Chromatographic separation and identification of conjugated linoleic acid isomers

Analytica Chimica Acta 465 (2002) 207–226

Review

Chromatographic separation and identification of conjugated linoleic acid isomers John A.G. Roach a , Magdi M. Mossoba a,∗ , M. Peter Yurawecz a , John K.G. Kramer b a

b

Food and Drug Administration, Center for Food Safety and Applied Nutrition, 5100 Paint Branch Parkway, College Park, MD 20740-3835, USA Food Research Program, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ont., Canada N1G 5C9 Received 31 October 2001; received in revised form 22 February 2002; accepted 1 March 2002

Abstract There are 56 possible geometric and positional isomers of conjugated octadecadienoic acids (18:2), better known as conjugated linoleic acid (CLA). Positive health benefits are ascribed to the consumption of the 9c,11t-18:2 and 10t,12c-18:2 isomers. The dietary significance of the other isomers is not known. Our understanding of the biological role of these acids relies on their proper identification and quantitation in complex biological extracts. Gas chromatography (GC) alone cannot completely separate the naturally occurring CLA isomers. The combination of silver ion high performance liquid chromatography (Ag+ HPLC) and GC offers the best separation of these isomers with complementary identification by GC–mass spectrometry (GC–MS) and GC–Fourier transform infrared (FTIR) analyses. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Review; Conjugated linoleic acid; CLA; Ag+ HPLC; GC–FID; Dimethyloxazoline; DMOX; Methyltriazolinedione; MTAD; GC–MS; GC–FTIR

1. Introduction Conjugated linoleic acid (CLA) isomers are reported to have anti-carcinogenic, -atherogenic, -diabetic and lean body mass-enhancing properties [1–4]. CLA was originally intended to describe only those isomers in fats from ruminants showing anti-carcinogenic properties [5]. They were thought to be similar to the commercially available conjugated octadecadienoic acid isomers obtained by alkali isomerization of linoleic acid (9c,12c-18:2). If the term is applied to all octadecadienoic acids with two conjugated double bonds, then there are 14 possible positional isomers counting from carbons 2,4 to carbons ∗ Corresponding author. Tel.: +1-301-436-1974; fax: +1-301-436-2655. E-mail address: [email protected] (M.M. Mossoba).

15,17. Each positional isomer has four geometric isomers (cis,cis; cis,trans; trans,cis; trans,trans) for a total of 56 possible isomers. The double bond positions of CLA isomers actually identified in rumen fat range from 6,8-18:2 to 12,14-18:2 in most of the possible geometric configurations for a total of about 20 isomers [6]. Data from animal models reportedly suggest that the 9c,11t-isomer is responsible for CLA anti-carcinogenic properties [7]. The 10t,12c-isomer is reported to be responsible for the re-partitioning of fat to muscle [7]. Dietary supplements enhanced in CLA are being developed and marketed in response to the reported physiological benefits found in animal models. Triacylglycerols (TAGs) containing CLA have recently been prepared by acidolysis of fish oils [8] and butterfat [9]. A patent has been issued for CLA

0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 0 1 9 3 - 9

208

J.A.G. Roach et al. / Analytica Chimica Acta 465 (2002) 207–226

esterification of glycerol to TAG containing more than 95% CLA [10]. The problem of determining the isomeric CLA composition of these products can be a challenging analytical task in spite of their enhanced CLA content. For example, butterfat is reported to be a mixture of some 100,000 TAG isomers containing some 400 different fatty acids [11]. It is essential that we know which isomers of CLA are contained in dietary supplements because our knowledge of the active isomers and modes of action of CLA is incomplete. The intracellular disposition of CLA isomers and their metabolites provides clues to the biochemical sites affected by CLA [12,13]. For example, the 11c,13t isomer is reported to selectively concentrate in pig heart phospholipids [14] when pigs are fed CLA derived from extensive alkali isomerization of linoleic acid. As a result of this finding, most commercial CLA preparations now exclude the 11c,13t isomer and consist primarily of the 9c,11t and 10t,12c isomers of CLA with lesser amounts of the 8t,10c and 11c,13t isomers [15]. This is because the biological effects of the relatively uncommon 11c,13t isomer remain to be explored. Further studies to deduce the true impact of CLA in the diet must rely on availability of pure CLA isomers and improved analytical procedures for the accurate identification of the CLA isomers. No gas chromatographic columns or conditions have been reported to date that will adequately resolve all the fatty acid methyl esters (FAMEs) of the isomers of CLA that have been identified in natural products and dietary supplements. The task requires complementary techniques. Current methods for CLA in biological samples consist of lipid extraction [16] and fractionation of lipid classes by thin layer chromatography (TLC) [16] or high performance liquid chromatography (HPLC) as a prelude to further sample workup and analysis by silver ion HPLC, gas chromatography (GC), mass spectrometry (MS), Fourier transform infrared (FTIR) spectroscopy [17] and nuclear magnetic resonance (NMR) [18]. 2. Sample preparation 2.1. Extraction and isolation of lipids All samples should be excised quickly and frozen at −70 ◦ C until analyzed. Use methods that rapidly

inhibit the action of lipases and phospholipases. Try to avoid mechanical grinders that may cause thermal lipolysis of the sample. Avoid extraction methods that involve acid digestion [19] and cause isomerization of CLA isomers [20]. Avoid non-polar solvents that only extract neutral lipids. Use chloroform/methanol/water in at least a 15:1 solvent to sample volume ratio to extract total milk, cheese or tissue lipids [21,22]. Commercial CLA preparations may be in acid, ester, or other forms. If the composition is unknown, alkali hydrolysis is recommended. Compare whatever method is selected or modified to a known standard technique such as that of Folch et al. [21] to insure reliability. Lipid separation by class is important because different lipids have different functions. HPLC is effective in the separation of selected lipid classes [23], but the separation of all tissue lipid classes is beyond the ability of a single HPLC column. TLC is currently the most reliable method for complete resolution of neutral and phospholipid classes. A three-directional TLC system effectively resolves 3–4 mg of total tissue lipids on silica gel H plates if the TLC plate is dried in a nitrogen chamber between each development [24]. Phospholipids and free fatty acids (FFAs) are scrapped off the plate after the second development. Neutral lipids are then separated in a third solvent system. Isolates from more than one plate are combined to obtain useful amounts of minor lipid components. 2.2. Methylation of fatty acids for further analysis The procedures that appear to be effective for the methylation of CLA in most matrices are sodium methoxide in methanol at 50 ◦ C for 15 min, or HCl/methanol at 80 ◦ C for 1 h. The acid catalyzed reaction alters CLA isomer composition and forms methoxy and hydroxy artifacts [20]. FFAs can be methylated under relatively mild conditions using trimethylsilyldiazomethane [25] or diazomethane. Sodium methoxide will not methylate sphingomyelins and plasmalogens. The vigorous acid conditions required to break the amide linkages of sphingomyelins adversely affects the intact CLA composition [14], and will also produce CLA isomers from unsaturated hydroxy fatty acids [26]. There is no method at this time to analyze for the intact CLA isomer distribution of sphingomyelin, except 13 C NMR that requires relatively large amounts of material.

J.A.G. Roach et al. / Analytica Chimica Acta 465 (2002) 207–226

209

Fig. 1. Silver ion HPLC chromatograms (using three columns in series) for (A) a commercial CLA FAME mixture, and (B) the same mixture co-injected with a 7t,9c-18:2 reference material. Often the cis,cis isomers are too weak to be observed.

