Accurate Mass for GC/MSD & LC/MSD

Accurate Mass for GC/MSD & LC/MSD A Presentation at Agilent Hospitality VIP Event Yongdong Wang, PhD Cerno Bioscience Norwalk, CT, USA June 6, 2018

Agenda Cerno s Novel MS Calibration: Why & How Application to GC/MSD Application to LC/MSD

Cerno/Agilent Startup in Norwalk, CT, USA MS calibration & analysis technology 14 granted/pending patents world-wide MassWorks software product: Standalone post acquisition application software V1.0, PittCon Bronze Medal (now V5.0) Supports nearly all MS systems but works the best on Agilent MSD Over 1,200 customers in US, Canada, Europe, Japan, China etc Agilent partnership Key function: the only approach for accurate mass formula determination / compound ID with a quadrupole MS Real key technical advance: Spectral Accuracy

Rel. Abundance Actual MS (Profile/Raw): C 25 H 23 N 2 OS + 0.8 Quadrupole, R = 800 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 398 400 402 404 406 m/z

Rel. Abundance After MS Centroiding: C 25 H 23 N 2 OS + 0.8 Quadrupole, R = 800 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 398 400 402 404 406 m/z

Rel. Abundance True Mass Spectrum: C 25 H 23 N 2 OS + 0.8 True Mass Spectrum @ R = Infinity 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 398 400 402 404 406 m/z

Rel. Abundance Enough Details Even at Low Res 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 Mass Spectra @ R=800 13 C 33 S 15 N 2 H C25H23N2OS C4H19N16OF2NaP C20H27N2FNaP2 C9H33N6O3S2P2 C10H27N10OS3 0.1 0 398 399 400 401 402 403 404 405 406 m/z

True Raw True m/z-only & Profile Fully Mass MS Mass Calibrated Calibration Spectrum and True MS MS Mass Accuracy Plus Spectral Accuracy 259 260 261 262 263 m/z 8

412.4835 413.4112 415.0452 411.4753 counts 410.4724 Mass Accuracy 50000000 Average of all RT Windows - C:\TestData\T42Desktop\BobStrife\MS-MS\101306_1.raw\_FUNC001.crn 45000000 40000000 Raw: mass accuracy ±0.2~0.5Da 35000000 30000000 Calibrated: mass accuracy ±0.002~0.005Da 25000000 20000000 100x better mass accuracy 15000000 10000000 5000000 0 409.5 410.0 410.5 411.0 411.5 412.0 412.5 413.0 413.5 414.0 414.5 415.0 415.5 m/z

±5mDa/400Da = ±12ppm?!

±5ppm Tough Even for TOF Row Formula Mono Isotope Mass Error (mda) Mass Error (ppm) Spectral Accuracy RMSE DBE 1 C19H15N10O 399.1425 0.0 0.0 95.2421 5.09E+05 17.5 2 C19H23N6S2 399.1420 0.5 1.2 95.3603 4.96E+05 11.5 3 C6H23N8O12 399.1430-0.5-1.2 85.5297 1.55E+06-0.5 4 C11H19N12O3S 399.1418 0.7 1.7 91.1774 9.43E+05 8.5 5 C14H27N2O9S 399.1432-0.7-1.7 90.9037 9.72E+05 2.5 6 C11H27N8O2S3 399.1414 1.1 2.9 88.0757 1.27E+06 2.5 7 C26H23O2S 399.1413 1.2 2.9 98.7355 1.35E+05 15.5 8 C22H23O7 399.1438-1.3-3.3 95.8267 4.46E+05 11.5 9 C18H19N6O5 399.1411 1.4 3.4 94.2692 6.13E+05 12.5 10 C15H31N2O4S3 399.1440-1.5-3.9 89.2286 1.15E+06 1.5 11 C18H27N2O4S2 399.1407 1.8 4.6 93.4288 7.02E+05 6.5 12 C7H19N12O8 399.1443-1.8-4.6 87.3267 1.35E+06 4.5 13 C10H23N8O7S 399.1405 2.0 5.0 89.1816 1.16E+06 3.5 14 C15H23N6O5S 399.1445-2.0-5.0 92.9282 7.56E+05 7.5 15 C23H27O2S2 399.1447-2.2-5.5 96.0715 4.20E+05 10.5 16 C23H19N4O3 399.1452-2.7-6.7 96.3067 3.95E+05 16.5 17 C17H23N2O9 399.1398 2.7 6.7 92.8259 7.67E+05 7.5 18 C8H23N12O3S2 399.1452-2.7-6.8 88.2131 1.26E+06 3.5 19 C16H27N6S3 399.1454-2.9-7.2 91.0878 9.53E+05 6.5 20 C8H15N16O4 399.1457-3.2-7.9 89.0377 1.17E+06 9.5 61 C17H27N4O3S2 399.1519-9.4-23.6 93.2796 7.18E+05 6.5

