procedures of the identied as acyclovir through comparison between RF of the
proposed methods are considered robust.
degradant and RF of the standard authentic acyclovir. About
45% of the drug was degraded in case of acidic degradation
while 52% of the drug was degraded in case of alkaline degra-
dation. On the other hand, the exposure to oxidation with 30%
v/v hydrogen peroxide and photodegradation with direct sun
light for ve days did not produce any signicant effect on the
drug chromatograms and only the peak of VAC was observed at
RF ¼ 0.55 (Fig. 7c and d). This gives an indication about the
3.5. Stability-indicating studies
VAC was exposed to different stress conditions including acid,
alkali, oxidation and photo-degradations. Aer applying the
degradation procedures, the proposed methods were applied
for determination of the extent of degradation of the cited drug.
Alkaline and Acidic induced hydrolysis resulted in the
Table 4 Analysis of containing 1110 mg of VAC using the proposed and reference methodsa,b
% Recovery ꢀ SD
Method
Proposed method
Reference method
t-value
F-value
TLC-densitometry
Spectroourimetry
100.13 ꢀ 0.33
98.5 ꢀ 1.75
99.44 ꢀ 0.62
99.44 ꢀ 0.62
1.68
0.31
3.70
1.08
a
Average of ve determinations. b Tabulated values for t and F are 2.306 and 6.338 respectively.
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stability of VAC against oxidation and photodegradation but its
liability of for basic and acidic hydrolysis.
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in tsetse ies (Glossina pallidipes) using LC-MSMS, J.
Chromatogr., B: Anal. Technol. Biomed. Life Sci., 2010, 878,
2384–2390.
The proposed methods were applied for determination of VAC
in the commercially available tablets. The mean recoveries
values were 100.13 ꢀ 0.33 and 98.5 ꢀ 1.75 for TLC-densito-
metric and spectrouorimetric methods, respectively. The
results of the proposed methods were statistically compared
with that of the reference method8 regarding t- and F- tests at
95% condence level. As shown in Table 4, there is no signi-
cant difference between the results of both proposed and
reported method, as the calculated values is less than the
tabulated one. This indicated that the proposed methods have
good accuracy and precision.
4. Conclusion
The proposed TLC-densitometric and spectrouorimetric
methods are accurate, sensitive and selective methods. They
allow the determination of VAC in pure form and in the pres-
ence of its degradation product, as well as in pharmaceutical
dosage form. So, it can be considered as a stability-indicating
one. The satisfactory sensitivity and simplicity make the
methods suitable for routine analysis of VAC in quality control
laboratories.
13 C. Pham-Huy, F. Stathoulopoulou and P. Sandouk, Rapid
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