Clinical Chemistry 45: 829-837, 1999;
(Clinical Chemistry. 1999;45:829-837.)
© 1999 American Association for Clinical Chemistry, Inc.
Falsely Increased Immunoassay Measurements of Total and Unbound Phenytoin in Critically Ill Uremic Patients Receiving Fosphenytoin
William L. Roberts1,a,
Barun K. De2,
John P. Coleman2 and
Thomas M. Annesley3
1
Department of Pathology, University of Utah Health Science Center, Salt Lake City, UT 84132.
2
Department of Pathology, University of Mississippi
Medical Center, Jackson, MS 39216.
3
Department of Pathology, University of Michigan Medical
Center, Ann Arbor, MI 48109.
a Address correspondence to this author at: ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108. Fax 801-584-5207; e-mail william.roberts{at}arup-lab.com
 |
Abstract
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Background: Fosphenytoin, a phosphate ester prodrug of phenytoin,
is metabolized to phenytoin in vivo. Phenytoin metabolites accumulate
in renal insufficiency and cross-react in some phenytoin immunoassays.
Our aim was to determine the accuracy of phenytoin immunoassays in
renal patients treated with fosphenytoin.
Methods: We measured phenytoin with HPLC and with the aca,
ACS:180, TDx phenytoin II, Vitros, and AxSYM methods. Specimens were
collected 2–120 h after fosphenytoin administration from 17 patients
with renal insufficiency.
Results: The AxSYM, TDx phenytoin II, ACS:180, and Vitros assays
displayed falsely increased phenytoin results up to 20 times higher
than the HPLC results. The aca Star results for these specimens were
comparable to the HPLC results. Although fosphenytoin can cross-react
with phenytoin immunoassays, no fosphenytoin was detected by a
sensitive HPLC method in any sample that was tested for its presence.
Conclusion: These results are consistent with the formation of
one or more novel metabolites or adducts of fosphenytoin that
accumulate in some critically ill patients with renal insufficiency and
that display significant cross-reactivity with some, but not all,
phenytoin immunoassay methods.© 1999 American Association for
Clinical Chemistry
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Introduction
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Fosphenytoin (Cerebyx®) is a phosphate
ester prodrug of phenytoin that can be administered either
intravenously or intramuscularly. This inactive prodrug is rapidly
metabolized to phenytoin by phosphatases present in liver, red blood
cells, and other tissues (1). The in vivo conversion
half-life is 5–15 min, and more rapid conversion is seen in patients
with renal or hepatic diseases, presumably because of decreased protein
binding of fosphenytoin (2)(3). The
cross-reactivity of fosphenytoin with phenytoin immunoassays is
variable and can be clinically significant(4)(5). Fosphenytoin is converted to phenytoin
in vitro in serum and heparin-treated plasma samples, but is resistant
to hydrolysis in EDTA plasma (6). For therapeutic drug
monitoring, phenytoin is quantified in serum or plasma. It is
recommended that serum phenytoin concentrations should not be measured
until at least 2 h after an intravenous (i.v.) dose of
fosphenytoin and at least 4 h after an intramuscular dose(4).
The phenytoin metabolites that accumulate in patients with renal
failure [primarily 5-(p-hydroxyphenyl)-5-phenylhydantoin
glucuronide (HPPH-G)] cross-react with some phenytoin immunoassays(7)(8). However, the Emit,
aca®, ACS:180, TDx phenytoin II, and Vitros
phenytoin assays are not affected by these metabolites(7)(8)(9)(10). We report here an index case and six additional
critically ill patients with renal insufficiency who received
fosphenytoin and subsequently displayed falsely increased free- and
total-phenytoin values by multiple immunoassay methods. These methods
included some of the methods listed above that previously had been
shown to be free of significant cross-reactivity with the phenytoin
metabolites that accumulate in patients with renal failure.
|
Materials and Methods
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subjects
Subject 1 was a 54-year-old man who had a cerebrovascular
accident, developed acute renal failure secondary to rhabdomyolysis,
and was admitted to an intensive care unit. Shortly after admission,
the patient had tonic-clonic seizures and was intubated, given an i.v.
