Journal of Chromatography A, 1150 (2007) 241251

Simultaneous analysis of glycolipids and phospholids molecular

species in avocado (Persea americana Mill) fruit
Deborah Pacetti a , Emanuele Boselli a , Paolo Lucci b , Natale G. Frega a,
a

Dipartimento di Scienze degli Alimenti, Universit`a Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy
Dottorato di Ricerca in Alimenti e Salute, Universit`a Politecnica delle Marche-A.C.R.A.F. Gruppo Angelini, Ancona, Italy
Available online 30 October 2006

Abstract
The molecular species of phospholipids (PLs) and glycolipids (GLs) were simultaneously characterized in the pulp and almond of the avocado
fruit (Persea americana Mill) of four varieties by means of high performance liquid chromatographyelectrospray ionisation ion-trap tandem
mass spectrometry. In the pulp, the predominant species of monoglycosyldiglycerides (MGD) were m/z 796.6 (oleic/linolenic and linoleic/linoleic
acids) and m/z 800.4 (stearic/linoleic and oleic/oleic acids). One of the main diglycosyldiglycerides (DGD) both in the pulp and almond was
m/z 958.5 (oleic/linolenic); however, the pulp was also rich of m/z 962.4 (oleic/oleic), whereas in the almond, m/z 934.5 (palmitic/linoleic and
palmitoleic/oleic) and m/z 960.5 (oleic/linoleic and stearic/linolenic) were more abundant. In the almond, the main PL classes (phosphatidic
acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI)) contained always palmitic/linoleic acids.
-Linolenic acid was contained as MGD (linolenic/linolenic) and DGD (linolenic/linolenic), more present in the pulp than in the almond.
The major molecular species of glycocerebrosides (GCer) in the pulp and almond carried hydroxy-palmitic acid (C16h:0 )/4,8-sphyngadienine
(d18:2 ).
2006 Elsevier B.V. All rights reserved.
Keywords: Persea americana Mill; Avocado fruit; High performance liquid chromatography; Mass spectrometry; Phospholipids; Glycolipids

1. Introduction
Glycolipids (GLs), phospholipids (PLs) and their breakdown
products are important components in the cell membranes of
animals and plants and are emerging as important second messengers for various cellular processes, such as cell cycle arrest,
differentation, senescence, apoptosis and others [14]. Glycosphingolipids and sphingomyelin together with cholesterol
are major components of specialised membrane microdomains
known as lipids rafts, which are involved in receptor aggregation and immune responses [511]. Moreover, GLs are naturally
occurring non-ionic surface active agents that have a wide variety of applications in the food, cosmetic, pharmaceutical and
agricultural industries. In many pharmaceutical preparations,
GLs have been used as stabilizers for mutually incompatible
ingredients in emulsions, when the latest cannot be successfully

Corresponding author. Tel.: +39 0712204924; fax: +39 0712204980.

E-mail address: n.g.frega@univpm.it (N.G. Frega).

0021-9673/$ see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2006.10.022

combined otherwise [1214]. In addition, the biocompatibility

of GLs derivates can also be used to synthesize niosomes that are
similar to natural liposomes of phospholipids assembly. These
glycolipids microspheres are highly versatile and have been used
in the encapsulation, transport and controlled release of flavours,
food products, agriculture and aquaculture products, human biologicals and pharmaceuticals, cosmetics and other personal care
products [15,16]. The increased commercial use of polar lipids
as novel food ingredients [17] and constituents of drugs has
resulted into working out of specific analytical methods to separate and identify the phospholipids added to drugs from the
endogenous phospholipids in blood, in order to perform pharmacokinetic and toxicokinetic analyses as part of product documentation [18]. Analyses of the different classes and species
of PLs and GSLs in biological material are thus an important
issue both for basic research and for the pharmaceutical industry
[1924].
The fruit of avocado (Persea americana Mill) is one of
the most important natural sources of monounsaturated food
lipids and essential fatty acids (-linoleic and -linolenic acid)

TL, total lipids; NL, neutral lipids; PoL, polar lipids (sum of glycolipids and phospholipids); SD, standard deviation; Cn:m = fatty acid (n = carbon number; m = number of double bonds); n.d., less than 0.1%; C14:0 , miristic acid; C15:0 , pentadecanoic
acid; C16:0 , palmitic acid; C16:1 , palmitoleic acid; C18:0 , stearic acid; C18:1 , oleic acid; C18:2 , linoleic acid; C18:33 , -linolenic acid; C20:1 , eicosenoic acid; C20:26 , eicosadienoic acid; C20:3 , eicosatrienoic acid; C24:0 , lignoceric acid.

