molecules

Review
Sample Preparation Methods for Lipidomics
Approaches Used in Studies of Obesity
Ivan Liakh 1,2 , Tomasz Sledzinski 1 , Lukasz Kaska 3 , Paulina Mozolewska 1
and Adriana Mika 1,4, *
1 Department of Pharmaceutical Biochemistry, Medical University of Gdansk, Debinki 1,
80-211 Gdansk, Poland; liakh_ivan@mail.ru (I.L.); tomasz.sledzinski@gumed.edu.pl (T.S.);
paulina.mozolewska@gumed.edu.pl (P.M.)
2 Department of Toxicology, Medical University of Gdańsk, Al. Gen. Hallera 107, 80-416 Gdańsk, Poland
3 Department of General, Endocrine and Transplant Surgery, Faculty of Medicine,
Medical University of Gdansk, Smoluchowskiego 17, 80-214 Gdansk, Poland; lukasz.kaska@wp.pl
4 Department of Environmental Analysis, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63,
80-308 Gdansk, Poland
* Correspondence: adrianamika@tlen.pl; Tel.: +48-585235190




Received: 30 September 2020; Accepted: 12 November 2020; Published: 13 November 2020


Abstract: Obesity is associated with alterations in the composition and amounts of lipids. Lipids have
over 1.7 million representatives. Most lipid groups differ in composition, properties and chemical
structure. These small molecules control various metabolic pathways, determine the metabolism of
other compounds and are substrates for the syntheses of different derivatives. Recently, lipidomics
has become an important branch of medical/clinical sciences similar to proteomics and genomics.
Due to the much higher lipid accumulation in obese patients and many alterations in the compositions
of various groups of lipids, the methods used for sample preparations for lipidomic studies of samples
from obese subjects sometimes have to be modified. Appropriate sample preparation methods allow
for the identification of a wide range of analytes by advanced analytical methods, including mass
spectrometry. This is especially the case in studies with obese subjects, as the amounts of some
lipids are much higher, others are present in trace amounts, and obese subjects have some specific
alterations of the lipid profile. As a result, it is best to use a method previously tested on samples
from obese subjects. However, most of these methods can be also used in healthy, nonobese subjects
or patients with other dyslipidemias. This review is an overview of sample preparation methods for
analysis as one of the major critical steps in the overall analytical procedure.

Keywords: sample preparation; obesity; lipids; protein precipitation; liquid–liquid extraction;

solid-phase extraction; biological samples

1. Introduction
Obesity remains one of the pressing problems of modern society, therefore, studies of the
mechanisms underlying its occurrence and the therapies used to treat it continue to be relevant.
Depending on the hypothesis, a wide range of research methods can be used, ranging from purely
assessing biochemical parameters to deep psychological research. However, research usually involves
standard procedures such as measuring body mass index (BMI) or fat content in human subjects.
Among the biochemical parameters, those that undergo the greatest changes with obesity (lipid profile,
fasting glucose, insulin, etc.) are first examined.
A large amount of accumulated data on obesity allows for their meta-analysis and underlies a
large number of systematic and retrospective reviews [1]. In particular, studies related to bariatric

Molecules 2020, 25, 5307; doi:10.3390/molecules25225307 www.mdpi.com/journal/molecules

Molecules 2020, 25, 5307 2 of 34

surgery are a powerful source of data because these types of surgery involve altering the stomach,
intestines, or both to induce weight loss [2–7]. In addition, a large number of studies of obesity are
associated with cardiovascular diseases [8–11], diabetes and metabolic syndrome [12–14]; many studies
are in the field of diets, psychology and neurology [15–23].
While the number of parameters in the study of obesity itself is limited only by the imagination of
scientists and the equipment available in the laboratory [24–29], the study of obesity in connection
with other diseases is strictly subordinate to the study area and is often limited to several parameters,
such as BMI and total fat content. [30–33]. In addition, determining the diagnosis of obesity is always
primary in that work; for this purpose, the most commonly used method is the calculation of BMI.
The World Health Organization used BMI to categorize humans into underweight (< 18.5), normal
weight (18.5–24.9), overweight (25–29.9) and obese (BMI ≥ 30) categories [34]. Since BMI may not
be a good indicator of obesity for bodybuilders and other groups of athletes [35,36], body fat [37–39]
and total body water [40,41] can be determined in these groups, as can concomitant states of lipid
alterations in blood (dyslipidaemia) [42], hyperinsulinemia [43], etc. Since obesity is directly related to
lipid metabolism, it is interesting to study not only standard plasma parameters but also alterations in
specific lipid groups in serum [44]. However, due to the much higher lipid accumulation in obesity
(Figure 1) and many alterations in the lipid composition, the methods used for sample preparations for
lipidomic studies in samples from obese subjects sometimes have to be modified. Thus, the purpose
of this review was to collect and systematize information on sample preparation for lipid analysis
in the study of obesity. We focused on both standard procedures and new promising methods.
However, most of these methods can be also used in healthy, nonobese subjects or patients with
other dyslipidemias.

Figure 1. Lipid alterations in obesity. Lipids in red are elevated in obesity, and lipids in green are reduced.
BCAA—branched chain amino acids; BCFA—branched chain fatty acids; DAG—diacylglycerols;
FFA—free fatty acids; HDL—high density lipoproteins; LDL—low density lipoproteins; LPP—lipid
peroxidation products; MUFA—monounsaturated fatty acids; OCFA—odd chain fatty acids;
SFA—saturated fatty acids; PUFA—polyunsaturated fatty acids; TAG—triacyclglycerols.

In the study of obesity, determining triglyceride (TG) and cholesterol levels is of great clinical
importance. Basic blood test results for total cholesterol (TC), TG and cholesterol in lipoprotein
fractions (low density lipoproteins (LDL) and high density lipoproteins (HDL)) should be considered
together. To study these indicators and related and often required for standard clinical practice
indicators (C-reactive protein, glucose, insulin levels, etc.), there are many standard methods and their
modifications that make these analyses routine in clinical practice, and they will not be presented in this
Molecules 2020, 25, 5307 3 of 34

review because of their routine nature. This work is focused more specifically on lipid groups, the study
of which is carried out much less frequently because it requires the use of complex extraction methods
and/or high-performance equipment. However, the alterations found in the extended lipidome
may provide much more additional information about metabolic disorders in obese subjects than
standard lipidograms.

2. Methods of Sample Preparation for Lipidomic Studies

2.1. Sample Collection and Storage

Fat metabolism disorders are detected by determining the lipid spectrum of the blood. Blood for
a study is taken from a vein, always on an empty stomach (12–14 h after eating); otherwise, the results
of the study are distorted, since 1-4 h after eating, alimentary hyperlipaemia occurs [45]. During blood
sampling, adverse events such as haemolysis, coagulation, and platelet activation should be avoided,
but the class of anticoagulants used should also be taken into account since calcium-chelating coagulants
(ethylenediaminetetraacetic acid (EDTA) and citrate) can cause the calcium-dependent formation or
degradation of certain classes of lipids ex vivo [46].
Different classes of lipids are subject to different changes during storage. Long-term storage of
plasma at room temperature (RT) leads to an increase in lysophosphatidylethanolamines (LPE),
lysophosphatidylcholines (LPC) and fatty acids (FAs), while phosphatidylethanolamines (PE)
and phosphatidylcholines (PC) decrease, which suggests the breakdown of ester bonds in these
phospholipids [47]. Avoiding freeze-thaw cycles is no less important because with their increase,
the number of lipid metabolites decreases significantly [48].

2.2. Pre-Extraction Additives

Additives used during or before extraction serve a variety of purposes. Internal standards are
a measure of extraction efficiency. In many cases, lipidomic studies of obesity are accompanied
by the determination of obesity-associated hormone levels (such as ghrelin, obestatin, glucagon,
leptin, and adiponectin); therefore, protease inhibitor cocktails are added to serum/plasma samples
to increase hormone stability [49]. In addition, various detergents serve to facilitate cell destruction
during homogenization, and buffers are used to maintain a stable pH. The most commonly added
substances to prevent oxidative processes during extraction are antioxidants and radical scavengers
such as butylated hydroxytoluene (BHT) [50,51]. This is especially important when studying unstable
compounds such as oxylipins [52–54], which are the metabolites of polyunsaturated fatty acids.

2.3. Sample Stability

It is generally assumed that lipids are highly stable at RT, while it is advisable to not allow them
to overheat during homogenization and to prevent oxidation by the addition of antioxidants [55].
Despite this, many studies consider the effects of storage conditions, the number of freeze/thaw cycles
and the behaviour of organic compounds in experimental conditions. Jiang et al. validated a liquid
chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of ceramides
(Cer) in human plasma and determined the stability of each analyte at low- and high-quality control
concentrations under long-term storage (39 days at -80 ◦ C), freeze/thawing (five times), tabletop mode
(14 h at RT before sample extraction) and autosampler conditions (3 days). The results showed that
Cer (22:0) and Cer (24:0) were stable in human plasma under all conditions [56]. Ferreiro-Vera et al.
assessed the stability of eicosanoids in serum under experimental conditions; every hour for 8 h,
they analysed samples spiked with eicosanoids, and no significant differences in analyte concentrations
were found [57]. Zeng and Cao also showed sufficient stability of short-chain FAs (SCFAs) and ketone
body derivatives during autosampler storage (5 ◦ C; 48 h), after 2 h at RT and after three freeze/thaw
cycles [58]. Klawitter et al. showed that freeze/thaw cycles and long-term storage of plasma (6 h; RT)
should be avoided to prevent changes in the composition of lipid classes of very low-density lipoprotein
Molecules 2020, 25, 5307 4 of 34

(VLDL) (loss of cholesterol esters and phospholipids), while free fatty acid (FFA) concentrations did not
change under the same conditions [59]. Oxylipins are especially unstable in this regard, and improper
collection and storage of samples can lead both to a significant decrease in their level and to an increase
in their content due to enzymatic and non-enzymatic oxidation [60]. Some oxylipins (resolvins and
prostanoids) are unstable even at −20 ◦ C [61], so the manufacturers of their standards recommend
storing them at −80 ◦ C, while the concentration of prostaglandins can significantly decrease with
prolonged storage at even −80 ◦ C [62].

