High resolution mass spectral data from the analysis of copper chlorophylls and copper chlorophyll degradation products in bright green table olives.

This publication reports high resolution mass spectral data for copper chlorophyll and copper chlorophyll degradation products extracted from bright green table olives. These data support analyte identifications made in "Quantitation of copper chlorophylls in green table olives by ultra-high-performance liquid chromatography with inductively coupled plasma isotope dilution mass spectrometry" in the Journal of Chromatography A (Petigara Harp et al., 2020 [1]). Table olive pigments, divided into lipophilic and hydrophilic fractions by liquid-liquid repartition, were separated by ultra-high-performance liquid chromatography and detected by visible wavelength absorbance and high resolution mass spectrometry, using an Orbitrap HF with positive electrospray ionization. Full-scan mass spectra were acquired to assign pigment chemical formulae. Fragment-rich higher-energy collisional dissociation tandem mass spectra were acquired to facilitate structural assignments. Extracted ion chromatograms, full-scan, and tandem mass spectra obtained from representative lipophilic and hydrophilic green table olive extracts are presented in Figures 1-6. Annotated mass spectra comparing experimental and calculated isotope distributions, .raw mass spectral data files, and experimental details linking .raw data files to annotated spectra are provided as Supplementary Material. Spectra extracted from these native data files can be added to mass spectral libraries for use in other studies. Access to native data files uniquely enables rigorous data examination (e.g., molecular ion isotopic distribution, effective mass resolution, presence of overlapping ion series) and use in ways that are not possible when spectra are otherwise reported in simple tables listing mono-isotopic peaks and mass errors. Mass spectra reported here can be used to design multiple-reaction monitoring methods to detect these bright green pigments in agricultural food commodities and finished products.

Oceanivirga miroungae sp. nov., isolated from oral cavity of northern elephant seal (Mirounga angustirostris).

Two independent strains of a Leptotrichia species (ES3154-GLUT and ES2714_GLU) were isolated from the oral cavity of northern elephant seals (Mirounga angustirostris) that were admitted to The Marine Mammal Centre facilities in California, USA. The strains were isolated from oral swabs by cultivation in PPLO broth supplemented with serum, penicillin and colistin in anaerobic conditions. The strains were Gram-negative, pleomorphic, indole-, oxidase- and catalase-negative, non-spore-forming, non-motile rods/coccobacilli in short chains. The 16S rRNA gene sequence of these strains shared 94.42 % nucleotide similarity with Oceanivirga salmonicida AVG 2115T but demonstrated ≤86.00-92.50 % nucleotide similarity to the 16S rRNA genes of other species of the family Leptotrichiaceae. The genome was sequenced for strain ES3154-GLUT. Average nucleotide identity values between strain ES3154-GLUT and 15 type strain genomes from the family Leptotrichiaceae ranged from 66.74 % vs. Sebaldella termitidis to 73.35 % vs. O. salmonicida. The whole genome phylogeny revealed that the novel species was most closely related to O. salmonicida AVG 2115T. This relationship was also confirmed by nucleotide similarity and multilocus phylogenetic analyses employing various housekeeping genes (partial 23S rRNA, rpoB, rpoC, rpoD, polC, adh, gyrA and gyrB genes). Chemotaxonomic and phenotypical features of strain ES3154-GLUT were in congruence with closely related members of the family Leptotrichiaceae, represented by similar enzyme profiles and fatty acid patterns. MALDI-TOF MS analysis was capable to clearly discriminate strain ES3154-GLUT from all currently described taxa of the family Leptotrichiaceae. Based on these data, we propose a novel species of the genus Oceanivirga, for which the name Oceanivirga miroungae sp. nov. is proposed with the type strain ES3154-GLUT (=DSM 109740T=CCUG 73653T=ATCC TSD-189T=NCTC 14411T) and one representative strain ES2714_GLU. The G+C content is 26.82 %, genome size is 1 356 983 bp.

