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ACTA FACULTATIS PHARMACEUTICAE UNIVERSITATIS COMENIANAE
Tomus LVII 2010
CHIRAL CAPILLARY ELECTROPHORESIS WITH ON-LINE SAMPLE
PRETREATMENT AND SPECTRAL DETECTION IN PHARMACEUTICAL
AND BIOMEDICAL ANALYSIS
Mikuš, P.
Department of Pharmaceutical Analysis and Nuclear Pharmacy, Faculty of Pharmacy, Comenius
University, Bratislava
The aim of this review is to present briefly the recent advances in the pharmaceutical and biomedical analysis.
Herein, we focused at the on-line coupled capillary electrophoresis separation methods hyphenated with UV-VIS
absorbance spectral detection for the enantioselective analysis of trace drugs (antihistaminics, cardiovascular drugs) in
solid dosage forms and body fluids. The drugs (pheniramine and its analogues) and matrices (tablets, urine) presented
here have been chosen as representatives illustrating remarkable potentialities of the proposed analytical approach
(isotachophoresis-capillary zone electrophoresis-diode array detection, ITP-CZE-DAD) in pharmaceutical and
biomedical field. The ITP-CZE-DAD methods were characterized by the favorable performance parameters and they
were applied for the enantiomeric purity control of the drugs, enantioselective pharmacokinetic and metabolic studies.
Key words: isotachophoresis – capillary zone electrophoresis – column-coupling – spectral detection – pheniramine
analogues – charged cyclodextrin – enantiomer purity – pharmaceuticals – clinical analysis – multicomponent mixture
INTRODUCTION
Nowadays, conventional single column separation techniques (chromatographic, electrophoretic) are
usually accompanied by off-line sample preparation procedures when analyzing trace analytes present in
complex matrices with variable qualitative and quantitative composition. An external sample handling has
several limitations and disadvantages which can be improved by using on-line sample preparation
approaches offering enhanced (i) reliability, (ii) automatization and (iii) miniaturization of the analysis.
Spectral detection is suitable for (i) evaluating of separation process and (ii) more or less detailed structural
characterization of separated compounds (depending on the type of the spectral method).
The aim of this review is to show that a hyphenation of these two particular approaches, i.e. the column
coupling electrophoresis with the spectral detection, creates a powerful tool for solving advanced analytical
problems. Column coupling electrophoresis methods with diode array detection (DAD) were developed for
the enantioselective analysis of trace drugs (pheniramine and its derivatives) in the directly injected
(unpretreated) pharmaceutical (tablets) and clinical samples (urine). The drugs and matrices presented here
[1-3] have been chosen as representatives illustrating potentialities of the proposed analytical approach in
pharmaceutical and biomedical field. Anyway, the importance of this approach is further highlighted by
other related applications (pharmacokinetic studies of various chiral cardiovascular drugs in urine) in this
area [4-10].
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EXPERIMENTAL
Material
Disophrol-repetabs tablets (3 mg of dexbrompheniramine in the surface layer of one tablet) were treated
in the following way: ten tablets were put into 100 ml of distilled water and vortex for 3 minutes at 2000
rpm. Rest of the tablets (cores) was removed from the solution after 3 minutes. The stock solution of tablet
extract was kept in the refrigerator until the use and it was filtered before the use through the disposable
membrane filter made of Nylon of a 1.2 µm pore size (Millipore, Molsheim, France).
Clinical sample was prepared by the following way: one dose of the commercial pharmaceutical
preparation Fervex (25 mg of pheniramine per dose) was administered per-orally to female volunteer. The
urine samples were taken in different time periods after Fervex was administered (8.5 hours, 16.17 hours
and 23.17 hours, resp.) to investigate the elimination of pheniramine and its metabolites in urine. Each
urine sample was frozen (-18°C) immediately after the sampling and kept in the refrigerator until the use.
