Isolation and characterization of a degradation product of deflazacort

Article abstract of DOI:10.1691/ph.2012.1141

Deflazacort (DFZ) is an oxazoline derivative of prednisolone with anti-inflammatory and immunosuppressive activity. The aim of this study was to investigate and to identify the main degradation product of DFZ, and to evaluate the anti-inflammatory effect of both DFZ and its major degradation product (namely DDP1). DFZ was subjected to alkaline and acid degradation. In 0.1N NaOH, DFZ was immediately degraded and 99.0% of product DDP1 was detected by high performance liquid chromatography (HPLC). The HPLC method was ideal to separate the primary and other minor degradation products and was carried out using C18 column, mobile phase consisting of water: acetonitrile: (60:40, v/v) with flow rate of 1.0 mL/min and detection at 244 nm. DDP1 was isolated and identified as 21-hydroxy deflazacort (21-OH-DFZ) by NMR, IR and LCMS. The in vivo pharmacological assays showed that both DFZ as 21-OH-DFZ are active in in vivo and in vitro inflammatory models, but 21-OH-DFZ is more potent than DFZ.

Full text of DOI:10.1691/ph.2012.1141

ORIGINAL ARTICLES
Universidade Federal de Santa Catarina1, Florianópolis; Universidade Federal de Santa Maria2; Universidade
Bandeirante de São Paulo3; Universidade Estadual de Campinas4, Campinas, SP, Brasil
Isolation and characterization of a degradation product of deflazacort
A. S. Paulino1, G. Rauber1, A. M. Deobald2, N. Paulino3, A. C. H. F. Sawaya4, M. N. Eberlin4, S. G. Cardoso1
Received August 20, 2011, accepted October 25, 2011
Amarilis Scremin Paulino, Universidade Federal de Santa Catarina. Centro de Ciências da Saúde. Programa de
Pós-Graduac¸ão em Farmácia. Campus Universitário - Trindade, Florianópolis, SC Brasil. CEP 80.040-970
amarilis paulino@yahoo.com.br
Pharmazie 67: 495–499 (2012)
doi: 10.1691/ph.2012.1141
Deflazacort (DFZ) is an oxazoline derivative of prednisolone with anti-inflammatory and immunosuppres-
sive activity. The aim of this study was to investigate and to identify the main degradation product of DFZ,
and to evaluate the anti-inflammatory effect of both DFZ and its major degradation product (namely DDP1).
DFZ was subjected to alkaline and acid degradation. In 0.1 N NaOH, DFZ was immediately degraded and
99.0% of product DDP1 was detected by high performance liquid chromatography (HPLC). The HPLC
method was ideal to separate the primary and other minor degradation products and was carried out using
C18 column, mobile phase consisting of water: acetonitrile: (60:40, v/v) with flow rate of 1.0 mL/min and
detection at 244 nm. DDP1 was isolated and identified as 21-hydroxy deflazacort (21-OH-DFZ) by NMR,
IR and LCMS. The in vivo pharmacological assays showed that both DFZ as 21-OH-DFZ are active in
in vivo and in vitro inflammatory models, but 21-OH-DFZ is more potent than DFZ.
1. Introduction
profile, including degradation products. The hydrolysis in an
aqueous environment of glucocorticoid that has a C-17 ester
has been reported (Teng et al. 2003). Considering the few
publications concerning stability studies of DFZ, the purpose
of this work was to isolate and to identify the main degradation
product of DFZ formed during hydrolysis conditions. In addi-
tion, anti-inflammatory effects of DFZ and major degradation
product were evaluated by means of inflammatory models.
Deflazacort (DFZ) (Scheme A) is an oxazoline (1-(1,16)-21-
(acetyloxy)-11-hydroxy-2-methyl-5H-pregna-1,4-dieno [17,
16-d]oxazole-3,20-dione) derivative of prednisolone with
anti-inflammatory and immunosuppressive activity (Joshi and
Rajeshwari 2009). This glucocorticoid has been prescribed for
the treatment of rheumatoid arthritis, asthma, and other applica-
tions such as myasthenia gravis, systemic lupus erythematosus,
thrombocytopenic purpura, Duchenne muscular dystrophy,
and kidney transplant patients (Angelini 2007; Biggar et al.