n.d.
n.d.
20.2 1.0
5.0 0.5
2.9 0.3
46.7 0.5
18.0 0.5
7.1 0.2
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
23.7 0.6
9.9 0.1
0.3 0.3
49.6 0.6
15.3 0.5
1.0 0.1
0.1 0.1
n.d.
n.d.
n.d.
n.d.
n.d.
23.2 0.0
9.7 0.3
0.5 0.1
49.8 0.7
15.6 0.0
1.1 0.1
0.1 0.1
n.d.
n.d.
n.d.
n.d.
n.d.
16.5 1.1
4.0 0.7
2.1 0.3
46.1 2.4
24.3 1.8
7.0 0.9
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
25.0 0.6
9.5 0.1
0.5 0.0
51.0 0.3
13.1 0.2
0.8 0.0
0.1 0.1
n.d.
n.d.
n.d.
n.d.
n.d.
25.4 0.1
9.7 0.0
0.5 0.0
50.3 0.0
13.2 0.1
0.8 0.0
0.2 0.0
n.d.
n.d.
n.d.
n.d.
n.d.
14.9 1.1
2.3 0.3
1.0 0.2
61.1 0.1
14.4 1.5
6.3 0.2
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
17.5 0.3
6.5 0.3
0.5 0.1
65.3 0.8
9.4 0.3
0.6 0.1
0.2 0.0
n.d.
n.d.
n.d.
n.d.
n.d.
17.4 0.3
6.3 0.0
0.5 0.0
65.8 0.3
9.3 0.0
0.5 0.0
0.2 0.0
n.d.
n.d.
n.d.
n.d.
n.d.
17.7 1.5
2.3 0.2
1.1 0.2
49.2 1.3
22.8 0.2
6.9 0.4
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
22.2 0.1
8.0 0.1
0.5 0.0
53.3 0.0
15.3 0.1
0.5 0.0
0.2 0.1
n.d.
n.d.
n.d.
n.d.
n.d.
22.1 0.4
8.0 0.1
0.5 0.0
53.3 0.1
15.4 0.2
0.5 0.0
0.1 0.0
n.d.
n.d.
n.d.
n.d.
n.d.
22.0 1.4
1.1 0.2
2.1 0.1
19.5 0.6
49.9 0.8
4.8 0.0
0.7 0.1
n.d.
n.d.
n.d.
1.9 0.1
0.9 0.0
18.6 1.5
2.8 0.3
2.0 0.2
23.9 2.1
38.0 2.1
7.3 0.5
0.7 0.0
0.7 0.1
1.0 0.5
2.2 0.9
1.2 0.3
0.9 0.0
23.6 0.4
2.2 0.2
2.0 0.1
22.7 2.3
36.4 3.0
5.9 0.3
0.7 0.0
0.5 0.2
1.2 0.2
2.7 0.3
0.7 0.0
0.5 0.2
15.2 0.4
2.4 0.0
1.9 0.1
25.8 0.3
44.9 0.4
5.1 0.2
0.8 0.0
n.d.
n.d.
2.7 0.5
0.8 0.2
0.4 0.0
21.7 1.2
1.7 0.2
1.0 0.1
32.0 1.4
37.2 1.8
4.2 0.0
0.8 0.2
0.2 0.2
n.d.
n.d.
C14:0
C15:0
C16:0
C16:1
C18:0
C18:1
C18:2
C18:3 3
C20:1
C20:2 6
C20:3
C24:0

n.d.
n.d.
21.3 1.2
0.8 0.0
1.3 0.2
22.4 1.4
51.0 1.6
2.5 0.4
0.7 0.2
n.d.
n.d.
n.d.

NL
Pinkerton

TL
PoL
NL

Rincon

TL
PoL
NL

Hass

TL
PoL
NL
TL

Reed

PoL
NL
TL

Hass

PoL
NL

The lyophilized fruit pulp (500 mg) was homogenized with

5 ml of acetone. After filtration on filter paper, the residue was
re-extracted with 5 ml of chloroform/methanol (2:1, v/v). The
acetone and chloroform/methanol extracts were then combined
and dried in a rotary evaporator.
The lipids from the almond of avocado fruits were extracted
using the method of Bligh and Dyer [30] with slight modifications: the almond (10 g) was finely ground in a mill and mixed
with 60 ml acetone; after extracting for 1 h at room temperature with intermittent mixing, the suspension was filtered and
the residue was re-extracted with 60 ml of chloroform/methanol
(2:1, v/v). The acetone and chloroform/methanol extracts were
then combined and dried using a rotary evaporator. The addition of 10 ml of chloroform, 10 ml of methanol and 9 ml of KCl
0.12 M resulted in a biphasic system with a final ratio chloroform/methanol/water of 1:1:0.9 (v/v/v). After separation, the
lower phase, containing the lipids (TL), was vacuum dried.

TL

2.2. Extraction of total lipids (TL)

Reed

HPLC-grade methanol, chloroform and water were purchased from Lab-Scan Analytical Sciences (Dublin, Ireland).
All other reagents were of analytical grade. Drupes of Persea
americana Mill, var. Hass, Pinkerton, Reed and Rincon were
supplied by a local distributor. Monogalactosyldiacylglycerols
(MGD) and digalactosyldiacyglycerols (DGD) from whole
wheat flour and PLs standards (purity greater than 99%), including phosphatidylethanolamine (PE), phosphatidylcholine (PC),
phosphatidylinositol (PI), N-palmitoyl-sphingomyelin and
1-oleoyl-glycero-3-phosphocholine (lysophosphatidylcholine,
LPC) were purchased from Sigma (St. Louis, MO, USA).

