Chip-Based P450 Drug Metabolism Coupled to Electrospray Ionization-Mass Spectrometry Detection

Article abstract of DOI:10.1021/ac030249+

A chip-based P450 in vitro metabolism assay coupled with ESI-MS and ESI-MS/MS detection is described in this paper. The chips were made of a cyclic olefin polymer using a hot embossing process. The introduction of reagent solutions into the chip was carried out using fused-silica capillaries coupled to two syringes with the flow rate controlled by a syringe pump. Initial experiments described here employed a small commercial guard column in an off-chip format to desalt and concentrate the products of the enzymatic reaction prior to ESI-MS analysis. The system was used both to yield the Michaelis constant (K_m) of the P450 biotransformation of imipramine into desipramine and to determine the IC50 value of a chemical inhibitor (tranylcypromine) for this CYP2C19-mediated reaction. The results demonstrated that the kinetics of the reaction inside the 4-μL volume within the channels of the cyclic olefin polymer chip provided results in agreement with those reported in the literature using conventional assays. The above reactions were carried out using human liver microsomes, and the metabolites were detected by ESI-MS showing the potential of the chip-based P450 reaction for metabolite screening studies as well as for P450 inhibition assays. A porous monolithic column was subsequently integrated into the chip to perform the reaction mixture cleanup process in an integrated fashion on the chip that is necessary for ESI-MS detection. The miniature monolithic SPE column was prepared in situ inside the chip via UV-initiated polymerization. The results obtained using the integrated system demonstrated the possibility of performing P450 enzymatic reactions in a microvolume reaction chamber coupled directly to ESI-MS detection and required less than 4 μg of HLM protein.

Full text of DOI:10.1021/ac030249+

Anal. Chem. 2003, 75, 6430-6436
Chip-Based P450 Drug Metabolism Coupled to
Electrospray Ionization-Mass Spectrometry
Detection
Salete Benetton,† Jun Kameoka,‡ Aimin Tan,† Timothy Wachs,† Harold Craighead,‡ and
Jack D. Henion*,†
Analytical Toxicology, College of Veterinary Medicine, Cornell University, 927 Warren Drive, Ithaca, New York 14850, and
School of Applied and Engineering Physics, Cornell University, 212 Clark Hall, Ithaca, New York 14850
reagent consumption, integration, and automation.1-8 Among the
various areas of application of these chip-based analytical systems
are enzymatic assays.9 Current on-chip enzymatic assays use
soluble or immobilized enzymes and rely on the conversion of a
nondetectable species to a detectable one.9-15 Fluorescence and
amperometry have been the main types of detection used in these
assays.9
In this work, we have studied the feasibility of performing
enzymatic reactions in a chip-based microfluidic format coupled
directly with electrospray ionization (ESI)-mass spectrometry
(MS) detection. The enzymes investigated here belong to the
cytochrome P450 system, which is a membrane-bound (water-
insoluble) enzymatic system responsible for the biotransformation
of xenobiotics.16
Cytochrome P450 enzymes have long been recognized as the
primary system responsible for human drug metabolism.16 The
P450 enzymatic system is a superfamily of heme-containing
monooxygenases located within the endoplasmic reticulum. The
enzyme system consists of two protein components: a hemopro-
tein called cytochrome P450 and a flavoprotein called NADPH-
cytochrome P450 reductase. Cytochrome P450 is the substrate-
and oxygen-binding site of the enzyme system, while the reductase
A chip-based P 4 5 0 in vitro metabolism assay coupled
with ESI-MS and ESI-MS/ MS detection is described in
this paper. The chips were made of a cyclic olefin polymer
using a hot embossing process. The introduction of
reagent solutions into the chip was carried out using
fused-silica capillaries coupled to two syringes with the
flow rate controlled by a syringe pump. Initial experiments
described here employed a small commercial guard
column in an off-chip format to desalt and concentrate the
products of the enzymatic reaction prior to ESI-MS
analysis. The system was used both to yield the Michaelis
constant (Km) of the P 4 5 0 biotransformation of imi-
pramine into desipramine and to determine the IC5 0
value of a chemical inhibitor (tranylcypromine) for this
CYP 2 C1 9 -mediated reaction. The results demonstrated
that the kinetics of the reaction inside the 4 -µL volume
within the channels of the cyclic olefin polymer chip
provided results in agreement with those reported in the
literature using conventional assays. The above reactions
were carried out using human liver microsomes, and the
metabolites were detected by ESI-MS showing the poten-
tial of the chip-based P 450 reaction for metabolite screen-
ing studies as well as for P 450 inhibition assays. A porous
monolithic column was subsequently integrated into the
chip to perform the reaction mixture cleanup process in
an integrated fashion on the chip that is necessary for ESI-
MS detection. The miniature monolithic SP E column was
prepared in situ inside the chip via UV-initiated polym-
erization. The results obtained using the integrated system
demonstrated the possibility of performing P 4 5 0 enzy-
matic reactions in a microvolume reaction chamber
coupled directly to ESI-MS detection and required less
than 4 µg of HLM protein.
