604
El-Fatatry et al.: Journal of AOAC International Vol. 99, No. 3, 2016
DRUG FORMULATIONS AND CLINICAL METHODS
A Validated Enantioselective HPLC Method for Determination
of Ibuprofen Enantiomers in Bulk and Tablet Dosage Form
Hamed M. El-Fatatry, Mokhtar M. Mabrouk, Sherin F. Hammad, and Samah F. El-Malla1
A new chiral reversed-phase (RP)-HPLC method
with UV detection was developed. Enantioselective
resolution of ibuprofen (IBP) was achieved using
(3R,4S)-4-(3,5-dinitrobenzamido)-3-(3-(trioxysilyl)propyl)-1,2,3,4-tetrahydro-phenanthrene
[(R,R)-Whelk-O2] chiral stationary phase (4.6 mm id
× 250 mm, 10 μm) with a mobile phase composed
of ethanol–water (30 + 70, v/v) containing 100 mM
ammonium acetate at a flow rate of 1.3 mL/min
using diode array detector at λ 220 nm. Calibration
curves were linear over the concentration range
of 20–180 μg/mL for both IBP enantiomers. Mean
% recoveries ±SD of 99.74 ± 1.73 and 99.60 ± 0.93
were obtained for dexibuprofen (dex-IBP) and
levoibuprofen (levo-IBP), respectively. Intra- and
interday precision calculated as RSD, % were not
more than 1.66% for dex-IBP and 1.93% for levo-IBP.
The detection limits were 2.09 and 2.06 μg/mL for
dex-IBP and levo-IBP, respectively. The method was
successfully applied for the determination of dex-IBP
in tablet dosage form.
I
buprofen [IBP; (2RS)-2-(4-(2-methylpropyl) phenyl)]
propanoic acid (MW 206.281), is a chiral nonsteroidal antiinflammatory drug (NSAID; Figure 1), cyclo-oxygenase
inhibitor, analgesic, and antipyretic drug. It is widely used in
the treatment of rheumatic arthritis and other inflammatory
conditions (1–4).
IBP belongs to the general category of 2-arylpropionic acids,
a group of NSAIDs. These compounds are characterized by
a chiral center adjacent to the carboxylic acid moiety. S-IBP
(dexibuprofen; dex-IBP) is >100-fold more potent as an inhibitor
of cyclo-oxygenase than R-IBP (levoibuprofen; levo-IBP).
There are also reports that the anti-inflammatory effect by the
levo-IBP might arise through a unique bioinversion at its chiral
center to the active dex-IBP. Therefore, dex-IBP is faster acting
than the racemic mixture. Dex-IBP shows an equipotency with
half of the racemic IBP dose, and the introduction of dex-IBP in
the market permits the prescription of lower doses. Therefore,
the stereoselective determination of the drug enantiomers is of
potential clinical importance (5–7).
IBP is marketed in Egypt either as a racemic mixture or as a
pure dexenantiomer.
Received November 5, 2015. Accepted by SW January 12, 2016.
1
Corresponding author’s e-mail: samah.elmalla@pharm.tanta.edu.eg;
elmallasfarouq@gmail.com
DOI: 10.5740/jaoacint.15-0273
Methods for analysis of IBP (3, 8) and IBP enantiomers
(5, 9, 10) were reported in various review articles. IBP
enantiomers were determined by indirect methods through
formation of diastereomeric derivatives (11–15). In spite of
being a versatile method for the separation of enantiomers
having reactive functional groups (12), most of these methods
require extensive sample preparation, long derivatization,
lengthy chromatography times, poor sensitivity, and late
eluting peaks. Formation of diastereomeric derivatives
may introduce inaccuracies into the determination of the
enantiomeric ratio because of chiral impurities in the
reagent or because of the racemization during the process of
derivatization (13).
Different types of chiral stationary phases (CSPs) have been
used for enantioseparation of IBP, e.g., α1-acid glycoprotein
(16, 17), ovoglycoprotein (18), cellulose derivatives (19, 20),
amylose (21, 22), and β-cyclodextrine (23). A comparison of
such methods appears in Table 1.
Direct separation of IBP using chiral mobile phase
additives has also been described using native and derivatized
β-cyclodextrins (24).
