LC-MS/MS Technology and Applications Transform your routine science

LC-MS/MS Technology and Applications Transform your routine science Jonathan Beck, Ph.D Marketing Scientist Environmental & Food Safety October 2th, 215 1 The world leader in serving science

Outline High Resolution Accurate Mass for Environmental Analysis EQuan MAX Plus online sample preconcentration and cleanup New Triple Quadrupole developments from Thermo Scientific Project with Duke University for Emerging contaminants 2

What is Environmental Analysis Challenge? Analysis of thousands of pollutants High diversity of chemical characteristics Different matrices Screening Workflows with Quan/Qual capabilities needed New issues: Hydraulic fracturing Regulatory EU Water Framework Directive Emerging Contaminants in The Environment USGS study F HO Pharmaceuticals, Pesticides, Endocrine Disruptors, O I I O N HN HN I OH O O Personal Care Products Cl N HO O O O O O O O O Cl O OH OH O O O N N NH N HO HO O OH O N N O OH OH O Contamination of ground and drinking water is a risk for human health 3

What s in our water supplies? Pharmaceuticals, Personal Care Products, Pesticides 4 *Image from National Geographic, April 21

Sample preparation/analyte detection strategy Clean-up efficiency Simple extraction LLE Dispersive SPE GPC SPE Detection selectivity HRAM-MS/MS MS/MS LC-MS HPLC-FLD GC-MS, ECD HPLC-UV 5

Major Challenges in EFS Analysis Low Concentration Being able to detect and identify trace level analytes in simple or complex samples. Large Sample Numbers High throughput for routine labs. Matrix Complexity Identifying low abundance analytes obscured by high abundance compounds. Small Sample Volumes Getting the most accurate information from precious samples 6

Analytical Challenges for Traditional Mass Spectrometry Detect and identify isobaric compounds Isolate compounds from matrix components Searching for unknowns Here targeted data acquisition (SIM, SRM ) fails Large number of compounds High Resolution High mass accuracy (HRAM) MS can do the job 7

Resolution (resolving power) Resolutio n m R m m (FWHM) m 1% m 5 Quadrupole MS R 833 m.6 m 5 Orbitrap MS R 1 m.5 8

Detection of analytes in heavy matrix Pirimicarb Pesticide in Matrix 1 239.15181 C 11 H 19 O 2 N 4 6.5 ppm Relative Abundance 8 6 4 2 R = 17,5 Error = 6.5 ppm 239. 239.5 239.1 239.15 239.2 239.25 m/z 239.1533 C 11 H 19 O 2 N 4.32 ppm R = 7, Error =.32 ppm 9

Resolution influence (17.5 vs. 7.) Resolution_17_5K #1823 RT: 2.3 AV: 1 NL: 1.6E6 Resolution_7K #123 RT:.54 AV: 1 NL: 1.8E6 T: FTMS + p ESI Full ms [12.-1.] T: FTMS + p ESI Full ms [12.-1.] 1 222.1263 1 222.1238 95 95 9 9 85 85 8 8 75 75 7 65 6 Name Molecular Formula 7 65 6 M+H Relative Abundance 55 5 45 4 35 55 Carbofuran C12H15NO3 5 222.1125 4 Formetanate C11H15N3O2 222.1237 35 Relative Abundance 45 3 3 25 25 222.1129 2 2 15 222.1463 15 222.1465 1 1 5 222.1838 222. 222.1 222.2 222.3 m/z 5 222.1854 222. 222.5 222.1 222.15 222.2 222.25 m/z 1

Mass accuracy Mass accuracy m m m/ z meas true 1 6 m true 5.1 5. Quadrupole MS m/ z 1 6 2ppm 5 5.1314 5.1214 Orbitrap MS m/ z 1 6 2ppm TOF MS 5.1314 11

