Chemico-Biological Interactions 220 (2014) 181–192

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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Mini-review

Ultra-performance liquid chromatography–mass spectrometry

as a sensitive and powerful technology in lipidomic applications
Ying-Yong Zhao a,b,⇑,1, Shao-Ping Wu c,1, Shuman Liu b, Yongmin Zhang c, Rui-Chao Lin d,⇑
a
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, The College of Life Sciences, Northwest University, No. 229 Taibai North Road,
Xi’an, Shaanxi 710069, PR China
b
Division of Nephrology and Hypertension, School of Medicine, University of California, Irvine, MedSci 1, C352, UCI Campus, Irvine, CA 92868, USA
c
Sorbonne Universités, UPMC Univ. Paris 06, CNRS UMR 8232, IPCM, 4 place Jussieu, 75005 Paris, France
d
School of Chinese Materia Medica, Beijing University of Chinese Medicine, No. 11 North Third Ring Road, Beijing 100029, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Lipidomics, the comprehensive illumination of lipid-based information in biology systems, involves in
Received 15 April 2014 identifying lipids and profiling lipids and lipid-derived mediators. The development of lipidomics enables
Received in revised form 31 May 2014 the characterization of lipid species and detailed lipid profiling in body fluid, tissue or cell, and allows for
Accepted 30 June 2014
a wider understanding of the biological roles of lipid networks. Lipidomic research has been greatly
Available online 9 July 2014
facilitated by recent advances in ultra-performance liquid chromatography–mass spectrometry
(UPLC–MS) and involved in lipid extraction, lipid identification and data analysis supporting applications
Keywords:
from qualitative and quantitative assessment of multiple lipid species. UPLC technique, different mass
Lipidomics
Lipid profiling
spectrometry technique, lipid extraction and data analysis in lipidomics are reviewed. Afterwards,
Ultra-performance liquid chromatography examples are provided on the use of UPLC–MS for finding lipid biomarkers in disease, drug, food, nutri-
Mass spectrometry tion and plant fields. We also discuss the UPLC–MS-based lipidomics for the future perspectives and their
Lipid biomarker potential problems.
Ó 2014 Elsevier Ireland Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
2. UPLC–MS techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
2.1. UPLC technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
2.2. Mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
3. Lipids and lipidomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
4. Lipid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
5. Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
6. UPLC–MS applications in lipidomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
6.1. UPLC–MS-based lipidomics in disease biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
6.2. Lipid biomarkers in drug research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
6.3. Nutrition and food of lipidomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
6.4. Lipidomics of plant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
7. Concluding remarks and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
8. Conflict of Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

⇑ Corresponding authors. Address: Key Laboratory of Resource Biology and

Biotechnology in Western China, Ministry of Education, The College of Life Sciences,
Northwest University, No. 229 Taibai North Road, Xi’an, Shaanxi 710069, PR China.
Tel.: +86 29 88304569; fax: +86 29 88304368 (Y.Y. Zhao). Tel.: +86 10 84738652;
fax: +86 10 84738653 (R.C. Lin).
E-mail addresses: zyy@nwu.edu.cn, zhaoyybr@163.com (Y.-Y. Zhao),
linrch307@sina.com (R.-C. Lin).
1
Ying-Yong Zhao and Shao-Ping Wu are co-first authors.

