Analysis of free drug fractions using near-infrared fluorescent labels and an ultrafast immunoextraction/displacement assay

Article abstract of DOI:10.1021/ac061215f

A chromatographic method was developed for measuring free drug fractions based on the use of an ultrafast immunoextraction/displacement assay (UFIDA) with near-infrared (NIR) fluorescent labels. This approach was evaluated by using it to determine the fre

Full text of DOI:10.1021/ac061215f

Anal. Chem. 2006, 78, 7547-7556
Analysis of Free Drug Fractions Using
Near-Infrared Fluorescent Labels and an Ultrafast
Immunoextraction/Displacement Assay
Corey M. Ohnmacht, John E. Schiel, and David S. Hage*
Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304
make it difficult to correlate the total concentration of a drug with
its free fraction and has created a need for new methods that can
routinely measure free drug fractions.
A chromatographic method was developed for measuring
free drug fractions based on the use of an ultrafast
immunoextraction/displacement assay (UFIDA) with near-
infrared (NIR) fluorescent labels. This approach was
evaluated by using it to determine the free fraction of
phenytoin in serum or samples containing the binding
protein human serum albumin (HSA). Items considered
in the design of this method included the dissociation rate
of HSA-bound phenytoin, the rate of capture of free
phenytoin by immunoextraction microcolumns, the be-
havior of NIR fluorescent labels in a displacement format,
and the overall response and stability of the resulting
assay. In the final UFIDA method, the free fraction of
phenytoin was extracted in ∼100 ms by a microcolumn
containing a small layer of anti-phenytoin antibodies. This
gave a displacement peak for a NIR-fluorescent-labeled
analogue of phenytoin that appeared within 2-3 min of
sample injection, creating a signal proportional to the
amount of free phenytoin in the sample. The UFIDA
method provided results within 1-5% of those deter-
mined by ultrafiltration for reference samples. The lower
limit of detection was 570 pM, and the linear range
extended up to 10 µM. This approach is not limited to
phenytoin but can be adapted for other analytes through
the use of appropriate antibodies and labeled analogues.
Phenytoin is an antiepileptic drug that has significant binding
in blood.6,7 This drug is mainly bound in blood to the protein
human serum albumin (HSA), with ∼90% of phenytoin being
complexed at therapeutic levels in adults.8-14 Two clinical situa-
tions in which the bound fraction of phenytoin might decrease
include infants with jaundice (due to the competition of bilirubin
with phenytoin for HSA) and patients that have low HSA levels
following trauma or surgery.9 It is also possible for a patient to
have an increased bound fraction of phenytoin, as can occur in
individuals with high HSA concentrations (i.e., hyperalbumin-
emia).9
There have been numerous methods developed for the
measurement of phenytoin’s free fraction in blood, plasma, or
serum.6,10,12,15-22 Examples include techniques based on equilib-
rium dialysis, ultrafiltration, and restricted access media (RAM)
columns.6,10,14,15,18,19,21 Other techniques such as CE can also can
be used to examine drug-protein interactions in standard
solutions,23-26 but many of these methods are not applicable for
(6) Bereczki, A.; Horvath, V. Anal. Chim. Acta 1999, 391, 9.
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G.; Rasmussen, K. E. J. Chromatogr., B 1995, 669, 281.
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Mitsuyama, Y. J. Clin. Pharm. Ther. 1998, 23, 361.
Many drugs exist in two forms in the circulation: a free fraction
and a fraction that is reversibly bound to serum proteins or other
agents in blood.1,2 The free fraction of a drug is generally thought
to represent its active form, since this is the form that can cross
cell membranes or bind to receptors.3 The binding of drugs to
blood or serum components is important in drug delivery.4
However, the extent of this binding can be affected by various
factors that can lead to individual variations in free drug fractions.5
For instance, the extent of a drug’s binding with serum proteins
can vary as a result of illness, trauma, surgery, or age.2 This can
(12) Roberts, W. L.; Annesley, T. M.; De, B. K.; Moulton, L.; Juenke, J. M.; Moyer,
T. Ther. Drug Monit. 2001, 23, 148.
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1986, 23, 603.
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Wang, C.-H. J.; Nystrom, D. D.; Keegan, C. L.; David, T. P.; Stroupe, S. D.
Clin. Chem. 1982, 28, 2278.
* Corresponding author. Phone: (402) 472-2744. FAX: (402) 472-9402.
E-mail: dhage@unlserve.unl.edu.
(18) McGregor, A. R.; Crookall-Greening, J. O.; Landon, J.; Smith, D. S. Clin.
Chim. Acta 1978, 83, 161.
(1) Clarke, W.; Schiel, J. E.; Moser, A.; Hage, D. S. Anal. Chem. 2005, 77,
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D. B. J. Pharm. Sci. 1985, 74, 692.
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10.1021/ac061215f CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/30/2006
Analytical Chemistry, Vol. 78, No. 21, November 1, 2006 7547

dissociation of this analyte from its binding agents in the sample
(e.g., HSA). At the beginning of this assay, shown in the upper
left of Figure 1a, an excess of a labeled analogue of the analyte is
injected onto the immunoextraction microcolumn in the presence
of an application buffer. Some of this labeled analogue will bind
to antibodies in the column while the remainder will be washed
away prior to sample injection. When a sample is later passed
through the column, the free fraction of the analyte will compete
with any labeled analogue that is momentarily dissociated from
the immobilized antibodies. This results in a displacement peak
for the labeled analogue, as shown in Figure 1b, and gives a signal
that is proportional to the analyte’s free fraction. The retained
analyte and labeled analogue can later be removed from the
column by using an elution buffer. The column is then allowed to
regenerate and the entire process is repeated for another sample.
Ultrafast immunoextraction and a displacement assay have
recently been used with chemiluminescence detection for the
measurement of free thyroxine in serum.1 However, this past work
involved the use of a complex postcolumn reaction and required
serum blanks to correct for matrix effects on the chemilumines-
cence signal.1 This current study will examine an alternative
approach using near-infrared (NIR) fluorescent cyanine dyes as
labels (see Figure 2). A major advantage of these labels is they
do not require any postcolumn reaction for detection. In addition,
these dyes have low background signals for many biological
samples and can provide detection limits in the femtomole range.29
These dyes have been used in DNA sequencing, traditional
immunoassays, and capillary electrophoresis,29,30 but they have
not yet been used in either chromatographic immunoassays or
the measurement of free drug fractions. Although more traditional
fluorescent labels have been employed in some types of chro-
matographic-based immunoassays,31-34 these other labels have
also not been used in UFIDA methods or free drug assays.