Resolution (Rs) of IBP enantiomers with TLC was also
achieved. The stationary phase was silica gel plates impregnated
with optically pure l-arginine (0.5%) as chiral selector, using
acetonitrile (ACN)–methanol–water (5 + 1 + 1, v/v/v) as the
solvent system (25).
An enantioselective supercritical fluid chromatographic
method was developed for the separation of IBP enantiomers.
Rs on different CSPs was evaluated using 7% isopropanol in
CO2 as a mobile phase (26).
Capillary zone electrophoresis was also used as an alternative
to HPLC as an adequately fast, accurate, and precise method
for quantification of IBP enantiomers using β-cyclodextrin
derivative in the background electrolyte (27, 28).
On the other hand, membrane electrodes were also developed
and used for determining drug enantiomers. Enantioselective
potentiometric membrane electrodes based on maltodextrins
were proposed for the assay of dex-IBP with potentiometric
selectivity coefficients <10−4 over levo-IBP (29).
To the best of our knowledge, no validated chiral chromato­
graphic method has been described in the literature for the
determination of IBP enantiomers using Whelk-O2 CSP by
reversed-phase (RP)-HPLC. Whelk-O2 is a type of Pirkle
CSP that contains both π-donor (tetrahydrophenanthren) and
π-acceptor (3,5-dinitrobenzoyl; DNB) groups, with amide
hydrogen. Immobilization of these groups to silica by a
trifunctional covalent bond increases the stability of the
Whelk-O2 column against hydrolysis while using strong
organic modifiers in the mobile phase (9).
Downloaded from https://academic.oup.com/jaoac/article/99/3/604/5658098 by Medical University of Plovdiv user on 04 April 2022
University of Tanta, Faculty of Pharmacy, Department of Pharmaceutical Analytical Chemistry, Al-Gaish St, Mail Code 31527,
Tanta, Egypt
El-Fatatry et al.: Journal of AOAC International Vol. 99, No. 3, 2016 605
NSAID and the carbonyl group of the 3,5-DNB carboxamide
in the CSP, steric interactions between aryl alkyl groups of
the NSAID and the CSP, and π–π donor–acceptor interaction
between the phenyl ring of the NSAID and the 3,5-DNB and
tetrahydrophenanthren groups of the CSP.
The present study describes an enantioselective, isocratic,
and validated RP-HPLC method for the separation and
quantitation of IBP enantiomers in bulk and tablet dosage
form.
Instrumentation
Figure 1. Chemical structures of S-(+)-IBP and R-(-)-IBP.
With respect to chiral NSAIDs, three simultaneous
interactions take place with Whelk-O2 (9): hydrogen bonding
between the hydroxyl moiety of the carboxylic acid group of the
HPLC measurements were performed on a Waters
instrument equipped with a model 600 pump, automatic
injector 2707, and 2996 diode array detector (DAD). Waters
Empower-II software was used as the data acquisition
program to collect and process all chromatographic data and
to measure retention time and system suitability parameters.
The HPLC column was (R, R)-Whelk-O2 (4.6 mm diameter
× 250 mm length, 10 μm particle size; Regis Technologies,
Inc., Morton Grove, IL). Sonicator (Ultrasons-Selecta) was
used for sonication and degassing of the mobile phase and the
preparation of dosage form solution.
Table 1. Different direct chromatographic methods for enantioselective determination of IBPa
Mobile phase
tRb
Sample
0.1 M Phosphate buffer (pH 7) containing
0.4% 2-propanol
3.9, 5.1
25
220 nm
1.5
Plasma
Yes
Yes
16
α1-Acid glycoprotein 0.5% 2-Propanol in 20 mM phosphate buffer
(pH 6.7) containing 5 mM dimethyloctyl amine
14, 16
40
220 nm
0.1
Plasma
No
Yes
17
Ovoglycoprotein
20 mM Phosphate buffer (pH 4)–ethanol
(90 + 10)
24, 44
5
220 nm
20
Bulk
Yes
No
18
Chiralcel OJ-H
n-Hexane–2-propanol–trifluoroacetic acid
(98 + 2 + 0.1)
8.5, 9.5
20
254 nm
27
Bulk
Yes
Yes
19
Methanol: 0.1 M–chlorate buffer (pH 2; 70:30); 10, 15
benzylamine is used for derivatization
20
254 nm
—
Bulk
No
No
20
α1-Acid glycoprotein
Cellulose
Inj. vol.c Detection
Baseline
Rse
Validation
QLd
Stationary phase
Reference
ChiralPak AD-RH
ACNf–water–phosphoric acid–TEAg
(35 + 64.85 + 0.1 + 0.05)
21.4,
23.4
50
220 nm
0.1
Rat
serum
Yes
Yes
21
ChiralPak AD-RH
Methanol–phosphoric acid solution
(pH 3; 75 + 25)
11, 13
50
230 nm
0.25
Urine
Yes
Yes
22
50
220 nm
1
Plasma
No
Yes
23
10
Urine
β-Cyclodextrin
a
ACN–0.1% Triethylammonium acetate buffer 27, 29.4
(pH 7.5; 30 + 70)
IBP = Ibuprofen.
