Determination of 21-hydroxydeflazacort in human plasma by high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry. Application to bioequivalence study

Article abstract of DOI:10.1002/(SICI)1096-9888(200003)35:3<440::AID-JMS955>3.0.CO;2-8

A liquid chromatographic atmospheric pressure chemical ionization tandem mass spectrometric method is described for the determination of 21-hydroxydeflazacort in human plasma using dexamethasone 21-acetate as an internal standard. The procedure requires a single diethyl ether extraction. After evaporation of the solvent under a nitrogen flow, the analytes are reconstituted in the mobile phase, chromatographed on a C₁₈ reversed-phase column and analyzed by mass spectrometry via a heated nebulizer interface where they are detected by multiple reaction monitoring. The method has a chromatographic run time of less than 5 min and a linear calibration curve with a range of 1-400 ng ml^-1 (r > 0.999). The between-run precision, based on the relative standard deviation for replicate quality controls, was ≤ 5.5% (10 ng ml^-1), 1.0% (50 ng ml^-1) and 2.7% (200 ng ml^-1). The between-run accuracy was ± 7.1, 3.8 and 4.8% for the above concentrations, respectively. This method was employed in a bioequivalence study of two DFZ tablet formulations (Denacen from Marjan Industria e Comercio, Brazil, as a test formulation, and Calcort from Merrell Lepetit, Brazil, as a reference formulation) in 24 healthy volunteers of both sexes who received a single 30 mg dose of each formulation. The study was conducted using an open, randomized, two-period crossover design with a 7-day washout interval. The 90% confidence interval (CI) of the individual geometric mean ratio for Denacen/Calcort was 89.8-109.5% for area under the curve AUC((0-24 h)) and 80.7-98.5% for C(max). Since both the 90% CI for AUC((0-24 h)) and C(max) were included in the 80-125% interval proposed by the US Food and Drug Administration, Denacen was considered bioequivalent to Calcort according to both the rate and extent of absorption. Copyright (C) 2000 John Wiley and Sons, Ltd.

Full text of DOI:10.1002/(SICI)1096-9888(200003)35:3<440::AID-JMS955>3.0.CO;2-8

JOURNAL OF MASS SPECTROMETRY
J. Mass Spectrom. 35, 440–445 (2000)
Determination of 21-hydroxydeflazacort in
human plasma by high-performance liquid
chromatography/atmospheric pressure chemical
ionization tandem mass spectrometry.
Application to bioequivalence study
Demian R. Ifa,1* Maria E. Moraes,2 Manuel O. Moraes,2 Vincenzo Santagada,3
Giuseppe Caliendo3 and Gilberto de Nucci1
1
Cartesius Analytical Unit, Department of Pharmacology, ICB-University of Sa˜o Paulo, Sa˜o Paulo, Brazil
Department of Pharmacology, Federal University of Ceara, Ceara, Brazil
Department of Pharmaceutical and Toxicological Chemistry, University of Naples Federico II, Naples, Italy
2
3
A liquid chromatographic atmospheric pressure chemical ionization tandem mass spectrometric method is
described for the determination of 21-hydroxydeflazacort in human plasma using dexamethasone 21-acetate
as an internal standard. The procedure requires a single diethyl ether extraction. After evaporation of the
solvent under a nitrogen flow, the analytes are reconstituted in the mobile phase, chromatographed on a C18
reversed-phase column and analyzed by mass spectrometry via a heated nebulizer interface where they are
detected by multiple reaction monitoring. The method has a chromatographic run time of less than 5 min and
a linear calibration curve with a range of 1–400 ng ml−1 (r > 0.999). The between-run precision, based on the
relative standard deviation for replicate quality controls, was Ä5.5% (10 ng ml−1), 1.0% (50 ng ml−1) and 2.7%
(200 ng ml−1). The between-run accuracy was ꢀ7.1, 3.8 and 4.8% for the above concentrations, respectively.
