A high-performance liquid chromatographic method for determination of
Valacyclovir in Pharmaceutical Dosage Forms and Rat Plasma
1
Suddhasattya Dey*, 1Anjan De, 1Arindam Sarkar, 2Prasana Pradhan, 2Shreya Shah, 2
Parmar Hardhik and 2Jayesh Thakar
1
Dr. B.C. Roy College of Pharmacy and Allied Health Sciences, Bidhan Nagar,
Durgapur-713206, West Bengal, India.
2
Sigma Institute of Pharmacy, At-Bakrol, Woghodia, Near Ajwa-Nimata Road,
Vadodara, Gujarat-390016, India.
Corresponding Author: Suddhasattya Dey
Phone no.: +91-9593469634/+91-9593469671
ABSTRACT
A simple, high performance liquid chromatographic method has been developed
for the determination of valacyclovir in pharmaceutical dosage forms and
rat plasma. The elution was performed using different mobile phase mixture
of acetonitrile: methanol in ratio of 15:85 for pharmaceutical dosage form
and acetonitrile: methanol: water in the ratio of 12:44:44 for plasma
samples at a flow rate of 1.2 ml min-1 on a Phenomenex C18 column (150 ×
4.6 mm, i.d., 5μm) at ambient temperature. The drugs were monitored at a
wavelength of 260 nm and were separated within 10 min. Marketed
formulations were prepared in suitable dilutions and plasma samples were
prepared by precipitating proteins with the help of 25% perchloric acid.
The method was successful in detecting the drugs at a concentration of less
than 0.05 μg/ml. %RSD for intra- and inter-day studies was found to be
within 8.83% for all the selected concentrations. Moreover, the method was
validated as per ICH guidelines and the results were found to be within the
acceptable range. Hence, the proposed method can be used for the routine
quality control of the drugs and can also be applied to pharmacokinetic
studies.
Keywords:
Valacyclovir, Reverse phase HPLC, Validation, Rat plasma
INTRODUCTION
Valacyclovir [1] (Fig 1) is, L-Valyl ester (9-[(2-hydroxy ethoxy) methyl]
guanine hydrochloride),
Figure1
: Structure of valacyclovir
of acyclovir, after oral administration is rapidly converted into acyclovir
which shows antiviral activity against herpes simplex virus type I (HSV-1)
and (HSV-2), Varicella Zoster Virus (VZV). Acyclovir oral bioavailability
was increased when administered in the form of valacyclovir [2] and
valacyclovir (VCV) rapidly converted to Acyclovir (ACV) in vivo which
inhibits DNA synthesis. Valacyclovir is available as tablet dosage form in
market and few HPLC [3-5] methods were reported for the estimation of
valacyclovir in pharmaceutical formulations and in biological fluids [6]
and one of spectrophotometric [7] method were also reported. There are two
stability indicating HPLC [8, 9] methods were developed for valacyclovir,
Several studies have reported HPLC determination of ACV in different
matrices viz. pharmaceuticals [10–14], human plasma [15–20], serum [21] and
maternal plasma, amniotic fluid, fetal and placental tissues [22].
High-performance capillary electrophoresis has also been used for the
determination of ACV (acyclovir) in urine [23] and plasma [24]. A sensitive
assay is reported by Jin et al. [25] to determine ACV in aqueous humor by
LC–MS. The response was linear over the concentration range of 5–50ng/ml.
