analysis of triacylglycerols— approaching the molecular composition of natural mixtures

This article was downloaded by: [Cornell University]On: 09 May 2012, At: 04:58Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Food Reviews InternationalPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/lfri20

Analysis of triacylglycerolsapproaching the molecular compositionof natural mixturesPivi Laakso aa Department of Biochemistry and Food Chemistry, University ofTurku, Turku, FIN20500, Finland

Available online: 03 Nov 2009

To cite this article: Pivi Laakso (1996): Analysis of triacylglycerols approaching the molecularcomposition of natural mixtures, Food Reviews International, 12:2, 199-250

To link to this article: http://dx.doi.org/10.1080/87559129609541075

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden.

The publisher does not give any warranty express or implied or make any representationthat the contents will be complete or accurate or up to date. The accuracy of anyinstructions, formulae, and drug doses should be independently verified with primarysources. The publisher shall not be liable for any loss, actions, claims, proceedings,demand, or costs or damages whatsoever or howsoever caused arising directly orindirectly in connection with or arising out of the use of this material.

Food Rev. Int., 12(2), 199-250 (1996)

ANALYSIS OF TRIACYLGLYCEROLSAPPROACHING THE MOLECULARCOMPOSITION OF NATURAL MIXTURES

PIVI LAAKSODepartment of Biochemistry and Food ChemistryUniversity of TurkuFIN-20500 Turku, Finland

ABSTRACTEdible fats and oils are mainly composed of triacylglycerols synthe-sized by plants and animals. Natural triacylglycerols are predominant-ly such complex mixtures that it is impossible to separate all moleculesby a single analytical technique. However, the molecular structure oftriacylglycerols is of great importance from the biochemical, nutri-tional, and technological points of view. This article reviews thechromatographic and mass spectrometric methods in the analysis oftriacylglycerols as well as the techniques for stereospecific analysis,with special attention to the analysis of milk fat and fish oils. Theprinciples of triacylglycerol separation with high-performance liquidchromatographic, gas chromatographic, and supercritical fluid chro-matographic methods are described. In general, chromatographictechniques offer a wide variety of possibilities for separation of mo-lecular species of triacylglycerols, but the identification of the com-ponents is often a problem. Mass spectrometry provides informationboth for structure elucidation and for quantitation purposes. Theadvantages of different ionization modes in the analysis of triacyl-glycerols are presented. In addition, the usefulness of tandem massspectrometric methods, yielding information on the molecular asso-ciation of fatty acids and the regiospecific distribution of fatty acids(sn-2 and sn-1/3 positions) is discussed. The traditional methods forthe complete stereospecific analysis of triacyl-sn-glycerols include the

199

Copyright 1996 by Marcel Dekker, Inc.

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012

200 LAAKSO

phosphorylation of diacylglycerols and their hydrolysis with phospho-lipases. Over the last few years methods for the separation of enantio-meric acylglycerols have been developed, and their capability for thestereospecific analysis of triacylglycerols is discussed in this review.

INTRODUCTION

The compositions of milk fat and fish oil triacylglycerols are extremely complex.Milk fat contains typically short-, branched-, and odd-chain fatty acyl residues, inaddition to most common fatty acids (Patton and Jensen, 1975; Jensen et al.,1991). Long-chain polyunsaturated fatty acids are characteristic to most fish oils(Padley et al., 1986). Furthermore, triacylglycerols are optically active moleculesin the case that the two primary hydroxyl groups of glycerol are esterified withdifferent fatty acids. In order to specify unambiguously the position at which thefatty acid is bound to the glycerol backbone, a stereospecific numbering (sn)*system, initially proposed by Hirschmann (1960), has been recommended by theIUPAC-IUB (1967) commission on the nomenclature of glycerolipids. When theglycerol molecule is drawn in a Fischer projection with the secondary hydroxylgroup to the left of the middle carbon atom (sn-2), the carbon atom above thisbecomes sn-1 and that below becomes sn-3.

The molecular structure of triacylglycerols is of great importance from the nu-tritional, biochemical, and technological points of view. During fat digestion, li-pases hydrolyze triacylglycerols predominantly into free fatty acids from sn-1/3positions and 2-monoacyl-s-glycerols which will be absorbed into the intestinalmucosal cells of the small intestine. The effect of the stereospecific structure onthe absorption and metabolism of triacylglycerols has never been thoroughly in-vestigated because of the diversity of dietary fats and oils (Small, 1991). Duringthe biosynthesis of triacylglycerols all glycerol positions are important. The majorroutes for triacylglycerol synthesis are the sn-glycerol-3-phosphate, the dihy-droxyacetone phosphate, and the monoacylglycerol pathways. In most cases, thesn-3 is the last position to be esterified. Moreover, the molecular structure oftriacylglycerols has an effect on the physical propertiessuch as crystal structure,melting point, solubility and viscosityof fats and oils (Larsson, 1986).

Fatty acid compositions alone do not yield enough information on the moleculestructures of triacylglycerols. Analytical methods which separate molecules ondifferent bases have therefore been proved useful. This article reviews the chro-matographic and mass spectrometric methods in the analysis of triacylglycerols aswell as the techniques for stereospecific analysis, with special attention to milk fatand fish oils.

* A list of abbreviations used is provided at the end of the text.

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012

ANALYSIS OF TRIACYLGLYCEROLS 201

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHYSilver Ion High-Pe\rformance Liquid ChromatographyThe capacity of silver ions to facilitate the separation of molecules according tounsaturation by chromatographic methods has been widely used for determiningthe triacylglycerol composition of fats and oils (reviewed by Morris, 1966; Litch-field, 1972; Nikolova-Damyanova, 1992; Christie, 1994; Dobson et al., 1995). Theseparation is based on the weak interaction between the n-electrons of the doubleand triple bonds and the silver ions. These interactions are weak enough to beaffected by normal chromatographic procedures, for example, changing the sol-vent composition. The separation of triacylglycerols depends on (1) the numberof double bonds, (2) the distribution of double bonds between the fatty acyl resi-dues within a single molecule, (3) the configuration and position of double bondswithin a fatty acyl residue, and (4) the position at which the fatty acid is bound tothe glycerol backbone. Most often silver ion chromatography has been performedby thin-layer chromatography (TLC) on silica gel plates impregnated with silverions, or by column chromatography. Methods for high-performance liquid chro-matography (HPLC) in silver ion mode have been developed during the last dec-ade and these are described in more detail.

Methods for Silver Ion HPLCThe first methods for silver ion HPLC were based on partition chromatography util-izing reversed-phase columns and mobile phases containing silver ions (Vonachand Schomburg, 1978; Plattner, 1981). The more polar molecules eluted beforethe less polar; for example, the retention increased in the order trilinolenin SSM > SMM > SSD > MMM > SMD > MMD > SDD = SST > SMT =MDD > MMT > SDT = DDD > MDT > STT > DDT > MTT > DTT > TTT, whereT = trienoic fatty acyl moiety and the positions of the fatty acids are not differen-tiated (Christie, 1988). This is comparable, though not identical, to the order ofelution by silver ion TLC reported by Gunstone and Padley (1965). One dienoicfatty acyl residue formed stronger complexes with silver ions than two monoenoic

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012


fatty acyl residues in the same molecule, and one trienoic acid was equivalent totwo dienoic acids. In addition to the number of double bonds, both the chainlength and the position of the double bond in the fatty acyl residues had an influ-ence on separation (Nikolova-Damyanova et al., 1990). The strength of interac-tion of conjugated double bonds with silver ions was weaker than that of noncon-jugated double bonds; therefore, the separation order of the main triacylglycerolsof Trichosanthes kirilowii seed oil was SDTcon, > MDTconj > DT^T^,, > DDTconj,where Tconj = conjugated trienoic fatty acyl residue (Joh et al., 1995). Triple bondshave been reported to form much stronger interaction with silver ions than doublebonds (Neff et al., 1994); for example, triacylglycerols containing one triple bondand five double bonds in the acyl chains (i.e. one crepenynic acid and two linoleicacid moieties) eluted before molecules containing two triple and two doublebonds in the acyl chains (i.e. two crepenynic acids and one saturated fatty acid).The capacity of silver ion TLC or silver ion column chromatography to separatetriacylglycerols differing in the configuration of monoenoic fatty acyl residues hasbeen investigated with standard compounds (DeVries, 1964; DeVries and Jur-riens, 1963; Wessels and Rajagopal, 1969; Hammond, 1981b; Aitzetmuller andGuaraldo Goncalves, 1990) and samples of food components such as milk fat(Breckenridge and Kuksis, 1968, 1969; Taylor and Hawke, 1975; Parodi, 1980,1981,1982; Arumughan and Narayanan, 1982; Myher et al., 1988), hydrogenatedfat (Dallas and Padley, 1977), and tallow (Chobanov et al., 1976). By means ofsilver ion HPLC, good separations of triacylglycerols of milk fat (Christie, 1991b;Briihl et al., 1993; Laakso et al., 1992; Laakso and Kallio 1993a, 1993b), sheepadipose tissue (Christie, 1988), and hardened/randomized palm fraction (Smithet al., 1994) having a cis-trans difference, have been obtained. Recently, Joh etal. (1995) reported the separation of triacylglycerols containing configurationalisomers of trienoic fatty acids by silver ion HPLC. In general, trans fatty acids formweaker complexes with silver ions than cis acids. According to Smith et al. (1994),the elution order of triacylglycerols was SSS > SSM' > SSMC > SM'M' > SMCM' >M'M'M' > SMCMC > M'M'M0 > M'MCMC > MCMCMC, where M' = fraras-monoenoicand Mc = cw-monoenoic fatty acyl residue. The separation of triacylglycerols hav-ing only a difference in the sn positions of fatty acyl residues has also been achievedon a silver-loaded cation-exchange column (Briihl et al., 1993): SMS eluted beforeSSM, and SMM before MSM. In general, the resolution of critical pairs may beimproved if analyses are performed at temperatures below ambient (Wessels andRajagopal, 1969; Smith et al., 1980). The amount of silver ions on the column alsohas an effect on separation (Adlof et al., 1980; Smith et al., 1980).

