Enantioselective analysis of ibuprofen enantiomers in mice plasma and tissues by high-performance liquid chromatography with fluorescence detection: Application to a pharmacokinetic study

Article abstract of DOI:10.1002/chir.22715

A direct fluorometric high-performance liquid chromatography (HPLC) method was developed and validated for the analysis of ibuprofen enantiomers in mouse plasma (100?μl) and tissues (brain, liver, kidneys) using liquid–liquid extraction and 4-tertbutylphenoxyacetic acid as an internal standard. Separation of enantiomers was accomplished in a Chiracel OJ-H chiral column based on cellulose tris(4-methylbenzoate) coated on 5?μm silica-gel, 250 x 4.6?mm at 22?°C with a mobile phase composed of n-hexane, 2-propanol, and trifluoroacetic acid that were delivered in gradient elution at a flow rate of 1?ml min^?1. A fluorometric detector was set at: λ_excit. = 220?nm and λ_emis. = 290?nm. Method validation included the evaluation of the selectivity, linearity, lower limit of quantification (LLOQ), within-run and between-run precision and accuracy. The LLOQ for the two enantiomers was 0.125 μg ml^?1 in plasma, 0.09?μg g^?1 in brain, and 0.25?μg g^?1 in for liver and kidney homogenates. The calibration curves showed good linearity in the ranges of each enantiomers: from 0.125 to 35?μg ml^?1 for plasma, 0.09–1.44?μg g^?1 for brain, and 0.25–20?μg g^?1 for liver and kidney homogenates. The method was successfully applied to a pharmacokinetic study of ibuprofen enantiomers in mice treated i.v. with 10?mg kg^?1 of racemate.

Full text of DOI:10.1002/chir.22715

Received: 4 December 2016
DOI: 10.1002/chir.22715
Revised: 23 April 2017
Accepted: 26 April 2017
R E G U L A R A R T I C L E
Enantioselective analysis of ibuprofen enantiomers in mice plasma
and tissues by high‐performance liquid chromatography with
fluorescence detection: Application to a pharmacokinetic study
Katarzyna Przejczowska‐Pomierny
| Monika Włodyka | Agnieszka Cios | Elżbieta Wyska
Department of Pharmacokinetics and
Physical Pharmacy, Jagiellonian University
Medical College, Cracow, Poland
Abstract
A direct fluorometric high‐performance liquid chromatography (HPLC) method was
developed and validated for the analysis of ibuprofen enantiomers in mouse plasma
(100 μl) and tissues (brain, liver, kidneys) using liquid–liquid extraction and
4‐tertbutylphenoxyacetic acid as an internal standard. Separation of enantiomers
was accomplished in a Chiracel OJ‐H chiral column based on cellulose tris(4‐
methylbenzoate) coated on 5 μm silica‐gel, 250 x 4.6 mm at 22 °C with a mobile
phase composed of n‐hexane, 2‐propanol, and trifluoroacetic acid that were deliv-
ered in gradient elution at a flow rate of 1 ml min−1. A fluorometric detector was
set at: λexcit. = 220 nm and λemis. = 290 nm. Method validation included the
evaluation of the selectivity, linearity, lower limit of quantification (LLOQ),
within‐run and between‐run precision and accuracy. The LLOQ for the two enantio-
mers was 0.125 μg ml−1 in plasma, 0.09 μg g−1 in brain, and 0.25 μg g−1 in for liver
and kidney homogenates. The calibration curves showed good linearity in the ranges
of each enantiomers: from 0.125 to 35 μg ml−1 for plasma, 0.09–1.44 μg g−1 for
brain, and 0.25–20 μg g−1 for liver and kidney homogenates. The method was
successfully applied to a pharmacokinetic study of ibuprofen enantiomers in mice
treated i.v. with 10 mg kg−1 of racemate.
Correspondence
K. Przejczowska‐Pomierny, Department of
Pharmacokinetics and Physical Pharmacy
Jagiellonian University Medical College,
Medyczna 9, 30‐688 Cracow, Poland.
Email: katarzyna.przejczowska@doctoral.uj.
edu.pl
KEYWORDS
(R,S)‐2‐(4‐isobutylphenyl)propionic acid, Chiracel OJ‐H, chiral separation, compartment modelling, normal
phase
1 | INTRODUCTION
pharmacodynamic, pharmacokinetic, and toxicological
properties of its enantiomers are different.2-5 Ibuprofen
Ibuprofen, (R,S)‐2‐(4‐isobutylphenyl)propionic acid, is a
nonsteroidal antiinflammatory drug (NSAID) that is widely
used for the treatment of acute and chronic pain, fever,
osteoarthritis, rheumatoid arthritis, and related diseases.1
It contains a chiral carbon atom located on its propionic
side chain and, therefore, it exists in two stereoisomeric
forms: (+)‐S and (−)‐R. Although ibuprofen is
commonly marketed as a racemic drug formulation, the
exerts antiinflammatory activity mainly by inhibiting cyclo-
oxygenase (COX)‐1 and COX‐2 and there is evidence that
(+)‐S‐ibuprofen is much more potent than its antipode.6
Recent studies have also demonstrated other mechanisms
of action of this drug that are independent of COX, such
as: an inhibition of leukocyte function and production, an
influence on nitric oxide (NO) production, an inhibition
of production of transcription factors, MAP kinase, nuclear
Chirality. 2017;1–12.
wileyonlinelibrary.com/journal/chir
© 2017 Wiley Periodicals, Inc.
1