2.4. Extraction Methods

2.4.1. Protein Precipitation

Protein precipitation (PPT) is used to remove protein from samples, therefore, when carrying out
PPT during sample preparation, it is important that the chosen solvent causes protein denaturation
and, at the same time, is a good solvent for lipids [55]. In addition, precipitation of proteins that make
up a large volume of the analysed matrix is necessary since some groups of lipids are present in the
matrix in trace amounts. This helps to minimize the risk of lack of detection or misidentification and to
release protein-bound compounds prior to target lipid extraction [63]. Most often, PPT is preceded by
subsequent solid-phase extraction (SPE) and liquid-liquid extraction (LLE). However, in the cases using
high-performance equipment or shotgun lipidomics, PPT alone may be sufficient. Bellissimo et al.
studied the metabolic profiles of obese individuals and used plasma acetonitrile (ACN) PPT before
liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis [64]. Jiang et al.
when developing a method for validating Cer in human plasma, optimized the PPT method using
3 organic solvents (methanol (MeOH), ACN and isopropanol (IPA)) separately and in combination with
chloroform. The mixture IPA:chloroform (9:1) was the most effective [56]. Drotleff et al. performed PPT
during lipidomics of murine plasma by adding 55 µL of IPA and 20 µL of MeOH to 25 µL of plasma [65].
Söder et al. performed liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS)-based
metabolomics analysis in overweight dogs using methanol extraction (5 µL of plasma:495 µL of MeOH),
which allowed the determination of 317 phospholipids in the plasma samples [66].

2.4.2. Liquid–Liquid Extraction

The high solubility of the hydrocarbon chains of lipids in organic solvents allows the use of
LLE for the separation of lipids in various immiscible liquids. Widely used methods such as those
of Folch [67] and Bligh and Dyer [68] have the drawback of using toxic solvents [69]; in addition,
some classes of lipids (for example, lysophospholipids (LPL)) can remain in the aqueous phase [70];
however, many proposed modifications of these methods can overcome the above disadvantages,
and these methods are still widely used in lipidomics of obesity samples [70–76]. Methyl tert-butyl
ether (MTBE) extraction, which has been popular recently, is undergoing various modifications and
shows very good efficiency over classical methods [69]. In the study of obesity, MTBE extraction is
used to isolate lipids from liver tissue [77], skeletal muscle [78], adipose tissue [79] and plasma [50,72].

2.4.3. Solid-Phase Extraction

SPE is more suitable than LLE for target lipidomics because it allows fractionation of specific
lipid classes after LLE [80,81]. Therefore, in a lipidomics study, SPE is resorted to when it is necessary
to isolate specific lipid groups or species that are present in the sample in a small amount, such as
eicosanoids [57], LPL [70], oxidized phospholipids [75], serum sterols [82] oxysterols, endocannabinoids,
and Cer [83], non-esterified FA and oxylipins [53,84–86]. Due to the wide variety of SPE protocols
and commercially available SPE columns, there are studies in which these parameters are compared,
for example, in studies of fatty acid esters of hydroxy fatty acids (FAHFAs) in serum [87] or oxylipins
in human plasma [88,89]. Additionally, SPE helps to separate lipids in complex matrices with a large
lipid abundance, such as adipose tissue [83,90] or brain tissue [80].
Molecules 2020, 25, 5307 5 of 34

Since most of the extraction protocols described below are based on the SPE and LLE methods,
the question arises—which of these protocols is more suitable for lipidomics studies. The advantages
and disadvantages of PPT, LLE and SPE have been reviewed comprehensively elsewhere [91–96] and
discussions on which of the methods is better are still ongoing. Among the main advantages of the LLE,
one can note its low costs and the presence of a large number of well-established protocols. In turn, SPE
is assumed to have a less pronounced matrix effect, there is less transfer of the aqueous phase, less toxic
solvents are used, and it is less labour and faster. However, the last 2 statements are controversial [91].
On the other hand, the advantage of SPE, such as the high selectivity can be considered a disadvantage
in the case when it is necessary to separate simultaneously several analytes with different physical
and chemical properties which would require several different SPE columns. In addition, the complex
structure of sorbents in SPE columns increases the risk of differences between individual bathes,
in contrast to LLE, which uses highly purified simple organic solvents. It can be difficult to achieve
high recovery efficiency with SPE since some analytes may partially elute in a different fraction during
separation. However, despite the complication of the process and sample preparation time, the most
comprehensive results can be obtained by combining LLE with SPE [70,79,80,83,87,97–101] or with
TLC [74,86,102–104] approaches in one study.

2.4.4. Other Extraction Methods

In addition to the well-established routine extraction methods described above, such as LLE,
SPE, and PPT, also more modern but at the same time rarer extraction methods are used in the
studies of obesity, such as solid-phase microextraction (SPME), stir bar sorptive extraction (SBSE),
dispersive liquid–liquid microextraction (DLLME) and their variants. The main disadvantage of the
above-listed solvent extraction is the use of organic solvents that have such disadvantages as toxicity
and harmfulness to the environment. In addition, they must be of high purity, which increases the cost
of analysis [105]. However, SPME, SBSE, DLLME are a solvent-free sample preparation method that is
easy to use, does not require preliminary sample preparation, and is easily automated [94].
The most widely used technique of above mentioned is SPME. In combination with gas
chromatography-mass spectrometry (GC-MS) it can be used not only for analysis of volatile organic
compounds, but also for the extraction of fatty acids and fatty acid esters from solid tissues and biofluids,
which requires a very small sample volume and reduces the matrix effect [106]. Although SPME
can be used for lipidomics studies, in obesity studies these methods are also used to study non-lipid
compounds. SPME followed by GC/MS was used to analyze aroma compound headspace release
from extra virgin olive oil after the interaction of saliva in obese and overweight individuals [107],
to evaluate volatile organic compounds of gut microbiota of obese patients [108,109], and for urinary
volatile organic compounds profiling in overweight children [110].
The SBSE method, like SPME, is a method of sample preparation without the use of solvents and
with the use of a solid sorbent for preliminary concentration of the analyte before analysis. The surface
area of the sorbing polymer is greater in SBSE than in SPME [111]. Eslami et al. used SBSE followed by
HPLC for quantification of ghrelin in human plasma [112].
The DLLME method is based on the rapid mixing of dispersing and extraction solvents with
an aqueous sample, resulting in the formation of an emulsion consisting of fine particles of the
extraction solvent dispersed in the aqueous phase, then the solvent is separated from the sample
by centrifugation [113]. Amin et al. used DLLME following GC/MS method for the evaluation of
urinary Bisphenol A in obese subjects [114]. Krawczyńska et al. applied DLLME technique for the
determination of vitamin D in obese patients plasma [115].
Thus, the relatively small number of studies in lipidomics using above methods is explained
by their recent appearance, while such advantages as relative easiness of implementation, accuracy,
small sample volume and lack of organic solvents make these extraction methods promising.
Molecules 2020, 25, 5307 6 of 34

3. Preparation of Different Sample Types

Most methods of lipid extraction from biological samples are based on the dissolution of
hydrocarbon chains in organic solvents mixed in various combinations (Folch [67] and Bligh and
Dyer [68] and modifications of their method). However, there is no unified methodology, and the
specific protocol should be selected depending on the lipid class being studied. Especially in studies in
obese subjects, when the amounts of some lipids are elevated, others are present in trace amounts and
obese subjects have some specific alterations of the lipid profile, it is best to use a method previously
tested on samples from obese subjects. Table 1 summarizing sample preparation methods for lipidomics
approaches described below is located at the end of this section.

3.1. Serum/Plasma Lipids

Plasma is the medium most responsive to changes in the body, which makes it one of the most
important sources of information. However, at the same time, a large number of metabolites in the
plasma and the fast rate of variation in their content can make it difficult to find specific markers of
diseases. Most often, in the study of obesity, an important parameter such as the fasting lipid profile,
which includes TC, HDL cholesterol, LDL cholesterol and TG, is determined for blood plasma [116].
A powerful indicator may be the content of FFAs in the blood, which reflects the influx of excess FAs
from visceral fat into the liver, which is observed in obesity [117,118]. However, advanced approaches
using lipidomics based on mass spectrometry can identify hundreds of lipid species belonging to
dozens of lipid classes in plasma [119,120] (Figure 2).

Figure 2. Lipid analysis in diagnostic laboratory vs. advanced lipidomic studies.

