Hongjing Fu

Wenhai Jin
Hua Xiao
Chuanhui Xie
Baochuan Guo
Hanfa Zou
National Chromatographic
R&A Center,
Dalian Institute
of Chemical Physics,
The Chinese Academy
of Sciences,
Dalian, China
Determination of basic pharmaceuticals in human
serum by hydrophilic interaction capillary
electrochromatography
Hydrophilic interaction capillary electrochromatography (HI-CEC) for the determination
of basic pharmaceuticals spiked in human serum is described. The organic modifier
content, ionic strength, and pHvalue of the mobile phase as well as the applied voltage
are optimized for separation and elution of these drug analytes. Excellent separation
was achieved for drugs using a mobile phase composition of 80% v/v acetonitrile in
100 mM triethylamine phosphate (TEAP) buffer at pH 2.8 with column efficiencies for
analytes more than 200000 plates/m. The samples of human serum spiked with basic
drugs were directly injected after a simple acetonitrile treatment. The linear range and
reproducibility of these basic drugs using an external and internal standard method
were compared. As a result, the reproducibility could be greatly improved by using
the internal standard method. Good calibration curves with regression coefficients
more than 0.998 in the range of 5160 mg/mL were observed with the internal standard
method. The limits of quantitation, based on standards with acceptable relative stand-
ard deviations (RSDs), were below 5 mg/mL. The intra- and inter-day precisions, deter-
mined as RSDs, were less than 4.57%.
Keywords: Basic pharmaceuticals / Capillary electrochromatography / Human serum / Hydro-
philic interaction / Strong-cation exchange DOI 10.1002/elps.200305757
1 Introduction
Basic substances compose a large category in pharma-
ceuticals. Their separation and analysis in body fluids are
important for therapeutic drug monitoring and for forensic
application [14]. High-performance liquid chromatogra-
phy (HPLC) has been used routinely for measuring the
level of basic drugs in serum [57] but it often involves
long method development times. Recently, capillary elec-
trophoresis (CE) and capillary electrochromatography
(CEC) have been found to be attractive approaches for
analysis of drugs in body fluids due to their shorter analy-
sis time, higher column efficiency, and much lower sam-
ple consumption [8, 9]. The main advantage of CEC over
CE is that both neutral and charged compounds can be
separated in a single run. Most separations of basic drugs
were carried out in reversed-phase CEC mode on silica-
based stationary phases [10, 11]. However, peak tailing
of basic compounds was frequently observed due to the
undesirable adsorption between basic compounds and
silanols on the surface of the stationary phases. Further-
more, many basic drugs with high polarity have weak
retention on the reversed-phase stationary phase. As the
addition of a competing amine to the mobile phase at low
pH is an effective approach to solve the peak tailing in
HPLC, Lurie et al. [12] first applied this method for
separation of basic solutes in reversed-phase CEC, and
both the peak shape and column efficiency for basic
solutes were significantly improved. Other reports also
confirmed their results [13, 14]. Alternative solutions to
the problem of tailing peaks are the use of other types
of stationary phases. The separations of basic solutes
carried out in CEC mode using a pure silica stationary
phase were reported by several groups, the addition of
competing amines being necessary in some cases [15
17]. Recently, strong ion-exchangers have attracted
much attention in the separation of basic solutes. In
addition to providing a substantial EOF even with low-
pH buffers, another attractive feature of strong cation
exchange capillary electrochromatography (SCX-CEC)
is that amine additives have not to be added to the
mobile phase to improve peak shape. A promising sepa-
ration of tricylic antidepressants (TCAs) and extreme
high column efficiency was obtained in CEC using a
silica-based strong cation exchange column [18]. The
peak compression effect on SCX-CEC was discussed
Correspondence: Dr. Hanfa Zou, National Chromatographic
R&A Center, Dalian Institute of Chemical Physics, The Chinese
Academy of Sciences, Dalian 116011, China
E-mail: zouhfa@mail.dlptt.ln.cn
Fax: 186-411-3693407
Abbreviations: HI, hydrophilic interaction; HILIC, hydrophilic
interaction chromatography; IS, internal standard; TEAP, triethyl-
amine phosphate
600 Electrophoresis 2004, 25, 600606
2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Electrophoresis 2004, 25, 600606 HI-CEC of basis pharmaceuticals 601
[19, 20]. Furthermore, types and pore size of strong cat-
ion exchange stationary phases affecting the separation
of basic solutes were also investigated [21, 22].
