American Journal of Analytical Chemistry, 2010, 2, 47-58
doi:10.4236/ajac.2010.12007 Published Online August 2010 (http://www.SciRP.org/journal/ajac)
A Validated Enantioselective Assay for the Determination
of Ibuprofen in Human Plasma Using Ultra Performance
Liquid Chromatography with Tandem Mass
Spectrometry (UPLC-MS/MS)
András Szeitz1*, Andrea Nicole Edginton2, Henry Tao Peng3, Bob Cheung3, Kenneth Wayne Riggs1
1Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, Canada
2School of Pharmacy, University of Waterloo, Waterloo, Canada
3Defence Research and Development Canada–Toronto, Toronto, Canada
Received April 8, 2010; revised July 13, 2010; accepted July 20, 2010
Abstract
A modified ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method
was developed and validated for the quantitation of ibuprofen enantiomers in human plasma. Ibuprofen and
flurbiprofen (internal standard) were extracted from human plasma at acidic pH, using a single-step liq-
uid-liquid extraction with methyl-tert-butyl ether. The enantiomers of ibuprofen and flurbiprofen were deri-
vatized to yield the corresponding diastereomers. Chromatographic separation was achieved using a phenyl
column with a run time of 20 min. (R)- and (S)-ibuprofen were quantitated at the multiple reaction monitor-
ing (MRM) transition of m/z 360.2  232.1, and (R)- and (S)-flurbiprofen were monitored at the MRM tran-
sition of m/z 398.3  270.1. The method was validated for accuracy, precision, linearity, range, limit of
quantitation (LOQ), limit of detection (LOD), selectivity, absolute recovery, matrix effect, dilution integrity,
and evaluation of carry-over. Accuracy for (R)-ibuprofen ranged between –11.8% and 11.2%, and for
(S)-ibuprofen between –8.6% and –0.3%. Precision for (R)-ibuprofen was ≤ 11.2%, and for (S)-ibuprofen ≤
7.0%. The calibration curves were weighted (1/X2, n = 7) and were linear with r2 for (R)-ibuprofen ≥ 0.988
and for (S)-ibuprofen ≥ 0.990. The range of the method was 50 to 5000 ng/mL with the LOQ of 50 ng/mL,
and LOD of 1 ng/mL, for (R)- and (S)-ibuprofen requiring 100 µL of sample. The method was applied suc-
cessfully to a pharmacokinetic study with the administration of a single oral dose of ibuprofen capsules to
human subjects.
Keywords: Ibuprofen Enantiomers, UPLC-MS/MS, Human Plasma, Method Validation, Pharmacokinetics
1. Introduction
Ibuprofen, a member of the 2-substituted arylpropionic
acid (2-APA) family, is a non-steroidal anti-inflammatory
drug (NSAID), which is used to treat moderate pain, fe-
ver, rheumatic disorders and related inflammatory dis-
eases. Ibuprofen, one of the most popular “profen” drugs,
was first marketed in the UK in 1969, and has been
commercialized as a racemate drug. In Switzerland and
Austria, in addition to the racemate drug, the (S)-enan-
tiomer is also sold.
Ibuprofen undergoes chiral inversion in vitro [1] and
in vivo, during metabolism in rats, mouse, rabbits [2-4]
and humans [5,6] and shows stereoselective pharmacol-
ogical effects [7,8] metabolism, pharmacokinetics [9-11]
and disposition [12,13]. It has been reported also that
ibuprofen is extensively bound to proteins in plasma in
humans [11,14,15].
Several methodologies are available for the determina-
tion of ibuprofen. These techniques include direct or in-
direct high performance liquid chromatography (HPLC)
methods. In the direct HPLC methods, a chiral stationary
phase is used and the enantiomers are analyzed without
sample derivatization. Certain direct HPLC methods em-
ploy ultra-violet (UV) detection using α1-acid glycopro-
tein [16-19], β-cyclodextrin [20], cellulose [21], poly-
saccharide [22], and amylose derivative [23] chiral sta-

48 A. SZEITZ ET AL.
tionary phases (CSPs). A cellulose derivative CSP using
radiometric and tandem mass spectrometric (MS/MS)
detection [24] and an amylose derivative CSP using
MS/MS [25] were also employed for the determination
of ibuprofen enantiomers.
The direct HPLC methods have the advantage that the
ibuprofen enantiomers can be analyzed without derivati-
zation, however, these assays have poor sensitivity, re-
producibility, require a large sample volume [16,18-20]
or lack the satisfactory chromatographic baseline separa-
tion of the stereoisomer peaks [20,25]. Some direct
HPLC methods achieved good sensitivity and use a small
sample volume, but due to the non-selective characteris-
tics of the UV detection they require extended analysis
time to avoid interferences between the ibuprofen enan-
tiomers and the co-eluting endogenous components from
the biological matrix [23]. Further direct HPLC assays
employ radiometric and selective mass spectrometric
detection, but they are reported for in vitro applications,
and not for the quantitation of the ibuprofen enantiomers
over a concentration range in body fluids [24].
In the indirect HPLC methods, a reverse-phase sta-
tionary phase is used and the enantiomers are analyzed
after sample derivatization to yield their corresponding
diastereomers. The indirect methods have improved sen-
sitivity and are better suited for the analysis of ibuprofen
enantiomers in a variety of complex biological matrices.
The indirect techniques include HPLC using UV [26,27],
fluorescence [28,29], and mass spectrometric detection
[30-33]. Gas chromatography-mass spectrometry (GC/
MS) methods have been reported as well [2,6,34-36].
