Triumphs and Challenges of High-Resolution Mass Spectrometry in Comprehensive Residue Screens Florida Department of Agriculture Mark Crosswhite, Ph.D.
outline ur Goal identify and quantify quickly and reliably UHPLC v HPLC easy and cost effective improvement ur Two Platforms rbitrap and Triple Quad advantages of high resolution MS on the Exactive high resolution and high mass accuracy allows for post acquisition data mining limitations of high resolution MS on the Exactive cannot separate compounds with same exact masses no mass filtering fragments can be produced from any ions in the cell Resolution elucidate fragment formulas and structures use trusted triple quad fragments even though their identities are often unknown Exactive Plus vs. ABSciex 5500 - real samples detection quantitation false positive realized Current and Future Strategies
outline ur Goal identify and quantify quickly and reliably UHPLC v HPLC easy and cost effective improvement ur Two Platforms rbitrap and Triple Quad advantages of high resolution MS on the Exactive high resolution and high mass accuracy allows for post acquisition data mining limitations of high resolution MS on the Exactive cannot separate compounds with same exact masses no mass filtering fragments can be produced from any ions in the cell Resolution elucidate fragment formulas and structures use trusted triple quad fragments even though their identities are often unknown Exactive Plus vs. ABSciex 5500 - real samples detection quantitation false positive realized Current and Future Strategies
UHPLC Capacities of orthogonal analyses are multiplicative, not additive. Especially helpful with duty cycle issues in MRM MS (triple quad) and high resolution MS where there may not be MS/MS capabilities. propazine vs sebuthylazine m = 229.1094 Dimethomorph (E and Z) Acquity UPLC BEH C18 Cl Cl 1.7 µm 2.1 x 150 mm Flow rate.200 ml/min 12.71 12.90 18.76 13.98 13.58 UHPLC HPLC UHPLC 12.0 13.0 18.0 14.0 19.0 20.0 12.0 14.0 16.0 Time (min) Time (min) Time (min)
outline ur Goal identify and quantify quickly and reliably UHPLC v HPLC easy and cost effective improvement ur Two Platforms rbitrap and Triple Quad advantages of high resolution MS on the Exactive high resolution and high mass accuracy allows for post acquisition data mining limitations of high resolution MS on the Exactive cannot separate compounds with same exact masses no mass filtering fragments can be produced from any ions in the cell Resolution elucidate fragment formulas and structures use trusted triple quad fragments even though their identities are often unknown Exactive Plus vs. ABSciex 5500 - real samples detection quantitation false positive realized Current and Future Strategies
representation of quadrupole in ms/ms mode Q1 Q2* Q3 mass filters fragments mass measures ion optics representation of rbitrap C-trap HCD cell* Sees what it looks for (MRM) ω=(qk/m) (1/2) orbitrap rbitrap sees everything all the time (full scan) Allows postacquisition data mining * fragments generated by colliding analyte with neutral gas
outline ur Goal identify and quantify quickly and reliably UHPLC v HPLC easy and cost effective improvement ur Two Platforms rbitrap and Triple Quad Advantages of High Resolution MS on the Exactive high resolution and high mass accuracy allows for post acquisition data mining limitations of high resolution MS on the Exactive cannot separate compounds with same exact masses no mass filtering fragments can be produced from any ions in the cell Resolution elucidate fragment formulas and structures use trusted triple quad fragments even though their identities are often unknown Exactive Plus vs. ABSciex 5500 - real samples detection quantitation false positive realized Current and Future Strategies