FAME%

2.1. Materials

Pulp

2. Experimental

Almond

[25,26]; the avocado oil is often seen as a substitute of olive oil

due to the similar composition in total fatty acids [27]. Since
the oil content reaches up to 20% in the pulp, the plant has been
cultivated since at least 10,000 years in Central America for its
food, medical and cosmetic applications [28]. The lipid fraction
of the pulp is rich of polar lipids, such as glycolipids and phospholipids [29]. Little is known on the polar lipid composition
of the almond, which is not taken in consideration for the oil
extraction.
The main objective of this study was to develop an effective
method for the simultaneous characterization of GLs and PLs
molecular species in pulp and almond of avocado fruit, as a
starting point for the development of new applications in the
cosmetic, nutraceutical and pharmaceutical industry. The use of
high performance liquid chromatography (HPLC) coupled online with ion-trap mass spectrometry of second order (MSMS)
was a very useful approach to increase the specificity of the GLs
and PLs analysis.
To our knowledge, this is the first time that the differences
between the almond and pulp polar lipids were discussed in
different varieties of avocado.

PoL

D. Pacetti et al. / J. Chromatogr. A 1150 (2007) 241251

Table 1
Average fatty acid (FAME) percentage composition (SD) of the almond and pulp of avocado fruits of four different varieties by GCFID

242

D. Pacetti et al. / J. Chromatogr. A 1150 (2007) 241251

2.3. Clean-up of polar and neutral lipids

The lipid fraction (10 mg) was dissolved in 200 l of chloroform/methanol (2:1, v/v) and subjected to silica gel column chromatography using LC-Si tubes, 6-ml volume, 1 g of
adsorbent (Supelclean, Supelco Bellefonte, USA) treated with
a sequential elution of 5 ml hexane/diethyl-ether (4:1, v/v),
5 ml hexane/diethyl ether (1:1, v/v), 5 ml of methanol and 5 ml
of chloroform/methanol/water (3:5:2, v/v/v). The fractions of
hexane and diethyl ether, containing neutral lipids (NL), were
combined, dried and used for fatty acid (FAME) analysis. The
fractions of methanol and chloroform/methanol/water, containing polar lipids (PoL), such as glycolipids and phospholipids,
were combined, dried and used for LC/MS and fatty acid
(FAME) analysis.
2.4. Fatty acids analysis
Fatty acids methyl esters (FAMEs) were obtained from total,
polar and neutral lipids according to the method of Christie [31].
Briefly, the transmethylation of the lipid fraction (50 mg) was
achieved in 1% sulfuric acid in methanol; after heating for 2 h in
an oven, FAMEs were extracted with n-hexane and analyzed.
The analysis was performed by means of gas chromatography using a CP-9003 apparatus (Chrompack Middelburg, NL),

243

equipped with a flame ionisation detector (FID) and a CP-Sil 88

fused silica capillary column (100 m 0.25 mm i.d., film thickness 0.2 m, Chrompack). The sample was injected on-column.
The carrier gas was helium at a flow rate of 1.6 ml min1 . The
temperature of the detector was 230 C; the injector temperature
was maintained at 60 C for 6 min and then raised to 225 C at a
rate of 20 C min1 . Temperature programming started at 55 C
for 3 min, then raised to 140 C at a rate of 4 C min1 , was held
for 1 min, increased again to 225 C at 2 C min1 and was held
at 225 C for 30 min. Peaks were identified by comparison with
known standards.
2.5. High performance liquid chromatographytandem
mass spectrometry (HPLCMSMS)
HPLCMSMS was carried out using a pump module
(Jasco PU-980) and a ternary gradient module (Jasco LG980-02, Tokyo, Japan). The column was a Polaris Si-A 3
150 mm 4.6 mm (Varian, Middelburg, NL) protected with a
silica precolumn (4 mm 3.0 mm i.d.) from Phenomenex (Torrance, USA). The glycolipid and phospholipid classes were
separated according to Malavolta et al. [32], however with a
modified flow rate, as reported below. Briefly, the mobile phase
was a gradient of solvent A [CHCl3 /MeOH/NH4 OH (30%)
80:19.5:0.5, v/v], and solvent B [CHCl3 /MeOH/H2 O/NH4 OH

Fig. 1. Positive ion HPLCESIMS analysis of glycolipid and phospholipids from avocado pulp with the MS operating in scan mode. MGD, monoglycosyldiacylglycerol; GCer, monoglycosylceramide; DGD, diglycosyldiacylglycerol; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PC, phosphatidylcholine; PA,
phosphatidic acid; LPC, lysophosphatidylcholine. Cn:m = fatty acid (n = carbon number; m = number of double bonds).

244

D. Pacetti et al. / J. Chromatogr. A 1150 (2007) 241251

Fig. 2. Positive HPLCESIMSMS product ion spectra of the glycolipids reported in Fig. 1 (with tentative identification of fragments): (a) MGD(C18:2 /C18:0 and
C18:1 /C18:1 ); (b) DGD(C18:1 /C18:3 ); (c) GCer (t18:1 /C22 h:0 ).

D. Pacetti et al. / J. Chromatogr. A 1150 (2007) 241251

245

Fig. 3. Positive HPLCESIMSMS product ion spectra of the phospholipids detected as [M + H]+ (with tentative identification of fragments): (a) PE(C18:2 /C18:2 );
(b) PC(C18:1 /C18:1 ); (c) LPC(C18:0 ).

