Qualitative and Quantitative Analysis of Lipids using SFC and LC combined with High Resolution Accurate Mass MS. Jim Lau, Ph.D. Agilent Technologies

Transcription

1 Qualitative and Quantitative Analysis of Lipids using SFC and LC combined with High Resolution Accurate Mass MS Jim Lau, Ph.D. Agilent Technologies

2 Collaborators and Acknowledgements Terry Berger, SFC Solutions, Inc. Jennifer Van Anda, Applications Scientist, SFC Joe Hedrick, Applications Scientist, LCMS and SFCMS Rick Wikfors, Director, R&D, SFC technologies Tony Brand, Applications Scientist, LCMS and SFCMS Jim Lau, Applications Scientist, LCMS and SFCMS 2 Confidentiality Label May 16, 2014

3 What is SFC? A separation technique similar to HPLC Same hardware AND software as HPLC Usually CO2 as the main component in the mobile phase Usually binary or ternary mixtures as mobile phase Predominantly polar packed columns. Highly non-polar Analytes amenable to Reverse Phase. Capillary SFC has almost disappeared Usually composition gradients. Seldom programmed pressure or density gradients SFC Solutions, Inc.

4 Practical Advantages of SFC? 3-5 X speed/throughput--more samples/day more rapid re-equilibration-- shorter cycle time compared to reversed phase HPLC 1/3 rd -1/5 th pressure drops Dramatically Lower operating cost Orthogonal to reversed phase HPLC Great for isomers Sensitivity, Dynamic Range, and Capacity like HPLC (Inj. Volumes) Low solvent consumption/low waste generation Green! SFC Solutions, Inc.

5 Where Does SFC Fit Relative to HPLC? Solute Families Normal Phase HPLC Reversed Phase HPLC Ion Pairing Ion Chromatogr. HILIC SFC w/ pure CO 2 CO 2 w/ organic modifiers CO2 + modifiers + additives + water CO 2 w/ modifiers + additives SFC Solutions, Inc.

6 1989 SFC Applications 91% non-pharma and chiral 9% Environmental Misc. Pharma + chiral Polymers Food Fuels Data based on the compendium from the International symposium on SFC/SFE,

7 Absorbance, mau SFC is the Premier Chromatographic Method for Chiral Separations. There is nothing that competes in terms of speed and resolution. Most large pharma have largely abandoned HPLC Most academics, and small pharma largely never heard of it Atenelol x250mm, 5µm Regispack Retention Time, min SFC Solutions, Inc.

8 SFC 2012: All, but Pharma, practically Non-Existent 10-15% Environmental Polymers Food Misc Pharma SFC-MS Achiral Fuels Chiral Anal. Pharma Prep 85-90% pharma 8

9 Cod Liver Oil by Capillary SFC FID detection-120 min A. Staby, C. Borch-Jensen, S. Balchen, J. Mollerup, Quantitative Analysis of Marine Oils by Capillary Supercritical Fluid Chromatography, (1994) Chromatographia complex density program; 45 Identified components; separated by mass compounds with same carbon number but different degrees of unsaturation co-eluted. SFC Solutions, Inc.

10 Rapeseed Oil: TRIGLYCERIDES 7-120mm columns in series (840mm total) Hypersil ODS (4.6 x 5µm), 3ml/min / 5.4% ACN. 0.6% MeOH, 94% CO2, 16 C, 100 bar, 210nm E. Lesellier, A. Tchapla, Subcritical Fluid Chromatography with Organic Modifiers on Octadecyl Packed Columns: Recent Developments for the Analysis of High Molecular Weight Organic Compounds, in Supercritical Fluid Chromatography with Packed Columns, K. Anton, C. Berger, Eds. Chromatographic Science Series Vol 75, Marcel Dekker, New York 1997 p. 210 SFC Solutions, Inc.

11 Soybean Oil by Packed Column SFC- 2002, APCI-MS 100 min. Sandra, P., Medvedovici, A, Zhao, Y., David, F., Characterization of triglycerides in vegetable oils by silver-ion packed-column supercritical fluid chromatography coupled to mass spectrometry with atmospheric pressure chemical ionization and chemical coordination ion spray, (2002)J. Chromatogr., A, UV 210nm APCI + Ag + 22 triglycerides identified Silver loaded Nucleosil SA strong cation exchanger 4.6x250mm, 5µm, pressure programming from 150 to 300 bar at 1.5bar/min % Modifier 1.2% for 2 min then 7.2% at 28 min, 12.2% at 37.3 min Modifier: acetonitrile:isopropanol 60:40; 1ml/min 65 C SFC Solutions, Inc.

12 Experimental Set-up Make-up Bin Pump restrictor 10:1 split SFC mod. ALS TCC DAD QTOF w/ ESI Agilent 6540 to BPR Agilent 1260 SFC SFC Solutions, Inc.

13 The SFC/LC System UHPSFC Mode Loop back Restrictor HPLC Waste SFC Waste BPR LC Pump out LC Pump: G1310B or G1311B or G1312B SFC Pump outlet Detector out SFC-Binary Pump Page 13

14 The SFC/LC System UHPLC Mode Restrictor Waste BPR LC Pump out LC Pump: G1310B or G1311B or G1312B SFC Pump outlet Detector out SFC Pump inlet SFC-Binary Pump Page 14

15 Direct vs. Split Flow to MS (QTOF) To QTOF To QTOF 15 Confidentiality Label May 16, 2014

16 Agilent Jetstream Technology (AJT) source Stainless from SFC Nebulizing gas: pressure *Sheath gas: flow and temperature *Nozzle voltage Standard Source used for SFCMS of Lipids The super-heated sheath gas collimates the nebulizer spray and presents more ions to the MS inlet. Drying gas: flow and temperature Resistive sampling capillary entrance: Capillary V Resistive sampling capillary exit: FragV *New parameters unique to AJT source 16

17 APCI Spray Chamber Stainless from SFC Nebulizer Pressure Corona current Heater Vcap Drying gas Temperature and Flow Fragmentor

18 Page 18 SFC RC.NET drivers

19 Fish Oil Analysis by SFC/QTOF

20 A General SFC Lipids Method Materials Carbon dioxide: Bone-dry grade (50lb steel cylinder, Linde) Modifier: MeOH and iproh, HPLC grade purchased (VWR) Additive: Ammonium acetate (>99%, Aldrich) Sample Preparation The fish oil was purchased as a commercial dietary supplement in gel capsule. The oil was sampled from capsule then diluted either 100:1 or 1000:1 with isopropyl alcohol. Chromatographic Method Column: Agilent Zorbax SB300 C18 4.6x150mm, 60 C Flow rate: 3 ml/min, outlet pressure = 140 bar Gradient: 0 mins 3% MeOH w/5mm NH4OAc 5.5 mins 3% MeOH w/5mm NH4OAc 11 mins 60% MeOH w/5mm NH4OAc 12 mins 60% MeOH w/5mm 12 mins, end of run

21 Extracted ion chromatogram of Fish Oil SFC/QTOF Agilent 1260SFC with Model 6450 QTOF with ESI 5mM ammonium acetate in MeOH 3mL/min 3% for 5.5min the 60% at 11 min 60 C, 140 bar outlet pressure SB100 C18 4.6x150mm, 3.5µm 10:1 split into QTOF w/ MeOH make-up 0.6min Commercial fish oil capsule 7.8min SFC Solutions, Inc.

22 Fish Oil Analysis by SFC/QTOF: Extracted Ion Chromatograms 2beta-methyl-1beta,25 dihydroxycholecalciferol DGs Vit D precursors TGs Commercial fish oil capsule

23 Reproducibility: Retention Time and Area Fish oil sample, EIC and , 3 replicates, MS QTOF data 0 mins 3% MeOH/5mM NH 4 OAc 5.5 mins 3% MeOH/5mM NH 4 OAc 11 mins 60% MeOH/5mM NH 4 OAc (hold for 1 minute) m/z RT Area Mean S. Dev % S. Dev m/z 5 RT Area Mean S. Dev % S. Dev

24 1600 Mass vs. Retention Time M a s s ( D a ) Retention Time (min)

25 M 950 a s s ( D a ) 900 Mass, Da time, min Retention Time (min)

26 TGs, C53H98O6, C53H96O6, C53H94O4 C53H94O6 C53H96O6 C53H98O6

27 DG separation with 1 degree of saturation difference C41H70O5 (M+H) ppm mass error C41H72O5 (M+H) , 0.7ppm mass error 0.6min 4.5min

28 # Double Bonds What defines Triglyceride Chromatographic Retention? C63 C65 C67 A C61 C55 C57 C59 C53 B Retention Time, min In these experiments, Retention is a Strong Function of Degree of Unsaturation and much Less according to Carbon Number

29 # Double Bonds Retention does not appear to be related to the Location of Double Bonds, but only the Number C61 C :0/20:X/22:6 16:0/22:X/22: :0/20:0/22:X 16:0/22:0/22:X Retention Time, min

30 Fish Oil: Individual lipids tentatively identified by exact mass 140 Largest TGs 42 Largest DGs

31 Full Results Set for Tripalmitin MS of M+NH4, M+Na and MSMS of each

32 Databases have revolutionized Lipid analysis Variants of Tripalmitin Loss of Neutral C18H34O2NH3 Loss of Neutral C16H30O2NH3 Loss of Neutral C18H36O2NH3 Loss of Neutral C16H32O2NH3 Sartain et.al. 32 Confidentiality Label May 16, 2014

33 13 possible C55H100O6 TAG s reduced to 1 possible by MSMS 13 possible TAG s MSMS defined Neutral Loss

34 Database Search Yields 502 precursors for all Lipids containing 9Z-Octadecenoyl (Could be DHA, Arachadonic Acid, etc.) Qual Utilities build Preferred Precursor List from Possible adducts and neutral masses

35 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (CAS ) Standard Switching to MS-MS as detector m/z=

36 PC Example from lecithin m/z =

37 Phosphatadyle cholines found in Lecithin Sample 11 to 54 Carbons FA up to C26 up to 6 double bonds per FA Mass: to

38 Achieving Ultra-High Performance SFC for Other Lipids (> 90% of Theoretical Efficiency) Back-pressure is not a dominant issue, plenty of headroom at 600 bar To maximize resolsution, smaller Sub-2µ particle columns (e.g. Rx-Sil 250x3.0mm, 1.8µ) Slightly easier to achieve higher efficiency with a 3.0mm ID column compared to a 2.1mm ID column, not to mention the better loadability. Are superficially porous columns really beneficial at the high diffusion rates for CO 2? Dispersion volume is an issue: Minimize tubing volume, preferred Minimize UV cell volume (new Agilent 2µL DAD cell) A 2µL flow cell with (125µm) produces >90% peak fidelity at > k= 2 from a 100x3.0mm 1.8µm column, clearly superior to a 3.5µm 150x4.6mm column with a 13µL flow cell with tubing. Page 38

39 Absorbance, mau Paprika Oleoresin: Natural red pigments Widespread concerns over artificial dyes in foods. Paprika produces natural red pigments. Extracted by SFE, by the ton! Pigments form many mono- and di- esters with fatty acids, complex chromatograms Saponification simplifies chromatograms Unsaponified SB-CN Retention Time, min Saponified SB-CN Retention Time, min

40 Rapid Separation of Fat Soluble Vitamins K3 E A D2 D3 E K1 K2 K3 40 K K2 D3 D2 10 A 0 Rs = 2.14, 2.48, 10.17, 18.21, 1.12 (NO A) 3MIN Separation of the 7 fat soluble Vitamins using MeOH as modifier. Conditions: 3ml/min of 5% methanol, 60 C, 150 bar. Column: 4.6x150mm, 3.5µm RX-Sil. 40

41 Cis-, Trans- Vitamin K1 Natural Vitamin K1 is primarily the trans-isomer of the 2,3 - double bond The cis-isomer is inert Synthetic Vitamin K1 has substantially higher cis- isomer Analyzing Vitamin K1 without separating the isomers may over-represent the nutritional value of Vit K1 added to processed foods. The isomers have never been resolved by SFC HPLC takes minutes 41

42 Absorbance, mau Absorbance, mau Separation of Cis-, Trans- Vitamin K X150mm, 3.5µ RX-Sil 4.6x150mm, 3.5µm RX-Sil cis- 3x100mm, 1.8µm RX-Sil trans x100mm, 1.8µm RX-Sil Retention Time, min Retention Time, min 42

43 Absorbance, mau Separation of 7 of 8 of Vitamin K1 Enantiomers nm * Retention Time, min Chiral separation of 7 of the 8 enantiomers of Vitamin K1, on a RegisPack 4.6 x 250mm, 5µm column with 2ml/min of 5% methanol, at 30 C, 150bar. Detection at 254nm. 43

44 Conclusions SFC/QTOF is remarkably effective at resolving complex mixtures of natural products in very short times. Interfacing MS to SFC is simple, sensitive and highly reproducible. Resolution is improved by orders of magnitude over chromatography and UV alone. Speed is improved by >10X compared to earlier chromatographic methods. Sensitivity was improved by at least 1000x over UV. Complex relationships rapidly become remarkably clear and unambiguous. SFC-QTOF has a bright future in lipidomics!

45 Acknowledgements Terry Berger, SFC Solutions, Inc. Jennifer Van Anda, Applications Scientist, SFC Joe Hedrick, Applications Scientist, LCMS and SFCMS Rick Wikfors, Director, R&D, SFC technologies Tony Brand, Applications Scientist, LCMS and SFCMS Jim Lau, Applications Scientist, LCMS and SFCMS 45 Confidentiality Label May 16, 2014