Selectivity increases with higher mass accuracy Beer extract, T-2 toxin 5 µg/l 1 RT: 8.45 NL: 1.2E5 m/z= 9 484.25313-484.2557 F: FTMS 8 {1,} + p APCI Full ms [3.-85.] 7 MS pivo_5_uh 6 Relative Abundance 5 4 3 2 1 Relative Abundance 2 ppm 7.26 7.41 7.49 7.76 7.91 7.99 8.55 7.67 9.46 1.16 1.31 1.5 1.61 7.2 7.4 7.6 7.8 8. 8.2 8.4 8.6 8.8 9. 9.2 9.4 9.6 9.8 1. 1.2 1.4 1.6 Time (min) RT: 8.45 NL: 1.2E5 1 m/z= 484.24441-9 484.26379 F: FTMS 8 {1,} + p APCI Full ms [3.-85.] 7 MS pivo_5_uh 6 5 4 3 2 ppm 2 1 Relative Abundance 8.21 7.59 7.62 7.85 7.37 7.4 7.81 7.92 7.99 8.25 7.26 7.3 8.61 8.67 8.88 9.11 9.46 1.9 1.16 1.31 1.5 1.61 7.2 7.4 7.6 7.8 8. 8.2 8.4 8.6 8.8 9. 9.2 9.4 9.6 9.8 1. 1.2 1.4 1.6 Time (min) 1 9 RT: 8.45 NL: 1.25E5 m/z= 484.2567-484.3253 F: FTMS 8 {1,} + p APCI Full ms [3.-85.] 7 MS pivo_5_uh 6 5 4 8.67 8.72 9.29 8.99 3 8.78 8.21 9.11 7.62 7.85 2 8.13 8.83 7.59 7.38 7.81 8.1 8.35 1 7.33 1 ppm 7.27 9.46 9.5 9.64 1.9 1.16 1.34 1.5 1.61 7.2 7. 7. 7.8 8. 8. 8. 8. 8. 9. 9. 9. 9. 9. 1. 1.2 1.4 1.6 4 6 2 4 6 8Time (min) 2 4 6 8 12 *Zachariasova M et al, Anal. Chim. Acta

Detection of Isobaric Compounds in mixtures Element Exact Mass H 1.7825 C 12. N 14.374 O 15.994915 Is a simultaneous measurement possible? Relative Abundance 1 9 8 7 6 5 4 3 2 1 289.1424 289.139 289.122 289.1314 289.1329 289.1547 289.514 288.441 C9H21O2P1S3 Terbufos 288.949 C13H21O3P1S1 Iprobenfos 289.514 288.1142 C15H17N4Cl1 Myclobutanil 288.1256 C11H2N4O3S1 Epronaz 289.513 289.121 289.122 289.1215 289.1215 289.1329 289.1424 289.1329 289.1424 288.1351 C11H21N4O3P1 289.1215 Pirimethaphos 288.1474 C16H2N2O3 Imazamethabenz R = 14, 289. 289.5 289.1 289.15 289.2 m/z.133 amu 13

The Exactive Plus Orbitrap LCMS system 14

Q Exactive: Orbitrap MS/MS system Higher ion transmission with rugged optics Increased sensitivity, selectivity, true MS/MS Directly interfaced to HCD increases spectrum quality Multiple fills for spectrum multiplexing increases duty cycle Predictive automatic gain control for parallel filling & detection brings more speed Advanced signal processing brings more resolution at same speed 15

What do we gain by using the quadrupole? 1 8 6 4 2 195.876 N=24842.81 NL: 1.94E8 [15.-2.] Full MS S/N = 745 Lowest detected signal/scan 2533 1 Relative Abundance 8 6 4 2 195.877 N=2741.58 NL: 1.12E8 [19.1-2.1] Lowest detected signal/scan SIM (1amu) S/N = 54 2824 6 5 Gain in sensitivity (7x) S/N (spectrum) 4 3 2 1 Caffeine 195.82 195.84 195.86 195.88 195.9 195.92 195.94 S/N (FMS) S/N (SIM1) Sensitivity gain 5 1 x with SIM mode 16

Data dependent MS/MS confirmation Relative Abundance 6.96 NL: 1.98E7 1 m/z= 297.541-297.571 F: FTMS Imazalil 5 + p ESI Full ms [5.-75.] MS 4xloq_2 7.35 NL: 4.67E6 1 m/z= 31.1163-31.1193 F: FTMS 5 Desmedipham + p ESI Full ms [5.-75.] MS 4xloq_2 7.35 NL: 8.97E7 1 m/z= 233.231-233.255 F: FTMS 5 + p ESI Full ms [5.-75.] Diuron MS 4xloq_2 7.44 NL: 1.15E7 1 m/z= 26.226-26.252 F: FTMS 5 + p ESI Full ms [5.-75.] Benoxacor MS 4xloq_2 7.55 NL: 2.21E7 1 m/z= 33.184-33.1118 F: FTMS 5 + p ESI Full ms [5.-75.] Fluridone MS 4xloq_2 7.58 NL: 1.42E7 1 m/z= 44.1221-44.1261 F: FTMS 5 Azoxystrobin + p ESI Full ms [5.-75.] MS 4xloq_2 7.8 NL: 4.23E7 1 m/z= 312.1149-312.1181 F: FTMS 5 + p ESI Full ms [5.-75.] MS 4xloq_2 Fenamidone 2 4 6 8 1 12 14 Time (min) Relative Abundance 1 5 1 5 1 5 1 5 1 5 1 5 158.9762 69.455 297.551 2.9867 19.763 255.82 72.451 316.1774 233.241 119.856159.1165 37.625 139.9895 112.395165.94 149.834 271.859 343.392 59.5 12.445 26.236 188.74 281.7393 344.126 329.791372.974 172.391216.655 316.178 81.75 45.2731 92.5 236.118 13.546165.481 267.951312.1149 1 2 3 4 m/z 17

