Separation and characterization of products from acidic methanolysis of plasmalogenic lipids by two-dimensional gas chromatography with online reduction

Separation and characterization of products from acidic 1 methanolysis of plasmalogenic lipids by two-dimensional gas 2 chromatography with online reduction. 3 4 5 6 Pierluigi Delmonte1*, Xabier Belaunzaran2, Clark Ridge1, Noelia Aldai2 and John 7 K.G. Kramer3 8 9 10 11 12 1 Office of Regulatory Science, Center for Food Safety and Applied Nutrition, Food and Drug 13 Administration, College Park, MD, USA14 2 Lactiker Research Group, Department of Pharmacy & Food Sciences, University of the Basque 15 Country (UPV/EHU), 01006, Vitoria-Gasteiz, Spain16 3Guelph Food Research Centre, Agriculture & Agri-Food Canada, Guelph, ON, Canada (retired) 17 18 19 20 21 22 23 24 25 ∗ Corresponding author at: HFS-717, US Food and Drug Administration, 5001 Campus Drive, 26 College Park, MD 20740, USA. Tel.: +1 240 402 1779; fax: +1 240 402 2622. 27 E-mail address: [email protected] 29 This is the accepted manuscript of the article that appeared in final form in Journal of Chromatography A 1619 : (2020) // Article ID 460955, which has been published in final form at https://doi.org/10.1016/j.chroma.2020.460955. © 2020 Elsevier under CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/) 1. Abstract 30 31 The complexity of determining the composition of animal tissue lipids is greatly increased by 32 the presence of plasmalogens in which the alkyl chain is linked to glycerol by a vinyl ether bond 33 instead of being esterified. Acidic methanolysis of animal tissue lipids provides the simultaneous 34 scission of acyl and alkenyl ether moieties, but the complexity of the products of reaction poses a 35 great challenge in their gas chromatographic analysis. Two-dimensional gas chromatography 36 with online reduction (GC-OR×GC) provided the resolution of all components contained in acid 37 methanolyzed animal lipids, taking advantage of the selective hydrogenation of alkenyl ether 38 methanolysis products prior to the second-dimension separation (2D). In this study, we also 39 studied the chemical transformations occurring during the acidic methanolysis of animal lipids 40 and the subsequent gas chromatographic analysis. In particular, we observed that using 41 methanolysis reagents contaminated with water resulted in the undesired formation of fatty 42 aldehydes, and we made recommendations on how to avoid these side reactions using proper 43 methanolysis conditions. Products of acidic methanolysis were studied by GC-OR×GC, GC-MS, 44 NMR spectroscopy, and GC with flame ionization detection (GC-FID). We defined the GC-FID 45 elution order of animal lipid acidic methanolysis products using 100 m x 0. 25 mm 100% 46 bis(cyanopropyl)siloxane columns and two different set of elution conditions: isothermal elution 47 at 180°C, and a temperature program optimized for dairy fats. A simple procedure for isolating 48 DMAs prior to GC analysis is also described. 49 50 Keywords: Dimethyl acetals; plasmalogens; fatty acids; GCxGC; animal lipids; two dimensional 51 gas chromatography. 52 53 2. Introduction 54 55 Most common lipids in animals consist primarily of long-chain aliphatic moieties linked to a 56 glycerol backbone by an acyl, alkenyl or alkyl linkage, and with (polar lipids) or without (neutral 57 lipids) a polar head group on the sn-3 position [1, 2]. Plants contain mainly acyl lipids, while 58 marine animals, insects and phyla contain significant proportions of alkenyl and alkyl ether lipids 59 [3]. These three functional groups show differences in their reactivity to acid and base catalysts, 60 which has been exploited in their analysis. While ester bonds are easily hydrolyzed using an 61 acidic or basic catalysts, vinyl ether linkages are stable under basic conditions but can be cleaved 62 by strong acids such as HI [4]. The aliphatic chains attached to these three functional groups are 63 generally similar in length but differ in unsaturation. The general method to determine the alkyl 64 chain composition of animal lipids is scission from glycerol by acid or base catalyzed 65 methanolysis, followed by GC analysis. As early as 1944, Klenk discovered that acid-catalyzed 66 methanolysis of acyl lipids yields fatty acid methyl esters (FAMEs), while for vinyl ether lipids 67 yields dimethylacetals (DMAs); ether lipids are not lyzed from the glycerol backbone [5]. 68 Available chromatographic techniques, primarily GC with packed columns, were not capable of 69 separating the complex animal lipid methanolysis products. Even modern long highly polar 70 capillary columns failed to provide complete resolution of these analytes [6, 7, 8, 9]. Despite 71 these limitations, some authors quantitated only the main DMAs and FAMEs in methanolyzed 72 lipids by GC [10, 11] or LC-MS/MS [12] and concluded that certain neurodegenerative diseases 73 were associated with a change in the alkenyl to acyl lipid ratio. 74 The need for more precise compositional information led to the isolation of the acyl, vinyl 75 and alkyl ether methanolysis products by thin-layer chromatography (TLC) prior to GC analysis, 76 or the application of selective chemical reactions. Alkenyl ethers produced fatty aldehydes when 77 exposed to HCl fumes [13, 14, 15], aqueous HCl [16, 17] or 90% acetic acid [18], which were 78 analyzed directly or converted to alcohol and acetate derivatives. Alkenyl ethers were also 79 converted to hydrazones by reaction with 2,4-dinitrophenylhydrazine in an acidic environment 80 and analyzed by HPLC [19]. Alkyl ethers were reduced with LiAlH4 to their alkyl glycerol ethers 81 and analyzed as their isopropylidene derivatives [14, 17]. Added to this difficulty of separating 82 these complex methanolyzed samples was the report that DMAs decompose to alk-1-enyl methyl 83 ethers (AME) when exposed to 200-300°C, as in the GC injection port [20, 21, 22]. In addition, 84 some authors have speculated that DMAs may be decomposed to fatty aldehydes [20], which 85 was later questioned by other researchers [22]. 