Blewett et al. used the modified Folch method to extract lipids from the plasma of obese rats,
after which phospholipids were separated on silica G plates; for this, they were visualized using
8-anilino-1-naphthalenesulfonic acid under ultraviolet (UV) light and compared with the corresponding
standards. The resulting phospholipid silica band was removed, and FA methyl esters (FAMEs) were
prepared for further analysis by gas chromatography (GC) [121]. Additionally, Choromańska et al.
used a combination of LLE and thin-layer chromatography (TLC) to extract lipid fractions (FFA and
triacylglycerols (TAG)) from plasma in women with morbid obesity. Lipid fractions were extracted
from 200 µL of plasma samples according to the method of Bligh and Dyer. Then, the samples
were separated by TLC on silica gel plates (Silica Plate 60, 0.25 mm; Merck, Darmstadt, Germany).
The separation was carried out in a solvent containing heptane, isopropyl and acetic acid (60:40:3;
Molecules 2020, 25, 5307 7 of 34

v/v/v) after methylation with boron in trifluoride (BF3)-MeOH. Than samples were analysed by gas
chromatography-mass spectrometry (GC-MS) [74].
Analysis of acyl-lysophosphatidic acids (LPAs) is of clinical importance in terms of prevention
of obesity [122]. Yoon et al. established a method of LPA determination in human plasma. For this,
extraction was performed with a MeOH/chloroform mixture (2:1) containing an internal standard
(LPA C14:0). After back extraction with chloroform and water, the centrifuged lower phase was
evaporated, redissolved in MeOH and analysed using ESI-MS-MS (directly injected into the ion
source) [122].
Im et al. used a modified Matyash method [123] to isolate sphingomyelins (SM) from the plasma
of men with abdominal obesity. Plasma was mixed with of 75% MeOH containing BHT and internal
lipid standards. After MTBE (1 mL) was added to the mixture, it was shaken, and water was added to
separate the phases. The mixture was then centrifuged, and the upper phase was dried and redissolved
in of chloroform/MeOH (1:9; v/v) for liquid chromatography-tandem mass spectrometry (LC–MS/MS)
analysis [50].
Wang et al. used a simple method of extraction in the study of the plasma lipidome in adults
with obesity. Plasma was mixed, sonicated and incubated with a solution of chloroform/MeOH (2:1)
containing internal standards after centrifugated and dried. The extracted lipids were resuspended in
butanol and 10 mM NH4 CHOO in MeOH. After liquid chromatography–electrospay ionization-tandem
mass spectrometry (LC–ESI-MS/MS) analysis, they quantified 328 lipid species from 24 lipid classes
and subclasses [124].
Misra et al. used untargeted metabolomics analysis of serum in high-fat and high-cholesterol
(HFHC) diet-fed baboons. Serum sample extraction was performed by sequential solvent extraction
of serum samples initially with an ACN/IPA/water (3:3:2) mixture (1 mL) and later with ACN/water
(1:1). The obtained extracts were mixed, dried and subjected to further chemical derivatization
(N-methyl-trimethylsilyl-trifluoroacetamide (MSTFA) and N-(t-butyldimethylsilyl)-N-methyltrifluoro-
acetamide (MTBSTFA) strategies), which allowed us to quantify 515 metabolites, many of which are
involved in lipid metabolism [125].
Wang et al. developed a method to perform plasma lipidomics in spontaneously obese rhesus
monkeys, based on cooling a plasma mixture with MeoH/ n-hexane with liquid nitrogen, followed
by incubation with acetyl chloride for 24 h, adding a K2 CO3 solution, and extracting the obtained
methylated FAs with hexane for further analysis using GC-MS. This method allowed the identification,
quantification and classification of 143 types of lipids [51].

3.1.1. Serum/Plasma Cholesterol

Dyslipidaemia that develops during obesity is reflected in the levels of both free cholesterol
and its individual fractions [42]. To measure indicators such as TC, cholesterol, HDL and TAG in
the blood, automated clinical systems based on enzymatic-calorimetric analysis are widely used,
while the determination of LDL was not possible until recently. Its value was calculated according to
the Friedewald formula [126]. At present, there are many methods for the direct measurement of LDL
levels with more accurate results than those obtained by calculation [127].
In the study of cholesterol metabolism in obese people, isotope methods are often used that
directly measure the fluxes of [2 H2 ]- and [U-13 C]-labelled metabolites [128], and isotope enrichment
can be estimated in lipid fractions extracted with organic solvents using the 13 C to 12 C ratio, which is
measured by gas chromatography continuous-flow isotope ratio-mass spectrometry (which is also
used for determining enrichment of [2 H2 ]) (Finnigan Incos-XL GC-MS) [129]. Cho et al. decided that
isotope-kinetic and sterile balance methods are not suitable for large-scale studies and developed
their own simple and less-expensive approach for measuring GC-MS serum sterol signatures [82].
For this, serum samples mixed with MeOH and spiked with internal standards were used for hybrid
solid-phase extraction-precipitation (H-PPT) and then eluted with MeOH three times. After drying,
the pool of eluates was derivatized, and 12 sterol signatures were measured using GC-MS [82].
Molecules 2020, 25, 5307 8 of 34

Recently, studies of fractions and subfractions of cholesterol have been carried out on the basis
of size using specialized systems based on gel chromatography. In the work of Lindqvist et al.
analysis of lipoprotein fractions during animal bariatric surgery modelling was performed using
the Quantimetrix Lipoprint® system (Quantimetrix Corporation, Redondo Beach, CA, USA) [130].
Additionally, Kwon et al. in studies of low-density lipoprotein subfractions in overweight and obese
women, also used this method to scan LDL subfractions. This system allowed them to divide LDL into
seven subfractions [131]. Doğan et al. also used this system in LDL and HDL subfraction analysis in
patients after laparoscopic sleeve gastrectomy [132].

3.1.2. Triglyceride Profiling

The study of blood TG levels is of great interest in the study of obesity because their level
quickly responds to dietary changes [133,134] and is associated with insulin resistance [135,136],
lipotoxicity [137] and dyslipidaemia [138]. Despite this, studies of the profile of individual FAs in plasma
TG are quite rare; this parameter is usually tested in adipose tissue, where TG accumulates [71,139],
or in breast milk [140]. Usually, the isolated plasma TAG fraction is examined as one of the components
in the study of the whole lipid profile [74,128,141].
Perreault et al. in a study of the inflammatory state of metabolically healthy obese individuals,
determined profiles of FAs from serum samples. They measured total FAs and fractionated FAs in
PL and TAG fractions. In both cases, isolated lipid fractions were obtained by incubation of samples
(45 min) on Silica-G TLC plates (Analtech, Newark, NJ, USA) with petroleum ether, ethyl ether and
acetic acid (80:20:1; v/v/v). After that, both the collected PL and TAG lipid bands and the samples
for the analysis of total FA were methylated for analysis on a GC with a flame ionization detector
(FID) [102].
Much work has been done by Klawitter et al. who compared 4 different approaches for
determining the plasma fatty acid desaturation index in response to a carbohydrate-rich diet.
Using ultracentrifugation, TLC, SPE, and saponification followed by ultra-high-performance liquid
chromatography (UHPLC)-MS for specific fatty acid analysis, they concluded that analysis of specific
FAs in the VLDL fraction best reflects the activity of the stearoyl-CoA desaturase 1 (SCD1) metabolic
pathway [59]. One of the approaches of UHPLC neutral loss MS was focused on analysing the fatty
acid composition of TAG directly from plasma extracts or from plasma VLDL extracts without prior
fractionation [59].

3.1.3. Fatty Acid Profiling

The FA profile is a rich source of information on dietary lipid intake and changes associated
with obesity. Changes in the FA blood profile affect the production and secretion of cytokines,
chemokines and eicosanoids, which can initiate inflammation of the whole body [102]. FFAs released
into the blood from adipose tissue during TAG lipolysis may act as a marker of FA adipose tissue
composition [142,143].
In our earlier study, we used high-performance liquid chromatography with a laser light scattering
detector (HPLC-LLSD) to separate lipid extracts obtained by the Folch method during the identification
of cyclopropane FA in the serum of obese people. The resulting FFA, TAG and PL were hydrolysed
with 0.5 M KOH in MeOH. After neutralization with 6 M HCl and addition of water (1 mL), triple
extraction of FA with n-hexane was performed, and the samples were dried again. Thereafter, FAMEs
and picolinyl esters of FA were obtained from FFA and FA from the hydrolysis of complex lipids [144].
This method of extraction and hydrolysis followed by FAME synthesis can also be used to determine
the levels of odd-chain fatty acids (OCFAs) and branched-chain fatty acids (BCFAs). The study of
OCFAs and BCFAs is of interest since changes in these FAs in serum may be associated with insulin
resistance in obese patients [145,146].
Kang et al. profiled FAs in the plasma of overweight subjects using a modified version of the
Bondia-Pons method [147] to prepare samples for GC analysis. For this, n-hexane containing internal
Molecules 2020, 25, 5307 9 of 34