2
PRZEJCZOWSKA‐POMIERNY ET AL.
receptors, heat shock proteins, cytokines and antibodies, a
reduction of apoptosis, and an increase of endogenous can-
nabinoids in the central nervous system7,8 having positive
effects on memory deficits in the Alzheimer's animal
model9 and reducing the risk of various human cancers.10
In the human body, 53–65% of (−)‐R‐ibuprofen is inverted
to the active form (+)‐S‐ibuprofen.2,5,6 This inversion is
enzymatic and proceeds via formation of thioester of
2 | MATERIALS AND METHODS
2.1 | Chemicals and reagents
Ibuprofen sodium salt, 4‐tertbutylphenoxyacetic acid, used as
an internal standard (IS), and trifluoracetic acid (TFA) were
purchased from Sigma‐Aldrich (Germany). n‐Hexane,
2‐propanol, ethyl acetate, methanol, and hydrochloric acid
were of HPLC grade and were obtained from Merck
(Germany). Deionized water used during the experiment
was prepared in‐house using a Hydrolab water purification
system (Poland) with a 0.2 μm microfiltration capsule. Blank
blood samples and tissues (brain, liver, and kidneys) used for
the validation of the analytical method were collected from
healthy CD‐1 mice. Plasma was obtained by centrifugation
(10 min at 3000 rpm) of blood containing heparin (Polfa,
Poland) as anticoagulant and all samples were stored at
−80 °C (Skadi Telstar, Spain) until the time of analysis.
(−)‐R‐ibuprofenyl adenylate with acyl coenzyme
A
(CoA).6,8,11,12 It has been reported that this biochemical
reaction is tissue‐ and species‐specific. It is both a
presystemic13 and systemic process, which takes place
mainly in the liver and kidneys. In general, in mammals
inversion of (−)‐R‐ibuprofen to (+)‐S‐ibuprofen is unidi-
rectional, although Chen et al. observed bidirectional chi-
ral inversion of ibuprofen in guinea pigs and, minimally,
in rats and rabbits.14 Due to differences in pharmacokinet-
ics and pharmacodynamics between both ibuprofen enan-
tiomers, it is necessary to measure their plasma
concentrations separately following administration of race-
mic ibuprofen.
2.2 | Instrumentation and chromatographic
conditions
There are several assays to determine ibuprofen enan-
tiomers in biological fluids described in the literature.
High‐performance liquid chromatography (HPLC) coupled
with UV detection is the most frequently analytical
technique used.11,13,15-24 In addition, HPLC methods with
fluorescence1,25 or mass spectrometry26-29 detection was
applied. Methods using HPLC with UV detection are
characterized by a relatively low cost, wide availability,
and high precision and accuracy, but they are the least
sensitive. Higher sensitivity may be achieved using fluores-
cence or mass spectrometry detectors. Unfortunately, the
HPLC/ tandem mass spectrometry (MS/MS) systems are
not available in many laboratories, whereas most of vali-
dated HPLC‐fluorescence methods for ibuprofen enantio-
mer quantification are indirect,1,25 i.e., they require a
derivatization process. This means that sample preparation
is extensive and long‐lasting. Furthermore, formation of
diastereomeric derivatives may lead to the obtaining of a
false concentration of enantiomers due either to chiral
impurities in the reagent or to the racemization during the
process of derivatization.30 To avoid these problems, direct
enantiomeric analysis using enantioselective chiral station-
ary phases may be advisable. In the literature, several direct
HPLC methods to quantify ibuprofen enantiomers in
biological fluids can be found but they were coupled with
UV detection.11,13,17-19,23,24
The analysis of ibuprofen enantiomers was performed with a
Hitachi HPLC system (Japan) consisting of a pump (model
L‐2130), an autosampler (model L‐2200), a column oven
(model L‐2350), a fluorometric detector (model L‐2485),
and a computer, Optiplex 745 (DELL) with EZChrom Elite
Client/Server v. 3.2 software for data collection and analysis.
Separation of both enantiomers and the IS was achieved at
ambient temperature (22 1 °C) using a Chiracel OJ‐H chi-
ral column based on cellulose tris(4‐methylbenzoate) coated
on 5 μm silica‐gel, 250 x 4.6 mm (Daicel Chemical Indus-
tries, Japan) protected with a guard column 10 x 4 mm
(Daicel Chemical Industries) with the same packing material.
The mobile phase consisted of n‐hexane with TFA mixed
in a 1000:0.664 (v/v) ratio (A) and pure 2‐propanol (B) that
were delivered in gradient elution at a flow rate of
1 ml min−1. Gradient elution for plasma samples was as
follows: 98% A for 9.5 min, 98–90% A from 9.5 to 13 min,
90–98% A from 13 to 16 min, and 98% A from 16 to
22 min. In turn, gradient elution for liver, kidneys, and brain
samples was as follows: 99% A for 9.5 min, 98.5–92% A
from 9.5 to 12 min, hold 90% A from 13 to 14 min,
90–99% A from 14 to 19 min, and 99% A from 19 to
23 min. The total time of analysis was 23 min. The injection
volume was 20 μl for plasma and 30 μl for tissue samples.
The spectrofluorimetric detector was operated at an
excitation wavelength of 220 nm and an emission wavelength
of 290 nm. These settings were developed before the
validation process. As the excitation wavelength, the maxi-
mum of absorbance of ibuprofen in mobile phase was
selected, whereas the emission wavelength was developed
experimentally.
The aim of this study was to develop and validate a
direct enantioselective HPLC method with fluorescence
detection for quantitative determination of ibuprofen enan-
tiomers in mouse plasma and tissues. Furthermore, the
method was applied to a pharmacokinetic study following
intravenous (i.v.) administration of a racemic drug in mice.

