Analytica Chimica Acta 520 (2004) 201–205

Improvement of a solid phase extraction method for analysis oflipid fractions in muscle foods

J. Ruiz∗, T. Antequera, A.I. Andres, M.J. Petron, E. Muriel

Food Science, Facultad de Veterinaria, Universidad de Extremadura, Campus Universitario s/n, 10071 Caceres, Spain

Received 16 December 2003; received in revised form 6 April 2004; accepted 19 April 2004

Available online 10 June 2004

Abstract

The most used method for muscle lipid fractionation into major lipid classes was modified for improving its separation efficiency. Extractedlipids from a masseter muscle of one Iberian pig were separated into neutral lipids (NL), free fatty acids (FFA) and polar lipids (PL) usingaminopropyl minicolumns, following the extensively used method of Kaluzny et al. [1] (old method-OM-) and a method based on that,developed by Pinkart et al. [2] with some (modifications modified method–MM). Obtained lipid classes were further analysed by TLC andlipid fractions were identified. TLC evidenced the presence of a certain amount of PL in the NL fraction obtained with the OM. On theother hand, using the MM only an almost undetectable presence of PL was evidenced in the NL fraction. Fatty acid composition of NL, PLand FFA obtained with each method was studied by gas chromatography. Fatty acid profile of NL was strongly influenced by the separationmethod used. Thus, NL obtained using the OM showed higher amounts of saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA)and lower of monounsaturated fatty acids (MUFA) than those obtained using the MM. Moreover, NL obtained using the OM showed thepresence of fatty alcohols, constituents of phospholipids (PhL) absent or present only in trace amounts in acylglycerols. This profile reflectsthe coelution of PL in the NL fraction. Fatty acid profile of FFA and PL fractions was also influenced by the solid phase extraction (SPE)method used, but to a lesser extent.© 2004 Elsevier B.V. All rights reserved.

Keywords: SPE; Muscle; Lipid fractions; Fatty acid profile

1. Introduction

Fractionation of muscle lipids into major lipids classes(neutral lipids (NL), free fatty acids (FFA) and polar lipids(PL)) by solid phase extraction (SPE) is a common analyticalprocedure for meat scientists[3]. The NL fraction of musclelipids is mainly constituted by triacylglycerols (TG) (morethan 90%), with small amounts of other glycerides (around5%), sterols, such as cholesterol and cholesteryl esters (lessthan 1%)[4]. Other compounds from the unsaponifiable mat-ter such as hydrocarbons or carotenes are in much lower con-centrations. A correct separation of lipid classes is importantto obtain a reliable fatty acid composition of each fraction,since fatty acid composition of lipid classes is very differentfrom each other[5], and coelution might lead to wrong con-

∗ Corresponding author. Tel.:+34 927 257122; fax:+34 27 257110.E-mail address: jruiz@unex.es (J. Ruiz).

clusions about the fatty acid profile. The most used methodfor lipid classes separation among meat researchers is thatdescribed by Kaluzny et al.[1]. However, this method seemsto be not accurate enough in separating lipid fractions fromother biological materials[6]. For example, Pinkart et al.[2] showed an almost 50% elution of phospholipids with theNL fraction using this method. Several researchers have alsofound inadequate elution of the acidic phospholipids withmethanol in the PL fraction[2,7,8]. Different authors haveproposed modifications of the Kaluzny method for improv-ing separation of major lipid classes in different biologicalsources[2,6–9], aimed to obtain an adequate separation ofPL and NL and to completely elute phospholipids. However,no study about improving the classical Kaluzny method forseparating major muscle lipid classes has been carried out.Therefore, our objective was to check the feasibility of twodifferent SPE methods for separating major muscle lipidclasses.

