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

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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)

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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