Research methods for lipid oxidation

CHAPTER 6

Research methods for lipid oxidation

Before advanced instrumental methods became available, conventional liquid adsorption and partition column chromatographic methods, as well as countercurrent distribution techniques, were generally used for the concentration and purification of fatty ester hydroperoxides to study the mechanism of lipid oxidation. Thin-layer chromatography was also developed as a useful tool in the separation and purification of mixtures of oxidation products, but its application was mainly qualitatitve. A number of powerful instrumental methods emerged later to analyse mixtures of complex products of lipid oxidation. These methods combined various improved chromatographic techniques that require sensitive detection systems with mass spectrometry (MS). They include high performance liquid chromatography (HPLC), and size-exclusion chromatography (HPSEC), combinations of GC and mass spectrometry (GC-MS), HPLC and 13C nuclear magnetic resonance (HPLC-13C NMR), HPLC-MS, chemical ionization MS (CI-MS), tandem-mass spectrometry or MS-MS or collision-induced dissociation (CID), electrospray MS (ESI-MS), and electro and coordination-ion spray MS (CIS-MS). The last section of this chapter deals with recently developed advanced techniques of direct MS, including atmospheric pressure chemical ionization-MS (APCI-MS) for volatile flavor analysis during food consumption. Significant improvements have been achieved in the separation of isomeric hydroperoxides and other labile oxidation products from complex lipids, and their direct characterization by various advanced MS techniques avoiding sample manipulation, problems of variable recoveries from biological samples and tedious derivatization. However, the direct analyses of lipid oxidation products achieved with many of these advanced MS techniques have not yet produced reliable quantitative information. Although a few synthetic and enzymatically-derived polyunsaturated lipid hydroperoxides have been described in the literature, they have not been generally used as standards for quantitative MS analyses. This chapter is organized in chronological order of method development.

A. Countercurrent distribution In early studies, although the hydroperoxides could be purified from autoxidized methyl oleate and linoleate by adsorption column chromatography or countercurrent distribution with a 29-tube apparatus, the same techniques failed with 129

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Figure 6.1. A. Separation of hydroperoxides by countercurrent distribution of autoxidized methyl linolenate (200 transfers between 8:2 water:ethanol and petroleum ether). B. Countercurrent distribution of hydroperoxide fraction from autoxidized methyl linolenate (600 transfers). From Frankel et al. (1961b). Courtesy of the American Chemical Society.

autoxidized linolenate. These results led to a controversy as to whether or not the oxidation of methyl linolenate produces hydroperoxides by the same mechanism as methyl linoleate. Later studies were more successful in isolating

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a hydroperoxide fraction partially separated from secondary oxidation products with a 200-tube countercurrent distribution apparatus (Figure 6.1A). By determining the partition coefficient of the linolenate hydroperoxide fraction (K = 0.35) and that of the secondary oxidation products (K = 0.12) it was calculated that 600 transfers were required to achieve complete separation of pure linolenate hydroperoxides (Figure 6.1B). Under the same conditions of oxidation the yield of pure linolenate hydroperoxides was much lower (36%) than that of linoleate hydroperoxides (90%) because linolenate produced much higher concentrations of secondary oxidation products. A major portion of these secondary products was later identified as a mixture of hydroperoxy epidioxides (Chapter 2.C).The yield of 2 g of pure hydroperoxides obtained by the countercurrent distribution technique permitted a detailed structural and quantitative study of the isomeric composition of linolenate hydroperoxides (Chapter 2, Figure 6.15).

B. Conventional column liquid chromatography Adsorption (solid–liquid) and normal-phase liquid partition (liquid–liquid) column chromatography run at low pressures were widely used to separate and analyse quantitatively hydroperoxides and secondary products of lipid oxidation. Adsorption column chromatography with silica and alumina used in early studies gave low yields of hydroperoxides due to various degrees of decomposition. Better results were obtained by combining adsorption and partition chromatography using a silicic acid column with methanol as a partial “stationary” phase to purify and analyse the hydroperoxides of fatty acids and esters, and dimeric and polymeric products in vegetable oils. By this procedure, silicic acid was treated with methanol as the stationary phase and benzene containing 2 to 4% by volume methanol as the mobile phase. The use of an insufficient amount of methanol to saturate the silicic acid column support resulted in a sharp gradient of methanol concentration in the eluate with a single mobile phase of methanolic benzene. Adsorption and partition effects were both indicated because the methanol concentration of the effluent was always lower than that of the eluent. This “semi-partition” chromatography was effective in yielding pure monomeric hydroperoxides efficiently separated from the unoxidized esters and secondary decomposition products (Figure 6.2). The chromatographic yields of hydroperoxides were significantly higher for methyl linoleate (89 to 95%) than for methyl linolenate (30 to 36%), and the relative concentration of secondary products was correspondingly higher for linolenate than for linoleate. This difference was in agreement with previous countercurrent distribution analyses (Section A) and was later accounted for by the significant formation of hydroperoxy epidioxides in linolenate, proportionately about 25% relative to the total monomeric hydroperoxides (Chapter 2. C). The same column chromatographic procedure was used to determine

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Figure 6.2. Chromatographic separation of autoxidized methyl oleate, linoleate and linolenate on a silicic acid column prepared with 2:8 methanol:benzene as immobile solvent and 2% methanol in benzene (v/v) as mobile solvent. Top graph: from Frankel et al. (1961a), courtesy of the American Oil Chemists’ Society, and bottom graph: Frankel et al. (1961b), courtesy of the American Chemical Society.

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compounds of average molecular weight corresponding to dimers in the free fatty acids obtained from deodorized vegetable oils (Figure 5.2), and thermally polymerized fatty acids and esters. The dimer contents of different vegetable oils varied from 1 to 3%. In oxidized–deodorized oils, a dimer concentration of 1% corresponded to a peroxide value of 40. The significant amounts of dimeric compounds produced after deodorization from hydroperoxides formed during processing was shown to decrease the flavor and oxidative stability of soybean oil (see Chapter 8). This determination is thus designated as “hidden oxidation”, and reflects the extent of oxidative abuse during processing (see Figure 5.2, Chapter 5.D). The use of small efficient chromatographic columns was later developed to separate and concentrate lipid oxidation products. Reproducible columns known as solid-phase extraction (SPE) became popular among lipid analysts when they became available commercially with a variety of polar and nonpolar adsorbents having different functionality by bonding to the silanol groups on the surface of silica gel. Pure hydroperoxides from autoxidized methyl linoleate, methyl linolenate, and hydroperoxy epidioxides from methyl linolenate were prepared in 20–40 mg quantities by a rapid and convenient chromatographic method using a silica cartridge (Table 6.1). These fractions of purified hydroperoxides were used to induce oxidation of n–6 polyunsaturated fatty acids in biological samples and compared with tert-butyl hydroperoxide. This synthetic hydroperoxide is commonly used as a convenient “oxidant” in biological oxidation studies because it is readily available commercially. The fatty acid hydroperoxides are, however, more appropriate and relevant sources of biological oxidants than the more stable and artificial tert-butyl hydroperoxide.

