Clinical Chemistry 44: 2516-2523, 1998;
(Clinical Chemistry. 1998;44:2516-2523.)
© 1998 American Association for Clinical Chemistry, Inc.
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Drug Monitoring and Toxicology |
Modified pentamer formation assay for measurement of tacrolimus and its active metabolites: comparison with liquid chromatography–tandem mass spectrometry and microparticle enzyme-linked immunoassay (MEIA-II)
Victor W. Armstrong1,a,
Ekkehard Schuetz1,
Qingling Zhang2,
Stephan Groothuisen1,
Christa Scholz1,
Maria Shipkova1,
Hoda Aboleneen2 and
Michael Oellerich1
1
Abteilung Klinische Chemie, Georg-August-Universitaet Goettingen, D-37075 Goettingen, Germany.
2
Abbott Diagnostics Division, Abbott Laboratories, Abbott
Park, IL 60064.
a Address correspondence to this author at: Abteilung Klinische Chemie, Zentrum Innere Medizin, Georg-August-Universitaet Goettingen, D-37075 Goettingen, Germany. Fax 49551-398551; e-mail varmstro{at}med.uni-goettingen.de.
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Abstract
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A modified pentamer formation assay (PFA) for quantification of
tacrolimus and active metabolites after extraction from whole blood is
described. The lower limit of detection was 2 µg/L. Intraassay
precision (CV) was 5.7–13.7%, and the interassay CV was 6.1–14.9%.
Tacrolimus trough concentrations in 104 whole blood specimens from
liver and kidney transplant recipients were compared with results from
HPLC–tandem mass spectrometry (LC/MS/MS) and microparticle enzyme
immunoassay (MEIA-II). Data were analyzed by difference plots and are
presented as median (95% confidence intervals) of the method
differences. MEIA-II results were on average 2.00 µg/L (range, -0.08
to 5.17 µg/L) higher than LC/MS/MS, whereas PFA results were only
1.07 µg/L (range, -2.62 to 5.33 µg/L) higher. Of 104 specimens
tested, 25 displayed differences 
3 µg/L between MEIA-II and PFA:
median difference, 4.65 µg/L (range, 3.01–8.79 µg/L). The
corresponding median difference between PFA and LC/MS/MS was -0.91
µg/L (range, -4.11 to 0.85 µg/L), and the difference between
MEIA-II and LC/MS/MS was 3.67 µg/L (range, 1.88–6.34 µg/L),
suggesting the presence of inactive metabolites that caused a positive
bias in the immunoassay. In contrast, similar median differences were
observed for the remaining 79 specimens: MEIA-II minus LC/MS/MS, 1.78
µg/L (range, -0.45 to 4.11 µg/L); PFA minus LC/MS/MS, 1.90 µg/L
(range, -1.70 to 5.50 µg/L). Active tacrolimus metabolites may have
contributed to the higher apparent tacrolimus concentrations in these
specimens.
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Introduction
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Because of the variability in the absorption and clearance of
tacrolimus, as well as its narrow therapeutic index, monitoring of
whole blood trough concentrations of tacrolimus (FK 506, Prograf) is
recommended (1) to achieve optimal therapeutic efficacy
while minimizing the risk of toxicity. Specific methods have been
described for quantification of the parent drug in whole blood, using
liquid chromatography–mass spectrometry
(LC/MS)1
(2)(3) or LC–tandem mass spectrometry
(LC/MS/MS) (4)(5). These methods are, however,
only available to a limited number of institutions, and most transplant
centers currently use one of the two commercially available
immunoassays (6). The latter are not entirely specific for
the parent drug, and cross-react with tacrolimus metabolites. This lack
of specificity for the parent drug is a major problem in the use of
immunoassays (7). Tacrolimus is extensively metabolized in
the liver by the cytochrome P450 3A4 pathway (8), and at
least nine metabolites have been described (6). These
metabolites display differing immunologic cross-reactivities with
respect to the tacrolimus monoclonal antibody used in the commercial
immunoassays. The pharmacological activity of the metabolites, however,
does not correlate with their immunologic cross-reactivities
(6).
The mechanism of action of tacrolimus involves formation of a
pentameric complex in which a dimer consisting of tacrolimus and a
12-kDa FK-binding protein (FKBP12) engages the trimer
calcineurin-calmodulin-Ca2+. This leads to inhibition
of the enzymatic activity of calcineurin, thereby blocking activation
of the nuclear factor of activated T cells and ultimately activation of
cytokine transcription. Asami et al. (9) demonstrated that
the pentameric complex could be detected by a simple binding assay
to polyethylenimine-treated glass filters. Tamura et al.