PRZEJCZOWSKA‐POMIERNY ET AL.
3
and column efficiency (N) for ibuprofen enantiomers and IS
in the eluting systems used for plasma and tissue samples.
The selectivity of the method was evaluated by analyzing
extracts of plasma and tissue homogenates (brain, liver, and
kidneys) from six different CD‐1 mice to investigate the
potential interference in the peak regions of ibuprofen
enantiomers and IS in chromatograms.
2.3 | Preparation of standard solutions
A
stock solution of racemic ibuprofen sodium salt
(500 μg ml−1 of each enantiomer) and IS (1 mg ml−1) were
prepared in methanol and were kept at 4 °C. The stock solution
of ibuprofen was subsequently diluted in methanol to prepare
working standard solutions in the ranges of each enantiomer:
0.125–30 μg ml−1 for plasma, 0.25–20 μg g−1 for liver and
kidneys, and 0.09–1.44 μg g−1 for brain. IS solutions
The calibration curves were prepared in the drug‐free plasma
or tissue homogenates by spiking them with 20 μl of each of the
standard working solutions to obtain the final concentrations of
ibuprofen enantiomers: 0.125, 0.25, 0.5, 2.5, 5, 10, 20, and
30 μg ml−1 for plasma samples; 0.09, 0.18, 0.36, 0.72, 1.08,
and 1.44 μg g−1 for brain samples; and 0.25, 0.5, 1, 5, 10, and
20 μg g−1 for liver and kidney samples. Three replicates were
prepared for each concentration, then the samples were submit-
ted to the extraction and analytical procedures described above.
The peak area ratios of ibuprofen enantiomers to IS were plotted
against spiked ibuprofen concentrations for the evaluation of lin-
earity and the coefficients of linear correlation. The regression
lines were used to calculate concentrations of ibuprofen enantio-
mers in the unknown plasma, brain, liver, and kidney samples.
The sensitivity of the method was characterized by the
lower limit of quantification (LLOQ) values, which were
established using five drug‐free samples of plasma or appro-
priate tissue homogenates spiked with a known amount of
ibuprofen racemate that after the extraction procedure were
analyzed with a coefficient of variation (CV) of ≤20% and
a relative error (RE) of ≤20%.
Precision and accuracy were determined within a run and
between runs for plasma samples and within a run for tissues
samples. Ibuprofen enantiomer quality control (QC) samples
were prepared at three concentrations: 0.5 μg ml−1 for plasma
samples, 0.36 μg g−1 for brain samples, and 0.5 μg g−1 for liver
and kidney samples as low quality control (LQC); 5 μg g−1 for
plasma samples, 0.72 μg g−1 for brain samples, and 5 μg ml−1
for liver and kidney as medium quality control (MQC); and
20 μg ml−1 for plasma samples, 1.44 μg g−1 for brain samples,
and 10 μg g−1 for liver and kidney samples as high quality con-
trol (HQC). In the interassay study each quality control sample
was analyzed in five replicates. In the intraassay study quality
control samples in duplicate were analyzed for 5 consecutive
days. The precision was expressed as the coefficient of varia-
tion and the accuracy as the relative error.
(IS1 = 125 μg ml−1, IS2 = 62 μg/ml, and IS3 = 31 μg ml−1
)
were prepared by diluting the stock solution with methanol.
2.4 | Sample preparation
Ibuprofen enantiomers were isolated from plasma and tissue
homogenates by liquid–liquid extraction. Frozen plasma sam-
ples were thawed at room temperature and vortex‐mixed briefly
(Reax top, Heidolph, Germany). Then 100 μl of the plasma sam-
ples was transferred to a glass tube and IS1 working solution
(125 μg ml−1, 20 μl) was added. The samples were acidified with
100 μl of 1 M HCl, mixed briefly on the vortex mixer, and
extracted with 3 ml of ethyl acetate/n‐hexane (30:70, v/v) mix-
ture for 10 min on a shaker (VXRVibrax, IKA, Germany). After
centrifugation (Universal 32, Hettich, Germany) at 3000 rpm for
15 min, the organic layers were transferred into conical glass
tubes and evaporated to dryness at 37 °C in the water bath under
a gentle stream of nitrogen. Frozen mouse tissues (brain, liver,
and kidneys) were thawed at room temperature, weighed, and
then homogenized (4 mg g−1) in distilled water with a tissue
homogenizer TH220 (Omni International, USA). The tissue
homogenates (200 μl for liver and kidneys and 500 μl for brain)
were transferred to the glass tubes. Then 20 μl of IS working
solution (IS1 for kidneys, IS2 for liver, and IS3 for brain) was
added and samples were acidified with 100 μl of 1 M HCl. After
vortex mixing for 15 s, 1 ml of NaCl solution (20 g/100 ml) was
added to clear samples from proteins. Next, the samples were
vortex mixed again and extracted with 4 ml of ethyl acetate/
n‐hexane (30:70, v/v) mixture for 10 min on a shaker. After cen-
trifugation at 3000 rpm for 15 min, the organic layers were
transferred into conical glass tubes and evaporated to dryness
at 37 °C under a gentle stream of nitrogen. The residues were
reconstituted in 100 μl of mobile phase components in
99(A):1(B) ratio, vortexed for 1 min, and 20 (plasma) or 30 μl
(tissue homogenates) were injected into the HPLC system.
The extraction recovery of analytes was determined by
comparing the peak area of analytes in the extracted QC
samples with mean peak area of analytes obtained by a direct
injection of standard solutions of analytes and IS at
corresponding concentrations.
Prestudy stability studies of ibuprofen enantiomers were
carried out in many laboratories in the past.20,29-31 In all these
experiments both (−)‐R‐ and (+)‐S‐ibuprofen was stable
in short and long stability studies and also during freeze/
thaw stability evaluations. Therefore, in these studies
2.5 | Method validation
The method validation was performed in accordance
to US Food and Drug Administration (FDA) Guidance
for Industry (www.fda.gov/downloads/drugs/.../guidances/
ucm368107.pdf). Throughout the study, the suitability of the
chromatographic system was monitored by calculating the
capacity factor (k'), the resolution (R), the selectivity (α),

4
PRZEJCZOWSKA‐POMIERNY ET AL.
postpreparative stability was only determined. To this end,
the QC samples after first injection into the chromatographic
system were maintained in the autosampler at 10 °C for 12 h
and after that time they were injected again.
2.6 | Pharmacokinetic study in mice
The validated method was applied to a pharmacokinetic
study in mice treated with a single dose of 10 mg kg−1 race-
mic ibuprofen i.v. The experimental study was approved by
the First Local Ethical Committee on Animal Testing of the
Jagiellonian University (Krakow, Poland).
Male CD‐1 mice weighing 25–30 g were used in this study.
Animals were housed under controlled environmental condi-
tions (temperature 22 1 °C) with a 12‐h dark/light cycle. They
had free access to water but 12 h before ibuprofen dosing they
were fasted. Racemic ibuprofen was diluted in 0.9% saline and
used within 1 day. Mice received the drug in a single i.v. dose
of 10 mg kg−1 mouse weight (4 ml kg−1). Animals were exsan-
guinated at 5, 15, 30, 45, 90, 120, 240, and 360 min postdose
(n = 3–4 per timepoint). Blood was collected into heparinized
tubes and plasma was separated by centrifugation at 3000 rpm
for 10 min and stored at −80 °C until analysis. Additionally, at
every timepoint brain, liver, and kidneys were harvested.
FIGURE
1
Proposed pharmacokinetic model for ibuprofen
enantiomers in mice plasma after i.v. administration of the racemic drug
at a dose of 10 mg kg−1. VCR and VCS are volumes of the central
compartments for (−)‐R‐ and (+)‐S‐ibuprofen, respectively; k10R and k10S
are the first‐order elimination rate constants of both enantiomers; k12, k21
and k34, k43 are the first‐order distribution rate constants of (−)‐R‐ and (+)‐
S‐enantiomer, respectively, and kRS is the first‐order rate constant
representing the unidirectional conversion of (−)‐R‐ to (+)‐S‐ibuprofen
Pharmacokinetic analyses were performed using Phoenix
WinNonlin program v. 6.3 (Pharsight, Certara, Mountain
View, CA). Concentration versus time profiles of both ibupro-
fen enantiomers in plasma were analyzed simultaneously
using a one‐ or two‐compartment model with unidirectional
conversion of (−)‐R to (+)‐S‐ibuprofen (Figure 1). The unidi-
rectional conversion has been confirmed in a study with the
use of perfused mouse liver performed in our laboratory (data
not shown). To reduce the number of model parameters, in the
first step the total elimination rate constant (k10Rt) for (−)‐R‐
enantiomer was estimated using a two‐compartment pharma-
cokinetic model. Then the obtained value of 0.094 min−1 was
used in pharmacokinetic modeling to calculate the first‐order
elimination rate constant k10R according to the equation:
k10R = 0.094–kRS (where kRS is the first‐order rate constant
representing the unidirectional conversion of (−)‐R‐ to (+)‐
S‐ibuprofen). The final model was selected on the basis of
visual inspection of the fitting, examination of residuals,
parameter precision, and Akaike and Bayesian information
criteria. Concentration versus time profiles of ibuprofen enan-
tiomers in plasma were also analyzed by means of a
noncompartmental approach using the same pharmacokinetic
program. This method was also used in relation to the concen-
tration–time relationships obtained in the studied tissues
calculated as ln2/λz. Area under the concentration–time curve
(AUC) from time zero to the last sampling time at which con-
centrations were at or above the LLOQ (AUC0–t), was calcu-
lated by the linear trapezoidal rule. Clearance was calculated
as D/AUC and mean residence time (MRT) as AUMC/
AUC, where AMUC is the area under the first moment curve.
3 | RESULTS AND DISCUSSION
In this study we developed and validated a sensitive
enantioselective HPLC method with fluorimetric detection.
Until now, the method where the Chiracel OJ‐H chiral
column was used to separate both enantiomers of ibuprofen
directly, and 4‐tertbutylphenoxyacetic acid was employed as
an internal standard, has not been described in the literature.
Moreover, the pharmacokinetics and tissue distribution of
ibuprofen enantiomers in mice was not extensively studied
in the past, whereas this species is frequently used to study
the pharmacodynamics of ibuprofen.9,10
3.1 | Chromatographic conditions
To avoid the long‐lasting derivatization process, we chose to
separate ibuprofen enantiomers using a chiral stationary phase
(brain, liver, and kidneys). The peak concentration (Cmax
)
and the time to reach the peak concentration (tmax) in tissues
were obtained directly from the concentration versus time
data. The terminal elimination rate constant (λz) was assessed
by linear regression and terminal half‐life (t0.5λz) was
column
Chiracel
OJ‐H
containing
cellulose
tris(4‐methylbenzoate) coated on 5 μm silica‐gel. This
column is recommended to separate ibuprofen enantiomers
on a producer's website (https://search.daicelchiral.com/name)