This method was employed in a bioequivalence study of two DFZ tablet formulations (Denacen from Marjan
Industria e Comercio, Brazil, as a test formulation, and Calcort from Merrell Lepetit, Brazil, as a reference
formulation) in 24 healthy volunteers of both sexes who received a single 30 mg dose of each formulation. The
study was conducted using an open, randomized, two-period crossover design with a 7-day washout interval.
The 90% confidence interval (CI) of the individual geometric mean ratio for Denacen/Calcort was 89.8–109.5%
for area under the curve AUC(0–24 h) and 80.7–98.5% for Cmax. Since both the 90% CI for AUC(0–24 h) and
Cmax were included in the 80–125% interval proposed by the US Food and Drug Administration, Denacen was
considered bioequivalent to Calcort according to both the rate and extent of absorption. Copyright  2000
John Wiley & Sons, Ltd.
KEYWORDS: deflazacort; metabolite; quantification; liquid chromatography/mass spectrometry
separation and UV detection,2–6 only the method
INTRODUCTION
proposed by Reynolds et al.5 has the sensitivity required
for pharmacokinetic studies, with low administration
doses. The minimum quantification limit is 1 ng mlꢀ1. For
routine analysis, this method has three inconveniences: it
employs large plasma sample volumes (2 ml) and has a
long extraction procedure (solid-phase extraction) and a
long retention time for 21-OH-DFZ (10.6 min).
Deflazacort, 11ˇ,21-dihydroxy-20-methyl-50ˇ,H-pregna-
1,4-dieno[17,16-d]oxazole-3,20-dione 21-acetate, (DFZ,
Fig. 1) is an inactive prodrug derivative of predinisolone
which is rapidly converted to the active metabolite
21-hydroxydeflazacort (21-OH-DFZ, Fig. 1) after oral
administration. 21-OH-DFZ has anti-inflammatory and
immunosuppressive activities. Deflazacort is employed in
the treatment of rheumatoid arthritis, Ducchene dystrophy,
systemic lupus erithematosus, uveitis and transplantation.1
Although several method have been reported for the
determination of 21-OH-DFZ in human plasma, all
using high-performance liquid chromatographic (HPLC)
The quantification of drugs in biological matrices
by liquid chromatography/tandem mass spectrometry
(LC/MS/MS) is becoming more usual, improving the sen-
sitivity and selectivity of methods.7 This paper describes
a fast, sensitive and specific LC/MS/MS method for
the quantification of 21-OH-DFZ using dexamethasone
21-acetate (21-Ac-DXM, Fig. 1) as an internal standard
(I.S.). The method was applied in a study of bioequiv-
alence of two commercial 30 mg tablets formulations
of DFZ:Calcort (Merrell Lepetit, Brazil) and Denacen
(Marjan Industria e Comercio, Brazil) in 24 healthy
volunteers.
* Correspondence to: D. R. Ifa, Jesuino Marcondes Machado 415,
Campinas, SP, Brazil.
E-mail: ifa@usp.br
Copyright 2000 John Wiley & Sons, Ltd.
Received 11 October 1999
Accepted 14 December 1999

DETERMINATION OF 21-HYDROXYDEFLAZACORT IN HUMAN PLASMA
441
2000 ng mlꢀ1 by diluting the 10 µg mlꢀ1 21-Ac-DXM
solution with mobile phase.
The quality control concentrations were fixed at 10, 50
and 200 ng mlꢀ1 (QCA, QCB and QCC, respectively) and
were taken directly from calibration standards.
Sample preparation
Aliquots (200 µl) of human plasma were employed for
extraction with addition of I.S. solution (50 µl). The tubes
were vortex-mixed briefly and allowed to stand at room
temperature for 5 min. Diethyl ether–dichloromethane
(3 ml, 80 : 20) was added and the samples were vortex-
mixed for 30 s. The tubes were centrifuged at 2000 rpm
for 10 min at 4 °C. The upper organic layer was removed
and transferred to new tubes. The solvent was evaporated
under a nitrogen flow in a dry-bath at 37 °C. The samples
were reconstituted with mobile phase (200 µl) and trans-
ferred into microvials, capped and placed in a Shimadzu
Avp10 autosampler rack.