This current study will explore the combined use of NIR
fluorescent dyes and UFIDA for measuring free phenytoin
fractions. One parameter that will be examined in this study is
the development of immunoextraction microcolumns that can bind
free phenytoin on the millisecond time scale. The creation and
use of NIR fluorescent labeled analogues of phenytoin for
displacement assays will also be considered. The final UFIDA
method will be evaluated in terms of its accuracy, precision, and
speed when analyzing both drug-protein mixtures and serum
samples. The advantages and limitations of this method will then
be considered, as well as its possible extension to other analytes.
Figure 1. (a) General scheme for an ultrafast immunoextraction/
displacement assay (UFIDA) and (b) a typical chromatogram for such
an assay. The example in (b) is based on the phenytoin system
described in the Experimental Section. In (b), the excess labeled
phenytoin was injected at 0 min and the sample containing the
unlabeled phenytoin was injected at 6 min. Symbols: (b), labeled
analogue of analyte; (O), unlabeled analyte from sample; (U), binding
agent in a sample for the analyte; (Y), immobilized antibody.
work with the small free fractions of drugs or the complex
matrixes that are found in clinical samples. Some previous
methods for free phenytoin measurements suffer from long
analysis times (e.g., equilibrium dialysis) or nonspecific adsorption
to membranes (e.g., ultrafiltration).19 Other techniques (e.g., RAM)
give only an incomplete separation of phenytoin’s free and bound
forms, making these methods difficult to use with real clinical
samples.1,2,16,27
This paper describes an alternative chromatographic-based
approach for measuring free drug fractions based on an ultrafast
immunoextraction/displacement assay (UFIDA), as illustrated in
Figure 1.28 This approach uses an immunoextraction microcolumn
that can bind a measurable amount of the free analyte (e.g.,
phenytoin) on a time scale that is sufficiently small to avoid
EXPERIMENTAL SECTION
Reagents. The phenytoin (99% pure), mouse IgG, human
plasma (lyophilized, pooled), and HSA (Cohn fraction V, 99% fatty
acid free) were from Sigma-Aldrich (St. Louis, MO). The IRDye
800 CW (N-hydroxysuccinimide, or NHS ester) was donated by
LI-COR Biosciences (Lincoln, NE). The monoclonal anti-phenytoin
(29) Reng, X.; Draney, D. R. LabPlus Int. 2004, 18, 8.
(30) Sowell, J.; Parihar, R.; Patonay, G. J. Chromatogr., B 2001, 752, 1.
(31) Miki, S.; Kaneta, T.; Imasaka, T. J. Chromatogr., A 2005, 1066, 197.
(32) Castle, P. E.; Phillips, T. M.; Hildesheim, A.; Herrero, R.; Bratti, C. M.;
Rodriguez, A. C.; Morera, L. A.; Pfeiffer, R.; Hutchinson, M. L.; Pinto, L. A.;
Schiffman, M. J. Biochem. Biophys. Methods 2003, 12, 1449.
(33) Dodge, A.; Fluri, K.; Verpoorte, E.; de Rooij, N. F. Anal. Chem. 2001, 73,
3400.
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(27) Wang, H.; Zou, H.; Zhang, Y. Anal. Chem. 1998, 70, 373.
(28) Nelson, M. A.; Reiter, W. S.; Hage, D. S. Biomed. Chromatogr. 2003, 17,
188.
(34) Phillips, T. M. J. Biochem. Biophys. Methods 2001, 49, 253.
7548 Analytical Chemistry, Vol. 78, No. 21, November 1, 2006

Figure 2. Scheme for the synthesis of a phenytoin conjugate containing a label based on a near-IR fluorescent dye. The first reaction (a)
shows the preparation of ADPH, while the second reaction (b) shows how ADPH was used to prepare the final labeled phenytoin. The dye
shown in this example is NHS-activated IRDye 700DX from LI-COR Biosciences, which is similar to the proprietary dye used in this current
study.29
antibodies (clone P0825.1, purified from ascites fluid; stored in
pH 7.2 phosphate-buffered saline plus 0.05% sodium azide) were
from Accurate Chemical (Westbury, NY). Nucleosil Si-300 (7-µm
particle size, 300-Å pore size) was purchased from P. J. Cobert
(St. Louis, MO). Reagents for the bicinchoninic acid (BCA) protein
assay were from Pierce (Rockford, IL). The phenytoin calibration
standards, free phenytoin calibrators, and control serum samples
(pooled serum containing known amounts of phenytoin) were
from Abbott Laboratories (Chicago, IL). Other chemicals were
reagent grade or better. All aqueous solutions were prepared using
deionized water from a Nanopure water system (Barnstead,
Dubuque, IA).
Technology International (Rockwood, TN), using an excitation
wavelength of 770 nm and an emission wavelength range of 780-
900 nm.
The HPLC system used to analyze free phenytoin fractions
after ultrafiltration consisted of a Jasco PU980 pump (Easton, MD),
an Alltech water jacket, and a LDC Analytical 3100 UV detector
(Riviera Beach, FL), along with a 2.1 mm i.d. × 4.5 cm HSA
column (prepared as described previously).8,35 Samples were
injected onto this system using a 20-µL sample loop and a
Thermoseparations AS3000 autosampler (Schaumberg, IL). The
same chromatographic system was used to determine the extrac-
tion efficiency of the anti-phenytoin immunoextraction microcol-
umns, with these columns or their controls being used in place
of the HSA column. The immunoextraction microcolumns were
packed according to a previous method.36,37
The chromatographic system used for the UFIDA method was
similar to that described in the previous paragraph but included
a second Jasco PU980 pump and a Rheodyne model EV700 six-
port switching valve (Cotati, CA) for alternating passage of the
application and elution buffers through the anti-phenytoin immu-
noextraction microcolumn. The labeled phenytoin was injected
onto this system in volumes ranging from 5 to 100 µL, followed
by 5-µL injections of the sample. Detection of the labeled
phenytoin was performed using a custom-built HPLC NIR fluo-
rescence detector from LI-COR. This detector was constructed
Apparatus. Mass spectra of phenytoin and its conjugates were
obtained using electron ionization mass spectrometry (EI-MS)
performed on a VG AutoSpec mass spectrometer from Fisons
1
(now Waters/Micromass) (Milford, MA). H NMR spectra were
acquired for phenytoin and its conjugates on a Bruker DRX Avance
500-MHz spectrometer (Billerica, MA) using samples dissolved
in deuterated dimethyl sulfoxide (DMSO). Ultrafiltration was
performed using Centrifree Micropartition Devices (MW cutoff,
30 kDa; sample capacity, 0.15-1.0 mL) from Amicon (Danvers,
MA), along with a fixed rotor basket centrifuge from Dynac
(Parsippany, NJ). The temperature during the ultrafiltration
experiments was controlled by placing the centrifuge in a Precision
P3 incubation cabinet from Expotech (Houston, TX). Samples for
the BCA protein assay were analyzed using a Shimadzu UV160U
absorbance spectrophotometer (Kyoto, Japan). The fluorescence
properties of the labeled phenytoin and serum samples were
examined using a QuantaMaster spectrofluorometer from Photon
(35) Ohnmacht, C. M.; Chen, S.; Tong, Z. J. Chromatogr., B 2006, 836, 83.