PRZEJCZOWSKA‐POMIERNY ET AL.
5
with the HPLC method coupled with UV detection and using
isocratic normal phase eluting (n‐hexane:2‐propanol:TFA,
98:2:0.1). Cellulose‐based chiral stationary phases (Chiralcel
OJ) have been used for the chiral separation of ibuprofen
enantiomers in one study, but they were analyzed after deriv-
atization into their amide.32 In turn, Ducret et al.33 used this
column for the resolution of ibuprofen esters. The Chiralcel
OJ stationary phase was also utilized for the thermodynamic
study of enantioseparation of arylpropionic acids.34 Tang
evaluated the influence of the mobile phase composition on
the enantioseparation of ibuprofen, ketoprofen, albuterol,
acebutolol, propafenone, betaxolol, methylphenidate, and
homatropine using a cellulose‐based chiral stationary phase.35
In turn, Valdermara et al. validated a direct enantioselective
method of ibuprofen using the Chiracel OJ column and UV
detection, although that study was not performed using bio-
logical fluids.23
In the present study, we used for the first time the
Chiracel OJ‐H column and a fluorometric detector to quan-
tify ibuprofen enantiomers in mouse plasma and tissues.
Because of the liquid–liquid extraction procedure applied
to isolate both analytes, we needed to add an internal stan-
dard to the analyzed samples. The internal standards, which
were used by other authors and HPLC/UV methods were
useless in the HPLC/fluorimetric assay. Among the different
compounds that were tested in the present study, for exam-
3.2 | Suitability of the method
Chromatographic parameters, such as resolution, selectivity,
capacity factor, and column efficiency for ibuprofen
enantiomers and IS in two eluting systems are listed in
Table 1. The calculated resolution values between each
peak‐pair were no less than 1.5 and selectivity was not less
than 1.1. k’ values were higher than 1 and lower than 10,
which indicates enough space between unretained
compounds and desired peaks and not too slow elution.
3.3 | Selectivity
Figure 2 shows the typical chromatograms of the blank
mouse plasma (A), brain (D), liver (G), and kidney (J)
homogenates. From this figure, no interfering peaks from
endogenous substances were observed at the retention times
of both enantiomers and the IS.
3.4 | Linearity
The linearity of the calibration curves for ibuprofen enan-
tiomers in mouse plasma was estimated using each enantio-
mer concentration in the range from 0.125 to 35 μg ml−1
.
As presented in Table 2, the calibration curves were linear
in the tested concentration range as indicated by high coef-
ficients of determination (R2
enantiomers.
=
0.999) for both
ple:
naproxen, ketoprofen, flurbiprofen, diclofenac, nimesulid, 4‐
aminosalicylic acid, p‐coumaric acid,
salicylic
acid,
acetylsalicylic
acid,
The linearity of the calibration curves for ibuprofen
enantiomers in mouse tissues was estimated using each
enantiomer concentration in the ranges: 0.09–1.44 μg g−1
for brain, and 0.25–20 μg g−1 for liver and kidney homog-
enates. Similarly, all of the calibration curves were linear
and the coefficients of determination ranged from
0.99755–0.9989 (Table 3).
p‐chlorophenoxyacetic acid, and p‐tolylacetic acid, only
one of them that is 4‐tertbutylphenoxyacetic acid was
extracted from plasma and tissue homogenates in acidic
conditions repetitively with a recovery of 68.89
(CV = 9.80%) for plasma and of 71.39
6.75%
0.46%
(CV = 0.65%), 76.53
6.54% (CV = 8.54%), and
78.72 4.17% (CV = 5.31) for brain, liver, and kidney
homogenates, respectively. In addition, it gave a satisfactory
detector response at an excitation wavelength of 220 nm and
an emission wavelength of 290 nm. Because the IS had a
long retention time under isocratic chromatographic condi-
tions, the gradient elution was applied leading to the total
runtime of 23 min per sample.
3.5 | Sensitivity
The LLOQ in plasma was 0.125 μg ml−1 (2.0 ng on‐column)
for the two enantiomers with the coefficient of variation (CV)
less than 20% (CV = 18.70% for (−)‐R and CV = 18.96% for
(+)‐S‐ibuprofen). In the case of tissue homogenates we
TABLE 1 System performance parameters of ibuprofen enantiomers and IS for two gradient eluting compositions: one used for plasma and the
second for tissue samples (n = 5)
*Values are means (RSD%). RSD% = (Standard Deviation/Mean) × 100.