246

D. Pacetti et al. / J. Chromatogr. A 1150 (2007) 241251

(30%) 60:34:5.5:0.5, v/v]. The gradient started at 100% of A,

decreased to 0% in 10 min, then was held for 15 min; and then
reached back 100% A in 5 min. The flow rate was 1.0 ml min1
and the injection loop was 5 l.
The HPLC system was coupled on-line to an LCQ ion-trap
mass spectrometer (Finnigan, San Jose, CA, USA) equipped
with an electrospray ionization source (ESI). The HPLC efflu-

ent was splitted and 0.4 ml min1 entered the MS through a

steel ionization needle set at 5.0 kV and a heated capillary set
to 200 C. The sheath gas flow was approx. 90 arbitrary units.
The ion source and the ion optic parameters were optimised
with respect to the positive molecular related ions of the glycolipids and phospholipids standards. The molecular mass peaks
from the HPLC effluent were detected using positive ion full-

Fig. 4. Positive HPLCESIMSMS product ion spectra of the phospholipids detected as [M + NH4 ]+ (with tentative identification of fragments): (a) PI(C16:0 /C18:2 );
(b) PA(C18:0 /C18:1 ).

D. Pacetti et al. / J. Chromatogr. A 1150 (2007) 241251

scan ESIMS analysis. Mass resolution was 0.1 Da. Tandem

mass (MS2 ) experiments were carried out with relative collision energy of 45%. The integration was performed with the
Interactive Chemical Information System (ICIS) peak detection
algorithm software provided by Finnigan, after correction for
the contribution from the 13 C isotope effect.
3. Results and discussion
3.1. Composition of neutral and polar lipids
The average content of polar lipids (phospholipids and glycolipids) of the almond (28.3 8.7 mg/100 mg lipids) was about
five-fold the content in the pulp (5.2 2.0), as calculated after
removing in vacuo the solvent eluate from the solid phase extraction.
In Table 1, the average fatty acid composition of the almond
and pulp of four different varieties are reported. In Reed and
Hass, -linoleic acid (C18:26 ) was the main fatty acid in the

247

almond, both in the neutral and polar lipids. This is probably

due to the fact that the almond accumulates reserve substances,
such as essential fatty acids, for the development of the new
plant. However, in the almond of the same varieties, -linoleic
acid was higher in the polar lipids with respect to neutral lipids
(containing more oleic acid). Unlike the almond, in all the varieties, the main fatty acid of the pulp was oleic acid (C18:1 ), as
already reported elsewhere [3335]. The third most abundant
fatty acid was palmitic acid (C16:0 ) (1425%) in all the samples. -Linolenic acid (C18:33 ) showed a peculiar distribution:
it was present (up to 7.1%) in the polar fraction of the pulp in
all varieties, as well as in the neutral lipids of the almond (up
to 7.3%), whereas it was very scarce in the neutral lipids of the
pulp (less than 1.1%). Eicosenoic acid (C20:1 ) was present in
the polar fraction of the almond, unlike the pulp. Palmitoleic
acid (C16:1 ) was more abundant in the pulp than in the almond,
particularly in the neutral lipids. Stearic acid (C18:0 ) was scarce
both in the pulp and in the almond; in the pulp, its content was
higher in the polar fraction than in the neutral lipids.

Table 2
Molecular species of glycolipids in the pulp
Relative abundance in percentage (mean SD)
GL molecular species
Ion (m/z)

Varieties
Fatty acids

REED

16:1/18:216:0/18:3
16:1/18:116:0/18:2
16:0/18:1
16:0/18:0
18:3/18:3
18:3/18:2
18:1/18:318:2/18:2
18:1/18:218:0/18:3
18:0/18:218:1/18:1
18:0/18:1

2.4
5.7
4.4
2.4
13.2
3.6
25.4
15.3
23.8
3.8

0.4
0.5
0.7
0.8
1.7
0.1
0.2
0.1
2.1
0.8

1.7
7.8
3.8
3.2
11.3
3.9
25.3
15.0
24.7
3.3

0.5
0.1
0.1
0.5
1.7
0.2
1.3
0.5
2.5
0.4

1.4
6.3
3.6
3.5
13.3
4.6
26.1
13.2
24.3
3.8

0.1
0.5
0.3
0.8
1.5
1.2
1.2
0.9
0.7
0.4

0.9
3.2
1.6
4.5
10.8
3.9
21.7
11.0
37.5
4.8

0.1
0.9
0.1
0.7
0.3
0.2
0.3
1.0
1.0
0.4

DGD ([M + NH4 ]+ )

930.6
932.6
934.5
936.5
938.4
954.6
956.6
958.5
960.5
962.4
964.4
966.5

16:1/18:3
16:1/18:216:0/18:3
16:1/18:116:0/18:2
16:1/18:016:0/18:1
16:0/18:0
18:3/18:3
18:3/18:2
18:1/18:3
18:1/18:218:0/18:3
18:1/18:118:0/18:2
18:0/18:1
18:0/18:0

1.7
4.4
8.4
6.4
0.5
9.0
4.3
26.6
11.5
23.0
3.8
0.4

0.3
0.8
0.2
0.7
0.2
1.1
1.0
1.3
0.1
1.8
0.1
0.2

2.8
4.2
8.5
8.0
1.2
7.7
4.7
21.3
13.6
23.0
4.5
0.6

1.2
0.9
1.9
0.5
0.1
0.1
0.4
0.7
0.8
1.3
1.1
0.2

1.8
4.2
7.5
5.5
1.0
11.0
7.2
23.0
14.4
18.2
4.7
1.5

0.2
0.1
0.2
0.6
0.2
0.9
0.5
0.4
0.8
1.3
0.7
0.5

1.3
3.5
6.0
8.1
1.0
8.1
3.0
25.0
9.5
28.7
5.4
0.4

0.2
0.2
0.1
0.3
0.1
0.1
0.1
0.6
0.6
0.2
0.7
0.1

Gcer ([M + H]+ )