(36) Clarke, W.; Beckwith, J. D.; Jackson, A.; Reynolds, B.; Karle, E. M.; Hage,
D. S. J. Chromatogr., A 2000, 888, 13.
(37) Clarke, W.; Hage, D. S. Anal. Chem. 2001, 73, 1366.
Analytical Chemistry, Vol. 78, No. 21, November 1, 2006 7549

with a 25-µL flow cell positioned at the interface of the laser and
detector focus, using the same optical system as described in ref
38, with the laser diode source and microscope detector being
positioned 90° with respect to the flow cell. The emitted wave-
length of the laser diode was 785 nm. Excitation wavelengths were
selected using a 20-nm band-pass filter centered at 820 nm. The
temperature of the immunoextraction microcolumn or control
column was maintained by using water jackets (Alltech) and a
Brinkmann circulating water bath (Westbury, MA). All chromato-
graphic data were collected using programs written in Labview
5.1 (National Instruments, Austin, TX). Retention times and areas
of the resulting chromatographic peaks were calculated using
PeakFit 4.12 (Systat Software, Richmond, CA).
assay. It was found later that the 38% of unconjugated dye in this
preparation did not create any noticeable interference due to
background signal or nonspecific binding in the final UFIDA assay.
The purity of this labeled phenytoin was measured by comparing
the NIR fluorescence for the retained peak (i.e., the labeled
phenytoin) and nonretained peaks (containing the unconjugated
dye) when a 5-µL sample of this preparation was injected in pH
7.4, 0.067 M potassium phosphate buffer at 0.5 mL/min onto a
2.1 mm i.d. × 4.5 cm immobilized HSA column, with pH 7.4, 0.067
M potassium phosphate buffer also being used as the mobile
phase. This HSA column was prepared as described earlier8,35,44,45
and had a protein content of 28 ((2 mg) of HSA/g of silica ((1
SD). As has been noted for phenytoin,8,35 this HSA column was
found to retain the labeled phenytoin while the free acid form of
the NIR fluorescent dye eluted near the column void volume of
this column.
A stock solution was made for the labeled phenytoin in pH
7.4, 0.067 M phosphate buffer. The concentration of labeled
phenytoin in this stock solution was estimated to be 558 ((20)
µM after a correction was made for unconjugated dye in the final
product. This concentration was determined by HPLC, as de-
scribed in the previous paragraph, using both this stock solution
and standards containing known concentrations of the NIR
fluorescent dye. When not in use, this stock solution was stored
in the dark in an amber vial at 4 °C. This stock solution was used
over the course of approximately four months during this study.
Preparation of Immunoextraction Microcolumn. Prior to
immobilization, the anti-phenytoin antibodies were transferred
from their original solution (pH 7.2 phosphate-buffered saline
containing 0.05% sodium azide) into pH 6.0, 0.10 M potassium
phosphate buffer. This transfer was accomplished by applying a
3.0-mL sample of these antibodies to a 10-mL Econo-PAC 10DG
column (exclusion limit, 6000 Da) from BioRad (Hercules, CA)
using pH 6.0, 0.10 M potassium phosphate buffer as the mobile
phase. The collected antibodies were stored at 4 °C in the same
pH 6.0, 0.10 M phosphate buffer.
Prior to its use in immobilization, Nucleosil Si-300 silica was
converted into a diol-bonded form according to a previous
method.44 The final diol coverage of this material was 306 ((3)
µmol/g of silica, as determined in triplicate by an iodometric
capillary electrophoresis assay.45 The anti-phenytoin antibodies in
pH 6.0, 0.10 M potassium phosphate buffer were immobilized onto
this diol-bonded silica by the Schiff base method.46 An inert control
support was prepared in an identical manner but with no
antibodies being added during the immobilization step (note: this
type of support was found to be an adequate control for this work
because of the specificity of the anti-phenytoin antibodies and
relatively low nonspecific binding of the analytes or labeled
analogues to the control support; however, a control support
containing immobilized nonspecific antibodies could also be used).
After immobilization, the anti-phenytoin support was washed three
times with pH 7.4, 0.067 M potassium phosphate buffer and stored
at 4 °C until use. A portion of this support was washed several
times with deionized water, dried, and analyzed in triplicate by a
BCA protein assay.47 This assay gave a protein coverage of 29
Preparation of Labeled Phenytoin. Phenytoin was converted
into an amine derivative (i.e., 3-N-amino-5,5-diphenylhydantoin, or
ADPH) by the reaction scheme shown in Figure 2.39-42 This
involved heating a mixture of 2.50 g of phenytoin (0.01 mol) with
3.90 mL of hydrazine hydrate (0.08 mol) at 135 °C for 5 h; no
other solvent was required for this reaction. ADPH was recrystal-
lized by placing 1 g of the solid product in ∼15 mL of ethanol.
Deionized water was then added, and this mixture was allowed
to stand undisturbed at 4 °C for 2 days. The resulting crystals
were filtered and dried at 80 °C under vacuum for 8 h. When
analyzed by EI-MS, the product of this reaction gave a molecular
ion with a mass-to-charge ratio of 267.1019 (mass accuracy, 4.2
ppm) and fragment ions with masses in agreement with those
previously reported for ADPH.39
A
1H NMR spectrum for this
substance also agreed with that expected for ADPH. Some
byproduct (i.e., 5,5-diphenyl-1,2,4-triazine-3,6-dione) was also noted
in this preparation, which was estimated to make up ∼40-50% of
the final product.
ADPH was conjugated to NHS-activated IRDye 800 CW by
using an approach similar to that shown in Figure 2.43 This
involved dissolving 9 µmol of NHS-activated IRDye 800 CW in
0.5 mL of DMSO, followed by the slow addition of 20 µmol of
ADPH in 0.5 mL of DMSO and 1 mL of pH 8.0, 0.10 M potassium
phosphate buffer. The resulting mixture was slowly stirred with
a magnetic stir bar for 4 h in an ice-water bath. This mixture
was then dried to remove all solvent, with the remaining residue
being dissolved in 10 mL of pH 7.4, 0.067 M potassium phosphate
buffer. This product was extracted three times with 10-mL portions
of ethyl acetate to remove any unconjugated reactants. The
remaining aqueous fraction was dried to remove any ethyl acetate;
this gave a residue containing the phenytoin-dye conjugate and
unconjugated dye. No further purification of the final labeled
phenytoin dye conjugate (i.e., “labeled phenytoin”) was required
for this study.
