Research article

Received: 26 March 2011, Accepted: 8 June 2011 Published online in Wiley Online Library: 21 July 2011

(wileyonlinelibrary.com) DOI 10.1002/bmc.1670

A chiral liquid chromatography method for the

simultaneous determination of oxcarbazepine,
eslicarbazepine, R-licarbazepine and other new
chemical derivatives BIA 2–024, BIA 2–059 and
BIA 2–265, in mouse plasma and brain
Ana Fortunaa,b, Gilberto Alvesb,c, Anabela Almeidaa,b, Bruno Lopesa,
Amílcar Falcãoa,b* and Patrício Soares-da-Silvad
ABSTRACT: Recently, in silico models have been developed to predict drug pharmacokinetics. However, before application,
they must be validated and, for that, information about structurally similar reference compounds is required. A chiral liquid
chromatography method with ultraviolet detection (LC-UV) was developed and validated for the simultaneous quantification
of BIA 2–024, BIA 2–059, BIA 2–265, oxcarbazepine, eslicarbazepine (S-licarbazepine) and R-licarbazepine in mouse plasma and
brain. Compounds were extracted by a selective solid-phase extraction procedure and their chromatographic separation was
achieved on a LiChroCART 250–4 ChiraDex column using a mobile phase of water–methanol (92:8, v/v) pumped at 0.7mL/min.
The UV detector was set at 235nm. Calibration curves were linear (r 2 ≥0.996) over the concentration ranges of 0.2–30mg/mL for
oxcarbazepine, eslicarbazepine and R-licarbazepine; 0.2–60mg/mL for the remaining compounds in plasma; and 0.06–15mg/mL
for all the analytes in brain homogenate. Taking into account all analytes at these concentration ranges in both matrices, the
overall precision did not exceed 9.09%, and the accuracy was within 14.3%. This LC-UV method is suitable for carrying out
pharmacokinetic studies with these compounds in mouse in order to obtain a better picture of their metabolic pathways and
biodistribution. Copyright © 2011 John Wiley & Sons, Ltd.

Keywords: chiral liquid chromatography; bioanalytical validation; mouse plasma and brain; oxcarbazepine; eslicarbazepine

Introduction low-toxicity properties, emphasizing the importance of this

At the initial phases of the drug discovery and development
(DDD) process, new chemical entities (NCEs) have traditionally
been screened over several decades based on their biological
activity and therapeutic effects using studies performed in
whole animal models (Nef, 2001). However, since the early
1990s, the challenges facing the pharmaceutical industries have
been tremendous at every step of DDD because of the exponen-
tial increase in the number of compounds emerging from com-
binatorial chemistry and high-throughput screening methods,
which has increased the demand for screening of NCEs using a
wide range of biological assays. A variety of in silico and
in vitro approaches have been developed and combined to iden-
tify the most potent and selective drug candidate, reducing the
costs and time required for new drugs to reach the market. Nev-
ertheless, this strategy of screening thousands of compounds
solely for potency and selectivity has not been as efficient as
expected, because a large number of NCEs fail during clinical
trials and do not reach the market. Problems with absorption,
distribution, metabolism and excretion (ADME) or toxicity have
been identified as the major reasons for failure of drug
candidates in clinical development. About 50% of promising
drug candidates do not progress through the DDD process
because they do not possess the required pharmacokinetic or
384
evaluation in the initial phases of DDD (Lipinski, 2000; Yengi
et al., 2007; Tsaioun et al., 2009). In this context, basic research
* Correspondence to: Amílcar Falcão, Pharmacology Department, Faculty of
Pharmacy, University of Coimbra Pólo das Ciências da Saúde, Azinhaga
de Santa Comba 3000-548 Coimbra, Portugal. E-mail:acfalcao@ff.uc.pt
a
Pharmacology Department, Faculty of Pharmacy, University of Coimbra,
3000-548 Coimbra, Portugal
b
Centre for Neurosciences and Cell Biology, University of Coimbra, 3004-517
Coimbra, Portugal
c
Health Sciences Research Centre (CICS), University of Beira Interior, 6200-506
Covilhã, Portugal
d
Department of Research and Development, BIAL, 4745-457 S. Mamede do
Coronado, Portugal
Abbreviations used: ADME, absorption, distribution, metabolism and ex-
cretion; BIA 2–024, 10,11-dihydro-10-hydroxyimino-5H-dibenz[b,f]azepine-
5-carboxamide; BIA 2–059, (+)-(R)-10-acetoxy-10,11-dro-5H-dibenz[b,f]
azepine-5-carboxamid; BIA 2–265, 10-(hydroxymethyl)-10-nitro-10,11-
dihydro-5H-dibenzo[b,f]azepine-5-carboxamide; DDD, drug discovery
and development; NCE, new chemical entity; OXC, oxcarbazepine; R-Lic,
R-licarbazepine; S-Lic, S-licarbazepine.