Recently a quantitative determination of ACV in plasma has been done by
near-infrared spectroscopy [26]. A sensitive and selective LC–MS/MS method
based on hydrophilic interaction liquid chromatography has been reported
for the determination of ACV in pregnant rat plasma and tissues [27] but
the reported methods were having disadvantages like high flow rate and high
retention time and more organic phase and the aqueous phase is not
compatible to LC-MS analysis. The simultaneous estimation of these drugs in
biological samples has been the subject of very few reports due to
structural similarity of VCV (valacyclovir) and ACV with the endogenous
components. Weller’s HPLC method [28], proposes a gradient mobile phase for
their simultaneous measurement in plasma. Pham-Huy et al. [29] have
developed a simple and specific HPLC–UV assay for VCV and ACV in human
serum, urine and dialysis liquids. The lower limits of quantification were
250 and 200 ng/ml for VCV and ACV respectively and the chromatographic run
time was 12 min. Recently a selective and rapid liquid
chromatography/negative-ion electrospray ionization mass spectrometry
method has been reported for the quantification of VCV and its metabolite
in human plasma [30]. A thorough and complete method validation of VCV in
rat plasma was done following the USFDA guidelines [31]. The analytes were
separated on a reversed-phase porous graphitized carbon column with a short
analytical run time of 4 min. The proposed HPLC method utilizes economical
solvent system as compared with the previous reported methods and is
compatible with LC-MS analysis. The proposed HPLC method leads to better
retention time, very sharp and symmetrical peak shapes. The aim of the
study was to develop a simple, precise and accurate reverse-phase HPLC
method for the estimation of valacyclovir in bulk drug samples, in
pharmaceutical dosage forms and rat plasma which can be effectively applied
for the pharmacokinetic study of the drug valacyclovir.
EXPERIMENTAL SECTION
1.
Chemicals and Reagents
Valacyclovir was obtained from Actavis Pharmaceutical Ltd., Chennai, India.
Acetonitrile, Methanol and Water (obtained from Merck chemicals, Worli,
Mumbai, India.) of HPLC grade were used. All the other reagents (70%
perchloric acid) used were of Development and Validation of RP-HPLC Method
for Determination of Valacyclovir was of analytical grade are also obtained
from Merck chemicals, Worli, Mumbai, India. Pharmaceutical formulations
were purchased from Cipla Pharmacy.
2. Instrumentation and Chromatographic Conditions
A high-performance liquid chromatography (Shimadzu, Kyoto, Japan) was
composed of a LC-20AT Prominence solvent delivery module, a manual rheodyne
injector with a 20-µl fixed loop and a SPD-20A Prominence UV–visible
detector. Separation was performed on a Phenomenex C18 column (particle
size 5µm; 250mm×4.6mm i.d.; Phenomenex, Torrance, USA) at an ambient
temperature. The data acquisition was made by Spinchrom Chromatographic
Station® CFR Version 2.4.0.195 (Spinchrom Pvt. Ltd., Chennai, India). The
mobile phase consisted of acetonitrile: methanol in ratio of 15:85 for
pharmaceutical dosage form and acetonitrile: methanol: water in the ratio
of 12:44:44 for plasma samples at a flow rate of 1.2 ml min-1.
3. Preparation of stock and standard solutions
Stock solution of 1mg/ml valacyclovir was prepared in methanol. Standard
solution of valacyclovir was prepared by mixing and diluting the
appropriate amounts from the individual stock solution. The final
concentrations of the standard solution were 1000, 900, 700, 500, 300, 100,
50, 25, 2.5 and 0.5µg/ml. Precision and accuracy standards with
concentrations of 900, 100, 25 and 0.5µg/ml were also prepared in the same
manner. Stock solutions were refrigerated when not in use and replaced on
bi-weekly basis. Fresh standard solutions were prepared for each day of
analysis or validation. For the analysis of pharmaceutical
formulations, ten tablets of valacyclovir were weighed and powdered
individually. The mixture of formulations was prepared by weighing amount
equivalent to labeled claim from the powdered formulations. To this, a
suitable amount of methanol was added. The mixture was subjected to
sonication for 30 min for a complete extraction of the drugs, and then
filtered through 0.2µm filter paper and diluted with methanol at a suitable
concentration range (15mcg/ml) and injected into HPLC system for the
analysis.
4. Calibration Curves
Pure drug calibration curve were prepared by mixing 20µl of the above
standard solutions and diluting it up to 200µl by methanol to obtain
calibration curve range of 0.05-100µg/ml. Plasma calibration points were
prepared by spiking 200µl of rat plasma with 20µl of above prepared
valacyclovir standard solutions. The calibration curves for rat plasma were
in the range of 0.05-100µg/ml. After each matrix was spiked, it was
subjected to further sample preparation before analysis.
