ORIGINAL PAPER Identification of budesonide metabolites in human urine after oral administration Xavier Matabosch & Oscar J. Pozo & Clara Pérez-Mañá & Magi Farré & Josep Marcos & Jordi Segura & Rosa Ventura Received: 16 February 2012 / Revised: 29 March 2012 / Accepted: 9 April 2012 / Published online: 10 May 2012 # Springer-Verlag 2012 Abstract Budesonide (BUD) is a glucocorticoid widely used for the treatment of asthma, rhinitis, and inflammatory bowel disease. Its use in sport competitions is prohibited when administered by oral, intravenous, intramuscular, or rectal routes. However, topical preparations are not pro- hibited. Strategies to discriminate between legal and forbid- den administrations have to be developed by doping control laboratories. For this reason, metabolism of BUD has been re-evaluated using liquid chromatography–tandem mass spectrometry (LC-MS/MS) with different scan methods. Urine samples obtained after oral administration of 3 mg of BUD to two healthy volunteers have been analyzed for metabolite detection in free and glucuronide metabolic frac- tions. Structures of the metabolites have been studied by LC-MS/MS using collision induced dissociation and gas chromatography–mass spectrometry (GC/MS) in full scan mode with electron ionization. Combination of all structural information allowed the proposition of the most comprehen- sive picture for BUD metabolism in humans to this date. Overall, 16 metabolites including ten previously unreported compounds have been detected. The main metabolite is 16α-hydroxy-prednisolone resulting from the cleavage of the acetal group. Other metabolites without the acetal group have been identified such as those resulting from reduction of C20 carbonyl group, oxidation of the C11 hydroxyl group and reduction of the A ring. Metabolites maintaining the acetal group have also been identified, resulting from 6-hydroxylation (6 α and 6 β -hydroxy-budesonide), 23-hydroxylation, reduction of C6-C7, oxidation of the C11 hydroxyl group, and reduction of the C20 carbonyl group. Metabolites were mainly excreted in the free fraction. All of them were excreted in urine during the first 24 h after administration, and seven of them were still detected up to 48 h after administration for both volunteers. Keywords Budesonide . Metabolism . Mass spectrometry . Doping analysis . Urine Introduction Budesonide (22(R,S)-16α,17α-butylidenedioxy-11β,21- dihydroxypregna-1,4-diene-3,20-dione, BUD, Fig. 1), a non-halogenated glucocorticoid, has been designed to ex- press high local effect with reduced systemic side effects. This was achieved by incorporating features into the drug Published in the special paper collection Progress on Environmental and Bioanalysis in Spain with guest editors Alfredo Sanz-Medel and Elena Domínguez. X. Matabosch : O. J. Pozo : J. Marcos : J. Segura : R. Ventura Bioanalysis Research Group, IMIM, Institut de Recerca Hospital del Mar, Doctor Aiguader 88, 08003 Barcelona, Spain C. Pérez-Mañá : M. Farré Human Pharmacology and Neurosciences Research Group, IMIM and UCIEC-IMIM-CAIBER, Doctor Aiguader 88, 08003 Barcelona, Spain C. Pérez-Mañá : M. Farré Department of Pharmacology, Therapeutics and Toxicology, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), 08193 Barcelona, Spain J. Marcos : J. Segura : R. Ventura (*) Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Doctor Aiguader 88, 08003 Barcelona, Spain e-mail: [email protected]Anal Bioanal Chem (2012) 404:325–340 DOI 10.1007/s00216-012-6037-0
Transcript
ORIGINAL PAPER
Identification of budesonide metabolites in human urineafter oral administration
Xavier Matabosch & Oscar J. Pozo & Clara Pérez-Mañá &
Magi Farré & Josep Marcos & Jordi Segura &
Rosa Ventura
Received: 16 February 2012 /Revised: 29 March 2012 /Accepted: 9 April 2012 /Published online: 10 May 2012# Springer-Verlag 2012
Abstract Budesonide (BUD) is a glucocorticoid widelyused for the treatment of asthma, rhinitis, and inflammatorybowel disease. Its use in sport competitions is prohibitedwhen administered by oral, intravenous, intramuscular, orrectal routes. However, topical preparations are not pro-hibited. Strategies to discriminate between legal and forbid-den administrations have to be developed by doping controllaboratories. For this reason, metabolism of BUD has beenre-evaluated using liquid chromatography–tandem massspectrometry (LC-MS/MS) with different scan methods.Urine samples obtained after oral administration of 3 mg
of BUD to two healthy volunteers have been analyzed formetabolite detection in free and glucuronide metabolic frac-tions. Structures of the metabolites have been studied byLC-MS/MS using collision induced dissociation and gaschromatography–mass spectrometry (GC/MS) in full scanmode with electron ionization. Combination of all structuralinformation allowed the proposition of the most comprehen-sive picture for BUD metabolism in humans to this date.Overall, 16 metabolites including ten previously unreportedcompounds have been detected. The main metabolite is16α-hydroxy-prednisolone resulting from the cleavage ofthe acetal group. Other metabolites without the acetal grouphave been identified such as those resulting from reductionof C20 carbonyl group, oxidation of the C11 hydroxyl groupand reduction of the A ring. Metabolites maintaining theacetal group have also been identified, resulting from6-hydroxylation (6α and 6β-hydroxy-budesonide),23-hydroxylation, reduction of C6-C7, oxidation of theC11 hydroxyl group, and reduction of the C20 carbonylgroup. Metabolites were mainly excreted in the free fraction.All of them were excreted in urine during the first 24 h afteradministration, and seven of them were still detected up to48 h after administration for both volunteers.
Budesonide (22(R,S)-16α,17α-butylidenedioxy-11β,21-dihydroxypregna-1,4-diene-3,20-dione, BUD, Fig. 1), anon-halogenated glucocorticoid, has been designed to ex-press high local effect with reduced systemic side effects.This was achieved by incorporating features into the drug
Published in the special paper collection Progress on Environmentaland Bioanalysis in Spain with guest editors Alfredo Sanz-Medel andElena Domínguez.
X. Matabosch :O. J. Pozo : J. Marcos : J. Segura : R. VenturaBioanalysis Research Group, IMIM,Institut de Recerca Hospital del Mar,Doctor Aiguader 88,08003 Barcelona, Spain
C. Pérez-Mañá :M. FarréHuman Pharmacology and Neurosciences Research Group,IMIM and UCIEC-IMIM-CAIBER,Doctor Aiguader 88,08003 Barcelona, Spain
C. Pérez-Mañá :M. FarréDepartment of Pharmacology, Therapeutics and Toxicology,Universitat Autònoma de Barcelona,Bellaterra (Cerdanyola del Vallès),08193 Barcelona, Spain
J. Marcos : J. Segura :R. Ventura (*)Department of Experimental and Health Sciences,Universitat Pompeu Fabra,Doctor Aiguader 88,08003 Barcelona, Spaine-mail: [email protected]
molecule that allow a pronounced hepatic metabolismresulting in low oral bioavailability and high hepatic clear-ance [1]. It is commonly used by inhalation in the treatmentof asthma and also intranasally in allergic rhinitis [2] or byoral route for the inflammatory bowel disease [3]. Its use insport practice is prohibited with some exceptions by theWorld Anti-Doping Agency (WADA). All glucocorticoste-roids are prohibited in-competition when administered byoral, intravenous, intramuscular, or rectal routes [4], but itsuse by other routes (e.g., inhalation) is allowed. In previousyears, BUD was the glucocorticosteroid with most numberof adverse analytical findings reported by laboratoriesaccredited by WADA, representing almost 50 % of reportedcases within this class of compounds [5].