Biomed. Chromatogr. 2012; 26: 384–392 Copyright © 2011 John Wiley & Sons, Ltd.
Determination of eslicarbazepine and derivatives in mouse

using in silico and in vitro assays with high throughput and low
costs must be extensively performed. These tools decrease the
turnaround time for delivery of information to medicinal che-
mists during the lead optimization phase. Such methodologies
should also be combined in an iterative way in order to prioritize
compounds with acceptable therapeutic potential and pharma-
cokinetic properties for advance to more expensive in vivo
testing phases (Pauli et al., 2008). Effectively, despite the consid-
erable changes recently introduced in the DDD process, in vivo
experimental studies using laboratory animal models remain de-
cisive in the development of new drugs, but they are now usu-
ally required only at later phases of pre-clinical development.
Pre-clinical studies in whole animal models are considered to
be essential for better assessing the pharmacology, toxicity
and pharmacokinetic properties of new drug candidates, with
the aim of extrapolating the results to humans. In fact, only com-
pounds demonstrating acceptable pharmacokinetic and safety
profiles in animals should progress to the first clinical trials for
in-depth studies (Bohets et al., 2001; Caldwell et al., 2001).
Accordingly, there is increasing interest in optimizing computer
and statistical models that simulate pharmacokinetics and toxicity
in the body (Schmitt and Willmann, 2004; Parrot et al., 2005;
Carlson and Fisher, 2008; Sui et al., 2008). However, for construc-
tion of such in silico pharmacostatistical models, adequate infor-
mation is required. In addition, before such tools can be routinely
used, they have to be validated by comparing the predicted
data with the information obtained experimentally for known Figure 1. Chemical structures of (+)-(R)-10-acetoxy-10,11-dihydro-
compounds. 5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-059), 10,11-dihydro-
Over the last 40years several dibenz[b,f]azepine-5-carboxa- 10-hydroxyimino-5Hdibenz[b,f]azepine-5-carboxamide (BIA 2-024),
mide derivatives have been empirically synthesized, and three 10-(hydroxymethyl)-10-nitro-10,11-dihydro-5H-dibenzo[b,f]azepine-
generations of structurally related but distinct antiepileptic 5-carboxamide (BIA 2-265), oxcarbazepine (OXC), eslicarbazepine
(S-licarbazepine; S-Lic), R-licarbazepine (R-Lic), and carbamazepine-10,
drugs are marketed: carbamazepine, oxcarbazepine (OXC) and
11-epoxide used as internal standard (IS).
eslicarbazepine acetate (Neels et al., 2004; Luszczki, 2009). How-
ever, many other dibenz[b,f]azepine-5-carboxamide derivatives
have been demonstrated to be devoid of antiepileptic potential chromatography elution conditions used, whenever possible,
after their evaluation against the gold standard seizure animal during the DDD in pharmacokinetic departments.
models. Thus, our research group aims to better understand the
reasons for the success or failure of many dibenz[b,f]azepine-
5-carboxamide compounds, taking advantage of in silico ap- Experimental
proaches specifically developed for these compounds. For this
purpose we aimed to obtain more pharmacokinetic data about Chemicals and reagents
some dibenz[b,f]azepine-5-carboxamide derivatives that have OXC (lot number FO40003), S-Lic (lot number 2070-2-1), R-Lic (lot num-
not been extensively evaluated: BIA 2–024 {10,11-dihydro-10- ber PC040414), BIA 2–059 (lot number PC040831), BIA 2–024 (lot number
hydroxyimino-5H-dibenz[b,f]azepine-5-carboxamide}, BIA 2–059 DL980709), BIA 2–265 (lot number PC050704) and carbamazepine-10,11-
{ ( + ) - ( R)-10-acetoxy-10,11-dihydro-5H-dibenz[b,f]azepine-5 - car- epoxide (lot number PC030916), used as internal standard (IS), were
boxamide}, and BIA 2–265 {10-(hydroxymethyl)-10-nitro-10,11- kindly supplied by BIAL (Porto, Portugal; Fig. 1). Methanol (LC gradient
dihydro-5H-dibenzo[b,f]azepine-5-carboxamide} (Fig. 1). However, grade) was purchased from Fisher Scientific (Leicestershire, UK) and
to date, there have been no bioanalytical methods developed to ultra-pure water (LC grade, >15MΩ) was obtained by means of a Milli-Q
simultaneously quantify these compounds. Therefore, the avail- water apparatus from Millipore (Milford, MA, USA). Ethyl acetate, sodium
dihydrogen phosphate dihydrate, di-sodium hydrogen phosphate dihy-
ability of a reliable bioanalytical method to quantify BIA 2–024,
drate and hydrochloric acid fuming 37% were purchased from Merck KGaA
BIA 2–059 and BIA 2–265 would be required. In addition, to
(Darmstadt, Germany).
get a better picture of the metabolic pathways of these com-
pounds and to assess their probable metabolism to well-known
compounds such as OXC, eslicarbazepine (S-licarbazepine; S-Lic), Chromatographic instrumentation and analytical conditions
and R-licarbazepine (R-Lic), we decided also to consider them as
The chromatographic analysis was carried out using a BAS-480 liquid
analytes (Fig. 1). Obviously, this fact prompted us to develop and
chromatograph equipped with a PM-80 pump, a Rheodyne manual
fully validate the first liquid chromatography (LC) method able
injector with a 20mL loop, a BAS UV-116 UV–vis detector, a BAS LC-
to quantify simultaneously BIA 2–024, BIA 2–059, BIA 2–265, 22 C temperature controller, a BAS DA-5 chromatography control
OXC, S-Lic and R-Lic, in mouse plasma and brain. Moreover, and a data system interface (all from Bioanalytical Systems, West
we chose to develop the new LC method employing ultra- Lafayette, IN, USA). Data collection and integration were achieved by
violet (UV) detection and isocratic conditions which are, means of BAS Chromgraph Control and Chromgraph Report software
respectively, the most usual detection system and the simplest version 2.30. Chromatographic separation of OXC, S-Lic, R-Lic, BIA
385

Biomed. Chromatogr. 2012; 26: 384–392 Copyright © 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc

2–059, BIA 2–024, BIA 2–265 and the IS was performed at 35 C on
a LiChroCART 250–4 ChiraDex (b-cyclodextrin, 5mm) column protected
by a LiChroCART 4–4 ChiraDex (b-cyclodextrin, 5mm) guard column
purchased from Merck KGaA (Darmstadt, Germany) and using a mobile
phase composed of water–methanol (92:8, v/v), which is delivered at a
flow-rate of 0.7mL/min. The mobile phase was filtered through a 0.45mm
filter and ultrasonically degassed prior to use. The wavelength of
detection was set at 235nm.
Preparation of stock solutions, calibration samples and
quality control samples
Stock solutions of OXC, S-Lic, R-Lic, BIA 2–059, BIA 2–024 and BIA 2–265
were individually prepared in acetonitrile at concentrations of 10mg/mL
and appropriately diluted to obtain solutions of 1mg/mL and 100mg/mL.
Considering the IS, its stock solution of 1mg/mL was prepared by dissol-
ving the appropriate amount in acetonitrile and the working solution of
200mg/mL was obtained daily by appropriately diluting the stock solu-
tion with acetonitrile. Drug working solutions were combined spiking
solutions obtained by mixing stock and dilution solutions with acetoni-
trile. The combined spiking solutions used for the preparation of calibra-
tion standards in plasma presented final concentrations of 6, 12, 36, 90,
300 and 900mg/mL for OXC, S-Lic and R-Lic, and 6, 12, 36, 150, 600 and
1800mg/mL for BIA 2–059, BIA 2–024 and BIA 2–265. On the other hand,
the corresponding combined spiking solutions used for the preparation
of calibration standards in brain homogenate supernatant were pre-
pared at 6, 12, 36, 140, 500 and 1500mg/mL for all the analytes. Stock, di-
lution and working solutions were all refrigerated at 4 C and protected
from light.
Six calibration standards were obtained spiking 10mL of the appropri-
ate working solution into 300mL of blank plasma or 1mL of brain homog-
enate supernatant and thereafter used for construction of the calibration
curves in each matrix. In plasma, the calibration ranges were 0.2–30mg/
mL for OXC, S-Lic and R-Lic, and 0.2–60mg/mL for BIA 2–059, BIA 2–024
and BIA 2–265, whereas in brain homogenate supernatant, the calibra-
tion range was 0.06–15mg/mL for all the analytes.
Quality control (QC) samples at low (QC1), middle (QC2) and high (QC3)
concentrations of the calibration ranges were also prepared indepen-
dently in the studied biological matrices. Thus, plasma (300mL) was
spiked to attain 0.5mg/mL for all the compounds in QC1; 15mg/mL for
OXC, S-Lic and R-Lic, and 30mg/mL for BIA 2–059, BIA 2–024 and BIA
2–265 in QC2; and 27mg/mL for OXC, S-Lic and R-Lic, and 54mg/mL for
BIA 2–059, BIA 2–024 and BIA 2–265 in QC3. Similarly, brain homogenate
supernatant (1mL) was spiked to attain all the compounds at 0.15, 7.5
and 13.5mg/mL in QC1, QC2 and QC3, respectively. QC samples were then
interpolated from the calibration curves to obtain the concentrations of
each compound.
Achievement of mouse plasma and brain homogenates
In order to obtain drug-free pools of mouse plasma and brain homoge-
nate, used as blank matrices during the validation studies, healthy adult
male CD-1 mice with 30–35g and obtained from HarlanTM (Udin, Italy)
were sacrificed by cervical dislocation followed by decapitation. Blood
was immediately collected into heparinized tubes, centrifuged at 4000
rpm for 10min (4 C) and the supernatant plasma stored at 30 C until
used. Brain tissues were quickly dissected, weighed, added to 0.1 M so-
dium phosphate buffer pH 5 (4mL to 1g of brain) and, thereafter, homo-
genised using a ThomasW teflon pestle tissue homogenizer. The
homogenates were transferred to 10mL glass centrifugation tubes and
centrifuged at 4800rpm for 15min (4 C); the supernatants produced
were stored at 30 C until used.
Mice were kept in local animal facilities under controlled environmen-
tal conditions (12h light/dark cycle, at 221 C and humidity 505%)
and received a regular standard chow diet (4RF21, Mucedola, Italy) and
tap water ad libitum until used. All the animal experiments were con-
ducted in accordance with the European Directive (86/609/EEC) for the
386
wileyonlinelibrary.com/journal/bmc Copyright © 2011 John Wiley & Sons, Ltd.
A. Fortuna et al.
accommodation and care of laboratory animals and the experimental
procedures were approved by the Portuguese Veterinary General
Division.
Extraction procedure
Samples were prepared by solid-phase extraction (SPE) process using the
OasisW HLB (30mg, 1mL) cartridges from Waters, Milford, MA, USA. At
the day of analysis, after plasma had been thawed, 300mL were added
to 700mL of 0.1 M sodium phosphate buffer (pH 5) and spiked with 10mL
of the IS working solution. Samples were mixed and loaded into the
SPE cartridges, previously conditioned with 1mL of methanol, 1mL of
acetonitrile and 1mL of water–acetonitrile (95:5, v/v). After sample elu-
tion, the loaded cartridges were submitted to 30 kPa and washed four
times with 1mL of water–methanol (90:10, v/v), then the sorbent was
dried under air flow for 5min. Thereafter, analytes were eluted with 1
mL of ethyl acetate under a gentle vacuum and the eluates were evapo-
rated to dryness under a nitrogen stream at 45 C. The residues were
reconstituted in 100mL of LC mobile phase, vortexed for approximately
1min and placed in an ultrasonic bath at room temperature for approx-
imately 1min. Following this, the reconstituted extracts were trans-
ferred to a 0.22mm Spin-X centrifugal filter, centrifuged at 13400rpm
for 2min and 20 mL of the final filtered samples were injected into the
LC system.
After thawing, 1mL of the brain homogenate supernatant was directly
spiked with 10mL of IS and then submitted to the SPE procedure previ-
ously described for plasma samples.
Assay validation
Considering the guidelines of one of the main regulatory agencies (US
DHHS et al., 2001) and the updated recommendations of the Conference
Report of the Washington Conference on ‘analytical methods validation:
bioavailability, bioequivalence and pharmacokinetic studies’ (Shah et al.,
2000), this method was validated for selectivity, linearity, intra- and inter-
day accuracy, intra and inter-day precision, lower limit of quantification
(LOQ), recovery and stability.
Selectivity is described as the ability of a method to discriminate the
analyte from all potentially interfering substances. It was investigated
by comparing the chromatograms obtained from blank plasma and
brain homogenate supernatant samples of six different mice with those
obtained from the correspondent spiked samples in order to analyze the
existence of peaks of endogenous substances interfering with those of
the analytes.
To evaluate the linearity of the analytical method, calibration curves
were prepared using six calibration standards and assayed on five sepa-
rate days (n=5). The sensitivity was evaluated by determining the LOQ,
which is defined as the lowest concentration of the calibration curve that
can be measured with acceptable precision and accuracy. It was estab-
lished using five extracted samples (n=5), ensuring a percentage of coef-
ficient of variation (CV%) of not more than 20% and a deviation from
nominal value in percentage (bias %) within20%.
The precision of an analytical procedure expresses the closeness of
agreement between a series of measurements obtained from multiple
determinations of the same homogeneous sample, while the accuracy
describes the closeness of experimental results to the nominal value of
the analyte. The intra-day accuracy and precision were determined by
analyzing five replicates (n=5) of each QC sample on the same day
whereas the inter-day studies were performed on five different days.
The acceptance criteria for intra- and inter-day precision (expressed as
CV%) and accuracy (expressed as percentage bias) were, respectively, a
CV not exceeding 15% and a bias within 15%.
The extraction recoveries of all the compounds were determined by
comparing the peak height ratio (analyte/IS) of extracted samples spiked
with known amounts of the analytes with the peak height ratio of analyt-
ical standards at corresponding concentrations. This procedure was
Biomed. Chromatogr. 2012; 26: 384–392
Determination of eslicarbazepine and derivatives in mouse

