Clinical Chemistry 43: 2083-2090, 1997;
(Clinical Chemistry. 1997;43:2083-2090.)
© 1997 American Association for Clinical Chemistry, Inc.
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 Albrecht1,
Jürg Reichen1,
Jan Visser2,
Dirk K. F. Meijer2 and
Wolfgang Thormann1,a
1
Department of Clinical Pharmacology, University of Bern, Murtenstr. 35, 3010 Bern, Switzerland.
2
Department of Pharmacokinetics and Drug Delivery,
University of Groningen, Antonius Deusinglaan 2, 9713 AV Groningen, The
Netherlands.
a Author for correspondence. Fax Int. +41 31 632 4997; e-mail thormann{at}ikp.unibe.ch
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Abstract
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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.
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Introduction
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Drug targeting is a promising approach in modern
pharmacotherapy. Cell-specific delivery of antitumor agents,
antivirals, and also antiinflammatory drugs have been described
(1). Naproxen (NAP),
(S)-6-methoxy-
-methyl-2-naphthaleneacetic acid (for structure see
Fig. 1
A), is a widely used nonsteroidal antiinflammatory
drug.1
Its antipyretic and analgesic effects are related to
the inhibition of cyclooxygenase, a major enzyme in the arachidonic
acid conversion pathway, resulting in a decrease of prostaglandin
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). Interestingly, this biochemical transformation
profoundly alters not only the pharmacokinetic behavior and cellular
distribution 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 pharmacokinetic 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 metabolite
naproxen–lysine (NAPLYS) is of high interest. No method has yet met
this challenge.
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Figure 1. Chemical structures of (A) NAP, (B)
NAP-HSA (NAP-HSA-MAN), and (C) NAPLYS, the primary
metabolite of (B).
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For the determination of NAP, its metabolites, and enantiomers in
plasma, synovial fluid, and urine, various methods based on HPLC have
been developed (5)(6)(7)(8)(9)(10)(11). Furthermore, capillary
electrophoretic techniques have been applied to the determination of
NAP in human serum and plasma (12)(13)(14)(15). In a previous
investigation from our laboratory, the possibility of monitoring total
plasma concentrations of NAP by using micellar electrokinetic capillary
chromatography (MECC) with direct plasma injection has been
demonstrated (15). In MECC, 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 electrophoresis, partitioning
between the two phases, and electrostatic 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 separated compounds as they pass an on-column absorbance
or fluorescence detector placed towards the other capillary end
(13)(14)(15)(16)(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 formulation (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 obtained.
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.
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Materials and Methods
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Drugs, chemicals, animal experiments, and plasma
samples.
All chemicals used were of analytical or research grade.
SDS was purchased from BHD Laboratory Supplies.
Na2HPO4,
Na2B4O7, 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 molecule of HSA, whereas the
NAP-HSA-MAN batch consisted 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
unreacted 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
experiments were performed at the Department of Pharmacokinetics and
Drug Delivery, Groningen, The Netherlands. Plasma samples were stored
at -18 °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 control of the capillary, data collection,
storage, and evaluation. Fused-silica capillaries (Polymicro
Technologies) of 50-µm 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 µA) 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 carousel 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 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 between 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 hydrogen 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 µmol/L)], NAP-HSA [1.5 g/L (20.9 µmol/L)], and
NAP-HSA-MAN [3.0 g/L (41.7 µmol/L)] as well as a methanolic
solution of NAPLYS [0.264 g/L (737.4 µmol/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 µL of plasma and IST
solution were pipetted into a 600-µL Eppendorf plastic vial and
vortex-mixed for ~2 s. Thereafter, the sample plastic vial was cut
down to half size and inserted into the vial holder of the P/ACE 5510.