Hydrophilic interaction capillary electrochromatography
(HI-CEC) is an alternative mode of electrochromatogra-
phy where separations are performed in aqueous-organic
mobile phases on polar stationary phases. This mode is
similar to normal-phase electrochromatography in which
polar compounds are more retarded than nonpolar com-
pounds. However, it is not conducted in pure organic
mobile phases, but water serves as the strong solvent
and the retention of analytes can be effectively manipu-
lated by varying the organic modifier concentration in the
mobile phase. HI-CEC has been demonstrated to be an
efficient method for the separation of polar compounds
including small molecules with different polarity and pep-
tides [23, 24].
In this paper, we present a rapid and simple procedure for
simultaneous screening of 13 basic drugs using HI-CEC.
Efforts were firstly focused on the optimization of analyti-
cal conditions, then the HI-CEC method was also applied
to determination of seven basic drugs in spiked human
serum samples.
2 Materials and methods
2.1 Instrumentation and materials
All CEC experiments were performed on a Beckman
P/ACE 5510 instrument (Beckman, Fullerton, CA, USA).
A Waters 510 pump (Waters, Milford, MA, USA) was
used to pack the capillary columns. Fused-silica capil-
laries (50 mm ID, 365 mm OD) were obtained from Yong-
nian Optic Fiber Plant (Hebei, China). PolySULFOETHYL
A (5 mm, 300 ) was a gift from PolyLC (Columbia, MD,
USA).
2.2 Chemicals, buffers, and standard solutions
Acetonitrile was of chromatographic grade, all other re-
agents used were of analytical reagent grade. Ultrapure
water used for the prepation of solutions was produced
by a Milli-Q water system (Millipore, Bedford, MA, USA).
The stock solution of triethylamine phosphate buffer
(TEAP, 1 M in phosphate), was prepared by addition of
triethylamine to a concentrated solution of phosphoric
acid until the desired pH was obtained, which was meas-
ured before mixing the buffer with the organic modifier.
The TEAP stock solution was filtered through a 0.45 mm
membrane. Mobile phases were prepared by addition of
the required volume of acetonitrile and TEAP buffer to a
volumetric flask, followed by addition of water to 1 mL
below the scale. Then the flask was placed in an ultra-
sonication bath for 5 min. Water was then added to the
mark after the solution reached roomtemperature. Before
the run, the mobile phase was further degassed in an
ultrasonic bath for 30 min. Blank human serum (collected
on sodium heparinate) was obtained from healthy volun-
teers free of drugs. A concentrated stock solution of basic
drugs except caffeine was prepared in acetonitrile at a
concentration of 1.6 mg/mL. A stock solution of caffeine
as internal standard (IS) was prepared in acetonitrile at
a concentration of 10 mg/mL. All stock solutions were
stored at 47C.
2.3 Column preparation and separation
conditions
CEC columns were packed in-house by a slurry packing
technique as reported in [25, 26]. Columns used for CEC
were 27 cm long with a packed length of 20 cm. Before a
CEC experiment, the columns were flushed with mobile
phase for 30 min using a syringe. The columns were then
conditioned in the CEC instrument with the mobile phase
for at least 2 h. The applied voltage was first ramped from
0 to 5 kV over 10 min and then operated at 10 kV. The
temperature was kept at 257C and the detection wave-
length was set at 214 nm. The separation voltage was
set at 10 kV if not otherwise stated. All CEC experiments
were performed in the isocratic elution mode. The in-
jections were performed hydrodynamically by applying a
pressure of 0.5 psi for 90 s.
