Investigation of Oxidative and Conjugative Metabolism Reactions with Liquid Chromatography / AccurateMass High Resolution Mass Spectrometry
Maciej Bromirski
Thermo Fisher Scientific (Bremen, Germany)
Denver, June 8, 2011
2
Why?
• In metabolic studies, the identification of metabolites in blood or urine samples as well as liver microsome incubations is relatively easy, but the detection of reactive species turns out to be tricky
• Trapping experiments deliver only indirect proof of identitiy
• Electrochemical simulations of metabolic processes, coupled to high performance liquid chromatography (HPLC), have been shown to be a simple and fast alternative to classical approaches
• Combination of electrochemistry with high resolution accurate mass (HRAM) mass spectrometry may deliver enhanced sensitivity and confidence in metabolite detection, especially for reactive metabolites
Methods
4
Experimental Setup
• For the shown experiments a ROXY™ potentiostat equipped with a Reactor Cell™ thin layer flowcell from Antec B. V. (Zoeterwoude, The Netherlands) was used, coupled to a Thermo Scientific Exactive Benchtop Orbitrap mass spectrometer
• Analyte solutions were provided in water / acetonitrile 1:1 with 0.1% formic acid
• Protein solutions for conjugation experiments were provided in 10 mM ammonium formiate solution, adjusted to pH 7.8
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The ROXY potentiostat
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Infusion experiments for Phase I and Phase II studies
Phase I studies
Phase II studies
only analyte passing EC cell is analysed
conjugation of reaction products with protein after EC cell
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Setup with chromatographic separation
reaction products are trapped in injection loop
content of injection loop is passed to column
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• Resolution100,000 at 1 scan per second 10,000 at 10 scans per second
• Mass accuracy ppm mass accuracy
• Sensitivity500 fg Buspirone > 10:1
• Dynamic Range>4000 within a spectrum
• Scan speedUp to 10 scans per second
• Mass Rangem/z 50 - 4000
• Polarity switchingYes, 1 full cycle <1 sec
Exactive™ - specification
Metabolites of a pesticide:Thiamethoxam
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Reaction products of Thiamethoxam
Voltammogramm Thiamethoxam 20110428 #625 RT: 7.85 AV: 1 NL: 1.88E8T: FTMS + p ESI Full ms [100.00-750.00]
200 210 220 230 240 250 260 270 280 290 300 310m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
247.04140
292.02628
220.09268
245.07022
249.03836 262.05215
294.02322
227.05960204.09781276.03137
230.01485309.05267264.04916
213.09799
243.05437
232.03033303.05478259.10352 267.07803 290.05493278.02841234.02750
274.04871251.03394 296.01865209.10304
218.07700
C8H10O3N5ClS
C8H10O4N5ClS
C7H13O5N3
C8H10O2N4S
C8H12O3N4S
C8H11ON4ClS
C8H12O1N5ClS
C8H10O2N5ClS
C8H11O5N5S
-H2?
Thiamethoxam
+O
11
Voltammogram of selected reaction products
RT: 0.00 - 11.00
0 1 2 3 4 5 6 7 8 9 10 11Time (min)
0
50
1000
50
1000
50
1000
50
1000
50
100
Re
lativ
e A
bu
nd
an
ce 0
50
1000
50
1000
50
10010.604.983.52 4.642.57 5.054.193.161.01 5.662.08 10.216.320.63 1.31 9.217.10 9.158.717.12
7.65 7.857.51 8.136.47 7.33
6.43 8.376.31 8.536.125.58 8.723.43 3.97 4.242.431.200.62 3.181.94 9.38
7.897.617.45 8.10 8.296.926.53
8.466.328.62 8.876.16 9.505.49
8.09 8.147.85
7.707.46 8.41
8.59 8.777.29 9.28 9.642.29 10.646.600.08 4.213.31 4.691.49 5.990.47 2.92 5.763.858.338.27
8.377.828.577.51 8.79
9.227.409.70 10.217.261.540.44 1.22 1.94 2.64 4.023.22 4.37 7.015.00 5.76
8.298.14 8.337.65
8.587.518.78 9.217.36 9.69 10.261.520.15 7.220.54 2.00 3.472.90 3.962.68 4.37 4.84 5.14 6.955.57
7.94 8.027.757.438.46
7.197.06 8.636.60 8.82
9.289.816.35 10.631.550.52 6.110.85 1.88 2.73 5.453.42 3.66 4.39
8.148.09 8.247.74
8.417.51 8.57
7.43 8.93 9.21 9.84 10.207.251.360.81 1.570.04