6
PRZEJCZOWSKA‐POMIERNY ET AL.
FIGURE 2 HPLC chromatograms of blank mouse plasma (a) brain (D), liver (G), and kidney (J) homogenates; plasma containing ibuprofen
enantiomers at concentrations of 10 μg ml−1 (B), brain (E), liver (H), and kidney (K) homogenates containing ibuprofen enantiomers at
concentrations of 0.36 μg g−1, 5 μg g−1, and 5 μg g−1, respectively; plasma (C), brain (F), liver (I), and kidneys (L) samples collected 15 min after i.v.
administration of racemic ibuprofen at a dose of 10 mg kg−1 to CD‐1 mice. Peaks: 1‐ (−)‐R‐ibuprofen, 2‐ (+)‐S‐ibuprofen, 3‐ IS
achieved an LLOQ of 0.09 μg g−1 for brain, and 0.25 μg g−1
for liver and kidneys, which gave about 2.5 ng for brain and
about 3 ng for liver and kidneys of each enantiomer on‐
column.
a value of 15%, which indicates that the validated
method has a good precision and accuracy in the tested
concentration ranges.
3.7 | Recovery
3.6 | Accuracy and precision
The liquid–liquid extraction from the acidified environment
was selected for isolation of both enantiomers from the
plasma and tissue samples. Several procedures of extraction
were examined. In the case of extraction of ibuprofen enan-
tiomers from plasma samples, 1 M HCl was selected from
a group of others acidifiers: 1 M H2SO4, 0.1 M HCl, and
0.1 M CH3COONa (pH = 4). A few extraction mixtures
The accuracy and precision of the method was deter-
mined in both within‐day and between‐day assays for
plasma and within‐day for brain, liver, and kidney
homogenates. Tables 2 and 3 demonstrate the results
obtained with three concentrations of both enantiomers.
As can be seen, neither CVs nor relative errors exceeded

PRZEJCZOWSKA‐POMIERNY ET AL.
7
TABLE 2 Validation parameters of the method for the enantioselective analysis of ibuprofen in mouse plasma
(−)‐R‐ibuprofen
(+)‐S‐ibuprofen
Recovery SD% (CV%)
0.5 μg ml−1
5.0 μg ml−1
20.0 μg ml−1
Linearity (0.125–35 μg ml−1
R2
81.17 7.37 (9.08)
68.71 8.32 (12.10)
65.10 2.26 (3.46)
y = 0.1476x + 0.0057
0.99912
74.15 8.66 (11.64)
67.85 9.55 (14.07)
65.00 2.50 (3.84)
y = 0.1476x – 0.0104
0.99901
)
LLOQ (μg ml−1
)
0.125
0.125
Precision (CV%, n = 5)
18.70
18.96
Accuracy (RE%)
10.22
12.61
Intraday precision (CV%, n = 5)
LQC (0.5 μg ml−1
MQC (5.0 μg ml−1
HQC (20.0 μg ml−1
Interday precision (CV%, n = 5)
LQC (0.5 μg ml−1
MQC (5.0 μg ml−1
HQC (20.0 μg ml−1
Intraday accuracy (RE%, n = 5)
LQC (0.5 μg ml−1
MQC (5.0 μg ml−1
HQC (20.0 μg ml−1
Interday accuracy (RE%, n = 5)
LQC (0.5 μg ml−1
MQC (5.0 μg ml−1
HQC (20.0 μg ml−1
)
3.21
13.45
5.32
4.24
4.13
5.11
)
)
)
9.30
4.04
3.72
6.53
3.76
5.94
)
)
)
6.44
3.89
6.13
13.31
6.58
2.96
)
)
)
10.25
6.10
1.28
12.43
5.67
0.34
)
)
were tested and different recoveries were achieved, whereby
the highest one was observed for extraction with dichloro-
methane, which was about 85% for both enantiomers and
n‐heptane/2‐propanol (95:5, v/v) or n‐hexane/2‐propanol
(95:5, v/v) mixtures, ranging about 80% for both enantio-
mers. Despite relatively high recoveries, these extraction
mixtures were not selected because of interfering peaks
observed at the retention times of the analytes or IS in chro-
matograms or a lengthy evaporation process when using the
n‐heptane/2‐propanol mixture. The lowest recovery was
observed during extraction with methyl‐tert‐butyl‐ether,
which in addition gave dirty samples. The best selectivity
was achieved when using ethyl acetate/n‐hexane (30:70,
v/v) as an extraction mixture with a good recovery of about
70%, and finally this mixture was selected for the extraction
process. To isolate both analytes from tissue homogenates,
several methods of sample purification were tested: proteins
precipitation with cooled methanol or acetonitrile and 10%
trifluoroacetic acid solution, but the best method for sample
purification was to vortex the samples with 1 ml of NaCl
solution in water (20 g/100 ml). The best selectivity was
achieved in the case of the double extraction process, where
in the first step homogenate was extracted from acidic sam-
ples by the ethyl acetate/n‐hexane (30:70, v/v) mixture and
then the organic phase was transferred to a new tube and
0.1 ml of 2 M NaOH was added. Then, after vortex mixing
and centrifugation, the water phase was acidified and
extracted with the same extraction mixture. Despite the good
selectivity, a small recovery was achieved (about 45%).
Because of the small volume of distribution of ibuprofen,
low concentrations of this drug are expected in tissues.
Therefore, the procedure with a single extraction with the
ethyl acetate/n‐hexane (30:70, v/v) mixture after NaCl pro-
tein precipitation was selected for tissue homogenates. The
selectivity was improved by starting the eluting process with
solvent A and B at a ratio: 99:1%. Tables 2 and 3 show the
obtained recoveries from plasma, brain, liver, and kidney
homogenates, respectively. The coefficients of variation were

8
PRZEJCZOWSKA‐POMIERNY ET AL.
TABLE 3 Validation parameters of the method for the enantioselective analysis of ibuprofen in mouse brain, liver,
and kidneys
(−)‐R‐ibuprofen
(+)‐S‐ibuprofen
BRAIN
Recovery SD % (CV%)
0.36 μg g−1
0.72 μg g−1
1.44 μg g−1
Linearity (0.09–1.44 μg g−1
R2
71.88 5.63 (7.84)
72.06 2.25 (3.12)
74.28 3.37 (4.53)
y = 1.5872x + 0.0012
0.99773
74.93 5.74 (7.66)
73.39 3.04 (4.14)
74.44 2.20 (2.95)
y = 1.5835x – 0.0395
0.99755
)
LLOQ (μg g−1
)
0.09
0.09
Precision (CV%, n = 5)
15.18
6.56
Accuracy (RE%)
11.42
11.79
Inter‐day precision (CV%, n = 5)
LOQ (0.36 μg g−1
MOQ (0.72 μg g−1
HQC (1.44 μg g−1
Inter‐day accuracy (RE%, n = 5)
LOQ (0.36 μg g−1
MOQ (0.72 μg g−1
HQC (1.44 μg g−1
LIVER
)
10.65
1.63
4.85
6.11
5.71
4.75
)
)
)
9.76
2.57
0.95
4.69
0.27
2.51
)
)
Recovery SD % (CV%)
0.5 μg g−1
5.0 μg g−1
10.0 μg g−1
Linearity (0.25–20 μg g−1
R2
79.87 2.85 (3.56)
76.99 5.82 (7.56)
75.19 3.96 (5.26)
y = 0.2015x + 0.0098
0.9989
78.32 6.31 (8.05)
75.31 6.60 (8.76)
72.38 2.13 (2.95)
y = 0.1965x – 0.0134
0.99856
)
LLOQ (μg g−1
)
0.25
0.25
Precision (CV%, n = 5)
9.34
5.38
Accuracy (RE%)
11.85
4.61
Inter‐day precision (CV%, n = 5)
LOQ (0.5 μg g−1
MOQ (5.0 μg g−1
HQC (10 μg g−1
Inter‐day accuracy (RE%, n = 5)
LOQ (0.5 μg g−1
MOQ (5.0 μg g−1
HQC (10 μg g−1
)
0.90
0.63
1.74
2.36
1.87
1.99
)
)
)
4.02
7.20
0.99
11.98
2.00
1.15
)
)
(Continues)