(1) Kricka, L. J. Clin. Chem. 1 9 9 8 , 44, 2008-2014.
(2) Waters, L. C.; Jacobson, S. C.; Kroutchinina, N.; Khandurina, J.; Foote, R.
S.; Ramsey, J. M. Anal. Chem. 1 9 9 8 , 70, 158-162.
(3) Wang, J.; Chatrati, M. P.; Tian, B.; Polsky, R. Anal. Chem. 2 0 0 0 , 72, 2514-
2518.
(4) Deng, Y.; Henion, J.; Li, J.; Thibault, P.; Wang, C.; Harrison, D. J. Anal.
Chem. 2 0 0 1 , 73, 639-646.
(5) Deng, Y.; Zhang, H.; Henion, J. Anal. Chem. 2 0 0 1 , 73, 1432-1439.
(6) Kameoka, J.; Craighead, H. G.; Zhang, H.; Henion, J. Anal. Chem. 2 0 0 1 ,
73, 1935-1941.
(7) Khandurina, J.; Guttman, A. J. Chromatogr., A 2 0 0 2 , 943, 159-183.
(8) Kameoka, J.; Orth, R.; Ilic, B.; Czaplewski, D.; Wachs, T.; Craighead, H. G.
Anal. Chem. 2 0 0 2 , 74, 5897-5901
(9) Wang, J. Electrophoresis 2 0 0 2 , 23, 713-718.
(10) Hadd, A. G.; Raymond, D. E.; Halliwell, J. W.; Jacobson, S. C.; Ramsey, J.
M. Anal. Chem. 1 9 9 7 , 69, 3407-3412.
(11) Hadd, A. G.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1 9 9 9 , 71, 5206-
5212.
Research on microfabricated analytical devices has grown
considerably in the last 10 years. Many publications have reported
results and accomplishments demonstrating the advantages of
these analytical microsystems, including speed, reduced sample/
(12) Duffy, M.; Gillis, H. L.; Lin, J.; Sheppard, N. F.; Kellog, G. J. Anal. Chem.
1 9 9 9 , 71, 4669-4678.
(13) Chen, C. B.; Chin-Dixon, E.; Jeong, S.; Nikiforov, T. Anal. Biochem. 1 9 9 9 ,
273, 89-97.
* Corresponding author: (e-mail) jdh4@cornell.edu; (phone) 607-253-3971;
(fax) 607-253-3973.
(14) Kerby, M.; Chien, R. L. Electrophoresis 2 0 0 1 , 22, 3916-3923.
(15) Mao, H.; Yang, T.; Cremer, P. S. Anal. Chem. 2 0 0 2 , 74, 379-385.
(16) Anzenbacher, P.; Anzenbacherova, E. Cell. Mol. Life Sci. 2 0 0 1, 58, 737-
747.
† College of Veterinary Medicine.
‡ School of Applied and Engineering Physics.
6430 Analytical Chemistry, Vol. 75, No. 23, December 1, 2003
10.1021/ac030249+ CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/30/2003

serves as an electron carrier, shuttling electrons from NADPH to
the cytochrome P450 substrate complex.17 The hepatic microsomal
cytochrome P450s catalyze the Phase I metabolism of xenobiotics,
the initial step in the biotransformation and elimination of a wide
variety of drugs and environmental pollutants.17,18
In the process of drug discovery, various in vitro methodologies
are routinely used in pharmaceutical companies to screen a vast
number of candidate drugs to determine rate of metabolism, to
predict drug-drug interactions and metabolic profiles, and to
determine the involvement of enzymes that present genetic
polymorphism.19 Traditionally, these in vitro methods use either
liver tissue slices, primary culture of hepatocytes, liver micro-
somes, or recombinant drug-metabolizing enzymes.19,20 Essential
characteristics desired for an analytical drug discovery method
include sensitivity, speed, automation, simultaneous testing of
microvolumes of candidate drug compounds, and use of minimum
quantities of costly material. In the case of P450 enzymatic
reactions, besides quantification, identification of the metabolites
generated by the candidate drug compounds is very important.
Hence, the combination of a chip-based P450 reaction with a more
universal detection system is highly desirable.
Figure 1. Design of the chip for the P450 reaction. The microchan-
nels before the reaction region are 100 µm wide and 100 µm deep.