The resolution limits have been tested by analyzing the polyunsaturated triacyl-glycerols of fish oils (Christie, 1988; Laakso et al., 1990). The resolution of theleast unsaturated molecules was excellent, but baseline separation of moleculescontaining trienoic or more highly unsaturated fatty acids could not be achieved dueto the presence of many overlapping components. However, fractions containing on

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012

204 LAAKSO

average as many as 14 double bonds in each molecule were obtained (Laakso etal., 1990). In spite of the complexity of fish oils, recognizable fractions of SMT,SMTe, SMP, and SMH molecular species (Te = tetraenoic, P = pentaenoic, andH = hexaenoic fatty acyl residue) were separated from three fish species studied.When compared with TLC analysis (Bottino, 1971; Kallio et al., 1991b), a muchbetter resolution offish oil triacylglycerols was obtained by silver ion HPLC. Over-all, silver ion HPLC with ion-exchange columns is a more reproducible, tidy, andefficient method for separation of triacylglycerols according to unsaturation thanTLC. Furthermore, oxidation of samples is minimized by HPLC and fractions freeof silver ions can be isolated. In addition to HPLC, this method has been adaptedto solid-phase extraction columns packed with a bonded sulfonic acid phase(Christie, 1989b) and applied to the separation of cocoa butter, palm oil, andsheep adipose tissue triacylglycerols (Christie, 1990) as well as to the fractionationof triacylglycerols of lipase-modified butterfat (Kalo and Kemppinen, 1993;Kemppinen and Kalo, 1993). The suitability of this method for supercritical fluidchromatography has been reported recently (Demirbuker and Blomberg, 1990,1991; Demirbuker et al., 1992,1993).

Reversed-Phase HPLC

Nonaqueous reversed-phase HPLC is a useful method for separation of triacyl-glycerols at moderate temperatures (reviewed by Aitzetmuller, 1982; Christie,1987a; Shukla, 1988; Kuksis, 1994), as is shown in the examples of milk fat andfish oils (Table 1). Generally, separations are performed almost exclusively oncolumns packed with chemically bonded octadecylsilyl (ODS) phases. The mobilephase usually consists of acetonitrile and acetone, although mixtures of acetoni-trile and propionitrile or acetonitrile and chlorinated solvents may be more effi-cient. The available detector often restricts the choice of suitable solvents. In ad-dition to ultraviolet (UV) and refractive index (RI) detection, more universalalternatives have been developed: flame ionization (FID) and evaporative light-scattering detectors (ELSD) (reviewed by Christie, 1987a, 1992b; Moreau, 1994).Excluding mass spectrometry, ELSD seems to be the best choice for triacyl-glycerols at present: it allows almost any solvent, including ketones, esters, andchlorinated and aromatic compounds, to be used in complex gradients. Moreover,it is sensitive and easy to use.

Separation of TriacylglycerolsSeparation of triacylglycerols by reversed-phase HPLC is based on both the com-bined chain lengths of the fatty acyl residues and on the total number of doublebonds in the molecule. Components elute in ascending order of combined chainlength but with each of the double bonds reducing the retention'time of the molecules

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012

Table 1. Examples of Analysis of Milk Fat and Fish Oil Triacylglycerols by Means of Reversed-Phase HPLC

ColumnTemperature Components of

(C) Mobile Phase Detector Ref.

LiChrosorb'" RP-18 5 ^ m, 200 X 2.0 mm IDLiChrospher1" 1000 CH-18/2 5 Aim,

250 x 4.0 mm IDLiChrospher 1000 CH-18/3 5 Aim,

2 X (250 X 4.0 mm ID)Nucleosir"-C18 5 Aim, 150 x 4.0 mm ID +

MicrospherT"-C18 3 Aim, 100 X 4.6 mm IDLiChrospher 100 CH-18/2 5 Aim,

2 x (250X4.0 mm ID)LiChrospher 100 RP-18 4 Aim,

250 x 4.0 mm IDSupelcosil LC-18 5 Aim,

2 x (150 mm x 4.6 mm ID)Supelcosil LC-18,5 Aim, 250 x 4.6 mm IDSpherisorb1" ODS-2,5 Aim,

2 X (150 x 4.6 mm ID)Supelcosil LC-18Hypersil ODS 5 Aim, 250 X 4.0 mm IDSupelcosil LC-18,250 X 4.6 mm IDSpherisorb ODS-2,3 Aim, 125 X 4 mm IDSupelcosil LC-18,250 X 4.6 mm IDSupelcosil LC-18,250 x 4.6 mm ID

Milk Fat Triacylglycerolsns

ns

ns

30,35

40

30,40

32

ns

25

3010-60ns

7-55ns

25

Acetonitnle, acetoneAcetonitrile, acetone

Acetonitrile, acetone





Acetonitrile, acetoneAcetonitrile, chloroform

Acetonitrile, propionitrileAcetonitrile, propionitrileAcetonitrile, propionitrile

Acetonitrile, propionitrileAcetonitrile, isopropanolAcetonitrile, propionitrile

ELSDELSD

ELSD

RI

FID

RI

RI

RIELSD

MS (PCI)RIMS (PCI)

RIELSDELSD, MS (PCI)

Stolyhwo et al., 1983Stolyhwo et al., 1984

Stolyhwo et al., 1984

Frede & Thiele, 1987

Nurmela & Satama, 1988

Maniongui et al., 1991;Gresti et al., 1993

Bornaz et al., 1992

Dotson et al., 1992Kermasha et al., 1993

Kuksis et al., 1986Weber et al., 1988a and bKuksis et al., 1989, Myher

et al., 1993Hinrichs et al., 1992Myher et al., 1993Marai et al., 1994

(continued)

o

205

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012

Table 1. Continued

ColumnTemperature Components of

Mobile Phase Detector Ref.

C18 5 Aim, 300 X 4.6 mm ID

Spherisorb ODS-25Aim,250 mm x 4.6 mm ID + Zorbax C185 Aim, 250 mm X 4.6 mm ID

LiChrospher 100 RP-18,250 X 4.6 mm IDSpherisorb ODS 5 Aim, 2 x (200 x

4.6 mm ID)Ultrasphere ODS-IP5Aim,

250 x 4.6 mm IDSpherisorb ODS2 5 Aim, 50 x 4.6 mm ID

+ 250 X 4.6 mm ID

RP18 5 Aim, 2 x (250 x 4.0 mm ID)RSil C18 HL 5 jam, 250 x 4.6 mm ID

C18 5 jam, 250 X 4.6 mm ID

Spherisorb ODS2 5 Aim, 250 x 5.0 mm ID

Zorbax ODS 5 Aim, 250 x 4.6 mm ID +Spheri-ODS 5 Aim, 250 x 4.6 mm ID

ns

ns

2230

25

Ambient

10-6020

ns

Ambient

Ambient

Acetonitrile, propionitrilePropionitrile, acetone,

dichloromethaneAcetonitrile, propionitrile,

dichloromethane

Acetonitrile, ethanol,Methyl-toY-butylether,

acetonitrileAcetonitrile, 2-propanol,

hexaneAcetonitrile, acetoneAcetonitrile, ethanol

Propionitrile, etherAcetonitrile, isooctane,

2-propanolAcetonitrile, waterAcetonitrile, acetoneAcetonitrile, acetoneAcetonitrile, 1,2-dichloro-

ethaneAcetonitrile, 1,2-dichIoro-

ethane, dichloromethane

MS (PCI)MS (NCI)

ELSD

ELSDUV 220 nm

UV 220 nm

RI, ELSDELSD, UV

225 nmRIUV 215 nm

UV 210 nmRIELSDELSD

ELSD

Kuksisetal., 1991a

Spanos et al., 1995

HersIof&Kindmark, 1985Barron et al., 1990

Gilkison, 1988

Robinson & Macrae, 1984

Frede, 1986Geeraert & De Schepper,

1983Lee, 1986

Christie, 1987a

Laakso et al., 1992; rLaakso & Kallio, 1993a, j *1993b O

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012

Zorbax ODS 6 Aim, 250 X 4.6 mm ID

AiOBondapak-C18LiChrospher 1000 CH-18/2 5 yum,

250 x 4.0 mm IDSupersphere RP-18 3 Aim, 250 x 4.0 mm ID AmbientSupelcosil LC-18 5 fim, 250 mm

Whatman Magnum-9 Partisil-10 ODS-3,500 X 9.4 mm ID

Hypersil ODS, 5 /um, 2 X (200 x 2.1 mm ID)+ (100 x 2.1 mm ID)

Supelcosil LC-18,5 /um, 240 mm

Supelcosil LC-18,250 x 4.6 mm ID

C18 5 jam, 300 X 4.6 mm ID

Spherisorb ODS25/*m, 2 x (250 X4.6 mm ID)

23

27Ambient

Ambientns

40

ns

ns

ns

Ambient

Fish Oil TriacylglycerolsAcetonitrile, tetrahydro-

furanMethanol, chloroformAcetonitrile, acetone

Acetonitrile, acetoneAcetonitrile, acetone


Acetonitrile, acetone,2-propanol

Acetonitrile, ethanol,tetrahydrofuran

Acetonitrile, propionitrile

Acetonitrile, propionitrilePropionitrile, acetone,

dichloromethaneAcetonitrile, dichloro-

methane, 1,2-dichloro-ethane

IR 5.75 Aim

RIELSD

RIRI

UV, MS (FAB)

RI

RI,UV225nm

MS (PCI)ELSDMS (PCI)MS (NCI)

ELSD

Parris, 1979

Wada et al, 1979a, 1979bStolyhwo et al., 1984

Takahashi et al., 1988Wojtusik et al., 1988a

Hori et al., 1994

Wojtusik et al., 1988b

Kuksis et al., 1987,1989Kuksis, 1994Kuksis etal., 1991a

Laakso & Christie, 1990

VI

>FTJ

r>

208 LAAKSO

by an equivalent of about two methylene groups (Christie, 1987a). To estimatethe elution order and to facilitate the identification of triacylglycerols, an equiva-lent carbon number (ECN) value (also called partition number) has been intro-duced and defined as the combined number of acyl carbon atoms minus twice thenumber of double bonds in the fatty acyl residues (Plattner et al., 1977). Moleculeshaving different ECN values are well separated, but those with identical ECNvalues (e.g., 16:0-18:1-18:1 and 16:0-16:0-18:1) have close retention properties onreversed-phase HPLC and thus are called critical pairs. Within each ECN value,the most unsaturated molecules elute first, and the least unsaturated last. Criticalpairs of triacylglycerols, which differ only in the position at which the fatty acid isbound to the glycerol backbone (also called reverse isomers), cannot be separatedas such. However, their resolution by reversed-phase HPLC becomes possibleafter halogen addition to the double bond (Podhala and Toregard, 1984,1989).Besides this, the chain-length asymmetry of triacylglycerols can have an effect onseparation: within each acyl carbon number triacylglycerols containing short-chain fatty acids elute later than those containing longer-chain fatty acids (Nur-mela and Satama, 1988; Kuksis et al., 1989; Maniongui et al., 1991; Myher et al.,1993; Marai et al., 1994). The residual polarity of the column support has beensuggested to affect the separation and elution order of isologous triacylglycerols(Myher et al., 1993). Triacylglycerols having only a cis-trans difference have beenseparated by reversed-phase HPLC using reference compounds (El-Hamdy andPerkins, 1981; Phillips et al., 1984; Gilkison, 1988; Podhala and Toregard, 1989)and milk fat (Laakso and Kallio, 1993a, 1993b). Furthermore, the position of thedouble bond in the fatty acyl residue has an influence on separation (Phillips etal., 1984).