5. Sample Preparation
The ten samples containing plasma spiked with valacyclovir of different
concentration was taken in 1.5ml micro-centrifuge tubes. To each 200µl of
sample was mixed with 45µl of 25% perchloric acid for 30 seconds. The
samples were centrifuged at 1200 g for 15min. 20µl of clear supernatant
liquid was transferred in Hamilton Syringe and injected into HPLC system
for analysis.
6. Sample Collection
The use of animals in this study was approved by GTU (Gujarat Technological
University, Ahmadabad, Gujarat, India) and CPCSEA (Committee for the
Purpose of Supervision on Experimental Animals). The rats were housed one
animal per cage in Sigma Institute of Pharmacy animal house. The
environment was controlled with daily feeding and water.
Blood samples were collected in 2ml micro-centrifuge tube from retro
orbital plexus of albino rats. The 2ml micro-centrifuge containing blood
was centrifuged at 15000 rpm for 15 min and the plasma was collected
carefully. Blood sample was collected on regular basis from different rats
and plasma was separated till the study is been completed so that the
analysis is unbiased in nature.
RESULTS AND DISCUSSION
1. Optimization of Chromatographic Conditions
The drugs were soluble in organic solvents like methanol and acetonitrile.
During the development phase, the mobile phase containing methanol-water in
different ratios and methanol-buffer solution resulted in peaks with poor
resolution and the acetonitrile-methanol-water and acetonitrile-methanol
resulted in good resolution of peaks. The successful use of both
acetonitrile and methanol along water reduced tailing and resulted in good
peak symmetry and resolution. The optimized mobile phase contained
acetonitrile: methanol in ratio of 15:85 for pharmaceutical dosage form and
acetonitrile: methanol: water in the ratio of 12:44:44 for plasma samples
flow rate of 1.2 ml min-1. The analytes were monitored at 260 nm and the
retention times were found to be 2.0min for valacyclovir in both the mo
bile phases, respectively. (Fig. 2, 3 and 4)
Figure 2:
Chromatogram of blank rat plasma
Figure 3:
Chromatogram of Valacyclovir in rat plasma 70mcg/ml
Figure 4:
Chromatogram of Pure Valacyclovir in methanol 30mcg/ml
1.1. Validation of the Developed Method
The proposed method was validated as per the guidelines in ICH for its
linearity, accuracy, precision, specificity and selectivity, robustness and
stability etc.
1.2. Linearity:
The linearity was tested for the concentration range of 100, 90, 70, 50,
30, 10, 5, 2.5, 0.25 and 0.05µg/ml the calibration curve was constructed
and evaluated by its correlation coefficient. The equation for plasma is Y=
82.09X + 56.90 and the equation for pure drug is Y= 38.22X + 3.598. (Table
1 & Fig. no.5 & 6)
Table 1
: Linear regression equations generated from validation for each matrix:
Slope, Intercept and Coefficient of determination
|
Analyte
|
Matrix
|
Concentration (µg/ml)
|
Area (mV.s)
|
Slope
|
Intercept
|
R2
|
|
Valacyclovir
|
Pure drug in plasma
|
0.05
|
5.08352
|
82.09
|
56.90
|
0.999
|
|
0.25
|
22.4121
|
|
2.5
|
247.176
|
|
5
|
438.352
|
|
10
|
922.704
|
|
30
|
2610.5
|
|
50
|
4223.52
|
|
70
|
5916.93
|
|
90
|
7290.29
|
|
100
|
8267.04
|
|
Pure drug in methanol
|
0.05
|
1.92097
|
38.22
|
3.598
|
0.999
|
|
0.25
|
8.73483
|
|
2.5
|
81.3546
|
|
5
|
187.232
|
|
10
|
394.193
|
|
30
|
1172.58
|
|
50
|
1947.09
|
|
70
|
2666.35
|
|
90
|
3411.74
|
|
100
|
3840.18
|
Figure 5:
Calibration curve of pure valacyclovir in methanol
Figure 6:
Calibration curve of pure valacyclovir in rat plasma
1.3. Accuracy:
The accuracy of a method is expressed as the closeness of agreement between
the value found and the value that is accepted as a reference value. It is
determined by calculating the percent difference (%bias) between the
measured mean concentrations and the corresponding nominal concentrations.