BUD is a C22 1:1 epimeric mixture, epimer 22R having2–3 times greater topical glucocorticoid potency than epi-mer (22S) [6]. Two major metabolites of BUD have beenidentified in man, 6β-hydroxy-budesonide, formed fromboth epimers, and 16α-hydroxy-prednisolone, formed onlyfrom (22R)-budesonide [7, 8]. Other metabolites had alsobeen identified in man, 5β-dihydrobudesonide, Δ6-budeso-nide, and 23-hydroxy-budesonide [9]. However, in dopinganalysis usually only the parent compound and 16α-hydroxy-prednisolone are monitored [10–13].
Former studies in glucocorticosteroids metabolism wereperformed using gas chromatography coupled to mass spec-trometry (GC/MS) [14–17]. In the last years, the use of
liquid chromatography coupled to tandem mass spectrome-try (LC-MS/MS) has demonstrated to be useful for theidentification of new metabolites of steroids, including glu-cocorticosteroids [18–21]. The use of this technology isespecially important for metabolic studies of glucocorticos-teroids due to the difficulties in obtaining adequate deriva-tives of polyoxygenated metabolites to make them amenableto GC/MS analysis [22]. Recent studies on methylprednis-olone and triamcinolone show that LC-MS/MS enables theidentification of unreported metabolites [18, 21].
The use of BUD in sports is prohibited when adminis-tered systemically. As it can be used by other routes (e.g.,inhalation), there is a need to develop strategies to distin-guish between different routes of administration through theanalysis of urine samples. Differences in the excretion ofmetabolites in urine depending on the route of administra-tion are used to discriminate between oral and inhaledadministrations for salbutamol [23–26]. Similarly, a betterknowledge on the metabolism of BUD may be useful in thefuture to investigate differences depending on the route ofadministration that can help antidoping laboratories to dis-criminate between its legal and forbidden use.
The goal of the present work was to perform a systematicstudy on BUD metabolism after oral administration of thedrug using LC-MS/MS technology. Open scan methods todetect metabolites sharing a common structure have beenapplied to urines samples obtained after administration of
2220
16
1514
1312
Budesonide
O
OH
OOH
OO
O
OH
OOH
OHOH
M-I
O
O
OOH
OHOH
M-IXa & M-IXb
O
OH
OOH
OO
OH
O
OH
OOH
OO
OH
M-IVa & M-IVb M-Va & M-Vb
M-VI
O
O
OOH
OO
M-VII
O
OH
OOH
OO
OH
M-III
O
OH
OOH
OO
O
OH
OOH
OO
OH
O
OH
OOH
OO
OH
M-XI
M-X
O
OH
OHOH
OHOH
O
OH
OOH
OO
OH
M-II
O
OH
OHOH
OO
M-VIII
O
O
OOH
OHOH
O
OH
OOH
OHOH
M-XII
M-XIII
1
2
3
45
6
7
89
10
19 11 17
18
21
23
24
25O
OH
OHOH
OHOH
i
ii
ii
ii
iiiii
iii
iv
iv
vv
vii
vi
vi
OH
O
OOH
OHOH
Fig. 1 Temptative structures of BUD metabolites detected after oral administration. Metabolic pathways: (i) acetal cleavage, (ii) hydroxylation, (iii)6,7-dehydrogenation, (iv) 11-oxydation, (v) 20-reduction, (vi) 4,5 hydrogenation, (vii) 3-reduction
326 X. Matabosch et al.
BUD to healthy volunteers. Characterization of the structureof the detected metabolites has been performed using LC-MS/MS and GC/MS analysis.
Experimental
Chemicals and reagents
Budesonide, ammonium formate, sodium borohydride, andmethoxyamine hydrochloride were obtained from Sigma(St. Louis, MO, US). 16α-hydroxy-prednisolone, 6α-hydroxy-budesonide, 6β-hydroxy-budesonide, budesonide-d8, and 16α-hydroxy-prednisolone-d5 were purchased fromToronto Research Chemicals (Toronto, Canada). Theβ-glucuronidase preparation (type Escherichia coli K12,140 U/mL) was purchased from Roche Diagnostics GmbH(Mannheim, Germany). Analytical grade di-sodium hydro-gen phosphate, sodium hydrogen phosphate, ethyl acetate,ammonium chloride, ammonia (25 % solution), cyclohex-ane, acetonitrile and methanol (LC gradient grade), formicacid (LC-MS grade) and pyridine were obtained from Merck(Darmstadt, Germany). Trimethylsilylimidazole (TMSI)was from Macherey-Nagel (Düren, Germany).
Ultrapurified water was obtained using a Milli-Q purifi-cation system (Millipore Ibérica, Barcelona, Spain).
LC-MS/MS instrumentation
LC-MS/MS analysis were carried out using a triple quadru-pole (Quattro Premier XE) mass spectrometer provided withan orthogonal Z-spray-electrospray interface (Waters Asso-ciates, Milford, MA, USA) interfaced to an ultraperform-ance liquid chromatographic (UPLC) system, Acquity(Waters Associates) for the chromatographic separation.Drying gas, nebulizing gas, cone gas as well as desolvationgas was nitrogen. The desolvation gas flow was set toapproximately 1,200 L/h and the cone gas flow to 50 L/h.The nitrogen desolvation temperature was set to 450 °C andthe source temperature to 120 °C. Different scan methodswere applied using electrospray ionization (ESI) in positiveor negative modes. A capillary voltage of 3.5 kV was usedin positive ionization mode. For precursor ion scan methods(PI), a cone voltage of 20 V and different collision energieswere used (PI of m/z 77 and 91, 60 eV; PI of m/z 105, 50 eV;PI of m/z 121 and 171, 40 eV; PI of m/z 147 and 237, 30 eV)and a window from m/z 300 to 600 was monitored. In thecollision-induced dissociation (CID) methods, two differentcollisions energies were used (20 and 30 eV) and the conevoltage was set to 20 V for metabolites with acetal group,and to 15 V for metabolites without acetal function. Acapillary voltage of 3.0 kV was used in negative ionizationmode. Two neutral loss scan methods (NL) were applied in
negative ion mode. In the NL of m/z 76, the collision energywas set to 10 eV and a window from m/z 300 to 500 wasmonitored, while in the NL of m/z 118, the collision energywas set to 20 eV and ions from m/z 350 to 600 weremeasured.
The LC separation was performed using an Acquity BEHC18 column (100×2.1 mm i.d., 1.7 μm particle size; WatersAssociates), at a flow-rate of 300 μL/min. Ammoniumformate 1 mM pH 3 (formic acid; solvent A) and acetonitrilewith formic acid (0.01 %; solvent B) were selected asmobile phase solvents. A first gradient program was usedfor detection of the metabolites using PI and NL methods;the percentage of organic solvent was linearly changed asfollows: 0 min, 17 % B; 0.5 min, 17 % B; 11 min, 62 % B;11.7 min, 90 % B; 11.8 min, 17 % B; 11.8 min, 17 % B.After detection of some possible metabolites, another pro-gram was used to improve chromatographic resolution; thepercentage of organic solvent was linearly changed as fol-lows: 0 min, 22 % B; 0.5 min, 22 % B; 2.8 min, 24 % B;3 min, 26 % B; 5.5 min, 29 % B; 6 min, 37 % B; 11.5 min,45 % B; 13 min, 90 %B; 13.1 min, 22 % B, 14.5 min22 %B.