The separation of triacylglycerols, including critical pairs, can be described withvalues which are related to the acyl carbon number of a hypothetical saturatedtriacylglycerol eluting with the same retention time as the unknown molecule.El-Hamdy and Perkins (1981) introduced the term "theoretical carbon number"(TCN) for this purpose. The TCN value is determined either from a plot of thelogarithm of the capacity factor (k') versus acyl carbon number of saturatedtriacylglycerols or calculated from the formula TCN = ECN - (E^Uj), where U;is a factor determined experimentally for each fatty acid. Podhala and Toregard(1982) used the term "equivalent carbon number," which is analogous to TCN.The determination of equivalent carbon numbers was based on the elution of twoknown saturated triacylglycerols under isocratic conditions. A similar approach,based on standard addition, was also used to determine retention indices fortriacylglycerols under gradient elutions (Herslof and Kindmark, 1985; Nurmelaand Satama, 1988; Laakso and Christie, 1991; Laakso et al., 1992; Laakso andKallio, 1993a, 1993b). Sempore and Bezard (1986) introduced an equation withwhich it is possible to calculate the equivalent carbon number (the same definitionas used by Podhala and Toregard 1982) from the experimentally determined log

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012


a value (a = selectivity factor). Takahashi et al. (1984,1985,1986) used a matrixmodel to enhance the identification of molecular species. This model was basedon the linear relationship between the logarithm of the relative retention timeversus acyl carbon number or the total number of double bonds.

In order to identify triacylglycerols unambiguously according to the numericalvalues described above, the chromatographic system has to be extremely well cali-brated using standard compounds. However, this is not possible when more com-plex mixtures such as milk fat and fish oil triacylglycerols are analyzed. Thus, thebest way to obtain exact information on the molecular weights and fatty acid com-positions of triacylglycerols is to use mass spectrometric detection.

An alternative to reversed-phase HPLC analysis is the separation of triacyl-glycerols on silica columns (Plattner and Payne-Wahl, 1979; Rhodes and Netting,1988). In contrast to reversed-phase HPLC resolution, molecules elute accordingto decreasing acyl carbon numbers, and saturated triacylglycerols elute before themore unsaturated ones. Silica columns are most often used for separation of dif-ferent lipid classes and very seldom for the analysis of triacylglycerol molecularspecies.

GAS CHROMATOGRAPHY

Determination of triacylglycerols by gas chromatography (GC) is an efficient al-ternative for reversed-phase HPLC analysis and therefore its principles are dis-cussed briefly. Triacylglycerols have been analyzed by GC on both packed andcapillary columns using stationary phases of various polarities (reviewed by Litch-field, 1972; Geeraert, 1987; Mares, 1988; Christie, 1989a; Kuksis, 1994).

Separation of Triacylglycerols

The separation coefficients, and thus separation, of triacylglycerols on nonpolarmethylsilicone type stationary phases are mainly based on the vapor pressure dif-ferences between analytes. In this case, the resolution obtained is due mainly tothe differences in molecular weights, that is, the combined number of acyl carbonatoms. Some separation of triacylglycerols according to the degree of unsaturationcan be achieved on more efficient nonpolar columns: unsaturated triacylglycerolselute before saturated ones. Instead of the number of double bonds, the numberof unsaturated fatty acyl moieties in a triacylglycerol molecule determines theorder of elution (Geeraert et al., 1983; Geeraert and Sandra, 1985). Moreover,separation of triacylglycerols based on chain-length asymmetry within a given acylcarbon number has been reported (Maniongui et al., 1991). The distribution oftriacylglycerols solely according to their total acyl carbon numbers is best achievedif the sample is completely hydrogenated before analysis.

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012

210 LAAKSO

Capillary columns coated with chemically bonded polarizable phenylmethyl-silicone type stationary phases separate triacylglycerols according to the numberof acyl carbon atoms and double bonds. The polarity and selectivity for doublebonds of the liquid phase seems to increase with increasing temperature (Geeraertand Sandra, 1984). At temperatures above 290C, within each acyl carbon number,the most unsaturated molecules have the longest retention time, and the mostsaturated the shortest. Furthermore, Geeraert and Sandra (1984) observed thatthe higher the polarity of the phase, the lower the elution temperature of thetriacylglycerols. In some cases, components having the same number of acyl car-bons and double bonds, but differing in fatty acid composition, are partially sepa-rated (Kuksis et al., 1973; Geeraert and Sandra, 1985; Myher et al, 1988): polaritydifferences between the fatty acyl residues result in a greater retention of triacyl-glycerols containing short-chain fatty acids than those containing longer-chainfatty acids. For example, triacylglycerols containing both short-chain and long-chain fatty acyl residues are separated in order of decreasing chain length of theshort-chain fatty acids; that is, the molecules eluted in the order of caprates fol-lowed by caprylates, caproates, butyrates, and acetates (Myher et al., 1988; Kuksis,1994). No separation has been reported on triacylglycerols differing only in theposition of fatty acid in the glycerol backbone. Likewise, triacylglycerols having acis-trans difference have not been separated by GC.

When compared with reversed-phase HPLC separation, triacylglycerols areclearly resolved by GC into acyl carbon number groups which do not overlap withthe preceding or following groups. The results are easier to interpret, althoughidentification of the components can be confirmed only by mass spectrometricdetection. One of the critical steps during the analysis of triacylglycerols by GC isthe sample introduction (Mares, 1988; Kuksis and Myher, 1989). Vaporizing in-jectors are not suitable, due to their discrimination of less volatile compounds(Grob, 1979). The most reproducible and representative results have been ob-tained by cold on-column injection (Grob, 1979) and on-column injection at highoven temperature (Geeraert et al., 1983). Another problem has been the stabilityof liquid phases. Nowadays polar stationary phases can be produced withoutbleeding problems at temperatures as high as 370C, which are necessary fortriacylglycerol analysis. The third problem is quantitative recovery of the sampleat the temperatures used for GC analysis. Correction factors are needed fortriacylglycerols having more than 54 acyl carbons due to on-column losses, de-pending on the length of the column (Kuksis, 1994). Unsaturated triacylglycerolsare lost primarily because of thermal degradation and to a lesser extent throughpolymerization (Grob, 1981). In order to minimize the thermal exposure time ofthe sample, analysis should be carried out as quickly as possible. However, triacyl-glycerols containing highly unsaturated fatty acids, such as fish oils, are not rec-ommended for GC analysis (Geeraert, 1987; Mares, 1988).

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012


SUPERCRITICAL FLUID CHROMATOGRAPHY

Supercritical fluid chromatography (SFC) is a method whereby a highly com-pressed gas above its critical temperature and critical pressure is used to eluteanalytes from a chromatographic column. The principles of SFC and some of itsapplications have been described thoroughly, for example, in the books edited byCharpentier and Sevenants (1988), Smith (1988) and White (1988). Liibke (1991)has reviewed the SFC analysis of low and medium molecular weight natural com-pounds; and Laakso (1992), the analysis of lipids by SFC.

Both packed (Carraud et al., 1987; Perrin and Prevot, 1988; Sakaki et al., 1988;Demirbuker and Blomberg, 1990,1991,1992; Demirbfiker et al., 1992; Taylor andChang, 1990; Kallio et al., 1991a) and capillary (Chester, 1984; White and Houck,1985; Proot et al., 1986; Sandra et al., 1986; Wright et al., 1986; Wright and Smith,1986; Cousin and Arpino, 1987; Hawthorne and Miller, 1987; Huopalahti et al.,1988; Khosah, 1988; Perrin and Prevot, 1988; Richter et al., 1988; Sandra et al.,1988; Kallio et al., 1989; Kallio and Laakso, 1990; Calvey et al., 1991; France etal., 1991; Kallio et al., 1991b; Baiocchi et al., 1993; Borch-Jensen et al., 1993; Stabyet al., 1994; Manninen et al., 1995a, 1995b) columns have been used in the analysisof triacylglycerols by SFC. Acylglycerols have usually been eluted with supercriti-cal carbon dioxide from capillary columns and detected by FID. In addition, otherdetectors have been utilized, for example, on-line Fourier-transform infraredwith FID for hydrogenated soybean oils (Calvey et al., 1991) and mass spectrome-try in both chemical ionization (Wright et al., 1986; Cousin and Arpino, 1987;Hawthorne and Miller, 1987) and electron impact ionization modes (Kallio et al.,1989). For elution of strongly retained analytes from the column, both pressureand temperature programming have been employed in order to increase the den-sity of supercritical fluid.

Separation of Triacylglycerols

Triacylglycerols have been separated according to the combined chain lengths ofthe fatty acyl residues using capillary columns coated with relatively nonpolarstationary phases (e.g., DB-1, DB-5, SE-54, SB-Octyl-50). The samplescomprised palm kernel oil and milk chocolate (Proot et al., 1986), soybean oil(Richter et al., 1988), butterfat or milk fat (Wright and Smith, 1986; Huopalahtiet al., 1988; Kallio et al., 1989; Manninen et al., 1995a; Makinen et al. 1995), fishoil (Huopalahti et al., 1988; Kallio et al., 1991b; Baiocchi et al., 1993; Borch-Jensenet al., 1993; Staby et al., 1994; Manninen et al., 1995a), vegetable oils (Baiocchi etal., 1993) and seed oils (Huopalahti et al., 1988; Kallio et al., 1991a; Manninen etal., 1995a, 1995b). The first report of glyceride analysis by capillary SFC using FIDfor detection was published by Chester in 1984. In general, SFC analysis on non-polar stationary phases separates triacylglycerols into groups according to the

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012

212 LAAKSO

combined number of acyl carbons. The separation of, for example, milk fat triacyl-glycerols on an SB-Octyl-50 stationary phase (Manninen et al., 1995a) resembledthat obtained by high-temperature GC on a nonpolar stationary phase (Kuksis,1994). The resolution in GC was better, resulting in enhanced separation oftriacylglycerols having odd and even numbers of acyl carbons. The quantitativeresults obtained by SFC and GC have been reported to be almost identical (Sandraet al., 1986). The most remarkable difference was in the duration of analysis: 30min for SFC and only 3 min for GC. However, a much faster separation waspossible by SFC with a shorter (1.5 m x 25 jum ID) fused silica capillary columncoated with the same phase and with rapid pressure programming at 100C(Wright and Smith, 1986).

