Kinetic study of alkaline induced hydrolysis of the skeletal muscle relaxant chlorzoxazone using ratio spectra first derivative spectrophotometry

Article abstract of DOI:10.1016/S0014-827X(02)00026-5

2-Amino-4-chlorophenol was found to be the alkaline induced degradation product and the synthetic precursor of chlorzoxazone. The aim of this work is to study different factors affecting the degradation process due to the high toxicity of 2-amino-4-chloro

Full text of DOI:10.1016/S0014-827X(02)00026-5

Il Farmaco 58 (2003) 337ꢀ342
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Kinetic study of alkaline induced hydrolysis of the skeletal muscle
relaxant chlorzoxazone using ratio spectra first derivative
spectrophotometry
M.M. Ellaithy, N.A. El-Ragehy *, M.A. El-Ghobashy
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini St., Cairo ET 11562, Egypt
Received 1 July 2002; accepted 25 October 2002
Abstract
2-Amino-4-chlorophenol was found to be the alkaline induced degradation product and the synthetic precursor of chlorzoxazone.
The aim of this work is to study different factors affecting the degradation process due to the high toxicity of 2-amino-4-
chlorophenol. Chlorzoxazone was found to follow pseudofirst order kinetics. Ratio spectra first derivative spectrophotometry
(DR1) was developed for monitoring the change in chlorzoxazone concentration during the degradation process. Kinetic parameters
(rate constant (K) and half-life (t0.5)) were calculated at different temperatures (40ꢀ
/
120 8C) and different sodium hydroxide
concentrations (3ꢀ10 M). Activation energy at 3 and 8 M sodium hydroxide concentration and alkaline induced catalysis constant
/
at 60, 70 and 80 8C were also calculated.
´
# 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Ratio spectra first derivative spectrophotometry; Chlorzoxazone; 2-Amino-4-chlorophenol; Stability study; Kinetics; Alkaline hydrolysis
1. Introduction
2-Amino-4-chlorophenol was found to be toxic and it
is classified in Aldrich catalogue as a harmful chemical
[5]. It was reported also that high levels of 2-amino-4-
chlorophenol could interfere with the ability of blood to
carry oxygen; causing headache, dizziness and methe-
moglobinemia. Higher levels can cause trouble breath-
ing, collapse and even death [6].
Several spectrophotometric methods such as absor-
bance ratio [7], difference spectrophotometry [8], second
derivative [9], zero crossing [10], multi-wave length
linear regression [11] and simultaneous equations [12]
were tried to resolve chlorzoxazone binary mixture with
paracetamol.
Investigation of the chemical stability of pharmaceu-
tical products is a matter of growing concern in many
analytical laboratories. Systematic kinetic studies of the
decomposition of drugs using stability testing techniques
is essential for the quality control of such products [1].
Chlorzoxazone, (5-chlorobenzoxazole-2(3H)-one), is
a centrally acting skeletal muscle relaxant with sedative
properties [2]. It contains a benzoxazolone ring system,
which is highly unstable due to presence of both lactam
and lactone functional groups in the fused ring system.
Both groups are subject to alkaline induced hydrolysis.
2-Amino-4-chlorophenol was found to be the alkaline
induced degradate of chlorzoxazone, as shown in Fig. 7
[3]. It is also one of the synthetic precursors of
chlorzoxazone as shown in Fig. 8 [4].
Various chromatographic techniques were also ap-
plied for the analysis of the same mixture in pharma-
ceutical dosage forms and in biological fluids such as
HPLC [13,14], GC [14,15], TLC densitometry [14,16]
and super critical fluid chromatography [17].
The aim of this work is to study different factors
affecting chlorzoxazone degradation. It is aimed also to
demonstrate the application of ratio spectra first deri-
vative spectrophotometry as a simple, rapid and in-
expensive method for determination of chlorzoxazone in
presence of 2-amino-4-chlorophenol.
* Corresponding author.
E-mail address: n.a.ragehy@mailcity.com (N.A. El-Ragehy).
´
0014-827X/02/$ - see front matter # 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(02)00026-5