2001; Biggar 2006). It is a corticosteroid with a lower risk
of side effects than other available steroids (Angelini 2007;
Ferraris et al. 2007; Gonzalez-Castanheda et al. 2007; Joshi
and Rajeshwari, 2009; Sousa et al. 2010). DFZ is currently
available in tablets and oral suspension. An official method
for determination of this drug in oral formulation has not
been described yet and literature survey reveals few analytical
methods reported for the quantitative estimation of DFZ (Gorog
2010). An HPLC method was developed for our group for the
determination of DFZ in pharmaceutical dosage forms (Corrêa
et al. 2007; Scremin et al. 2010). To verify the specificity of the
method, forced degradation studies were performed (hydrolysis
and oxidation). In these studies, we have observed the instability
of DFZ under acidic and basic conditions. The stability of DFZ
in solid state was also investigated by Cuffini et al. 2007 using
differential scanning calorimetry (DSC), thermogravimetry
(TG) and thin layer chromatography (TLC). However, in these
studies, the degradations products were heights isolated has
characterized. According to ICH guidelines, stress studies
should be carried out on a drug substance to establish its
inherent stability, leading to the identification of degradation
products (ICH 2006). It is also important to evaluate the
biological activity of an individual impurity or a given impurity
2. Investigations and results
In this study, hydrolytic stability of DFZ in aqueous solution
was carried out under stress conditions. In preliminary forced
stress testing, we have observed the instability of DFZ under
acid and basic conditions, using a HPLC method developed for
the assay of the DFZ in pharmaceutical dosage forms (Corrêa
et al. 2007; Scremin et al. 2010). In this method, DFZ eluted
at about 3.4 min and one detected unknown impurity eluted at
about 2.8 min under acid and alkaline stress. To achieve better
retentionandresolution, themobilephasecompositionoftheour
previously (80:20 (v/v), acetonitrile:water) method was modi-
fied to 60:40 (v/v) water:acetonitrile. In the optimized method
the conditions were ideal to separate the primary and other minor
degradation products and DFZ eluted at about 11.6 min. The
method was validated according to ICH (2005) with a correla-
tion coefficient >0.99 (in the range of 5.0–50.0 ␮g/mL), good
specificity (resolution > 2), accuracy (mean recovery: 100,2%),
and precision (RSD < 2%). In alkaline and acid conditions, the
main peak was observed at a retention time of 4.7 min, and was
namely as DDP1. Figure 1 represents the chromatograms show-
ing the decomposition of DFZ in alkaline and acid conditions. In
0.1 N NaOH DFZ was immediately degraded and about 99% of
product DDP1 was initially detected. DDP1 was degraded into
other products after 24 h. In 0.1 N HCl relatively less degrada-
Pharmazie 67 (2012)
495

ORIGINAL ARTICLES
Fig. 1: Chromatograms showing decomposition of DFZ in alkali-induced degradation product (0.1 N NaOH), time zero (A); in acid-induced degradation product (0.1 N HCl) -
time zero (B) and 24 hours (C), C18 (250 × 4.6 mm, i.d., 4 ␮m) column, mobile phase consisted of water:acetonitrile (60:40, v/v), applied at a flow rate of 1.0 mL/min,
injection volume was 20 ␮L and elution of peaks was monitored at 244 nm
tion was observed, and about 39% of the major product DDP1
was detected after 24 h. Due to the extensive decomposition in
alkaline solution, the DDP1 product was isolated from this con-
dition by crystallization in methanol. So, the identification was
possible. The structure of DDP1 was proposed according to the
results obtained by NMR spectroscopy. The 1H and 13C NMR
spectra were interpreted by comparing the chemical shifts of
DFZ reference standard with those of degradation product, as
shown in the Table. The NMR assignment of DFZ carbons is
in accordance with data reported by Cuffini et al. (2007). The
comparison of chemical shifts of DPP1 indicates that structure is
similar, with a short modification. In the 13C NMR spectrum of
DFZ we observed the signal of a methyl (␦ 20.3 ppm) and a car-
bonyl (␦ 170 ppm). Differences in the relationship of the carbon
spectrum between DFZ and DDP1 include the absence of signal
of a methyl (␦ 20.3 ppm) and of a carbonyl (␦170 ppm). The
chemical shift of CH2 at position 21 was less shielded on DFZ
(␦ 66.68 ppm) than on DDP1 (␦ 65.67 ppm). In the proton NMR
spectrum of DFZ, we observed the signals of four methyl (sin-
glet) with displacement in 0.90, 1.38, 1.91, and 2.10 ppm. For
DDP1 we observed a minor electronic effect of an OH group on
C21, with doublets that are more shielded (␦ 4.3 ppm). There are
three methyl with displacement in 0.89, 1.37, and 1.86 ppm, and
the singlet in 2.10 ppm disappears. The effect of an ester group
on the DFZ molecule is higher than that of an alcohol group, and
the ester is less shielded than the doublets (␦ = 4.8 ppm). The IR
spectrum of DFZ show main absorptions centered at 1749, 1730,
and 1652 cm−1, and were assigned to the carbonyl group in posi-
tions 20 and 22, and the cyclic ␣-␤ unsaturated 3-ketone (3-CO),
respectively, as described by Cuffini et al. (2007). The spec-
trum of DDP1 revealed an absence of absorption at 1749 cm−1
relating to carbonyl group in position 22.