By selecting a more polar stationary phase, triacylglycerols have been separatedaccording to the degree of unsaturation. A baseline separation of tristearin, tri-olein, trilinolein and trilinolenin has been reported with a 25% cyanopropyl-25%phenyl-50% methyl polysiloxane (White and Houck, 1985; Sandra et al., 1988) aswell as with a 50% cyanopropyl-50% methyl polysiloxane stationary phase (Calveyet al., 1991). A good separation of triacylglycerols differing by only one doublebond, for example, triolein and linoleoyldiolein, was achieved in the analysis ofsoybean oil on a polyethylene glycol stationary phase (Richter et al., 1988) andberry pulp and seed oils on an SB-Cyanopropyl-25 column (25% cyano-propyl-25% phenyl-50% methyl polysiloxane) (Manninen et al., 1995a, 1995b).A baseline separation of a- and y-linolenic acid containing triacylglycerols withan identical number of acyl carbons and degree of unsaturation on an SB-Cyano-propyl-25 stationary phase has been reported recently (Manninen et al., 1995b).The method was applied to the analysis of triacylglycerols of black currant andalpine currant seed oils containing both a- and y-linolenic acid. Polar stationaryphases, such as DB-225 and SB-Cyanopropyl-25, have also been applied to theanalysis of fish oils; however, the resolution achieved was not adequate for ana-lytical purposes (Borch-Jensen et al., 1993; Manninen et al., 1995a). The principleof separation by SFC resembles that by GC due to the similarity in the polar sta-tionary phases. The molecules are firstly separated according to their acyl carbonnumbers and secondly according to unsaturation within each carbon numbergroup, with the result that the most unsaturated molecules have the strongestretention to the stationary phase. However, the resolution obtained by SFC is notas good as that obtained by GC (Christie, 1989a), mainly because of less efficientseparation by carbon number. When SFC is compared with reversed-phase HPLCanalysis, there are advantages in each method as they separate triacylglycerols ondifferent bases. As an example, molecules with the same equivalent carbonnumber, for example, 16:0-18:1-18:1 and 18:1-18:1-18:1, tend to elute close to-gether by reversed-phase HPLC (Christie, 1987a) whereas an improved separa-tion is possible by SFC.

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012


SFC has been applied to silver ion chromatography of triacylglycerols byDemirbiiker and Blomberg (1990,1991) and Demirbuker et al. (1992,1993). Theyused a short piece of fused silica capillary tubing filled with Nucleosil 5SA, acation-exchange packing material, loaded with silver ions for the separation oftriacylglycerols of vegetable and fish oils as well as standard compounds accordingto the total number of double bonds. Molecules were eluted with a mixture ofcarbon dioxide-acetonitrile-isopropanol, and were detected either by an UVspectrophotometer at 210 nm or by an evaporative light-scattering detector. Insome applications simultaneous gradients for temperature decrease and pressureincrease were utilized. Separations comparable to those by silver ion HPLC(Christie, 1988) were obtained; however, the elution order of components wasdifferent. In addition to the degree of unsaturation and the nature of doublebonds, silver ion SFC separated molecules according to the chain lengths of fattyacyl residues, which is not always a desired phenomenon. The major advantageof silver ion HPLC is the possibility for semipreparative fractionation of a samplefor further analyses (Laakso and Christie, 1991, Laakso and Kallio, 1993a, 1993b).Recently, Demirbuker and Blomberg (1992) introduced the separation of triacyl-glycerols according to unsaturation with an anion-exchange and silica gel station-ary phase impregnated with permanganate ions. The separations were not as goodas those obtained by silver ion chromatography. Further studies are required inorder to explain the mechanism of interactions and prove the value of this methodfor lipid analysis.

Combining SFC with a mass spectrometer is one potential method to get infor-mation on both the degree of unsaturation and carbon number distribution (Kal-lio et al., 1989). The proportions of butterfat triacylglycerols differing in unsatu-ration within each carbon number group were determined by recording themolecular ions M+* with a sector mass spectrometer in selective ion monitoringmode using electron impact ionization (70 eV). A slightly polar stationary phasewas utilized to group butterfat molecules according to their carbon numbers.

Differentiation of fatty acyl residues between the sn-1/3 and sn-2 positions of atriacylglycerol molecule was demonstrated by SFC-MS with reference com-pounds by recording the [M - RCO2CH2]+ ions, where R is an aliphatic hydrocar-bon chain (Kallio et al. 1989). The fragments of interest were generated only fromthe fatty acids attached to the primary hydroxyl groups of the glycerol moiety. Posi-tions sn-1 and sn-3 cannot be distinguished from each other by physical methods.

The effects of temperature (Proot et al., 1986) and linear velocity of supercriti-cal carbon dioxide (Kallio and Laakso, 1990) on the separation of triacylglycerolsby SFC have been studied. The separation efficiency increased with increasingtemperatures, reaching a maximum at 150 to 170C. Temperatures above 230Ccannot be recommended because peak splitting and broadening may occur. In thetemperature range of 150-230C, degradation or polymerization did not appearto happen with the polyunsaturated triacylglycerols. In GC analysis, higher tem-

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012

214 LAAKSO

peratures are needed to volatilize triacylglycerols. Although analyses at ambienttemperatures can be performed by HPLC, a major drawback has until recentlybeen the lack of a universal detector suitable for HPLC. The practical optimumlinear velocity (juopt) of supercritical carbon dioxide for the separation of triacyl-glycerols was determined to be around 0.3 to 0.4 cm/sec (Kallio and Laakso, 1990).However, capillary SFC analyses are very seldom carried out under such condi-tions, because working close to the juopt can result in an analysis lasting of severalhours (Sandra et al, 1988). Linear velocities of 5 to 10 times ^ opt are often used atthe expense of efficiency, especially at high pressures (Leyendecker, 1988).

The repeatability of retention times and the accuracy of the quantitative databy SFC have also been examined. Proot et al. (1986) reported relative standarddeviations of less than 0.16% for the retention times and less than 0.8% for thequantitation data for a mixture of four triacylglycerols. Relative standard devia-tions calculated from raw peak areas of 9.1% to 11.1% (n = 4) for diacylglycerolsand 7.9% to 10.1% (n = 4) for triacylglycerols from bayberry wax were reported(Hawthorne and Miller, 1989). The relative standard deviations varied between0.9% and 2.5% when one of the wax components was used as an internal standard.According to Lee et al. (1991), the precision of the SFC analyses indicated by therelative standard deviations of the peak areas was less than 5% (n = 4) for un-derivatized mono- and diacylglycerol samples, and less than 2.5% (n = 4-6) forderivatized samples. Manninen et al. (1995a, 1995b) have analyzed the triacyl-glycerols of berry seed oils, milk fat, and fish oil on an SB-Cyanopropyl-25 station-ary phase with good retention time repeatability (relative standard deviation ) and secondary (sn-2) glycerolpositions, can be obtained. Various ionization and sample introduction tech-niques have been applied to the analysis of triacylglycerols by MS (Table 2).

Ionization and Fragmentation of Triacylglycerols

Electron Impact IonizationThe first mass spectrometric studies of triacylglycerols were performed using elec-tron impact (El) ionization. Molecular weight can be determined from the lowabundance M+" or [M - 18]+ ions (Barber et al., 1964). The abundances of theseions depend on the structure of molecules: triacylglycerols containing short-chainfatty acyl moieties do not have a molecular ion in their mass spectra; however, theabundance of the M+" is generally enhanced with increasing chain length of thefatty acyl residues (Lauer et al., 1970). Furthermore, an increased degree of un-saturation results in reduced abundance of [M -18]+ (Hites, 1975). The combinednumber of carbon atoms and double bonds in the acyl chains of a triacylglycerolcan be calculated, if the molecular weight of the molecule is known. In additionto M+' and [M -18]+ ions, an El spectrum consists of several other ions useful forstructure elucidation, such as [M - RCO2]+, [M - RCO2H]+, [M - RCO2CH2]+,[RCO + 128]+, [RCO + 74]+ and RCO+, where R = aliphatic hydrocarbon chain(Ryhage and Stenhagen, 1960; Barber et al., 1964). In addition to [RCO + 115]+ions, Lauer et al. (1970) and Aasen et al. (1970) revealed a series of ions [RCO+ 128 + 14n]+ (n = 1, 2, 3,...) to determine the location of double bonds in thefatty acyl residues after addition of deuterium. Generally, the most abundantpeaks in the El spectra correspond to [M - RCO2]+ and RCO+ ions, which can beused for fatty acid identification. However, the abundances of most ions dependon the structure of triacylglycerols. The abundance of [M - RCO2]+ ions is thegreater, the longer the chain length of the cleaving saturated acyloxy group (Laueret al., 1970). On the other hand, the abundance of [M - RCO2CH2]+ ion decreaseswith increasing chain length. In addition, this ion is only formed by the cleavageof RCO2CH2from thesn-1 andsn-3 positions (Ryhage and Stenhagen, 1960; Bar-ber et al., 1964). Increased degree of unsaturation results in reduced abundanceof RCO+ and [M - RCO2]+ ions (Wakeham and Frew, 1982) and increased abun-dance of [RCO - 1]+ and [M - RCO2H]+ ions (Lauer et al., 1970; Hites, 1975).

Chemical IonizationAs compared with El spectra, greater abundance of molecular or quasi-molecularions and less fragmentation are obtained by soft ionization methods, chemicalionization (CI) being that mostly used (Table 2) (reviewed by Lin and Smith,1984). Triacylglycerols are chemically ionized by reactant gas ions (NHt+, CH5+,C4H9+) to form [M - H]+, [M + H]+ or [M + X]+ adduct ions. Components of an

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012

Table 2. Examples of Analysis of Triacylglycerols by Mass Spectrometry

Sample

Reference TAGsReference TAGsSeed oilReference TAGs

Reference TAGsVegetable oils, ref.

TAGSVegetable oilsSeed oilRat adipose tissueMilk fat, e.g.Milk fatButter fatButter fat,

zooplanktonOlive oilGreen algaButteroilVegetable oilsCeramic potsherdsButter fatButter fatVegetable oilsBlood

Inlet

DirectDirectDirectDirect

DirectDirect

DirectDirectHPLC, belt interfaceGCGCGCGC

GCGCGCGCGCGCSFCDirectDirect

MS instrument

NoncommercialA.E.I. MS9A.E.I. MS9Hitachi RMU6D, A.E.I. MS9

A.E.I. MS902Nuclide Corp. 12-90-DF

CEC 21=110Finnigan TSQ 4600VG 7070FShimadzu LKB 9000Shimadzu LKB 9000VarianMAT112Finnigan 3200

Shimadzu QP1000Finnigan Mat 1020BHewlett Packard 5985BShimadzu QP1000VG7070HFinnigan MAT INCOS 50VG 7070EShimadzu LKB 9000Biospect

Ionization"

ElElElEl (70 eV)

El (70 eV)El (70 eV)

ElEl (70 eV)El (20 eV)El (20 eV)El (20 eV)ElEl (70 eV)

El (70 eV)El (70 eV)El (70 eV)El (70 eV)El (70 eV)El (70 eV)El (70 eV)CI (ammonia)CI (ammonia)

Ion sourcetemp. (C)ns

170-200ns

ns

250250

ns

150260330310290ns

300ns

230330ca. 300190280220150-250

Ref.