The purity of the labeled phenytoin in the final product was
estimated to be 62%. This purity was sufficient for use in this study,
since an excess of labeled phenytoin was used in the displacement
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A.; Osterman, H.; Peterson, R.; Rada, J.; Raghavachari, R.; Roemer, S. In
Near-infrared dyes for high technology applications; Daehne, S., Ed.; Kluwer
Academic: Dordrecht, The Netherlands, 1998; p 21.
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(42) Shaffer, J. W.; Sheasley, R.; Winstead, M. B. J. Med. Chem. 1967, 10, 739.
(43) Hermanson, G. T. Bioconjugate Techniques; Academic Press: San Diego,
1996.
(44) Ruhn, P. F.; Garver, S.; Hage, D. S. J. Chromatogr. 1994, 669, 9.
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7550 Analytical Chemistry, Vol. 78, No. 21, November 1, 2006

((7) mg of antibodies/g of silica when using mouse IgG as the
standard and the control support as the blank.
performed on control serum samples containing known concentra-
tions of phenytoin and on various HSA/phenytoin mixtures in pH
7.4, 0.067 M potassium phosphate buffer. Each of these samples
was centrifuged in the presence of an ultrafiltration membrane at
37 °C for 45 min at 1500g. After centrifugation, ∼0.5 mL of the
filtrate was collected and stored at 4 °C until further use. The
concentration of free phenytoin in the filtrate was measured by
using HPLC along with the same HSA column described earlier
for examining the purity of the labeled phenytoin. Samples
containing 20 µL of the filtrate were injected onto this column in
triplicate at 0.5 mL/min and at room temperature. The elution of
phenytoin was monitored at 205 nm and gave a retention factor
of 2.3 on this column. No peaks from other sample components
were noted in the vicinity of the retention time for phenytoin. A
linear response was obtained on this column for phenytoin
standards containing 2-15 µM phenytoin in pH 7.4, 0.067 M
phosphate buffer (correlation coefficient, 0.9998 for n ) 5).
Nonspecific binding by phenytoin in the ultrafiltration device
was measured by using a series of standards that contained
phenytoin in pH 7.4, 0.067 M phosphate buffer. The amount of
phenytoin in the recovered filtrate was then determined by HPLC,
as described previously. These experiments indicated that pheny-
toin had 7.6 (( 0.1)% nonspecific binding to the ultrafiltration
membrane, in agreement with earlier studies.19 All phenytoin
results obtained by ultrafiltration were corrected for nonspecific
binding based on this value.
Ultrafast Immunoextraction/Displacement Assay. The
labeled phenytoin and immunoextraction microcolumn developed
in this work were used in an UFIDA method according to the
scheme given earlier in Figure 1a. At the beginning of this assay,
20 µL of a 55.8 µM sample of the labeled phenytoin was applied
to the immunoextraction microcolumn at 0.8 mL/min using pH
7.4, 0.067 M potassium phosphate buffer as the mobile phase.
Once a baseline had been established, the flow rate was increased
to 1.2 mL/min. A 5-µL sample containing a phenytoin/HSA
mixture or phenytoin in human serum was injected at 6 min after
application of the labeled phenytoin. Once the resulting displace-
ment peak had eluted, the retained phenytoin and any remaining
labeled phenytoin were removed from the column by applying a
pH 2.5, 0.067 M phosphate solution as the elution buffer at 1.2
mL/min for 5 min. The column was then regenerated by applying
pH 7.4, 0.067 M phosphate buffer at 0.8 mL/min for 5 min prior
to the next application of the labeled phenytoin. All experiments
measuring free phenytoin concentrations on this system were
performed in triplicate at 37 °C. Further details on how the
conditions for this final method were selected can be found later
in this study.
The anti-phenytoin support was used to prepare immunoex-
traction microcolumns,2,37 which had an inner diameter of 2.1 mm
and a total length of 0.5 cm. The central layer of these columns
was ∼1 mm thick (940 µm) and contained the anti-phenytoin
support, while the remainder of the columns contained the inert
control support. These columns were prepared by making fifteen
50-µL injections at 3 mL/min of a 4.2 mg/mL slurry of the control
support to one end of the column in the presence of pH 7.4, 0.067
M phosphate buffer. This was followed by application of the same
buffer at 5 mL/min for 5 min to stabilize this layer to a thickness
of ∼0.20 cm. A small layer of the anti-phenytoin support was next
placed within this column by making fourteen 50-µL injections
of a 2.1 mg/mL slurry of this material in pH 7.4, 0.067 M
phosphate buffer and at 3 mL/min; this layer was also stabilized
by later increasing the flow rate to 5 mL/min for 5 min. The
remainder of this column was filled in the same manner with the
inert control support. Each immunoextraction microcolumn was
stored in pH 7.4, 0.067 M phosphate buffer at 4 °C when not in
use. The typical back pressure of these columns at 0.5-1.6 mL/
min was 130-420 psi (0.9-2.9 MPa) and increased by only 19%
over four months of regular use.
Characterization of Immunoextraction Microcolumn. The
amount of active anti-phenytoin antibodies in the immunoextrac-
tion microcolumns was determined by frontal analysis.47,48 This
was performed using solutions that contained 2-40 µM phenytoin
in pH 7.4, 0.067 M phosphate buffer and that were applied at 1.2
mL/min. The breakthrough curves for phenytoin were monitored
at 205 nm, with all runs being conducted in triplicate at 37 °C.
Elution of the retained phenytoin was accomplished by applying
pH 2.5, 0.067 M phosphate buffer to the immunoextraction
microcolumns. Sample application and column regeneration were
performed by using pH 7.4, 0.067 M phosphate buffer as the
mobile phase. Corrections for the system void time and nonspe-
cific binding of phenytoin to the column were made by conducting
identical experiments using a column with the same dimensions
as the immunoextraction microcolumn but containing only the
control support. The extent of phenytoin binding to this control
support was less than 8% of the total binding measured for the
immunoextraction microcolumn.8,35
The extraction efficiency of the immunoextraction microcol-
umn was determined by making 20-µL injections of a 6 µM
phenytoin standard at 37 °C in pH 7.4, 0.067 M phosphate buffer;
these injections were made onto both a column containing only
the control support and onto the immunoextraction microcolumn.
The amount of nonretained phenytoin was measured on each
column at 205 nm and at flow rates ranging from 0.6 to 1.6 mL/
min. Similar experiments were conducted when no column was
present. The difference between the total peak areas measured
with the control column and with no column present was less
than 3%.
RESULTS AND DISCUSSION
Selection of Conditions for Ultrafast Immunoextraction.