The sample was thawed out just before the manipulation and preparation of the sample. Each sample was
10 times diluted with pure water and immediately injected into the sampling loop of the electrophoretic
analyzer.
Methods
An ITAChrom EA-101capillary electrophoresis analyzer (J&M, Aalen, Germany), assembled in the
column-coupling configuration of the separation unit, was used in this work for performing the ITP-CZE
runs. The samples were injected by a 30 µl internal sample loop of the injection valve of the analyzer. An
ITP column was provided with an 800 µm I.D. capillary tube made of FEP (fluorinated ethylene-propylene
copolymer) and an on-column conductivity sensor. Its total length was 90 mm. A CZE column was
provided with a 320 µm I.D. capillary tube made of fused silica (J&W, Folsom, Canada) of a 240 mm total
length (180 mm to the detection cell).
A TIDAS, multiwavelength photometric absorbance diode array detector (J&M) was connected to an
on-column photometric detection cell, mounted on the CZE column, via optical fibers (J&M). The detector
operated under the following conditions: (1) scanned wavelength range 200 – 400 nm; (2) integration time
15 ms; (3) scan interval 0.225 s; (4) number of accumulations 15. The spectral data were acquired and
processed by the Spectralys program (version 1.82, J&M).
RESULTS AND DISCUSSION
On-line coupled isotachophoresis-capillary zone electrophoresis (ITP-CZE) separation methods with
diode array detection (DAD) were developed (optimized, validated and applied) for the enantioselective
analysis of trace drugs (pheniramine and its analogues) in pharmaceutical (tablets) and clinical samples
(urine).
Benefits of the on-line coupled isotachophoresis (ITP) stage in isotachophoresis-capillary zone
electrophoresis (CZE) combination presented in our work were (i) high sample load capacity, (ii)
preseparation and purification of drugs, (iii) preconcentration of sample constituents. In this way ITP
served as an ideal injection technique of on-line pretreated samples for the CZE stage.
The CZE stage provided final analysis of the drugs and their enantiomers. Here the enhanced
(enantio)separation selectivity was achieved by a ionizable chiral selector, carboxyethyl-β-cyclodextrin.
Potentialities of this chiral buffer additive were demonstrated in the recognition between the enantiomers of
the drugs on one hand as well as between the drugs and sample matrix constituents (including also
accompanied drugs in dosage forms, e.g. pseudoephedrine, paracetamol, vitamin C) on the other hand. This
(enantio)separation selectivity enabled to obtain pure zones of the analytes, suitable for their detection and
quantitation.
DAD in comparison with single wavelength UV detection can enhance the value of analytical
information when analytes and interferents have different spectra. In this context DAD was used to
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examine the purity of analytes zones. Obtained results indicated the pure zones confirming effective ITPCZE (enantio)separation process. Moreover, distinguishing the trace analytes signals superposed on the
baseline noise was provided with sufficient reliability (for this purpose the background correction and
smoothing procedure had to be applied to the raw DAD spectra).
The proposed ITP-CZE-DAD methods were characterized by favorable performance parameters
(sensitivity, linearity, precision, recovery, accuracy, robustness, selectivity). Validated methods were
successfully applied to (i) enantiomeric purity testing of dexbrompheniramine in commercial
pharmaceutical tablets and (ii) an enantioselective metabolic study of pheniramine (and its metabolites) in
human urine. Minimized sample handling / pretreatment makes this analytical approach suitable for (i) a
wide scale of samples and/or analytes, (ii) routine use, and (iii) analyses where enhanced reliability of
results is required.