what high resolution MS will do Phosmet [M + H + ] + = 318.0018 RT = 13.63 min required resolution = 28909 m/(δm) Azinphos methyl [M + H + ] + = 318.0130 RT = 13.29 min S S P S S P Triazophos [M + H + ] + = 314.0723 RT = 14.92 min required resolution = 31407 Hexaconazole [M + H + ] + = 314.0821 RT = 14.96 min Cl S P H Cl
resolving power simazine (202.0854) carbaryl (202.0863) RP required = 224444 thiabendazole (202.0433) resolvable with new rbitrap RP required = 4698 carbaryl (202.0863) 202.07 202.08 202.09 m/z 202.02 202.07 202.12 m/z
314.00 314.08 314.16 m/z 314.00 314.08 314.16 m/z simulation v observed of a 1:10 mixture triazophos [M + H + ] + = 314.0723 hexaconazole [M + H + ] + = 314.0821 simulation resolution observed 314.0835 25,000 314.0707 314.0822 50,000 314.0732 314.0825 100,000 314.0734
outline ur Goal identify and quantify quickly and reliably UHPLC v HPLC easy and cost effective improvement ur Two Platforms rbitrap and Triple Quad advantages of high resolution MS on the Exactive rbitrap high resolution and high mass accuracy allows for post acquisition data mining limitations of high resolution MS on the Exactive cannot separate compounds with same exact masses no mass filtering fragments can be produced from any ions in the cell Resolution elucidate fragment formulas and structures use trusted triple quad fragments even though their identities are often unknown Exactive Plus vs. ABSciex 5500 - real samples detection quantitation false positive realized Current and Future Strategies
what high resolution MS alone will not do Secbumeton [M + H + ] + = 226.1662 RT 8.23 min Prometon [M + H + ] + = 226.1662 RT 8.21 min Terbumeton [M + H + ] + = 226.1662 RT 8.32 min Prometryn [M + H + ] + = 242.1434 RT 10.78 min S Terbutryn [M + H + ] + = 242.1434 RT 10.86 min S
outline ur Goal identify and quantify quickly and reliably UHPLC v HPLC easy and cost effective improvement ur Two Platforms rbitrap and Triple Quad advantages of high resolution MS on the Exactive rbitrap high resolution and high mass accuracy allows for post acquisition data mining limitations of high resolution MS on the Exactive cannot separate compounds with same exact masses no mass filtering fragments can be produced from any ions in the cell Resolution elucidate fragment formulas and structures use trusted triple quad fragments even though their identities are often unknown Exactive Plus vs. ABSciex 5500 - real samples detection quantitation false positive realized Current and Future Strategies
Extracted Ion Chromatogram (upper) Mass Spectra (lower) 8.21 PRMET STADARD Θ * 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min) 226.1660 0 V Collision Energy 15 V Collision Energy 184.1190 Θ 226.1658 45 V Collision Energy * 142.0723 100 120 140 160 180 200 220 240 m/z
Extracted Ion Chromatogram (upper) Mass Spectra (lower) 8.32 TERBUMET STADARD Θ * 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min) 226.1660 0 V Collision Energy 15 V Collision Energy 170.1033 Θ 226.1657 45 V Collision Energy 142.0723 * 100 120 140 160 180 200 220 240 m/z
trusted triple quad fragments real detect.040 ug/g ABSciex 5500 azoxystrobin frag 372.9 intensity (arb) azoxystrobin frag 344.1.041 ug/g 0 V collision energy 15 V collision energy 45 V collision energy Exactive Plus Time extracted ion chromatogram azoxystrobin + H + extracted ion chromatogram mass = 372.9560 extracted ion chromatogram mass = 344.1010
