A Presentation at Agilent Hospitality VIP Event

Yongdong Wang, PhD

Cerno Bioscience

Norwalk, CT, USA

June 6, 2018

Accurate Mass for GC/MSD & LC/MSD

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

Actual MS (Profile/Raw): C25H23N2OS+

398 400 402 404 4060

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Quadrupole, R = 800

After MS Centroiding: C25H23N2OS+

398 400 402 404 4060

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Quadrupole, R = 800

True Mass Spectrum: C25H23N2OS+

398 400 402 404 4060

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eTrue Mass Spectrum @ R = Infinity

Enough Details Even at Low Res

398 399 400 401 402 403 404 405 4060

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eMass Spectra @ R=800

C25H23N2OS

C4H19N16OF2NaP

C20H27N2FNaP2

C9H33N6O3S2P2

C10H27N10OS3

“33S”“15N”“2H”

“13C”

8

259 260 261 262 263m/z

Raw Profile Mass SpectrumRaw Profile MS and True MSm/z-Only CalibrationTrue Mass SpectrumTrue & Fully Calibrated MS

Mass Accuracy

Plus

Spectral Accuracy

410.4

724

411.4

753

412.4

835

413.4

112

415.0

452

0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

40000000

45000000

50000000

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

Average of all RT Windows - C:\TestData\T42Desktop\BobStrife\MS-MS\101306_1.raw\_FUNC001.crn

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Mass Accuracy

Raw: mass accuracy ±0.2~0.5Da

Calibrated: mass accuracy ±0.002~0.005Da

100x better mass accuracy

±5mDa/400Da = ±12ppm ?!

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

±5p

pm

Enters Spectral Accuracy…

399.1

433

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403.9

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397.0

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396 397 398 399 400 401 402 403 404 405

Average of Scans 14 thru 49 (0.25 to 0.87)

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Spectral Accuracy

Calibrated MS

Theoretical MS

Unparalleled

Spectral Accuracy

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

GC/MS: Readily Available Cal Gas

• Available on all GC/MS systems

• Computer-controllable & user-programmable

• Fragment ions covering 45-550Da

• Even internal to a GC/MS run

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CF3+

C9F20N+

AutoCal™: Out-of-Box Accurate Mass

Mass accurate to 0.00x Da

Unique Specifications for 5977

Mass 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

Spectral Accuracy 1-uL injection of a 100-pg/uL OFN standard scanning from

50-300 u will give 99.0% spectral accuracy

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

Unique Specifications for LC/MSD

5991-7829EN: InfinityLab LC/MSD SeriesAgilent LC/MSDFor OpenLAB CDS and OpenLAB CDS ChemStation Edition

5991-7830EN:InfinityLab LC/MSD SeriesAgilent LC/MSD XTFor OpenLAB CDS and OpenLAB 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 FormulaSpectral

Accuracy (%)

27 C20O2 90.62

Rank FormulaSpectral

Accuracy (%)

21 C2HN2O3F9 93.34

Rank FormulaSpectral

Accuracy (%)

16 C15N2O4 94.61

Rank FormulaSpectral

Accuracy (%)

10 C4N8O7 96.82

Rank FormulaSpectral

Accuracy (%)

5 C7HN4O7F 98.17

Rank FormulaSpectral

Accuracy (%)

4 C10N4O6 98.38

Rank FormulaSpectral

Accuracy (%)

3 C9H2N2O6F2 99.04

Rank FormulaSpectral

Accuracy (%)

2 C10N2O3F4 99.14

Rank FormulaSpectral

Accuracy (%)

1 C10F8 99.72

Forensics Chemistry Unknown

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GC/MS Calibration

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A v er age of S c ans 785 t hr u 799 ( 8. 66 t o 8. 74)

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MassWorks calibrates {m/z, peak shape} to improve

mass accuracy by up to 100x

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A v er age of S c ans 785 t hr u 799 ( 8. 66 t o 8. 74)

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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 #1Impurity #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

Collaterals/Support/Contact Info• Cerno web

– 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-series-

cerno-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-training-

video-gcmsd/

– info@cernobioscience.com – live demos / visits, anyone?