338
M.M. Ellaithy et al. / Il Farmaco 58 (2003) 337ꢀ342
/
2. Experimental
2.1. Equipment
The pH of the solution was then adjusted to pH 7ꢀ8 by
/
addition of 6 M hydrochloric acid. Ether was used for
extraction using 10 ml portions, the process was
repeated five times. The pH was then readjusted to pH
ꢀ
/
Shimadzu 1601 PC double beam UVꢀVis spectro-
/
7ꢀ8 and the process was repeated till no pH rise was
/
photometer with fixed slit width (2 nm) connected to
an IBM computer loaded with Shimadzu UV PC
software was used for all absorbance measurements
and data treatment.
Bruker FT-IR, Infrared spectrophotometer.
Oakton, pH meter.
observed after ether extraction. Ethereal extract was
collected, filtered and evaporated using rotavap at
ambient temperature. The obtained residue was purified
by re-dissolving in ether, filtration and re-evaporation
till pale brown flakes were obtained. Purity of the
prepared 2-amino-4-chlorophenol was confirmed by
evaluation of its melting point (m.p.) and comparing it
to that found in literature [5].
ꢀ
/
ꢀ
/
2.2. Pure samples
The identity of 2-amino-4-chlorophenol was con-
firmed by interpretation of the characteristic peaks
and comparing the IR spectra of both the drug and
the degradate. Figs. 1 and 2 show the IR spectra of
chlorzoxazone and the prepared 2-amino-4-chlorophe-
nol, respectively. The peak at 1772 cmꢁ1 shown in Fig. 1
is characteristic for the carbonyl group in chlorzoxazone
(keto form). This peak has disappeared in the spectrum
of the degradate as shown in Fig. 2. This goes with the
hydrolysis reaction shown in Fig. 7. Appearance of a
doublet peak at 3382 and 3314.3 cmꢁ1 in the spectrum of
the degradate indicates formation of primary amino
group that was absent in chlorzoxazone [19].
Chlorzoxazone pure substance was obtained from
Glaxo Wellcome, Egypt. Its purity was found to be
(100.02%90.422) using direct spectrophotometry [18].
/
2.3. Degraded sample
2.3.1. Preparation of the degradate; 2-amino-4-
chlorophenol
Weigh accurately 0.5 g of chlorzoxazone into a 100 ml
flat bottom rounded flask, add 50 ml of aqueous 6 M
sodium hydroxide and reflux for 2 h. Completion of
hydrolysis was checked by recording the absorption
spectrum of diluted solution of the reaction mixture
against a blank of 0.1 M sodium hydroxide. This process
was repeated till disappearance of the absorption band
characteristic for chlorzoxazone (at 288 nm) and ap-
pearance of that characteristic for 2-amino-4-chlorophe-
nol (at 307 nm). The reaction flask was then cooled and
the contents were transferred into a separating funnel.
2.4. Stock solutions
Stock solutions 400 mg mlꢁ1 of chlorzoxazone and of
2-amino-4-chlorophenol were prepared in 0.1 M sodium
hydroxide. These solutions were used to prepare the
synthetic mixtures shown in Table 1.
Fig. 1. IR spectrum of chlorzoxazone.

M.M. Ellaithy et al. / Il Farmaco 58 (2003) 337ꢀ
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339
Fig. 2. IR spectrum of 2-amino-4-chlorophenol.
2.5. Laboratory prepared mixtures
zoxazone from its aqueous alkali standard solution into
a series of 25 ml volumetric flasks. Complete to volume
using 0.1 M sodium hydroxide, to obtain chlorzoxazone
Into a series of 25 ml volumetric flasks transfer
accurately aliquot portions equivalent to (80ꢀ
chlorzoxazone from its stock solution. Add aliquot
portions of 2-amino-4-chlorophenol stock solution
/
800 mg)
series (3.2ꢀ
40 mg mlꢁ1). Transfer accurately aliquot
/
portions equivalent to (160 mg) of 2-amino-4-chlorophe-
nol from its aqueous alkali stock solution into a 25 ml
volumetric flask, complete to volume using 0.1 M
sodium hydroxide. Record the absorption spectra of
chlorzoxazone series and that of the degradate against
0.1 M sodium hydroxide as a blank. Divide the
chlorzoxazone series by the prepared degradate divisor
(6.4 mg mlꢁ1). Record the first derivative of the ratio
spectra obtained for chlorzoxazone series (Fig. 4). Plot
the calibration curve using the response obtained at
equivalent to (80ꢀ800 mg) to prepare the binary mixtures
/
shown in Table 1. Complete to volume using aqueous
0.1 M sodium hydroxide.
2.6. Procedures
2.6.1. First derivative of ratio spectra (DR1) for
determination of chlorzoxazone
272.2 nm using instrumental parameters Dlꢂ
scaling factor Fꢂ10. Linear relation between instru-
mental response (DR1) and concentration was obtained
/
4 and
2.6.1.1. Linearity of DR1 method. Transfer accurately
/
aliquot portions equivalent to (80ꢀ1000 mg) of chlor-
/
Table 1
List of concentrations of synthetic binary mixtures of chlorzoxazone with 2-amino-4-chlorophenol in 0.1 M sodium hydroxide
Mixture number Chlorzoxazone (mg mlꢁ1
)
2-Amino-4-chlorophenol (mg mlꢁ1
)
Percent of degradate (%) Recovery(%) Reference method a
1
2
3
4
5
32.00
6.40
6.40
3.20
3.20
6.40
3.20
16.67
33.33
50.00
83.33
90.91
100.19
101.41
99.69
99.69
101.88
5
6.40
16.00
32.00
n
5
Mean recovery (%)
SD
RSD
100.34
1.068
1.064
0.550
0.16
100.04
0.422
0.420
t (2.776)
F (6.39)
Figures in parentheses are the corresponding theoretical t and F values (Pꢂ0.05) [21].
/
a
Direct spectrophotometry [18].

