Differentiation between naproxen, naproxen-protein conjugates, and naproxen-lysine in plasma via micellar electrokinetic capillary chromatography - A new approach in the bioanalysis of drug targeting preparations

Abstract

Pharmacotherapy through the targeting of drugs is a promising new approach that requires adequate analytical methods capable of differentiating between the free drug, the drug carrier, and metabolites. Using micellar electrokinetic capillary chromatography (MECC), we report the separation of naproxen (NAP) from NAP covalently coupled to human serum albumin or to mannosylated serum albumin and the metabolite naproxen-lysine. An assay for selective analysis of the different forms of NAP by direct plasma injection was developed with salicylate as internal standard and solute detection by laser-induced fluorescence. Compared with previously applied techniques, including HPLC and total plasma fluorescence, MECC offers the advantage that free and covalently bound NAP can be differentiated in one run and can be accurately monitored in microliter quantities of plasma. Summation of all NAP equivalents determined by MECC revealed data that compare well with those produced by total plasma fluorescence and HPLC.

Full text

Differentiation between naproxen,
naproxen–protein conjugates, and naproxen–lysine
in plasma via micellar electrokinetic capillary
chromatography—a new approach in the
bioanalysis of drug targeting preparations
Christiane Albrecht,
1
Ju¨ rg Reichen,
1
Jan Visser,
2
Dirk K.F. Meijer,
2
and
Wolfgang Thormann
1
*
Pharmacotherapy through the targeting of drugs is a prom-
ising new approach that requires adequate analytical
methods capable of differentiating between the free drug,
the drug carrier, and metabolites. Using micellar electro-
kinetic capillary chromatography (MECC), we report the
separation of naproxen (NAP) from NAP covalently cou-
pled to human serum albumin or to mannosylated serum
albumin and the metabolite naproxen–lysine. An assay for
selective analysis of the different forms of NAP by direct
plasma injection was developed with salicylate as internal
standard and solute detection by laser-induced fluores-
cence. Compared with previously applied techniques, in-
cluding HPLC and total plasma fluorescence, MECC offers
the advantage that free and covalently bound NAP can be
differentiated in one run and can be accurately monitored
in microliter quantities of plasma. Summation of all NAP
equivalents determined by MECC revealed data that com-
pare well with those produced by total plasma fluores-
cence and HPLC.
Drug targeting is a promising approach in modern phar-
macotherapy. Cell-specific delivery of antitumor agents,
antivirals, and also antiinflammatory drugs have been
described [1]. Naproxen (NAP), (S)-6-methoxy-
a
-methyl-
2-naphthaleneacetic acid (for structure see Fig. 1A), is a
widely used nonsteroidal antiinflammatory drug.
3
Its
antipyretic and analgesic effects are related to the inhibi-
tion of cyclooxygenase, a major enzyme in the arachidonic
acid conversion pathway, resulting in a decrease of pros-
taglandin formation. Coupling NAP to human serum
albumin (HSA) or mannosylated HSA (Fig. 1B) as carriers
provides a means for specific delivery of this compound
to endothelial and Kupffer cells of the liver [2]. Interest-
ingly, this biochemical transformation profoundly alters
not only the pharmacokinetic behavior and cellular dis-
tribution of the parent drug [1], but also has a marked
influence on its pharmacological effects in acute [3] and
chronic [4] liver disease. With regard to in vitro and in
vivo results demonstrating successful targeting of NAP to
the liver, growing interest emerges concerning the phar-
macokinetic properties of the different NAP conjugates
and their metabolites in biological fluids and organs. For
this purpose, the simultaneous determination of free
NAP, protein conjugates of NAP, and the primary metab-
olite naproxen–lysine (NAPLYS) is of high interest. No
method has yet met this challenge.
For the determination of NAP, its metabolites, and
enantiomers in plasma, synovial fluid, and urine, various
methods based on HPLC have been developed [5–11].
