1. Mass Spectrometry II

2. Ion trap

3. Magnetic Sector FBFB

6. Time of Flight MS In a quadrupole MS masses are separated based on their flight path In a time-of-flight MS masses are separated based on their inertia detector Ion-source + + + + + + + + + + + + + + + + + + + + + + + + + + + Field-free flight zone Mass is determined by the time it takes the ion to move from source to detector t = L (length) (velocity) = 2V (m/z) √ + - (acceleration voltage) (mass-to-charge) Acceleration zone

7. 235.16 to 235.19 amu 281.13 to 281.15 amu 193.12 to 193.14 amu 179.10 to 179.12 amu 169.09 to 169.08 amu 301.14 to 301.16 amu

8. MS Application

9. Environmental Issue: Presence of bioloigcally-active compounds (pharmaceuticals…) that are not degraded in a wastewater treatment plant Compound of interest is trimethoprim, an anti-microbial used to treat infectious diseases in humans Analytical - want to analyze for trimethoprim LC/GC? Ionization source? LC – why? ESI – why? Hypothesis Primary treatment of wastewater with nitrifying activated sludge will degrade trimethoprim MS Application

10. Hypothesis Primary treatment of wastewater with nitrifying activated sludge will degrade trimethoprim Experiment Small scale laboratory batch reactor was used to degrade trimethoprim LC-ESI coupled to a single quadrupole MS to determine the unit mass of degradation products LC-ESI coupled to an ion trap MS to determine structural features of the degradation products LC-ESI coupled to a quadrupole time of flight MS to determine the accurate mass of the degradation products to get a chemical formula MS Application

11. Hypothesis Primary treatment of wastewater with nitrifying activated sludge will degrade trimethoprim Experiment Small scale laboratory batch reactor was used to degrade trimethoprim LC-ESI coupled to a single quadrupole MS to determine the unit mass of degradation products LC-ESI coupled to an ion trap MS to determine structural features of the degradation products LC-ESI coupled to a quadrupole time of flight MS to determine the accurate mass of the degradation products to get a chemical formula MS Application

12. Data from LC-ESI-single quad MS Parent [M+1] MW: 290.32 g mol -1 Degradation products Anything strange… Total ion chromatogram Single ion chromatogram MS Application

13. Experiment LC-ESI coupled to a single quadrupole MS to determine the unit mass of degradation products 2 degradation products [M+H] of 307 m/z and 325 m/z Increased mass but earlier LC elution Suggests oxidation Mass difference between 291 m/z and 307 m/z is 16 m/z (oxygen) Mass difference between 291 m/z and 325 m/z is 34 m/z (2O and 2 H) MS Application

14. Data from LC-ESI-Ion trap MS (MS 2 ) LC mobile phase is H 2 O and CH 3 OH 291 (trimethoprim) trapped in ion trap Accelerated/dissociated and the products detected [M+H] is 291 291 m/z → products MS Application

15. MS Applications LC-ESI coupled to an ion trap MS to determine structural features of the degradation products Parent Demonstrates that the phenyl rings are recalcitrant to dissociation Useful information for the ion trap analysis of the degradation products Experiment LC-ESI coupled to a single quadrupole MS to determine the unit mass of degradation products 2 degradation products [M+H] of 307 m/z and 325 m/z Increased mass but earlier LC elution Suggests oxidation Mass difference between 291 m/z and 307 m/z is 16 m/z (oxygen) Mass difference between 291 m/z and 325 m/z is 34 m/z

16. Data from LC-ESI-Ion trap MS (MS 2 ) Degradation product 307 m/z trapped in ion trap Accelerated/dissociated and the products detected D 2 O and CD 3 OD 307-18 Loss of water [M + H – H 2 O] + 307 m/z → products MS Application

17. Data from LC-ESI-Ion trap MS (MS 3 ) Degradation product 307 m/z trapped in ion trap Accelerated/dissociated and 289 m/z trapped in ion trap Accelerated/dissociated and the products detected D 2 O and CD 3 OD Can be formed from the dissociation of 307m/z or 289 m/z 307 m/z → 289 m/z → products MS Application

18. Data from LC-ESI-Ion trap MS (MS 3 ) Degradation product 307 m/z trapped in ion trap Accelerated/dissociated and 274 m/z trapped in ion trap Accelerated/dissociated and the products detected D 2 O and CD 3 OD 307 m/z → 274 m/z → products Can be formed from the dissociation of 289 m/z and 274 m/z MS Application

19. Products Use MS 3 data to propose a fragmentation pathway for 307 m/z degradation product Structure confirmation LC-ESI coupled to an ion trap MS to determine structural features of the degradation products Parent Demonstrates that the phenyl rings are recalcitrant to dissociation Useful information for the ion trap analysis of the degradation products Experiment LC-ESI coupled to a single quadrupole MS to determine the unit mass of degradation products 2 degradation products [M+H] of 307 m/z and 325 m/z Increased mass but earlier LC elution Suggests oxidation Mass difference between 291 m/z and 307 m/z is 16 m/z (oxygen) Mass difference between 291 m/z and 325 m/z is 34 m/z MS Application

20. - H, - OH, - CH 3 Data from LC-ESI-Ion trap MS (MS 2 and MS 3 ) Propose fragment structures MS Application

21. Hypothesis Primary treatment of wastewater with nitrifying activated sludge will degrade trimethoprim Experiment Small scale laboratory batch reactor was used to degrade trimethoprim LC-ESI coupled to a single quadrupole MS to determine the unit mass of degradation products LC-ESI coupled to an ion trap MS to determine structural features of the degradation products LC-ESI coupled to a quadrupole time of flight MS to determine the accurate mass of the degradation products to get a chemical formula Answer Degradation of trimethoprim is possible using nitrifying activated sludge Degradation products identified using several MS techniques MS Application

22. Data from LC-ESI-Ion trap MS LC mobile phase is D 2 O and CD 3 OD 296 (d-trimethoprim) trapped in ion trap Accelerated/dissociated and the products detected [M+D] is 296 MS Application

23. Data from LC-ESI-Ion trap MS D 2 O and CD 3 OD H 2 O and CH 3 OH MS Application