0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.aca.2004.04.059

202 J. Ruiz et al. / Analytica Chimica Acta 520 (2004) 201–205

Table 1Scheme of the two SPE methods used for fractionation of muscle lipid into major lipid clases: Kaluzny et al.[1] OM using 100 mg aminopropylminicolumns and Pinkart et al.[2] MM using 500 mg aminopropyl minicolumns

Solvent Eluted lipids

OM MM

Activation 3 mL chloroform 7.5 mL hexane –Sample 100 mg in 1 mL chloroform 20 mg in 150�l hexane:chloroform:methanol (95:3:2) –Fraction 1 3 mL chloroform:2-propanol (2:1) 5 mL chloroform Neutral lipidsFraction 2 3 mL diethylether:acetic acid (98:2) 5 mL diethylether:acetic acid (98:2) Free fatty acidsFraction 3 3 mL methanol 2.5 mL methanol:chloroform (6:1) Polar lipidsFraction 4 – 2.5 mL 0.05 M sodium acetate in methanol:chloroform (6:1) Polar lipids

2. Experimental

The left masseter muscle from one Iberian pig wasdissected 24 h after slaughter and stored at−80◦C until anal-ysis. Sample was ground using a commercial grinder imme-diately before fat extraction. Intramuscular total lipids wereextracted and quantified with chloroform:methanol (1:2)according to the method described by Bligh and Dyer[10].

Two different SPE methods for separating major musclelipid classes (NL, FFA and PL) were followed to comparetheir effectiveness in obtaining pure lipid fractions. Theschemes used to separate the lipid classes are shown inTable 1. The method developed by Kaluzny et al.[1] wasnamed “old method” (OM). Briefly, 100 mg of intramus-cular fat dissolved in 1 mL of chloroform was added toa 100 mg aminopropyl minicolumn (Varian, Harbor City,USA), which was previously activated with 3 mL of chloro-form. NL were eluted with 3 mL of chloroform:2-propanol(2:1). FFA were eluted with 3 mL of diethyl ether:aceticacid (98:2), and PL were subsequently, eluted with 3 mL ofmethanol. A SPE method based on that developed by Pinkartet al. [2] was named “modified method” (MM), but modi-fied in order to obtain the FFA fraction, was also followedfor separating lipid fractions. Briefly, 20 mg of intramuscu-lar fat dissolved in 0.15 mL of hexane:chloroform:methanol(95:3:2) was added to a 500 mg aminopropyl minicolumn(Varian, Harbor City, USA), which have been previously ac-tivated with 7.5 mL of hexane. NL were eluted with 5 mL ofchloroform and FFA with 5 mL of diethyl ether:acetic acid(98:2). PL were eluted in two different fractions. The firstone with 2.5 mL methanol:chloroform (6:1) and the secondone with 2.5 mL of sodium acetate in methanol:chloroform(6:1). The two fractions containing PL obtained with thismethod were mixed and analysed together. Regardless themethod, the vacuum was adjusted to generate a flow of1 ml min−1.

In one of the samples, after evaporation of solvents un-der nitrogen, each fraction was dissolved in 0.3 mL of chlo-roform and separated by TLC on 20 cm× 20 cm platesof silica gel 60 F254 (1 mm thickness) (Merck) developedtwice in the same direction with a solvent mixture of chlo-roform:methanol:acetic acid (100:5:2)[11]. Lipid classeswere identified by running different standards (either tri-olein, oleic acid or dioleyl phosphatidylcholine, Sigma) in

a TLC plate under exactly the same conditions. Lipid frac-tions were evidenced by using UV light. For obtaining thephotographs, spots in the TLC plates were visualized by ex-posing the plates to iodine vapour in a chamber for 30 min.

After solvent evaporation, fatty acids from each lipid frac-tion were subjected to acidic trans-esterification in presenceof sulphuric acid (5% sulphuric acid in methanol) to obtainfatty acid methyl esters (FAMEs) following the method-ology described by Sandler and Karo[12]. FAMEs wereanalysed by gas chromatography using a Hewlett-PackardHP-5890A gas chromatograph (GC), equipped with aflame ionisation detector. Separation was carried out ona polyethylene glycol-TPA modified fused silica semi-capillary column (Hewlett-Packard, 30 m× 0.53 mm i.d.,1�m film thickness). GC oven temperature was isothermal(225◦C) and the chromatographic run lasted 30 min. Injec-tor and detector temperatures were 230◦C. Carrier gas wasnitrogen at a flow rate of 1.8 mL/min. Individual FAMEpeaks were identified by comparing their retention timeswith those of standards (Sigma, St. Louis, MO, USA).Results are expressed as percentage of total fatty acidsdetected.