,
In order to confirm the structure of DDP1, MS measurements
were performed. The analysis was conducted using the direct
insertion technique, without coupling with LC method. The
mass spectrum of DFZ had an ion at m/z 442 (mass 441 + H),
and m/z value for DDP1 was 399 (m/z 400). Results of LC-MS
of DFZ (A) and DDP1 (B) are show in Fig. 2.
In order to evaluate the pharmacological effects of DFZ and
DDP1, in vivo and in vitro studies were performed. In a paw
edemamodel, 1or10 mg/kgofeitherDFZorDDP1(i.p.)signifi-
cantly inhibited edema induced by carrageenan, with a maximal
average inhibition, respectively, of 24 2% and 29 3%, or
39 3% and 68 4% after 360 min.
In a carrageenan-induced model of peritonitis, when the animals
were treated with DFZ (1 mg/kg) or DDP1 (0.1, 1, or 10 mg/kg,
i.p), astatisticallysignificantdecreaseinthetotalnumberofcells
in the peritoneal cavity was observed. DFZ (1 mg/kg) decreased
the number of neutrophils to 46 3%, while 1 or 10 mg/kg
of DDP1 produced a mean inhibition of 65 3% or 79 4%,
respectively, with an estimated IC50 value of 0.77 (0.70–0.89)
mg/kg.
When assessed in vitro, treatment with 1, 10, or 100 ␮g/ml of
either DFZ or DDP1 led to a decrease in nitrite concentra-
tion in the supernatant of RAW 264.7 macrophages stimulated
with LPS with respective percentiles of 5 1%, 21 2%, and
496
Pharmazie 67 (2012)

ORIGINAL ARTICLES
Fig. 2: LC-MS of DFZ (A) and DDP1 (B)
24 2%, or 11 1%, 46 2%, and 61 3%, without interfere
with the viability of cells, as shown by the MTT test.
degradation products, establish the degradation pathways and
the intrinsic stability of the molecule. In general, these goals
achieved in forced degradation studies should be conducted,
in most cases, under conditions that induce thermal, alkaline,
acid, oxidative and photolytic drug decomposition (Breier et al.
2008; Garcia et al. 2008; Mendez et al 2008). Teng et al. (2003)
demonstrated that chemical stability of mometasone in aque-
ous systems was significantly dependent on pH, with maximum
stability under weakly acidic conditions. Another study also
demonstrated that halobetasol propionate is unstable in basic
environment (Cravotto et al. 2007). DFZ, like these corticoids
showed to be unstable under basic and acid conditions.
3. Discussion
According to ICH (2006), pharmaceutical impurities are compo-
nents found in a drug substance or drug product that are neither
the drug substance nor excipients. Degradation products are a
type of impurity (Li et al. 2008). The stress testing is the first
part of the stability evaluation and can help to identify the likely
Table: Comparative 1H and 13C NMR assignments for deflaza-
cort and DDP1
The degradation levels under hydrolysis conditions exceed the
threshold indicated by ICH (2006) and the identification of
DDP1 was necessary. The complete examination of the NMR,
IR and MS spectra of the degradation product suggest deacety-
lation of DFZ. Degradation reactions of steroids primarily occur
at the C-17 and C-21 side-chains, and this reaction is catalyzed
by a proton, hydroxide, and trace metal ions (Amin et al. 1976).
Studies have demonstrated that C-17 and/or C-21 esterified cor-
ticosteroids undergo hydrolysis in aqueous and biological media
(Alman et al. 2004). DFZ is a C-21 esterified corticosteroid
and the loss of acetyl group, resulted in 21-hydroxy-deflazacort
(Scheme B). This compound is reported as an active metabolite
of DFZ and its formation occurs when the DFZ is hydrolyzed
by seric esterases in blood (Ifa et al. 2000; Santos-Montes et al.
