INTRODUCTION:
Aripiprazole; 7-[4-[4-(2,3-dichlorophenyl)-1-piperazinyl]butoxy] 3,4-dihydro-2 (1H)-quinolinone,
(ARP, Fig.1) is a recent atypical antipsychotic drug that is effective for the
treatment of patients with schizophrenia
or schizoaffective disorder1-3. Some methods have been described for
the analysis of ARP in human plasma or serum 4-11. The analysis is
mainly carried out by means of HPLC with UV 6, 11 or mass
spectrometry detection4, 7, 8 or by gas-chromatography-mass
spectrometry (GC-MS)9. Methods of Liquid chromatography-tandem mass
spectrometry (LC-MS/MS)5, 10 and capillary electrophoresis 11
were also developed. Some HPLC-UV12,13 and spectrophotometric
14-16 methods have been described for the determination of ARP in
pharmaceutical preparations. Use of chromatographic methods4-11 is
justified when sample matrix is rather complex and the drug concentration is
low, as is usually the case with clinical samples and in biological fluids,
e.g., human plasma.
However, in pharmaceutical analysis, where the sample
matrix is usually less complex and analyte
concentration levels are fairly high. The main aim is to develop fast, simple,
inexpensive methods that can readily be adapted for routine analysis at
relatively low cost to the different requirements of analytical problems. Many
drugs are easy to be determined by spectrophotometric
methods based on formation of colored charge-transfer (CT) complexes between
electron acceptors, either π or σ acceptors and drugs as electron
donors17-20 or formation of colored compounds with a number of
organic acid dyes 20-23. To my best knowledge, no more attempts have
been made to determine aripiprazole in tablets by
colorimetric method and the literature are still poor in such analytical procedure.The methods are based on the ability of the cited
drug to form ion associations with DDQ, I2, BCG and BCP. The
reaction conditions and the application of the methods to the determination of aripiprazole in tablets have been established. In addition,
the spectral characteristic and the stability of the formed ion associate were
also included.
Fig.1. Chemical structure of
ARP.
MATERIAL AND METHODS:
Instrumentation:
A Shimadzu 1601 PC double beam UV-Vis spectrophotometer
with 1-cm quartz cuvettes, a fixed slit width (2 nm),
connected to an IBM-PC computer loaded with Shimadzu UVPC software was equipped
with HP desk jet printer and used for all the absorbance measurements and
treatment of data.
Reagents and Chemicals:
ARP (99.97%) and its pharmaceutical preparation (Aripiprex) containing 10 mg or 30 mg of aripiprazole
per tablet were kindly supplied by Al-Andalous
Medical Co., Egypyt. All solvents used were of
analytical grade. DDQ (Sigma Chemical Co. USA) 5x10-3 M was freshly
prepared in acetonitrile. Iodine, resublimed
(Riedel-De-Haen AG, Germany) was 4x10-3 M
in 1,2-dichloroethane. The solution was found to be stable for at least 1 week
at 4 °C. BCG or BCP (Aldrich Co., USA) was 1x10-3 M in
1,2-dichloroethane . The solutions were stored at 4 °C in PVC containers. Stock
solutions of ARP containing 500 µg mL-1 in acetonitrile
(DDQ method) and in 1,2-dichloroethane (I2, BCG and BCP methods)
were also prepared. Whenever required dilute solutions were obtained by
appropriate dilution with the same solvent. Another stock solution of drug
(1x10-2 mol L-1) in 1,2-dichloroethane or acetonitrile was also prepared for the stoichiometric
study. The stock solution of drug was stable for at least 3 days when kept in
the refrigerator (at about 4 °C). N.B. 1,2-Dichloroethane was always dried over
anhydrous sodium sulphate.