GC/MS instrumentation
GC/MS analyses were carried out on a 7890N gas chro-matograph coupled with a 5975 C mass spectrometer and a7693 autosampler (Agilent Technologies, Palo Alto, CA,USA). The steroids were separated on a HP-Ultra1 cross-linked methyl-silicone column, 16.5 m×0.2 mm i.d., filmthickness 0.11 μm (J&W Scientific, Folsom, CA, USA).Helium was used as the carrier gas at a constant pressureof 5 psi. The system was operated in splitless mode (valveopened at 2 min). The GC temperature was ramped asfollows: initial 50 °C, held for 3 min, increased to 230 °Cat 30 °C min−1, thereafter increased to 285 °C at 2 °C min−1.The injector and transfer line are kept at 280 °C. Whenscanning, the mass range scanned was from 100 to 850 Da.
Sample preparation
Analysis of the free fraction
Internal standards (ISTD) solutions (20 ng of budesonide-d8and 40 ng of 16α-hydroxy-prednisolone-d5) were added to2 ml of urine, followed by the addition of 200 μl ammoniumchloride/ammonia 5 M buffer at pH 9.5. Liquid–liquid ex-traction was performed by shaking for 20 minutes with6 mL of ethyl acetate. After centrifugation (5 min at1,400×g) the organic layer was transferred into a new tubeand evaporated to dryness under nitrogen stream in a waterbath at 50 °C.
Budesonide metabolites in human urine after oral administration 327
For LC-MS/MS analysis, the residue was reconstitutedinto 100 μl of a mixture of water/acetonitrile (50:50, v/v)and 10 μl were directly injected into the system.
For GC/MS analysis, methoxylation of the ketone groupsand trimethylsilylation of the hydroxyl groups was per-formed to obtain the methyloxime–trimethylslyl derivatives(MO-TMS). The residue was dissolved in 100 μl of a 2 %(w/v) methoxyamine hydrochloride solution in pyridine, andheated for 60 min at 60 °C. The pyridine was blown off and50 μl of TMSI were added. The silylation proceeded for16 h at 80 °C. The involatile reagents were removed prior toGC/MS analysis by a cyclohexane–water extraction. Theorganic layer was transferred to injection vial. A 2-μl aliquotof the final derivatized extract was injected into the GC/MSsystem.
Analysis of the combined fraction
ISTD solutions were added to 2 ml of urine, followed by theaddition of 500 μl phosphate buffer 1 M pH 7. Then,β-glucuronidase from E. coli was added (30 μl) and hydro-lysis was carried out for 1 h at 55 °C. The buffered solutionwas alkalinized with 150 μl of 25 % potassium carbonatesolution to pH 8–9 and the steroids were extracted with6 mL of ethyl acetate. After centrifugation, the procedurewas analogous to the described for the free fraction analysis.
LC fractionation
In order to isolate the metabolites, urine samples (30 ml)were extracted as described above for the combined frac-tion. The extract was reconstituted in 250 μl of a mixture ofwater/acetonitrile (50:50, v/v). The UPLC system abovedescribed was used. In this case, the mobile phase flow-rate was 500 μL/min and the following gradient programwas used; 0 min, 16 % B; 0.5 min, 16 % B; 6 min, 17 % B;6.5 min, 18.5 % B; 15 min, 20 % B; 15.5 min, 37 % B;20 min, 39 % B; 22 min, 90 %B; 22.1 min, 16 % B, 24 min16%B. Four consecutive injections of 10 μL of the urineextracts were performed for the fractionation of each me-tabolite. All the fractions containing the same metabolitewere combined.
The LC fractions were treated as follows, 4 mL of phos-phate buffer 0.2 M pH 7 and 250 μL potassium carbonate5 % were added to each tube, and metabolites wereextracted with 6 mL of ethyl acetate. Solvent was removedunder nitrogen stream and samples were processed as pre-viously described for GC/MS analysis.
Chemical reduction
Chemical reduction of the C20 carbonyl group of budeso-nide and 16α-hydroxy-prednisolone was performed in order
to compare authentic materials with the putative 20-hydroxy-metabolites detected in post-administration urines.A method previously described was applied [27]. Reductionwas performed by adding 100 mg of NaBH4 to a solution ofbudesonide or 16α-hydroxy-prednisolone (60 ng) in 6 ml ofmethanol and 1 mL of water. After stirring the mixture atroom temperature for 5 h, a few drops of acetic acid wereadded. Then, the mixture was diluted with ethyl acetate(5 mL), washed with a saturated NaHCO3 solution andwater; washing was performed by adding 3 mL of solution,vortexing for 3 min, centrifuging 5 min at 1,400×g anddiscarding the aqueous layer. The organic layer was evapo-rated to dryness, and the mixture of products obtainedanalyzed by LC-MS/MS.
Administration study samples
A single oral dose of 3 mg of racemic budesonide (sustainedrelease capsules: Entocord/Entocort EC, AstraZeneca, Lon-don, UK) was administered to two healthy male volunteers(both 21 years old with weights of 77.5 kg (Vol1) and 55 kg(Vol2)). Urine samples were collected from 0–8 h, 8–24 h,24–36 h, and 36–48 h, 48–72 h, 72–96 h, 96–120 h and120–144 h and stored at −20 °C until analysis.
Ethical approval for the study had been granted by theEthical Committee of our Institute (Comité Ètic d’Investi-gació Clínica CEIC-Parc de Salut Mar, Barcelona, Spain)and the Spanish Medicines Agency (EudraCT protocolnumber 2010-021237-31). Both subjects participating inthe study gave their written informed consent prior inclusionand underwent a general physical examination, routine lab-oratory test, urinalysis, and a 12-lead electrocardiogram.
Results and discussion
Detection of budesonide metabolites by LC-MS/MS
Identification of BUD metabolites was accomplished byanalysis by LC-MS/MS of urine samples obtained afteradministration of the drug. Several methods based on bothPI and NL scans in positive and negative modes wereapplied. These methods allow for the detection of unknownmetabolites of corticosteroids that share common structuralfeatures [18]. After combining the information of all PI andNL methods, and contrasting the results between post- andpre-administration samples, sixteen candidates weredetected (Tables 1 and 2). These compounds were consid-ered as potential BUD metabolites.
In positive ionization mode, compounds are ionized atthe conjugated 3-keto function forming the [M + H]+ ion.Therefore, only metabolites containing this function areionized [18]. In negative ionization mode, the compounds
328 X. Matabosch et al.
are ionized forming the adduct ion [M + HCOO]− formed byinteraction between the formate and the carbonyl and hy-droxyl groups in C20 and C21, respectively. Only metabo-lites containing a keto group in C20 and two oxygen atomsin C17 and C21 are ionized in negative mode [18]. Com-bining the information obtained in the different ionizationmodes, the molecular mass (MM) of each metabolite wascalculated (Tables 1 and 2).
Characterization of BUD and three of the metabolites(16α-hydroxy-prednisolone M-I, 6β-hydroxy-budesonideM-II, and 6α-hydroxy-budesonide M-III) was performedby comparison with commercially available standards. Forthe rest of metabolites, structures were proposed based onLC-MS/MS analysis using product ion scan methods and,for some of them, GC/MS analysis after LC isolation of themetabolite and MO-TMS derivatization was also used. Theions produced for each metabolite were compared withthose obtained for the parent drug and the commerciallyavailable metabolites. Characteristic ions of the CID massspectra of the metabolites are listed in Tables 1 and 2, andthe proposed structures are depicted in Fig. 1.