loading dose of both fosphenytoin and phenytoin, and started on i.v.
phenytoin. He was dialyzed every other day beginning on hospital day 4
through hospital day 11. On hospital day 7, he was switched to i.v.
fosphenytoin. This patient came to the laboratory's attention on
hospital day 9, when it was noted that the concentration of
non-protein-bound phenytoin (i.e., free phenytoin) was nearly equal to
the total-phenytoin concentration. Within the clinical laboratory,
total phenytoin was measured with the Emit method on the aca Star
analyzer because of its reported specificity for phenytoin. In the
absence of a commercially available Emit free-phenytoin assay, free
phenytoin was measured on the Abbott TDx analyzer with TDx/FLx
phenytoin or phenytoin II reagents. Although no longer commercially
available, the phenytoin II reagents were used during this period
because they had also been shown to be free from significant
cross-reactivity with the HPPH-G metabolite of phenytoin. The temporal
association of the discrepancy between total- and free-phenytoin
measurements and the switch from phenytoin to fosphenytoin prompted us
to evaluate several immunoassay methods for measuring total and free
phenytoin in patients with renal insufficiency who were receiving
fosphenytoin.
Subject 2 was a 68-year-old woman with a history of diabetes mellitus,
hypertension, and degenerative joint disease who was admitted
emergently for a thoracoabdominal aortic aneurysm repair. After
surgery, she developed acute renal failure, seizures, hypotension, and
metabolic acidosis. Subject 3 was a 55-year-old woman with a history of
diabetes mellitus, hypertension, and end-stage renal disease who
developed a subarachnoid hemorrhage and was admitted to the intensive
care unit for mechanical ventilation. Subject 4 was a 70-year-old woman
with a history of hypertension, end-stage renal disease, and a seizure
disorder who was admitted with a Staphylococcus aureus graft
infection; she subsequently developed sepsis, hypotension, and
refractory seizures. Subject 5 was a 54-year-old man with a history of
diabetes mellitus, hypertension, and end-stage renal disease who was
admitted with orbital cellulitis; he subsequently developed sepsis and
seizures, and required enucleation of the affected eye on hospital day
16. Subject 6 was a 31-year-old woman who developed gram-negative
sepsis, alveolar hemorrhage, and acute renal failure 1 month post renal
transplant. Subject 7 was a 33-year-old man with a history of
hypertension, end-stage renal disease, and seizures who was admitted
with a subarachnoid hemorrhage and hypertension.
apparatus
The TDx and AxSYM analyzers were from Abbott Laboratories, the aca
Star analyzer was from Dade Behring, the Vitros 950 analyzer was from
Ortho Clinical Diagnostics, and the ACS:180 analyzer was from Chiron
Diagnostics. HPLC analyses for phenytoin were performed on a
Hewlett-Packard series 1050 system. Centrifree®
micropartition devices with YMT membranes (Amicon) were used for
ultrafiltration.
specimen collection and preparation
Fosphenytoin (50 g/L phenytoin equivalent) was obtained from
Parke-Davis. All doses of fosphenytoin administered to patients are
expressed in phenytoin equivalents. Serum or EDTA plasma samples were
collected at least 2 h after an i.v. dose of fosphenytoin.
Fosphenytoin was administered solely by the i.v. route in all study
patients. All studies with human subjects were approved by the
Institutional Review Board of the University of Mississippi Medical
Center. Ultrafiltrates were prepared by centrifugation of ~1 mL of
serum or plasma in a Centrifree device at 2000g for 20 min
at ambient temperature.
assay procedures
Total and free phenytoin were measured by reversed-phase HPLC
using a validated procedure developed for the analysis of multiple
anticonvulsant drugs (11). Briefly, 0.1 mL of serum or 0.3
mL of ultrafiltrate was added to a conical polypropylene tube. Internal
standard (alphenal in 100 µL of acetonitrile) was added, and the tube
was vortex-mixed briefly. After the addition of 100 µL of 0.5 mol/L
sodium phosphate buffer, pH 6.0, and 750 µL dichloromethane, tubes
were vortex-mixed for 10 s. After centrifugation, the upper
aqueous layer was aspirated to waste and the solvent was evaporated
under nitrogen. After the extract was reconstituted with 75 µL of
methanol and 500 µL of water, 20 µL was analyzed using a
C8 reversed-phase column with detection at
214 nm. The limit of quantification for phenytoin in serum was
0.2 mg/L.
