Kinetic spectrophotometric methods for the determination of isatin

Article abstract of DOI:10.14233/ajchem.2013.14067

Simple, rapid and sensitive kinetic spectrophotometric methods are described for the analysis of isatin in pure form and in mixture. The procedures were based on the kinetic investigations and studies were done upon the oxidation of isatin with potassium

Full text of DOI:10.14233/ajchem.2013.14067

Asian Journal of Chemistry; Vol. 25, No. 8 (2013), 4563-4568
http://dx.doi.org/10.14233/ajchem.2013.14067
Kinetic Spectrophotometric Methods for the Determination of Isatin
1
1,*
1
ZEID A. ALOTHMAN , MASOOM RAZA SIDDIQUI , SAIKH MOHAMMAD WABAIDUR ,
2
2
2
HAMAD A. AL-LOHEDAN , MOHD. SAJID ALI and M.Z.A. RAFIQUEE
1
2
Advanced Materials Research Chair, Chemistry Department, College of Science, King Saud University, Riyadh, Saudi Arabia
Surfactant Research Chair, Department of Chemistry, College of Science, King Saud University, Riyadh, Saudi Arabia
*
Corresponding author: E-mail: siddiqui124@gmail.com
(
Received: 19 May 2012;
Accepted: 15 February 2013)
AJC-13013
Simple, rapid and sensitive kinetic spectrophotometric methods are described for the analysis of isatin in pure form and in mixture. The
procedures were based on the kinetic investigations and studies were done upon the oxidation of isatin with potassium permanganate in
basic medium. Various parameters affecting the colour developments were investigated and parameters were optimized. The reliability
and the analytical performance of the proposed methods were validated for its linearity, range, accuracy and precision. The calibration
-1
curves were linear over the range of 5-50 µg mL . The limit of detection and limit of quantitation of the two methods ranges from
3
-4
-3
.4 × 10 -0.878 and 1.05 × 10 -2.663, respectively. The possible mechanism of the reaction is also proposed.
Key Words: Isatin, Spectrophotometry, Method development, Method validation.
1
2
13
15
graphic method , hyphenated techniques such as LC-MS
INTRODUCTION
14
GC-MS have their role in estimation of isatin. Fluorimetry
Chemically, isatin is known as 1H-indole-2,3-dione. It
1
was discovered by Erdman and Laurent and is reported to be
and colour reaction was also exploited for the determination
16
of isatin . Survey of the literature reveals that till date there is
present in the mammalian tissues and fluids and also found in
2
various plants . Owing to remarkable antibacterial and anti-
no analytical method based on kinetic spectrophotometry for
the determination of isatin. There is, therefore, a need for a
simple and sensitive kinetic spectrophotometric method for
the determination of isatin.
3
fungal properties this compound has a great significance in
medicinal chemistry. Various biological activities are asso-
ciated with isatin that include analgesic, CNS activities and
anticonvulsant, antidepressant, antiinflammatory, antimicro-
bial and also reported to be capable of crossing the blood-
Kinetic methods have been applied here for the estimation
owing to certain advantages in pharmaceutical analysis,
regarding selectivity and elimination of additive interferences,
which affect direct spectrophotometric methods. During the
method developmental process it was found that the reaction
of potassium permanganate with isatin in alkaline medium
resulted in green colour product and this reaction has not been
used in past to estimate the drug spectrophotometrically.
This paper describes a simple and sensitive kinetic
spectrophotometric method for the determination of isatin. The
method involves the oxidation of drug with alkaline potassium
permanganate at 25 ± 1 ꢀC and subsequent measurement of
absorbance at 605 nm. The initial rate and fixed time methods
are adopted for its determination in synthetic mixture.