Furthermore, capillary electrophoretic techniques have
been applied to the determination of NAP in human
serum and plasma [12–15]. In a previous investigation
from our laboratory, the possibility of monitoring total
plasma concentrations of NAP by using micellar electro-
kinetic capillary chromatography (MECC) with direct
plasma injection has been demonstrated [15]. In MECC,
1
Department of Clinical Pharmacology, University of Bern, Murtenstr. 35,
3010 Bern, Switzerland.
2
Department of Pharmacokinetics and Drug Delivery, University of Gro-
ningen, Antonius Deusinglaan 2, 9713 AV Groningen, The Netherlands.
* Author for correspondence. Fax Int. 141 31 632 4997; e-mail
thormann@ikp.unibe.ch.
3
Nonstandard abbreviations: NAP, naproxen; HSA, human serum albu-
min; NAPLYS, naproxen–lysine; MECC, micellar electrokinetic capillary chro-
matography; SDS, sodium dodecyl sulfate; MAN, mannose; DSI, direct sample
injection; LIF, laser-induced fluorescence; IST, internal standard; and LOD,
limit of detection.
Received January 17, 1997; revision accepted June 24, 1997.
Clinical Chemistry 43:11
2083–2090 (1997)
Drug Monitoring and
Toxicology
2083

surfactants [e.g., sodium dodecyl sulfate (SDS)] above
their critical micelle concentrations are added to the
running buffer, permitting the separation of uncharged
solutes on the basis of differential partitioning. For
charged components, separation is governed by electro-
phoresis, partitioning between the two phases, and elec-
trostatic interactions and solute complexation with the
surfactant. One interesting and appealing feature of
MECC with dodecyl sulfate micelles is the possibility of
directly injecting a tiny (few nanoliters) amount of a
proteinaceous body fluid (e.g., plasma) onto an uncoated
fused-silica capillary at one end and detecting the sepa-
rated compounds as they pass an on-column absorbance
or fluorescence detector placed towards the other capil-
lary end [13–17].
Previously, plasma concentrations of NAP-HSA and of
NAP conjugated to mannosylated HSA (NAP-HSA-
MAN) have been determined as total plasma fluorescence
with excitation and emission wavelengths of 330 and 360
nm, respectively [18]. Although this is a rapid and simple
approach, it does not differentiate between the protein
conjugate, free NAP originating from the drug formula-
tion (up to ;20 g/kg), and the metabolite NAPLYS. By
using HPLC, free NAP and NAPLYS were determined by
analyzing plasma extracts and extracts of hydrolyzed
plasma [2, 19]. Through subtraction, indirect information
on the plasma concentration of the conjugate was ob-
tained. MECC with its capability of handling low- and
high-molecular-mass compounds appears to be the ideal
analytical free solution approach for the simultaneous
determination of carriers and metabolites used in drug
targeting. In this paper, analysis of different forms of
NAP-containing drugs in rat plasma by using MECC with
direct sample injection (MECC-DSI) is discussed, and data
obtained are compared with those produced by HPLC
and a fluorometric assay. MECC-DSI with on-column
laser-induced fluorescence (LIF) detection of solutes is
shown to be a selective, simple, and economical approach
for the assessment of the distribution and metabolism of
NAP–protein conjugates.
Materials and Methods
Drugs, chemicals, animal experiments, and plasma samples. All
chemicals used were of analytical or research grade. SDS
was purchased from BHD Laboratory Supplies.