While the indirect HPLC methods using UV and fluo-
rescence detection are suitable for the enantioselective
determination of (R)- and (S)-ibuprofen in biological
samples, they still lack the satisfactory sensitivity (e.g.,
limit of quantitation, LOQ ≥ 0.1 µg/mL) and use a large
sample volume (i.e., 0.5 mL plasma or serum) [26,27,
29], therefore, they may not be used satisfactorily for
detailed pharmacokinetic studies over prolonged time
periods. GC/MS methods can be stereoselective, but they
use a larger sample size for analysis (i.e., 0.8 mL plasma
or 1 mL of serum) [34,6], and their sensitivity is insuffi-
cient (i.e., 0.25 µg/mL or LOQ 5 µg/mL) [34,35], or they
are not stereoselective [35,36]. Several indirect HPLC/
MS/MS methods were reported for various in vitro
[30-32] and in vivo [33] analyses of ibuprofen, but these
methods are not stereoselective and do not represent sig-
nificant improvement in comparison to the methodolo-
gies reported above.
In the present study, an enantioselective ultra per-
formance liquid chromatography-tandem mass spec-
trometry (UPLC-MS/MS) method with improved sensi-
tivity is presented for the quantitation of (R)- and
(S)-ibuprofen enantiomers in human plasma using (R)-
and (S)-flurbiprofen as internal standards. The assay was
validated and applied successfully to the pharmacoki-
netic study of orally administered ibuprofen capsules to
humans.
2. Experimental
2.1. Chemicals and Standards
(R)-(-)-ibuprofen (purity 98%), and (S)-(+)-ibuprofen (pu-
rity 98%), were purchased from Toronto Research Chem-
icals Inc. (North York, ON, Canada). (S)-(+)-Flurbiprofen
(purity 98%), (R)-(-)-Flurbiprofen (purity 97%), internal
standards, N-(3-Dimethylaminopropyl)-N’-ethyl-carbod-
iimide hydrochloride (CDI) (purity >98%), (R)-(+)-1-(1-
Naphtyl)-ethylamine ((R)-NEA) (purity > 99%) were
purchased from Sigma-Aldrich (St. Louis, MO, USA),
and 1-hydroxybenzotriazole (HOBt·H2O) was purchased
from AnaSpec, Inc. (San Jose, CA, USA). Methyl-tert-
butyl ether, acetonitrile, methanol (HPLC grade) were
purchased from Fisher Scientific (Fair Lawn, NY, USA),
and dichloromethane (HPLC Grade) was obtained from
Acros Organics (Geel, Belgium). Ammonium Acetate
(AnalaR grade) was obtained from BDH Inc., (Toronto,
ON, Canada), formic acid (puriss. p.a. for mass spec-
troscopy) was purchased from Fluka (Steinheim, Ger-
many), hydrochloric acid (1.0 N) was purchased from
VWR (West Chester, PA, USA). Blank human plasma
(Sodium EDTA-treated) was purchased from Biorecla-
mation, Inc. (Westbury NY, USA). Ultra pure water was
prepared in our laboratory using a Milli-Q Synthesis sys-
tem (Millipore, Billerica, MA, USA). Ibuprofen capsules
(AdvilTM ibuprofen 400 mg Liquid Filled Capsules, Extra
Strength Liqui-Gels, Wyeth Consumer Healthcare Inc.,
Mississauga, ON, Canada) were obtained from Shoppers
Drug Mart, Toronto, ON, Canada.
2.2. Instrumentation and Experimental
Conditions
The UPLC-MS/MS system consisted of a Waters Ac-
quity UPLC Binary Solvent Manager and a Waters Ac-
quity UPLC Sample Manager connected to a Waters
Quattro Premier XE triple quadrupole mass spectrometer.
The mass spectrometer was operated in electrospray
positive ionization (ES+) mode, and data were acquired
using a MassLynx v. 4.1 software on a Microsoft Win-
dows XP Professional operating platform.
Chromatographic separation was achieved using a
Waters Acquity UPLC BEH Phenyl 1.7 µm, 2.1 × 150
mm column maintained at 30˚C, and the autosampler
tray temperature was maintained at 10˚C. Solvent a was
water containing 10 mM ammonium acetate and 0.1%
formic acid, and solvent b was a mixture of 64% ace-
tonitrile/36% methanol containing 10 mM ammonium
acetate and 0.1% formic acid. The mobile phase initial
conditions were solvent a (35%) and solvent b (65%)

A. SZEITZ ET AL.
49
with a flow rate of 0.2 mL/min, which were maintained
for 12 min (0-12 min). At 12.1 min, solvent b was in-
creased to 100% and the flow rate was gradually in-
creased to 0.5 mL/min by 14 min (12.1-14 min) and held
for 1 min (14-15 min). At 15.1 min, the initial conditions
were set and the column was equilibrated for 4.9 min
(15.1-20 min). The total run time was 20 min, the injec-
tion volume was 15 µL.
Mass spectrometric conditions were as follows: capil-
lary voltage 3 kV, cone voltage 30 V, source temperature
120˚C, desolvation gas temperature 300˚C, desolvation
gas flow 1000 L/hour. Diastereomeric amide derivatives
of (R)- and (S)-ibuprofen were quantitated in multiple
reaction monitoring (MRM) using the transition of m/z
360.2  232.1 at collision energy (CE) 10 eV. The dwell
time was set to 25 ms. Diastereomeric amide derivatives
of (R)- and (S)-flurbiprofen were monitored in MRM
using the transition of m/z 398.3  270.1 at CE 10 eV.
The dwell time was set to 50 ms. To protect the mass
spectrometer from contamination from the samples and
to reduce the solvent load in the source, the mobile phase
flow was diverted to the waste before 7 min and after 12
min during the chromatographic run.
2.3. Preparation of Stock Solutions and
Calibration Standards
Two separate master stock solutions of (R)-ibuprofen
(100 µg/mL), and (S)-ibuprofen (100 µg/mL) enanti-
omers were prepared in methanol. The (R)- and (S)-ibu-
profen master stock solutions were combined in equal
parts into a mixed working stock solution of (R) and
(S)-ibuprofen (50 µg/mL, each). The mixed working
stock solution was further diluted with water to yield a
series of diluted working stock solutions. The series of
diluted working stock solutions were used to prepare the
calibration standards in human plasma. The calibration
standards were prepared by spiking 10 µL aliquots of
appropriately diluted working stock solutions into 90 µL
aliquots of blank human plasma yielding the final vol-
ume of 100 µL calibration standards in human plasma.
