LC/MS Chad Borges, Matthew Slawson, James Taccogno, and Dennis Crouch Center for uman Toxicology University of Utah Salt Lake City, UT USA John M. ughes Agilent Technologies, Inc. Pleasanton, CA USA (oa-tof) Agilent LC/MSD TOF 1 2 ng/ml 4 1 TOF (2 ppm ) (EIC) LC/MSD TOF EIC 1.8 C18 [1 3] ( BALCO TG ) / (GC/MS) [2] EI GC/MS [4] GC/MS [5] GC/MS GC/MS GC/MS (SRM) (MRM) / (LC/MS) [6]
API LC/MS 40 GC/MS LC/MS [7] 2 -ng/ml [ (WADA) (MRPL)] WADA MRPL Agilent LC/MSD TOF 1 (ISTD) (ESI) APCI APCI ESI (Na + K + ) APCI 1 APCI ESI O Cl 2 N Cl N 3 C C 3 C 3 O Clenbuterol 19-norandrosterone N N 3 C 3 C C 3 O C 3 O C 3 3'- stanozolol 3 C 3 C Methyltestosterone (internal standard) Methyltestosterone metabolite (17α-methyl-5β-androstane-3α,17β-diol) C 3 O O Epitestosterone Epimetendiol (methandrostenolone metabolite) 1. 2
[M+] + m/z 121.050873 922.009798 2 ( P-921) LC MS APCI 1 GC/MS the Center for uman Toxicology, Sports Medicine Research and Testing Laboratory LC/MS GC/MS 3 ml (10 ng/µl 20 µl) 0.15 M 1mL p 5 Extrelut-3 (MerckVMR 48219-494 100 ) 1 g SPE (J.T.Baker VMR J7088-3 50 ) 8 9 ml 13 x 100 mm LC/MS 40 Agilent laboratory (Pleasanton, CA) -10 100 µl LC LC/MS API-TOF Agilent 1100 LC ( UV-VIS ) G1969A LC/MSD TOF ESI APCI Agilent 1. LC : Agilent ZORBAX RRT SB-C18 2.1 50 mm 1.8 µm (Agilent 822700-902) : A = 0.1% / B = : 0.4 ml/min : 55 ºC : 55% B 5 55% 75% B (5 9 ) : 14 : 5 : 4 µl MS : APCI () : 3,500 V : 450 ºC : 4 µa : : 60 psig 5 L/min : 350 ºC : : : : LC/MS m/z 100 1000 10,000 / (0.89 /) 121 922 (5 µl/min 10 µm ) 150 V [ (CID) ] 60 V ( ) RF: 250 V ( ) 3
API-TOF LC/MS GC/MS LC/MS m/z 7,000 2 ppm MRM-MS/MS MS/MS / () ESI 2 ppm [M+] + m/z APCI APCI APCI ESI (Na + K + ) 2 (3 ml 16 ng/ml ) 15 / Base Peak Chrom. of +TOF MS: from steroid2tofap42.wiff Intensity, cps 1.3e5 1.2e5 1.1e5 1.0e5 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e4 0.530 Clenbuterol 3 stanozolol 2.620 5.478 Epitestosterone MeTestosterone (IS) 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 Time, min 5.749 6.625 8.086 19-norandrosterone MeTestosterone metabolite Epimetendiol 11.064 2. ( 16 ng/ml ()) 4
(CID) CID 50V LC/MSD TOF 3 APCI [M+] + [M+-18] + [M+] + -1.04 ppm 3 [M+] + 6,890 [M+] + m/z 290 13 C m/z 289.2159 (R)m/z (FWM w 1/2 Da) R = M/w 1/2 M = 289.2159M ( ) w 1/2 = 0.042 Da R = 289.2159/0.042 = 6890 13 C [M+] + Intensity, counts 1.10e5 1.00e5 9.00e4 8.00e4 7.00e4 6.00e4 5.00e4 4.00e4 3.00e4 2.00e4 1.00e4 0.00 +TOF MS: 5.474 to 5.547 min from steroid2tofap42.wiff Agilent, subtracted (4.696 to... 120 Epitestosterone, MW 288, Expected (M+) + 289.2162 Observed (M+) + 289.2159 Mass accuracy (mass error) = (mass obs _ massexp )/mass exp ) 10 6 (289.2159 _ 289.2162)/289.2162) 10 6 = _ 1.04 ppm 271.2051 269.1893 289.2159 287.1997 Max. 1.2e5 counts. +TOF MS: 5.474 to 5.547 min from steroid2tofap42.wiff 140 160 180 200 220 240 260 280 300 320 340 360 380 400 m/z, amu Intensity, counts 1.10e5 1.00e5 9.00e4 8.00e4 7.00e4 6.00e4 5.00e4 4.00e4 3.00e4 2.00e4 1.00e4 0.00 289.2159 289.4107 Max. 1.2e5 counts. Resolution 6890 [(m/z)/w 1/2 ] 290.2190 288.0 288.5 289.0 289.5 290.0 290.5 291.0 291.5 292.0 m/z, amu 3. APCI TOF [M+] + = 289.2162 m/z 271.2051 5