3. Silver ion (Ag+ ) HPLC of CLA The complementary use of Ag+ HPLC (Fig. 1) with GC (discussed later) is currently the most effective way to separate and quantitate individual isomers of CLA. Unambiguous identification of individual isomers of CLA relies on additional supporting data obtained by MS and NMR, or GC–MS and GC–FTIR. Reports that concentrations of CLA in commercial CLA samples were not accurately described by manufacturers’ assays led to the first use of Ag+ HPLC [25] to complement CLA analysis by GC. Our HPLC method for separating CLA methyl esters using commercially available silver ion-impregnated silica columns was a modification of the work of Adlof [27] on TAG. The report described use of an acetonitrile/hexane mobile phase to separate CLA FAME using two Ag+ HPLC columns connected in series. CLA FAME are selectively detected by their characteristic UV absorbance at 233 nm. Isomer identities in HPLC chromatograms are based on co-injections of known reference materials obtained from commercial sources or synthesized [28]. GC–MS and GC–FTIR confirmation of these tentative co-injection assignments is carried out on fractions collected from the

Ag+ HPLC separations. The lack of commercially available standard CLA reference materials hinders this work. 3.1. CLA reference materials Only the limited number of CLA isomers given below is commercially available. A CLA FAME mixture was purchased from Nu-Chek-Prep (Elysian, MN, USA). Pure isomers (9c,11t-18:2, 10t,12c-18:2, 9c,11c-18:2, and 9t,11t-18:2) were obtained as free acids from Matreya Inc (Pleasant Gap, PA, USA). Catalytic isomerization using iodine and light or p-toluene sulfinic acid [28] was used to prepare solutions containing all of the geometric isomers of selected positional isomers of CLA. The 7t,9c-18:2 methyl ester was a generous gift from Dr. Lutz Lehman, University of Hamburg, Germany. 3.2. Ag2+ HPLC operating conditions for CLA separations The HPLC system used for isocratic separations of CLA FAME was equipped with a 100 ␮l injection loop and between one and six ChromSpher five lipids silver

210

J.A.G. Roach et al. / Analytica Chimica Acta 465 (2002) 207–226

impregnated 4.6 mm × 250 mm columns (Chrompack, Bridgewater, NJ, USA) in series. The 0.1% acetonitrile in hexane mobile phase was prepared fresh daily and stirred while in use. Mobile phase flow was 1.0 ml/min. The column was equilibrated with mobile phase for at least 30 min prior to each injection. The column was flushed on a weekly basis with 1% acetonitrile in hexane for 30–60 min to remove buildup of extraneous co-extractives. A commercial CLA FAME reference was injected two to three times to check column performance prior to any injections of test portions. Typical injections were 5–15 ␮l representing less than 250 ␮g lipid. Column head pressure increased from 23 to 133 bar as the number of analytical columns in series increased from one to six. The retention volumes of the CLA isomers change with time but with little loss of resolution. Fresh mobile phase preparation and constant mobile phase mixing reduce, but do not eliminate, drift in CLA retention volumes. Resolution within the t,t and c,t groups of CLA isomers is greatly improved using three columns in series (Figs. 1 and 2). Use of more than three columns in series provides diminishing benefits.

FFA are also efficiently separated by Ag+ HPLC. Ostrowska et al. report a separation of CLA as FFAs using a single Ag+ HPLC and a ternary mobile phase of 2.5% acetic acid and 0.025% acetonitrile in hexane [29]. They repeated each separation four times or until repeatable retention times were obtained. The column must be conditioned with freshly prepared mobile phase for at least 30 min prior to sample injection. Nikolova-Damyanova et al. report Ag+ HPLC resolution of the phenacyl and p-methoxyphenacyl esters of CLA superior to that obtained for FAME [30]. Changing the mobile phase composition from 40:60:0.2 (v/v/v) hexane/dichloromethane/acetonitrile to 100:0.1 (v/v) dichloromethane/acetonitrile or 50:50:0.3 (v/v/v) hexane/dichloromethane/isopropanol alters the elution order of the CLA isomers. This is advantageous in certain applications. All phenacyl esters are detected at 245 nm and all p-methoxyphenacyl esters are detected at 270 nm. The non-specificity of UV detection of these esters presents a problem. All esters in the sample will be detected with the same sensitivity as esters of CLA. Test portions will require cleanup after ester preparation and

Fig. 2. Silver ion HPLC chromatograms (using three columns in series) for human milk FAME, isomerized [28] Nu-Chek-Prep FAME, and a co-injection of both.

J.A.G. Roach et al. / Analytica Chimica Acta 465 (2002) 207–226

isolation of CLA esters prior to Ag+ HPLC in order to detect CLA esters without interference. The choice of derivative for Ag+ HPLC analysis may be affected by subsequent analyses. FFAs are preferable to FAME for Ag+ HPLC separation prior to conversion to dimethyloxazoline derivatives. Use of FFAs avoids problems with acid catalysts in the synthesis of FAME from FFAs [31]. Preparation of FAME is almost always necessary to determine the amount of CLA and other fatty acids in a food, dietary supplement, or reference standard material by GC. The retention volumes and peak shapes of individual CLA isomers are influenced by column preconditioning, column temperature, eluant composition, conjugated double bond position and geometry, and sample loading [27,29,30,32]. The sample load of non-CLA FAME strongly affects resolution of CLA isomers by Ag+ HPLC. Non-conjugated FAME respond poorly at 233 nm. Their buildup from injection to injection is not readily apparent. It is prudent to monitor column effluent at 200 nm for co-extractives. Allow these materials to elute after each analysis and periodically rinse the column with 1% acetonitrile in hexane to avoid co-extractive buildup. These steps will improve CLA isomer resolution and avoid subsequent misidentifications by GC. Our understanding of the biological role of CLA relies on the proper identification and quantitation of CLA in complex mixtures of fatty acids obtained from chemical and biological matrices. Matrices of interest include commercial CLA mixtures (Fig. 1), milk fats (Fig. 2) and tissue extracts.

4. CLA analysis by capillary gas chromatography Although the information on CLA isomeric composition provided by GC is incomplete, GC is often the only method used in the analysis of fatty acids for CLA. Identifications are then limited to comparisons of retention times with a limited number of standards. The fatty acids in tissue lipids present a relatively simple profile in comparison to milk fats. Milk fats may contain up to 400 different fatty acids [33] differing in chain length, branching, unsaturation, geometric and positional configuration, and functional groups. Several GC parameters should be considered in the

211

analysis of CLA co-extracted with other fatty acids of milk and tissue lipids. The GC should be equipped for capillary column use with an FID, a split/splitless injection system, an autosampler, and computer software to acquire, integrate and transform the resultant data. The autosampler is highly recommended for consistency of injection, better reproducibility of retention times and the benefit of continuous analysis. The split and splitless modes of injection should be compared for given types of sample to insure that representative mixtures are analyzed. For example, a splitless injection of 0.1 min gives a more representative profile of total milk FAME ranging from C4 to C26 than a 20:1 split injection, but the split mode gives more satisfactory results for total tissue FAME ranging from 14:0 to 22:6n-3. Each injection mode requires unique inlet inserts. Use a cup splitter for split injection and a single Gooseneck insert (neck to column side) for splitless injection. Check the purity of all gases used for sample preparation and analysis. Use a process blank to check for analytical artifacts arising from the gas or apparatus used to evaporate test portions. Do not proceed with FID analysis unless a steady baseline is obtained at a high sensitivity setting of the detector with the GC oven about 10 ◦ C above the highest temperature reached during the analysis. Use hydrogen as a carrier gas to obtain optimal resolution. Connect the split vents to an exhaust and routinely check for leaks when using hydrogen. Check all solvents used in the preparation of GC samples for the absence of peaks in the region of interest. Prepare process blanks and solvent blanks to check the suitability of all reagents and to guard against cross-contamination. Routinely replace the wash solvents in the autosampler. 4.1. Choice of capillary column Non-polar capillary columns such as methylsilicone or phenylmethylsilicone phases fail to resolve the isomers of CLA. A polar capillary GC column is absolutely mandatory for the analysis of the closely related geometric and positional isomers of CLA. Supelcowax 10 was used in the separation of CLA isomers in 1989 [5] and remains in use today [15] in spite of the lack of adequate GC–MS data on the