340
M.M. Ellaithy et al. / Il Farmaco 58 (2003) 337ꢀ342
/
Table 2
Assay validation for DR1 method for determination of chlorzoxazone
in presence of 2-amino-4-chlorophenol at (272.2 nm)
2.6.2. Stability study of chlorzoxazone
Weigh accurately 0.1 g of chlorzoxazone into a 100 ml
flat bottom rounded flask. Add 50 ml aqueous sodium
hydroxide of the specified molarity and attach a water
reflux condenser. The above set was maintained in a
thermostated brine water bath with continuous stirring
of flask contents using magnetic stirrer. At the specified
time intervals, 0.5 ml aliquots were transferred accu-
rately into 25 ml volumetric flasks and completed to
volume using aqueous 0.1 M sodium hydroxide to stop
the reaction. The rate of degradation was found to be
negligible in cold 0.1 M sodium hydroxide. Immediately
record the absorption spectrum against aqueous 0.1 M
sodium hydroxide as a blank. The obtained spectrum
was divided by the chosen 2-amino-4-chlorophenol
divisor spectrum. First derivative of the ratio spectrum,
obtained using the same experimental conditions de-
scribed under linearity (Section 2.6.1.1). Chlorzoxazone
concentration was calculated from the regression equa-
tion of first derivative of the ratio spectra method
(DR1). Data are then manipulated to obtain various
kinetic parameters studied. Table 3 shows conditions
and the calculated kinetic parameters for each run.The
procedure described under Section 2.6.1.2 was used for
Parameter
Value
40 mg mlꢁ1
0.4191
0.002
0.3367
0.038
0.9999
0.568
0.910
Range
Slope
3.2ꢀ
/
SE of slope
Intercept
SE of intercept
Correlation coefficient (r)
RSD (nꢂ
/
9) a
9) b
RSD (nꢂ
/
a
RSD is intra-day precision applied on mixtures number 1, 3 and 5
(Table 1) on three different days.
b
RSD is inter-day precision applied on mixtures number 1, 3 and 5
(Table 1) on three different days.
as described by the following regression equation:
Y ꢂ0:4191X ꢃ0:3367
where (Y) is the DR1 response and (X) is concentration
in mg mlꢁ1
.
different temperatures (40ꢀ
boiling point of the reaction mixture (reflux tempera-
ture) and different alkali concentrations (3ꢀ10 M).
Reaction rate constant (K) and half-life (t0.5) were
calculated for each run as tabulated in Table 3.
/
120 8C), where 120 8C is the
2.6.1.2. Validation of DR1 method. The DR1, at 272.2
nm and 6.4 mg mlꢁ1of 2-amino-4-chlorophenol as a
divisor, was applied for determination of chlorzoxazone
in presence of 2-amino-4-chlorophenol in their labora-
tory prepared mixtures. DR1 was found suitable for
chlorzoxazone determination in presence of its degra-
date up to (90%). Average recovery percent was found
/
to be (100.579
/
1.008). Statistical analysis of the results
3. Discussion
obtained as compared with reference method is shown in
Table 1. Inter-day and intra-day repeatability was
performed by applying the proposed procedures on
mixtures number 1, 3 and 5 shown in Table 1. Results
are shown in Table 2.
Zero order spectra of each of chlorzoxazone and 2-
amino-4-chlorophenol show significant overlap that
hinders tracing of the remaining amount of chlorzox-
azone during degradation process as shown in Fig. 3.
Table 3
List of conditions and calculated kinetic parameters used in the kinetic study of the base induced chlorzoxazone degradation
Temperature (8C)
Alkali strength (M)
Reaction rate constant (K) (minꢁ1
)
Half life (t0.5) (min)
40
60
60
60
70
70
70
75
80
80
80
90
120
120
10
3
No visible change
No visible change
495.35
177.69
130.83
289.96
93.65
1.399ꢄ
3.9ꢄ
10ꢁ3
5.297ꢄ
2.390ꢄ
7.400ꢄ
0.011
/
10ꢁ3
6
/
8
/
10ꢁ3
10ꢁ3
10ꢁ3
3
/
6
/
8
63
231.46
57.75
3
2.994ꢄ
/
10ꢁ3
6
0.012
0.018
0.044
8
38.50
15.75
10
3
9.440ꢄ
/
10ꢁ3
73.41
16.5
3
0.042
0.119
8
5.82