Calibration standards were prepared freshly on the day of
a batch analysis in the concentration range of 50 ng/mL
to 5000 ng/mL for (R)- and (S)-ibuprofen. The solutions
were stored at 4˚C until analysis.
The internal standard solutions were prepared as fol-
lows. Two separate master stock solutions of (R)-flurbi-
profen (100 µg/mL), and (S)-flurbiprofen (100 µg/mL)
enantiomers were prepared in methanol. The (R)- and
(S)-flurbiprofen master stock solutions were combined in
equal parts and diluted with water into a mixed working
stock solution of (R)- and (S)-flurbiprofen (10 µg/mL,
each). The mixed working stock solution was further
diluted with water to yield the diluted working stock so-
lution of (R)- and (S)-flurbiprofen (100 ng/mL, each).
The solutions were stored at 4˚C until analysis.
2.4. Preparation of Quality Control Samples
Quality control (QC) samples were prepared as QC-Low
(80 ng/mL), QC-Mid (250 ng/mL), and QC-High (2500
ng/mL) samples in human plasma. A volume of 5 mL of
QC samples at each concentration level was prepared.
The QC-High samples were prepared by spiking appro-
priate volumes of each of the master stock solutions of
(R)-ibuprofen (100 µg/mL), and (S)-ibuprofen (100 µg/mL)
enantiomers in methanol into blank human plasma, and
the QC-Mid, and QC-Low samples were prepared by
spiking the appropriately diluted mixed working stock
solutions of (R)- and (S)-ibuprofen into blank human
plasma. The QC samples were dispensed in equal ali-
quots (approx. 130 µL) into vials and stored at –80˚C
until use. For each batch analysis, fresh aliquots of
QC-Low, QC-Mid, and QC-High samples were thawed,
analyzed, and then discarded.
2.5. Preparation of Reagent Solutions
Stock solutions of CDI and (R)-NEA (1 mg/mL) were
prepared in dichloromethane. Stock solution of HOBt
Hydrate (1 mg/mL) was prepared in dichloromethane
containing 10% acetonitrile. The stock solutions were
stored at 4˚C until use.
2.6. Sample Preparation
Human plasma samples were stored at –80˚C until analy-
sis. On the day of sample analysis, the samples were
thawed at room temperature and processed with the
method. All human plasma samples, with the exception
of the 0-min time point samples, were diluted into the
range of the calibration curve by mixing 10 µL aliquots
of human plasma samples with 90 µL aliquots of blank
human plasma yielding a final volume of 100 µL of
sample (i.e., 10-fold dilution). In disposable borosilicate
glass tubes, to 100 µL of samples, aliquots of 100 µL of
the diluted working stock solution of the internal stan-
dards ((R)- and (S)-flurbiprofen, 100 ng/mL, each) were
added followed by the addition of 200 µL of 1 N HCl
solution. The samples were vortex-mixed for at least 15
seconds, and 3.0 mL of methyl-tert-butyl ether was added
to the tubes. The samples were vortex-mixed for at least
45 seconds, and the tubes were placed at –80˚C for at
least 10 min. The tubes were removed from the –80˚C
freezer and the top layers were transferred to a new set of
tubes. The organic layer was brought to dryness in a
sample evaporator, under nitrogen, at ca. 35˚C, and the
dried residues were derivatized according to a previously
reported procedure [29] with modification. In short, to
the dried residues, aliquots of 100 µL of CDI solution (1
mg/mL), 100 µL of HOBt Hydrate solution (1 mg/mL),
and 100 µL of (R)-NEA solution (1 mg/mL) were added,
and the mixtures were kept in a dark environment, at

50 A. SZEITZ ET AL.
room temperature, for 2 hours. The samples were then
brought to dryness in a sample evaporator, under nitro-
gen, at ca. 35˚C, and the dried residues were reconsti-
tuted with 200 µL of acetonitrile/water : 50/50 mixture
containing 0.1% formic acid. The samples were analyzed
with UPLC-MS/MS.
2.7. Method Validation
The method was validated for accuracy, precision, line-
arity, range, limit of quantitation (LOQ), limit of detec-
tion (LOD), selectivity, absolute recovery, matrix effect,
dilution integrity, and evaluation of carry-over in human
plasma using 100 µL of sample. Calibration curves in-
cluded six to seven calibration levels and were prepared
on each day of batch analysis with a matrix blank.
QC-Low, QC-Mid, and QC-High samples were freshly
thawed on each day of batch analysis and 100 µL ali-
quots were used during the validation process.
2.7.1. Accuracy
Six replicate spiked samples of QC-Low, QC-Mid, and
QC-High were prepared in human plasma, and analyzed.
Accuracy was expressed as the percentage deviation of
the measured (R)- and (S)-ibuprofen concentration
against the added concentration, according to the fol-
lowing formula: %Deviation = [(measured amount/added
amount) × 100]-100 with negative %Deviation represent-
ing under-estimation, and positive %Deviation repre-
senting over-estimation of the true value. The acceptance
criteria for accuracy were %Deviation ± 20% for the
QC-Low samples, and ± 15% for QC-Mid, and QC-
High samples.
For intra-day accuracy, six replicates of QC-Low, QC-
Mid, and QC-High samples were prepared and analyzed
on the same day. Intra-day accuracy experiments were
repeated for three separate days. For inter-day accuracy,
six replicates of QC-Low, QC-Mid, and QC-High were
prepared and analyzed on three separate days.
2.7.2. Precision
A single spiked sample for QC-Low, QC-Mid, and
QC-High (5 mL each) was prepared in human plasma.