4
brain-barrier . Several other pharmacological importance of
isatin is highlighted in the literature including the induction
of arousal, reduction in duration of slow-wave sleep and
increase of the seizure threshold in rats. The main purpose of
the discovery of the isatin was to serve as an inhibitor of
monoamine oxidase (MAO) and subsequently identified as
5
,6
selective inhibitor of MAO B . Further study on isatin
revealed the fact that it acts as an antagonist of both arterial
7
natriuretic peptide- stimulated and nitric oxide-stimulated
8
guanylatecyclase activity. Isatin is also considered as an
9
endogenous marker of stress and anxiety . In view of such an
important pharmacological role of isatin, there are needs to
develop a cost effective, simple and sensitive method for its
quality control. There are several analytical methods reported
for the quantitative analysis of isatin which include electro
EXPERIMENTAL
Evolution 300, thermo UV-visible spectrophotometer,
with matched quartz cells was used to measure the change in
absorbance with time. Waters Aquity UPLC system (waters
1
0,11
analytical methods , high performance liquid chromato-

4
564 Alothman et al.
Asian J. Chem.
Manchester UK) was employed to validate mass of the
oxidation product.
mixture (active isatin + placebo) equivalent to 5-50 µg/mL of
isatin was transferred to a 10 mL conical flask and measured
according to either of the proposed methods mentioned above.
Interference studies: The possible interferences of amino
acids and other oxidant were checked with the currently
developed methods. It was found that oxidant such as chlora-
mine T and sodium metavanadate interfere with the determi-
nation process while no interference was found from the
oxidant such as potassium iodate. The effect of amino acids
on the determination process was checked and it was found
that amino acids such as proline, L-tryptophan interfere with
the reagent in the determination process.
All reagents and chemicals used were of analytical or
pharmaceutical grade and the solutions were prepared in Milli
Q water. The isatin was obtained from Merck, Germany and
was used without further purification. Aqueous solutions of
-
1
-4
2
.0 × 10 M sodium hydroxide and 1.26 × 10 M potassium
permanganate (GR Grade, Merck Limited, Mumbai, India)
were prepared in doubly distilled water. Potassium perman-
ganate (GR Grade, Merck Limited, Mumbai, India) solution
should be freshly prepared and its apparent purity be assayed
17
by titrimetric method .
Standard drug solution: The standard test solution of
Evaluation of bias: The evaluation of bias has been done
2
0,21
0
5
.05 % isatin(Merck, Germany)was prepared by dissolving
0 mg of isatin in 100 mL Milli Q water.
by point and interval hypothesis tests . In the interval
hypothesis test the proposed method (method 2) is acceptable
when the true mean is within ± 2 % of that of reference method
Recommended procedure for the determination of isatin
(
method 1). i.e.,
-4
Initial-rate method:Aliquots of 1.26 × 10 M potassium
-1
1 2 1 1
-0.02µ < (µ - µ ) < 0.02µ
permanganate solution (2.5 mL) and 2.0 × 10 M NaOH (2.5
mL) were transferred into a series of 10 mL volumetric flask.
An accurate volume of the working solution of isatin (0.1-1.0
mL) was added and diluted up to the mark with the Milli Q
water. The content of the mixtures were mixed well and trans-
ferred immediately to the spectrophotometric cell. The increase
in absorbance was recorded as a function of time at 605 nm.
The initial rate of the reaction (ν) at different concentration
was obtained from the slope of the tangent of the absorbance-
time curve. The calibration curve was constructed by plotting
the log of initial rate of reaction (log ν) against the log of
molar concentration of isatin (log C). The content of the drug
was calculated either from the calibration graph or the linear
regression equation.
the above equation can also be written as
µ2
0
.98 <
< 1.02
µ1
This can be generalized to
θL < µ2
µ1 < θ
U
where, θ
L
U
and θ are lower and upper acceptance limits,
respectively. The limits of this confidence interval can be
calculated as the two roots of the following quadratic equation:
2
θ (x − S t / n ) − 2θx x + θ (x − S t / n ) = 0
2
1
2 2
p tab
2
2
2
2 2
p tab
1
1
2
2
Fixed-time method: The absorbance of each drug sample
solution was measured at 605 nm against a reagent blank
prepared similarly at a preselected fixed time of 20 min. The
calibration curve was constructed by plotting the absorbance
against the final concentration of the drug. The amount of the
drug was computed either from calibration curve or regression
equation.