In the reaction region, the channels are 200 µm wide and 100 µm
deep with a total volume from the Y-junction to the outlet of 4 µL. (A)
Enzyme inlet; (B) substrate + cofactor inlet; (C) Nanoport connection;
(D) reaction region.
EXPERIMENTAL SECTION
Chemicals. Imipramine, desipramine, doxepin, amitriptyline,
tranylcypromine, â-nicotinamide adenine dinucleotide phosphate
(NADPH), magnesium chloride, and Tris base were purchased
from Sigma Chemical Co. (St. Louis, MO). Pooled human liver
microsomes (HLM) containing 20 mg of microsomal protein/ mL
were purchased from Gentest Corp. (Woburn, MA). Ethylene
dimethacrylate (EDMA), butyl methacrylate (BMA), 2, 2-dimethoxy-
2-phenylacetophenone (DAP), 2-acrylamido-2-methyl-1-propane-
sulfonic acid (AMPS), and hexane were all obtained from Aldrich
Chemical Co. (Milwaukee, WI). EDMA and BMA were filtered
through a 0.45-µm PTFE syringe filter (13-mm diameter, Nalge
Nunc International, Rochester, NY) filled with fresh basic alumina
powder (mesh 60-325, Fisher, Pittsburgh, PA). Other reagents
were hydrochloric acid (EM Science, Gibbstown, NJ) and HPLC
grade acetonitrile (J. T. Baker, Phillipsburg, NJ). Zeonor polymer
was purchased from Zeon Chemicals (Louisville, KY). The 2.5
and 25 mM Tris buffer was prepared by dissolving Tris base in
deionized water with the pH adjusted to 7.4 using hydrochloric
acid. All solutions in this work were prepared using NANOpure
deionized water (Barnstead, Boston, MA).
Chip Fabrication. Figure 1 shows the layout and exterior
dimensions of the polymer microfluidic chip used in this report.
The dimensions of these chips were 4 cm × 6 cm (3.7 mm thick)
with two inlet channels meeting at a Y-junction and forming the
mixing region (reaction region). The dimensions of the inlet
channels are 100 µm × 100 µm. The microchannels in the reaction
region are 200 µm wide and 100 µm deep with a total volume of
4 µL from the Y-junction to the outlet.
ESI-MS is a sensitive and selective detection system that is
compatible with chip flow rates and can also provide structural
information that is essential for metabolite screening studies. The
advantage of miniaturizing P450 enzymatic reactions includes the
low sample volumes and hence the use of minimum quantities of
costly materials and the potential for development of high-
throughput assays.
To monitor an on-chip reaction by ESI-MS, the reaction product
mixture must be desalted in addition to removal of the enzyme
molecules. Also, preconcentration of metabolites is desirable to
bring them to a higher concentration and thus increase the
sensitivity of the assay. Initial experiments described here
employed a small commercial guard column in an off-chip format
to desalt and concentrate the products of the enzymatic reaction
prior to ESI-MS analysis. A porous monolithic column was later
integrated into the chip to perform an integrated solid-phase
extraction (SPE) cleanup process necessary for ESI-MS detection.
The miniature monolithic SPE columns were prepared in situ
inside the chip via UV-initiated polymerization.
Using imipramine, doxepin, and amitriptyline (antidepressant
drugs) as test small-molecule drug compounds, we investigated
the biotransformation of these compounds in a chip-based format.
The system was also used both to yield the Michaelis constant
(Km) of the P450 biotransformation of imipramine into desipramine
and to determine the IC50 value of a chemical inhibitor (tranyl-
cypromine) for this CYP2C19-mediated reaction. The chips were
made of Zeonor (a cyclic olefin) polymer that has already been
shown to be suitable for making chips because it is readily
embossed and bonded as previously reported.6 This report
describes the successful on-chip P450 in vitro metabolism of three
representative drugs followed by on-chip SPE sample cleanup and
direct electrospray analysis of the resulting samples.
The fabrication process for the polymeric microfluidic chip,
including the fabrication of etched silicon wafer masters, was as
described previously.6
Chip Operation. The introduction of reagent solutions into
the chip was carried out using fused-silica capillaries coupled to
two syringes with the flow rate (1 µL/ min) controlled by a two-
channel infusion pump (Harvard Apparatus, South Natick, MA).
The fused-silica capillaries are connected to the chip using
commercial microconnectors (N-124S, Nanoports from UpChurch
Scientific, Oak Harbor, WA) attached to the two inlets and to the
outlet of the chip by an epoxy-based glue (DP420, 3M Engineered
Adhesives Division, St. Paul, MN). In Figure 2 is shown a
(17) Meyer, U. A. J. Pharmacokinet. Biopharm. 1 9 9 6 , 24, 449-459.