Ryhage & Stenhagen, 1960Barber et al., 1964Sprecher et al., 1965Aasen et al., 1970; Lauer

et al., 1970Klein, 1971Hites, 1970,1975

Merritt et al., 1982Demirbuker et al., 1992Rawle et al., 1990Murata & Takahashi, 1973Murata, 1977Schmid et al., 1979Wakeham & Frew, 1982

Murata & Nakamura, 1985Rezanka et al., 1986Myher et al., 1988Ohshima et al., 1989

.Evershedetal., 1990aKalo and Kemppinen, 1993Kallio et al., 1989Murata and Takahashi, 1977Bose et al., 1978

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012

Butter fat, e.g.Vegetable oilsGreen algaTurnip rapeseed oilBlood plasmaPlant oils and fatsReference TAGReference TAGCoconut oilVegetable oils,

lard, e.g.

Menhaden oil, e.g.

Rearrangedbutteroil

Milk fats

Butteroil, plasmalipids, fish oil

Soybean oilEvening primrose oilReference TAGsReference TAGsVegetable oilsButteroilButter fat

DirectDirectDirectDirectDirectDirectHPLC, DLIHPLC, belt interfaceHPLC, belt interfaceHPLC, DLI

HPLC, DLI

HPLC, DLI

HPLC, DLI

HPLC, DLI

HPLC, belt interfaceHPLC, frit interfaceSFCSFCGCGCGC

VarianMAT212MAT 312Varian MAT 411VG 7070EFinnigan MATFinnigan MAT 90Hitachi RMH-2Biospect 7401BFinnigan 3200Hewlett Packard 5985B

Hewlett Packard 5985B




Biospect 7501JEOLJMS-AX505HExtranuclear LaboratoriesNermag SQ 156Shimadzu LKB 9000Hewlett Packard 5985BVG7070H

CI (ammonia)CI (ammonia)CI (methane)CI (ammonia)CI (ammonia)CI (ammonia)CI (methanol)6CI (methane)CI (methane)CI (ACN +

PCN)6

CI (ACN +PCN)6

CI (ACN +PCN)b

CI, NCI(ACN +PCN)*

NCI (CH1C12)'>CI (ACN +

PCN)6CI (methane)CI (ammonia)CI (methane)CI (ammonia)CI (ammonia)CI (methane)NCI (ammonia)

200ns

220180200200ns

ns

ns

ns

ns

ns

ns

150200

265350-450ns

ns

230230300

Schulte et al., 1981Merritt et al., 1982Rezanka et al., 1986Kallioetal., 1991aMares et al., 1991Rezanka & Mares, 1991McLafferty et al., 1975Erdahl & Privett, 1977McFadden et al., 1977Kuksis et al., 1983; Marai

et al., 1983; Myher etal., 1984

Kuksis et al., 1987

Marai et al., 1994; Myheret al., 1993

Kuksis et al., 1986

Kuksis e tal , 1991a, 19911

Erdahl & Privett, 1985Hori et al., 1991Wright et al., 1986Cousin & Arpino, 1987Murata, 1977Myher et al., 1988Evershed et al., 1990b

(continued)

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012

Table 2. Continued

Sample

Waxes

Reference TAGs

Vegetable oilsReference TAG

Reference TAGs

Fish oil

Edible oilsReference TAGsReference TAGs

Reference TAGs,e.g.

TAG mixture

Trilaurin

Inlet

Direct

GCDirect

DirectContinuous flow

probeDirect

HPLC, frit-FABinterface

DirectHPLC, thermosprayHPLC, thermospray/

plasmaspray probeFlow injection via

ion spray interface

HPLC (silver ioncolumn)

SFC, API interface

MS instrument

FinniganMAT731

FinniganMAT212Varian CH4 or LKBA.E.I. MS9Varian LH4Varian LH4Varian CH5DKratos MS50FS-TC

VG ZAB-2F

JEOLJMS-AX505H

Finnigan MAT 90Extrel 400-2VGTrio3

SciexTAGA6000E

Fisons Trio-2000

SciexAPIIII

Ionization"

FIFDFAB (Xe)FI, El (70 eV)El (70 eV)CI (isobutane)FIFDFDFAB (Xe)

FAB, NFAB(Xe)

(FAB or NFAB)+ CIT>C

FAB, NFAB

FAB (Xe)thermosprayplasmaspray

ESI (Na+ orNH4+)6

ESI + CID (Ar)cESI (Na+)fc

APCI

Ion sourcetemp. (C)250ca. 50

275100,200200Ambientns

ns

ns

ns

ns

300280

ns

ns

ns

Ref.

Schulten et al., 1987

Fales et al., 1975

Evans et al., 1974Barber et al., 1987

Evans et al., 1991

Hori et al., 1994

Lamberto & Saitta, 1995Kim & Salem, 1987Valeur et al., 1993

Duffin et al., 1991

Schuyl et al., 1995

Tyrefors et al., 1993

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012

Reference TAGs HPLC, API interface Finnigan MAT SSQ 710C

Soybean TAGs HPLC, API interface Finnigan MAT SSQ 710C

VG 70-250 SEQ

VG 70-250 SEQ

VG 70-250 SEQ

Castor bean neutral Directlipids

Embryos of Brassica Directnapus

Columbia seed oil Direct

Milk fat TAGs Direct

Vernolia galamensis Directoil

Various seed oils, Direct

Baltic herring oil,human milk TAGs

Butter fat Direct

Berry pulp and seed Directoils

Colostrum milk fat Direct

JEOLJMS-HX110HF

Finnigan MAT TSQ 46

Finnigan MAT TSQ-70

Finnigan MAT TSQ-700



APCI (PCN + nshexane)6

APCI (PCN + nshexane)6

CI (ammonia) nsEl + CID (Ar)cEl (70 eV) 250El + CID (Ar)cCI (ammonia) 250El nsCI/EI + CID

(Xe)cCI (isobutane) 150CI + CID (He)cCI (methane or ns

isobutane)CI + CID (Xe)cNCI (ammonia) 215NCI + CID

(Xe or Ar)c

NCI (ammonia) 180NCI + CID

(Ar)cNCI (ammonia) 200

NCI (ammonia) 200

Byrdwell & Emken, 1995

Neff & Byrdwell, 1995

Hogge et al., 1991

Taylor et al., 1991

Taylor et al., 1995

Spanos et al., 1995

Anderson et al., 1993

Kallio, 1992; Kallio &Currie, 1991

Kallio & Currie, 1993a,1993b; Currie & Kallio,1993; Kallio & Rua, 1994

Laakso & Kallio, 1993a,1993b

Manninen et al., 1995a,1995b

Makinen et al., 1995

00O

O

n

(continued)

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012

Table 2. Continued

Sample Inlet MS instrument Ionization"Ion sourcetemp. (C) Ref.

Spruce and pineseed oils

Reference TAGs,cocoa butter

Microbial TAGs

Direct

Direct

Direct

Finnigan MATTSQ-700

Finnigan MATTSQ-70

VarianMATCH-5DF

NCI (ammonia)

NCI (CH4/N2O,75/25 v/v)

NCI + CID

El (70 eV) +MDC

200

230

250

Tillman-Sutela et al., 1995

Stroobant et al, 1995

Batrakov et al., 1983

Abbreviations. ACN, acetonitrile; APCI, atmospheric pressure chemical ionization; API, atmospheric pressure ionization; CI, chemical ionization; ESI, electrosprayionization; NCI, negative ion Cl; DLI, direct liquid inlet; CID, collision induced dissociation; El, electron impact ionization; FAB, fast atom bombardment; NFAB,negative ion FAB; FD, field desorption; FI, field ionization; MD, metastabile defocusing technique; ns, not specified; PCN, propionitrile; TAG, triacylglycerol." Positive ions were analyzed unless otherwise stated.

Ionizing reagents are present in the HPLC eluent or sample solvent.c MS/MS analysis.

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012


HPLC eluent or a sample solvent can also act as reactant gases. In addition topure chemical ionization, some charge exchange due to CO2+* may occur duringSFC-MS analysis, as CO2 is used as a carrier fluid (Cousin and Arpino, 1987).Murata and Takahashi (1977) and Murata (1977) reported the formation of abun-dant [M + NH4]+ ions and [MH - RCO2H]+ fragment ions of triacylglycerols usingeither a direct inlet system or a GC for sample introduction and ammonia as areactant gas. These ions are useful for the determination of both molecularweights and combinations of fatty acids in triacylglycerol molecular species. Abun-dant [M + NH4]+ ions were also formed by introducing the sample via a solidsprobe into the ion source followed by ammonia CI of the vaporized triacylglycerols(Taylor et al., 1995). Desorption CI of triacylglycerols with isobutane (Spanos etal., 1995) and ammonia (Anderson et al., 1993) yielded mass spectra exhibiting[M + H]+ and [MH - RCO2H]+ as well as some low molecular weight fragmentions. Marai et al. (1983) and Kuksis et al. (1983) introduced the formation ofprominent [M + H]+ and [MH - RCO2H]+ ions of triacylglycerols by combiningreversed-phase HPLC separation with MS detection, using acetonitrile andpropionitrile as eluents and reactant gases. Low-abundance adduct ions of ace-tonitrile [M + 41]+ and propionitrile [M + 55]+ as well as weak acyl ions RCO+were recorded, but these ions were not used for identification. A fairly largeamount of sample was needed because only 1% of HPLC eluent was introducedin the MS via a direct liquid inlet interface. Detection sensitivity was greatly im-proved by negative ion CI with dichloromethane, which yielded solely [M + CI]"ions (Kuksis et al., 1991a, 1993b). The quasi-molecular ions of both chloride isotopes35C1 and 37C1 were detected (Kuksis et al., 1991b). As no fragmentation duringchloride attachment negative ion CI occurs, information regarding molecularweights only is obtained. The formation of abundant [M - H]~ and RCO2~ ions oftriacylglycerols by negative ion CI with ammonia was first introduced by Kallioand Currie (Kallio and Currie, 1991,1993a, 1993b; Kallio, 1992). Similar ioniza-tion of triacylglycerols has been obtained by using a mixture of CH4/N2O (75:25,v/v) as a reactant gas (Stroobant et al., 1995). On the contrary, no molecular orquasi-molecular ions were detected using ammonia negative ion CI according toEvershed et al. (1990b); instead, RCO2", [RCO2 - H2O]' and [RCO2 - H2O - H]"fragment ions were recorded.