http://dx.doi.org/10.1016/j.cbi.2014.06.029
0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
182
Transparency Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
1. Introduction
The identification and detection of disease biomarkers play an
important role in drug and medicine development. The biological
samples including urine, plasma, serum and tissue extracts have
been analyzed using several techniques such as proton nuclear
magnetic resonance (1H NMR) spectroscopy, gas chromatogra-
phy–mass spectrometry (GC–MS), liquid chromatography–mass
spectrometry (LC–MS) and capillary electrophoresis–mass spec-
trometry (CE–MS) [1,2]. Such biomarker identification can be a
time-consuming process requiring sample reanalysis. The analysis
of global profiling of small molecules from complex biological
samples provides a significant analytical challenge for system
biology. Recently, the introduction and development of two novel
analytical platforms including ultra performance liquid chroma-
tography (UPLC) and mass spectrometry, especially mass spec-
trometryElevated Energy (MSE, where E represents collision energy),
has increased the volume of metabolic information obtained from
any single sample compared with current other analytical systems.
2. UPLC–MS techniques
2.1. UPLC technique
UPLC operates with sub-2 lm chromatographic particles and
this liquid chromatographic system can operate at pressures in
the 6000–15,000 psi range, providing an increased chromato-
graphic resolution compared with conventional HPLC with larger
particles. UPLC provides a wider range of linear velocities while
maintains good chromatographic resolution and therefore can pro-
vide more rapid analysis time. The high chromatographic resolu-
tion, which results in an increased signal/noise and narrow peak
width compared with conventional HPLC, is benefit for metabolic
profiling to allow the determination of an enormous number of
metabolites at physiological level. The previous literature reported
that UPLC offered significant advantages over conventional
reversed phase HPLC amounting to a more than twofold peak
capacity, an almost ten-fold increase in speed and a 3–5-fold
increased sensitivity compared with a conventional 3.5 lm sta-
tionary phase [3]. Another study reported that UPLC for separation
of human serum metabolites resulted in 20% more detected com-
ponents compared with HPLC [4]. UPLC–MS has become a main-
stay of proteomics and metabolomics for protein identification
and metabolite identification, respectively [5]. A greater number
of UPLC applications have been described in recent reviews and
research articles [2,6–12].
2.2. Mass spectrometry
Technological advances in MS play an important role in increas-
ing lipidomics. Traditionally, lipid is analyzed using GC–MS, The
two soft-ionization techniques including electrospray ionization
MS and matrix-assisted laser desorption/ionization MS have been
introduced until the late 1980s and revolutionized MS [13,14].
The development of matrix-assisted laser desorption/ionization
and electrospray ionization has significantly extended the range
of lipids that can be analyzed by MS [15,16]. LC–MS has greatly
increased the number of lipid classes that can be analyzed in a
single run. Generally speaking, Identification biomarker needs
two experimental procedures including low collision energy MS
Y.-Y. Zhao et al. / Chemico-Biological Interactions 220 (2014) 181–192
conditions first to get molecular ions and then higher collision
energy MS2 conditions to get the fragment ions. Fragment ions
can be acquired by various different ways and the simplest method
is the selection of a precursor ion and then fragmentation in the
collision cell monitored using a scanning mode (tandem mass
spectrometer). MS/MS instrument includes two mass analysers
separated by a collision cell either in space or time (ion-traps).
Beam-based MS/MS instruments include triple quadrupoles MS,
quadrupole-time-of-flight MS and time-of-flight–time-of-flight
MS. The crude lipid extracts can produce complex mass spectra
with many different ionized lipids and many isobaric species in
untargeted lipidomics. The fragment ions of lipids allow the use
of precursor ion and neutral loss scans on triple quadrupole MS
to observe molecules with common structural information such
as lipid classes and subclasses in a single spectrum with signifi-
cantly reduced chemical noise. Triple quadrupole MS was demon-
strated as a powerful tool for analysis of complex lipid extracts by
the use of specific precursor ion and neutral loss scans [17,18]. Ion-
trap MS can conduct each of the steps of MS2. The advantage of
ion-traps MS can perform not only MS2 but also MS3 and up to
MS10. However, the disadvantage of ion-traps MS is the loss of
the bottom third of the spectrum. This is a characteristic of MSn
when performed on ion-traps MS. The second major disadvantage
of ion-traps is their poor mass accuracy. These approaches need
know prior information of the indentified ions from full-scan MS.
Alternatively, data-dependent analysis can switch from full-scan
MS mode to MS2 mode when an eluting peak rises above a prede-
fined threshold by mass spectrometer [19]. However, data-depen-
dent analysis results in a loss of fragment ion information in the
MS mode when fragment ion information of MS2 are being
acquired and poor duty cycles, thus make it less than ideal for fast
analysis and narrow peaks. Therefore, these approaches are per-
haps less efficient that would be desired for the rapid analysis of
complex biological samples. However, the ever elevating require-
ment for increased throughput, higher sensitivity and higher chro-
matographic resolution remain a key goal for research. To avoid
triggering of data-dependent acquisition experiments, full-scan
MS and MS2 (without specific precursor ion selection) data are col-
lected from a single injection. MSE technique was produced and
introduced to metabolomics in 2005 [20]. This type of experiment
uses an orthogonal hybrid quadrupole time-of-flight mass spec-
trometer that leverages the fast scanning capability and non-
resolving Q1 in a unique manner. Briefly, two interleaved scan
functions Q1 and Q2 are used for data acquisition such that the
first scan function Q1 acquires a wide mass range (m/z 50–1200)
using low collision energy between Q0 and the pusher region.
Function Q1 collects information on the intact ions from the sam-
ple. The second scan function Q2 now has a high collision energy
that fragments all of the ions transmitted through function Q1. This
function acquires data over the same mass range (m/z 50–1200);
however, the collision energy is ramped from low energy to high
energy (15 to 50 eV). This scan function allows for the collection
of fragment information from the ions in the second scan function
Q1, which is equal to a non-selective tandem mass spectrometric
scan. In this way, two chromatograms are generated, one with
information on the intact molecules from the first function Q1,
and the other with the fragmented ion information from the sec-
ond function Q2. A variety of data-processing algorithms can be
used to extract metabolite information from these data [21,22].
In other words, MSE can provide parallel alternating scans for
 Y.-Y. Zhao et al. / Chemico-Biological Interactions 220 (2014) 181–192
Fig. 1. Low-collision energy and high-collision energy exact mass spectra of PC (16:0/18:2). The low-energy spectrum only contains the precursor ion at m/z 758.5697
whereas in the high-energy spectrum various fragments appear as the loss of the various fatty acid chains at 496.3410 and 520.3407 and their respective water losses. Also,
the major fragment ion at m/z 184.0733 can be seen corresponding to the polar head group.
acquisition at either low collision energies to obtain precursor ion
information or high collision energies to obtain full-scan accurate
mass fragment, precursor ion and neutral loss information
(Fig. 1). MSE involves in a simultaneous acquisition through alter-
nating between high and low collision energies during a single
chromatographic run and Fig. 2 shows reconstructed ion chro-
matograms of UPLC–MSE data with low and high energy acquisi-
tions from human plasma [23]. This ability provides the
structural information required for the identification of unknown
biomarkers in the context of untargeted analyses. Recently,
UPLC–MSE techniques have proved to be powerful tools for the
identification of trace components of complex mixtures and for
confirming their presence in proteomics, metabolomics and
lipidomics [2,4,24–29].
3. Lipids and lipidomics
Lipidomics, a branch of metabolomics, was first put forward by
Han and Gross in 2003 [30]. Lipidomics has been defined as ‘‘the
full characterisation of lipid molecular species and of their biolog-
ical roles with respect to expression of proteins involved in lipid
metabolism and function, including gene regulation’’ [31]. The
metabolome is a diverse, complex array of compounds that display
varying physiochemical characteristics. Various proteins and
metabolites play a fundamental role in the disease process.
Proteomics focuses on characterizing and quantifying various
proteins involved in gene expression. On the other hand, lipido-
mics is at the opposite end of the ‘omics’ spectrum. The main dif-
ference between lipids and carbohydrates, proteins and nucleic
acids is their solubility in organic solvents. Historically, lipids are
defined either by these physical properties-specifically their solu-
bility in non-polar solvents or by the presence of long hydrocarbon
chains; however, not all lipids satisfy both definitions. Recently,
183
investigators have attempted to refine this definition. A new
nomenclature system has been proposed for lipids based on lipid
biosynthesis, namely ‘‘hydrophobic or amphipathic small mole-
cules that may originate entirely or in part by carbanion-based
condensations of thioesters and/or by carbocation-based conden-
sation of isoprene units’’. As a result, lipids were regrouped under
the eight categories that cover eukaryotic and prokaryotic sources
(Table 1) [32]. Each category contains distinct classes, subclasses,
subgroups, and subsets of lipid molecules. Unlike other biomole-
cules, complex lipids such as sphingolipids and glycerophospholip-
ids include a wide range of building blocks that can give rise to a
bewildering array of combinations. Permutations that may arise
only from common eukaryotic lipid motifs give rise to more than
180,000 theoretical phospholipid structures that could be present
in a given cell or tissue extract [33]. Note that this number does
not include complexity that may arise from consideration of iso-
meric lipids that differ only in double-bond position, backbone
substitution or stereochemistry [34]. Although once considered
merely a structural component of cells, it is now realized that
Lipids play an important role in biological system including
composing membrane bilayer, storing energy, producing signal
transduction, providing functional implementations of membrane
proteins and their interactions, etc. [35]. Lipidomics involves in
mapping of the entire spectrum of cellular lipids in biological sys-
tems, including metabolic pathways and lipid–lipid interactions
[36]. Therefore, Lipidomics complements genomics, proteomics
and metabolomics to provide a more complete understanding of
system biology, which enables us to understand at a fundamental
level the various molecules implicated in health and disease [37].
Traditional methodologies include biochemical assays, chromatog-
raphy and various imaging techniques. However, such methods
were limited in their sensitivity and accuracy. Recently, an increas-
ing number of publications have described lipidomic studies using
various techniques including GC–MS, LC–MS and 1H NMR [38,39].
184 Y.-Y. Zhao et al. / Chemico-Biological Interactions 220 (2014) 181–192