An underlying requirement for free drug measurements by
ultrafast immunoextraction is that this method must be able to
extract a representative portion of a drug’s free fraction while
avoiding any appreciable release of the same drug from its bound
form in the sample. The time scale needed for this can be
estimated by using the dissociation rate constant of a drug from
Ultrafiltration of Phenytoin Samples. Ultrafiltration was
used as a reference method for validating the free fraction
measurements made for phenytoin in this study.4,19,21,49 This was
(47) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.;
Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D.
C. Anal. Biochem. 1985, 150, 76.
(49) Argyle, C. J.; Kinniburgh, D. W.; Costa, R.; Jennison, T. Ther. Drug Monit.
1984, 6, 117.
(48) Hage, D. S. J. Chromatogr., B 2002, 768, 3.
Analytical Chemistry, Vol. 78, No. 21, November 1, 2006 7551

its carrier agents in a sample along with the extent of this binding
in typical samples.1,2,36 Several previous reports have examined
the binding of phenytoin to HSA.8,35 Two specific regions on HSA
that are involved in this binding are the indole-benzodiazepine
site (Sudlow site II) and the digitoxin site of HSA. These sites
have been reported to have association equilibrium constants for
phenytoin of 1.04 × 104 and 6.5 × 103 M-1, respectively, at pH 7.4
and 37 °C. It has also been found that phenytoin has allosteric
plus possible direct interactions with the warfarin-azapropazone
site (Sudlow site I) and tamoxifen site of HSA.8,35
Table 1. Ultrafast Immunoextraction of Phenytoin at
Various Flow Rates in a Sample Containing a Fixed
Total Amount of Phenytoin and HSAa
residence time
in antibody layer of extraction
measured free
fraction phenytoin
(%)
flow rate
immunoextraction
microcolumn (ms)
efficiency
(%)
(mL/min)
0.6
0.8
1.2
1.4
1.6
207
155
103
89
100 ((10)
98 ((2)
95 ((3)
92 ((3)
87 ((5)
16.7 ((0.6)
16.1 ((0.5)
15.8 ((0.5)
15.8 ((0.4)
15.8 ((0.2)
78
Through previous kinetic measurements using HSA columns,
the overall dissociation rate constant for phenytoin from these
binding sites of HSA has been estimated to be 10.8 ((0.05) s-1 at
pH 7.4 and 37 °C.50 Based on this result and previous work
performed in the ultrafast immunoextraction of warfarin and
thyroxine,1,2 it was originally estimated that the analysis of free
phenytoin by UFIDA would require a sample residence time in
an immunoextraction microcolumn of less than 200 ms. To obtain
these conditions, a sandwich microcolumn was used that con-
tained a 2.1 mm i.d. × 940 µm thick layer of an anti-phenytoin
support. This gave an expected residence time for samples in the
immunoextraction layer of roughly 100 ms at a flow rate of 1.2
mL/min.
The binding capacity of the immunoextraction microcolumn
was estimated by frontal analysis to be 67 ((3) pmol of phenytoin
at pH 7.4 and 37 °C. It was further found from protein assays that
this column contained ∼290 ((90) pmol of antibodies. This gave
an effective concentration of active antibody binding sites in the
immunoextraction layer of 3.4 ((0.2) µM and a relative activity
of the antibodies for phenytoin of 12 ((3)% (assuming there were
two accessible binding sites per antibody). Although this relative
activity was low, the total column binding capacity was sufficient
for this work since it was roughly two times higher than the
amount of free phenytoin expected at the high end of this drug’s
therapeutic range in a 5-µL sample (i.e., based on a 90% bound
fraction and a total phenytoin concentration of 80 µM). If desired,
this relative activity and binding capacity could be improved by
employing more site-selective techniques for antibody immobiliza-
tion; examples include the use of hydrazide-activated supports plus
antibodies that have been oxidized in their carbohydrate regions,
or the use of biotin tags in these same regions along with avidin
or avidin-containing supports.51-53
a These values were determined using a sample that contained 550
µM HSA and 35.0 µM phenytoin. The numbers in parentheses
represent a range of (1 SD.
librium constant and the binding capacity of the immunoextraction
microcolumn, the retention factor for phenytoin on this column
at pH 7.4 and 37 °C was estimated to be 348 ((26). This value
was calculated by using the relationship k ) KAmL/VM, where KA
is the association equilibrium constant for phenytoin with the
antibodies, mL is the moles of active antibodies in the column,
VM is the void volume of the immunoextraction layer, and (mL/
VM) is the effective concentration of active antibodies in this layer.48
Further examination of the frontal results, as described in ref 28,
gave a measured association rate constant (ka) for phenytoin with
the immobilized anti-phenytoin antibodies of 2.4 ((0.4) × 106 M-1
s-1 at pH 7.4 and 37 °C. From the relationship KA ) ka/kd, the
dissociation rate constant (kd) for phenytoin from the immobilized
antibodies was also determined, giving a value of 4.4 ((0.8) ×
10-3 s-1. All of these results indicated that any phenytoin extracted
by the immobilized antibodies would bind tightly to this column
and only slowly dissociate from these antibodies in the presence
of a pH 7.4 buffer.
The ability of this microcolumn to extract phenytoin was
measured by comparing injections of phenytoin standards made
onto this column versus injections made onto an inert control
column. Table 1 summarizes the results that were obtained. The
concentration and quantity of phenytoin that was injected (20 µL
of a 6 µM solution, or 120 pmol of phenytoin) corresponded to
the free amount of this drug that would be expected in serum at
typical therapeutic levels. It was found that 95% or more of the
phenytoin was extracted when using residence times of 100 ms
or greater in the immunoextraction layer, with 98% being extracted
at residence times of 150 ms or greater. These results are in
agreement with previous observations made for the ultrafast
immunoextraction of thyroxine and warfarin.1,2
The association equilibrium constant for phenytoin with the
immobilized anti-phenytoin antibodies was determined by frontal
analysis to be 5.4 ((0.3) × 108 M-1 at pH 7.4 and 37 °C (note: no
significant change in this value was noted at lower flow rates).
Although this result represents reasonably strong binding, this
association equilibrium constant is ∼20-fold lower than a solution-
The immunoextraction microcolumn was also used in studies
examining the effect of flow rate and sample residence on the
apparent free fraction that was obtained for phenytoin in a
displacement assay. It was found that a flow rate of 1.2 mL/min
or greater (i.e., a sample residence time in the immunoextraction
layer of 103 ms or less) gave a consistent measured free phenytoin
fraction of 15.8% for the test mixture in Table 1. However, slower
flow rates and longer sample residence times resulted in a greater
phase equilibrium constant of 1 × 1010
M
-1 that was provided by
the manufacturer of these antibodies. This difference is not
surprising because immobilized antibodies can often have lower
binding constants than their soluble forms.54,55 Using this equi-
(50) Ohnmacht, C. M. Ph.D. Dissertation, University of NebraskasLincoln,
Lincoln, NB,2006.
