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Determination of Phospholipids in Food Samples
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DOI: 10.1080/87559129.2011.563398
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Food Reviews International, 28:1–46, 2012
Copyright © Taylor & Francis Group, LLC
ISSN: 8755-9129 print / 1525-6103 online
DOI: 10.1080/87559129.2011.563398
Determination of Phospholipids in Food Samples
DONATELLA RESTUCCIA1, U. GIANFRANCO SPIZZIRRI1,

FRANCESCO PUOCI1, GIUSEPPE CIRILLO1,
GIULIANA VINCI2, AND NEVIO PICCI1
1
Dipartimento di Scienze Farmaceutiche, Università della Calabria, Edificio
Polifunzionale, Rende (CS), Italy
2
Dipartimento per le Tecnologie le Risorse e lo Sviluppo, Sapienza Università di
Downloaded by [Universita Studi la Sapienza] at 08:38 05 October 2012
Roma, Rome, Italy
Phospholipids (PLs), the most important class of polar lipids in foods, are ubiquitous
compounds because they are the major components of animal and plant cell mem-
branes. Their technological and biological properties therefore make them important
in human nutrition. The efficient separation and accurate quantification of PLs can
be achieved with high-performance liquid chromatography–evaporative light scatter-
ing detection (HPLC-ELSD) because ELSD is a quasi-universal detector for liquid,
countercurrent, and supercritical fluid chromatography and can detect any analyte
less volatile than the mobile phase. In this article, the principles of ELSD operation
(i.e., nebulization of the chromatographic effluent, evaporation of the mobile phase, and
measurement of the scattered light) as well as the application of LC-ELSD to determine
the amount of PLs in different food matrices are reviewed. Food matrices containing
PLs include milk and dairy products, meat, fish, eggs, cereals, and oils. Both the techno-
logical aspects and analytical parameters affecting the concentration and quantitative
determination of PLs in food matrices are evaluated. In particular, different extraction,
purification, and chromatographic conditions were extensively investigated; moreover,
wherever possible, the results obtained are reported and compared with other detection
methods.
Keywords phospholipids, HPLC, ELSD, food analysis
Introduction
Phospholipids (PLs) are amphiphilic molecules with lipophilic acyl chains and a
hydrophilic head (Fig. 1). They are divided into two main groups: glycerolphospholipids
(GPLs) and sphingolipids (SLs). GLPs are derived from glycerol with a polar head group
(choline, ethanolamine, inositol, and serine) and two fatty acids esterified at the sn-1 and
sn-2 positions of the glycerol backbone.(1) They principally include phosphatidylcholine
(PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine
(PS).(2) SLs are derived from sphingosine; sphingomyelin (SM) is the dominant type
and it is composed of a phosphorylcholine head group and a fatty acid linked to the
amide nitrogen of the sphingoid long base chain.(3) Lysophospholipids (LPLs) are GLPs
in which one acyl chain is lacking and so only one hydroxyl group of the glycerol
Address correspondence to Donatella Restuccia, Dipartimento di Scienze Farmaceutiche,

Università della Calabria, Edificio Polifunzionale, Arcavacata di Rende (CS), Cosenza 87036, Italy.
E-mail: donatella.restuccia@unical.it
1
2 Restuccia et al.
Figure 1. Molecular structure of different phospholipid classes.
backbone is acylated. 1-Lysophospholipids maintain the acyl chain in position 2, whereas

2-lysophospholipids are only acylated at position 1. Lysophosphatidylcholine (LPC) is the
most widely researched LPL, being the most abundant in nature. All plant and animal
foods contain some PLs as part of their cell membranes and the phosphate-containing
Determination of Phospholipids in Food Samples 3
component and fatty-acid composition vary according to the source. Recent studies have
shown that PLs can have a positive nutritional effect on human health, such as reducing
the risk of cardiovascular disease,(4–6) reducing blood cholesterol levels,(7) and enhancing
brain function.(8) Moreover, their antioxidative properties,(9) bacteriostatic properties,(10)
and the inhibitory effect of SL on colon cancer have been intensively studied.(11) However,
recently PLs have also been linked to adverse effects on human health. Apoptosis involves
an alteration in the plasma membrane.(12) Furthermore, alterations in phospholipid com-
position have been noted in certain diseases, such as decreased plasmalogens in the gray
matter of patients with Alzheimer’s disease(13) and decreased cardiolipin (CL) in the red
blood cells of Barth syndrome patients.(14) Another recently recognized lipid feature is
the occurrence of lipid rafts, which are an assembly of SL, phospholipids, and choles-
terol. It has been suggested that lipid rafts form within the plasma membrane of cells to
produce regions with different phospholipid compositions from that of the surrounding
plasma membrane.(15) Finally, endogenous and exogenous oxidation of phospholipids can

lead to the production of biologically active molecules.(16–18)
The importance of PLs is also related to their many industrial applications principally
based on their surfactant properties; in particular, in the food industry, when combined
with proteins, PLs are used as emulsifiers or emulsion stabilizers.(19–26) In addition to their
biological and technological role, PLs are used for several biomedical applications, such
as emulsification in pharmaceuticals and the preparation of liposomes for cosmetics and
drug delivery.(27,28)
One of the problems in lipid analysis is the lack of a satisfactory detection method that
enables the quantification of all of the major classes of lipids present in food matrices. On
the other hand, the demand for fast and reliable quantitative screening methods of com-
plex food matrix extracts has led to the development of more widely applicable detection
strategies. With regard to this, the analysis of PLs has been performed by several differ-
ent methods, including thin-layer chromatography (TLC),(29–32) high-performance liquid
chromatography (HPLC),(32–37) and solid-phase extraction (SPE).(38–40) In earlier articles,
the lipid quantification had been performed exclusively by TLC, but the method has several
disadvantages; namely, the separation of individual classes is very difficult and time con-
suming, and the technique is not always accurate. Over the course of the past few decades,
HPLC has become the preferred method for determining PLs, because a quantitative and
qualitative analysis can readily be obtained at a relatively low cost.
Detection by liquid chromatography can be carried out in different ways, and in the
case of phospholipids this has been a major problem.(41) Although lipids lack specific
absorption peaks, ultraviolet (UV) detection has been mostly used(42–47) as well as mass
spectrometers (MS)(48–51) or refractive index (RI) detectors.(52–54) Detectors based on
ultraviolet-visible (UV-Vis) absorbance and fluorescence provide selective responses for
appropriate compounds and this response may be extended to low concentrations. The
strong absorption at 200–210 nm is caused by the presence of unsaturated bonds and
functional groups such as carbonyl, carboxyl, and amino groups. However, because the
extinction coefficient depends on the unsaturation degree, UV detection does not allow a
quantitative estimation. Furthermore, the mobile phase must be UV-transparent, whereas
gradients cause baseline drift. Mass spectrometers are sensitive and universal detectors,
but they are expensive; moreover, both UV and MS detectors suffer from nonuniform
responses due to differences in absorptivities and ionization efficiencies as a function of
chemical structure, respectively. Nevertheless, HPLC technology for PL analysis has, over
the past decade, been significantly extended by the progress achieved by mass spectrom-
eters. Most types of MS can be used to analyze PLs, including triple quadrupole,(55–57)
quadrupole time-of-flight,(58,59) LTQ Orbitrap,(58) ion trap,(59) and magnetic sector(60)
4 Restuccia et al.
mass spectrometers. Moreover, coupling an electrospray ionization (ESI) source with

a mass spectrometer capable of performing collision-induced dissociation revolution-
ized how LPs were identified and quantified.(61) The combination of collision-induced
dissociation and ESI allowed the determination of all of the molecular species in a
class(62,63) and the shotgun analysis (shotgun lipidomics(64)) of complex lipid mixtures.
In addition, matrix-assisted laser desorption/ionization (MALDI) for PL analysis has also
emerged.(65–68) The problems related to buffer and salt contamination are minimized,
and the sensitivity is high for the detection of PLs because MALDI is often coupled
to a time-of-flight analyzer, which can collect virtually all of the ions formed after the
laser burst.
Others, such as RI detectors, provide a more universal response but only for
moderately high concentrations. Moreover, RI detectors are relatively insensitive and
incompatible with gradient elution and difficult to stabilize.
In recent years, HPLC coupled to an evaporative light-scattering detector (ELSD)

represented a useful alternative. The use of an ELSD approach for spectrophotomet-
ric derivatization (i.e., insertion of chromophoric groups) is feasible and therefore the
drawbacks of derivatization (dependence on experimental parameters, incompleteness of
derivatization reaction, use of salt-laden mobile phases, prolonged analysis time, additional
cost for derivatization system and reagents) can be eliminated. In particular, the use of an
ELSD for lipid analysis has become essential, because it is a universal detector that is
compatible with a broad range of solvents and gradient elution (unlike the refractive index
detector) and the signal is independent of the degree of saturation and chain length of
an acyl chain (unlike the UV detector).(69–73) The main advantages and limitations of the
detectors are summarized in Table 1.
Up-to-date data on the PL fraction in food products are scarce. The various food com-
position databases do not mention the content of bioactive components such as SM and
other PLs. Only the Souci et al.(74) database sporadically reports the content of some PLs.
As the technological and nutritional claims for PLs emerge, the PL content of various food
products is of great importance. In this article the most recent achievements in determining
PLs in various food matrices by HPLC-ELSD are compared with data obtained by other
LC techniques.
ELSD Principles and Applications

Evaporative light-scattering detection was developed in 1966 as an alternative to liquid
chromatography detectors available at the time (UV-Vis, fluorimetric, or RI). Despite
its potential, the new type of detector only started to be widely used at the end of the
1970s,(75) and from the beginning, the intrinsic characteristics of ELSD restricted its scope
of application to liquid chromatography although it now also appears to be compatible with
countercurrent(76,77) and supercritical fluid chromatography.(78,79)
ELSD measurement is based on the amount of light scattered by analyte particles
created by the evaporation of a solvent as it passes through a light beam. Therefore, the
resulting signal corresponds to the body of compounds present in the sample that do
not evaporate or decompose during the evaporation in the solvent or mobile phase.(80–82)
Essentially, an evaporative light-scattering detector operates in three consecutive, well-
defined steps, namely: (a) nebulization of the chromatographic effluent, (b) evaporation of
the mobile phase, and (c) detection of the nonvolatile residual particles by means of the
measurement of the scattered light (Fig. 2). In the nebulisation step, the effluent from the
column is merged, usually in a coaxial manner, with a gas stream (nitrogen or compressed
Table 1
Main limitations and advantages of ELSD over mass spectrometry and
refractive index detectors
Advantages of ELSD over
Limitations of ELSD Refractive index Mass spectrometry

Low identification potential Insensibility to ambient Low cost
temperature
Low selectivity Compatibility with gradient Easy operation
elution
Requirement for highly Better detectability No solvent front peaks
volatile mobile phases

Destructive detector No negative peaks Operation at
atmospheric pressure
Quantitation limit usually No solvent front peak
above 0.1 µg/mL−1
Nonlinear response Calibration curves show
smaller deviation from
linearity
Low sensitivity/detectability Wider dynamic range
for volatile and
thermosensitive analytes
Sensitive to column bleed Compatibility with much
(e.g., silica-based amino wider range of solvents and
column in the presence of modifier
an aqueous mobile phase)
Similar response factor for
molecules with similar
structure
Shorter equilibration time
No sensibility to pulsations of
HPLC pump
air) to obtain an aerosol of small drops. Distribution and mean values of droplet diam-
eter are considered to be very critical parameters that strongly influence the analytical
characteristics (i.e., detectability, sensitivity, and repeatability) of the ELSD methods.(80)
The aerosol is then driven by a gas stream to the evaporation chamber, where the solvent
or mobile phase evaporates to form analyte particles. Ideally, the purpose of this stage
is to completely vaporise the mobile phase, without any analyte loss (due to evapora-
tion or thermal decomposition). The completeness of the mobile phase evaporation and
the extent of loss of analyte is mainly determined by the evaporation temperature, which
should be selected according to the mobile phase and analyte volatility, mobile phase
flow rate, and ELSD type (i.e., aerosol or nonaerosol splitting). Inappropriate selection
of the evaporation temperature leads, in the case of low temperature, to an excessive
noise or baseline with pointed peaks or, in the case of high temperature, to reduced
sensitivity. Finally, the solid particles coming from the evaporation chamber are driven
6 Restuccia et al.
Figure 2. Schematic representation of the evaporative light-scattering detector. (color figure

available online.)
by the previous gas stream to an optical cell that is crossed by a (usually polychromatic)
light beam. The particles scatter light, which is then measured with a photomultiplier tube.
Depending on their size, the particles can cause Rayleigh-Debye scattering, Mie scattering,
or reflection/refraction. The type of interaction between the light and the particles depends
on the size, shape, and surface properties of the particles and the wavelength (λ) of the inci-
dent light. Rayleigh-Debye scattering occurs with the smallest particles (D/λ < 0.1), Mie
scattering becomes the predominant mechanism for 0.1 < D/λ < 1, and the refraction–
reflection mechanism occurs in cases when the particle size is greater than the wavelength
(D/λ > 1). Actually, in most cases, more than one scattering mechanism occurs in
the ELSD optical cell, due to (a) variations in the aerosol droplet diameter D, which
is dependent on the nebulization and evaporation processes; (b) the polychromatism
of the light source; and (c) dependence of the mean droplet diameter on the sample
concentration.
In most ELSD applications, the relationship between the concentration of the analyte
and the signal it produces does not obey Beer’s law but rather an exponential equation. The
area of the chromatographic peak (A) appears in good correlation with the analyte mass (m)
according to the exponential relationship (1):
A = a · mb (1)
where a and b are coefficients depending on the ELSD instrumentation and on the neb-
ulisation and evaporation processes (flow rates of the nebulization gas and mobile phase,
composition of the mobile phase, evaporation temperature, etc.). Coefficient b generally
varies between 0.9 and 2.(83,84) In the case that b is close to unity (b ≈ 1; mainly observed
for high water content in the mobile phase), a linear calibration curve can be constructed;
otherwise, it is necessary to use double logarithmic coordinates in order to obtain a linear
calibration curve (2), the slope of which is equal to b:
log A = b · log m + log a (2)