outline ur Goal identify and quantify quickly and reliably UHPLC v HPLC easy and cost effective improvement ur Two Platforms rbitrap and Triple Quad advantages of high resolution MS on the Exactive rbitrap high resolution and high mass accuracy allows for post acquisition data mining limitations of high resolution MS on the Exactive cannot separate compounds with same exact masses no mass filtering fragments can be produced from any ions in the cell Resolution elucidate fragment formulas and structures use trusted triple quad fragments even though their identities are often unknown Exactive Plus vs. ABSciex 5500 - real samples detection quantitation false positive realized Current and Future Strategies
B 3688 compound matrix ABSciex 5500 quantitation (μg/g) Exactive plus quantitation (μg/g) percent difference exact mass (H+ adduct, Da) measured mass (Da) mass error (ppm) azoxystrobin eggplant 0.034 0.034 0.0 404.12410 404.12393 0.0 pyraclostrobin blueberry 0.016 0.016 0.0 388.10586 388.10571 0.0 dimethoate blueberry 0.083 0.096 14.5 230.00690 230.00676 0.1 thiabendazole pear 0.240 0.230 4.3 202.04334 202.04333 0.0 pyrimethanil strawberry 0.016 0.016 0.0 200.11822 200.11809 0.1 flonicamid strawberry 0.068 0.068 0.0 230.05357 230.05344 0.1 fluopicolide onion 0.120 0.120 0.0 382.97271 382.97256 0.0 azoxystrobin onion 0.027 0.026 3.8 404.12410 404.12399 0.0 thiabendazole apple 0.250 0.250 0.0 202.04334 202.04329 0.0 imazalil banana 0.130 0.130 0.0 297.05560 297.05548 0.0
B 3704 compound matrix ABSciex 5500 quantitation (μg/g) Exactive plus quantitation (μg/g) percent difference exact mass (H+ adduct, Da) measured mass (Da) mass error (ppm) methamidophos collards 1.2 0.99 19.2 142.00861 142.00830 0.2 * acephate collards 0.10 0.10 2.0 142.99260 142.99245 0.1 dinotefuran collards 0.43 0.40 7.2 203.11387 203.11356 0.2 thiabendazole papaya 0.070 0.13 60.0 202.04334 202.04294 0.2 chlorantraniliprole collards 1.5 1.1 30.8 481.97807 481.97757 0.1 chlorantraniliprole peas 0.25 0.20 22.2 481.97807 481.97710 0.2 azoxystrobin papaya 0.018 0.019 5.4 404.12410 404.12360 0.1 boscalid blackberries 0.012 0.014 15.4 343.03995 343.03931 0.2 boscalid papaya 0.034 0.030 12.5 343.03995 343.03946 0.1 * possible suppression in source or interference in mass measurement
B 3706 compound matrix ABSciex Exactive 5500 plus quantitation quantitation (μg/g) (μg/g) percent difference exact mass (H+ adduct, Da) measured mass (Da) mass error (ppm) methamidophos acephate bell peppers 0.10 0.10 0.0 142.00861 142.00847 0.1 bell 142.99260 peppers 0.28 0.31 10.2 142.99245 0.1 * carbendazim okra 0.0080 0.011 31.6 192.07675 192.07654 0.1 azoxystrobin bell peppers 0.040 0.041 2.5 404.12410 404.12393 0.0 azoxystrobin bell peppers 0.083 0.086 3.6 404.12410 404.12357 0.1 boscalid tomato 0.015 0.020 28.6 343.03995 343.03943 0.2 methoxyfenozide bell peppers 0.028 0.031 10.2 313.15410 313.15399 0.0 bifenazate bell peppers 0.057 0.054 5.4 301.15467 301.15442 0.1 difenoconazole bell peppers 0.043 0.045 4.5 406.07197 406.07214 0.0 * remember the extracted ion chromatograms using trusted triple quad fragments
B 3707 compound matrix ABSciex Exactive 5500 plus quantitation quantitation (μg/g) (μg/g) percent difference exact mass (H+ adduct, Da) measured mass (Da) mass error (ppm) phorate sulfoxide potatoe 0.059 0.058 1.7 277.01502 277.01483 0.1 trifloxystrobin strawberry 0.056 0.056 0.0 409.13697 409.13663 0.1 pyrimethanil strawberry 0.19 0.20 5.1 200.11822 200.11784 0.2 novaluron strawberry 0.12 0.12 0.0 493.01959 493.01907 0.1 propamocarb squash 0.11 0.11 0.0 189.15975 189.15959 0.1 pyraclostrobin bell pepper 0.029 0.029 0.0 388.10586 388.10522 0.2 pyraclostrobin tomato 0.081 0.08 1.2 388.10586 388.10489 0.3 pyraclostrobin strawberry 0.39 0.39 0.0 388.10586 388.10483 0.3 penthiopyrad bean 0.063 0.063 0.0 360.13519 360.13446 0.2 propamocarb cucumber 0.26 0.26 0.0 189.15975 189.15948 0.1