Total fosphenytoin was measured by reversed-phase HPLC, as described
previously (12). Briefly, equal parts of internal standard
[5-(p-methylphenyl)-5-phenylhydantoin] in water and
concentrated phosphoric acid were added to serum or plasma.
Fosphenytoin was extracted with diethyl ether, and the extract was
dried under nitrogen and reconstituted with HPLC mobile phase. HPLC was
performed using a 150 x 3.9 mm C18 column
with a mobile phase consisting of 200 mL/L acetonitrile in deionized
water containing 5 mmol/L tetrabutyl ammonium adjusted to pH 2.2–2.5
with phosphoric acid. The flow rate was 2.0 mL/min.
Abbott TDx/FLx phenytoin and phenytoin II reagents and calibrators were
used to measure total and free phenytoin with a TDx analyzer. Abbott
AxSYM reagents and calibrators were used to measure total phenytoin
with an AxSYM analyzer. Chiron ACS:180 reagents and calibrators were
used to measure total phenytoin with an ACS:180 analyzer. Dade/Behring
aca phenytoin analytical test packs and calibrators were used to
measure total phenytoin with an aca Star. Vitros reagent slides and
calibrators from Ortho Clinical Diagnostics were used to measure total
phenytoin with a Vitros 950 analyzer. All reagents were used according
to manufacturers' instructions.
data analysis
EP Evaluator, release 3, software (David G. Rhoads Associates) was
used for Deming regression analysis and calculation of r and
Sy|x.
|
Results
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Subject 1, whose phenytoin results first called the laboratory's
attention to the discrepancy between immunoassays, had a free-phenytoin
concentration measured by one immunoassay method (TDx phenytoin II)
that was greater than the total measured by another immunoassay (aca
Star). The dose and timing of phenytoin and fosphenytoin administration
were reviewed, and it was noted that the free-phenytoin concentration
was nearly equal to the total-phenytoin concentration 1 day after
fosphenytoin administration was restarted. To further evaluate this
unusual phenomenon, we quantified phenytoin concentrations in numerous
serum or plasma samples by HPLC and five immunoassay methods. We also
assayed fosphenytoin in some of these samples. The results, which are
summarized in Table 1
, showed that the aca Star phenytoin results agreed with those
obtained by HPLC, whereas the other immunoassays methods overestimated
the true phenytoin concentration in samples collected after the
administration of fosphenytoin. For the six additional patients who had
renal insufficiency (creatinine >25 mg/L) with a concurrent major
illness and who also received fosphenytoin, the phenytoin
concentrations measured by several immunoassays were found to be
significantly higher than the HPLC values (Tables 1–4
). Of note, only four of these seven subjects survived their
hospital stay.
Regression analysis of the immunoassay results vs HPLC are shown in
Fig. 1
. The aca Star method compared well with HPLC (Fig. 1A
), whereas
the other four immunoassay methods had high slopes and/or poor
correlations, suggesting the presence of one or more cross-reacting
substances in the samples. In an attempt to quantify the magnitude of
cross-reactivity in each immunoassay that was affected, we subtracted
the HPLC result from each immunoassay result (designated 
) except
for the aca Star and the results were subjected to regression analysis.
There was poor correlation between values except for the
comparisons between the TDx phenytoin II and ACS:180 methods and the
ACS:180 and Vitros methods. The elimination half-lives of any possible
cross-reacting substances were estimated in subjects 1, 3, and 5 after
the discontinuation of fosphenytoin administration by plotting log
vs time. The results are summarized in Table 5
. For each immunoassay method, the apparent half-life varied
significantly between patients. For each individual patient, the
half-life also varied, depending on the immunoassay method used.