(10) adapted the original assay to the ELISA technique,
using recombinant human FKBP12 coated to a microtiter plate. They
investigated the ability of tacrolimus and seven of its metabolites to
sustain pentamer formation. Although only a poor correlation was found
between FKBP12 binding and pentamer formation, a good correlation was
observed between the ability of the metabolite to form the pentamer and
its activity in the mixed lymphocyte response assay. These authors did
not, however, investigate tacrolimus concentrations in whole blood
specimens. We now describe an extraction procedure and pentamer
formation assay (PFA) for quantification of the parent drug tacrolimus
and those of its metabolites that are capable of forming the pentameric
complex in whole blood specimens. The results obtained with the
pentamer assay were compared with those obtained with a specific
LC/MS/MS assay (4) and a microparticle enzyme immunoassay
(MEIA-II).
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Materials and Methods
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reagents
Human recombinant FKBP12, calcineurin (bovine brain),
streptavidin-alkaline phosphatase conjugate,
3-[N-morpholino]propane sulfonic acid (MOPS),
p-nitrophenyl phosphate, and dithiothreitol were from
Sigma-Aldrich. Biotinylated calmodulin was obtained from
Calbiochem-Novabiochem. Acetonitrile (gradient grade), methanol
(gradient grade), and KH2PO4 were from Merck,
and Tween 20 was from Bio-Rad. The tacrolimus was a kind gift of
Fujisawa Pharmaceuticals (Osaka, Japan).
blood specimens
For the method comparison, 104 EDTA whole blood specimens from
kidney and liver transplant recipients who were receiving tacrolimus as
part of their immunosuppressive regimen were investigated. The
specimens had been sent to our routine laboratory for monitoring of
whole blood trough tacrolimus concentrations. Specimens were stored
frozen at -70 °C until analysis. EDTA whole blood specimens from
healthy subjects not on any drug therapy were used for calibration of
the PFA.
extraction
EDTA whole blood (200 µL) was mixed with 200 µL of saturated
KH2PO4 solution, pH 4.4, followed by
addition of 600 µL of acetonitrile. The mixture was vigorously mixed
by vortex-mixing for 30 s and was then centrifuged for 10 min at
10 000g. A 600-µL aliquot of the organic upper layer was
removed and evaporated to dryness at 32 °C in a vacuum centrifuge.
The residue was reconstituted in 20 µL of methanol and 200 µL of
MOPS, pH 6.6, containing 1 mmol/L CaCl2. For calibration of
the PFA, tacrolimus from a stock solution in methanol was added to
drug-free whole blood to give the following final concentrations: 2.5,
5.0, 10.0, 20.0, and 30 µg/L.
pfa
Maxisorp C8 microtiter plates (Nunc) were coated overnight at
4 °C with FKBP12 (1.7 µg/well) in coating buffer (0.2 mol/L
Na2CO3–0.2 mol/L NaHCO3, pH
9.5). All subsequent steps were carried out at room temperature
(20–22 °C). After the wells were washed (50 mmol/L MOPS, pH 7.4,
containing 0.5 g/L Tween 20 and 100 mmol/L NaCl) to remove unbound
protein, they were blocked by incubating the plates with 1 g/L bovine
serum albumin in coating buffer for 1 h. After an additional
washing step, 100 µL of the reconstituted samples was added to each
well, followed by 50 µL of a solution of calmodulin-biotin (0.175
µg/well) and calcineurin (0.11 µg/well) in 50 mmol/L MOPS, pH 6.6,
containing 1 mmol/L CaCl2, 0.5 mmol/L dithiothreitol, 5 g/L
bovine serum albumin, and 0.5 g/L Tween 20 (buffer A). The plates were
incubated for 1 h and then washed. This was followed by a 30-min
incubation with 0.1 mL of streptavidin-alkaline phosphatase conjugate
(1:200 dilution in buffer A). After another washing procedure, 100 µL
of 10 mmol/L p-nitrophenyl phosphate in 1 mmol/L
diethanolamine, pH 9.8, 0.5 mmol/L MgCl2 was added to each
well. Color development was stopped after a 45-min incubation by the
addition of 50 µL of 2 mol/L NaOH, and the absorbance was read at 405
nm (490 nm reference wavelength).
