lc-ms/ms analysis of three antibiotics used in swine ...lc-ms/ms analysis of three antibiotics used...
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![Page 1: LC-MS/MS analysis of three antibiotics used in swine ...LC-MS/MS analysis of three antibiotics used in swine production Jonathan Bailey Dani Degenhardt Allan J. Cessna. Environment](https://reader030.vdocuments.mx/reader030/viewer/2022040813/5e57c96c39bb64416c52ebf3/html5/thumbnails/1.jpg)
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LC-MS/MS analysis of three antibiotics used in swine
production
Jonathan Bailey Dani Degenhardt Allan J. Cessna
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A study was established to determine whether penicillin, tiamulin and oxytetracycline in
raw manure may leach to ground water and contribute to antimicrobial resistance in soil indicator microorganisms
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Figure 2: 9-ha tile drained fieldFigure 2: 9-ha tile drained field
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Study SiteStudy Site
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Sprinkler IrrigationSprinkler Irrigation
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An LC-MS/MS analytical method was needed for the determination of oxytetracycline, penicillin and
tiamulin in the following matrices: 1) tile-drain effluent
2) ground water
3) soil
4) liquid hog manure
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Method Development Strategy• Review Literature• Establish LC-MS/MS separation &
detection parameters• Tile-Drain Effluent and Ground Water:
fortify & determine SPE recoveries• Soil: freeze dry, fortify & use PLE• Liquid Hog Manure: centifuge, dilute &
carry out SPE on liquid component; use PLE on solid component
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Oxytetracycline Literature
1. “Quantitative analysis of oxytetracycline and its impurities by LC-MS-MS” Journal of Pharmaceutical and Biomedical Analysis 34 (2004) 325–332
2. “Aqueous oxytetracycline degradation and the toxicity change of degradation compounds in photoirradiation process” Journal of Environmental Sciences 20 (2008) 806–813
3. “Pre-study and in-study validation of an ultra-high pressure LC method coupled to tandem mass spectrometry for off-line determination of oxytetracycline in nasal secretions of healthy pigs” Journal of Chromatography B 877 (2009) 2349–2357
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Tiamulin Literature
1. “Simultaneous determination of sulfonamides, tetracyclines and tiamulin in swine wastewater by solid-phase extraction and liquid chromatography–mass spectrometry” Journal of Chromatography A 1202 (2008) 173–180
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Penicillin Literature
1. “A versatile liquid chromatography–tandem mass spectrometry system for the analysis of different groups of veterinary drugs” AnalyticaChimica Acta 529 (2005) 137–144
2. “High-throughput analysis of tetracycline and penicillin antibiotics in animal tissues using electrospray tandem mass spectrometry with selected reaction monitoring transition” Journal of Chromatography A 1100 (2005) 193–199
3. “Determination of penicillin G and its degradation products in a penicillin production wastewater treatment plant and the receiving river”Water Research 42 (2008) 307 – 317
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Penicillin
N
S
OOH
H
HN
O
O
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Tiamulin
OH
O
HO
O
SN
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Oxytetracycline
OH
H
O
N
OHO
H
OH
OH
OHHO
NH2
O
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LC-MS/MS
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ESI(+) Collision-induced Dissociation
7.203030
3030
335.0 > 160.0335.0 > 176.0
Penicillin
6.543030494.6 > 192.3Tiamulin
2.373030
3030
461.0 > 426.1461.0 > 443.9
Oxytetracycline
Retention Time(min)
CollisionEnergy
(eV)
ConeVoltage
(V)
MRM TransitionMonitored
(m/z)
Antibiotic
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Transitions Used for Quantification
1. Oxytetracycline (461>426) + (461>444) 2. Tiamulin (494.6>192.3) 3. Penicillin (335>160) + (335>176)
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Oxytetracycline [M+H]+ Precursor ion (461.0)
HO H
O NH+
HO
O
H
HO
OH
OH
OH
NH2
O
Product Ion 1[M+H]+- [NH3] = (444)
Product Ion 2[M+H]+- [NH3] - [H2O]
= (426)
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Tiamulin (percursor ion m/z 494.6) fumarate
+NHS
O
O
HO
O
O
OHO
-O
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Collision-Induced Fragmentation(494.6 > 192.3)
+NHS
O
HO
HO
O
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ESI(+) Fragmentation of Penicillin
N
S
OOH
H
HN
O
O
m/z 160
m/z 176
H+
H+
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MS/MS Parameters
• Instrument: Micromass Quattro Ultima• Source temp: 90°C• Electrospray Interface: ESI(+)• Cone Voltage: 30 V• Capillary voltage setting: 3.02 kV• Hexapole 1: 7.9 V• Hexapole 2: 0 V• Aperture Voltage: 0 V• Desolvation temp: 220°C
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MS/MS Parameters Cont.