Structural Determination and Occurrence in Ahiflower Oil of Stearidonic Acid Trans Fatty Acids.

Several marine oils and seed oils on the market contain relevant quantities of stearidonic acid (18:4n-3, SDA). The formation of 18:4n-3 trans fatty acids (tFA) during the refining of these oils necessitates the development of a method for their quantification. In this study, 18:4n-3 was isolated from Ahiflower and isomerized to obtain its 16 geometric isomers. The geometric isomers of 18:4n-3 were isolated by silver ion HPLC (Ag+ -HPLC) and characterized by partial reduction with hydrazine followed by gas chromatography analysis. The elution order of all 16 isomers was established using a 100 m × 0.25 mm 100% poly(biscyanopropyl siloxane) capillary column and at the elution temperature of 180 °C. The 4 mono-trans-18:4n-3 isomers produced during the refining of oils rich in 18:4n-3 were chromatographically resolved from each other, but c6,t9,c12,c15-18:4 coeluted with the tetra-cis isomer. These 2 fatty acids (FA) were resolved by reducing the separation temperature to 150 °C, but this change caused tetra-cis-18:4n-3 to coelute with t6,c9,c12,c15-18:4. Combining the results from 2 isothermal separations (180 and 150 °C) was necessary to quantify the 4 mono-trans 18:4n-3 FA in Ahiflower oil.

Neisseria zalophi sp. nov., isolated from oral cavity of California sea lions (Zalophus californianus).

Three independent strains of Neisseria sp. were isolated from the oral cavity of California sea lions (Zalophus californianus) that were admitted to The Marine Mammal Center facilities in California, USA. The strains were isolated from oral swabs by cultivation on Trypticase Soy agar with 5% sheep blood under aerobic conditions. The 16S rRNA gene sequence of these three strains shared 99% similarity, but demonstrated only 97-98% nucleotide similarity to the phylogenetically closest relatives such as N. canis, N. zoodegmatis, N. animaloris, and N. dumasiana. These three strains also shared 99% sequence similarity of their rplF, rpoB, and gyrB gene sequences. Based on the biochemical tests alone (i.e., without genetic analysis of housekeeping genes), it is difficult to discriminate this novel species from N. canis; however, it can be easily discriminated from all phylogenetically closely related species using the sequencing analysis of its housekeeping genes (e.g., rplF, rpoB, or gyrB genes). Thus, genetic testing is indispensable for accurate identification of this species in a routine laboratory practice. The species is an obligate aerobe and able to grow in Mueller-Hinton broth supplemented with 6% NaCl, but the phylogenetically closely related species (N. canis, N. zoodegmatis, N. animaloris, and N. dumasiana) were not. Based on these phenotypic and genotypic characteristics and phylogenetic data, we conclude that these new strains represent a novel species of the genus Neisseria, for which the name Neisseria zalophi sp. nov. is proposed. The type strain is CSL 7565T (= ATCC BAA2455T = DSM 102031T).

Evaluation of poly(90% biscyanopropyl/10% cyanopropylphenyl siloxane) capillary columns for the gas chromatographic quantification of trans fatty acids in non-hydrogenated vegetable oils.

Current gas chromatographic (GC) methods for the analysis of fatty acids (FA) were optimized primarily for the quantification of the trans 18:1 FAs (18:1 tFAs) produced during the partial hydrogenation of fats and oils. Recent regulatory action regarding the application of partial hydrogenation in the processing of edible fats and oils may reshape the FA composition of these products. The higher content in 18:3 tFAs compared to 18:1 tFAs of most refined non-hydrogenated vegetable oils (RNHVO), and the challenge in their quantification applying current methods, suggest the need for new methodologies. This manuscript describes a simple GC method for the analysis of FAs in RNHVOs utilizing a 100m (0.25mm I.D.) capillary column coated with poly(90% biscyanopropyl/10% cyanopropylphenyl siloxane) (90% BCS). The optimization of the chromatographic conditions and the detection of co-eluting compounds were carried out by applying comprehensive two dimensional gas chromatography with online reduction (GC-OR×GC). Results showed that 90% BCS capillary columns operated at the elution temperature of 162°C provide the separation of the 18:1, 18:2 and 18:3 tFAs, contained in RNHVOs, from other components. A minor constituent of Canola oil, 16:3n-3, partially co-eluted with trans-18:1 FAMEs. This simple GC method showed the ability to measure trans-fat in RNHVOs at the level of 0.5g/100g, providing comparable quantitative results to the more complex GC×GC methodology.