M.M. Ellaithy et al. / Il Farmaco 58 (2003) 337ꢀ
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341
Fig. 3. Spectral characteristics for chlorzoxazone (*
NaOH.
/
) and 2-amino-4-chlorophenol (ꢀ ꢀ
) 40 mg mlꢁ1 each, both are dissolved in aqueous 0.1 M
/
/
First derivative of the ratio spectra (DR1) at 272.2 nm
proposed procedure as compared with those of the
reference direct spectrophotometric method [18]. The
calculated t and F values are less than their correspond-
ing tabulated ones, indicating non significant difference
between the suggested procedure and the reference one.
Inter-day and intra-day precision was also performed
according to USP [20] on mixtures number 1, 3 and 5.
RSD obtained are shown in Table 2.
Reaction order was determined by plotting the
logarithm of chlorzoxazone concentration remaining
versus time. Linear relationship suggests first order
kinetics as shown in Fig. 5. Alkaline catalyzed hydro-
lysis is a bimolecular reaction, but one of the reactants is
as shown in Fig. 4 was found suitable for tracing
chlorzoxazone remaining during degradation since this
method was proved to be able to determine chlorzox-
azone in presence of 2-amino-4-chlorophenol up to 90%
degradate as presented in Table 1. Different divisor
concentrations (6.4, 16, 25.6 mg mlꢁ1) were tried. The
first divisor (6.4 mg mlꢁ1) was found the best regarding
average recovery percent when the model was used for
prediction of chlorzoxazone concentration in its labora-
tory prepared mixtures. Statistical analysis of the results
obtained by the suggested DR1 method has been carried
out. Table 1 shows the results obtained by adopting the
Fig. 4. First derivative of the ratio spectra (DR1) for chlorzoxazone series (3.2ꢀ
divisor.
/
40 mg mlꢁ1) using (6.4 mg mlꢁ1) of 2-amino-4-chlorophenol as a

342
M.M. Ellaithy et al. / Il Farmaco 58 (2003) 337ꢀ342
/
Fig. 8. Preperation of chlorzoxazone by treating 2-amino-4-chloro-
phenol with phosgene in ethyl acetate.
Fig. 5. First order plot for the alkaline degradation of chlorzoxazone
(2 mg mlꢁ1) starting concentration, 8 M sodium hydroxide and 80 8C.
water, which is present in large excess, so the change in
its concentration is negligible. Such reactions where one
of the reactants is present in large excess are considered
to follow pseudofirst order kinetics and all equations
describing first order can be applied for it [1]. Reaction
rate constant (K) and half-life (t0.5) for each run are
calculated and tabulated in Table 3. Effect of tempera-
ture on degradation rate was studied at sodium hydrox-
ide concentration of 3 and 8 M by plotting of the
logarithm of the reaction rate constant (log K) versus
the reciprocal of the absolute temperature (1/T). This
plot is called Arrhenius plot and is shown in Fig. 6.
Temperature was found to accelerate the reaction as
proved by higher reaction rate constants and smaller
half-lives as shown in Table 3. Activation energy at 3
and 8 M sodium hydroxide were found to be 62 829.43
and 54 393.2 kcal molꢁ1, respectively.
Fig. 7. Hydrolysis of chlorzoxazone to give 2-amino-4-chlorophenol
and sodium carbonate.
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