Table 6.1. Chromatographic separation of hydroperoxides and hydroperoxy epidioxides on solid-phase extraction (SPE) column a Autoxidized methyl esters (200 mg)b

Eluting solvents, ml (ether:hexane, v/v) c

Methyl linoleate Methyl linolenate Methyl linolenate

4, 4 (5:95) 4 (6:94), 3.5 (12:88) 4 (12:88), 4 (25:75)

a

Hydroperoxide Hydroperoxy absorptivity (Anm) d epidioxides (Anm) d A231: 28,640 A235: 23,772

A235: 24,975

From Frankel et al. (1989), using a Waters Sep-Pak silica cartridge. Methyl esters were shaken with oxygen at 40°C until 20–25% oxidation was obtained on the basis of conjugated diene absorptivity. c Isolated pure fractions are underlined. d Literature values: 26,000–28,600 for pure cis,trans and trans,trans isomers of methyl linoleate 9- and 13-hydroperoxides (Chan and Levett, 1977); 24,600 for isomeric mixture of cis,trans and trans,trans methyl linolenate hydroperoxides (Frankel et al., 1961a), and 24,200–28700 for pure cis,trans and trans,trans isomers of methyl linolenate hydroperoxy epidioxides (Neff et al., 1981). b

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Figure 6.3. Normal phase HPLC separation of the four cis,trans and trans,trans isomeric 9- and 13hydroperoxides as the hydroxyoctadecadienoate derivatives: 1, methyl 13-hydroxy-cis-9, trans-11-; 2, methyl 13-hydroxy-trans-9, trans-11-; 3, methyl 9-hydroxy-trans-10, cis-12-; 4, methyl 9-hydroxytrans-10, trans-12-octadecadienoate, from autoxidized methyl linoleate with a microporous 5 μm silica (Partisil-5) column and 0.75% ethanol in hexane (v/v) as eluting solvent, UV detector at 234 nm. From Chan and Levett (1977a). Courtesy of the American Oil Chemists’ Society.

C. High performance liquid chromatography (HPLC) When more advanced HPLC systems became available, the four cis,trans and trans,trans isomeric 9- and 13-hydroperoxides were partially separated from autoxidized methyl linoleate by adsorption chromatography with a microporous 5 μm silica acid column. The corresponding hydroxyoctadecadienoate isomers obtained by sodium borohydride reduction were better and completely separated with the same silica column (Figure 6.3). Similarly, the isomeric hydroperoxides from autoxidized methyl linolenate were not resolved with the same adsorption column used with autoxidized methyl linoleate. However, after reduction, the resulting hydroxyoctadecatrienoate derivatives were completely separated into the expected eight cis,trans and trans,trans isomers (Figure 6.4). The corresponding hydroxyoctadecanoate derivatives obtained by catalytic hydrogenation of different mixtures of isomeric hydroperoxides were sufficiently separated with a 10 μm silica column to permit quantitative analyses of the isomeric composition, in samples of autoxidized and photo-

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Figure 6.4. Normal phase HPLC of hydroxyoctadecatrienoate isomers from autoxidized methyl linolenate as described in Figure 6.3: 1, 13-cis, trans-; 2, 12-cis, trans-; 3, 12-trans,trans-; 4, 13trans,trans-; 5, 16-cis,trans-; 6, 9-cis,trans-; 7, 16-trans,trans-; 8, 9-trans,trans. From Chan and Levett (1977b). Courtesy of the American Oil Chemists’ Society.

sensitized-oxidized methyl linoleate and linolenate (Figure 6.5). The isomers of hydroperoxy epidioxides from oxidized linolenate (Figure 6.6) and from photosensitized-oxidized linoleate (Figure 6.7) were effectively separated by HPLC on microporous 5 or 10 μm silica columns. Partial resolution of stereoisomers of linolenate hydroperoxyepidioxides is indicated in peaks III and IV (Figure 6.6). The same HPLC systems were used to demonstrate the effect of α-tocopherol in inhibiting the formation of trans,trans-hydroperoxides and hydroperoxy epidioxides in autoxidized methyl linolenate (Figure 6.8) (Chapter 2, B,C). Autoxidized trilinolein was separated into mono-, bis- and tris-hydroperoxides by preparative reversed phase HPLC using a 5 μm C-18 column with UV at 235 nm and refractive index detectors (Chapter 2, F; Figure 6.9). The monohydroperoxides of trilinolein were further resolved into the positional isomers components by normal phase HPLC on a 5 μm silica column with UV detection (Figure 6.10). Complete and partial separation of various cis,trans- and trans,trans-2- and 1(3)-mono-13- and 9-hydroperoxides were identified by lipolysis and capillary GC. The ratio of 1(3)- to 2-monohydroperoxides estimated by normal phase HPLC averaging 2.0 indicated that oxidation of trilinolein was not selective toward either the 1(3)- or 2-position. Analyses of oxidized trilinolenin by reversed phase HPLC with a 5 μm C-18 column showed that monohydroperoxides and hydroperoxy epidioxides are the only products formed initially. Bis- and tris-hydroperoxides were detected

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Figure 6.5. Normal phase HPLC of hydroxyoctadecanoate derivatives of isomeric hydroperoxides of autoxidized and photosensitized-oxidized methyl linoleate and linolenate with a 10 μm silica Porasil column and 0.25% isopropanol in hexane (v/v), variable wavelength detector at 212 nm. A. photosensitized-oxidized methyl linoleate; B. photosensitized-oxidized methyl linolenate; C. autoxidized methyl linoleate; D. autoxidized methyl linolenate. From Neff and Frankel (1980). Courtesy of the American Oil Chemists’ Society.

at peroxide values above 30, and hydroperoxy bicycloendoperoxides and mono-dihydroperoxides at peroxide values above 75 (Chapter 2, Figure 6.11). The monohydroperoxide mixture of oxidized trilinolenin was resolved by analytical normal phase HPLC into six peaks identified as 1,3- and 2,3dilinolenoyl glycerol with 2-and 1(3)-substituted [12(13)]-, 2-(16)-, and 9-hydroperoxides (Figure 6.12). These HPLC analyses enabled to estimate the relative triacylglycerol positions of the hydroperoxides isomers. The cis,trans16-hydroperoxides favored the 1(3)- over the 2-triacylglycerol positions by a ratio of 2.3, compared to 1.8 for the cis,trans-9-hydroperoxides (Chapter 2, F).

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Figure 6.6. Normal phase HPLC isomers of hydroperoxy epidioxide isomers from autoxidized linolenate on microporous 10 μm silica Partisil 10 column, 0.3% ethanol in hexane (v/v) as eluting solvent, variable wavelength detector at 212 nm. From Frankel et al. (1981). Courtesy of the American Oil Chemists’ Society.