A linear calibration curve (3) can also be obtained by raising the area of the chromato-
graphic peak to 1/b(85) :
A1/b = a1/b · m = k · m (3)
Equation (3) provides a linear calibration curve passing through the origin and can be used
for routine quantifications using the single-point calibration approach, provided that the
coefficient b has been previously determined from Eq. (2), using a series of standards.
Apart from the exponential relationship, second- and third-order polynomial regressions
have also been utilized to achieve a good correlation between the peak area and the analyte
mass.(86)
ELSD has been effectively used for the determination of a wide variety of com-
pounds in various synthetic or natural matrices. The main areas of application of ELSD are
pharmaceuticals,(87–92) foods and beverages,(38,54),(71,93–96) natural products, biological

samples,(97–102) and polymers.(103–106) Moreover, in recent years, new, unconventional uses
have arisen. They exploit the capabilities of ELSD for the development of rapid-response
analytical systems enabling sample classification/qualification based on a previously
established cutoff value or the determination of overall responses for compound families,
facilitated by the ability to obtain universal responses without the need for chromatographic
separation of individual compounds.(80)
Analysis of Food PLs by HPLC-ELSD
Milk and Dairy Products

Bovine milk PLs usually make up approximately 1% (w/w) of the total lipids. The actual
amounts appear to vary, probably depending on the nutritional status of the cow, the state
of lactation, and other on-farm factors.(107) The most prevalent classes of bovine milk PLs
are PC, PE, and SM, which account for about 90% (w/w) of the total.(108) The remain-
der consists of 3–5% (w/w) each of PS and PI and trace amounts of glucosylceramide
(GluCer) and lactosylceramide (LacCer). LPC and PA are only rarely reported in dairy
products and are probably attributed to poor sample storage and handling or to the activity
of phospholipases.(109,110) In Table 2 the experimental conditions applied for determining
the PLs in milk and dairy samples by HPLC-ELSD are summarized.
In dairy products, the polar lipids are mainly situated in the milk fat globule membrane
(MFGM), stabilizing the fat globule in the milk serum. The lipid composition of the
MFGM has been determined by Lopez et al.,(111) considering both GPL and SL. Folch’s
procedure was applied for lipid extraction and a silica column for HPLC-ELSD separa-
tion of PL classes. The elution program mixed isocratic conditions with a linear gradient
of chloroform–methanol–buffer for a total run time of 36 minutes per sample. Isocratic
conditions did not allow the elution of any polar lipids, so the linear gradient was nec-
essary to elute the polar lipids. The experiments were performed with two groups of six
cows feeding on (1) maize silage ad libitum (+grassland hay, mixture of cereals, soya bean
meal) or (2) the maize silage-based diet supplemented with extruded linseed (meaning a
lipid proportion of 5% of dry matter). As shown by HPLC-ELSD experiments, MFGM in
milk from cows fed the diet rich in polyunsaturated fatty acids (PUFA) resulted in a higher
amount of PLs (+18%). The significantly greater surface area, which was related to the
smaller milk fat globule size measured for milk from cows fed the linseed-supplemented
diet, may be responsible for the greater amount of PL-containing membrane per unit of
Table 2
Experimental conditions applied for the analysis of PLs in milk and dairy products
Extraction/ ELSD
Sample PLs purification HPLC Elution parameters Ref.
Milk PE, PI, PS, PC, MeOH/CH3 Cl Silica (150 × 3 mm ID) Isocratic conditions with Nebulizing gas: dried and (111)
SM precolumn in silica with CH3 Cl, MeOH, and buffer filtered compressed air
the same packing and (1 M formic acid, Flow rate: 1.7 L/min
ID neutralized to pH 3 with Nebulizing T: 50◦ C
Flow rate: 0.5 mL/min triethylamine) 87.5:12:0.5 Evaporating T: 85◦ C
column T: 40◦ C (v/v/v) then linear
Injection volume: 10 µL gradient of same eluents
to 28:60:12 (v/v/v)
8
Milk PE, PI, PS, PC, MeOH/CH2 Cl2 PVA-Sil (150 × 3.0 mm (1) Preconcentration and Nebulizing gas: N2 (113)
SM, LPC ID) with one of the analysis on PVA-Sil guard Flow rate 1.5 L/min
following guard and analytical columns Nebulizing T: 40◦ C
columns: (1) PVA-Sil with eluents: CH2 Cl2 ; Evaporating T: 100◦ C
(23 × 4.0 mm ID); (2) 2-propanol with 14.3 mM
Resolve Pak guard triethylamine and formic
insert acid; 2,2,4-tri
(3.0 × 4.6 mm ID); (3) methylpentane with
Conosil 10-µm guard 7.2 mM triethylamine and
cartridge (7.5 × 4.6 mm HCOOH; MeOH with
ID). Flow rate: 1.0 14.3 mM triethylamine
mL/min and HCOOH
Injection vol:
1007–300 µL
(2) Preconcentration and