General analytical procedures:
(a) Charge-transfer method using DDQ: Into 10- mL
volumetric flasks were transferred 0.2-2.4 mL of 500
µg mL-1 of drug or sample solution in acetonitrile
and 2 ml of 5x10-3 mol L-1 DDQ solution was added. The
mixture was mixed and allowed to stand for 20 min at 25±1°C. The volume was
made up to 10- mL with acetonitrile
and the absorbance was measured at 457 nm, against a reagent blank prepared and
treated similarly.
(b) Charge-transfer method using iodine: Into 10- mL
volumetric flasks were placed 0.2-2.8 mL of 100 µg mL-1
of drug or sample solution in 1,2-dichloroethane and 2 mL
of 4x10-3 mol L-1 iodine in the same solvent was added.
The reaction mixture was mixed and allowed to stand in the dark at 25±1°C for
30 min, then the solution was diluted to volume with 1,2-dichloroethane. The
absorbance was measured at 364 nm, against a reagent blank similarly prepared.
(C)
Ion-pair complexation reaction with BCG and BCP. Aliquots of solution of the drug in 2.0 mL of 1,2-dichloroethane in the concentration range 20-240
µg (for BCG method) or 20-200 µg (for BCP method) were transferred into
separate 10- mL volumetric flasks. To each flask, 2.0
mL of BCG or BCP (1x10-3mol L-1)
solution in 1,2-dichloroehane was added and mixed well and the solution was
diluted to volume with
1,2-dichloroethane. The absorbance of the resultant complexes was
measured instantaneously at 413 and 400 nm for BCG and BCP, respectively,
against blank similarly prepared. Under experimental conditions above
described, standard calibration graphs for ARP were constructed by plotting the
absorbance versus concentration and the regression equations were computed and
recorded in Table 2.
Procedure for assay of Aripiprex
tablets:
Firstly, an accurately weighed amount of the finely
powdered tablets equivalent to 50 mg of drug was washed several times with
distilled water on G4 funnel to remove povidone excipient in the powdered tablets. The residue was dried
under vacuum and then dissolved in acetonitrile (DDQ
method) or 1,2-dichloroehane (I2, BCG and BCP methods). The solution
was filtered into a 100-mL volumetric flask and the volume was made up to 100 mL using acetonitrile or
1,2-dichloroehane for DDQ or I2, BCG and BCP, respectively, these
solutions contain on 500 µg mL-1 ARP. A suitable amount of filtrate
was then taken and analyzed as described under General analytical procedures.
For the proposed methods, the content of a tablet was calculated using the
corresponding regression equation of the appropriate calibration graph.
Stoichiometric relationship
Jobs method of continuous variation24 was
employed to establish the stoichiometry of the colored
products. In this method, a series of solutions was prepared by mixing equimolar solutions (5x10-4mol L-1)
of drug and DDQ, I2, BCG and BCP in varying proportions while
keeping the total molar concentration constant at 1x10-4mol L-1.
Then the General analytical procedures were followed.
Stability constant and free energy change
Serial volumes of 0.5-5.0 mL
of 10-3 mol L-1 drug solution (in 0.5 mL
steps) in acetonitrile (DDQ method) or in
1,2-dichloroehane (I2, BCG and BCP methods) were transferred into
10-mL volumetric flasks. To each flask, 1 mL of
reagent (1x10-4 mol L-1) for DDQ and I2,
(0.5x10-4 mol L-1) for BCG and BCP in the same solvent
was added and continued as directed under General analytical procedures.
RESULTS AND DISCUSSION:
The studied drug has high electron density sites, so it
may act as a powerful electron donor. The structure of ARP is shown in Fig.1.
As can be seen the existence of a piperazine ring in
the structure of ARP acts as a base and n-donor to form a charge-transfer
complex with an acceptor. Spectrophotometric properties of the colored CT
complexes as well as the different parameters affecting the color development
between the different acceptors and drug were extensively studied to determine
the optimal conditions for the assay procedure. The reaction was studied as a
function of the volume of reagent, nature of solvent, time and stoichiometry.