Figure 1. Structures of deflazacort (DFZ), 21-hydroxydeflaza-
cort (21-OH-DFZ) and the Internal standard, dexamethazone
21-acetate (21-Ac-DXM).
EXPERIMENTAL
Materials
HPLC-grade solvents and analytical-grade reagents were
purchased from Mallinckrodt (St Louis, MO, USA) and
Nuclear (Sao Paulo, Brazil). 21-OH-DFZ was prepared
from DFZ (acquired from Marjan Industria e Comer-
cio) by treatment with methanol saturated with anhydrous
hydrochloric acid. The reaction mixture was stirred for
1 h, keeping the temperature at 0 °C, and then the reac-
tion mixture was stirred overnight at room temperature.
After the solvent had been removed on a rotary evap-
orator, water was added and the mixture was adjusted
to pH 10 with 1 M NaOH, and the aqueous layer was
extracted three times with dichloromethane. The com-
bined dichloromethane layers were washed with brine and
dried over anhydrous sodium sulfate and evaporated in
vacuum. The residue was purified by chromatography on
a silica gel column (eluent diethyl ether–ethanol (9 : 1)).
The crude 21-OH-DFZ was recrystallized from diethyl
ether to yield a colorless crystalline solid. Homogene-
ity of the purified compound was assessed by analytical
Chromatographic conditions
An aliquot (80 µl) of the plasma extract was injected
into a Supelcosil C18 column, 150 ð 4.6 mm i.d., 5 µm
(Supelco, Bellefonte, PA, USA) using a Shimadzu AVP
LC system. Separation and elution were achieved using
70% of acetonitrile (ACN) and 30% of 0.5 mM acetic
acid in water as the mobile phase at a flow-rate of
0.60 ml minꢀ1. The oven temperature was 40 °C and the
time between injections was 5 min.
Mass spectrometric conditions
Mass spectrometric detection was performed using a
MicroMass (Manchester, UK) Quatro LC triple-quadru-
pole mass spectrometer, equipped with a heated nebulizer
as the APCI source. The temperatures of the atmospheric
pressure chemical ionization (APCI) probe and source
block were 350 and 120 °C, respectively. Nitrogen was
used as nebulizer gas (70 l hꢀ1) and desolvation gas
(699 l hꢀ1). The corona was set to 2.9 kV. The APCI
source was operated in the positive ionization mode
(APC), and multiple reaction monitoring (MRM), m/z
400.12 ! 124.13 and m/z 435.19 ! 397.29, was used
for quantification of 21-OH-DFZ and 21-Ac-DXM respec-
tively. The dwell time, the cone voltage, the collision
energy and collision gas (argon) pressure were 0.1 s,
45 V, 40 eV and 3 ð 10ꢀ3 bar, respectively, for 21-OH-
DFZ, and 0.1 s, 25 V, 10 eV and 3 ð 10ꢀ3 bar, respec-
tively, for 21-Ac-DXM. Data were acquired by MassLynx
software (version 3.2, MicroMass) and calibration curves
for the analyte were constructed using the calibration sam-
ples using 21-OH-DFZ to I.S. peak-area ratios (PARS) via
a weighted (1/x) least-squares linear regression. Unknown
sample PARS were then interpolated from the calibration
curve to provide concentrations of 21-OH-DFZ.
˚
reversed-phase HPLC with a µBondpack C18 (125 A) col-
umn, 10 mm, 3.9 ð 300 mm, spherical, and the purity was
>98%. The melting point (216–217 °C) was determined
using a Kofler melting point apparatus and is uncorrected.
1H and 13C NMR spectra were recorded on a Bruker AMX
500 MHz spectrometer with CDCl3 as solvent. The NMR
data were consistent with the structure of 21-OH-DFZ.