212

J.A.G. Roach et al. / Analytica Chimica Acta 465 (2002) 207–226

Fig. 3. Partial GC chromatogram of the CLA region from a commercial CLA mixture consisting of four major cis,trans positional CLA isomers (as FAME) analyzed using a 100 m CP-Sil 88 capillary column that was operated at an isothermal temperature of 125 ◦ C. In the lower panel the same CLA mixture was analyzed using a 60 m Supelcowax 10 capillary column [16].

elution order and separation of CLA isomers with this column. On the other hand, many separations have been published using 100 m cyanopropylsilicone capillary columns in which the identities of many peaks have been confirmed by GC–MS and GC–FTIR. The 100 m cyanopropyl polysilicoxane capillary columns (CP-Sil 88, Varian, Palo Alto, CA; SP 2560, Supelco Inc., Bellefonte, PA; BPX70, SGE, Melbourne, Australia) are the best columns available for CLA analysis (Fig. 3). 4.2. Identity of GC peaks Establish the identity of most of the analytes that give rise to GC peaks in a representative chromatogram with GC–MS (see later) after conversion to 2-alkenyl-4,4-dimethyl oxazoline (DMOX) [34]. This will also establish the purity of many of the peaks. Use a reported set of GC conditions for similar samples, but remember that slight differences in columns, temperature program, and concentration affect the relative retention times of FAME.

4.3. Quantitative internal standards The internal standard should be eluted in a region that causes minimum interference and have a similar FID response as the sample constituents of interest. Odd chain fatty acids are typically used as internal standards because their natural abundance is low. In the case of milk and tissue lipids, 19:0 and 21:0 should not be used because 19:0 is eluted among 18:1 and 18:2 isomers [20], while 21:0 is eluted among the CLA isomers [28,35,36] using either CP-Sil 88 or SP 2560 capillary columns. Add the internal standard during the methylation stage to insure representative sampling and add an amount that will give a final relative concentration of about 10% of the total FAME. 4.4. Interferences in the CLA region A shorter (50 or 60 m) column is more prone to interferences than a 100 m column. Methoxylation artifacts elute after the CLA region on a 100 m CP-Sil 88

J.A.G. Roach et al. / Analytica Chimica Acta 465 (2002) 207–226

213

Fig. 4. Partial GC chromatogram of the CLA region from total cheese fat FAME (a) analyzed using a 100 m CP-Sil 88 capillary column, and (b) analyzed by GC–MS using high-resolution selected-ion recording at m/z 294.2559 [33]. An asterisk denotes non-CLA responses. The (A∗ ) denotes 21:0 FAME (reproduced with kind permission from authors and Lipids).

or SP 2560 column [20] but they may be eluted with CLA using a 50 m column [37]. The CLA region of milk and tissue lipid extracts lies between the elution of 18:3n-3/20:1 and 20:2n-6. The region is essentially free of other fatty acids except for 21:0 FAME using a 100 m column (Fig. 4). This is in part due to the low occurrence of potentially interfering non-conjugated 20:2 FAME in these extracts [35]. 4.5. CLA isomer separation There are about 20 different CLA isomers in natural milk fat based on Ag+ HPLC [6]. Their total fat content of less than 1% is dominated by the

9c,11t-18:2 isomer which constitutes about 80% of CLA in milk fat [38,6]. Many of the CLA isomers in milk fat are not resolved with the GC columns currently in use. For example, the 7t,9c-18:2 isomer is the second most abundant CLA isomer in dairy and human fat at about 7% of total CLA [38]. This isomer is not resolved from 9c,11t-18:2 by GC using a 100 m capillary column. Its discovery in the leading edge of the 9c,11t-18:2 peak was by Ag+ HPLC and GC–MS rather than GC–FID [38]. Isomerized mixtures [28] containing comparable amounts of eight isomers of CLA are partially resolved by GC (Fig. 5), but such a separation is not apparent when one isomer predominates. If the ana-

214

J.A.G. Roach et al. / Analytica Chimica Acta 465 (2002) 207–226

Fig. 5. Partial GC chromatogram of the CLA region from a commercial CLA mixture consisting of four major cis,trans positional CLA isomers (as FAME) analyzed using a 100 m CP-Sil 88 capillary column (a) and the same CLA mixture after isomerization that yielded eight cis,trans CLA isomers (b) [28] (reproduced with kind permission from authors and Lipids).

lyte is a commercial CLA mixture of only two major positional isomers (9c,11t-18:2 and 10t,12c-18:2), one should be able to resolve these two isomers and their opposite isomers (10c,12t-18:2 and 9t,11c-18:2) with a 100 m GC column. In commercial CLA preparations containing four major positional isomers of CLA, the 8t,10c-18:2 isomer is a tailing shoulder on the 9c,11t-18:2 peak, while the 10t,12c-18:2 and 11c,13t-18:2 isomers are well separated (Figs. 3 and 5). However, the minor CLA isomers present in most natural matrices are not sufficiently resolved for FID analysis, and GC–MS is required. The elution order by GC on a 100 m CP-Sil 88 column is: all the cis,trans followed by all the cis,cis and finally, all the trans,trans CLA positional isomers (Table 1) [15,6]. Overlap occurs with some of the cis,trans and cis,cis CLA isomers. The elution order decreases within the cis,trans isomers as the ∆ value of the cis double bond increases. If a pair of cis,trans isomers has the same ∆ cis value, the isomer with the lower ∆ trans value elutes first. This means that for the same positional isomer, the cis,trans will elute before the trans,cis geometric isomer. The elution times of cis,cis isomers increase with increasing ∆ values.

The elution times of trans,trans isomers decrease with increasing ∆ values. These elution assignments are based on natural and synthetic mixtures in which the isomer composition varied sufficiently to detect and identify specific isomers. It is important to remember that a number of CLA isomers will be concealed by the predominant CLA isomers in a mixture of unequal proportions. Proper FID analysis of CLA isomers must rely on resolution of the isomers by Ag+ HPLC as either methyl esters [25] or FFAs [29], and in combination with GC–MS and GC–FTIR analyses to validate the FID data.

5. Capillary GC–MS analysis of CLA isomers Capillary GC–MS combines an efficient separation technique with a sensitive detector that can provide elemental composition, double bond equivalents and mass spectra indicating the location of double bonds for CLA and related compounds. Methyl esters of CLA gas chromatograph well, but the electron impact (EI) mass spectra of individual CLA methyl esters are

J.A.G. Roach et al. / Analytica Chimica Acta 465 (2002) 207–226 Table 1 Elution ordera [6,15] of positional and geometric CLA isomers on a 100 m CP-Sil 88 capillary gas chromatographic column cis,trans-18:2b

cis,cis-18:2c

trans,trans-18:2d

7c,9t (6t,8c) 8c,10t 7t,9c 9c,11t 8t,10c 10c,12t 9t,11c 11c,13t 10t,12c 12c,14t

(7c,9c)e 8c,10c 9c,11c 10c,12c 11c,13c 12c,14c

12t,14t 11t,13t 10t,12t 9t,11t 8t,10t 7t,9t

a The elution order is: all the cis,trans, followed by all the cis,cis, followed by all the trans,trans CLA positional isomers. Several cis,trans CLA isomers overlap with some cis,cis geometric isomers. b The observed elution time of cis,trans CLA isomers increase as the ∆ value of the cis double bond increases in the molecule. For a pair of cis,trans isomers in which the cis double bond has the same ∆ cis value, the isomer with the lower ∆ trans value elutes first. Therefore, it follows that for the same positional isomer, the cis,trans elutes before the trans,cis geometric isomer. c The observed elution time of cis,cis CLA isomers increases with increased ∆ values. d The observed elution time of trans,trans CLA isomers increases with decreased ∆ values. e CLA isomers shown in parentheses are predicted.

