In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (13). Sport und Buch Strauß - Köln 2005
Elzbieta Palonek, Fredrik Österwall, Mats Garle
Screening for Corticosteroids in Urine with on-line Extraction and LC-MSMS Detection
Karolinska University Hospital, Department of Clinical Pharmacology, Doping Control Laboratory, C2-66, SE-141 86 Stockholm, Sweden
Introduction A new method has been developed for routine analysis of corticosteroids in urine. This assay for corticosteroids and their metabolites is performed by on-line solid phase extraction, liquid chromatography separation and tandem mass spectrometry detection, so called XLC-MSMS technology – automated from sample list to MSMS results. The glucocorticosteroids are on the IOC/WADA Prohibited List1 and the doping control laboratories are obliged to analyse them in the samples taken during competition. They are prohibited when administered orally, rectally, intravenously or intramuscularly. Their use requires a Therapeutic Use Exemption approval. There is a list of glucocorticosteroids to be detected by IOC/WADA accredited laboratories: beclomethasone, betamethasone, budesonide, desonide, dexamethasone, fludrocortisone, flumethasone, flunisolide, fluticasone, fluocortolone, methylprednisolone, prednisolone, prednisone, triamcinolone. There are several methods available to analyse this group of compounds using different techniques and instrumentation which have been published4-5 and presented6-9 during annual meetings in Cologne. Gas chromatography mass spectrometry has never become a good method for analysing corticosteroids since it requires derivatisation which is both time consuming and occasionally incomplete for these compounds7. To solve this problem liquid chromatography mass spectrometry LC-MS as well as LC-MSMS technique4-6, 8-9 have been employed. In this paper we present our way of dealing with the problem of analysing corticosteroids using the LC-MSMS technique with the implemented on-line solid phase extraction procedure to diminish the workload in the laboratory. At this stage the method is designed for screening of glucocorticosteroids only.
47 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (13). Sport und Buch Strauß - Köln 2005
Experimental 20 µL of urine sample is extracted by solid phase extraction (SPE) using the Symbiosis Pharma instrument from Spark Holland interfaced to the Finnigan TSQ Quantum Discovery tandem mass spectrometer. Confirmation of corticosteroids can be performed with the same extraction and chromatography system utilizing a modified mass spectrometry method to register at least two ion transitions for each compound (Tab. 1).
Sample pre-treatment
Urine samples are centrifuged after adding 50 ng/mL of methyltestosterone and D4- cortisol (internal standards) and transferred to vials, which are placed in a cooled chamber of the automatic injector, a so called Reliance of Symbiosis Pharma instrument. For screening purposes an unhydrolyzed urine fraction is used. If necessary (e.g. confirmation of budesonide) the sample is hydrolyzed with β-glucuronidase from E.Coli at pH 7 using our standard procedure for androgenic anabolic steroids. After hydrolysis and centrifugation, the sample is applied directly to XLC-MSMS on-line solid phase extraction.
Symbiosis Pharma Spark Holland on – line extraction 20 µL of urine sample is injected in partial loopfill mode. Total sample consumption for one injection is 65 µL. The cartridge, which is used for corticosteroid extraction is a Sorbent HySphere C2 8 µm silica based ethyl phase. Sample Extraction Procedure - Conditioning of Cartridge o Solvation 1 mL of methanol o Equilibration 1 mL of water - Sample loading 0.5 mL of water - Cartridge washing o 1 mL of 5% methanol o 1 mL of water - Analyte Elution Mobile phase A/B (70:30) o Elution time 6 min.
48 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (13). Sport und Buch Strauß - Köln 2005
As soon as the cartridge is eluted the next sample is prepared. As long as the SPE time is shorter than the time required for LC-MSMS, the total process time is determined by the LC- MSMS time only.
Chromatographic conditions Column Phenomenex Luna 3u C18(2), 100 x 2.0 mm Mobile phase: A: Water / Acetonitril (95:5), 1% Acetic Acid B: Acetonotril / Water (95:5), 1% Acetic Acid Flow: 0.3 mL/min. Gradient: A/B (70:30) 1 min., A/B (70:30) to A/B (35:65) 9 min., A/B (35:65) 1 min., A/B (35:65) to A/B (70:30) 1 min., A/B (70:30) 4 min.