Q Exactive Quanfirmation what is it? High performance HRAM Quantitation and Confirmation bench top LCMS system, capable of : Multi-residue quan performance similar to mid-high end Triples Ideal for targeted and general unknown screening Highest confidence confirmation with R = 14K, and MS/MS UHPLC compatible Let s use the QExactive to combine these 2 experiments into one. 18

Outline What is EQuan MAX Plus? EQuan MAX Plus How can EQuan MAX Plus help your workflow? Customer testimonials Who uses EQuan MAX Plus? 19

Low Sample Concentration Challenge Demanding assays which require the absolute lowest limit of detection TSQ Quantiva Powered by AIM technology, the TSQ Quantiva MS is the world s most sensitive triple quadrupole MS, detecting compounds at the ppt level. 2

Environmental Analysis (Water) EQuan MAX Plus For targeted quantitation (TSQ) or Targeted/non-targeted screening and quantitation using High Resolution Accurate Mass (Orbitrap platform) 21

EQuan MAX Plus: What is it? Turnkey method for assaying environmental water samples (pesticides, antibiotics, etc.) at low ppt levels On-line sample clean-up and preconcentration 2 Columns : Loading and Analytical 2 pumps High injection volumes 1mL-2mL Standard injection volumes 1-1 ul 22

EQuan MAX Plus: Targeted Quantitation Couple EQuan MAX Plus with any TSQ Quantum from Thermo Scientific for the most sensitive and selective experiments. Fast Positive Negative Switching TraceFinder Software Built in SRM parameters Built in EQuan Methods Quantum Access MAX Quantum Endura Quantum Quantiva 23

EQuan MAX Plus: Non-targeted screening and Quantitation Couple EQuan MAX Plus with the Exactive Orbitrap instruments (Exactive Plus or Q- Exactive). High Resolution Accurate Mass (HRAM) All ions are collected in every experiment. Re-interrogate your data at a later time Quantitation and screening methods are easy to set up since there are no compound dependant parameters to optimize. 24

SPE - standard enrichment procedure Filtration 1 Sample preparation: 1L sample filtration internal Std. ~ 5h 4 samples 2 Conditioning: MeOH / H 2 O Enrichment 3 Extraction: 4 Washing: 6mg Oasis H 2 O/MeOH (95/5) ~ 2d 5 Drying: air 6 Elution: MeOH LC-MS 7 Detection: LC-MSMS ~ 2d 25 Slide Courtsey Heinz Singer eawag, Zurich Switzerland

EQuan MAX Plus Solution Gain Vs. Conventional Injections TriazinePestM ix_1ppt_1ul_4 3/24/25 1:1:1 AM RT : 5.98-8. RT: 5.98-8.2 1 NL: 8.1E4 TIC F: + c ESI RT: 6.91 NL: 8.1E4 AA: 319999 TIC F: + c ESI sid=-1. 95 sid=-1. SRM m s2 216.13@ -18. [ 174.] M S 1 SN: 116RMS SRM m s2 216.13@ -18. [ 174.] M S ICIS TriazinePestM ix_1ppt_1 9 triazinepestm ix_1ppt_ 1ul_5 95 ul_4 9 85 85 8 8 75 75 7 7 65 6 1 ul injection 65 1 ul injection 6 Relative Abundance 55 5 45 Relative Abundance 55 5 45 4 4 35 35 3 3 25 25 2 2 15 15 1 1 5 5 6. 6.5 7. Time (m in) 7.5 8. 6. 6.5 7. 7.5 8. Time (min) 1 ppt Atrazine in ground water 26

Sensitivity, Speed & Robustness for EFS high throughput laboratories TSQ Quantiva Mass Range 1-185 1-34 TSQ Endura TSQ Quantiva Extreme quantitative performance Designed for the most challenging assays. For scientists needing to stay at the forefront of analytical technology Max SRM Number 3, SRMs 3, SRMs SRM/Sec 5 SRMs/sec 5 SRMs/sec Ion Optics Quadrupole Design Active Ion Management (AIM) Ion Max NG source Electrodynamic ion funnel ion beam guide with neutral blocker 6 mm HyperQuad quadrupoles with asymmetric RF drive S- LENS with Beam Blocker Technology 4mm Quadrupoles with Asymetric RF TSQ Endura Extreme quantitative value Designed for non-stop operation. For scientist who need to run routine samples day in and day out. Reserpine Specification 1, :1 S/N for 1 pg Reserpine 1, :1 S/N for 1 pg Reserpine 27