where,
2
S t
p tab
2
2
1
a = x −
n1
b = −2x1x
2
2
S t
p tab
2
Determination procedure for isatin in synthetic
mixture and application of the developed method: Synthetic
mixtures have been used in the past for the determination of
pharmaceutical compounds and check for the applicability of
2
2
c = x −
n2
The values of θ
obtained as:
L
and θ
U
of the confidence interval can be
1
8,19
the proposed method . The proposed method is aimed to
estimate isatin in pure form as well in dosage form. Since,
there was no drug formulation of isatin available in the local
market the determination procedure was done on the synthetic
mixture which was prepared by adding isatin to placebo. The
mixture containing isatin was prepared with excipients com-
monly used in solid dosage forms and analyzed to check the
applicability of the proposed method. The following excipients
were used to prepare a synthetic mixture for solid dosage
forms: isatin (100 mg), lactose (280 mg), silicon dioxide (40
mg), corn starch (360 mg), glucose (100 mg) and magnesium
stearate (25 mg). In the synthetic mixture, the mass ratio of
isatin to lactose, silicon dioxide, glucose and magnesium
stearate was 1:2.8:0.4:3.6:1:0.25, respectively, where the
content of lactose, silicon dioxide, corn starch, glucose and
magnesium stearate forms the placebo.A portion of the synthetic
2
b − b − 4ac
−
−
θL =
2a
2
b + b − 4ac
θU
=
2
a
RESULTS AND DISCUSSION
Potassium permanganate acts as strong oxidizing agent,
has been exploited in oxidimetric analytical method for deter-
mination of isatin. During the sequence of the reaction in basic
medium the heptavalent manganese ion changes to the green
colour (Mn VI). The absorption spectrum of isatin solution in
doubly distilled water shows two absorption bands peaking at
2
18 nm and 242 nm, while that of potassium permanganate

Vol. 25, No. 8 (2013)
Kinetic Spectrophotometric Methods for the Determination of Isatin 4565
solution in the alkaline medium exhibits an absorption band
peaking at 530 nm. The addition of potassium permanganate
to the solution of pure drug produces a new characteristics
band at 605 nm (Fig. 1). This band is attributed to the formation
of isatinic acid, which resulted due to the oxidation of isatin
in alkaline medium by potassium permanganate. The intensity
of the green coloured product increases with time and there-
fore, a kinetic method was developed for the determination of
isatin in pure form and in synthetic mixture.
Optimization of variables: The influence of the concen-
tration of NaOH solution on the rate of reaction was studied
-4
by keeping the constant concentrations of isatin (3.40 × 10
-5
4
M) and KMnO (3.16 × 10 M) and varying the concentration
-3
-2
of NaOH (2.0 × 10 -5.0 × 10 M) in a final volume of 10 mL
solution. Fig. 3 shows that the absorbance of reaction increased
-
2
up to 4.6 × 10 M NaOH; beyond this concentration the
absorbance remained constant. Therefore, a concentration of
-2
5 × 10 M NaOH was used throughout the experiment. In the
similar way the effect of the concentration of KMnO solution
on the change in absorbance was studied in the range of 1.27
4
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
a
b
c
-
6
-5
×
10 -3.16 × 10 M. The absorbance in due course of reaction
(
Fig. 4) increased with increasing the concentration of KMnO
4
-5
and became constant at 2.65 × 10 M. Thus, a concentration
-5
4
of 3.16 × 10 M KMnO in the final solution proved to be
sufficient for the maximum concentration of isatin used in the
determination process.
1
.0
.9
0
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
50
300
450
Wavelength (nm)
600
750
-4
Fig. 1. Absorption spectra of (a) 3.4 × 10 M isatin in Milli Q water (b)
-2
-
4
+
-4
3
4
.16 × 10 M KMnO + 5.0 × 10 M NaOH 3.4 × 10 M isatin (c)
-4
-2
3
4
.16 10 M KMnO + 5.0 × 10 M NaOH. The absorbance of each
((b) and (c)) was taken 20 min of mixing of the solution
Reaction mechanism: To validate the reaction product
formed, reaction product was infused into the electrospray
ionization-mass spectrometry (ESI-MS) in negative mode. The
mass spectrum has shown that the major component is having
parent mass of 164.18 (Fig. 2) which indicates the formation
0
.0
0.5
1.0
1.5
2.0
2.5
3.0
Volume of NaOH (mL)
22
of isatinic acid . On the basis of the mass spectra and the
literature following mechanism could be proposed.