Structural elucidation of metabolites with acetal moiety
Besides the parent compound BUD, nine metabolites main-taining the acetal moiety were identified in urine samples(metabolites M-II to M-VIII). Metabolites M-III, M-Va,M-Vb, M-VII and M-VIII were not described before. CIDspectra of [M + H]+ and [M + HCOO]− of BUD andmetabolites M-II to M-VII are depicted in Fig. 2, andCID spectra of [M + H]+ of metabolite MV-III is shownin Fig. 3.
In LC-MS/MS analysis, two chromatographic peaks wereobtained for BUD due to the presence of two enantiomers, 22Rand 22S (Table 1). After analysis of the post-administrationurine samples, the two peaks were also observed with differentabundances: the peak at 9.7 min was around 10 times higherthan the peak at 9.4 min. According to bibliography, theformation of the main BUD metabolite, 16α-hydroxy-prednisolone (M-I) is stereoselective for the 22R epimer [8].Assuming equal intensity for both isomers, and absence ofstereoselective reactions for 22S epimer, the most abundantpeak in post-administration samples, eluting at 9.7 min wasproposed to be the 22S epimer. This is in agreement withprevious studies where the epimers were separated in similarchromatographic conditions (column and mobile phase), andthe 22R epimer eluted first than the 22S epimer [28].
Due to presence of the two epimers of BUD and thepossibility of additional chiral centers in the side chain, thereis also the possibility of different isomers for the metaboliteswith acetal chain. During the description of these metabo-lites, the epimers have been named using the same Romannumeric code, but adding a Latin letter (a, b) to distinguishT
able
1Metabolitesof
budesonide
(BUD)with
acetal
grou
pidentifiedafteroral
administration
Com
pound
MM
RT
(min)
ESIpo
sitiv
emod
eESInegativ
emod
e
[M+
H]+
CID
massspectrum
of[M
+H]+
[M+HCOO]−
CID
massspectrum
of[M
+HCOO]−
[M+H-nH2O]+
1,4-dienea
[M+H-H
2O-
CH3CH2CHXCHO]+
b[M
+H-CO-
nH2O]+
Other
[M+HCOO-H
COOH-
CH3CH2CHXCHO]−
b
Bud
(22R
/22S
)43
09.4/9.7
431
413,
395,
377
121,
147,
171
341(N
L90)
–173
475
357(N
L118)
M-II(22R
/22S
)44
66.0/6.2
447
429,
411,
393,
375
121,
147,
171
357(N
L90)
–173
491
373(N
L118)
M-III(22R
/22S)
446
4.4/4.7
447
429,
411,
393,
375
121,
147,
171
357(N
L90)
–173
491
373(N
L118)
M-IV
(a/b)
446
5.1/5.2
447
429,
411,
393,
375
121,
147,
171
341(N
L106)
–173
491
357(N
L134)
M-V
(a/b)
444
4.9/5.0
445
427,409,
391,
373
–339(N
L106)
–171
489
355(N
L134)
M-V
I42
89.6
429
411,
393,
375
–321(N
L90)
–171
473
355(N
L118)
M-V
II42
89.2
429
411,
393,
375
121,
147,
171
339(N
L90)
401,38
3,311
173
473
355(N
L118)
M-V
III
432
7.3
433
415,
397,
379
121,
147,
171
325(N
L90)
–173
––
Molecular
mass(M
M),retentiontim
es(RT)ob
tained
byLC-M
S/M
S,p
rotonatedmolecules
[M+H]+
andions
oftheCID
massspectrum
inpo
sitiv
eESI,andform
ateaddu
ctions
[M+HCOO]−
andions
oftheCID
massspectrum
innegativ
eESImod
eaIons
indicatin
gintact
1,4-dienestructure
bX
0H
orOH,themassof
theneutralloss
(NL)isindicatedin
brackets
Budesonide metabolites in human urine after oral administration 329
between them. As the 22R epimer of BUD is mainly me-tabolized to M-I, we suggest that for the metabolites withacetal moiety the epimer giving higher signals in urinesamples will be the 22S. In some cases, only one peak isdetected for these metabolites. This fact might be caused bythe low concentration of the second epimer, falling under thelimit of detection of the method or due to a coelution of bothepimers.
The presence of the two epimers was observed for metab-olites M-II and M-III, in both the reference materials andthe urine extracts (Table 1). As expected, the abundanceratio of the two epimers was different between the standardand the post-administration samples. The most abundantpeak in urine extracts was assigned to the 22S isomer(Table 1).
Characteristic ions of the CID spectra of the metaboliteswith acetal chain are listed in Table 1. In positive ionization,ions resulting from the loss of water molecules were alwayspresent in the spectrum at 20 eV, collision energy. Thenumber of losses of water is related with the number ofhydroxyl groups and the unconjugated keto groups [29].Neutral losses related the acetonide chain are also observed:a neutral loss of 90 Da can be explained by the loss of theacetal chain and, therefore, in metabolites with an hydroxylgroup located in the acetal chain, an equivalent loss of106 Da is observed.
At 30 eV collision energy, the most abundant ions werefound at m/z 121, 147, and 171 characteristic of 1,4-dienecorticosteroids [18]. Any change in this pattern, or the lackof some of these ions means a change in either A or B rings.Particularly a major m/z 171 ion is seen is 6-ene-metabolites[21, 30, 31]. This ion may result from the fragmentation ofthe C ring with loss of a water molecule, equivalent to ionm/z 173 for BUD. A loss of 28 Da observed for somemetabolites is related to the presence of a keto function inC11 [18].
CID mass spectrum of [M + HCOO]− shows neutrallosses related with the acetonide chain (Table 1). A neutralloss of 118 Da is the result of the loss of formic acid andbutanal coming from the acetal and when an hydroxyl groupis located at the acetonide chain, an equivalent loss of134 Da is observed.
M-IV
In LC-MS/MS analysis, two peaks with the same CIDspectra were found at 5.1, 5.2 min and a small peak at5.4 min. The MM of this metabolite is 446 Da, 16 Da morethan BUD, suggesting a hydroxylation. Taking into accountthe MS data (Table 1) the additional hydroxyl group islocated at the side chain. Edsbäcker et al. described a me-tabolite of BUD hydroxylated in the acetal side chain [9].Using NMR studies, they suggested that the hydroxyl groupT
able
2Metabolitesof
budesonide
(BUD)with
outaceton
idegrou
pidentifiedafteroral
administration
Com
poun
dMM
RT
(min)
ESIpo
sitiv
emod
eESInegativ
emod
e
[M+
H]+
CID
massspectrum
of[M
+H]+
[M+HCOO]−
CID
massspectrum
of[M
+HCOO]−
[M+H-nH2O]+
1,4-dienea
[M+H-CO-
nH2O]+
Other
[M+HCOO-H
COOH-
H2CO]−
[M+HCOO-H
COOH-H
2CO-
H2O]−
M-I
376
2.7
377
359,
341,
323,
305
121,
147,
171
–17
342
134
532
7
M-IX
(a+b)