21-Ac-DXM was purchased from Sigma (St Louis, MO,
USA). Blank human blood was collected from healthy,
drug-free volunteers. Plasma was obtained by centrifu-
gation of blood treated with the anticoagulant sodium
heparin. Pooled plasma was prepared and stored at approx-
imately ꢀ70 °C until needed.
Calibration standards and quality control
Solutions of 21-OH-DFZ and 21-Ac-DXM were pre-
pared in methanol–water (50 : 50) at a concentration of
10 µg mlꢀ1. Calibration standards of 21-OH-DFZ were
prepared at concentrations of 400.0, 200.0, 100.0, 50.0,
20.0, 10.0, 7.5, 5.0, 3.5, 2.0 and 1.0 ng mlꢀ1 with an ini-
tial dilution of 0.4 ml of 10 µg mlꢀ1 to 9.6 ml of blank
plasma (final concentration 400 ng mlꢀ1). The subsequent
concentrations were obtained by serial dilutions with the
same blank plasma. An I.S. solution was prepared at
Bioequivalence study
The method was applied to evaluate, in human volun-
teers, the performance of one DFZ tablet formulation
Copyright 2000 John Wiley & Sons, Ltd.
J. Mass Spectrom. 35, 440–445 (2000)

442
D. R. IFA ET AL.
(Denacen, tablets, 30 mg, Marjan Brazil, Lot Number
0068) against one standard DFZ tablet formulation (Cal-
cort, tablets, 30 mg, by Merrell Lepetit Brazil, Lot Num-
ber 9808521/3). The comparison was done through the
quantification of 21-OH-DFZ, the active metabolite of
DFZ. The bioequivalence between the two formulations
hospitalized at 11:00 p.m., having already had a normal
evening meal, and after an overnight fast they received
at 7:00 a.m. a single 30 mg dose of the appropriate DFZ
formulation along with 200 ml of tap water. No food was
allowed during 4 h following drug administration, after
which a standard lunch was consumed and an evening
meal was provided 10 h after dosing. No other food was
permitted during the ‘in-house’ period. Liquid consump-
tion was permitted ad libitium after lunch but xanthine-
containing drinks, including tea, coffee, and cola, were
avoided.
Blood samples (10 ml) from a suitable antecubital vein
were collected in EDTA-containing tubes before and 0.5,
1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, 10.0, 12.0 and 24.0 h
after the administration of each dose of DFZ. The blood
samples were centrifuged at 4000 rpm for 10 min at room
temperature and the plasma was decanted and stored at
ꢀ20 °C until assayed. All samples from a single volunteer
were analyzed on the same day to avoid inter-assay
variation.
was assessed by calculating individual Cmax, AUC.0–24 h/
,
AUC.0–1/ and Cmax/AUC.0–24 h/ ratios (test/reference)
together with their mean and 90% confidence intervals
(CI) after logarithmic transformation of the data (additive
model8). The inclusion of the 90% CI for the ratio in the
80–125% range was analyzed by a parametric (analysis
of variance (ANOVA)) method.
Twenty-four healthy volunteers of both sexes (12 fe-
males and 12 males) who were between the ages of
18 and 35 years (mean š SD, 25.2 š 5.5 years), who
had heights between 153.5 and 181.0 cm (mean š SD,
164.9 š 7.8 cm) and who weighed between 55.2 and
82.6 kg (mean š SD, 64.0 š 9.2 kg) and within 15% of
their ideal body weight were enrolled in the study. All
subjects gave written informed consent, and the Ceara
Federal University Hospital Ethics Committee of Clinical
Investigation approved the clinical protocol.
The volunteers were free from significant cardiac, hep-
atic, renal, pulmonary, neurological, gastrointestinal and
hematological disease, as assessed by physical investiga-
tion, EGC and the following laboratory tests: blood pres-
sure, urea, creatinine, AST, ALT, alkaline phosphatase,
ꢀ-GT, total bilirubin, uric acid, total cholesterol, triglyc-
erides, albumin and total protein, hemoglobin, hematocrit,
total and differential white cell counts, erythrocyte sedi-
mentation rate and routine urinalysis. All subjects were
negative for HIV, HBV and HCV.
