Rapid sample analyses for environmental toxicants Erin Shammel Baker - PowerPoint PPT Presentation

Rapid sample analyses for environmental toxicants Erin Shammel Baker Xueyun Zheng, Noor A. Aly, Francesca B. Smith, Kristin E. Burnum-Johnson, Samuel H. Payne, Matthew E. Monroe, Richard D. Smith Pacific Northwest National Laboratory

Understanding health risks

Understanding health risks

Understanding health risks

Understanding health risks

Understanding health risks

Multi-omic analyses • Human genome project illustrated that >90% of diseases are not due solely to genetics MS-based Measurements

Main challenges with small molecule measurements 1. Small molecules occur from very low to high concentrations (fM-mM) so high dynamic range and sensitivity measurements are essential 2. Biological changes are best understood when both endogenous metabolites and xenobiotics are analyzed 3. Untargeted measurements covering thousands of small molecules are desired for time course studies and large cohort analyses 4. Many small molecules have the same masses but a different chemical makeup so distinguishing them with MS-based approaches can be difficult or impossible Testosterone In the NIST database there are Exact mass = 288.2089188 18 different options with exact mass = 288.2089188

Ion mobility concept E in out Drift Time Pulse of 2 ions with Different conformers separate same m/z but different in time with peak heights representing shape the amount of each Drift Cell velocity is constant v = K E K = ion mobility

Ion-neutral collision cross section (CCS) 1. Value related to the size and shape of an ion 2. Corresponds to the area that collides with the drift gas 3. Robust physicochemical property 4. Can easily be compared between labs 5. Varies depending on drift gas Slide courtesy of Professor Kevin Pagel and Waters

Ion mobility concept LC (minutes) MS (~100 µs) IMS (~60 ms) IMS MS Elution Time Drift Time m/z 1100 1100 1100 m/z m/z m/z 100 20 30 40 50 60 100 Drift Time (ms) 100 20 30 40 50 60 20 30 40 50 60 Drift Time (ms) Drift Time (ms) Intensity 0 10 20 30 40 50 60 Elution Time (minutes)

Isomers difficult to separate with hydrophobic interaction liquid chromatography (HILIC) H H D-Fructose-6-phosphate (F6P) D-Glucose-6-phosphate (G6P) α-D-Glucose-1-phosphate (G1P) G6P G1P F6P Deprotonated form [M - H] - m/z = 259.02

Isomeric xenobiotic separations Collaboration with Keri Hornbuckle ’ s Group

Isomeric xenobiotic separations Collaboration with Keri Hornbuckle ’ s Group

IMS collision cross section (CCS) precision • Compare CCS accuracy across 4 international labs • Analyze 80 molecules (metabolites, lipids, peptides and proteins) to determine CCS agreement Pacific Northwest National Laboratory, Richland Agilent Technologies, Inc., Santa Clara Vanderbilt University, Nashville BOKU – Univ. of Natural Resources and Life Sciences, Vienna S. M. Stow, et al., Anal. Chem. 2017 , 89, 9048-9055.

Interlab precision comparison _ m/z x MTHF 95% CI [M+H] + & [M+Na] + Cortisol[M+Na] Cortisol Metabolites Pyr5P[M+Na] Cyst[M+Na] Pyr5P • Ran triplicate injections at L-cystine GLC[M+Na] all 4 labs L-tyrosine L-arginine L-phenylalanine L-histidine Creat[M+Na] • Analyzed in positive and H-Cyst L-threonine negative ion mode L-proline Creat MTHF Metabolites Pyr5P • Mean %RSD of 0.24% L-cystine L-tyrosine [M-H] - L-arginine Uric Acid L-phenylalanine L-histidine L-glutamic acid L-aspartic acid Creat C20:1 C20:2 C20:3 C20:4 Lipids [M-H] - C18:0 C18:1 C18:2 C18:3 C17:0 C16:0 C16:1 C15:0 C12:0 0.00% 0.10% 0.20% 0.30% 0.40% 0.50% %RSD

Interlab mixture reproducibility Sample 1 Sample 2 Sample 3 Collaboration with Ivan Rusyn ’ s Group

1. Inject Sample 2. Wash Cartridge 3. Reverse Flow Automated SPE Send to MS system 10 sec analyses MS SPE Cartridge SPE Cartridge SPE Cartridge wash 6 sample injections/min 3 counts (a.u.) 2 1 12 24 36 48 60 72 84 96 108 120 Time (s)

SPE-IMS-MS analyses of biological samples Polar molecules extracted from mouse plasma Polar molecules extracted from human urine Isomeric Separation: Same nominal mass: m/z=327.197 138.3 m/z=138.054, 138.129 328.5 138.0 328.0 138.1 330.5 327.5 Isomeric Separation: 138.2 m/z=330.228 330.4 327.0 137.9 330.3 18.0 18.5 19.0 19.5 20.0 13.0 13.4 13.8 14.2 330.2 330.1 330.0 19.5 20.0 20.5 21.0 21.5 ~1400 features with S/N > 5 ~1000 features with S/N > 5 X. Zhang, et al., Clin. Mass Spectrom. 2016 , 2, 1-10.

Calibration curve for xenobiotics in plasma 6 100 nM [Imazaquin+H]+ 5.5 [Hexaconazole+H]+ [Thiabendazole+H]+ [Metribuzin+H]+ 5 [Napropamide+H]+ [Flumeturon+H]+ Log10 (Intensity) 4.5 [Isoxaben+H]+ [Oryzalin+H]+ [Fluroxypyr-1-methylheptyl ester+Na]+ 4 [Resmethrin+Na]+ 500 pM [Minocycline+H]+ 3.5 [Fenamidone+H]+ [Fenamiphos+H]+ 3 2.5 2.5 3 3.5 4 4.5 5 5.5 Log10 (Concentration in pM) X. Zhang, et al., Clin. Mass Spectrom. 2016 , 2, 1-10.

Small molecule pipeline Extraction Instrumental Analysis Data Processing & Analysis ID Mass Intensity ---- ---- ---- m/z ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- Methanol/Chloroform SPE-IMS-MS Database Matching & Extraction False Discovery Assessment (100 samples/day) (8200 samples/day) (? days)

Collision cross section database 1000 800 Lipids 600 m/z 400 200 0 100 150 200 250 300 DT CCS N2 (Å 2 )

Collision cross section database 1000 800 Lipids 600 m/z 400 200 >600 small molecules 0 100 150 200 250 300 DT CCS N2 (Å 2 ) X. Zheng, et al., Chem. Sci. 2017 , ASAP.

Metabolic pathways

Primary metabolite trend lines Protonated form 800 600 m/z 400 Amino Acids 200 Nucleotides Steroids 120 160 200 240 DT CCS N2 (Å 2 )

Primary metabolite trend lines Protonated form Deprotonated form 800 800 600 600 m/z m/z 400 400 Amino Acids Fatty Acids Lipid Mediators Amino Acids Nucleotides 200 200 Nucleotides Steroids Steroids Sugars 120 160 200 240 120 160 200 240 DT CCS N2 (Å 2 ) DT CCS N2 (Å 2 )

Xenobiotic trend lines

Primary metabolite versus xenobiotic L-Tyrosine (amino acid) Glufosinate (herbicide) Exact mass: 181.0738896 Exact mass: 181.0503922 134.7 Å 2 147.5 Å 2 1 [Tyrosine - H]- [Glufosinate - H]- Normalized Intensity 0 16 17 18 19 20 Arrival time (ms) Deprotonated form

Website - http://panomics.pnnl.gov/metabolites/ X. Zheng, et al., Chem. Sci. 2017 .

Summary • Ion mobility spectrometry enables rapid structural analyses • Combining multiple separations and methods enables faster and better small molecule identifications (i.e. SPE-IMS-MS) Phenomics

Acknowledgements • Agilent Technologies • PNNL Laboratory Directed Research and Development Program • NIEHS R01ES022190 • Environmental Molecular Sciences • NIEHS Superfund Research Program P42 Laboratory ES027704 • NIH General Medical Sciences P41 GM103493-11 Noor Aly Ion Mobility R&D Group