been reported in which microsomal UDPGTs were immobilized
on chromatographic supports to create immobilized enzyme
reactors (IMERs).10-13 The on-line use of UPDGT-IMERs typically
reduced analysis time and labor and allowed for the reuse of the
UDPGTs, but required a number of steps to prepare. In addition,
in order to separate substrates and products, it was necessary to
couple the UDPGT-IMERs to analytical columns.
tris(hydroxymethyl)aminomethane, magnesium chloride hexahy-
drate (MgCl ‚6H O), and uridine 5′-diphosphoglucuronic acid
(UDPGA) were purchased from Sigma-Aldrich (St. Louis, MO).
2
2
Ammonium persulfate, (γ-methacryloxypropyl)trimethoxysilane,
polyacrylamide (average M
w
∼10 000), acrylamide, and TEMED
were purchased from Aldrich (Milwaukee, WI). Methanol (HPLC
grade) was from Fisher Scientific (Pittsburgh, PA). Protease
inhibitor cocktail set III was obtained from Calbiochem (San
Diego, CA). Reagents for the bicinchoninic acid (BCA) protein
assay were obtained from Pierce Chemical Co. (Rockford, IL).
All other chemicals were of the highest purity available. All
aqueous solutions were prepared using water from a Millipore
Milli-Q water system (Billerica, MA) and filtered using Osmonics
0.22-µm nylon filters purchased from Fisher Chemical Co.
Capillary Electrophoresis. CE was performed on a Beckman
P/ACE MDQ capillary electrophoresis system (Beckman Coulter,
Inc., Fullerton, CA) controlled by 32 Karat software (Version 5.0,
Beckman Coulter, Inc.). The CE instrument was equipped with
PDA detector. The electrophoretic separations were performed
in a 100-µm-i.d., 40-cm-total length (30 cm to the detector window)
neutrally coated columns. The preparation of neutrally coated
The use of capillary electrophoresis (CE) is an alternative
approach to the on-line formation and monitoring of glucuronida-
tion. CE generally requires small amounts (nL range) of reagents
and provides relatively fast analysis times with highly efficient
separations. An on-column immobilized enzyme microreactor has
1
4-16
been used with CE in several studies.
Sakai-Kato et al. have
reported a miniaturized on-line glucuronidation system based on
encapsulation and immobilization of UDPGTs on monolithic
17
capillary columns. The columns were placed in a CE system and
used to carry out enzymatic glucuronidations. However, this
method required an on-line incubation of 10-90 min, which was
accomplished by stopping the electroosmotic flow. Similar ap-
proaches were also reported by Righetti and co-workers using
enzymes that were isoelectrically trapped in membranes and
enzymatic products were later migrated to the detection window
2
0
capillary (i.e., linear polyacrylamide coating) was followed. The
bare silica capillary was obtained from Polymicro Technologies
(Phoenix, AZ). The capillary was cut with capillary cutting tool,
and a capillary window (0.3 cm) was prepared by burning with
flame and removing burned residues. The capillary was filled with
0.1 M NaOH for 3 h and rinsed with water to maximize free
silanols on capillary surface. The capillary was again filled with
0.1 M HCl for 5 min and rinsed with water. Finally the capillary
was filled with 3% (v/v) (γ-methacryloxypropyl)trimethoxysilane
in 60% (v/v) acetone/water mixture for overnight to neutralize
the capillary surface. The silane reagent attached to the capillary
was further reacted with acrylamide (3%, w/v) in the presence of
TEMED (0.8 µL/mL) and ammonium persulfate (2 mg/mL) in
water. The capillary was sealed for 8 h after which the unreacted
reagents were removed with water. CE Separation polymeric run
buffer was prepared by mixing 80% (v/v) polyacrylamide, 0.1%
1
8,19
by differential charges.
In these reports, the reactions were
not carried out under the conditions of an applied electric field or
continuous flow.