Na
2
HPO
4
,Na
2
B
4
O
7
, and sodium salicylate were obtained
from Merck. Synthesis of NAP-HSA and NAP-HSA-MAN
was performed according to Franssen et al. [18]. NAPLYS
was synthesized as described by Grolleman et al. [20] and
Franssen et al. [18]. The batch of NAP-HSA used in the
present study contained 23 molecules of NAP per mole-
cule of HSA, whereas the NAP-HSA-MAN batch con-
sisted of 9 mol of NAP and 10 mol of MAN bound per
mole of HSA (determined by analysis of drug and protein
content [21, 22]). Both preparations contained some unre-
acted free NAP. NAP–protein conjugates were freshly
dissolved in saline and administrated to male Wistar rats
(250–350 g, anesthetized with pentobarbital 50 mg/kg)
via intravenous bolus injection. Plasma samples were
withdrawn over a period of 3 h thereafter. Animal exper-
iments were performed at the Department of Pharmaco-
kinetics and Drug Delivery, Groningen, The Netherlands.
Plasma samples were stored at 218 °C until analysis.
Electrophoretic instrumentation and running conditions for
MECC. MECC was performed on a P/ACE 5510 capillary
electrophoresis system (Beckman Instruments) featuring
automated capillary rinsing, sampling, temperature con-
trol of the capillary, data collection, storage, and evalua-
tion. Fused-silica capillaries (Polymicro Technologies) of
50-
m
m i.d. were used. The effective capillary length was
20 cm (total length of 27 cm). A constant voltage of 8 kV
was applied (current ;30
m
A) and the anode was on the
sampling side. Sample injection was effected by applying
positive pressure at 3448 Pa (0.5 psi) for 1 s. The capillary
temperature was maintained at 20 °C. The sample carou-
sel was at ambient temperature. Solute detection was
effected by LIF with an air-cooled 10 mV HeCd laser
(Liconix), which emits at 325 nm. A 366-nm emission filter
Fig. 1. Chemical structures of (
A
) NAP, (
B
) NAP-HSA (NAP-HSA-MAN),
and (
C
) NAPLYS, the primary metabolite of (
B
).
2084 Albrecht et al.: Differentiation via micellar electrokinetic capillary chromatography

was used. The photomultiplier tube gain was set to 1.
Data were evaluated with the Gold Software package
version 8.1 (Beckman). Capillaries were conditioned be-
tween runs by application of positive pressure [34.48 kPa
(5 psi)] with 0.1 mol/L NaOH (3 min), water (3 min), and
running buffer (3 min). The running buffer consisted of 10
mmol/L sodium tetraborate, 6 mmol/L disodium hydro-
gen phosphate, and 75 mmol/L SDS (pH ;9.2). The
buffer vials were replenished every 5–6 runs.
Calibrator solutions, preparation of samples, and principle of
quantification by MECC. Aqueous calibrator solutions of
NAP [10 mg/L (43.4
m
mol/L)], NAP-HSA [1.5 g/L (20.9
m
mol/L)], and NAP-HSA-MAN [3.0 g/L (41.7
m
mol/L)]
as well as a methanolic solution of NAPLYS [0.264 g/L
(737.4
m
mol/L)] were prepared and diluted with blank rat
plasma. Salicylate (0.5 or 1 mmol/L), diluted from a stock
solution of 16.6 g/L (100 mmol/L) sodium salicylate, was
used as internal standard (IST). Aliquots of 50
m
Lof
plasma and IST solution were pipetted into a 600-
m
L
Eppendorf plastic vial and vortex-mixed for ;2 s. There-
after, the sample plastic vial was cut down to half size and
inserted into the vial holder of the P/ACE 5510. Quanti-
fication was based upon internal, multilevel calibration by
using the peak area ratio of the compound to the IST.