-1
Fig. 3. Effect of volume of NaOH (2.0 × 10 ) M keeping constant the
-
4
-4
concentration of KMnO (3.16 × 10 ) and isatin (3.40 × 10 M)
4
Analytical parameters and method validation: Under
the optimized experimental conditions, the high difference in
4
the concentration of potassium permanganate (KMnO ) and
sodium hydroxide solution which established a pseudo-order
reaction condition with respect to the reagents concentration.
Therefore, on the basis of experimental observations, kinetic
equation for the reaction may be written as:
n
rate = k [C] [KMnO
m
] [NaOH]
l
4
-
4
-1
For [KMnO
M at 25 ꢀC.
The above equation reduces to:
4
] ≥ 1.265 × 10 M and [NaOH] ≥ 2.0 × 10
Fig. 2. Mass spectrum of the oxidized isatin in ESI-MS, using negative
ionization mode
n
ψ
rate = k [C]
O
where k
ψ
is the pseudo-order rate constant, C is the concen-
OH
tration of isatin and n is the order of reaction. The logarithmic
form of the equation may be written as:
C
C
KMnO
4
C
C
O
N
NaOH
log (rate) = log k + n log C
ψ
NH
2
The initial rates of reaction were determined at different
concentrations of isatin by measuring the slopes of the initial
H
Isatin
Isatinic acid

4
566 Alothman et al.
Asian J. Chem.
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
log (rate) = 1.898 + 1.029 log C
with coefficient of correlation, r = 0.9999. The value of n in
the equation confirmed that the reaction is first order with
-2
-1
respect to isatin and rate constant is 1.26 × 10 s .
A calibration curve was constructed by plotting the initial
rate of reaction versus initial concentration of isatin, which
showed a linear response over the concentration range of 5-50
-
1
µg mL . The linear regression analysis of calibration data
(n = 6) was made to evaluate slope, intercept and correlation
coefficient. The regression of rate versus concentration of isatin
-
1
(
µg mL ) gave the following linear regression equation:
-6
-2
Rate = -4.125 × 10 + 1.151 × 10 C
with a correlation coefficient (r) of 0.9999. The values of
confidence limit for the slope (b ± tS ) and intercept (a ± tS
of the calibration line were computed and found to be 1.151 ×
b
a
)
-2
-5
-6
-6
1
0 ± 5.007 × 10 and -4.125 × 10 ± 1.009 × 10 , respec-
tively for n-2 degrees of freedom at 95 % confidence level.
The values of the confidence limit are appreciably low thus
indicating the high reproducibility of the initial rate method.
The limits of detection (LOD) and quantitation (LOQ) were
evaluated using the following equation:
0
.0
0.5
1.0
1.5
2.0
(mL)
2.5
3.0
Volume of KMnO
4
-4
4
Fig. 4. Effect of volume of KMnO (1.26 × 10 ) M keeping constant the
-1
-4
concentration of NaOH (2.0 × 10 ) and isatin (3.40 × 10 M)
tangent to the absorbance (at 605 nm)-time curves during the
first 20 min of the reaction (Fig. 5) and are summarized in
Table-1. The plot of log rate versus log C gave the following
linear equation:
S0
b
S0
b
LOD = 3.3×
LOQ = 10×
and
where S
0
is the standard deviation of the calibration line and b
-3
is the slope and found to be 3.4 × 10 and 1.05 × 10 µg mL
-4
-
1
,
tion:
respectively. The variance was calculated using the equa-
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
a
b
c
d
e
f
2
∑(logνexpt.
− logνreg. )
2
S0
=
n − 2
-
12
-1
and found to be 1.46 × 10 µg mL . The low value of
variance indicated negligible scattering of the experimental
data points around the line of regression. The results are
summarized in Table-2.
