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MICROFLUIDICS LIPIDOMICS USING A NOVEL INTEGRATED MASS SPECTROMETRY TECHNOLOGY Giuseppe Astarita1, Michael Jones1, Angela Doneanu1, Jay Johnson1, Jim Murphy1, Robert Plumb1, James Langridge1 1. Waters Corporation, Milford, MA, USA

METHODS Lipids were extracted from mouse plasma as previously reported [1-4]. Untargeted analysis were performed with ionKey/MS system comprised of an ACQUITY UPLC M-Class, the ionKey source and an iKey C18 130 Å, 1.7µm particle size, columns (Waters Corporation, Milford, Massachusetts, USA). Flow rates were 2-3 µl/min and the gradient as previously reported [2,4]. MS detection was conducted using a Synapt G2-S HDMS and a Xevo TQ-S (Waters Corporation, Manchester, UK) operated in both negative and positive ES modes. Progenesis QITM informatics solution and TargetLynxTM Application Manager were used to analyze the data.

References

1. Strassburg K, et al., “Targeted lipidomics of oxylipins (oxygenated fatty acids)”. Waters App note. 2013. 720004664en.

2. Targeted Lipidomics Using the ionKey/MS System” Waters App note. 2014. 720004968EN.

3. Broccardo et al., Multiplexed Analysis of Steroid Hormones Using ionKey/MS “ Waters App note. 2014. 720004956en.

4. Astarita G, et al., “A protective lipidomic biosignature associated with a balanced omega-6/omega-3 ratio in fat-1 transgenic mice” PlosOne April 2014

5. Isaac G, McDonald S, and Astarita G. “Lipid Separation using UPLC with Charged Surface Hybrid Technology”. Waters App note. 2011. 720004107en.

INTRODUCTION Lipidomics aims to measure the wide array of lipid species in biological samples to offer a better understating of their roles in health and disease. Alterations in lipid pathways have been associated with many diseases including cardiovascular diseases, obesity, and neurodegenerative disorders.

The ability to measure the wide array of lipid species in biological samples could help our understanding of their roles in health and disease. The need for a fast, comprehensive and sensitive analysis of the hundreds of lipid species challenges both the chromatographic separation and mass spectrometry.

Here we used a novel microfluidics platform— the ionKey/MS System— which integrates the UPLC separation into the source of the mass spectrometer. The iKey contains the fluidic connections, electronics, ESI interface, heater, e-cord, and the 1.7 um particles for fast and robust analysis. Inserting the iKey simultaneously engages both electronic and fluidics connections eliminating the need to make manual connections, delivering exceptional reproducibility and simplifying the user experience. Such integrated platforms are suitable for lipidomics analyses with performance comparable to analytical scale LC-MS analysis.

UNTARGETED LIPIDOMICS

CONCLUSIONS The novel ionKey/MS system leads to highly efficient LC separation of lipid molecules. Chromatographic results were equivalent to using analytical-scale columns [1-4], bringing considerable advantages: >200x decrease in solvent consumption, making it convenient for the arge-scale analysis and screenings of hundreds or thousands samples. >10x increase in sensitivity, which could facilitate the detection of

low abundance metabolites. low volumes injection (e.g., 0.2 µl), which makes it ideal when

sample limited studies or when multiple injections are required. Flexibility for both Proteomics and Lipidomics experiments on a

single platform.

OVERVIEW A novel microfluidics platform — the ionKey/MS — for fast and robust lipidomics analyses with considerable reduction in solvent consump-tion and increase in sensitivity. Potential applications include large-scale lipid profiling and low-abundance lipids analyses in biological materials.

TARGETED LIPIDOMICS

UPLC-like Chromatographic Performance for Free Fatty Acids sep-aration [4,5].

The novel ionKey/MS System.

Increase in Sensitivity [1,3,5].

Increase in Speed of Analysis.

Peer-Reviewed Publications and Application Notes.

UPLC-like Separation of Isomeric Phospholipids and Sphingolipids.

Pea

k A

rea

PC 34:

0

PC 36:

6

PC 36:

5

PC 36:

4

PC 36:

3

PC 36:

2

PC 36:

1

PC 38:

7

PC 38:

6

PC 38:

5

SM d

18:1

/16:

0

SM d

18:1

/18:

0

SM d

18:1

/20:

0

SM d

18:1

/24:

1

SM d

18:1

/26:

1

SM d

18:1

/26:

0

SM d

18:1

/24:

0

SM d

18:1

/22:

0

SM d

18:1

/22:

1

HexCer

d18

:1/h

24:1

HexCer

d18

:1/h

24:0

HexCer

d18

:1/2

6:1

HexCer

d18

:1/2

6:0

HexCer

d18

:1/2

4:1

HexCer

d18

:1/2

4:0

HexCer

d18

:1/1

8:

HexCer

d18

:1/h

18:0

HexCer

d18

:1/2

0:0

HexCer

d18

:1/2

2:1

HexCer

d18

:1/h

24:1

HexCer

d18

:1/h

22:0

Cer d

18:1

/16:

0

Cer d

18:1

/18:

0

Cer d

18:0

/18:

0

Cer d

18:1

/20:

0

Cer d

18:1

/22:

0

Cer d

18:0

/22:

0

Cer d

18:1

/24:

1

PEp 40:

4

PE 40:

5

PE 40:

3

PE 40:

6

PE 40:

7

LPE 18:

0

PE 38:

6

PE 38:

5

PE 38:

4

PEp38 :4

PEp38:5

PEp 36:

4

PE 36:

5

PE 36:

4

Concentration (pmol)

Pea

k A

rea

0 2 4 6 80

1000000

2000000

3000000

R2 = 0.9980

PC (18:0/20:4)

Wide Dynamic Range and Linearity.

Microfluidics Electrospray increases Sampling Efficiency.

Increase in Peak Capacity.

High Reproducibility.

Packed with 1.7 m Particles

Workflow lipidomic analyses of biological samples.

Lipid Class No. MRMs Cone Voltage Collision Energy

PE 45 26 18

Lyso PE  18 26 18

PC 44 42 26

Lyso PC 19 42 26

Ceramide 19 20 30

Sphingomyelin 20 36 24

HexosylCeramide 19 20 26

LactosylCeramide 16 20 30

Cholesteryl Ester  15 36 24

  MRM transitions for over 200 lipid species.

Microfluidics Regular UPLC

PC 14:0/14:0 iKey0.15x100 CSH 18 2.1x100 CSH C18

fmol on column Area Area Fold Difference

36.85 540214 48504 11

18.42 328347 22217 15

9.21 146452 14395 10

4.61 83265 5233 16

2.30 40832 2823 14

1.15 19403 1287 15

0.58 8512 701 12

0.29 4333 377 11

0.14 2108 236 9

0.07 839 98 9

0.04 429 114 NA