Other Ionization MethodsIn addition to CI, other soft ionization techniques, i.e., field ionization (FI), fielddesorption (FD), fast atom bombardment (FAB), and thermospray (TSP) (re-viewed by Games, 1978; Jensen and Gross, 1988; Matsubara and Hayashi, 1991;Kim and Salem, 1993), have been applied to the analysis of triacylglycerols (Ta-ble 2). Similarly to CI, these techniques produce mass spectra containing abun-dant ions consisting of the intact molecule and a few characteristic fragment ions.In addition to M+* and [M + H]+ ions, interfering adduct ions such as [M + Li]+,

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012

222 LAAKSO

[M + Na]+ and [M + K]+ may be formed if metal salts are present in the sampleduring FD ionization. The advantage of cationization during FAB ionization oftriacylglycerols in forming [M + Na]+ ions has been reported (Evans et al., 1991)and applied to the characterization of various edible oils (Lamberto and Saitta,1995). TSP mass spectrum of triacylglycerols consisted of abundant [M - RCO2]+ions in addition to less abundant [M + NH(]+ and/or [M + H]+ions and monoacyl-glyceryl fragment ions (Kim and Salem, 1987). Discharge-assisted TSP (plas-maspray) ionization of triacylglycerols yielded solely abundant [M - RCO2]+ ionswithout the formation of [M + H]+ ions (Valeur et al., 1993).

Mass spectrometric techniques utilizing ionization at atmospheric pressure,that is, electrospray ionization (ESI) and atmospheric pressure chemical ioniza-tion (APCI), have been of great interest, especially for interfacing MS with HPLC.Duffin et al. (1991) have published the only application, so far, on the analysis oftriacylglycerols by ESI-MS. A continuous flow of sample (2 /iL/min), withoutchromatography, was introduced into the ionization chamber. Triacylglycerolsyielded abundant [M + NH4]+ and [M + Na]+ ions in the presence of ammoniumions. The use of electrospray ionization in the MS analysis of lipids has been re-viewed recently (Myher and Kuksis, 1995). Contrary to nonpolar triacylglycerols,ESI is an ideal technique for ionization of phospholipids, which are ionizablealready in solution. In APCI, neutral molecules are introduced into the gas phaseat atmospheric pressure followed by corona discharge ionization. No buffers insolution are needed; therefore, the technique is suitable for the ionization of non-polar molecules as well. One of the first studies on the analysis of triacylglycerolsby APCI-MS has been reported by Tyrefors et al. (1993). The model componentswere separated by capillary supercritical fluid chromatography which was con-nected to the APCI source. The mass spectrum of trilaurin exhibited abundant[M + H]+ and [M + H2O]+ ions and a weaker [M - RCO2H]+ fragment ion.Byrdwell and Emken (1995) combined reversed-phase HPLC separation oftriacylglycerols, using a gradient of propionitrile and hexane, with APCI-MS. Themass spectra of triacylglycerols consisted of abundant [M + H]+ and [M - RCO2]+ions. In addition, adduct formation with water and propionitrile was recorded toproduce weak [M + H + H2O]+, [M - RCO2 + H2O]+ and [M + H + 55]+ ions.Recently, reversed-phase HPLC-APCI-MS has been applied to the analysis oftriacylglycerols of genetically modified soybean oils (Neff and Byrdwell, 1995).

Tandem Mass Spectrometric AnalysisDuring the last few years, analyses of triacylglycerols utilizing tandem mass spec-trometric (MS/MS) techniques have been published (Table 2). In general, triacyl-glycerols are ionized followed by collision-induced dissociation (CID) of the ionsconsisting of the intact molecule, such as [M + H]+, [M + NHt]+ and [M - H]~,into characteristic fragment ions. In addition to molecular weight distribution,these analyses provide information concerning the fatty acid combinations of each

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012


molecular weight species of triacylglycerols without chromatographic separations.Batrakov et al. (1983) used metastabile defocusing technique for each prominent[M - RCO2]+ ion to find the corresponding molecular ion. According to these dataone of the fatty acyl residues of a triacylglycerol was identified. The daughter ionspectra of [M - RCO2]+ or [M - RCO2CH2]+ ions were examined in order todetermine the remaining two fatty acyl residues. Duffin et al. (1991) introducedthe sample dissolved in solvent containing ammonium acetate into the ion sourcevia an ion spray interface. The mass spectra exhibited abundant [M + NH4]+ and[M + Na]+ ions. Low-energy CID fragmentation of the [M + NH4]+ ions withargon yielded useful [M - RCO2]+ and RCO+ fragment ions for structure eluci-dation. Dissociation of the [M + NH4]+ ions was preferred to the [M + Na]+ ions:high-quality daughter spectra of the sodiated ions were difficult to obtain, becausethe [M + Na]+ ions were very stable and required extreme conditions for colli-sional activation. Hogge et al. (1991) analyzed castor bean neutral lipids, rich inricinoleic acid (12-hydroxy-9-octadecenoic acid), as their trimethylsilyl derivativesby MS/MS. Molecular weights were determined according to [M - CH3]+ ionsproduced by El. Collision-induced dissociation of the [M - CH3]+ ions yieldedfragment ions of diagnostic value such as [RCO + 74]+, [M - RCO2]+, [M -RCO2H]+, [M - CH3 - RCO2H]+ and [(M - RCO2H) - 16]+. Similarly, collision-induced dissociation of the M+" ions, produced by El (Taylor et al., 1991), and the[M + NH4]+ ions, produced by ammonia desorption CI (Taylor et al., 1995), re-sulted in the formation of [M - RCO2]+ and [RCO + 74]+ daughter ions. Theseions have been used for structural assignment of the triacylglycerols of the em-bryos of Brassica napus (Taylor et al., 1991) and the seeds of Arabidopsis thaliana(Taylor et al., 1995). Spanos et al. (1995) have investigated the molecular speciescomposition of milk fat triacylglycerols by MS/MS. The selected parent ions, [M+ H]+, produced by isobutane desorption CI, were collisionally activated withhelium in the first field free region, followed by linked scanning at constant mag-netic field strength/electrostatic analyzer voltage. The CID mass spectra exhibitedabundant [MH- RCO2H]+ ions. Anderson et al. (1993) reported also the forma-tion of abundant [MH - RCO2H]+ daughter ions by low-energy collision of the[M + H]+ parent ions, which were produced by methane CI. This method wasused for characterization of the triacylglycerols of Vemonia galamensis oil rich invernolic acid (c/s-12,13-epoxy-cw-9-octadecenoic acid). Evans et al. (1991) re-ported the formation of [M - RCO2 + Na]+ and [M - RCO2]+ fragment ions from[M + Na]+ ions, which were produced by FAB ionization. Kallio and Currie (1991,1993a, 1993b) and Kallio (1992) utilized negative ion CI with ammonia (NH2") tof orm [M - H]~ ions. Collision of selected [M - H]~ ions with xenon or argon resultedin the formation of abundant [M - H - RCO2H -100]" and RCOf ions, and weak[M - H - RCO2H]-, [M - H - RCO2H - 56]~ and [M - H - RCO2H - 74]- ions.Similar CID fragmentation of the [M - H]- ions of triacylglycerols has been reportedby using a mixture of CH4/N2O (75:25, v/v) for chemical ionization (Stroobant et

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012

224 LAAKSO

al., 1995). The m/z values of the RCO2" ions define the fatty acid moieties, that is,the number of acyl carbons and double bonds, of the selected parent ion, withoutinformation on the positions of the double bonds or the configuration of fattyacids. The abundances of the RCO2" ions can be used for calculation of the pro-portions of different combinations of the three fatty acids in triacylglycerols. Inaddition to the data on fatty acid composition, the abundances of the [M - H -RCO2H - 100]- ions of the CID mass spectra of the [M - H]" ions of the tri-acylglycerols provide information on the distribution of fatty acids between pri-mary (sn-1/3) and secondary (sn-2) positions.

Quantitative Analysis of Triacylglycerols

Quantitation by MS is often puzzling, because the abundances of many ions de-pend on both the structure of triacylglycerols and the MS conditions used. Hites(1970) summed the abundances of M+- and [M -18]+ ions, produced by El, foreach type of triacylglycerol for quantitative analysis of several vegetable oils. Inaddition, he determined correction factors for triacylglycerols because of differ-ences in their molecular distillation from the direct inlet probe, and applied cor-rections for heavy isotopes. The importance of summing all mass spectra obtainedduring the evaporation of samples was reported by Schulte et al. (1981). Never-theless, correction factors were needed because the abundances of [M + NH4]+ions decreased with increasing total number of acyl carbon atoms. According toSchulte et al. (1981), the responses were almost independent from the degree ofunsaturation. This is unlike the results of Myher et al. (1984), Kallio et al. (1991a),and Rezanka and Mares (1991), which showed that increased unsaturation oftriacylglycerols results in decreased responses of quasi-molecular ions. Byrdwelland Emken (1995) and Neff and Byrdwell (1995) reported that the unsaturationof triacylglycerols has a strong effect on the relative abundances of the [M + H]+ions produced by APCI-MS: [M + H]+ was the base peak in the mass spectrumof trilinolenin whereas the mass spectrum of tristearin showed no [M + H]+. Inaddition, Duffin et al. (1991) reported that [M + Na]+ and [M + NH4]+ ions oftriacylglycerols, produced by ESI-MS, containing short-chain or unsaturated fattyacyl residues were observed in greater abundance than those of triacylglycerolscontaining long-chain and saturated fatty acyl moieties. In order to obtain greaterquantitation reliability, Myher et al. (1984) used the total ion current responseunder positive ion CI conditions, as this varied less according to triacylglycerolstructure than does the abundance of molecular ions. However, the total ion cur-rent response cannot be used for quantitation of chromatographically unresolvedtriacylglycerols. Instead, the responses of [M + H]+ ions, if molecules differ inmolecular weight, and [MH - RCC^H]* ions, if molecules have identical molecu-lar weights, are useful for quantitation. More unambiguous quantitation is possibleby negative ion CI with chloride attachment which gives nearly correct proportions

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012


of molecular ions with both saturated and unsaturated triacylglycerols of short-chain and long-chain fatty acyl residues (Kuksis et al., 1991a, 1991b). The 13C and18O corrected abundances of the [M - H]~ ions of triacylglycerols, produced byammonia negative ion CI, characterize the proportions of different molecularweight species. Recently, the mass spectrometric conditions as well as the sampletreatment have been optimized for the direct probe analysis of triacylglycerols(Laakso, 1995; Laakso and Kallio, 1996). Under well-controlled and optimizedconditions, semiquantitative analysis of triacylglycerols, consisting of 50 to 56 acylcarbon atoms, is possible with this technique without the use of correction factorscaused by differences in molecular size and unsaturation.