repeated five times (n=5) for the three different concentration levels of
QC samples. Recovery of IS was determined in the same way.
Experiments were also performed in order to evaluate the stability of
the compounds in mobile phase, plasma and brain homogenate under
different temperature and time conditions. The data of QC1 and QC3
samples were analyzed before (reference samples) and after being ex-
posed to the conditions for stability assessment (stability samples) and
a stability/reference samples ratio of 85–115% was accepted as the sta-
bility criterion (n=5). The short-term (bench-top) temperature stability
was evaluated by keeping the QC samples at room temperature for a pe-
riod of 2h and also at 4 C for 24h. The long-term stability was assessed
at low temperature ( 30 C) for a period of 30days. The post-preparative
stability was measured by reanalyzing extracted QC samples kept at 4 C
for 24h to simulate the time that the sample can be in the auto-sampler.
Finally, the stability was also evaluated after three freeze–thaw cycles.
For that, the aliquots of QC1 and QC3 were stored at 30 C for 24h,
thawed unassisted at room temperature and, when completely thawed,
refrozen for 24h under the same conditions until three cycles had been
completed.

Results and Discussion

Figure 2. Dependence of chromatographic resolution of pairs of
analytes as a function of temperature (BIA 2-059/OXC, OXC/BIA 2-024,
and S-Lic/R-Lic). BIA 2-059, (+)-(R)-10-acetoxy-10,11-dihydro-5H-dibenz
[b,f]azepine-5-carboxamide; BIA 2-024, 10,11-dihydro-10-hydroxyimino-
5Hdibenz[b,f]azepine-5-carboxamide; BIA 2-265, 10-(hydroxymethyl)-
10-nitro-10,11-dihydro-5H-dibenzo[b,f]azepine-5-carboxamide;
OXC, oxcarbazepine; R-Lic, R-licarbazepine; S-Lic, eslicarbazepine
(S-licarbazepine).
LC-UV optimization
A simple and reliable LC method with UV detection was de-
veloped for the simultaneous quantification of BIA 2–024,
BIA 2–059, BIA 2–265, OXC, S-Lic and R-Lic, in plasma and brain
of mouse. As we aim to obtain pharmacokinetic information to
construct and validate in silico models that are useful at early

Figure 3. Typical chromatograms of extracted mouse samples. Blank plasma sample (a), spiked plasma at levels of the limit of quantification (c) and
spiked plasma at levels of the upper limit of the calibration ranges (e); blank supernatant of brain homogenate (b), spiked supernatant of brain homog-
enate at levels of the limit of quantification (d) and spiked supernatant of brain homogenate at levels of the upper limit of the calibration ranges (f). BIA
2-059, (+)-(R)-10-acetoxy-10,11-dihydro-5H-dibenz[b,f]azepine-5-carboxamide; BIA 2-024, 10,11-dihydro-10-hydroxyimino-5Hdibenz[b,f]azepine-5-
carboxamide; BIA 2-265, 10-(hydroxymethyl)-10-nitro-10,11-dihydro-5H-dibenzo[b,f]azepine-5-carboxamide; OXC, oxcarbazepine; R-Lic, R-licarbazepine;
S-Lic, eslicarbazepine (S-licarbazepine); IS, internal standard.
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 A. Fortuna et al.