b
tR = Retention time (min).
c
Inj. vol. = Injection volume (μL).
d
QL = Quantitation limit (μg/mL).
e
f
g
Rs = Resolution.
ACN = Acetonitrile.
TEA = Triethylamine.
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Experimental
606
El-Fatatry et al.: Journal of AOAC International Vol. 99, No. 3, 2016
Chemicals and Reagents
Standard Solutions and Calibration
Stock standard solution of 1 mg/mL IBP was prepared in
ethanol (EtOH). This solution contained 500 μg/mL of each
enantiomer. Different aliquots from the stock solution were
taken and diluted with the mobile phase to obtain working
standard solutions containing from 40 to 360 μg/mL of IBP (i.e.,
they contained from 20 to 180 μg/mL of each IBP enantiomer).
Similarly, stock standard solution of 1 mg/mL dex-IBP was
prepared in EtOH. Different aliquots from stock solution were
taken and diluted with the mobile phase to obtain working
standard solutions containing between 20 and 180 μg/mL of
dex-IBP.
A calibration curve of each IBP enantiomer was constructed
by plotting the average peak area versus its corresponding
concentration (μg/mL) and the regression equation was
computed.
Chromatographic Conditions
A chromatographic separation assay was performed on
a chiral (R, R)-Whelk-O2 (250 × 4.6 mm id, 10 μm particle
size) column at ambient temperature (25 ± 1°C). The optimum
mobile phase consisted of EtOH–water (30 + 70 v/v) containing
100 mM AmAc. The mobile phase was prepared by dissolving
7.708 g of ammonium acetate (MW 77.08) in 700 mL of distilled
water, and then filtering through a 47 mm nylon membrane
filter (0.45 μm) under vacuum, mixing with 300 mL EtOH,
and then degassing in a sonicator before use. The mobile phase
was prepared daily, and delivered isocratically at a flow rate of
1.30 mL/min. The column was equilibrated for 45 min before
assay. The injection volume was 20 μL and UV detection was
performed at 220 nm using DAD.
Application to Pharmaceutical Dosage Form
Brufen tablets (200 mg IBP/tablet).—The colored sugar
coating of ten tablets was first washed with distilled water and
then was allowed to dry. Tablets were weighed, ground, and an
accurately weighed amount of the powdered tablets equivalent
to 200 mg IBP was transferred into 100 mL volumetric flask,
then, dissolved in 50 mL EtOH, sonicated for 15 min and
then completed to volume with the same solvent and filtered
to prepare stock solution of IBP containing 2000 μg/mL (i.e.,
1000 μg/mL of dex-IBP). An aliquot of 0.5 mL of this stock
solution was diluted with the mobile phase to 10 mL to prepare
assay solution containing 100 μg/mL of IBP (i.e., 50 μg/mL of
dex-IBP). A 20 μL volume of the final solution was injected in
Results and Discussion
Method Development
The method was developed using the Whelk-O2 CSP and
changing the mobile phase composition to achieve higher Rs
with short retention time values, i.e., better separation.
A solution representing a racemic mixture, 50 + 50 of each
IBP enantiomer, was used for the method development. The
order of elution was determined by injecting dex-IBP solution
under the same conditions. To evaluate the chromatographic
separation, Rs was calculated and compared.
Separation in normal phase (NP) mode was first tried using
n-hexane–isopropyl alcohol (IPA; 90 + 10) in the presence
of 10 mM AmAc. Enantioseparation was achieved with
excellent Rs, however repeating the experiment produces
nonreproducible results concerning retention times and
Rs values, which may be a result of low solubility of IBP in
n-hexane. The separation conditions were switched to the RP
mode to improve reproducibility of the results, and the scheme
for method development recommended by the column supplier
was followed (data not shown).