From Identification...???????????????????? Challenge Orbitrap V elos MS Confidently identify unknown emerging contaminants High-Resolution Accurate Mass for identification of unknown emerging contaminants. 28

To Routine Quantitation Challenge EQuan MAX Plus and TSQ Quantiva MS Followed by routine quantitation. Online sample prep LC/MS for quantitation of emerging contaminants identified by the Orbitrap Velos 29

Analysis of Targeted and Non-targeted Contaminants in Storm Water Retention Ponds Emerging Contaminants, from ID to Quantitation. The Total Thermo Scientific Solution Gordon Getzinger Duke University Jonathan Beck, PhD Thermo Fisher Scientific

UHPLC-HRMS: An Emerging Technique for Helping Contaminants Emerge Target screening: Monitor known contaminants using reference standards. Suspect screening Screen high-resolution accurate-mass data against molecular databases of suspected contaminants. Non-target screening Assign molecular and structural formula to chromatographic features without previous knowledge of contaminant presence or identity. Krauss et al. LC-high resolution MS in environmental analysis: from target screening to the identification of unknowns. (21) Anal Bioanal Chem. 397 [3]. 31

Emerging Contaminant ID to Quantitation Workflow Reclaimed waste water used for irrigation on a golf course for emerging contaminants (PPCPs) Lee Ferguson and Gordon Getzinger, Environmental Chemistry, Duke University, USA Acquire Fast Identify Develop Routine HRAM Data Contaminants Quantitative Quantitation Assay ExactFinder Software Etofenprox Benzotriazole Acephate Propanamide Allethrin 32

Kiawah Island, SC, USA Land application of WW for golf course irrigation WWTP Question: Which microcontaminants are entering the aquatic environment as a result of wastewater reuse practices? 33

Typical Sampling Site 34

Variety of Sampling Sites Sample Site Pond 5 Pond 25 Pond 43 Wastewater lagoon Wastewater Composite Well 1 Well 7 Inputs Golf course runoff Golf course runoff Residential stormwater Treated municpal wastewater 24hr composite effluent Infiltration from pond 25 Infiltration from pond 5 35

Quantitation of Samples Use a triple quad for quantitation. Analyze samples directly, no offline SPE to save time and money. Remove variables and sources of experimental error by automated extraction and preconcentration. An ideal solution for this challenge is 36

What Did We Want to See in the Quantitation Experiment? Quantitative results at low concentrations low and sub PPT levels Calibration range:.6 pg/ml 1 pg/ml RT :4.-13.57 With a 1mL injection, that s 6 fg 1 pg on column. 1 9 9.68 NL: 1.7E6 TIC MS 1,75pgmL 8 7 TIC of 1.75 pg/ml injected standard 5 1.75 pg on column Relative Abunduance 6 4 9.79 3 6.87 1.26 1.37 2 11.26 9.46 1 8.17 6.63 7.85 6.1 4.34 5.84 11.51 12.42 4 5 6 7 8 9 1 11 12 13 Time (min) 37

Calibration range,.6-1 pg/ml - Flutolanil 6fg On Column Ion Ratio overlay 4.5 orders of magnitude 38

39 Results of Compounds Detected in a WWTP Sample

Identification of Emerging Contaminants Data Analysis Workflow Target screening Are compounds x, y, & z present in this sample? Suspect screening Which compounds of a defined list are present in this sample? Non-target screening Which compounds are present in this sample? 4

LC/MS Workflows for Environmental and Food Safety Tools to Develop methods for Known and Emerging contaminants Accelerated Solvent Extraction (ASE) Raw Sample Dionex ASE Dionex UHPLC 3 Equan MAX Plus online sample prep LCMS EQuan MAX Plus Exactive Plus and Q Exactive Mass Spectrometers: High Resolution Accurate Mass to identify, quantify and confirm Customized Reporting TSQ Series Mass Spectrometers: triple stage quadrupole LC-MS/MS and GC MS/MS for high sensitivity quantitation Custom software to meet the needs of your workflow Orbitrap HRAM TraceFinder, SIEVE, mzcloud TSQ Quantiva TSQ Endura Orbitrap Fusion 41

42 Transform Your Science