The use of HPLC with post-column chemiluminescence (CL) detection permits more sensitive and direct analyses of hydroperoxides from oxidized complex lipids in biological samples. By this technique, the HPLC effluent is mixed with different CL cocktails at a post column tee and is monitored by a CL detector measuring light emitted by the reaction of hydroperoxides with a heme

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Figure 6.7. HPLC of hydroperoxy epidioxide isomers from photosensitized-oxidized linoleate with a microporous 10 μm silica Partisil 10 column and 6:4:1 hexane:methylene chloride:ethyl acetate (v/v) as mobile solvent, refractive index detector. From Neff et al. (1982). Courtesy of the American Oil Chemists’ Society.

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Figure 6.8. Normal phase HPLC of hydroxyoctadecatrienoate isomers to study the effect of α-tocopherol in inhibiting the formation of trans,trans-hydroperoxides and hydroperoxy epidioxides in autoxidized methyl linolenate with a 5 μm Partisil-5 column, and 0.4% ethanol in hexane (v/v) as eluting solvent, UV detector at 234 nm. (i) no α-tocopherol added, (ii) + 0.05% α-tocopherol, (iii) + 0.5% α-tocopherol, (iv) + 5% α-tocopherol. From Peers et al. (1981). Courtesy of Society of Chemical Industry.

protein (cytochrome c or peroxidase) and an oxidized dye (luminol or isoluminol). A cytochrome c and luminol CL cocktail was used to analyse phospholipid hydroperoxides in human plasma, human blood LDL, and animal liver and brain tissues. With this mixture, spectrophotometric evidence supports the formation of singlet oxygen in the decomposition of peroxyl radicals by the Russell mechanism (Chapter 1, A.3). Although the HPLC-CL approach allows the detection of hydroperoxides at the picomole levels in oxidized lipid extracts from complex biological samples, many problems arise in recovery of different hydroperoxides, in differences in substrate specificities between CL cocktails, mistaken structural assignment for different HPLC peaks, and spurious results resulting from some artificial

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Figure 6.9. Preparative reversed-phase HPLC of autoxidized trilinolein with a 5 μm C-18 column, 30:70 methylene chloride:acetonitrile (v/v) as eluting solvent; broken line, UV detection at 235 nm; solid line, refractive index detection. From Neff et al. (1990). Courtesy of the American Oil Chemists’ Society.

oxidizing agents. Less polar hydroperoxides derived from triacylglycerols and cholesterol esters can be effectively extracted with hexane and methanol, but more polar phosphatidylcholine (PC) hydroperoxides require Folch extraction with chloroform and methanol. The cytochrome c–luminol cocktail is 100 times more responsive in the CL detection of PC hydroperoxides than the peroxidase-isolumionol CL cocktail. A peak assigned erroneously to cholesteryl ester hydroperoxides in blood plasmas by using the microperoxidase-isoluminol CL reagent was shown to be actually due to the hydroquinone of ubiquinol-10. Further analyses by LC-thermospray MS revealed that PC samples oxidized with the free radical initiator azobis-dimethylvaleronitrile (AMVN) produced AMVN-derived hydroperoxides that were not formed when the same samples were photooxidized with the sensitizer methylene blue (Table 6.2, Section F). Although this study demonstrated that the use of AMVN produced misleading results, this and other artificial free radical azo initiators are unfortunately commonly used to promote oxidation in biological systems (see Chapter 13).

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Figure 6.10. Analytical normal phase HPLC separation of monohydroperoxide isomers of autoxidized trilinolein with a 5 μm silica column, 0.5:99.5 2-propanol:hexane (v/v) as eluting solvent, UV detection at 235 nm. Lo, linoleoyl residue; ct 13-OOH, cis,trans linoleic 13-hydroperoxides; tt 13-OOH, trans,trans linoleic 13-hydroperoxides; ct 9-OOH, cis,trans linoleic 9-hydroperoxides; tt 9-OOH, trans,trans linoleic 9-hydroperoxides. From Neff et al. (1990). Courtesy of the American Oil Chemists’ Society.

An HPLC-fluorescence method using post-column detection was effectively applied in the analyses of hydroperoxide mixtures containing conjugated and non-conjugated diene structures. The fluorescent reagent diphenyl-1pyrenylphosphine (DPPP) reacts with lipid hydroperoxides to form DPPP oxide with exciting (352 nm) and emission (380 nm) absorptions. This reagent allowed the detection of both conjugated 9- and 13-hydroperoxides and the non-conjugated 10- and 12-hydroperoxides produced by photosensitized oxidation of methyl linoleate (Figure 6.13). This method was also applied to the detection of conjugated and non-conjugated hydroperoxides produced by photosensitized oxidation of methyl linolenate and the non-conjugated hydroperoxides from autoxidized methyl oleate. Other post-column detection systems

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Figure 6.11. Autoxidation of trilinolenin at 40°C. Analyses by reversed-phase HPLC as described in Figure 6.10. Mono-OOH, monohydroperoxides; OOH Epi, hydroperoxy epidioxides; di-OOH, dihydroperoxides; Bis-OOH, bis-hydroperoxides; Tris-OOH, tris-hydroperoxides; OOH Bicyclic, hydroperoxy bicycloendoperoxides. From Frankel et al. (1990). Courtesy of the American Oil Chemists’ Society.

Table 6.2. Analyses of hydroperoxide in phosphatidyl choline (PC) oxidized with azobis-dimethylvaleronitrile (AMVN) and photooxidized with methylene blue a Type of oxidation

Substrate

Photosensitized Photosensitized AMVN AMVN AMVN AMVN

di-18:2 PC 18:0, 18:2-PC di-18:2 PC 18:0, 18:2-PC di-18:2 PC 18:0, 18:2-PC

PCOOH (pmole) 18.0 18.6 5.1 4.8 29.4 27.5

AMVN-OOH b Other (pmole) X-OOH – – 61 54 36 51

2.0 1.4 34.9 41.2 34.6 21.5

% of PC-OOH 90.0 93.0 5.1 4.8 29.4 27.5

a From Zhang et al. (1995). PCOOH were determined by HPLC-CL comparing PC-OOH peak with standard purified from PC photosensitized oxidized with methylene blue. AMVN-OOH were determined by HPLC-CL comparing the peak with standard AMVN-OOH. Identification of peak due to AMVNOOH was based on thermospray LC-MS showing ions at m/z 238, 270, 302 and 334 absent in the MS of PC-OOH showing m/z 814,818 or 846. X-OOH = other hydroperoxides, remaining from total hydroperoxide subtracted by PCOOH and AMVN-OOH. b AMVN-OOH are very responsive to the microperoxidase–isoluminol but not to the cytochrom c–luminol CL cocktail, which in turn is more sensitive to PC-OOH.