86 There is a need to accurately characterize the reaction products formed during acid-catalyzed 87 methanolysis of products containing plasmalogenic lipids. Ignoring the plasmalogenic content of 88 animal tissues is not an option, since they can contribute up to 15% of the total lipids in some 89 tissues [6]. In this study, we investigated the chemical compounds originating from the acidic 90 methanolysis of animal tissue lipids using GC-FID and our recently developed two-dimensional 91 gas chromatography with online reduction (GC-OR×GC). The chemical hydrogenation applied 92 between 1D and 2D of the GC-OR×GC permitted the separation in the two dimensional space of 93 complex samples as FAMEs prepared from marine oils [23], DHA biohydrogenation products 94 [24], and seed oils [25]. GC/quadrupole time-of-flight (GC/Q-TOF) mass spectrometry and 95 nuclear magnetic resonance (NMR) spectroscopy were additionally used to confirm which 96 compounds were produced by the acidic methanolysis, and the reactions taking place in the GC 97 injection port. we proposed a simple procedure to isolate DMAs from FAMEs prior to GC 98 analysis to simplify their routine quantification and investigate the formation of fatty aldehydes 99 during sample preparation. 100 101 3. Materials and Methods 102 103 All chemicals and solvents were of analytical or ACS grade. Anhydrous methanol was 104 prepared by adding Molecular Sieve 5A (Sigma Aldrich St. Louis, MO, USA) to methanol, and 105 let the molecular sieve to settle overnight. Sulfuric acid (98%) and octadecane were purchased 106 from Sigma Aldrich. Pure FAMEs and GLC 463 reference mixture were purchased from Nu 107 Chek Prep Inc. (Elysian, MN). Cis and trans mono-unsaturated fatty acids were synthesized in a 108 previous study along with other reference solutions [26]. Fatty aldehydes 16:0 and 18:0 were 109 purchased from TCI America (Boston, MA). Beef heart samples were purchased in retail stores 110 in College Park (MD, USA) and horse meat samples were collected during study conducted in 111 Spain [27]. 112 113 3.1 Preparation of lipid extracts. Samples of fresh meat (5 g) were cut into narrow slices to 114 which 60 mL of chloroform/methanol (1:1) was added, and then homogenized (IKA, T25 digital 115 ULTRA TURRAX, Atkinson, NH, USA) for one min at 1500 rpm. Ten milligrams of 116 octadecane and glycerol tritridecanoate (1 mL of 250 mg each in 25 mL tert-butyl methyl ether) 117 were added as internal standards, followed by 27 mL of aqueous KCl (0.88% by weight) and 4 118 drops of 6N HCl to achieve the final solvent ratio of chloroform:methanol:water of 1:1:0.9 [28]. 119 The mixture was shaken and separated into two layers by centrifugation at 1000 rpm for 5 min. 120 The bottom layer was collected and the extraction was repeated by addition of another 30 mL of 121 chloroform [6, 29]. The combined chloroform extracts were passed through 1 g of anhydrous 122 Na2SO4 in an SPE tube, and the solvent was removed under a mild stream of argon at the bottom 123 of a pre-weighed 20 mL screw cap glass container. The neat lipids were weighed and stored at -124 20°C diluted in 15 mL chloroform. 125 3.2 Acid-catalyzed methanolysis of animal lipids. Approximately 40 mg of animal lipids 126 were dried under a stream of argon at the bottom of a 15 mL screw-capped test tube. Lipids were 127 reconstituted in 1 mL of toluene, followed by 2 mL of anhydrous methanol containing 2% H2SO4 128 (v/v) and purged with argon. The tube was mixed by vortex and heated at 80°C for 1 hour with 129 occasional mixing. The tube was chilled to room temperature, and 2 mL of hexane were added 130 followed by hand mixing. The H2SO4 was neutralized by filling the test tube (~10 mL) with 131 aqueous buffer solution (150 g/L sodium hydrogen citrate and 100 g/L NaCl) and mixed 132 carefully. The two layers were separated by centrifugation, and the upper organic phase was 133 transferred into a second 15 mL screw-caped test tube after passing the content through 1 g of 134 anhydrous Na2SO4. The hexane extraction was repeated, and the extracts combined. Half of the 135 methanolysis products were placed into a 2 mL auto-sampler vial and analyzed by GC. 136 3.3 Isolation of DMAs from FAMEs. The second part of the methanolysis products still in 137 the 15 mL screw-capped test tube were taken to dryness, and 2.85 mL of ethanol were added 138 followed by 150 µL of a 15% (w/w) aqueous KOH solution. The sample was purged with 139 nitrogen, mixed and kept 48 hours in the dark at room temperature. Hydrolysis was terminated 140 by addition of 2 mL of hexane and 10 mL of water. The tube was mixed by vortex and 141 centrifuged at 1500 rpm for 5 min to achieve two discrete layers. The upper organic layer 142 (containing DMAs) was moved to a separate test tube, and the lower layer was extracted again 143 with 2 mL of hexane. The combined extracts were washed with 10 mL deionized water and a 144 few drops of the 15% KOH solution, and passed through 1 g of anhydrous Na2SO4 prior to GC 145 analysis. 146 147 3.4 Preparation of aldehydes from the DMAs. After GC analysis, the isolated DMAs 148 were taken to dryness under a gentle stream of nitrogen at the bottom of the GC vial. Toluene 149 (500 μL) and water (100 μL) were added in sequence, followed by mixing and addition of 40 μL 150 of 37% HCl. The vial was then heated for 2 h at 100ºC. The reaction mixture was extracted with 151 1 mL of pentane. Aldehydes were dried at the bottom of a second vial under a stream of argon 152 and reconstituted in 1 mL of isooctane for GC analysis. 153 154 3.5 GC analysis of FAMEs and DMAs. Analyses were made with an Agilent 6890N gas 155 chromatograph (Wilmington, DE, USA) equipped with a FID and a Supelco SP-2560 capillary 156 column (100 m × 0.25 mm i.d. × 0.20 μm film thickness; Bellefonte, PA). Two different elution 157 temperature conditions were used: 1)180°C isothermal; 2) a temperature program optimized for 158 dairy fats (45°C for 4 min, ramped at 13°C/min to 175°C, maintained at 27 min, ramped 4°C/min 159 to 215°C, and maintained 45 min) [30, 31]. The injector and detector temperatures were set at 160 250°C. Hydrogen was used as carrier gas at a flow rate of 1 mL/min, and 1 μL of sample was 161 injected with a split ratio of 100:1. 