714.5
716.5
816.6
818.5
830.5
844.5
858.5

d18:2/16 h:0
d18:1/16 h:0d18:0/16 h:1
t18:1/22 h:0
t18:0/22 h:0
t18:1/23 h:0
t18:1/24 h:0
t18:1/25 h:0

40.4
5.7
18.6
3.0
6.9
18.4
7.0

1.5
1.2
0.9
0.7
1.5
0.1
0.3

52.9
7.2
15.1
2.2
4.2
11.3
7.1

1.7
0.5
0.2
0.1
1.0
1.1
0.4

46.4
10.1
14.4
3.0
4.6
14.4
7.0

2.5
0.6
2.5
0.5
1.1
0.6
1.9

51.3
6.4
14.2
3.3
6.8
12.0
6.0

4.0
1.4
0.2
0.1
1.1
1.5
1.9

MGD ([M + NH4

770.5
772.5
774.6
776.6
792.7
794.6
796.6
798.6
800.4
802.5

RINCON

PINKERTON

HASS

]+ )

Fatty acids: total fatty acid carbon number:number of double bonds. 16 h:0, 2-hydroxy fatty acids having carbon chain length 16; t18:1, 4-hydroxy-8-sphingenine;
d18:2, 4,8-sphingadienine; d18:1, 8-sphingenine; d18:0, sphinganine; SD, standard deviation of three samples.

248

D. Pacetti et al. / J. Chromatogr. A 1150 (2007) 241251

3.2. Simultaneous HPLC of the glycolipid and phospholipid

classes and characterization of their molecular species
The HPLC simultaneous separation of the glycolipid and
phospholipid classes is reported in Fig. 1. The different polar
lipid classes were eluted within 18 min; the HPLC retention
time increased in the following order: MGD < Gcer < DGD <
PE < PI < PC < PA < LPC.
The pseudomolecular mass peaks of the different glycolipid and phospholipid classes were detected using positive ion
full-scan ESIMS analysis. The mass spectra were acquired
under peaks obtained from the reconstructed positive ion chromatogram in the time expected for the elution of each molecular
species. In order to achieve the characterization of each glycolipid class, positive ion fragments (MS2 ) formed after collision
activated dissociation (CAD) were investigated. The fragmentations were compared with those obtained from known standards
and previous literature data [19,32,36].

3.2.3. Phosphatidylcholine
The species of PC were detected as [M + H]+ as well. The
product ion spectra of PC(C18:1 /C18:1 ) at m/z 786.6 showed a
major fragment ion (Fig. 3b) at m/z 726.4 corresponding to the
loss of trimethylamine and the loss of one fatty acid (m/z 505.2).
In addition, the spectrum showed a fragment at m/z 522.4 resulting from the loss of an acyl group and a second fragment at m/z
602.5, resulting from the loss of phosphocholine (184.1 Da), as
already reported elsewhere [37].
3.2.4. Lysophosphatidylcholine
An example of the fragmentation of LPC, detected as
[M + H]+ , was reported in Fig. 3c. The main fragments are given
by the loss of one and two water molecules (m/z 505.3 and m/z
487.4), the polar headgroup (m/z 184.1), an aliphatic moiety

Table 3
Molecular species of glycolipids in the almond

3.2.1. Monoglycosyldiglycerides, diglycosyldiglycerides

and glycocerebrosides (Gcer)
The species of MGD and DGD were all detected as their
adducts with ammonium ([M + NH4 ]+ ), whereas Gcer were
detected as their [M + H]+ . The product ion spectrum from MS2
of MGD and DGD yielded fragments resulting from the loss
of an acyl group ([M RCO + 2H]+ ) and to the neutral loss of
the glycosyl moiety ([M RCO Glycosyl]+ ). In Fig. 2a, the tandem mass spectrum of the pseudomolecular ion at m/z 800.4,
i.e. MGD (C18:0 /C18:2 and C18:1 /C18:1 ) was reported together
with the fragment structures. Two fragment ions at m/z 521.1
and 519.2 resulted from the loss of a linoleic or an oleic moiety,
respectively, whereas the fragment at m/z 337.3 resulted from
the simultaneous loss of a stearic group (stearic acid, C18:0 ) and
the glycosyl group [C6 H11 O6 ].
In Fig. 2b, the fragmentation of the pseudomolecular peak at
m/z 958.4, i.e. DGD (C18:1 /C18:3 ) was reported. The main fragment was given by the loss of linolenic acid (m/z 681.3) and
the diglycosyl moiety (m/z 617.3). The fragment at m/z 339.2
aroused from the loss of both the linolenic and diglycosyl moieties. In Fig. 2c, the fragmentation of the Gcer(t18:1 /C22 h:0 ) with
pseudomolecular ion at m/z 816.6 was reported; the main fragments corresponded either to the loss of the glycosyl moiety
(m/z 654.3 and 636.5), or to the acyl moiety (m/z 339.7) or the
sphingoid moiety (m/z 283.9).