(51) Kim, H. S.; Hage, D. S. In Handbook of Affinity Chromatography, 2nd ed.;
Hage, D. S., Ed.; CRC Press: Boca Raton, FL, 2005; Chapter 3.
(52) Hermanson, G. T.; Mallia, A. K.; Smith, P. K. Immobilized Affinity Ligand
Techniques; Academic Press: New York, 1992.
(54) Van Regenmortel, M. H. V. Structure of Antigens, Vol. 1; CRC Press: Boca
Raton, FL, 1992.
(53) Hage, D. S.; Phillips, T. M. In Handbook of Affinity Chromatography, 2nd
ed.; Hage, D. S., Ed.; CRC Press: Boca Raton, FL, 2005; Chapter 6.
(55) Butler, J. E. Immunochemistry of Solid-Phase Immunoassay; CRC Press: Boca
Raton, FL, 1991.
7552 Analytical Chemistry, Vol. 78, No. 21, November 1, 2006

apparent free fraction (1.9% higher at 0.8 mL/min and 5.7% higher
at 0.6 mL/min). As indicated in previous simulations performed
for warfarin and thyroxine,1,2 this increase in the apparent free
fraction is believed to reflect dissociation of the analyte from
proteins or other binding agents in the sample. Thus, as a
compromise between extraction efficiency and accuracy, an
injection flow rate of 1.2 mL/min (i.e., a sample residence time
of roughly 100 ms in the immunoextraction layer) was used in all
later experiments for free phenytoin measurements.
Behavior of NIR Fluorescent Label under Assay Condi-
tions. The next item considered was the signal intensity and
behavior of the labeled phenytoin under the conditions to be used
for ultrafast immunoextraction. It was found in pH 7.4, 0.067 M
phosphate buffer that the NIR fluorescence of the labeled
conjugate gave a linear response over a broad range of concentra-
tions. This linear range extended from approximately 1.7 fmol to
2.1 pmol (i.e., 0.33 to 412 nM for a 5-µL injection, with a lower
limit of detection of 1.4 nM at a signal-to-noise ratio of three).
Although this amount is less than the free phenytoin levels in 5
µL of serum (i.e., 20-40 pmol or 4-8 µM), this UFIDA format
only requires that a small, representative amount of the labeled
phenytoin be displaced by an injected analyte. Thus, as will be
seen later, the response for the NIR fluorescent label was more
than adequate for analyzing the displacement peaks that were
created during the detection of free phenytoin fractions in such
samples.
Another item examined was the effect of biological samples
on the fluorescence of the labeled phenytoin. This was of interest
since it has been noted in previous work with chemiluminescent
labels that up to a 30% change in signal can be seen for a labeled
analyte in the presence of serum versus buffer.1 To study this
effect, the emission spectrum of the labeled phenytoin was
obtained in the presence and absence of human plasma (i.e.,
serum plus clotting factors), as shown in Figure 3a. It was found
that there was no appreciable reduction in signal intensity (i.e.,
less than 3% change) between a buffered standard and plasma
containing labeled phenytoin. Similar results were obtained when
comparing plasma and buffered solutions containing 0.33-412 nM
labeled phenytoin, which gave less than a 17% difference in signal
at all concentrations examined. The similarity of the plasma and
buffer results indicated that there was either only a small amount
of binding between the labeled phenytoin and HSA in plasma or
that this binding did not have any appreciable affect on the NIR
fluorescence of the dye in this conjugate.
Figure 3. (a) Emission spectrum for human plasma and labeled
phenytoin in the presence of plasma or pH 7.4, 0.067 M phosphate
buffer, and (b) chromatograms for a UFIDA method performed for
samples containing a free phenytoin concentration of 4-8 µM injected
at 6 min after the application of labeled phenytoin. A chromatogram
is also shown (b) that was obtained under the same conditions for a
5-µL, 550 µM sample of HSA (representing the main protein of serum
or plasma and a nonretained component in the UFIDA method), as
monitored using absorbance detection at 205 nm. The emission
spectra in (a) were obtained using a 100 µM solution of labeled
phenytoin in plasma or buffer at an excitation wavelength of 770 nm.
signal could be expected from such a sample. This spectrum is
also shown in Figure 3a and gave no detectable signal at the
emission wavelengths that were monitored. From this result, as
well as the difference in sample and displacement peak elution
times noted in Figure 3b, it was concluded that no significant
background signal should have been present under the conditions
used in this study for detection of the labeled phenytoin in the
displacement peaks.
Optimization of UFIDA Method. After the initial conditions
for ultrafast immunoextraction and detection of the labeled
phenytoin had been selected, these components were combined
and optimized for use in an UFIDA assay for measuring free
phenytoin fractions. One item considered was the effect of varying
the amount of labeled phenytoin that was applied to the system
for analyte detection. This item was studied by applying 5-100
µL of a 0.558-55.8 µM solution of the labeled phenytoin (i.e., 2.8
pmol-5.6 nmol) at 0.8 mL/min to the immunoextraction micro-
column prior to the injection of a 5-µL sample of 35 µM phenytoin
(i.e., a typical therapeutic concentration expected for free pheny-
toin in serum). The amount of labeled phenytoin used under these
conditions ranged from 0.04 to 84 times the binding capacity of
the immunoextraction microcolumn. The area of the displaced
peak gave less than a 6% change when the amount of labeled
The actual extent of interferences from the sample matrix
would be expected to be even smaller than 3-17% when the
labeled phenytoin is used in a displacement assay, since the peak
for the displaced conjugate elutes slightly after the nonretained
fraction of the sample. This is demonstrated in Figure 3b, where
even the highest concentration standards gave displacement peaks
with a mean elution time that occurred 1 min after the nonretained
components of a serum sample (as represented by HSA). The
overlap of the sample and displacement peaks was estimated to
be less than 5% for this assay, which would further reduce any
effects of the sample matrix on the fluorescent signal of the NIR
dye used in the labeled phenytoin.
The emission spectrum for a sample of human plasma with
no NIR dye added was acquired to see what type of background
Analytical Chemistry, Vol. 78, No. 21, November 1, 2006 7553

injection should be performed in 2-6 min after application of the
labeled phenytoin to give less than 2% variation in the displacement
peak’s size. These conditions minimized loss of the retained
labeled phenytoin while also allowing sufficient time for excess
labeled conjugate (and any associated contaminants) to be washed
from the column.
The next study considered the use of sequential sample
injections during the UFIDA method. This was done to determine
whether it was possible to perform more than one analysis per
column loading of the labeled phenytoin. As shown in Figure 4b,
the use of two sequential sample injections gave less than a 5%
change in displacement peak area and less than a 3% change in
peak height. The intensity of the displacement peaks then began
to decrease with further sample injections, giving signals that were
36 and 93% lower (versus the first sample) for the third and fourth
injections. In the remainder of this study, only one sample injection
was performed after each application of the labeled phenytoin.