analysis on PVA-Sil guard
and analytical columns
with N-ethyl
morpholine/glacial
CH3 COOH modifiers in
the MeOH. CH2 Cl2 ;
2,2,4-tri methyl
pentane/2-propanol
(98:2); MeOH with 24
mM N-ethyl morpholine,
16.4 mM glacial
CH3 COOH; MeOH
9
Milk and PE, PS, PI, PC, MeOH/CH3 Cl Prevail silica (150 × 3.2 Linear gradient of CH3 Cl, Nebulizing gas: N2 (117)
dairy GluCer, mm ID) with precolumn MeOH, and buffer (1 M Flow rate:1.4 L/min
products LacCer, SM, with the same packing HCOOH, neutralized to Evaporating T: 85◦ C
SPH and ID. Flow rate: 0.5 pH 3 with triethylamine)
mL/min from 87.5:12:0.5 (v:v:v)
T column: 40◦ C to 28:60:12 (v:v:v)
Injection volume: 25 µL
Milk and GluCer, LacCer, MeOH/CH3 Cl Prevail silica (150 × 3.2 Linear gradient of Nebulizing gas: N2 (110)
dairy PA,PE, PI, PS, mm ID) with precolumn (A) CH3 Cl, (B) MeOH, Flow rate: 1.4 L/min
products PC, SM, LPC with the same packing and (C) buffer (1 M Evaporating T: 85◦ C
and ID. HCOOH, neutralized to
Flow rate: 0.5 mL/min pH 3 with triethylamine)
T column: 40◦ C from 87.5:12:0.5 (v/v/v)
Injection volume: 25 µL to 28:60:12 (v/v/v)
(Continued)
Table 2
(Continued)
Extraction/ ELSD
Milk and PE, PI, PS, PC, (1) Digestion: NH3 ; Silica normal-phase Linear gradient of Nebulizing gas: dried and (38)
dairy SM extraction: diethyl Zorbax Rx-SIL CH3 Cl/MeOH/NH4 OH filtered compressed air
products ether (250 × 4.6 mm ID) (80:19.5:0.5, v/v/v) and Evaporating T: 50◦ C
(2) MeOH/CH3 Cl Flow rate: 0.5 mL/min CH3 Cl/MeOH/NH4 OH/
(3) SPE: (a) silica gel Injection volume: 10 µL H2 O (60:34:0.5:5.5,
10
bonded column v/v/v)
Recovery: MeOH and
mixture
CH3 Cl/H2 O/MeOH
(3:5:2, v/v/v); (b)
C8 phase bonded
column
Recovery: (95)
PE = phosphatidylethanolamine; PI = phosphatidylinositol; PS = phosphatidylserine; PC = phosphatidylcholine; SM = sphingomyelin; LPC = lyso-220
phosphatidylcholine; GluCer = glucosylceramide; LacCer = lactosylceramide; PA = phosphatidic acid.
milk fat. The major milk phospholipids were PE (19.8–42.0%, w/w), PC (19.2–37.3%,
w/w), PS (1.9–10.5%, w/w), and PI (0.6–11.8%, w/w), and the major sphingolipids were
SM (18.0–34.1%, w/w). The concentration of all of the individual phospholipids was sig-
nificantly higher in the milk from cows fed the linseed-supplemented diet compared to the
regular diet, with the following percentages: PE + 19.8%, PI + 10.8%, PS + 18.6%, and
PC + 11.7%. The content of SM was significantly higher for milk from cows fed the diet
supplemented with linseed, with an increase of 30%. The given explanation was that dur-
ing the secretion of milk fat globules from mammary cells, the droplets of triacylglycerols
were enveloped by the plasma membrane, thereby incorporating SM, which is primarily
found in the outer leaflet of the bilayer in the MFGM.(112) The obtained results confirmed
those of Fagan and Wijesundera,(113) who analyzed 21 samples of fresh milk from different
dairy farms in Victoria, Australia for PL composition. Extraction was obtained by the Bligh
and Dyer method for the analysis of PL classes and the authors developed two methods of
normal phase (NP)-HPLC with ELSD. Different elution gradients and eluent modifiers
have been evaluated depending on the particular experiment (Table 2). In the first method,
a polyvinylalchol-Sil guard column was used for the rapid determination of the major milk
PLs, PE, PC, and SM. In the second method, the guard column was used to preconcentrate
the PL, which was then transferred on-line to a polyvinylalchol-Sil analytical column by
the use of column switching valves. This enabled the separation of complete milk PLs,
including PI, PS, and LPC. The results showed the following distribution in the samples:
PE (%) 38.6 ± 1.7, PC (%) 32.2 ± 1.3, SM (%) 29.2 ± 1.7, and the total PL content was
23.5 mg ± 0.3/100 g milk. The use of on-line removal of simple lipids and simultane-
ous preconcentration of PLs has been shown to be useful for the analysis of milk lipids.
Byrdwell and Perry(114) analyzed SL first by employing an NP chromatographic system
to separate the classes of dihydrosphingomyelin (DSM) and SM, followed by detection
using two complementary atmospheric pressure ionization (API)-MS techniques, atmo-
spheric pressure chemical ionization (APCI)-MS and ESI-MS. APCI-MS mass spectra
exhibited mostly ceramide-like fragment ions, [Cer-H2 O + H]+ and [Cer-2H2 O + H]+ ,
which were used to identify individual molecular species of bovine SL according to fatty
acyl chain length: degree of unsaturation and long-chain base. ESI-MS was used to confirm
the molecular weights of species of both SM and DSM. Molecular species were identified,
with DSM species constituting 20% of bovine SL. In 1994, Valeur et al.(115) reported the
use of HPLC/thermospray ionization mass spectrometry for the analysis of bovine milk
and other sphingomyelin species. They used reverse phase (RP)-HPLC for separation of
the SLs, so the SLs were not separated by class but were instead separated by carbon chain
length and degree of unsaturation. Only SM species, with no DSM species, were identi-
fied. In 1998, Karlsson et al.(116) reported the use of HPLC-APCI-MS and HPLC-ESI-MS
for the analysis of bovine milk SM and other commercially available sphingolipids. APCI-
MS-MS was used to obtain structural information to identify the long-chain base and fatty
amide portions of the molecules. An up-front collision-induced dissociation (CID) voltage
of 30 V was used. The authors employed a diol column to perform the NP separation of
SL species and showed elution of three SL peaks for bovine milk, which were identified as
SM1, saturated long-chain base with hydroxy fatty acids (saturated and unsaturated); SM2,
unsaturated long-chain base with long-chain fatty acids (saturated and unsaturated); and
SM3, unsaturated long-chain base with short chain fatty acids (saturated and unsaturated).
Byrdwell and Perry(114) showed that the APCI-MS data of SL can be complex and require
careful interpretation using supplemental MS-MS data. Also, the very simple ESI-MS data
are invaluable to provide confirmation of types identified by APCI-MS. Because APCI-MS
produces some in-source fragmentation, some cases of isobaric fragments were observed.
12 Restuccia et al.
APCI-MS-MS data and ESI-MS data were valuable for eliminating any ambiguity and
for apportioning peaks arising from isobaric molecular species. Because of the fragments
that are already produced in the APCI-MS source, additional fragmentation by application
of an up-front CID voltage between the capillary outlet and the skimmer or between the
skimmer and the first multipole ion-focusing element should be avoided. The nonspecific
fragmentation caused by up-front CID can complicate spectra and lead to misinterpretation.
Dairy products have also received attention in recent years in relation to phospho- and
sphingolipid content. Rombaut et al.(117) evaluated 31 commercial products by HPLC-
ELSD after extraction with chloroform/methanol. In particular, they considered milk,
cream, condensed milk, butter, fermented products, cheeses, and whey. Values were given
for different PLs (PE, PI, PS, PC) and SL (GluCer, LacCer, SM). The sum of phospho-
lipid (PE, PI, PS, PC) and sphingolipid (GluCer, LacCer, SM) concentration was regarded
as total PL concentration. A silica column was used for the separation of PL classes and
the elution program was a linear gradient of chloroform : methanol : buffer (1 M formic
acid, neutralized to pH 3 with triethylamine) from 87.5:12:0.5 (v/v/v) to 28:60:12 (v/v/v)
in 16 minutes. PL values ranged from 3.6 to 479.5 mg/100 g product, 0.01 to 2.19% of
the dry matter, and 0.1 to 24.7% of the lipid fraction. PL types expressed as a percentage
of total PLs ranged from 1.3 to 5.9% for GluCer, 3.7 to 11.7% for PI, 5.3 to 12.7% for
LacCer, 5.6 to 10.5% for PS, 12.1 to 25.7% for SM, 15.8 to 24.2% for PC, and 24.6 to
45.4% for PE. In milk for consumption, the PL content varied from 12.8 to 9.4 mg/100
g milk, depending on the fat content. These values are in accordance with the 22.1, 18.5,
10.7, and 10.3 mg/100 g reported by Christie et al.(109) for, respectively, raw, whole, semi-
skimmed, and skimmed milk. However, they reported a seasonal range of 13.5–22.9 mg
PLs/100 g whole milk within a half-year time span. Other variations were published by
Bitman and Wood,(118) who reported a PL concentration of 12.4–34.3 mg/100 g raw milk
based on the stage of lactation of the cow, and considerable differences of PLs on a fat basis
could also be noted between different products. No correlation between fat and PL content
was observed and the conclusion was that an estimation of the PL content based on the fat
content, as is sometimes done due to lack of data on PL content, may give rise to erroneous
results. Moreover, the influence of thermal treatment and other processing techniques on
PLs should be considered. It is, however, impossible to solely attribute the reported differ-
ences in PL concentration and profile to different processing methodologies, because the
original raw milk, from which all other dairy products are derived, was not identical. The
same authors reported a new HPLC method, using an ELSD that enabled the analysis of
the PL content of raw milk, cream, butter, buttermilk, cheddar whey, skimmed Quarg, and
cheddar cheese.(110) The new method enabled the excellent separation of GluCer, LacCer,
PA, PE, PI, PS, PC, SM, and LPC in less than 21 minutes, including the regeneration of
the column. No loss in column performance was observed after 1500 runs because an acid
buffer was used. The output signal of the ELSD was highly dependent on the flow of the
carrier gas and the temperature of the nebulizer and was maximized by means of a response
surface experimental design. The analysis of variance revealed a significant difference in
the relative composition of PL classes among the dairy samples. PE was the most predom-
inant PL (36.5–42.9%), followed by PC (18.7–20.7%) and SM (12.8–20.2%). These three
species represented more than 70% of total PLs in dairy samples. PA and LPC were not
observed in detectable amounts. Fat-rich products were high in PL, because they are situ-
ated in the milk fat globule membrane, which surrounds the fat globule. However, during
churning, the milk fat globule membrane is ruptured and migrates substantially toward the
water phase,(119) resulting in a low-fat product (buttermilk) rich in PLs. The evaluation
of the performance of different methods for both the extraction of lipids from milk and
the isolation of PLs has also been investigated.(38) Two different fat extraction procedures
were tested to evaluate the influence of the solvent polarity on the PL recovery (PC, PE, PI,
PS, and SM) and the quantification was performed by HPLC coupled with ELSD detector.
It was found that the Rose-Gottlieb (RG) modified procedure was not able to extract PS
and PI. The presence of ammonia in the RG reagents, such as MFGM dissociating agent,
probably increased the water solubility of PS and PI, due to their acidic characteristics. On
the contrary, the Folch extraction method has been demonstrated as being the most reliable
method to extract PLs from the milk matrix. Moreover, the performance of different SPE
cartridges and solvent programs to isolate PLs from the other lipids was investigated. The
best recovery was obtained by the silica column, eluting the PLs by 2 mL of methanol
followed by 2 mL of a mixture of chloroform–methanol–water (3:5:2, v/v/v). The incom-
plete recovery of PLs from the hydrophobic reverse-phase liquid chromatography column
(C8) was probably due to an interaction between the PLs diacylglycerol group and the
alkyl chain of the SPE solid phase, as was previously reported by Caboni et al.(120) After
the optimization of extraction and SPE procedure, the analytical method showing the best
performance was applied to dairy products having different fat contents. A silica column
was used and the chromatographic separation was carried out using a linear binary gradient
of chloroform–methanol–ammonium hydroxide (80:19.5:0.5, v/v/v) and of chloroform–
methanol–ammonium hydroxide–water (60:34:0.5:5.5, v/v/v). Total chromatographic run
time was 40 minutes per sample, which consisted of a 23-minute analysis, 12 minutes to
restore initial conditions, and 5 minutes for re-equilibration. The quantification of PC, PE,
PI, PS, and SM showed that the PL content of the natural cream was higher than that of
cream obtained by centrifugation. This result has been explained by the incorporation, dur-
ing the natural creaming, of a large number of small fat globules, leading to a higher ratio
between the MFGM and the fat content. Once again the high PL content of buttermilk and
the corresponding low content of butter was shown; the churning process, usually applied
for butter production, is responsible for the disruption of MFGM, resulting in an important
increase in membranous material in buttermilk.
Meat–Fish
The lipid composition of meat depends on several factors, such as the animal species,
rearing conditions, composition of the feed, and also some intrinsic factors, such as the
kinds of muscle and muscle fibre. Red muscle usually contains higher amounts of PLs
and polyunsaturated fatty acids with respect to white fibre.(121) Moreover, the quantity and
quality of the polar lipid fraction is affected by technological factors,(122) such as the fat
melting point and the rheology of both minced(123) and nonminced(124) products, emul-
sion characteristics,(125) oxidative stability,(126) and therefore the shelflife of the finished
product, as well as the sensory properties and the nutritional quality.(127) In 2008, Boselli
et al.(51) proposed an analytical method for the simultaneous determination of both PL
classes and molecular species within each class in raw and cooked pork meat. The consid-
ered PLs were PC, plasmalogen (pPE)+PE, cardiolipin (CL), SM, PI, PS, and LPC. The
lipid fraction was extracted according to the Folch method; after purification of the polar
lipid fraction by SPE, the PL classes were separated with HPLC coupled on-line with two
detection systems: an ion trap equipped with electrospray ionization for tandem mass spec-
trometry (MS-MS) and an ELSD. A silica column was used for separation using a gradient
of chloroform–methanol–ammonium hydroxide (30%; 70:25:1, v/v/v), and chloroform–
methanol–ammonium hydroxide (30%; 60:40:5.5:0.5, v/v/v) for elution. The results were
as follows: PC (42.9% ± 4.5 for raw vs. 42.6% ± 8.0 for cooked meat), plasmalogen
14 Restuccia et al.
(pPE)+PE (26.7% ± 3.1 vs. 28.5% ± 2.3), cardiolipin (CL; 8.3% ± 2.9 vs. 6.3% ± 0.7),
SM (7.5% ± 0.9 vs. 8.3% ± 2.1), PI (6.8% ± 0.7 vs. 6.5% ± 2.1), PS (4.9% ± 0.5
vs. 4.6% ± 1.4), and LPC (2.9% ± 1.3 vs. 3.3% ± 2.6). It was found that the main PL
classes were PC and pPE+PE, as in the intramuscular fat of pigs(128) and in beef.(120) No
significant differences were found in the composition of the different PL classes between
raw and cooked pork using HPLC-ELSD, whereas using LC-MS significant differences
were found only for the molecular species of PI and LPC. Boselli et al.(129) also studied
the effects of a dietary administration of high-oleic sunflower oil (about 85% oleic acid)
to pigs, either in combination with or not in combination with α-tocopheryl acetate on
the composition of the triacylglycerols, PLs and the level of vitamin E. Using the same
experimental conditions as previously reported,(51) the quantification of the PL classes was
accomplished by HPLC-ELSD, and LC-MS was applied for the characterization of PL
molecular species. Four different diets with the same lysine–calorie ratio were adminis-
tered to castrated males and intact females until slaughtering (160–170 kg) and the lipids
of the loin (longissimus lumborum) were analyzed. The dietary supplement of high-oleic
sunflower oil in association with vitamin E acetate affected the neutral lipid fraction. The
triacylglycerols containing saturated fatty acids (palmitic and stearic) decreased by about
14.0%, whereas the content of triacylglycerols containing two oleic moieties increased
(116.0%); the content of triolein increased by 40.0%. Except for PC and PS, the polar lipid
fraction was not affected by the diet, probably due to the homeostatic regulation, which
was significant in heavy pigs slaughtered at 160–170 kg, unlike in animals slaughtered
at 105 kg of another genotype,(130,131) or because the lysine/calorie ratio was constant in
the present diets, unlike in previous works dealing with heavy pigs.(132) The effect of the
diet (acorn and grass vs. oleic acid–enriched concentrates) on the amount of individual PL
classes (i.e., PC, PE, PS, and PI) in muscle from Iberian pigs as well as their fatty acid
composition have also been recently investigated by Perez-Palacios et al.(133) The lipid
fraction was extracted according to the Folch method using NP-HPLC-ELSD for quanti-
tative analysis. A linear gradient was used for elution consisting of chloroform, methanol,
and triethylamine buffer (pH 3, 1 M formic acid), for a total run time of 25 minutes. Firstly,
it was found that PC was the major PL, followed by PE, PS, and PI in decreasing order,
and each PL class showed a different lipid profile. Secondly, the feeding regimen influ-
enced the quantity and the fatty composition of the individual PL classes, implying that
PL profiles could be used as tools for differentiating meat from Iberian pigs with dif-
ferent feeding backgrounds. The quantity of PC, PE, and PI was significantly higher in
meat from pigs fattened with oleic acid–enriched concentrates than in meat from pigs fat-
tened with acorn and grass, whereas PS was not influenced by pig feeding. Meat from
pigs fattened with high oleic acid concentrates also had higher amounts of polyunsatu-
rated fatty acids. This difference could make the meat from animals fattened with high
oleic acid concentrates more prone to lipid oxidation because PLs are very sensitive to
oxidation, mainly due to their high polyunsaturated fatty acid content.(134–140) Changes in
intramuscular PLs (PE, PC, PS, PI, SM, and LPC) and free fatty acids were tracked dur-
ing the processing of Nanjing dry-cured duck by Xu et al.(141) After chilling (2 hours),
duck carcasses were dry-cured (salt content: 7% of carcass weight, 24 hours), marinated
in brine (saturated salt solution, 20 hours), piled (48 hours), and then dried at 17–18◦ C
in a well-ventilated room for 5, 10, and 15 days, respectively. At the end of each pro-
cessing stage (including raw), six carcasses were selected for the biochemical analyses.
PLs were extracted using chloroform–methanol, purified by aminopropyl–silica minicol-
umn, and then identified and quantified by NP-HPLC combined with UV and ELSD
installed in series. A gradient elution lasting 70 minutes was carried out using different
ratios of solutions: n-hexane/ 2-propanol (3:2, v/v), n-hexane/2-propanol/25 M ammo-