propoxur (internal control) from B 3707 measured mass of propoxur +H + ppm error average error 210.11224 0.11 0.14 210.11227 0.10 210.11227 0.10 210.11221 0.12 210.11220 0.13 210.11227 0.10 210.11234 0.06 210.11217 0.14 210.11223 0.11 210.11223 0.11 210.11221 0.12 210.11226 0.10 210.11212 0.17 210.11221 0.12 210.11214 0.16 210.11217 0.14 210.11215 0.15 210.11209 0.18 210.11203 0.21 210.11206 0.20 210.11201 0.22 210.11208 0.19 210.11215 0.15 210.11208 0.19 210.11214 0.16 210.11206 0.20
summary information of real samples f ~ 125 samples screened for ~ 250 pesticides per sample on the Exactive Plus we found: no false positives when compared to ms/ms analysis on an ABSciex 5500 (everything confirmed) no false negatives when compared to an ABSciex 5500 (Exactive Plus did not miss anything at the established LDs) the largest percent difference and average percent difference in quantitation between the Exactive Plus and the ABSciex 5500 was 60 and 4 respectively. one false positive on the ABSciex 5500 (not their fault)
real sample false positive realized 302.1/97.2 fenhexamid B3716_R1_01_57782 intensity (arb) 302.1/55.0 fenhexamid B3716_R1_01_57782 2.745 E 5 2.837 E 6 extracted ion chromatogram of fenhexamid +H + on Exactive Plus B 3716_R1_01_57782 Time
outline ur Goal identify and quantify quickly and reliably UHPLC v HPLC easy and cost effective improvement ur Two Platforms rbitrap and Triple Quad advantages of high resolution MS on the Exactive rbitrap high resolution and high mass accuracy allows for post acquisition data mining limitations of high resolution MS on the Exactive cannot separate compounds with same exact masses no mass filtering fragments can be produced from any ions in the cell Resolution elucidate fragment formulas and structures use trusted triple quad fragments even though their identities are often unknown Exactive Plus vs. ABSciex 5500 - real samples detection quantitation false positive realized Current and Future Strategies
our current strategy and flow if no detections screen with Exactive Plus for ~250 compounds if analyte found above LD report report using ABSciex 5500 identification and quantitation using ms/ms, ion ratios and a 6 point calibration curve
our next strategy and flow if analyte found at <.5 tolerance screen with Exactive Plus for ~500 compounds if analyte found at >.5 tolerance report report using ABSciex 5500 estimate and characterization- 1 point calibration standard, and characteristic fragments for confirmation identification and quantitation using ms/ms, ion ratios and a 6 point calibration curve
our following strategy and flow if analyte found at < tolerance screen with Exactive Plus for ~750 compounds if analyte found at > tolerance Exactive Plus report using ABSciex 5500 quantitation and characterization- 6 point calibration curve, and characteristic fragments for confirmation identification and quantitation using ms/ms, ion ratios and a 6 point calibration curve
our ideal strategy and flow routine screen with Exactive Plus for ~1000 compounds if needed/special case Exactive Plus report using ABSciex 5500 quantitation and characterization- 6 point calibration curve, and characteristic fragments for confirmation identification and quantitation using ms/ms, ion ratios and a 6 point calibration curve
acknowledgements Walter Hammack Ghislain Gerard Casey Burrows Erin McAllister Jon Pelt