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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

In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

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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

In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

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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

In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

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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

In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006