162 163 3.6 GC-OR × GC. Two-dimensional separations were carried out as previously described 164 [23] with modifications. An Agilent 7890A GC with a FID and a split/splitless injection port was 165 equipped with a ZX2 dual stage thermal modulator (Zoex, Houston, TX). The column set was 166 sequentially composed of a SP-2560 capillary column (100 m × 0.25 mm i.d. × 0.25 μm film 167 thickness), a capillary tube coated with palladium (0.20 m × 0.18 mm i.d.), a deactivated 168 uncoated capillary tube (2 m × 0.10 mm, modulator loop), and a SLB-IL111 capillary column 169 (2.5 m × 0.10 mm i.d.). The modulation spots were set at 0.05 m after the beginning of the 170 modulation loop and 0.05 m from the beginning of 2D. The modulation time was set to 3 s. The 171 oven was maintained at the constant temperature of 180°C, and the injector port at 300°C. 172 Hydrogen was used as carrier gas at a constant pressure of 55 psi, and the injection volume was 1 173 μL. The acquisition rate was set to 200 Hz, and data were processed with the GC Image GC × 174 GC software (Version 2.1, GC Image, LLC, Lincoln, NE). All capillary connections were made 175 with Micro Unions (SGE Analytical Science, Australia). 176 177 3.7 GC/MS separations with soft positive chemical ionization (CI). An Agilent 7200A 178 GC/Q-TOF was equipped with a SP-2560 capillary column (100 m × 0.25 mm, 0.20 μm film 179 thickness) followed by a 2 m × 0.10 mm deactivated retention gap. The injection volume was 1 180 μL, the split ratio was set to 100:1, and He was used as carrier gas. The oven temperature was 181 maintained at 180°C, the injection port and transfer line at 250°C, the ion source at 300°C. The 182 mass spectrometer was operated in CI+ mode with isobutane as chemical ionization reagent with 183 the filament current set to 100 μA. 184 185 3.8 NMR analyses. NMR spectra were collected with a 600 MHz Bruker Avance III 186 spectrometer with a standard 5mm BBO probe. Samples were prepared in deuterated chloroform 187 (“100%” CDCl3, Cambridge Isotope Laboratories, Andover, MA). Standard sequences were used 188 to obtain 1H, 13C, COSY, HSQC and HMBC spectra. All spectra were referenced to the 189 chloroform signals: 7.26 ppm for 1H and 77.23 for 13C. Spectral integration and other data 190 processing were performed with the MestReNova 10.0 software. 191 192 4. Results 193 194 4.1 Analysis of animal tissue lipids by GC-FID. 195 In this study animal tissue lipids were extracted using the method of Bligh and Dyer [28], 196 and successively methanolized with 2% H2SO4 in anhydrous methanol. Beef heart lipids were 197 selected as reference because of their high content in plasmalogens. Methanolyzed beef heart 198 lipids were separated by GC-FID applying the temperature program optimized for dairy lipids 199 (Fig. 1A). While DMAs were eluted on the GC capillary column as AMEs, we have chosen to 200 label them as DMAs in Figure 1 and elsewhere in the manuscript, since they were introduced in 201 this form into the GC injection port. Two internal standards were added prior to lipid extraction: 202 glycerol tritridecanoate for the comprehensive direct analysis of the methanolysis products, and 203 octadecane to quantitate only the DMAs after their isolation. Half of the methanolysis product 204 was used for direct GC-FID analysis (Fig. 1A), the remaining portion was taken to dryness and 205 saponified with 95:5 (v/v) ethanol/aqueous KOH. After hydrolysis, the neutral components 206 including DMAs were extracted with hexane, while the K+ salts of fatty acids (FAs) repartitioned 207 in the aqueous phase. The isolated DMAs were analyzed by GC (Fig. 1C) and the comparison 208 with the original methanolyzed sample (Fig. 1A) showed that FAMEs were quantitatively 209 removed. 210 211 The reference solution of fatty aldehydes was prepared by reacting the isolated DMAs with 212 water in presence of concentrated HCl, and was analyzed applying the same elution conditions 213 (Fig. 1D). FAMEs from beef heart lipids with no DMAs were prepared by alkaline trans-214 esterification with 0.5M NaOH in methanol, and also analyzed applying the same elution 215 conditions (Fig. 1B). The components of the acid methanolyzed bovine heart lipids (Fig. 1A) 216 were identified by comparison of the solutions containing only FAMEs (Fig. 1B), only DMAs 217 (Fig. 1C), and only fatty aldehydes (Fig. 1D). FAMEs were identified using available standards 218 and previously synthetized reference solutions [26]. Since individual DMA reference materials 219 were not available, identification of geometric/positional isomers of unsaturated DMAs was 220 made by comparison with the elution profile of FAMEs. In addition, acidic methanolysis of 221 bovine heart lipids was repeated by spiking the methanolysis reagent (2% H2SO4 in anhydrous 222 methanol) with 100 L of water (Fig. 1E). The comparison between the acidic methylation 223 products prepared with and without spiked water (Fig. 1E, 1A) showed that the addition of water 224 caused the formation of 16:0 and 18:0 fatty aldehydes, identified by comparison with the fatty 225 aldehydes reference solution (Fig. 1D). Identification of 16:0 and 18:0 fatty aldehydes was also 226 confirmed by using reference standards. The addition of water also resulted in a higher content of 227 c/t-18:2 FAMEs evidenced by comparing Figure 1E with 1A and 1B. 228 229 Separations stacked in Figure 1 show that most FAMEs, DMAs and fatty aldehydes 230 occurring in acid methanolyzed beef heart lipids could be resolved utilizing a long highly polar 231 capillary column (100 m x 0.25 mm, SP2560) and a temperature program optimized for the 232 short-medium chain FAMEs of methanolyzed dairy fats [30]. The DMAs (as AMEs) showed 233 retention times close to their equivalent FAME one methylene group shorter. Saturated DMAs 234 eluted slightly after the FAME with one less methylene, and were all baseline resolved. 235 However, the mono-unsaturated 18:1 DMAs were eluted with the 17:1 FAMEs, and the 18:2n-6 236 DMA was eluted among the 18:1 FAMEs. Fatty aldehydes eluted slightly after their equivalent 237 FAME. The 18:1 fatty aldehydes showed slightly higher retention time compared to 18:1 238 FAMEs, but if present in the same sample they would not be resolved from them. Saturated fatty 239 aldehydes provided similar retention of DMAs with one more methylene group, for example 240 15:0 fatty aldehyde provided same retention time of 16:0 DMA (Fig 1C, 1D). 