Data concerning proportion of individual FAMEs in theNL, FFA and PL fractions of both methods were subjected toa one-way analysis of variance (ANOVA) using the generallinear model (GLM) procedure of the SPSS package (v. 11.0)to determine the effect of the SPE method (OM versus MM)on the proportion of each FAME detected.

3. Results and discussion

Examination of TLC plates of the lipid fractions obtainedwith the two studied SPE methods (Fig. 1) showed thatfraction 1 obtained with the OM was not constituted only byNL (mainly triacylglycerols) but it also contained a certainamount of phospholipids. On the other hand, only an almostundetectable presence of phospholipids was evidenced inthe fraction 1 of the MM, evidencing that this fraction wasconstituted practically only by NL. The fraction 2 of the OMwas mainly constituted by FFA, with a small contaminationof PL. Fractions 2 and 3 of the OM showed a small spotof NL. In contrast, fraction 2 of the MM was exclusivelyconstituted by FFA and showed no evident presence of NL

J. Ruiz et al. / Analytica Chimica Acta 520 (2004) 201–205 203

Fig. 1. TLC of eluted lipid fractions from the masseter muscle of pigs obtained following two different SPE methods (OM vs. MM), triacylglycerols(TG); phospholipids (PhL); free fatty acids (FFA).

nor of PL, while fractions 3+ 4 obtained with this SPEmethod showed exclusively a spot of PL.

Problems in separating NL in aminopropyl minicolumnsusing clhoroform:isopropanol as states the method ofKaluzny et al. [1] has been evidenced by other authors.Thus, Pinkart et al.[2] showed an almost 50% elution ofphospholipids in the NL fraction using this method forstudying microbial lipids. However, other authors have de-tected no cross-contamination between fractions using thismethod, even if columns were intentionally overloaded[8].Nevertheless, Juaneda and Rocquelin[13] advised that theamount of lipids should be carefully controlled, with riskof cartridge saturation. These contradictions in the abilityof the SPE method for separating NL and PL could be dueto the nature of the NL and PL in each study. Thus, Pinkartet al. [2] claimed that such a problem in correctly elutingNL and PL was due to the low polarity of microbial lipidsstudied, due to their lower degree of unsaturation. However,Bateman and Jenkins[8] also carried out their study on mi-crobial lipids. In our study, we have followed the most usedmodification of the Kaluzny et al.[1] procedure for musclelipids. It could be that a lower ratio between the amount ofadded muscle lipids and the size of the column resulted in

a better separation of lipid classes. However, an inadequateseparation of NL and PL has also been evidenced by Pinkartet al. [3] using standards and being very far from cartridgesaturation.

Fatty acid profiles of NL, FFA and PL obtained usingboth methods were analysed by gas chromatography. As aconsequence of the wrong separation of major muscle lipidclasses obtained with the OM, proportions of several indi-vidual FAMEs from NL were significantly different to thoseobtained with the MM (Table 2). Thus, fraction 1 (whichwas theoretically constituted only by NL) obtained using theOM showed higher amounts of saturated fatty acids (SFA)(P < 0.001) and polyunsaturated fatty acids (PUFA) (P <

0.001) and lower of monounsaturated fatty acids (MUFA)(P < 0.001) than those obtained using the MM. This was aconsequence of significant differences in most major fattyacids, such as palmitic acid (C16:0) (P < 0.001) and spe-cially oleic acid (C18:1 n-9) (P < 0.001) and linoleic acid(C18:2 n-6) (P < 0.001). Muscle phospholipids show highproportions of different PUFA, linoleic acid (C18:2 n-6) be-ing the major fatty acid in masseter muscle phospholipids,while oleic acid (C18:1 n-9) is the major one in NL[3,5].Therefore, the presence of phospholipids in the fraction 1

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Table 2Fatty acid and fatty alcohol composition (average of % of total fattyacids and alcohols detected together with the relative standard deviationbetween brackets) of the neutral lipid fraction of pig masseter musclesobtained following two different SPE methods (OM vs. MM)