Six aliquots were removed from each of the QC-Low,
QC-Mid, and QC-High samples and analyzed. The rela-
tive standard deviation (%RSD) of the (R)- and (S)-ibu-
profen concentrations measured in each QC sample were
calculated. The acceptance criteria for precision were
%RSD ≤ 20% for the QC-Low samples, and %RSD ≤
15% for the QC-Mid, and QC-High samples.
For intra-day precision, six aliquots were removed
from each of the QC-Low, QC-Mid, and QC-High
spiked samples and analyzed on the same day. Intra-day
precision experiments were repeated for three separate
days. For inter-day precision, six aliquots were removed
from each of the QC-Low, QC-Mid, and QC-High
spiked samples and analyzed on three separate days.
2.7.3. Linearity and Range
Calibration curves were prepared for each batch analysis
in the following concentrations: 50, 100, 200, 300, 400,
1000, 5000 ng/mL in human plasma. Calibration curves
were constructed by plotting the concentrations of (R)-
and (S)-ibuprofen on the X-axis, vs. the chromatographic
peak area ratio of (R)- and (S)-ibuprofen to (R)- and
(S)-flurbiprofen internals standards on the Y-axis. Linear
regression analyses were performed using the calibration
curve data. At least six out of seven of the calibration
standards were used to construct the calibration curves.
Using the y = mx + b equation, the y-intercept (b), slope
(m) and correlation coefficient (r) were calculated. The
calibration curves were weighted using the weighting
factor of 1/X2. The acceptance criterion for linearity was
the coefficient of determination r2 ≥ 0.980 for (R)- and
(S)-ibuprofen after weighting with 1/X2. (R)- and
(S)-ibuprofen concentrations were calculated by the
MassLynx software using the following formula: x =
(y-b)/m, where y = (R)- and (S)-ibuprofen to IS peak area
ratio, b = weighted y-intercept, m = weighted slope. The
range of the assay was established as the section of the
calibration curve where the curve was linear, i.e., r2 ≥
0.980, the calibration levels were accurate (%Deviation ±
15%) and precise (%RSD ≤ 15%).
2.7.4. Limit of Quantitation and Limit of Detection
To determine LOQ, six replicates of the 50 ng/mL cali-
bration standard were prepared in human plasma and
analyzed. The mean response (i.e., signal-to-noise ratio,
S/N), accuracy and precision were determined from the
samples. The LOQ was determined as the lowest con-
centration of the calibration curve which met the follow-
ing acceptance criteria. The mean response of (R)- and
(S)- ibuprofen peaks in the samples was at least 5-times
the response compared to the blank sample (the response,
S/N, was calculated using MassLynx software). The (R)-
and (S)-ibuprofen peaks were identifiable, discrete, and
reproducible, with an accuracy (%Deviation) of ±20%
and precision (%RSD) ≤ 20%. To determine LOD, trip-
licate samples of 1 ng/mL (R)- and (S)-ibuprofen were
prepared in human plasma and analyzed. The mean re-
sponse was determined from the samples. The LOD was
determined as the lowest concentration of (R)- and (S)-
ibuprofen in the samples where the mean response of the
(R)- and (S)-ibuprofen peaks was at least 3-times the
response compared to the blank sample.
2.7.5. Selectivity
Selectivity was determined in pooled human plasma
samples made from three separate lots spiked at the LOQ
level. Triplicate samples were prepared. Blank human
plasma samples without (R)- and (S)-ibuprofen and (R)-

A. SZEITZ ET AL.
51
and (S)-flurbiprofen were also prepared using the pooled
human plasma samples made from the three lots. The
blank samples were visually compared to the LOQ sam-
ples for any significant interference at the retention times
of (R)- and (S)-ibuprofen and (R)- and (S)-flurbiprofen.
The acceptance criteria for selectivity were that the mean
response (i.e., S/N) of (R)- and (S)-ibuprofen in the LOQ
samples were at least 5-times the response compared to
the blank samples, and there was no significant matrix
interference at the retention times of (R)- and (S)-ibu-
profen and (R)- and (S)-flurbiprofen when the blank sam-
ples were compared with the LOQ samples.
2.7.6. Absolute Recovery
Samples of QC-Low, QC-Mid, and QC-High were pre-
pared in human plasma and analyzed. The (R)- and (S)-
ibuprofen peak area counts of the extracted samples were
compared to the (R)- and (S)-ibuprofen peak area counts
of directly injected standards of the same concentration.
Six determinations per concentration were performed
and absolute recovery was calculated according to the
following formula: %Absolute recovery = (extracted (R)-
and (S)-ibuprofen peak area counts/unextracted (R)- and
(S)-ibuprofen peak area counts) × 100.
2.7.7. Matrix Effect
Matrix effect, which may cause ionization suppression or
enhancement of the analytes, was determined in three
blank human plasma lots. Samples of QC-Low, QC-Mid,
and QC-High were prepared in human plasma and in
water and analyzed. The (R)- and (S)-ibuprofen peak area
counts of the plasma samples were compared to that ob-
tained in samples prepared in water. Samples were ana-
lyzed in triplicates and the matrix effect was calculated
according to the following formula: %Matrix Effect =
peak area counts in plasma—peak area counts in water/
peak area counts in water × 100. Matrix effect was con-
sidered negligible if no more than 10% difference in the
peak area counts of (R)- and (S)-ibuprofen was observed
in the human plasma samples compared to the samples
prepared in water. Negative %matrix effect represented
ionization suppression, and positive %matrix effect rep-
resented ionization enhancement.
2.7.8. Dilution-Integrity
Six aliquots of QC-High (2500 ng/mL) samples were
diluted 10-fold with blank human plasma and analyzed.
The acceptance criteria for (R)- and (S)-ibuprofen were
the accuracy (%Deviation) ± 15% from the actual value
(250 ng/mL), and precision (%RSD) ≤ 15% from the six
determinations.