PRZEJCZOWSKA‐POMIERNY ET AL.
9
TABLE 3 (Continued)
(−)‐R‐ibuprofen
(+)‐S‐ibuprofen
KIDNEY
Recovery SD % (CV%)
0.5 μg g−1
5.0 μg g−1
10.0 μg g−1
Linearity (0.25–20 μg g−1
R2
79.88 1.39 (1.73)
74.43 5.03 (6.75)
75.43 2.05 (2.72)
y = 0.0932x + 0.0298
0.9985
78.38 0.69 (0.88)
75.39 4.96 (6.58)
74.32 5.03 (7.25)
y = 0.0923x + 0.0164
0.99875
)
LLOQ (μg g−1
)
0.25
0.25
Precision (CV%, n = 5)
12.11
14.14
Accuracy (RE%)
3.99
2.00
Inter‐day precision (CV%, n = 5)
LOQ (0.5 μg g−1
MOQ (5.0 μg g−1
HQC (10 μg g−1
Inter‐day accuracy (RE%, n = 5)
LOQ (0.5 μg g−1
MOQ (5.0 μg g−1
HQC (10 μg g−1
)
1.43
6.12
8.96
4.31
7.83
9.23
)
)
)
6.09
3.92
3.52
12.39
5.81
4.10
)
)
lower than 15%, thus confirming the repeatability of the
extraction procedures.
3.8 | Postprocessing stability
The results of postprocessing stability studies revealed that
there was no change in the area of the peaks of both ibuprofen
enantiomers and IS after keeping samples in the autosampler
at 10 °C for 12 h, provided that samples were tightly closed to
prevent evaporation of the solvent.
FIGURE
3
The mean observed (symbols
SD) and
3.9 | Pharmacokinetics of ibuprofen
pharmacokinetic model predicted (lines) plasma concentration–time
profiles of ibuprofen enantiomers after intravenous administration of
racemic drug at a dose of 10 mg kg−1 to mice
enantiomers in mice
The developed method was applied for the determination of
ibuprofen enantiomers in the pharmacokinetic study per-
formed in CD‐1 mice, which were given 10 mg kg−1 of
racemic ibuprofen i.v. Based on the standard goodness‐of‐
may be further confirmed by the relatively low CV
values presented in Table 4.
fit criteria,
a
two‐compartment model presented in
The volumes of distribution of both enantiomers were
comparable (188.01 and 190.94 ml kg−1 for (−)‐R‐ and
(+)‐S‐ibuprofen, respectively). Similarly, no differences
between volumes of distributions of ibuprofen enantiomers
were observed in rats16 and dogs36 after i.v. administration
of one enantiomer or the racemic drug. In turn, Cheng et al.
noticed some differences in healthy humans after i.v. admin-
istration of each enantiomer (Vss was 7.4 and 8.8 L for (−)‐
R‐ and (+)‐S‐ibuprofen, respectively).5 The inversion of
Figure 1 best describes the pharmacokinetics of the studied
enantiomers.
Figure 3 presents the mean observed and pharmacoki-
netic model‐predicted plasma concentration–time profiles
of ibuprofen enantiomers and Table 4 shows the esti-
mated pharmacokinetic parameters and their respective
CVs. From this figure, the model very well captured
the observed concentrations of both enantiomers that

10
PRZEJCZOWSKA‐POMIERNY ET AL.
TABLE 4 Pharmacokinetic parameters of ibuprofen enantiomers
estimated following i.v. administration of a single dose of 10 mg kg−1 of
racemic ibuprofen to mice
Parameters
Final estimate
CV [%]
VCR [ml kg−1
]
188.01
190.94
4.86
5.92
VCS [ml kg−1
k10R [1 min−1
k10S [1 min−1
kRS [1 min−1
]
]
0.038 (0.094–0.056)
0.018
‐‐‐
]
20.37
58.82
39.64
52.43
47.43
34.14
]
0.056
K12 [1 min−1
K21 [1 min−1
K34 [1 min−1
K43 [1 min−1
]
0.027
]
]
]
0.037
0.018
0.016
VCR volume of thecentral compartment for (−)‐R‐ibuprofen, VCS volume of the
central compartment for (+)‐S‐ibuprofen; k10R and k10S first order elimination rate
constants; kRS first‐order inversion rate constant; k12, k21, k34, and k43 distribution
rate constants.
(−)‐R‐ to (+)‐S‐enantiomer (kRS = 0.056 min−1) was 3½
times
(k10S
faster
=
than
0.018
the
elimination
) and (−)‐R‐enantiomer
of
(+)‐S‐
min−1
(k10R = 0.038 min−1) from the central compartment. The rate
of this biochemical process in rats was almost the same
(kRS = 0.06 min−1) but the elimination rate constants were
higher (k10R = 0.05 min−1 and k10S = 0.1 min−1) than those
observed in the present study in mice.16 A relatively large
difference was observed in the elimination half‐lives of
both enantiomers in mice that were 7.37 (t0.5R = 0.693/
(k10R + kRS)) and 38.5 min (t0.5S = 0.693/k10S) for (−)‐
R‐ and (+)‐S‐ibuprofen, respectively. Slightly higher
differences were observed by Wang et al. in rats, who
found that t0.5 values of both enantiomers differed about
6 times after i.v. administration, but only 3 times after
oral drug intake.11 Smaller differences were observed
in dogs, where after i.v. administration of individual enan-
FIGURE 4 Mean brain, liver, and kidney concentration–time
profiles of ibuprofen enantiomers following a single i.v. administration
of 10 mg kg−1 racemic drug to mice. Symbols represent the means SD
of 3–4 mice
tiomer or racemic ibuprofen the ratio of t0.5 (+)‐S‐ibuprofen
/
(Figure 4, Table 5). Moreover, AUC0‐t of (+)‐S‐ibuprofen
in plasma and tissues was considerably higher than that
of its optical antipode. (+)‐S‐ to (−)‐R‐ibuprofen AUC
ratios were 8.98, 9.10, 27.56, and 19.14 in plasma, brain,
liver, and kidneys, respectively. The values of t0.5λz
assessed in plasma, brain, liver, and kidneys were 4‐,
4‐, 6‐, 2.5‐times higher, respectively, for (+)‐S‐enantiomer
in comparison to (−)‐R‐enantiomer. Tissue‐to‐plasma
AUC ratios of (−)‐R‐ibuprofen were 0.03, 0.18, and
0.32 for brain, liver, and kidneys respectively, whereas
for (+)‐S‐enantiomer these values were 0.03, 0.54, and
0.69. The main reasons for all these differences are the
unidirectional inversion of (−)‐R‐ to (+)‐S‐enantiomer in
the body and a slower elimination of (+)‐S‐antipode from
plasma and all studied tissues. Taken together, these
t0.5 (−)‐R‐ibuprofen was only 2.36 In human, there were small
differences in t0,5 values of each enantiomer after oral
intake2,13,37 or no differences after i.v.5 or oral administra-
tion of ibuprofen.38 The distribution rates constants were
also different for both enantiomers. As a result, Vdss
calculated as: (1
+
k12/k21)Vc were 325.21 and
405.75 ml kg−1 for (−)‐R‐ and (+)‐S‐ibuprofen,
respectively.
Figure 4 shows the concentration versus time profiles
of both enantiomers in studied tissues, and pharmacoki-
netic parameters estimated based on the noncompartmental
analysis are listed in Table 5. The collected data indicate
that the highest concentrations of both enantiomers were
observed in plasma and the lowest in brain tissue