241 242 4.2 Analysis by GC-OR × GC. 243 GC-OR × GC separations were achieved utilizing a previously described instrumental 244 configuration consisting of a 100% bis(cyanopropyl siloxane) capillary column for 1D and a 245 short SLB-IL111 for 2D, both maintained at 180°C. The capillary tube coated with Pd (reducer) 246 was put in front of the cryogenic modulator [24]. The separation of beef heart lipids is presented 247 divided in four 1D retention time intervals: from 12:0 to 16:0 (Fig. 2); from 16:0 to 18:0 (Fig. 3); 248 from 18:0 to 18:3n-3 (Fig. 4); from 18:3n-3 to 22:6n-3 (Fig. 5). Methanolized animal lipids were 249 also analyzed by GC-FID with the same 1D column maintained at 180°C (Fig. 3-5, lower panel 250 in each figure). As previously reported [23, 24] the FAMEs eluted on straight lines parallel to the 251 1D time axis, each defined by the 2D retention time of a fully saturated FAME. Saturated DMAs 252 eluted on their own straight line bisecting the plane, characterized by a lower angular coefficient 253 with the x-axis compared to the one of saturated FAMEs. Similarly to FAMEs, DMAs differing 254 for the number/position of double bonds were eluted on the same straight line parallel to the 1D 255 time axis, defined by the 2D retention time of the saturated form. Saturated DMAs eluted at 256 significantly lower 2D retention time relative to the saturated FAME diagonal line at their 1D 257 retention time, indicating that the lower 2D retention was caused by chemical reduction of the 258 analyte functional group prior to 2D. FAMEs and DMAs eluted as two independent sets of 259 analytes with no co-elutions, and DMAs eluted below the separation space characteristic of 260 FAMEs. 261 262 Fatty aldehydes eluted in the separation space below DMAs, and slightly above the line 263 defining the 2D dead volume. The 18:0 fatty aldehyde eluted at the same 2D retention time as the 264 internal standard octadecane. In some separations obtained with a reducer that was aged, but still 265 providing full reduction of FAMEs and DMAs, both reduced and unreduced 16:0 and 18:0 fatty 266 aldehydes were detected (Fig. 3, Fig. 4). The 16:0 and 18:0, in trace amounts, were the only fatty 267 aldehydes detected in the samples methanolyzed with H2SO4 in anhydrous methanol. Applying 268 the 180°C elution temperature, unreduced fatty aldehydes eluted slightly after their equivalent 269 FAME in 1D and slightly before of it in 2D, indicating different interactions with the two liquid 270 phases. For each alkyl structure, 2D eluted in sequence: the reduced fatty aldehyde (an alkane), 271 the DMA (an alkyl ether), the unreduced fatty aldehyde, and finally the FAME. 272 273 lipids are saponified or trans-esterified with alkali, then reacted (i.e. esterified) with a Brønsted-460 Lowry acid in methanol without a lipid extraction and dehydration step in between. The use of a 461 Lewis acid such as BF3 instead of a mineral acid to methylate the saponified fatty acids may 462 result in the formation of additional side products [4]. In this study, the addition of water to the 463 acid methanolysis reagent also resulted in the formation of c/t-18:2 trans fatty acids (Fig. 1E), 464 which were present only in trace amounts in the same sample methanolyzed with the anhydrous 465 reagent (Fig. 1A), and absent when methylation was carried out by alkaline trans-esterification 466 (Fig. 1B). 467 468 Regardless of which reagent is used for the acidic methanolysis, either HCl or H2SO4 in 469 methanol, it is strictly necessary to analyze the acid methanolysis products (or isolated DMAs) in 470 the shortest possible time. We observed that the neat DMAs stored in the freezer while waiting 471 for the NMR analysis partly converted to fatty aldehydes, which was confirmed by re-analyzing 472 the isolated DMAs by GC after the NMR spectrum acquisition. Also, a large portion of DMAs 473 had converted to fatty aldehydes in the methanolyzed horse meat samples transported from 474 Spain. The absorption of small amounts of atmospheric moisture is hard to avoid, and can 475 provide the formation of fatty aldehydes if residual traces of acid are present and methanol is 476 absent. 477 478 5.3 GC-OR × GC separation of methanolyzed animal lipids. 479 The GC-OR × GC methodology we developed for the analysis of FAMEs prepared from 480 complex samples such as fish oils and vegetable oils provided the separation of all components 481 contained in the acid methanolyzed bovine heart lipids, and critical information for their 482 identification. The reducer placed in front of the GC × GC modulator played a central role for 483 achieving these separations. The resolution of FAMEs, DMAs (as AMEs) and fatty aldehydes in 484 the 2D plane was achieved by taking advantage of the different reactivity of these compounds 485 toward catalytic hydrogenation: AMEs (R-HC=CH-O-R’) were reduced to their alkyl ether (R-486 CH2-CH2-O-R’) homologs, fatty aldehydes to their alkane homologs, while FAMEs were left 487 unaltered. The compounds originally introduced in the GC injection port as DMAs were eluted 488 by 1D as AMEs, and by 2D as alkyl methyl ethers. The hydrogenation of the AME vinyl ether 489 double bond reduced the dipole-induced dipole interactions with the 2D phase, providing the 490 drop in 2D retention time responsible for the resolution of DMAs from FAMEs. Similarly, the 491 reduction of the aldehyde functional group to alkanes eliminated all the fatty aldehydes dipole-492 induced dipole interactions with the 2D phase, resulting in their elution slightly above the 2D 493 dead volume. These chemical transformations resulted in the elution of FAMEs, DMAs and fatty 494 aldehydes in 3 different distinct areas of the separation space with no overlaps. DMAs (as 495 AMEs) showed the same elution order as FAMEs using the SP2560 capillary column, but 496 occurred in different relative amounts. Substitution of the methyl ester group of FAMEs with the 497 methyl vinyl ether of DMAs did not alter the interactions of the analyte carbon chain with the 498 bis(cyanopropyl)siloxane phase, providing the same elution pattern for the two groups of 499 derivatives. 