We now report the development of an on-line capillary
electrophoresis method for the rapid production and separation
of glucuronides that does not require an incubation period. In this
approach, rat liver microsomes were used with a replaceable
polymeric run buffer to create a pseudostationary phase in linear
polyacrylamide-coated capillary column. The substrates and neces-
sary cofactors were then injected onto the system and the
glucuronides were formed and separated. The results of this study
demonstrate that this technique can be applied to the screening
of new drug candidates and should reduce overall analysis time
and increase throughput. This approach can be expanded to other
microsomal and membrane-bound enzymes.
(
w/v) acrylamide, 10% (w/v) tris(hydroxymethyl)aminomethane,
EXPERIMENTAL SECTION
and 10% (w/v) boric acid in water.
Microsomal Fraction Preparation. The UDPGT-containing
microsomal fraction of rat liver was isolated using a previously
Reagents. Morphine, morphine glucuronides (3 and 6),
codeine, codeine 6-glucuronides, 4-nitrophenol (4NP), 4-nitrophe-
nol â-
hydroxycoumarin, 4Me7OHC), 4-methylumbelliferone â-
ronide (4Me7OHCG), boric acid, Trizma hydrochloride (Tris-HCl),
D-glucuronide (4-NPG), 4-methylumbelliferone (4-methyl-7-
21
reported method, which was modified to meet the experimental
D-glucu-
requirements of this study. All steps in the microsomal fraction
preparation were done at 4 °C unless stated otherwise. Briefly,
fresh liver from an 8-week-old Sprague-Dawley rat was minced
and rinsed several times with 50 mL of ice-cold potassium
phosphate buffer (homogenized buffer, 250 mM, pH 7.4) contain-
ing 150 mM KCl to remove excess blood. The minced liver was
placed in 30 mL of ice-cold homogenized buffer with 1 mL of
protease inhibitor cocktail set III. The minced liver mixture was
homogenized for 30 s using a Polytron PT 2100 homogenizer
(Kinematica AG, Luzern, Switzerland) at setting 11. The homo-
genate was centrifuged at 14500g for 20 min using a Beckman
XL 90 Ultracentrifuge (Beckman Coulter, Inc.), the supernatant
was recovered, centrifuged again at 14500g for 20 min, the
resulting supernatant was further centrifuged at 100000g for 60
(
8) Qian, M. R.; Zeng, S. Acta Pharmacol. Sin. 2006, 27, 623-628.
(
9) Bogan, D. P.; Killard, A. J.; O’Kennedy, R. J. Capillary Electrophor. 1995,
2, 241-245.
(
(
(
10) Kim, H. S.; Wainer, I. W. J. Chromatogr., B: Anal. Technol. Biomed. Life
Sci. 2005, 823, 158-166.
11) Zihnioglu, F.; Telefoncu, A. Artif. Cells, Blood Substitutes. Immobilization
Biotechnol. 1995, 23, 533-543.
12) Zihnioglu, F.; Telefoncu, A. Artif. Cells, Blood Substitutes. Immobilization
Biotechnol. 1995, 23, 545-552.
(
(
13) Zihnioglu, F.; Telefoncu, A. Biochim. Biophys. Acta 1995, 1244, 291-294.
14) Belenky, A.; Hughes, D.; Korneev, A.; Dunayevskiy, Y. J. Chromatogr., A
2004, 1053, 247-251.
(15) Tang, Z. M.; Kang, J. W. Anal. Chem. 2006, 78, 2514-2520.
(16) Jin, Z.; Chen, R.; Colon, L. Anal. Chem. 1997, 69, 1326-1331.
(17) Sakai-Kato, K.; Kato, M.; Toyo’oka, T. J. Chromatogr., A 2004, 1051, 261-
2
66.
(
18) Righetti, P. G.; Bossi, A. Electrophoresis 1998, 19, 1075-1080.
(
19) Castelletti, L.; Bossi, A.; Righetti, P. G. Biotechnol. Bioeng. 2000, 69, 39-
(20) Kim, H. S.; Austin, J.; Hage, D. S. Electrophoresis 2002, 23, 956-963.
(21) Schenkman, J. B.; Jansson, I. Methods Mol. Biol. 1998, 107, 55-67.
4
6.
7072 Analytical Chemistry, Vol. 78, No. 20, October 15, 2006