Mobile phases that contain water and different organic
modifiers (ACN, MeOH, or EtOH) in the ratio 50 + 50 were
tested. Enantioseparation was achieved only by using EtOH as
an organic modifier, but with very low Rs. The effect of adding
HAc to the mobile phase was studied. The addition of 0.1%
HAc to the mobile phase containing EtOH–water (50 + 50)
improved Rs. This may be attributed to the presence of IBP
in the nonionized form in the acidic environment produced by
HAc, so the available –COOH proton produces more H-bonding
with Whelk-O2 CSP enabling more enantioselectivity but with
increased retention times (data not shown).
The effect of EtOH, % is also tested. It was observed that
increasing the EtOH, % in the mobile phase decreases both
retention time and Rs. This may be attributed to increased
solubility of IBP in mobile phase with higher EtOH, %, which
rapidly elutes IBP from the column, thus decreasing Rs. To
increase Rs, the EtOH, % in the mobile phase was decreased.
Different mobile phases with decreased EtOH, % were tried.
Rs increased to 1.8 using 30% EtOH, but run time was severely
prolonged (≈80 min; unpublished data).
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IBP was kindly donated by Sigma Pharmaceutical Industries
(Quesna, Egypt). Dex-IBP was purchased from Hubei Biocause
Pharmaceutical Co., Ltd (Hubei, China). Methanol (MeOH)
HPLC grade, ethanol (EtOH) HPLC grade, and ACN HPLC
grade were purchased from Sigma-Aldrich (St. Louis, MO).
Ammonium acetate (AmAc) was purchased from Oxford
Laboratory (Mumbai, India). Glacial acetic acid (HAc) HPLC
grade was purchased from Honil Ltd (London, UK). Brufen
200® and dexa-ibufen 400® tablets were purchased from a local
pharmacy.
triplicate and chromatographed under the previously mentioned
chromatographic conditions.
Dexa-ibufen tablets (400 mg dex-IBP/tablet).—The sugar
coating of 10 tablets was first washed with distilled water and
then was allowed to dry. Tablets were weighed and ground,
and an accurately weighed amount of the powdered tablets,
equivalent to 400 mg dex-IBP, was transferred into a 100 mL
volumetric flask, dissolved in 50 mL EtOH, sonicated for
15 min, completed to volume with the same solvent, and
then filtered to prepare stock solution of dex-IBP containing
4000 μg/mL. A 5 mL aliquot of this stock solution was diluted
to 50 mL with mobile phase to obtain a 400 μg/mL working
solution. A 2.5 mL aliquot of this working solution was diluted
to 10 mL with the mobile phase to obtain assay solution
containing 100 μg/mL of dex-IBP. A 20 μL volume of the
final solution was injected in triplicate under the previously
mentioned chromatographic conditions.
El-Fatatry et al.: Journal of AOAC International Vol. 99, No. 3, 2016 607
However, increasing Rs was achieved by the use of HAc;
retention time values were also increased, which makes the use
of HAc unreliable in enantioseparation of IBP. Replacement
of HAc with AmAc was attempted. The addition of 10 mM
AmAc to the mobile phase [MeOH–water (50 + 50)] results
in increased Rs with no effect on retention time. This mobile
phase was set as the initial chromatographic condition to begin
the optimization procedures for RP-HPLC enantioseparation
of IBP.
Different factors affecting the enantioseparation were
thoroughly studied and optimized. Enantiomeric separation
was evaluated by measuring retention time of dex-IBP (tR1).
The aim of the optimization process was to achieve the best
enantioselectivity with higher Rs and shorter retention time.
Effect of MeOH Ratio
Variations in the MeOH ratio were investigated from 20 to
60%. It was found that tR1 and Rs increased with decreasing
MeOH ratio. The optimum separation was achieved at 30%
MeOH. A further decrease in the MeOH ratio would significantly
increase retention time with decreased Rs (Figure 2), which
might be attributed to peak tailing, which results in an increasing
width of peaks, thus decreasing Rs.
Effect of Flow Rate
Flow rate of 1.3 mL/min was selected as an optimum flow
rate with respect to retention time and Rs values (Figure 3).
Figure 3. Effect of flow rate on retention time tR1 and Rs of IBP.
Effect of Type of Organic Modifier
The effect of EtOH on enantioseparation of IBP was studied.