378
2.1
379
361,
343,
325,
307,
289
121,
147,
171
–17
3–
––
M-X
374
3.0
375
357,
339,
321,
303
121,
147,
171
329,
311,
293,
275
173
419
343
325
M-X
I37
84.4
379
361,
343,
325,
307,
289
––
121
423
347
329
M-X
II37
64.7
377
359,
341,
323,
305,
287
–33
1,29
512
142
134
532
7
M-X
III
378
4.8
––
––
–42
334
732
9
Molecularmass(M
M),retentiontim
es(RT)ob
tained
byLC-M
S/M
S,protonatedmolecules
[M+H]+
andions
ofits
CID
massspectrum
inpo
sitiv
eESI,andform
ateaddu
ctions
[M+HCOO]−
and
ions
ofits
CID
massspectrum
innegativ
eESImod
eaIons
indicatin
gintact
1,4-dienestructure
330 X. Matabosch et al.
was located in C23. The presence of a hydroxyl group inC23 creates a new chiral center and, therefore, four stereo-isomers are expected. Due to stereoselective transformationof BUD to M-I, it is suggested that the two major peaks (at5.1 and 5.2 min) correspond to the R and S isomers of C23,with 22S configuration. The smaller peak at 5.4 min maycorrespond to one of the C23 isomers with a 22R configu-ration. Based on these data, the structure 23(R/S)-hydroxy-budesonide is suggested for metabolites M-IV.
M-V
Two peaks with same intensity and the same CID spectrawere detected at 4.9 and 5.0 min after LC-MS/MS analysis.The MM of metabolites M-V is 444 Da, suggesting ahydroxylation and an oxidation with respect to BUD. MSdata (Table 1) suggest that the extra hydroxyl group is alsolocated in the side chain, and the extra double bond islocated in position 6 (see Fig. 1). 6-ene-metabolites have
100 150 200 250 300 350 400 4500
147
121
173
171
161
413
323277225
187
263
237251
305
295
307 341
m/z
Rel
ativ
e A
bund
ance
(%)
100
431395377
475
339
295187
357
411391
100 150 200 250 300 400 450 500350
Rel
ativ
e A
bund
ance
(%)
100
Rel
ativ
e A
bund
ance
(%)
100 357
339
236 263 321
491
447408 465
m/z100 150 200 250 300 400 450 500350
147
121135
173
171
323265
263
225
185211
237249
305
277
295
287
429
341367
359 411447
m/z100 150 200 250 300 350 400 450
Rel
ativ
e A
bund
ance
(%)
100
393375
m/z100 150 200 250 300 350 400 450
Rel
ativ
e A
bund
ance
(%
)
100 265263
171121
147
135161
237
211173
223
321
275
303
291
309
339
411
357373 399
447429393
100 150 200 250 300 350 400 450 500R
elat
ive
Abu
ndan
ce(%
)
100373
355
337203 327
491
445
m/z
m/z100 150 200 250 300 350 400 450
265263
261
237171
147121
135
159223
211173
249
321
275293
303
339
323
411357
345 447393373
381 399 429
Rel
ativ
e A
bund
ance
(%
)
100
m/z100 150 200 250 300 350 400 450 500
Rel
ativ
e A
bund
ance
(%)
100
A
C
D
373
355
311203 327337
491
B
Fig. 2 From A toG, CID mass spectra of compounds BUD,M-II, M-III, M-IVb, M-Vb, M-VI and M-VII, respectively. Left, CID mass spectrum of[M + H]+ obtained in ESI positive (20 eV); right, CID mass spectrum of [M + HCOO]− in ESI negative (20 eV)
Budesonide metabolites in human urine after oral administration 331
been also described for anabolic steroids and other cortico-steroids [21, 30, 31]. Due to the presence of two chiral centers,four isomers ofM-V were expected. Analogously to metabo-lite M-IV, it is suggested that the two detected peaks corre-spond to the R and S isomers of C23, with a 22S configuration.A small peak was also observed at 5.3 min that may corre-spond to one of the C23 isomers (23R or 23S) with a 22Rconfiguration. 22(S),23(R/S)-16α,17α-butylidenedioxy-11β,21,23-trihydroxypregna-1,4,6-triene-3,20-dione is thesuggested structure for metabolites M-Va and MVb.
M-VI
MetaboliteM-VI exhibits a MM of 428 Da, 2 Da lower thanthat of BUD. Therefore, an additional double bond isexpected. The MS data (Table 1) suggest that the extradouble bond is located in position 6 (see Fig. 1). 16α,17
α-butylidenedioxy-11β,21-dihydroxypregna-1,4,6-triene-3,20-dione (Δ6-budesonide) is suggested as plausible struc-ture for M-VI. This compound was described as BUDmetabolite by previous authors [9].
M-VII
Metabolite M-VII exhibits a MM of 428 Da, 2 Da less thanBUD, suggesting an oxidation. Several ions related to lossesof CO, observed in CID spectrum in positive mode (Table 1),suggest the presence of a keto function in C11 [18]. CIDspectrum at high collision energy, with ions m/z 121, 147,171 and 173 but with different profile compared to BUD(Fig. 2), also suggest a modification in the A, B, or C rings.According on these data, 11-oxo-budesonide (16α,17α-butylidenedioxy-21-hydroxypregna-1,4-diene-3,11,20-trione) is suggested as structure for M-VII.
F
m/z100 150 200 250 300 350 400 450
171
211
173
237
225
279263 303
293321
429411375
Rel
ativ
e A
bund
ance
(%)
100
393
187147
171159
161
173
311
213
293
275265241225
235
253
283
303
339
321
329
429
355357383
373 411393
m/z100 150 200 250 300 350 400 450
Rel
ativ
e A
bund
ance
(%)
100
171
145127
121
263211 237225 251
249
303279275
291
321
311
445427339
331
m/z100 150 200 250 300 350 400 450
Rel
ativ
e A
bund
ance
(%)
100 E 413355
337
130 327265 295
489
451 471
100 150 200 250 300 400 450 500350
Rel
ativ
e A
bund
ance
(%)
100
m/z
473
435337185
319215355 391 417
100 150 200 250 300 400 450 500350R
elat
ive
Abu
ndan
ce(%
)
100
m/z
185
413
355337309
191
273
389
473
437427
100 150 200 250 300 400 450 500350m/z
Rel
ativ
e A
bund
ance
(%)
100G
409391373
401
Fig. 2 (continued)
332 X. Matabosch et al.
M-VIII
Metabolite M-VIII exhibits a MM of 432 Da, 2 Da morethan the parent drug, suggesting a reduction. The metabolitewas not detected in negative ionization (Table 1), and forthis reason, the reduction of the ketone in C20 is the mostfeasible metabolic pathway to generate M-VIII. The pres-ence of a hydroxyl group in C20 creates a new chiral center,so four isomers are expected. In order to synthesize metab-olite M-VIII, BUD standard was reduced with NaBH4. Anisocratic method (with 23 % of organic solvent and a flow-rate of 500 μL/min) was applied to separate the four iso-mers. Peaks at 9.5, 9.8, 10.6, and 12.0 min were obtainedwith similar CID mass spectra (Fig. 3b). However, theabundances of the chromatographic peaks were different,being peaks at 9.5 and 9.8 min less abundant than the othertwo. It is known that the C20 carbonyl group present in sidechains of the pregnane and 21-hydroxypregnanes series isreduced preferentially to the 20β configuration [27]. So, theless abundant peaks obtained after reduction were the 20α-hydroxy-metabolites of the 22R and 22S enantiomers ofBUD, that are present in equal amounts in the BUD stan-dard. Extracts of urine samples were analyzed using theseconditions, and metabolite M-VIII eluted at 9.8 min. Noneof the other three isomers was detected. It is suggested thatmetabolite M-VIII eluting at 9.8 min is a 20α-hydroxy-metabolite with a 22S configuration. The CID mass spectraof metabolite M-VIII obtained in urines (Fig. 3a) is similarto that of the synthetized compound (Fig. 3b). The structure
22(S)-16α,17α-butylidenedioxy-11β,20α,21-trihydroxy-pregna-1,4-diene-3-one is proposed for metabolite M-VIII.
Structural elucidation of metabolites without acetal moiety
The main metabolite without acetal moiety was 16α-hydroxy-prednisolone (M-I). Moreover, six additionalmetabolites were identified (metabolites M-IX to M-XIII,Table 2) and only metabolite M-X was previously described[32]. The higher polarity of these metabolites compared tometabolites with acetal moiety, due to the presence of ahigher number of hydroxyl groups, results in lower retentiontime after LC analysis (Table 2). CID spectra of thesemetabolites are shown in Fig. 3 and most important frag-ment ions for identification purposes are summarized inTable 2. In positive mode, the same fragmentation behavioras metabolites with acetal chain was observed. In negativemode, neutral losses of 76 Da, resulting from the loss offormic acid and formaldehyde [18], and 94 Da, resultingfrom the loss of formic acid, formaldehyde and water, wereonly observed.
GC/MS analysis after MO-TMS derivatization was alsoperformed. The number of MO groups incorporated to themolecules indicates the number of ketone groups, and TMSgroups indicate the number of hydroxyl moieties. It has tobe taken into account that a ketone in C11 do not form MOderivative due to sterical impediments [33]. Electron ioni-zation mass spectra of the MO-TMS derivatives are alsopresented in Fig. 4.
D
150100 150 200 250 300 350 400 450
265
147
121
173
171
161225
211237
415
325307 433
100 200 250 300 350 400
265
147
121
135225173
237307
283289
361325
379
100 150 200 250 300 350 400
265
147
121135
173
171
225
211 237
361325289 307
100 150 200 250 300 350 400440
450
265
147
121
107
135
173
171
161225
185 211 237
415
307289 325
433
107
379
211
m/z
m/z m/z
m/z
AR
elat
ive
Abu
ndan
ce(%
)100
B
CR
elat
ive
Abu
ndan
ce(%
)
100
Rel
ativ
e A
bund
ance
(%)
100
Rel
ativ
e A
bund
ance
(%)
100
397379
397379343
171
343
343
Fig. 3 CID mass spectra (ESI positive, 20 eV) of metabolites M-VIII (A) and M-IX (C) in urine samples. CID mass spectra (ESI positive, 20 eV)of compounds obtained after NaBH4 reduction of commercially BUD (B) and 16α-hydroxy-prednisolone (D)
Budesonide metabolites in human urine after oral administration 333
M-IX
Metabolite M-IX exhibits a MM of 378 Da (Table 2), 2 Damore than M-I. It was not detected in negative ionization,indicating the reduction of the ketone group in C20.
To obtain the C20 reduced metabolite, authentic16α-hydroxy-prednisolone (M-I) was reduced usingNaBH4. Reduction in C20 creates a chiral center andtwo isomers are expected, however only one peak wasobtained after LC-MS/MS analysis. An isocratic methodwas developed to separate the two synthetized isomers(using 12 % of organic solvent and a flow-rate of500 μL/min). The isomers eluted at 8.9 and 9.5 minin these conditions, with relative abundance 1:9, andboth of them had similar CID spectrum (Fig. 3d). Asthe C20 carbonyl group is reduced preferentially to the20β configuration [27], and the 20β-hydroxy steroidsnormally elute later than the 20α isomers in the LC-MS analysis, it is suggested that the peak at 8.9 min isthe isomer 20α-hydroxy and the peak at 9.5 min, theisomer 20β-hydroxy. When the isocratic method wasapplied to extracts of urine samples two peaks wereobtained: the most abundant at 8.9 min (20α-hydroxy)and a second peak, representing a 20 % of the majorcompound, at 9.5 min (20β-hydroxy). Relative abun-dances of ten selected ion transitions were the same forthe synthetic compound and for the metabolitesdetected in urine. Therefore, it is concluded that thepeak detected using the first applied gradient, containsboth isomers.
Based on these data, 11β,16α,17α,20α,21-pentahydrox-ypregna-1,4-diene-3-one and 11β,16α,17α,20β,21-penta-hydroxypregna-1,4-diene-3-one are suggested as possiblestructures for metabolites M-IXa and M-IXb, respectively.Reduction of the keto group in C20 has also been describedfor other corticosterois such as methylprednisolone. In thatcase the isomer 20α-hydroxy was also found to be moreprominent than the 20β-hydroxy [21].
M-X
The MM of metabolite M-X is 374 Da, 2 Da less than M-I,suggesting an oxidation with respect to M-I. MS data(Fig. 4b, Table 2) suggest an oxidation in C11, as severalions related with losses of CO were observed. The GC/MSspectrum of the MO-TMS derivative of metabolite M-X(Fig. 4b) showed a molecular ion at m/z 648, indicatingthe presence of two MO groups (ketones in C3 and C20)and three TMS moieties (hydroxyl groups in C16, C17 andC21). According to these data, the structure of 16α-hydroxy-prednisone (16α,17α,21-trihydroxypregna-1,4-di-ene-3,11,20-trione) is suggested for M-X. This metabolitehas been recently described by other authors [32].
M-XI
Metabolite M-XI exhibits a MM of 378 Da, suggesting areduction with respect to metabolite M-I. The mass spectraat high collision energy show different profile than that ofmetabolite M-I (Fig. 4c, Table 2), suggesting some modifi-cation in A or B rings. The MO-TMS derivative of M-XIshows a molecular ion at m/z 724, indicating the incorpora-tion of two MO groups and four TMS moieties. Accordingto this data, the metabolite has two ketone groups and fourhydroxyl groups, so the reduction was not produced in theketone groups present in metabolite M-I. As the CID spec-trum indicates modification of the A or B rings structure, itis suggested that one of the double bonds presents in the Aring was reduced. Mass spectrometric data available doesnot indicate which one of the two double bonds in the A ringis reduced. However, it is known that other compounds witha 1,4-diene structure (boldenone, metandienone) are metab-olized by reduction of double bond in C4-C5 producing a5β-reduced metabolite [34, 35]. Based on these data11β,16α,17α,21-tetrahydroxy-1-pregnene-3,20-dione isproposed as potential structure for M-XI.
M-XII
Metabolite M-XII exhibits a MM of 376 Da, the samemolecular mass than M-I, suggesting an oxidation and areduction compared to this metabolite, and an oxidationcompared to metabolite M-XI. The mass spectra at highcollision energy also suggest a modification in A or B rings(Fig. 4d, Table 2). Some ions related with losses of CO,indicate the presence of a ketone group in C11. The MO-TMS derivative of M-XII (Fig. 4D) shows a molecular ionat m/z 650, indicating the presence of two MO groups andthree TMS moieties. This data is consistent with the pres-ence of only three hydroxyl groups and an underivatizedketone at C11. Therefore, 16α,17α,21-trihydroxy-1-pre-gnene-3,11,20-trione is proposed as potential structure formetabolite M-XII.
M-XIII
Metabolite M-XIII exhibits a MM of 378 Da, suggesting areduction with respect to M-I, or an oxidation and tworeductions. It was not detected in positive ionization, indi-cating that the keto function in C3 is not present. For this
Fig. 4 CID mass spectra obtained by LC-MS/MS (left) and electronionization spectra of the MO-TMS derivatives by GC/MS (right) ofmetabolites M-I (A), M-X (B), M-XI (C), M-XII (D), and M-XIII (E).CID mass spectra in ESI positive (20 eV) are shown for metabolites M-I, M-X, M-XI, and M-XII; CID mass spectra in ESI negative (20 eV) isshown for metabolite M-XIII
Budesonide metabolites in human urine after oral administration 335
reason, reduction to 3-hydroxy group is the most feasiblestructure for M-XIII.
The metabolite was ionized in negative mode (Fig. 4e,Table 2) indicating no modification on the C20-C21 chainwith respect to M-I. The MO-TMS derivative of M-XIII(Fig. 4e) showed a molecular ion at m/z 695, indicating thepresence of only one MO group, four TMS moieties and anunreacted ketone in C11. 3ξ,16α,17α,21-tetrahydroxy-1-pregnen-11,20-dione is proposed as potential structure forM-XIII.
Phase II metabolism
Extraction procedures applied in this work are able todetect BUD metabolites in free form as well as conju-gated with glucuronic acid. BUD, M-VI and M-VIIwere not detected in the free fraction. The rest ofmetabolites identified were detected in both fractions(free and combined) at similar concentrations, indicat-ing that they are mainly excreted as free metabolites.This could be explained by the high hydrophilic prop-erties of most of these metabolites due to the presenceof a high number of hydroxyl groups. BUD, M-VI andM-VII contain only one or two hydroxyl groups andconjugation with a more hydrophilic compound, such asglucuronic acid, is needed to eliminate those metabo-lites in urine, whereas the higher polarity of the othermetabolites allow for their direct excretion in urine. Ithas been reported that BUD suffers sulfatation [36],however sulfated metabolites were not evaluated inthe present study.
Metabolic pathways of BUD
As already reported, in a first step, the acetonide group ofBUD is hydrolyzed to 16α-hydroxy-prednisolone (M-I) [8].As mentioned, this reaction is stereoselective for 22R-BUD.From this point, further metabolism starts with 16α-hydroxy-prednisolone, 22S-BUD and, to a lesser extent,22R-BUD.With these three compounds, some of the principalcorticosteroids biotransformations were confirmed. It is well-known that 11β-hydroxysteroid dehydrogenase type 2 presentin the kidney and other tissues is responsible for C11 oxidationof cortisol to its inactive metabolite cortisone [37]. Similarly,from BUD, metabolite M-VII is formed, and from 16α-hydroxy-prednisolone, metabolitesM-X,M-XII, andM-XIIIare formed.
6-Hydroxylation is an important pathway in BUD, as dem-onstrated by the formation of 6β-hydroxy-budesonide (M-II)and 6α-hydroxy-budesonide (M-III). 6-hydroxylation ofBUD has been extensively studied being the enzyme respon-sible of the reaction, cytochrome P450 3A (CYP3A) [38].Although, 6α-hydroxylation was not previously demonstrated
for BUD, 6α-hydroxy-metabolites have been described forother glucocorticosteroids. For deflazacort, 6α-hydroxy-metabolite was detected as minor metabolite, while 6β-hydroxy-metabolite was the major metabolite in urine ofcynomolgus monkeys [39], and in vitro metabolism of dexa-methasone in human liver also showed the presence of a 6α-hydroxy-metabolite [40].
Formation of Δ6-budesonide (M-VI) has also been de-scribed as a secondary reaction of CYP3A [9]. Neither 6-hydroxy norΔ6-metabolite were detected for 16α-hydroxy-prednisolone in the present study.
Hydroxylation in C23 has already been reported [9]. Twoout of the four possible metabolites (metabolites M-IVa andM-IVb) have been detected, and another peak with very lowabundance that could be another of the steroisomers. Metab-olites M-V are formed by hydroxylation of C23 and Δ6-
Table 3 SRM transitions used for the detection of budesonide metab-olites by LC-MS/MS. CV, cone voltage; CE, collision energy
Metabolite Ionization mode Transition CV (V) CE (eV)
BUD Positive 431>147 20 24
431>173 20 26
431>323 20 12
M-I Positive 377>226 15 26
377>159 15 28
377>253 15 14
M-II Positive 447>339 20 14
M-III 447>171 20 35
M-IV 447>173 20 28
447>147 20 32
M-V Positive 445>171 20 30
445>321 20 16
M-VI Positive 429>147 20 31
M-VII 429>171 20 26
429>211 20 16
429>311 20 16
M-VIII Positive 433>265 20 20
433>147 20 34
M-IX Positive 379>265 15 16
379>147 15 28
379>283 15 18
M-X Positive 375>147 15 30
375>171 15 30
375>225 15 20
M-XI Positive 379>325 15 15
379>283 15 18
M-XII Positive 377>329 15 15
377>359 15 19
M-XIII Negative 423>347 20 10
BUD-d8 Positive 439>173 20 20
MI-d5 Positive 382>229 15 26
336 X. Matabosch et al.
unsaturation; in this case, two of the four possible isomershave been detected.
20α and 20β-hydroxysteroid dehydrogenases are twoenzymes that usually play an important role in corticosteroidmetabolism. Identification of M-VIII and M-IX confirmedthat C20 reduction take place. The low concentrations ofthese compounds in urine samples suggest that theseenzymes are not very important in BUD metabolism. Thelow activity could be consequence of a lower affinity forsterical reasons due to the presence of the acetal moiety.
Reduction of the A ring is the first stage of the majormetabolic pathway for the inactivation of endogenous cor-ticosteroids and it is catalyzed by two enzymes, the 4-ene-5β-reductase and the 4-ene-5α-reductase. The second stageutilizes the enzymes 3α- and, to a much lesser extent, 3β-hydroxysteroid dehydrogenases; thus, for endogenous cor-ticosteroids the major products are A ring tetrahydro-
reduced metabolites [41]. By following the same pathway,metabolites M-XI, M-XII and M-XIII are formed. Howev-er, whereas the 5β/5α ratio for endogenous corticosteroidmetabolites is about 3:1, according to results obtained in invitro experiments BUD is more efficiently metabolized via5α [42].
Detection time of the metabolites in urine
Once the metabolites were identified, all urine samplescollected after the administration of an oral dose of BUDto healthy volunteers were analyzed. An LC-MS/MS methodin SRM mode was developed for this purpose, includingspecific transitions for all detected metabolites. Table 3 sum-marizes the most critical parameters for BUD and the metab-olites identified. The optimized SRM method enabled thedetection of BUD, M-I, M-II, and M-III at concentrations
Time5.00 5.50 6.00
%
0
100 447 > 1734.31e5
5.22
5.09
4.695.39
Time4.50 5.00 5.50
%
0
100 445 > 1712.40e5
5.00
4.88
5.26
Time9.00 10.00
%
0
100 429 > 1713.46e5
9.58
9.14
Time8.00 9.00 10.00
%
0
100 429 > 3114.03e4
9.14
8.18 8.729.62
Time7.00 7.50 8.00 8.50
%
0
100 433 > 2655.32e4
7.24
6.98
7.30
8.067.72 8.34
Time2.00 3.00
%
0
100 379 > 2652.52e5
2.05
1.21
1.90
2.362.68
Time2.00 3.00 4.00
%
0
100 375 > 1471.27e6
2.99
Time4.50 5.00 5.50
%
0
100 379 > 3253.27e4
4.39
4.37
4.07 4.534.97 5.445.40
Time4.50 5.00 5.50
%
0
100 377 > 3291.11e5
4.68
Time4.50 5.00 5.50
%0
100 423 > 3471.28e4
4.81
4.37
4.174.65
Time9.00 9.50 10.00
%
0
100 439 > 1733.08e5
9.33
9.52
Time9.00 9.50 10.00
%
0
100 431 > 1472.11e6
9.64
9.43
Time5.00 6.00
%
0
100 447 > 3392.59e5
6.24
4.68
4.466.05
5.22
Time3.00 4.00
%
0
100 377 > 2261.30e3
2.19
2.27
2.742.68
2.34
3.11 3.19
3.503.55
3.613.88
3.94
Time5.00 5.50 6.00
%
0
100 447 > 1734.25e3
5.05
4.744.78
5.29
5.085.98
5.955.33
5.61
Time4.50 5.00 5.50
%
0
100 445 > 1713.76e3
4.96
4.384.35
4.03
4.06
4.854.55
5.28
5.225.31
5.49
5.54
Time9.00 10.00
%
0
100 429 > 1713.28e310.23
9.06
8.98
9.2410.20
9.36
9.83
9.38
9.60
10.57
Time8.00 9.00 10.00
%
0
100 429 > 3111.93e3
8.528.16
8.239.38
8.909.09 9.57 9.79
9.839.95
9.99
Time7.00 7.50 8.00 8.50
%
0
100 433 > 2656.82e3
6.99
8.39
7.038.30
7.178.08
7.607.43 8.03
7.68
8.43
8.45
8.488.52
Time1.00 2.00 3.00
%
0
100 379 > 2653.92e4
2.37
1.212.26
1.96
1.901.30
1.64
2.78
2.44
2.85
Time2.00 3.00 4.00
%
0
100 375 > 1477.70e3
3.87
3.202.71
2.682.44
2.40
3.04
3.74
3.68
3.90
Time4.50 5.00 5.50
%
0
100 379 > 3259.05e3
5.40
4.98
4.96
4.56
4.064.274.60
4.63
5.02
5.25
5.46
5.53
Time4.50 5.00 5.50
%
0
100 377 > 3291.28e3
4.81
4.76
4.03 4.72
4.67
4.634.42
4.08
5.00
5.215.33
5.51
Time4.50 5.00 5.50
%
0
100 423 > 3471.11e3
4.66
4.64
4.404.37
4.06
4.81
5.235.195.385.49
Time9.00 9.50 10.00
%
0
100 431 > 1476.29e3
9.11
9.17
9.3710.04
9.41 9.61
9.89
10.15
10.17
10.19
Time9.00 9.50 10.00
%
0
100 439 > 1732.07e5
9.33
9.53
Time5.00 6.00
%
0
100 447 > 3391.69e3
5.82
4.404.51
5.304.86
6.76
6.68
6.34
6.03
Time2.00 3.00 4.00
%
0
100 377 > 2263.49e6
2.69
BUD-d8 BUD M-I M-III M-II M-IV M-V M-VI
BUD-d8 BUD M-I M-III M-II M-IV M-V M-VI
M-VII M-VIII M-IX M-X M-XI M-XII M-XIII
M-VII M-VIII M-IX M-X M-XI M-XII M-XIII
Fig. 5 Chromatograms of the transitions monitored for BUD-d8, BUD and all metabolites (M-I–M-XIII) in a sample collected after oraladministration of BUD (two rows on top) and in a blank sample (two rows on bottom)
Budesonide metabolites in human urine after oral administration 337
338 X. Matabosch et al.
around 0.5 ng/ml. Themethodwas found to be selective for allthe selected analytes as no interferences were found in any ofthe ten blank samples analyzed (Fig. 5). In Fig. 5, chromato-grams of a sample collected from 8 to 24 h after administrationof the drug.
The excretion profiles of the different metabolites arepresented in Fig. 6. Even though amounts of all metabolitesare higher in volunteer 1, excretion profile was similar forboth volunteers. Differences in amounts of metabolites seemto be due to the different volume of urine collected for eachvolunteer. For example, in the interval from 8 to 24 h, thevolume from volunteer 1 was 155 mL, while for volunteer 2was 800 mL. The maximum amount for all metabolites wascollected in fraction from 8 to 24 h, and an importantdecrease of the amounts excreted was observed after 36 hafter administration. However, it is possible to identifyseven of the metabolites in the samples collected 48 h afteradministration at least, two of the metabolites in samplescollected up to 72 h.
Conclusions
Metabolism of BUD was studied using LC-MS/MS tech-nology, combined with GC/MS for identification of some ofthe metabolites. By combining all the information collected,the overall metabolism for BUD has been re-evaluated.Sixteen metabolites were detected in urine samples afteroral administration of BUD. Structures of metabolites result-ing from the cleavage of the acetal chain (M-I, 16α-hydroxy-prednisolone) and 6-hydroxylation (M-II and M-III, 6β-hydroxy-budesonide, and 6α-hydroxy-budesonide)were confirmed by comparison with commercially availablestandards. Plausible structures for the rest of metaboliteswere proposed based on mass spectrometric data.
Some metabolites maintaining the acetal chain were gen-erated through hydroxylation in the acetal chain (M-IVa,M-IVb, M-Va, and M-Vb), reduction of C6-C7 (M-Va, M-Vb, andM-VI), oxidation of the hydroxyl group in C11 (M-VII), or reduction in the C20 carbonyl group (M-VIII).Other metabolites were produced by cleavage of the acetalchain, asM-I, and additional modifications (reduction in theC20 carbonyl group, M-IXa and M-IXb; oxidation of thehydroxyl group in C11, M-X; or reductions in the A ring,M-XI to M-XIII). Ten of these metabolites were notreported before for BUD (M-III, M-Va, M-Vb, M-VII,MVIII, MIXa, MIXb, MXI, MXII, and MXIII).
A better knowledge on the metabolism of BUD may beuseful to investigate differences on the metabolites excretedin urine samples depending on the route of administration.These differences could be used in the future to defineanalytical strategies to discriminate between legal and for-bidden use of BUD in doping control analyses.
Acknowledgments Grant from Ministerio de Ciencia e Innovación(Spain) (DEP2009-11454), Generalitat de Catalunya (Consell Catalàde l’Esport and DIUE 2009SGR4929) and support by ISCIII-FIS-CAIBER CAI08/01/0024 are gratefully acknowledged. The authorswould like to thank Esther Menoyo and Núria Renau for their collab-oration in the excretion studies and management of samples.
References
1. Clissold SP, Heel RC (1984) Drugs 28:485–5182. Brogden RN, McTavish D (1992) Drugs 44:375–4073. McKeage K, Goa KL (2002) Drugs 62:2263–22824. The World Anti-Doping Code. The 2012 Prohibited List. International
Standard (2011) World Anti-Doping Agency, Montreal, Canada. http://www.wada-ama.org/Documents/World_Anti-Doping_Program/WADP-Prohibited-list/2012/WADA_Prohibited_List_2012_EN.pdf
Fig. 6 Excretion profile of BUD metabolites after oral administration:ratios of the areas of the metabolites against internal standard (formetabolites with ketal moiety BUD-d8 and for metabolites withoutketal moiety 16α-hydroxy-prednisolone-d5 were used), and period oftime after administration at which metabolites are detected
R
Budesonide metabolites in human urine after oral administration 339