(m/z 271 ) 19- [ 4] m/z 259.2049 241.1949 1 2 ESI 4 121 922 LC T MS 2 P-921 (Agilent API ) LC/MSD TOF 5.0e4 4.5e4 4.0e4 +TOF MS: 6.596 to 6.650 min from steroid2tofap42.wiff Agilent, subtracted (7.374 to... 241.1949 Observed (M+ _ 2 2 O) + m/z 241 Resolution 6403 _ 0.73 ppm 19-norandrosterone, MW 276 3.5e4 259.2049 Intensity, counts 3.0e4 2.5e4 2.0e4 Ref mass 8000 counts Observed (M+ _ 2 O) + m/z 259 Resolution 6464 _ 2.86 ppm 1.5e4 1.0e4 121.0502 Ref mass 8000 counts 922.0080 5000.0 0.0 200 300 400 500 600 700 800 900 1000 m/z, amu 4. 19-276 2 m/z 121 922 6
19-50 ng/ml ISTD 66 ng/ml 5 3' - (EIC) Base Peak Chrom. of +TOF MS: from steroid2tof092007.wiff Intensity, counts 3.0e6 2.8e6 2.6e6 2.4e6 2.2e6 2.0e6 1.8e6 1.6e6 1.4e6 1.2e6 1.0e6 8.0e5 6.0e5 4.0e5 2.0e5 0.0 0.535 4.207 Epitestosterone+IS 19-norandrosterone 4.958 6.333 MeTestosterone metabolite 11.352 Epimetendiol 0 2 4 6 8 10 12 14 Time, min 5. 50 ng/ml 7
2 ng/ml 19-1 ng/ml (MRPL) EIC 6 EIC 50,000 100,000 S/N EIC TOF EIC 1 mda ( 3 ppm) 19-241.1949 [M+ - 2 2 O] + EIC 241.1944 241.1954 TOF S/N 19-6. 2 ng/ml(19-1 ng/ml) EIC (1 mda ) 8
6 7 TOF 100% 240 pg (19-120 pg) 2 ppm (IS): -0.33 ppm : 1.59 ppm 19- : 1.99 ppm : 1.84 ppm : 1.78 ppm 7. 2 ng/ml (19-1 ng/ml) ( 240/120 pg) [ 6 ] 9
LC/MSD TOF 19- ( 3 ml 0.16 ng/ml ) 20 pg 4 µl (1 mda ) EIC 8 S/N 1.8 -µm 3.5 -µm 1.8 -µm 19-8. 0.16 ng/ml ( 20 pg) EIC (1 mda ) 10
GC/MS () API-TOF LC/MS 1 2 ng/ml LC/MSD TOF EIC S/N 100 µl 4 µl 1 EIC (m/z 1 mda EIC) ng/ml 2 ppm 1. A. Leinonen, T. Kuuranne, T. Kotiaho, and R. Kostiainen, Screening of unconjugated anabolic steroids in urine by liquid chromatography/ mass spectrometry. In W. Schanzer,. Geyer, A. Gotzmann, and U. Mareck, Recent advances in doping analysis. (2003) Sport und Buch Straub, Koln, 11, 163. 2.. Pereira, M. Marques, I. Talhas, and F. Neto, Analysis of androgenic steroids, beta-2-agonists and other substances by GC-MS-ITD. In W. Schanzer,. Geyer, A. Gotzmann and U. Mareck, Recent advances in doping analysis. (2003) Sport und Buch Straub, Koln, 11, 259. 3. T. uynh, G. Trout, and R. Kazlauskas, The detection of low level anabolic agents in bovine and human urine using LC-ESI-MS-MS. In Recent advances in doping analysis. (2003) Sport und Buch Straub, Koln, 11, 271. 4. G. Trout, S. Soo, and R. Kazlauskas, Single screen for steroids using RMS. In Recent advances in doping analysis, (2003) Sport und Buch Straub, Koln, 11, 249. 5. I. Ojanpera, A. Pelander, S. Laks, M. Gergov, E. Vouri and M. Witt, Application of accurate mass measurement to urine drug screening. (2005) J Anal Tox, 29, 34. 6. L. Politi, A. Groppi, and A. Polettini, Application of liquid chromatography-mass spectrometry in doping control. (2005) J Anal Tox, 29, 1. 7. M. Gergov, I. Ojanpera and E. Vuori, Simultaneous screening for 238 drugs in blood by liquid chromatography-ionspray tandem mass spectrometry with multiple reaction monitoring. (2003) J Chrom B, 795, 41. Agilent Technologies, Inc. John ughes Agilent (www.agilent.com/chem/jp) 11
www.agilent.com/chem/jp Agilent 0120-477-111 Agilent Technologies, Inc. 2006 Printed in Japan March 20, 2006 5989-4738JAJP