2.7.9. Evaluation of Carry-over
An aliquot of a QC-Mid sample was prepared and three
injections were made, immediately followed by three
injections of a blank human plasma sample. Carry-over
was expressed as the percentage difference between the
mean (R)- and (S)-ibuprofen or (R)- and (S)-flurbiprofen
peak area counts in blank human plasma samples and the
mean (R)- and (S)-ibuprofen or (R)- and (S)-flurbiprofen
peak area count in the QC-Mid sample. Carry-over was
considered negligible if no more than 5% of (R)- and
(S)-ibuprofen or (R)- and (S)-flurbiprofen was observed
in the blank plasma. The percentage carry-over was cal-
culated as follows: %Carry-over = (BL/QC-Mid) × 100,
where BL = Mean peak area count in the blank samples
(at retention times of (R)- and (S)-ibuprofen or (R)- and
(S)-flurbiprofen), QC-Mid = Mean (R)- and (S)-ibupro-
fen or (R)- and (S)-flurbiprofen peak area count in the
QC-Mid sample.
3. Results and Discussion
The objective of this study was to develop and validate a
sensitive and enantioselective UPLC-MS/MS method for
the quantitation of the (R)- and (S)-ibuprofen in human
plasma. A method was needed, which had a simple sam-
ple preparation step including derivatization, so that the
resulting (R)- and (S)-ibuprofen diastereomers could be
analyzed with conventional reverse-phase chromato-
graphic conditions.
3.1. Method Development and Optimization
The present analytical procedure is based upon a previ-
ously reported assay [29] with modifications to the
method of detection, chromatography and sample prepa-
ration as described below.
3.1.1. Mass Spectrometry
The spectrofluorometric detection [29] was replaced with
mass spectrometric detection. Mass spectrometry is a
much improved detection technique, because it allows
for the selective monitoring of the MRM transitions of
(R)- and (S)-ibuprofen and (R)- and (S)-flurbiprofen,
therefore, eliminating any interferences with co-eluting
endogenous components from human plasma. In order to
monitor (R)- and (S)-ibuprofen and (R)- and (S)-flurbi-
profen using a non-chiral chromatographic system, the
compounds were derivatized as described above to yield
the corresponding diastereomeric amides. The amide
derivatives contained a nitrogen atom, which could be
ionized efficiently using ES + mode. The molecular ions
were determined by the direct injection of about 100
µg/mL solutions of the analytes in water/methanol: 50/50
into the mobile phase flow of the same composition in
the absence of a chromatographic column. The molecular
ions observed for (R)- and (S)-ibuprofen were m/z 360.2,
and m/z 398.3 for (R)- and (S)-flurbiprofen. The cone
voltage value was optimized with the most intense signal

52 A. SZEITZ ET AL.
obtained at a cone voltage of 30 V for each analyte.
Source temperature, desolvation temperature, and desol-
vation gas flow values were optimized for the highest
signal and are reported in Subsection 2.2. The product
ions of the analytes were determined and representative
product ion mass spectra obtained in ES+ for (R)- and
(S)-ibuprofen, and for (R)- and (S)-flurbiprofen are pre-
sented in Figure 1. The CE values were optimized to
obtain the most abundant signals for the product ions.
The proposed fragmentation pattern of the diastereo-
meric amide derivatives of (R)- and (S)-ibuprofen, and
(R)- and (S)-flurbiprofen obtained in ES+ is presented in
Figure 2. MRM transitions were created and (R)- and
(S)-ibuprofen was detected at m/z 360.2 232.1 (CE 10
eV), and (R)-and (S)-flurbiprofen at m/z 398.3  270.1
(CE 10 eV). The dwell time was set to 25 ms for (R)-and
(S)-ibuprofen as this provided sufficient sampling points
during the chromatographic peaks to achieve reliable inte-
gration and, therefore, reproducible results. The dwell
time for (R)-and (S)-flurbiprofen was set to 50 ms.
3.1.2. Liquid Chromatography
The HPLC system using a conventional C18 column was
replaced with a UPLC system using a narrow-bore Ac-
quity UPLC ethylene bridged hybrid (BEH) phenyl
column, and the mobile phase system containing a mix-
ture of phosphate buffer and acetonitrile was replaced
with a mobile phase system containing a mixture of wa-
ter, ammonium acetate, formic acid, methanol and ace-
tonitrile.
For our initial studies, an Acquity UPLC BEH C18 1.7
µm, 2.1 × 100 mm column was tested for the separation
of (R)- and (S)-ibuprofen, and (R)- and (S)-flurbiprofen.
The mobile phase composition was solvent a: water with
0.1% formic acid and solvent b: methanol with 0.1%
formic acid with the flow rate set at 0.2 mL/min. A series
of isocratic chromatographic experiments were per-
formed which are summarized as follows. Ammonium
acetate (10 mM) was added to the mobile phase, differ-
ent mobile phase compositions were tested, methanol
was replaced with acetonitrile in solvent b, various sol-
vent b compositions were prepared with different pro-
portions of methanol and acetonitrile mixed in solvent b.
While it was found that methanol was a selective solvent
for the separation of (R)- and (S)-ibuprofen and acetoni-
trile for (R)- and (S)-flurbiprofen, no baseline separation
could be achieved for these compounds with any addi-
tional changes made to the chromatographic conditions
when the C18 chromatographic column was used.
