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Figure 3. The UPC2 BEH provided the best initial selectivity and peak shape

for the intra-class lipid separation. The lipid classes were identified using

MS ES+. The optimized gradient was 10-50% at 1.9 mL/min over 10 min-

utes with a 2 minute hold at 50%B and 2 minute re-equilibration at initial

conditions before the next injection.

Lipidomic Profiling of Rat Adipose Tissue after Treatment with PPAR-pan

Agonist using Sub-2μm Particle CO2 Based Supercritical Chromatography

Giorgis Isaac2, Michael D. Jones

2, James Langridge

3, John P. Shockcor

1,2, Julian L. Grifiin

1

1Department of Biochemistry, University of Cambridge, Cambridge, UK ;

2Waters Corporation, Milford, MA, 01757, USA;

2Waters Corporation, Manchester, UK.

METHODS

Sample preparation

Lipids are commonly extracted from a complex biological

matrices using chloroform/methanol (2/1) as an extraction solvent. In a typical reversed phase (RP) chromatographic

analysis the organic extracts containing all the lipids have to go through a phase transfer to put the lipids into a less organic

phase to be able to be injected onto RP systems. This phase transfer process is eliminated by injecting directly the organic

extract containing lipids onto a UPC2 system showing a significant saving in solvent, cost and sample preparation time.

Experimental

Final UPC2 Conditions Instrument: ACQUITY UPC2 coupled to Xevo G2 QTof and

SYNAPT G2 Mobile Phase A: CO2 (tank, medical grade)

Modifier B: Various (see figure caption)

Column: ACQUITY UPC2 BEH 3.0x100mm, 1.7µm ACQUITY UPC2 HSS C18 SB 2.1x100mm,

1.8µm Needle Wash: 7:3 Methanol:Isopropanol

Seal Wash: Methanol Flow Rate: Various (see figure caption)

CCM pressure: 1600psi Column Temp.: 60 C

Injection Vol.: 1.0 µL

RESULTS

1. LIPID CLASS SEPARATION METHOD

Column Screening for selectivity

Optimizing for UPC2 BEH

CONCLUSIONS

The organics phase lipid extract can be directly injected into

the UPC2 system saving sample prep time and reducing cost

per analysis.

Significant saving in chromatographic separation time for

both FFA and TAG (10X faster compared to GC/MS).

No derivitization required which results in easier and fast

sample prep and eliminated of artifact formation.

UPC2 provides an alternative method for lipid class

separation.

UPC2 provide a single technique for the separation of polar

and non-polar lipids with a simple switch of the column and

gradient, thus enabling to combine two or more techniques into one.

The developed method can be applied for lipid profiling in

pharmaceutical, food and chemical samples.

3. BIOLOGICAL APPLICATIONS

The developed UPC2 BEH and HSS C18 method was applied for

the analysis of biological samples (Figures 6-9). The UPC2 BEH

method separates lipid by class in a similar fashion to normal phase chromatography. The UPC2 HSS C18 SB provides a

simple, fast and effect means to separate free fatty acids and neutral lipid molecular species with in lipid class.

INTRODUCTION

Traditionally, lipids are only associated with cellular roles involving energy storage and used as structural building

blocks. Recent developments in lipid research have identified the important role of lipids in modulating cellular signaling and

cellular trafficking. Alterations in lipid metabolites are associated with various human diseases including obesity,

heart disease, and diabetes mellitus. Hence, there is a need to develop comprehensive analytical approaches that allow for

the automatic analysis and identification of lipids in complex

biological mixtures.

The discovery of novel alterations in lipid levels related to human diseases could lead to the development of novel

biomarkers and future diagnostic testing.

Utilizing a single chromatographic technique for the separation of complex lipids lacks feasibility due to the diversity of lipid

polarity and the large range of concentrations of thousands of lipid species that are present in biological samples. Recent

advances in technology have revived the exploration of supercritical fluid chromatography (SFC) as a viable analytical

technique, especially for hydrophobic molecules.

Ultra Performance Convergence Chromatography (UPC2)

is a chromatographic system that utilizes liquid CO2 as primary solvent and co-solvents such methanol as a mobile phase to

leverage the chromatographic principles and selectivity of normal phase chromatography while providing the ease-of-use

of reversed-phase LC. It is a category of separation science that provides orthogonal and increased separation power,

compared to liquid or gas chromatography, to solve separation challenges. In convergence chromatography, the separation is

achieved by manipulating the density of the mobile phase. Due to the very low viscosity of the technique, a high separation

efficiency can be achieved. Schematic of the UPC2 system is shown in figure 1.

In this study, UPC2 was investigated for the separation of free

fatty acids, neutral and polar lipids. The method was initially

developed using lipid standard mixtures and then applied to different biological matrices.

Figure 2: A 12 minute 10-50%B screening method was used. The 2-EP

and PFP gradients were modified 10-30%B to adjust for comparative use of

the separation space. Column Screen Utilizing UPC2 available stationary

phases performed by injection of the PL-1A mixture. The mobile phase B

was 2g/L ammonium formate in MeOH based on previous reports from

Bamba et al 1 and MS ionization purposes.