2.4 Clean-up procedures for spiked serum
samples
The clean-up procedure for spiked serum samples was
carriedout accordingtotheprocedurereportedby Shihabi
et al. [27] as follows: 100 mL of human serum spiked with
basic drugs and 100 mL IS, (10 mg/mL caffeine in aceto-
nitrile) were vortex-mixed for 30 s with 300 mL acetonitrile
for theinternal standardmethod. For the external standard
method, 100 mL volume of human serumspiked with basic
drugs were vortex-mixed for 30 s with 400 mL acetonitrile
without IS. The mixture was centrifuged at 11006g for
10 min and the supernatant was prepared for injection.
2.5 Method validation
Calibration standards were prepared at concentrations of
6.25, 12.5, 25, 50, 100, 200, 400, 800, and 1600 mg/mL
by diluting appropriate amounts of the stock solution
with acetonitrile. For spiked serum standards [28], 100 mL
2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
C
E
a
n
d
C
E
C
602 H. Fu et al. Electrophoresis 2004, 25, 600606
of the above standards ranging from 6.25 mg/mL to
1.6 mg/mL was added to 0.9 mL of normal human serum
to yield the spiked calibration standards ranging from
0.625 to 160 mg/mL. New standard curves were per-
formed with each sample batch to minimize the analytical
variability. Calibration curves were generated by plotting
the ratio of the peak height of the compound and IS
against the theoretical concentrations for the internal
standard method, while the calibration curves with the
external standard method were generated by plotting the
peak height of a compound against theoretical concen-
trations. Correlation coefficients describing the calibra-
tion curves were determined by the linear regression anal-
ysis. The quality control sample was prepared at concen-
tration of 30 mg/mL. The recoveries of basic drugs spiked
in human serum were estimated using standard samples
at a concentration of 30 mg/mL by comparing the peak
height ratios from pretreated serum standard samples
with those from a calibration curve prepared from
analytes. The intra-day and inter-day precisions of the
method were also determined from the experimental
data. The limit of quantification was set at the lowest con-
centration of the calibration curve. The limit of detection,
defined as three times the signal-to-noise ratio, was
determined by injecting diluted solutions of a mixture of
basic drugs.
3 Results and discussion
Generally, in CEC samples are injected electrokinetically
or hydrodynamically [29, 30]. In order to estimate the
effect of different injection techniques on the reproducibil-
ity, 150 mg/mL caffeine as the test sample was performed
by electrokinetic injection at 5 kV65 s and hydrodynamic
injection at 0.5 psi690 s, respectively. The results of
RSDs of the peak height were 3.9% (n = 10) and 1.5%
(n = 10) in two methods, respectively. Although an on-line
concentration technique was also described in CEC [31]
by electrokinetic injection to enhance the detection sensi-
tivity, unfortunately the reproducibility was not satisfying.
Furthermore, the hydrodynamic injection showed a rela-
tively better reproducibility and loss of resolution was not
observed. Thus, hydrodynamic injection was chosen for
further experimental studies.
In CEC, the electroosmotic flow (EOF) is the main driving
force responsible for the movement of analytes and
mobile phase through the columns. Acidic mobile phases
are advantageous to the separation of basic compounds
on silica-based stationary phases because of the same
direction of electrophoresis and EOF. However, conven-
tional silica-based stationary phases cannot produce
enough EOF and lead to long elution times for basic ana-
lytes under acidic eluent conditions. Thus, ion-exchange
CEC has been reported for the separation of basic com-
pounds [1822] because the ion-exchange stationary
phases can assure substantial EOF in a much wider pH
range than conventional reversed-phase packing. Poly-
SULFOETHYL A, a stationary phase frequently used in
hydrophilic interaction chromatography (HILIC) [32] has
been chosen as the stationary phase for the HI-CEC
experiments, which is prepared by chemically bonding a
polypeptide, poly(2-sulfoethyl aspartamide), on a silica
gel matrix and proved to be very hydrophilic, particularly
when compared to other silica-based and nonsilica-
based matrices [33]. Moreover, the sulfonic groups on
the surface of the stationary phase can easily produce
substantial EOF in a wide pH range [32].