1994). However, at this moment, the 21-hydroxy-deflazacort
(21-OH-DFZ) had not been reported in the literature as a degra-
dation product of DFZ from a stability study.
According to Ifa et al. (2000), DFZ is an inactive prodrug which
is rapidly converted into the active metabolite 21-OH-DFZ after
oral administration, and this metabolite has anti-inflammatory
and immunosuppressive activities. To the best our knowledge,
no comparative studies of anti-inflammatory activity of DFZ and
21-OH-DFZ by in vivo and in vitro anti-inflammatory models
are reported in the literature.
a
Position
Deflazacort
DDP1
1
13
1
13
H (ppm), multiplicity
C(ppm)
H (ppm), multiplicity
C (ppm)
1
2
3
4
5
6
7
8
7.30 (d)
6.18 (d)
–
5.91 (s)
–
2.30 (m)
1.70 (m)
2.05 (m)
0.95 (m)
–
4.30 (s)
1.75 (m)
–
1,10 (m)
1.95 (m)
5.15 (m)
–
0.90 (m)
1.38 (s)
–
4.84 (dd)
–
2.10 (s)
–
156.0
127.0
185.0
122.0
169.5
31.0
33.5
30.0
55.0
43.0
68.5
41.0
47.0
50.0
34.0
84.0
94.0
18.0
21.0
200.9
66.6
170.0
20.3
165.0
14.0
7.30 (d)
6.18 (d)
–
5.91 (s)
–
2.28 (m)
1.68 (m)
2.0 (m)
0.95 (m)
–
4.25 (m)
1.75 (m)
–
1.10 (m)
1.96 (m)
5.15 (m)
–
0.89 (m)
1.37 (s)
–
4.30 (m)
–
–
157.0
127.0
185.0
122.0
170.0
31.0
33.5
30.0
55.0
44.0
68.5
41.0
47.0
50.0
34.0
84.0
94.0
18.0
21.0
207.0
65.7
–
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
From these results it is possible to conclude that DFZ is unstable
under basic conditions, leading to the formation of 21-OH-DFZ.
It is demonstrated in the literature that stability of mometa-
sone furoate also is pH dependent (Sahasranaman et al, 2004;
Teng et al. 2003). It was also shown, at least in part, that
both DFZ and 21-OH-DFZ are active in inflammatory mod-
els, whereas 21-OH-DFZ is more potent. Our results agree with
those described by Omote et al. (1994), indicating that while
DFZ and 21-desacetyl-DFZ have stronger anti-allergic effects
–
–
165.0
14.0
1.91 (s)
1.86 (s)
s singlet; d. doublet; m. multiplet; dd. double doublet
a
Refer structures (Fig. 1) for numbering
Pharmazie 67 (2012)
497

ORIGINAL ARTICLES
Scheme: Chemical structure of DFZ (DFZ) (A) and its degradation product- DDP1: 21-hydroxy deflazacort (B)
than prednisolone, they seem to have little acute effect on mast
cell degranulation or on chemical mediators at the receptor site.
through quantitative paper. The methanol was evaporated in desiccators, and
the identification of obtained crystal was carried out by NMR, IR and MS
spectroscopy.
4. Experimental
4.5. In vivo and in vitro assay
In vivo anti-inflammatory activity was evaluated by measurement of paw
edema as described by Paulino et al. (2008), following Guidelines of Insti-
tutional Review Board of UNIBAN Ethical Committee 047/2009. In vitro
assay was performed by nitric oxide determination and cell viability quan-
tification as described by Paulino et al. (2006).
4.1. Materials and reagents
DFZ was obtained from Pharma Nostra (São Paulo, Brazil). Water was
purified using a Millipore system Milli-Q Gradient. Sodium hydroxide,
hydrochloric acid, and anhydrous sodium sulfate were analytical grade
and were obtained from Vetec (São Paulo, Brazil). Dimethyl sulfoxide and
tetramethylsilane deuterated were purchased from sigma. Chromatographic
grade acetonitrile and methanol were purchased from Merck (São Paulo,
Brazil). All chemicals used in in vivo and in vitro studies were acquired
from Sigma Chemical Co. (St Louis, MO, USA).
Acknowledgments: The authors thank CAPES for the scholarship.
References
4.2. Instrumentation
Alman BA, Raza SN, Biggar WD (2004) Steroid treatment and the devel-
opment of scoliosis in males with duchenne muscular dystrophy. J Bone
Joint Surg Am 3: 519–524.