All four patients who had free-phenytoin concentrations measured had at
least one result that was greater than the corresponding total result
measured by either the HPLC or aca Star methods. When the
free-phenytoin values obtained by HPLC were compared with those
obtained with the TDx phenytoin II method, the immunoassay method was
found to significantly overestimate the true free-phenytoin
concentration (Fig. 2
). Furthermore, the correlation between the two methods was poor
(r = 0.559). In subject 7, free phenytoin was measured
with the TDx phenytoin assay (Table 4
). As was observed for the TDx
phenytoin II, the free-phenytoin concentration measured with this
method was greater than or equal to the total phenytoin measured by the
HPLC or aca Star methods.
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Figure 2. Comparison of TDx free-phenytoin II immunoassay with HPLC.
Samples collected from subjects 1 and 3 were analyzed for free
phenytoin by HPLC and TDx phenytoin II assays. Deming regression
analysis was performed (solid line); the dashed
line represents an ideal comparison with a slope of 1.00 and an
intercept of 0 (n = 15; slope = 3.01 ± 1.21;
intercept = 2.14 ± 1.22; r = 0.559;
Sy|x = 2.86).
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To confirm that the phenytoin metabolites that usually accumulate in
renal insufficiency were not responsible for these falsely increased
results, 24 samples from patients receiving phenytoin with renal
insufficiency (creatinine, 15–92 mg/L) were assayed by the ACS:180,
AxSYM, HPLC, and Vitros methods. The ACS:180 and Vitros methods
provided results that were very close to HPLC values (Fig. 3
, A
and C
). These results are consistent with the previously
reported minimal cross-reactivity of phenytoin metabolites with these
two immunoassays. The AxSYM assay did demonstrate moderately increased
results for some of these specimens (Fig. 3B
), presumably because of
the known cross-reactivity of HPPH-G. However, the increases noted were
not as great as those observed for the patients receiving fosphenytoin
and could not account for all of the falsely increased results shown in
Tables 1–4
.
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Figure 3. Comparison of three phenytoin immunoassays and HPLC, using
samples from uremic patients.
Samples were collected from 24 patients with uremia (creatinine >14
mg/L) who were receiving phenytoin and analyzed by HPLC and three
immunoassay methods. Deming regression analysis was performed
(solid lines); the dashed lines represent
an ideal comparison with a slope of 1.00 and an intercept of 0.
(A), results for the ACS:180 method (slope =
0.98 ± 0.04; intercept = 0.15 ± 0.33;
r = 0.983; Sy|x = 0.90).
(B), results for the AxSYM method (slope =
1.14 ± 0.06; intercept = 1.96 ± 0.51;
r = 0.970; Sy|x = 1.40).
(C), results for the Vitros method (slope =
1.03 ± 0.04; intercept = 0.42 ± 0.32;
r = 0.987; Sy|x = 0.82).
|
|
Although all samples from the seven patients were collected at least
2 h after an i.v. dose of fosphenytoin and no residual
fosphenytoin was detected in any of the samples tested, the
cross-reactivity of fosphenytoin with each of the immunoassays used in
this study was determined (Table 6
). The aca Star and Vitros assays demonstrated similar low
fosphenytoin cross-reactivity; the TDx, AxSYM, and ACS:180 showed
intermediate cross-reactivity; and the TDx phenytoin II showed very
high fosphenytoin cross-reactivity.
To determine whether all patients with renal insufficiency who received
fosphenytoin had falsely increased phenytoin immunoassays results, we
analyzed samples from 10 additional patients with renal insufficiency
(creatinine, 24–91 mg/L) who had received fosphenytoin, several of
whom were critically ill. Comparison of the results obtained with
AxSYM, Vitros, or ACS:180 with those obtained with HPLC or aca Star did
not reveal any significant falsely increased results (data not shown).
This demonstrated that the effect was observed only in select patients
receiving fosphenytoin.