(18) Guengerich, F. P. J. Pharmacokinet. Biopharm. 1 9 9 6 , 24, 521-533.
(19) Venkatakrishnan, K.; Von Moltke, L. L.; Greenblatt, D. J. J. Clin. Pharmacol.
2 0 0 1 , 41, 1149-1179.
(20) Rodrigues, A. D. Biochem. Pharmacol. 1 9 9 4 , 48, 2147-2156.
Analytical Chemistry, Vol. 75, No. 23, December 1, 2003 6431

incubation, the reaction mixture was pumped to and collected in
a C18 guard column (15 mm long, 1 mm i.d., Opti-Guard column,
Optimize Technologies, Inc. Oregon City, OR) at a flow rate of 2
µL/ min to desalt and preconcentrate the sample before mass
spectrometric analysis. After washing the column with Tris buffer
2.5 mM (ph 7.4) for 10 min, the column was connected to an PE
Sciex API 3000 tandem triple quadrupole mass spectrometer, and
the reaction mixture was eluted from the column with acetonitrile
containing 0.1% formic acid at a flow rate of 10 µL/ min using an
infusion pump. The mass spectral data were acquired across the
range m/ z 250-350. A control experiment was performed follow-
ing the same procedure described above but using inactivated
human liver microsomes (microsomes previously kept at 100 °C
for 5 min).
Figure 2. Schematic representation of on-chip P450 reaction using
an off-chip guard column to clean up and concentrate the reaction
mixture before detection by ESI-MS. (A) Zeonor chip; (B) fused-silica
capillary connecting the chip to the guard column (flow rate, 2 µL/
min); (C) fused-silica capillary connecting the infusion pump delivering
the elution solvent to the guard column (flow rate, 10 µL/min); (D)
infusion pump for delivering the elution solvent; (E) C18 guard column;
(F) ion spray unit; (G) PE Sciex API 3000 mass spectrometer.
Enzyme Assay. The biotransformation of imipramine into
desipramine was used as a model to study a P450 enzymatic assay
in the described chip-based format. The activity of an enzyme can
be measured by determining the rate of product formation.21 The
velocity of the substrate conversion to product versus substrate
concentration can be described by the Michaelis-Menten kinetics
that gives hyperbolic kinetics in which the Km and Vmax are
determined by the following equation:
schematic representation of a coupled on-chip P450 metabolism
region, an off-chip guard column to desalt and concentrate the
reaction mixture, followed by detection via ESI-MS. The guard
column was connected directly to the turbo ion spray unit of the
LC-MS/ MS system (Figure 2). When the guard column was used
for the sample desalting step, a fused-silica capillary was also
connected to the outlet of the chip to transfer the product reaction
mixture to the guard column. A 22.3 × 50.8 mm Thermal-Clear
Heater (Minco Prod., Inc. Minneapolis, MN) is positioned under
the chip to maintain the reaction region temperature at 37 °C.
The temperature of the Zeonor polymer chip is monitored using
a reversible surface thermometer (Omega Eng., Inc. Stanford,
CT).
MS/ MS Conditions. The MS/ MS system was a PE Sciex API
3000 tandem triple quadrupole mass spectrometer (Concord, ON,
Canada) equipped with a turbo ion spray LC/ MS interface
operated in the ion spray mode at 3300 V in the positive ion mode.
Operating conditions, optimized by infusion of a mixture of
imipramine and desipramine (both at 1 µg/ mL in acetonitrile that
contained 0.1% formic acid) at a flow rate of 10 µL/ min, were
determined as follows: the nebulizer, curtain, and collision gases
were set to 4, 12, and 4, respectively. The declustering potential
was set to 26 V and the ring voltage at 220 V. Selected reaction
monitoring (SRM) in the positive ionization mode was performed
using a dwell time of 200 ms per transition to detect transitions
at m/ z 281.2 f 86.2 (imipramine), and m/ z 267.2 f 72.0
(desipramine) using a collision energy of 28 eV and nitrogen as
the collision gas.