HPLC assay. For the determination of total NAP concen-
trations, the method of Franssen et al. [19] was used with
some minor modifications. Briefly, plasma samples (50
m
L) were first subjected to alkaline hydrolysis for 72 h at
80 °C by using 1 mL of 5 mol/L NaOH. Then, the samples
were acidified with 5 mol/L HCl to pH 1.5, 100
m
LofIST
solution [flurbiprofen, 10 mg/L (40.9
m
mol/L)] was
added, and NAP was extracted with 6 mL of dichlo-
romethane. After evaporation of the organic phase under
a steady stream of nitrogen, the residue was redissolved
in 300
m
L of mobile phase and 100
m
L was injected into
the HPLC column. The mobile phase consisted of water:
acetonitrile:acetic acid (60:40:1 by vol) and the flow rate
was 1.5 mL/min. Separation was done on a reversed-
phase C18 column (Nucleosil C18 ET 250/8/4, Macherey
Nagel). NAP was detected by fluorometry at excitation
and emission wavelengths of 334 and 360 nm, respec-
tively (Perkin-Elmer Fluorescence Spectrophotometer
204). The IST was monitored simultaneously by UV
detection at 254 nm with a Spectroflow 773 (Kratos
Analytical) and a D-2000 Chromato-Integrator (Merck-
Hitachi). Retention times for NAP and IST were deter-
mined to be 8.2 and 14.6 min, respectively: The limit of
detection (LOD) for NAP was found to be 100
m
g/L.
Calibration graphs for NAP [1.25–25.0 mg/L (5.4–108.5
m
mol/L)] were constructed by adding calibrator solutions
to whole heparinized rat plasma.
Plasma fluorescence assay. Plasma concentrations of NAP–
protein conjugates were also estimated via total plasma
fluorescence according to Franssen et al. [18] on a fluores-
cence spectrophotometer Aminco SLM SPF 500 (SLM
Instruments) by using excitation and emission wave-
lengths of 330 nm and 360 nm, respectively. Plasma
samples (20
m
L) were diluted with Krebs buffer (118
mmol/L NaCl, 5.0 mmol/L KCl, 1.1 mmol/L MgSO
4
z 7
H
2
O, 2.5 mmol/L CaCl
2
z 2H
2
O, 1.2 mmol/L KH
2
PO
4
)to
a final volume of 2 mL and vortex-mixed for 5 s. Calibra-
tion graphs for NAP-HSA [100 –1500 mg/L (1.4–20.9
m
mol/L)] and NAP-HSA-MAN [250 –3000 mg/L (3.5–
41.7
m
mol/L)] were constructed by adding calibrator
solutions to rat blood plasma.
Results and Discussion
Data obtained with plasma samples containing NAP-HSA
and salicylate as IST are depicted in Fig. 2. For the blank
rat plasma (panel A), no peak was detected. A typical
electropherogram containing blank rat plasma supple-
mented with NAP-HSA, NAPLYS, and IST is depicted in
panel B. The NAP detected stems from the NAP-HSA
calibrator. Panel C shows an electropherogram of a
plasma sample drawn 120 min after intravenous admin-
istration of 22 mg/kg NAP-HSA to an anesthetized rat. In
addition to NAP-HSA and free NAP, NAPLYS (metabo-
lite of NAP-HSA) could be clearly detected. The low-
molecular-mass substances are shown to form sharp
peaks, whereas NAP-HSA is registered as broader peak
(see below). All analytes are completely separated, reveal-
ing retention times of 4.4, 5.4, 8.1, and 9.4 min for NAP,
IST, NAP-HSA, and NAPLYS, respectively. Correspond-
ing data with plasma samples containing the mannosy-
lated conjugate NAP-HSA-MAN are presented in Fig. 3.
Panels B and C depict data obtained with a calibrator
plasma sample and with rat plasma drawn 180 min after
intravenous injection of 48 mg/kg NAP-HSA-MAN, re-
spectively. As with NAP-HSA, NAP-HSA-MAN appears
as a broad peak. It is important to note that LIF solute
detection as used here selectively visualizes the compo-
nents of interest only. Endogenous substances are not
detected. This is similar to the conditions used for MECC-
based immunochemical drug assays [16].
In MECC-DSI, proteins are solubilized by dodecyl
sulfate and elute as broad peaks [14, 15]. NAP and
NAPLYS elute in front and after the rat plasma proteins,
respectively, thereby producing sharp peaks. The protein
conjugates, however, do not completely separate from
other proteins and therefore appear as broad peaks.