Enters Spectral Accuracy

396.4632 397.0712 401.1496 402.1394 403.0832 403.9872 405.0512 400.1520 counts 399.1433 Spectral Accuracy Average of Scans 14 thru 49 (0.25 to 0.87) 35000000 30000000 Calibrated MS 25000000 Theoretical MS 20000000 15000000 10000000 Unparalleled Spectral Accuracy 5000000 0 396 397 398 399 400 401 402 403 404 405 m/z

Technology: Patented, Peer-Reviewed & Published Wang, Y.; Gu, M. Anal. Chem. 2010, 82, 7055-7062 (17).

Agilent/Cerno Partnership MassWorks for GC/MSD, Agilent Part # G6851AA MassWorks for LC/MSD, Agilent Part # G6887AA Specially configured version with highly attractive pricing when bundled with new MSD purchase Unique ease of use: GC/MSD-only AutoCal Unique spec among all SQ systems: Mass accuracy: ±0.005 Da Spectral accuracy: 99%

GC/MSD: Hardware Advantages Heated gold-coated ceramic quads Superb stability and reproducibility /how to quantify? High mass accuracy demonstratable w/ RIGHT calibration Raw Scan (Profile) mode readily available! None of detrimental on-board processing/filtering Compromised spectral accuracy Wide (5-6 orders of) linear dynamic range High spectral accuracy

counts relative abundance PFTBA Valve On PFTBA Valve Off GC/MS: Readily Available Cal Gas E:\Mulligan\06-25-2009\TETSR3.D\DATA.MS 1.0 0.9 0.8 GC Peak of Unknown 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 14000 Average of Scans 1398 thru 1439 (12.23 to 12.47) 5 6 7 8 9 10 11 12 13 minutes CF 3 + 12000 10000 8000 6000 4000 2000 C 9 F 20 N+ 0 50 100 150 200 250 300 350 400 450 500 m/z Available on all GC/MS systems Computer-controllable & user-programmable Fragment ions covering 45-550Da Even internal to a GC/MS run

AutoCal : Out-of-Box Accurate Mass Mass accurate to 0.00x Da

Unique Specifications for 5977 EI SIM IDL (Helium Carrier gas with Auto Liquid Sampler) 10 fg or less IDL for Extractor ion source, turbo molecular pump system 24 fg or less for Inert EI source, turbo molecular pump system 30 fg or less for Inert EI source, diffusion pump system IDL statistically derived at 99% confidence level from the area precision of eight sequential splitless injections of 100 fg OFN,monitoring m/z 272. EI scan S/N (Helium carrier gas manual injection) 1500:1 or higher S/N for Extractor ion source, turbo molecular pump system 600:1 or higher for Inert EI Ion Source, turbo molecular pump system 300:1 or higher for Inert EI Ion Source, diffusion pump system These numbers will be given by 1-μL injection of 1 pg/μl OFN standard scanning from 50 to 300 u at nominal 272.0 u ion. PCI scan S/N (Methane) 125:1 S/N will be given by 1-μL injection of 100-pg/μL BZP2 standard scanning from 80 to 230 u at nominal 183 u ion NCI scan S/N (Methane) 600:1 S/N will be given by 2-μL injection of 100 fg/μl OFN standard scanning from 50 to 300 u at nominal 272 u ion Mass Accuracy Spectral Accuracy 1-uL injection of a 100-pg/uL OFN standard scanning from 50-300 u will give its monoisotope at m/z 271.987 ± 0.005 1-uL injection of a 100-pg/uL OFN standard scanning from 50-300 u will give 99.0% spectral accuracy

Unique Specifications for LC/MSD 5991-7830EN: 5991-7829EN: InfinityLab LC/MSD Series Agilent LC/MSD XT For For OpenLAB CDS CDS and and OpenLAB CDS CDS ChemStation Edition

Applications Elemental composition determination (research/education/teaching) Forensics chemistry Pharma impurity ID Spectral accuracy for mixture analysis M+ and (M-H)+ in EI Labeled isotopes LC or MS peak purity: deamidation of biologics Metal organics, catalyst etc