Mass spectrometer Finnigan TSQ Quantum Discovery tandem mass spectrometer with electrospray ionization ESI ion source is operated in the positive ionization mode with SRM monitoring and 0.20s dwell time. All compounds produce [M+H]+ ions as listed in Table 1. ESI voltage used is 4800V, capillary temperature 350oC and collision gas pressure 1.5 mTorr.
Results and discussion The presented XLC-MSMS on-line solid phase extraction method is used in our laboratory for screening and confirmation of glucocorticosteroids (Tab.1, Fig.1). The method has been accredited by the Swedish Accreditation Body (SWEDAC) according to the standard ISO 17025.
Table 1 – Screening and confirmation of glucocorticosteroids, mass spectrometry parameters, retention time (RT) and limit of detection (LOD)*
Compound Precursor Product ion Collision RT LOD ion screening Energy min ng/mL (confirmation) 279 21 6.66 0.8 Beclomethasone 409 (279, 147, 171) 279 20 6.18 0.2 Betamethasone 393 (279, 147, 171) 147 41 8.88 0.9 Budesonide 431 (147, 173, 225) 225 28 6.93 0.5 Desonide 417 (147, 173, 225)
49 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (13). Sport und Buch Strauß - Köln 2005
Compound Precursor Product ion Collision RT LOD ion screening Energy min ng/mL (confirmation) 237 25 6.28 0.6 Dexamethasone 393 (237, 147, 279) 239 27 5.07 1.5 Fludrocortisone 381 (239, 91, 128) 253 18 6.37 1.5 Flumethasone 411 (253,121, 235) 263 22 7.03 0.8 Flunisolide 435 (171, 223, 263) 303 10 7.50 0.2 Fluocortolone 377 (303, 171, 121) 161 19 6.00 2.8 Methylprednisolone 375 (161, 185, 253) 147 29 4.88 1.3 Prednisolone 361 (147, 171, 121) 147 28 4.98 1.6 Prednisone 359 (147, 171, 237) 225 22 3.41 1.6 Triamcinolone 395 (225, 237, 147) Triamcinolone 213 31 6.94 1.4 435 acetonide (213, 225, 275) 253 18 7.23 0.5 Fluocinolone acetonide 453 (253, 121, 279) 147 32 3.31 1.9 16α-OH-Prednisolone 377 (147, 173, 226) 253 22 6.47 1.3 Betamethasone M** 391 (253, 147, 237) 147 32 7.53 2.0 Desoximethasone 377 (171,147,128,91) Fluticasone 275 18 12.01 0.2 501 propionate *** (275, 293, 205) 163 27 5.17 Cortisone 361 Endogenous (163, 105, 121) 121 42 4.97 Cortisol 363 Endogenous (121, 91, 145) 239 30 2.75 6β-OH-Cortisol 379 Endogenous
121 42 4.95 Internal D4-Cortisol ISTD 367 standard Methyltestosterone 97 32 8.98 Internal 303 ISTD standard *LOD is appraised at a signal to noise ratio of 3:1 for all substances ** Betamethasone M – (16β)-9-Fluoro-17,21-dihydroxy-16-methylpregna-1,4-diene-3,11,20-trione. *** Fluticasone propionate is used as indicator of properly working chromatography.