Quantification 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
concentrations, the method of Franssen et al. (19) was
used with some minor modifications. Briefly, plasma samples (50 µL)
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 µL of IST solution [flurbiprofen, 10 mg/L
(40.9 µmol/L)] was added, and NAP was extracted with 6 mL of
dichloromethane. After evaporation of the organic phase under a steady
stream of nitrogen, the residue was redissolved in 300 µL of mobile
phase and 100 µL 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,
respectively (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 determined to be
8.2 and 14.6 min, respectively: The limit of detection (LOD) for NAP
was found to be 100 µg/L. Calibration graphs for NAP [1.25–25.0
mg/L (5.4–108.5 µmol/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
fluorescence spectrophotometer Aminco SLM SPF 500 (SLM Instruments) by
using excitation and emission wavelengths of 330 nm and 360 nm,
respectively. Plasma samples (20 µL) were diluted with Krebs buffer
(118 mmol/L NaCl, 5.0 mmol/L KCl, 1.1 mmol/L MgSO4 · 7
H2O, 2.5 mmol/L CaCl2 · 2 H2O,
1.2 mmol/L KH2PO4) to a final volume of 2 mL
and vortex-mixed for 5 s. Calibration graphs for NAP-HSA
[100–1500 mg/L (1.4–20.9 µmol/L)] and NAP-HSA-MAN [250–3000
mg/L (3.5–41.7 µmol/L)] were constructed by adding calibrator
solutions to rat blood plasma.
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Results and Discussion
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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 supplemented 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 administration of 22
mg/kg NAP-HSA to an anesthetized rat. In addition to NAP-HSA and free
NAP, NAPLYS (metabolite 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, revealing retention times of 4.4, 5.4, 8.1, and
9.4 min for NAP, IST, NAP-HSA, and NAPLYS, respectively. Corresponding
data with plasma samples containing the mannosylated 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, respectively. 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 components of
interest only. Endogenous substances are not detected. This is similar
to the conditions used for MECC-based immunochemical drug assays
(16).
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Figure 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
µmol/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.
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Figure 3. Electropherograms obtained with direct plasma injection
with (A) rat plasma blank; (B) rat plasma
supplemented with 500 mg/L (6.95 µmol/L) NAP-HSA-MAN, 4.17 mg/L (11.6
µmol/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.
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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 NAP-HSA, whereas NAP-HSA-MAN is substituted with only
nine NAP groups.
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Figure 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.
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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 µmol/L), 25–600 mg/L
(0.35–8.38 µmol/L), 25–1000 mg/L (0.35–13.9 µmol/L), and 3–60
mg/L (8.38–167.6 µmol/L) concentration ranges for NAP, NAP-HSA,
NAP-HSA-MAN, and NAPLYS, respectively. 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.
With MECC-DSI it was possible to assess the elimination 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 proteins, are determined (15). The
proteinaceous material can be applied without any sample pretreatment,
i.e., no time-consuming sample cleanup (e.g., extraction,
derivatization) 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 attractive for the determination of NAP, NAP–protein
conjugates, and their primary metabolite NAPLYS.
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Figure 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.
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To demonstrate the efficacy of the electrokinetic assay, MECC and HPLC
data of 61 plasma samples were assessed. The data presented in Fig. 6
A 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 concentrations 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 µmol/L, and 0.910, respectively. These data
indicate a good linear relation. Furthermore, plotting the difference
against the mean of the corresponding data pairs according 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 (-3.95 µmol/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 ± 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.
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Figure 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 ± 2 SD is
marked by the broken lines.
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Comparative data obtained by the fluorometric assay and MECC-DSI of 50
plasma samples are presented in Fig. 7
A. Linear regression analysis revealed a linear relation
(r = 0.884) with a calculated intercept of -0.155
µmol/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 difference against the mean of the
corresponding data pairs is plotted. The mean difference between the
MECC and fluorometric assay data was 0.306 µmol/L. Only one of 50
data points was outside the region defined by the mean difference
± 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 min after drug administration and are
subsequently increasing.
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Figure 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 ± 2 SD is
marked by the broken lines.
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In conclusion, MECC with direct plasma injection is shown to
provide a selective, simple, rapid, and attractive approach for the
simultaneous assessment of newly developed NAP–protein drugs used in
drug targeting, their primary metabolite NAPLYS, and free NAP. No
expensive 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 superior 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. Although 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.
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Acknowledgments
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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 European Union through the
framework of BIOMED Project BMH1-CT93–1436.
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Footnotes
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1 Nonstandard abbreviations: NAP, naproxen; HSA, human
serum albumin; NAPLYS, naproxen–lysine; MECC, micellar electrokinetic
capillary chromatography; SDS, sodium dodecyl sulfate; MAN, mannose;
DSI, direct sample injection; LIF, laser-induced fluorescence; IST,
internal standard; and LOD, limit of detection. 
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Abstract
Introduction