RESULTS AND DISCUSSION
Method development
Full-scan positive-ion mass spectra of 21-OH-DFZ and the
I.S. showed the protoned molecular ion [M C H]C at m/z
400 and 435, respectively. The most abundant ion in the
product ion spectra was at m/z 124 for 21-OH-DFZ and at
m/z 397 for 21-Ac-DXM (Fig. 2). From these results, the
mass spectrometer was set as follows: m/z 400 for 21-OH-
DFZ and m/z 435 for 21-Ac-DXM as the precursor ions
and m/z 124 for 21-OH-DFZ and m/z 397 for 21-Ac-DXM
as product ions in the MRM mode.
The study was conducted in a open, randomized, two-
period crossover fashion with a 7-day washout period
between doses. During each period, the volunteers were
Figure 2. APCI product ion spectra of the protonated molecular ion of 21-OH-DFZ (top) and 21-Ac-DXM (bottom). The structure of the
molecular ion and neutral losses are shown, proposing an explanation of base peaks.
Copyright 2000 John Wiley & Sons, Ltd.
J. Mass Spectrom. 35, 440–445 (2000)

DETERMINATION OF 21-HYDROXYDEFLAZACORT IN HUMAN PLASMA
443
at final concentrations of 10, 50 and 200 ng mlꢀ1, were
68 š 8.6, 70 š 6.2 and 71 š 7.8% (mean š RSD, n D 3),
respectively. For the I.S. (2000 ng mlꢀ1) the recovery was
73 š 6.8% (mean š RSD, n D 3).
Neutral losses of 20 and 18 Da were found for 21-Ac-
DXM, as in a previous report on the collision-induced dis-
sociation of betamethasone (geometric isomer of DXM9).
From Fig. 2, we propose, for 21-OH-DFZ, a cleav-
age point and subsequent loss of water generating the
base peak.
Diethyl ether, chloroform, hexane, dichloromethane and
binary mixtures of these solvents in different propor-
tions were tested for extraction. The best results were
obtained with diethyl ether–dichloromethane (80 : 20).
The recoveries of 21-OH-DFZ, based on peak area ratios
of extracted plasma mobile phase, both previously spiked
No peak was observed in the mass chromatogram
of human blank plasma serum under the LC/APCI-
MS/MS conditions described above, as shown in Fig. 3(a)
and (b).
The mass chromatograms of a sample are shown
in Fig. 4(a) and (b), in which the retention times of
21-OH-DFZ and 21-Ac-DXM were 3.3 and 3.9 min,
respectively.
Figure 3. MRM chromatograms of blank human plasma: (A) 21-Ac-DXM and (B) 21-OH-DFZ. MRM chromatograms of spiked blank
human plasma at a final concentration of 1 ng mlꢀ1: (C) 21-Ac-DXM and (D) 21-OH-DFZ.
Figure 4. MRM chromatograms of plasma sample collected 1 h after administration of DFZ: (A) 21-Ac-DXM and (B) 21-OH-DFZ.
Copyright 2000 John Wiley & Sons, Ltd.
J. Mass Spectrom. 35, 440–445 (2000)