Profile of trans fatty acids (FAs) including trans polyunsaturated FAs in representative fast food samples.

The content of trans fat in foods is most commonly determined by summing the levels of individual trans fatty acids (FAs), analyzed as FA methyl esters (FAME) by gas chromatography. Current Official Methods of the American Oil Chemists' Society (AOCS) enable quantitation of total trans fat in foods but were not designed for the determination of transFA isomeric compositions. In the present study, the content of trans fat in 32 representative fast food samples ranged from 0.1 to 3.1 g per serving, as determined according to AOCS Official Method Ce 1j-07. Further analysis of FAME using the 200 m SLB-IL111 ionic liquid column yielded quantitative results of total, trans, saturated, and cis unsaturated fat that were comparable to those of Method Ce 1j-07 and also allowed for the complementary determination of individual trans 18:1, trans 18:2, and trans 18:3 FA isomeric compositions under conditions suitable for routine sample analysis.

Evaluation of highly polar ionic liquid gas chromatographic column for the determination of the fatty acids in milk fat.

The SLB-IL111, a new ionic liquid capillary column for gas chromatography available from Supelco Inc., was recently shown to provide enhanced separation of unsaturated geometric and positional isomers of fatty acid (FAs) when it was compared to cyanopropylsiloxane (CPS) columns currently recommended for the analysis of fatty acid methyl esters (FAMEs). A 200 m SLB-IL111 capillary column, operated under a combined temperature and eluent flow gradient, was successfully used to resolve most of the FAs contained in milk fat in a single 80 min chromatographic separation. The selected chromatographic conditions provided a balanced, simultaneous separation of short-chain (from 4:0), long-chain polyunsaturated fatty acids (PUFAs), and most of the unsaturated FA positional/geometric isomers contained in milk fat. Among the monounsaturated fatty acids (MUFAs), these conditions separated t11-18:1 and t10-18:1 FAs, the two most abundant trans fatty acids (t-FA) contained in most dairy products. These t-FAs reportedly have different biological activities. The conjugated linoleic acid (CLA) isomers commonly found in dairy products were separated from each other, including t7,c9-18:2 from c9,t11-18:2, which eliminated the need for their complementary silver ion HPLC analysis. The application of the SLB-IL111 column provided a complementary elution profile of FAMEs to those obtained by CPS columns, allowing for a more comprehensive FA analysis of total milk fat. The FAMEs were identified by the use of available reference materials, previously synthesized and characterized reference mixtures, and prior separations of the milk fat FAMEs by silver ion chromatography based on the number/geometry of double bonds.

Comparison of separations of fatty acids from fish products using a 30-m Supelcowax-10 and a 100-m SP-2560 column.