nium acetate (120:80:11, v/v/v), and n-hexane/2-propanol/water (120:80:11, v/v/v). PE
and PC were found to be major components of PLs in the duck meat (17.4 and 24.7%,
respectively) and PI the smallest (only 0.1%). The contents of PLs, fatty acids, and triacyl-
glycerols varied greatly during the processing of Nanjing dry-cured duck, which indicated
that they underwent hydrolysis, especially enzymatic lipolysis.(142) For PE, PI, and PS,
the lipolysis occurred primarily at the marinating and piling stages because they had been
completely hydrolyzed by the piling stage. The quantities of SM declined at the dry-salting
stage and also at the final period of the drying stage, whereas the decrease in the quantity
of LPC mainly took place during the dry-salting and marinating stages. During the pro-
cessing, the percentage of PE decreased by 50.0%, whereas that of PC declined by 30.0%,
which indicated that PE was more susceptible to hydrolysis or oxidation than PC. This
was ascribed by Hernandez et al.(143) to the greater susceptibility of PE to oxidation for its
high content of polyunsaturated fatty acids and plasmalogen, which are very sensitive to
hydrolysis.
Wang et al.,(144) by applying the same analytical procedure, also investigated the
changes in intramuscular PLs (PE, PC, PS, PI, SM, and LPC) and free fatty acids and
their interrelationships during the processing of other Chinese duck products (i.e., raw, dry-
cured, roasted, and water-boiled salted ducks). They confirmed that in raw duck meat, PLs
accounted for 46.7% of total lipids. The PE and PC accounted for the largest quantities of
PLs (13.1 and 30.2% of lipids, respectively) and PI the smallest one (only 0.1% of lipids).
The PS, SM, and LPC accounted for 1.4, 1.2, and 0.6% lipids, respectively. Cooked duck
(roasted and water-boiled salted duck) had significantly smaller decreases in PLs com-
pared with dry-cured duck. Also in this case, all six PLs decreased markedly at the end
of dry-cured duck processing, which indicated that they underwent hydrolysis, especially
enzymatic lipolysis.(142) Significant differences were found only in the content of PE and
SM after the processing of water-boiled salted duck. Meanwhile, Boselli et al.(51) found
no significant differences between raw and cooked pork meat in the composition of the
different classes of phospholipids; these different findings are probably due to the differ-
ent processing methods for different meat products. During the roast duck processing, the
percentage of PE decreased by 21.8%, whereas that of PC declined by 9.8%, which indi-
cated that cooking damaged PE more seriously than PC, as was also reported by Gandemer
et al.(145) Moreover, a significant change in fatty acids during the processing of dry-cured
duck was observed. The results supported the hypothesis that free fatty acids come mostly
from the hydrolysis of PLs. Moreover, a selective degradation in the fatty acid distributions
of PLs was found during the dry-cured duck processing, but lipolysis of PLs was not spe-
cific to fatty acid chain length or unsaturation through the roasted and water-boiled duck
processing.
Wang et al.(146) determined intramuscular phospholipid classes (PC, PE, PC, PI, PS,
SM, and LPC) and molecular species of PC and PE in Gaoyou duck. After extraction by the
Folch method and purification of the PLs by an aminopropyl–silica minicolumn, classes
of PLs were identified and quantified by NP-HPLC combined with UV and ELSD. The
authors designed and optimised a new HPLC method consisting of a ternary column with
a UV detector at 205 nm that could only detect PE and PC. The same sensitive chromatog-
raphy system with an ELSD could detect low quantities of PI, PS, and LPC. A gradient
elution during 70 minutes was carried out using different ratios of solutions: n-hexane/2-
propanol (3:2, v/v), n-hexane/2-propanol/25 M ammonium acetate (120:80:11, v/v/v),
and n-hexane/2-propanol/water (120:80:11, v/v/v). In Gaoyou duck meat, PLs accounted
for 46.7% of total lipids, and the proportions of PC, PE, PI, PS, SM, and LPC were 64.7,
16 Restuccia et al.
28.1, 0.3, 3.1, 2.5, and 1.4%, respectively. Molecular species of PC and PE were charac-
terised by reverse-phase HPLC, which was coupled in parallel with both an ESI-MS and
an ELSD; a gradient elution was carried out using various ratios of chloroform–methanol
(1:17.5, v/v), and acetonitrile–water (1:1, v/v). There were different results for PC and
PE; in this case, the use of ELSD gave a more precise relative content of the PL molec-
ular species; in contrast, the quantification of the molecular species with mass detection
with an ion trap proved to be affected by the different ionization potentials of the analytes.
Arachidonic acid was mainly present in PE and formed molecular species containing a sat-
urated fatty acid, such as stearic or myristic acid; however, oleic acid together with palmitic
or stearic acid formed the main molecular species in PC. The content of the molecular
species with polyunsaturated fatty acids in PE accounted for 98.3%, whereas that in PC
only 46.2%.
Far less information is available on the analysis of PL classes by HPLC-ELSD in fish
samples. Silversand and Haux(147) developed a binary gradient solvent system for HPLC
able to separate and quantify the major classes present in fish, extracted from samples
using the method of Bligh and Dyer.(148) The HPLC methods developed were highly repro-
ducible and allowed baseline separation of PA, CL, PE, PC, SM, PS, PI, and LPC as well
as neutral lipid classes. Using a diol column and a binary gradient of hexane–isopropanol–
acetic acid (82:17:1.0, v/v/v) and propanol–water–acetic acid (85:14:1.0, v/v/v), adding
triethylamine (0.08%, v/v) as a modifier, the separation of polar lipids could be carried
out in 44 minutes. All lipid classes present in liver and eggs were well separated with
minor molecular species separation. Polar lipid classes were the major constituents of lipids
from the liver of Atlantic salmon, whereas eggs of the same species contained approxi-
mately equal amounts of polar and neutral lipids. The observed lipid class composition of
Atlantic salmon eggs was similar to that analysed previously by TLC and flame ioniza-
tion detection.(149) Chromatography of polar lipids (% of total lipid) revealed that liver and
eggs contained, respectively, PE (16.0 ± 2.0; 4.3 ± 0.7), PC (47.0 ± 5.0; 44.0 ± 4.4), and
PI (6.0 ± 0.8; 2.3 ± 0.3). The liver also contained minor amounts of PS (4.5 ± 0.6) and
SM (2.6 ± 0.4). In the present study, the livers of Atlantic salmon contained considerably
more polar lipids (80.0% wt of total lipid) than were found in an earlier study on farmed
Atlantic salmon (54.0% wt).(150) This difference was explained by their different diets.(151)
More recently, Ong et al.(152) presented a metabolomic study to investigate the bio-
chemical profiles of livers from male and female zebrafish (Danio rerio) using a multiple
platform approach, incorporating 1 H-NMR, GC-MS, and LC-MS. LPC and PC, analyzed
by LC-MS, were extracted by chloroform–methanol. The HPLC separation was performed
on a C18 column and the gradient elution involved a mobile phase consisting of water
(0.1% formic acid) and acetonitrile (0.1% formic acid). The initial condition was set at
5% of acetonitrile, gradually increasing to 100% in 10 minutes and returning to the initial
condition for 5 minutes. A wide range of metabolites and phospholipids could be quanti-
fied; it was observed that many target analytes were found to elute close to each other. In
order to increase the specificity, extracted ion chromatograms of selected metabolites were
used for comparison between the two classes. Based on a comparison using reference stan-
dards and the retention behavior on an RP column, LPC and PC were identified. After data
normalization, the peak intensities were subjected to multivariate analysis. Data for the
male and female specimens clearly scattered into two distinct regions, and metabolites that
were observed to be significantly different in the liver tissue extracts of male and female
zebrafish included choline, LPC C18:2, LPC C16:0, LPC C18:1, and LPC C18:0.
Wang et al.(153) reported PL classes (PE, PI, PS, PC, SM, LPC, and CL) and fatty acid
compositions in the egg of the squid Sthenoteuthis oualaniensis, which is commercially
fished near Okinawa (in the Ryukyu chain), Taiwan, and Hawaii for tuna bait and for human
consumption.(154) Total lipids were extracted using the method of Bligh and Dyer(148) and
the PLs purified using a silica gel column. NP-HPLC-ELSD was then applied for sepa-
ration and quantification of major PL classes. A gradient of chloroform–methanol–25%
ammonium hydroxide (80:19.5:0.5, v/v/v) and chloroform–methanol–25% ammonium
hydroxide (60:34:0.5:5.5, v/v/v) was used and the total chromatographic run time was
40 minutes per sample, which consisted of a 23-minute analysis, 12 minutes to restore ini-
tial conditions, and 5 minutes to re-equilibrate. It has been reported that, in general, fish
roe contains large amounts of eicosapentaenoic acid (C20:5n-3) and docosahexaenoic acid
(C22:6n-3),(155–157) which play an important role in the prevention and treatment of cardio-
vascular diseases(158) and in the improvement of learning ability.(159,160) Compared to that
of fish, squid lipid tends to be lower in triglycerides and higher in PLs.(161) In this study,
the phospholipid and fatty acid composition of the eggs of S. oualaniensis were analyzed
to determine whether the egg could be a new resource of docosahexaenoic acid–containing