444
D. R. IFA ET AL.
Table 1. Between-run plasma calibration quality report
Concentration
ꢀ1
(ng ml
)
1.00
2.00
3.50
5.00
7.50
10.0
20.0
50.0
100
200
400
Slope
Intercept
r
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Mean
SD
1.25
0.99
1.02
1.10
1.27
1.08
1.11
0.12
2.48
2.43
1.95
2.08
1.84
2.09
2.14
0.26
3.59
3.90
3.40
3.57
3.22
3.41
3.51
0.23
6.60
0.38
5.04
5.25
5.27
4.98
4.77
5.02
5.05
0.19
3.68
7.14 11.1
7.51 10.6
7.56 10.0
7.36 10.3
20.0
19.4
21.1
20.6
47.5
53.7
49.0
50.7
49.6
51.9
50.4
2.19
4.34
0.80
98.4
206
397
0.99751 0.18110 0.99984
0.98866 0.82410 0.99994
0.98752 0.96504 0.99966
0.99763 0.16925 0.99999
1.00303 ꢀ0.23020 0.99998
1.00223 0.01984 0.99998
0.99610 0.32152 0.99990
101
102
99.1
101
98.5
100
1.63
198
207
200
198
201
202
3.66
1.81
0.86
396
391
399
402
401
398
7.21
7.47
9.80 19.8
9.99 19.7
7.37 10.3
20.1
0.63
3.14
0.43
0.17
2.32
0.48
4.69
3.02
3.80 0.00661 0.46992 0.99989
0.96
ꢀ0.60
RSD (%)
RE (%)
10.5
11.8
12.0
7.17
1.62
0.10
1.07 ꢀ1.71
Assay performance
between-run precision was estimated as 0.96–12.0%, and
accuracy as 0.10–11.8% over the calibration range.
Table 2 summarizes the accuracy and precision for
quality controls. The within-run accuracy was š11.0, 2.2
and 7.4% for QCA, QCB and QCC, respectively, and the
within-run precision was Ä8.7, 5.9 and 6.7%, respectively.
The between-run precision was Ä7.1, 3.8 and 4.8% for
QCA, QCB and QCC, respectively, and the between-run
accuracy was š5.6, 1.0 and 2.8%, respectively.
The calibration curves showed good linearity throughout
the range 1.0–400.0 ng mlꢀ1. The slopes, intercepts and
coefficients of determinations from the validation analyses
are reported in Table 1.
The between-run precision and accuracy were deter-
mined from the calibration standards and quality con-
trols samples. The within-run precision and accuracy were
determined from the quality control samples. Calibration
curve experiments were performed in duplicate. A qual-
ity control sample was analyzed after a sequence of 10
unknown samples.
The limit of quantification was 1 ng mlꢀ1 (RSD D
10.5% and RE D 11.8%) [Fig. 3(c) and (d)].
Bioequivalence study
The assay precision was determined as the relative stan-
dard deviation, RSD (%) D 100(SD/M), and accuracy as
the percentage relative error, RE (%) D .E ꢀ T/.100/T/,
where M is the mean, SD is the standard deviation of M,
E is the experimentally determined concentration and T
is the theoretical concentration.
Both DFZ formulations were well tolerated at the dose
administered. No adverse effects were reported, and the
biochemical parameters remained unchanged and within
the reference range. The maximum concentration reached
(Cmax) and the areas under the curves (AUC.0–24 h/) were
compared. For Denacen 30 mg tablets the Cmax geometric
mean ratio was 89.2% (90% CI D 80.7–98.5) of Cal-
cort. The Denacen 30 mg tablets AUC.0–24 h/ geometric
mean ratio was 98.2% (90% CI D 89.8–109.5) of Cal-
cort. Pharmacokinetic parameters are given in Table 3 and
mean plasma concentrations of 21-OH-DFZ are shown in
Fig. 5. These results are consistent with those reported by
Mollmann et al.4 in a pharmacokinetic/pharmacodynamic
evaluation of DFZ in comparison with methylprednisolone
and prednisolone.
Table 1 summarizes the between-run accuracy and pre-
cision for calibration standards. The calibration curve
Table 2. Within-run and between-run plasma quality control
report
Sample
n
Mean
SD
RSD (%) RE (%)
QCA
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
7
11.3 0.97 8.72 11.0
(10 ng mlꢀ1
)
5
6
7
8
3
11.0 0.87 7.92
10.4 0.42 4.06
10.3 0.67 6.53
10.3 0.62 5.96
10.1 0.32 3.22
9.70
4.30
2.90
3.25
0.80
5.56
Since the 90% CI for both Cmax and AUC ratio were
inside the 80–125% interval proposed by the US Food
and Drug Administration,10,11 it is concluded that Denacen
30 mg tablets are bioequivalent to Calcort 30 mg tablets
for both the rate and the extent of absorption.