Commercial fish oils and foods containing fish may contain trans and/or isomerized fatty acids (FA) produced during processing or as part of prepared foods. The current American Oil Chemists' Society (AOCS) official method for marine oils (method Ce 1i-07) is based on separation by use of poly(ethylene glycol) (PEG) columns, for example Supelcowax-10 or equivalent, which do not resolve most unsaturated FA geometric isomers. Highly polar 100-m cyanopropyl siloxane (CPS) columns, for example SP-2560 and CP Sil 88 are recommended for separation of geometric FA isomers. Complementary separations were achieved by use of two different elution temperature programs with the same CPS column. This study is the first direct comparison of the separations achieved by use of 30-m Supelcowax-10 and 100-m SP-2560 columns for fatty acid methyl esters (FAME) prepared from the same fish oil and fish muscle sample. To simplify the identification of the FA in these fish samples, FA were fractionated on the basis of the number and type of double bonds by silver-ion solid-phase extraction (Ag⁺-SPE) before GC analysis. The results showed that a combination of the three GC separations was necessary to resolve and identify most of the unsaturated FA, FA isomers, and other components of fish products, for example phytanic and phytenic acids. Equivalent chain length (ECL) values of most FAME in fish were calculated from the separations achieved by use of both GC columns; the values obtained were shown to be consistent with previously reported values for the Supelcowax-10 column. ECL values were also calculated for the FA separated on the SP-2560 column. The calculated ECL values were equally valid under isothermal and temperature-programmed elution GC conditions, and were valuable for confirmation of the identity of several unsaturated FAME in the fish samples. When analyzing commercially prepared fish foods, deodorized marine oils, or foods fortified with marine oils it is strongly recommended that quantitative data acquired by use of PEG columns is complemented with data obtained from separations using highly polar CPS columns.

Employing ultra high pressure liquid chromatography as the second dimension in a comprehensive two-dimensional system for analysis of Stevia rebaudiana extracts.

Stevia rebaudiana extracts and plant materials are increasingly used as natural sweeteners. Polyphenolic and stevioside compounds contained in S. rebaudiana extracts were separated by comprehensive LC. A polyamine column operated in normal phase mode was used for the first dimension separation (D1), and a UHPLC C18 column operated in reversed phase mode was used for the second dimension separation (D2). The sub-2 µm column (2.1 mm × 30 mm, maintained at 70°C) and the UHPLC pump employed for D2 elution allowed a separation/cycle time of 20 s, with a backpressure oscillating between 805 and 922 bar at 3.4 mL/min. The reduced D2 cycle time allowed 3-12 D2 samplings for each peak eluted by D1. Polyphenolic and stevioside compounds were identified by combining the information coming from the position of the compounds in the 2D plot and UV spectra with that of reference materials.

Separation characteristics of fatty acid methyl esters using SLB-IL111, a new ionic liquid coated capillary gas chromatographic column.

The ionic liquid SLB-IL111 column, available from Supelco Inc., is a novel fused capillary gas chromatography (GC) column capable of providing enhanced separations of fatty acid methyl esters (FAMEs) compared to the highly polar cyanopropyl siloxane columns currently recommended for the separation of cis- and trans isomers of fatty acids (FAs), and marketed as SP-2560 and CP-Sil 88. The SLB-IL111 column was operated isothermal at 168°C, with hydrogen as carrier gas at 1.0 mL/min, and the elution profile was characterized using authentic GC standards and synthetic mono-unsaturated fatty acids (MUFAs) and conjugated linoleic acid (CLA) isomers as test mixtures. The SLB-IL111 column provided an improved separation of cis- and trans-18:1 and cis/trans CLA isomers. This is the first direct GC separation of c9,t11- from t7,c9-CLA, and t15-18:1 from c9-18:1, both of which previously required complimentary techniques for their analysis using cyanopropyl siloxane columns. The SLB-IL111 column also provided partial resolution of t13/t14-18:1, c8- from c6/c7-18:1, and for several t,t-CLA isomer pairs. This column also provided elution profiles of the geometric and positional isomers of the 16:1, 20:1 and 18:3 FAMEs that were complementary to those obtained using the cyanopropyl siloxane columns. However, on the SLB-IL111 column the saturated FAs eluted between the cis- and trans MUFAs unlike cyanopropyl siloxane columns that gave a clear separation of most saturated FAs. These differences in elution pattern can be exploited to obtain a more complete analysis of complex lipid mixtures present in ruminant fats.

Dietary trans fatty acid isomers differ in their effects on mammary lipid metabolism as well as lipogenic gene expression in lactating mice.