Replacement of MeOH with EtOH resulted in increasing Rs
between IBP enantiomers.
Figure 5a and b shows the chromatograms obtained for IBP
and dex-IBP, respectively, at the optimum chromatographic
conditions: EtOH–water (30 + 70), 100 mM AmAc, flow rate
1.3 mL/min, temperature 25°C, λ 220 nm, and injection volume
20 μL. The retention times of dex-IBP and levo-IBP were
approximately 12.4 ± 0.07 and 16.1 ± 0.1 min, respectively.
Method Validation
The concentration of AmAc was an important optimization
parameter. Different molarities (10–150 mM) were tried, with
a significant improvement in Rs being achieved with 100 mM
AmAc. A further increase in AmAc concentration produced an
adverse effect on Rs, which may be a result of increased peak
tailing (Figure 4).
The validation of the chiral analytical method is similar to
that of any achiral method, but it includes additional specificities
(30). Every step of a classical method validation would have to
be followed for three chemical entities, the two enantiomers, and
the racemate. The International Conference on Harmonization
(ICH) guidelines for method validation (31) were followed.
Because levo-IBP is not available, method validation would
have to be followed for dex-IBP and IBP only. The validation
parameters that were addressed follow.
Figure 2. Effect of MeOH ratio on retention time tR1 and Rs of IBP.
Figure 4. Effect of AmAc concentration (mM) on retention time and
Rs of IBP.
Effect of Ammonium Acetate Concentration
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Method Optimization
608
El-Fatatry et al.: Journal of AOAC International Vol. 99, No. 3, 2016
Linearity and Range
As for any achiral method, linearity will be checked from
standard curves constructed from data points covering the
expected range of concentrations. The measured signal can be
either peak height or area. Because enantiomers possess identical
physicochemical properties, their UV absorption will be identical.
However, separate injections of the racemate and the individual
enantiomers may have relatively different retention times, leading
to alterations in peak shapes and thus an adverse effect on peak
heights. Therefore, in chiral chromatography, peak areas tend to
afford more reproducible results (30).
For greater soundness in method validation, calibration
curves were prepared with IBP as well as with the available dexIBP. So calibration curves for dex-IBP were available from two
sources; IBP and dex-IBP, whereas linear regression analysis
data for levo-IBP were available only from IBP.
Calibration curves were linear in the range of 20 to 180 μg/mL
for each enantiomer. The quantitative statistical parameters
for the determination of IBP enantiomers are summarized in
Table 2. The high values of correlation coefficients (r) with
small intercepts indicate good linearity of the method.
The slopes of the calibration curves might differ for both
enantiomers. This could result from different peak integrations
(peak shapes, tailing, etc.; 30).
Accuracy
The accuracy of the method was tested by analyzing freshly
prepared solutions of IBP in triplicate at concentrations of 80,
200, and 320 μg/mL (i.e., 40, 100, and 160 μg/mL for each dexIBP and levo-IBP). Similar procedures were achieved using
dex-IBP solutions prepared at the same concentrations (i.e., 40,
100, and 160 μg/mL).
The accuracy of the method was determined by calculating
the mean % recovery of triplicate determination for each IBP
enantiomer at three concentrations within the linearity range
as shown in Table 3. Good % recoveries ±SD indicates the
accuracy of the method.
Precision
The precision of an analytical method provides information
on the random errors. The precision expresses the closeness of
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Figure 5. HPLC chromatogram of (a) 200 μg/mL IBP (1: dex-IBP; 2: levo-IBP), (b) 100 μg/mL dex-IBP, (c) Brufen tablet, 100 μg/mL, (d) Dexaibufen tablet, 100 μg/mL, using the proposed HPLC method.
El-Fatatry et al.: Journal of AOAC International Vol. 99, No. 3, 2016 609
Table 2. Quantitative statistical parameters for the determination of IBPa enantiomers by the proposed method.
Linearity,
μg/mL
rb
a, 105c
b, 105d
Sy/x, 105e
Sa, 105f
Sb, 103g
DL, μg/mLh
QL, μg/mLi
Dex-IBP in
racemic IBP
20−180
0.9999
−0.23253
0.44802
0.39105
0.28409
0.25243
2.09
6.34
Levo-IBP in
racemic IBP
20–180
0.9999
−0.19031
0.44452
0.38277
0.27808
0.24708
2.06
6.26
Pure Dex-IBP
20–180
0.9999
1.52857
0.34723
0.37813
0.27471
0.24408
2.61
7.91
Enantiomer
a
IBP = Ibuprofen.
r = Correlation coefficient.
c
a = Intercept.
d
b = Slope.
e
Sy/x = Residual SD of the regression line.
f
Sa = Standard error of intercept.
g
Sb = Standard error of slope.
h
i
DL = Detection limit (calculated).