The accuracy of the proposed method was tested by recovery experiments by
adding known amounts of valacyclovir corresponding to 80, 100 and 120% of
the label claim from the respective standard solution. The accuracy was
then calculated as the percentage of valacyclovir recovered by the assay
(Table 2). The precision of the proposed method was assayed by replicate
injections of valacyclovir.
Table 2
: Accuracy Data Obtained by Recovery Studies of valacyclovir in plasma and
adding pure valacyclovir 80%, 100% and 120% in methanol.
|
Analyte
|
Matrix
|
Nominal concentration (µg/ml)
|
Mean Concentration
Found (µg/ml)
|
S.D.
|
Precision
(%RSD)
|
Mean accuracy (%)
|
|
Valacyclovir
|
Pure drug in plasma
|
Intra-day
|
|
|
|
|
|
90
|
90.272
|
0.707805
|
0.784080
|
100.201
|
|
10
|
10.084
|
0.436433
|
4.32
|
100.84
|
|
2.5
|
2.53733
|
0.153221
|
6.038
|
101.49
|
|
0.05
|
0.052
|
0.003356
|
6.45
|
104
|
|
Inter-day
|
|
|
|
|
|
90
|
90.466
|
0.70734
|
0.78188
|
100.417
|
|
10
|
9.798
|
0.383627
|
3.915
|
97.98
|
|
2.5
|
2.594
|
0.187697
|
7.23
|
103.76
|
|
0.05
|
0.0516
|
0.004561
|
8.83
|
103.2
|
|
Pure drug in methanol
|
No. of Preparations
|
Amount Added
|
Recovery (%)
|
|
S1:80%
|
10
|
8
|
100.25
|
|
S2:80%
|
10
|
8
|
101.75
|
|
S3:80%
|
10
|
8
|
101.5
|
|
S1:100%
|
10
|
10
|
101
|
|
S2:100%
|
10
|
10
|
100.5
|
|
S3:100%
|
10
|
10
|
101.2
|
|
S1:120%
|
10
|
12
|
99.54
|
|
S2:120%
|
10
|
12
|
97.87
|
|
S3:120%
|
10
|
12
|
100.793
|
1.4. Precision:
The method was validated using four QC point for each calibration curve.
Five replicates of each QC points were analyzed every day to determine the
intra-day precision. This process was repeated three times over three days
in order to determine the inter-day precision. The concentration of the QC
points for plasma and pure drug were 90, 10, 2.5 and 0.05µg/ml. (Table 3)
Table 3
: Inter-day (n=5) and Intra-day (n=5) precision (%R.S.D.) measured for QC
points for valacyclovir from plasma and pure drug.
|
T.C.
Conc.
|
Day 1
|
Day 2
|
Day 2
|
Inter-day
|
|
Plasma
|
µg/ml
|
E.C.
|
%R.S.D.
|
E.C.
|
%R.S.D.
|
E.C.
|
%R.S.D.
|
E.C.
|
%R.S.D.
|
|
1.
|
90
|
90.46
|
0.6044
|
90.34
|
0.7061
|
90.06
|
0.1042
|
90.466
|
0.78188
|
|
2.
|
10
|
9.96
|
3.91
|
10.35
|
3.499
|
9.942
|
5.604
|
9.798
|
3.915
|
|
3.
|
2.5
|
2.486
|
5.68
|
2.53
|
8.32
|
2.596
|
4.163
|
2.594
|
7.23
|
|
4.
|
0.05
|
0.056
|
6.27
|
0.052
|
6.082
|
0.048
|
7.064
|
0.0516
|
8.83
|
|
Pure Drug
|
µg/ml
|
E.C.
|
%R.S.D.
|
E.C.
|
%R.S.D.
|
E.C.
|
%R.S.D.
|
E.C.
|
%R.S.D.
|
|
1.
|
90
|
90.36
|
1.24
|
90.94
|
1.24
|
90.98
|
0.907
|
90.808
|
1.4944
|
|
2.
|
10
|
9.88
|
2.35
|
10.01
|
1.01
|
10.07
|
2.837
|
10.094
|
2.8130
|
|
3.
|
2.5
|
2.516
|
1.27
|
2.49
|
1.53
|
2.494
|
2.7866
|
2.494
|
2.4814
|
|
4.
|
0.05
|
0.049
|
0.32
|
0.05
|
2.65
|
0.048
|
1.839
|
0.0496
|
3.05846
|
T.C. denotes theoretical concentration and E.C. denotes experimental
concentration.