Distribution of Fatty Acids in Triacylglycerols

Instead of the average distribution of fatty acids in a triacylglycerol sample ob-tained by enzymatic methods, the differentiation of fatty acids in primary (sn-1/3)and secondary (sn-2) glycerol positions in single molecular species by MS has beenof interest. Information concerning molecular species of triacylglycerol mixturesmay be obtained if MS, in combination with chromatographic methods or MS/MSanalysis, is utilized. The [M - RCO2CH2]+ ions (Ryhage and Stenhagen, 1960;Barber et al., 1964; Lauer et al., 1970; Hasegawa and Suzuki, 1973; Myher et al.,1978; Kallio et al., 1989) and less frequently RCO2CH2+ ions (Sprecher et al., 1965)have been used to distinguish the fatty acids in sn-1/3 and sn-2 positions under Elconditions. According to Myher et al. (1984), the cleavage of a fatty acyl residuefrom the sn-1 or sn-3 positions, under CI conditions, is about four times moreabundant than that from the sn-2 position. The position of a fatty acid rather thanits structure affects the abundance of [MH - RCO2]+ ion. This is in contrast to thefindings of Lauer et al. (1970), who reported that the yield of [M - RCO2]+ ions,obtained by El MS, depends on the fatty acyl chain length rather than its sn posi-tion. Recently, Kallio and Currie (1991,1993a, 1993b) showed the dependence ofthe abundances of RCO2" fragment ions, not only on the sn position of fatty acids,but also on the collision energy used to dissociate [M - H]~ ions during MS/MSanalysis. By selection of a proper collision energy, the effect of sn position on theabundance of RCO2~ ions can be minimized. Alternatively, preferential cleavageof fatty acyl residues from the primary glycerol positions resulting in greater abun-dances of [M - H - RCO2H - 100]" ions than from the sn-2 position can be usedto distinguish between sn-1/3 and sn-2 positions (Kallio and Currie, 1991,1993a,1993b; Kallio, 1992). Recently, Stroobant et al. (1995) have published the frag-mentation pathways of triacylglycerols. The pathway that leads to the formationof ketone enolate, that is, [M - H - RCO2H -100]" ion, is sensitive to the positionsof fatty acyl residues (sn-1/3 vs. sn-2) on the molecular backbone. The techniquehas been applied to the regiospecific analysis of triacylglycerols of, for example,seed oils (Kallio and Currie, 1993a, 1993b), human milk (Currie and Kallio, 1993;

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012

226 LAAKSO

Kallio and Currie, 1993b; Kallio and Rua, 1994), and Baltic herring (Kallio, 1992;Kallio and Currie, 1993b). So far, similar information on the regiospecific distri-bution of fatty acyl residues has not been achieved with other MS/MS methodsapplied to the analysis of triacylglycerols. In addition to triacylglycerols, the dis-tribution of fatty acids in diacylglycerols, especially 1,2-diacyl-sn-glycerols derivedfrom phospholipids, has been investigated by mass spectrometric methods. Unliketriacylglycerols, diacylglycerols lose the fatty acyl residue more easily from the sn-2position than from the sn-1/3 positions (Myher et al., 1978; Pind et al., 1984; Mat-subara and Hayashi, 1991). Although a lot of specific structural information isproduced by MS, the identification of single molecular species is not easilyachieved due to the complexity of natural mixtures of triacylglycerols.

STEREOSPECIFIC ANALYSIS

Generally, the optical activity of natural triacylglycerols is too weak to be meas-ured directly. Only highly asymmetric molecules, such as those present in milk fat,may display measurable optical activity (Schlenk, 1965; Anderson et al., 1970).Thus, the methods for the stereospecific analysis of triacylglycerols are nearlyalways based on the formation and separation of diacyl-sn-glycerols. The tradi-tional methods include the phosphorylation of diacylglycerols and their hydrolysiswith phospholipases (reviewed, e.g., by Brockerhoff, 1971; Litchfield, 1972; Chris-tie, 1982,1986; Bhati, 1987). Over the last few years methods for the separationof enantiomeric acylglycerols have been developed and their capability for thestereospecific analysis of triacylglycerols has been investigated (reviewed by Tak-agi, 1990; Christie, 1992a, 1994). In general, there are two principal ways to sepa-rate enantiomers: (1) By the reaction with an achiral reagent, enantiomers formenantiomeric derivatives which may be separated by chiral stationary phases. (2)By the reaction with a chiral reagent, enantiomers form diastereomeric derivativeswhich may be separated by achiral stationary phases.

Preparation of Acylglycerols

Partial deacylation of triacylglycerols is performed by the action of lipase or Grig-nard reagent (reviewed by Litchfield, 1972). The most important requirements arethat diacylglycerols should be formed without acyl migration in equimolar propor-tions, and that during deacylation no discrimination according to the structure of thefatty acids should occur. Pancreatic lipase (EC 3.1.1.3) is usually used to determinethe fatty acids in the sn-2 position (Luddy et al., 1964). It catalyzes the hydrolysisof the primary ester bonds of triacylglycerols, producing free fatty acids, 2-mono-acyl-sn-glycerols, and 1,2- and 2,3-diacyl-sn-glycerols. Unfortunately, the esterbonds are not hydrolyzed equally with the action of pancreatic lipase (Brockerhoff,

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012


1968) and representative diacylglycerols are not produced: for example, long-chain polyunsaturated fatty acids such as eicosapentaenoic (20:5n-3) and docosa-hexaenoic (22:6n-3) acids are hydrolyzed more slowly (Bottino et al., 1967) andshort-chain fatty acids more rapidly than others (Sampugna et al., 1967; Franzkeet al., 1973).

At present, partial deacylation of triacylglycerols with Grignard reagent is thepreferred method for the production of representative diacyl-sn-glycerols becauseit has no fatty acid specificity (Franzke et al., 1973) and causes less acyl migrationthan other methods (Yurkowski and Brockerhoff, 1966). Both primary and sec-ondary ester bonds are degraded equally with Grignard reagent (ethyl or methylmagnesium bromide), resulting in a mixture of 1,2-, 2,3-, and 1,3-diacyl-stt-glycer-ols; 1-, 2-, and 3-monoacyl-sn-glycerols; and tertiary alcohols derived from theliberated fatty acyl groups (Yurkowski and Brockerhoff, 1966).

After deacylation of triacylglycerols, the separation of 1,3- and 1,2(2,3)-diacyl-sn-glycerols can easily be accomplished by TLC or HPLC using silica gel as sta-tionary phase. In addition, separation of diacylglycerols based on the chain lengthsof the fatty acyl resides is possible (Itabashi et al., 1993): for example, diacyl-glycerols produced by Grignard degradation of the hydrogenated acetyl-enrichedtriacylglycerol fraction of milk fat were separated by TLC in the order of 1,2(2,3)-diacyl-5-glycerols containing two long-chain fatty acids, 1,3-diacyl-sn-glycerolscontaining one acetyl and one long-chain acyl chain, followed by l,2(2,3)-diacyl-5-glycerols containing one acetyl and one long-chain fatty acid residue. With theexception of the rotation direction of plane polarized light, the physical and chemi-cal properties of enantiomers are identical. Thus 1,2- and 2,3-diacyl-sra-glycerolshave as such been considered inseparable by chromatographic means. However,Traitler et al. (1990) reported that 1,2- and 2,3-diacyl-sn-glycerols were partiallyresolved by planar chromatography with silica gel using two elution steps. In theirstudy, no boric acid was used to stabilize diacyl-sn-glycerols and the mobile phasecontained formic acid. Generally, 1,2- and 2,3-diacyl-s-glycerols have been dis-tinguished by the action of enzymes or separation of their enantiomeric or dias-tereomeric derivatives by chromatography.

Methods Based on Phosphorylation of Diacylglycerols and Phospholipases

Brockerhoff (1965) introduced the first method for the stereospecific analysis oftriacylglycerols. The diacylglycerols were produced by partial hydrolysis of triacyl-glycerols with pancreatic lipase (later with Grignard reagent). Purified 1,2(2,3)-diacyl-sn-glycerols were phosphorylated to their phosphatidylphenols. Phospholi-pase A2 (EC 3.1.1.4) was used to hydrolyze the sn-2 ester bond of sn-1,2-diacylglycerol phosphatidylphenol, producing a lysophosphatidylphenol with thefatty acid in thesn-l position, free fatty acid from the positionsn-2, and unaffected5n-2,3-diacylglycerol phosphatidylphenols. Thus the fatty acids in the sn-1 position

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012

228 LAAKSO

are obtained by analysis of lysophosphatidylphenols. The composition of the sn-2position is determined either from free fatty acids or from 2-monoacyl-sn-glycer-ols. The fatty acids in the position sn-3 can only be determined indirectly by cal-culation.

In an alternative method, Brockerhoff (1967) described the direct analyses ofthe fatty acids in each position. Isolated 1,3-diacyl-sn-glycerols prepared withGrignard reagent were converted to their phosphatidylphenols followed by phos-pholipase A2 cleavage of the fatty acid from the sn-1 position producing alysophosphatidylphenol with the fatty acid in the sn-3 position. Thus, the compo-sitions of the sn-1 and sn-3 positions were determined as the methyl esters by GCfrom the released fatty acids and lysophosphatidylphenols, respectively. The fattyacids in the sn-2 position were obtained by pancreatic lipase hydrolysis. Unfortu-nately representative 1,3-diacyl-sn-glycerols are difficult to obtain due to acyl mi-gration (Litchfield, 1972), and therefore the results are not always reliable. Chris-tie and Moore (1969) modified Brockerhoff s original method (1965) making itsuitable for 10-40 mg triacylglycerols. This method was subjected to extensiveinvestigation comparing its accuracy with Brockerhoff s (1965,1967) methods.

Instead of chemical synthesis, Lands et al. (1966) introduced an enzymatic deri-vatization of diacylglycerols. In this method diacylglycerol kinase (EC 2.7.1.107)from Escherichia coli phosphorylates selectively the 1,2-diacyl-sn-glycerols. Thecompositions oisn-\, sn-2, and sn-3 positions were determined by comparing thefatty acids of the intact triacylglycerols, 2-monoacyl-sn-glycerols produced by pan-creatic lipase, and 1,2-diacyI-sn-glyceroI phosphatidic acids.

