Experiences with application of full scan high resolution MS for pesticide residue analysis Marc Tienstra, Hans Mol, Paul Zomer? EPRW; Cyprus; 25-May-216
Current routine practice Chromatography with mass spectrometric detection Pesticide Residue analysis only GC amenable both GC and LC amenable only LC amenable Pesticide Manual: 163 entries Current golden standard: Targeted quantitative measurement by LC-MS/MS and GC-MS/MS Multi-residue methods, ~15-25 analytes/method Emerging: Non-targeted measurement by LC and GC + full scan MS for better coverage of pesticide scope and easier measurement
Selectivity using HR-MS Extract signal of exact mass ± x Da (ppm), e.g. Dimethoate [M+H] + 23.69 ± 5 ppm (±.12 Da) 15.67 7.53 3 m/z 23.69 T C 5 H 12 NO 3 PS 2 5 9 p dimethoate [1 1 8 6.57 Q 2 5 7 9.44 13.55 14.2 m/z 23.536 C 9 H 6 F 3 N 3 O flonicamid Relative Abundance 6 5 4 9.3 9.88 11.25 1.19 12.24 13.42 m/z 365.1449 C 19 H 25 ClN 2 OS pyridaben 3 2 1 2.49 5.43 5.5 7.1 6.66 8.73 7.92 9.13 12.32 15.2 2 4 6 8 1 12 14 16 Time (min) Leek spiked @ 1 ppb, Full scan m/z 135-; Res = 7,
Work flow LC-ESI-Q-Orbitrap-MS Sample Extract QuEChERS extraction (215) GC-EI-Q-Orbitrap-MS Quan (usual suspects): XICs, MEW ± 5 ppm, 2 ions Manual verification of all targets in all samples Extensive AQC: Linearity, LOQ recoveries/rsds Identification: peaks 2 ions fully overlap RT ±.1 min from sequence, ion ratio ±3% (rel) Raw data Qual (others): XICs, MEW ± 5 ppm, 2 ions Fully automated by software Detection: RT ±.5 min from database, peaks detected for both ions
LC-HRMS: Identification LC-HRMS LC-ESI-MS: soft ionisation no fragments Generation of fragments: 1) needed for identification, 2) improve screening selectivity For optimum detection and identification: full scan acquisition without and with fragmentation in 1 run Non-targeted fragmentation: two options: AIF and vdia
Combined Full scan + vdia acquisition FS: m/z - Ϟ: -2 Ϟ: 2-3 Ϟ: 3-4 Ϟ: 3-4 Ϟ: 5- LC-HRMS FS: m/z - Upper & lower m/z cut-off m/z isolation window
Combined Full scan + vdia acquisition FS: m/z - Ϟ: -2 Ϟ: 2-3 Ϟ: 3-4 Ϟ: 3-4 Ϟ: 5- LC-HRMS FS: m/z - Upper & lower m/z cut-off m/z isolation window
Combined Full scan + vdia acquisition FS: m/z - Ϟ: -2 Ϟ: 2-3 Ϟ: 3-4 Ϟ: 3-4 Ϟ: 5- LC-HRMS FS: m/z - Upper & lower m/z cut-off m/z isolation window
Scan speed sufficient (FS + vdia)? LC-HRMS full scan: no fragmentation m/z 135-@7K Fragments of 95-25@35K Fragments of 195-35@35K Fragments of 295-45@35K Fragments of 395-55@35K Fragments of 495-5@35K 978 ms RT: 6.87-7.47 Relative Abundance 5 ppb Clothiadinin (C 6 H 8 ClN 5 O 2 S) in lettuce 9 8 7 6 5 4 3 2 1 9 8 7 6 5 4 3 2 1 7.17 7.16 7.14 7.13 7.12 7.15 7.13 7.16 7.19 7.18 7.21 7.2 7.22 7.24 7.21 7.23 [M+Na]+ m/z 271.9979 6.9 7. 7.1 7.2 7.3 7.4 Time (min) vdia m/z 195-35 fragment C4H3NCl5 m/z 131.96692 RT: 11.94-12.54 Relative Abundance 9 8 7 6 5 4 3 2 1 9 8 7 6 5 4 3 2 1 5 ppb Profenofos (C 11 H 15 BrClO 3 PS)in lettuce 12.22 12.2 12.23 12.25 12.26 12.28 12.18 12.3 12.24 12.22 12.21 12.19 12.17 12.26 12.27 12.29 1 12. 12.1 12.2 12.3 12.4 Time (min) [M+H]+ m/z 372.9424 vdia m/z 295-45 Fragment C 6 H 6 81 BrClO 3 PS m/z 34.86212