However, as illustrated with the data presented in Fig. 4,
the two NAP–protein conjugates could be separated in the
presence of the plasma proteins. NAP-HSA-MAN eluted
in front of NAP-HSA. Interestingly, application of the
NAP-HSA-MAN calibrator dissolved in water produced a
rather sharp peak (see left inset in Fig. 4), whereas a
relatively broad peak was observed for NAP-HSA that
was sampled in water (right inset in Fig. 4). This is likely
to be due to the difference and possible variation in drug
loading of the carriers: HSA contains 23 molecules of the
hydrophobic NAP per protein molecule in the case of
Clinical Chemistry 43, No. 11, 1997 2085

Fig. 2. Electropherograms obtained with direct plasma injection with (
A
)
rat plasma blank; (
B
) rat plasma supplemented with 70 mg/L (977.6
nmol/L) NAP-HSA, 3.82 mg/L (10.7
m
mol/L) NAPLYS, and 44.4 mg/L
(0.28 mmol/L) IST; and (
C
) rat plasma withdrawn 120 min after
injection of 22 mg/kg NAP-HSA.
Conditions are as described in
Materials and Methods
.
Fig. 3. Electropherograms obtained with direct plasma injection with (
A
)
rat plasma blank; (
B
) rat plasma supplemented with 500 mg/L (6.95
m
mol/L) NAP-HSA-MAN, 4.17 mg/L (11.6
m
mol/L) NAPLYS, and 26.7
mg/L (0.17 mmol/L) IST; and (
C
) rat plasma withdrawn 180 min after
injection of 48 mg/kg NAP-HSA-MAN.
Conditions are as described in
Materials and Methods
.
2086 Albrecht et al.: Differentiation via micellar electrokinetic capillary chromatography

NAP-HSA, whereas NAP-HSA-MAN is substituted with
only nine NAP groups.
Quantification of NAP and its derivatives was based
upon internal, four to six-level calibration by using the
peak area ratio of the compound to the IST and having
0.5–25 mg/L (2.17–108.6
m
mol/L), 25– 600 mg/L (0.35–
8.38
m
mol/L), 25–1000 mg/L (0.35–13.9
m
mol/L), and
3–60 mg/L (8.38 –167.6
m
mol/L) concentration ranges for
NAP, NAP-HSA, NAP-HSA-MAN, and NAPLYS, respec-
tively. Calibration graphs were linear, with F-values for
all compounds .200 (P ,0.0001). The y-intercepts were
significantly smaller than the smallest calibrator values
and were thus negligible. For all compounds, intra- and
interday CVs were ,10% (n $5). Table 1 summarizes the
analytical characteristics of the assay.
Fig. 4. Separation of different NAP–protein conjugates after direct
injection of rat plasma suplemented with NAP-HSA (50 mg/L), NAP-
HSA-MAN (125 mg/L), and IST (80 mg/L).
Data obtained via injection of calibrators (without plasma) are depicted in the
insets
.
Fig. 5. Elimination curves of NAP and NAP–protein conjugates [(
A
)
NAP-HSA and (
B
) NAP-HSA-MAN] in anesthetized rats during 180 min
after drug administration together with the temporal increase of the
primary metabolite NAPLYS.
Conditions are the same as those of Figs. 2C and 3C, respectively.
Table 1. Characteristics of the MECC assay with direct
injection of rat plasma
Compound
LOD,
nmol/L
CV, %
Intraday n Interday n
NAP 140 1.92 7 4.83 5
NAP-HSA 7 1.43 7 7.68 6
NAP-HSA-MAN 140 8.36 7 4.67 6
NAPLYS 70 0.63 6 1.90 5
LOD is based on a signal/noise ratio 5 3.
