ABSTRACT: Commercial cheese products were analyzed for their composition and content of conjugated linoleic acid (CLA) isomers. The total lipids were extracted from cheese using pe- troleum ether/diethyl ether and methylated using NaOCH 3 . The fatty acid methyl esters (FAME) were separated by gas chro- matography (GC), using a 100-m polar capillary column, into nine minor peaks besides that of the major rumenic acid, 9c,11t-octadecadienoic acid (18:2), and were attributed to 19 CLA isomers. By using silver ion–high performance liquid chro- matography (Ag + –HPLC), CLA isomers were resolved into seven trans,trans (5–9%), three cis/trans (10–13%), and five cis,cis (<1%) peaks, totaling 15, in addition to that of the 9c,11t-18:2 (78–84%). The FAME of total cheese lipids were fractionated by semipreparative Ag + –HPLC and converted to their 4,4-dimethyl- oxazoline derivatives after hydrolysis to free fatty acids. The geometrical configuration of the CLA isomers was confirmed by GC–direct deposition–Fourier transform infrared, and their dou- ble bond positions were established by GC-electron ionization mass spectrometry. Reconstructed mass spectral ion profiles of the m + 2 allylic ion and the m + 3 ion (where m is the position of the second double bond in the parent conjugated fatty acid) were used to identify the minor CLA isomers in cheese. Cheese contained 7t,9c-18:2 and the previously unreported 11t,13c- 18:2 and 12c,14t-18:2, and their trans,trans and cis,cis geomet- ric isomers. Minor amounts of 8,10-, and 10,12-18:2 were also found. The predicted elution orders of the different CLA isomers on long polar capillary GC and Ag + –HPLC columns are also presented. Lipids 33, 963–971 (1998). Conjugated linoleic acid (CLA) has been reported to lead to reduced carcinogenesis (1–5) and atherosclerosis (6,7), in- creased bone mass (8) and muscle mass (9–11), and to have antidiabetic properties (12) in laboratory animals. The term CLA refers to a mixture of positional and geometric conju- gated octadecadienoic acid (18:2) isomers. The active isomer has been assumed to be 9c,11t-18:2 (also called rumenic acid) because it is the major CLA isomer present in milk (13–20), cheese (15,21–27), and meat (14,15,28) from ruminant ani- mals. It is formed as an intermediate in the biohydrogenation of linoleic acid in the rumen (29–31), and possibly by ∆9 de- saturation of vaccenic acid (11t-18:1) (32). Besides the major 9c,11t-18:2 established by Parodi (13), there are a number of minor CLA isomers in cheese and milk. To date, their reported identities and levels remain question- able, because of inadequate chromatographic separations and confirmatory methods used to establish the double bond posi- tions and configurations. Ha et al. (21) were the first to report six additional CLA peaks in cheese separated on a 60-m Su- pelcowax-10 gas chromatographic capillary column. Based on comparisons of gas chromatographic equivalent chain lengths with published data (33), they reported the presence of 10c,12t-18:2, 10t,12c-18:2, 11c,13c-18:2, 9c,11c-18:2, 10c,12c-18:2, 9t,11t-18:2, and 10t,12t-18:2 as fatty acid methyl esters (FAME) after BF 3 methylation. They deter- mined the molecular weight of the CLA isomers by gas chro- matography (GC)–chemical ionization mass spectrometry (MS), but were unable to identify CLA positional isomers by GC–MS using 4-phenyl-1,2,4-triazoline-3,5-dione deriva- tives. Nevertheless, their BF 3 methylation procedure, GC sep- aration conditions, and equivalent chain length comparisons were subsequently used by many investigators (17,23,24,34), with variation in the methylation catalyst, i.e., HCl (15,22), tetramethylguanidine (25), or NaOCH 3 (35). Acid (BF 3 or HCl)-catalyzed methylations (8,15,17,21–24,34) were shown to lead to isomerization of cis/trans to trans,trans CLA iso- mers and the formation of methoxy artifacts (19), as well as CLA artifacts from allylic hydroxy fatty acids (36). Acid fat extraction procedures (22,25) may also lead to CLA isomer- Copyright © 1998 by AOCS Press 963 Lipids, Vol. 33, no. 10 (1998) 1 On sabbatical leave at the U.S. Food and Drug Administration, Washington, DC. *To whom correspondence should be addressed at Office of Food Labeling (HFS-175), U.S. Food and Drug Administration, 200 C St., S.W., Washing- ton DC 20204. E-mail: mpy@cfsan.fda.gov Abbreviations: c; cis; cis/trans, refers to the same positional isomers that have either a cis,trans or a trans,cis configuration; CLA, conjugated linoleic acid; DMOX, 4,4-dimethyloxazoline; FAME, fatty acid methyl esters; GC–DD–FTIR, gas chromatography–direct deposition–Fourier transform in- frared; GC–EIMS, gas chromatography–electron ionization mass spectrome- try; HPLC, high performance liquid chromatograph(y); t, trans. Identification of Conjugated Linoleic Acid Isomers in Cheese by Gas Chromatography, Silver Ion High Performance Liquid Chromatography and Mass Spectral Reconstructed Ion Profiles. Comparison of Chromatographic Elution Sequences Najibullah Sehat a , John K.G. Kramer b,1 , Magdi M. Mossoba a , Martin P. Yurawecz a, *, John A.G. Roach a , Klaus Eulitz a , Kim M. Morehouse a , and Youh Ku a a U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Washington, DC 20204, and b Southern Crop Protection, Food Research Center, Agriculture and Agri-Food Canada, Guelph, Ontario N1G2W1, Canada