PRZEJCZOWSKA‐POMIERNY ET AL.
11
TABLE 5 Plasma, brain, liver, and kidney pharmacokinetic parame-
ters of ibuprofen enantiomers in mice after a single i.v. dose of 10 mg
kg−1 of racemic drug using noncompartmental analysis
applied to the pharmacokinetic study of ibuprofen enantio-
mers after i.v. administration of the racemic drug to
CD‐1 mice, where a small volume of plasma (100 μl) and
tissue homogenates (0.2–0.5 ml) was used for the analysis.
Pharmacokinetic parameters (−)‐R‐ibuprofen (+)‐S‐ibuprofen
PLASMA
REFERENCES
tmax [min]
C0 [μg ml−1
AUC0‐t [μg min ml−1
CL [ml min−1 kg−1
5.0
14.32
239.88
20.16
22.65
24.39
5.0
25.23
1. Canaparo R, Muntoni E, Zara GP, et al. Determination of ibuprofen
in human plasma by high‐performance liquid chromatography:
Validation and application in pharmacokinetic study. Biomed
Chromatogr. 2000;14:219‐226.
]
]
2155.91
2.20
]
t0.5λz [min]
89.39
2. Lee E, Williams K, Day R, Graham G, Champion D. Stereoselective
disposition of ibuprofen enantiomers in man. Br J Clin Pharmacol.
1985;19:669‐674.
MRT [min]
113.83
BRAIN
3. Evans AM. Enantioselective pharmacodynamics and pharmacoki-
netics of chiral non‐steroidal antiinflammatory drugs. Eur J Clin
Pharmacol. 1992;54:237‐256.
tmax [min]
Cmax [μg g−1
AUC0‐t [μg min g−1
5
15
]
0.51
7.03
10.09
15.94
1.15
64.04
42.95
60.48
4. Evans AM. Comparative pharmacology of S(+)‐ibuprofen and
(RS)‐ibuprofen. Clin Rheumatol. 2001;20(Suppl 1):S9‐S14.
]
]
]
t0.5λz [min]
5. Cheng H, Rogers JD, Demetriades JL, Holland SD, Seibold JR,
Depuy E. Pharmacokinetics and Bioinversion of ibuprofen enantio-
mers in humans. Pharm Res. 1994;11:824‐830.
MRT [min]
LIVER
6. Davies NM. Clinical pharmacokinetics of ibuprofen. The first 30
years. Clin Pharmacokinet. 1998;34:101‐154.
tmax [min]
Cmax [μg g−1
AUC0‐t [μg min g−1
5
5
]
2.11
42.38
14.08
21.70
12.34
7. De La Cruz JP, Reyes JJ, Ruiz‐Moreno MI, Lopez‐Villodres JA,
Jebrouni N, Gonzalez‐Correa JA. Differences in the in vitro anti-
platelet effect of dexibuprofen, ibuprofen, and flurbiprofen in
human blood. Anesth Analg. 2010;111:1341‐1346.
1169.19
90.35
t0.5λz [min]
MRT [min]
113.82
8. Rainsford KD. Ibuprofen: Pharmacology, efficacy and safety.
Inflammopharmacology. 2009;17:275‐342.
KIDNEYS
tmax [min]
Cmax [μg g−1
AUC0‐t [μg min g−1
5
5
9. McKee AC, Carreras I, Hossain L, et al. Ibuprofen reduces Aβ,
hyperphosphorylated tau and memory deficits in Alzheimer mice.
Brain Res. 2008;1207:225‐236.
]
3.95
77.29
24.06
27.96
12.01
1479.43
64.31
10. Xie G, Sun Y, Nie T, et al. Phospho‐ibuprofen (MDC‐917) is a novel
agent against colon cancer: Efficacy, metabolism, and pharmacoki-
netics in mouse models. J Pharmacol Exp Ther. 2011;337:876‐886.
t0.5λz [min]
MRT [min]
115.68
11. Wang Y. Pharmacokinetics of ibuprofen enantiomers in rats after
intravenous and oral administration of ibuprofen arginate. Acta
Pharm Sin. 2012;47:88‐93.
results indicate that after i.v. administration of racemic
ibuprofen to mice a stereoselective disposition of this
drug occurs, which is in agreement with the data
from pharmacokinetic studies in other species and
humans.5,11,16,19,36,39
12. Hao H, Wang G, Sun J. Enantioselective pharmacokinetics of
ibuprofen and involved mechanisms. Drug Metab Rev. 2005;
37:215‐234.
13. Ding G, Liu Y, Sun J, et al. Effect of absorption rate on pharmaco-
kinetics of ibuprofen in relation to chiral inversion in humans.
J Pharm Pharmacol. 2007;59:1509‐1513.
14. Chen C‐S, Shieh W‐R, Lu P‐H, Harriman S, Chen C‐Y. Metabolic
stereoisomeric inversion of ibuprofen in mammals. Biochim Biophys
Acta ‐ Protein Struct Mol Enzymol. 1991;1078:411‐417.
4 | CONCLUSION
In the present study, a direct enantioselective HPLC method
coupled with fluorescence detection for quantitative determi-
nation of ibuprofen enantiomers in mouse plasma and tissues
was developed and validated. The proposed method is simple,
sensitive, and selective, meaning that ibuprofen enantiomers
could be measured in biological samples with acceptable
accuracy and precision. Finally, this method was successfully
15. Bannwarth B, Lapicque F, Pehourcq F, et al. Stereoselective dispo-
sition of ibuprofen enantiomers in human cerebrospinal fluid. Br J
Clin Pharmacol. 1995;40:266‐269.
16. Itoh T, Maruyama J, Tsuda Y, Yamada H. Stereoselective phar-
macokinetics of ibuprofen in rats: Effect of enantiomer‐
enantiomer interaction in plasma protein binding. Chirality.
1997;9:354‐361.