standards was added to plasma samples and then mixed with MeOH, and acetyl chloride was slowly
added. After incubation 6% potassium carbonate was added to each tube, and after centrifugation, a
transparent top layer of n-hexane with FAMEs was taken for GC-MS analysis [148]. Later, Lee et al.
used this method to determine circulating fatty acid profiles in overweight individuals [149].
Wijayatunga et al. used improved direct synthesis of fatty acid methyl ester to measure serum
FAs in patients after bariatric surgery. For this, serum was mixed with MeOH containing internal
standard, water solution of 10 N KOH and MeOH. After incubation and shaking, samples were cooled,
and 24 N H2 SO4 in water was added and shaken. After that, the obtained FAMEs were extracted with
n-hexane for subsequent analysis by GC-MS [7].
Aslan et al. studied changes in plasma polyunsaturated FAs (PUFAs) after bariatric surgery.
For this, plasma was mixed with an internal standard solution (arachidonic acid (AA)-d8). After a
mixture of ACN/37% hydrochloric acid (4:1; v/v) was added, the samples were hydrolysed, after which
extraction was carried out with n-hexane. The upper phase, containing FFA, was dried, dissolved in
MeOH-water (180:20; v/v) and filtered for LC-MS/MS analysis [150]. Later, Badoud et al. used this
method to determine the profile of FAs in obese individuals. In both works, the authors were able to
isolate, extract and quantify 28 FAs [151].
Ma et al. analysed serum FFA profiles to examine the relationship between FFA and the metabolic
phenotype of obesity of two ethic groups in China. For this, a sample was mixed with ACN and
incubated. After centrifugation, derivatives and 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide were
added to improve detection sensitivity, degree of separation, and binding efficiency, which allowed
them to quantify 34 types of FFAs in serum [152].
Itariu et al. determined the profiles of FA in plasma phospholipids in severely obese nondiabetic
patients. The main lipids were separated by TLC on silica gel plates (Merck) using n-hexane/diethyl
ether/acetic acid (80:30:1; v/v) and BHT as the mobile phase and 11,2-dioleoyl-sn-glycero-
3-phosphocholine (as the standard). After a solution (100 mg of berberine chloride in 100 mL
of ethanol) was sprayed, lipid spots were visualized in ultraviolet light. Phospholipids were scraped
into glass tubes, followed by methanolysis with a solution of MeOH/toluene (4:1). After 6% K2 CO3
was added, the organic phase was collected and analysed by GC-MS [86].
Given the effect of high plasma NEFA levels on insulin resistance and insulin secretion, Nemati et al.
studied changes in NEFA in patients with type 2 diabetes after bariatric surgery [153]. Dole extraction
was performed to separate NEFAs. For this, serum was mixed with an internal standard (heptadecanoic
acid in IPA and a modified Dole’s mixture containing IPA/n-heptane/phosphoric acid (2 mol/L; 40/10/1;
v/v/v). After incubation, n-heptane and water were added, and the samples were mixed and centrifuged.
Then, the upper organic layer was dried and dissolved in IPA for analysis by LC-MS, which quantified
5 NEFAs (palmitic, stearic, oleic, palmitoleic and linoleic acid) [153].
Lin et al. described a procedure for FFA analysis in serum samples of patients after bariatric
surgery in which a blood serum sample was evaporated in a nitrogen atmosphere and then addedd
triheptadecanoin as an internal standard. After drying, the samples were subjected to direct
esterification by dry 2.5 M HCl in methanol. The obtained FAMEs were extracted twice with
isooctane, and a total of 16 FAs were determined using a GC instrument equipped with a FID [154].
Ramos-Molina et al. used two specific lipid extraction protocols to obtain the serum lipidome in
obese subjects after bariatric surgery. Two separate ultrahigh-performance liquid chromatography
(UPLC)-MS-based platforms analysing MeOH and chloroform/MeOH serum extracts were combined.
In the first protocol, PPT was carried out by adding MeOH to serum. After vortexing and incubation,
the supernatants of the samples were centrifuged, dried and restored in MeOH to determine FAs,
bile acids (BA), steroids and LPL. A second protocol was used to determine glycerolipids, cholesterol
esters, sphingolipids and phospholipids. For this, serum extracts were mixed with sodium chloride
(50 mM) and chloroform/MeOH (2:1). After stirring and incubation, the samples were centrifuged, and
the organic phase was collected, dried and restored in ACN/IPA (1: 1). Both extraction protocols used
Molecules 2020, 25, 5307 10 of 34

internal standards for each class of lipids. After that, two separate UPLC-MS-based platforms were
used for analysis, allowing the identification of nearly 300 lipids present in human plasma [155].
Recently, new studies on FAHFAs that are related to diabetes and obesity have
appeared [73,79,87,156]. Based on the fact that low concentrations of FAHFA are present in blood
serum, López-Bascón et al. developed an automated online SPE LC-MS/MS method for sensitive
and selective analysis of FAHFA [87]. Optimization of the SPE protocol included tests with four
commercial sorbents (C8, C18, C18HD and Resin SH) packed with the same technology and based on
nonpolar interactions. After testing the retention/elution ability of these sorbents using the generic
reversed-phase protocol, the greatest retention/elution of FAHFAs was exhibited by C8. This sorbent
was used in further optimization of the protocol, which involved varying the composition, volume
and flow rate of the tested solvents; the gradient and the elution time [87].
Short-chain fatty acids (SCFAs) are saturated aliphatic FAs with fewer than six carbon atoms that
can be produced by the anaerobic intestinal microbiota or by catabolism of branched-chain amino
acids. The important role that SCFAs play in homeostasis and data indicating the association of
SCFAs with multiple metabolic diseases make them an important object of research [58,157,158].
Other research methods targeting SCFAs are commonly based on extraction with a complex
derivatization procedure for subsequent GC–MS analysis [158–160]. Zeng and Cao developed
a novel LC-MS/MS method using fast derivatization with O-benzylhydroxylamine (O-BHA) and
N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDC) in combination with LLE
with dichloromethane for detecting SCFAs in the serum of obese and lean mice [58].
When describing FA profiling, it is important to mention such important metabolites of PUFAs
such as oxylipins and endocannabinoids. It has long been known that endocannabinoids are key
components of systems that regulate both nutrition and body mass, and oxylipins are also closely
associated with obesity due to the wide range of their biological effects [53,161]. In our previous works,
the features of sample preparation and methods for the analysis of oxylipins and endocannabinoids
in biological samples are described in sufficient detail [60,162]. Compared to other classes of lipids,
the analysis of oxylipins can be complicated by their low content in samples, susceptibility to oxidation,
and ability to be synthesized de novo during sample preparation and extraction, all of which should
be taken into account in the analysis of oxylipins.
Astarita et al. proposed an SPE method to fractionate lipid classes in which in one part of the
organic phase after solvent extraction, high-abundance positively charged lipids were analysed by
LC/MS. In the second part, the lipids were fractionated according to their relative polarities by serial
elution with chloroform/MeOH (9:1 and 1:1; v/v) mixtures on open-bed silica gel columns (silica gel
60 230–400 mesh). Moreover, for further chromatographic separation, two different octadecyl (C18)
columns were used [99]. Later, Argueta et al. used this protocol for the extraction of endocannabinoids
from the plasma and jejunum of mice with western diet-induced obesity and their analysis by
UPLC-MS/MS [100]. Additionally, Perez et al. used the method of Astarita et al. when studying the
endocannabinoid system in plasma, pancreas and jejunum by UPLC-MS/MS in offspring obtained
from obese mice fed a Western diet during pregnancy [101].
Ferreiro-Vera et al. developed a fast automatic method for quantitative analysis of oxylipins in
serum samples from obese individuals. The approach is based on online SPE–LC–MS/MS method in
which a sequence of automatic operations was performed on injected human serum. Further steps
included rinsing the cartridge with MeOH, conditioning with water, loading sample into the cartridge
with water and, after washing with 20% MeOH in water, switching a valve to elute through the
cartridge into the chromatographic column using a mobile phase containing MeOH/water/ACN/acetic
acid (76:22:2:0.02; v/v) [57].
Pickens et al. studied plasma lipid FA profiles in subjects with various BMIs and performed
nonesterified plasma PUFA and oxylipins extraction and isolation. Oxilipids were isolated on SPE
Phenomenex Strata-X columns (60 mg/3 mL, Phenomenex, Torrance, CA, USA) [163]. Later, Pickens et al.
used this method for the extraction and isolation of nonesterified PUFAs and oxylipins [53].
Molecules 2020, 25, 5307 11 of 34

Hernandez-Carretero et al. investigated changes in oxylipins, endocannabinoids, and Cer in

mouse plasma after weight loss using a 96-well Ostro™ Pass Through Sample Preparation Plate
(Waters Corp, Milford, MA, USA) to remove proteins and phospholipids. [52].
Azar et al. performed LC-MS/MS analysis of endocannabinoids in the serum of patients undergoing
bariatric surgery. For this, after PPT with a mixture of acetones and Tris buffer, serum samples were
homogenized in a mixture of MeOH and Tris buffer with the addition of an internal standard.
The resulting homogenates were extracted with a mixture of chloroform/MeOH (2:1; v/v), washed three
times with chloroform, dried and redissolved with MeOH [164].
Fan et al. investigated the production of unesterified oxylipins and endocannabinoids in mice
fed a high-fat diet. To extract oxygenated PUFA metabolites, plasma samples were mixed with
antioxidants (BHT/EDTA in MeOH:H2 O; 1:1) and deuterated surrogates in MeOH. Then, the proteins
were precipitated by adding a mixture of MeOH/ACN (1:1). After centrifugation, the supernatants
were filtered and quantified using UPLC-MS/MS] [54].

3.1.4. Ceramides and Sphingolipids

Sphingolipids are one of the most complex and structurally diverse families of compounds.
Sphingolipids are not only components of biological structures such as membranes and lipoproteins
but also highly biologically active compounds that affect dozens of biological processes [165]. Ceramides
are sphingolipids that promote insulin resistance and are associated with the distribution of body fat,
obesity, and type 2 diabetes [74]. Brozinick et al. used the method of single-phase extraction with
MeOH-dichloromethane for the quantitative determination of seven types of Cer in rhesus macaque
plasma on a Western-style diet [166].
León-Aguilar et al. used the method described by Croyal et al. [167] to quantify plasma Cer in
offspring born to obese women. The method consisted of preparing seven standard dilutions (1, 5, 10,
50, 100, 250, and 500 nM) of 10 types of Cer in MeOH. Then, standard solutions and plasma samples
(25 µL) were extracted using the Bligh and Dyer method with the addition of Cer (d18:1/17:0) as an
internal standard for further UPLC-MS/MS analysis [76]. Neeland et al. when studying the role of Cer
in the development of insulin resistance, used a method based on comparing total acyl carbon content
and degrees of saturation between samples and a deuterated internal standard Cer (d18: 1) to identify
13 different types of Cer in plasma by UPLC-MS [168].
Özer et al. were able to quantify five Cer and three SM using successive chloroform/MeOH and
chloroform/water extractions before ultrafast liquid chromatography (UFLC)-MS/MS analysis when
studying changes in serum SM and Cer after bariatric surgery [169].