lc/ms/ms
The procedure for the measurement of tacrolimus in human whole
blood by LC/MS/MS has been described in detail elsewhere
(4). Human whole blood specimens (1.2 mL) were shipped to
Abbott Park from Goettingen on dry ice. The investigators at Abbott
were not aware of the results of the PFA or the MEIA-II, which were
determined independently at the laboratory in Goettingen.
meia-ii
Whole blood tacrolimus concentrations were measured on an IMx
System using the Tacrolimus-II assay based on MEIA technology,
according to the manufacturer's instructions. The interassay CVs were
12.9%, 9.9%, and 7.3% at 5.0, 11.0, and 22 µg/L tacrolimus,
respectively.
calibrator cross-check
To compare calibrator assignments between the three methods, the
following experiments were carried out. Calibrators for the LC/MS/MS
procedure were prepared by adding tacrolimus stock solution in methanol
to whole blood-based calibrator diluent (4). Calibrators
with the assigned tacrolimus concentrations of 3, 6, 12, and 20 µg/L
were shipped to Goettingen on dry ice. After thawing, these calibrators
were measured in replicate (n = 4), using the MEIA-II method,
which had been calibrated using the calibrators provided in the kit by
the manufacturer. Likewise, whole blood calibrators from the PFA, with
assigned tacrolimus concentrations of 2.5, 5.0. 10.0, and 20.0 µg/L,
were also measured in replicate (n = 4), using the MEIA-II
procedure.
other analytical methods
Plasma concentrations of bilirubin and creatinine as well as
plasma catalytic concentrations (25 °C) of alanine aminotransferase,
aspartate aminotransferase, and 
-glutamyltransferase were determined
by routine laboratory procedures.
statistics
The nonparametric regression procedure developed by Passing and
Bablok (11) was used for the method comparison. (EVAPAK,
Ver. 2.08; Boehringer Mannheim). The 95% confidence intervals for the
estimates of slope and intercept were given, together with the 68%
median distance of the residuals. For comparison, the more familiar SD
of the residuals, Sy|x, was also calculated, using
the standard principal component procedure. Agreement between the
methods was also assessed by plotting the method differences against
the method means as recommended by Bland and Altman (12).
The median difference and 95% limits of agreement were also included
in the plots. Differences between continuous variables were compared
using the Mann–Whitney U-test.
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Results
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extraction efficiency
In a set of separate experiments, tacrolimus at concentrations
ranging from 2.5 to 30 µg/L was added to whole blood. Samples (200
µL) were extracted and reconstituted as described in Materials
and Methods. Tacrolimus concentrations in the reconstituted
samples were determined relative to samples of reconstitution solution
to which tacrolimus had been added in concentrations corresponding to
those used in the MEIA-II procedure. The results are presented in Table 1
. The recovery of tacrolimus using this extraction procedure was
relatively constant over the concentration range tested.
performance characteristics of the pfa
A calibration curve for the PFA is shown in Fig. 1
. The lower limit of detection, determined as the mean + 3 SD of
20 replicates of drug-free EDTA blood, was 2 µg/L. The lower limit of
quantification was designated as 2.5 µg/L, the value of the lowest
calibrator. The intraassay CV was calculated by replicate analysis
(n = 20) of four samples in a single assay. At mean tacrolimus
concentrations of 3.5, 6.0, 10.5, and 20.2 µg/L, the intraassay CVs
were 13.7%, 8.0%, 5.7%, and 11.2%, respectively. The interassay CVs
were determined by duplicate determinations of three samples in six
separate assays. At mean tacrolimus concentrations of 4.1, 8.4, and
13.7 µg/L, the interassay CVs were 14.9%, 6.1%, and 8.0%,
respectively.
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Figure 1. Calibration curve for the PFA.
The results represent the mean values (± SD) from six separate
experiments.
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method comparison
The results of the calibrator cross-check are presented in Table 2
. Close concordance was observed between the assigned values of
the calibrators used for the LC/MS/MS and the PFA and the values
obtained with the MEIA-II procedure. Tacrolimus concentrations were
measured in 104 whole blood specimens from kidney and liver transplant
recipients. A total of 55 specimens from 19 liver recipients and 49
specimens from 29 kidney recipients were investigated. The results of
the regression analysis for the method comparisons are presented in
Table 3
for both liver and kidney samples separately, as well as for
all 104 specimens. The data were also analyzed (Fig. 2
) by plotting the differences in the tacrolimus concentrations
between the two methods against the mean of the tacrolimus
concentration from the two methods (12). The slope of the
regression line for comparison of LC/MS/MS (x) and PFA
(y) was significantly >1. The statistical data were similar
when liver and kidney specimens were analyzed separately or together.
