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Simultaneous analysis of glycolipids and phospholids molecular species in avocado (Persea americana Mill) fruit
Journal of Chromatography A, 1150 (2007) 241–251
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
b
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 chromatography–electrospray 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 [1–4]. 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 [5–11]. 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: [email protected] (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 [12–14]. 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 [19–24]. 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 (MS–MS) 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) 241–251
Table 1 Average fatty acid (FAME) percentage composition (±SD) of the almond and pulp of avocado fruits of four different varieties by GC–FID
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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),
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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 min−1 . 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 min−1 . Temperature programming started at 55 ◦ C for 3 min, then raised to 140 ◦ C at a rate of 4 ◦ C min−1 , was held for 1 min, increased again to 225 ◦ C at 2 ◦ C min−1 and was held at 225 ◦ C for 30 min. Peaks were identified by comparison with known standards. 2.5. High performance liquid chromatography–tandem mass spectrometry (HPLC–MS–MS) HPLC–MS–MS 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 HPLC–ESI–MS 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).
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Fig. 2. Positive HPLC–ESI–MS–MS 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) 241–251
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Fig. 3. Positive HPLC–ESI–MS–MS 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 ).
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(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 min−1 and the injection loop was 5 l. The HPLC system was coupled on-line to an LCQ ion-trap mass spectrometer (Finnigan, San Jos´e, CA, USA) equipped with an electrospray ionization source (ESI). The HPLC efflu-
ent was splitted and 0.4 ml min−1 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 HPLC–ESI–MS–MS 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) 241–251
scan ESI–MS 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
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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 [33–35]. The third most abundant fatty acid was palmitic acid (C16:0 ) (14–25%) 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
16:1/18:2–16:0/18:3 16:1/18:1–16:0/18:2 16:0/18:1 16:0/18:0 18:3/18:3 18:3/18:2 18:1/18:3–18:2/18:2 18:1/18:2–18:0/18:3 18:0/18:2–18:1/18:1 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 16:1/18:2–16:0/18:3 16:1/18:1–16:0/18:2 16:1/18:0–16:0/18:1 16:0/18:0 18:3/18:3 18:3/18:2 18:1/18:3 18:1/18:2–18:0/18:3 18:1/18:1–18:0/18:2 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:0–d18:0/16 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.
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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 ESI–MS 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 16:1/18:2–16:0/18:3 772.5 16:1/18:1–16:0/18:2 774.6 16:0/18:1 792.7 18:3/18:3 794.6 18:3/18:2 796.6 18:1/18:3–18:2/18:2 798.6 18:1/18:2–18:0/18:3 800.4 18:0/18:2–18:1/18:1 802.5 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 16:1/18:2–16:0/18:3 934.5 16:1/18:1–16:0/18:2 936.5 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 18:1/18:2–18:0/18:3 962.4 18:1/18:1–18:0/18:2 964.4 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:0–d18:0/16 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) 241–251
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 GC–FID 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
16:0/18:2–16:1/18:1 16:0/18:1 18:2/18:2 18:1/18:2 18:1/18:1–18:0/18:2 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 16:0/18:2–16:1/18:1 16:0/18:1 16:0/18:0 18:1/18:3–18:2/18:2 18:1/18:2–18:0/18:3 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 18:2/18:2–18:1/18:3 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 18:2/18:2–18:1/18:3 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) 241–251
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 16:0/18:1–16:1/18:0 18:2/18:2 18:1/18:2 18:1/18:1–18:0/18:2 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 18:1/18:1–18:0/18:2 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 16:0/18:1–16:1/18:0 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 16:0/18:2–16:1/18:1 692.5 16:0/18:1–16:1/18:0 694.5 16:0/18:0 714.5 18:2/18:2–18:1/18:3 716.5 18:1/18:2 718.5 18:1/18:1–18:0/18:2 720.5 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 42–49% 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 20–23% 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 (39–42%) than in the pulp (10–17%), 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) 241–251
spectrometry using electrospray nebulization (ESI(+)-MS–MS). 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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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
b
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 chromatography–electrospray 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 [1–4]. 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 [5–11]. 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: [email protected] (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 [12–14]. 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 [19–24]. 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 (MS–MS) 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) 241–251
Table 1 Average fatty acid (FAME) percentage composition (±SD) of the almond and pulp of avocado fruits of four different varieties by GC–FID
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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),
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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 min−1 . 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 min−1 . Temperature programming started at 55 ◦ C for 3 min, then raised to 140 ◦ C at a rate of 4 ◦ C min−1 , was held for 1 min, increased again to 225 ◦ C at 2 ◦ C min−1 and was held at 225 ◦ C for 30 min. Peaks were identified by comparison with known standards. 2.5. High performance liquid chromatography–tandem mass spectrometry (HPLC–MS–MS) HPLC–MS–MS 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 HPLC–ESI–MS 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).