50 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (13). Sport und Buch Strauß - Köln 2005
Fig.1 - Screening of glucocorticosteroids
C:\Xcalibur\...\Q09-050225 2005-02-25 10:36:42 Q09-050225
RT: 7.00 - 11.00 SM : 7G RT: 3.00 - 7.00 SM : 7G RT: 1.00 - 6.00 SM : 7G RT: 8.98 N L: 3 . 11E 4 RT: 4.95 NL: 9.14E3 RT: 3.31 NL: 10 0 10 0 10 0 TIC F: + c SRM TIC F: + c sid=-18.00 5.9 5E3 M ETHYLTESTOSTERONE ms2 D4-CORTISOL SRM ms2 16a-OH-PREDNISOLONE TIC F: + c SRM 80 [email protected] 80 [email protected] [ 80 ms2 [ 96.61-97.41] 120.53-121.33] MS [email protected] M S Genesis Genesis Q09-0502 25 [ 60 Q09-050225 60 60 146.64-147.44] MS Q09-050225 40 40 40 Relative Abundance Relative Abundance Relative Abundance 20 20 20
1. 6 6 2.30 5.14 0 0 0 7 8 9 10 11 3 4 5 6 7 1 2 3 4 5 6 Time (min) Time (min) Time (min)
RT: 2.00 - 5.00 SM : 7G RT: 3.00 - 7.50 SM : 7G RT: 3.60 - 6.60 SM : 7G RT: 3.41 NL: RT: 4.88 NL: RT: 4.98 NL: 10 0 2.28E3 10 0 6.38E3 10 0 4.65E3 TRIAM CINOLONE TIC F: + c SRM PREDNISOLONE TIC F: + c SRM PREDNISONE TIC F: + c SRM 80 ms2 80 ms2 80 ms2 [email protected] [email protected] [email protected] [ [ [ 60 224.57-225.37] 60 146.54-147.34] 60 146.67-147.47] MS MS MS Q09-050225 Q09-050225 Q09-050225 40 40 40 Relative Abundance Relative Abundance Relative Abundance 6.17 20 20 20 2.31 5.71 4.41 4.78 3.59 3.96 6.89 7.26 4.33 0 0 0 2.0 2.5 3.0 3.5 4.0 4.5 5.0 3 4 5 6 7 4.0 4.5 5.0 5.5 6.0 6.5 Time (min) Time (min) Time (min)
RT: 3.60 - 6.60 SM : 7G RT: 4.50 - 7.50 SM : 7G RT: 4.50 - 8.00 SM : 7G RT: 5.07 NL: RT: 6.00 NL: RT: 6.18 NL: 10 0 4.87E3 10 0 6.69E3 10 0 6 . 11E 3 FLUDROCORTISONE TIC F: + c SRM M ETHYLPREDNISOLONE TIC F: + c SRM BETAMETHASONE TIC F: + c SRM 80 ms2 80 ms2 80 ms2 [email protected] [email protected] 393.10@- [ [ 20.00 [ 60 238.64- 60 160.56-161.36] 60 278.44- 239.44] MS MS 279.24] MS Q09-050225 Q09-050225 Q09-050225 40 40 40
Relative Abundance 3.79 4.06 Relative Abundance Relative Abundance 6.64 6.08 20 20 7.00 20 4.90 4.62 5.17 5.72 0 0 0 4.0 4.5 5.0 5.5 6.0 6.5 4.5 5.0 5.5 6.0 6.5 7.0 7.5 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 Time (min) Time (min) Time (min)
RT: 4.50 - 8.50 SM : 7G RT: 4.50 - 9.00 SM : 7G RT: 4.00 - 9.00 SM : 7G RT: 6.28 NL: RT: 6.37 NL: RT: 6.47 NL: 1.24E4 10 0 10 0 10 0 5.4 7E3 5.19E3 TIC F: + c SRM DEXAM ETHASONE TIC F: + c SRM FLUM ETHASONE TIC F: + c SRM BETAMETHASONE M ms2 80 ms2 80 ms2 80 [email protected] [ [email protected] 4 11. 10 @ - 18 . 0 0 [ 252.67-253.47] [ 252.53-253.33] M S Genesis 60 236.48- 60 MS 60 Q09-050225 237.28] MS Q09-050225 Q09-050225 40 40 40 Relative Abundance Relative Abundance Relative Abundance
20 6.64 20 20
4.81 5.27 5.73 7.75 8.30 5.005.6 4 7.2 9 7.75 8.39 0 0 0 5 6 7 8 5 6 7 8 9 4 5 6 7 8 9 Time (min) Time (min) Time (min)
C:\Xcalibur\...\Q09-050225 2005-02-25 10:36:42 Q09-050225
RT: 4.50 - 8.50 SM : 7G RT: 5.50 - 8.50 SM : 7G RT: 5.50 - 8.50 SM : 7G RT: 6.66 NL: RT: 6.93 NL: RT: 6.94 NL: 10 0 3 .75E3 10 0 5.77E3 10 0 4.41E3 BECLOM ETHASONE TIC F: + c SRM DESONIDE TIC F: + c SRM TRIAM CINOLONE TIC F: + c SRM ACETONIDE 80 ms2 80 ms2 80 ms2 [email protected] [email protected] [email protected] [ [ [ 60 2 78.59-279.39 ] 60 224.67-225.47] 60 212.56-213.36] MS MS MS Q09-050225 Q09-050225 Q09-050225 40 40 40 Relative Abundance Relative Abundance Relative Abundance 20 20 20
4.82 7.67 6.29 5.75 6.20 0 0 0 5 6 7 8 5.5 6.0 6.5 7.0 7.5 8.0 8.5 5.5 6.0 6.5 7.0 7.5 8.0 8.5 Time (min) Time (min) Time (min)