Clinical Chemistry 43, No. 11, 1997 2087

With MECC-DSI it was possible to assess the elimina-
tion of free and conjugated NAP and to register the
appearance and temporal increase of the metabolite
NAPLYS (Fig. 5). It is important to note that with this
assay, total plasma concentrations of NAP, i.e., free NAP
together with NAP noncovalently bound to plasma pro-
teins, are determined [15]. The proteinaceous material can
be applied without any sample pretreatment, i.e., no
time-consuming sample cleanup (e.g., extraction, deriva-
tization) before analysis is necessary. As the separation of
the analytes by MECC-DSI can be performed in one single
run, the load of samples and consumption of organic
solvents is minimized. Furthermore, automation and run
times of #10 min make this method effective and attrac-
tive for the determination of NAP, NAP–protein conju-
gates, and their primary metabolite NAPLYS.
To demonstrate the efficacy of the electrokinetic assay,
MECC and HPLC data of 61 plasma samples were as-
sessed. The data presented in Fig. 6A represent total NAP
concentrations determined after hydrolysis and extraction
with HPLC. These values were compared with the total
NAP equivalents calculated from the various drug con-
centrations that were determined by MECC-DSI. Linear
regression analysis of the data pairs (Fig. 6A) revealed a
correlation coefficient, y-intercept, and slope of 0.860, 9.49
m
mol/L, and 0.910, respectively. These data indicate a
good linear relation. Furthermore, plotting the difference
against the mean of the corresponding data pairs accord-
ing to Bland and Altman [23] provided a better insight
into the equality of the two sets of data (Fig. 6B). In
relation to the calibration range, the mean difference
between MECC and HPLC data (23.95
m
mol/L) was
small and the data appear to be evenly distributed,
indicating that the two methods provide comparative
total plasma concentrations of NAP. Fifty eight of the 61
data points are within the region defined by the mean
difference 6 2 SD (region bracketed by broken lines). It is
important to realize that with HPLC the concentrations of
NAP conjugates can be determined only indirectly, i.e.,
via hydrolysis of the NAP–lysine bond. The possibility of
distinguishing between all NAP-containing compounds
with the MECC assay therefore is a clear advantage.
Comparative data obtained by the fluorometric assay
and MECC-DSI of 50 plasma samples are presented in Fig.
7A. Linear regression analysis revealed a linear relation
(r 5 0.884) with a calculated intercept of 20.155
m
mol/L
and a slope close to one (0.958). These results indicate that
both methods provide comparable drug concentrations.
This is further underlined in Fig. 6B, in which the differ-
ence against the mean of the corresponding data pairs is
plotted. The mean difference between the MECC and
fluorometric assay data was 0.306
m
mol/L. Only one of 50
data points was outside the region defined by the mean
difference 6 2 SD. The fluorometric assay is based on the
measurement of total plasma fluorescence, and the data
are not corrected via use of an IST. The different NAP
compounds are not distinguished. Drug concentrations
assessed with total fluorometric assay can only be used as
a first estimate to characterize plasma clearance of the
conjugate in the initial (30 min) period after injection, in
which metabolism of the conjugate plays only a minor
role because of a lag time in the lysosomal degradation of
the HSA conjugate. After this period they not only include
contributions from small amounts of free NAP, which is
coadministrated with NAP-HSA and NAP-HSA-MAN
(typically ,20 g/kg), but also from the metabolite
NAPLYS. As is shown with the data presented in Fig. 5,
concentrations of free NAP and NAPLYS are not constant:
The concentrations of NAP are slowly decreasing with
time, whereas those of NAPLYS could only be detected 20
Fig. 6. Comparative (
A
) total NAP plasma concentrations in 61 samples
determined by MECC and HPLC and (
B
) difference vs mean of each
data pair.
The region covered by the mean of the differences 6 2 SD is marked by the
broken lines
.