500 501 The extension of GC-OR × GC (originally developed for FAMEs) to the analysis of DMAs 502 and fatty aldehydes required studying the hydrogenation efficiency of the capillary reducer for 503 the new analytes. In the case of samples consisting only of FAMEs, the wide difference in Pd 504 reactivity for the hydrogenation of olefins and ester groups allowed to use reducers of various 505 efficiencies, and for prolonged time [23]. Enol ethers require more energy than olefins for their 506 complete hydrogenation, and aldehydes are only slightly more reactive than esters. To study the 507 effect of temperature on the reduction efficiency, we placed the capillary reducer in an auxiliary 508 oven and tested hydrogenation temperatures from 140 to 230°C (Figure 6). Isolated double 509 bonds and the double bond of vinyl ethers were already quantitatively reduced at 140°C, while 510 the complete reduction of aldehydes (except when present in trace amounts) required at least 511 210°C. Maintaining the reducer at 210°C also resulted in a minor reduction of esters (FAME) to 512 alkanes. We preferred to trade the full reduction of fatty aldehydes with the simplification of the 513 system by maintaining the entire column set (including the reducer) in the main GC oven at 514 180ºC. Each capillary reducer provided the desired reduction efficiency for only 5-10 days prior 515 to showing incomplete reduction of DMAs. 516 517 5.4 GC-FID analysis of methanolyzed animal lipids. 518 In this study, GC-FID analyses were carried out applying two different elution conditions 519 widely applied in these types of research studies and routine analyses [30, 31, 45, 46]. The first 520 set of elution conditions, based on the 180°C isothermal elution (Fig. 2-5, lower panel) was 521 developed to determine the fatty acid composition of vegetable oils and was optimized for the 522 separation of unsaturated C18 FAME geometric isomers [45, 46]. This method was later 523 extended to the analysis of generic food lipid extracts by increasing the elution temperature after 524 the elution of 18:3n-3 [47]. The second method was optimized for the analysis of FAMEs 525 prepared from dairy lipids, and is based on a temperature program (Fig. 1) that focused on the 526 separation of short-chain and mid-chain FAMEs, while still providing the separation of most C18 527 FAME geometric isomers [26, 30, 31, 38]. 528 529 The application of the isothermal 180°C method (Fig. 2-5, lower panel) to the analysis of 530 acid methanolyzed beef heart lipids provided the expected separation of C18 FAME geometric 531 isomers as reported in literature [26, 46], but unfavorable resolution of DMAs from mid chain 532 FAMEs (Fig. 2-5). All the linear and the isomid chain saturated DMAs were eluted with the 533 FAMEs with one less carbon (i.e., 16:0 DMA with 15:0 and c9-14:1 FAMEs), while the isoand 534 linear 18:0 DMA were eluted alone. Regarding unsaturated DMAs, c9-16:1 DMA was resolved 535 from FAMEs and c11-18:1 DMA was eluted with c9-17:1 FAME. The lack of separation 536 between DMAs and FAMEs, in particular the saturated ones, also affects the accurate 537 quantification of fatty acids. The exclusion of the FAME-DMA mixed peaks from the fatty acid 538 quantification results in an under-estimation of the content of total fat as well as total unsaturated 539 fatty acids, and their inclusion provides the opposite result (over-estimation). While fatty 540 aldehydes were eluted after their homolog FAMEs and DMAs (i.e., 16:0 fatty aldehyde in Fig. 3, 541 and 18:0 fatty aldehydes in Fig. 4) and resolved from them, they may be mis-identified as ethyl 542 esters if identifications are based uniquely on retention time comparisons. Based on these results, 543 GC-FID method that relies on an isothermal 180°C elution and a 100% bis(cyanopropyl)siloxane 544 capillary column [45, 47] should not be used for the analysis of samples containing acid 545 methanolyzed animal lipids, unless DMAs are isolated from FAMEs prior to GC analysis. If only 546 the quantification of acyl lipids is desired, samples may be prepared for GC-FID analysis by 547 applying alkaline trans-esterification with no prior acid digestion. 548 549 The separations of acid methanolyzed beef heart lipids, the isolated DMAs, and FAMEs from 550 alkaline trans-esterification applying the dairy temperature program [30, 31], are shown in Figure 551 1A, 1C and 1B, respectively. The separation of fatty aldehydes prepared from beef heart DMAs 552 (Fig. 1D) was included to allow for the identification of these side products when plasmalogens 553 are improperly methanolyzed. The magnitude of the fatty aldehydes formation instead of DMAs 554 may be monitored by quantifying the 18:0 fatty aldehydes, since the co-eluting 19:0 DMA was 555 not detected in any sample. While DMAs are characterized by very close retention to FAMEs 556 with one less carbon, all the saturated DMAs were baseline resolved from FAMEs using this 557 temperature program. However, the 18:1 DMAs co-eluted with the c9-17:1 FAME. 558 Unfortunately, fatty aldehydes were eluted with saturated DMAs with more carbon, an 559 inconvenience may lead to incorrect DMA quantification if samples are improperly 560 methanolyzed or are not timely analyzed. 561 562 5.5 Development of a simple procedure to isolate DMAs. 563 We developed a simple procedure to isolate DMAs from FAMEs using well known reactions 564 and partitioning principles. The DMAs were prepared along with FAMEs by acid catalyzed 565 methanolysis. FAMEs were subsequently hydrolyzed to free fatty acids under alkaline conditions 566 and repartitioned as K+ salts in a water layer, while DMAs remained in the organic phase. 567 FAMEs may be quantified without DMAs following two simple approaches: 1) neutralize the 568 aqueous solution containing the K+ salts of the fatty acids, recover the free FAs with organic 569 solvent (i.e. 50:50 diethyl ether/petroleum ether) and re-prepare FAMEs with using 2% H2SO4 or 570 BF3 in anhydrous methanol; or 2) trans-esterify another portion of animal lipids by alkaline 571 methanolysis, which converts acyl lipids to FAMEs but does not affect alkenyl ethers [6, 7]. 