3.2. Adipose Tissue

In the study of obesity, adipose tissue is more useful than just its basic physiological functions
(storage and release of fat); adipose tissue plays an important role in insulin sensitivity and is the site
of the synthesis of many hormones and other signaling molecules associated with obesity [71,170,171].
Moreover, adipose tissue is relatively easily available as a research material and can be obtained during
bariatric surgery. Most lipid extractions are carried out at RT, which is associated with poor lipid
solubility in cold conditions; however, overheating can occur (during homogenization or sonication)
and lead to the release of acyl FAs and the formation of lysolipid species [55]. For these reasons
(in addition to reducing oxidation), antioxidants (BHT) and metal chelators (EDTA) are added to
samples [52,83,103].
Roberts et al. described several approaches based on Folch extraction from white adipose tissue.
The described methods, including GC–MS analysis of total fatty acid composition and intact lipid and
acylcarnitine profiling by LC–MS, make it possible to obtain wide lipid profiles. At the same time,
the authors suggest using SPE with LC–MS/MS analysis for eicosanoids from adipose tissue [90].
Kunešová et al. studied the fatty acid composition of TG in adipose tissue after weight loss.
For this, the lipid fraction was extracted from a fat cake obtained during the extraction of RNA and
Molecules 2020, 25, 5307 12 of 34

then transmethylated to FAMEs (1 M sodium methoxide; RT; 60 min). After neutralization with 1 M
acetic acid, the FAMEs were extracted twice with n-hexane, passed through a column (5 × 20 mm) of
anhydrous sodium sulfate, mixed together and dried for further analysis on a GC instrument equipped
with an FID [172]. Later, Montastier et al. also used this method (lipid fraction extraction from fat cakes
produced during RNA extraction) for the analysis of FAs in the adipose tissue of obese women [173].
Hu et al. used a three-phase MTBE/MeOH/water extraction procedure for quantification of FAHFAs
in a WAT sample of hamsters fed an HFD. In addition, the samples were subjected to additional
removal neutral lipids by subsequent SPE, which allowed the identification of 64 FAHFAs [79].
Okada et al. measured the concentrations of lipid mediators in adipose tissue of obese mice.
For this, the samples were extracted using an automated system (RapidTrace Biotage). Samples were
pumped onto C18 cartridges, washed with water, n-hexane was added, and oxylipins were eluted with
methyl formate [85].
Similar to plasma, when the levels of oxylipins are being examined in adipose tissue, additional
purification with SPE is usually required. For example, Itariu et al. when studying chronic inflammation
of adipose tissue in patients with severe obesity, used SPE (Oasis HLB Extraction Cartridge; Waters) to
extract lipid mediators, which was followed by LC-MS [86].
Simple LLE extraction without SPE can also be quite effective in lipidomics. Therefore,
Al-Sulaiti et al. successfully used the Bligh and Dyer method to extract lipids for further nontarget
LC-MS determination of 76 TAG species from subcutaneous adipose tissue and omental depots from
obese individuals [71]. Additionally, using a two-step chloroform/MeOH extraction, Grzybek et al.
identified more than 300 lipids in different AT types of mice fed a high-fat diet (a rodent model of
obesity) [174]. It is interesting that although all stages of fluid processing were performed using the
Hamilton Robotics STARlet robotic platform with the Anti Droplet Control function, the authors
encountered difficulties pipetting reproducible amounts of homogenized tissues due to the presence of
large amounts of fat droplets when using an aqueous buffer. The problem was solved by homogenization
of AT in 50 vol.% ethanol, followed by dilution in pure ethanol [174].
Tomášová et al. described their modification of the Folch lipid extraction method, which solved
the problem of the high content of TAG in adipose tissue. They used TLC for separating lipid classes
before LC-MS analysis, which allowed them to detect 37 lipids that were below the detection limit
without TLC [103]. Similarly, Choromańska et al. applied the previously described method (Bligh and
Dyer LLE with TLC on silica gel plates) for TAG, CER and DAG separation in adipose tissue of women
with morbid obesity [74].
Depending on the studied lipid class and the selected extraction method, an appropriate organic
solvent/mixture of solvents is added during or after homogenization. Mutemberezi et al. used
dichloromethane/MeOH/water (8:4:2) extraction with subsequent SPE in LC-MS analysis of oxysterols,
Cer, and endocannabinoids involved in obesity and metabolic syndrome [83]. The authors paid special
attention to the elimination of the cholesterol oxidation during extraction. To minimize this process, they
chose solvents with low prooxidant properties, and to remove cholesterol from the sample, they created
an SPE procedure where three different solvent mixtures, n-hexane/IPA (99:1; v/v), n-hexane-diethylether
(90:10; v/v) and n-hexane-dichloromethane (80:20; v/v), were tested. Based on their ability to selectively
elute cholesterol and their tendency to oxidize cholesterol (5α,6α-epoxycholesterol,27-oxysterol),
they chose the n-hexane-IPA mixture as the best for this purpose [83].
Serbulea et al. developed a targeted LC-MS approach to analyse oxidized phospholipids (OxPL) in
lean and obese mice. OxPL was extracted by a modified Bligh and Dyer method. The method consisted
in the fact that the aqueous phase remaining after the first extraction with a chloroform/MeOH mixture
was extracted with chloroform, and the organic layer of the second extraction was combined with the
first, dried, and resuspended in 300 µL of the mobile phase for further LC-MS analysis. In addition,
phospholipids were separated on an EVO C18 column (Kinetex 5 µm; 100 × 4.6 mm; Phenomenex)
using a binary gradient [75].
Molecules 2020, 25, 5307 13 of 34

Hanzu et al. when studied visceral (VIS) and subcutaneous (SC) adipose tissue from obese subjects,
examined not the tissue itself but the adipose tissue-conditioned medium samples, which were obtained
by 24-h incubation of intact fat pads in serum-free medium, and further annotation and identification
of metabolites was performed using GC-MS [175].

3.3. Liver
Although in the study of obesity, the liver is a pivotal tissue associated with lipid metabolism,
similar to adipose tissue, there are few studies describing the extraction and identification of lipids
in the liver. Undoubtedly, it is associated with difficulties in obtaining samples from this organ from
humans. Most often, liver samples from rodent models of obesity are used. In various studies based
on animal model liver tissue, lipids such as oxysterols, endocannabinoids, Cer [83], FAHFAs [79],
SM, Cer [176], and glucosylceramides [177] have been studied. As mentioned above, Fan et al. used
simple MeOH extraction for the analysis of oxylipins and endocannabinoids from the livers of mice
fed a HFD [54].
Pakiet et al. studied the effect of a western diet on mouse brain lipid composition, but they
also examined the liver; after a Folch extraction, they used two SPE procedures to separate lipid
species [80]. In the first procedure, various eluents were used during SPE with chloroform/IPA (2:1;
v/v) that allowed neutral lipids (NL) to be obtained; diethyl ether with 2% acetic acid (v/v) was used to
elute FFAs, and MeOH was used to extract PLs. After that, neutral lipids were dissolved in n-hexane,
fractionated on a new SPE cartridge and eluted, and cholesterol esters (with 6 mL n-hexane) were
discarded. TAG (elution with methylene chloride/diethyl ether/n-hexane; 10:1:89; v/v/v), cholesterol
(elution with 5% ethyl acetate in n-hexane (v/v), diacylglycerols (DAG) (elution with 15% ethyl acetate in
n-hexane (v/v) and monoacylglycerols (MAG) (elution with chloroform-MeOH (2:1; v/v)) were obtained.
After that, the MAG, DAG and TAG fractions were mixed together into a mixture of acylglycerols [80].
Using the second method, from the lipid extracts recovered in chloroform, the following were obtained
by extraction: NL (15% ethyl acetate in n-hexane (v/v) was eluted), Cer (chloroform/MeOH; 23:1,
v/v), FFA and α-hydroxy-FFA (5% acetic acid in diisopropyl ether (v/v)), glycosphingolipids (GSPL)
(acetone-MeOH (9:1.35; v/v), and SM (chloroform/MeOH (2:1; v/v)). After hydrolysis of all collected
lipid fractions and derivatization, the obtained FAMEs were determined using GC-MS [80].
Lytle et al. used chloroform/MeOH (2:1) extraction followed by TLC to fractionate total lipids
in a study of steatohepatitis in obese mice; after that, the lipids were saponified, and the FAs were
extracted with n-hexane and identified using RP-HPLC with a UV detector. Additionally, for some
other types of lipids from saponified FAs, FAMEs were prepared, which were extracted in n-hexane for
subsequent quantification by GC with an FID [104].
Du et al. studied the hepatic expression of sirtuin 5 (SIRT5) in obese mice and determined the
liver TAG content. For this, Soxhlet extraction of liver lipids using diethyl ether as a solvent was
performed; in addition, to study changes in the size and number of lipid droplets (LD) in hepatocytes,
neutral lipids were stained with probe-lipid TOX Green, and haematoxylin and eosin staining was
performed [178].
Yetukuri et al. performed lipid profiling in the liver in obese mice with hepatic steatosis. For this,
20–30 mg tissue sample was subjected to chloroform/MeOH extraction with an internal standards
mixture (diacylglycerophosphocholine (GPCho) (17:0/17:0), diacylglyceroethanolamine (17:0/17:0),
GPCho (17:0/0:0), Cer (d18:1/17:0), and TG (17:0/17:0/17:0)) after which to the separated lower phase,
10 µL of a labelled standard mixture was added, and the sample was analysed by UPLC-MS [179].
Wang et al. when analysing obese mouse liver by shotgun lipidomics, developed an approach that
solves the problem of analysing lipids such as LPL that remain in the aqueous phase and are usually
discarded after the classical extraction procedure of Bligh and Dyer (because they cannot be directly
used for MS analysis due to a high salt content). The method is based on the purification of the aqueous
phase solution remaining after LLE extraction. For this purpose, the aqueous phase was loaded onto a
Hybrid SPE cartridge that had been twice washed with MeOH; next, lysophosphatidylinositol (LPI),
Molecules 2020, 25, 5307 14 of 34