Selection of the suitable wavelength:
DDQ method:
Recently, the reaction of DDQ with some pharmaceutical
compounds has been reported 15-17, 21. In acetonitrile,
the reaction of DDQ with ARP results in the formation of an intense orange-red
product which exhibits three maxima at 457, 548 and 587 nm, respectively. These
new broad absorptions in the visible region after addition of ARP to a fixed
concentration of DDQ indicate the formation of electron donor-acceptor complex.
The intensities of the three absorption bands were nearly equal; the absorbance
readings for the first band were more stable and reproducible. Therefore, the measurements were performed at
457 nm.The interaction of ARP with DDQ in non-polar
solvents, such as dioxane and halogenated solvents
was found to produce colored charge-transfer (CT) complexes with low molar absorptivity values. In polar solvents, such as acetonitrile and alcohols, complete electron transfer from
donor to acceptor moiety takes place with the formation of intensely colored
radical ions with high molar absorptivity values,
according to the following Scheme;
The dissociation of the DA complex is promoted by the
high ionizing power of the acetonitrile. Further
support for the assignment was provided by comparison of the absorption bands
with those of the DDQ˙Æ radical anions produced by the
iodide reduction method. Acetonitrile was considered
an ideal solvent as it afforded maximum sensitivity, due to its high dielectric
constant (37.5)25 that promotes maximum yield of radical anions in
addition to the high solvating power of the reagent and drug.
Iodine method:
The immediate change of the violet color of iodine in
1,2-dichloroethane (500, 290 and 247 nm) to a lemon yellow upon reaction with
ARP was taken as suggestive of CT complex formation which exhibited absorption
bands at 292 and 364 nm (Fig.2). The complex formation is distinguished from other
slow oxidation or substitution reactions of the halogen with ARP, by being
practically instantaneous, in analogy to ionic reactions.
The high intensity of the CT bands is common to
complexes of n-donors with iodine26. The appearance of absorption
peaks at 292 and 364 nm was attributed to the formation of a CT complex between
ARP and iodine, having an ionized structure DI+
I3-,
taking into account that the spectrum of I3- in
1,2-dichloroethane shows two absorption maxima at 292 and 364 nm (Fig.2). This
complex should originate from an early intermediate outer- complex D
..I2,
as in the following Scheme:
Measurements were carried out at 364 nm due to the
interference from the native UV absorption of drug at 286 nm (shoulder band) as
indicated in Fig.2
Fig.2.
Absorption spectra of (a) 20 μg mL -1 ARP, (b) 10-3 mol L-1
iodine, (c) CT-ARP ( 20 μg mL
-1)- iodine complex in 1,2- dichloroethane
vs. reagent blank.
1,2-Dichloroethane was found to be an ideal solvent for
the formation of a tri-iodide ion pair, dichloromethane and chloroform produced
lower absorbance readings. Polar solvents such as acetonitrile
and alcohols were found to be unsuitable as their blanks with iodine gave high
absorbance. It is obvious that, the rate of transformation of outer complex to
inner complex is in the order of
1,2-dichloroethane>dichloromethane>chloroform27. There is
actually a considerable decrease in the energy of activation along with an
increased dielectric constant εr of
the medium; in 1,2-dichloroehane (εr
= 10.2) the transformation of inner complex
proceeds much faster than that in dichloromethane (εr
= 9.1) and chloroform (εr
= 4.8). This is in support of the proposed three-steps
mechanism. In fact, the resulting charged transition states in going from the
outer complexes to the inner ones (as the rate determining step of the
mechanism) are expected to be more stabilized in 1,2-dichloroethane because of
higher solvating ability and relative permittivity than dichloromethane and
chloroform28.
BCG and BCP methods:
The absorption spectrum of solution containing ARP and
BCG or BCP exhibits new absorption at longer wavelength than that drug and dye
alone. The new broad absorption band in the visible region (yellow color) after
addition of drug to a fixed concentration of dye indicates the formation of DA
complex or ion associate. The maximum wavelengths of ARP-BCG and ARP-BCP
associates are located at 413 and 400 nm, respectively.
The type of solvent employed affects both wavelength
and intensity of maximum absorption. The effect of 1,2-dichloroethane,
dichloromethane, chloroform and dioxane were
examined. 1,2-Dichloroethane was considered to be an ideal solvent for the
color reaction as it offers excellent solvent capacity for drug and reagents
and gives the highest yield of ion associates.
Effect of reaction time and temperature:
The optimum reaction time was determined by following
up the color development at ambient temperature (25±1°C). Complete color
development was attained after 20 min in case of DDQ and remained stable up to
2 h. In case of iodine, the yellow color is gradually increased till 30 min and
then remained stable at least a further 1 h in the dark. In case of BCG or BCP,
complete color development was attained instantaneously and was stable for more
than 2 h, thus permitting quantitative analysis to be carried out with good
reproducibility. The intensity of the complex in case of BCG and BCP is stable
within the temperature 20-40 °C, but in case of DDQ, the CT complex was
gradually decreased with increasing temperature, hence, ambient temperature
(25±1°C) was found to be suitable to carry out the study.
Effect of reagent concentration:
When various concentrations of DDQ, I2, BCG
and BCP were added to a fixed concentration of ARP (120 µg mL-1 for
DDQ and 20 µg mL-1 for I2, BCG and BCP), 2.0 mL from each 5x10-3 mol L-1 DDQ, 4x10-3
mol L-1 I2 and 1x10-3 mol L-1 BCG
or BCP in the total volume of 10 mL were found to be
sufficient for the production of maximum reproducible color intensity. Higher
concentration of reagent did not affect the color intensity. The higher
concentration of the reagents may be useful for rapidly reaching equilibrium,
thus minimizing the time required to attain maximum absorbance readings at the
corresponding maxima.
Stoichiometry of the reaction:
The molar ratio of the studied drug with DDQ, I2,
BCG and BCP, using Jobs method of continuous variation24, it was
found to be 1:1 ion associate for all acceptors (Fig.3). This finding was
anticipated by the presence of one basic center or electron-donating center (piperazine ring) in the drug studied.
Fig. 3. Continuous variation
plots for ARP associates with
(a) DDQ, (b) I2,
(c) BCG and (d) BCP; λ= 457, 364, 413 and 400 nm, respectively. Total
molar concentration = 1 x 10-4 mol L-1.
Association
constant and free energy change:
The association
constant was calculated for the interaction of drug with either DDQ, I2,
BCG or BCP to give CT- complex using Benesi-Hildebrand
equation 29:
Where [A0] and [Do] are the total
concentration of the acceptor and donor, respectively, AλAD
is the absorbance of the complex, ελAD is
the molar absorptivity of the complex and KcAD is the association constant of
the complex (L.mol-1). From the above equation (1), on plotting the
values of [Ao] / AλAD
versus 1/ [Do] a straight line was obtained (Fig.4), from which the
association constants and the molar absorptivities
were obtained (Table 1 ). The standard free energy changes of complexation
(∆G°) were calculated by the following equation 30:
Fig.4.
Benesi- Hildebrand plots for ARP with (a) DDQ, (b) I2,
(c) BCG, (d) BCP.
ΔG˚=
-2.303 RT log KcAD ------(2)
Where ΔG˚ is the free energy change of the
complex (K cal.mol-1), R the gas constant (1.987 cal.mol-1
deg-1), T the temperature in Kelvin (273+ 25°C) and KCAD
is the association constant of drug-acceptor complex (L.mol-1). The
results are recorded in Table 1. The high values of association constants are
common in n-electron donors where the intermolecular overlap may be considered 26.