1.5. Stability:
The stability of the drugs extracted from the plasma was subjected to
short-term stability by keeping at -20°C for 30 days. The study indicated
that the samples were stable where the percent ratios were within the
acceptable limits of 90-110%. (Table 4)
Table 4
: Storage stability data of valacyclovir in plasma at concentrations 90 and
10µg/ml
|
Matrix
|
Time (months)
|
Conc. Added (µg/ml)
|
Concentration Measured (µg/ml)
|
Mean
|
S.D.
|
%Dev
|
|
Plasma
|
Long stability
|
|
Assay 1
|
Assay 2
|
Assay 3
|
|
|
|
|
1
|
90
|
90.56
|
90.44
|
89.65
|
90.21
|
0.49
|
+0.23
|
|
10
|
10.54
|
11.02
|
9.65
|
10.40
|
0.69
|
+4
|
|
2
|
90
|
90.45
|
90.65
|
89.65
|
90.25
|
0.52
|
+0.17
|
|
10
|
10.47
|
10.02
|
10.41
|
10.3
|
0.24
|
+3
|
|
3
|
90
|
90.56
|
90.78
|
88.25
|
89.86
|
1.40
|
-0.255
|
|
10
|
10.89
|
11.25
|
9.65
|
10.59
|
0.83
|
+5.9
|
|
5
|
90
|
90.17
|
88.98
|
88.17
|
89.10
|
1.00
|
-1.099
|
|
10
|
9.59
|
9.89
|
9.45
|
9.643
|
0.22
|
-3.57
|
|
Freeze stability
|
|
|
|
|
|
|
|
|
|
90
|
91.25
|
90.56
|
89.59
|
90.46
|
0.83
|
+0.41
|
|
10
|
10.65
|
11.14
|
10.59
|
10.79
|
0.30
|
+7.9
|
|
Matrix
|
Time (months)
|
Conc. Added (µg/ml)
|
Concentration Measured (µg/ml)
|
Mean
|
S.D.
|
%Dev
|
|
Drug in methanol
|
Long stability
|
|
Assay 1
|
Assay 2
|
Assay 3
|
|
|
|
|
1
|
90
|
90.14
|
90.43
|
90.57
|
90.38
|
0.21
|
+0.32
|
|
10
|
10.51
|
10.14
|
10.23
|
10.29
|
0.19
|
+2.9
|
|
2
|
90
|
90.54
|
90.05
|
90.58
|
90.39
|
0.29
|
+0.33
|
|
10
|
10.51
|
10.11
|
10.58
|
10.4
|
0.25
|
+4
|
|
3
|
90
|
90.23
|
90.15
|
88.89
|
89.75
|
0.75
|
-0.377
|
|
10
|
10.48
|
10.47
|
10.12
|
10.35
|
0.20
|
+3.5
|
|
5
|
90
|
90.57
|
90.43
|
90.12
|
90.37
|
0.23
|
+0.31
|
|
10
|
10.04
|
9.54
|
9.15
|
9.576
|
0.44
|
-4.24
|
|
Freeze stability
|
|
|
|
|
|
|
|
|
|
90
|
90.54
|
90.47
|
90.57
|
90.52
|
0.05
|
+0.477
|
|
10
|
10.51
|
10.11
|
10.78
|
10.46
|
0.33
|
+4.6
|
1.6. Robustness:
The robustness of the proposed method was found after altering the
parameters deliberately: the mobile phase ratio variants: acetonitrile 14%
and 16% for plasma and 10% and 12% in pure methanol, flow rate variants:
1.3 and 1.1 ml min-1for both. The retention time of the compound was
evaluated, and the resolution had no significant changes when the
parameters were changed. However, there was a change in the retention times
with a change in flow rate, but this did not affect the peak symmetry. Each
mean value was compared with the mean value obtained by the optimum
conditions. A solution of 50 μg ml-1 of all the drugs extracted from the
plasma was used for the study. The relative standard deviation (%RSD) was
found to be within the limit. (Table 5 & 6)
Table 5
: Robustness for valacyclovir in methanol
|
Samp-le I.D.