3.1 Optimization of separation conditions for
HI-CEC
In order to find the optimal conditions for the separation of
basic drugs, the effects of the content of organic modifier,
pH, ionic strength, as well as the applied voltage were
investigated. As demonstrated in the separation of pep-
tides in both HILIC [32] and HI-CEC [24], the content of
organic modifier in the mobile phase has a great influence
on the resolution and selectivity of polar compounds, and
hydrophilic interactions are promoted by increasing the
organic modifier content. Therefore, the influence of the
acetonitrile content on the retention of eight basic drugs
Figure 1. Effect of acetonitrile concentration on the
retention time of basic drugs in HI-CEC. Experimental
conditions: column packed with 5 mm PolySULFOETHYL
A, 27 cm (packed length, 20 cm)650 mm ID; separation
voltage, 10 kV; injection, 0.5 psi690 s. Mobile phase,
acetonitrile concentration from 50% to 80% v/v in
100 mM TEAP buffer (pH 2.8). Solutes: (1) amobarbital;
(2) phenobarbital; (3) barbital; (4) caffeine; (5) sulfanil-
amide; (6) theophylline; (7) 2,4-dimethylquinoline; (8) pro-
pranolol.
2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Electrophoresis 2004, 25, 600606 HI-CEC of basis pharmaceuticals 603
Figure 2. Effect of the eluent ionic strength on the reten-
tion time of basic drugs in HI-CEC. Experimental condi-
tions: mobile phase, 80% v/v acetonitrile in TEAP buffers
with varying concentrations from 40 to 150 mM (pH 2.8).
Other experimental conditions and tested solutes are the
same as in Fig. 1.
in HI-CEC was investigated (Fig. 1). As a result, the higher
the acetonitrile content, the better the separation
amongst the basic drugs will be. Thus, the volume ratio
of acetonitrile was kept at 80%in the TEAP buffer. Gener-
ally, an acidic eluent was advantageous in CEC separa-
tions of basic compounds due to the same direction of
both electrophoresis and EOF. So pH 2.8 was selected
as the TEAP buffer pH.
The effect of the ionic strength of the mobile phase on the
retention of basic drugs is shown in Fig. 2. It can be seen
that the retention time of basic drugs was decreased with
the increasing ionic strength of the mobile phase. Al-
though the difference in retention time for different drugs
was found to increase using a mobile phase with lowionic
strength, the peak shape in high ionic strength was supe-
rior to that in low ionic strength. However, too high ionic
strength of mobile phase will cause Joule heating and
lead to bubble formation and current breakdown. So a
relatively optimal phosphate concentration was selected
at 100 mM in the mobile phase.
The effect of the applied voltage on basic drug separa-
tion is shown in Fig. 3. The higher the applied voltage,
the shorter the retention time of the drugs. However,
too high voltage also leads to Joule heating. Thus, a rela-
tively optimal applied voltage of 10 kV was selected in
this work.
Figure 3. Effect of the applied voltage on retention
time of basic drugs in HI-CEC. Experimental conditions:
mobile phase, 80%v/v acetonitrile in 100 mM TEAP buffer
(pH 2.8); applied voltages varied from 5 to 17.5 kV. Other
experimental conditions and solutes are the same as in
Fig. 1.
The optimized operational parameters are summarized in
Table 1 for HI-CEC separation of basic drugs. Figure 4
shows a typical electrochromatogram of the separation
of 13 standard basic drugs. The average of the column
efficiency of these compounds is more than 200000 the-
oretical plates per meter (N/m). For most of the tested
solutes, the order of elution follows their hydrophilicity.
For example, the analytes were eluted in the order of
amobarbital , phenobarbital , barbital ,caffeine , sul-
fanilamide , theophylline. However, the elution order
was not always consistent with the hydrophilicity of ana-
lytes. For example, 2,4-dimethylquinoline, which is much
more hydrophobic than the solutes eluted before it, was
more retarded on the HI-CEC column. This is because
Table 1. Optimized operational parameters for HI-CEC
separation of basic drugs
Column Fused-silica capillary, 27 cm (20 cm packed
with 5 mm, 300 PolySULFOETHYL A
particles)650 mm ID
Mobile phase 80% v/v acetonitrile in 100 mM TEAP buffer
TEAP buffer pH 2.8
Applied voltage 10 kV
Injection 0.5 psi for 90 s
Temperature 257C
Detection UV at 214 nm
2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
604 H. Fu et al. Electrophoresis 2004, 25, 600606
Figure 4. HI-CEC separation of standard basic drugs.
For experimental conditions see Table 1. Solutes: (1) amo-
barbital; (2) phenobarbital; (3) barbital; (4) caffeine; (5) sul-
fanilamide; (6) theophylline; (7) 2,4-dimethylquinoline;
(8) propranolol; (9) metoprolol; (10) gramine; (11) atenolol;
(12) nicotine; (13) quinoline.
that besides the hydrophilic interaction, both the electro-
phoretic mechanism and electrostatic interaction are
responsible for the retention of the solutes. It can also
be seen in Fig. 4 that peak tailing which is often
observed in HPLC for basic compounds, is not ob-
served in HI-CEC. HI-CEC was also applied to the
separation of seven basic drugs extracted from spiked
human serum (Fig. 5). It can be seen that the compo-
nents in human serum didnt interfere the separation of
basic drugs after the serum samples were treated with
acetonitrile.
Figure 5. HI-CEC separation of basic drugs spiked in
human serum. For experimental conditions see Table 1.
Concentration of basic drugs, 40 mg/mL. Solutes: (1)
amobarbital; (2) phenobarbital; (3) barbital; (4) caffeine;
(5) sulfanilamide; (6) theophylline; (7) 2,4-dimethylquino-
line; (8) propranolol.
3.2 Calibration curves
The calibration curves were made on three different days
and used to determine the sample concentration. Table 2
gives the analytical parameters of this method. The linear-
ities of amobarbital, phenobarbital, and barbital were
found in the range of 5160 mg/mL. The linear calibration
range of sulfanilamide was 2.5160 mg/mL, of theophyl-
line 0.625160 mg/mL, of 2,4-dimethyl quinoline and pro-
Table 2. Analytical parameters for the determination of basic drugs spiked in human serum by
HI-CEC
Basic drugs Detection
limit
a)
(mg/mL)
Calibration
range
(mg/mL)
Correlation
coefficient
Repeatability
(RSD, %)
b)
Amobarbital 5 5160 0.9990
c)
0.9975
d)
1.75
c)
3.43
d)
Phenobarbital 5 5160 0.9994
c)
0.9989
d)
1.81
c)
3.55
d)
Barbital 5 5160 0.9993
c)
0.9984
d)
1.79
c)
4.01
d)
Sulfanilamide 2.5 2.5160 0.9990
c)
0.9983
d)
2.15
c)
3.15
d)
Theophylline 0.625 0.625160 0.9988
c)
0.9981
d)
2.61
c)
3.19
d)
2,4-Dimethyl quinoline 1.25 1.25160 0.9989
c)
0.9979
d)
2.25
c)
3.53
d)
Propranolol 1.25 1.25160 0.9992
c)
0.9979
d)
2.91
c)
4.51
d)
a) Detection limit based on S/N = 3
b) n = 5
c) Data obtained with the internal standard method
d) Data obtained with the external standard method
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Electrophoresis 2004, 25, 600606 HI-CEC of basis pharmaceuticals 605
pranolol 1.25160 mg/mL. The determination coefficients
were 0.99750.9989 with the external standard method.
However, better correlation coefficients between 0.9988
0.9994 were obtained with the internal standard method
though the calibration ranges of these drugs with both
internal and external standard methods were almost the
same. The RSDs of the correlation coefficients for the
calibration curves with the internal standard method
were also much better than those with the external stand-
ard method. The limits of detection of these basic drugs,
which are defined as three times the baseline noise level,
were , 5 mg/mL in both external and internal standard
method. The results indicate that the reproducibility of
HI-CEC method could be partly improved by adding an
internal standard. The developed HI-CEC method may
be used for therapeutic monitoring of drugs with narrow
therapeutic windows, because the linear ranges of the
present method covers the therapeutic window of some
basic drugs. For example, the therapeutic window of
theophylline is in the range of 1020 mg/mL and that of
phenobarbital between 15 and 40 mg/mL [34].
3.3 Recovery, accuracy, and precision
The recoveries of the basic drugs from normal serum
were in the range of 97.86100.21% at a concentration
of 30 mg/mL (n = 5). The RSDs were between 2.28 and
4.75%. The results from the analysis of basic drugs at a
concentration of 30 mg/mL to estimate the accuracy and
precision of the method are presented in Table 3. The
inter-day precisions were estimated in the range of 2.28
4.57%. The intra-day precisions were estimated in the
range of 1.301.95%.
Table 3. Recovery of the method
Drugs Amount
added
Recovery Relative
standard
deviation
(mg/mL) (n = 5, %) (n = 5, %)
Amobarbital 30 97.86 4.57
Phenobarbital 30 98.67 3.64
Barbital 30 98.35 3.18
Sulfanilamide 30 99.70 2.89
Theophyline 30 99.32 2.65
2,4-Dimethylquinoline 30 100.21 2.66
Propranolol 30 98.49 2.28
4 Concluding remarks
HI-CEC has proved to be an efficient tool to separate
basic drugs, especially for those with high polarity. More-
over, obvious peak tailing of basic compounds was not
Table 4. Precision of the method
Drugs Amount
added
(mg/mL)
Intra-day (n = 5) Inter-day (n = 5)
Amount
determined
(mg/mL)
RSD
(%)
Amount
determined
(mg/mL)
RSD
(%)
Amobarbital 30 29.5660.47 1.95 29.3661.06 4.57
Phenobarbital 30 29.7460.45 1.39 29.6460.99 3.64
Barbital 30 29.6260.30 1.31 29.5160.87 3.18
Sulfanilamide 30 29.9260.34 1.35 29.9160.76 2.89
Theophylline 30 29.7360.39 1.49 29.7960.64 2.65
2,4-Dimethyl-
quinoline
30 30.0660.41 1.41 30.0260.70 2.66
Propranolol 30 29.5860.34 1.30 29.5560.64 2.28
observed in this CEC mode. Based on the results ob-
tained in a preliminary validation study, HI-CEC appears
to be suitable for the quantitative determination of basic
drug mixtures spiked in human serum with a single
extraction step. Good calibration curves covering the
range of 5160 mg/mL with correlation coefficients .
0.9988 were obtained with the internal standard method.
The limits of quantitation, based on standards with
acceptable RSDs, were , 5 mg/mL. The intra- and inter-
day precisions, determined as RSDs, were acceptable.
Financial supports from the National Natural Sciences
Foundation of China (No.20075032) and the Knowledge
Innovation programof DICP to Dr. Hanfa Zou are gratefully
acknowledged.
Received July 7, 2003
5 References
[1] Manetto, G., Crivellente, F., Tagliaro, F., Ther. Drug Monit.
2000, 22, 8488.
[2] Gram, L. F., Int. Congress Series 2001, 1220, 117 123.
[3] Gergov, M., Robson, J. N., Ojanper, I., Heinonen, O. P.,
Vuori, E., Forensic Sci. Int. 2001, 121, 108115.