Figure 7. TIC of cotton seed extract using UPC2 BEH column showing lipid

class separation. When exploring a sample known to have more acylglyc-

eride content, the gradient can be manipulated to be CO2 rich at the begin-

ning of the analysis to better separate the non-polar/neutral intra-class

moieties, then the gradient can be ramped up with co-solvent to elute and

separate the more polar constituents of interest. The column was re-

equilibration for 2 min at initial conditions before the next injection. Top

panel shows the low energy precursor ion and lower panel shows corre-

sponding high energy fragment ion for lipid identification.

Figure 6. TIC of mouse heart extract using UPC2 BEH column showing lipid

class separation.

Coupled to Mass Spectromet

Figure 1. Schematic of the UPC2™ configuration.

2. FREE FATTY ACID AND NEUTRAL LIPID METHOD

The typical chromatographic methods for analyzing fatty

acids are gas chromatography/mass spectroscopy (GC/MS)

after derivitization and liquid chromatography-tandem mass

spectrometry (LC/MS/MS). However, there are shortcomings

associated with each of these methods. For example, GC

methods require derivatization of the fatty acids to methyl esters (FAME), which is burdensome, time consuming, and

there is a risk of re-arrangement of the fatty acids during derivitization which leaves doubt as to whether the esters

formed are from free fatty acids or intact complex lipids. In

LC/MS/MS methods, the runs typically involve labor intensive and time consuming sample preparation, and utilize toxic

organic solvents, which are expensive to purchase and dispose. We have developed rapid, high throughput and

Figure 5. The separation depends on both chain length and degree of un-saturation (top). Extracted ion chromatogram showing the separation of iso-baric lipid species based on the position of the double bond. Gradient: 5-25% Methanol in 0.1% FA over 5min, flow 0.6mL/min.

Standard Mixtures

Mixtures for UPC2 BEH Method Development: Total lipid

class extract were purchased from Avanti Polar Lipids

(Alabaster, AL). All were Brain (porcine) extracts except for LPC and PG which were Egg (chicken). Stocks were prepared

in 50:50 chloroform:methanol. Working mixes were prepared to specified concentration by dilution in methanol.

Phospho- Mix: Ceramide, SM, (0.05mg/mL) PG, PE, PC,

(0.1mg/mL) Lyso- Mix: LPC, LPE, (0.05mg/mL)

Sphingo Mix: Cerebrosides (0.1mg/mL); Sulfatides (0.05mg/mL)

MetDev Mix(PL-1A): 1:1 of Phospho- mix and Lyso- mix

Mixtures for UPC2 HSS C18 SB Method Development: A complex model mixture of 32 different FFA standards (GLC-85 in FFA

form) and 15 saturated triaclglyceride standards C8-C22 (GLC-768) was pur-

chased from Nu-Check prep. A 1 mg/mL stock solution was prepared in

chloroform and 0.1 mg/mL working lipid mixtures were prepared in chloro-

form and injected onto the UPC2/Xevo G2 QToF system.

Biological Sample Preparation: Lipid extraction from mouse heart, cotton seed and mouse adipose tissue was performed

according to the procedure described by Folch et al (2). The lower organic phase containing lipids was collected and directly

injected to the UPC2 system.

Inte

nsity

0.0

7.0x107

1.4x108

2.1x108

2.8x108

Inte

nsity

0.0

5.0x107

1.0x108

1.5x108

2.0x108

Inte

nsity

0.0

8.0x107

1.6x108

2.4x108

3.2x108

Minutes

0.00 1.20 2.40 3.60 4.80 6.00 7.20 8.40 9.60 10.80 12.00

UPC2 HSS PFP

UPC2 BEH 2-EP

UPC2 BEH

Inte

ns

ity

0.0

8.0x107

1.6x108

2.4x108

3.2x108

Minutes

0.00 1.20 2.40 3.60 4.80 6.00 7.20 8.40 9.60 10.80 12.00

CER

PG PE

PC

SM

LPC

LPE

PC

SMLPC

PE

TAG

50%

5%

Figure 8. TIC of mouse adipose tissue extract using UPC2 HSS C18 SB col-

umn showing triacylglycerides molecular species separation. The co-solvent

IPA/ACN(90/10) provided better separation and chromatographic resolution

compared to 100% MeOH.

100% MeOH

IPA/ACN(90/10)

Low energy

High energy

PC

TAG

DAG??

NAPE

2%

50%

Figure 4. UPC2 provides a single technique for high throughput separation

of free fatty acids and neutral lipids. Top panel shows 32 different free

fatty acid analysis without derivatization and lower panel shows the

separation of 15 triacylglycerides C8-C22 molecular species.

Figure 9. TIC of algae extract using UPC2 HSS C18 SB column showing free

fatty acids (C8-C34) separation. The co-solvent gradient can be optimized to

CL