The biological activities and mechanisms of action of individual transoctadecenoic acids (trans-18:1 FA) have not been completely elucidated. We examined the effects of several individual trans-18:1 FA isomers and trans-10, cis-12 conjugated linoleic acid (CLA) on fat synthesis, and expression of lipogenic genes in mammary and liver tissue in lactating mice. From d 6 to 10 postpartum, 30 lactating C57BL/6J mice were randomly assigned to either a control (CTR) diet containing 20 g/kg oleic acid or diets in which the oleic acid was either completely replaced by partially hydrogenated vegetable oil (PHVO), trans-7 18:1 (T7), trans-9 18:1 (T9), or trans-11 18:1 (T11) or partially replaced with 6.66 g/kg trans-10, cis-12 CLA. Milk fat percentage was decreased by CLA (44%), T7 (27%), and PHVO (23%), compared with CTR. In the mammary gland, CLA decreased the expression of genes related to de novo FA synthesis, desaturation, triacylglycerol formation, and transcriptional regulation. PHVO and T7 diets decreased the expression of 1-acylglycerol-3-phosphate O-acyltransferase and thyroid hormone responsive SPOT14 homolog (THRSP) mRNA. In contrast, dietary trans FA (tFA) did not affect hepatic lipogenic gene expression. However, mice fed CLA, T7, and PHVO diets had increased liver weights due to hepatic steatosis. Trans-7 18:1 was extensively desaturated to trans-7, cis-9 CLA in mammary and liver tissues. Dietary trans-7 18:1 could lead to milk fat depression in lactating mice, possibly through its desaturation product trans-7, cis-9 CLA. Also, the differences between the effects of trans-10, cis-12 CLA and other tFA could be attributed to its effects on carbohydrate response element binding protein and PPARgamma, in addition to sterol regulatory element binding transcription factor 1c and THRSP.

Review of methods for preparation and gas chromatographic separation of trans and cis reference fatty acids.

In recent years, several countries have implemented new regulations regarding the limitation or labeling of the trans fatty acid (TFA) content of foods and dietary supplements. GC methods for fatty acid (FA) analysis have been updated by improving the separation of TFAs from other FAs, especially trans- and cis-18:1, and by focusing more attention on the FAs contained in fats and oils in lower amounts. FA analysis is affected by the limited availability of reference materials. Identifications are frequently made simply by comparison with separations reported in the literature. This report describes the preparation of mixtures containing fatty acid methyl esters (FAMEs) that are not available as reference materials. These mixtures can be used for FAME identifications. The prepared mixtures are analyzed under the experimental conditions of the American Oil Chemists' Society (AOCS) Official Method Ce 1h-05 and AOCS Recommended Practice Ce 1j-07.

Recent studies on selected botanical dietary supplement ingredients.

The market for botanical dietary supplements in the US has grown rapidly during the last 15 years. Use of newly introduced botanical ingredients has often outpaced an adequate scientific understanding of the ingredients themselves. This may lead to problems, including misidentification, mislabeling, adulteration, and toxicity related to the intended ingredient or one substituted for it. This article reviews recent work with several botanical ingredients (Ephedra, Citrus species, Hoodia gordonii, Teucrium, isoflavones) that illustrates the complexity of the current situation and approaches that contribute to ensuring the quality of botanical ingredients. Recent work with contamination of botanical products by mycotoxins is also reviewed. The need for tools for botanical authentication and methods for reproducible extraction of bioactive constituents is critical. Such tools, and improved analytical techniques for identifying potentially bioactive constituents in fresh plant material and in concentrated extracts and for detection of hazardous contaminants, are expected to improve the overall quality and safety of botanical dietary supplement ingredients.

Analysis of isoflavones in foods and dietary supplements.