Relative abundance in percentage (mean SD)

3.2.2. Phosphatidylethanolamine
The species of PE were all detected as [M + H]+ . CAD spectra
of PE molecular species displayed a fragment resulting from
the loss of the polar headgroup ([M NH3 (CH2 )2 OPO3 H]+ ) and
a second fragment resulting from the loss of one acyl group
([M RCO]+ ), as reported in Fig. 3a. The product ion spectra of
PE (C18:2 /C18:2 ) at m/z 740.4 showed a major fragment ion at
m/z 599.3 ([M NH3 (CH2 )2 OPO3 H]+ ) and two minor fragment
ions at m/z 476.2 and m/z 336.9, corresponding to the loss of the
linoleic acyl group and the simultaneous loss of both the polar
headgroup and the linoleic acyl group, respectively.

GL molecular species

Varieties

Ion (m/z)

REED

Fatty acids

HASS

]+ )

MGD ([M + NH4

770.5
16:1/18:216:0/18:3
772.5
16:1/18:116:0/18:2
774.6
16:0/18:1
792.7
18:3/18:3
794.6
18:3/18:2
796.6
18:1/18:318:2/18:2
798.6
18:1/18:218:0/18:3
800.4
18:0/18:218:1/18:1
802.5
18:0/18:1
804.5
18:0/18:0

1.8
3.2
1.3
4.6
9.9
40.4
29.3
7.8
1.2
0.7

0.3
0.1
0.3
0.7
0.8
2.8
1.0
1.2
0.3
0.5

3.2
6.1
2.0
4.2
13.3
42.0
21.6
5.0
1.4
4.2

0.2
0.6
0.2
1.2
0.8
0.8
0.4
1.1
0.2
1.2

DGD ([M + NH4 ]+ )

932.6
16:1/18:216:0/18:3
934.5
16:1/18:116:0/18:2
936.5
16:1/18:0-16:0/18:1
938.4
16:0/18:0
954.6
18:3/18:3
956.6
18:3/18:2
958.5
18:1/18:3
960.5
18:1/18:218:0/18:3
962.4
18:1/18:118:0/18:2
964.4
18:0/18:1
986.4
18:3/20:1
988.4
18:2/20:1
990.3
18:1/20:1
992.4
18:0/20:1

2.3
23.6
15.9
2.1
0.2
2.5
20.9
21.2
8.4
1.4
0.6
0.5
0.2
0.2

0.4
0.6
1.8
0.2
0.1
0.2
1.9
0.8
0.3
0.1
0.1
0.1
0.1
0.1

3.1
20.6
10.9
0.8
0.5
3.6
22.3
26.6
6.7
1.1
1.5
1.1
0.7
0.4

0.1
1.6
0.4
0.3
0.1
0.2
1.6
1.7
1.5
0.5
0.1
0.7
0.2
0.1

Gcer ([M + H]+ )

714.5
d18:2/16 h:0
716.5
d18:1/16 h:0d18:0/16 h:1
816.6
t18:1/22 h:0
818.5
t18:0/22 h:0
830.5
t18:1/23 h:0
844.5
t18:1/24 h:0
858.5
t18:1/25 h:0

54.3
2.3
17.7
3.7
5.0
13.7
3.4

0.7
0.1
0.8
0.6
0.1
1.4
0.8

54.5
6.0
16.3
2.8
4.6
12.3
3.4

5.2
0.3
1.4
0.1
1.3
3.1
0.3

Fatty acids: total fatty acid carbon number:number of double bonds. 16 h:0,
2-hydroxy fatty acids having carbon chain length 16; t18:1, 4-hydroxy-8sphingenine, d18:2, 4,8-sphingadienine; d18:1, 8-sphingenine; d18:0, sphinganine; SD, standard deviation of three samples.

D. Pacetti et al. / J. Chromatogr. A 1150 (2007) 241251

249

resulting from stearic acid (m/z 169.0) and the loss of the polar
headgroup (m/z 356.8).

carbon chain of the fatty acid at the carbon C1 (m/z 463.4) and
C2 of the chain (m/z 477.5).

3.2.5. Phosphatidylinositol
PI was reported as an adduct with ammonium [M + NH4 ]+ .
The CAD of PI (palmitic/linoleic acid) (Fig. 4a) yielded the
molecular ion m/z 833.6 and a fragment obtained from the loss
of palmitic acid (m/z 577.4).

3.3. Molecular species composition of polar lipids in the

pulp and almond

3.2.6. Phosphatidic acid

PA was also detected as its adduct with ammonium
[M + NH4 ]+ . Its CAD (Fig. 4b) produced the loss of the hydro-

3.3.1. Glycolipids
The molecular species composition of glycolipids from the
pulp and almond of different varieties of avocado is reported
in Tables 2 and 3, respectively, giving further information
with respect to the GCFID determination of total fatty acids
(Table 1).

Table 4
Molecular species of phospholipids in the pulp
Relative abundance in percentage (mean SD)
PL molecular species
Ion (m/z)

Varieties
Fatty acids

REED

RINCON

PINKERTON

HASS

PE
716.6
718.8
740.4
742.5
744.7
746.6

16:0/18:216:1/18:1
16:0/18:1
18:2/18:2
18:1/18:2
18:1/18:118:0/18:2
18:0/18:1

18.6
46.4
8.6
13.8
11.2
1.3

1.9
1.7
0.4
0.2
0.2
0.2

25.2
23.2
16.7
23.5
10.6
0.7

1.2
3.8
2.2
1.6
0.9
0.2

18.7
31.9
11.3
22.6
13.9
1.6

0.2
0.1
1.1
1.4
0.2
0.1

13.2
28.9
5.5
24.3
25.7
2.5

1.0
0.9
0.3
0.3
0.1
0.2

PI ([M + NH4 ]+ )
828.5
852.4
854.4
856.5
876.4
878.4
880.4
882.4
884.5

16:0/16:0
16:0/18:216:1/18:1
16:0/18:1
16:0/18:0
18:1/18:318:2/18:2
18:1/18:218:0/18:3
18:1/18:1
18:0/18:1
18:0/18:0

2.3
24.6
38.2
4.1
5.2
8.6
12.5
3.2
1.4

0.9
2.5
1.8
0.5
2.9
0.4
1.0
1.1
0.3

0.9
30.8
31.1
3.7
4.3
12.0
13.4
2.1
1.7

0.2
2.4
2.3
1.0
1.9
2.2
1.0
0.1
0.3

1.5
34.9
32.1
3.6
2.6
10.2
12.4
1.7
1.0

0.4
1.9
1.7
0.4
0.3
0.8
0.6
0.5
0.4

0.7
22.4
39.6
4.0
1.6
9.0
18.4
3.4
1.0

0.1
1.6
0.1
0.1
0.1
0.8
0.7
0.7
0.4

PC ([M + H]+ )
734.6
758.6
760.6
762.6
780.6
782.6
784.6
786.6
788.6

16:0/16:0
16:0/18:2
16:0/18:1
16:0/18:0
18:2/18:3
18:2/18:218:1/18:3
18:1/18:2
18:1/18:1
18:0/18:1

1.0
1.5
35.8
6.2
2.3
5.3
4.0
39.5
4.4

0.3
0.7
0.1
2.1
0.1
0.4
1.0
2.4
0.1

1.6
1.9
34.2
5.8
2.9
4.2
6.0
39.4
4.0

1.0
0.4
1.0
0.9
1.8
0.9
1.1
2.0
0.5

1.0
1.1
33.5
4.7
1.5
3.9
5.0
45.1
4.5

0.2
0.5
1.1
0.3
1.1
0.9
1.1
4.4
0.5

0.8
0.6
36.6
4.9
0.5
2.6
2.1
47.5
4.4

0.3
0.3
1.6
0.1
0.3
1.2
0.1
3.7
0.1

PA ([M + NH4 ]+ )
690.5
692.5
694.5
714.5
716.5
718.5
720.5

16:0/18:2
16:0/18:1
16:0/18:0
18:2/18:218:1/18:3
18:1/18:2
18:1/18:1
18:0/18:1

11.5
26.2
3.0
4.4
22.0
32.9
2.9

1.9
0.6
0.9
0.9
1.6
0.9
0.5

12.9
25.7
2.0
9.4
24.8
25.3
3.0

1.8
0.6
0.1
2.7
1.7
2.4
0.4

11.9
24.5
2.0
5.8
25.0
30.8
3.3

0.1
1.8
0.3
0.5
0.2
0.7
0.7

9.5
23.1
1.9
3.5
18.7
43.4
3.5

0.5
1.6
0.4
0.1
0.5
1.3
0.1

LPC ([M + H]+ )

496.3
520.3
522.3
524.5

16:0
18:2
18:1
18:0

36.6
4.8
55.8
2.8

3.4
1.4
2.9
1.4

28.8
21.9
41.5
7.7

2.6
3.9
2.4
1.7

14.7
16.6
63.9
4.8

1.3
2.4
4.1
1.2

16.0
5.8
75.7
2.4

3.9
1.2
4.2
0.9

([M + H]+ )

Fatty acids, total fatty acid carbon number:number of double bonds; SD, standard deviation of three samples.

250

D. Pacetti et al. / J. Chromatogr. A 1150 (2007) 241251

The main molecular species of both MGD and DGD in the

pulp of all varieties (Table 2) were those containing combinations of oleic/linolenic (C18:1 /C18:3 ) or linoleic/linoleic acids
(C18:2 /C18:2 ), oleic/oleic (C18:1 /C18:1 ) or stearic/linoleic acids
(C18:0 /C18:2 ), and oleic/linoleic (C18:1 /C18:2 ) or stearic/linolenic
(C18:0 /C18:3 ) acids; the sum of these species accounted for more
than 60% of all species.
In the almond (Table 3), the difference in the composition
between MGD and DGD was remarkable, unlike the pulp. The
main MGD and DGD species were those with C18:1 /C18:3 or
Table 5
Molecular species of phospholipids in the almond
Relative abundance in percentage (mean SD)
PL molecular species

Varieties

Ion (m/z)

Fatty acids

REED

PE
716.6
718.8
740.4
742.5
744.7
768.6
770.6
772.6

16:0/18:2
16:0/18:116:1/18:0
18:2/18:2
18:1/18:2
18:1/18:118:0/18:2
18:3/20:1
18:2/20:1
18:1/20:1

41.3
11.6
27.6
13.6
2.9
0.6
1.2
1.2

0.1
0.7
3.0
1.1
0.9
0.1
0.2
0.1

41.6
8.1
30.9
13.1
2.9
1.1
1.6
0.7

3.1
2.1
1.6
1.2
1.2
0.6
0.5
0.1

PI ([M + NH4 ]+ )
852.4
854.4
856.5
876.4
878.4
880.4
882.4
884.5

16:0/18:2
16:0/18:1
16:0/18:0
18:2/18:2
18:1/18:2
18:1/18:118:0/18:2
18:0/18:1
18:0/18:0