Amin M I, Koshy KT, Bryan JT (1976) Stability of aqueous solutions of
mibolerone. J Pharm Sci 65: 1777–1779.
The HPLC system consisted of a Shimadzu LC-10 ADVP pump, an SPD-
10AV VP UV-Vis Detector, an SCL-10 AVP system controller, and a
degasser module; data were acquired and processed by Shimadzu CLASS-
VP5.032software(Shimadzu, Kyoto, Japan). ThecolumnusedwasanRP18
(250 × 4.6 mm i.d., 4 ␮m). The mobile phase consisted of water:acetonitrile
(60:40, v/v) at a flow rate of 1.0 ml/min. The injection volume was 20 ␮l,
and the detection was set at a wavelength of 244 nm.
Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker
500 MHz spectrometer using dimethyl sulfoxide (DMSO-d6) as solvent and
tetramethylsilane (TMS) as internal standard. The infrared spectroscopy was
recorded in Shimadzu FT-IR instrument using 1% (w/w) pressed discs with
KBr.
A Waters HPLC system was used for the LC-MS analysis. The previously
described chromatography conditions were employed, with minor changes;
the mobile phase consisted of water with 0.1% formic acid and acetonitrile
(60:40, v/v). An Analytical Biosystems, Q-TRAP was used for the mass
spectrometric analysis. The ESI source was operated in positive ionization
modeanditsparameterswereasfollows:temperature, 300 ◦C;capillaryvolt-
age + 5500 V, DP + 50 V. Nitrogen was used as the nebulizer and collision
gas. A Micromass Waters Q-TOF spectrometer was used for the ESI-MS/MS
spectra under the following conditions: temperature 100 ◦C, 300 ◦C; capil-
lary voltage + 3000 V, cone + 40 V. Nitrogen was used as the nebulizer gas
and argon as the collision gas, collision 30 V.
Angelini C (2007) The role of corticosteroids in muscular dystrophy: a
critical appraisal. Muscle nerve 36: 424–435.
Biggar WD, Gingras M, Fehlings DL, Harris V, Steele C (2001) Deflazacort
treatment of Duchenne muscular dystrophy. J Pediatr 138: 45–50.
Biggar WD, Harris VA, Eliasoph L, Alman B (2006) Long-term benefits
of deflazacort treatment for boys with Duchenne muscular dystrophy in
their second decade. Neuromuscul Disord 16: 249–255.
Breier AR, Nudelman NS, Steppe M, Schapoval EE (2008) Isolation and
structure elucidation of photodegradation products of fexofenadine. J
Pharm Biomed Anal 46: 250–257.
Corrêa GM, Bellé LP, Bajerski L, Borgmann SHM, Cardoso SG (2007)
Development and validation of a reversed-phase HPLC method for the
determination of deflazacort in pharmaceutical dosage forms. Chro-
matographia 65: 591–594.
Cravotto G, Giovenzana GB, Masciocchi N, Palmisano G, Volante P (2007)
A degradation product of halobetasol propionate: characterization and
structure. Steroids 72: 787–791.
Cuffini SL, Ellena JF, Mascarenhas YP, Ayala AP, Sielser HW, Filho JM,
Monti, GA, Aiassa V, Sperandeo NR (2007) Physicochemical characteri-
zation of deflazacort: thermal analysis, crystallographic and spectroscopic
study. Steroids 72: 261–269.
4.3. Degradation procedure
Forbasicdegradation, 12.5 mgofDFZwastransferredtoa50 mLvolumetric
flask and dissolved with 4.0 ml of acetonitrile. The volume was made up to
volume with 0.1 N sodium hydroxide (NaOH). The flask was sealed and
placed at 37 ◦C in bath, then cooled to room temperature at different times
(0, 1, 2, 4, and 24 h). The pH of the solution was adjusted to neutrality by
adding 0.1 N hydrochloric acid (HCl). The solution was filtered, diluted to
32 ␮g/mL and evaluated by HPLC. For acid degradation, the same basic
degradation procedure was used, using 0.1 N HCl for degradation and 0.1 N
NaOH for adjusted the pH to neutrality.
Ferraris JR, Pasqualini T, Alonso G, Legal S, Sorroche P, Galich AM, Jasper
H (2007) Effects of deflazacort vs. methylprednisone: a randomized study
in kidney transplant patients. Pediatr Nephrol 22: 734–741.