H
O
H H
O
Cholesteryl ester (22 6)

H
O
H H
Cholesteryl ester (20 4) O

H
O
H H
Cholesteryl ester (20 5) O
Low energy
H

H
H H
O
Cholesteryl ester (18 1)
O

H
H H
Cholesteryl ester (18 2) O
O H

H H
O
O
Cholesteryl ester (18 3)

High energy H

m/z 369.3515 HC
H H

Fig. 2. Reconstructed ion chromatograms of UPLC–MSE data with low and high energy acquisitions from human plasma. The high energy reconstructed ion chromatogram of
the key fragment ion from the cholesteryl (m/z 369.3515) is used to identify the possible presence of cholesteryl esters, and subsequently at the indicated in the low energy
UPLC–MS trace as the key fragment ion from the cholesteryl (m/z 369.3515). Following alignment with the low energy trace it is possible to extract the unfragmented ion
information, which corresponds to the cholesteryl esters. The structures shown are examples of possible structures.

Table 1
Lipid categories and typical classes.

Categories Typical classes

Fatty acyls Fatty acids and conjugates, fatty esters, fatty alcohols, fatty amides, eicosanoids
Glycerophospholipids Phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines phosphatidylglycerols, phosphatidic acids, phosphatidylinositols,
cardiolipinds
Glycerolipids Monogalactosyldiacylglycerol, diacylglycerides, digalactosyldiacylglycerol, triacylglycerides
Sphingolipids Ceramides, sphingosines, sphingosine-1-phosphates, ganglioside mannoside 3, sphingomyelins
Prenol lipids Polyprenols, quinines, hydroquinines, isoprenoids
Sterol lipids Cholesterols, cholesteryl esters, cholesteryl sulfates
Saccharolipids Acylaminosugars, acylaminosugar glycans, acyltrehaloses, acyltrehaloses glycans
Polyketides Macrolide polyketides, aromatic polyketides, non-ribosomal peptide/polyketide hybrids
 Y.-Y. Zhao et al. / Chemico-Biological Interactions 220 (2014) 181–192
Workflow for setting up the system
Prepare solvents
Calibrate UPLC Prepare instrument
Create a MassLynx project Creating a project
Create an LC method Instrument methods
Create an MS method MS and MSE method
Prepare QC samples
Create a sample list Sample lists
Acquire data
Fig. 3. A typical example work flow of lipidomics using UPLC–QTOF/MS and a MSE data collection technique as research tools for discovering lipids in complex mixtures.
Among the analytical techniques, LC–MS is recognized as one of
the best analytical techniques in selectivity, sensitivity and repro-
ducibility [40]. Furthermore, among the various LC–MS platforms,
UPLC–MS/MS and UPLC–MSE are considered as the most powerful
tools for high-throughput lipidomic analysis, especially for large-
scale untargeted lipid profiling due to its enhanced reproducibility
of retention time. Fig. 3 shows a sample workflow of UPLC–QTOF/
MS and a novel MSE data collection technique in lipidomics.
4. Lipid extraction
Because lipids embedded in complex biological matrixes, but
not appear in their free form, extraction procedures are necessary
in analysis of lipids to remove other compounds such as sugars,
proteins or other small molecules from the biological samples that
would interfere with the chromatographic separation. The general
procedures are lipids separation from the matrix; removal of any
non-lipid components, such as saccharides, proteins or other small
molecules; and fractionation and isolation of lipids from the
extract. Generally, there are mainly two extraction methods to
extract lipids from samples including liquid–liquid extraction
and solid–phase extraction. The most popular methods of
liquid–liquid extraction for lipids were proposed by Folch and
co-workers [41] using CCl3: CH3OH (2:1, v/v) as an extraction
method, and by Bligh and Dyer [42] using incorporated water as
185
Workflow for analyzing results
Import data
Peak detection
Data preprocessing
Denoising and deconvolution
Pattern recognition
Statistical analysis
PCA, LASSO
Lipid database Lipid identification
Lipids of interest
Pathway analysis
extraction component. Different proportions of CCl3, CH3OH and
H2O depend on the moisture content of the complex biological
samples. The addition of salt in the extraction solvent has its origin
in the fact that the formation of emulsions was one of the major
disadvantages of liquid–liquid extraction method. Liquid–liquid
extraction allows an instant partition of the lipids without an
additional step. Other commonly method was reported using hex-
ane–isopropanol (3:2, v/v) extraction method [43], which is a less
toxic extractant. Most methods still carry out the above-mentioned
lipid extraction procedure. Recently, Löfgren and co-workers
developed a mixture of CH3CH2CH2CH2OH and CH3OH extraction
method for total lipid extraction from human plasma [44]. The
results showed this method was more suitable for total lipid
extraction than the Folch’s method. In addition, the CH3CH2CH2-
CH2OH and CH3OH extraction method was rapid and high through-
put for lipidomics. A methyl tert-butyl ether liquid–liquid
extraction method to extract lipids and different classes of metab-
olites was developed by Chen and co-workers in 2013 [45]. Methyl
tert-butyl ether extraction method achieved a complete analysis of
lipids and other metabolites after a single extraction. Besides the
extraction of blood, tissue or cells, a full fecal lipidome liquid–
liquid extraction method was reported by Gregory and co-workers
in 2013 [46]. The method utilizes two separate, complementary
extraction chemistries, CH2Cl2 and a methyl tert-butyl ether/
hexafluoroisopropanol mixture, alone or with high pressure
cycling. Extracts were assessed by LC–MS method. 304 endogenous
186 Y.-Y. Zhao et al. / Chemico-Biological Interactions 220 (2014) 181–192
lipid species were identified in feces, which covered six categories
from LIPID MAPS as well as various related classes and subclasses.
In addition, fecal lipidome liquid–liquid extraction method was
developed to provide fecal lipidomics for both animal models
and clinical applications [47].
Solid-phase extraction is a rapidly extraction method and can
minimize degradation and set up automatic pre-analytical facilities
for a simultaneous preparation of numerous samples. This method
did not require for partition the solvent/water mixture and reduced
the consumption of solvents and time [48]. Solid-phase extraction
has been proposed for lipid extraction using aminopropyl car-
tridges as reported for neutral and acidic phospholipids [49], phos-
phatidylcholines, non-esterified fatty acids, cholesterol esters and
triacylglycerols from plasma [50]. Liquid–liquid extraction and
solid-phase extraction have also been carried out with phospholip-
ids from human serum by Ferreiro-Vera and co-workers [51]. They
compared the extraction efficiency of liquid–liquid extraction using
different nonpolar-polar solvents with that of solid-phase extrac-
tion using three elution solvent. The results showed that the highest
sensitivity and selectivity was acquired by solid-phase extraction
with CH3OH as the optimum elution solvent for lipid extraction.
Solid–phase extraction method is preferred in lipidomics analysis,
with CH3OH, hexane, and CHCl3 as elution solvents. Compared with
liquid–liquid extraction method, solid–phase extraction method
reduces the consumption of time and solvents. However, when a
large volume of samples were prepared, the recovery will be dra-
matically decreased due to the low peak capacity of solid–phase
extraction method.
Recently, many new extraction methods have been used for lip-
idomic analysis including ultrasound-assisted extraction, disper-
sive liquid–liquid microextraction, pressurized fluid extraction
and solid–phase microextraction [52–55].
Generally speaking, extraction of organic solvents is usually the
first step for the lipid analysis, so results from different groups may
be variable from different methods of lipid isolation. Liquid–
liquid extraction method may be preferred to extract more
comprehensive lipids and it may be more suitable for nontargeted
Fig. 4. Typical base peak chromatograms from extracted heart tissue lipids for transgenic (A) and wild type (B) mice using UPLC–QTOF/MS with a Waters ACQUITY UPLC HSS
T3 column (2.1 cm  100 mm, 1.8 lm), eluted with 40–100% linear gradient of acetonitrile/water (40/60, v/v) with 10 mM ammonium acetate and acetonitrile/isopropanol
(10:90, v/v) with 10 mM ammonium acetate over 10 min at a flow rate of 0.4 mL/min.
lipidomics. When the researchers focus on all the simple and com-
plex lipids from a tissue in a near quantitative manner, they nor-
mally use the liquid–liquid extraction method. For targeted
lipidomics, solid–phase extraction method may perform better
specificity and extraction efficiency.
5. Data analysis
Fig. 3 represents a typical UPLC–MS-based lipidomic workflow
and shows that the initial step in processing raw data is lipid iden-
tification. Several lipid databases and software packages to achieve
this goal have been developed including LMSD (LIPIDMAPS), Lipid-
View, MZmine 2 and LipidBank. Software of metabolomics may
also be employed for lipid identification and quantification
[56,57]. The second step of data processing is to normalize the data
via a set of internal standards. Once these calculations have been
carried out with raw data, the identified lipids can then be quanti-
fied by comparison to appropriate internal standards [58]. The
third step is performing statistical analysis of the complex data
sets. Principal component analysis (PCA) is an unsupervised multi-
variate data analysis method and it gives the comprehensive view
of the clustering trend for the multidimensional data in lipidomics.
PCA can help to visualize correlated variations in more than two
dimensions. This test describes data in the form of a linear combi-
nation of scores containing information on the samples and load-
ings containing information on the variables. The advantage of
PCA is that the outcomes are straightforward and intuitively
understandable because of the graphical representation [59]. A
score plot provides an indication of the clustering of observations
as function of the treatment. The distance between clusters is a
measure of the differential efficiency of the treatments. The greater
the similarity between samples, the smaller the separation appears
between labels in the score plots. A loading plot can be helpful to
locate the concerted lipid variations. The load plots are also used
to illuminate the compositional trends. Two lipid species that point
in the same direction show a positive correlation, whereas lipid
 Y.-Y. Zhao et al. / Chemico-Biological Interactions 220 (2014) 181–192
species pointing in opposite directions reveal a negative
correlation.
6. UPLC–MS applications in lipidomics
Lipids are involved in various biochemical and signaling
pathways, cell structure and function of health and diseases.
UPLC–MS-based lipidomics are suitable for lipid analysis from a
variety of samples and appropriate extraction, sample clean-up
and derivatization procedures are followed [60–67]. Fig. 4 displays
typical UPLC–QTOF/MS chromatograms from extracted heart tis-
sue lipids of transgenic and wild type mice in positive ion mode.
Lipidomics has been applied to disease biomarker discovery, drug
development, drug safety assessment, nutritional supplementation
assessment and plant research. The following section gives an
overview of such studies focusing on recent reports discussing
lipid biomarkers, disease biomarker discovery, drug discovery,
nutritional supplementation assessment as well as some examples
relevant to plant research. Table 2 displays UPLC-MS-based
lipidomics applications for discovering biomarkers in the above-
mentioned fields.
6.1. UPLC–MS-based lipidomics in disease biomarkers
UPLC-based lipidomics was applied for differential phenotyping
from pet dogs developing spontaneous malignant mammary
tumors and health controls. A biological signature related to cancer
was successfully revealed from this lipidome analysis [68]. Type 2
diabetes is a multi-factorial disease with a complex pathogenic
mechanism and a combination of metabolomic and lipidomic
approach was employed to analyze serum samples from Type 2
diabetes. The results demonstrated significant perturbations in
amino acid metabolism, TCA cycle and glycerol-phospholipid
metabolism had an important effect on the overall glucose homeo-
stasis in Type 2 diabetes [69]. Another study indicated that serum
lipids were associated with progression to type 2 diabetes in the
METSIM study [70]. The analyses of serum lipids from type 1 dia-
betes were performed using UPLC–MS-based lipidomics platform
[71]. Type 1 diabetes is characterized by a distinct cord blood lip-
idomic profile that includes reduced major choline-containing
phospholipids (sphingomyelins and phosphatidylcholines). Reduc-
tion in choline-containing phospholipids in cord blood therefore is
specifically related to progression to type 1 diabetes but not with
development of b-cell autoimmunity.
A lipidomics platform using UPLC–MS was applied for the anal-
ysis of serum samples from schizophrenia in twin pairs discordant
for schizophrenia as well as unaffected twin pairs [72].
Lysophosphatidylcholines are preferred carriers of polyunsatu-
rated fatty acids across the blood–brain barrier. Decreased
lysophosphatidylcholines indicated that patients might be more
susceptible to infections. Their association with cognitive speed
supports the opinion that altered neurotransmission in schizo-
phrenia may be in part mediated by reactive lipids. UPLC–MS
was employed to analyze serum samples collected from Lamin A/
C gene carriers, dilated cardiomyopathy patients without Lamin
A/C gene mutation and controls. The analysis helped us to obtain
novel insights into how the affected lipids might relate to cardiac
shape and volume changes [73]. In addition, lipidomic profiling
of hepatocellular carcinoma in human and animal studies was
investigated by UPLC approach and lysophosphatidylcholine
(24,0.0) was identified as a discriminatory biomarkers [74,75].
Non-alcoholic fatty-liver disease, as with the metabolic syndrome,
is approaching epidemic proportions in many countries. Patients
with non-alcoholic fatty-liver disease had increased triacylglyce-
rols with low carbon number and double-bond content while
187
lysophosphatidylcholines and ether phospholipids were dimin-
ished in those with non-alcoholic fatty-liver disease [76]. Lipido-
mic approach was also applied to characterization of alcohol
induced metabolic changes in mouse liver [77,78].
6.2. Lipid biomarkers in drug research
UPLC–MS lipidomics was undertaken on changes of the glycer-
olipids upon b-amyloid peptide-induced neurotoxicity and the
neuroprotective effect of (-)-epigallocatechin gallate in PC12 cells.
The glycerolipids were significantly elevated in Ab-treated cells,
but were restored near to normal levels after (-)-epigallocatechin
gallate treatment. The increased phosphatidylcholines were
associated with the reduced phospholipase A2 activity and the
enhanced activity of lysophospholipid acyltransferases. In addition,
an increased arachidonic acid was observed as another important
response of PC12 cells to the b-amyloid peptide aggregates, indi-
cating an active inflammatory process occurring in b-amyloid pep-
tide induced neurotoxicity. (-)-epigallocatechin gallate treatment
can reverse the deregulated metabolism of phosphatidylcholines,
which might be one of the biochemical mechanisms contributing
to its neuroprotective effect [79]. A lipidomics approach using
UPLC–MS investigated the anti-depressive effect of the traditional
Chinese medicine Allium macrostemon in a rat model of depression
[80]. Several increased LPC (18:1 ? :2), LPC (O-16:2), LPC (20:1),
and LPC (O-18:3) were observed in rat plasma, while some PC
(32:1), PC (37:4), PC (36:4 ? :5), PC (38:4 ? :6), PC (O-36:4), PC
(40:6), and PC (O-38:5) and TG (60:12), TG (58:12), and TG
(62:13 ? :14) were decreased in depressed rats. These alteration
showed that depression were related to inflammatory conditions
and an incomplete b-oxidation of fatty acids [81,82]. Most of these
abnormal lipids were returned to their normal levels after treat-
ment with A. macrostemon. UPLC–MS lipidomic analysis was
undertaken on plasma from rosuvastatin treated patients [83].
The results demonstrated that the overall lipids in plasma
decreased by drug response.
6.3. Nutrition and food of lipidomics
The evidence of the multiple beneficial health effects of fish, fish
oil, dietary fiber and whole grain consumption are well reported.
Lipidomics of the effect of fish oil supplementation was performed
using UPLC–MS, where 568 lipids were detected and 260 identi-
fied. The intervention groups were well separated after three
weeks. Several lipid classes including phosphatidylethanolamine,
phosphatidylcholine, lysophosphatidylcholine, phosphatidylserine,
sphingomyelin, triglycerides and phosphatidylglycerol contributed
strongly to this separation. Significantly decreased 23 lipids were
observed in the fish oil group compared with the high oleic sun-
flower oil group, whereas 51 were significantly increased including
selected phospholipids and triglycerides of long-chain polyunsatu-
rated fatty acids [84]. The effects and possible mechanisms of fatty
fish or lean fish against coronary heart disease were studied by
UPLC–MS and GC lipidomic approach [85]. Lysophosphatidylcho-
lines, ceramides and diacylglycerols were significantly decreased
in the fatty fish group, whereas cholesterol esters and specific
long-chain triacylglycerols were significantly increased in the lean
fish group. Diacylglycerol is the mediator of lipid-induced insulin
resistance via activation of novel protein kinase C, which inhibits
insulin action and is also associated with inflammatory response
[86]. Ceramides are suggested to attenuate insulin signaling
through multiple pathways [86–88]. Lysophosphatidylcholine is
the main bioactive lipids of oxidized low-density lipoprotein and
may be related to many of the inflammatory effects of oxidized
low-density lipoprotein [89], which may be associated with
anti-inflammatory effects of n-3 fatty acids [90]. The eight-week
188 Y.-Y. Zhao et al. / Chemico-Biological Interactions 220 (2014) 181–192

consumption of fatty fish decreased lipids, which are potential
mediators of lipid-induced insulin resistance and inflammation,
and may be associated with the therapeutic effects of fatty fish
on the progression of insulin resistance or atherosclerotic vascular
diseases. An ultra-performance convergence chromatography cou-
pled with Q/TOF–MS was utilized to analyze triacylglycerols and
diacylglycerols in cow milk fat [91]. Forty-nine triacylglycerols
and seven diacylglycerols were identified in cow milk fat. The lipid
class composition of the ordinary and dark muscles of chub mack-
erel were compared by thin-layer chromatography and UPLC–MS
[92]. Diacylglycerol (18:0/18:1 and 16:0/16:0) and triacylglycerol
(22:0/22:1/22:3 or 22:0/22:0/22:4) were observed in neutral
lipids. Neutral and acidic glycosphingolipids were observed in
the glycolipids. Phosphatidylinositol (18:0/20:5 or 18:1/20:4) and
phosphatidylserine (20:5) were present in the phospholipids in
thin-layer chromatography. UPLC–MS analysis found a difference
in the neutral lipid fraction between the ordinary and dark muscles
but the glycolipid and phospholipid patterns were similar in both
muscles [92]. Lipidomic analysis was performed on the consump-
tion of high-fiber rye bread or white-wheat bread modifies the
plasma lipid profiles in postmenopausal women using UPLC–MS
[93]. There were no changes in plasma lipidomic profiles during
the rye bread or white-wheat bread intervention periods. The
results suggest that eight-week consumption of high-fiber rye
bread increases metabolites that might mediate positive effects
of rye bread on satiety and weight maintenance. Other evidence
indicated that lysophosphatidylcholines was increased in the oat
and wheat bread and potato group, while in the rye bread and
pasta group docosahexaenoic acid (22:6n-3) increased and isoleu-
cine decreased [94]. The lipid profiles had the correlation with the
alterations in the adipose tissue differentiation pathway when
using the elastic net regression model of the lipidomic profiles
on selected pathways.
6.4. Lipidomics of plant
The snow alga Chlamydomonas nivalis (C. nivalis) is a typical mic-
roalgal species that can adapt and resist to natural habitats in the
polar region and similar extreme environments. The snow alga C.
nivalis was subjected to nitrate or phosphate deprivation to study
its stress responses in lipid profiles analyzed by UPLC–MS [95].
Three clusters were distinguished as the control, nitrate-deprived
and phosphate-deprived groups. The lipidomic approach identified
monogalactosyldiacylglycerols, digalactosyldiacylglycerols, phos-
phatidylethanolamine, phosphatidylglycerols, sulfoquinovosyl-
diacylglycerols and phosphatidylinositiol as differentiating lipid
biomarkers. The changes of these lipid biomarkers provided new
insights into the lipid metabolism of the snow alga in response to

Table 2
UPLC–MS-based lipidomic applications for discovering biomarkers.

Application Specimen types Lipid classes or metabolites References

Disease biomarkers
Canine mammary cancer Serum PC, PE, LysoPC and LysoPE [68]
Type 2 diabetes Serum Glycerophospholipid [69,70]
Type 1 diabetes Serum SM, PC, PE, TG and LysoPC [71]
Schizophrenia patients Patient serum LysoPC and TG [72]
Dilated cardiomyopathy Patient serum PC (38:5e), PS (38:2), TG (49:2)/TG (49:1), TG (49:3), TG (50:10), TG (50:2)/TG (50:1), TG [73]
(54:3)/TG (54:2)
Hepatocellular carcinoma Human and animal LysoPC (24,0.0) [74,75]
blood
Non-alcoholic fatty-liver disease Serum Odd- and short-chain TG, ether lipids, LysoPC, SM, PC, PE, PUFA-containing PC, PE and TG [76]
Drug research
Neuroprotective effect of PC12 cells PC, Glycerolipids and arachidonic acid [79]
epigallocatechin gallate
Anti-depressive effect of Allium Rat plasma LysoPC (18:1 ? :2), LysoPC (O-16:2), LysoPC (20:1), LysoPC (O-18:3), PC (32:1), PC (37:4), [80]
macrostemon PC (36:4 ? :5), PC (38:4 ? :6), PC (O-36:4), PC (40:6), PC (O-38:5), TG (60:12), TG (58:12)
and TG (62:13 ? :14)
Rosuvastatin Human plasma PS, PG, PE, PC, TG, PI, SM, LysoPC and lysophosphatidic acid [83]
Nutrition and food
Fish oil supplementation Healthy subjects PE, PC, LysoPC, PS, SM, TG and PG [84]
plasma
Fatty fish or lean fish against coronary Patient plasma LysoPC, DAG, ceramides, cholesterol esters and specific long-chain TAG [85]
heart disease
Cow milk Cow milk fat 7 DAG and 49 TAG [91]
Chub mackerel Ordinary and dark DAG (18:0/18:1 and 16:0/16:0), TAG (22:0/22:1/22:3 or 22:0/22:0/22:4), PI (18:0/20:5 or [92]
muscles 18:1/20:4), PS (20:5), neutral GSL, acidic GSL and glycolipids
High-fiber rye bread or white-wheat Plasma of SM (d18:1/25:1) and SM (d18:1/25:3) [93,94]
bread postmenopausal
women
Plant lipidomics
Snow alga C. nivalis under nitrate or Tissue MGDG, DGDG, PE, PG, PI and SQDG [95]
phosphate deprivation conditions
Snow alga C. nivalis treated with Tissue TG, PC, PG, SQDG, MGDG, LPG, LysoPC, LMGDG and lysoSQDG [96]
different NaCl concentrations
Nitzschia closterium Tissue PG, TAG, SQDG, MGDG, DGDG, lysoMGDG, lysoDGDG, free fatty acid, harderoporphyrin [97]
and cholesterol
Stephanodiscus sp. under cold exposure Tissue TAG, PC, PG, LPG, LysoPC and lysoMGDG [98]
Dunaliella tertiolecta Tissue DGDG and DGTS [99]
Stephanodiscus sp. Tissue PG, DGDG, MGDG and SQDG [100]