Myher and Kuksis (1979) synthesized phosphatidylcholines of a purified mix-ture of 1,2- and 2,3-diacyl-sn-glycerols prepared by Grignard degradation. Theirmethod was based on the action of phospholipase C (EC 3.1.4.3), which released1,2-diacyl-sn-glycerols completely in 2 min whereas 2,3-diacyl-sn-glycerols re-quired 4 hr. With this method representative 1,2- and 2,3-diacyl-sn-glycerols canbe isolated. The fatty acid compositions of the molecular species were determinedby GC-MS after conversion of diacylglycerols to their f-butyldimethylsilyl ethers.The composition of position sn-2 was obtained by pancreatic lipase hydrolysis. Inaddition to stereospecific distribution, this method provides information concern-ing the molecular association of acyl groups in triacylglycerols. The disadvantageis that the fatty acids in the sn-1 and sn-3 positions are obtained indirectly.

Later Kuksis, with his research group (Kuksis et al., 1983), introduced a methodwhereby digestion with phospholipase A2 converts natural sn-l,2-diacylglycerolphospholipids into the corresponding lysophospholipids, with a release of the fattyacids from the sn-2 position. A subsequent treatment of the reaction mixture withphospholipase C converts lysophospholipids into the corresponding monoacyl-glycerols. The liberated fatty acids and 1-monoacyl-sn-glycerols can be deter-mined by GC as their trimethylsilyl derivatives.

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012


Methods Based on Separation of Enantiomeric AcylglycerolsSeparation of Enantiomers with Chiral Stationary Phases

Separation ofMonoacyl-sn-Glycerols: Takagi and Itabashi (1985,1986) sepa-rated 1- and 3-monoacyl-sn-glycerols as well as 1- and 3-O-monoalkyl-5-glycerols(Itabashi and Takagi, 1986) as their 3,5-dinitrophenyl urethane (DNPU) deriva-tives on a chiral stationary phase, Sumipax OA-2100, A^-(5)-2-(4-chlorophenyl)isovaleroyl-D-phenylglycine chemically bonded to y-aminopropyl silanized silica.An improved separation of enantiomeric monoacylglycerols was obtained with aSumipax OA-4100 stationary phase, iV-(i?)-l-(a-naphthyl)-ethylaminocar-bonyl-(S)-valine chemically bonded to silanized silica (Takagi and Ando, 1990b).With both stationary phases the derivatized l-monoacyl-sn-glycerol eluted earlierthan the corresponding 3-monoacyl-j'n-glycerol. The order of elution was reversedwhen a stationary phase with a reverse chirality was used (Ando et al., 1992).

Based on the resolution of enantiomeric 1- and 3-monoacyl-.s7z-glycerol deriva-tives, Takagi and Ando (1990a) introduced an alternative method for thestereospecific analysis of triacylglycerols. Monoacylglycerols were prepared fromtriacylglycerols with ethyl magnesium bromide followed by the isolation of 1(3)-monoacyl-jn-glycerols by TLC on boric acid impregnated silica gel plates. Thel(3)-monoacyl-5-glycerols were separated into saturated and unsaturated frac-tions by silver ion TLC followed by derivatization with 3,5-dinitrophenyl isocy-anate. The 1- and 3-monoacyl-sn-glycerol derivatives were separated on a Sumi-pax OA-2100 column with a mixture of hexane/dichloroethane/ethanol (40:10:1,v/v/v). Fatty acids of the original triacylglycerols, 1(3)- and 2-monoacylglycerols,and 1- and 3-monoacyl-src-glycerols were determined by GC as their methyl esters.The positional distribution of fatty acids in the sn-1, sn-2, and sn-3 positions wascalculated from the data. A more efficient separation of 1- and 3-monoacyl-sn-glycerol fractions, without a need for silver ion prefractionation, was possible usinga Sumipax OA-4100 column (Takagi and Ando, 1991a, 1991b). In addition to seedoils (Takagi and Ando, 1991b; Taylor et al., 1994,1995), stereospecific analysesof several fish oil triacylglycerols using this method have been published (Andoet al., 1992; Ota et al., 1994). The procedure has been modified for stereospecificanalysis of small amounts (1-10 mg) of triacyl-sn-glycerols (Ando and Takagi,1993) and applied to the analysis of cocoa butter (Takagi and Ando, 1995). Inorder to minimize acyl migration, monoacylglycerols were converted directly totheir 3,5-DNPU derivatives after Grignard degradation, followed by isolation ofthe DNPU-derivatives of 1- and 2-monoacylglycerols by HPLC on a silica column.This is the same approach as that introduced by Christie et al. (1991) to preventisomerization. Still, the main problem of the procedure lies in the preparation ofrepresentative monoacyl-sn-glycerols: Yurkowski and Brockerhoff (1966) re-ported that representative 1(3)- and 2-monoacyl-.sn-glycerol.s cannot be producedby Grignard degradation. According to Takagi and Ando (1991b) acyl migration

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012

230 LAAKSO

is not a problem, because only 2.5-3.0% of the acyl groups of 1- and 3-monoacyl-sn-glycerols originated from the sn-2 position. The microscale procedure has beenreported to reduce the acyl migration to 1.7-1.8% (Ando and Takagi, 1993).

Separation of Diacyl-sn-Glycerols: In addition to monoacylglycerols, 3,5-DNPU derivatives of enantiomeric diacyl-sn-glycerols were separated with a Su-mipax OA-2100 stationary phase (Itabashi and Takagi, 1987). Improved separa-tions of enantiomeric l,2(2,3)-diacyl- (Takagi and Itabashi, 1987; Takagi andSuzuki, 1990) and l,2(2,3)-dialkyl-sn-glycerol derivatives (Takagi and Itabashi,1987), andrac-l-alkyl-2-acyl- andrac-l-alkyl-3-acylglycerol derivatives (Takagi etal., 1990) were obtained with a chiral Sumipax OA-4100 stationary phase. Theelution order was 1,3-, followed by 1,2- and 2,3-diradyl-sn-glycerols. A l-alkyl-3-acyl-SH-glycerol eluted earlier than the corresponding l-acyl-3-alkyl-sn-glycerol.An almost baseline separation of saturated diacyl-sn-glycerols, differing by twomethylene groups, was achieved within 1,2- and 2,3-diacyl-s-glycerols (Takagiand Suzuki, 1990). The groups of 1,2- and 2,3-diacyl-sn-glycerols were well sepa-rated if the difference of the total acyl carbon numbers of the molecular specieswas 6 or less. So far the best separation of 3,5-DNPU derivatives of enantiomericdiacylglycerols has been obtained with a YMC-Pack A-KO3 stationary phase,(i?)-(+)-l-(l-naphthyl)ethylamine polymer covalently bonded to silica gel(Itabashi et al., 1990a, 1990b, 1991; Yang and Kuksis, 1991). Diacylglycerol de-rivatives elute from the column in order of increasing number of double bondsand decreasing number of acyl carbon atoms. The 1,2- and 2,3-diacyl-s/i-glycerolsare resolved into two distinguishable groups, although some peak overlappingbetween the most unsaturated 1,2-diacyl-sn-glycerols and the least unsaturated2,3-diacyl-srt-glycerols is possible, for example, with linseed and menhaden oildiacylglycerols (Itabashi et al., 1990a).

In order to obtain information concerning molecular species, Itabashi et al.(1990b) isolated the groups of 1,2- and 2,3-diacyl-sn-glycerol urethanes by HPLCwith a YMC-Pack A-KO3 column. The 3,5-DNPU derivatives of diacylglycerolswere converted to trimethylsilyl ethers which were suitable for separation by GCwith a polar stationary phase. Enantiomeric diacylglycerols generated by Grignarddegradation of corn oil, cocoa butter, and lard triacylglycerols were identified andquantified with this method. Recently, molecular species of enantiomeric diacyl-glycerols originating from triacylglycerols of cocoa butter, corn oil, and hydrogen-ated butteroil distillate containing short-chain triacylglycerols were analyzed witha combination of chiral-phase HPLC and mass spectrometry (Itabashi et al., 1991,1993). The molecular weights and pairs of fatty acids of molecular species over-lapping between enantiomers and within each enantiomer class were determinedaccording to [M - DNPU]+ and [RCO + 74]+ ions using positive ion chemicalionization. DNPU derivatives of diacylglycerols have also been studied by nega-tive ion chemical ionization mass spectrometry (Marai et al., 1992). The chloride

Dow

nloa

ded

by [C

orne

ll Univ

ersity

] at 0

4:58 0

9 May

2012


attachment fragment ions, [M - DNPU + Cl]~, provided a sensitive method foridentification and quantitation of the molecular species of the diacylglycerols.Since negative ion chemical ionization MS does not yield a detectable signal formonoacylglycerol type of fragment ions, it is advisable to parallel record the posi-tive ion chemical ionization mass spectra, yielding prominent ions for both mono-and diacylglycerol types of fragments (Marai et al., 1992). The separation of 1,2-and 2,3-diacyl-sra-glycerols as their DNPU-derivatives by chiral-phase HPLC com-bined with MS detection has been utilized, for example, for investigation of thestereospecificity of the membrane-bound and solubilized triacylglycerol syn-thetase from rat intestinal mucosa (Lehner et al., 1993).

The potential of separating 1,2- and 2,3-diacyl-5-glycerol groups as their 3,5-DNPU derivatives for the stereospecific analysis of triacylglycerols has been re-ported (Yang and Kuksis, 1991). Prior to derivatization and HPLC separation,1,3- and l,2(2,3)-diacyl-5-glycerols, prepared from the triacylglycerols of rat chy-lomicrons, were separated by TLC on boric acid silica gel plates. The fatty acidsin the sn-1, sn-2, and sn-3 positions were calculated from the GC data. In addition,the composition of the sn-2 position was determined by pancreatic lipase hydroly-sis. A similar approach has been used to determine the positional distribution ofshort-chain fatty acids in bovine milk fat (Itabashi et al., 1993). Recently, Yang etal. (1995) have studied the positional distribution and molecular association offatty acids in the triacylglycerols of rat liver and rat plasma very low density lipo-proteins by analyzing 1,2- and 2,3-diacyl-stt-glycerol derivatives by chiral-phaseHPLC-MS and reversed-phase HPLC-MS.

Sempore and Bezard (1991a, 1991b, 1991c) separated 3,5-DNPU derivativesof l,2(2,3)-diacyl-5-glycerols according to chain length and degree of unsatura-tion by reversed-phase HPLC prior to enantiomeric separation by chiral-phase(Sumipax OA-4100). By means of silver ion TLC and reversed-phase HPLC,triacylglycerols containing three known fatty acyl residues were isolated from pea-nut and cottonseed oils prior to Grignard degradation. According to the propor-tions of l,2(2,3)-diacyl-5rt-glycerol molecular species, and 1,2- and 2,3-diacyl-5in-glycerols and their fatty acid compositions, the individual molecular species of onecottonseed oil

analysis of triacylglycerols— approaching the molecular composition of natural mixtures

Documents