indistinguishable. Spitzer recently reviewed derivatization techniques that produce structurally useful EI data from CLA [39]. Satisfactory derivatives are of two types, those that fix the location of double bonds, such as 4-methyl-1,2,4-triazoline-3,5-dione (MTAD),

215

and those that control fragmentation by retention of the positive charge, like the DMOX derivative. Diels–Alder adducts of conjugated dienes with MTAD produce intense diagnostic ions in their EI mass spectra that identify the positional isomers of CLA [40]. The conjugated diene is incorporated into a six-member heterocyclic ring that dominates fragmentation of the molecule. Information about the geometry of the diene system is destroyed, but the location of the diene is easily determined from the EI mass spectrum. The most abundant ions are diagnostic of the diene’s position in the carbon chain. These ions arise as preferential cleavages on either side of the ring and provide complementary data for the location of the conjugated system in the carbon chain [41]. The MTAD derivative is used for the analysis of CLA standard materials and CLA isolates from biological materials in conjunction with other derivatives such as DMOX that do not destroy chromatographic separations derived from the geometry of the diene system [42]. The reaction of the carboxylic acid functionality with 2-amino-2-methylpropanol forms a DMOX derivative that directs EI fragmentation by retaining the positive charge on the nitrogen of the derivative [34]. The DMOX derivative is strongly preferred for two reasons: general applicability and superior chromatography. The DMOX reagent reacts with the acid functionality. This permits GC–MS DMOX identification of most fatty acids including CLA (Fig. 6). MTAD CLA derivatives separate by GC on the basis of the position of the MTAD adduct. DMOX CLA separations are influenced by both the geometry and

Fig. 6. EI mass spectrum of DMOX derivative of 10t,12c-18:2 isomer. Molecular ion is m/z 333. Characteristic loss sequence of 12, 14 and 12 is at m/z 248–236 (12 amu), 236–222 (14 amu), and 222–210 (12 amu). Abundant allylic cleavage ions at m/z 196, 276 and 290 flank the characteristic loss sequence.

216

J.A.G. Roach et al. / Analytica Chimica Acta 465 (2002) 207–226

position of the double bonds of the CLA isomers with the result that DMOX CLA derivatives provide superior CLA isomer resolution compared to MTAD derivatives. The chromatographic separations observed for FAME are retained or improved upon by the separations observed for the corresponding DMOX derivatives [43,44]. This makes it possible to combine DMOX or FAME GC–FTIR double bond geometry data (see later sections) with DMOX GC–MS double bond position data (Fig. 6), and assign an identity to individual isomers of CLA in a complex mixture [25]. The excellent chromatography of DMOX derivatives and their applicability to the structural elucidation of fatty acids containing a wide variety of functional groups make them the best choice for the GC–MS analysis of CLA isomers, metabolites, and related compounds. 5.1. GC–MS conditions for CLA methyl esters and DMOX CLA This laboratory uses a high-resolution doublefocusing mass spectrometer for most GC–MS CLA analyses. The instrument can provide accurate mass measurement data if needed and it is more sensitive than a bench top GC–MS. Detector sensitivity is important because the isomers of CLA typically represent less than 1% of a biological extract. A sensitive detector can yield satisfactory data for CLA from small extract portions that do not overload the capillary column with co-extractives that may degrade the chromatography. The gas chromatographic conditions include using hydrogen carrier gas at 26 psi (180 kPa) and a 100 m ×0.25 mm, 0.2 ␮m film cyanopropyl polysiloxane phase fused silica capillary column (CP-Sil 88, Varian, Raritan, NJ, USA). Split flow vent flow is 100 ml/min, and septum purge flow is 3 ml/min. Injections are manual, splitless, into a double-gooseneck glass injector insert at an injection port temperature of 220 ◦ C. The oven temperature program for isooctane solutions is: 75 ◦ C for 2 min after injection, 5 ◦ C/min to 170 ◦ C, held at 170 ◦ C for 40 min, 5 ◦ C/min to 220 ◦ C and hold at 220 ◦ C for 20 min. The transfer line temperature is 220 ◦ C, and the ion source temperature is 250 ◦ C. Filament trap current is 200 ␮A at 70 eV. The positional isomers of DMOX CLA spread

out over a retention time window (57–63 min) of approximately 5 min under these conditions. A 50 m column supplied with helium carrier gas at a head pressure of 13 psi (90 kPa) will elute the analytes in half the time with greater sensitivity because the reduced peak width of the shorter retention times increases the effective analyte concentration in the ion source. The price for this improved sensitivity is a loss of isomer separation as the retention time window (28–31 min) shrinks to approximately 3 min. 5.2. Electron impact fragmentation of DMOX CLA The positive ions analyzed under EI conditions are formed when a molecule loses an electron to form an odd-electron species known as the molecular ion. The unpaired electron gives a net positive charge to the molecular ion. The positive charge of the unpaired electron localizes in the non-bonding orbitals of the nitrogen. A hydrogen atom in the chain may then move to the nitrogen creating a radical site in the chain that leads to fragmentation. If no other functional groups are present in the chain, the probability of hydrogen migration to the nitrogen from the carbon chain is about equal for all of the hydrogens. This creates the appearance of stepwise scissions of the carbon chain. The resulting DMOX CLA spectra (Fig. 6) are characterized by a loss of 15 from the molecular ion and successive losses of 14 except for the conjugated diene system, which may be located in the chain by its characteristic loss sequence of 12, 14 and 12. Carbons in the chain allyl to the conjugated diene system are favored radical sites and produce more abundant fragmentation than other positions in the chain [45]. These abundant fragment ions flanking a loss sequence of 12, 14 and 12 facilitate assignment of the positions of the double bonds in the carbon chain [34,46]. Diagnostic DMOX fragment ions for CLA positional isomers with double bond positions from C6 to C15 are listed in Table 2. The predictable pattern of these fragment ions makes it possible to search GC–MS data records of DMOX derivatized extracts for previously undetected CLA isomers [38]. The identification of a specific CLA isomer as its DMOX derivative requires sufficient chromatographic separation from other congeners and co-extractives to obtain a representative mass spectrum of the isomer.

J.A.G. Roach et al. / Analytica Chimica Acta 465 (2002) 207–226 Table 2 Diagnostic DMOX CLA fragment ions for CLA isomers with double bond positions from C6 to C15 CLA isomer

Allylic ion

Losses of 12, 14 and 12

Allylic ion

Allylic ion

6,8a 7,9b 8,10 9,11 10,12 11,13 12,14 13,15

140 154 168 182 196 210 224 238

154, 168, 182, 196, 210, 224, 238, 252,

220 234 248 262 276 290 304 318

234 248 262 276 290 304 318 332

166, 180, 194, 208, 222, 236, 250, 264,

180, 194, 208, 222, 236, 250, 264, 278,

192 206 220 234 248 262 276 290

Molecular ion (m/z 333) should be part of the data record. a 154 is weak, 220 is more abundant than 234 in spectrum of 6,8-18:2 isomer. b 154 is weak, 234 is more abundant than 248 in spectrum of 7,9-18:2 isomer.

217

The abundant allylic cleavage ions listed in Table 2 can be used to determine the locations of the CLA isomers in the GC–MS data record [6]. Signal contributions of adjacent gas chromatographic peaks are then subtracted to obtain representative mass spectra. The pattern in Table 2 is less distinct for isomers in which the diene is near either end of the carbon chain. One allylic cleavage predominates over the other allylic cleavages. Additional useful ions appear in the spectra. This is because the relative probability of the possible fragmentation pathways shifts with the position of the conjugated diene in the carbon chain. Published spectra of DMOX derivatives of monoenes and polyenes with isolated double bonds at C4, C5 or C6 show intense odd-electron ions (m/z 139,

Fig. 7. EI mass spectra of DMOX derivatives of (a) 6,8-18:2, (b) 7,9-18:2 and (c) 13,15-18:2 CLA isomers.