lysophosphatidylserine (LPS) and lysophosphatidylglycerol (LPG) species were eluted with a 10%
solution of MeOH in ammonia, and the LPAs were eluted with a solution of 20% ammonia in MeOH.
Furthermore, SPE eluents were dried and reduced in MeOH prior to analysis by MS [70].
One of the most comprehensive approaches to liver lipidomics was shown by Garcia-Yaramillo
et al. when studying Western diet-induced nonalcoholic steatohepatitis in mice. They used GC
analysis of free and saponified FAs converted to FAMES to quantify SFA, MUFA, and ω3, and ω6
PUFA. For quantification of DAG, TAG, PC, phosphatidylserines (PS), phosphatidylinositols (PI),
phosphatidylglycerols, PE, LPL and SM, they used an untargeted LC/MS approach. For this, extraction
was performed with methylene chloride/IPA/MeOH (25:10:65; v/v/v; -20 ◦ C). In addition, they quantified
ω3 and ω6 PUFA and PUFA-derived oxylipins using targeted LC/MS. For this, MeOH extraction
with additional SPE on Strata-X columns was used. Both targeted and nontargeted analyses were
performed on the same UHPLC system (a Shimadzu Nexera system coupled to a triple time-of-flight
(TOF) 5600 mass spectrometer) and the same column (Waters Acquity (UPLC); CSH C18) [98].
In their studies of obesity, Preuss et al. drew attention to the composition of LDs in various tissues,
including the liver. To separate LDs from the cytosolic and membrane fractions in homogenates,
the authors used 1 h of centrifugation (100,000× g; 4 ◦ C), after which LDs formed a clear top layer.
Then, lipids from LDs were extracted according to the Folch method. The authors focused on the use
of a Centri-Tube slicer (Beckman Coulter, Brea, CA, USA) that increases the purity of the collected
LD fraction. Subsequent SPE (Sep Pak Diol Cartridges; Waters, MA, USA) followed by LC-MS/MS
analysis allows the determination of diacylglycerols and Cer in all cell fractions [180]. The authors
noted that compared with other tissues, the liver had a high level of lipids; therefore, 10 mL of buffer
was used to homogenize 20 mg of liver tissue, whereas in the case of muscle and cardiac tissue, 50 mg
was homogenized in 500 µL of buffer [180].

3.4. Brain
Due to the difficulties of collecting material for research in humans, the study of lipid metabolism
in the brain tissue most often occurs with material obtained from animals with obesity induced by a
high-energy diet. In addition to difficulties with biopsy, in cases of animals with large brains as in
humans, additional difficulties arise due to the heterogeneity of cell populations in grey and white
matter, which can lead to different ratios of cell populations in samples [181].
Yang et al. in a study of circulating Cer in HFD-fed mice, used the methodology of Bielawski et al. [182].
After homogenization of frozen tissues with a buffer (0.25 M sucrose, 25 mM KCl, 50 mM Tris, and 0.5 mM
EDTA, pH 7.4) in a 1:10 (w/v) ratio, homogenate was filtered through layers of gauze. After that, tissue
homogenates were spiked with IS solution and extracted twice with IPA/water/ethyl acetate (30:10:60;
v/v/v) mixture with subsequent vortexing, sonication and centrifugation. The combined upper layers
were dried and reconstituted in the mobile phase for LC-MS analysis [183]. Later, Gao et al. used this
approach when exploring ceramide metabolism in the hypothalamus of mice in a model of leptin
hypothalamic control of feeding [184].
Rutkowsky et al. performed metabolic analysis of complex lipids in the brains of mice fed
a western diet. For this, they used the Matyash protocol [123] based on methyl tert-butyl ether
(MTBE) extraction. [185]. Additionally, LLE protocol with MeOH/ethyl acetate in this work was used,
for analysis of non-esterified oxylipins and endocannabinoids in the brain was carried out [185].
Rawish et al. performed hypothalamic lipid analyses in mice fed a high-fat diet. For this,
they combined several approaches, including using fluorescence microscopy to quantify neutral lipids
in the LD hypothalamus (staining LD with lipophilic dye (LD540)) and studying the metabolism of FAs
in slices of the hypothalamus using the tracer alkyne oleate; lipidomics analysis of the hypothalamus
(analysis of neutral glycerolipids, phosphoglycerol, sphingolipids and acyl-carnitine lipids) was also
performed by LC-MS [186].
Kirkham et al. studied the effects of diet on endocannabinoid levels in the rat forebrain and
hypothalamus using 5 v of chloroform/MeOH/50 mM Tris HCl (2:1:1) for endocannabinoid extraction
Molecules 2020, 25, 5307 15 of 34

from tissue homogenates. After consequent double extraction with 1 v of chloroform, organic phases
were pooled, dried and resuspended in chloroform/MeOH (99:1; v/v) for GC-MS analysis, but before this,
the obtained solutions were purified by open bed chromatography on silica and further fractionated
by normal-phase high-pressure liquid chromatography (NP-HPLC) on a silica column (Spherisorb
S5W; Phase Sep, Queensferry, Clwyd, UK) using a 40 min linear gradient [161].

3.5. Skeletal Muscle

Gudbrandsen et al. analysed lipid metabolism in rats after bariatric surgery and extracted FAs
from skeletal muscle. They used the Bligh and Dyer method with the addition of heneicosanoic acid as
an internal standard. Then, the extracts were methylated in anhydrous MeOH containing 2.5 M HCl
(100 ◦ C; 2 h) and extracted twice with isooctane, and methyl esters were quantified using GC with an
FID [187].
The most commonly studied classes of muscle lipids in obesity are TAG and CER since
the accumulation of these lipids in muscle tissue is associated with the development of IR [188].
The aforementioned Preuss et al. also examined muscles when studying the composition of LDs and
determined DAG and Cer in cell fractions [180].
Van Hees et al. when checking the “lipid overflow” hypothesis, did not limit their analysis to
TAG content in muscles during the study of IR because they also divided the total amount of lipids
obtained after extraction by chloroform-MeOH into FFA, DAG, TAG and PL by TLC before further
GC-MS analysis [129].
Laurentius et al. developed a new method to identify and classify FAMEs by GC-MS in HFD-fed
rats. At the initial stage, CE, TAG and GPL fractions were extracted from muscle homogenates using
tert-butyl methyl ether (90%, tert-BME) and MeOH, and the FFA fraction was extracted using a
chloroform/MeOH (2:1) mixture. In the further separation of lipid classes, they used SPE during which
they sequentially eluted the CE fraction (elution 1% methyl acetate/n-hexane; v/v) and TAG fraction
(elution of 2.5% methyl acetate/n-hexane; v/v), and after the column was washed with acetone, the GPL
fraction was eluted with MeOH. FFA were extracted with 80% n-hexane/diethyl ether (v/v) on separate
SPE columns, followed by incubation with 1% sulfuric acid in MeOH, addition of 5% sodium chloride,
and three washes of the n-hexane layer with water [97].
One of the latest approaches developed by Eum et al. allows the identification of hundreds of
individual lipid species in the muscle tissue of mice with HFD [78]. A two-stage extraction method
was performed using MTBE for the first extraction and subsequent secondary extraction of lipids
from the lower aqueous layer with MeOH. After mixing, the organic layers were dried and reduced
with a mixture of chloroform/MeOH (1:9; v/v) for subsequent nanoflow ultrahigh-performance liquid
chromatography with tandem mass spectrometry (nUHPLC-ESI-MS/MS) analysis [78].

3.6. Heart
The study of lipid metabolism in heart tissue is of interest primarily due to the strong influences of
obesity and a high-fat diet, particularly the influences on the phospholipid composition of mitochondrial
membranes of cardiomyocytes [189]. Due to the unique lipid composition of mitochondrial membranes,
they are very sensitive to high levels of FAs and their oxidation products in the blood [189].
Harmancey et al. when studying changes in the composition of cardiac acyl-CoA in obese rats,
used the Bligh and Dyer method to extract cardiac lipids from heart tissue; then, extraction was
repeated three times to complete lipid recovery for further quantification of Cer and diacylglycerols by
HPLC-UV [190]. When the cardiac ceramide content in rats fed a high-fat diet was studied, the Bligh
and Dyer method was also used to extract total cardiac lipids [191]. Later, they used the extraction
method described by Merrill et al. [165], which is based on extraction with a mixture of MeOH
and chloroform, followed by sonication (48 ◦ C; overnight), which is necessary for the extraction of
sphingolipids, followed by incubation (37 ◦ C; 1 h) to remove interfering glycerolipids (in particular
PC). After another extraction with chloroform, ceramide species were detected by LC-MS [165].
Molecules 2020, 25, 5307 16 of 34

Table 1. Sample preparation methods for lipidomics approaches used in the studies of obesity.