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Fig. 2. Positive HPLC–ESI–MS–MS 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) 241–251
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Fig. 3. Positive HPLC–ESI–MS–MS 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 ).
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(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 min−1 and the injection loop was 5 l. The HPLC system was coupled on-line to an LCQ ion-trap mass spectrometer (Finnigan, San Jos´e, CA, USA) equipped with an electrospray ionization source (ESI). The HPLC efflu-
ent was splitted and 0.4 ml min−1 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 HPLC–ESI–MS–MS 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) 241–251
scan ESI–MS 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
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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 [33–35]. The third most abundant fatty acid was palmitic acid (C16:0 ) (14–25%) 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
16:1/18:2–16:0/18:3 16:1/18:1–16:0/18:2 16:0/18:1 16:0/18:0 18:3/18:3 18:3/18:2 18:1/18:3–18:2/18:2 18:1/18:2–18:0/18:3 18:0/18:2–18:1/18:1 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 16:1/18:2–16:0/18:3 16:1/18:1–16:0/18:2 16:1/18:0–16:0/18:1 16:0/18:0 18:3/18:3 18:3/18:2 18:1/18:3 18:1/18:2–18:0/18:3 18:1/18:1–18:0/18:2 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:0–d18:0/16 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.
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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 ESI–MS 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 16:1/18:2–16:0/18:3 772.5 16:1/18:1–16:0/18:2 774.6 16:0/18:1 792.7 18:3/18:3 794.6 18:3/18:2 796.6 18:1/18:3–18:2/18:2 798.6 18:1/18:2–18:0/18:3 800.4 18:0/18:2–18:1/18:1 802.5 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 16:1/18:2–16:0/18:3 934.5 16:1/18:1–16:0/18:2 936.5 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 18:1/18:2–18:0/18:3 962.4 18:1/18:1–18:0/18:2 964.4 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:0–d18:0/16 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) 241–251
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 GC–FID 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
16:0/18:2–16:1/18:1 16:0/18:1 18:2/18:2 18:1/18:2 18:1/18:1–18:0/18:2 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 16:0/18:2–16:1/18:1 16:0/18:1 16:0/18:0 18:1/18:3–18:2/18:2 18:1/18:2–18:0/18:3 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 18:2/18:2–18:1/18:3 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 18:2/18:2–18:1/18:3 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.
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D. Pacetti et al. / J. Chromatogr. A 1150 (2007) 241–251
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 16:0/18:1–16:1/18:0 18:2/18:2 18:1/18:2 18:1/18:1–18:0/18:2 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 18:1/18:1–18:0/18:2 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 16:0/18:1–16:1/18:0 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 16:0/18:2–16:1/18:1 692.5 16:0/18:1–16:1/18:0 694.5 16:0/18:0 714.5 18:2/18:2–18:1/18:3 716.5 18:1/18:2 718.5 18:1/18:1–18:0/18:2 720.5 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 42–49% 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 20–23% 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 (39–42%) than in the pulp (10–17%), 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) 241–251
spectrometry using electrospray nebulization (ESI(+)-MS–MS). The positive ionization was more suitable than the negative ionization for the simultaneous determination of all the polar lipids, particularly glycolipids.
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