Enzymatic Reaction. The on-chip metabolic reaction was
performed by pumping enzyme (3.3 mg of microsomal protein/
mL in 25 mM Tris buffer, pH 7.4) and substrate solution (20 µM
in 25 mM Tris buffer, pH 7.4) through the reaction region (mixing
section of the chip). After the channels of the reaction region were
filled, the flow was stopped and the mixed solution was allowed
to incubate for 30 min at 37 °C. Pumping enzyme and substrate
solution at the same flow rates (1 µL/ min) resulted in the 1:1
dilution of the sample in the reaction region. Other components
of the reaction, including NADPH and MgCl2, were premixed off-
chip with the substrate. A mixture of the three antidepressant
drugs, imipramine, doxepin, and amitriptyline, all at an on-chip
concentration of 10 µM, was incubated with HLM (6.6 µg of
microsomal protein) in the 4-µL channel (reaction region) on the
chip. The incubation took place in 25 mM Tris buffer at a pH of
7.4, with 1 mM NADPH and 3 mM MgCl2. After 30 min of
v ) VmaxS/ (Km + S)
(1)
where S is the substrate concentration, v is the velocity of the
reaction, Vmax is the velocity of the enzyme reactions when all
the substrates are at saturating concentrations, Km is the concen-
tration of substrate required to achieve half the maximum velocity
of the enzyme. Km, and Vmax are the parameters that define a
rectangular hyperbola relating v to s. The derivation of the
equation relies on three assumptions: (1) the rate of the reverse
reaction is negligible during the measurement period, (2) the
measurement is made in the steady state when the concentration
of enzyme-substrate complex is unchanging, and (3) the forma-
tion of enzyme-substrate complex does not significantly deplete
the concentration of free substrate.21 The measured velocity should
be linearly proportional to enzyme concentration and to the time
of reaction, both requirements to ensure the assumptions underly-
ing the Michaelis-Menten relationship. Hence, following the same
procedure described above, the standard incubation conditions
were chosen on the basis of the results of preliminary experiments
that showed that the reaction for the biotransformation of
imipramine to desipramine was linear with respect to the incuba-
tion time (20 min) and the amount of enzyme protein (6.6 µg),
and it gave sufficient response by ESI-MS/ MS detection to
proceed with the planned kinetic and inhibition experiments. To
determine the value of Km for the enzymatic formation of
desipramine, enzyme kinetic experiments were performed (in
duplicate on-chip incubations) with four concentrations of imi-
pramine ranging from 5 to 100 µM. The metabolite formation (area
of the metabolite’s SRM ion current)-substrate concentration data
were fitted to the Michaelis-Menten equation, and the Km value
was determined by nonlinear regression analysis (SigmaPlot 2000,
Version 6.0).
(21) Eisenthal, R., Danson, M. J., Eds. Enzyme Assays. A Practical Approach;
Oxford University Press: New York, 1992.
6432 Analytical Chemistry, Vol. 75, No. 23, December 1, 2003

cover plate. For the two embossed inlet channels, two connecting
capillaries (360-µm o.d., 50-µm i.d., 280 mm long) were inserted
into each channel until 10 mm of each of the two capillaries was
inside the embossed channels. Another connecting capillary (360-
µm o.d., 50-µm i.d., 180 mm long) was inserted into the embossed
outlet channel until 10 mm of the capillary was inside the
embossed channel. Then, the embossed substrate plate (Figure
3B) and the embossed cover plate (Figure 3C) were aligned and
thermally bonded for 10 min. After this thermal bonding process,
epoxy glue was applied to the edges of the bonded chip around
the connecting capillaries to reinforce the bonding in that area.
P reparation of the Monolithic SP E Column. The monomer
and porogen mixture was similar to those reported by others.27-29
A typical formulation of monomer and porogen mixture was as
follows. Five milligrams of DAP and 3 mg of AMPS were dissolved
in 500 µL of methanol and 250 µL of hexane, to which 150 µL of
BMA and 100 µL of EDMA were added. After being purged with
nitrogen for 5 min, the mixture was aspirated into the chip using
a syringe. A mask containing one layer of black tape and one layer
of aluminum foil was placed over the chip with the 25-mm-long
embossed channel in the right side of the chip uncovered. The
polymerization was initiated by UV irradiation for 20 min under
UV lamp (UVL-28, 365 nm, VWR, West Chester, PA). When the
polymerization process was complete, the remaining monomer
mixture inside the connecting capillaries and nonpolymerized
monomer in the chip were removed with methanol. In Figure 3A
is shown a schematic representation of the completed chip with
the integrated monolithic column.
Figure 3. (A) Schematic diagram of the Zeonor polymeric chip (Z)
containing an incorporated SPE monolithic column. C1, C2, inlet
connecting capillaries; C3, outlet connecting capillary; RC, reaction
channels; M, monolithic SPE column. (B) The substrate plate with
embossed reaction channels (RC). (C) The cover plate embossed
against three fused-silica capillaries (EC1, EC2, EC3) to make channels
for connecting capillaries and monolithic SPE column (see text for
further details).