The laboratory and clinical characteristics of this latter group of
patients were compared with those of the seven patients who had falsely
increased free and total phenytoin results. No drug that was
unique to all seven affected patients could be identified. Several
laboratory values, including arterial pH, arterial
PCO2, serum anion gap,
glucose, lactate, bilirubin, and amino transferases were examined. No
pattern of abnormalities unique to the affected patients could be
identified.
|
Discussion
|
|---|
In the present study, seven hospitalized patients with renal
insufficiency who were receiving i.v. fosphenytoin had falsely
increased total- and free-phenytoin results with several immunoassay
methods. These falsely increased results could not be explained by the
accumulation of phenytoin metabolites in patients with renal
insufficiency because previous studies have shown that three of the
affected phenytoin assays (TDx phenytoin II, ACS:180, and Vitros) do
not exhibit this problem (7)(8)(10).
These earlier studies included samples from critically ill patients,
but no samples from patients receiving fosphenytoin. We also confirmed
that the ACS:180 and Vitros assays do not cross-react with the
phenytoin metabolites that accumulate in uremia, whereas the AxSYM
method shows significant constant and proportional positive bias (Fig. 3
).
We believe the falsely increased results may be caused by one or more
substances that cross-react with some assay antibodies, and not
chemical interference. The reason is that each one of the identified
immunoassays utilizes a distinct analytical methodology. The Abbott
AxSYM and TDx II methods are homogeneous assays that use fluorescence
polarization. The ACS:180 method is a heterogeneous method that uses
chemiluminescence. The Vitros method is heterogeneous and uses an
enzymatic rate reaction monitored at 540 nm by reflectance
spectrophotometry. The discrepant results between these immunoassays
and HPLC are not caused by interference with phenytoin recovery in the
HPLC. In the HPLC assay, which has been in clinical use for a long
time, solvent extraction is used to isolate phenytoin from other
potential interferences before analysis. The recovery of drugs using
solvent extraction rarely is affected by the presence of other
components in serum. The fact that the HPLC results correlate well with
one immunoassay also argues against this because one would have to
postulate identical decreases in two methodologically distinct assays.
The falsely increased results in the present study could not be
explained by the cross-reactivity of residual fosphenytoin for the
following reasons. First, samples were collected at least 2 h
after i.v. administration of fosphenytoin, a more than adequate period
of time for complete conversion of fosphenytoin to phenytoin. It is
known that the half-life of fosphenytoin in patients with low albumin
and uremia is decreased compared with healthy individuals; therefore,
no residual fosphenytoin should be present in any samples collected in
our study (2). Second, no fosphenytoin was detected in any
samples that were analyzed by a sensitive HPLC method (Tables 1
and 2
).
Third, the minimal degree of fosphenytoin cross-reactivity was similar
for the aca Star and Vitros assays; however, the aca Star did not show
falsely increased results, whereas the Vitros method did. If phenytoin
metabolites and fosphenytoin are eliminated as causes of the falsely
increased phenytoin immunoassay results, the presence of one or more
novel cross-reacting fosphenytoin metabolites must be considered. These
falsely increased results do not appear to be caused by an interfering
substance because multiple immunoassays that use different
methodologies (i.e., fluorescence polarization, chemiluminescence, and
heterogeneous enzymatic methods) all show similar effects. Rather, one
or more substances cross-react with each antibody used in the affected
assays. Although we have yet to identify any novel cross-reacting
substances, our data suggest that such substances could be metabolites
or conjugates derived from fosphenytoin. The measured free-phenytoin
concentration did not become greater than the total phenytoin in
subject 1, who had been critically ill with renal failure, until 2 days
after he was switched from phenytoin to fosphenytoin. We had been using
the same total- and free-phenytoin assays for >2 years in critically
ill patients and had not had any reports of problems until the pharmacy
switched its i.v. phenytoin product to fosphenytoin. The first subject
was identified ~1 month after this change.
Fosphenytoin is thought to be dephosphorylated by tissue phosphatases
to an intermediate that spontaneously hydrolyzes to phenytoin(13). One possibility is that the intermediate forms an
adduct with a metabolic product present in abnormally high
concentrations in some critically ill patients with renal
insufficiency. This hypothetical adduct is ultrafilterable and can
cross-react with the free-phenytoin assays that were investigated.