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Figure 6.12. Analytical normal phase HPLC separation of monohydroperoxide isomers of autoxidized trilinolenin, as described in Figure 6.10. Ln, linolenic acid residue; ctc 12(13)OOH, cis,trans,cis linolenic 12+13-hydroperoxides; cct 16-OOH, cis,cis,trans linolenic 16-hydroperoxide; tcc 9-OOH, trans,cis,cis linolenic 9-hydroperoxide. From Frankel et al. (1990). Courtesy of the American Oil Chemists’ Society.

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Table 6.3. Quantitative analyses of monohydroperoxides (mg/g) isolated by reversedphase HPLC from trilinolein oxidized at different levels a PV (meq/kg) 2.6 8.3 22.0 47.4 63.2

CL detector

UV detector

ELS detector

0.10 0.32 0.93 2.04 2.42

0.55 0.74 1.82 4.38 6.39

0.55 1.00 1.75 3.50 4.46

a

From Mäkinen et al. (1996). Abbreviations: PV, peroxide value; CL, chemiluminescence, UV, ultraviolet; ELS, evaporative lightscattering. Analytical conditions: The polar hydroperoxides were first separated from non-polar material by solidphase extraction on ammonium columns, followed by reversed phase HPLC on C-18 columns using either methanol:2-propanaol (90:10, v/v) or methanol-2-propanol: dichloromethane (80:10:10, v/v). The post-column CL reagent contained isoluminol and microperoxidase in borate buffer (pH 10)–methanol (30:70, v/v). Methyl linoleate hydroperoxides were used as external standards for quantification by CL and UV detectors, and 1,3-diolein by ELS detector.

Figure 6.13. Normal phase HPLC analyses of hydroperoxide with a 5 μm silica column, 34:500 diethyl ether:hexane (v/v) as eluting solvent. A, photosensitized oxidized methyl linoleate detected by postcolumn fluorescent reagent diphenyl-1-pyrenylphosphine (DPPP), exciting (352 nm) and emission at (380 nm) absorptions and B, by UV detection at 234 nm, C, Autoxidized methyl linoleate detected DPPP fluorescence, and D, UV detection at 234 nm. See Figure 6.10 caption for abbreviations. From Ohshima et al. (1996). Courtesy of the American Oil Chemists’ Society.

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Table 6.4. 13C NMR and preparative HPLC analyses (rel %) of dienol isomers from hydroperoxides of methyl linoleate oxidized at different temperatures a Temperature and method

13-OHc-9,t-11

13-OHt-9,t-11

9-OHt-10,c-12

9-OHt-10,t12-

Ratio c,t/t,t

25°C NMR HPLC Average

31.6 30.5 31.0

18.9 21.4 20.2

31.7 27.6 29.7

17.8 20.5 18.9

1.72 1.39 1.56

50°C NMR HPLC Average

18.2 20.8 19.5

28.1 27.8 28.8

23.5 22.3 22.9

30.2 29.1 29.7

0.72 0.76 0.74

65°C NMR HPLC Average

17.7 19.5 18.4

34.0 33.0 33.5

18.1 16.9 17.5

30.2 30.6 30.4

0.56 0.57 0.57

a

From Frankel et al. (1990) Abbreviations: c, cis; t, trans Analytical conditions: Hydroperoxide isomeric mixtures from autoxidized methyl linoleate are reduced to hydroxydiene derivatives by sodium borohydride, and separated by preparative TLC on plates coated with silica gel treated with UV marker and developed with diethyl ether–hexane (6:4, v/v) and UV active hydroperoxides are eluted with diethyl ether. The dienol isomers are separated by HPLC on a preparative 6 μm porous silica column with ethanol–hexane (0.5:99.5, v/v) as mobile phase with a UV detector set at 234 nm. The weight percent composition is based on the weight of each fraction collected.

varied significantly for the quantitative analyses of triacylglycerol hydroperoxides separated by reverse phase HPLC. UV and CE detectors were more sensitive and selective than the evaporative light-scattering (ELS) detector for measuring the hydroperoxides from trilinolein at peroxide values ranging between 3 and 8 (Table 6.3). At higher peroxide values (22–63), detection by CE and ELS was more sensitive than by UV. The separation of conjugated diene hydroperoxides or the hydroxydiene derivatives by HPLC and their quantification by UV detection requires calculations based on the different molar absorptivities of each isomer, which are lower for the cis,trans (27,200–28,300) than the trans,trans (30,500–31,600) isomers. In contrast, separation of the hydroxydiene derivatives by preparative HPLC allows direct quantification based on the weight of fractions. 13C NMR studies of the four geometric hydroxydiene isomers isolated by preparative HPLC showed characteristic differences in resonance for their olefinic carbons and for the methylene carbons allylic to the diene systems. 13C NMR analyses based on the integrals of the olefinic carbons were in good agreement with the gravimetric HPLC analyses on samples of methyl linoleate oxidized at different temperatures (Table 6.4). The ratios of cis,trans- to trans,trans-hydroxydiene isomers decreased from 1.7 at 25°C to 0.56 at 65°C.

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D. High performance size exclusion chromatography (HPSEC) Separation by HPSEC (also known as gel permeation chromatography) is based on the differences in effective diameter of molecules and is not affected by their functionality. Different solutes are separated by their physical restriction in column packing consisting of crosslinked copolymers of styrene divinyl benzene. This stationary phase discriminates molecules by eluting the larger ones first and separating the smaller ones by mechanical diffusion into pores of different sizes. Ideally, the separation is only affected by the size of the molecules without the interactions that occur with adsorption, partition and ion exchange chromatography. Several detection systems are used for lipid oxidation products based on UV absorption for conjugated dienes, infrared, refractive index, flame ionization, or evaporative light scattering. Detection and quantitative analyses of hydroperoxides by post-column reactions include the use of luminol with a catalyst that generates chemiluminescence, reduction with potassium iodide into I3– detected spectrophotometrically, by the ferric thiocyanate complex formation and reduction with triphenyphosphine. An important application of HPSEC includes analyses of dimers, trimers, oligomers, partial glycerides and cyclic fatty acids in heated, thermally oxidized and frying fats and fish oils. This technique has become an accepted method for evaluating the quality of frying fats (Chapter 12), crude and refined oils and the effectiveness of processing. Analyses by HPSEC showed significant increases in oxidized triglyceride monomers, triglyceride dimers and triglyceride polymers in extracts from potato chips fried in sunflower oil after storage at room temperature, but none in the corresponding potato chips fried in high-oleic sunflower oil and palmolein (Table 6.5). Table 6.5. High performance size exclusion chromatography (HPSEC) analyses of oils extracted from potato chips after storage at room temperature (wt%) a Oils Sunflower HiOlSun PalmOl