Quantitative data review LC-HRMS Tracefinder (Thermo Scientific TM software) Review by pesticide (compound view): For each quan pesticide: click through the samples and check asignment/integration of quantifier (main adduct) and qualifier (fragment), adjust when needed Quantifier OK Qualifier OK
Quantitative data review LC-HRMS delete
Qualitative screening: method set up LC-HRMS Same raw data, different data review High number of target pesticides, low probability of detection Manual verification of all XICs too time consuming Automated pesticide detection by the software (Tracefinder; Thermo Scientific ) Default settings for pesticide detection (quan module used without quantification) Mass extraction window: Time window: Requirement: Output: exact m/z ±5 ppm database RT ±.5 min signal found for pre-set adduct AND fragment ion report of samples showing only pesticides found
Screening: data review LC-HRMS Review by sample (sample view): For each sample, click through the pesticides found: Check: 2 peaks present? Matching peak profile/rt? Optional: isotope pattern, additional fragments 12.17 Prosulfocarb? Reject X
Screening: data review LC-HRMS Terbutylazine? (upon quantification: << 1 ppb)
GC-HRMS: introduction Features/specifications: Q Exactive GC Resolution FWHM @ m/z 2 (scan speed) 15,; 3,; 6,; 12, ~ 18 Hz 3 Hz m/z 3-3 Acquisition options: Non-targeted (full scan) / Targeted (SIM, MS/MS, data-dependent MS/MS) Mass accuracy: internal < 1 ppm RMS; external < 3 ppm RMS Dissociation in source EI / PCI / NCI
Identification GC-HRMS GC-EI-MS: hard ionisation techique, multiple fragments => one acquisition event suffices 13April15_6K_W O_LM _1 #3316 RT: 16.68 AV: 1 SB: 142 16.42-16.63, 16.76-17.4 NL: 1.3E6 T: FTMS + p EI Full m s [5.-5.] Relative Abundance 9 8 7 6 5 4 3 2 1 159.9841 169.9684 194.93433 23.92947 23.9118 236.8476 215.88751 249.84859 186.98241 266.88776 Mw 44 338.87242 142.9311 276.87214 12.9638 71.8554 85.1118 118.9312 294.88255 57.6989 322.87736 357.84631 373.1763 415.2886 242.91116 beta-endosulfan QEGC 6 8 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 42 m/z beta-endosulfan NIST Mw 44
Scanspeed and XICs vs TIC Lindane C6Cl3H4 1 ppb in leek Pendimethalin C11H14N3O4 1 ppb in leek GC-HRMS TIC RT: 12.2-12.12 9 8 Relative Abundance 7 6 5 4 3 2 1 18.93731 NL: 1.31E m/z= 18.9364 18.9382 p EI Full m [5.-5 3April15 252.9788 191.6893 12.5 12.1 Time (min) 6K 5 Hz 162.7876
Method (datahandling) GC-HRMS Thermo Scientific TM Software: XCalibur TraceFinder 3.3 EFS NIST library Pesticide database with t r and exact masses of (fragment) ions On-going (currently 574 pesticides) Method development: Default MEW: ± 5 ppm Assess sensitivity/selectivity of the different ions available for each pesticide CompoundNa RT Q;q1;q2;q3;q4;q5;q6 Q Q q1 q1 q2 q2 q3 q3 q4 q4 q5 q5 2-phenylphenol 9.47 17;141;115 C12H9O 169.6479 C12H1O 17.7262 C11H9 141.6988 C9H7 115.5423 Azoxystrobin 22.76 344;388;329;172;36;43 C2H14N3O3 344.1297 C21H14N3O5 388.928 C19H11O3N3 329.7949 C1H6O2N 172.3931 C2H14O4N3 36.9788 C22H17N3O5 43.11627 Bifenthrin 18.51 181;166;141;165;153 C14H13 181.1118 C13H1 166.777 C13H9 165.6988 C12H9 153.6988 Biphenyl 7.88 154;153;152;76 C12H1 154.777 C12H9 153.6988 C12H8 152.625 Bitertanol 2.11 17;168;112;(141;152) C12H1O 17.7262 C8H14ON3 168.11314 C4H6ON3 112.554 C11H9 141.6988 C12H8 152.625 