Imipramine is converted to its N-demethylated metabolite
primarily by CYP2C19,22 which may be inhibited by the action of
tranylcypromine.23,24 Tranylcypromine was therefore used as a
chemical inhibitor to determine its IC50 value for the CYP2C19
imipramine N-demethylation-mediated reaction. The concentra-
tions of tranylcypromine (0, 5, 10, and 15 µM) used for the
calculation of the IC50 were chosen according to those previously
shown for this compound to inhibit CYP2C19-mediated reac-
tions.25,26 Tranylcypromine was dissolved in 50% acetonitrile (final
concentration of organic solvent inside the chip was e0.5% v/ v)
and was premixed off-chip with the substrate. For the determi-
nation of the IC50, the following equation was used to determine
the formation of metabolite: Am/ Am+Ad (where Am and Ad are
the area of the metabolite’s and the area of the parent drug’s SRM
ion current, respectively). Comparison was made to experimental
control incubations (0 µM inhibitor) and the enzyme activity
expressed as the percentage of control remaining. The IC50 value
was determined by graphical linear interpolation.
Integration of a P orous Monolithic SP E Column on the
Chip. Figure 3A shows the layout of the polymer microfluidic chip
containing an incorporated monolithic SPE column. Zeonor
polymer plate was cut into 4 cm by 6 cm pieces. The substrate
plate was embossed against an etched silicon wafer master to
obtain reaction channels (Figure 3B). The cover plate was then
embossed against three conventional fused-silica capillaries (360-
µm o.d., 50-µm i.d.) as shown in Figure 3C. After completing this
embossing process, the three capillaries were peeled off and three
corresponding embossed channels (18 mm long for the two inlet
channels, 35 mm long for the outlet channel) were left in this
Using the chip with the integrated monolithic column, the P450
enzymatic reaction was performed as described above using 10
µM imipramine as the parent drug. A control experiment was also
performed following the same procedure described above but
using inactivated human liver microsomes.
RESULTS AND DISCUSSION
This work focused on demostrating the feasibility of performing
P450 enzymatic reactions using human liver microsomes in a chip-
based format coupled with ESI-MS detection. The chip was
designed to permit the mixing of the reagents, notably the enzyme
and substrate, by a molecular diffusion process in a pressure-
driven laminar flow. Experiments using a fluorescent substrate
were performed to confirm the mixing process under a fluorescent
microscope (data not shown). A pressure-driven flow inside the
chip was selected because the reactions of P450 enzymes take
place in a complex environment (two membrane-bound protein
components, cofactor, MgCl2). Although electrokinetic driven flow
is easy to implement and is used in most of the current on-chip
enzymatic reactions, it is also sensitive to the physicochemical
properties of the fluid being pumped (pH, ionic strength, viscosity)
and to bubbles in the channel, and it is surface sensitive. To ensure
adequate mixing and preclude these problems, a syringe pump
was used to provide pressure-driven flow.
(22) Madsen, H.; Rasmussen, B. B.; Brosen, K. Clin. Pharmacol. Ther. 1 9 9 7 ,
61, 319-324.
(23) Bu, H. Z.; Knuth, K.; Magis, L.; Teitelbaum, P. J. Pharm. Biomed. Anal.
Enzymatic Reaction. In vitro metabolism using the described
system was demonstrated by the transformation of a mixture of
2 0 0 1 , 25, 437-442.
(24) Hartter, S.; Tybring, G.; Friedberg, T.; Weigmann, H.; Hiemke, C. Pharm.
Res. 2 0 0 2 , 19, 1034-1037.
(25) Yu, C.; Shin, Y. G.; Kosmeder, J. W.; Pezzuto, J. M.; van Breemen, R. B.
Rapid Commun. Mass Spectrom. 2 0 0 3 , 17, 307-313.
(26) Dierks, E. A.; Stams, K. S.; Lim, H.; Cornelius, G.; Zhang, H.; Ball, S. E.
Drug Metab. Dispos. 2 0 0 1 , 29, 23-29.
(27) Yu, C.; Davey, M. H.; Svec, F.; Frechet, J. M. J. Anal. Chem. 2 0 0 1 , 73,
5088-5096.
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2 0 0 1 , 22, 3959-3967.
(29) Tan, A.; Benetton, S.; Henion, J., Anal. Chem., submitted.
Analytical Chemistry, Vol. 75, No. 23, December 1, 2003 6433

Figure 5. Michaelis-Menten curve showing the results of the
kinetics of on-chip desipramine formation. The results are the average
of duplicate measurements. The Km value was found to be 46 µM.
Figure 6. Inhibition of desipramine formation by tranylcypromine.