53.8
21.0
1.8
7.2
9.9
4.8
0.9
0.7

1.3
0.4
0.3
0.7
0.7
0.8
0.1
0.3

54.2
20.1
1.4
4.2
12.8
4.9
1.7
0.6

3.3
3.1
0.8
0.6
1.4
0.7
0.7
0.3

PC ([M + H]+ )
732.6
734.6
758.6
760.6
762.6
780.6
782.6
784.6
786.6
788.6

16:0/16:1
16:0/16:0
16:0/18:2
16:0/18:116:1/18:0
16:0/18:0
18:2/18:3
18:2/18:2
18:1/18:2
18:0/18:2
18:0/18:1

0.5
1.3
28.9
17.5
1.2
1.2
21.5
19.6
7.5
0.7

0.2
0.1
2.7
0.9
0.5
0.7
0.5
1.6
1.8
0.2

5.2
2.9
34.9
16.0
1.5
2.6
17.8
14.3
4.6
0.3

1.2
1.1
2.9
1.1
0.1
0.1
1.9
2.0
0.9
0.1

PA ([M + NH4 ]+ )
690.5
16:0/18:216:1/18:1
692.5
16:0/18:116:1/18:0
694.5
16:0/18:0
714.5
18:2/18:218:1/18:3
716.5
18:1/18:2
718.5
18:1/18:118:0/18:2
720.5
18:0/18:1

29.7
13.6
1.2
28.7
19.0
6.8
1.0

0.7
2.4
0.1
1.2
1.5
0.2
0.3

33.1
12.8
0.9
27.5
18.7
6.1
0.9

4.9
1.0
0.2
0.9
1.8
0.6
0.3

LPC ([M + H]+ )

496.3
520.3
522.3
524.5

21.5
53.2
23.5
1.8

0.1
2.4
2.0
0.3

24.4
50.5
22.9
2.2

1.6
1.9
0.6
0.4

HASS

([M + H]+ )

16:0
18:2
18:1
18:0

Fatty acids, total fatty acid carbon number:number of double bonds; SD, standard
deviation of three samples.

C18:2 /C18:2 and C18:1 /C18:2 or C18:0 /C18:3 ; however, these two
classes represented almost 70% of MGD, but only 4249% of
DGD. In fact, DGD contained high amount of the species with
C16:0 /C18:2 or C16:1 /C18:1 . The predominant species of DGD
were the same combinations of MGD; in addition, DGD also
contained 2023% of the species with C16:0 /C18:2 or C16:1 /C18:1 .
Regarding other differences among the composition of
pulp and almond, it was reported that MGD (C18:0 /C18:2 or
C18:1 /C18:1 ) was high in the pulp (up to 37.5% in Hass), but
low in the almond (5% in Hass) and that the content of both
species of MGD and DGD containing C18:3 /C18:3 in the pulp
was at least twice the content in the almond of all the samples.
Eicosenoic acid was only present in the almond (Table 1) as
a component of DGD and phosphatidylethanolammine (PE).
The composition of the glycocerebrosides (containing
hydroxy fatty acids) of the pulp was similar to that of the almond;
GCer(d18:2 /C16 h:0 ) was the main component in both extracts.
3.3.2. Phospholipids
The compositions of PL are reported in Tables 4 and 5. In the
almond, all the PL classes contained the C16:0 /C18:2 as the main
molecular species; the predominant species of LPC contained
C18:2 in the almond. In the pulp, the main molecular species of
PE and PI was the less unsaturated C16:0 /C18:1 . In PC and PA,
a prevalence of C18:1 /C18:1 was detected; thus LPC (C18:1 ) prevailed in the pulp with respect to LPC (C18:2 ), mostly present in
the almond. From this data it can be also concluded that palmitic
acid is preferentially bound to PL and DGD with respect to MGD
and GCer in the almond. In the pulp, more palmitic acid was
present in the PL than in glycolipids.
4. Conclusions
The lipid composition of the almond and pulp of the avocado
fruits are different and reflect the different biological functions
of the parts of the drupe. The high content of polar lipids of
the almond would be interesting for a selective extraction and
use in the cosmetic, pharmaceutical and food industry. Oleic
acid represented the main fatty acid in the pulp, but not in the
almond.
Looking at the essential fatty acids (C18:26 and C18:33 ),
they are more abundant in the almond (3942%) than in the pulp
(1017%), but their distribution in the different lipid classes is
different. In the almond, the percentage of linoleic acid is higher
in the polar lipids than triacylglycerols, unlike -linolenic acid.
In the pulp, both essential fatty acids are higher in the polar lipids
than in the triacylglycerols; -linolenic acid was contained as
MGD (C18:3 /C18:3 ) and DGD (C18:3 /C18:3 ), more present in the
pulp than in the almond.
Regarding saturated fatty acids, palmitic acid was predominant in the main molecular species of all the PL present in the
pulp and in the almond, whereas it was not always present in the
preponderant molecular species of the glycolipids; stearic acid
showed a variable presence.
The chromatographic separation of the polar lipid classes
with NP-HPLC was achieved in only 18 min and the characterization of the molecular species could be performed with mass

D. Pacetti et al. / J. Chromatogr. A 1150 (2007) 241251

spectrometry using electrospray nebulization (ESI(+)-MSMS).

The positive ionization was more suitable than the negative ionization for the simultaneous determination of all the polar lipids,
particularly glycolipids.

[18]

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[20]

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