|
Analytical Method
|
Valacyclovir Input (mg)
|
Valacyclovir Recovery (mg)
|
Valacyclovir Recovery (%)
|
Mean Recovery (%)
|
S.D.
|
%R.S.D.
|
|
1.
|
Flow rate 1.1ml/min
Mobile Phase: 16:84
Column: Phenomenix
|
10
|
10.12
|
101.2
|
101.2
|
0.282843
|
0.27948
|
|
2.
|
Flow rate 1.2ml/min
Mobile Phase: 15:85
Column: Phenomenix
|
10
|
10.14
|
101.4
|
|
3.
|
Flow rate 1.3ml/min
Mobile Phase: 15:85
Column: Phenomenix
|
10
|
10.08
|
100.8
|
|
4.
|
Flow rate 1.2ml/min
Mobile Phase: 17:83
Column: Phenomenix
|
10
|
10.14
|
101.4
|
Table 6
: Robustness for valacyclovir in plasma
|
Samp-le I.D.
|
Analytical Method
|
Valacyclovir Input (mg)
|
Valacyclovir Recovery (mg)
|
Valacyclovir Recovery (%)
|
Mean Recovery (%)
|
S.D.
|
%R.S.D.
|
|
1.
|
Flow rate 1.1ml/min
Mobile Phase: 12:44:44
Column: Phenomenix
|
10
|
9.26
|
92.6
|
96.025
|
2.564339
|
2.6704
|
|
2.
|
Flow rate 1.2ml/min
Mobile Phase: 10:45:45
Column: Phenomenix
|
10
|
9.56
|
95.6
|
|
3.
|
Flow rate 1.3ml/min
Mobile Phase: 14:43:43
Column: Phenomenix
|
10
|
9.75
|
97.5
|
|
4.
|
Flow rate 1.2ml/min
Mobile Phase: 12:44:44
Column: Phenomenix
|
10
|
9.84
|
98.4
|
1.7. Assay of Pharmaceutical Formulations:
The method developed was sensitive and specific for the
quantitative determination of Valacyclovir and also was subjected to
validation for different parameters; hence, it was applied
for the estimation of drug in pharmaceutical formulations.
Drug quantity equivalent to the labeled claim was weighed
accurately and used for the assay. Each sample was
analyzed in triplicate after extracting the drug as was
mentioned above in the experimental section (2.3). The
amounts of drugs were found to be within the range of
96-102%. None of the tablet excipients were found to
interfere with the analyte peak as shown in Fig. 7.
Figure 7:
Chromatogram of Marketed Valacyclovir tablet 15mcg/ml
CONCLUSIONS
A simple, specific, selective and precise method was developed for the
determination of anti-viral drugs valacyclovir. The mobile phase was easy
to prepare with little or no variation with-out the involvement of buffers
and was economical. The analysis time was found to be less than 4 min. The
recovery from formulations and rat plasma were in good agreement and they
suggested no interference in the estimation. Hence, this method can be
easily and conveniently used for the routine quality control of the drugs
in pharmaceutical dosage forms, can also be applied to clinical studies and
pharmacokinetic study of the drug valacyclovir.
ACKNOWLEDGEMENTS
I Mr.Suddhasattya Dey and other authors are very much thankful to Dr.
(Prof.) U.M. Upadhayay, Principal, Sigma Institute of Pharmacy, Vadodara,
India for providing the necessary chemicals for our work. I am also
thankful to the management of Sigma Institute of Pharmacy, Vadodara, India
for providing the facilities and instruments for this research work to be
carried out.
REFERENCES
[1] Martindale - The Complete Drug Reference, 33rd Edition. London,
Pharmaceutical Press, 1999; p.643
[2] ICH, Stability Testing of New Drug Substances and Products;
International Conference on Harmonization, IFPMA, Geneva, 2003.
[3] A.S. Jadhav, D.B. Pathare, M.S. Shingare, J. Pharmaceutical Biomedical
Analysis, 2007; 43, supplement 4: 1568-72.