Storage (weeks) 0 15 0 15 0 15

Total polar Ox TG compounds monomers 5.2 7.5 4.9 4.8 8.9 8.6

15.3 37.3 12.4 12.4 11.4 11.6

TG dimers

TG polymers

DG

FA

15.0 16.9 10.3 10.0 12.5 12.2

1.7 1.9 1.0 1.3 1.0 1.2

12.7 12.9 18.9 18.1 60.3 58.2

6.9 6.2 5.9 5.8 3.4 3.1

a From Marquez-Ruiz et al. (1996) Abbreviations: OxTG, oxidized triglycerides; DG, diglycerides; FA, fatty acids; HiOlSun, high-oleic sunflower; PalmOl, palmolein. Analytical conditions: A solid-phase extraction silica cartridge is used to separate the nonpolar components by elution with petroleum ether:diethyl ether (90:10) and the polar components with diethyl ether. The polar components are resolved by HPSEC on 2 columns in series of highly cross-linked styrenedivinylbenzene copolymers, using tetrahydrofuran as mobile phase and a refractive index detector.

9.60

15.9

20

40

46.1

40

1.03

29.5

20

0

2.45

0

MW Conc. MW Conc. MW Conc.

MW Conc. MW Conc. MW Conc. nd nd 3230 0.17 2980 0.21

nd nd 2475 1.79 6315 4.51

Fraction 1

1860 0.13 3030 0.46 2820 0.49

3465 0.17 2220 4.02 4960 6.65

Fraction 2

1285 0.52 2235 1.50 2140 1.32

2450 0.90 1900 15.3 2390 15.9

Fraction 3

1110 1.59 1325 3.80 1285 2.87

1740 4.20 990 5.44 2310 4.39

Fraction 4

1010 0.30 915 0.69 910 0.38

1190 7.07 925 0.84 1615 0.57

Fraction 5

660 0.27 nd nd nd nd

nd 1.69 nd 0.30 nd nd

Fraction 6

From Abidi and Warner (2001) Oils were used to fry French fried potatoes and tortilla chips at 190°C. Polar compounds were determined by column chromatography. Abbreviations: MW, molecular weight; Conc, concentrations; nd, not determined. Compositions: soybean oil (SBO): contained 13.2% 16:0+18:0, 26.2% 18:1, 53.1% 18:2, 5.90% 18:3; high-oleic soybean oil (HOSBO) contained 10.0% 16:0+18:0, 84.0% 18:1, 1.60% 18:2, 2.40% 18:3. Analytical conditions: The polar fraction from frying oils was separated by silica gel chromatography by removing the nonpolar components with petroleum ether:diethyl ether (87:13 v/v) followed by chloroform:methanol (1:1). The polar fraction was fractionated by HPSEC with 3 columns in series packed with high-efficiency mixed-bed PLGEL columns, eluting with THF as mobile phase. The chromatograph was interfaced with a differential viscometer and a refractometer connected in parallel.

a

HOSBO

SBO

Mol.Wt./ Conc. (g/100g oil)

Molecular weight and concentration distribution of decomposition products in vegetable oils used for frying a

Frying Frying Polar oil time compounds (h) (%)

Table 6.6.

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The molecular weight distribution of macromolecular triglyceride polymers formed in frying oils was recently analysed by HPSEC with viscometric and refractometric detection. The molecular weight (MW) distribution is determined by plotting the log of (intrinsic viscosity × MW) vs. retention volume using fatty acids, di- and triglycerides and several polystyrene standards. By this novel technique, the nonvolatile polar components of heated and frying fats may be better represented by their molecular weight distribution rather than by the oversimplified and ambiguous assignments as monomer, dimer, trimer, tetramer and highly polymeric triglycerides. As expected, the molecular weight distribution was a function of frying time and vegetable oil type (Table 6.6). There was no evidence of formation of distinct dimer, trimer and tetramer species in the oils used in these frying experiments. As expected, soybean oil showed a higher degree of polymerization than high-oleic soybean oil and other oils containing no linolenate. This novel method may be useful to clarify the chemical nature of high molecular weight carbonyl compounds produced during oxidation followed by polymerization during the processing of polyunsaturated vegetable and fish oils (Chapter 8).

E. Gas chromatography-mass spectrometry (GC-MS) The combination of GC and MS has been used to analyse qualitatively and quantitatively various derivatives of oxidation products that can be separated in the vapor phase and emerge from fused silica capillary columns, either directly into the ion source of the MS or through membranes or molecular jet separators to concentrate the components of each GC peak. In the mass spectrometer, the organic molecules in the vapor state are bombarded by electron-impact ionization (EI) to form positively charged ions, which fragment into smaller ions. The fragmentation patterns obtained by the “hard” EI method are most useful to characterize the volatile compounds of low-molecular weight separated by capillary GC. Much work has been published to characterize qualitatively by GC-MS hydroperoxides and secondary oxidation products from various unsaturated lipids. Quantitative analyses by GC-MS require however careful standardization with stable derivative compounds of well-defined structures such as hydroxy fatty acid derivatives that can be readily synthesized unambiguously. The EI method is not suitable for molecules of larger molecular weight because they produce excessive fragmentation of molecular ions. Chemical ionization (CI) and other “soft” ionization MS methods cause less fragmentation with lipid hydroperoxides and other labile oxidation products. More advanced MS techniques described in Section F permit the direct qualitative analysis of hydroperoxides with less fragmentation, but their quantification is still elusive. Allylic unsaturated hydroperoxides and the corresponding hydroxy esters cannot be analysed directly by GC and GC-MS. The thermally labile

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hydroperoxides are readily decomposed, and EI-MS provides no useful molecular weight information. The allylic hydroxy derivatives are also labile and thermally dehydrated into conjugated polyenes under the GC conditions. The trimethylsilyl (TMS) derivatives of hydroxyoctadecenoates, hydroxyoctadecadienoates and hydroxyoctadecatrienoates from the corresponding hydroperoxides are most suitable for GC and GC-MS. These derivatives produce intense ions due to α-scission (a) and a much less intense fragment (b) allylic to the single double bond of oleate (1), or the conjugated diene systems of linoleate (2) and linolenate (3):

(1)

(2)

(3) The relative intensity of the two fragments varies because of 1,3-rearrangement of the allylic TMS ethers (4) under the conditions of GC-MS: (4) For quantitative MS analyses, the confounding effect of 1,3-rearrangement of allylic TMS ethers can be completely avoided by catalytic hydrogenation of the allylic alcohols. The resulting saturated hydroxy compounds are suitable derivatives for direct quantitative GC-MS analysis of the positional isomers of the hydroperoxides because they produce two intense fragments by α-scissions at both sides (a) and (b) of the TMS ether position: (5)

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Figure 6.14. Mass chromatography of TMS ethers of methyl hydroxy octadecanoate isomers from autoxidized methyl oleate. From Frankel et al. (1977a). Courtesy of the American Oil Chemists’ Society.