218

J.A.G. Roach et al. / Analytica Chimica Acta 465 (2002) 207–226

153 and 167, respectively) adjacent to expected cleavages at m/z 140, 152 and 166, respectively [47]. A suggested fragmentation pathway rationalizing these odd-electron ions is based on interaction of the unpaired electron of the nitrogen with a nearby double bond [46]. It is reasonable to suspect that CLA with double bonds at the C4, C5 or C6 positions would fragment differently from other CLA. Our recent synthesis, separation, and purification of all of the isomers of CLA listed in Table 2 permit a detailed comparison of the mass spectra of the DMOX derivatives of the 6,8-18:2, 7,9-18:2, and 13,15-18:2 positional isomers of CLA with the data in Table 2. The 6,8-18:2 isomer (Fig. 7a) fragments to produce a single abundant allylic cleavage ion at m/z 220. The other allylic cleavage ions at m/z 140 and 234 are significantly less abundant than 220. The conjugated diene in the 6,8 position preferentially forms m/z 166 rather than the odd-electron m/z 167 ion formed by an isolated C6 double bond. The 154 diagnostic ion in Table 2 is so low relative to m/z 152 that 154 appears to be absent in the 6,8 mass spectrum. An odd-electron ion at m/z 127 is present in the 6,8 spectrum at an abundance that makes it possible to screen for this isomer by looking for the co-elution of m/z 127 and 220 in the GC–MS data. The measured mass of 127 is within −0.4 ppm of C7 H13 NO indicating an interesting, but not totally unexpected origin. The ions observed for the 7,9-18:2 isomer (Fig. 7b) conform completely to those listed in Table 2 in that they all occur in the mass spectrum of the 7,9-18:2 isomer. However, the abundance of m/z 154 in the 7,9-18:2 spectrum is also so low relative to m/z 152 as to make 154 appear absent. The allylic cleavage ion, m/z 234, is much more abundant than the other diagnostic ions in the 7,9-18:2 spectrum. A comparative ion profile of m/z 234 and 220 may be used to distinguish 7,9-18:2 from 6,8-18:2 isomers by GC–MS. The 13,15-18:2 isomer (Fig. 7c) is best identified by its diagnostic losses of 12, 14 and 12 observed between m/z 290 and 252. An allylic cleavage ion flanks the end of the diagnostic losses at m/z 238. Allylic cleavage ions are also present at m/z 318 and 332, but only m/z 318 is significantly more abundant than adjacent ions in the 13,15 mass spectrum. Loss of a hydrogen radical to form m/z 332 is clearly a

less favorable decomposition pathway than the other allylic cleavages.

6. Confirmation of CLA geometric isomers by GC–FTIR The infrared characterization of trace amounts of CLA relies upon gas chromatographic separation of its isomers followed by their sub-ambient deposition onto a cryogenically-cooled moving surface. The trail of frozen GC components captured on the cold surface retains the separation achieved by the capillary column. Each component is then scanned either on-the-fly or post-GC separation by FTIR. The scans are summed to provide useful IR data from even minor components of a biological extract. Infrared spectroscopy differentiates CLA isomers by the geometry of their double bonds. Dispersive infrared spectra were reported about half a century ago [48] to distinguish between the cis and trans double bond configurations in fats and oils. These included transmission infrared absorption spectra for conjugated diene fatty acid isomers that were also used for the quantitative determination of conjugated cis,trans and trans,trans geometric isomers [49,50]. In recent years, GC–FTIR [51] hyphenated techniques allowed the on-line measurement of vibrational spectra for individual analytes eluting from a gas chromatograph. Hence, CLA isomers from complex chemical and biological mixtures could be separated as FAME or DMOX derivatives, and measured by FTIR [52–59]. Capillary GC–FTIR spectroscopy can confirm the double bond configuration, cis,trans, cis,cis, or trans,trans, for the resolved components of complex mixtures of CLA geometric isomers (Figs. 8 and 9). It can also provide structural information on methoxy fatty acids, furan fatty acids and other mixtures of unknown reaction products. 6.1. Capillary GC–FTIR interfaces Capillary GC–FTIR is a hyphenated tool that has provided structural information about double bond configuration and functional groups for fats and oils, including CLA isomers. It was first demonstrated on complex mixtures of cyclic fatty acid monomers isolated from heated linseed and sunflower oils in

J.A.G. Roach et al. / Analytica Chimica Acta 465 (2002) 207–226

219

Fig. 8. GC–DD–FTIR spectra observed at 8 cm−1 resolution can readily discriminate between CLA geometric isomers as shown for DMOX derivatives (reproduced with kind permission from authors and AOCS Press).

1987 [60]. The ring size of monounsaturated cyclic fatty acids could be determined by differentiating between a cis double bond in a five-membered ring (712 cm−1 ) and one in a six-membered ring (660 cm−1 ) using a light-pipe (LP) GC–FTIR interface. The LP interface [51] consists of a heated cylindrical tube that is coated internally with gold and sealed with IR-transparent (alkali halide) windows. As the separated analytes exit the GC column and travel through the LP, IR spectra are measured continuously on-the-fly. Some mixing of the chromatographic components unavoidably takes place in the flow-through cell causing degradation of resolution. The signal-to-noise ratio (SNR) is limited by the relatively long path length (10–20 cm) of the LP. Its length limits the transmission of IR light, and its narrow aperture (1 mm i.d.) increases spectral noise. The LP minimum identifiable quantity (MIQ) is in the range of

5–25 ng per analyte. This is usually inadequate for fatty acid research. The matrix isolation (MI) and the direct deposition (DD) interfaces [51] used in our laboratory are better suited for CLA analysis [54]. They provide improvements in MIQ by about an order of magnitude down to the subnanogram levels. The GC effluent is trapped with MI in the solid phase for subsequent off-line measurement by FTIR. During a chromatographic run, the GC effluent is sprayed onto the outer rim of a slowly rotating gold-plated disk held at 12 K under vacuum. The individual analytes are frozen on the moving collection disk as they exit the uncoated capillary transfer line. The GC carrier gas consists of a mixture of 98.5% helium and 1.5% argon. At 12 K, helium is evacuated while the argon atoms are deposited with the separated analytes on the cryogenic disk. Hence, a solid matrix is formed in which analyte molecules are isolated from each other by a large excess of IR-transparent

220

J.A.G. Roach et al. / Analytica Chimica Acta 465 (2002) 207–226

Fig. 9. Expanded GC–DD–FTIR hydrocarbon stretch spectral region, observed at 8 cm−1 resolution for DMOX conjugated 18:2 geometric isomers, exhibited unique differences in the number, position and relative intensity of infrared bands (reproduced with kind permission from authors and AOCS Press).

argon atoms. Each analyte peak is deposited on a small area (diameter 0.5 mm), thus resulting in greater IR absorbance. For individual analytes, post-GC run FTIR data acquisition is then carried out for several minutes, as opposed to about 2 s with the LP. This improves the SNR. The DD is another sensitive mobile-phase elimination interface. In this case, the analytes are directly deposited (without an argon matrix) on a zinc selenide IR-transparent window cooled with liquid nitrogen to about 77 K. During a chromatographic run, the window is moved in small increments using a motorized x–y stage. Each frozen analyte is measured about 15 s after deposition on the window. The transmitted IR light is collected and focused by microscope objectives onto a remote mercury–cadmium–telluride detector operating at liquid nitrogen temperature. One advantage that the DD interface offers is that the on-the-fly

measurements are sufficiently sensitive for most CLA research [54]. 6.2. CLA geometric isomers in chemical and biological matrices CLA geometric isomers have a similar chain length and degree of unsaturation, yet their double bonds have different configurations. Conjugated fatty acids give rise to characteristic bands (Figs. 8 and 9) that make their geometric isomers readily distinguishable [52,54]. Data obtained for CLA isomers with GC–MI–FTIR were first reported for partially hydrogenated vegetable oils more than 10 years ago [54]. The characteristic GC–FTIR bands that allow the differentiation of FAME isomers are usually much weaker than most other features observed in these spectra [52]. Most FAME spectra exhibit similar