QL = Quantitation limit (calculated).
agreement between a series of measurements obtained from
multiple samplings of the same homogenous sample under
prescribed conditions. It includes repeatability (intraday) and
intermediate (interday) precision.
Precision was evaluated by assaying freshly prepared
solutions of IBP in triplicate at concentrations of 80, 200, and
320 μg/mL (i.e., 40, 100, and 160 μg/mL for each dex-IBP
and levo-IBP). Measurements were performed either on the
same day (repeatability), or on 3 successive days (intermediate
precision). Similar procedures were achieved using dex-IBP
solutions prepared at the same concentrations (i.e., 40, 100, and
160 μg/mL).
Repeatability and intermediate precision were determined by
calculating the SD and RSD, % for triplicate determinations of
three concentrations of each IBP enantiomer within the linearity
range (Table 3). Accepted values of SD and RSD, % indicate
reproducibility of the method.
Specificity
According to ICH, specificity is the ability to assess
unequivocally the analyte in the presence of components that
may be expected to be present. Typically these might include
impurities, degradation product, matrix, etc.
Table 3. Evaluation of accuracy, intra- and interday precision for the determination of IBP enantiomers by the proposed
method.
Accuracy and intraday precision
Concn taken, μg/mL
b
Dex-IBP in racemic IBP
Mean concn
found, μg/mLa
SD
RSD, %
Mean concn
found, μg/mL
SD
RSD, %
40
39.27
0.25
0.64
98.17
40.27
0.67
1.66
101.59
0.55
0.54
101.59
100.46
1.25
1.24
160
159.14
0.57
0.36
99.46
161.39
1.93
1.20
40.09
0.54
1.34
99.74 ± 1.73
RSD, %
1.73
40
39.50
0.76
1.93
100
99.45
0.78
0.78
99.45
98.86
0.52
0.52
160
160.95
0.72
0.45
100.59
161.09
0.17
0.11
39.78
0.31
0.78
Mean recovery, % ±SD
98.76
99.60 ± 0.93
RSD, %
Pure Dex-IBP
Recovery, %
100
Mean recovery, % ±SD
Levo-IBP in racemic IBP
Interday precision
0.93
40
39.31
0.28
0.70
98.28
100
100.39
0.71
0.70
100.39
99.51
0.85
0.85
160
160.14
0.94
0.59
100.09
159.38
0.95
0.60
Mean recovery, % ±SD
RSD, %
a
Mean of three determinations, n = 3.
b
IBP = Ibuprofen.
99.58 ± 1.14
1.14
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b
610
El-Fatatry et al.: Journal of AOAC International Vol. 99, No. 3, 2016
DL and QL
The ICH guidelines (31) for calculation of DL and QL were
followed. The method was based on SD of the response and the
slope. These may be expressed as DL = 3.3 σ / S and QL = 10 σ / S,
in which σ is the SD of the y-intercept of the regression line, and
S is the slope of the calibration curve. Calculated DL and QL of
the proposed methods are shown in Table 2.
The slopes of the calibration curves may differ for both
enantiomers. This could result from different peak integrations
(peak shapes, tailing, etc.; 30). As a result, calculated detection
and QLs were significantly different between both enantiomers.
System Suitability Testing
System suitability tests (32) are an integral part of the
chromatographic method. These tests are used to verify that
Rs and reproducibility of the chromatographic system are
adequate for analysis purposes. During chromatographic
analysis, different system suitability parameters were calculated
(Table 4). Such parameters include Rs, number of theoretical
plates (N), height equivalent to a theoretical plate (HETP),
tailing factor (T), capacity factor (k′), and selectivity factor (α).
Robustness
The optimum HPLC conditions set for this method have been
slightly modified as a means to evaluate the method robustness.