Isoflavones are phytochemicals found in many plants. Because of their structural similarity to beta-estradiol, health benefits of isoflavones have been evaluated in age-related and hormone-dependent diseases. Daidzein, genistein, and glycitein are present as free forms or derivatives in foods containing soy or soy protein extracts. The analysis of isoflavones has become more complex, because preparations contain isoflavones from multiple sources (e.g., red clover, kudzu). Red clover contains primarily formononetin and biochanin A, while kudzu extracts, which are becoming increasingly common in dietary supplements, contain puerarin and daidzein, among other components. Isoflavones are present in foods and dietary supplements as free compounds, glucoside derivatives, 6"-O-malonyl-7-O-beta-D-glucoside derivatives, and 6"-O-acetyl-7-O-beta-D-glucoside derivatives. High-performance liquid chromatography (HPLC)/tandem mass spectrometry has been applied to the identification of isoflavone derivatives based on the fragmentation pattern of the parent ion, providing high selectivity and sensitivity in the quantitation of isoflavones in complex mixtures. HPLC with ultraviolet detection is often chosen for routine analysis, but a preliminary acid or basic hydrolysis of isoflavone derivatives is often required for the investigation of samples containing extracts from multiple sources. Several internal standards have been used in the analysis of isoflavones from a single botanical source (e.g., soy, red clover), but the identification of a general internal standard remains a challenging process.

Determination of isoflavones in dietary supplements containing soy, Red Clover and kudzu: extraction followed by basic or acid hydrolysis.

Isoflavones are phytochemicals found in many plants. Because of their structural similarity to beta-estradiol, health benefits of isoflavones have been evaluated in age-related and hormone-dependent diseases. Dietary supplement preparations contain extracts from soy, Red Clover and kudzu. Soy products contain primarily genistein, daidzein, and glycitein, while Red Clover products contain primarily formononetin and biochanin A. Kudzu extracts contain puerarin and daidzein among other components. Previous methods of analysis focused on the determination of isoflavones from a single botanical source, while dietary supplements are often a blend of extracts from different plants. We developed a method for the analysis of isoflavones in dietary supplements regardless of their botanical composition, using HPLC-PDA because of its applicability to routine analysis. Isoflavones are found as free compounds, glucoside derivatives, 6''-O-malonyl-beta-d-glucoside and 6''-O-acetyl-beta-d-glucoside derivatives. In this study, the samples were extracted at room temperature with 50:50 (v/v) MeCN/water, and then analyzed before and after hydrolyzing the isoflavones by acid or basic digestion. 2'-Methoxy-flavone and 6-methoxy-flavone were used as internal standards and were added together to every sample. Daidzein, glycitein, genistein, puerarin, calycosin, pratensein, pseudobaptigenin, formononetin, biochanin A and prunetin were among the isoflavones determined.

Relative retention order of all isomers of cis/trans conjugated linoleic acid FAME from the 6,8- to 13,15-positions using silver ion HPLC with two elution systems.

CLA, defined as one or more octadecadienoic acids (18:2) with conjugated double bonds, has been reported to be active in a number of biological systems. GC and silver ion HPLC (Ag(+)-HPLC) have been the primary techniques for identifying specific CLA isomers in both foods and biological extracts. Recently, GC relative retention times were reported for all c,c, c/t (c,t and tc), and t,t CLA FAME from the 6,8- to the 13,15-positions in octadecadienoic acid (18:2). Presented here is the relative retention order of the same CLA FAME using Ag(+)-HPLC with two different elution systems. The first elution system, consisting of 0.1% acetonitrile/0.5% diethyl ether (DE)/hexane, has been used previously to monitor CLA composition in foods. Also presented here is the retention order of CLA FAME using 2% acetic acid/hexane elution solvent, which has advantages of more stable retention volumes and a complementary elution order of CLA FAME isomers. The data are reported using retention volumes (RV) adjusted for toluene, an estimator for dead volume, and relative to c9,t11-18:2. Measurement of relative RV in the analysis of 88 samples of cow plasma, milk, and rumen fluids using Ag(+)-HPLC is also presented here. The % CV ranged from 1.04 to 1.62 for t,t isomers and from 0 to 0.48 for c/t isomers.