J.A.G. Roach et al. / Analytica Chimica Acta 465 (2002) 207–226

features, except for a few characteristic differences due to the number, position and configuration of double bonds. For long hydrocarbon chain FAME, the common bands obtained by GC–MI–FTIR at 4 cm−1 resolution are due to the CH3 asymmetric (2961 cm−1 ) and symmetric (2880 cm−1 ), and CH2 asymmetric (2935 cm−1 ) and symmetric (2863 cm−1 ) stretching vibrations, the CH2 in-plane bend (1463 cm−1 ), the CH3 symmetric scissors (1381 cm−1 ), the CH3 in-plane rock (1123 cm−1 ) and CH2 rock (727 cm−1 ) deformation vibrations, the ester symmetric C–O stretch (1176 cm−1 ), and the strong ester carbonyl stretch at 1754 cm−1 . The intensity of the strong CH2 asymmetric stretch band usually decreases relative to that of the ester carbonyl stretch, as the degree of unsaturation increases. With DMOX derivatives, the oxazoline ring gives rise to several features (Fig. 8) common to all fatty acid DMOX spectra [54,56]. These are usually the ring C=N stretching vibration at 1678 cm−1 , the C–O cyclic ether band, which is split with a maximum at 1002 cm−1 , and three weaker

221

components near 1018, 979, and 954 cm−1 . Several other minor bands in the fingerprint region attributed to the ring skeletal vibrations are found. Most importantly, the position of the discriminating MI–FTIR bands observed for the unsaturated group H–C=C–H are at 3028 and 3009 cm−1 (conjugated cis and trans double bonds), and 3023 and 3005 cm−1 (two conjugated trans double bonds) [52]. The corresponding DD–FTIR data for CLA DMOX derivatives [61] (Fig. 9) are: cis,trans 3020 and 3002 cm−1 ; cis,cis 3037 and 3005 cm−1 ; and trans,trans 3017 cm−1 . In contrast, bands for non-conjugated FAME [52] were observed at 3035 and 3005 cm−1 (one or two trans double bonds), 3010 cm−1 (cis double bond), 3018 cm−1 (two or three cis double bonds), 3035, 1010 and 3005 cm−1 (cis and trans double bonds separated by more than one methylene group), 3035, 3018 and 3005 cm−1 (cis and trans double bonds separated by a single methylene group). Using GC–MI–FTIR, the C–H out-of-plane deformation vibrations in H–C=C–H groups exhibit highly

Fig. 10. Flame ionization detector profile for the GC separation of the geometric isomers of the 9c,11t-18:2 FAME positional isomer. Methoxy FAME products (peaks 1–4) were obtained due to methylation with BF3 . Autoxidation of the same CLA mixture gave rise to the corresponding furan FAME 9,12-epoxy-9,11-octadecadienoic (F9,12 ). F9,12 elutes in the same retention time range as methoxy FAME products, but can be identified by FTIR (Fig. 12) (reproduced with kind permission from authors and AOCS Press).

222

J.A.G. Roach et al. / Analytica Chimica Acta 465 (2002) 207–226

characteristic band positions for CLA FAME isomers [54]: 986 and 950 cm−1 (conjugated cis and trans double bonds), and 990 cm−1 (two conjugated trans double bonds). The corresponding GC–DD–FTIR spectra for CLA DMOX derivatives [61] are given in Fig. 8. For non-conjugated FAME [52], GC–MI–FTIR bands were found at 971 cm−1 (one or two trans double bonds), 730 cm−1 (one or two cis double bonds), 721 cm−1 (three cis double bonds), 971 and 730 cm−1 (cis and trans double bonds interrupted by one or more methylene groups). GC–DD–FTIR spectroscopy was used to identify CLA isomers obtained commercially (chemical suppliers, dietary supplements) [25], or found in foods (cow milk, cheese, beef) and biological matrices [6,14,38]. The latter included human milk, adipose tissue and organs of pig that were fed CLA diets.

6.3. Conjugated octadecatrienes in edible fats and oils An investigation of edible fats and oils [62] showed that the dehydration of secondary oxidation products of linoleic acid led to the formation of conjugated octadecatriene (COT) fatty acids at levels of about 0.2% or less during the processing of vegetable oils. COTs reportedly have physiological effects [63] at low levels [64]. When coriolic acid (13-hydroxy,9cis,11trans-octadecadienoic acid) was exposed to acidic conditions, it converted to COT. Transmethylation with sodium methoxide/methanol has been recommended [62] to avoid the formation of COT artifacts. ␤-Eleostearic acid (9cis,11trans,13trans-octadecatrienoic acid) and ␣-eleostearic acid (all-trans-9,11,13-octadecatrienoic acid) were extracted from heated cottonseed oil,

Fig. 11. GC–DD–FTIR spectra observed at 8 cm−1 resolution confirmed the identity of the methoxy FAME products (peaks labeled 1–4 in Fig. 10). The inset (upper left corner) shows the corresponding GC trace measured by FTIR. The double bond configuration was determined to be trans (974 cm−1 ) for compounds 1–3, and cis ( 3004 and 729 cm−1 ) for compound 4 (reproduced with kind permission from authors and AOCS Press).

J.A.G. Roach et al. / Analytica Chimica Acta 465 (2002) 207–226

separated and characterized as FAME by GC–MI– FTIR spectroscopy [62]. Characteristic =C–H stretching vibration bands were found at 3026 and 3005 cm−1 for ␣-eleostearic acid, and at 3020 and 3002 cm−1 for ␤-eleostearic acid. Bands at 995, 968 and 730 cm−1 for ␣-eleostearic acid, and at 998 cm−1 for ␤-eleostearic acid were attributed to =C–H out-of-plane deformation. COT derived from sunflower and tung oils were also characterized [62]. 6.4. CLA artifacts and methoxy fatty acid artifacts Acid catalyzed methylation procedures have been found to lead to the formation of CLA artifacts from allylic hydroxy monounsaturated fatty acids [26] and result in the isomerization of cis,trans to trans,trans CLA isomers and the formation of methoxy fatty

223

acid artifacts (Fig. 10). Confirmatory GC–DD–FTIR bands were observed for methoxy fatty acids at 2829 and 1094 cm−1 due to the CH3 symmetric stretch in –O–CH3 and the C–O–C asymmetric stretch, respectively (Fig. 11). 6.5. CLA oxidation products Furan fatty acid (FFA) are reportedly formed by the reaction of singlet oxygen and conjugated dienes [65]. The biological activities of FFA have attracted the attention of many researchers [66–68]. The FFA 9,12-epoxy-9,11-octadecadienoic (F9,12 ) is a plant lipid found in Exocarpus cupressiformis [66] and has been the subject of oxidation studies [67,68]. Interestingly, similar FFA oxidation products were also found in the autoxidation of CLA [69].

Fig. 12. GC–DD–FTIR spectra observed at 8 cm−1 resolution near the GC retention time of one of the methoxy FAME products (peak 3, Figs. 10 and 11) before (bottom spectrum) and after (upper spectrum) autoxidation of the 9,11-18:2 positional isomer to the corresponding furan FAME 9,12-epoxy-9,11-octadecadienoic (F9,12 ). The inset shows the corresponding GC trace as detected by FTIR (reproduced with kind permission from authors and AOCS Press).

