Chapter 18
Errors in the Identification by Gas-Liquid Chromatography of Conjugated Linoleic Acids in Seafoods
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Robert G . Ackman Canadian Institute of Fisheries Technology, DalTech, Dalhousie University, P.O. Box 1000, Halifax, Nova Scotia B 3 J 2X4, Canada
Several papers have appeared in which cis-9,trans-11 -octadecadienoic acid (cis-9, trans-11 conjugated linoleic acid, CLA) has been stated to be present in raw seafoods. The trans10-cis-12 isomer was listed as not detected in these reports. The errors lie in the presence of two fatty acids, 18:4n-3 and 18:4n-1, in most seafoods. When the methyl esters of fatty acids are examined on polyglycol- based capillary GLC columns the cis9,trans-11 CLA isomer usually coincides with the natural 18:4n3 and the trans-10,cis-12 CLA isomer with the 18:4n- 1. Thus 18:4n-3 was reported as cis-9,trans-11CLA, but relative to 18:4n-3 the 18:4n-1 is usually very small, and would not be reported as trans-10,cis-12 CLA. More polar liquid phases present a different set of identification challenges. The increasing use of vegetable proteins and oils in feeds for fish farming could create new problems and misunderstandings in respect to CLAin marine-based foods. Analyses of deep-fried fish and seafood dishes with substantial pastry or vegetable components could also be confusing. Fortunately, silver nitrite thin layer chromatography (AgNO -TLC) is a solution to this problem. 3
© 2001 American Chemical Society
In Omega-3 Fatty Acids; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.
235
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236 Conjugated linoleic acids (CLA) are currently a subject of intense interest among nutritionists, biochemists and medical researchers (7). In brief, one of the two cis ethylenic bonds of cis-9,cis-12-linoleic (octadecadienoic) acid migrates to a position adjacent to the other and in the process will be converted about 75 percent of the time to the lower energy trans configuration (2). In one common enzyme system the direction of this reaction is position-specific (3) and for that reason the dominant isomer found in ruminant fats will be the cis-9,trans-l 1-18:2. If chemical isomerization is promoted by alkali catalysis the two most likely CLA isomers (cis-9,trans-11 and trans-10-cis-12) will be found in equal proportions (4). The reaction conditions to prevent an excessive number of minor components were described in a thorough investigation by Mounts et al. in 1970 (5), and basically suggest optimum isomerization conditions of 90 °C or less (6). In the last decade CLA has achieved some notoriety and this has led to the recording of several sets of analytical conditions for the GLC (gas-liquid chromatography) analyses of frying oils (7) or of dairy products (8). The most recent publication, that of Jung, and Ha (9) records the result of non-selective and selective hydrogénation conditions on the formation of CLA isomers. Table I gives the ECL (equivalent chain lengths) on SUPELCOWAX-10 for seven peaks accounting for over a dozen possible isomers. The two 18:2 components commonly expected are identifiable but the unusual feature of Table I, observed under non-selective conditions, was the very large GC peak (No.7) for three trans, trans isomers. This superfluity of isomers is accounted for by the relatively harsh reaction conditions, and the catalyst of hydrogénation. Much gentler conditions are preferred as noted above. Highly polar liquid phases have been used in recent CLA studies and the elution order of CLA isomers on CP-Sil 88 has been established (Table II). An alternative cyanosilicone phase is SP-2560, used by Kramer et al. (10) to supplement the earlier work of Shantha et al. (11) on the loss of CLA during esterifications. Both groups carefully checked the production of artifacts or loss of CLA during various esterification procedures. Acid-catalysed methylation resulted in "the loss of 12% total CLA, 42% recovery of A-9c, 11-18:2, and a fourfold increase in Δ-9*,11Μ8:2", as well as the introduction of methoxy artifacts. However these reports do not deal with the possible coincidence of CLA with 18:4n-3 or other fatty acids as discussed below for one case of the analysis of marine food lipids by GLC. In a study of the uptake of CLA by fish (13) the GLC column was Omegawax 320 (a polyglycol similar to SUPELCOWAX-10) and six CLA peaks could be detected in an alkali-catalyzed preparation of CLA from safflower seed oil. The analysis was facilitated by AgN0 -TLC (silver nitrate-thin layer chromatography) clean-up of the methyl esters of the fatty acids of the fish lipids with development in benzene. The CLA band will lie below that for saturated fatty acids and above that for cis-monoethylenic fatty acids. 3
In Omega-3 Fatty Acids; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.
237 Table I. Equivalent chain length (ECL) of C L A isomers (methyl esters) on a SUPELCOWAX10 fused-silica capillary column (60 m χ 0.25 mm, 0.25 film thickness)
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Peak no.
1 2 3 4 5 6 7
CLA isomer"
From Ref. 9
c-9,Ml/c-8,M0 c-10,M2/i-9,c-ll MO, c-12 c - l l , M 3 or M l , c-13 c-9, c-ll/c-8, c-10 c-10, c-12/c-ll, c-13 ί-9,Μ1/Μ0,Μ2/ί-8, MO
19.51 19.56 19.63 19.71 19.84 19.88 20.16
From Ref. 12
19.49 19.53 19.62 19.67 19.80 18.82 20.01
aPeak identification was based on the previously reported in ref. 12 and ref. 8.
It was involvement in thisfish-orientedresearch that led to our surprise at the table published in 1998 by O'Shea et al. (15), listing seafood as having, 0.5 mg, CLA/g of fat, with no detectable cis-9,trans-11 isomer. The reference for this data was to a definitive paper on CLA in foods by Chin et al. (16), when two tables listed 19 seafoods (also including lake trout) with totals of