The question may be posed as to why other institutions have not
observed this same effect in patients receiving fosphenytoin. The
answer may partly lie in the fact that during the time that we observed
this novel cross-reactivity, different assays were in use for free
phenytoin (fluorescence polarization immunoassay) and total phenytoin
(Emit). The more-specific TDx phenytoin II assay was chosen for
free-phenytoin measurement instead of the more widely used TDx/FLx
phenytoin assay, which exhibits cross-reactivity with phenytoin
metabolites. Many laboratories that perform both free- and
total-phenytoin determinations are not likely to do this, choosing
instead to use the same method for both free- and total-drug assays. A
review of a recent College of American Pathologists proficiency testing
survey revealed that 48% of respondents used either the Abbott AxSYM
or TDx–TDx/FLx methods for phenytoin (14). The Abbott TDx
free-phenytoin assay was used by 82% of respondents. Both of these
methods are subject to the cross-reactivity we have described.
Therefore, falsely increased free-phenytoin results may not be readily
recognized.
Overestimation of total- and/or free-phenytoin concentrations in the
serum by immunoassay methods can lead to significant underdosing of
fosphenytoin, with consequent seizure activity. In fact, in our study,
subjects 1, 3, 6, and 7 appear to have had their dosages of
fosphenytoin reduced in response to falsely increased free-phenytoin
concentrations measured by immunoassay. Of the immunoassays we
investigated, only the aca Star total-phenytoin method, which is based
on the Emit assay, correlated well with HPLC and was apparently
unaffected.
It is noteworthy that different immunoassays quantify the
cross-reacting substance or substances quite differently, as seen in
Fig. 1
. The highly variable method-dependent half-lives observed after
discontinuation of fosphenytoin (Table 5
) might be explained by
multiple cross-reacting species with different cross-reactivities in
different phenytoin immunoassays. Possible alternative explanations
might be that the concentration-response curve for a single
cross-reacting species has a markedly different shape for each
phenytoin immunoassay, or that the cross-reactivity might also depend
on the concentrations of phenytoin present at the same time, as has
been described for fosphenytoin and phenytoin metabolites(4)(8). Some of the falsely increased results
for subjects 1–7 in the AxSYM method can be attributed to the apparent
cross-reactivity of HPPH-G with the AxSYM method in uremic patients
(Fig. 3B
). This HPPH-G cross-reactivity leads to overestimation of the
apparent cross-reactivity of any hypothetical fosphenytoin metabolites
measured by the AxSYM method. The falsely increased results seen with
the other phenytoin immunoassays presumably arise primarily from
fosphenytoin metabolite cross-reactivity. Because our data demonstrate
a lack of cross-reactivity with the Emit antibody, this method is
clearly preferred for total-phenytoin measurements. An investigation of
the Emit reagents for the determination of free phenytoin as an
alternative to HPLC is also warranted.
The experiments reported here, using assays that do not cross-react
with phenytoin metabolites that accumulate in uremia (primarily
HPPH-G), demonstrate that one or more novel immunoreactive compounds
can be found in serum specimens from some, but not all, patients with
renal insufficiency receiving fosphenytoin. On the basis of our
analysis of multiple samples from seven patients, the source of the
cross-reactivity does not appear to be the prodrug fosphenytoin, but
one or more as yet unidentified metabolites or adducts of fosphenytoin.
None of the affected immunoassays require specimen extraction.
Therefore, experimental protocols will have to be designed to isolate
and identify any novel cross-reacting substances. Purification
protocols using solid-phase extraction, solvent extraction, or
antigen-antibody complex isolation may be successful approaches. Once
any novel compounds are identified and assays for their quantification
are developed, pharmacologic studies can be designed to assess their
pharmacokinetic and pharmacodynamic properties. It may then also be
possible to evaluate why one or more cross-reacting substances are
observed only in certain patients, what additional factors might
contribute to their effects, and whether these are all-or-none
phenomena. At the present time, however, any such novel compounds are
of interest primarily because of the analytical effects described in
this report.
|
Acknowledgments
|
|---|
We thank Johnson & Johnson Clinical Diagnostics and Chiron
Diagnostics for providing some of the phenytoin reagents and
calibrators.
|
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910 - 918.
[Abstract]
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Abstract
Introduction