572 Another benefit of the milder alkaline process is that it does not alter the content of conjugated 573 FAs such as conjugated linoleic acid [38], but it does not methanolyze N-acyl lipids such as 574 sphingomyelin [6, 38, 39]. The isolation of DMAs by selective removal of FAMEs is less time 575 consuming than separating FAMEs and DMAs by thin-layer chromatography [6, 7, 33, 39, 48, 576 49], or conducting separate analyses for FAMEs and DMAs [14, 15, 22, 19]. In this study, the 577 isolated DMAs were quantified using the octadecane internal standard. A practical approach is to 578 analyze the acid methanolyzed sample before and after the DMA isolation, quantify all DMAs in 579 the isolated DMA solution relative to 16:0 DMA, and use the 16:0 DMA concentration in the 580 original sample to calculate the content in the other DMAs. 581 582 6. Conclusions 583 584 The GC-OR × GC method provided the resolution of all components contained in the acid 585 methanolyzed animal lipids. Based on these results, and other evidences collected by GC-QTOF-586 MS and NMR, we concluded that acidic methanolysis of the plasmalogens contained in animal 587 lipids yields DMAs if no water is present in the reaction solution. The presence of water causes 588 the formation of fatty aldehydes. Our results also support that the DMAs are quantitatively 589 pyrolyzed to AMEs in the GC injection port at 250°C, and are eluted in this form. The GC-FID 590 separation using bis(cyanopropyl)siloxane columns and 180°C elution temperature provided 591 highly unfavorable separation of DMAs from FAMEs. The temperature program optimized for 592 dairy fat analysis provided the separation of almost all DMAs with only few minor coelutions of 593 DMAs and FAMEs. Pure DMAs can be prepared by saponification of the acid methanolyzed 594 extract followed by removal of hydrolyzed fatty acids. FAMEs (from only acyl lipids) with no 595 DMAs may be prepared by base-catalyzed trans-esterification of the original lipid extract. 596 Hydrochloric acid in methanol should not be used for acidic methanolysis of plasmalogens 597 unless it is prepared onsite shortly prior to use, and in this study, we replaced it with 2% H2SO4 598 in anhydrous methanol. Sequential acidic digestion, saponification and acid-catalyzed 599 esterification of samples containing animal lipids should be avoided because the acid-base 600 neutralization reactions produce water. Regardless to what reagent is used for acid methanolysis, 601 samples must be analyzed quickly to avoid degradation of DMAs to fatty aldehydes during 602 storage. 603 604 7. Acknowledgements 605 606 Dr. X. Belaunzaran thanks the Department of Economic Development & Competitiveness of the 607 Basque Government (Spain) for financing his research, and the US Food and Drug 608 Administration for his internship at the Center for Food Safety and Applied Nutrition. 609 610 8. References 611 612 1) L.A. Horrocks, Content, composition, and metabolism of mammalian and avian lipids that 613 contain ether groups. In: F. Snyder (Ed), Ether lipids. Academic, New York, 1972, 177-272. 614 615 2) D.A. White, The phospholipid composition of mammalian tissue. In: G.B. Ansell, J.N. 616 Hawthorne, R.M.C. Dawson (Eds), Form and function of phospholipids, Elsevier, Amsterdam, 617 1973, 441–482. 618 619 3) T. Sugiura, T. Fukuda, T. Miyarnoto, K. Waku, Distribution of alkyl and alkenyl ether-linked 620 phospholipids and platelet-activating factor-like lipid in various species of invertebrates, 621 Biochim. Biophys. Acta 1126 (1992) 298-308. 622 623 4) W.W. Christie, Lipid Analysis: Isolation, Separation, Identification and Structural Analysis of 624 Lipids, 3rd Edn, The Oily Press, Barnes & Associates, Bridgewater, UK, 2003. 625 626 5) E. Klenk, Eine Methode zur Herstellung der Acetalphosphatide des Gerhirns und ihrer 627 Aldehydkomponenten in Form der Dimethylacetale, Hoppe-Seyler Z. physiol. Chem. 281 (1944) 628 25-28. 629 630 6) C. Cruz-Hernandez, J.K.G. Kramer, J. Kraft, V. Santercole, M. Or-Rashid, Z. Deng, M.E.R. 631 Dugan, P. Delmonte, M.P. Yurawecz, Systematic analysis of trans and conjugated linoleic acids 632 in the milk and meat of ruminants. In: M.P. Yurawecz, J.K.G. Kramer, O. Gudmundsen, M.W. 633 Pariza, S. Banni (Eds), Advances in Conjugated Linoleic Acid Research, 3rd ed, AOCS Press, 634 Urbana, IL, 2006, 45-93. 635 636 7) V. Santercole, R. Mazzette, E.P. De Santis, S. Banni, L. Goonewardene, J.K.G. Kramer, Total 637 lipids of Sarda sheep meat that include the fatty acid and alkenyl composition and the CLA and 638 trans-18:1 isomers, Lipids 42 (2007) 361-382. 639 640 8) V. Santercole, P. Delmonte, J.K.G. Kramer, Comparison of separations of fatty acids from 641 fish products using a 30-m Supelcowax-10 and a 100-m SP-2560 column, Lipids 47 (2012) 329–642 344. 643 644 9) P. Delmonte, A.R. Fardin-Kia, N. Aldai, M.M. Mossoba, J.K.G. Kramer, Analysis of 645 Conjugated and Other Fatty Acids in: Sels B, Philippaerts A (eds), Conjugated Linoleic Acids 646 and Conjugated Vegetable Oils, The Royal Society of Chemistry, 2014, 172-231. 647 648 10) I. Björkhem, L. Sisfontes, B. Bostrom, B.F. Kase, R. Blomstrand, Simple diagnosis of the 649 Zellweger syndrome by gas-liquid chromatography of dimethylacetals, J. Lipid Res. 27 (1986) 650 786-791. 651 652 11) G. Dacremont, G. Vincent, Assay of plasmalogens and polyunsaturated fatty acids (PUFA) 653 in erythrocytes and fibroblasts, J. Inher. Metab. Dis. 18 Suppl. 1 (1995) 84-89. 654 655 12) D.B. Goodenowe, L.L. Cook, J. Liu, Y. Lu, D.A. Jayasinghe, P.W. Ahiahonu, D. Heath, Y. 656 Yamazaki, J. Flax, K.F. Krenitsky, D.L. Sparks, A. Lerner, R.P. Friedland, T. Kudo, K. Kamino, 657 T. Morihara, M. Takeda, P.L. Wood, Peripheral ethanolamine plasmalogen deficiency: a logical 658 causative factor in Alzheimer's disease and dementia, J. Lipid Res. 48 (2007) 2485-2498. 659 660 13) H.H.O. Schmid, H.K. Mangold, Alkoxylipids. II. "Neutral plasmalogens" in the liver oil of 661 the ratfish (Hydrolagus colliei). Bichim. Biophys. Acta 125 (1966) 182-184. 662 663 14) H.H.O. Schmid, T. Takahashi, The alk-i-enyl ether and alkyl ether lipids of bovine heart 664 muscle, Biochim. Biophys. Acta 164 (1968) 141-147. 665 666 15) R.L. Wolff, Characterization of trans-monounsaturated alkenyl chains in total plasmalogens 667 (1-O-alk-1′-enyl-2-acyl glycerophospholipids) from sheep heart, Lipids 37 (2002) 811-816. 668 669 16) R.E. Anderson, R.D. Garrett, M.L. Blank, F. Snyder, The quantitative production of 670 aldehydes from O-alk-1-enyl glycerols, Lipids 4 (1969) 327-330. 