Elemental Composition Determination Rank Formula Spectral Accuracy (%) 21 16 10 27 51 24 3 C9H2N2O6F2 C2HN2O3F9 C10N2O3F4 C7HN4O7F C15N2O4 C10N4O6 C4N8O7 C20O2 C10F8 93.34 94.61 98.17 99.72 99.14 96.82 98.38 99.04 90.62

counts relative abundance PFTBA Valve On PFTBA Valve Off Forensics Chemistry Unknown E:\Mulligan\06-25-2009\TETSR3.D\DATA.MS 1.0 0.9 0.8 GC Peak of Unknown 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 14000 Average of Scans 1398 thru 1439 (12.23 to 12.47) 5 6 7 8 9 10 11 12 13 minutes CF 3 + 12000 10000 8000 6000 4000 2000 C 9 F 20 N+ 0 50 100 150 200 250 300 350 400 450 500 m/z

2 4 1. 0 0 0 3 2 4 1. 9 9 9 4 2 4 2. 9 5 0 0 c ount s c ount s 2 4 0. 0 1 2 7 GC/MS Calibration A v er age of S c ans 785 t hr u 799 ( 8. 66 t o 8. 74) 35000 A v er age of S c ans 785 t hr u 799 ( 8. 66 t o 8. 74) 30000 35000 25000 30000 25000 20000 20000 15000 15000 10000 10000 5000 5000 0 0 238. 5 239. 0 239. 5 240. 0 240. 5 241. 0 241. 5 242. 0 242. 5 243. 0 243. 5 238. 5 239. 0 239. 5 240. 0 240. 5 m / z 241. 0 241. 5 242. 0 242. 5 243. 0 243. 5 m / z MassWorks calibrates {m/z, peak shape} to improve mass accuracy by up to 100x

GC/MS Formula ID Correct identification w/ Spectral Accuracy

NIST Library Search w/same Data Acquisition

A Highly Toxic Compound Not Found in NIST Library

A Highly Toxic Compound Not Found in NIST Library

LC/MSD MassWorks Calibration Agilent tune solution: Universal standard (wide m/z range, ESI+ and ESI-) Stable and readily available External acquisition preferred Calibration drift: not significant (~5mDa/day) Known internal standards in sample, e.g., API Conveniently found within same sample Internal within same run No extra experimental work/acquisition Limited m/z range coverage: effective ±100Da

Agilent Tune Solution Calibration 10uL loop injection

Drug Impurity ID: Parent Drug as Calibration Standards Parent Impurity #1 Impurity #2 RT Window Name Formula Entry Net Formula Charge Mono Isotope Centroid Left Edge Right Edge 1 0: Scans 1792-1844 Simvastatin C25H39O5 C25H39O5 +1 419.2797 419.5751 417.280 423.280 2 1: Scans 1830-1845 NH4 Adduct C25H42NO5 C25H42NO5 +1 436.3063 436.6056 434.306 440.306 3 0: Scans 1792-1844 K Adduct C25H38O5K C25H38O5K +1 457.2356 457.6654 455.236 461.236 4 All

Drug Impurity ID #1

Drug Impurity ID #2 Impurity in impurity

[C14] Ring Labeled Compounds: Diclofenac Experimental * Diclofenac Sodium C14H10Cl2NNaO2 [14C] labeled SA=62.7 mci/mmol 7 possible isotopes Native, 1[14C] - 6[14C]

Results of Spectral Accuracy Mixture Analysis

GC/MSD RCM Paper: Flavor

GC/MSD RCM Paper: Forensics

LC/MS Paper: HDX Kinetics

LC/MS: Environmenal

Application Areas Pharmaceutical impurity ID: must have Small molecule or large biologics Leachables / Extractables Food / Beverages / Beer / Wine / Liquor Flavor / Fragrances / Cosmetics / Natural products Academic / Government Labs: Organic synthesis or other research support/acs journal accepted Novel tool to teach MS and accurate mass concept Forensics / Crime labs / Anti-doping / Homeland security / Environmental / Fine chemical / Petrochemical / Drug testing / Art museums / Paint / Automobile / Energy / Materials etc QA / QC labs Any GC/MSD with NIST library - need for compound ID

Cerno web Collaterals/Support/Contact Info Tech demo: http://cernobioscience.com/truecaltm/ Publications: http://cernobioscience.com/publications/ App Notes: http://cernobioscience.com/application-notes/ Agilent web Dedicated page: http://www.agilent.com/en-us/infinitylab-lc-msd-seriescerno-bioscience-massworks (another one for GC/MSD) Flyer: 5991-4788EN, 5991-8085EN App Notes: 5990-4966EN, 5991-8065EN, 5991-8976EN (polymer), 5991-8977EN (food) Support and Contacts Data acquisition setup: http://cernobioscience.com/faqs/ Training video: http://cernobioscience.com/video-massworks-trainingvideo-gcmsd/ info@cernobioscience.com live demos / visits, anyone?