224

J.A.G. Roach et al. / Analytica Chimica Acta 465 (2002) 207–226

The oxidation of a commercial reference mixture (Nu-Chek-Prep Inc., Elysian, MN) that consisted of four CLA positional isomers, produced four FFA that were well resolved as FAME by GC [70]. They were determined to be 8,11-epoxy-8,10-octadecadienoic (F8,11 ); 9,12-epoxy-9,11-octadecadienoic (F9,12 ); 10, 13-epoxy-10,12-octadecadienoic (F10,13 ); and 11,14epoxy-11,13-octadecadienoic (F11,14 ) acids. Unique furan ring bands were observed by GC–MI–FTIR [27,28] at 3111, 3031 and 3000 cm−1 (=C–H stretch); 1574 cm−1 (C=C stretch); 1013 cm−1 (C–O stretch) and 780 cm−1 (=C–H out-of-plane bend) and were consistent with furan FAME (Fig. 12).

7. Conclusions GC alone is not capable of separating the isomers of CLA known to occur in chemical and biological matrices. Ag+ HPLC is a necessary complementary tool in the isolation and separation of individual isomers of CLA; see Table 3. Identification of trace amounts of CLA found in biological extracts must rely on MS and IR data in addition to GC and HPLC retention time data. GC–FID is effective in the quantitation of CLA and associated components as FAME. GC–MS of the DMOX derivatives unambiguously identifies positional isomers of CLA with double bonds located at carbons 6,8 through 13,15. GC–FTIR can confirm the double bond configuration (cis,trans; cis,cis; trans,trans) of CLA geometric isomers. GC–FTIR and GC–MS provide structural information for fatty acid reaction products as well as CLA. GC–MI–FTIR Table 3 Selected applications of separation techniques to different CLA matrices Matrix

Technique

Year

Reference

Edible fats and oils Adipose tissue Dairy products Human milk

GC

1991, 1997

[71,72]

GC–Ag+ HPLC GC–Ag+ HPLC GC–Ag+ HPLC

1998 1998 1999,

[38,73] [6,20,38,74] [38,75,76]

GC–Ag+ HPLC GC–Ag+ HPLC

1997, 1997, 1998, 2000 1998 1998,

1999

[14] [25,77]

GC–Ag+ HPLC

1999

Animal organs Dietary supplements Food products

[17]

and GC–DD–FTIR have the requisite sensitivity for capillary GC analyses of biological extracts containing CLA isomers. References [1] M.A. Belury, Nutr. Rev. 53 (1995) 83. [2] J.A. Scimeca, in: M.P. Yurawecz, M.M. Mossoba, J.K.G. Kramer, M.W. Pariza, G. Nelson (Eds.), Advances in Conjugated Linoleic Acid Research, Vol. 1, AOCS Press, Champaign, IL, 1999, pp. 420–443. [3] H.B. MacDonald, J. Am. Coll. Nutr. 19 (2000) 111. [4] M. Pariza, Y. Park, M.E. Cook, Prog. Lipid Res. 40 (2001) 283–298. [5] Y.L. Ha, N.K. Grimm, M.W. Pariza, J. Agric. Food Chem. 37 (1989) 75. [6] N. Sehat, J.K.G. Kramer, M.M. Mossoba, M.P. Yurawecz, J.A.G. Roach, K. Eulitz, K.M. Morehouse, Y. Ku, Lipids 33 (1998) 963. [7] Y. Park, J.M. Storkson, K.J. Albright, W. Liu, M.W. Pariza, Lipids 34 (1999) 235. [8] H.S. Garcia, J.A. Arcos, D.J. Ward, C.G. Hill Jr., Biotechnol. Bioeng. 70 (2000) 587. [9] H.S. Garcia, K.J. Keough, J.A. Arcos, C.G. Hill Jr., J. Dairy Sci. 83 (2000) 371. [10] F. Trimmerman, R. Gaupp, J. Gierke, R. von Kries, W. Adams, A. Sander, German DE 19,718,245 (1997). [11] D. Marini, in: L.M.L. Nollet (Ed.), Food analysis by HPLC, 2nd Edition, Marcel Dekker, New York, 2000, pp. 160–249. [12] S. Banni, E. Angioni, V. Casu, M.P. Melis, G. Carta, F.P. Corongiu, H. Thompson, C. Ip, Carcinogenesis 20 (1999) 1019. [13] S. Banni, E. Angioni, G. Carta, V. Casu, M. Deiana, M.A. Dessi, L. Lucchi, M.P. Melis, A. Rosa, Vargiolu, F.P. Corongiu, in: M.P. Yurawecz, M.M. Mossoba, J.K.G. Kramer, M.W. Pariza, G. Nelson (Eds.), Advances in Conjugated Linoleic Acid Research, Vol. 1, AOCS Press, Champaign, IL, 1999, pp. 307–318. [14] J.K.G. Kramer, N. Sehat, M.E.R. Dugan, M.M. Mossoba, M.P. Yurawecz, J.A.G. Roach, K. Eulitz, J.A. Aalhus, A.L. Schaefer, Y. Ku, Lipids 33 (1998) 549. [15] J.K.G. Kramer, N. Sehat, J. Fritsche, M.M. Mossoba, K. Eulitz, M.P. Yurawecz, Y. Ku, in: M.P. Yurawecz, M.M. Mossoba, J.K.G. Kramer, M.W. Pariza, G.J. Nelson (Eds.), Advances in Conjugated Linoleic Acid Research, Vol. 1, AOCS Press, Champaign, IL, 1999, pp. 83–109. [16] J.K.G. Kramer, J. Zhou, Eur. J. Lipid Sci. Technol. 103 (2001) 594, 600. [17] J. Fritsche, R. Rickert, H. Steinhart, M.P. Yurawecz, M.M. Mossaba, N. Sehat, J.A.G. Roach, Y. Ku, Fett/Lipid 101 (1999) 272. [18] A.L. Davis, G.P. McNeill, D.C. Caswell, Chem. Phys. Lipid 97 (1999) 155. [19] N.C. Shantha, E.A. Decker, Z. Ustunol, J. Am. Oil Chem. Soc. 69 (1992) 425.

J.A.G. Roach et al. / Analytica Chimica Acta 465 (2002) 207–226 [20] J.K.G. Kramer, V. Fellner, M.E.R. Dugan, E.D. Sauer, M.M. Mossoba, M.P. Yurawecz, Lipids 32 (1997) 1219. [21] J. Folch, M. Lees, G.H. Sloane Stanley, J. Biol. Chem. 226 (1957) 497. [22] E.G. Bligh, W.J. Dyer, Can. J. Biochem. Physiol. 37 (1959) 911. [23] W.W. Christie, High-Performance Liquid Chromatography and Lipids. A Practical Guide, Pergamon Press, Oxford, UK, 1987. [24] J.K.G. Kramer, R.C. Fouchard, E.R. Farnworth, Lipids 18 (1983) 896. [25] N. Sehat, M.P. Yurawecz, J.A.G. Roach, M.M. Mossoha, J.K.G. Kramer, Y. Ku, Lipids 33 (1998) 217. [26] M.P. Yurawecz, J.K. Hood, J.A.G. Roach, M.M. Mossoba, D.H. Daniels, Y. Ku, J. Am. Oil Chem. Soc. 71 (1994) 1149. [27] R.O. Adlof, in: R.E. McDonald, M.M. Mossoba (Eds.), New Techniques and Applications in Lipid Analysis, AOCS Press, Champaign IL, 1997 pp. 256–265. [28] K. Eulitz, M.P. Yurawecz, N. Sehat, J. Fritsche, J.A.G. Roach, M.M. Mossoba, J.K.G. Kramer, R.O. Adlof, Y. Ku, Lipids 34 (1999) 873. [29] E. Ostrowska, F.R. Dunshea, M. Muralitharan, R. Cross, Lipids 35 (2000) 1147. [30] B. Nikolova-Damyanova, S. Momchilova, W.W. Christie, J. High Resolut. Chromatogr. 23 (2000) 348. [31] M.P. Yurawecz, J.K.G. Kramer, Y. Ku, in: M.P. Yurawecz, M.M. Mossoba, J.K.G. Kramer, M.W. Pariza, G.J. Nelson (Eds.), Advances in Conjugated Linoleic Acid Research, Vol. I, AOCS Press, Champaign, IL, 1999, pp. 64– 82. [32] B. Nikolova-Damyanova, B.G. Herslof, W.W. Christie, J. Chromatogr. 609 (1992) 133. [33] R.G. Jensen, A.M. Ferris, C.J. Lammi-Keefe, J. Dairy Sci. 74 (1991) 3228. [34] J.Y. Zhang, Q.T. Yu, B.N. Liu, Z.H. Huang, Biomed. Environ. Mass Spectrom. 15 (1988) 33. [35] J.A.G. Roach, M.P. Yurawecz, J.K.G. Kramer, M.M. Mossoba, K. Eulitz, Y. Ku, Lipids 35 (2000) 797. [36] D. Precht, J. Molkentin, Milchwissenschaft 55 (2000) 687. [37] F. Dionisi, P.A. Golay, M. Elli, L.B. Fray, Lipids 34 (1999) 1107. [38] M.P. Yurawecz, J.A.G. Roach, N. Sehat, M.M. Mossoba, J.K.G. Kramer, J. Fritsche, H. Steinhart, Y. Ku, Lipids 33 (1998) 803. [39] V. Spitzer, in: M.P. Yurawecz, M.M. Mossoba, J.K.G. Kramer, M.W. Pariza, G.J. Nelson (Eds.), Advances in Conjugated Linoleic Acid Research, Vol. 1, AOCS Press, Champaign, IL, 1999, pp. 110–125. [40] G. Dobson, J. Am. Oil Chem. Soc. 75 (1998) 137. [41] D.C. Young, P. Vouros, B. Decosta, M.F. Holick, Anal. Chem. 59 (1987) 1954. [42] G.N. Smith, M. Taj, J.M. Braganza, Free Radic. Biol. Med. 10 (1991) 13–21. [43] J.L. Sebedio, P. Juaneda, G. Dobson, I. Ramilison, J.C. Martin, J.M. Chardigny, W.W. Christie, Biochim. Biophys. Acta 1345 (1997) 5.