RT: 5.50 - 8.50 SM : 7G RT: 6.00 - 9.00 SM : 7G RT: 7.00 - 11.00 SM : 7G RT: 7.03 NL: RT: 7.50 NL: 7.50 NL: 10 0 4.69E3 10 0 1. 10 E4 10 0 1.83E4 FLUNISOLIDE TIC F: + c SRM FLUOCORTOLONE TIC F: + c SRM TIC F: + c SRM 80 ms2 80 ms2 80 ms2 [email protected] 3 77. 10 @ - 10 . 0 0 BUDESONIDE [email protected] [ [ [ 60 262.53- 60 302.71-303.51] 60 RT: 8.88 146.59-147.39] 263.33] MS MS MS Q09-050225 Q09-050225 Q09-050225 40 40 40 Relative Abundance Relative Abundance Relative Abundance 20 20 20 8.05 10 . 3 4 7.95 6.49 8.14 0 0 0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 7 8 9 10 11 Time (min) Time (min) Time (min)
RT: 6.00 - 9.00 SM : 7G RT: 6.00 - 9.00 SM : 7G RT: 10 . 50 - 13 . 50 SM : 7G RT: 7.53 NL: RT: 7.23 NL: RT: 12.01 NL: 10 0 3.07E3 10 0 3.17E3 10 0 8.83E3 DESOXIMETHASONE TIC F: + c SRM FLUOCINOLONE TIC F: + c SRM FLUTICASONE TIC F: + c SRM PROPIONATE 80 ms2 80 ACETONIDE ms2 80 ms2 [email protected] [email protected] [email protected] [ [ [ 6.40 60 146.64-147.44] 60 2 52.59-253.39 ] 60 274.57-275.36] MS MS MS Q09-050225 Q09-050225 Q09-050225 40 40 40 Relative Abundance Relative Abundance Relative Abundance 20 20 20 6.15 6.52 8.88 8.33 10 . 8 1 11. 2 7 12 . 9 2 0 0 0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 10 . 5 11. 0 11. 5 12 . 0 12 . 5 13 . 0 13 . 5 Time (min) Time (min) Time (min)
RT: 3.50 - 6.50 SM : 7G RT: 1.50 - 4.00 SM : 7G RT: 4.00 - 7.00 SM : 7G RT: 4.97 NL: RT: 2.75 NL: 5.31E2 RT: 5.17 NL: 10 0 10 0 10 0 4.76E3 TIC F: + c 1. 0 7E 4 CORTISOL TIC F: + c SRM 6B-OH-CORTISOL sid=-18.00 SRM CORTISONE TIC F: + c SRM 80 ms2 80 ms2 80 ms2 363.10@- [email protected] [ [email protected] 3.87 42.00 [ 238.61-239.41] [ 60 120.58-121.38] 60 MS Q09-050225 60 162.65-163.45] MS MS Q09-050225 Q09-050225 40 40 40 Relative Abundance Relative Abundance Relative Abundance 20 20 20 3.76 5.80 1. 74 2.11 6.18 0 0 0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 1. 5 2.0 2.5 3.0 3.5 4.0 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Time (min) Time (min) Time (min)
51 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (13). Sport und Buch Strauß - Köln 2005
When applying this method we use a negative control urine and two quality control samples (QC) which are analysed together with every batch of samples. The negative control urine samples are spiked with 30ng/mL of different corticosteroids. Two different QC samples are used to avoid coelution of compounds in each of them, for example betamethasone and dexamethasone are in different QC samples. Although bethamethasone and dexamethasone are only slightly separated in our screening method, they could be properly recognized. The fragment ions obtained from both stereoisomers are similar but their relative abundances are different, (Tab.1 and Tab.2). In a situation were both of them are present in the same sample, the chromatographic gradient is changed to allow separation. However, if only one of the compounds is present it is possible to prove its presence using difference in MSMS fragmentation (see Table 2). O O OH CH3 OH OH CH3 OH OH OH CH CH 3 H 3 H CH3 CH3 F F O H O H
Betamethasone Dexamethasone MW = 392 MW = 392
Table 2 - Sample positive for betamethasone Betamethasone Betamethasone Dexamethasone Method [M+H]+ RT min. Relative RT min. Relative Abundance Abundance 393 (% of base peak) (% of base peak) 147 6.18 100 6.28 100 279 93* 48** 171 62 52 *See difference in relative abundance for transition m/z 393 - 279 which is 93% if betamethasone is present. **The relative abundance is 48% for m/z 393 - 279 in this case because there is dexamethasone in the sample. To show this difference both betamethasone and dexamethasone were analysed with the confirmation method for betamethasone. For fragment ions used in respective methods (see Table 1).