Small changes in methanol ratio, flow rate, and ammonium
acetate concentration were applied (Table 5). It was found that
the Rs of IBP enantiomers was good under most conditions and it
remains unaffected by small deliberate changes of experimental
parameters. Variation in the experimental parameters without
Table 4. Results of system suitability tests for
determination of IBP enantiomers by the proposed HPLC
method
Parameter
Retention times,
tR; min
Capacity factor, k′
Dex-IBP
Levo-IBP
Reference value (33)
12.4 ± 0.07
16.1 ± 0.1
—
3.92
5.37
>2
Selectivity factor, α
1.37
Theoretical
plates, N
1835.06
HETP, mm
0.14
Tailing factor, T
0.86
Resolution, Rs
1574.26
0.16
0.80
2.64
Table 5. Robustness results for the HPLC methoda
Parameters
Methanol ratio, %
Flow rate,
mL/min
Ammonium acetate
concn, mM
29
1.47
30
1.47
31
1.45
1.2
1.52
1.3
1.53
1.5
1.52
102
1.87
100
1.88
98
1.86
Effect of experimental parameters on the Rs of IBP enantiomers.
b
Rs = Resolution.
change in Rs of IBP enantiomers indicates reliability and
robustness of the proposed method.
Application to Pharmaceutical Dosage Forms
The method was successfully applied for the determination
of dex-IBP in two commercial dosage forms, namely Brufen
tablets (200 mg IBP/tablet) and dexa-ibufen tablets (400 mg
dex-IBP/tablet; Figure 5c and d). The % recovery was calculated
as follows:
The concentration of dex-IBP in dosage form
(Cu) = Cs * Au/As
(1)
where Au and As are peak areas of dex-IBP in assay and standard
solutions, respectively, and Cs is the concentration of dex-IBP in
a standard solution.
% Recovery = (Cu/Cc) * 100 (2)
where Cc is the claimed concentration of dex-IBP in tablet dosage
form. The mean % recoveries ±SD for dex-IBP were calculated
as presented in Table 6. In summary, the developed RP-HPLC
method was stereospecific, reproducible, accurate, and sensitive. It
has been validated and successfully applied for the determination
of IBP enantiomers in bulk and pharmaceutical dosage form.
The present method offers significant advantages compared
to previously reported indirect chiral HPLC methods (11–15),
because most of them require lengthy precolumn derivatization
time. Different CSPs are reported in the literature for direct
enantioseparation and quantitation of IBP by HPLC (16–23).
The proposed method applies to the RP mode, which is
considered more ecologically friendly than the NP mode (19). It
also separates IBP in a relatively short period compared to other
Table 6. Determination of dex-IBPa in pharmaceutical
products by the proposed HPLC method
Claimed concn,
μg/mL
Found concn
(µg/mL)a ± SD
% Recovery
± SDb
Brufen tablet
50
48.06 ± 0.81
96.14 ± 1.89
Dexa-ibufen
tablets
100
98.14 ± 1.80
98.14 ± 1.80
Dosage form
≥2000
—
≤2
Rsb
a
>1
>1.5
Modification
a

b
IBP = Ibuprofen.
Mean of three determinations, n = 3.
Downloaded from https://academic.oup.com/jaoac/article/99/3/604/5658098 by Medical University of Plovdiv user on 04 April 2022
According to United States Pharmacopeia, a compound with the
same two-dimensional chemical structure as the drug substance
but differing in the three-dimensional orientation of substituents
at a chiral center within that structure is termed a stereomeric
impurity (32). So the specificity of the method was illustrated by
the complete separation of dex-IBP from levo-IBP as shown in
Figure 5a and b. The Rs value from levo-IBP was 2.64.
The method was applied for the determination of dex-IBP in
two pharmaceutical preparations. The peak of dex-IBP in the
chromatograms of tablet assay preparation (Figure 5c and d)
is typical to that obtained from standard solution of dex-IBP
at the same concentration without interferences, indicating the
specificity of the method.
El-Fatatry et al.: Journal of AOAC International Vol. 99, No. 3, 2016 611
reported methods (17, 18, 21, 23). Using the present method, large
numbers of samples may be analyzed in a relatively short period.
Moreover, several reported methods achieved poor baseline Rs
of IBP enantiomers (17, 20, 23). Others use achiral derivatization
before direct separation on CSP (20). Few of the reported direct
HPLC methods were not validated (18, 20).
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Downloaded from https://academic.oup.com/jaoac/article/99/3/604/5658098 by Medical University of Plovdiv user on 04 April 2022
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