DGDG: digalactosyldiacylglycerols; DGDG: digalactosyldiacylglycerols; DGTS: diacylglyceryltrimethylhomoserines; LPG: lysophosphatidylglycerols; lysoMGDG: lysomo-
nogalactosyldiacylglycerols; LysoPC: lysophosphatidylcholines; MGDG: monogalactosyldiacylglycerols; PC: phosphatidylcholines; PE: phosphatidylethanolamines; PG:
phosphatidylglycerols; PI: phosphatidylinositols; PS: phosphatidylserines; SM: sphingomyelins; SQDG: sulfoquinovosyldiacylglycerols; TAG: triacylglycerols; TG:
triacylglycerides.
 Y.-Y. Zhao et al. / Chemico-Biological Interactions 220 (2014) 181–192
nitrate or phosphate deprivation stress condition. UPLC–MS
approach was developed for lipidomic profiling from C. nivalis trea-
ted with different NaCl concentrations. Seven types and 35 kinds of
polar lipid molecules were selected and identified as biomarkers
[96]. Su and co-workers developed an UPLC–MS method to investi-
gate the lipid changes in different growth phases of Nitzschia closte-
rium. Thirty-one lipids were selected and identified as putative
biomarkers. Further analysis on the putative biomarkers demon-
strated that nitrate starvation played an important role in the tran-
sition from exponential phase to stationary phase in Nitzschia
closterium [97].
UPLC–MS-based approach was developed for investigating the
lipid changes during cold exposure in Stephanodiscus sp. [98].
Thirty-eight lipid molecules were selected and identified as puta-
tive biomarkers, including triacylglycerol, phosphatidylcholine,
phosphatidylglycerol, lyso-phosphatidylglycerol, lysophosphati-
dylcholine, lyso-sulfoquinovosyldiacylglycerol, etc. These metabo-
lites have been shown previously to function in energy storage,
membrane stability and photosynthesis efficiency. A total of 29 lip-
ids and 7 carotenoids were detected in a Dunaliella tertiolecta under
light intensity and nitrogen starvation condition [99]. Alterations
of digalactosyldiacylglycerol and diacylglyceryltrimethylhomoser-
ine species were observed under stress conditions. The total carot-
enoid content was decreased under stress conditions, while
ã-carotene was increased under nitrate-deficient cultivation. The
highest productivity of carotenoid was attained under high light
and nitrate sufficiency condition, which result from the highest
level of biomass under high light and nitrate sufficiency. In addi-
tion, a comprehensive characterization of the photosynthetic
glycerolipids of the diatom Stephanodiscus sp. was carried out by
UPLC–MS [100]. Four classes of photosynthetic glycerolipid
including 9 digalactosyldiacylglycerols, 16 monogalactosyldiacyl-
glycerols, 8 phosphatidylglycerols and 23 sulfoquinovosyldiacyl-
glycerols were identified in Stephanodiscus sp.
Arabidopsis thaliana has a wide geographical range throughout
the Northern Hemisphere with significant natural variation in
freezing tolerance. Hummel and co-workers developed an UPLC–
based method for the comprehensive profiling of more than 260
polar and non-polar lipids from Arabidopsis thaliana leaf [101].
Accumulated evidence indicated that the relative abundance of
several lipid species was related to the freezing tolerance of the
accessions, allowing the identification of possible marker lipids
for plant freezing tolerance [102].
7. Concluding remarks and future perspectives
Recently, the rapid development of UPLC–MS led to significant
advances within the field of lipidomics. A sub-2 lm packing particles
combined with an UPLC system enabled significantly increases in LC
performance over conventional HPLC system, mainly with enhanced
peak resolution, increased sensitivity and speed [4,5,103]. The
improved resolution led to a reduction in the number of co-eluting
components and hence a decrease in ion suppression resulting in
an increase in the number of compounds detected by MS. UPLC
showed better reproducibility and signal/noise ratios over HPLC
and this technology was more suitable for untargeted lipidomics.
Several publications have reported sensitivity of UPLC–MS and this
approach which exhibits chromatographic peak area associated
with a high mass accuracy allowed to lipid study in complex biolog-
ical samples [51,59]. UPLC–MS had the flexibility of separating lipids
from any sample type into individual lipid classes or separated lipids
within the same class based on the used chromatographic phase,
though choice of solvent also played a critical role.
Although UPLC has high separation and quantification power,
UPLC–MS may be less suitable for some class of compounds.
189
Suitability of UPLC may be increased by combining hydrophilic
interaction chromatography separation methods for untargeted
lipid profiling to get comprehensive information on lipidome. How-
ever, other techniques may have to be employed for obtaining a
comprehensive investigation of the lipidome. GC–MS may be com-
plementary to UPLC–MS. Thus, the technological breakthroughs of
UPLC have provided researchers with the capacity to measure hun-
dreds or even thousands of various lipids in as little as a few min-
utes per sample, paving the way for complex diseases, drug, food
and nutrition. One of the great benefits of the lipidomic application
is the fact that groups of lipid biomarkers are eager for high sensi-
tivity and specificity than other biomarkers such as gene, protein or
metabolite, promoting the direct comparison of animal models
with human diseases, which enhances the potential of the tech-
nique to rapidly transfer laboratory research into clinical applica-
tion. The current UPLC–MS approach can allow high-throughput,
sensitivity and resolution of lipid analysis and can identify lipid
structures. However, continued incremental developments of new
analytical techniques along with data-handling routines are still
necessary for data preprocessing, data mining, statistical analysis,
biomarker identification and interpretation of biochemical path-
ways and achieve more breakthroughs in lipid research. Better
and more data on very low abundant lipids and better predicting
data models will reveal more abnormalities in lipid metabolism
associated with diseases, drug, food and nutrition.
8. Conflict of Interest
The authors declare that there are no conflicts of interest.
Transparency Document
The Transparency document associated with this article can be
found in the online version.
Acknowledgements
This study was supported by the Program for New Century Excel-
lent Talents in University, China (No. NCET-13-0954) and Changji-
ang Scholars and Innovative Research Team in University, China
(No. IRT1174), National Natural Science Foundation of China, China
(Nos. J1210063, 81202909, 81274025, 81001622), As a Major New
Drug to Create a Major National Science and Technology Special,
China (No. 2014ZX09304-307-02), China Postdoctoral Science
Foundation, China (No. 2012M521831), National Innovation Train-
ing Plan Program (201310697004), Key Program for the Interna-
tional S&T Cooperation Projects of Shaanxi Province, China (No.
2013KW31-01), Natural Science Foundation of Shaanxi Provincial
Education Department, China (No. 2013JK0811) and Administration
of Traditional Chinese Medicine of Shaanxi, China (No. 13-ZY006).
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