phospholipids. The total lipid content of the squid egg was 6.2 ± 0.4% (w/w), higher than
that of the integument of squid O. bartrami (2.5%, w/w).(162) PLs were found as the major
lipid class, 72.0 ± 3.2% of total lipids (w/w), which was lower than the PL content of
the integument of squid O. bartrami (80.0–85.0% of total lipids, w/w).(163) PE, PI, PC,
SM, and LPC were separated but no PS was detected. The predominant phospholipid of
the sample was PC (67.5 ± 0.6% of total PLs, w/w), which is the main component in
the phospholipid class of fish roe products Ikura (78.0 ± 0.1%), Tarako (68.6 ± 0.5%),
Tobiko (76.7 ± 0.9%), and Kazunoko (65.3 ± 5.8%).(162) In Table 3, the experimental
conditions applied for the HPLC-ELSD determination of PLs in meat and fish samples are
summarized.
Egg
Eggs are a good source of PLs, which represent approximately 10.0% of the wet weight
of egg yolk. This is equivalent to about 22.0% of the total egg yolk solid. PLs in common
egg yolk are composed of 60.0–73.0% PC, 15.0–26.0% PE, 2.5–4.8% SM, 4.1–5.8% LPC,
2.1% LPE, 0.6% PI, and traces of phosphatidylglycerol (PG) and PS.(164,165)
PE, PI, PS, PC, SM, and PG were successfully extracted by Boselli and Caboni(166)
from dried egg yolk using neat supercritical carbon dioxide (SC-CO2 ) without organic
cosolvent. The spray-dried egg yolk powder was extracted with SC-CO2 at a density of
1.06 g/mL (517 bar, 40◦ C). The extraction yield obtained (67.0 g of extract per 100 g
of sample) proved to be comparable with the conventional Bligh and Dyer(148) extraction
method (63.0 g of extract per 100 g of sample), which involves a chloroform : methanol :
water mixture as an extraction solvent. The solvent extract contained 29.0% of PLs, and the
neat SC-CO2 extract consisted of 26.0% PLs. The purification of PLs was carried out by
SPE using C8 as a stationary phase and quantification was effected by NP-HPLC-ELSD. A
gradient of CH3 Cl-MeOH-30% NH4 OH (80:19.5:0.5, v/v/v) to CH3 Cl-MeOH-H2 O-30%
NH4 OH (60:34:5.5:0.5, v/v/v) was used for elution, with each analysis lasting 50 min-
utes. Chromatograms showed that the neat CO2 extract has the same qualitative PL pattern
obtained for the solvent extract and the major PLs were PC, PE, SM, and PI.
More recently, Palacios and Wang(167) developed an effective method for extracting
a sufficient amount of lecithin from fresh egg yolk and then evaluating its functional
properties. Ethanol was used to dehydrate and partially extract the PLs, followed by
hexane to extract the total lipids. A phase separation of the combined extracts resulted in
neutral and polar lipid fractions. An acetone precipitation of PLs (PE and PC) from the final
Table 3
Experimental conditions applied for the analysis of PLs in meat and fish samples
Extraction/ ELSD
Raw and CL, PC, MeOH/CH3 Cl Polaris Si-A Gradient of CHCl3 , Nebulizing gas: (51)
cooked LPC, SPE: silica gel bonded 150 × 4.6 mm ID MeOH/NH4 OH dried and filtered
pork pPE, PE, column with silica precolumn 30% (70:25:1, compressed air
meet PI, PS, Recovery: Flow rate: 1.0 mL/min v/v/v) to CHCl3 / Flow rate: 7.5 L/min
SM CH3 Cl/H2 O/MeOH Injection volume: 5 µL MeOH/H2 O/ Evaporating T: 60◦ C
(3:5:2, v/v/v) NH4 OH 30%
(60:40:5.5:0.5,
v/v/v)
Raw pork CL, LPC, MeOH/CH3 Cl Polaris Si-A Gradient of Nebulizing gas: (129)
meat as PC, pPE, SPE: silica gel bonded 150 × 4.6 mm ID CHCl3 /MeOH/ dried and filtered
affected PE, PI, column with silica precolumn NH4 OH 30% compressed air
18
by diet PS, SM Recovery: MeOH plus Flow rate:1.0 mL/min (70:25:1, v/v/v) Flow rate: 7.5 L/min
mixture Injection volume: 5 µL to CHCl3 / Evaporating T: 60◦ C
CH3 Cl/H2 O/MeOH MeOH/ H2 O/
(3:5:2, v/v/v) NH4 OH 30%
(60:40:5.5:0.5,
v/v/v)
Fresh hams PC, PE, PS, MeOH/CH3 Cl Prevail silica Gradient of CHCl3 , Nebulizing gas: N2 (133)
from pigs PI SPE: NH2 -aminopropyl 150 × 3.0 mm ID MeOH, and Flow rate: 1.6 L/min
fattened mini-columns Flow rate: 0.7 mL/min triethylamine Evaporating T: 65◦ C
with Recovery: PC with T column: 40◦ C buffer (pH 3, 1 M
different CH3 CN/n-propanol (2:1, Injection volume: HCOOH) from
diets v/v), PE with MeOH, PS 10 µL 87.5:12:0.5 to
with iso-propanol/HCl 2:90:8 (v/v/v)
methanolic 3N (4:1,v/v), PI
with CH3 Cl/MeOH/37%
HCl (200:100:1)
Nanjing PE, PI, PS, MeOH/CH3 Cl SI 60-5 silica gel Gradient of Nebulizing gas: N2 (141)
dry-cured PC, SM, SPE: NH2 -aminopropyl column (n-hexane/2- Flow rate: 1.8 L/min
duck LPC mini-columns 250 × 4.0 mm ID propanol, 3:2, Evaporating T: 70◦ C
Recovery: MeOH Flow rate: v/v), (n-hexane/2-
1.0 mL/minT propanol/25
column: 30◦ C mmol/L of
NH4 Ac
120/80/11,
v/v/v), and
(n-hexane/2-
propanol/H2 O
19
120/80/11,
v/v/v)
Raw duck, PE, PI, PS, MeOH/CH3 ClSPE: SI 60-5 silica gel Gradient of Nebulizing gas: N2 (144)
dry-cured PC, SM, NH2 -aminopropyl column (n-hexane/ Flow rate: 1.8 L/min
duck, LPC mini-columnsRecovery: 250 × 4.0 mm ID 2-propanol, 3:2, Evaporating T: 70◦ C
roasted MeOH Flow rate: 1.0 mL/min v/v), (n-hexane/2-
duck, T column: 30◦ C propanol/25
water- mmol/L of
boiled NH4 Ac,
salted 120/80/11,
duck v/v/v), and
(n-hexane/2-
propanol/H2 O,
120/80/11,
v/v/v)
(Continued)
Table 3
(Continued)
Extraction/ ELSD
Gaoyou PE, PI, PS, MeOH/CH3 Cl SI 60-5 silica gel Gradient of Nebulizing gas: N2 (146)
duck PC, SM, SPE: NH2 -aminopropyl column (n-hexane/2- Flow rate: 1.8 L/min
LPC mini-columns 250 × 4.0 mm ID propanol, 3:2, Evaporating T: 70◦ C
Recovery: MeOH Flow rate: 1.0 mL/min v/v), (n-hexane/2-
T column: 30◦ C propanol/25 mmol/L
of NH4 Ac,
120/80/11,
v/v/v), and
(n-hexane/2-
20
propanol/H2 O,
120/80/11,
v/v/v)
Livers and PA, PE, PI, MeOH/CH3 Cl LiChrosphere 100 Diol Gradient of Nebulizing gas: (147)
eggs of PS, 250 × 4.0 mm ID n-hexane/ dried and filtered
Atlantic PCSM, Flow rate: 1.0 mL/min isopropanol/ compressed air
salmon LPC, T column: CH3 COOH Flow rate: 7.5 L/min
DPG 45◦ CInjection (82:17:1.0, v/v/v) Evaporating T: 45◦ C
volume: 20 µL to isopropanol/
H2 O/ CH3 COOH
(85:14:1.0,
v/v/v).
Triethylamine
(0.08%, v/v)
Squid eggs PE, PI, PS, MeOH/CH3 Cl Normal-phase Zorbax Gradient of Nebulizing gas: N2 (153)
21
PC, SPE: silica gel column Rx-SIL CH3 Cl/MeOH/25% Evaporating T: 65◦ C
SM,LPC, Recovery: MeOH 250 × 4.6 mm ID NH4 OH
DPG Flow rate: 1.0 mL/min (80:19.5:0.5,
Injection volume: v/v/v) and
20 µL CH3 Cl/MeOH/25%
NH4 OH
(60:34:0.5:5.5)
CL = cardiolipin; pPE = plasmalogen; PE = phosphatidylethanolamine; PI = phosphatidylinositol; PS = phosphatidylserine; PC = phosphatidylcholine;
SM = sphingomyelin; LPC = lysophosphatidylcholine; PA = phosphatidic acid; DPG = diphosphatidylglycerol.
22 Restuccia et al.
polar lipid fraction was necessary to remove residual neutral lipids, especially cholesterol.
The PL purity of the lecithin product was 95.0% and PL distribution in the neutral and PL
fractions was about 4.0 and 96.0%, respectively, showing good values of the total PL recov-
ery achieved by this fractionation procedure. The quantification of PLs in samples was
done by HPLC-ELSD in 35 minutes using a diol column and a gradient program with two
mobile phases consisting of chloroform–methanol–ammonium hydroxide (80:19:1, v/v),
and chloroform–methanol–water–ammonium hydroxide (50:48:1:1, v/v). On a dry weight
basis, the examined yolk material contained about 11.0% PLs (about 80.0% PC, about
20.0% PE). Moreover, surface tension reduction, emulsion stability, and oxidative stabil-
ity studies were conducted to characterize the functional properties of egg yolk lecithin.
The results showed that egg yolk lecithin and soy lecithin had similar surface activity as
evaluated by surface tension reduction in an aqueous system and the critical micelle con-
centration. Soybean lecithin created a more stable emulsion than egg yolk lecithin, and egg
yolk lecithin was more oxidatively stable than soybean lecithin.

Further insight regarding separation and identification by LC-ESI-MS-MS of molec-
ular species of PC was achieved by Le Grandois et al.(168) Lipid extraction was accom-
plished using a chloroform–methanol mixture and a preparative flash chromatography with
silica cartridge was used for purification of the total lipid extract. NP-HPLC-ELSD was
used for PC purification and to check the purity of the isolated PC. Separations were per-
formed using a 20-minute linear gradient ranging from chloroform–methanol (88:12, v/v)
to chloroform–methanol–formic acid (1 M) adjusted to pH 3 with triethylamine (28:60:12,
v/v/v). Molecular species of PC were detected as m/z of lithium adducts [M+Li]+ .
Lithium was added to the chromatographic mobile phase and was shown not to interfere
with the separation, which allowed for the combination of an effective chromatographic
separation and an improved structural identification. The major molecular species in egg
yolk PC were identified as (16:0–18:1) PC (39.7%), followed by (16:0–18:2) PC (20.2%),
(18:0–18:2) PC (8.9%), and (18:0–18:1) PC (8.3%). Minor species included (18:2–18:2)
PC (3.3%), (18:1–18:2) PC (3.1%), (16:0–22:6) PC (2.8%), and (18:0–22:6) PC (1.1%).
Table 4 contains the experimental conditions applied for the HPLC-ELSD determination
of PLs in egg samples.
Sala-Vila et al.(169) developed a sensitive and precise method for determining PLs in
infant formulas and phospholipid sources of long-chain LC-PUFAs by NP-HPLC-ELSD.
The samples studied were an infant formula supplemented with LC-PUFAs, which derives
around 10% of its fat from egg PLs, and Ovothin® (Lueas Meyer, Hamburg, Germany),
which is used as a source of LC-PUFAs (mainly arachidonic acid (AA) and docosa-
hexaenoic acid (DHA)) in some infant formulas. Ovothin is a natural, highly viscous
mixture of egg PLs and egg oil that contains around 30% of PLs, mainly PC and PE. The
compounds (PC, PE, PI, PS, and SM) studied were extracted by dichloromethane–absolute
methanol and separated in less than 25 minutes using a silica column by isocratic elution
with a mixture of isopropanol–hexane–water. Several chromatographic conditions were
tried to optimize the method (solubilization of PLs, mobile phase, column and nebulization
temperature), whose suitability is shown by the detection limits obtained, linearity ranges,
and precision rates.
Cereals and Oils