This also held true for the method comparisons of LC/MS/MS vs MEIA-II
and MEIA-II vs PFA. The median difference between tacrolimus
concentrations determined with PFA and those determined with LC/MS/MS
was 1.07 µg/L, with 95% limits of agreement of -2.62 to 5.33 µg/L
(Fig. 2A
). Thus the apparent tacrolimus concentrations measured with
the PFA tend to be higher when compared with LC/MS/MS, although there
is some scatter in the data. In the case of the comparison between
LC/MS/MS and MEIA-II, the slope of the regression line was also
significantly >1. The median difference in tacrolimus concentrations
between the two methods was 2.00 µg/L, with 95% limits of agreement
of -0.08 to 5.17 µg/L (Fig. 2B
). In this patient collective, MEIA-II
values for the tacrolimus concentrations were in general greater than
those obtained with the reference procedure specific for the parent
drug. However, there was less scatter of the data when compared with
the comparison between LC/MS/MS and PFA, as shown by the lower 68%
median distance and Sy|x as well as the narrower
95% limits of agreement in Fig. 2B
. The greatest scatter was seen in
the comparison between MEIA-II and PFA. The slope of the regression
line did not differ significantly from unity (Table 3
) although there
was a significant positive intercept. The difference plot (Fig. 2C
)
revealed a tendency to higher tacrolimus concentrations with the
MEIA-II, with a median difference of 0.52 µg/L and 95% limits of
agreement of -3.02 to 5.98 µg/L.
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Table 3. Structural relationship between the methods as assessed by
the nonparametric regression analysis of Passing and Bablok
(11).
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Figure 2. Plot of the method differences in tacrolimus
concentrations vs the mean tacrolimus concentration from the two
methods.
(A) PFA vs LC/MS/MS; (B) MEIA-II vs LC/MS/MS;
(C) MEIA-II vs PFA. The solid line represents the
median deviation of the two methods, and the dotted lines
represent the 95% confidence intervals.  , tacrolimus trough blood
concentrations in samples from liver graft patients;  , tacrolimus
trough blood concentrations in samples from kidney graft patients.
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Inspection of the data in Fig. 2C
revealed that 25 specimens exhibited
a difference of 3 µg/L or greater between MEIA-II and PFA. We
therefore plotted the method differences for these 25 specimens and the
remaining 79 specimens separately. The plots are illustrated in Fig. 3
. Remarkably, the 25 specimens displaying a discrepancy 3
µg/L between MEIA-II and PFA (Fig. 3A
; median difference, 4.65
µg/L; 95% limits of agreement, 3.01–8.79 µg/L), revealed
relatively good agreement between PFA and LC/MS/MS (Fig. 3B
; median
difference, -0.91 µg/L; 95% limits of agreement, -4.11 to 0.85
µg/L). On the other hand, the tacrolimus concentrations of these 25
specimens as measured with the MEIA-II showed a marked positive
deviation (Fig. 3C
) from the LC/MS/MS results, with a median difference
of 3.67 µg/L and 95% limits of agreement of 1.88–6.34 µg/L. In
the remaining 79 specimens there was good agreement between the MEIA-II
and PFA results (Fig. 3D
), whereas both MEIA-II (Fig. 3E
) and PFA (Fig. 3F
) displayed generally higher tacrolimus concentrations than the
LC/MS/MS.
To determine if these findings were related to liver or renal function,
we compared the plasma concentrations of total bilirubin and creatinine
as well as the plasma catalytic concentrations of aspartate
aminotransferase, alanine aminotransferase, and -glutamyltransferase
in the 25 plasma specimens with the corresponding values in the other
79 plasma specimens (Table 4
). As can be seen, bilirubin and alanine aminotransferase values
were significantly greater in the group of 25 plasma specimens that
were associated with a marked bias between MEIA-II and PFA, whereas
there was no difference in creatinine concentrations between the two
groups. Furthermore, the median time after transplantation in which
these 25 plasma specimens were taken was significantly shorter than
that of the remaining 79 plasma specimens.