[4] M.L. Palacios, G. Demasi, M.T. Pizzono, A.I. Seall, J. Liquid
chromatography Related Technology 2005; 28, supplement 5: 751-62.
[5] D.N. Fish, V.A. Vidaurri, R.G. Deeter, American Journal of Health
system pharmacy1999; 56(19):1957-1960.
[6] L. Jan, M. Clas. Antimicrobial Agents. Chemotherapy 2003; 47: 2438-41.
[7] G. Srinu Babu, I. Sarat babu, N. Kiran kumar, N.M. Yurandhar, CHAI.
Raju, Asian J. Chem 2007; 19 (2):1642-44.
[8] E.G. Gladys, L.A. Gordon, International Journal of Pharmaceutics 2006;
317: 14-18.
[9] K. Srinivasa Rao, M. Sunil, International Journal of Chem Tech Research
2009; 1(3): 702-708.
[10] M.M. Ayad, H.E. Abdellatef, M.M. El-Henawee, H.M. El-Sayed,
Spectrochimica Acta Part A 66 (2007) 106.
[11] I.A. Darwish, A.S. Khedr, H.F. Askal, R.M. Mahmoud, II Farmaco, 60
(2005) 555.
[12] K. Basavaiah, H.C. Prameela, U. Chandrashekar, II Farmaco 58 (2003)
1301.
[13] K. Basavaiah, H.C. Prameela, II Farmaco 57 (2002) 443.
[14] M. Sultan, II Farmaco 57 (2002) 865.
[15] D. Teshima, K. Otsubo, T. Yoshida, Y. Itoh, R. Oishi, Biomed.
Chromatogr. 17 (2003) 500.
[16] J.M. Poirier, N. Radembino, P. Jaillon, Ther. Drug Monitoring 21
(1999) 129.
[17] M. Fernandez, J. Sepulveda, T. Aranguiz, C. von Plessing, J.
Chromatogr. B 791 (2003) 357.
[18] R.A. Bangaru, Y.K. Bansal, A.R.M. Rao, T.P. Gandhi, J. Chromatogr. B
739 (2000) 231.
[19] K.K. Peh, K.H. Yuen, J. Chromatogr. B 693 (1997) 241.
[20] R. Boulieu, C. Gallant, N. Silbersrein, J. Chromatogr. B 693 (1997)
233.
[21] G. Bahrami, S. Mirzaeei, A. Kiani, J. Chromatogr. B 816 (2005) 327.
[22] S.D. Brown, C.A.White, C.K. Chu, M.G. Bartlett, J. Chromatogr. B 772
(2002) 327.
[23] S.S. Zhang, H.X. Liu, Y. Chen, Z.B. Yuan, Biomed. Chromatogr. 10
(1996) 256.
[24] H.C. VO, P.A. Henning, D.T. Leung, S.L. Sacks, J. Chromatogr. B 772
(2002) 291.
[25] L. Jin, G.Wei, L.U.Wei-Yue, X.U. Ling-Jie, J. Pan, Chromatographia 63
(2006) 239.
[26] L. Yu, B. Xiang, Microchem. J. 90 (2008) 63.
[27] S.D. Brown, C.A.White, M.G. Bartlett, Rapid Commun. Mass Spectrom. 16
(2002) 1871.
[28] S. Weller, M.R. Blum, M. Doucette, T. Burnette, D.M. Cederberg, P. de
Miranda, M.L. Smiley, Clinical Pharmacol. Ther. 54 (1993) 595.
[29] C. Pham-Huy, F. Stathoulopoulou, P. Sandouk, J.-M. Scherrmann, S.
Palombo, C. Girre, J. Chromatogr. B 732 (1999) 47.
[30] M. Kasiari, E. Gikas, S. Georgakakou, M. Kazanis, I. Panderi, J.
Chromatogr. B 864 (2008) 78.
[31] Guidance for Industry: Bioanalytical Method Validation, U.S.
Department of
Health and Human Services, Food and Drug Administration Centre for Drug
Evaluation and Research (CDER), Centre for Veterinary Medicine (CVM) 2001.