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Figure 6.15. Reaction schemes and different methods used to determine the isomeric composition of hydroperoxides from autoxidized methyl linolenate. From Frankel (1999).

However, quantitative studies required standardization with authentic synthetic standards of the positional hydroxyoctadecanoate isomers. The TMS ethers of the four positional hydroxy isomers from autoxidized methyl oleate were analysed quantitatively by standardizing the intensity of both α-scission fragments (A and B) obtained with synthetic methyl 8-, 9-, 10-, and 11-hydroxyoctadecanoates after computer summation of all the spectra within the GC peak due to hydroxyocadecanoate (Figure 6.14). A similar GC-MS computer summation method was standardized with synthetic 9- and 13hydroxyoctadecanoate. Equal amounts of 9- and 13-hydroperoxides were found in all samples of methyl linoleate oxidized at different temperatures and peroxide levels (Table 2.2, Chapter 2, A, B). With methyl linolenate oxidized at different temperatures and peroxide values, the standardized GC-MS computer summation method applied on the hydroxyoctadecanoate isomers showed that the proportion of 9- and 16-hydroxy esters was significantly higher (75– 82%) than that of the 12- and 13-hydroxy esters (18–25%) (Table 2.3). These GC-MS results were in complete agreement with earlier studies based on analyses of cleavage products of monoenes derived by dehydration of hydroxyoctadecanoates derivatives with boric acid, and HPLC of the hydroxyoctadecatrienoate derivatives (Figure 6.15). The uneven distribution of isomeric hydroperoxides of methyl linolenate was later accounted for by the significant formation of hydroperoxy epidioxides derived by cyclization of the 12- and 13-hydroperoxides (Chapter 2, C). Solid phase microextraction coupled to gas chromatography-mass spectrometry (SPME-GC/MS) has been used as a sensitive method for measuring volatiles in the headspace of milk powders. Headspace measurements on two samples of infant milk powders gave values of hexanal initially of 466 and 1181 parts per billion (ppb), and 617 to 3430 after accelerated storage at 37°C for 1 to 4 weeks (Table 6.7). These levels are far above the hexanal perception thresholds of 4.5 ppb in water and 50 ppb in homogenized whole milk.

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Table 6.7. Analyses of hexanal by solid-phase microextraction (SPME) /MS and by isotope dilution in milk powders after accelerated storage a Storage (weeks)

Hexanal (ppb) Sample A

0 1 3

1181 1848 3430 Sample B

0 2 4

466 617 1022

a From Fenaille et al. (2003). Infant formula milk powder samples were stored ungassed in the dark at 37°C. Analytical conditions: Milk powder was reconstituted in bidistilled water and an aliquot was sealed in a glass vial and equilibrated 1 h at 25°C, before analysis by SPME inserting a PDMS/DVB fiber and volatiles sampled during 10 min at 25°C, followed by desorption in the GC inlet interfaced with a mass selective detector. Quantification was carried out by isotope dilution using deuterated d2-hexanal (m/z 58). Sample A at 0-time gave a value of 1316 ppb by standard addition using non-deuterated hexanal (m/z 56).

F. High performance liquid chromatography and mass spectrometry (HPLC-MS) MS coupled with normal phase and reversed-phase HPLC is a powerful tool that permits the direct characterization of thermally labile hydroperoxides and nonvolatile high molecular weight secondary oxidation products of triacylglycerols, cholesterol and phospholipids without the necessity of prior derivatization. The problems of removing the solvent from these labile compounds emerging from an HPLC column prior to MS have been overcome by using thermospray, or electrospray ionization (ESI), or atmospheric-pressure chemical ionization (APCI) interfaces. The thermospray interface consists of a rapidly heated capillary tube connecting the HPLC column and the MS that produces a supersonic vapor jet carrying particles or droplets of solute. By traveling at high speed through a lens system, the droplets acquire an electronic charge and the high density ions are ejected into the MS. With the more recently developed ESI interface, a high electric field is applied to nebulize the sample solution and impart a negative or positive charge to the droplets that builds up as the solvent evaporates. This relatively mild and sensitive “soft” ionization technique provides useful structural information on molecular ions with little fragmentation based on both positive and negative ions spectra obtained with sodium or ammonium salts. With the APCI technique, the sample is ionized either similarly to the ESI technique, or by a charge-transfer method. The ESI technique was applied to the analyses of synthetic isomers of triacylglycerol hydroperoxides of eicosapentaenoic acid, and other oxidation products, including hydroxides, epoxides and triglyceride core aldehydes

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(aldehydo glycerides, Chapters 4, 5, 7 and 11, also called “2½ glycerides”). The APCI technique was used to analyse qualitatively oxidation products from triacylglycerols separated by reversed-phase HPLC. Triacylglycerol hydroperoxides, epoxides, bishydroperoxides, epidioxides and diepoxides from oxidized triolein, trilinolein and trilinolenin could be identified from the protonated molecular ions, near-molecular ions and molecular ion adducts, and from characteristic diacylglycerol fragment ions. Analyses of oxidized sunflower oil by reverse-phase HPLC/ESI/MS showed the presence of a mixture of triglyceride core aldehydes, dialdehydes, hydroxy and epoxy aldehydes. HPLC-CL (Section C) and HPLC-thermospray MS were used to analyse hydroperoxides generated from PC with either the free radical initiator azobisdimethylvaleronitrile (AMVN), or by photooxidation sensitized with methylene blue. Mono-and di-PC hydroperoxides were established by HPLC-CL in the sample produced by photosensitized oxidation. Over 90% of the hydroperoxide peak was shown to be PC hydroperoxides and reacted preferentially with the cytochrome c–luminol CL cocktail (Section C). In contrast, the sample prepared with AMVN showed that 70–95% of the hydroperoxides was not PC-OOH, but was largely due to spurious AMVN-derived hydroperoxides (Table 6.2). Lipid hydroperoxides in phospholipid vesicles prepared from egg lecithin enriched with arachidonylstearoylphosphatidylcholine (ASPC) were analysed directly by ESI-MS. The positive ESI of phospholipid vesicles obtained in the presence of excess sodium chloride showed mass 832 corresponding to the sodiated form M+23 of ASPC. Oxidation with a mixture of tert-butyl hydroperoxide and Fe2+ produced new species at mass 864, 896, and 928, corresponding to the addition of one (+32), two (+64) and three (+96) dioxygen molecules to ASPC, consistent with the formation of mono-, di-, and trihydroperoxides. Various lipid oxidation products have been separated by HPLC and the purified compounds characterized by chemical ionization mass spectrometry (CI-MS) with a direct exposure probe. CI-MS yields the protonated molecular ion [M+H]+ with isobutane and the adduct ion [M+NH4]+ with ammonia as the reagent gases. Because the ionic species formed by chemical ionization have much less energy than the molecular ion formed by electron impact ionization, fragmentation is greatly reduced and provides useful structural information and more intense mass fragments with intact hydroperoxides, mono- and di-hydroxy fatty acids without derivatization. The CI-MS technique produces simpler and more straightforward fragmentation patterns that may be easier to interpret than by GC-electron ionization MS. With the isomeric methyl 9- and 13-hydroperoxides of linoleate purified by HPLC, the direct exposure CI technique with isobutane as reagent gas provided structural information on the location of the hydroperoxide group and fragmentation patterns that could be explained by the homolytic and heterolytic