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Table 4. Comparison of time after transplantation and
clinical chemical indices of liver and kidney function between the 25
plasma specimens with a difference 3 µg/L and the 79 plasma
specimens with a difference <3 µg/L in the tacrolimus values
determined by MEIA-II and PFA.
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Finally, we examined whether there was any correlation between the
method differences in tacrolimus concentrations. When the method
differences between the PFA and LC/MS/MS tacrolimus concentrations were
plotted against the method differences between MEIA-II and PFA
tacrolimus concentrations (Fig. 4
A), a significant negative correlation (r =
-0.82; P <0.001) was observed. A significant although
weaker correlation was also seen (r = 0.54;
P <0.001) between the differences in the MEIA-II and
LC/MS/MS measurements and the differences in the MEIA-II and PFA
measurements (Fig. 4B
). No correlation was found between the method
differences for PFA minus LC/MS/MS and those for MEIA-II minus LC/MS/MS
(Fig. 4C
).
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Figure 4. Correlation of the method differences in tacrolimus
concentrations.
(A) Difference between MEIA-II and PFA (x) vs the
difference between PFA and LC/MS/MS (y); r =
-0.820; P <0.001. (B) Difference between
MEIA-II and PFA (x) vs the difference between MEIA-II and
LC/MS/MS (y); r = 0.543; P
<0.001. (C) Difference between MEIA-II and LC/MS/MS
(x) vs the difference between PFA and LC/MS/MS
(y); r = 0.035; P = 0.727.
, tacrolimus trough blood concentrations in samples from liver graft
patients; , tacrolimus trough blood concentrations in samples from
kidney graft patients.
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Discussion
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Several assay methods have been developed for the measurement of
tacrolimus and its metabolites in biological specimens (6).
The most convenient methods for therapeutic drug monitoring of
tacrolimus are undoubtedly immunoassays such as the ProTrac ELISA
(13) and MEIA-II, which allow a rapid determination of
tacrolimus in whole blood. These assays have the disadvantage that the
anti-tacrolimus monoclonal antibody also exhibits substantial
cross-reactivity with tacrolimus metabolites, in particular
31-O-demethyl (M-II), 15-O-demethyl (M-III), and
15,31-O-didemethyl (10). Of these three
metabolites, only M-II has similar immunosuppressive activity
(10) to the parent drug, although Lhoest et al.
(14) have shown that ring- and open-chain tautomerism
affects the immunosuppressive activity of M-III.
Methods have therefore been sought that would allow measurement of
either the state of immunosuppression or a biological marker that has
been shown to correlate with immunosuppression (6). The
immunosuppressive efficacy of tacrolimus or its metabolites depends on
the ability to form a pentameric complex with FKBP12, calcineurin,
calmodulin, and Ca2+. Tamura et al. (10) first
described a PFA based on recombinant FKBP12 immobilized to microtiter
plates. We have modified this assay to allow a photometric
quantification of tacrolimus and its metabolites. In place of
calmodulin we used calmodulin-biotin. The pentameric complex could then
be detected using a streptavidin-alkaline phosphatase conjugate
followed by color development with p-nitrophenyl phosphate
and measurement of color intensity at 405 nm. By using a simple
extraction procedure, we were able to use this PFA to quantify
tacrolimus in whole blood samples from transplant recipients. The
results obtained with the PFA were then compared with those from a
specific LC/MS/MS procedure and a semiautomated immunoassay (MEIA-II).
In a study into the metabolite patterns in blood from liver and kidney
transplant recipients, Gonschior et al. (15) reported the
main metabolites to be demethyl, demethylhydroxy, didemethyl,
didemethylhydroxy, and hydroxy tacrolimus. On average, all metabolites
accounted for 42% (range, 0–145%) of the tacrolimus concentration in
liver transplant patients and 44.8% (range, 16–152%) in kidney
recipients. In the present investigation, tacrolimus concentrations
determined by the MEIA-II were on average 25% (range, 0–71%) higher
in liver recipients and 23% (range, -7% to 67%) higher in kidney
recipients when compared with the actual parent drug concentrations
determined with a specific LC/MS/MS method. A calibrator cross-check
revealed that these differences were not attributable to differences in
calibrator assignment. This discrepancy could therefore be caused by
the presence of cross-reactive tacrolimus metabolites in these patient
specimens that may or may not be immunosuppressive. When compared with
the LC/MS/MS results, the tacrolimus concentrations determined by the
PFA showed more scatter than those observed with the MEIA-II,
reflecting the somewhat poorer precision of the PFA. However, the
difference between the PFA and LC/MS/MS was on average only about
one-half that seen for the MEIA-II: liver 11% (range, -36% to 67%);
kidney 14% (range, -31% to 58%). The higher values observed with
the PFA compared with LC/MS/MS may reflect the presence of tacrolimus
metabolites that are capable of forming the pentameric complex. The
fact that there was such a wide scatter of differences between the
results from the MEIA-II and the PFA may in part be attributable to the
lower precision of the latter. However, those specimens exhibiting a
large positive difference (3µg/L) between MEIA and PFA, i.e.,
specimens containing relatively large amounts of inactive metabolites,
were found to display a fairly close agreement between their PFA and
LC/MS/MS results. In contrast, the MEIA-II overestimated the tacrolimus
concentrations in these 25 specimens (from 15 liver and 10 kidney
transplant patients) by 33% compared with LC/MS/MS. These specimens
tended to be found in those patients with evidence of liver dysfunction
in the early posttransplant phase. We surmise that they contained
predominantly inactive tacrolimus metabolites that cross-react with the
antibody in the MEIA-II. In the present investigation, no
quantification of tacrolimus metabolites was undertaken by LC/MS/MS. A
qualitative review of the chromatograms, however, revealed that in all
cases the M-I (13-O) and M-III (15-O) desmethyl
metabolites were the most abundant metabolites. M-II (31-O)
was either not present or present in only very small amounts relative
to the other two. The results of the tacrolimus measurements from the
remaining 79 specimens showed close agreement between PFA and MEIA-II;
however, the results were higher than those seen with LC/MS/MS. The
mean overestimation compared with LC/MS/MS was 20% and 21% for PFA
and MEIA-II, respectively. Most probably these specimens contain
predominantly tacrolimus metabolites that are active in pentamer
formation. A qualitative inspection of these chromatograms revealed
that in almost every sample both M-I, which displays some ability to
form the pentamer complex (10), and M-II were again the most
abundant metabolites. The signal for M-I was generally greater than
that for M-III, whereas the metabolite M-II was present in much lower
amounts relative to the other two. Because inspection of the
chromatograms did not reveal any major qualitative differences in the
metabolite patterns of the three desmethyl compounds between the two
groups of 25 and 79 specimens, the explanation for the differences in
the tacrolimus assays may be quantitative differences in the
metabolites relative to tacrolimus, the presence of tautomeric forms
(14) of M-III in vivo, and/or the presence of other unknown
metabolites that cross-react with the MEIA (7).
Noteworthy was the observation that there was a strong negative
correlation between the difference in the PFA and LC/MS/MS results,
which should be a measure of the concentration of active tacrolimus
metabolites in the specimens, and the difference in the MEIA-II and PFA
results, a measure of the inactive metabolites. In other words, the
higher the concentrations of inactive metabolites were in a sample, the
lower the concentrations of active metabolites were in the same
specimen. Because the metabolite M-III is able to bind to FKBP12 but
cannot support pentamer complex formation (10), it can be
speculated that high concentrations of M-II would suppress the binding
of tacrolimus and its active metabolites and might, therefore, explain
the lower values observed in the PFA compared with LC/MS/MS (Fig. 4B
).
In vitro experiments with purified metabolites will be required to
prove this hypothesis.
In conclusion, the results of this investigation have shown that an
assay based on pentamer formation can be applied to the quantification
of tacrolimus in whole blood samples from transplant recipients on
immunosuppression with tacrolimus. In ~76% of the specimens studied
from liver and kidney recipients, the results from the PFA and the
MEIA-II were in good agreement, whereas both assays displayed a
positive bias compared with LC/MS/MS. In the remaining 24% of
specimens, the LC/MS/MS and PFA results were in close agreement,
whereas the tacrolimus concentrations determined with the MEIA-II
were on average one-third higher.
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Acknowledgments
|
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We thank Huaiquin Wu and Janice Simpson for
assistance in the qualitative assessment of the MS chromatograms.
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Footnotes
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1 Nonstandard abbreviations: LC/MS, liquid chromatography–mass spectrometry; LC/MS/MS, liquid chromatography–tandem mass spectrometry; FKBP12, 12-kDa tacrolimus (FK-506)-binding protein; PFA, pentamer formation assay; MEIA, microparticle enzyme immunoassay; and MOPS, 3-[N-morpholino]propane sulfonic acid. 
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Abstract
Introduction