From the above, the molar absorptivities are equal to
2.94x103, 1.33x104, 1.67x104 and 2.22x104
L.mol-1 cm-1 for DDQ, I2, BCG and BCP complexes,
respectively, which are comparable with those obtained from the regression line
equation of Beers law (Table 2).
Spectral characteristics of the CT complex:
The experimental oscillator strength (), which is a
dimensionless quantity used to express the transition probability of the CT
band and the transition dipole moment (µEN) of the CT complexes were
calculated from the following expression 31:
= 4.32x10-9
[ε max Δn1/2] (3)
µEN = 0.0958 ε max Δn1/2 1/2 (4)
nmax
Where Δn1/2 is the band width at half intensity, εmax and nmax are the extinction coefficient and wavenumber
at the absorption maximum of the complex, respectively, where nmax has been expressed in cm-1 unit; the
results are shown in Table 1. Except in DDQ complex the values of the
calculated oscillator strength are rather relatively large indicating a strong
interaction between the donor-acceptor pair with relative high probabilities of
CT transitions 31. This is also supported by the large transition
dipole moment (µEN﴿.
The RN is the resonance energy of the
complex in the ground state, which is obviously a contributing factor to the
stability constant of the complex (a ground state property), can be determined
by the following equation 32:
ε max = 7.7x104 (5)
(hnCT/
RN) -3.5
Where nCT
is the frequency of the complex at the maximum of the CT absorption. Resonance
energies for the studied CT complexes of ARP are given in Table 1.
The ionization potential (Ip)
of the donor was determined from the CT energies of the CT-band of its
complexes with DDQ, I2, BCG and BCP making use of the following
relationship33:
Ip (eV) = 5.76 + 1.52 x 10-4nmax
(6)
Where nmax
was included in Table 1. The values of ionization potentials of drug (Ip) thus determined are given in Table 1. It has
been reported that the ionization potential of the electron donor may be
correlated with the charge transfer transition energy of the complex 26.
Comparison of the transition energies of the CT-complex with the ionization
potential values of the electron donor in the same solvent reveals a regular
relationship, which is in accordance with the results obtained by McConnell et
al. 34.
Validation of the proposed methods:
Analytical data:
The linear calibration graphs were obtained under the
optimum experimental conditions. The analytical results obtained from this
investigation are summarized in Table 2. The calibration data were fitted by
least square treatment and a linear relationship was found between absorbance
and concentration in the ranges of 10-120, 2-28, 2-24 and 2-20 µg mL-1
ARP for DDQ, I2, BCG and BCP, respectively. The very small values of
intercepts (0.0004-0.0060), indicate that there is no differences between the
determined and expected concentration within the investigated range using the
presented methods. The correlation coefficients were between 0.9997-0.9999,
indicate good linearity of the present methods. For accurate determination,
Ringbom35 concentration range was calculated by plotting log
concentration of drug in µg mL-1 against transimittance
% from which the linear portion of the curve gives accurate range of microdetermination of ARP and represented in Table 2. The
high molar absorptivity and lower Sandell
sensitivity values reflect the good and high sensitivity of the methods.
According to the International Conference on Harmonization (ICH) Recommendation36,
the approached based on the standard deviation (SD) of the response and the
slope (b) of the calibration curve, was used for determination the limits of
detection and quantitation of drug, the results are
included in Table 2.
Precision and accuracy:
In order to study the accuracy and precision of the
proposed methods, three concentration levels of ARP within the linearity range
were selected. The within day precision (intraday precision) was performed by
taking five independent analyses at each concentration level within 1 day
during the stability time period. The daily precision (interday
precision) was measured by assaying a single sample of each concentration on
five consecutive days within the stability time period. The mean recovery and
RSD values are included in Table 2. The results obtained in Table 2, show that
no significant difference for the assay, which tested within-day
(repeatability) and between-day (reproducibility). The RSD values were less
than 1% which indicates high degree of precision of the proposed methods.