671 672 17) R. Wood, R.D. Harlow, Gas-liquid chromatographic analysis of long-chain aldehydes, J. 673 Lipid Res. 10 (1969) 463-465. 674 675 18) G.M. Gray, The separation of the long chain fatty aldehydes by gas-liquid chromatography, 676 J. Chromatogr. 4 (1960) 52-59. 677 678 19) D. Dannenberger, S. Lorenz, G. Nuernberg, N. Scollan, K. Enger, K. Nuernberg, Analysis of 679 fatty aldehyde composition, including 12-methyltridecanal, in plasmalogens from Longissimus 680 muscle of concentrateand pasture-fed bulls, J. Agric. Food Chem. 54 (2006) 182-188. 681 682 20) R.A. Stein, V. Slawson, A model for fatty aldehyde dimethyl acetal gas-liquid 683 chromatography. The conversion of octadecanal dimethyl acetal to methyl 1-octadecenyl ether. J. 684 Chromatogr. 25 (1966) 204-212. 685 686 21) V. Mahadevan, C.V. Viswanathan, F. Phillips, Conversion of fatty aldehyde dimethyl acetals 687 to the corresponding alk-1-enyl methyl ethers (substituted vinyl ethers) during gas-liquid 688 chromatography, J. Lipid Res. 8 (1967) 2-6. 689 690 22) A. Hara, T. Taketomi, Chemical study of the mechanism for conversion of dimethylacetal 691 obtained by methanolysis of plasmalogen to alkenylmethylether, J. Biochem. 104 (1988) 1011-692 1015. 693 694 23) P. Delmonte, A.R. Fardin-Kia, J.I. Rader, Separation of Fatty Acid Methyl Esters by GC-695 Online Hydrogenation × GC, Anal. Chem. 85 (2013) 1517–1524. 696 697 24) N. Aldai, P. Delmonte, S.P. Alves, R.J.B. Bessa, J.K.G. Kramer, Evidence for the Initial 698 Steps of DHA Biohydrogenation by Mixed Ruminal Microorganisms from Sheep Involves 699 Formation of Conjugated Fatty Acids, J. Agric. Food Chem. 66 (2018) 842-855. 700 701 25) P. Delmonte, Evaluation of poly(90% biscyanopropyl/10% cyanopropylphenylsiloxane) 702 capillary columns for the gas chromatographic quantification of trans fatty acids in non-703 hydrogenated vegetable oils, J. Chromatogr. A 1460 (2016) 160-172. 704 705 26) P. Delmonte, A.R. Fardin-Kia, Q. Hu, J.I. Rader, Review of methods for preparation and gas 706 chromatographic separation of trans and cis reference fatty acids,. J AOAC Int. 92 (2009) 1310–707 1326. 708 709 27) X. Belaunzaran, P. Lavín, L.J.R. Barron, A.R. Mantecón, J.K.G. Kramer, N. Aldai, An 710 assessment of the fatty acid composition of horse-meat available at the retail level in northern 711 Spain, Meat Sci. 124 (2017) 39-47. 712 713 28) E.G. Bligh, W.J. Dyer, A rapid method of total lipid extraction and purification, Can. J. 714 Biochem. Physiol. 37 (1959) 911–917. 715 716 29) N. Aldai, J.K.G. Kramer, C. Cruz-Hernandez, V. Santercole, P. Delmonte, M.M. Mossoba, 717 M.E.R. Dugan, Appropriate extraction and methylation techniques for lipid analysis, in: G. 718 Cherian, R. Poureslami (eds), Fats and Fatty Acids in Poultry Nutrition and Health, 2012, 249-719 290. 720 721 30) J.K.G. Kramer, C.B. Blackadar, J. Zhou, Evaluation of two GC columns (60-m 722 SUPELCOWAX 10 and 100-m CP Sil 88) for analysis of milkfat with emphasis on CLA, 18:1, 723 18:2 and 18:3 isomers, and shortand long-chain FA, Lipids 37 (2002) 823–835. 724 725 31) J.K.G. Kramer, M. Hernandez, C. Cruz‐Hernandez, J. Kraft, M.E.R. Dugan, Combining 726 results of two GC separations partly achieves determination of all cis and trans 16:1, 18:1, 18:2, 727 18: 3 and CLA isomers of milk fat as demonstrated using Ag‐ion SPE fractionation, Lipids 43 728 (2008) 259-273. 729 730 32) A.R. Fardin‐Kia, P. Delmonte, J.K.G. Kramer, G. Jahreis, K. Kuhnt, V. Santercole, J.I. 731 Rader, Separation of the fatty acids in menhaden oil as methyl esters with a highly polar ionic 732 liquid gas chromatographic column and identification by time of flight mass spectrometry, 733 Lipids, 48 (2013) 1279–1295. 734 735 33) W.R. Morrison, L.M. Smith, Preparation of fatty acid methyl esters and dimethylacetals from 736 lipids with boron fluoride-methanol, J. Lip. Res. 5 (1964) 600–608. 737 738 34) T.H. Chan, Formation and addition reactions of enol ethers, in: B.M. Trost, (Ed.), 739 Comprehensive Organic Synthesis, Pergamon Press: New York, 1991, Vol. 2, 595-628. 740 741 35) D.G.W. Bradley, A. Cullen, A. Fourie, H. Henning, M. Lawton, W. Mommsen, P. Nangu, J. 742 Parker, A. Renison, Mild water-promoted selective deacetalisatison of acyclic acetals, Green 743 Chem., 12 (2010) 1919–1921. 744 745 36) P.W. Atkins, The Elements of Physical Chemistry (3rd ed.). Oxford University Press, 1993. 746 747 37) W. Stoffel, F. Chu, E.H. Ahrens, Analysis of long-chain fatty acids by gas-liquid 748 chromatography. Micromethod for preparation of methyl esters, Anal. Chem. 31 (1959) 307-308. 749 750 38) J.K.G. Kramer, V. Fellner, M.E.R. Dugan, F.D. Sauer, M.M. Mossoba, M.P. Yurawecz, 751 Evaluating acid and base catalysts in the methylation of milk and rumen fatty acids with special 752 emphasis on conjugated dienes and total trans fatty acids, Lipids 32 (1997) 1219–1228. 753 754 39) C . Cruz-Hernandez, Z. Deng, J. Zhou, A.R. Hill, M.P. Yurawecz, P. Delmonte, M.M. 755 Mossoba, M.E.R. Dugan, J.K.G. Kramer, Methods for analysis of conjugated linoleic acids and 756 trans-18:1 isomers in dairy fats by using a combination of gas chromatography, silver-ion thin-757 layer chromatography/gas chromatography, and silver-ion liquid chromatography, J. AOAC 758 Internat. 87 (2004) 545–562. 759 760 40) J. Kraft, J.K.G. Kramer, F. Schoene, J.R. Chambers, G. Jahreis, Extensive analysis of long-761 chain polyunsaturated fatty acids, CLA, trans-18:1 isomers, and plasmalogenic lipids in different 762 retail beef types, J. Agric. Food Chem. 56 (2008) 4775-4782. 763 764 41) Y. Kishimoto, N.S. Radin, A reaction tube for methanolysis; instability of hydrogen chloride 765 in methanol, J. Lipid Res. 6 (1965) 435-436. 766 9 10 11 12 13 14 15 16 Min. 0.00 0.15 0.30 0.45 Sec. 0.60 14:0 15:0 i15:0 ai15:0 i16:0 16:0 13:0 i14:0 14:0 15:0 i15:0 ai15:0 i16:0 16:0 c914:1 17:0 ai17:0 i17:0 12:0 i13:0 i-14:1 16:1 pA 1.5 2.5 3.5 4.5 5.5 Min. 9 10 11 12 13 14 16:0 14:0 15:0 i-15:0 ai15:0 i-16:0 13:0 i-14:0 12:0 i-13:0 c9-14:1 (17:0) ai17:0 i17:0 16:0 (15:0) (i-15:0) ai15:0 (i-16:0) i-14:1 14:0 c916:1 DMAs FAMEs Figure 2 Figure 2 15.6 16.6 17.6 18.6 19.6 20.6 Min. 0.00 0.20 0.40 0.60 Sec. 0.80 1.00 16:0 18:0 17:0 ai17:0 i17:0 i18:0 17:0 18:1 18:0 c9 trans c11 17:1 c9 c11 16:1 c9 c7 c11 trans c12 c13 c14 c15 16:0 ald. 16:0 (17:0) 18:0 17:1 c9 c11 18:1 18:0 c13 c9 trans c11 c13 i18:0 17:0 ai17:0 i17:0 16:1 c9 c7 c11 c12 trans c14 16:0 ald. Min. 