Between-run 36 10.6 0.75 7.12
QCB
(50 ng mlꢀ1
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
7
5
6
7
8
3
51.1 3.04 5.94
2.17
)
49.7 1.70 3.42 ꢀ0.59
50.6 1.64 3.25
50.4 2.36 4.68
50.6 1.21 2.39
50.1 0.79 1.59
1.25
0.90
1.22
0.12
1.01
Table 3. Pharmacokinetic parameters from 24 human volun-
teers following oral administration of a single 30 mg
DFZ tablet
Between-run 36 50.5 1.92 3.80
Calcort
Denacen
Parameter
Mean
SD
Mean
SD
QCC
(200 ng mlꢀ1
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
7
5
6
7
8
3
215
204
202
204
206
196
10.7
4.96
7.42
2.04
0.87
1.83
3.24
)
8.01 3.92
2.18 1.08
4.38 2.15
AUCall.0
336
344
97.8
143
1.0
120
122
322
343
93.9
129
1.0
100
103
24 h/
AUC1
AUCall.0
Cmax.ng
Tmax.h/
24 h//1
ꢀ1
13.9
6.74
44
0.5 2.5
0.3
40
0.5 3.0
1.5
ml
/
1.01 0.51 ꢀ1.88
.range/
Between-run 36 206
9.90 4.81 2.79
T1/2.h/
1.3
1.7
Copyright 2000 John Wiley & Sons, Ltd.
J. Mass Spectrom. 35, 440–445 (2000)

DETERMINATION OF 21-HYDROXYDEFLAZACORT IN HUMAN PLASMA
445
CONCLUSION
A fast and reliable LC/APCI-MSMS method for the deter-
mination of 21-OH-DFZ in human plasma was devel-
oped and validated. The validation results indicated that
the method is rugged, precise and accurate and is suit-
able for routine determination of 21-OH-DFZ in human
plasma.
Acknowledgement
Figure 5. Mean plasma concentration of 21-OH-DFZ in 24 human
volunteers following oral administration of a single 30 mg DFZ
tablet.
Demian R. Ifa is supported by the Fundac¸a˜o de Amparo a` Pesquisa
do Estado de Sa˜o Paulo (FAPESP).
REFERENCES
1. Markham A, Bryson HM. Drugs 1995; 50: 317.
2. Rao N, Eller M, Arumugham T, Weir S. Eur. J. Drug Metab.
Pharmacol. 1996; 21: 241.
3. Rao N, Bhargava VO, Reynolds DL, Eller MG, Weir SJ. Bio-
pharm. Drug Dispos. 1996; 17: 753.
7. Huang EC, Wachs T, Conboy JJ, Henion JD. Anal. Chem.
1990; 62: 713.
8. Marzo A. J. Pharm. Pharmacol. 1997; 49: 1259.
9. Polettini A, Bouland GM, Montagna M. J. Chromatogr B
1998; 713: 399.
4. Mollmann H, Hochhaus G, Rohatagi S, Barth J, Derendorf H.
Pharm. Res. 1995; 12: 1096.
5. Reynolds DL, Burmaster SD, Eichmeier LS. Biomed. Chro-
matogr. 1994; 8: 230.
6. Hirata H, Kasama T, Sawai Y, Fike RR. J. Chromatogr. B
1994; 658: 55.
10. Federal Register Part 320: Bioavailability and Bioequivalence
Requirements. Food and Drug Administration: Washington,
DC, 1985; 154.
11. Food and Drug Administration. Pharmacopeial Forum 1993;
19: 6501.
Copyright 2000 John Wiley & Sons, Ltd.
J. Mass Spectrom. 35, 440–445 (2000)