boscalid 21.3 14;142;342;344;112 C6H3ONCl 139.98977 C6H3ONCl(37) 141.98682 C18H12Cl2N2O 342.3212 C18H12ClCl(37)N2O 344.2917 C5H3NCl 111.99485 Bupirimate 16.3 273;193;28;316;166 C1H17N4O3S 273.1159 C1H17N4 193.14477 C11H18N3O 28.14444 C13H24N4O3S 316.15636 C8H12ON3 166.9749 Captan 15.9 79;149;17;8 C6H7 79.5423 C8H7NO2 149.4713 C7H7O 17.4919 C6H8 8.625 chlorfenvinphos 15.8 267;269;323;295;325 C8H6O4Cl2P 266.93753 C8H6O4ClCl(37)P 268.93458 C12H14O4Cl2P 323.13 C1H1O4Cl2P 294.96883 C12H14O4ClCl(37)P 324.99718 Chlorobenzilate 16.71 251;139;253;141;111 C13H9OCl2 251.25 C7H4ClO 138.99452 C13H9OClCl(37) 252.99955 C7H4Cl(37)O 14.99157 C6H4Cl 11.9996 Chloropropylate 16.71 251;139;253;141;111 C13H9OCl2 251.25 C7H4ClO 138.99452 C13H9OClCl(37) 252.99955 C7H4Cl(37)O 14.99157 C6H4Cl 11.9996 Chlorothalonil 12.46 266;264;268;124;229 C8Cl3Cl(37)N2 265.8786 C8Cl4N2 263.8811 C8Cl2Cl(37)2N2 267.87511 C8N2 124.56 C8N2Cl3 228.91216 Chlorpropham 11.5 127;171;213;154 C6ClH6N 127.1833 C7H6NO2Cl 171.816 C1H12ClNO2 213.5511 C8H9NCl 154.418 Chlorpyrifos-ethyl 14.22 197;199;258;313;125 C5Cl3H2NO 196.91965 C5H2Cl2Cl(37)NO 198.9167 C5H3Cl2NO3PS 257.89428 C9H11Cl2NO3PS 313.95688 C2H6O2PS 124.9826 Chlorpyrifos-methyl 13.28 286;288;125 C7H7Cl2NO3PS 285.92558 C7H7ClCl(37)NO3PS 287.92263 C2H6O2PS 124.9826 coumaphos 2.28 226;362;21;19;182 C1H7O2ClS 225.98498 C14H16ClO5PS 362.1391 C1H7O3Cl 21.782 C2H6O3P 19.491 C9H7O2Cl 182.1291 Cyhalothrin I 19.31 197;141;28;181 C8H9ClF3 197.3394 C8H7F2 141.513 C14H1ON 28.7569 C13H9O 181.6479 C14H11NO 29.8352 Cyhalothrin-lambda 19.48 197;141;28;181 C8H9ClF3 197.3394 C8H7F2 141.513 C14H1ON 28.7569 C13H9O 181.6479 C14H11NO 29.8352 Cypermethrin I 21.5 163;127;29;181 C7H9Cl2 163.758 C7H8Cl 127.39 C14H11NO 29.8352 C13H9O 181.6479 C12H8 152.625 Cypermethrin II 21.17 163;127;29;181 C7H9Cl2 163.758 C7H8Cl 127.39 C14H11NO 29.8352 C13H9O 181.6479 C12H8 152.625 Cypermethrin III 21.21 163;127;29;181 C7H9Cl2 163.758 C7H8Cl 127.39 C14H11NO 29.8352 C13H9O 181.6479 C12H8 152.625 Cypermethrin IV 21.26 163;127;29;181 C7H9Cl2 163.758 C7H8Cl 127.39 C14H11NO 29.8352 C13H9O 181.6479 C12H8 152.625 DDD p,p' 16.85 235;237;199;212;165 C13Cl2H9 235.758 C13H9ClCl(37) 237.463 C13H8Cl 199.39 C14H9Cl 212.3873 C13H9 165.6988 DDE p,p' 16.8 245;248;318;316;176;21 C14Cl2H8 245.99976 C14H8ClCl(37) 247.99681 C14H8Cl3Cl(37) 317.93451 C14H8Cl4 315.93746 C14H8 176.625 C14H7Cl 21.238 DDT o,p 16.87 235;237;199;212;165 C13Cl2H9 235.758 C13H9ClCl(37) 237.463 C13H8Cl 199.39 C14H9Cl 212.3873 C14H8Cl2 245.99976 C13H9 165.6988 DDT p,p' 17.52 235;237;199;212;165 C13Cl2H9 235.758 C13H9ClCl(37) 237.463 C13H8Cl 199.39 C14H9Cl 212.3873 C14H8Cl2 245.99976 C13H9 165.6988 Deltamethrin cis 22 43 253;174;172;251;255;181;29 C7H9BrBr(81) 252 945 C7H9Br(81) 173 98616 C7H9Br 171 98821 C7H9Br2 25 9655 C7H9Br(81)2 254 9246 C13H9O 181 6479
Quantitative data processing method GC-HRMS Data processing by TraceFinder + manual verification of peak assignment/integration Q q1 q2 q3 alpha-hch 1 µg/kg, XICs exact mass ±5ppm
Quantitative data processing method GC-HRMS