The results are the average of triplicate measurements (error bar is
the standard error of the mean). The IC50 value, calculated by
graphical linear interpolation, is 8 µM.
triptyline with a [M + H]+ ) 278.2, doxepin with a [M + H]+
)
280.2, and imipramine with a [M + H]+ ) 281.2) compared to
the control baseline ion current, which is from an incubation with
inactivated enzyme (dotted traces). In Figure 4B is shown the
formation of the N-demethylated metabolites of amitriptyline with
a [M + H]+ ) 264.2, doxepin with a [M + H]+ ) 266.2, and
imipramine with a [M + H]+ ) 267.2 (solid traces) compared to
control baseline ion current (dotted line). In Figure 4C is shown
the formation of the monohydroxylated metabolites of amitrip-
tyline with a [M + H]+ ) 294.2, doxepin with a [M + H]+
)
296.2, and imipramine with a [M + H]+ ) 297.2 (solid traces)
compared to control baseline ion current (dotted line). Imipramine
is reported to be hydroxylated in more than one position,30 and
these isomers are not resolved under the conditions described
here. Although, under the described conditions, both the N-
demethylated and hydroxylated metabolites of imipramine have
the interference of an isotope of amitriptyline, it was still possible
to observe the formation of these metabolites (Figure 4B,C).
Enzyme Assay. On-chip experiments were performed to
characterize the enzyme kinetics of the N-demethylation of
imipramine with pooled human liver microsomes, and the results
were plotted in a Michaelis-Menten curve (Figure 5). Imipramine
was chosen as a model compound for the enzymatic assay because
the kinetics of its transformation into desipramine is described
in the literature along with the enzyme responsible for this
biotransformation.31,32 The Km value was found to be 46 µM, which
Figure 4. On-chip incubation of doxepin, amitriptyline, and imi-
pramine with HLM. Preconcentrantion of sample using a 1-mm C18
guard (Optiguard) and detection by Q1 full-scan (m/z 250-350) ESI-
MS (PE Sciex API 3000). Solid line, results from incubation; dotted
line, results from blank (incubation with inactive HLM). (A) Consump-
tion of amitriptyline (m/z 278.1), doxepin (m/z 280.1), and imipramine
(m/z 281.1); (B) formation of N-demethylated metabolites of amitrip-
tyline (m/z 264.2), doxepin (m/z 266.2), and imipramine (m/z 267.2),
respectively; (C) formation of OH-amitriptyline (m/z 294.2), OH-
doxepin (m/z 296.2), and OH-imipramine (m/z 297.2).
imipramine, doxazepin, and amitriptyline into their respective
metabolites inside a 4-µL volume within the channel of a polymer
chip by the action of human liver microsomes (Figure 4). The
identities of the metabolites were confirmed by the mass spectra
obtained under the abbreviated full-scan MS analysis. Figure 4A
shows the consumption (solid traces) of the parent drugs (ami-
(30) Chen, A. G.; Wing, Y. K.; Chiu, H.; Lee, S.; Chen, C. N.; Chan, K. J.
Chromatogr., B: Biomed. Sci. Appl. 1 9 9 7 , 693, 153-158.
(31) Obach, R. S.; Reed-Hagen, A. E. Drug Metab. Dispos. 2 0 0 2 , 30, 831-837.
6434 Analytical Chemistry, Vol. 75, No. 23, December 1, 2003

Figure 7. ESI-MS results from the incubation of imipramine with HLM in a chip with on-chip sample cleanup employing a monolithic SPE
column. The data were acquired with Q1 scan across the range m/z 250-350. The spectrum shows the formation of the N-demethylated
metabolite, desipramine, with a [M + H]+ ) 267.2, the formation of the monohydroxylated isomeric metabolites of imipramine with [M + H]+
297.2, and the remaining parent drug imipramine with [M + H]+ ) 281.2.
)
is an acceptable result33 compared to those reported in the
literature, 10-20 µM.31 On-chip incubation of imipramine with
different concentrations of tranylcypromine allowed the determi-
nation of the IC50 value for the inhibition of the CYP2C19-
mediated formation of desipramine. The value of the IC50 for
tranylcypromine was found to be 8 µM, which is in agreement
with values (8.9, 9 µM) found for the inhibition of other CYP2C19-
mediated reactions that show correlation with the N-demethylation
of imipramine.25,26,32 The experiments were carried out in triplicate
on three different days and the precision of the assay, expressed
as coefficient of variation (CV), varied from 7 to 23% at all
concentrations used, which demonstrated the acceptable repro-
ducibility of the on-chip experiments under the conditions
described in this work (Figure 6).
Integrated P orous Monolithic Column. The mass spectral
results obtained using the monolithic column integrated on the
chip are shown in Figure 7. In this experiment, imipramine was
incubated on-chip in the described P450 in vitro mixture followed
by on-chip SPE desalting employing the monolithic column. The
identity of the metabolites was confirmed by the mass spectra
obtained under the mass range m/ z 250-350. Figure 7 shows
the formation of the N-demethylated metabolite, desipramine, with
[M + H]+ ) 267.2, and the formation of the monohydroxylated
isomeric metabolites of imipramine with [M + H]+ ) 297.2 with
a good response by ESI-MS. These results demonstrate the
possibility of performing P450 enzymatic reactions in a microvol-
ume and detecting the metabolic products of the reaction using
ESI-MS with a fully integrated on-chip sample preparation pro-
cedure.