Optimal separation conditions
Table 1. Electrolyte systems
Parameter
ITP1
Solvent
water
ITP2
water
Parameter
solvent
CZE1
water
CZE2
water
leading cation
concentration [mmol/l]
Na+
10
Na+
10
carrier cation
concentration [mmol/l]
glycine
25
EACA
25
counter ion
concentration [mmol/l]
acetate
20
acetate
20
counter ion
concentration [mmol/l]
acetate
100
acetate
20
pH
EOF suppressor
concentration [%, w/v]
terminating kationt
concentration [mmol/l]
4.75
HEC
0.1
Glycine
5
4.75
HEC
0.1
EACA
5
pH
EOF suppressor
concentration [%, w/v]
complexing agent
concentration [mg/ml]
3.1
HEC
0.1
CE-β-CD
5
4.5
HEC
0.1
CE-β-CD
5
counter ion
Acetate
acetate
concentration [mmol/l]
10
10
pH
3.5
4.5
Abbreviation: EOF – electroosmotic flow; HEC – hydroxyethylcellulose;
CE-β-CD – carboxyethyl-β-cyclodextrin; EACA – ε-aminocaproic acid
Figure 1. Direct ITP-CZE analyses of trace drugs enantiomers in unpretreated model samples. (a) Enantioseparation of
dexbrompheniramine standard in electrolyte system ITP1-CZE1 (Tab.1). LBP:DBP ratio was 0.16:99.84 and concentration of
trace enantiomer (impurity), LBP, was 9.10-8 mol.l-1 in the injected sample. (b) Enantioseparation of pheniramine racemic
standard in electrolyte system ITP2-CZE2 (Tab.1). Concentration of PHM was 7.10-8 mol.l-1 in the injected sample. The
detection wavelength in CZE step was 261 nm. The driving currents in the ITP and CZE stages were 200 µA and 80 µA,
respectively. Other working conditions are in the part Experimental.
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Validation parameters
Table 2. Performance parameters of ITP-CZE methods applied for enantioseparations of racemic drugs
Parameter
t [min]
st [min]
a [mAU.s.min-1]
sa [mAU.s.min-1]
b [mAU.s.min-1.mg-1.l]
sb [mAU.s.min-1.mg-1.l]
RSS
R
r2
QC
LOD [µg.l-1]
LOQ [µg.l-1]
N
H [µm]
R
repeatability (RSD) [%]
recovery [%]
accuracy (RE) [%]
robustness (∆R) [%]
a
ITP1-CZE1, water sample
LBP
DBP
8.022
8.340
0.0356
0.0359
0.3166
0.3385
0.0369
0.0643
48.8466
50.4837
0.26191
0.4570
0.0162
0.0494
0.99993
0.99980
0.99990
0.99960
0.88267
1.48727
2.5
4.2
7.5
12.7
33400
30450
5.5
6.1
2.94
0.99
1.65
98.1
97.8
-1.9
-2.2
< 3.9
ITP2-CZE2, urine samplea
PHM 1
PHM 2
27.67
29.95
0.360
0.400
-0.191
-0.287
0.061
0.081
74.47
74.96
0.607
0.811
0.0625
0.1116
0.99980
0.99965
0.99960
0.99930
1.62825
2.19733
5.2
6.8
7.7
10.1
17600
17600
10.2
10.2
4.4
1.30
1.33
98.5
98.3
-1.5
-1.7
< 2.7
Comparable data were obtained also for PHM dissolved in demineralized water.
Figure 2. Spectral evaluation of analytes zones using ITP-CZE-DAD method. DAD spectra of the drugs enantiomers, (a) BP
and (b) PHM, were taken from the concentration maxima of their zones (peaks). Upper traces illustrate raw (unprocessed)
spectra while lower traces final (processed) spectra (passed through the background correction and smoothing procedure).
Samples, separation and other working conditions as in Fig. 1.