225

[44] M.M. Mossoba, R.E. McDonald, J.A.G. Roach, D.D. Fingerhut, M.P. Yurawecz, N. Sehat, J. Am. Oil Chem. Soc. 74 (1997) 125–130. [45] J.A.G. Roach, in: M.P. Yurawecz, M.M. Mossoba, J.K.G. Kramer, M.W. Pariza, G. Nelson (Eds.), Advances in Conjugated Linoleic Acid Research, Vol. 1, AOCS Press, Champaign, IL, 1999, pp. 126–140. [46] L. Fay, U. Richli, J. Chromatogr. 541 (1991) 89. [47] V. Spitzer, Prog. Lipid Res. 35 (1997) 387. [48] J.E. Jackson, R.F. Paschke, W. Tolberg, H.M. Boyd, D.H. Wheeler, J. Am. Oil Chem. Soc. 29 (1952) 229. [49] J.R. Chipault, J.M. Hawkins, J. Am. Oil Chem. Soc. 36 (1959) 535. [50] C.R. Scholfield, J. Am. Oil Chem. Soc. 51 (1974) 33. [51] R. White, Chromatography/Fourier transform infrared spectroscopy and its applications, Practical Spectroscopic Series, Vol. 10, Marcel Dekker, New York, 1998. [52] M.M. Mossoba, R.E. McDonald, J.Y.T. Chen, D.J. Armstrong, S.W. Page, J. Agric. Food Chem. 38 (1990) 86. [53] M.M. Mossoba, R.E. McDonald, D.J. Armstrong, S.W. Page, J. Agric. Food Chem. 39 (1991) 695. [54] M.M. Mossoba, R.E. McDonald, D.J. Armstrong, S.W. Page, J. Chromatogr. Sci. 29 (1991) 324. [55] M.M. Mossoba, R.E. McDonald, A.R. Prosser, J. Agric. Food Chem. 41 (1993) 1998. [56] M.M. Mossoba, M.P. Yurawecz, J.A.G. Roach, H.S. Lin, R.E. McDonald, B.D. Flickinger, E.G. Perkins, Lipids 29 (1994) 893. [57] M.M. Mossoba, M.P. Yurawecz, B.D. Flickinger, H.S. Lin, R.E. McDonald, E.G. Perkins, American Laboratory, September 1995, p. 16K. [58] M.M. Mossoba, M.P. Yurawecz, R.E. McDonald, B.D. Flickinger, E.G. Perkins, J. Agric. Food Chem. 44 (1996) 3193. [59] M.M. Mossoba, M.P. Yurawecz, J.K.G. Kramer, K.D. Eulitz, J. Fritsche, N. Sehat, J.A.G. Roach, Y. Ku, in: M.P. Yurawecz, M.M. Mossoba, J.K.G. Kramer, M.W. Pariza, G. Nelson (Eds.), Advances in Conjugated Linoleic Acid Research, Vol. 1, AOCS Press, Champaign, IL, 1999, pp. 141–151. [60] J.L. Sebedio, Le Quere, E. Semon, O. Morin, J. Prevost, A. Grandgirard, J. Am. Oil Chem. Soc. 64 (1987) 1324. [61] J. Fritsche, M.M. Mossoba, M.P. Yurawecz, J.A.G. Roach, N. Sehat, Y. Ku, H. Steinhart, Z. Lebensm. Unters. Forsch. A 205 (1997) 415. [62] M.P. Yurawecz, A.A. Molina, M.M. Mossoba, Y. Ku, J. Agric. Food Chem. 70 (1993) 1093. [63] D.H. Nutgeren, E. Christ-Hazelhof, Prostaglandins 33 (1987) 403. [64] V. Spitzer, J.G.S. Maia, K. Pfeilstiker, J. Am. Oil Chem. Soc. 68 (1991) 183. [65] R. Ideses, A. Shani, J.T. Klug, Chem. Ind. 19 (1982) 409. [66] L.J. Morris, M.O. Marshall, V. Kelly, Tetrahedron Lett. 36 (1996) 4249. [67] G. Rosenblat, M. Tabak, M.S.F. Lie Ken Jie, I. Neeman, J. Am. Oil Chem. Soc. 70 (1993) 501.

226

J.A.G. Roach et al. / Analytica Chimica Acta 465 (2002) 207–226

[68] R.F. Boyer, D. Litts, J. Kostishak, R.C. Wijesundera, F.D. Gunstone, Chem. Phys. Lipids 25 (1979) 237. [69] M.P. Yurawecz, J.K. Hood, M.M. Mossoba, J.A.G. Roach, Y. Ku, Lipids 30 (1995) 595. [70] M.P. Yurawecz, N. Sehat, M.M. Mossoba, J.A.G. Roach, Y. Ku, in: R.E. McDonald, M.M. Mossoba (Eds.), New Techniques and Applications in Lipid Analysis, AOCS Press, Champaign, IL, 1997, pp. 183–215. [71] M.M. Mossoba, R.E. McDonald, D.J. Armstrong, S.W. Page, J. Chromatogr. Sci. 29 (1991) 324. [72] D. Precht, J. Molkentin, Fett/Lipid 99 (1997) 319.

[73] J. Fritsche, M.M. Mossoba, M.P. Yurawecz, J.A.G. Roach, N. Sehat, Y. Ku, H. Steinhart, Z. Lebensm. Unters. Forsch. A 205 (1997) 415. [74] F. Lavillonniére, J.C. Martin, P. Bougnoux, J.-L. Sébédio, J. Am. Oil Chem. Soc. 75 (1998) 343. [75] D. Precht, J. Molkentin, Nahrung 43 (1999) 233. [76] D. Precht, J. Molkentin, Eur. J. Lipid Sci. Technol. 102 (2000) 102. [77] M.P. Yurawecz, N. Sehat, J.A.G. Roach, M.M. Mossoba, J.K.G. Kramer, Y. Ku, Fett/Lipid 101 (1999) 277.