12
PRZEJCZOWSKA‐POMIERNY ET AL.
17. Fornasini G, Monti N, Brogin G, et al. Preliminary pharmacokinetic
study of ibuprofen enantiomers after administration of a new oral
formulation (ibuprofen arginine) to healthy male volunteers.
Chirality. 1997;9:297‐302.
29. Cavalcanti JL, Lanchote VL, Pereira MPM, Moraes NV, Lepera JS.
Analysis of ibuprofen enantiomers in rat plasma by liquid chroma-
tography with tandem mass spectrometry. J Sep Sci. 2014;
37:944‐949.
30. Ahn HY, Shiu GK, Trafton WF, Doyle TD. Resolution of the enan-
tiomers of ibuprofen; comparison study of diastereomeric method
18. Główka FK. 1998HPLC methodology for determination of ibupro-
fen enantiomers to be used in pharmacokinetics. Chem Anal.
1998;43:79‐84.
and chiral stationary phase method.
J
Chromatogr B.
1994;653:163‐169.
19. Vermeulen B, Remon JP. Validation of a high‐performance liquid
chromatographic method for the determination of ibuprofen
enantiomers in plasma of broiler chickens. J Chromatogr B.
2000;749:243‐251.
31. Główka FK, Karaźniewicz M. High performance capillary electro-
phoresis method for determination of ibuprofen enantiomers in
human serum and urine. Anal Chim Acta. 2005;540:95‐102.
32. Van Overbeke A, Baeyens W, Van Den Bossche W, Dewaele C.
Enantiomeric separation of amide derivatives of some 2‐
arylpropionic acids by HPLC on a cellulose‐based chiral stationary
phase. J Pharm Biomed Anal. 1994;12:911‐916.
20. Teng XW, Wang SW, Davies NM. Stereospecific high‐performance
liquid chromatographic analysis of ibuprofen in rat serum.
J Chromatogr B. 2003;796:225‐231.
21. Zhao X, Chen D, Li K, Wang D. Sensitive liquid chromatographic
assay for the simultaneous determination of ibuprofen and its
prodrug, ibuprofen eugenol ester, in rat plasma. Yakugaku Zasshi.
2005;125:733‐737.
33. Ducret A, Trani M, Pepin P, Lortie R. Chiral high performance liq-
uid chromatography resolution of ibuprofen esters. J Pharm Biomed
Anal. 1998;16:1225‐1231.
34. Kang GW, Ho Ko J, Jo CW. Thermodynamic study of
enantioseparation of arylpropionic acids with a chiralcel OJ‐H sta-
tionary phase. J Liq Chromatogr Relat Technol. 2005;28:513‐526.
22. De Oliveira ARM, Cesarino EJ, Bonato PS. Solid‐phase
microextraction and chiral HPLC analysis of ibuprofen in urine.
J Chromatogr B. 2005;818:285‐291.
35. Tang Y. Significance of mobile phase composition in
enantioseparation of chiral drugs by HPLC on a cellulose‐based
chiral stationary phase. Chirality. 1996;8:136‐142.
23. Valderrama P, Romero AL, Imamura PM, Poppi RJ. Figures of merit
in the quantification of ibuprofen enantiomers by chiral HPLC.
J Chromatogr Sci. 2009;47:777‐780.
36. Hae‐Young A, Amidon GL, Smith DE. Stereoselective systemic
disposition of ibuprofen enantiomers in the dog. Pharm Res.
1991;8:1186‐1190.
24. Ye J, Yu W, Chen G, Shen Z, Zeng S. Enantiomeric separation of
2‐arylpropionic acid nonsteroidal antiinflammatory drugs by HPLC
with hydroxypropyl‐β‐cyclodextrin as chiral mobile phase additive.
Biomed Chromatogr. 2010;24:799‐807.
37. Zheng C, Hao H, Wang G, et al. Chiral separation of ibuprofen and
chiral pharmacokinetics in healthy chinese volunteers. Eur J Drug
Metab Pharmacokinet. 2008;33:45‐51.
25. Tan SC, Jackson SHD, Swift CG, Hutt AJ. Enantiospecific analysis
of ibuprofen by high performance liquid chromatography: Determi-
nation of free ant total drug enantiomer concentrations in serum and
urine. Chromatographia. 1997;46:23‐32.
38. Geisslinger G, Schuster O, Stock KP, Loew D, Bach GL, Brune K.
Pharmacokinetics of S(+)‐ and R(−)‐ibuprofen in volunteers and
first clinical experience of S(+)‐ibuprofen in rheumatoid arthritis.
Eur J Clin Pharmacol. 1990;38:493‐497.
26. Bonato PS, Del Lama MPFM, De Carvalho R. Enantioselective
determination of ibuprofen in plasma by high‐performance liquid
chromatography‐electrospray mass spectrometry. J Chromatogr B.
2003;796:413‐420.
39. Knihinicki RD, Day RO, Graham GG, Williams KM.
Stereoselective disposition of ibuprofen and flurbiprofen in rats.
Chirality. 1990;2:134‐140.
27. Szeitz A, Edginton AN, Peng HT, Cheung B, Riggs KW. A vali-
dated enantioselective assay for the determination of ibuprofen in
human plasma using ultra performance liquid chromatography with
tandem mass spectrometry (UPLC‐MS / MS). Am J Anal Chem.
2010;2:47‐58.
How to cite this article: Przejczowska‐Pomierny K,
Włodyka M, Cios A, Wyska E. Enantioselective
analysis of ibuprofen enantiomers in mice plasma and
tissues by high‐performance liquid chromatography
with fluorescence detection: Application to a
pharmacokinetic study. Chirality. 2017;1‐12. https://
doi.org/10.1002/chir.22715
28. Ogawa S, Tadokoro H, Sato M, Higashi T. Enantioselective determi-
nation of ibuprofen in saliva by liquid chromatography/tandem mass
spectrometry with chiral electrospray ionization‐enhancing and sta-
ble isotope‐coded derivatization.
2014;98:387‐392.
J
Pharm Biomed Anal.