Garcia CV, Nudelman NS, Steppe M., Schapoval EE (2008). Structural
elucidation of rabeprazole sodium photodegradation products. J Pharm
Biomed Anal 46: 88–93.
Gonzalez-Castan˜eda RE, Castellanos-Alvarado EA, Flores-Marquez MR,
Gonzalez-Perez O, Luquin S, Garcia-Estrada J, Ramos-Remus C (2007).
Deflazacort induced stronger immunosuppression than expected. Clin
Rheumatol 26: 935–940.
Görög S (2010) Advances in the analysis of steroid hormone drugs in phar-
maceuticals and environmental samples (2004–2010). J Pharm Biomed
Anal 55: 728–743.
ICH (2005) Validation of Analytical Procedures Text and Methodology.
Q2(R1). ICH Harmonised Tripartite Guideline 1–11.
4.4. Isolation and characterization of degradation product
DFZ (100 mg) was dissolved in 4.0 mL acetonitrile followed by addition
of 25 mL of 0.1 N NaOH. The pH was adjusted to 6.5–7.0 with 1 N HCl.
The solvent was evaporated in a Savant SPD 1010 Speed Vac Concentrator,
at ambient temperature. The residue was dissolved in methanol and filtered
498
Pharmazie 67 (2012)

ORIGINAL ARTICLES
ICH (2006) IMPURITIES IN NEW DRUG PRODUCTS Q3B(R2). ICH
Harmonised Tripartite Guideline 1–11.
Paulino N, Teixeira C, Martins R, Scremin A, Dirsch V M, Vollmar A M,
Abreu SR, de Castro SL, Marcucci MC (2006) Evaluation of the analgesic
and anti-inflammatory effects of a Brazilian green propolis. Planta Med
72: 899–906.
Sahasranaman S, Issar M, Tóth G, Horváth G, Hochhaus G (2004) Charac-
terization of degradation products of mometasone furoate. Pharmazie 59:
367–373.
Ifa DR, Moraes ME, Moraes MO, Santagada V, Caliendo G, de Nucci
G (2000) Determination of 21-hydroxydeflazacort in human plasma by
high-performance liquid chromatography/atmospheric pressure chemi-
cal ionization tandem mass spectrometry. Application to bioequivalence
study. J Mass Spectrom 35:440–445.
Joshi N, Rajeshwari K (2009) Deflazacort. J Postgrad Med 55: 296–300.
Li M, Lin M, Rustum A (2008) Application of LC-MS(n) in conjunction
with mechanism-based stress studies in the elucidation of drug impurity
structure: rapid identification of a process impurity in betamethasone 17-
valerate drug substance. J Pharm Biomed Anal 48: 1451–1456.
Mendez A, Chagastelles P, Palma E, Nardi N, Schapoval EE. (2008) Thermal
and alkaline stability of meropenem: degradation products and cytotoxi-
city. Int J Pharm, 350: 95–102.
Omote M, Sakai K, Mizusawa H (1994) Acute effects of deflazacort and
its metabolite 21-desacetyl-deflazacort on allergic reactions. Arzneimit-
telforschung 44: 149–153.
Paulino N, Abreu SRL, Uto Y, Koyama D, Nagasawa H, Hori H, Dirsch VM,
Scremin A, Bretz WA (2008) Anti-inflammatory effects of a bioavail-
able compound, Artepillin C, in Brazilian propolis. Eur J Pharmacol 587:
296–301.
Santos-Montes A, Gonzalo-Lumbreras R, Gasco-Lopez AI, Izquierdo-
Hornillos R (1994) Extraction and high-performance liquid chro-
matographic separation of deflazacort and its metabolite 21-
hydroxydeflazacort. Application to urine samples. J Chromatogr B
Biomed Appl 657: 248–253.
Scremin A, Piazzon M, Silva AMS, Kuminek G, Correa GM, Paulino
N, Cardoso SG (2010). Spectrophotometric and HPLC determination
of deflazacort in pharmaceutical dosage forms. J Pharm Sci 46: 281–
287.
Sousa NG, Faria E, Carrapatoso I, Almeida E, Geraldes L, Chieira C (2010)
Deflazacort: a possible alternative in corticosteroid allergy. J Investig
Allergol Clin Immunol. 20: 449–451.
Teng XW, Cutler DC, Davies NM (2003) Degradation kinetics of mometa-
sone furoate in aqueous systems. Int J Pharm, 259: 129–141.
Pharmazie 67 (2012)
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