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Table 3. Proof of selectivity of the proposed methods. Pearson ' s correlation coefficients for DAD
spectra of the drugs in real samplesa
Raw spectrumc
Corrected spectrumc
Analyteb
LBP
0.8125
0.98958
DBP
0.8347
0.99825
PHM1
0.9419
0.9971
PHM2
0.8853
0.9995
M1
0.8507
0.9942
M2
0.8945
0.9950
I
0.0827
0.1153
a
DAD spectra of real samples were compared with DAD spectra of reference samples (racemic standards).
b
LBP, DBP in pharmaceutical tablets and PHM1, PHM2, M1, M2, I in clinical urine samples;
c
Average values from 5 measurements of pharmaceutical tablets Disophrol Repetabs (batch №.1) and clinical
urine samples taken 16.67 hours after Fervex per oral application
Application examples
Figure 3. Electropherograms from CZE step of ITP-CZE-DAD combination during the chiral analyses of the drugs in real
pharmaceutical and clinical samples. (a) Enantiomeric purity testing of BP in tablets Disophrol Repetabs (batch №.1, obtained
data are in Tab. 4). (b) Enantioselective metabolic study of PHM in clinical urine sample taken 16.67 h after Fervex was
administered to female volunteer (obtained data are in Tab. 5). For the samples preparations see the part Experimental.
Separation, detection and other working conditions as in Fig.1.
Table 4: Enantiomeric purity testing of DBP in pharmaceutical samples Disophrol tablets
Batch №.
Content (mg per tablet)
RSD %, n=5
Enantiomeric ratio %
DBP
LBP
DBP
LBP
DBP:LBP
1
5.82
0.128
0.81
2.84
97.85 : 2.15
2
5.77
0.111
1.15
3.57
98.11 : 1.89
3
5.71
0.089
0.96
3.31
98.47 : 1.53
4
5.78
0.098
0.91
3.02
98.33 : 1.67
5
Table 5: Enantioselective metabolic study of PHM in clinical urine samplesa
Time [hours]
8.50
16.67
23.83
M1
0.773
0.812
0.274
Concentration level [mg.l-1]
PHM1
M2
1.087
0.630
0.483
0.692
0.295
0.229
PHM2
0.959
0.371
0.293
a
Urine samples taken at different times after using 1 dose of Fervex (equivalent to 25 mg PHM) by female volunteer.
Clinical urine samples significantly differed from each other in qualitative and quantitative composition of sample matrix
constituents.
CONCLUSION
The performance parameters of the ITP-CZE-DAD methods (limit of detection/quantification, linearity, precision,
accuracy, recovery, robustness, selectivity, separation efficiency), as well as the application examples (enantiomeric
purity control of the commercial drugs, enantioselective pharmacokinetic and metabolic studies) clearly illustrated
potentialities of this unique analytical approach in the pharmaceutical and biomedical field. Hence, it can be concluded
that the ITP-CZE-DAD methods are suitable for a routine use in advanced analytical applications, i.e. ultratrace
determinations, complex matrices, enantioselective separations, direct injections of unpretreated samples, basic
structural characterization of separated analytes and spectral information in composition of separated zones, as well as
combinations of these particular tasks.
Acknowledgment: This work was supported by the grant from the Slovak Grant Agency for Science under the project
VEGA №. 1/0003/08.
REFERENCES
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electrophoresis provided with a fiber-based diode array detection and an on-line sample pretreatment by
capillary isotachophoresis. Electrophoresis 28, 2007, p. 2738-2747.
MARÁK, J. – MIKUŠ, P. – MARÁKOVÁ, K. – KANIANSKY, D. – VALÁŠKOVÁ, I. – HAVRÁNEK, E.:
Potentialities of ITP-CZE method with diode array detection for enantiomeric purity control of
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MIKUŠ, P. – MARÁKOVÁ, K. – MARÁK, J. – KANIANSKY, D. –VALÁŠKOVÁ, I. – HAVRÁNEK, E.:
Possibilities of column coupling electrophoresis provided with a fiber-based diode array detection in
enantioselective analysis of drugs in pharmaceutical and clinical samples. J. Chromatogr. A 1179, 2008, p. 916.
MIKUŠ, P. – MARÁKOVÁ, K. – MARÁK, J. – NEMEC, I. – VALÁŠKOVÁ, I. – HAVRÁNEK, E.: Direct
quantitative determination of amlodipine enantiomers in urine samples for pharmacokinetic study using online coupled isotachophoresis-capillary zone electrophoresis separation method with diode array detection. J.