However, the findings in Figure 4b indicate that multiple injections
could be used in such an assay to further increase sample
throughput in the UFIDA method.
Elution of the retained phenytoin and labeled phenytoin in the
UFIDA method was accomplished by using a pH 2.5, 0.067 M
phosphate buffer applied at 1.2 mL/min for 5 min. Regeneration
of the immunoextraction microcolumn was conducted by applying
pH 7.4, 0.067 M phosphate buffer for at least 5 min prior to a new
injection of labeled phenytoin. The UFIDA method was found to
be quite stable under these conditions, allowing a reproducible
response to be obtained on a single column over at least 250
injections and four months of regular use.
Validation of UFIDA Method. The final conditions used in
the UFIDA assay for free phenytoin measurements are sum-
marized in the Experimental Section. An example of a typical run
obtained under such conditions is given in Figure 1b. The
displacement peak for this assay occurred within 2-3 min of
sample injection, and the total assay time (i.e., one injection,
elution, and regeneration cycle) was 20 min. However, it was found
that the total time to detect and analyze free phenytoin fractions
could be reduced to less than 10 min per sample by using multiple
sample injections after each application of the labeled phenytoin
(see previous section).
A typical calibration curve obtained for the UFIDA method is
shown in Figure 5. The lower limit of detection for this method
was 570 pM phenytoin (S/N ) 3) for a 5-µL sample (i.e., 2.9 fmol).
The linear range (i.e., the range of analyte concentrations giving
a response within 10% of the best-fit line) extended from the lower
limit of detection up to ∼10 µM phenytoin (∼50 pmol). This linear
response covered the entire range of free phenytoin concentrations
that would be expected at normal therapeutic levels of this drug.
The calibration curve for this assay leveled off as phenytoin
concentrations above 10 µM were injected. This behavior is
probably due to saturation of the immunoextraction microcolumn,
since these high sample concentrations gave rise to amounts of
phenytoin that approached or exceeded the binding capacity of
this system (i.e., 50 pmol of phenytoin in 5 µL of a 10 µM sample
versus a 67-pmol binding capacity for the column).
Figure 4. Effect of (a) injection time on the application of phenytoin
samples to the UFIDA system and (b) the use of sequential sample
injections in this assay. The experiments with the injection time were
performed using a 5-µL, 4 µM phenytoin sample. The sequential
injection studies were performed using a sample containing 30 µM
phenytoin and 550 µM HSA. In both (a) and (b), the column was
loaded with 20 µL of a 5.58 µM solution of labeled phenytoin at 0.8
mL/min, with the flow rate being changed to 1.2 mL/min after 1.5 min
into each run and before sample injection.
phenytoin was at least 110 pmol (e.g., a 20-µL injection of 5.58
µM labeled phenytoin), or conditions in which the amount of the
labeled phenytoin was present in more than a 1.6-fold excess
versus the column binding capacity.
The only change noted when using larger amounts of labeled
phenytoin was a slight increase in the time it took to wash the
excess, nonretained labeled conjugate from the column. When
using small amounts of this conjugate, it took ∼3 min to remove
99.9% of the nonretained labeled phenytoin from the column.
However, it took ∼5.5 min to wash off the excess conjugate when
using the highest amounts of labeled phenytoin that were
examined in this work. Based on these results, a 20-µL injection
of a 5.58 µM preparation of the labeled phenytoin (i.e., 110 pmol)
was used along with a wash time of 3 min in the final UFIDA
method as a compromise between assay speed and response.
Another item considered in developing the UFIDA method was
the effect of varying the time between injection of the labeled
phenytoin and the injection of a sample. This was examined to
see if overlap of the sample peak with the remaining nonretained
labeled phenytoin (at small injection times) or loss of the retained
labeled phenytoin (at long injection times) gave a significant
change in response for the displacement peak. The results of these
studies are shown in Figure 4a. Using the displacement peak
obtained with sample injection at 2 min as the reference, the
changes in area noted for displacement peaks measured after
sample injections at 4, 6, or 12 min were 1.7, 1.9, and 4.8%,
respectively. It was concluded from these data that sample
The precision of this assay was determined by making replicate
injections of standards, phenytoin/HSA mixtures and spiked
control serum samples. A relative standard deviation of (0.5% or
7554 Analytical Chemistry, Vol. 78, No. 21, November 1, 2006

Table 3. Determination of Free Phenytoin Fractions in
Serum Samples by Ultrafiltration or a UFIDA Methoda
measured free
phenytoin concn (µM)
total concn
phenytoin (µM)
ultrafiltration
UFIDA
10
20
40
1.32 ((0.34)
2.81 ((0.42)
6.11 ((0.44)
1.27 ((0.05)
2.70 ((0.12)
5.99 ((0.14)
a All measurements were performed in pH 7.4, 0.067 M potassium
phosphate buffer and 37 °C. The values for ultrafiltration have been
corrected for nonspecific binding to the membrane. The values in
parentheses represent (1 SD.
Figure 5. (a) Calibration curve based on displacement peak area
for phenytoin in the UFIDA method. The experimental conditions are
given in the text. The best-fit line over the linear range in (a) was y )
5.58 ((0.02)× + 0.010 ((0.106), with a correlation coefficient of
0.9999 (n ) 10).
of the UFIDA and ultrafiltration results overlapped within 1 SD
of their values and had differences in these values of only 1.6-
5.4% (average, 2.9%). Although there was a small apparent bias in
the ultrafiltration results compared to UFIDA, the size of this bias
was strongly linked with experimental uncertainly in the nonspe-
cific binding measured for the ultrafiltration membranes.
Another comparison was made between UFIDA and ultrafil-
tration in terms of their ability to determine free phenytoin
concentrations in human serum. This was conducted with stan-
dards and samples used in a commercial immunoassay kit for free
phenytoin.12,15,21 The four standards supplied with this kit (total
phenytoin concentration, 1.98-12 µM in phosphate buffer) gave
a linear response for the UFIDA method, with a best-fit line of y
) 4.85 ((0.04)x + 2.9 ((3.0) and a correlation coefficient of 0.9932
(n ) 4). The results for control serum samples are shown in Table
3. Comparison of the UFIDA and ultrafiltration results for these
samples again resulted in statistically identical values for the
measured free fractions of phenytoin. When using ultrafiltration,
control sera containing 10-40 µM concentrations of total pheny-
toin gave free phenytoin concentrations of 1.32-6.11 µM (or 13.4-
15.4% free fractions), while analysis of the same samples using
UFIDA gave free phenytoin concentrations of 1.27-5.99 µM (or
free fractions of 12.2-15.1%). The difference between the results
of these two assays was less than 2-5%, with all differences being
within 1 SD of the measurements.
