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TARGETED AND UNTARGETED LIPIDOMICS USING AN INTEGRATED MICROFLUIDICS MASS SPECTROMETRY TECHNOLOGY Steven Lai; Paul Rainville; Angela Doneanu; Jay Johnson; James Murphy; Robert Plumb; Giuseppe Astarita

Waters Corporation, Milford, MA

Untargeted data were processed and analyzed using Progenesis QI in-

formatics, which allowed peak picking, multivariate statistical analy-

METHODS

A microfluidic-MS device was optimized for MS analysis of lipids in

complex biological extracts. The integrated microfluidic device was

fabricated from resistant ceramic materials that permit operation at

high pressure with sub 2 µm particles, leading to highly efficient LC

separations of lipid molecules. Lipids were separated using 150 µm ID

x 100 mm devices packed with reversed phase C18, 1.7 µm particles

at flow rates of 3 µl/min. Analyses were performed using both TOF

and TQ operated in both negative and positive ES modes. Ion mobility

was integrated in the TOF platform to provide collision cross section

measurements for lipids for lipid identification.

References

1. Targeted Lipidomics Using the ionKey/MS System” Waters App

note. 2014. 720004968EN.

2. Broccardo et al., Multiplexed Analysis of Steroid Hormones

Using ionKey/MS “ Waters App note. 2014. 720004956en.

3. 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

4. Isaac G, McDonald S, and Astarita G. “Lipid Separation using UPLC with Charged Surface Hybrid Technology”. Waters App

note. 2011. 720004107en.

INTRODUCTION

Lipidomics - the screening of lipid species in biological samples - aims to

offer a better understating of health and disease. The need for a fast,

comprehensive and sensitive analysis of hundreds of lipid species

challenges both the chromatographic separation and mass spectrometry.

Here we used a novel microfluidics platform, which integrates the UPLC

separation into the source of the mass spectrometer. The platform

contains the fluidic connections, electronics, ESI interface, heater, and 1.7

um particles. Such integrated platforms are suitable for lipidomics

analyses with performance comparable to analytical scale LC-MS

analysis.

UNTARGETED LIPIDOMICS

OVERVIEW

A fast and robust microfluidics platform for lipidomics analyses with

considerable reduction in solvent consumption and increase in sensi-

tivity.

TARGETED LIPIDOMICS

Increase in sensitivity using the ionKey / Ion Mobility MS Sytem [1,2,4].

Representative extracted ion chromatograms of isobaric glycerophospholip-ids in mouse plasma. Samples were analyzed using ionKey MS system with

150µm device .

UPLC-like Separation of Isomeric phospholipid and sphingolipid species.

Microfluidics electrospray increases sampling efficiency.

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 analyzed

Targeted lipidomics analysis were conducted using internal stan-

dards and TargetLynx for the identification and quantification of se-lected lipid molecules.

CONCLUSIONS

The ionKey/MS system leads to highly efficient LC separation of lipid molecules extracted from biological samples. 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. The information obtained can be integrated with clinical data to

generate testable hypotheses on the functional significance of the lipid abnormalities observed in brains from subjects with Alzheimer’s disease.

Advantages of microfluidics compared to nanofluidics.

Easy method transfer from 2.1mmID to a 150um column ID method.

Analysis of a selected phosphatidylcholine molecule (PC 14:0/14:0) using ei-

ther the ionKey-MS systemor a regular ACQUITY UPLC system coupled with a Synapt G2-S HDMS operated in MSE mode. Volume injected was the same on

both systems (i.e., 0.2 µl).

Linearity of response and dynamic range of detection of

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1 0 2

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C o n c e n tra t io n (p m o l)

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