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Figure 6.16. Mass fragment ions obtained by chemical ionization-mass spectrometry (CI-MS) analysis of 9- and 13-hydroperoxides from oxidized methyl linoleate, from Plattner and Gardner (1985) (note: both hydroperoxide isomers produced intense ions at m/z 309 and 311 in the isobutane spectrum), and 9hydroperoxy epidioxide and 9,16-dihydroperoxide from oxidized methyl linolenate, from Frankel et al. (1986).

decomposition pathways established by GC-MS (Chapter 4, D) (Figure 6.16). With ammonia as the CI gas, both 9- and 13-hydroperoxides showed a more stable molecular adduct ion (M+NH4)+ at m/z 344. The 9-hydroperoxide

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produced methyl 9-oxononanoate (m/z 204) and decadienal (m/z 170), and the 13-hydroperoxide produced methyl 13-tridecadienoate (m/z 256), expected by homolytic cleavage. The same analytical approach was applied to analyse hydroperoxy epidioxides and dihydroperoxides obtained from methyl linoleate and linolenate (Figure 6.16). The fragmentation patterns observed under CIMS conditions were in complete agreement with those established by decompositions under GC-electron ionization MS conditions (Chapter 4, D). Similarly, the sites of hydroperoxy substitution in isomeric hydroperoxides of methyl oleate were identified directly by CI-MS (Figure 6.17). The fragmentation ions (m/z 213, 199, 155 and 169) were attributed to scission α to the hydroperoxy group with loss of water. However, previous CI-MS analyses of the hydroperoxides of linoleate and secondary oxidation products gave ions with isobutane for protonated fragments, produced by either homolytic β-scission or heterolytic (also referred to as Hock cleavage) decomposition pathways established under thermal and acid conditions (Chapter 4, D). Thus, the fragmentation ion m/z 213 from the 11-oleate hydroperoxide can be attributed to the protonated ion (212+H+) due to methyl 11-oxo-9-undecenoate, the ion m/z 199 from the 10-oleate hydroperoxide to the protonated ion (198+H+) due to methyl 10-oxo-8-decenoate, the ion m/z 155 from the 9-oleate hydroperoxide to the protonated ion (154+H+) due to 2-decenal, and the ion m/z 169 from the 8-oleate hydroperoxide to the protonated ion (168+H+) due to 2-undecenal (Chapter 4, D). The corresponding fragmentation patterns obtained from linoleate hydroperoxides were also supported unambiguously by tandem MS discussed below. By tandem mass spectrometry or MS/MS a specific parent ion is ionized again into “daughter” ions, which are separated and monitored with higher sensitivity of detection. Fragments arising from CI of intact hydroperoxides were thus isolated and studied by tandem spectrometry. Although both the 9and 13-hydroperoxides gave intense CI ions at m/z 309 and 311 with isobutane (Figure 6.16), the daughters of these ions obtained by MS/MS were clearly different. From the ion at m/z 309 relatively intense daughter fragments were obtained at m/z 99 (apparently due to hexanal–H+) in the 13-hydroperoxide and at m/z 185 (apparently due to methyl 9-oxononanoate–H+) in the 9-hydroperoxide. Similar information was obtained from the CI ion at m/z 328 with ammonia giving intense daughter fragments at m/z 185 and 99 from the 9- and 13-hydroperoxides, respectively. Tandem mass spectrometry and CI-MS can thus be used as powerful tools for establishing the position of hydroperoxides and other complex oxygenated functions in isomeric mixtures of oxidized unsaturated lipids without the tedious problems of derivatization. Although valuable and useful, the structural information from the advanced MS techniques APCI, CI and other “soft” MS techniques has been mainly qualitative. For quantitative analyses, more careful work is needed to standardize spectra with authentic reference compounds and to better

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Figure 6.17. Mass fragment ions obtained by chemical ionization-mass spectrometry (CI-MS) analysis of 8-, 9-, 10- and 11-hydroperoxides from oxidized methyl oleate. From Porter et al. (1994). Fragmentation ions m/z 213, 199, 155 and 169 were attributed to scission α to the hydroperoxy group with loss of water (see discussion in text).

interpret the complicated fragmentation patterns obtained by these MS techniques. Although these methods are useful for the direct MS identification of intact labile polyunsaturated hydroperoxides and other oxidation products without the necessity of derivatization, authentic standards of these compounds required for quantification are very difficult to synthesize and therefore not readily available.

G. Coordination ion-spray-mass spectrometry (CIS-MS) This new technique of chemical ionization is based on the formation of charged complexes with silver or other transition metal ions, producing stable coordination compounds with olefinic compounds that can be detected by MS. The use of silver ions with CIS-MS proved to be a useful technique for the analysis of complex lipid oxidation products, which readily form Ag+ coordination

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compounds. Lipid hydroperoxides and cyclic peroxides form stable Ag+ adducts producing dominating molecular ions [Mg+Ag]+ and fragmentation ions by heterolytic decomposition pathways. These pathways were previously established by GC and GC-MS (Chapter 4) under either thermal or acid conditions and by CI-MS (Section F); they are also referred to as Hock fragmentation (see Chapter 4). The CIS-MS technique was used to study the hydroperoxides produced from cholesterol linoleate, cholesterol arachidonate and the corresponding polyunsaturated fatty esters of phospholipids, by introducing samples either by direct liquid infusion or by normal phase HPLC. Analyses by direct liquid injection of solutions of 13- and 9-hydroperoxides containing AgBF4 produced molecular ions [M+Ag]+ adducts from the Ag isotopes 107 and 109. Collisioninduced dissociation experiments (also known as tandem MS, or MS-MS, section F) on the Ag adduct ions gave fragment ions (m/z 687, 689) corresponding to 12-oxo-9-dodecenoate cholesteryl ester for the 13-hydroperoxides of linoleate, and fragment ions (m/z 647, 649) corresponding to 9-oxononanoate cholesteryl ester for the 9-hydroperoxides of linoleate (Figure 6.18). The same analytical approach was used to identify the corresponding aldehyde esters from the 9- and 13-hydroperoxides of linoleoyl phosphatidylcholine and from the 9- and 12-hydroperoxides of arachidonyl phosphatidylcholine (Figure 6.19). The same aldehyde esters (also known as core aldehydes) were previously identified among the thermal decomposition products of methyl linoleate hydroperoxides (Chapter 4). The CIS-MS technique was also used to characterize the fragment ions corresponding to aldehydes and epoxides from cholesterol arachidonate hydroperoxides as well as the cholesteryl 7α - and 7βhydroperoxides (Chapter 2, Section D) produced by direct oxidation of the cholesterol backbone. How much competition occurs between oxidation of the side chain of arachidonate esters and the cholesterol backbone is not established. More quantitative analyses are needed to clarify the mechanism of lipid hydroperoxide decomposition under CIS-MS conditions.

