Small Molecule Metabolite Profiling and Characterization in Complex Biological Samples by GCxGC-TOFMS

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1 Small Molecule Metabolite Profiling and Characterization in Complex Biological Samples by GCxGC-TOFMS John Heim and Jeff Patrick Life Science and Chemical Analysis Centre LECO Corporation St. Joseph, MI 1

2 Metabolomics is the "systematic study of the unique chemical fingerprints that specific cellular processes leave behind" - specifically, the study of their small-molecule metabolite profiles. 2

3 Metabolic Pathways 3

4 LARGE DATA SETS 4

5 Techniques for Metabolomics NMR, IR spectroscopy, thin layer chromatography, CE with various detectors, HPLC with various detectors, GC-MS Compromise between speed, selectivity and sensitivity GC-MS and LC-MS in general provide good selectivity and sensitivity. GC-TOFMS and LC-TOFMS add the speed needed for high sample throughput often more complex to run GCxGC-TOFMS adds the additional separation needed when matrix interference is present GC-HR-MS and LC-HR-MS provide specific molecular identification 5

6 Comprehensive Two Dimensional Gas Chromatography (GCxGC) 1 st Dimension Column Long (non-polar) 2 nd Dimension Column Short (polar) Connected via a Modulator Thermal or Valve Based Comprehensive All sample injected reaches the detector D TIC 2D Plot 3D View 6

7 LECO s Dual Stage Quad Jet Thermal Modulator Stage 2 COLD JETS HOT JETS Stage 1 7

8 GCxGC-TOFMS Schematic GCxGC-TOFMS ATTRIBUTES Continous non-skewed full mass range spectra Full mass range sensitivity Fast acquisition up to 500 spectra per second enabling narrow peak identification TOFMS Delivers the data density needed for True Signal Deconvolution GCxGC is comprehensive (All of the sample injected is detected) A 10 fold increase in signal intensity is commonly obtained in a GCxGC analysis. Kinetic energy KE = ½ mv 2 Ions are pulsed into the field free region, flight times are related to their mass-to-charge ratios. 8

9 Human Breath Analysis Noninvasive technique Alveolar Breath Pulmonary Aveolar Membrane Signature markers linked to metabolic states Acetone for diabetes Isoprene for hyperchlosterolemia Straight chain hydrocarbons related to lipid peroxidationof polyunsaturated fatty acids found in cellular membranes Increased hydrocarbons related to pulmonary, liver, bowel and neurological diseases 9

10 Experimental Overview Sample Preparation 3-Liter Tedlar Bags TenaxGR TDU Tubes ATEX TDU/CIS 4 Inlet Sample Analysis GCxGC-TOFMS Data Analysis Data Mining 10

11 Experimental Conditions (GCxGC-TOFMS) Column 1: GERSTEL-MACH LTM 10 m x 0.18 mm I.D. x 0.2 μm RTX-5 Column 2: In GC Oven (No Secondary Oven) 1.0 m x 0.10 mm I.D. x 0.1 μm DB-17 Temperature Program 40 C 5 min 240 C 2 min (+ 15 C Column Offset) Modulation Period 6 seconds Flow 1.50 ml/min Transfer Line Temperature 280 C Data Acquisition Rate 200 spectra/sec 11

12 Human Breath 12

13 13

14 Creating a Reference 14 14

15 Creating a Reference 15 15

16 Results for Sample REFERENCE of Breath Out of Tolerance 16

17 Human Breath Summary Human Breath is a very complex sample, 100 s 1000 s of compounds that vary diversely from person to person Human Breath Analysis is an excellent diagnostic tool for human metabolomics Detection of biomarkers is key for early disease state identification Personalized medicine will include Human Breath Analysis GCxGC-TOFMS is an excellent analytical tool for breath screening and trace level identification and quantification 17

18 Diabetes Worldwide In 2000, according to the World Health Organization, at least 171 million people worldwide suffer from diabetes, or 2.8% of the population. Its incidence is increasing rapidly, and it is estimated that by 2030, this number will almost double. Prevalence of diabetes worldwide (per 1000 inhabitants) Diabetes mellitus occurs throughout the world, but is more common (especially type2) in the more developed countries. The greatest increase in prevalence is, however, expected to occur in Asia and Africa, where most patients will probably be found by The increase in incidence of diabetes in developing countries follows the trend of urbanization and lifestyle changes, perhaps most importantly a "Westernstyle" diet. This has suggested an environmental (i.e., dietary) effect, but there is little understanding of the mechanism(s) at present, though there is much speculation, some of it most compellingly presented. 18