52 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (13). Sport und Buch Strauß - Köln 2005
Fluticasone can not be found using our screening method. Fluticasone is poorly absorbed and undergoes extensive first-pass metabolism. The only known metabolite in humans is a 17β-carboxylic acid metabolite. Less than 5% of a dose is excreted in urine as the metabolite with remainder being excreted in faeces as the parent drug (up to 75%) and the metabolite2. We included fluticasone propionate in our method (as the only available standard at the time of validation of the method) and decided to keep it as it appeared to be good indicator of properly working chromatography and SPE extraction as it elutes late in our system.
Confirmation of budesonide Budesonide is a mixture of two isomers, the content of the S-isomer in the mixture varies between 40 – 51% (Merck Index). It is available on the market as bidien, budeson, cortivent, micronyl, preferid, pulmicort, rhinocort, spirocort, symbicort. The metabolism of budesonide is well known and published3. For screening purposes 16α-OH-prednisolone, which is a metabolite of budesonide is used3. It was observed in the unconjugated fraction (Fig. 2). Small amounts of budesonide could be found in conjugated fraction (Fig. 3).
O O CH OH CH3 OH OH 3 OH O OH CH CH 3 CH3 3 H H H O OH
H H O H O H
Budesonide 16α-OH-Prednisolone MW = 430 MW = 376
53 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (13). Sport und Buch Strauß - Köln 2005
Fig. 2 - Unconjugated fraction: sample, (A), quality control at MRPL level 30 ng/mL, (B)
A* B 16α-OH-Prednisolone Budesonide isomers 16α-OH-Prednisolone Budesonide isomers RT=3.13 RT=3.16 RT=10.09 / 10.33
C:\Xcalibur\Data\20050020\FF-20050020 2005-02-23 18:46:20 FF-20050020 C:\Xcalibur\Data\20050020\FF-Q050222-20 2005-02-23 19:05:56 FF-Q050222-20
RT: 2.50 - 3.80 SM : 7G RT: 9.40 - 11.00 SM : 7G RT: 2.50 - 3.80 SM : 7G RT: 9.40 - 11.00 SM : 7G RT: 3.13 NL: 4.66E4 NL: 2.05E3 RT: 3.16 NL: 1.12E4 RT: 10.09 NL: 3.81E4 10 0 10 0 10 0 10 0 TIC F: + c sid=-14.00 TIC F: + c sid=-14.00 TIC F: + c sid=-14.00 TIC F: + c sid=-14.00 SRM ms2 SRM ms2 SRM ms2 SRM ms2 90 90 90 90 [email protected] [ [email protected] [ [email protected] [ RT: 10.33 [email protected] [ 146.64-147.44] M S 146.59-147.39] M S 146.64-147.44] M S 146.59-147.39] M S 80 FF-20050020 80 Genesis FF-20050020 80 FF-Q050222-20 80 Genesis FF-Q050222-20 70 70 70 70
60 60 60 60
50 50 50 50
40 40 40 40 Relative Abundance Relative Abundance Relative Abundance Relative Abundance 30 30 30 30
20 20 20 20
10 10 10 10
2.74 3.74 2.80 3.51 3.65 0 0 0 0 RT: 3.12 NL: 1.86E4 NL: 1.02E3 RT: 3.15 NL: 5.80E3 RT: 10.10 NL: 1.93E4 10 0 10 0 10 0 10 0 TIC F: + c sid=-14.00 TIC F: + c sid=-14.00 TIC F: + c sid=-14.00 TIC F: + c sid=-14.00 SRM ms2 SRM ms2 SRM ms2 SRM ms2 90 90 90 90 [email protected] [ [email protected] [ [email protected] [ RT: 10.33 [email protected] [ 172.57-173.37] M S 172.62-173.42] M S 172.57-173.37] M S 172.62-173.42] M S 80 FF-20050020 80 Genesis FF-20050020 80 FF-Q050222-20 80 Genesis FF-Q050222-20 70 70 70 70
60 60 60 60
50 50 50 50
40 40 40 40 2.62 Relative Abundance Relative Abundance Relative Abundance Relative Abundance 30 30 30 2.77 30
20 20 20 20
3.53 3.74 10 2.54 10 10 10 2.77 3.56 0 0 0 0 RT: 3.12 NL: 2.28E4 9.84 NL: 2.12E2 RT: 3.15 NL: 6.27E3 RT: 10.10 NL: 1.14E4 10 0 10 0 10 0 10 0 TIC F: + c sid=-14.00 TIC F: + c sid=-14.00 TIC F: + c sid=-14.00 TIC F: + c sid=-14.00 SRM ms2 SRM ms2 SRM ms2 SRM ms2 90 [email protected] [ 90 [email protected] [ 90 [email protected] [ 90 [email protected] [ 225.81-226.61] M S 224.46-225.26] M S 225.81-226.61] M S RT: 10.34 224.46-225.26] M S 80 FF-20050020 80 Genesis FF-20050020 80 FF-Q050222-20 80 Genesis FF-Q050222-20 70 70 70 70 10 . 5 1 60 60 60 60
50 50 50 50 10 . 10 40 40 10 . 7 2 40 40 Relative Abundance Relative Abundance Relative Abundance Relative Abundance 30 30 30 30 10 . 3 1 20 20 20 20
10 10 10 10
2.74 3.56 3.76 2.77 3.50 0 0 0 0 2.6 2.8 3.0 3.2 3.4 3.6 9.4 9.6 9.8 10 . 0 10 . 2 10 . 4 10 . 6 10 . 8 11. 0 2.6 2.8 3.0 3.2 3.4 3.6 9.4 9.6 9.8 10 . 0 10 . 2 10 . 4 10 . 6 10 . 8 11. 0 Time (min) Time (min) Time (min) Time (min) *Only 16α-OH-prednisolone is found in unconjugated fraction
Fig. 3 - Conjugated fraction after E.Coli hydrolysis: sample, (A), quality control at MRPL level 30 ng/mL, (B)
A B 16α-OH-Prednisolone Budesonide isomers* 16α-OH-Prednisolone Budesonide isomers* RT=3.19 RT=10.12 / 10.36 RT=3.19 RT=10.15 / 10.36
C:\Xcalibur\Data\20050020\EC-20050020 2005-02-24 09:33:30 EC-20050020 C:\Xcalibur\Data\20050020\EC-Q050222-20 2005-02-24 09:53:05 EC-Q050222-20
RT: 2.50 - 3.80 SM : 7G RT: 9.40 - 11.00 SM : 7G RT: 2.50 - 3.80 SM : 7G RT: 9.40 - 11.00 SM : 7G RT: 3.19 NL: 2.46E4 RT: 10.36 NL: 1.51E4 RT: 3.19 NL: 4.35E3 RT : 10.15 NL: 2.88E4 10 0 10 0 10 0 10 0 TIC F: + c sid=-14.00 TIC F: + c sid=-14.00 TIC F: + c sid=-14.00 TIC F: + c sid=-14.00 SRM ms2 SRM ms2 SRM ms2 SRM ms2 90 [email protected] [ 90 [email protected] [ 90 [email protected] [ 90 [email protected] [ RT: 10.36 146.64-147.44] M S 146.59-147.39] M S 146.64-147.44] M S 146.59-147.39] M S 80 EC-20050020 80 EC-20050020 80 EC-Q050222-20 80 EC-Q050222-20
70 70 70 70
60 60 60 60
50 50 50 50
40 40 40 40
Relative Abundance Relative Abundance RT: 10.12 Relative Abundance Relative Abundance 30 30 30 30
20 20 20 20