The rate of the P450 enzymatic reaction, as with any enzymatic
reaction, depends on the temperature and the pH. P450 enzymatic
reactions were performed at a controlled temperature of 37 °C,
and heating of the chip was achieved with the use of a 22.3 ×
50.8 mm Thermal-Clear heater positioned under the chip. The
actual temperature inside the chip was not determined, but it was
inferred to be in the vicinity of 37 °C by monitoring the
temperature of the upper part of the Zeonor polymer chip using
a reversible surface thermometer. The optimal pH value of 7.4
used in P450 assays is not favorable for the retention of basic
drugs and their respective metabolites in a C18 SPE column. The
pKa of imipramine, for example, is 9.4, and making a direct
connection between the reaction region and the SPE column, as
shown in Figure 3, does not permit adjustment of the pH for an
efficient extraction of these compounds, which would lead to the
prospect of using less enzyme or a smaller volume. Despite
working with a nonfavorable pH for extraction, the retention of
metabolites was enough for determining the feasibility of the
process. In future work, the integration of pH adjustment before
introducing the enzymatic mixture into the column could poten-
tially lead to a more efficient protocol.
Another characteristic of a microscale device is the increase
in the surface-to-volume ratio, sometimes by orders of magnitude.34
A well-known problem due to this phenomenon is that surface
adsorption of biological macromolecules and small molecules can
increase. Since the on-chip enzymatic reaction described in this
work takes place in the whole volume of the chip, enzyme
adsorption to the walls of the channels did not appear to impede
the formation of the products. Additionally, P450 reactions are
traditionally carried out in an open environment so that the
(32) Chiba, K.; Saitoh, A.; Royana, E.; Tani, M.; Hayashi, M.; Ishizaki, T. Br. J.
Clin. Pharmacol. 1 9 9 4 , 37, 237-242.
(33) Tucker, G. T.; Houston, J. B.; Huang, S. M. Br. J. Clin. Pharmacol. 2 0 0 1 ,
(34) Locascio, L. E.; Perso, C. E.; Lee, C. S. J. Chromatogr., A 1 9 9 9 , 857, 275-
52, 107-117.
284.
Analytical Chemistry, Vol. 75, No. 23, December 1, 2003 6435

reaction is exposed to atmospheric oxygen. Although this work
presents only preliminary results on the feasibility of performing
P450 enzymatic reactions on a chip format, it demonstrated that
P450 biotransformation reactions can occur in a closed compart-
ment.
Generally, the amount of enzyme and the volume used for an
assay will depend on several factors. These include the follow-
ing: (1) the sensitivity of the analytical assay for the metabolites;
(2) the kinetic parameters of enzyme catalysis for a particular
substrate and P450; (3) the incubation period of the assay; and
(4) the solubility of the test compound or substrate. Using three
antidepressant drugs, we have demonstrated that, even using
these microvolumes, the P450 enzymatic reaction can be per-
formed for these compounds and form sufficient quantities of
metabolites to permit their detection by ESI-MS.
order to perform, directly on the chip, the cleanup and concentra-
tion process necessary for ESI-MS detection. This combined
system allowed the P450 enzymatic reactions to occur in a
microvolume and the detection of expected metabolic products
of the reaction using ESI-MS in chip-based integrated process. A
key advantage of miniaturizing P450 enzymatic reactions is the
use of low sample volumes, hence the use of minimum quantities
of costly material. Furthermore, the use of ESI-MS provides a
sensitive, selective detection strategy, which also can provide
structural information. We suggest this strategy may present an
important advance for both P450 inhibition assays and for
metabolite screening studies in the future.
ACKNOWLEDGMENT
The authors thank Merck Research Laboratories (MRL) for
financial support of the research project and Dr. Richard King
from MRL for the helpful discussions. We also thank PE Sciex
for the generous loan of the API 3000 mass spectrometer system.
CONCLUSIONS
In this work, we have shown the feasibility of performing P450
enzymatic reactions inside a 4-µL volume housed in a cyclic olefin
polymer chip by the action of human liver microsomes. A
commercial guard column was initially used to desalt the products
of the enzymatic reaction and preconcentrate the analytes. A
porous monolithic column was also integrated into the chip in
Received for review July 2, 2003. Accepted August 28,
2003.
AC030249+
6436 Analytical Chemistry, Vol. 75, No. 23, December 1, 2003