[4] Bengtsson, F., Lundmark, J., Nordin, C., Reis, M., Wlinder,
J., Eur. Neuropsychopharmacol. 1997, 7, S191S192.
[5] Minkler, P. E., Hoppel, C. L., J. Chromatogr. 1988, 428, 388
394.
[6] Olesen, O. V., Plougmann, P., Linnet, K., J. Chromatogr. B
2000, 746, 233239.
[7] Olesen, O. V., Linnet, K., J. Chromatogr. B 1996, 675, 8388.
[8] Hiroyuki, N., Electrophoresis 1999, 20, 32373258.
[9] Altria, K. D., J. Chromatogr. A 1999, 856, 433463.
[10] Enlund, A. M., Hagman, G., Isaksson, R., Westerlund, D.,
Trends Anal. Chem. 2002, 21, 412427.
[11] McKeown, A. P., Euerby, M. R., Lomax, H., J. Sep. Sci. 2002,
25, 12571268.
2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
606 H. Fu et al. Electrophoresis 2004, 25, 600606
[12] Lurie, I. S., Conver, T. S., Ford, V. L., Anal. Chem. 1998, 70,
45634569.
[13] Dittmann, M. M., Masuch, K., Rozing, G. P., J. Chromatogr.
A 2000, 887, 209221.
[14] Hilhorst, M. J., Somsen, G. W., de Jong, G. J., J. Chroma-
togr. A 2000, 872, 315321.
[15] Lai, E. P. C., Dabek-Zlotorzynska, E., Electrophoresis 1999,
20, 23662372.
[16] Wei, W., Luo, G., Hua, G., Yan, C., J. Chromatogr. A 1998,
817, 6574.
[17] Gillott, N. C., Euerby, M. R., Johnson, C. M., Barrett, D. A.,
Shaw, P. N., Chromatographia 2000, 51, 167174.
[18] Smith, N. M., Evans, M. B., Chromatographia 1995, 41, 197
203.
[19] Enlund, A. M., Andersson, M. E., Hagman, G., J. Chroma-
togr. A 2002, 979, 335344.
[20] Sthlberg, J., Anal. Chem. 1997, 69, 38123821.
[21] Smith, N., Evans, M. B., J. Chromatogr. A 1999, 832, 4154.
[22] Enlund, A. M., Isaksson, R., Westerlund, D., J. Chromatogr.
A 2001, 918, 211220.
[23] Ye, M., Zou, H., Kong, L., Lei, Z., Wu, R., Ni, J., LC?GC2001,
19, 1076, 1078, 1080, 1082, 1084, 1086.
[24] Fu, H., Jin, W., Xiao, H., Huang, H., Zou, H., Electrophoresis
2003, 24, 20842091.
[25] Ye, M., Zou, H., Liu, Z., Ni, J., Zhang, Y., Anal. Chem. 2000,
72, 616621.
[26] Zhang, Y., Shi, W., Zhang, L., Zou, H., J. Chromatogr. A
1998, 802, 5971.
[27] Shihabi, Z. K., Hinsdale, M. E., J. Chromatogr. B 1996, 683,
115118.
[28] Chou, C., Brown, M. P., Merritt, K. A., J. Chromatogr. B2000,
742, 441445.
[29] Coln, L.A., Reynolds, K. J., Alicea-Maldonado, R., Fermier,
A. M., Electrophoresis 1997, 18, 21622174.
[30] Pyell, U., J. Chromatogr. A 2000, 892, 257278.
[31] Chien, R. L., Burgi, D. S., J. Chromatogr. 1991, 559, 141
152.
[32] Alpert, A. J., J. Chromatogr. 1990, 499, 177196.
[33] Burke, T. W. L., Mant, C. T., Black, J. A., Hodges, R. S.,
J. Chromatogr. 1989, 476, 377389.
[34] http://www.med.uni-heidelberg.de/med/klinpharm/klin-
pharm_e/TDM/Allgemein/tdmberei.htm
2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim