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Qualitative and Quantitative Analysis of Lipids using SFC and LC
combined with High Resolution Accurate Mass MS
Jim Lau, Ph.D.
Agilent Technologies
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
May 16, 2014
Confidentiality Label
2
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.
Practical Advantages of SFC?
3-5 X speed/throughput--more samples/day more rapid re-equilibration--shorter cycle time compared to reversed phase HPLC
1/3rd-1/5th 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.
SFC w/ pure CO2
CO2 w/ organic modifiers
CO2 w/ modifiers + additives
CO2 + modifiers +
additives + water
Normal Phase HPLC
Reversed Phase HPLC
Ion Pairing
HILIC
Ion
Chromatogr.
Where Does SFC Fit Relative to HPLC?
Solute Families
SFC Solutions, Inc.
Data based on the compendium from the
International symposium on SFC/SFE, 1989.
Polymers
Fuels Food
Environmental
Misc. Pharma
+ chiral
1989 SFC Applications
≈ 9%
91% non-pharma and chiral
6
-50
0
50
100
150
200
250
300
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Retention Time, min
Ab
so
rb
an
ce
, m
AU
Atenelol
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
4.6x250mm, 5µm Regispack
SFC Solutions, Inc.
Pharma Prep
Chiral Anal.
Fuels Pharma
SFC-MS
Misc
Environmental
Food
Achiral
Polymers
SFC 2012:
All, but Pharma, practically Non-Existent 10-15%
85-90% pharma
8
A. Staby, C. Borch-Jensen, S. Balchen, J. Mollerup, “ Quantitative Analysis of Marine Oils by Capillary Supercritical
Fluid Chromatography”, (1994) Chromatographia 39 697-705
Cod Liver Oil by Capillary SFC- 1994 FID detection-120 min
complex density program; 45 Identified components; separated by mass
compounds with same carbon number but different degrees of unsaturation co-eluted.
SFC Solutions, Inc.
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.
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, 974 231-241.
Soybean Oil by Packed Column SFC- 2002, APCI-MS 100 min.
Silver loaded Nucleosil 100-5 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
22 triglycerides identified
UV 210nm
APCI + Ag+
SFC Solutions, Inc.
QTOF w/ ESI Agilent 6540
SFC Solutions, Inc.
Experimental Set-up
Make-up
DAD
TCC
ALS
Bin Pump
SFC
mod.
Agilent 1260 SFC
to BPR
restrictor
≈10:1 split
HPLC
Waste
1 2 3
4
5
6 7
8
9
10
SFC-Binary Pump
BPR
Loop back
Restrictor
SFC Pump outlet
LC Pump:
G1310B or
G1311B or
G1312B LC
Pu
mp
ou
t D
ete
cto
r ou
t SFC
Waste
The SFC/LC System UHPSFC Mode
Page 13
Waste
1 2 3
4
5
6 7
8
9
10
BPR
Restrictor
SFC Pump inlet
LC
Pu
mp
ou
t D
ete
cto
r ou
t
SFC Pump outlet
The SFC/LC System UHPLC Mode
Page 14
SFC-Binary Pump
LC Pump:
G1310B or
G1311B or
G1312B
Direct vs. Split Flow to MS (QTOF)
May 16, 2014
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To QTOF
To QTOF
Agilent Jetstream Technology (AJT) source
The super-heated sheath gas collimates the nebulizer spray and presents more ions to the MS inlet.
*Nozzle voltage
Resistive sampling capillary exit: FragV
Nebulizing gas: pressure
*Sheath gas: flow and temperature
Drying gas:
flow and temperature
Resistive sampling capillary entrance: Capillary V
16
*New parameters unique to AJT source
Standard Source used for
SFCMS of Lipids
Stainless from SFC
Vcap
Corona
current
Nebulizer
Pressure
Fragmentor
Drying gas
Temperature
and Flow
Heater
APCI Spray Chamber
Stainless from SFC
Page 18
SFC RC.NET drivers
Fish Oil Analysis
by
SFC/QTOF
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, 3.5µm* @ 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 NH4OAc @ 12 mins, end of run
Extracted ion chromatogram of Fish Oil
SFC/QTOF
Commercial fish oil capsule 7.8min 0.6min
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
SFC Solutions, Inc.
Fish Oil Analysis by SFC/QTOF: Extracted Ion Chromatograms
Commercial fish oil capsule
DGs Vit D precursors
2beta-methyl-1beta,25 dihydroxycholecalciferol
TGs
Reproducibility: Retention Time and Area Fish oil sample, EIC 561.448 and 1039.8695, 3 replicates, MS QTOF data
m/z 561.448
RT Area
1.155 195
1.154 191
1.151 192
Mean 1.153 192.67
S. Dev. 0.002 2.08 % S. Dev. 0.0018 0.0108
m/z 1039.869
5
RT Area
7.015 575
7.018 571
7.015 590
Mean 7.016 578.67
S. Dev. 0.002 10.02 % S.
Dev. 0.0002 0.0173
0 mins
3% MeOH/5mM NH4OAc
5.5 mins
3% MeOH/5mM NH4OAc
11 mins
60% MeOH/5mM NH4OAc
(hold for 1 minute)
0
200
400
600
800
1000
1200
1400
1600
M a
s s (
D a )
Mass vs. Retention Time
0 1 2 3 4 5 6 7 8
Retention Time (min)
2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 Retention Time (min)
700
750
800
850
900
950
M a s s ( D
a )
750
900
Mass, Da
2.2 2.4 time, min
TGs, C53H98O6, C53H96O6, C53H94O4
C53H98O6
C53H96O6
C53H94O6
DG separation with 1 degree of saturation
difference
C41H70O5 (M+H)+ 643.5281 1.5ppm mass error
C41H72O5 (M+H)+ 645.5422, 0.7ppm mass error
4.5min 0.6min
0
2
4
6
8
10
12
14
16
18
20
2 3 4 5 6 7 8
Retention Time, min
# D
ou
ble
Bo
nd
s
C53
C55 C57 C59
C61
C63
C65
C67
What defines Triglyceride Chromatographic Retention?
A.
B.
In these experiments, Retention is a Strong Function of Degree of Unsaturation and
much Less according to Carbon Number
0
2
4
6
8
10
12
3 4 5 6 7 8
Retention Time, min
# D
ou
ble
Bo
nd
s
16:0/22:X/22:6
16:0/22:0/22:X
16:0/20:0/22:X
16:0/20:X/22:6
C61
C63
Retention does not appear to be related to the
Location of Double Bonds, but only the Number
Fish Oil: Individual lipids tentatively identified by exact mass
140 Largest TGs
42 Largest DGs
Full Results Set for Tripalmitin
MS of M+NH4, M+Na and MSMS of each
Databases have revolutionized Lipid analysis
Variants of Tripalmitin
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Loss of Neutral
C18H34O2NH3 Loss of Neutral
C16H30O2NH3
Loss of Neutral
C18H36O2NH3 Loss of Neutral
C16H32O2NH3
Sartain et.al.
13 possible C55H100O6 TAG’s reduced to 1
possible by MSMS
13 possible TAG’s
MSMS defined Neutral Loss
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
1,2-Dimyristoyl-sn-glycero-3-phosphocholine (CAS 18194-24-6)
Standard
m/z= 184.0736
Switching to MS-MS as detector
PC Example from lecithin
m/z = 184.0730
Phosphatadyle cholines found in Lecithin Sample
11 to 54 Carbons
FA up to C26
up to 6 double bonds
per FA
Mass: 300.1472
to 948.8231
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 CO2?
Dispersion volume is an issue:
Minimize tubing volume, 0.005” preferred
Minimize UV cell volume (new Agilent 2µL DAD cell)
A 2µL flow cell with 0.005” (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 0.007” tubing.
Page 38
• 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
0
100
200
300
400
Unsaponified SB-CN
2 4 6 8 10
Retention Time, min
Ab
sorb
ance
, mA
U
Paprika Oleoresin: Natural red pigments
0
200
400
600
800
SB-CN
2 4 6 8 10
Retention Time, min
Saponified
-10
0
10
20
30
40
50
60
0 0.5 1 1.5 2 2.5 3 3.5
AD2D3EK1K2K3
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.
K3
K1
K2
E
A
D2
D3
Rapid Separation of Fat Soluble Vitamins
40
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 ≈ 12-14 minutes
41
-50
0
50
100
150
200
250
300
350
400
0 1 2 3 4 5
Retention Time, min
Ab
sorb
ance
, mA
U
-200
0
200
400
600
800
1000
1200
0 1 2 3 4 5
Retention Time, min
Ab
sorb
ance
, mA
U4.6X150mm, 3.5µ RX-Sil
3x100mm, 1.8µm RX-Sil
Separation of Cis-, Trans- Vitamin K1
4.6x150mm, 3.5µm RX-Sil
3x100mm, 1.8µm RX-Sil
cis-
trans-
42
-20
0
20
40
60
80
100
120
140
0 5 10 15 20
Retention Time, min
Ab
so
rb
an
ce
, m
AU
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.
1 2*
3
4
5 6 7
Separation of 7 of 8 of Vitamin K1 Enantiomers
43
254nm
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!
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
May 16, 2014
Confidentiality Label
45