Sample Preparation Method Analysis

Lipid Class(es) Matrix References
Pre-Preparation Extraction Method Derivatization Step Method
modified Folch sodium
PL Plasma - GC Blewett et al. [121]
method methoxide—FAME
Bligh and Dyer’s 14% BF3 - MeOH - Choromańska et al.
FFA, TAG Plasma - GC-MS
method FAME [74]
hydrochloric acid +
LPAs Plasma - MeOH/chloroform - ESI-MS –MS Yoon et al. [122]
(2:1)
modified Matyash
SM Plasma MeOH - LC-MS/MS Im et al. [50]
method
lipidomic profile (328 lipid species from
24 lipid classes: dhCer, Cer. MHC, DHC,
chloroform/MeOH
THC, GM3, SM, PC, PC(0), PC(P), LPC, Plasma MeOH - LC ESI-MS/MS Wang et al. [124]
(2:1)
PE, PE(0), PE(P), LPE, PI, LPI, PS, PG,
CE, COH, DG, TG)
untargeted metabolomics analysis ACN: isopropanol:
Serum - MSTFA + MTBSTFA 2D GC-ToF-MS Misra et al. [125]
/lipidomic profile? (515 metabolites) water (3:3:2)
lipidomic profile (143 lipid species from
acetyl chloride + 6%
lipid classes: FA, FFA, PC, PE, PI, PS, PG, Plasma - MeOH/n-hexane (4:1) GC- FID/MS Wang et al. [51]
K2 CO3
LPC, LPA, SM)
solid-phase extraction MSTFA/ammonium
(hybrid solid-phase iodide (NH 4
sterols Serum MeOH GC-MS Cho et al. [82]
extraction-precipitation I)/dithioerythritol
(H-PPT) cartridge) (DTE) (500:4:2)
chloroform/MeOH methylation (100 ◦ C;
total FAs + circulating PL, TG Serum - GC- FID Perreault et al. [102]
(2:1) 1.5 h)
FFA, TAG, PL Serum - Folch method 10% BF3 - MeOH GC–MS Śledziński et al. [144]
acetyl chloride + 6%
SFA, MUFAs, PUFAs Plasma - MeOH GC-MS Kang et al. [148]
K2 CO3
Molecules 2020, 25, 5307 17 of 34

Table 1. Cont.

Sample Preparation Method Analysis

Lipid Class(es) Matrix References
Pre-Preparation Extraction Method Derivatization Step Method
10M KOH in MeOH + 24 N
MCFAs, NEFAs Serum MeOH - GC-MS Wijayatunga et al. [7]
H2SO4
ACN/37%
PUFAs Plasma hydrochloric acid n-hexane - LC-MS/MS Aslan et al. [150]
(4:1)
chloroform/MeOH
MUFAs, PUFAs, OCFAs Serum ACN - UHPLC-MS Ma et al. [152]
(2:1)
PUFAs Plasma MeOH - acetyl chloride + 6% K2 CO3 LC-MS Itariu et al. [86]
NEFAs Plasma - Dole extraction - LC-MS Nemati et al. [153]
chloroform/MeOH
MUFAs, PUFAs, SFA Serum - HCl in MeOH GLC-FID Lin et al. [154]
(2:1)
For glycerolipids,
For FAs, bile cholesterol esters,
FAs, bile acids (BA), steroids,
acids (BA), sphingolipids and Ramos-Molina et al.
LPL, glycerolipids, cholesterol Serum - UPLC-MS
steroids and LPL phospholipids - NaCl [155]
esters, SPL, PL
- MeOH + chloroform/MeOH
(2:1)
chloroform/MeOH Ramos-Molina et al.
Serum - - UHPLC-MS
(2:1) [155]
solid-phase extraction
López-Bascón et al.
FAHFAs Serum MeOH (hysphere C8 - LC-MS/MS
[87]
cartridges)
0.1 M O-benzylhydroxylamine
(O-BHA) in MeOH and 0.25 M
SCFAs Plasma MeOH dichloromethane N-(3-dimethylaminopropyl)- LC-MS/MS Zeng and Cao [58]
N0 -ethylcarbodiimide
hydrochloride (EDC) in MeOH
Molecules 2020, 25, 5307 18 of 34

Table 1. Cont.

Sample Preparation Method Analysis

Lipid Class(es) Matrix References
Pre-Preparation Extraction Method Derivatization Step Method
MAG, FAE, oxFAE, oxMAG, FA, oxFA,
Plasma - Chloroform - LC/MS Astarita et al. [99]
DAG, TAG, PE, PI, NAPE, LNAPE, PC
endocannabinoids Plasma - Chloroform - UPLC-MS/MS Argueta et al. [100]
endocannabinoids Plasma - Chloroform - UPLC-MS/MS Perez et al. [101]
solid-phase extraction online Ferreiro-Vera et al.
oxylipins Serum - -
(C18 cartridges) SPE–LC–MS/MS [57]
MeOH + formic solid-phase extraction
nonesterified PUFAs and oxylipins Plasma - UHPLC-MS/MS Pickens et al. [163]
acid (Strata-X)
IPA with 10 mM
ammonium Hernandez-Carretero
oxylipins, endocannabinoid, Cer Serum - - UPLC-MS/MS
formate + 1% et al. [52]
formic acid
acetones + Tris
chloroform/MeOH
endocannabinoids Serum buffer (50 mM, - LC-MS/MS Azar et al. [164]
(2:1)
pH 8.0)
unesterified oxylipins, solid-phase extraction
Plasma MeOH/ACN (1:1) - UPLC-MS/MS Fan et al. [54]
endocannabinoids (BEH C18 colum)
Bligh and Dyer León-Aguilar et al.
Cer Plasma - - UPLC-MS/MS
method [76]
chloroform/MeOH
Cer, SM Serum - - (UFLC)-MS/MS Özer et al. were [169]
(2:1)
Adipose MeOH/chloroform
FA - 10 % BF3 - MeOH GC–MS Roberts et al. [90]
tissue (2:1)
Adipose MeOH/chloroform
acylcarnitines - - LC–MS Roberts et al. [90]
tissue (2:1)
Adipose GC–FID/MS
SFA, MUFAs, TFA, PUFAs - n-hexane sodium methoxide Kunešová et al. [172]
tissue analysis
Molecules 2020, 25, 5307 19 of 34

Table 1. Cont.

Sample Preparation Method Analysis

Lipid Class(es) Matrix References
Pre-Preparation Extraction Method Derivatization Step Method
Adipose
FAHFAs - MTBE/MeOH/water - UPLC-MS/MS Hu et al. [79]
tissue
Adipose
oxylipins MeOH RapidTrace Biotage - LC-MS-MS Okada et al. [85]
tissue
Adipose Bligh and Dyer
TAG - - LC-MS Al-Sulaiti et al. [71]
tissue method
more than 300 lipid species from lipid
two-step
classes: CL, Cer, ST. HexCer, LPA, LPC, Adipose
- chloroform/MeOH - MS Grzybek et al. [174]
LPE, LPG, LPI, LPS, SM, TAG, CE, DAG, tissue
extraction
PA, PC, PE, PG, PI, PS
TAG, MAG, DAG, LysoPC, PC, LysoPE, Adipose modified Folch
- - LC-MS Tomášová et al. [103]
PE, Cer, SM, PI, PS, FA tissue method
dichloromethane/MeOH/water
Adipose (8:4:2) + solid-phase Mutemberezi et al.
oxysterols, Cer, endocannabinoids - - LC-MS
tissue extraction (C18 [83]
colum)
Adipose chloroform/MeOH
OxPL - - LC-MS Serbulea et al. [75]
tissue (3:1) + BHT
MAG, DAG, TAG, NL, Cer, FFA,
Liver - Folch extraction 10% BF3 - MeOH GC-MS Pakiet et al. [80]
GSPL, SM
chloroform/MeOH
STA, MUFA, PUFA HepG2 cells - hexane + 0.05% BHT GC- FID Lytle et al. [104]
(2:1) + BHT
Cer, SM, GPCho, GPEtn, GPSer, GPA, chloroform/MeOH
Liver - - UPLC-MS Yetukuri et al. [179]
GPGro, DG, TG (2:1)
modified method of
4% formic acids Bligh and Dyer +
LPL (LPS, LPA, LPI, LPG, LPC, LPE) Liver - ESI-MS Wang et al. [70]
in MeOH solid-phase extraction
(HybridSPE cartridge)
Molecules 2020, 25, 5307 20 of 34

Table 1. Cont.

Sample Preparation Method Analysis

Lipid Class(es) Matrix References
Pre-Preparation Extraction Method Derivatization Step Method
chloroform:MeOH (2:1) Garcia-Yaramillo et al.
SFA, MUFA, PUFA Liver - 1% H2SO4 in MeOH GC–FID
plus 1 mM BHT [98]
solid-phase extraction targeted Garcia-Yaramillo et al.
PUFA, PUFA-derived oxylipins Liver MeOH -
(Strata-X) UPLC-TOF-MS/MS [98]
methylene
untargeted Garcia-Yaramillo et al.
DAG, TAG, PC, PS, PI, PG, PE, LPL, SM Liver - chloride/IPA/MeOH -
UPLC-TOF-MS/MS [98]
(25:10:65)
Folch method +
DAG, Cer Liver - solid-phase extraction (Sep - LC-MS/MS Preuss et al. [180]
Pak Diol Cartridges)
IPA/water/ethyl acetate
Brain - - LC-MS Yang et al. [184]
(30:10:60)
non-esterified oxylipins, Rutkowsky et al.
Brain MeOH MTBE - UHPLC-QTOF-MS
endocannabinoids, PUFAs [185]
CSH-ESI QTOF Rutkowsky et al.
Cer, DG, ClcCer, LPC, PC, PE, FA, PI, SM Brain - MeOH/ethyl acetate -
MS/MS [185]
chloroform/MeOH/50 mM MSTFA + 1%
endocannabinoid Brain - GC-MS Kirkham et al. [161]
Tris HCl (2:1:1) trimethylchlorosylane
anhydrous MeOH
Skeletal Gudbrandsen et al.
SFA, MUFA, PUFA - Bligh and Dyer method containing 2.5 M HCl GC-FID
muscle [187]
(100 ◦ C; 2 h)
Skeletal
FFA, DAG, TAG, PL - chloroform/MeOH (2:1) 14% BF3 - MeOH GC-MS Van Hees et al. [129]
muscle
for CE, TAG and GPL
fractions - tert-butyl methyl
Skeletal ether (90%, tert-BME) and 2 M sodium
CE, TAG, GPL - GC-MS Laurentius et al. [97]
muscle MeOH; for FFA fraction methoxide solution
chloroform/MeOH (2:1) +
solid phase extraction
Molecules 2020, 25, 5307 21 of 34

Table 1. Cont.