19 Diabetes Experimental Approach Morning fast urine was collected from four subjects 2 non-diabetic (control) 1 type I diabetic and 1 type II diabetic Samples were stored under refrigeration prior to liquid/liquid extraction and derivatizationwith BSTFA Six 10mL aliquots were prepared by acidification with H2SO4(pH of 2) 10mL aliquots were extracted with 2mL of methylene chloride into a 20mL scintillation vial containing 5mg sodium sulfate Derivatization was carried out by adding 200μL of extract into a sealed 2mL auto sampler vial containing 0.5mg sodium sulfate 30μL of dry pyridine was added 200μL of BSTFA was added to each vial Samples were heated to 60 o C for 1 hour and then analyzed 19

20 Type I vs. Type II Diabetic Urine Analysis GCxGC-TOFMS Analysis 30m x.25mm x.25μm Rtx-5ms 1.5m x.18mm x 0.18μm Rtx ºC (1 min) 6º C/min to 190º C (10 min.) Secondary Oven: +10ºC μ at 200 spectra / s Type I Diabetic Healthy Type II Diabetic Contour plot of Healthy Individual Urine 20

21 21

22 Diabetes Summary GCxGC-TOFMS is an excellent analytical tool for Human Urine analysis Advanced Data processing tools are available for sample comparison in complex matrixes and larger sample groups Key biomarkers for type I and type II diabetes are under investigation 22

23 The Detection of Anabolic Steroid metabolites in Urine A number of the drugs have common metabolic pathways, and their excretion profiles may overlap those of the endogenous steroids, making interpretation of testing results a very significant challenge to the analytical chemist. STEROID GLUCURONIDE HYDROLYSIS SAMPLE PREPARATION LABOR INTENSIVE POOR IONIZATION LOW DETECTABILITY ENDOGENOUS STEROID INTERFERENCE 23

24 Highlights from: The Detection of Anabolic Steroid metabolites in Urine Experimental Methods: Sample Extraction and Derivatization Sample Preparation in 2mL urine Add Sodium Phosphate buffer to ph 6 Add ISTD Methyltestosterone at 200ng/mL Acid Hydrolysis with β-glucuronidase Hydrolysis at 50ºC for 1 hour Add potassium carbonate solution to ph 9 and shake at 3000rpm for 5 minutes Extract with 5mL of Methyl-tert-butyl-ether (MTBE) Transfer organic phase to new clean glass vial Evaporate to dryness with Nitrogen Derivatize dry extracts with 100µL MSTFA-NH4I-ethanethiol for 30 minutes at 60ºC. 24

25 Sample preparation Steroid Standards were prepared, derivatized and spiked into 2mL aliquots of urine at the concentrations of 2, 10, 20, 50 and 100ng/mL. On column autosampler injections were made at 3uL each. STEROID TARGET ANALYTES 19-Norandrosterone (Metabolite of Nandrolone) Boldenone (Anabolic steroid developed for veterinary use) 17α-Methylandrostan - 3α, 17β-diol (Metabolite of Methyltestosterone) 3-Hydroxystanozolol (Metabolite of Stanozolol) H 3 C 4-Hydroxystanozolol (Metabolite of Stanozozlol) Methyltestosterone (Used as an Internal Standard ISTD) References

26 Instrumental Method: GCxGC-TOFMS Analysis GC: Agilent 7890 equipped with a LECO GCxGC secondary oven and dual stage thermal modulator GERSTEL MPS2 autosampler GC Primary Column: GC Secondary Column: 30m x 0.25mm id x 0.25µm film thickness, Rxi-5MS (Restek Corp.) 1.2m x 0.18mm id x 0.20µm film thickness, BPX-50 (SGE Analytical Science) Carrier gas: 1.5mL / min constant flow Injection port temperature: 280 C Injection mode: Pulsed Splitless Transfer line temperature: 280 C Primary column GC temperature program: Initial temperature 140 C for 0.2 min. 20 C/min. to 170 C then 5 C/min. to 260 C for 2.0 min., then 10 C/min. to 315 C held for 12.0 minutes. Secondary oven GC temperature program: Initial temperature 145 C for 0.2 min. 20 C/min. to 175 C then 5 C/min. to 265 C for 2.0 min., then 10 C/min. to 320 C held for 12.0 minutes Total run time: minutes Mass spectrometer: LECO Pegasus 4D Mass range: m/z Acquisition rate: 100 spectra / sec Detector Voltage: 1950 V Acquisition delay: 400 sec Ion Source temperature: 230ºC 26

27 3D Plot of 20ng/mL TMS-derivatized Steroids in urine 19-Norandrosterone-2TMS Stanozolol-1TMS 3-Hydroxystanozolol-2TMS Stanozolol-2TMS 4-Hydroxystanozolol-2TMS Boldenone-2TMS 4-Hydroxystanozolol-3TMS 3-Hydroxystanozolol-3TMS Methyltestosterone-2TMS (ISTD) 17-alpha-Methylandrostan-3-alpha, 17-beta-diol (2TMS) 1 st Dimension Retention Time 27

28 ENHANCED PEAK CAPACITY AND RESOLUTION OF GCxGC 2-TMS 1-TMS 3-TMS 2-TMS 2-TMS STANOZOLOL 3-TMS 3-HYDROXY-STANOZOLOL 4-HYDROXY-STANOZOLOL 28

29 Achieving WADA cut-off guidelines: Steroids in Urine 2ng/mL (2ppb) Standard with Quantitative S/N Ratios 29

30 17-alpha-Methylandrostan-3-alpha, 17-beta-diol (2TMS) Quantitative Calibration Linearity 4-Hydroxystanozolol (3TMS) 100pg/µL 100pg/µL 50 pg/µl 50 pg/µl 20pg/µL 10pg/µL 2pg/µL r = Concentration y= x Norandrosterone, 3-trimethylsilyl ether, 17-TMS enol ether 20pg/µL 10pg/µL 2pg/µL r = Concentration y= x '-Hydroxystanozolol, N,O.O-tris(trimethylsilyl) (3TMS) 100pg/µL 100pg/µL 50 pg/µl 50 pg/µl 20pg/µL 10pg/µL 2pg/µuL r = Concentration y= x pg/µL 10pg/µL 2pg/µL r = Concentration y= x

31 GCxGC-TOFMS Steroid Urine Analysis Conclusions Limits of Detection were shown that meet or in most cases exceed the cut-off limits set by WADA. Successful calibration curve development for 5 steroids over the concentration range of 2 to 100ng/mL. Calibration linearity achieved greater than 99.9%. GCxGC-TOFMS demonstrates improved detectability and enhanced chromatographic resolution for anti-doping screening control. The use of GCxGC-TOFMS for anti-doping control is proving to be a valuable asset for this instrumental analysis. 31

32 Amino Acid Analysis in Human Blood 10 blood plasma samples and 2 blanks were provided for analysis by GCxGC-TOFMS All samples and blanks were extracted and dried. Samples were prepared by the addition of a 9 component internal standard followed by trimethylsilyl derivatization using BSTFA. Kevin King, vice president of operations for the Physician s Plasma Alliance, collects a blood sample from Randy Jones on Thursday at the center in Gray, Tenn. Jones has Crohn sdisease and agreed to donate his blood for diagnostic study.photo by Adam Brimer 32

33 Experimental Goals and Analytical Conditions Identification of Amino Acids Improve separation of C-18 Fatty acids Identify designated trace level compounds Experimental Conditions Column 1: Rtx-1MS 30m x 250μm id x 0.25μm film thickness Column 2: BPX m x 100μm id x 0.10μm film thickness Column 1 Temperature Program: Initial Temp: 60 C for 0.2min. Ramped at 13 C/min. to Final Temp of 310 C Column 2 Temperature Program: Initial Temp: 65 C for 0.20min Ramped at 13 C/min. to Final Temp. of 310 C Total Runtime: 19.20min. TOFMS Acquisition Delay: 267sec. Ion Source Temperature: 200 C Mass Range: amu Electron Energy: -70eV Acquisition Rate: 200spectra/sec. Detector Voltage: 1850V 33

34 34

35 Amino Acid Identification 35

36 C-18 Fatty Acid Separation 1. Linolenic acid TMS ester 2. 9,12-Octadecadienoic acid (Z,Z) TMS ester 3. trans-9-octadecenoic acid -TMS ester 4. cis-11-octadecenoic acid TMS ester 5. (cis-9-octadecenoic acid) Oleic acid TMS ester 6. Octadecanoic acid TMS ester 36

37 Results Conclusions The value of GCxGC-TOFMS was illustrated identification 16 different amino acids in the blood plasma extracts. Separation of fatty acid isomers in the second dimension illutrates the resolving power of comprehensive two dimensional gas chromatography. GCxGC-TOFMS proves to be a valuable analytical tool for metabolomics. 37

38 GCxGC-TOFMS Metabolite Comparison in Pooled Plasma Samples from Lean, Fat, and Obese Diabetic Zucker Rats 38

39 Sample Preparation Proteins were removed from 30 samples of lean, fat and obese Zucker rat plasma with a 5000 MWCO filter. 100uL samples were dried in a Savant SpeedVac. The dried residue was suspended in 100uL BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) and incubated overnight at 60ºC. GCxGC-TOFMS analysis was performed on the pooled samples from various states of health, lean, fat and diabetic obese. 39

40 GCxGC-TOFMS Instrumental Conditions Pooled Rat Plasma Comparison GC: Agilent 6890 equipped with a LECO GCxGC secondary oven and dual stage thermal modulator GERSTEL MPS2 autosampler GC Primary Column: 30m x 0.25mm id x 0.25µm film thickness, Rxi-5silMS (Restek Corp.) GC Secondary Column: 1.24m x 0.15mm id x 0.15µm film thickness, Rxi-17SilMS (Restek Corp.) Carrier gas: 1.5mL / min (corrected constant flow via pressure ramps) Injection port temperature: 260 C Injection mode: Splitless Injection Volume: 1µL Transfer line temperature: 260 C Primary column GC temperature program: Initial temperature 70 C for 0.5 min. 6 C/min. to 305 C held for 5.0 minutes. Secondary oven GC temperature program: Initial temperature 75 C for 0.5 min. 6 C/min. to 310 C held for 5.0 minutes GCxGC Parameters: Modulator temperature offset: 20 C Column offset: 20 C Modulation period (2nd dimension separation time): 4 sec. Hot pulse time: 0.60sec. Cool time: 1.40sec. Total run time: minutes Mass spectrometer: LECO Pegasus 4D Mass range: m/z Acquisition rate: 150 spectra/s Detector Voltage: 1650 V Acquisition delay: 450 sec Ion Source temperature: 240ºC 40

41 Obese Pooled Plasma Sample GCxGC-TOFMS Analysis OBESE DIABETIC POOLED PLASMA SAMPLE GCxGC-TOFMS ANALYSIS 41

42 Leucine, N,O- TMS LEUCINE, N,O-TMS-B LEUCINE, N,O-TMS-B st Time (s) 2nd Time (s) Lean Pooled Lean Pooled OP - Obese Pooled:1 158 FP - Fat Pooled:1 158 LP - Lean Pooled:1 Fat Pooled Obese Pooled Sample Pool Name Peak # Name R.T. (s) Area Height UniqueMass Similarity S/N Library Lean pooled 0 NOT FOUND Fat pooled 260 LEUCINE, N,O-TMS , Max Planck1 Obese pooled 254 LEUCINE, N,O-TMS , Max Planck1 42

43 Glycine, N-(1-oxobutyl)-trimethylsilyl ester Obese Pooled GLYCINE, N-(1-OXOBUTYL)-, TRIMETHYLSILYL ESTER GLYCINE, N-(1-OXOBUTYL)-, TRIMETHYLSILYL ESTER 200 1st Time (s) 2nd Time (s) FP - Fat Pooled:1 202 LP - Lean Pooled:1202 OP - Obese Pooled: Lean Pooled Fat Pooled Obese Pooled Sample Pool Name Peak # Name R.T. (s) Area Height UniqueMass Similarity S/N Library Lean pooled 0 NOT FOUND Fat pooled 265 Glycine, N-(2-methyl-1-oxopropyl)-, trimethylsilyl ester , mainlib Obese pooled 260 GLYCINE, N-(1-OXOBUTYL)-, TRIMETHYLSILYL ESTER , Wiley7 43

44 Glucopyranose Penta-TMS GLUCOPYRANOSE PENTATMS-B GLUCOPYRANOSE PENTATMS-B -0 1st Time (s) 2nd Time (s) LP - Lean Pooled: FP - Fat Pooled:1204 OP - Obese Pooled: Lean Pooled Fat Pooled Obese Pooled Sample Pool Name Peak # Name R.T. (s) Area Height UniqueMass Similarity S/N Library Lean pooled 300 GLUCOPYRANOSE, 1,2,3,4,6-PENTAKIS-O-(TRIMETHYLSILYL)-, D , Wiley7 Obese pooled 350 GLUCOPYRANOSE, 1,2,3,4,6-PENTAKIS-O-(TRIMETHYLSILYL)-, D , Wiley7 Fat pooled 356 GLUCOPYRANOSE, 1,2,3,4,6-PENTAKIS-O-(TRIMETHYLSILYL)-, D , Wiley7 44

45 Conclusions This GCxGC-TOFMS study of pooled samples shows promise as an analytical screening tool for evaluating metabolite differences in pooled sample groups from various states of health. Potential biomarker discovery can be performed quickly for large groups of pooled samples. Valuable instrument & laboratory staff time can be saved providing a cost efficient solution for small metabolite profiling. 45

46 Metabolomic Analysis of Rat and Mouse Using High Performance Time of Flight Mass Spectrometry with FFP Technology Fat, Diabetic and Control Zucker Rats; Lipids in Plasma and More by UHPLC and GC Jeffrey S. Patrick 46

47 Key Messages The benefit of complementary techniques in metabolomic analysis The potential for improved information content through HPLC with High Performance Mass Spectrometry GC with High Performance Mass Spectrometry Screening with GCxGC with Time-of-Flight MS Qualitative analysis of the response of metabolic markers Lean, Fatty and Obese Zucker Rats Liver stress Conditioning The capabilities of two new high performance mass spectrometers in addressing the needs of metabolomic analysis Emphasizing the benefit of high performance mass spec 47

48 Ultra-High Resolution High Resolution Nominal LECO LC-HRT Using FFP A D Mirr ors Lens es Mirr ors A D A D m/z R=100,000 4:1 mass Range m/z R=50,000 m/z = m/z R=2,500 m/z = Folded Flight Path of up to 40 m yields ultra-high resolution Vernchikov et.al US Patent Allows ultra-fast capture of high resolution spectra The new LECO HRT platform uses Folded Flight Path (FFP ) technology. This has been translated into three modes of operation which permit user flexibility and routine high performance. 48

49 LECO s High Performance HRTs Citius LC-HRT Pegasus GC- HRT CITIUS 49

50 System Under Study Zucker Rats 3 Phenotypes/Strains w/ Animals bred to be Lean (n=10), Fatty (n=9), Obese (n=10) 7-9 Weeks old (terminal bleed) Disodium EDTA as anti-coagulant 0.1 µm filtered Objectives Identify analytes which are up or down regulated with phenotype using high performance MS Test the capabilities of HRTs Fit Husky?? 50

51 Sample Preparation Plasma (purchased from Bioreclamation) Remove Protein With Centrifuge LC-HRT HPLC TOF-MS SpeedVac BSTFA GC-HRT GC TOF-MS Pool by Condition SpeedVac BSTFA GCxGC - Comparison with precipitation with acetonitrile and methanol was made 51

52 HPLC-MS Conditions LECO LC-HRT 8 spectra/sec 4 in low energy (parent ions) 4 in high energy (fragmentation) High Resolution Mode LECO Electrospray Ionization Source Agilent 1290 UHPLC HPLC at 0.2 ml/min A = 0.1% Formic Acid B = 0.1% Formic Acid in AcN C18 2.1x100 mm Column 52

53 Data Extraction Capability of Accurate Mass TIC for 4 Animals Buried in the baseline 4 Animals TIC Each XIC Each TIC and XIC for 4 Animals Analyte Response * ±

54 Response Ratios for Selected Analytes 3-way comparisons = (Analyte Area(Fatty))/(Analyte Area(Lean)) Response Ratio Fatty:Lean Fatty:Obese Obese:Lean Response Ratio Fatty:Lean Fatty:Obese Obese:Lean Analyte Analyte 54

55 Response Ratios for Selected Analytes Response Ratio Fatty:Lean Fatty:Obese Obese:Lean J. Nutrition, March, Targeted Analyte 55

56 Analyte for Identification C 11 H 21 NO (0.77 ppm) Butyryl Carnitine in Rat Plasma -(CH3)N Carnitine M+H + core Obese Fatty High Energy Fragment Channel Lean CITIUS 56

57 kv In-Source CID (iscid) Alternative to MS/MS Acquire in separate data channels +Maintains TOF acquisition speed +Maintains mass accuracy - Lose direct link to parent ion Higher Pressure Voltage Difference Large Fragment Ions CITIUS 57

58 10 ev MS/MS Validity of iscid 58

59 Relative Isotope Abundance - Butyryl Carnitine Relative Isotope Abundance Formula Sample 12 C 1* 13 C 2* 13 C 3* 13 C A B C* E-05 D E* C 11 H 21 NO 4 Calculated C 12 H 17 N 5 Calculated Option # Accurate Mass Match Fragmentation Match Relative Isotope Abundance Match % Theoretical; All 12 C C + 3* 13 C Sample A Sample B Sample C Sample D Sample E Average Theoretical CITIUS 59

60 Trends for Butyryl Carnitine Carnitine in Zucker Rats FATTY LEAN OBESE ALL ALL OBESE LEAN FATTY 60

61 Rat Plasma Analysis with Speed Same Mass Resolution and Accuracy Analysis of Rat Plasma in < 3 min at 40 spectra/second (split iscid) Analysis of Human Plasma in < 3 min at 40 spectra/second (split iscid) 100x Signal from x Signal from

62 Dynamic Range- ButyrylCarnitine Linearity of ButyrylCarnitine on LC-HRT Beyond 500 nm Up to 2500 nm) > 4 logs Linearity of ButyrylCarnitine on LC-HRT Area Response Analyte Concentration (nm) Area Response LOQ ca nm (200 fg on column) Analyte Concentration (nm) GRATEFULLY ACKNOWLEDGE C. AGUER 1, O. FIEHN 2, S. ADAMS 3, M-E. HARPER 1- U OTTAWA 1, UC DAVIS 2, WHNRC DAVIS 3 FOR STANDARDS 62

63 LC-HRT Performance During Rat Plasma Study Mass Error (ppm) Average Mass Error ppm From Real Analytes Sorbitol Analyte m/z IMP Note sorbitol and IMP elute at the solvent front Average Mass Error vs m/z Study Progressed over three days Included issu es External Calibration Achieved at a Resolution (FWHH) of better than across the range Resolution m/z Asymptotic Resolution of

64 Sample Preparation Plasma (purchased from Bioreclamation) Remove Protein With Centrifuge LC-HRT HPLC TOF-MS SpeedVac BSTFA GC-HRT GC TOF-MS HPLC at 0.1 ml/min 4 spectra/sec m/z High Resolution Mode Pool by Condition SpeedVac - Comparison with precipitation with acetonitrile and methanol was made GCxGC TOF-MS 64

65 GC-MS Conditions LECO GC-HRT 20 spectra/sec High Resolution Mode (R = 25,000) Electron Impact ionization Agilent 7890 GC 1 ml/min Split 100:1 Temperature Ramp o C in 20 min Column Rxi-5MS (Restek) 0.25mm x 30m 65

66 Representative Chromatogram of Rat Plasma XIC for 10 mmu(x2 66

67 8.00E E E+06 Ave. Area 5.00E E E E E E+00 l-alanine, ditms Lean Fatty Obese Uric acid, N,O,O',O''-tetrakis(trimethylsilyl)- 3.00E E Ave. Area 2.00E E E E E+00 Lean Fatty Obese 67

68 Percent Theoretical Isotopes Relative Isotope Abundance Relative Isotope Abundance Creatinine enol (TMS) C13H31N3OSi3 Urea (tri-tms) C10H28N2OSi % At low S/N L-Valine (TMS) C7H18NSi (Fragment ion) Resolving Power High Res Mode Resolution FWHH) Resolution Performance of the Leco GC-HRT Throughout the Study >25000 for all PFTBA m/z 80.0 Mono 1 st 2 nd Mono 3 rd Mono 3 rd m/z 68

69 Complementary Nature of Approaches For analytes observed to modulate with phenotype in each experiment GC/ GCxGC overlaps in amino acids, fatty acids and carbohydrates GC/GCxGC Overlap 22 GC-HRT GCxGC LC-HRT LC/GCxGC Overlap LC and GC/ GCxGC overlaps in amino acids 69

70 KEGG Pathway Lipid Synthesis Acyl Variations Chain length Unsaturation Branching Combinations 70

71 Structures of Representative Lipids Phosphatidyl Ethanolamines Acyl Phosphoglycerates N-Methyl Phosphatidyl Ethanolamines Acyl Phosphatidyl Inositols Phosphatidyl Cholines Chemical and Physical Similarity Charged Headgroups Homologous Series hp 71

72 LIPID PROFILE AND LIVER STRESS Rough Sample Preparation (Univ. of Louisville) Rats were fed a high fat diet (F) and a high fat diet and simultaneously dosed with PCB Livers were removed and homogenized Extracted with methanol (80%) Analyzed using: Advion Nanomate Triversa direct analysis (after centrifugation) HILIC UHPLC 72

73 HILIC Analysis of Liver Extracts PCB Treated (Conditions from UC-Davis) Base Peak Chromatogram XIC (PCs and PC ) 2.5e6 2.0e6 1.5e ±0.03 (Precursor Ions) LC ±0.01 (Fragment Ions) LC ±0.03 (Precursor Ions) LC ±0.01 (Fragment Ions) LC ±0.03 (Precursor Ions) LC ±0.01 (Fragment Ions) LC ±0.03 (Precursor Ions) LC ±0.01 (Fragment Ions) LC Caliper - sample "LC ", s to s - ( s to s s to s ) e e4 2.5e4 2.0e4 1.5e4 1.0e4 5.0e3-0.0e0 M/Z Caliper - sample "LC ", s to s - ( s to s s to s ) Intensity(counts) Intensity(counts) 3.0e3 2.0e3 1.5e e2-0.0e0 M/Z Caliper(Cid) - sample "LC ", s to s - ( s to s s to s ) 1.0e6 Intensity(counts) 7.0e5 5.0e5 3.0e e5 0.0e0 M/Z Caliper(Cid) - sample "LC ", s to s - ( s to s s to s ) 5.0e5 0.0e0 Time (s) Intensity(counts) 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e4-0.0e0 M/Z

74 Changes in select PCs with Treatment by HILIC Name Formula Expected m/z Observed m/z Area Ratio (T/N) PC 36:4 C44H81NO8P PC 36:3 C44H83NO8P SM 40:2 C45H90N2O6P n.d. n.d. PC 36:2 C44H85NO8P PC 36:1 C44H87NO8P PE 42:6 C45H79NO8P n.d. n.d. PC 38:6 C46H77NO8P PC 38:4 C46H81NO8P PC 38:2 C46H85NO8P

75 ANIMAL MODELS CELINE AGUER 1, OLIVER FIEHN 2, SEAN ADAMS 3, MARY-ELLEN HARPER 1 U OTTAWA 1, UC DAVIS 2, WHNRC DAVIS 3 Endurance training is well known to enhance fatty acid oxidation (for review see Achten et al., Nutrition, 2004) Training protocol: - Running on a treadmill - 14 m / min - 1 h / day - 5 days / week - 5 weeks (from Leick et al., AJP Endocr Metab, 2008) 75

76 The utility of high resolving power R =

77 Molecular Ion Region of 16:0 and 16:1 LysoPhosphatidylCholines Caliper - sample "503_U", s to s - ( s to s s to s ) 1.4e Caliper - sample "503_U", s to s - ( s to s s to s ) 4.0e4 M+2 Isotope of LysoPC 16:1 Intensity(counts) 1.2e6 1.0e6 8.0e5 6.0e5 4.0e Intensity(counts) 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e4 5.0e3-0.0e0 M/Z e5-0.0e0 M/Z M+1 M

78 Mass and Chromatographic Resolution of lysopcs Partially Unsaturated or Isotope Peak 238 C23H47ON8P 503_U Peak 17 C27H54ON3Na2 503_U Peak 52 C22H49N7PNa2 503_U Peak 3 C23H41N8P2 503_U Peak 234 C23H43O2N8P 503_U Peak 217 C29H47O4NNa 503_U Peak 233 C23H45O2N8P 503_U Peak 57 C13H25O7N3P3Na3 503_U Peak 55 C13H13O10N10P 503_U Peak 175 C21H10ON6P3Na2 503_U Peak 50 C22H49O2N9P 503_U Peak 236 C31H51O3NNa 503_U Partially Reduced? 78

79 Mass Resolution of Assignment of Unsaturated PCs (16:0 and 16:1) Phenotype A Phenotype B Physiologically relevant relative amounts 20-60:1 (16:0 to 16:1) Here it is 45:1 Physiologically relevant Relative amounts 20-60:1 (16:0 to 16:1) Here it is 25:1 79

80 Lipid Analysis by Nano-infusion Advion Nanomate Citius LC-HRT CITIUS 80

81 NANOMATE Analysis of Polar Lipids from Liver (methanol) Caliper - sample "869FP-2", s to s Caliper - sample "869FP-2", s to s Intensity(counts) 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e Intensity(counts) 2.0e4 1.8e4 1.5e4 1.3e4 1.0e4 7.5e3 5.0e3 2.5e3 0.0e0 M/Z e0 M/Z

82 Caliper - sample "873FP-2", s to s 3.5e3 3.0e3 Comparison of Polar Lipid Profile in Mouse Liver Extracts PE 38:6 PE 38:8 PC 38:2 PC 38:1 PC 36:2 PC 36:1 PC 38:2 PC 38:1 Treated Caliper - sample "876FP", s to s 8.0e3 7.0e3 6.0e3 PE 38:6 PE 38:8 PC 38:2 PC 38:1 PC 36:2 PC 36:1 PC 38:2 PC 38:1 Treated Intensity(counts) 2.5e3 2.0e3 1.5e3 Intensity(counts) 5.0e3 4.0e3 3.0e3 1.0e3 2.0e3 5.0e2 1.0e3 0.0e0 M/Z e0 M/Z Caliper - sample "852F", 0 s to s 1.4e4 1.2e4 PE 38:6 PE 38:8 PC 38:2 PC 38:1 PC 36:2 PC 36:1 PC 38:2 PC 38:1 Untreated Caliper - sample "854F", s to s 1.4e4 1.2e4 PE 38:6 PE 38:8 PC 36:2 PC 36:1 PC 38:2 PC 38:1 PC 38:2 PC 38:1 Untreated Intensity(counts) 1.0e4 8.0e3 6.0e3 Intensity(counts) 1.0e4 8.0e3 6.0e3 4.0e3 4.0e3 2.0e3 2.0e3 0.0e0 M/Z 0.0e M/Z Great Qualitative and Relative Quantitative Information 82

83 Summary and Conclusions The potential for improved, complementary information content through a multi-faceted approach Model system is Lean, Fatty and Obese Zucker Rats >50 analytes identified as modulated between the three approaches HPLC with High Performance Mass Spectrometry GC with High Performance Mass Spectrometry Screening with GCxGC with Time-of-Flight MS Signaling molecules and lipid metabolism Substantial Information from Direct Nano-infusion The capabilities of two new high performance mass spectrometers are highlighted in addressing the needs of metabolomic analysis Emphasizing the benefit of high performance MS Showing the capability of two Prototype systems using FFP, Resolution, Speed Mass. Accuracy No Compromise 83

84 For More Information Contact LECO at: Life Science & Chemical Analysis Centre Telephone: