A Semi-Automated Lipid Extraction
Protocol Using the Agilent Bravo
Automated Liquid Handling Platform

Application NoteAuthors

Abstract

Magendran Muniandy,

Automated protocols to facilitate sample preparation in lipidomics have been

Pradeep Narayanaswamy,

previously developed. The aim of these procedures is to minimize humanFederico Torta, and Markus R. Wenk

errors resulting from handling large quantities of samples, and to decrease theSLING, National University ofamount of time spent in sample preparation. This application note describes theSingapore, Singapore

semi-automated extraction of phospholipids and sphingolipids from human plasma

Matthew Foong
Agilent Technologies
Singaporeusing the Agilent Bravo Automated Liquid Handling Platform. This procedureincludes the transfer of the starting material (plasma is usually delivered in
1.5-mL tubes) to 96-well plates, which can be either stored or used for the lipidextraction. In the latter case, a butanol/methanol (BuMe) mixture is added to thesample. Although the samples are still transferred manually to the sonicator andthe centrifuge during the lipid extraction procedure, the reported protocol allows
a high number of samples to be processed uniformly, decreasing the sample
preparation time and yielding high reproducibility according to the results obtained
after analysis of the extracted lipids with an Agilent UHPLC/MS/MS system.

Introduction
Lipidomics is the branch of metabolomics
that seeks to identify all the lipid
species present in living organisms andunderstand their synthesis, regulation,and effects.
Mass spectrometry-based lipidomics
is increasingly popular due to theconstant development in liquid
chromatography-mass spectrometry
systems. A large number of methods havebeen developed, and many biologicallyimportant lipids can now be routinely
analyzed and quantiﬁed1. However, thesample preparation techniques used forlipid extraction from biological samples
are often long and tedious, and still relyon those techniques established in thelate 1950s2.
Usually, lipidomics labs have toprepare large numbers of samples(for example, in large epidemiologicalstudies), making the use of standard
manual preparation impractical. Thisapplication note describes use of theAgilent Bravo Automated Liquid Handling
Platform to generate a semi-automated,
high-throughput method for extracting
lipids from human plasma. The results
show that this procedure can give
precision comparable to, or better than,that achieved using traditional manual
methods, while reducing experiment
time and costs. Moreover, lipid extraction
protocols involve the use of organic
solvents that are highly volatile and toxic,
and the use of a robotic station canimprove safety in the lab.

Instruments and materials•

96-well polypropylene conicalbottom (V-bottom) plate,(250 μL well capacity)•24-well 1.5 mL Eppendorf tube
plate (G5498B#572)•

Agilent 96LT 250 μL sterile, ﬁlteredpipette tips (19477-022)•

Agilent VWorks Automation
Control software•

Butanol:methanol (1:1) withammonium formate 10 mM(extraction solvent)•

Empty tip box (tip waste)•Corning Axygen, Multi-Well
12 Channel Reservoir(plate volume 21 mL)•

Branson Ultrasonic Bath andSorvall RT legend centrifuge•••

1,2-dimyristoyl-sn-glycero-3phosphoethanolamine (DMPE)•

N-heptadecanoyl-D-erythrosphingosine (Cer)•N-lauroyl-D-erythrosphingosylphosphorylcholine (SM)•1-arachidoyl-2-hydroxy-sn-glycero3-phosphocholine (LPC)

LC/MS/MS analysis•

Agilent ZORBAX Eclipse Plus C18
RRHD 2.1 × 50 mm, 1.8 μm

Seralab Human Plasma EDTA K2,
not ﬁltered•

Mobile phase A: 60/40
water/acetonitrile in 10 mM
ammonium formate•

Mobile phase B: 90/10
isopropanol/acetonitrile in 10 mM
ammonium formate

LC/MS/MS method
LC conditions
Column temperature

40 °C

Injection volume

2 µL
0 minutes (A:B = 80:20), 2 minutes (A:B = 40:60),
7 minutes (A:B = 0:100), 9 minutes (A:B = 0:100),9.01 minutes (A:B = 80:20), 10.50 minutes (A:B = 80:20)•96-channels LT Disposable Tip
Head (G5498B#042)

MS conditions

VarioMag Teleshake Plate Shaker(Bravo deck)

1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC)

Thermo Scientiﬁc PCR tube caps,
skirtedGradient••

Data were acquired in both positive
and negative dynamic MRM modes
using an Agilent 1290 Inﬁnity
Series LC interfaced to an Agilent
6460 Triple Quadrupole Mass
Spectrometer.

Agilent Bravo Automated Liquid
Handling Platform (G5523A)

CPAC Ultraﬂat heating and coolingplate (Bravo deck)

Purchased from Avanti Polar Lipids:•••

Lipid standards

Ionization mode

ESI

Ionization polarity

positive and negative

Drying gas ﬂow

5 L/min

Drying gas temperature

300 °C

Nebulizer pressure

45 psiSheath gas temperature

250 °C

Sheath gas ﬂow

1 L/minNozzle voltage

500 V

Capillary voltage

1,000 V2

Protocol workﬂow

Manual extraction

Semi-automated extraction

Aliquoting samples

Samples aliquoted manually fromoriginal tubes into smaller fractions

Samples automatically aliquoted fromoriginal tubes into smaller fractions

Extraction solvent additionby manual transfer

Extraction solvent addition
by automated transferSonication and centrifugation

Sonication and centrifugation(Manual transfer to equipment)

Supernatants manually transferred
to vials for MS analysis

Supernatants automatically transferred
to 96-well plates for MS analysis

Total time ~8 hours

Total time ~2 hours

As standard procedure in most labs,
original samples are initially split intosmaller aliquots for long-term storage oranalyte extraction. This protocol starts
with the transfer of 10-µL aliquots of theoriginal human plasma samples from
1.5-mL tubes into a 96-well plate forsample storage or lipid extraction.
1.

2.

3.

The positions of the plates for thesample transfer step are shown inFigure 2A. The 24-well holder for
1.5-mL Eppendorf tubes and the96-well destination plate are kept on
the CPAC Ultra-ﬂat cooling stations(Station 4) to maintain the sample
temperature at 4 °C.
A method generated with VW
software can aliquot 24 samples
from Station 4 to the 96-wellplates in Station 6. The dispensing
destination, head mode, and tips
ﬁlled at Station 1 have to be changedin the method according to Table 1.
Once all the wells needed for thesample extraction procedure havebeen ﬁlled with sample, the lipid
extraction protocol can be started
separately. We chose to extract thelipids in a butanol/methanol (BuMe)
mixture to be able to work witha single organic phase, avoiding
the need for phase separation,
as reported in previous manual
methods. Since the presence of twophases would make sample recovery
with the Bravo platform problematic,
we optimized this method forsingle-phase extractions.

Figure 1. Workﬂow for manual and semi-automated procedures.

AB
24-Well
Eppendorf
tube plate

New pipette tips
96-Well sample
stock plate

Reservoir for
extraction
solvent

New pipette tips
Samplestock plate

Samplepreparation
platform

Used pipette tips

Used pipette tips
Final destination
plate for MS injection

Figure 2. A) Sample transfer conﬁguration. B) Lipid extraction conﬁguration.

Table 1. Head mode, dispensing destination, and pipette tips to be ﬁlled at Station 1. Parameters used for
initial sample transfer.
Head mode

Tips to be ﬁlled at Station 1

Subset mode

Subset orientation

ColumnRow

Full column

Front left2, 4, 6, 8, 10

A, C, E, G

Dispense location
Dispense: 1 Selection column: 1

Full row

Back left

2, 4, 6, 8, 10

A, C, E, G

Dispense: 1 Selection row: 1

Full column

Front left

2, 4, 6, 8, 10

A, C, E, G

Dispense: 1 Selection column: 2Single barrel

Back left

2, 4, 6, 8, 10

A, C, E, G

Dispense: 1 Selection: B2

Lipid extraction
1.

The plate orientation for the lipid
extraction is shown in Figure 2B. The
96-well plate containing the samples
and the multiwell 12-channel
reservoir containing BuMe + internalstandard (IS) mix for subsequent
lipid quantiﬁcation, are kept on
the CPAC Ultra-ﬂat cooling plates(Station 4 and Station 6) to maintaina constant temperature of 4 °C.

2.

Samples are extracted at Station 8
on the VarioMag Teleshake Plate
Shaker as the protocol requires
uniformly mixing the plasma andBuMe before the sonication step.

3.

The tips are taken from Station 1and primed with the extraction
solvent in Station 6. Then, 100 µLof the extraction solvent witha post-aspiration air-volume of3

10 µL (to prevent the solvent fromleaking while transferring) aredelivered to the plate containing
the samples. Due to differencesin liquid vapor pressure andsurface tension, the BuMe mixture
needs to be characterized as aseparate liquid class, based on
solvent-speciﬁc aspiration anddispensing volumes. We empiricallyvalidated these values following the4.

5.

After dispensing the extraction
solvent + lipid standards mixture tothe destination plate in Station 8, theplate wells are individually cappedwith PCR tube caps and shaken for
60 seconds. After this step, the plate
has to be removed from the Bravo
platform and put in a sonication bathfor 60 minutes at room temperature.
The plate is then centrifuged in aSorvall RT centrifuge at 4,200 rpm for
20 minutes to precipitate the protein
fraction.
The plate is again placed in Station 8of the Bravo platform, and 70 µL of
the supernatant (with a post-aspirate
volume of 10 µL) are then transferredto the collection plate at Station 9.
This plate can then directly be
used in the Agilent 1290 Inﬁnity
Autosampler for UHPLC/MS/MS
analysis.

Results and Discussion
Experiments were performed to compare
the efﬁciency of the manual extractionof lipids from commercial human plasma
with automated extraction using theAgilent Bravo platform. Phospholipids
and sphingolipids from different aliquots(n = 12) of the same human plasma
were extracted and quantiﬁed using an
in-house developed LC/MS/MS method,
based on reversed-phase separationand multiple reaction monitoring (MRM)
detection on a triple quadrupole mass

spectrometer. The aim was to evaluate if
the manual and automated methods could
produce comparable results. One hundred
ﬁfteen different lipid molecular species
were analyzed, and the mean, standarddeviation (SD), and percent relative
standard deviation (% RSD) for eachspecies concentration were compared.
The mean lipid concentrations measured
by manual and automated methods
showed high correlation (R = 0.9964),
indicating good reproducibility between
the two methods (Figure 3).

140

120

Bravo extraction of plasma (nmol/mL)

procedure reported in a dedicatedApplication Note3. By aspirating
and dispensing different volumesof the speciﬁc solvent, a volume
correction factor table is generated
to ensure accuracy. This procedure
is essential, and its effect on thereduction of the error associatedwith dispensing small volumes oforganic solvents was signiﬁcant.

10080

y = 0.9076x + 0.3895
R² = 0.9964

6040200020406080
100
Manual extraction of plasma (nmol/mL)

120140160Figure 3. Accuracy and precision of the semi-automated preparation method compared with the manualmethod. Correlation between the the concentration (nmol/mL of plasma) for the endogenous lipids
prepared by the conventional manual method (n = 12) and the semi-automated method (n = 12).

4The variation measures for the lipid
standards in each class (Table 2)demonstrates that the automated methodon the Bravo provides lower %RSDs
compared to the manual method.

Conclusion
The results demonstrate that theAgilent Bravo Automated Liquid
Handling Platform can implement any
single phase-based lipid extraction
method, generating samples suitablefor LC/MS/MS analysis in lipidomics.
Automated processing using the Agilent
Bravo improved the throughput of
the method and showed a consistentanalytical precision. While thepreparation time for 96 samples with themanual method can take approximately
8 hours, the automated approach requires
only 2 hours. Since, by some estimates,
60–80 % of the work activity andoperating cost in an analytical lab is spent
preparing samples for introduction into
an analytical device, this experimental
protocol can signiﬁcantly reduce thecost/sample for lipid analysis.

Table 2. Coefﬁcients of variation of the peak areas of the lipid standards, representative of each class,used in the reported experiments .
Lipid standard% RSD semi-automated% RSD manual

PE 14:0/14:0

4.80

7.13PC 14:0/14:0

5.85

5.72

C17 Ceramide

7.32

11.97

LysoPC 20:0

5.27

5.05

SM 30:1

3.79

4.96

GluCer d18:1/8:0

6.81

6.34

References
1. Wenk, M. R. Lipidomics: New Tools
and Applications. Cell 2010, 143(6),pp 888-895.
2. Bligh, E. G; Dyer, W. J. A Rapid
Method of Total Lipid Extraction andPuriﬁcation. Can. J. Biochem. Physiol.
1959, 37(8), pp 911-917.
3. Albert, K. J. Optimizing Accuracy
Performance on an Agilent Bravo
platform using the Artel MVS, Artel
Application Note 12A6480A, 2013.

5www.agilent.com/chem
This information is subject to change without notice.
© Agilent Technologies, Inc., 2015
Published in the USA, July 24, 2015
5991-5724EN