463
Lance Brooker1,2, Catrin Goebel1, Ray Kazlauskas1, Graham Trout1 and Adrian George2
Confirmation of Triamcinolone Acetonide Use By LC-MS/MS
1. Australian Sports Drug Testing Laboratory, National Measurement Institute, Pymble,
Australia.
2. School of Chemistry, University of Sydney, Sydney, Australia
Triamcinolone acetonide (TA) is a commonly prescribed synthetic glucocorticosteroid
used in the treatment of minor, acute and chronic inflammatory conditions such as eczema,
sports injuries, arthritis and asthma. For doping control purposes, WADA restricts
administration to dermatological preparations or with appropriate therapeutic use exemption,
local forms such as inhalation and intra-articular injection. To enable the confirmation of
synthetic corticosteroid use, the excretion and metabolism of TA was investigated.
Quantification of Triamcinolone Acetonide
A quantitative ESI-LC-MS/MS assay was developed for the analysis of TA in urine
utilizing a stable isotopically-labelled processed internal standard or surrogate (Figure 3).
After addition of d6-TA surrogate and enzymatic hydrolysis, 2.5 mL urine aliquots were
prepared by liquid-liquid extraction with ethyl acetate. Fludrocortisone was added as internal
standard, and then the extract was dried and reconstituted in 200 uL of mobile phase ready for
analysis. HPLC separation was performed on a Waters Alliance 2795 Separations Module,
using a C18 column and a gradient elution program (2% Formic Acid/H2O:Acetonitrile).
Mass spectrometric detection was performed on a Waters Micromass Quattro Micro, with
electrospray ionisation.
Calibration standards (0 - 100 ng/mL) were accurately prepared from stock solutions in
methanol, evaporated and reconstituted in 200 uL of mobile phase for LC-MS/MS analysis.
Calibration curves (y = ax2 + bx + c) were created from the calibration standards, by plotting
the analyte/surrogate peak area ratio (y) vs. the nominal concentration of the standards (x).
Method validation was undertaken and the results from accuracy and precision testing are
shown below (Figure 1, accuracy and precision are represented as percentages).1 A total of
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60 samples were analysed, including replicate and random blank urine aliquots (SG 1.006 -
1.028) spiked with multiple analytes at two concentrations (12 and 60 ng/mL).
Accuracy and Precision (n = 15)
0
20
40
60
80
100
120
Triamcinolone Acetonide Triamcinolone
Acc
urac
y (%
)
12 ng/mL Duplicates 60 ng/mL Duplicates 12 ng/mL Random 60 ng/mL Random
Triamcinolone Acetonide Excretion Profiles
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
0 3 6 9 12 15 18 21 24 27 30 33 36
Hours (after administration)
Con
cent
ratio
n(n
g/m
L, S
G C
orre
cted
)
Gel 1.4 mg Puffer 2 x 400 ug Nasal Spray 220 ug
Heel Injection 10 mg Oral 8 mg WADA MRPL
Figure 1: Method Validation Results Figure 2: TA Excretion Profiles The related analyte triamcinolone (Figure 3) was quantified without the use of a stable
isotopically-labelled surrogate (i.e. with internal standard, fludrocortisone). A marked
decrease in the accuracy and precision of the quantification can be observed with comparison
to triamcinolone acetonide. The use of appropriate surrogates (such as stable isotopically-
labelled analogues) is extremely important in LC-MS/MS quantitation of corticosteroids.
This is due to the pronounced matrix effects observed to influence absolute analyte response
in biological matrices (such as human urine).2
The validated method was then applied to five TA excretion studies from WADA
permitted and prohibited modes of administration (Figure 2).3 Results from four different
forms of topical/local administration showed maximum urinary concentrations below the
WADA specified (laboratory) minimum required performance limit (MRPL) of 30 ng/mL.
An oral administration of TA (8 mg) produced a maximum urinary concentration of 77
ng/mL. For all studies, excretion was complete in 24-36 hours, except for the heel injection
for which a prolonged excretion was observed (> 3 days).
Metabolism of Triamcinolone Acetonide
In humans, TA is predominantly metabolised to 6β-hydroxy TA (Figure 3).4 6β-
hydroxylation is a common metabolic route for steroid hormones, both endogenous and
synthetic.5,6
2nd Inhalation
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O
HO
O
OH
O
O
F
O
HO
OOH
OO
F
OH
O
HO
O
OH
OH
OH
F
O
HO
OOH
OHOH
F
OHO
O
OOH
OHOH
F
O
HO
OOH
OO
F
CD3
CD3
O
HO
OOH
OH
F
triamcinolone acetonideES+: 435>415.2, ES+: 435>397.1
triamcinoloneES-: 393.1>345.2, ES-: 393.1>363.2
d6-triamcinolone acetonideES+: 441>421.2
fludrocortisoneES-: 425.1>349.1
6β-hydroxy triamcinolone acetonideES+: 451>386.8
6β-hydroxy triamcinoloneES+: 411>347, ES+: 411>215
11-dehydro-triamcinoloneES-: 391.1>343.2, ES-: 391.1>361.2
Figure 3: Corticosteroid Surrogate, Internal Standard, Analytes and Proposed Metabolites
For metabolite investigation, full scan LC-MS/MS analysis was performed on TA
excretion study extracts. Extracted ion chromatograms corresponding to the proposed TA
metabolite ([M+H]+ = 451), from before and after administration are shown in Figure 4.
Figure 4: EIC of m/z = 451 from TA excretion study extracts (T0 and T15) The cone voltage was then optimised for the chromatographic peak located at the
retention time of 6.69 minutes. Daughter scan MS/MS analysis was undertaken to obtain a
mass spectrum of the proposed TA metabolite (Figure 5, Cone 18V, Collision 15eV). Using
this data, a multiple reaction monitoring (MRM) window was incorporated into the ASDTL
LC-MS/MS screening method for corticosteroids. In order to confirm the identity of this
metabolite, work has begun on the synthesis and certification of an appropriate reference
material.7,8 6β-hydroxy triamcinolone acetonide has been synthesized, isolated and partially
characterized - its MS/MS spectrum is consistent with the proposed TA metabolite (Figure 5).
Using the above LC-MS/MS methodology, MRM transitions have also been developed for
two possible triamcinolone metabolites (Figure 3). As also observed with the TA metabolite,
excretion of these metabolites follows a similar concentration-time profile to the parent
compound (Figure 6).
Peak observed at RT: 6.69 mins Background Noise
T0 T15
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Figure 5: Daughter Scan Mass Spectrum of proposed TA metabolite peak at RT = 6.69 mins
Triamcinolone and Metabolites
0
10
20
30
40
50
60
0 3 6 9 12 15 18 21 24 27 30 33 36Hours (after administration)
Ana
lyte
/IS
Peak
Are
a R
atio
(SG
Cor
rect
ed)
Triamcinolone 4 mg 11-dehydro-triamcinolone 6β-hydroxytriamcinolone
Figure 6: Triamcinolone and Metabolites Excretion Profiles and HPLC Chromatogram (T14) References
1. Eurachem Guide, The Fitness for Purpose of Analytical Methods: A Laboratory Guide to Method Validation and Related Topics. 1998. 2. Stokvis, E; Rosing, H. and Beijnen, J.H. Rapid Commun. Mass Spectrom. 2005, 19, 401-407. 3. Southern Cross University Human Ethics Committee (Approval N0 ECN-05-24). 4. Argenti, D; Jensen, B.K; Hensel, R; Bordeaux, K; Schleimer, R; Bickel, C. J Clin Pharm, 2000, 40, 770-780. 5. Burstein, S; Dorfmann, R and Nadel, E. Arch Biochem Biophys, 1954, 53, 307-308. 6. Schanzer, W. Clinical Chemistry, 1996, 42 (7), 1001-1020. 7. Dusza, J.P and Bernstein, S. C-6 Hydroxylated Steroids. V. J Org Chem, 1963, 28, 760–3. 8. Thalén, A and Wickström, L-I. Steroids, 2000, 65, 16–23.
O
HO
O
OH
O
O
F
OH
T14
Res
pons
e (a
rbitr
ary
units
)
Retention Time (mins)
6β-hydroxy triamcinolone
triamcinolone
11- dehydro-
triamcinolone
cortisone d6-TA Surrogate
Cone 18v Collision 15eV