Qualitative screening Two approaches: GC-HRMS 1. Library: match of EI-spectra deconvolution of HR spectra cleaned spectrum Library search (NIST, PEST library) Software: Report if match (SI) > user threshold Analyst: manual review hits 2. Database: RT + 2 exact masses RT ±.5 min XICs ± 5 ppm 2 ions (w/o ratio criterion) Software: Report if signal is found for both ions Analyst: manual review hits Only briefly assessed, preliminary results
Qualitative screening: approach-1 GC-HRMS Orange spiked @ 1 μg/kg: 73% automatically found
Qualitative screening GC-HRMS 16111_66 #2682-2685 RT: 14.98-14.99 AV: 4 SB: 5 14.96, 15.1-15.2 NL: 3.79E5 T: FTMS + p EI Full lock ms [5.-5.] 117.3669 Relative Abundance 9 8 7 6 5 4 3 2 1 97.6488 129.3666 166.98564 135.1168 185.15336 chlorpyrifos present in orange at 1.4 μg/kg 196.91959 3 313.25574 257.89434 27.94813 285.92578 12 14 16 18 2 22 24 26 28 3 32 m/z Not automatically found by spectral match... 1616_6 #237-2373 RT: 15.2-15.3 AV: 4 SB: 4 14.99-15., 15.5 NL: 1.84E7 T: FTMS + p EI Full lock ms [5.-5.] 196.91955 Relative Abundance 9 8 7 6 5 4 2 1 96.9585 168.92464 27.9489 114.96135 243.88834 257.89431 285.92556 313.9568 5 15 2 25 3 35 m/z E:\GC-Orbitrap\...\16111_66 1/13/16 3:3:14 RT: 14.72-15.37 SM: 3B 14.92 15.13 14.75 14.81 14.86 14.88 14.97 15. 15.9 15.2 15.28 15.35 14.98 117.3669 313.25574 14.98 14.98 14.98 14.97 14.97 TIC 196.91965 198.9167 257.89428 313.95688 14.8 14.9 15. 15.1 15.2 15.3 Time (min) easily found through approach-2
Qualitative screening: prelim. conclusion GC-HRMS Two approaches: 1. Library: match of EI-spectra deconvolution of HR spectra cleaned spectrum Library search (NIST, PEST library) 2. Database: RT + 2 exact masses RT ±.5 min XICs ± 5 ppm 2 ions (w/o ratio criterion) Software: Report if match (SI) > user threshold Analyst: manual review hits + allows screening for analytes not (yet) included in dedicated HR/AM database - low μg/kg levels in complex matrices are challenging Software: Report if signal is found for both ions Analyst: manual review hits + better screening sensitivity - needs database with t r / exact masses
Conclusions For pesticide residue analysis, both LC and GC with MS detection is required Full scan HRMS is more straightforward than targeted triple quad MS/MS and better suited for simultaneous detection of high numbers of analytes (retrospective data evaluation possible) LC: multiple scan events required to obtain fragments; vdia advantagous for sensitivity/selectivity; GC: EI => one scan event suffices Orbitrap MS + use of resolving powers of 5-7K @ m/z 2 both for LC and GC: Enables use of MEWs of 5 ppm, => high selectivity (similar to MS/MS) Acquisition speed adequate for routine chromatography Analytica Sensitivity/quantitative Chimica Acta performance fit-for-purpose ( 1 µg/kg) Hans G.J. Mol, Marc Tienstra, Paul Zomer More efficient & effective: LC-Q-Orbitrap-HRMS published 215 Evaluation of gas chromatography - electron ionization - full scan high resolution Orbitrap mass spectrometry for pesticide residue analysis 1 injection LC-HRMS, 1 injection GC-HRMS for both quan and qual screening better coverage of pesticides (and more...) for pesticides amenable to both LC-MS and GC-MS: GC-HRMS and LC-HRMS analysis complement each other GC-Q-Orbitrap-HRMS submitted 216
Acknowledgement RIKILT: Hans Mol, Paul Zomer Thermo: Dominic Roberts, Cristian Cojocariu, Paul Silcock (Runcorn, UK) Thank you for your attention!