Sample Preparation Method Analysis

Lipid Class(es) Matrix References
Pre-Preparation Extraction Method Derivatization Step Method
two-stage extraction
LPC, LPE, PI, PG, Cer, PC, PE, PS, TG, Skeletal
- method using - nUHPLC-ESI-MS/MS Eum et al. [78]
HexCer, SM muscle
MTBE/MeOH
Bligh and Dyer Harmancey et al.
DAG, Cer, acyl-CoA Heart - - HPLC-UV
method [190]
sphingolipids Heart - MeOH/chloroform - LC-MS/MS Merrill et al. [165]
chloroform/MeOH
SFA, MUFA, PUFA Heart - (2:1) + solid-phase 10% BF3—MeOH GC–MS Pakiet et al. [192]
extraction (Strata)
ACQUITY UPLC
SM, PC, PE, PG, PI, PS Urine - - UPLC-QTOF-MS/MS Feng et al. [193]
HSS-T3 C18 column
for cholesterol and
7-ketocholesterol
chloroform/MeOH
Araujo and Santos
Cholesterol, 7-ketocholesterol Saliva - (2:1); for - HPLC-DAD
[194]
25-hydroxyvitamins
D2 and D3
MeOH/IPA
Follicular
IPA/acetonitrile/water
FFA, PI, PC, LPC, PS, PE, TG fluid and - MSTFA + 1% TMCS GC-MS Ruebel et al. [195]
(3:3:2) or MTBE
serum
Follicular untargeted
IPA/acetonitrile/water
FFA, PI, PC, LPC, PE, PS fluid and - - CSH-ESI QTOF Ruebel et al. [195]
(3:3:2) or MTBE
serum MS/MS
Molecules 2020, 25, 5307 22 of 34

Table 1. Cont.

Sample Preparation Method Analysis

Lipid Class(es) Matrix References
Pre-Preparation Extraction Method Derivatization Step Method
CTC Combipal 3 autosampler
Faecal Cuesta-Zuluaga et al.
SCFAs - - in HS/SPME mode equipped GC-MS
samples [196]
with a gray fibe
2D GC-ToF-MS: two-dimensional gas chromatography time-of-flight mass spectrometer; ESI-MS–MS: turbo electrospray ionization tandem mass spectrometry; GC- FID/MS: gas
chromatography-flame ionization detector-mass spectrometer; GC-FID: gas chromatography-flame ionization detection; GLC-FID: gas liquid chromatography-flame ionization
detector; HPLC-UV: high-performance liquid chromatography with UV-detection; LC-HRMS/MS: liquid-chromatography high-resolution tandem mass spectrometry; LC-MS-MS liquid
chromatography with tandem mass spectrometry; nUHPLC-ESI-MS/MS: nanoflow ultrahigh performance liquid chromatography with tandem mass spectrometry; UHPLC/Q-TOF-MS:
ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry; UPLC-QTOF-MS: ultra-performance liquid chromatography quadrupole time-of-flight
mass spectrometry; UPLC-TOF-MS/MS: ultra-high performance liquid chromatography coupled to a Triple time-of-flight mass spectrometer; PL: phospholipids; FFA: free fatty
acids; TAG: triacylglycerol; LPA: lysophosphatidic acid; SM: sphingomyelin; dhCer: dihydroceramide; Cer: ceramides; MHC: monohexosylceramide; DHC: dihexosylceramide;
THC: trihexosylceramide; GM3:ganglioside; PC: phosphatidylcholine; PC(0): alkylphosphatidylcholine; PC(P): phosphatidylcholine plasmalogen; LPC: lysophosphatidylcholine; PE:
phosphatidylethanolamine, PE(0): akylphosphatidylethanolamine; PE(P): phosphatidylethanolamine plasmalogen; LPE: lysophosphatidylethanolamine; PI: phosphatidylinositol; LPI:
lysophosphatidylinositol, PS: phosphatidylserine; PG: phosphatidylglycerol; CE: chofesterol ester; COH: free cholesterol; DG: diacylglycerol; TG: triacylglycerol; FA: fatty acid; SFA:
saturated fatty acid; MUFAs: monounsaturated fatty acids; PUFAs: polyunsaturated fatty acids; NEFAs: non-esterified fatty acids; OCFAs: odd-carbon fatty acids; SPL: sphingolipids;
FAHFAs: fatty acid esters of hydroxy fatty acids; SCFAs: short-chain fatty acids; MAG: monoacylglycerol; FAE: fatty-acid ethanolamides; oxFA: oxygenated FAE; oxMAG: oxygenated
MAG; oxFA: oxygenated fatty acids; NAPE: N-acyl-phosphatidylethanolamine; LNAPE: lyso-NAPE; TFA: trans fatty acids; CL: cardiolipin; ST: cholesterol; HexCer: hexosylceramide; LPG:
lysophosphatidylglycerol; LPI: lysophosphatidylinositol; LPS: lysophosphatidylserine; PA: Phosphate; LysoPC: Lysophosphatidylcholines; LysoPE: Lysophosphatidylethanolamines; OxPL:
oxidized phospholipids; NL: neutral lipids; GSPL: glycosphingolipids; GPCho: diacylglycerophosphocholine; GPEtn: glycerophosphoethanolamines; GPSer: glycerophosphoserine;
GPGro: Glycerophosphoglycerol; GlcCer: Glucosylceramides; Acyl:CoA:dihydroxyacetone phosphate acyltransferase; CSH-ESI QTOF MS/MS: charged-surface hybrid column-electrospray
ionization quadrupole time-of-flight tandem mass spectrometry.
Molecules 2020, 25, 5307 23 of 34

The abovementioned protocol of Pakiet et al. with the use of LLE and two SPE methods for
extracting lipids from the brains of mice was also successfully used to extract lipids from the hearts of
mice fed a high-fat diet [192].

3.7. Other Biological Materials

3.7.1. Urine
Feng et al. used an approach based on UPLC-MS to study the effects of HFD on glycolipid
metabolism in young obese men. Metabolomic profiling was performed on urine samples that were
centrifuged (14,000× g; 4 ◦ C; 10 min) and then analysed using UPLC-QTOF-MS/MS [193]. However,
to obtain a more complete urinary lipid profile, extraction with organic solvents was necessary [197].

3.7.2. Saliva
Araujo and Santos used high performance liquid chromatography–diode array detector
(HPLC–DAD) Shimadzu analytical system to show the possibility of using saliva as a biomarker.
The authors used chloroform/MeOH extraction for measurements of cholesterol and 7-ketocholesterol,
and MeOH/IPA extraction was used for measurements of 25-hydroxyvitamins D2 and D3. The authors
claim that saliva, with its simplicity and non-invasiveness of collection, can be an important source
of markers of nutritional status [194]. This may indeed be true since the study of changes in the
metabolism of vitamin D associated with obesity in adipose tissue, in addition to requiring invasive
intervention, requires complex MTBE extraction followed by SPE for further liquid-chromatography
high-resolution tandem mass spectrometry (LC-HRMS/MS) determination of 25-hydroxyvitamin D
and 1,25-dihydroxyvitamin D in adipose tissue [198].

3.7.3. Follicular Fluid

Ruebel et al. used two untargeted metabolomics approaches for primary metabolism analyses
and metabolomics assessment of complex lipids in the study of follicular fluid (FF) in obese women.
The first approach included GC-MS analysis of FF samples after extraction and derivatization by
silylation/methyloximation [195]. During the second approach after MTBE extraction, FF samples were
analysed using CSH-ESI QTOF MS/MS; negative ion MS was used for FFA and PI analysis, while PC,
LPC, PE and PS were analysed in positive ion mode [195].

3.7.4. Faecal samples

Regarding nonstandard approaches, it is worth mentioning that de la Cuesta-Zuluaga et al.
developed a method for determining SCFAs in faecal samples from obese subjects. They determined
volatiles using a CTC Combipal 3 autosampler in HS/SPME mode equipped with a grey fibre
(Carboxen/DVB/PDMS–ref. SU57329U; Supelco) in water extracts heated to 80 ◦ C, which was followed
by desorption of the fibre (at 250 ◦ C) and GC-MS analysis [196].

4. Conclusions
Using lipidomics in the study of obesity is very important to understanding the regulatory and
diagnostic value of changes in the lipidome associated with obesity. Depending on the studied tissue,
the role of changes in specific lipids may have different levels of importance (CER and TAG in muscles,
fatty acid composition of TG in adipose tissues, etc.). Despite the fact that new modifications of
extraction methods such as those of Bligh and Dyer and new methods (e.g., MTBE) are constantly
emerging, none of the extraction methods allows for quantitative extraction of all types of lipids due to
the huge differences in their chemical characteristics. This may explain the great popularity of the
targeted approach and the SPE method, which can reduce lipid degradation during extraction and
can also be automated. At the same time, LLE using phase separation is a more suitable method
for nontarget lipidomics, where fractionation is not required. Thus, the study of changes in lipid
Molecules 2020, 25, 5307 24 of 34

metabolism in obesity contributed to the development of new methods for sample preparation,
extraction and quantification, not only to improve the accuracy and sensitivity of existing methods but
also to develop methods for detecting new specific markers among a huge variety of lipid compounds.

Author Contributions: Conceptualization, I.L. and A.M.; resources, I.L.; writing—original draft preparation,
I.L.; writing—review and editing, A.M., L.K., and T.S.; visualization, P.M. and A.M.; supervision, A.M. and T.S.;
funding acquisition, T.S., L.K. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Medical University of Gdansk, grant number ST40 and S89; and by the
National Science Centre of Poland, grant number NCN 2016/21/D/NZ5/00219.
Conflicts of Interest: The authors declare no conflict of interest.

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