2088 Albrecht et al.: Differentiation via micellar electrokinetic capillary chromatography

min after drug administration and are subsequently in-
creasing.
In conclusion, MECC with direct plasma injection is
shown to provide a selective, simple, rapid, and attractive
approach for the simultaneous assessment of newly de-
veloped NAP–protein drugs used in drug targeting, their
primary metabolite NAPLYS, and free NAP. No expen-
sive column and no sample preparation are required to
separate the different NAP compounds in a single run.
The direct application of proteinaceous material and the
high degree of automation combined with short run times
of maximal 10 min make this MECC method highly
effective and economic. The MECC-DSI method is supe-
rior to the total plasma fluorescence method, which lacks
specificity, and to HPLC, which requires time-consuming
sample preparation and more than one run per sample for
the estimation of the plasma concentration of a conjugate.
Finally, the fact that only microliter quantities of plasma
are required for MECC-DSI is an important advantage for
kinetic investigations in small laboratory animals. Al-
though this paper reports data obtained with rat plasma,
the same assay could be used for monitoring the NAP
compounds in human plasma [24]. These promising drug
targeting preparations are not yet used in human studies;
however, they represent an exciting new approach in
pharmacotherapy.
We gratefully acknowledge technical assistance in the
animal experiments provided by Barbro Melgert, Leonie
Beljaars, Roelof Oosting, and Klaas Poelstra. The skillful
analytical assistance of Lone Steinmann and Franz von
Heeren was greatly appreciated. This work was partly
sponsored by the Swiss National Science Foundation
(grants 32–45349.95 and 31–32428.91) and by the Euro-
pean Union through the framework of BIOMED Project
BMH1-CT93–1436.
References
1. Meijer DKF, Molema G. Targeting of drugs to the liver. Semin Liver
Dis 1995;15:203–56.
2. Franssen EJF, Jansen RW, Vaalburg M, Meijer DKF. Hepatic and
intrahepatic targeting of an antiinflammatory agent with human
serum albumin and neoglycoproteins as carrier molecules. Bio-
chem Pharmacol 1993;45:1215–26.
3. Lebbe C, Reichen J, Wartna E, Sa¨gesser H, Poelstra K, Meijer DKF.
Targeting naproxen to non parenchymal liver cells protects against
endotoxin induced liver damage. J Drug Target 1997;4:303–10.
4. Albrecht C, Meijer DKF, Sa¨gesser H, Reichen J. Naproxen targeted
to endothelial and Kupffer cells protects against endotoxin in
biliary cirrhosis in the rat. J Hepatol 1996;25(Suppl 1):130.
5. Karidas T, Avgerinos A, Malamataris S. Extractionless HPLC
method for the determination of naproxen in human plasma and
urine. Anal Lett 1993;26:2341– 8.
6. Singh AK, Jang Y, Mishra U, Granley K. Simultaneous analysis of
flunixin, naproxen, ethacrynic acid, indomethacin, phenylbuta-
zone, mefenamic acid and thiosalicylic acid in plasma and urine by
high-performance liquid chromatography and gas chromatography
–mass spectrometry. J Chromatogr 1991;568:351– 61.
7. Andersen JV, Hansen SH. Simultaneous determination of (R)- and
(S)-naproxen and (R)- and (S)-6-
O
-desmethylnaproxen by high-
performance liquid chromatography on a chiral-AGP column. J
Chromatogr 1992;577:362–5.
8. Blagbrough IS, Daykin MM, Doherty M, Pattrick M, Shaw PN. High
performance liquid chromatographic determination of naproxen,
ibuprofen and diclofenac in plasma and synovial fluid in man. J
Chromatogr 1992;578:251–7.
9. Vree TB, van den Biggelaar-Martea M, Verwey-van Wissen CPWGM.
Determination of naproxen and its metabolite
O
-desmethyl-
naproxen with their acyl glucuronides in human plasma and urine
by means of direct gradient high-performance liquid chromatogra-
phy. J Chromatogr 1992;578:239 – 49.