OM MM P

C12:0 0.05 (6.07) 0.00 0.000C14:0 1.09 (1.77) 0.93 (5.17) 0.000C15:0 0.03 (4.23) 0.02 (25.82) 0.011C16:0 ol 0.34 (9.08) 0.01 (10.83) 0.000C6:0 22.62 (0.44) 21.96 (0.88) 0.000C16:1 3.82 (1.76) 3.77 (1.67) 0.117C17:0 0.21 (9.19) 0.21 (0.90) 0.975C17:1 0.26 (5.66) 0.26 (0.63) 0.668C18:0 ol 0.14 (22.29) 0.00 0.000C18:1 ol 0.06 (15.54) 0.00 0.000C18:0 10.00 (1.65) 10.01 (0.85) 0.899C18:1 51.53 (0.44) 54.74 (0.40) 0.000C18:2 7.14 (1.34) 5.92 (1.35) 0.000C18:3 0.67 (37.45) 0.34 (2.39) 0.001C20:0 0.10 (63.01) 0.22 (8.89) 0.001C20:1 1.19 (1.84) 1.34 (1.98) 0.000C20:4 0.75 (6.67) 0.27 (1.10) 0.000� Fatty acids 99.45 (0.07) 99.99 (0.00) 0.000� Fatty alcohols 0.55 (12.63) 0.01 (63.61) 0.000SFA 34.09 (0.48) 33.35 (0.46) 0.000MUFA 56.80 (0.40) 60.11 (0.31) 0.000PUFA 8.56 (2.53) 6.53 (1.27) 0.000

obtained with the OM explains the higher proportion oflinoleic acid (C18:2 n-6) using this method than with theMM, in which phospholipids were practically absent in thefraction containing the NL. As a consequence of this higheramount of linoleic acid (C18:2 n-6) (and other PUFA), theproportion of oleic acid (C18:1 n-9) was lower in the NLfraction using the OM method. Other minor fatty acids alsoevidenced the coelution of phospholipids with the NL frac-tion using the OM. For example, linolenic acid (C18:3 n-3)showed values nearly twice higher in the NL fraction usingthe OM than with the MM, and arachidonic acid (C20:4 n-6)showed proportions almost three times higher.

Therefore, as a consequence of wrong separation of NLand PL from muscle lipids using the Kaluzny procedure,the obtained fatty acid profile does not accurately reflect theactual fatty acid composition of NL, with an overestimationof PUFA and underestimation of MUFA. On the other hand,the modified Pinkart method, due to a better separation ofthese two lipid classes, allowed obtaining a more accuratefatty acid profile of the NL fraction.

Moreover, NL obtained using the OM showed the pres-ence of three different fatty alcohols:hexadecanol (C16:0 ol),octadecanol (C18:0 ol) and octadecenol (C18:1 ol) (Table 2).These molecules are linked to the sn-1 position of somephospholipids through an ether linkage[14,15]. However,such ether linkages binding a fatty alcohol to a glycerolbackbone are absent or only in trace amounts in triacylglyc-erols [16]. Therefore, the presence of fatty alcohols in theNL fraction obtained using the OM confirms the coelution ofphospholipids in such fraction. On the other hand, by using

Table 3Fatty acid and fatty alcohol composition (average of % of total fattyacids and alcohols detected together with the relative standard deviationbetween brackets) of the free fatty acids fraction of pig masseter musclesobtained following two different SPE methods (OM vs. MM)

OM MM P

C14:0 0.47 (3.62) 0.69 (11.56) 0.000C15:0 0.05 (22.75) 0.08 (14.30) 0.015C16:0 ol 0.09 (104.24) 0.10 (15.65) 0.840C16:0 17.72 (1.76) 19.28 (3.48) 0.000C16:1 2.35 (3.53) 2.68 (6.97) 0.000C17:0 0.34 (5.60) 0.34 (5.65) 0.945C17:1 0.26 (4.54) 0.29 (5.57) 0.000C18:0 ol 0.09 (126.58) 0.05 (4.79) 0.537C18:1 ol 0.01 (51.06) 0.00 0.206C18:0 13.84 (3.31) 13.77 (5.55) 0.808C18:1 43.07 (3.20) 44.29 (4.40) 0.131C18:2 16.31 (6.06) 14.05 (21.16) 0.036C18:3 0.75 (20.76) 0.55 (12.72) 0.003C20:0 0.15 (26.55) 0.00 0.000C20:1 1.12 (5.08) 1.36 (61.61) 0.377C20:4 3.37 (8.20) 2.44 (10.75) 0.000� Fatty acids 99.81 (0.27) 99.85 0.19) 0.754� Fatty alcohols 0.19 (145.73) 0.15 (123.18) 0.754SFA 32.58 (1.89) 34.18 (3.31) 0.001MUFA 46.80 (2.91) 48.63 (5.09) 0.059PUFA 20.44 (5.61) 17.04 (18.73) 0.006