NL: 2.32E8m/z= 292.02479-292.02771 F: FTMS + p ESI Full ms [100.00-750.00] MS Voltammogramm Thiamethoxam 20110428
NL: 1.13E7m/z= 308.01961-308.02269 F: FTMS + p ESI Full ms [100.00-750.00] MS Voltammogramm Thiamethoxam 20110428
NL: 4.91E6m/z= 290.05255-290.05545 F: FTMS + p ESI Full ms [100.00-750.00] MS Voltammogramm Thiamethoxam 20110428
NL: 2.80E7m/z= 276.03002-276.03278 F: FTMS + p ESI Full ms [100.00-750.00] MS Voltammogramm Thiamethoxam 20110428
NL: 7.41E7m/z= 262.05093-262.05355 F: FTMS + p ESI Full ms [100.00-750.00] MS Voltammogramm Thiamethoxam 20110428
NL: 2.27E8m/z= 247.04016-247.04264 F: FTMS + p ESI Full ms [100.00-750.00] MS Voltammogramm Thiamethoxam 20110428
NL: 9.55E7m/z= 245.06907-245.07153 F: FTMS + p ESI Full ms [100.00-750.00] MS Voltammogramm Thiamethoxam 20110428
NL: 2.37E7m/z= 230.01365-230.01595 F: FTMS + p ESI Full ms [100.00-750.00] MS Voltammogramm Thiamethoxam 20110428
Thiamethoxam
M1 m/z 308
M2 m/z 290
M3 m/z 276
M4 m/z 262
M5 m/z 247
M6 m/z 245
M7 m/z 230
12
Reaction product 1: Oxygenated Thiamethoxam?
YES!
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... and it is not Glutathion
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Reaction product 2: Dehydrogenated Thiamethoxam?
NO!
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... but what else is it???
• Dehydrogenated Thiamethoxam: C8H8O3N5ClS
• exact mass 290.01091
• Measured mass: 290.05487• on an Exactive, this is a clear mismatch
• Proposed elemental composition: C8H11O5N5S
• Proposed structure:N
SNO
N N
NO O
O
OH
N
SNO
N N
NO O
Cl
Metabolites of a drug:Ticlopidin
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Reaction products of Ticlopidin
260 262 264 266 268 270 272 274 276 278 280 282m/z
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
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Re
lativ
e A
bu
nd
an
ce
264.0598
266.0567
265.0632
267.0600
262.0441
264.0594
260.0290
278.0390266.0566263.0477
265.0630 280.0359261.0324
279.0423
267.0601
NL: 2.49E6data06#130-145 RT: 1.14-1.27 AV: 16 T: FTMS {1,1} + p ESI Full ms [50.00-1000.00]
NL: 1.46E6data06#663 RT: 5.76 AV: 1 T: FTMS {1,1} + p ESI Full ms [50.00-1000.00]
0.0 V EC potential
2.5 V EC potential
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Metabolites confirmed by microsome incubation1
• 264.0594: Ticlopidin• 262.0441: Ticlopidin – 2H• 260.0290: Ticlopidin – 4H
• 278.0390: Ticlopidin – 2H + O
• 280.0544: Ticlopidin + O
1 Davie et al., DMD 32:49–57, 2004
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A closer look on the oxygenation products
data06 #663 RT: 5.76 AV: 1 NL: 3.17E5T: FTMS {1,1} + p ESI Full ms [50.00-1000.00]
278.0 278.5 279.0 279.5 280.0 280.5 281.0 281.5 282.0m/z
0
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60
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95
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Re
lativ
e A
bu
nd
an
ce
278.0390
280.0359
279.0423
282.0515281.0392
C14H12ONClSTiclopidin – 2H +OΔm 1.3 ppm
C14H14ONClSTiclopidin +OΔm 1.1 ppm
data06 #663 RT: 5.76 AV: 1 NL: 1.03E5T: FTMS {1,1} + p ESI Full ms [50.00-1000.00]
279.9 280.0 280.1 280.2m/z
0
10
20
30
40
50
60
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80
90
100
110
Re
lativ
e A
bu
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280.0359
280.0544
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Summary
• Metabolites can be detected with high sensitivity and specificity
• Ultra high resolution provides clear separation from interferences
• Unique scan-to-scan mass accuracy provides clear distinction of false positives from correct signals
• High confidence in identification is provided
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Acknowlegements
• Antec B. V., Zouterwoude, The Netherlands• Agnieszka Kraj
• University of Muenster, Germany• Uwe Karst
• Anne Baumann
• Thorsten Vielhaber
• Thermo Fisher Scientific, Germany• Olaf Scheibner
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