Based on these findings, the C18 column was replaced
with a Waters Acquity UPLC BEH Phenyl 1.7 µm 2.1 ×
150 mm column. With the phenyl column, when solvent
a was water with 10 mM ammonium acetate and 0.1%
formic acid, and solvent b was a mixture of acetonitrile/
methanol: 64/36 containing 10 mM ammonium acetate
and 0.1% formic acid, and using an isocratic run with a
Figure 1. Representative product ion mass spectra of the
diastereomeric amide derivatives of (a) (R)- and (S)-ibu-
profen, and (b) (R)- and (S)-flurbiprofen obtained in ES+
with a collision energy (CE) 10 eV.
flow rate of 0.2 mL/min, and a mobile phase composition
of solvent a/solvent b: 35/65, baseline separation was
achieved for (R)- and (S)-ibuprofen, and (R)- and (S)-
flurbiprofen. The final gradient programming is detailed
in Subsection 2.2. With this chromatographic column,
mobile phase composition and gradient programming,
(R)- and (S)-flurbiprofen eluted at a retention time of 8.82

A. SZEITZ ET AL.
53
Figure 2. Proposed fragmentation pattern of diastereomeric
amide derivatives of (1) (R)- and (2) (S)-ibuprofen, (3) (R)-
and (4) (S)-flurbiprofen obtained in ES+.
min and 10.03 min, respectively, and (R)- and (S)-ibu-
profen eluted at a retention time 10.12 min and 11.07
min, respectively. A representative chromatogram of the
plasma sample taken from a human volunteer 120 min
after the oral administration of a 400 mg ibuprofen cap-
sule is presented in Figure 3.
3.1.3. Sample Preparation
The sample volume required for analysis was reduced
from 0.5 mL [29] to 100 µL, and the sample preparation
was simplified. The buffering step with sodium phos-
phate buffer was removed and the extraction solvent di-
ethylether was replaced with methyl-tert-butyl ether
which is less flammable, less toxic and easier to use. The
mixing time for sample preparation was reduced, and the
transfer of the organic layer was made quicker and more
efficient by freezing the bottom aqueous layer in the
tubes and decanting the top organic layer into a clean set
of tubes. After derivatization, the step of passing the
samples through solid-phase extraction silica cartridges
was eliminated.
3.2. Method Validation
3.2.1. Accuracy and Precision
The results for accuracy and precision are presented in
Table 1. Accuracy was expressed as the %Deviation for
the QC-Low, QC-Mid and QC-High samples. Intra-day
accuracy for (R)-ibuprofen ranged between -11.8% and
11.2%, and for (S)-ibuprofen between -8.6% and -0.3%.
Inter-day accuracy for (R)-ibuprofen ranged between
–10.4% and 4.5%, and for (S)-ibuprofen between –4.8%
and –0.6%. Precision was expressed as the %RSD for the
QC-Low, QC-Mid and QC-High samples. Intra-day pre-
cision for (R)-ibuprofen ranged between 2.2% and 10.3%,
and for (S)-ibuprofen between 1.7% and 7.0%. Inter-day
precision for (R)-ibuprofen ranged between 5.9% and
11.2%, and for (S)-ibuprofen between 4.0% and 4.8%.
The method met the acceptance criteria for accuracy of
%Deviation ± 20% for the QC-Low samples, and ± 15%
for the QC-Mid, and QC-High samples, and for precision
of % RSD  20% for QC-Low samples, and  15% for
the QC-Mid, and QC-High samples. This indicated that
the method was accurate and precise over the range of
the assay.
3.2.2. Linearity and Range
Linearity of the calibration curve was evaluated in seven
batches over the course of the validation. The coefficient
of determination (mean ± SD, n = 7) for (R)-ibuprofen
was r2 = 0.995 ± 0.004, and for (S)-ibuprofen r2 = 0.996
± 0.004. The accuracy (%Deviation) of the calibration
curve levels for (R)-ibuprofen ranged between -12.9%
and 5.5%, and for (S)-ibuprofen between -4.6% and
3.4%. The precision (%RSD) of the calibration curve
levels for (R)-ibuprofen ranged between 2.3% and 4.4%,
and for (S)-ibuprofen between 1.2% and 10.2%. The
calibration curve met the acceptance criterion for linear-
ity of r2 ≥ 0.980 after weighting with 1/X2. The range of
the method was established as 50 ng/mL to 5000 ng/mL
where the calibration levels met the acceptance criteria
of accuracy (%Deviation) ± 15%, and precision (%RSD)
≤ 15%. The results indicated that the calibration curve
was linear, accurate, and precise over the range of the
method.

A. SZEITZ ET AL.
54
Table 1. Accuracy (intra- and inter-day) and precision (intra-and inter-day) for (R)- and (S)-ibuprofen in human plasma.
(R)-ibuprofen (S)-ibuprofen
Accuracy Intra-Day Inter-Day Accuracy Intra-Day Inter-Day
QC-Low (80 ng/mL) Day 1
(n = 5)
Day 2
(n = 6)
Day 3
(n = 6)
Day 1-3
(n = 17) QC-Low (80 ng/mL) Day 1 (n = 5)
Day 2
(n = 6)
Day 3
(n = 6)
Day 1-3
(n = 17)
Mean (ng/mL) 76.7 82.2 81.1 80.2 Mean (ng/mL) 79.7 79.1 79.6 79.5
SD (ng/mL) 2.38 5.11 3.63 4.38 SD (ng/mL) 4.99 5.21 3.69 4.36
%Deviation –4.1 2.7 1.3 0.2 %Deviation –0.3 –1.1 –0.5 –0.6
QC-Mid (250 ng/mL) QC-Mid (250 ng/mL)
Mean (ng/mL) 242 261 278 261 Mean (ng/mL) 244 244 249 246
SD (ng/mL) 22.6 10.8 14.5 18.9 SD (ng/mL) 6.72 7.82 8.47 7.62
%Deviation –3.4 4.4 11.2 4.5 %Deviation –2.3 –2.4 –0.5 –1.7
QC-High (2500 ng/mL) QC-High (2500 ng/mL)
Mean (ng/mL) 2205 2246 2267 2241 Mean (ng/mL) 2428 2285 2433 2379
SD (ng/mL) 18.1 71.0 43.9 54.0 SD (ng/mL) 37.7 98.4 85.7 104
%Deviation –11.8–10.2 –9.3 –10.4 %Deviation –2.9 –8.6 –2.7 –4.8
(R)-ibuprofen (S)-ibuprofen
Precision Intra-Day Inter-DayPrecision Intra-Day Inter-Day
QC-Low (80 ng/mL) Day 1
(n = 6)
Day 2
(n = 5)
Day 3
(n = 6)
Day 1-3
(n = 17) QC-Low (80 ng/mL) Day 1 (n = 6)
Day 2
(n = 5)
Day 3
(n = 6)
Day 1-3
(n = 17)
Mean (ng/mL) 78.6 89.6 96.7 88.2 Mean (ng/mL) 89.5 90.0 89.0 89.4
SD (ng/mL) 4.88 8.29 5.73 9.87 SD (ng/mL) 3.40 6.28 2.36 3.92
%RSD 6.2 9.2 5.9 11.2 %RSD 3.8 7.0 2.6 4.4
QC-Mid (250 ng/mL) QC-Mid (250 ng/mL)
Mean (ng/mL) 268 298 274 279 Mean (ng/mL) 271 267 252 263
SD (ng/mL) 9.25 12.5 10.3 16.4 SD (ng/mL) 4.48 5.20 8.58 10.5
%RSD 3.4 4.2 3.7 5.9 %RSD 1.7 1.9 3.4 4.0
QC-High (2500 ng/mL) QC-High (2500 ng/mL)
Mean (ng/mL) 1912 2121 2040 2018 Mean (ng/mL) 2219 2121 2074 2139
SD (ng/mL) 81.7 218 44.5 149 SD (ng/mL) 79.6 110 68.4 103
%RSD 4.3 10.3 2.2 7.4 %RSD 3.6 5.2 3.3 4.8
SD = Standard Deviation; %Deviation = [(measured amount/added amount) × 100]-100 [%]; %RSD = Relative Standard Deviation [%].