10 10 10 10 9.80 9.95 9.65 10 . 6 2 3.45 9.65 2.72 2.89 3.51 9.45 2.69 10 . 8 6 0 0 0 0 RT: 3.18 NL: 1.14E4 RT : 10.36 NL: 8.59E3 RT: 3.18 NL: 1.92E3 RT : 10.16 NL: 1.44E4 10 0 10 0 10 0 10 0 TIC F: + c sid=-14.00 TIC F: + c sid=-14.00 TIC F: + c sid=-14.00 TIC F: + c sid=-14.00 SRM ms2 SRM ms2 SRM ms2 SRM ms2 90 [email protected] [ 90 [email protected] [ 90 [email protected] [ 90 [email protected] [ RT: 10.36 172.57-173.37] M S 172.62-173.42] M S 172.57-173.37] M S 172.62-173.42] M S 80 EC-20050020 80 EC-20050020 80 EC-Q050222-20 80 EC-Q050222-20
70 70 70 70
60 60 60 60
50 50 50 50
40 40 40 40
Relative Abundance Relative Abundance RT : 10.13 Relative Abundance Relative Abundance 30 30 30 30 2.71 2.89 20 20 20 20 2.62 9.80 3.59 10 2.89 10 9.98 10 10 9.80 3.51 10 . 6 0 10 . 7 4 9.51 9.63 9.57 10 . 6 6 10 . 7 7 10 . 9 2 0 0 0 0 RT: 3.18 NL: 1.28E4 RT : 10.37 NL: 3.80E3 RT: 3.18 NL: 1.32E3 RT : 10.13 NL: 8.60E3 10 0 10 0 10 0 10 0 TIC F: + c sid=-14.00 TIC F: + c sid=-14.00 TIC F: + c sid=-14.00 TIC F: + c sid=-14.00 SRM ms2 SRM ms2 SRM ms2 SRM ms2 90 [email protected] [ 90 [email protected] [ 90 [email protected] [ 90 [email protected] [ 225.81-226.61] M S 224.46-225.26] M S 225.81-226.61] M S 224.46-225.26] M S 80 80 80 80 EC-20050020 Genesis EC-20050020 Genesis RT : 10.37 Genesis EC-Q050222-20 EC-Q050222-20 70 70 70 70
60 60 60 60
50 50 50 50
40 40 40 40 RT : 10.13 Relative Abundance Relative Abundance Relative Abundance Relative Abundance 30 30 30 30
20 20 20 20 9.81 10 10 10 10 10.57 10.69 2.74 3.53 3.62 9.60 0 0 0 0 2.6 2.8 3.0 3.2 3.4 3.6 9.4 9.6 9.8 10 . 0 10 . 2 10 . 4 10 . 6 10 . 8 11. 0 2.6 2.8 3.0 3.2 3.4 3.6 9.4 9.6 9.8 10 . 0 10 . 2 10 . 4 10 . 6 10 . 8 11. 0 Time (min) Time (min) Time (min) Time (min) *Different composition of budesonide isomers found in the sample (A) than that from spiked urine (B)
54 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (13). Sport und Buch Strauß - Köln 2005
Conclusions - Screening method for Corticosteroids and their metabolites in urine based on XLC-ESI- MSMS was developed; - Conjugated fraction after hydrolysis with β-glucuronidase from E.Coli (or Helix Pomatia) could be analysed with the same XLC-ESI-MSMS system; - The method can be extended for more compounds e. g. new corticosteroids, unconjugated steroids, …; - Confirmation of corticosteroids is performed with the same XLC-system and ESI-MSMS methods modified for respective compounds; - All compounds analysed produce at least two transitions, which is sufficient for confirmation purposes; - If coeluting compounds are present in the sample (e.g. betamethasone and dexamethasone) the LC gradient is modified. - There is a drawback in the on-line extraction system in comparison to the off-line extraction. The limiting factor is the elution of the compounds from the cartridge, which is done with mobile phase.
55 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (13). Sport und Buch Strauß - Köln 2005
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