Identification and characterization of conjugated fatty acid methyl esters of mixed double bond geometry by acetonitrile chemical ionization tandem mass spectrometry.

Fatty acids with conjugated double bonds have attracted great interest because of their reported potent bioactivities. However, there are currently no rapid methods for their structural characterization. We report here a convenient mass spectrometry-based strategy to establish double bond geometry by analysis of collisional dissociation products of cis/trans and trans/cis conjugated linoleic acids (CLAs), as methyl esters, and to distinguish CLAs from homoallylic (methylene-interrupted) fatty acids in a single-stage mass spectrum. A series of CLA standards with double bond positions 6,8; 7,9; 8,10; 9,11; 10,12; 11,13; 12,14; and 13,15, with all four possible geometries (cis/trans; trans/cis; cis/cis; trans/trans) were analyzed. The m/z 54 (1-methyleneimino)-1-ethenylium ion, generated by self-reaction of acetonitrile under chemical ionization conditions, reacts with unsaturated fatty acids to yield an [M + 54]+ ion, which decomposes in the single-stage mass spectrum by loss of neutral methanol to form [M + 54 - 32]+. The ratio of [M + 54]+/[M + 54 - 32]+ in the single-stage mass spectra of CLA isomers is 1 order of magnitude less than for homoallylic diene FAME. Collisional dissociation of the [M + 54]+ ion yields two diagnostic ions that contain the alpha- and omega-carbon atoms and is characteristic of double bond position in the analyte. The fragment vinylic to the trans double bond is significantly more abundant than that for the cis double bond, revealing double bond geometry. The ratio of alpha to we diagnostic ion abundances is >4.8 for cis/trans isomers, <0.5 for trans/cis isomers, and 0.7-3.2 for cis/cis and trans/trans isomers. This method provides a rapid alternative to conventional conjugated fatty acid analysis and, together with complementary elution time information provided by gas chromatography, enables rapid, positive identification of double bond position and geometry in most CLA FAME.

Trans-7,cis-9 CLA is synthesized endogenously by delta9-desaturase in dairy cows.

Cis-9,trans-11 and trans-7,cis-9 CLA are the most prevalent CLA isomers in milkfat. The majority of cis-9,trans-11 CLA is synthesized endogenously by delta9-desaturase. We tested the hypothesis that trans-7,cis-9 CLA originates from endogenous synthesis by inhibiting delta9-desaturase with a source of cyclopropene FA (sterculic oil: SO) or with a trans-10,cis-12 CLA supplement. Experiment 1 (four cows; Latin square) involved four treatments: control, SO, partially hydrogenated vegetable oil (PHVO), and PHVO + SO. Milk, plasma, and rumen fluid were collected. Experiment 2 treatments (four cows) were 0 or 14.0 g/d of 10,12 CLA supplement; milk and plasma were collected. Samples were analyzed by GC and Ag+-HPLC to determine FA. In Experiment 1, SO decreased milkfat content of trans-7,cis-9 CLA by 68 to 71% and cis-9,trans-11 CLA by 61 to 65%. In Experiment 2, the 10,12 CLA supplement decreased milkfat content of trans-7,cis-9 CLA and cis-9,trans-11 by 44 and 25%, respectively. Correcting for the extent of treatment-induced inhibition of delta9-desaturase based on changes in myristic and myristoleic acids, endogenous synthesis of trans-7,cis-9 CLA represented 85 and 102% in Experiments 1 and 2, respectively. Similar corrected values were 77 and 58% for endogenous synthesis of cis-9,trans-11 CLA. Thus, milkfat cis-9,trans-11 CLA was primarily from endogenous synthesis with a minor portion from rumen escape. In contrast, trans-7,cis-9 CLA was not present in rumen fluid in significant amounts. Results indicate this isomer in milkfat is derived almost exclusively from endogenous synthesis via delta9-desaturase.