Although present in small amounts in wheat, PLs have an important effect on the final tex-
ture of wheat-based products. Polar lipids such as PLs and glycolipids interact primarily
with the wheat gluten protein network that is the backbone of wheat flour dough mix-
ing properties and gas retention.(170) Interactions between gluten and PLs are sensitive
Table 4
Experimental conditions applied for the analysis of PLs in egg and egg-based products
Extraction/ ELSD
Dried egg PE, PI, PS, (1) Supercritical CO2 Luna silica gel column Gradient of Nebulizing gas: dried (166)
yolk PC, SM, extraction 250 × 4.6 mm ID CH3 Cl/MeOH/ and filtered
PG (2) MeOH/CH3 Cl/H2 O Flow rate: 1.6 mL/min 30% NH4 OH compressed air
(3) SPE: C8 cartridge Injection volume: 20 µL (80:19.5:0.5, Flow rate: 1.8 L/min
Recovery: MeOH v/v/v) to Evaporating T: 60◦ C
CH3 Cl/MeOH/
H2 O/30%NH4 OH
(60:34:5.5:0.5,
v/v/v)
23
Fresh egg PE, PC Multistep liquid–liquid Diol normal-phase silica Gradient of Nebulizing gas: N2 (167)
yolk extraction with ethanol, column 250 × 4.6 mm ID, CH3 Cl/MeOH/ Flow rate: 1.7 L/min
hexane, and precipitation with integral guard column. NH4 OH (80:19:1, Evaporating T: 60◦ C
with acetone Flow rate: 1.0 mL/min v/v), to
CH3 Cl/MeOH/
H2 O/30%
NH4 OH
(50:48:1:1, v/v/v)
Infant PE, PI, PS, MeOH/CH2 Cl2 Extrasilica silica 150 × 4.0 Isocratic elution Nebulizing gas: dried (169)
formulae, PC, SM mm ID with precolumn with isopropanol/ and filtered
Ovothin® Flow rate: 1.0 mL/min hexane/H2 O compressed air
T column: 35◦ C (55:37.2:7.8, Flow rate: 10 L/min
Injection volume: 20 µL v/v/v) Evaporating T: 85◦ C
PE = phosphatidylethanolamine; PI = phosphatidylinositol; PS = phosphatidylserine; PC = phosphatidylcholine; SM = sphingomyelin; PG = phosphatidylglycerol.
24 Restuccia et al.
to temperature and mechanical work and facilitate the ability of gluten to form the net-
work needed for effective dough formation. The extraction, separation, and quantification
of the main wheat flour PLs were carried out by Nèron et al.(171) Total lipids (2.0% dry
mass of wheat flour) were extracted from flour or dough by a mixture of chloroform–
methanol–water (1:1:1, v/v). PE, PC, PG, LPC, PI, N-acyllysophosphatidylethanolamine
(NALPE), and N-acylphosphatidylethanolamine (NAPE) were separated from the lipid
extract on a silica cartridge using an SPE procedure under vacuum. The recovery of the
lipid extract was 100%, whereas the SPE yield for the PLs was 50.0%. The resulting
fraction was then submitted to HPLC-ELSD on a diol stationary phase, allowing the sep-
aration and quantification of each class of PLs in less than 16 minutes using a gradient
of n-hexane/2-propanol/acetic acid/triethylamine (81.42:17.00:1.50:0.08, v/v/v/v) and
2-propanol/H2 O/acetic acid/triethylamine (84.42:14.00:1.50:0.08, v/v/v/v). The method
allowed the quantification of the PL amounts from eight wheat flours as well as their evo-
lution during mixing in the presence of phospholipase. For the eight flours, LPC was the
main phospholipid (29.0–48.0%) followed by PC (26.0–37.0%), NAPE (4.0–10.7%), and
NALPE (4.2–8.7%) in decreasing order, in agreement with Morrison(172) and Berger.(173)
Model doughs composed of water and wheat flour were then mixed with and without phos-
pholipase. Aliquots of dough were collected after 5, 15, and 60 minutes of mixing, and
their PL contents were analyzed. The amount of polar lipids extracted remained constant
during mixing without phospholipase. When 10 phospholipase units per mL (PLU) g−1
of Lecitase 10 L were added, 95% of NAPE and PC were hydrolyzed after 30 minutes
and only traces of these PLs were present after 60 minutes, and an increase in the lyso
forms of PLs (NALPE and LPC) was observed. A similar mixing experiment, which lasted
30 minutes, was carried out with 5 PLU g−1 of Lecitase 10 L added to the dough. In this
case, only 30.0% of the PC was hydrolyzed after 30 minutes of mixing with a concomitant
increase in LPC, whereas the amount of NAPE remained almost constant during mixing
(less than 10% hydrolyzed). Therefore, it was concluded that Lecitase 10 L is more efficient
on PC than on NAPE.
More recently, Pelillo et al.(174) determined the PL profile of lipids extracted from the
whole grains of 10 genotypes of tetraploid wheats (Triticum dicoccon Schrank and Triticum
durum Desf.) and 10 genotypes of hexaploid wheats (Triticum spelta L. and Triticum aes-
tivum L.). The first aim of the study was to determine the levels of different PL classes in
the lipid fraction of durum, soft wheats (Triticum durum Desf. and Triticum aestivum L.),
and their hulled ancestors (Triticum dicoccon Schrank and Triticum spelta L.). Lipids were
extracted using the Folch method, and PC, PE, PI, PG, and LPC were determined by
NP-HPLC-ELSD without further purification of the extracts. Gradients of chloroform–
methanol–30% ammonium hydroxide were used for elution and the total run time was 42
minutes. The data regarding the total PL amount were then compared with those derived
from spectrophotometric determination of organic phosphorus. The lipid amount was in
the range 2.5–2.7% (w/w) and lipids detected in ancient spelt were at levels about 7.0%
higher than those observed in common naked wheat, even though no consistent difference
was assessed. As for PL, they represented an important element in lipid matter, varying
from 5.0 to 17.0% of lipids. PLs represented about 10.0% of total lipids and were detected
in naked wheats at levels of 10.1 and 10.4% of lipids (T. durum and T. aestivum, respec-
tively), whereas in the corresponding ancestors T. dicoccon and T. spelta, PL amounts
were 8.4 and 9.1% of lipids, respectively. The PL amount in naked wheats was about
17.0% higher than that found on average in hulled wheats: 10.2 ± 2.8 vs. 8.7 ± 2.2%
of lipids, respectively. No significant difference was found between the data obtained by
HPLC-ELSD and organic phosphorus assay concerning total PL content. PE, PI, and PC
were the major PLs, accounting for more than 70.0% of total PLs, whereas PS and SM
were not detected. Finally, a first attempt to determine different molecular species for each
PL class was carried out by an HPLC system coupled on-line with a mass spectrome-
ter working in negative atmospheric pressure ionization-electrospray source mode. In this
case, although chromatographic conditions were optimized, the PC was poorly retained
and probably co-eluted with other nonretained species. The PE gave a broad band that par-
tially co-eluted with the L-PC band. Moreover, various unknown peaks were determined
and they could be attributed to low-molecular-weight LPL.
Soy lecithin, a general term for total soybean PLs, is widely used in the food, cosmetic,
and pharmaceutical industries as an emulsifier, lubricant, and release agent.(175) It is a
by-product of soybean oil processing; during degumming, water is mixed into soybean oil,
and PLs hydrate and settle out as gum. Thus, the physical stability of the degummed oil
is improved and lecithin is produced by further drying of the gum. PLs in soy lecithin are
approximately 55.3% PC, 26.3% PE, and 18.4% PI.(176)

Supercritical carbon dioxide is very effective in removing oils from a variety of seed
matrices, devoid of any appreciable amount of PL content.(177) However, the limited sol-
ubility of PLs in SC-CO2 leaves behind a potentially valuable by-product in the spent
seed matrix. The potential use of SC-CO2 -ethanol mixture for extraction and fractiona-
tion of PLs (PC, PE, PI, and PA) from defatted flaked soybean seeds in comparison with
liquid–liquid extraction has been investigated by Montanari et al.(177) An initial SC-CO2
extraction of soybean flakes was performed at 32 MPa and 80◦ C to extract the oil, leaving
the PLs in the defatted soybean flakes. A second step was performed using 10% ethanol,
varying the pressure from 16.6 to 68.9 MPa and the temperature from 60 to 80◦ C. For all
SC-CO2 -ethanol extractions, a fraction rich in PLs was obtained. A quantitative and qual-
itative analysis of PLs in soybean seeds, defatted soybean flakes, and different extracted
phospholipid fractions was carried out by NP-HPLC-ELSD to investigate combinations
of extraction pressure and temperature. A gradient of chloroform–methanol–30% ammo-
nium hydroxide was applied and each analysis lasted 34 minutes. The results showed that,
comparing the PLs present in soybean flakes and in defatted soybean flakes, no PLs were
extracted by supercritical fluid extraction with net CO2 . The highest amount of PLs was
extracted at 68.9 MPa, 74.8% of the total present in the defatted soybean flokes (DSF)
according to the extraction performed using the liquid–liquid extraction. At this pressure,
no enrichment of any PL was obtained and the relative percentages of PE, PC, PI, and
PA were the same (25.1, 68.8, 3.6, and 2.5%, respectively) as obtained using liquid–liquid
extraction. Decreasing the pressure decreased the amount of PLs extracted, but the rela-
tive percentage of each PL changed. In particular, at 19.4 MPa, PC represented 80.1% of
the total PLs extracted, whereas with liquid–liquid extraction it represented only 64.7%.
At 68.9 MPa, decreasing the temperature increased the amount of extracted PLs. At lower
pressures, reducing the temperature gave an overall reduction in the total extractable PLs
and the most interesting enrichment in PC was achieved at 40.7 MPa and 70◦ C.
Taylor et al.(178) proposed a new method in which a supercritical fluid extraction has
been combined with supercritical fluid chromatography in a preparative mode to develop
a system for fractionating and enriching high-value PLs (PE, PI, PA, PC) contained in
soyflakes. Soyflakes were initially extracted with neat carbon dioxide at 680 bar and
80◦ C to remove the available oil. The defatted flakes were then extracted with 15 mol%
ethanol-modified CO2 at 655 bar and 80◦ C to obtain the PLs. This phospholipid-enriched
extract was then delivered to the head of a chromatographic column containing neutral
alumina. The modifier was then changed to ethanol : water (9:1) and supercritical fluid
extraction was performed at 350 bar and 50◦ C, and fractions were collected at equal
26 Restuccia et al.
volume intervals of CO2 . The supercritical fluid extraction modifier was added, apply-
ing an increasing step-wise gradient to effect elution of the PLs. The resultant fractions
analyzed by NP-HPLC-ELSD in 30 minutes and using a gradient of chloroform–tert–butyl
methyl ether (75:25, v/v) and methanol–ammonium hydroxide–chloroform (920:70:10,
v/v/v) showed PL enrichment factors relative to the defatted soyflakes, from a 2.3-fold
(PI) to 19.9-fold (PA) increase depending on the individual PL.
Another study on the content of SL and PL during soybean seed development hypoth-
esized that the contents of PE, PI, PC, GluCer, and Cer would decrease during seed
development due to a reduction in the relative proportion of membrane components in
the seed.(179) Three soybean cultivars that differed in seed size and protein content were
selected for this study. IA1008 was a cultivar with the seed size and protein content of
conventional soybeans, IA1010 was a cultivar with the large seeds preferred for con-
sumption as a green vegetable, and IA1014 was a cultivar with large seeds and high
protein content for use in making tofu and other soy foods. Differences were consid-
ered in nine seed development stages as measured in days after flowering. Extraction was
accomplished by methanol : chloroform and separation was made with a column packed
with silica gel. NP-HPLC-ELSD was used for the chromatographic determination of each
class of PLs with a gradient of chloroform–methanol–30% ammonium hydroxide (80:19:1,
v/v/v) and chloroform–methanol–30% ammonium hydroxide–water (50:48:1:1, v/v/v/v)
in 42 minutes. Significant differences were observed for the PL percentage of the total lipid
in the seed development stages but not among cultivars. Averaged across cultivars, the PL
percentage in the total extractable lipid decreased from 9.1% at 28 days after flowering to
3.5% at 68 days after flowering as the neutral oil content increased from 81.5% at 28 days
after flowering to 92.9% at 68 days after flowering. The PL content on a dry weight basis
decreased by 44.4% from 28 to 68 days after flowering. Moreover, significant differences
for PL subclasses were observed among cultivars and seed development stages. PC was the
predominant PL component, followed by PE and PI in both immature and mature seeds.
Significant differences were also observed for Cer content among the three cultivars but not
for GlcCer. GlcCer and Cer contents decreased significantly during seed development. The
authors concluded that there was a significant decrease in SL and PL during seed devel-
opment, which supported the initial hypothesis. The results also indicated that immature
soybeans harvested at their heaviest fresh weight had high SL and PL contents.
The same authors(180) developed effective methods for the separation and quantifi-
cation of SL and to determine the relationship between palmitate and SL contents of
mature soybean seeds. Methods using column chromatography and NP-HPLC-ELSD
were developed to separate and quantify GlcCer and Cer in 15 soybeans lines in
which palmitate content ranged from 3.7 to 40.7%. Extraction and separation of SL
were achieved as previously reported.(181) Each analysis lasted 59 minutes, with dif-
ferent solvents in the gradient applied for the separation of Cer and GluCer (Table 5).
There were significant differences among the lines for GlcCer (83.4–397.6 nmol/g)
and major Cer contents (8.4–20.7 nmol/g) on a dry weight basis, whereas palmitate
content did not have a significant influence on GlcCer or Cer content in the soybean
seed because genetic factors seemed to be responsible for the significant differences
among lines for the two SL components. The PC, PE, PI, LPC, and LPI contents
of the 12 genetically modified (GM) soybean cultivar lines have been analyzed with
RP-HPLC-MS-MS by Lee et al.(182) Chloroform–methanol (1:1, v/v) was used for
extraction and a mobile phase of water–methanolic ammonium formate (5 mM and pH
4.0) was applied for chromatographic separation, which was carried out in 43 min-
utes. The LC-MS-MS method operating in the multiple reaction monitoring mode, with
Table 5
Experimental conditions applied for the analysis of PLs in cereals, oils, and oil by-product samples
Extraction/ ELSD
Wheat PE, PC, Successive additions of LiChrospher 100 Diol, Gradient of Nebulizing gas: (171)
flours PG, chloroform 10 mL (100 × 4 mm ID) n-hexane/2-propanol/ dried and filtered
LPC, containing butyl- Flow rate: gradient CH3 COOH/ compressed air,
PI, hydroxy-toluene from 0.8 to triethylamine 2.5 atm
NALPE, 0.01%, chloroform 1.5 mL/min. (81.42:17.00:1.50: Evaporating T: 50◦ C
NAPE 10 mL, and NaCl T column: 0.08, vol) and
SPE: Sep-Pak 50◦ CInjection 2-propanol/H2 O/acetic
normal-phase volume: 20 µL acid/triethylamine
cartridges (84.42:14.00:1.50:
Recovery: MeOH 0.08, vol)
Hexaploid, PC, PE, MeOH/CH3 Cl Luna silica 2 Gradient of CH3 Cl/ Nebulizing gas: (174)
27
tetraploid PI, PG, (250 × 4.6 mm ID) MeOH/30% dried and filtered
wheats, LPC with precolumn in NH4 OH compressed air,
and their silica (80/19.5/0.5, vol) 2 atm
respec- Flow rate: 1.5 mL/min and CH3 Cl/MeOH/ Evaporating T: 60◦ C
tive Injection volume: H2 O/30% NH4 OH
hulled 20 µL (60/34/5.5/
ancestors 0.5, vol)
Soybean PE, PI, (1) Supercritical Silica µPorasil Gradient of Nebulizing gas: (177)
flakes, PA, PC CO2 /ethanol (300 × 3.9 mm ID) CH3 Cl/MeOH/30% dried and filtered
defatted (2) Benzene/ethanol/ with precolumn in NH4 OH compressed air
soybean petroleum ether 1:1:1 silica (60:34:5.5:0.5, Flow rate: 3.5 L/min
flakes Flow rate: 1.0 mL/min vol), and Evaporating T: 80◦ C
Injection volume: CH3 Cl/MeOH/30%
10 µL NH4 OH
(80:19.5:0.5, vol)
(Continued)
Table 5
(Continued)
Extraction/ ELSD
Defatted PE, PI, Supercritical Licrosphere Si 60/11 Gradient of CH3 Cl/ Nebulizing gas: N2 (178)
soybean PA, PC CO2 /ethanol mixture (250 × 4 mm ID) tert-butyl methyl Flow rate: 1.7 L/min
flakes Flow rate: 0.7 mL/min ether (75:25, vol) Evaporating T: 90◦ C
T column: 35◦ C and MeOH/NH4
Injection volume: OH/CH3 Cl
15 µL (920:70: 10, vol)
Soybean PE, PI, MeOH/CH3 Cl Pholipidec silica Gradient of Nebulizing gas: N2 (179)
seeds PC, Separation with column column CH3 Cl/MeOH/30% Flow rate: 1.7 L/min
GluCer, packed with silica gel (250 × 4.6 mm ID) NH4 OH (80:19:1, Evaporating T: 55◦ C
Cer Recovery with guard column vol) and
28
CH3 Cl/MeOH (SL), (20 × 4 mm) CH3 Cl/MeOH/30%
MeOH (PL), and Flow rate: 1.0 mL/min NH4 OH/H2 O
MeOH/H2 O (PL) Injection volume: (50:48:1:1, vol)
10 µL
Soybean GluCer, MeOH/CH3 Cl Cer: Chromegasphere Cer: gradient of Cer: nebulizing gas: (180)
seeds Cer Separation with Si60 column hexane, and N2
column packed with (150 × 3.2 mm ID) 2-propanol/ethyl Flow rate: 0.8 L/min
silica gelRecovery GluCer: (155) acetate/88% Evaporating T: 60◦ C
CH3 Cl/MeOH (SL) HCOOH GluCer: nebulizing
(50:50:0.5, vol) gas: N2
GluCer: gradient of Flow rate: 1.4 L/min
hexane/THF Evaporating T: 60◦ C
(99:1, vol) and
MeOH/methyl
tert-butyl ether
(75:25, vol)
Crude and PE, PI, Oil fractionation on a Normal-phase column, Linear gradient Not reported (184)
degummed PC, PA, silica gel Lichrosorb Si-60 elution
oils from columnRecovery: (250 × 4.6 mm ID from CH3 Cl/
soybeans MeOH and 0.1% Flow rate: 1.0 mL/min tetrahydrofuran
subjected phosphoric acid in Injection volume: (1:1, vol) to
to storage MeOH 10 µL MeOH/30%
at high NH4 OH/H2 O
moisture (92:7:1, vol)
content
Soybean PC, PE Oil fractionation on a AsahiPak with octa Isocratic elution with Not reported (186)
and silica gel decanoyl ACN/MeOH/water
canola oil columnRecovery: p(vinylalcohol) (47.5:47.5:5)
29
MeOHl and 0.1% (ODPVA) packing
phosphoric acid in 250 × 4.6 mm ID
MeOHl Flow rate: 1.0 mL/min
Injection volume:
10 µL
Lecithins PE, PI, Extrusion-expelling Silica column Gradient of CH3 Cl/ Nebulizing gas: N2 (189)
from four PC and conventional (250 × 2.1 mm ID) MeOH/ NH4 OH Flow rate: 1.6 L/min
GM solvent extraction Flow rate: 0.3 mL/min (80:19.5:0.5, vol) Evaporating T: 50◦ C
soybeans with hexane and CH3 Cl/
and one MeOH/ H2 O/
commod- NH4 OH
ity (60:34:5.5:0.5,
soybean vol)
(Continued)
Table 5
(Continued)
Extraction/ ELSD
Soybean PE, PC, Acetone to obtain Silica column Ternary gradient of Nebulizing gas: N2 (192)
degummed PI, SM, lecithins; then ethanol (4.6 × 250 mm ID) (A) hexane, (B) Flow rate: 2.0 L/min
oil LPC and separation with Flow rate: gradient isopropanol, and Evaporating T:
residue silica gel preparative from 0.8 to (C) H2 O 63.5◦ C
chromatographic 1.2 mL/min
column T column: 35◦ C
Recovery:
hexane/isopropanol/
H2 O (1:1:0.175, vol)
30
Palm- PE, PG, Hexane or 95% LiChrosorb Gradient of hexane, Nebulizing gas: (195)
pressed PC, PA, ethanolSPE: Florisil (250 × 4.6 mm ID) 2-propanol/CH3 Cl dried and filtered
fiber Recovery: MeOH Flow rate: 0.9 mL/min (4:1, v/v), and compressed air
Injection volume: 2-propanol/H2 O Flow rate: 1.5 L/min
100 µL (1:1, vol)
Palm- PE, PG, MeOH/CH3 Cl SPE: Silica Lichrocart Si 60 Gradient of Nebulizing gas: N2 (196)
pressed PC, (1) Silica SPE (250 × 4.6 mm ID) hexane/isopropanol/ Flow rate: 2.0 L/min
fiber LPC Recovery: MeOH, then column H2 O (53:42:5, Evaporating T:
a mixture of MeOH Flow rate: gradient vol), 63.5◦ C
and from 0.5 to hexane/isopropanol/
MeOH/CH3 Cl/H2 O 0.8 mL/min Injection H2 O (36:54:10,
(3:5:2, vol) volume: 20 µL vol), and
hexane/isopropanol/
H2 O (17:66:17,
vol)
(2) Aminopropyl SPE