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 pA 2 4 6 8 c12 c13 c12 c10 DMAs FAMEs Figure 3 17:1 c9 c11 Figure 3 0.0 0.4 0.8 1.2 1.6 Sec. 20 22 24 26 28 30 32 34 36 Min. 18:0 18:1 18:2 c/t c9,c12 17:1 19:0 19:1 c9 c11 c12 c13 t6-11 t12 c9 c11 20:0 20:1 c9 c11 c7 18:3n-6 18:3n-3 19:2 c9,t11 18:2 c15 c14, t16 c13 c15 18:1 19:0 20:0 18:2 18:0 ald. t13-t14 t13 t14 18:1 18:2 c/t c9,c12 19:0 19:1 c9 c11 c13 t6-11 (c9) c11 20:0 20:1 18:3n-6 18:3n-3 c9,t11 18:2 c11 c13 c,c 18:0 c9 c6-c8 c15 c14, t16 20:0 18:0 ald. t12 c7 Min. 18 20 22 24 26 28 pA 1.5 2.5 3.5 4.5 5.5 18:1 Figure 4 Figure 4 0.00 0.60 1.20 1.80 Sec. 2.40 3.00 35 50 65 80 95 110 min 125 22:1 22:0 24:0 18:3n-3 c9,t11-18:2 23:0 24:1 pA 1.4 1.8 2.2 2.6 3.0 min 30 40 50 60 70 80 90 100 20:5 n-3 20:4 n-6 20:3 n-6 22:5n-3 22:4n-6 22:6n-3 22:5n-6 n-9 n-3 22:5n-3 22:4n-6 23:0 24:0 20:5 n-3 20:4 n-6 20:3 n-6 n-9 n-3 c9,t1118:2 22:6n-3 22:5n-6 (24:1) 22:0 20:2 c1322:1 20:2 Figure 5 Figure 5 Figure 6 40 45 50 55 60 Min. 35 0.00 1.00 2.00 Sec. 1.50 0.50 40 45 50 55 60 Min. 35 0.00 1.00 2.00 Sec. 1.50 0.50 170°C 230°C 16:0 DMA 16:0 FAME t9 c9 16:1 FAME 16:0 DMA 16:0 FAME t9 c9 16:1 FAME 40 45 50 55 60 Min. 35 0.00 1.00 2.00 Sec. 1.50 0.50 140°C 16:0 DMA 16:0 FAME t9 c9 16:1 FAME 40 45 50 55 60 Min. 35 0.00 1.00 2.00 Sec. 1.50 0.50 210°C 16:0 DMA 16:0 FAME t9 c9 16:1 FAME 16:0 Ald. 16:0 Ald. 16:0 Ald. 16:0 Ald. 16:0 Ald. Red. 16:0 Ald. Red. 16:0 Ald. Red. 16:0 Ald. Red. Figure 6 4 x10 0 1 +CI EIC(283.3056) 4 x10 4 +CI EIC(285.3215) 0 4 x10 0 1 +CI EIC(269.2889) 5 x10 0 2 +CI EIC(271.3058) 4 x10 0 1 +CI EIC(299.3390) 3 x10 0 2 +CI EIC(267.2682) 4 x10 0 1 2 +CI BPC(150.0000-450.0000) Counts vs. Acquisition Time (min.) 32 33 34 35 36 37 38 39 40 41 42 43 44 3 x10 0 5 +CI EIC(281.3264) 18:0 16:0 16:0 18:0 i-18:0 17:0 i-17:0 ai-17:0 17:0 18:0 18:1 c9 c11 c12 c13 c15 c14 t11 16:1 c9 c7 c11 c10 c12 c13 17:1 c9 c11 17:1 c9 c11 trans (18:0) i-18:0 17:0 17:0 i-17:0 ai-17:0 c9 (c7) c11 c10 trans 16:1 18:0 18:1 c9 c11 (c15) 17:1 c9 c11 c12 t11 c13 (c13) (c12) Figure 7 Figure 7 Figure 8 Figure 8 ΔT GC injection port phosphatidylethanolamine plasmalogen H+ H2O + H+ CH3OH H+ CH3OH H2O H2O CH3OH CH3OH CH3OH H+ H+ H2O H+ CH3OH H2O H+ H2O Figure 9 Figure 9 FAME & DMAs togeth er mg/100g meat i-14:0 14:0 i-15:0 15:0 i-16:0 16:0 i-17:0 ai-17:0 Dir. Isol. % Diff. Dir. Isol. % Diff. Dir. Isol. % Diff. Dir. Isol. % Diff. Dir. Isol. % Diff. Dir. Isol. % Diff. Dir. Isol. % Diff. Dir. Isol. % Diff. 1 0.61 0.61 0.78 0.38 0.38 -0.66 - 0.39 - 0.64 0.65 -1.0 1.44 1.45 -1.0 33.82 34.62 -2.3 0.42 0.45 -7.0 1.75 1.78 -1.5 2 - 0.25 - 0.40 0.41 -2.4 - 0.00 - 0.40 0.42 -4.6 0.62 0.63 -2.2 19.92 20.45 -2.6 - 0.00 - 0.51 0.53 -3.0 3 - 0.18 - 0.25 0.26 -4.0 - 0.00 - 0.52 0.53 -1.8 1.01 1.05 -3.0 26.44 27.40 -3.6 0.21 0.22 -5.4 1.05 1.10 -4.3 4 - 0.52 - 0.56 0.57 -1.8 - 0.00 - 0.67 0.66 0.95 1.20 1.22 -0.92 35.02 35.71 -1.9 - 0.00 - 1.18 1.28 -8.2 5 - 0.25 - 0.39 0.40 -3.1 - 0.25 - 0.54 0.57 -5.4 0.81 0.83 -2.0 24.69 25.45 -3.1 - 0.00 - 0.66 0.67 -1.6 6 - - - 0.51 0.54 -5.8 - 0.00 - 0.50 0.54 -7.7 0.69 0.69 -0.90 21.74 22.53 -3.5 - 0.00 - 0.78 0.81 -2.6 7 0.20 0.22 -11.4 0.43 0.46 -7.1 - 0.00 - 0.40 0.43 -6.2 0.87 0.89 -3.1 26.31 27.14 -3.1 0.22 0.22 4.1 0.98 1.02 -4.4 8 - - - 0.39 0.41 -3.7 - 0.00 - 0.45 0.46 -2.2 0.98 1.04 -5.6 24.85 25.70 -3.4 0.20 0.21 -4.1 1.03 1.08 -5.4 9 - - - 0.32 0.34 -4.4 - 0.00 - 0.33 0.35 -3.7 0.41 0.42 -3.3 17.10 17.53 -2.5 0.16 0.15 4.8 0.68 0.70 -2.6 10 - - - 0.51 0.54 -7.0 - 0.00 - 0.48 0.50 -4.5 0.82 0.81 1.5 33.23 34.12 -2.6 0.00 0.00 - 0.75 0.73 3.2 11 - - - 0.57 0.59 -3.3 - 0.00 - 0.42 0.45 -7.0 0.67 0.67 0.12 23.13 24.00 -3.7 0.24 0.24 0.70 0.98 1.01 -3.4 12 - - - 0.49 0.51 -3.2 - 0.15 - 0.38 0.38 0.68 0.62 0.65 -3.4 26.19 27.13 -3.5 0.21 0.23 -7.4 0.16 0.17 -4.2 13 - - - 0.48 0.51 -5.2 - 0.00 - 0.51 0.52 -0.59 0.87 0.91 -4.4 25.33 25.97 -2.5 0.28 0.32 -13 1.17 1.23 -4.7 14 - 0.10 - 0.54 0.55 -1.4 - 0.00 - 0.32 0.35 -8.0 0.75 0.78 -3.9 27.70 28.57 -3.1 0.20 0.20 -0.26 0.89 0.93 -4.6 15 0.14 0.14 -4.7 0.33 0.35 -4.3 - 0.00 - 0.24 0.25 -4.5 0.56 0.60 -7.6 17.30 17.79 -2.8 0.16 0.17 -1.0 0.82 0.85 -3.7 FAME & DMAs togeth er mg/100g meat 17:0 i-18:0 18:0 c9-18:1 c11-18:1 18:2n-6 18:3n-3 Total Dir. Isol. % Diff. Dir. Isol. % Diff. Dir. Isol. % Diff. Dir. Isol. % Diff. Dir. Isol. % Diff. Dir. Isol. % Diff. Dir. Isol. % Diff. Dir. Isol. % Diff. 1 1.49 1.55 -3.6 1.38 0.65 72 11.53 11.93 -3.4 13.25 13.08 1.3 0.99 1.05 -5.9 2.73 1.34 68 - 0.92 - 70.43 70.85 -0.58 2 0.81 0.84 -3.8 0.45 0.23 65 10.75 11.19 -4.0 9.83 9.62 2.2 0.66 0.69 -5.1 1.02 0.91 11 - 0.62 - 45.37 46.79 -3.1 3 1.09 1.15 -4.9 0.73 0.56 26 10.00 10.38 -3.7 6.47 6.26 3.3 0.53 0.56 -5.8 0.79 0.44 57 - 0.68 - 49.09 50.75 -3.3 4 1.60 1.65 -2.8 1.22 - - 16.76 16.98 -1.3 15.57 15.06 3.3 0.85 0.93 -9.1 2.89 1.46 66 - 1.56 - 77.52 77.59 -0.09 5 1.03 1.08 -4.9 0.70 0.31 77 11.63 12.04 -3.4 11.47 11.01 4.1 0.54 0.56 -2.3 1.87 1.11 51 - 1.01 - 54.34 55.53 -2.2 6 0.74 0.79 -6.1 0.74 0.28 92 9.07 9.44 -4.0 5.23 5.08 3.0 0.60 0.62 -1.9 0.80 0.34 81 - 0.60 - 41.41 42.23 -2.0 7 0.79 0.83 -5.1 0.54 0.34 44 12.34 12.77 -3.5 9.31 9.12 2.1 0.81 0.86 -5.7 1.34 0.72 60 - 0.95 - 54.52 55.96 -2.6 8 1.12 1.20 -7.0 0.75 0.46 48 13.29 13.79 -3.7 8.77 8.72 0.6 0.61 0.64 -3.8 1.13 0.89 23 - 1.31 - 53.57 55.91 -4.3 9 0.87 0.68 26 0.41 0.27 42 7.80 8.05 -3.2 5.49 5.16 6.1 0.59 0.62 -5.2 0.78 0.53 38 - 0.69 - 34.95 35.50 -1.6 10 0.98 1.01 -2.9 0.74 0.47 44 13.46 13.84 -2.8 9.94 9.77 1.7 0.95 0.98 -3.1 1.01 0.80 23 - 0.99 - 62.87 64.57 -2.7 11 0.96 0.97 -1.4 0.47 0.37 24 11.09 11.50 -3.6 7.50 7.34 2.1 0.81 0.88 -8.4 0.60 0.53 12 - 0.96 - 47.44 49.52 -4.3 12 0.79 0.80 -1.1 0.45 0.39 14 10.28 10.70 -3.9 9.26 9.04 2.4 0.90 0.92 -2.1 0.68 0.55 21 - 0.82 - 50.42 52.42 -3.9 13 0.92 0.97 -5.1 0.73 0.54 30 12.77 13.27 -3.8 7.55 7.28 3.8 1.01 1.02 -1.4 0.93 0.57 47 - 1.09 - 52.56 54.17 -3.0 14 0.80 0.84 -4.0 0.46 0.46 -0.36 11.05 11.47 -3.7 6.81 6.80 0.20 0.74 0.78 -4.3 0.69 0.58 17 - 1.05 - 50.96 53.45 -4.8 15 0.71 0.75 -5.2 0.43 0.38 14 8.38 8.71 -3.8 6.10 5.83 4.6 0.66 0.68 -3.8 0.45 0.37 21 - 0.56 - 36.29 37.42 -3.1 Table 1 Table 1