10. Andersen JV, Hansen SH. Simultaneous quantitative determina-
Fig. 7. Comparative (
A
) total NAP plasma concentrations of 50
samples determined by MECC and a fluorometric assay (FLUO) and (
B
)
difference vs mean of each data pair.
The region covered by the mean of the differences 6 2 SD is marked by the
broken lines
.
Clinical Chemistry 43, No. 11, 1997 2089

tion of naproxen, its metabolite 6-
O
-desmethylnaproxen and their
five conjugates in plasma and urine samples by high-performance
liquid chromatography on dynamically modified silica. J Chro-
matogr 1992;577:325–33.
11. Shimek JL, Rao NGS, Wahba Khalil SK. An isocratic high-pressure
liquid chromatographic determination of naproxen and desmeth-
ylnaproxen in human plasma. J Pharm Sci 1982;71:436 –9.
12. Soini H, Novotny MV, Riekkola M-L. Determination of naproxen in
serum by capillary electrophoresis with ultraviolet absorbance and
laser-induced fluorescence detection. J Microcol Sep 1992;4:
313– 8.
13. Zhang C-X, Thormann W. Determination of drug levels in human
serum by micellar electrokinetic capillary chromatography with
direct sample injection using different quantitation strategies. J
Capillary Electrophor 1994;1:208 –18.
14. Caslavska J, Gassmann E, Thormann W. Modification of a tunable
UV-visible capillary electrophoresis detector for simultaneous
absorbance and fluorescence detection: profiling of body fluids for
drugs and endogenous compounds. J Chromatogr A 1995;709:
147–56.
15. Schmutz A, Thormann W. Assessment of impact of physico-
chemical drug properties on monitoring drug levels by micellar
electrokinetic capillary chromatography with direct serum injec-
tion. Electrophoresis 1994;15:1295–303.
16. Steinmann L, Thormann W. Characterization of competitive bind-
ing, fluorescent drug immunoassays based on micellar electroki-
netic capillary chromatography. Electrophoresis 1996;17:1348 –
56.
17. Thormann W, Zhang C-X, Schmutz A. Capillary electrophoresis for
drug analysis in body fluids. Ther Drug Monit 1996;18:506 –20.
18. Franssen EJF, Koiter J, Kuipers CAM, Bruins AP, Moolenaar F, de
Zeeuw D, et al. Low molecular weight proteins as carriers for renal
drug targeting. Preparation of drug–protein conjugates and drug–
spacer derivatives and their catabolism in renal cortex homoge-
nates and lysosomal lysates. J Med Chem 1992;35:1246 –59.
19. Franssen EJF, van Amsterdam RGM, Visser J, Moolenaar F, de
Zeeuw D, Meijer DKF. Low molecular weight proteins as carriers
for renal drug targeting: naproxen–lysozyme. Pharm Res 1991;8:
1223–30.
20. Grolleman CWJ, Visser AC, Wolke JGC, Timmerman H. Studies on
a bioerodable drug carrier system based on polyphosphazene.
Part 1. Synthesis. J Contr Rel 1986;3:143.
21. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measure-
ment with the Folin phenol reagent. J Biol Chem 1951;93:265–
75.
22. Habeeb AFSA. Determination of free amino groups in proteins by
trinitrobenzenesulfonic acid. Anal Biochem 1966;14:328 –36.
23. Bland JM, Altman DG. Statistical methods for assessing agree-
ment between two methods of clinical measurement. Lancet
1986;i:307–10.
24. Thormann W. Drug monitoring by capillary electrophoresis. In:
Wong SHY, Sunshine I, eds. Handbook of analytical therapeutic
drug monitoring and toxicology. Boca Raton, FL: CRC Press,
1997:1–19.
2090 Albrecht et al.: Differentiation via micellar electrokinetic capillary chromatography