Table 1. Calibration curve parameters for BIA 2–265, eslicarbazepine (S-licarbazepine, S-Lic), R-licarbazepine (R-Lic), BIA 2–059,
oxcarbazepine (OXC) and BIA 2–024 in mouse plasma and brain homogenate (n=5). The equation of the calibration curve
is y=mx+c, where x is the drug concentration, expressed in mg/mL, and y is the drug to internal standard peak height ratio,
expressed in arbitrary height units. Slope, intercept values and regression coefficients are means

Matrix/drug Calibration standards (µg/mL) Slope (m) Intercept (c) Regression coefficient (r2)
Plasma
BIA 2-265 0.2, 0.4, 1.2, 5, 20, 60 0.1341 0.0030 0.996
S-Lic 0.2, 0.4, 1.2, 3, 10, 30 0.1642 0.0030 0.999
R-Lic 0.2, 0.4, 1.2, 3, 10, 30 0.1266 0.0031 0.997
BIA 2-059 0.2, 0.4, 1.2, 5, 20, 60 0.0887 0.0043 0.999
OXC 0.2, 0.4, 1.2, 3, 10, 30 0.1554 0.0002 0.999
BIA 2-024 0.2, 0.4, 1.2, 5, 20, 60 0.1377 0.0023 0.998
Brain
BIA 2-265 0.06, 0.12, 0.36, 1.4, 5, 15 0.4982 0.0035 0.999
S-Lic 0.06, 0.12, 0.36, 1.4, 5, 15 0.4554 0.0001 0.999
R-Lic 0.06, 0.12, 0.36, 1.4, 5, 15 0.5268 0.0028 0.998
BIA 2-059 0.06, 0.12, 0.36, 1.4, 5, 15 0.3033 0.0002 0.999
OXC 0.06, 0.12, 0.36, 1.4, 5, 15 0.5823 0.0029 0.998
BIA 2-024 0.06, 0.12, 0.36, 1.4, 5, 15 0.4166 0.0007 0.998

saving time and allowing easy switching from one compound

Table 2. Values of precision, expressed by coefficient varia- to another.
tion (CV%), and accuracy, expressed by percentage bias, at As S-Lic and R-Lic are enantiomers, chiral separation has to be
the limit of quantification (LOQ) of BIA 2–265, eslicarbazepine performed either indirectly using derivatization reagents or di-
(S-licarbazepine, S-Lic), R-licarbazepine (R-Lic), BIA 2–059, rectly using chiral selectors as additives in mobile phase or using
oxcarbazepine (OXC) and BIA 2–024 in mouse plasma and chiral stationary phases. The last option was chosen because it is
brain homogenate (n=5) the most convenient and it has been the most frequently used
despite the higher cost and lesser robustness in comparison to
CNominal CExperimentala CV (%) Bias (%)
achiral stationary phases (Alves et al., 2008a). ChiradexW chiral
Matrix/drug (mg/mL) (mg/mL)
stationary phase was chosen and consists of spherical silica par-
Plasma ticles covalently bonded to b-cyclodextrins, which interact differ-
BIA 2-265 0.2 0.2080.013 6.41 3.87 ently with each enantiomer. Indeed S-Lic and R-Lic have been
S-Lic 0.2 0.1890.007 3.51 5.67 separated using this column (Hainzl et al., 2001; Alves et al.,
R-Lic 0.2 0.1830.008 4.87 8.41 2007a, 2007b), but none of the techniques was developed to
BIA 2-059 0.2 0.2180.004 1.66 8.90 separate simultaneously all the analytes considered herein. Tak-
OXC 0.2 0.1850.011 6.35 7.65 ing into account the column manufacturer's recommendations
BIA 2-024 0.2 0.2280.019 8.61 14.3 for the use of methanol as the most powerful organic modifier,
Brain and also the chromatographic method previously reported by
BIA 2-265 0.06 0.0660.006 9.09 10.0 our research group (Alves et al., 2007a), the first experiments
S-Lic 0.06 0.0560.003 5.48 6.67 were performed at 30 C using a mixture of water and methanol
R-Lic 0.06 0.0660.002 2.40 9.94 (88:12, v/v) as mobile phase and a flow-rate of 0.7mL/min. Under
BIA 2-059 0.06 0.0640.005 7.64 7.15 these conditions, BIA 2–059, OXC and BIA 2–024 did not sepa-
OXC 0.06 0.0580.004 6.90 3.33 rate adequately and the percentage of methanol had to be di-
BIA 2-024 0.06 0.0640.003 5.30 7.30 minished. Thus a mobile phase composed of water–methanol
(92:8, v/v) pumped at 0.7ml/min was demonstrated to best
CNominal, Nominal concentration; CExperimental, experimental
correlate retention times and resolution of the peaks. In addition
concentration.
a it was also decided to evaluate the effects of temperature in the
Meanstandard deviation.
technique, as it can have a huge impact on peak resolution. The
effects of temperature ranging from 30 to 45 C on the resolution
of S-Lic, R-Lic, BIA 2–059, OXC and BIA 2–024 are shown in Fig. 2.
The chromatographic resolution was determined according to
phases of DDD, the major characteristic of this bioanalytical as- the method of Budakova et al. (2008), who recommend a resolu-
say is to provide sample analysis quickly, with high accuracy, tion factor higher than 1.5 as this corresponds to greater than
precision and selectivity. Moreover, considering that some of 90% separation between two peaks. As can be seen, the resolu-
the compounds under study are metabolites of each other, the tion of BIA 2–059 with OXC and OXC with BIA 2–024 was highest
analytical method has to be able to accurately distinguish all of at 30 and 35 C. Considering that at 30 C the run time was sub-
them. It was preferred to use a unique sample preparation and stantially longer than at 35 C, it was decided to use 35 C. These
analysis technique for all the compounds in both matrices, simple and economical chromatographic conditions with a
388

wileyonlinelibrary.com/journal/bmc Copyright © 2011 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2012; 26: 384–392
Determination of eslicarbazepine and derivatives in mouse

Table 3. Intra- and inter-day accuracy (expressed as a percentage of nominal concentration) and precision (expressed as coeffi-
cient of variation, CV %) for BIA 2–265, eslicarbazepine (S-licarbazepine, S-Lic), R-licarbazepine (R-Lic), BIA 2–059, oxcarbazepine
(OXC) and BIA 2–024 in mouse plasma and brain homogenate quality control samples (n=5)

Matrix/ CNominal Intra-day Inter-day

drug (mg/mL) a
CExperimental (mg/mL) CV (%) Bias (%) a
CExperimental (mg/mL) CV (%) Bias (%)
Plasma
BIA 2-265 0.5 0.4760.01 3.37 4.75 0.5010.02 4.56 0.31
30 29.840.96 3.23 0.50 30.331.69 5.59 1.11
54 54.561.33 2.43 1.04 53.491.36 2.55 0.95
S-Lic 0.5 0.5310.02 4.41 6.19 0.4780.02 4.68 4.41
15 15.960.20 1.24 6.41 15.990.11 0.67 6.57
27 29.100.27 0.94 7.79 29.200.80 2.73 8.17
R-Lic 0.5 0.5120.02 4.57 2.33 0.5100.02 3.47 1.90
15 15.690.27 1.70 4.59 15.550.10 0.62 3.68
27 27.940.33 1.19 3.50 28.941.11 3.85 7.17
BIA 2-059 0.5 0.5210.02 4.84 4.14 0.5290.02 4.48 5.80
30 31.270.61 1.96 4.23 31.880.36 1.14 6.26
54 56.611.32 2.33 4.84 58.062.14 3.70 7.53
OXC 0.5 0.5060.01 1.93 1.13 0.4840.10 2.80 3.19
15 15.660.29 1.86 4.40 15.420.29 1.83 2.79
27 27.700.89 3.66 2.61 26.420.97 3.21 2.14
BIA 2-024 0.5 0.4860.02 3.92 2.87 0.4990.01 2.81 0.29
30 29.730.76 2.56 0.90 30.240.38 1.25 0.78
54 54.961.70 3.09 1.77 56.542.83 5.01 4.70
Brain
BIA 2-265 0.15 0.1470.01 5.68 1.80 0.1600.01 5.82 6.54
7.5 7.4940.31 4.15 0.08 7.4750.25 3.88 0.33
13.5 14.250.78 5.50 5.53 14.200.48 3.38 5.15
S-Lic 0.15 0.1470.01 3.69 1.81 0.152.001 5.60 1.45
7.5 7.2200.32 4.39 3.73 6.9610.31 4.44 7.19
13.5 13.390.69 5.16 0.80 13.120.53 4.01 2.83
R-Lic 0.15 0.1420.00 3.23 5.34 0.1560.01 7.01 4.22
7.5 7.2390.33 4.55 3.48 7.3280.34 5.43 2.29
13.5 13.680.72 5.28 1.30 13.970.31 2.22 3.46
BIA 2-059 0.15 0.1460.00 2.25 2.70 0.1570.01 5.79 4.48
7.5 7.3010.30 4.10 2.65 7.3070.32 4.35 2.57
13.5 13.950.71 5.06 3.31 14.140.61 4.31 4.71
OXC 0.15 0.1460.01 5.48 2.83 0.1540.01 5.99 2.72
7.5 7.1820.22 3.11 4.24 7.0600.54 7.72 5.87
13.5 13.300.67 5.01 1.49 13.630.67 4.89 0.97
BIA 2-024 0.15 0.1520.01 4.99 1.17 0.1590.01 3.67 5.70
7.5 7.2230.17 2.38 3.69 7.3770.44 5.91 1.64
13.5 13.010.77 5.93 3.60 13.350.50 3.71 1.10
CNominal, Nominal concentration; CExperimental, experimental concentration.
a
Meanstandard deviation.

mobile phase essentially composed of water are major advan-
tages of this technique, especially when applied in the initial
phases of DDD.
Unfortunately, LC instrumentation and conditions are not the
only limiting factor in high-throughput analysis and sample
preparation must also be considered. Indeed, because of the
complex nature of plasma and brain homogenate, direct sample
injection could not be considered a rugged approach and a pre-
treatment procedure to remove proteins and other potential
matrices interferences was needed prior to LC analysis. Protein
precipitation was the first extraction procedure tested,
performing the precipitation with methanol, acetonitrile and tri-
chloroacetic acid (20%). The use of methanol and acetonitrile
was not possible as they had very low selectivity. Trichloroacetic
acid (20%) guaranteed optimum sample clean-up; however its
acidic properties easily degraded BIA 2–265 and the IS. Thus,
a SPE procedure was developed, testing first the conditions
reported by Alves et al. (2007a). As under these conditions recov-
eries of analytes and IS were low, new conditions in washing
steps were tried and the best ones were obtained by washing
the loaded cartridges four times with 1mL of water–methanol
(90:10, v/v). No endogenous interferences at the retention times
389

Biomed. Chromatogr. 2012; 26: 384–392 Copyright © 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc
 A. Fortuna et al.

of the analytes and IS were found with the SPE procedure, allow- namely the fact that it is faster, less cumbersome and gives bet-
ing an accurate and selective sample preparation. Moreover, this ter extraction yields than the liquid–liquid extraction procedures
sample pre-treatment presents other important advantageous, reported in the literature (Pienimäki et al., 1995; Levert et al., 2002),

Table 4. Recovery of BIA 2–265, eslicarbazepine (S-licarbazepine, S-Lic), R-licarbazepine (R-Lic), BIA 2–059, oxcarbazepine (OXC)
and BIA 2–024 from plasma and brain homogenate supernatant of mouse (n=5)

Matrix
Drug Plasma Brain
a
CNominal (mg/mL) Recovery (%) CV (%) CNominal (mg/mL) Recoverya (%) CV (%)
BIA 2-265 0.5 91.041.81 1.99 0.15 100.23.10 3.09
30 87.984.70 5.34 7.5 98.115.01 5.10
54 85.624.16 4.85 13.5 103.12.71 2.63
S-Lic 0.5 107.76.69 6.21 0.15 107.54.69 4.37
15 109.35.35 4.89 7.5 100.94.68 4.64
27 107.44.52 4.21 13.5 108.33.65 3.37
R-Lic 0.5 101.13.55 3.51 0.15 103.43.42 3.31
15 108.94.98 4.58 7.5 101.94.94 4.85
27 109.30.88 0.80 13.5 108.53.27 3.01
BIA 2-059 0.5 103.58.78 8.48 0.15 109.86.35 5.78
30 105.05.43 5.18 7.5 99.564.72 4.74
54 104.92.41 2.30 13.5 106.44.41 4.13
OXC 0.5 97.287.23 7.43 0.15 96.822.65 2.74
15 95.844.95 5.17 7.5 93.795.49 5.86
27 96.473.85 3.99 13.5 98.273.19 3.24
BIA 2-024 0.5 84.605.39 6.35 0.15 81.711.85 2.27
30 85.004.26 5.01 7.5 86.693.94 4.54
54 86.484.19 4.84 13.5 93.292.78 2.98
CNominal, Nominal concentration.
a
Meanstandard deviation.

Table 5. Stability (values in percentage) of BIA 2–265, eslicarbazepine (S-licarbazepine, S-Lic), R-licarbazepine (R-Lic), BIA 2–059,
oxcarbazepine (OXC) and BIA 2–024 under conditions mimicking the entire assay process in plasma and brain homogenate super-
natant of mouse: after 30days at 30 C, 24h at 4 C, 2h in plasma and 2h in brain homogenate supernatant at room temperature
(RT), three freeze–thaw cycles and in mobile phase at 4 C for 24h

BIA 2-265 S-Lic R-Lic BIA 2-059 OXC BIA 2-024

Plasma CNominal 0.5 54 0.5 27 0.5 27 0.5 54 0.5 27 0.5 54
(mg/mL)
30 C, 30days 86.8a 87.8a 95.5 89.0 98.3 95.7 92.8a 88.8a 85.4 89.9 89.4 96.2

4 C, 24h 87.4 91.6 106.5 98.4 90.7 95.3 86.3a 85.7a 88.7 85.8 97.9 100.7
RT; 2h 87.0a 85.4a 95.2 97.1 92.1 109.7 86.1a 87.8a 85.2 90.1 89.3 103.1
Three freeze–thaw 89.4a 87.0a 93.3 103.2 88.0 89.1 87.3a 90.1a 102.2 99.8 108.9 108.1
cycles
Mobile phase 4 C, 95.0 98.8 103.9 99.9 96.5 98.5 99.2 102.3 99.7 102.2 94.0 101.5
24h
Brain CNominal 0.15 13.5 0.15 13.5 0.15 13.5 0.15 1.35 0.30 13.5 0.30 13.5
(mg/mL)
30 C, 30days 100.8 97.9 109.5 98.2 109.7 99.2 105.8 96.8 106.6 94.5 106.6 98.3

4 C, 24h 96.1 91.6 93.2 88.8 93.4 92.3 88.8 86.5 93.8 88.9 96.7 86.3
RT; 2h 90.9 92.9 93.9 93.2 92.1 95.2 87.4 88.5 89.5 85.9 90.6 90.9
Three freeze–thaw 99.2 101.6 102.6 99.4 100.3 101.3 96.3 96.1 95.9 96.2 94.3 91.1
cycles
Mobile phase 4 C, 101.6 101.1 103.5 100.5 106.6 100.5 103.9 99.8 100.4 100.4 100.7 94.4
24h
CNominal, Nominal concentration.
a
Results found at 30 C for 15days; at 4 C for 8h; at RT for 1h; and after two freeze–thaw cycles.
390

wileyonlinelibrary.com/journal/bmc Copyright © 2011 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2012; 26: 384–392
Determination of eslicarbazepine and derivatives in mouse
and it is also suitable for automation with an on-line SPE and
injection device.
Finally, as we intended to use UV detection as it is the most
usual detection system, several wavelengths were analyzed
and 235nm was chosen as the best compromise in terms of sen-
sitivity and selectivity.
Method Validation
Considering the internationally accepted guidelines (Shah et al.,
2000; US DHHS et al., 2001), two independent validation
studies were carried out for plasma and brain homogenate su-
pernatant of mouse. The selectivity of the developed method
was demonstrated by representative chromatograms from
extracts of drug-free plasma and brain homogenates of mice
and those from the correspondent extracts of samples spiked
with OXC, S-Lic, R-Lic, BIA 2–059, BIA 2–024, BIA 2–265 and IS
(Fig. 3). No interferences exist at the retention times of analytes,
showing the ability of the method to measure the compounds
unequivocally in the corresponding biological matrices. The
run time was 30min and the order of elution was BIA 2–265,
S-Lic, R-Lic, BIA 2–059, OXC, BIA 2–024 and IS.
The peak height ratios of analyte with IS in mouse plasma and
brain homogenate were linear in the concentration ranges used;
however the variance was not constant across the calibration
range (heteroscedasticity was found) and hence a weighting fac-
tor was used to improve residuals and to counter the variance
(Almeida et al., 2002). The best fit for the calibration curve was
achieved by a linear equation of y=mx+c with 1/x2 as weighing
factor. The calibration parameters for each compound, including
slope, interception and regression coefficient (r 2) are shown in
Table 1. Good linearity was found for all analytes in both matri-
ces (r 2 ≥0.996). In addition, for all the compounds, LOQ values
were established at 0.2 and 0.06mg/mL in plasma and brain
homogenate, respectively. By evaluating five samples of blank
plasma and blank brain homogenate supernatant spiked with
each analyte at those concentrations, the values estimated for
bias and CV were within the acceptance criteria (Table 2). At this
point it is important to highlight that the LOQ and the upper
limits of the concentration ranges were defined considering
not only the instrumental capabilities and pre-validation results
but also the previous concentrations found for the analytes in
some in vivo studies. There are several techniques for quantifi-
cation of OXC and/or racemic licarbazepine in biological ma-
trices, but few of them are able to distinguish S-Lic and R-Lic
(Flesch et al., 1992; Volosov et al., 2000a, 2000b; Hainzl et al.,
2001; Alves et al., 2007a, 2007b; Mazzucchelli et al., 2007).
Among these, only two are able to simultaneously separate and
quantify OXC, S-Lic and R-Lic in human plasma (Alves et al.,
2007b; Mazzucchelli et al., 2007) and only one in the plasma
and brain of mouse (Alves et al., 2007b). In comparison to the
technique developed by Mazzucchelli et al. (2007), the LOQ
values reported herein are higher probably because we based
our method on UV detection at 235nm instead of 210nm as
Mazzucchelli et al. (2007). Although the use of shorter wave-
lengths allows a lower LOQ for OXC, S-Lic and R-Lic, this was
not possible in our method because endogenous interferences
were detected. The choice of 235nm was a compromise to ob-
tain good selectivity for all analytes evaluated. However, the
LOQ of the analytes obtained with the method developed was
often lower than those previously reported (Alves et al., 2007a,
2007b). On the other hand, the concentrations of OXC, R-Lic
Biomed. Chromatogr. 2012; 26: 384–392 Copyright © 2011 John Wiley & Sons, Ltd.
and S-Lic found in plasma and brain homogenates after their ad-
ministration to mice (Hainzl et al., 2001; Alves et al., 2008b) are
likely to be determined using our methodology, because all con-
centrations are included in our calibration ranges. Regarding BIA
2–059 and BIA 2–024, only two in vivo studies have been per-
formed in order to trace their pharmacokinetic profiles after
drug administration to mice (Hainzl et al., 2001, 2002; Ambrósio
et al., 2002). Once again, our calibration ranges include the brain
and plasma concentrations found in vivo. As no in vivo studies
have been performed for BIA 2–265 and it is structurally related
to the other compounds, the authors defined its calibration
range considering those of the remaining analytes, using the
largest ranges.
Table 3 depicts the intra- and inter-day precision and accuracy
of each of QC samples (QC1, QC2 and QC3) in mouse plasma and
brain homogenates. Considering the validation performed for
plasma samples, the intra- and inter-day precision did not ex-
ceed 5.59% and the accuracy varied from 4.75 to 8.17%, while
for brain homogenate, the intra- and inter-day precision did not
exceed 7.72% and the accuracy varied from 7.19 to 6.54%.
These data demonstrate that both precision and accuracy were
within the acceptable limits in all matrices, being the method
precise and accurate to determine these drugs in mouse plasma
and brain samples.
The relative drug recoveries from plasma and tissue homoge-
nates were estimated and are presented in Table 4. The mean
relative recoveries, taking the compounds into account in all ma-
trices, ranged from 81.71 to 109.8%. The CV values were also de-
termined and demonstrated to be low (≤ 8.48%), suggesting
consistent average recoveries over the evaluated concentration
range. In plasma, the absolute recovery of the IS was 73.7% with
a CV value of 2.77%, while in brain homogenate it was found to
be 84.6% with a CV of 4.46%.
From the drug stability data obtained in mouse brain homog-
enate under the conditions stated in the previous section, all the
compounds were demonstrated to be stable with no significant
losses (Table 5). Considering the stability results obtained in
plasma, also presented in Table 5, all the compounds were sta-
ble at all studied conditions except BIA 2–265 and BIA 2–059. In-
deed, BIA 2–265 and BIA 2–059 were stable at 30 C for only 15
days, at room temperature for 1h, and for only two freeze–thaw
cycles. BIA 2–059 was also stable in plasma for only 8h at 4 C. Al-
though these compounds are less stable, this will have no im-
pact on application if the stability results are taking into account.
Conclusion
A rapid and sensitive chiral LC-UV method was developed and vali-
dated for the simultaneous determination of OXC, S-Lic, R-Lic, BIA
2–059, BIA 2–024 and BIA 2–265 in the matrices of mouse
plasma and brain homogenate. The adequate selectivity, sensi-
tivity, precision and accuracy, with high recovery yields and ap-
propriate run time, make it suitable for pharmacokinetic
studies and it will be easy to change from one compound to an-
other. In addition, because of the simple LC conditions and
straightforward sample pre-treatment procedure, the method
is appropriate for application to elucidate the metabolic path-
ways and pharmacokinetics of BIA 2–024, BIA 2–059 and BIA
2–265. In addition, such pharmacokinetics data will be a useful
contribution to the development of an in silico approach that
helps to understand the reasons for the success or failure of
dibenz[b,f]azepine-5-carboxamide compounds.
391
wileyonlinelibrary.com/journal/bmc

Acknowledgements
The authors would like to thank the Fundação para a Ciência e a
Tecnologia (SFRH/BD/31390/2006), Portugal and BIAL, Portugal.
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