Comparison of UFIDA and Ultrafiltration. Although UFIDA
and ultrafiltration gave comparable results for the samples in
Tables 2 and 3, they did differ in terms of their precision and
speed. For instance, the relative precision of the UFIDA results
in Table 2 was 2.4-4.8%, while the relative precision of the
ultrafiltration results for the same samples was over 2-fold larger,
ranging from 8.1 to 9.7%. A similar trend was noted for the control
serum results in Table 3. The worse precision of the ultrafiltration
results is thought to be the combined result of (1) the multiple,
manual steps that are involved in this method and (2) the random
experimental variations that were noted in nonspecific binding of
phenytoin to the ultrafiltration membrane. In contrast to this, the
UFIDA method was performed as an automated system that did
not require any sample pretreatment steps, which probably
contributed to the better precision of this method versus ultra-
filtration.
Table 2. Determination of Free Phenytoin Fractions in
Phenytoin/HSA Mixtures by Ultrafiltration or a UFIDA
Methoda
measured free
phenytoin concn (µM)
total concn
total concn
HSA (µM)
phenytoin (µM)
ultrafiltration
UFIDA
550
550
550
550
550
650
650
650
650
650
750
750
750
750
750
30
35
40
45
50
30
35
40
45
50
30
35
40
45
50
4.9 ((0.4)
5.7 ((0.5)
6.6 ((0.6)
7.5 ((0.7)
8.3 ((0.7)
4.2 ((0.4)
4.9 ((0.4)
5.6 ((0.5)
6.4 ((0.6)
7.1 ((0.6)
3.7 ((0.3)
4.3 ((0.4)
4.9 ((0.4)
5.6 ((0.5)
6.2 ((0.6)
4.7 ((0.2)
5.5 ((0.2)
6.4 ((0.2)
7.2 ((0.2)
8.1 ((0.2)
4.1 ((0.1)
4.8 ((0.1)
5.5 ((0.1)
6.2 ((0.2)
6.9 ((0.2)
3.5 ((0.1)
4.2 ((0.2)
4.8 ((0.2)
5.4 ((0.2)
6.1 ((0.2)
a All measurements were performed at 37 °C in pH 7.4, 0.067 M
phosphate buffer. The values for ultrafiltration have been corrected
for nonspecific binding to the membrane. The numbers in parentheses
represent a range of (1 SD.
less was seen for standards containing free phenytoin concentra-
tions of 1.24 nM-2.02 µM. As will be shown later, the precision
was ∼2-5% for serum and HSA samples that contained phenytoin
at typical therapeutic concentrations.
The accuracy of the UFIDA assay was first assessed by
comparing it to ultrafiltration in the analysis of phenytoin/HSA
mixtures that had been prepared in pH 7.4, 0.067 M potassium
phosphate buffer (see Table 2). The sample concentrations used
in this study were chosen to match the levels of HSA and
phenytoin that would be expected in serum samples containing
typical therapeutic concentrations of phenytoin. These samples
gave average free phenytoin fractions of 15.9, 13.7, and 12.0% at
HSA concentrations of 550, 650, and 750 µM, respectively (i.e.,
results similar to literature values reported for free phenytoin
fractions in serum).11,14 All of the 25 phenytoin/HSA samples that
were examined gave statistically identical results at the 95%
confidence level for UFIDA versus ultrafiltration. In addition, all
In terms of speed, UFIDA gave results for each sample within
2-3 min of injection, with a total run time of 20 min per cycle.
For ultrafiltration, the minimum time required for one sample was
∼1 h, which included 45 min to perform the ultrafiltration and 15
Analytical Chemistry, Vol. 78, No. 21, November 1, 2006 7555

min to conduct the HPLC analysis of the filtrate’s free phenytoin
content. For UFIDA, a total of 15 h was required for a triplicate
analysis of all the samples and standards used to generate the
data in Table 2. Although ultrafiltration has a much higher analysis
time per sample, up to 15 samples could be centrifuged simulta-
neously in this approach, giving it an overall sample throughput
comparable to that of the UFIDA method (i.e., 14 h for analysis
of all samples and standards used in Table 2). However, it should
be kept in mind that the throughput of the UFIDA method could
be increased by almost 2-fold by using sequential injections of
samples after each application of the labeled phenytoin, as
demonstrated earlier in Figure 4b. This throughput could be
improved even further through the use of multiple immunoex-
traction columns and a multiport valve for flow-splitting. Thus,
UFIDA has the capability of not only providing a faster analysis
per sample than ultrafiltration but also providing higher sample
throughput in some applications.
The UFIDA method developed in this study gave good
correlation versus ultrafiltration for the measurement of free
phenytoin fractions in serum and phenytoin/HSA samples. This
new method allowed free phenytoin fractions to be measured
within 2-3 min of sample injection, while giving a precision and
analysis time per sample that was much better than that obtained
for ultrafiltration. This method is also much faster than the
technique of equilibrium dialysis and, unlike methods based on
capillary electrophoresis and RAM columns, can easily be used
for free analyte measurements in real clinical samples. Although
this method does require appropriate antibodies and labeled
analogues for the drug or analyte of interest, this is not a major
limitation since these reagents can be obtained or modified from
those used for the same analytes in other types of immunoassays.
These properties should make the UFIDA method useful in a
variety of areas, including therapeutic drug monitoring, pharma-
cological studies, and measurements of drug-protein bind-
ing.1,2,5,6,13-15,20,21,27,56,57
CONCLUSIONS
This study examined the development and validation of NIR
ACKNOWLEDGMENT
fluorescent labels and an ultrafast immunoextraction/displacement
assay for free fraction measurements of the drug phenytoin. The
final system used an immunoextraction microcolumn that allowed
for the extraction and analysis of free phenytoin when using a
sample residence time as low as 100 ms. Phenytoin that had been
labeled with a NIR fluorescent dye was then combined with this
microcolumn to provide detection limits for phenytoin down to
the picomolar range without any observable background signals
or matrix effects from serum samples.
The authors thank Sara Basiaga and Dipanjan Nag for their
help with the NMR studies and Carita Kordik for assisting with
the mass spectrometric analysis. The authors also thank LI-COR
Biosciences for the donation of the NIR dyes and detector and
Chuck Prescott and Dan Draney for their helpful discussions. This
work was supported by the National Institutes of Health under
grant R01 GM044931 and was conducted in facilities that were
renovated under NIH grant RR015468-01.
(56) Beck, D. E.; Farringer, J. A.; Ravis, W. R.; Robinson, C. A. Clin. Pharm.
Received for review July 5, 2006. Accepted August 27,
2006.
1987, 6, 888.
(57) Oates, M. R.; Clarke, W.; Zimlich, A. I.; Hage, D. S. Anal. Chim. Acta 2002,
470, 37.
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