Chromatogr. B 875, 2008, p. 266-272.
MIKUŠ, P. – MARÁKOVÁ, K. – MARÁK, J. – NEMEC, I. – VALÁŠKOVÁ, I. - HAVRÁNEK, E.: Direct
determination of celiprolol in human urine using on-line coupled isotachophoresis-capillary zone
electrophoresis method with fiber-based diode array detection. Electrophoresis 29, 2008, p. 4561-4567.
MIKUŠ, P. – MARÁKOVÁ, K. – MARÁK, J. – NEMEC, I. – VALÁŠKOVÁ, I. – HAVRÁNEK, E.:
Determination of amlodipine enantiomers in pharmaceuticals using capillary electrophoresis separation and
diode array detection. Pharmazie 64, 2009, p. 76-79.
MIKUŠ, P. – MARÁKOVÁ, K. – VALÁŠKOVÁ, I. – HAVRÁNEK, E.: Enantiomeric purity control of
levocetirizine in pharmaceuticals using anionic cyclodextrin mediated capillary electrophoresis separation
and fiber-based diode array detection. Pharmazie 64, 2009, p. 423-427.
MIKUŠ, P. – MARÁKOVÁ, K. – MARÁK, J. – NEMEC, I. – VALÁŠKOVÁ, I. – HAVRÁNEK, E.:
Pharmacokinetic study of amlodipine in human urine using on-line coupled isotachophoresis-capillary zone
electrophoresis with diode array detection. Curr. Pharm. Anal. 5, 2009, p. 171-178.
MIKUŠ, P. – MARÁKOVÁ, K.: Advanced capillary electrophoresis for chiral analysis of drugs, metabolites
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10. MIKUŠ, P. – MARÁKOVÁ, K.: Chiral capillary electrophoresis with on-line sample preparation. Curr.
Pharm. Anal. 6, 2010, p. 76-100.
Registred: October 12, 2010
Accepted: November 29, 2010
doc. RNDr. Peter Mikuš, PhD.
Faculty of Pharmacy
Comenius University
Odbojárov 10
832 32 Bratislava
Slovak Republic
CHIRÁLNA KAPILÁRNA ELEKTROFORÉZA S ON-LINE PREDÚPRAVOU VZORKY
A SPEKTRÁLNOU DETEKCIOU VO FARMACEUTICKEJ A BIOMEDICÍNSKEJ ANALÝZE
Mikuš, P.
Department of Pharmaceutical Analysis and Nuclear Pharmacy, Faculty of Pharmacy, Comenius University, Bratislava
Cieľom tohto review je stručne prezentovať najnovšie pokroky vo farmaceutickej a biomedicínskej analýze. Tu
sme zamerali pozornosť na on-line spojené separačné metódy kapilárnej elektroforézy v kombinácii s UV-VIS
absorpčnou spektrálnou detekciou pre enantioselektívne analýzy stopových liečiv (antihistaminiká, liečivá s
kardiovaskulárnym účinkom) v pevných liekových formách a telových tekutinách. Liečivá (feniramín a jeho analógy)
ako aj matrice (tablety, moč) prezentované v tejto práci boli vybrané ako reprezentatívne na ilustráciu pozoruhodných
možností navrhnutého analytického prístupu (izotachoforéza-kapilárna zónová elektroforéza-diode array detekcia, ITPCZE-DAD) vo farmaceutickej a biomedicínskej oblasti. ITP-CZE-DAD metódy sú charakterizované priaznivými
validačnými parametrami a boli aplikované na kontrolu enantiomérnej čistoty komerčných liekov, ako aj
enantioselektívne farmakokinetické a metabolické štúdie.
Acta Facult. Pharm. Univ. Comenianae 57, 2010, p. strana-strana.
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