TABLE-2
OPTICAL CHARACTERISTICS AND ANALYTICAL DATA
FOR THE INITIAL RATE AND FIXED TIME METHODSFOR
THE DETERMINATION OF ISATIN
Values
Parameters
Initial rate
8 min
Fixed time
20 min
Time
-1
-1
Linear dynamic range
Regression equation
5-50 µg mL
5-50 µg mL
-3
5.028 × 10 +
0.0186 × C
0.9999
-6
-4.125 × 10 +
0.0115 × C
0.9998
Correlation coefficient
0
0
5
10
15
20
-
6
-3
Sa
1.01 × 10
4.08 × 10
Time (min)
-5
-4
Sb
5.01 × 10
-12
1.46 × 10
1.21 × 10
-5
Fig. 5. Initial rate of reaction at different concentration of isatin
Variance
2.46 × 10
-
4
Limit of detection
Limit of quantitation
3.4 × 10
0.878
2.663
-3
1.05 × 10
TABLE-1
INITIAL RATE OF REACTION AT DIFFERENT
CONCENTRATION OF ISATIN
Solution stability: The solution stability of isatin was
checked by observing UV spectra of isatin for 5 days. The
aqueous solution of the drug having two λmax: 218 and 242
nm, showing no change in the absorption spectra of standard
and sample solutions of drug for at least 5 days, when the
solutions were stored at room temperature. To check the
stability of the isatin solution thin layer chromatography was
C [Isatin]
(mol L )
Initial rate of
reaction (ν)
-2.49757
-1
log C
log ν
-
5
3.398 × 10
1.020 × 10
2.719 × 10
3.059 × 10
3.398 × 10
-4.4687
-3.9916
-3.8664
-3.5145
-3.4687
-4.4687
-3.9916
-3.8641
-3.5145
-3.4687
-
4
-2.03058
-
4
-1.91080
-
4
-1.57060
-
4
-1.52400

Vol. 25, No. 8 (2013)
Kinetic Spectrophotometric Methods for the Determination of Isatin 4567
-
4
also performed. The standard solution, sample solution of isatin
was applied on TLC plates coated with silica gel and deve-
loped in chloroform-methanol (9.0:1.5 v/v) solvent system.
The plates were air-dried and spots were detected in the iodine
parameters investigated were: volume of 1.26 × 10 M KMnO
4
-1
(± 0.2 mL), volume of 2.0 × 10 M NaOH (± 0.2 mL).
Under these conditions a synthetic mixture solution
-1
containing 6.0 µg mL synthetic mixture of isatin was assayed
five times by the initial rate and fixed time methods. The values
of mean recovery, standard deviation and relative standard
deviation represent good reliability of the proposed methods.
Applicability of the proposed methods: The proposed
methods were successfully applied to the determination of
isatin in synthetic mixtures prepared by adding active drug to
placebo. The results of the methods proposed were compared
chamber. A single spot was observed up to 5 days with R
.83 corresponding to isatin proving the stability of solution
up to 5 days.
f
=
0
Accuracy and precision: The accuracy and precision of
the proposed methods were established by measuring the
content of isatin in pure form at three different concentration
levels (low, medium and high). The short-term (intra-day assay)
and the daily precisions (inter-day assay) were performed by
16
with those of the reference method using point and interval
hypothesis tests and are summarized in Table-5. The calcu-
lated t- (paired) and F-values at 95 % confidence level do not
-1
measuring five independent analyses at 5, 40 and 50 µg mL
concentration levels within one day and on five consecutive
days, respectively (Table-3). The standard deviation, relative
standard deviation and mean percent recoveries obtained by
both the initial rate and fixed time methods can be considered
to be very satisfactory.