H. Advanced instrumental techniques for volatile flavor analysis Important advances have been achieved recently in developing very sensitive methods for analysing volatile flavor compounds to link their relative release to sensory perception in foods. Volatile compounds can now be measured “innose” by introducing the whole mixture into the mass spectrometer and resolving them entirely by mass followed by APCI-MS based on proton transfer. This powerful technique also referred to commercially as the “MSNose”, allows in vivo sampling of moist air from the panelist to the source and characterizing the effect of lipds and food emulsions on flavor release and delivery from food matrixes during consumption.

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Figure 6.18. Coordination (Ag+) ion spray-mass spectrometry (CIS-MS) of oxidized cholesteryl linoleate showing heterolytic cleavage of silver adducts of cholesteryl linoleate hydroperoxides. Adapted from Havrilla et al. (2000).

Monitoring flavor release from foods during eating requires a rapid and sensitive response in the range of ppb. Direct MS techniques have been developed for this purpose by sampling directly into the source and resolving the ions by mass alone. The current MS-nose method used for sensitive flavor analysis by APCI or proton transfer reaction (PTR) followed by MS, can tolerate water and air, produce single ions for most compounds and operate at pressures that allow interfacing between people and the ion source. This powerful technique, also referred to as “nosepace analysis”, can measure the actual concentration of compounds close to the olfactory receptors, and allows the study of relationships between concentration of volatile compounds and perceived intensity and any synergy or masking between compounds during food consumption.

Figure 6.19. Coordination (Ag+) ion spray mass spectrometry (CIS-MS) of oxidized 1-palmitoyl -2-linoleoyl-sn-glycero-3-phosphatidylcholine and 1-stearoyl 2-arachidonyl-sn-glycero-3-phosphatidylcholine showing heterolytic cleavage of silver adduct of linoleoyl and arachidonyl hydroperoxides (CID = collision induced dissociation). Adapted from Milne and Porter (2001). See Figure 6.18.

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Table 6.8. Nosespace release (NR) and perceived intensity (PI) of hexanal in four different matrices at three different concentrations a Matrix

Water Skim milk 2.7% Fat milk 3.8% Fat milk

Low hexanal (1 mg/l)

Medium hexanal (4 mg/l)

High hexanal (16 mg/l)

NR

PI

NR

PI

NR

PI

6.3 6.3 5.4 5.9

29 31 5 6

33.0 28.5 33.4 26.1

52 49 38 28

131 110 114 81

84 86 65 48

a From Roberts et al. (2003). NR, nosespace release in nl/l; PI, perceived intensity in line scale value, 0–100. Nosespace analysis: The air exhaled through the nose was drawn up into a special heated stainless steel tube and split into two fractions. One fraction was analysed by proton reaction mass spectrometry, and the remainder was released into the laboratory air. Perceived intensity determination: Five experienced panelists rated the intensity of the added hexanal with the sample’s background of milk flavor. They marked their PI on a line anchored at either end with a line scale of 0 (weak) to 100 (strong).

Nosespace analyses can measure the actual compound concentration that comes out of the nose after passing through the nose cavity. Nosespace release analysis of hexanal was compared with perceived intensity by trained panelists at three concentrations to determine the effect of fat concentration on flavor absorption (Table 6.8). This analysis showed only significant differences between water and whole milk at the high hexanal concentration. The perceived intensity by the sensory panel was significantly different over water with whole milk and low-fat milk but not with skim milk, decreasing in the order: 3.8% fat milk < 2.7% fat milk < skim milk = water. This reduction in nosespace release with increasing fat concentration was lower than that observed by static headspace-GC. This difference was attributed to several possible factors, including the physiology of swallowing liquid samples, dilution with saliva, transfer of the volatile compound from the sample to the air and through the retronasal route to the nasal cavity and final exhalation through the nostrils. These matrix effects on relative perceived intensity are consistent with the well-recognized lower threshold values of flavor volatile compounds in oil than in aqueous systems (Chapter 11, C.4 ). The same phenomenon, referred to as “flavor paradox”, is reflected by the higher impact of flavor volatiles in lowfat foods than in higher fat foods, due to their higher headspace concentration in the former than in the latter foods (Chapter 11, C.4). Comparison of the amounts of volatiles in the headspace above a solution and the breath volatile concentration after consumption of the solution showed that the volatiles exhaled from the mouthspace and nosespace had significantly lower intensities relative to those for headspace analysed by APCI-MS (Table 6.9). Breath exhaled from the mouth contained 8-fold more volatiles than

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Table 6.9. Breath analysis of mouthspace and nosespace signal intensities relative to headspace intensities by atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) a Compound Acetaldehyde Butanal Hexanal 2-Hexenal Octanal Decanal

Mouthspace (%) 19 13 14 13 5.4 2.1

Nosespace (%) 3.0 3.4 3.2 3.4 0.80 0.38

a From Linforth et al. (2002) Headspace analysis: The headspace of volatile solutions (0.1 to 5 ppm) were analysed by APCI-MS by drawing an aliquot into the heated sampling line of the mass spectrometer and determining the maximum signal within 20 seconds. Breath analysis: Aliquots (15 ml) of the solutions were placed in-mouth and swallowed immediately. Breath was exhaled through the nose or mouth through a tube connected to the end of the mass spectrometer sampling line and the maximum signal intensity was compared with the signal during headspace analysis.

breath exhaled from the nose. This difference was not due to dilution of the sample by saliva in the mouth. The most important factor appeared to be mass transfer equilibration affecting volatile delivery from the solution to the gas phase where it is diluted through the upper airway. The concentration of volatiles exhaled from the nose was affected by absorption to the nasal epithelia, which increased with lower air/water partition coefficients. The studies published in this promising area of flavor chemistry and physiology have been limited to a few volatile compounds. They need to be extended to the multitude of other volatile compounds derived from lipid oxidation. Many of these volatiles are known to have a significant impact on quality and acceptability of lipid-containing foods. The complex volatiles produced by food lipids containing n–3 polyunsaturated fatty acids reported to impart nutritional and health benefits are especially important because they develop undesirable fishy odors and flavors at extremely low levels of oxidation (Chapter 4.D; Chapter 5.F; Chapter 11.E).

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