Recovery: 1 mL MeOH
(four times)
(3) Diol SPE
Recovery: MeOH
containing ammonia
solution
Palm- PE, PC Ultrasonic Merck Silica Lichrocart Gradient of Nebulizing gas: N2 (197)
pressed extractionSPE: Diol Si 60 (4.6 × 250 mm hexane/isopropanol/ Flow rate: 2.0 L/min
fiber column ID) column H2 O (53:42:5, Evaporating T:
31
Recovery: MeOH Flow rate: gradient vol), 63.5◦ C
containing ammonia from 0.5 to hexane/isopropanol/
solution 0.8 mL/min H2 O (36:54:10,
Injection volume: vol), and
20 µL hexane/isopropanol/
H2 O (17:66:17,
vol)
PE = phosphatidylethanolamine; PI = phosphatidylinositol; PC = phosphatidylcholine; SM = sphingomyelin; LPC = lysophosphatidylcholine; GluCer =
glucosylceramide; LacCer = lactosylceramide; PA = phosphatidic acid; PG = phosphatidylglycerol; Cer = ceramide; NALPE
= N-acyllysophosphatidylethanolamine; NAPE = N-acylphosphatidylethanolamine.
32 Restuccia et al.
two transitions monitored for each PL, provided good selectivity and sensitivity for
determination of PLs. In the LC, the ammonium formate in methanol (0.1 M) was essen-
tial to obtain good peak separation with minimum tailing. The best results in terms of
sensitivity were always obtained with ESI in the positive ionization mode, using the
[M+H]+ as the precursor ion for PC, PE, and PI. LPC and LPI were detected as their
[M+Na]+ ; moreover, ammonium formate suppressed the production of adduction ions
such as [M+Na]+ and [M+NH3 ]+ . The method validation showed good results in terms
of linearity, limit of determination, limit of quantification, and recovery values. The appli-
cation of the optimized method to real samples showed that the average content of total
PLs in the 12 soybean cultivars was about 75.7 mg/kg. The PC content in all of the soy-
beans investigated was much greater than the other PL types, ranging from 16.1 ± 1.1 to
49.0 ± 0.2 mg/kg. The LPC content of the PLs investigated was lowest in all 12 cultivars,
ranging from 0.1 to 1.3 mg/kg. No LPI was detected in the studied cultivars.
Le Grandois et al.(168) noted that fragmentation of dithiated adducts allowed for the
identification of fatty acids linked to the glycerol backbone in soybean samples. It was
found that soy PC was particularly rich in species containing essential fatty acids, such
as (18:2–18:2) PC (34.0%), (16:0–18:2) PC (20.8%), and (18:1–18:2) PC (16.3%). Soy
lecithin, mainly made of PC and already in use as a food additive and a nutritional com-
plement, was therefore shown to be highly rich in essential FAs but not in their LC-PUFA
metabolites.
PA, PC, PE, PI, PS, NAPE, lysophosphatidic acid (LPA), LPC, lysophos-
phatidylethanolamine (LPE), lysophosphatidylinositol (LPI), and SM were determined
qualitatively and quantitatively in eight commercial lecithins by TLC, HPLC, and
31
P-NMR.(183) NP-HPLC-UV with a mobile phase of 2-propanol/n-hexane/aqueous
formic acid (0.1% w/w) was used. For the detection of PL classes, the eluent of the HPLC
was transferred into an electrospray ionization MS running in the APCI mode and ana-
lyzing negatively charged ions. The total amounts of PLs, as well as the amounts of PL
classes, were comparable but depended on the method used for quantification, although the
ratio of the phospholipid classes was comparable for each of the three methods. In most of
the samples, PC was the major phospholipid, followed by PI and PE with concentrations
that were in the same range. Within the PLs, PA had the lowest amount and LPL and PE
derivatives were only minor components. HPLC-UV showed low selectivity, because LPL,
except LPE, could not be determined. Moreover, HPLC quantification gave substantially
higher amounts of PC. This might be due to the broad HPLC signal, possibly including
further compounds, and the low selectivity of the HPLC method due to the UV detection
at wavelengths between 203 and 210 nm. Unfortunately, this poor selectivity may disturb
the quantitative assay, leading to differences in the amounts of particular phospholipids in
comparison to those obtained by the other methods.
Soybean oil can be extracted by two methods: mechanical pressing, such as extrusion-
expelling, and solvent extraction. Solvent extraction processing uses hexane and is a
common practice for conventional oil processing plants. Extrusion-expelling technology
uses a dry autogenous extruder that generates pressure and heat, disrupting the cellular
structure of the seed. A screw press is then used to press the oil out. Mounts et al.,(184) char-
acterized the PL fraction (PE, PI, PC, PA) isolated from crude soybean oils extracted by
solvents from soy flakes stored at high moisture content and their degummed oils prepared
by laboratory simulations of commercial degumming procedures. An NP-HPLC-ELSD
35-minute analysis, with linear gradient elution of chloroform–tetrahydrofuran (1:1, v/v)
and methanol–30% ammonium hydroxide–water (92:7:1, v/v) showed peak shapes and
phospholipid elution profiles that were noticeably better than those previously reported
with UV detection.(185) Comparing analyses of the PL from unaged 10.0% and 14.0%
moisture soy flakes indicated that 10.0% of the PL was decomposed during the ini-
tial extraction process. After 10 days’ storage, nearly 40.0% of the PL was destroyed.
A decrease in the total phosphorus content of crude oil is indicative of PL destruction
through the action of phospholipases. Analysis of the phospholipid classes indicated that
PE and PC decreased from 41.1 and 31.9% to 12.9 and 12.3%, respectively, whereas
PA increased significantly, both initially and during storage. PC and PE were separated
in soybean and canola oils, after oil fractionation on a silica gel column, on an octade-
canoyl poly(vinyl alcohol) column by reverse-phase HPLC with UV and ELSD.(186) The
HPLC-UV analysis of the polar lipids yielded components with peak intensities some-
what different from those obtained by HPLC-ELSD despite a discernible similarity in
the peak profiles observed in the two detection systems. The application of the octade-
canoyl poly(vinyl alcohol) column to the HPLC-ELSD for the analysis of PC and PE in
vegetable oil samples derived from various genetically modified oil seeds confirmed the
potential utility of the new column technique in practical sample assays. The distribu-
tion patterns of PL molecular species varied considerably among different oil samples.
From reported data, it is noteworthy that compositions of some corresponding molecu-
lar species were quite different in PC and PE derived from the same sample. The HPLC
method developed in this study was particularly useful in the PL analyses of canola oils
due to the low concentrations of PC and PE in these oils (PL contents of canola oils are
generally about three times lower than those of soybean oils).(186) More recently, Harrabi
et al.(187) applied LC-ESI-MS for the analysis of glyceropospholipids (PA, PE, PG, PC,
and PI) in sweet and dent corn oils. The Folch method was used for lipid extraction and
the PL fraction was separated by hydration with 3% water and precipitation with acetone
at 0◦ C. LC analysis (40 minutes total run time) was performed using a diol column; the
binary solvent gradient consisted of a solvent mixture of hexane–isopropanol–acetic acid–
triethylamine (82:17:1:0.08, v/v/v/v) and isopropanol–water–acetic acid–triethylamine
(85:14:1:0.08, v/v/v/v). PC was found to be the most abundant class of phospholipid,
accounting for 57.0–68.0% of the total. PI (14.5–19.7%) and PE (10.2–13.8%) ranked sec-
ond and third, respectively; PA and PG were the minor classes, accounting together for less
than 10.0% of the total phospholipids. Various molecular species within each class were
detected; in particular, the most abundant PC species contained polyunsaturated fatty acids
(C18:2).
The PL class profile of lecithin could be changed by different oil extraction
methods.(188) To this end, Wu and Wang(189) determined whether and how soybean
lecithins from the two oil processing methods, as well as from the genetically modified soy-
bean seeds, were different with regard to their PL class (PE, PI, PC) and FA compositions.
Crude oil extracted by extrusion-expelling and SE methods from the five types of soybean
seeds was filtered and then 3% of water was stirred into the oil to hydrate the PL. The mix-
ture was then centrifuged and the oil and gum were separated. Crude gum obtained from
degumming contains a high proportion of neutral oil soluble in acetone, whereas PLs are
insoluble,(190) so acetone treatment was used to de-oil the crude lecithins.(191) NP-HPLC
with an ELSD analysis with a gradient of chloroform–methanol–30% ammonium hydrox-
ide (80:19.5:0.5, v/v/v) and chloroform–methanol–water–30% ammonium hydroxide
(60:34:5.5:0.5, v/v/v/v) demonstrated that the relative proportions of PL classes for five
soybean seeds and two extraction methods were different. Extrusion-expelling lecithin con-
tained significantly more PC and less PE than SE lecithin. Extrusion-expelling processing
may result in a superior PL profile of lecithin. Statistical analysis also showed that soybean
seed type significantly affected the PL composition. The PC and PE contents of commodity
34 Restuccia et al.
soybean and lipoxygenase-free soybean lecithin obtained from both extrusion-expelling