20
exceed the theoretical ones which indicates that there are no
significant differences between the performance of the
proposed methods and the reference method. A bias of ± 2 %,
which is based on recovery experiments, is permissible by the
21
The validity of the proposed methods was also checked
by performing recovery experiments through standard addition
method. For this, a known amount of the pure drug was added
to synthetic mixture and then the total amount of isatin was
determined following the recommended procedures and refe-
rence method. The results are summarized in Table-4, which
showed recoveries in the range of 99.96-100.87 %, 100.03-
Canadian Health Protection Branch . Therefore, the accept-
able limit lies within θ = 0.98 and θ = 1.02. It is evident
from Table-5 that the true bias of all samples of drug is smaller
than ± 2 % thus, confirming that the proposed methods are
reliable with acceptable recovery.
L
U
Conclusion
1
00.18 % for initial rate and fixed time methods, respectively.
Initial rate and fixed time methods are applied for the
routine quality control analysis of isatin in pharmaceutical
formulations. The determination of isatin in the literature shows
that the estimation procedures need more pretreatment/sepa-
ration procedure when relying on higher instrumentation meth-
ods. Conventional extractive spectrophotometric procedures
No interference from the common excipients was observed.
Robustness: The conditions are robust for the application
of the proposed methods to determine the active drug in
pharmaceutical formulations. Each operational parameter was
checked for the robustness of the methods. The operational
TABLE-3
EVALUATION OF ACCURACY AND PRECISION FOR THE PROPOSED METHODS BY INTRA-DAY AND INTER-DAY ASSAY
-1
Amount (µg mL )
b
c
Proposed methods
RSD (%)
SAE
CL
a
Taken
Found ± SD
Initial rate
5
5.01 ± 0.09
40.03 ± 0.13
49.76 ± 0.34
4.98 ± 0.09
40.07 ± 0.17
49.94 ± 0.41
1.69
0.32
0.68
1.80
0.42
0.82
0.04
0.06
0.15
0.04
0.08
0.18
0.11
0.16
0.42
0.11
0.21
0.51
Intra-day assay
Inter-day assay
40
50
5
40
50
Fixed time
5
5.00 ± 0.06
40.05 ± 0.04
50.13 ± 0.26
5.02 ± 0.07
40.04 ± 0.07
50.04 ± 0.33
1.18
0.11
0.52
1.39
0.16
0.66
0.03
0.02
0.12
0.03
0.03
0.15
0.17
0.06
0.32
0.09
0.08
0.41
Intra-day assay
Inter-day assay
40
50
5
40
50
TABLE-4
STANDARD ADDITION METHOD FOR THE DETERMINATION OF ISATIN IN SYNTHETIC MIXTURE
-
1
Proposed methods for
determination of isatin
Amount of isatin (µg mL )
Recovery (%)
RSD (%)
SAE
a
Taken
Added
5
Found ± SD
10
15.13 ± 0.11
29.99 ± 0.42
49.87 ± 0.31
15.02 ± 0.22
30.01 ± 0.35
50.10 ± 0.31
100.87
99.96
0.74
1.40
0.62
1.14
1.15
0.61
0.05
0.18
0.14
0.10
0.16
0.14
Initial rate method
Fixed time method
10
20
40
5
1
0
0
99.73
1
100.16
100.03
100.18
10
20
40
10

4
568 Alothman et al.
Asian J. Chem.
TABLE-5
POINT AND INTERVAL HYPOTHESIS TEST: EVALUATION OF APPLICABILITY OF THE PROPOSED METHOD
16
AND OBSERVATION OF RESULTS OF CURRENT METHOD WITH THE EXISTING METHOD
Recovery
%)
Reference method
Methods
RSD
θL
θU
t
F
(
Recovery
RSD
1.80
1.75
Initial rate
Fixed time
100.14
100.41
1.65
1.44
0.999
0.997
1.008
1.004
1.58
0.70
0.17
0.19
99.74
100.28
7
.
.
A.E. Medvedev,A. Clow, M. Sandler andV. Glover, Biochem. Pharmacol.,
2, 385 (1996).