3.2.3. Limit of Quantitation, Selectivity, Limit of
Detection
LOQ was determined in six replicates of the 50 ng/mL
calibration standard. The mean response of the (R)- and
(S)-ibuprofen peaks in the samples was higher than
5-times the response obtained in the blank sample. The
accuracy (%Deviation) for (R)-ibuprofen ranged between
–7.1% and 7.3% and for (S)-ibuprofen between –0.4%
and 5.2%. The precision (%RSD) for (R)-ibuprofen was
5.8% and for (S)-ibuprofen 2.0%. Results for the deter-
mination of LOQ met the acceptance criteria of the mean
response which was at least 5-times the response com-
pared to the blank sample, accuracy (%Deviation) ± 20%
and precision % RSD ≤ 20%, and the (R)- and (S)-ibu-
profen peaks were identifiable, discrete, and reproducible.
The method was accurate and precise with an established
LOQ of 50 ng/mL of (R)- and (S)-ibuprofen requiring
100 µL of sample. The selectivity of the method was
investigated in triplicate samples of pooled human
plasma made from three separate lots spiked at LOQ
level. Blank human plasma samples from three separate
lots were also prepared. The selectivity met the accep-
tance criteria that the mean response for (R)- and (S)-
ibuprofen was at least 5-times the response compared to
the blank samples, and there was no significant interfer-
ence at the retention times of (R)- and (S)-ibuprofen and

A. SZEITZ ET AL.
55
(R)- and (S)-flurbiprofen when the blank human plasma
samples were compared to the LOQ samples. The results
indicated that the method was selective for these analytes.
LOD was determined in triplicate samples of 1 ng/mL
(R)- and (S)-ibuprofen prepared in human plasma. The
mean response (i.e., S/N) of the (R)- and (S)-ibuprofen
peaks in the samples was higher than 3-times the re-
sponse obtained in the blank sample. The method had an
established LOD of 1 ng/mL of (R)- and (S)-ibuprofen
requiring 100 µL of sample. Representative chroma-
tograms of LOD (1 ng/mL), and blank human plasma
samples are presented in Figure 4.
3.2.4. Absolute Recovery and Matrix Effect
The mean %Absolute recovery values (n = 6) for
(R)-ibuprofen for QC-Low, QC-Mid and QC-High were
81.9%, 86.9% and 84.5%, respectively, and for (S)-ibu-
profen 82.2%, 83.4% and 83.7%, respectively. The re-
sults indicated that (R)- and (S)-ibuprofen were extracted
relatively uniformly over the concentration range of the
assay. The mean %Matrix effect values (n = 9) for
(R)-ibuprofen for QC-Low, QC-Mid, and QC-High were
Figure 3. A representative chromatogram of the plasma
sample taken from a human volunteer 120 min after the
oral administration of a 400 mg ibuprofen capsule. The
MRM signal of m/z 360.2  232.1 for (R)- and (S)-ibupro-
fen is on the top, and m/z 398.3  270.1 for internal stan-
dards (R)- and (S)-flurbiprofen is on the bottom (the meas-
ured (R)- and (S)-ibuprofen concentrations were 803 ng/mL
and 1087 ng/mL, respectively).
1.3%, 2.2% and –2.5%, respectively, and for (S)- ibu-
profen 2.1%, –6.2%, and –2.0%, respectively. The re-
sults met the acceptance criterion of %Matrix effect of
no more than 10%. This indicated that the matrix effect
from human plasma contributing to the ionization sup-
pression or enhancement of (R)- and (S)-ibuprofen was
negligible.
3.2.5. Dilution Integrity and Evaluation of Carry-over
After the 10-fold dilution of the six aliquot of the QC-
High samples, the accuracy (%Deviation, n = 6) for
(R)-ibuprofen ranged between –5.5% and 4.1%, and for
(S)-ibuprofen between –7.9% and 5.6%. The precision
(%RSD, n = 6) for (R)-ibuprofen was 3.7% and for
(S)-ibuprofen 4.5%. The results met the acceptance crite-
ria of accuracy (%Deviation) ±15% from the actual value
(250 ng/mL), and precision (%RSD) ≤ 15%. This indi-
cated that samples exceeding the calibration curve con-
centrations could be diluted 10-fold to bring them into
the range of the assay with accuracy and precision.