and solvent extraction were similar for two of the seeds, but they were significantly differ-
ent from those of the other three seeds (low-linolenate soybean, high-oleate soybean, and
low-saturates soybean). Apparently, the genetic modification of lipoxygenase-free soybean
did not affect relative PC and PE contents, probably because the lipoxygenase-free soybean
seeds were modified to remove the lipoxygenases. The PC contents of low-linolenate soy-
bean, high-oleate soybean, and low-saturate soybean from both extrusion-expelling and
solvent extraction lecithins were all higher than extrusion-expelling and solvent extraction
lecithins from commodity soybean, whereas the PE contents were all lower. It appears
that the genetic modification of certain seeds would not only improve the oxidative sta-
bility of their oils but also increase the PC content in their lecithins, adding value to their
lecithin products. Finally, the critical micelle concentration was also determined to examine
the functionality of soy lecithin. Statistically, there were no correlations between critical
micelle concentration and the composition of individual PL class and FA.

Most of the PLs used in industry are obtained from a by-product of vegetable oil (soy-
bean degummed oil residue and palm-pressed fiber). An HPLC method with ELSD for
the separation and accurate quantification of the most important soybean PLs in soybean
degummed oil residue was described by Zhang et al.(192) This method is based on normal-
phase chromatography with 5-mm silica gel as stationary phase and a ternary gradient with
n-hexane, isopropanol, and water as mobile phase. Not only neutral lipids, PE and PI, but
also SM and LPC were well resolved from PC. The method has good repeatability and
accuracy from the viewpoint of high recovery and low coefficients of variance in retention
time and peak area for each phospholipid. In addition, a comparison between three detec-
tors was made. The UV-absorption of a hexane–isopropanol–water mobile phase system
at 203–210 nm decreases the response of the UV detector. Using the RI detector, accurate
quantification of the PE is difficult, due to the partial overlapping of the solvent mixture,
neutral lipids, and PE peaks, leading the authors to conclude that ELSD can be considered
the best in the HPLC analysis of natural PLs.
The increased production of oil palm also resulted in an increased production of
by-products as well as waste. Palm-pressed fiber constitutes about 15% by weight of the
fresh fruit bunches(193) and it has been reported that large amounts of PLs still remain
in palm-pressed fiber and sludge.(194) The high level of PLs remaining in palm-pressed
fiber is understandable because, unlike seed oils, which are solvent-extracted, palm oil is
mechanically extracted without solvents and is separated from an aqueous slurry during
milling. Thus, it can be expected that considerable amounts of PLs still remain in the fiber.
Choo et al.(195) studied the recovery of PLs (PE, PG, PC, PA) from palm-pressed fiber.
Samples were extracted successively with hexane and 95% ethanol and purified by Florisil
(Sigma Chemical Company, St. Louis, Mo), obtaining a concentrate of PLs based on their
different solubility in hexane and ethanol. The extracts were analyzed by HPLC-ELSD
with a gradient of hexane, 2-propanol/chloroform (4:1, v/v), and 2-propanol/water (1:1,
v/v), showing a concentration of 46.8 mg/kg of PLs in the ethanolic extract, whereas only
1.4 mg/kg of PLs and mostly neutral lipids were present in the hexane extracts. The PL
composition was found to be predominantly PC, PE, PG, and PA. PC, PE, and PG were
also found in crude palm oil,(194) whereas PI, another major component of PLs previously
found in crude palm oil, was not observed. The major PLs of ethanol extract were some-
what similar to those of soybean, which consists of 35.0–46.0% PC and 25.0–27.0% PE,
whereas the hexane extract contained a higher amount of PC. A noticeable amount of PA in
ethanolic extract is likely due to hydrolysis during palm oil milling or to prolonged heating
during the removal of solvents by rotary evaporation.
More recently, Chua et al.(196) evaluated the best SPE method to obtain PE, PG,
PC, and LPC from palm-pressed fiber extracted by the Folch method, to be successively
analyzed using HPLC-ELSD. Different solvent phases and normal phase SPE cartridges
(silica, aminopropyl-bonded silica, and diol-bonded silica) at the same ratio of extract–
sorbent mass were used to study the separation of PL in a model oil system. The diol
phase cartridge gave the best qualitative and quantitative PLs recovery and was there-
fore selected for the SPE purification of PLs in palm-pressed fiber. An NP-HPLC-ELSD
analysis was performed with a gradient of n-hexane/isopropanol/water (53:42:5, v/v/v),
hexane–isopropanol–water (36:54:10, v/v/v) and hexane–isopropanol–water (17:66:17,
v/v/v) over 65 minutes. Values of 9.0, 2.2, 7.5, and 0.7 g/kg were found for PE, PG,
PC, and LPC, respectively. The same authors(197) developed a central composite design
to study the effect of ultrasound-assisted extraction conditions (amplitude 10–90%, cycle
0.1–1.0 W/s, and sonication time 5–30 minutes) on the extraction yield of PLs from
palm-pressed fiber; SPE purification and NP-HPLC-ELSD analysis were the same as the
previous study.(196) Overall extraction efficiency and individual extraction yield of PE and
PC were considered as response variables. The obtained results showed that the extraction
efficiency of PL increased with increasing sonication time and decreasing amplitude and
cycle. The optimum operating ultrasound condition, resulting in maximum PL extraction
efficiency, was found to be provided by 30 minutes’ sonication time, 20% of amplitude,
and 0.2 W/s cycle with a solid–liquid ratio of 1:4 (g/mL). In optimum conditions, the
response values obtained for overall extraction efficiency and individual extraction yield
of PE and PC were 110.0, 12.6, and 5.4 mg/g, respectively. Moreover, from the SEM
microscopic images, ultrasound-treated palm-pressed fiber showed more fractured sections
on the surface structure when compared to palm-pressed fiber without ultrasound treat-
ment. This observation confirmed that ultrasonic treatment caused the vegetal cell walls to
disaggregate and rupture, thereby facilitating the extraction of PL from palm-pressed fiber.
Table 5 summarizes the experimental conditions for the HPLC-ELSD determination
of PLs in cereals and oils.
Conclusions
The critical factors in the analysis of PLs in food products are the methods of extrac-
tion, separation, and detection because the majority of PLs are present in membranous
structures, interacting with compounds of a complex food matrix. The standard extrac-
tion methods, such as Rose-Gottlieb and Werner-Schmidt, are used extensively for food
products. Because these methods use a base or acid in combination with heat, they can
lead to the oxidation and hydrolysis of PL. Therefore, the extraction procedures by using
chloroform–methanol or n-hexane-isopropanol mixtures should be alternatively employed.
The analysis of relatively low concentrations of PL (with particular reference to milk and
oils) requires concentration and fractionation prior to HPLC. Solid–liquid column chro-
matography or SPE can be used to concentrate the PLs. In general, SPE cartriges with
silica, aminopropyl-bonded silica, diol-bonded silica, C8 and C18 as stationary phase are
the most popular in food analysis because they provide more reproducible results and
cleaner extracts for instrumental measurement. However, even these techniques do not
ensure the complete purification of PL from less polar compounds such as partial glyc-
erides and free fatty acids. In recent years, HPLC coupled to ELSD has become a standard
methods for the determination of PLs in food matrices. ELSD affords advantages over UV
detection, RI, and flame ionization in that with it there is no baseline drift. Moreover, it is
not sensitive to the flow rate of the solvent and quantifies any solute that is less volatile than
36 Restuccia et al.
the solvent. However, the droplet size (and thus the response) is highly dependent on the
flow of the nebulizing gas, temperature of the evaporating tube, flow rate, and composition
and physical characteristics of the mobile phase. Therefore, working conditions should be
optimized to ensure the highest possible detector sensitivity and should be reproduced rig-
orously each time. Moreover, HPLC-ELSD enables a quantification of different PL classes
but fails to determine different PL molecular species. The fatty acid composition of the PL
fraction is highly related to food quality because it influences important parameters of cell
membranes. With regard to this, the high specificity and separation power of HPLC-MS
for PL fatty acid profile can be employed to determine PL molecular species in foods (in
particular, meat and cereals).
Future Trends
PLs possess nutritional and therapeutic properties as well as technological features that are
very promising in the food sector. From a nutritional point of view, the detailed quantita-
tive determination of PLs in various food products could be of great importance for the
creation of a food PL database. As far as food technology is concerned, because many
natural and processed foods are dispersions or have been in the dispersed state at some
time during their formation (i.e., bakery, confectionery, meat products, ice cream, dress-
ings, and/or new products such as low-fat and instant foods, high- or low-alcohol food
formulations, and functional foods), interest in PL analysis could be related to their abil-
ity in forming self-assembling supramolecular structures. This is a key component of the
bottom-up nanoscience approach for the separation of immiscible phases vital for food dis-
persions and cosmetic formulations. It follows that this synergy between physiology and
technology justifies the interest in PLs for the formulation of functional foods.
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Determination of Phospholipids in Food Samples

Article in Food Reviews International · January 2012

Donatella Restuccia Giancarlo Spizzirri

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Francesco Puoci Giuseppe Cirillo

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Determination of Phospholipids in Food Samples

DONATELLA RESTUCCIA1, U. GIANFRANCO SPIZZIRRI1,

Roma, Rome, Italy

Keywords phospholipids, HPLC, ELSD, food analysis

Address correspondence to Donatella Restuccia, Dipartimento di Scienze Farmaceutiche,

Figure 1. Molecular structure of different phospholipid classes.

backbone is acylated. 1-Lysophospholipids maintain the acyl chain in position 2, whereas

plasma membrane.(15) Finally, endogenous and exogenous oxidation of phospholipids can

mass spectrometers. Moreover, coupling an electrospray ionization (ESI) source with

In recent years, HPLC coupled to an evaporative light-scattering detector (ELSD)

ELSD Principles and Applications

Advantages of ELSD over

Limitations of ELSD Refractive index Mass spectrometry

volatile mobile phases

Figure 2. Schematic representation of the evaporative light-scattering detector. (color figure

log A = b · log m + log a (2)

A1/b = a1/b · m = k · m (3)

pharmaceuticals,(87–92) foods and beverages,(38,54),(71,93–96) natural products, biological

Analysis of Food PLs by HPLC-ELSD

Milk and Dairy Products

(2) Preconcentration and

ratios of solutions: n-hexane/ 2-propanol (3:2, v/v), n-hexane/2-propanol/25 M ammo-

to determine whether the egg could be a new resource of docosahexaenoic acid–containing

yolk lecithin was more oxidatively stable than soybean lecithin.

Cereals and Oils

approximately 55.3% PC, 26.3% PE, and 18.4% PI.(176)

(2) Aminopropyl SPE

soybean and lipoxygenase-free soybean lecithin obtained from both extrusion-expelling

micelle concentration and the composition of individual PL class and FA.

36. Christie, W.W. Separation of phospholipid classes by high-performance liquid chromatography.

Analytical Chemistry 1978, 50, 1414–1420.

chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS2) of molec-

Céréales; Lavoisier Tec. Doc.: Paris, France, 1991.

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