the MM the NL fraction showed only an almost insignificantamount of hexadecanol (C16:1 ol).

Differences found in the fatty acid profile of FFA obtainedwith each method were smaller than those found in the NLfraction (Table 3). As in the latter fraction, FFA obtainedwith the OM showed lower proportions of MUFA and higherof PUFA than those obtained with the MM. On the otherhand, SFA were higher in FFA obtained using the MM.These differences between both methods are most likelydue to coelution of phospholipids with the FFA fraction inthe OM. In fact, a small amount of phospholipids can beobserved in the FFA fraction (fraction 2) obtained with theOM in the TLC shown inFig. 1.

Finally, PL showed a much lesser influence of the SPEmethod used (Table 4). Thus, only palmitic acid (C16:0),stearic acid (C18:0) and arachidonic acid (C20:4 n-6) amongmajor fatty acids showed significant differences betweenmethods. These differences between methods could be dueto either the elution of less polar phospholipids with theNL fraction when the OM is used, or the inadequate elutionof acidic phospholipids in this method, due to the use ofmethanol as the only solvent for eluting PL. Both problemshave been previously reported[2]. The incorrect elution ofdifferent classes of phospholipids could easily lead to a dif-ferent fatty acid profile, since fatty acid composition of dif-ferent muscle phospholipids classes is very different[17].

It can be concluded that the Pinkart et al.[2] SPE lipidseparation method is more convenient for separating mus-cle lipids into major lipid classes and obtaining an accuratefatty acid profile of neutral lipids. The widely used Kaluznyet al. [1] SPE method leads to coelution of PL with the NL

J. Ruiz et al. / Analytica Chimica Acta 520 (2004) 201–205 205

Table 4Fatty acid and fatty alcohol composition (average of % of total fattyacids and alcohols detected together with the relative standard deviationbetween brackets) of the polar lipid fraction of pig masseter musclesobtained following two different SPE methods OM vs. MM)

OM MM P

C16:0 ol 6.02 (29.45) 10.38 (26.87) 0.002C16:0 9.66 (6.49) 14.04 (1.93) 0.000C16:1 0.69 (9.70) 0.71 (7.87) 0.469C17:0 0.26 (7.33) 0.30 (2.47) 0.000C17:1 0.24 (30.43) 0.25 (19.31) 0.777C18:0 ol 5.44 (19.73) 6.04 (28.36) 0.360C18:1 ol 3.38 (21.26) 3.71 (26.76) 0.407C18:0 17.05 (6.89) 14.71 (2.67) 0.000C18:1 14.30 (12.01) 14.16 (8.03) 0.833C18:2 24.75 (4.56) 23.96 (13.13) 0.464C18:3 0.45 (8.83) 0.45 (10.59) 0.887C20:1 0.35 (10.01) 0.29 (9.06) 0.103C20:4 17.44 (7.01) 11.02 (9.82) 0.000� Fatty acids 85.17 (5.02) 79.88 (6.87) 0.027� Fatty alcohols 14.83 (29.19) 20.12 (27.26) 0.027SFA 26.96 (6.41) 29.04 (1.43) 0.002MUFA 15.57 (10.99) 15.40 (7.35) 0.796PUFA 42.64 (4.61) 35.43 (12.02) 0.000

fraction, which in turn leads to obtain a wrong NL, FFA andPL fatty acid profile.

Acknowledgements

The authors are grateful to Ana Galaz and NatividadHurtado for their technical assistance. This study was

partially supported by the FEDER project “Optimisationof Genetic Evaluation of Iberian swine including qual-ity parameters in both raw meat and processed products”(1FD97-1252-C02-01).

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