Carry-over between injections of the QC-Mid and blank
plasma samples for (R)-ibuprofen was 0.4%, for
(S)-ibuprofen 1.0%, for (R)-flurbiprofen 0.1%, and for
(S)-flurbiprofen 0.1%; all met the acceptance criteria of
no more than 5%.
3.2.6. Pharmacokinetics of (R)- and (S)-ibuprofen in
humans
Healthy human volunteers were involved in a pharma-
cokinetic study following the administration of a single
oral dose of 400 mg ibuprofen capsules. The detailed
pharmacokinetic results of this study will be published
elsewhere. Here we report the results of four randomly
selected subjects to demonstrate the applicability of the
developed and validated method to determine the (R)-
and (S)-ibuprofen concentrations in human plasma.
Plasma samples were collected at various time intervals
up to 300 min following the ibuprofen dose. A represen-
tative plasma concentration vs. time plot obtained from
one subject is presented in Figure 5. (R)- and (S)-ibu-
profen concentrations increased during the first 90 min
reaching a peak value of 15,026 ng/mL for (R)-ibuprofen,
and 18,915 ng/mL for (S)-ibuprofen. After that time, the
drug concentrations decreased gradually and remained
above 3100 ng/mL for (R)-ibuprofen, and 5800 ng/mL
for (S)-ibuprofen at the final sampling time of 300 min.
Pharmacokinetic analysis of the plasma concentration
data obtained from the four subjects was performed us-
ing a non-compartmental analysis (PK Solutions 2.0,
Montrose, CO, USA). The results were normalized to
body weights. The estimated pharmacokinetic parame-
ters for (R)-ibuprofen were as follows (mean ± SD, n =
4): terminal elimination half-life (t1/2): 83.8 ± 8.62 min,
Tmax: 37.5 ± 8.66 min, Cmax: 14,076 ± 3160 ng/mL, ap-
parent volume of distribution (V/F): 350 ± 50.4 mL/kg,
apparent oral clearance (CL/F): 2.91 ± 0.519 mL/min/kg,

56 A. SZEITZ ET AL.
Figure 4. Limit of detection (LOD, 1 ng/mL) and blank
human plasma samples. (a) shows the MRM m/z 360.2 
232.1 for (R)- and (S)-ibuprofen in blank human plasma
(top), and in the LOD sample (bottom) (the signal-to-noise
[S/N] values are reported on the top of the (R)- and
(S)-ibuprofen peaks). (b) shows the MRM m/z 398.3 
270.1 for (R)- and (S)-flurbiprofen, internal standard, in
blank human plasma (top), and in the LOD sample (bot-
tom).
Figure 5. Concentration of (R)- and (S)-ibuprofen vs. time
profile obtained from a volunteer following a single oral
dose of a 400 mg ibuprofen capsule.
and mean residence time (MRTINF): 142 ± 23.4 min; and
for (S)-ibuprofen (mean ± SD, n = 4): terminal elimina-
tion half-life (t1/2): 108 ± 14.6 min, Tmax: 41.3 ± 7.50 min,
Cmax: 15,779 ± 4720 ng/mL, apparent volume of distribu-
tion (V/F): 325 ± 91.1 mL/kg, apparent oral clearance
(CL/F): 2.06 ± 0.303 mL/min/kg, and mean residence
time (MRTINF): 173 ± 16.1 min.
In summary, the results show that following the oral
administration of 400 mg racemic ibuprofen, both enan-
tiomer showed rapid absorption and stereoselective dis-
position. For (S)-ibuprofen, the plasma concentrations
peaked at 41.3 min, reached higher levels (i.e., 15,779
ng/mL), had higher terminal elimination half life (108
min), apparent volume of distribution (325 mL/kg), mean
residence time (173 min), and lower apparent oral clear-
ance (2.06 mL/min/kg) than those values for (R)-ibuprofen
(i.e., Tmax: 37.5 min, Cmax: 14,076 ng/mL, t1/2: 83.8 min,
V/F: 350 mL/kg, MRTINF: 142 min, and CL/F: 2.91 mL/
min/kg, respectively). These findings are consistent with
the data of in vivo chiral inversion of (R)-ibuprofen to
(S)-ibuprofen reported in the literature [9-11].
4. Conclusions
An enantioselective modified UPLC-MS/MS method
was developed and validated for the quantitation of (R)-
and (S)-ibuprofen enantiomers in human plasma. Com-
pared to previously published assays, sample preparation
was simplified without the need of post-derivatization
sample clean-up [29], sample volume required for analy-
sis was reduced at least 5-fold [6,25,29], the range of the
calibration curve was extended into the three-digit range
[23,25,29], baseline separation of (R)- and (S)-ibuprofen
was achieved [20,25] and the sensitivity was increased
2-fold [23-25,29]. The assay was validated with a LOQ
of 50 ng/mL, however, the sensitivity of the assay proved
to be significantly higher with a LOD of 1 ng/mL. It was
not necessary to validate the method at a LOQ less than
50 ng/mL, because of the high concentration of the sam-
ples analyzed during this study. However, the LOQ of
the present assay can be easily extended to the low
ng/mL levels if future studies require a methodology
with significantly higher sensitivity. The assay demon-
strated good reproducibility and was applied to investi-
gate the pharmacokinetics of ibuprofen enantiomers fol-
lowing oral administration of the racemate drug.
5. Acknowledgements
The authors would like to thank Dr. Cathy Boscarino for
her valuable work with the design of the human study;
Mrs. Debbie Kerrigan-Brown, Ingrid Smith, Christina
Powlesland, and Mr. Kevin Hofer, Robert Limmer, Doug
Saunders, Jan Pope for their excellent technical support
of the human study. This work was funded by a research
contract with Defence Research and Development Can-
ada; contract number W7711-088126.

A. SZEITZ ET AL.
57
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