A. Medvedev, O. Bussygyna, N. Pyatakova, V. Glover and I. Severina,
for determination of isatin are effective but are tedious, time-
consuming and require a large amount of sample and reagents
and sometimes vulnerable to contamination and losses of
analyte. Therefore, the current methods could be a better
alternate for the determination of isatin in pure form as well
as in mixtures (active drug + placebo) as these methods are
simple and sensitive and do not require any laborious cleanup
procedure prior to analysis and therefore, can be frequently
used in the laboratories of research, hospitals and pharma-
ceutical industries.
5
8
Biochem. Pharmacol., 63, 763 (2002).
9. S.K. Battacharya, A. Clow, A. Prareski, J. Halket,V. Glover and M.
Sandler, Neurosci. Lett., 132, 44 (1991).
0. H. Xu, D. Wang, W. Zhang, W. Zhu, K. Yamamoto and L. Jin, Anal.
Chim. Acta, 577, 207 (2006).
1
1
1
1
1
1. V.C. Diculescu, S. Kumbhat andA.M. Oliveira-Brett, Anal. Chim. Acta,
575, 190 (2006).
2. V. Manabe,Q. Gao, J. Yuan, T. Takahashi and A. Ueki, J. Chromatogr. B:
Biomed. Sci. Appl., 691, 197 (1997).
3. M. Unger, W. Jacobsen, U. Holzgrabe and L.Z. Benet, J. Chromatogr. B,
7
67, 245 (2002).
4. J.M. Halket, P.J. Watkins, A. Przyborowska, B.L. Goodwin, A. Clow,
ACKNOWLEDGEMENTS
V. Glover and M. Sandler, J. Chromatogr. B: Biomed. Appl., 562, 279
(
The authors (Z.A.O, M.R.S, S.M.W) extended their
appreciation to the Deanship of Scientific Research at King
Saud University for funding the work through the research
group project No. RGP-VPP-043.
1991).
5. K.-I. Mawatari, M. Segawa, R. Masatsuka, Y. Hanawa, F. Iinuma and
M. Watanabe, Analyst, 126, 33 (2001).
1
1
6. Z. Xiang-Hui, X. Wei and L. Jun, J. Shanxi Normal Univ., [DOI]:
CNKI:SUN:SFDX.0.2008-02-018.http://en.cnki.com.cn/Article_en/
CJFDTOTAL-SFDX200802018.htm.
REFERENCES
1
1
1
2
2
7. Vogel's Textbook of Quantitative Chemical Analysis, Pearson Education,
Singapore, edn. 6, p. 420 (2002).
1
2
.
.
P. Zou and H.L. Koh, Rapid Commun. Mass Spectrom., 21, 1239 (2007).
Silva J.F.M. Da, S.J. Garden and A.C. Pinto, J. Braz. Chem. Soc., 12,
8. S. Riahi, F. Hadiloo, S.M.R. Milani, N. Davarkhah, M.R. Ganjali, P.
Norouzi and P. Seyfi, Drug Testing Anal., 3, 319 (2011).
9. N. Rahman, M.R. Siddiqui and S.N.H. Azmi, J. Chin. Chem. Soc., 53,
273 (2001).
3
4
5
6
.
.
.
.
N. Hamaue, N.Yamazaki, M. Minami, M. Endo, M. Hirahuji,Y. Monma
and H. Togashi, Gen. Pharmacol., 30, 387 (1998).
B. Bhrigu, D. Pathak, N. Siddiqui, M.S.Alam andW.Ahsan, Int. J. Pharm.
Sci. Drug Res., 2, 229 (2010).
735 (2006).
0. C. Hartmann, J. Smeyers-Verbeke, W. Penninck, Y.V. Hayden, P.
Venkeerberghen and D.L. Masart, Anal. Chem., 67, 4491 (1995).
1. Canada Health Protection Branch, Drug Directorate Guidelines, Ac-
ceptable Methods, Ministry of National Health and Welfare Draft
V. Glover, S.K. Bhattacharya, A. Chakrabarti and M. Sandler, Stress
Med., 14, 225 (1998).
V. Glover, M. Reveley and M. Sandler, Biochem. Pharmacol., 29, 467
(1992).
2. A.M. Ismail and A.A. Zaghloul, Int. J. Chem. Kinet., 30, 463 (1998).
2
(
1980).