• Desolvation gas: N2 @ 488 L h-1
• Cone gas: N2 @ 154 L h-1
• Quadrupole 1: unit mass resolution • Quadrupole 3: ~2.0 m/z resolution• Photomultiplier voltage: 689 V• Collision gas: Argon• Collision gas pressure: 2.04 x 10-4 mbar• Collision energy: 30 eV
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Liquid Chromatographic Parameters• LC Instrument: Waters 2695 Alliance HPLC
System• Column: Waters Xterra: C18: 2.1mm x 150mm• Particle size: 3.5 µ• Column temp: 30oC• Solvent A: 10:90 (Acetonitrile / H2O)• Solvent B: 90:10 (Acetonitrile / H2O)• Modifier: 0.1 % formic acid• Flow rate: 200 µL/min• Separation mode: Gradient• Injection volume: 20 µL
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LC Gradient Table
1109020015.006
1109020010.105
61000.020010.004
61000.02001.103
1110902001.002
110902000.001
CurveB (%)A (%)Flow(uL min-1)
Time(min)
Gradient Step
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2.40Oxytetracycline
6.54Tiamulin
7.20Penicillin
Retention Time (min)
Antibiotics
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SRM Chromatogram of Antibiotics
Oxytetracycline2.40 min
Time1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50
%
0
100
1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50
%
0
100
1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50
%
0
100
461 > 426.1
April16_10_STD3
494.6>192.3
April16_10_STD3
335 > 160
6.718
3.1417
1.568
2.179
Tiamulin6.54 min
Penicillin7.20 min
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Tiamulin Oxytetracycline
Penicillin
Structures of Antibiotics
OH
O
HO
O
SN
OH
H
O
N
OHO
H
OH
OH
OHHO
NH2
O
N
S
OOH
H
HN
O
O
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Method Development Progress Report
• ☑Review Literature• ☑Establish LC-MS/MS separation &
detection parameters• Fortify & determine SPE recoveries in
tile-drain effluent and ground water
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Solid-Phase Extraction Recoveries
124.5 (+/-) 10.7
33.2 (+/-) 5.7
28.0 (+/-) 16.1
HLBSpike (1000 ng L-1)%Recovery + RSD
3
3
3
Number of Replicates
0.0 (+/-) 0.0
74.9 (+/-) 5.0
52.6 (+/-) 11.7
WCX+HLBSpike (1000 ng L-1)%Recovery + RSD
Penicillin(335 > 160) + (335 > 176)
Tiamulin(494.6 > 192.3)
Oxytetracycline(461 > 444) + (461 > 426)
Antibiotic
Citrate Buffer
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Penicillin Stability Study
662747742652887-20oCMethanol (1 ppm)
531142213356364oCMethanol (1 ppm)
7888857837429064oCWater (1ppm)
1004
1093
880
Day 4(ppb)
1000
1001
729
Day 3(ppb)
1003
922
697
Day 2(ppb)
1000
1107
835
Day 1(ppb)
-20oC
4oC
-20oC
Temperature
1001
984
805
Day 5(ppb)
Acetonitrile (1 ppm)
Acetonitrile (1 ppm)
Water (1ppm)
Solvent
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Penicillin procaine (MeOH)
O
O
+NH
NH2
N
S
O
O-
H
NH
O
O
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Protonation of Carbonyl Oxygen
O
O
+NH
NH2
N
S
O
O-
H
NH
O
H+
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MeOH attacks Carbonyl Group
N
S
O
O-
H
NH
O
O
H+
H3C
OH
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Methanolysis results in Methyl Ester
H2N+
S
O
OH
H
HN
O
O OCH3
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Fullscan 4oC MeOH Standard (m/z 367)
Penicillin MEOH_1000ppb_April17_20uL
m/z326 328 330 332 334 336 338 340 342 344 346 348 350 352 354 356 358 360 362 364 366 368 370 372 374
%
0
100April17_10_test2a 235 (1.579) Cm (226:369) Scan ES+
8.19e7367
368
369
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Fullscan 4oC H20 standard (m/z 353)
Penicillin H2O_1000ppb_20uL
m/z324 326 328 330 332 334 336 338 340 342 344 346 348 350 352 354 356 358 360 362
%
0
100Scan ES+
335
353
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Hydrolysis leads to formation of Penicillinoic Acid [M+H]+ (m/z 353)
H2N+
S
O
OH
H
HN
O
O OH
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Conclusions:
• Penicillin procaine salt is not stable in Methanol or H2O at 4oC
• Oxytetracycline will likely prove to be unstable in aqueous solution
• Tiamulin is stable in MeOH
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Next Steps
• Determine SPE recoveries from Tile-drain effluent & Ground Water
• Determine extraction efficiencies from fortified soil using PLE
• Develop method to analyze liquid swine manure
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Acknowledgements
• NSERC for funding from Yost, (Biology U. of R.)
• AAFC & Canada/Saskatchewan Irrigation Diversification Centre
• WCTOW Organizing Committee
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The End
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“A human being is a part of a whole, called by us the Universe - a part limited in time and space. He experiences himself, his thoughts and feelings as something separated from the rest ... a kind of optical delusion of his consciousness. This delusion is a kind of prison for us, restricting us to our personal desires and to affection for a few persons nearest to us. Our task must be to free ourselves from this prison by widening our circle of compassion to embrace all living creatures and the whole of nature in its beauty.” Albert Einstein
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