Determination of trans octadecenoic acids by silver-ion chromatography-gas liquid chromatography: an intercomparison of methods

Several silver-ion chromatography-gas liquid chromatography (GLC) techniques for the determination of trans octadecenoic acids in partially hydrogenated vegetable fats were collaboratively evaluated. Twelve laboratories participated in the study. All collaborators used high polarity fused silica capillary columns for the separation of fatty acid methyl esters by GLC; 7 collaborators isolated trans monoenes by silver-ion liquid chromatography (Ag-LC) and the remainder used silver-ion thin-layer chromatography (Ag-TLC). Eight artificially prepared materials [soybean oil spiked with either methyl elaidate, trielaidin, or trans octadecenoates isolated from partially hydrogenated sunflower oil (PHSO)] and 2 matrix materials (PHSO and a blend of PHSO and palm oil) served as test samples. Ag-TLC and Ag-LC proved to be equivalent techniques for the prefractionation of trans monoenes. Recovery of methyl elaidate, trielaidin, or trans octadecenoates isolated from PHSO varied between 97.9-103.7% over a concentration range of 1 to 30 g trans fatty acids/100 g. Reproducibility relative standard deviation (RSDR) for the spiked samples were in the range of 3.1-8.6% for 30-1% trans monoene content. For the 2 matrix samples (mean 3.75 and 19.08% trans monoene content) RSDR was 13.2 and 3.6%. The hyphenated techniques tested proved to be highly accurate and sufficiently precise methods for the determination of trans monoenes in partially hydrogenated vegetable fats. Procedural variations of the silver-ion chromatography prefractionation step (separation mode, mobile phase, and detection systems) did not significantly influence the results of the test. Therefore, silver-ion chromatography is a robust method, which does not need rigorous standardization to achieve high precision of test results. A further benefit of the hyphenated technique is that any type of efficient polar capillary column can be used.     

Methods for analysis of conjugated linoleic acids and trans-18:1 isomers in dairy fats by using a combination of gas chromatography, silver-ion thin-layer chromatography/gas chromatography, and silver-ion liquid chromatography

Conjugated linoleic acids (CLA) are octadecadienoic acids (18:2) that have a conjugated double-bond system. Interest in these compounds has expanded since CLA were found to be associated with a number of physiological and pathological responses such as cancer, metastases, atherosclerosis, diabetes, immunity, and body fat/protein composition. The main sources of these conjugated fatty acids are dairy fats. Rumen bacteria convert polyunsaturated fatty acids, especially linoleic and linolenic acids, to CLA and numerous trans- containing mono- and diunsaturated fatty acids. It has been established that an additional route of CLA synthesis in ruminants and monogastric animals, including humans, occurs via delta9 desaturation of the trans-18:1 isomers. To date, a total of 6 positional CLA isomers have been found in dairy fats, each occurring in 4 geometric forms (cis,trans; trans,cis; cis,cis; and trans,trans) for a total of 24. All of these CLA isomers can be resolved only by a combination of gas chromatography (GC), using 100 m highly polar capillary columns, and silver-ion liquid chromatography, using 3 of these 25 cm columns in series. Complete analysis of all the trans-18:1 isomers requires prior isolation of trans monoenes by silver-ion thin-layer chromatography (TLC), followed by GC analysis using the same 100 m capillary columns operated at low temperatures starting from 120 degrees C. These analytical techniques are required to assess the purity of commercial CLA preparations, because their purity will affect the interpretation of any physiological and/or biochemical response obtained. Prior assessment of CLA preparations by TLC is also recommended to determine the presence of any other impurities. The availability of pure CLA isomers will permit the evaluation and analysis of individual CLA isomers for their nutritional and biological activity in model systems, animals, and humans. These techniques are also essential to evaluate dairy fats for their content of specific CLA isomers and to help design experimental diets to increase the level of the desired CLA isomers in dairy fats. These improved techniques are further required to evaluate the CLA profile in monogastric animals fed commercial CLA preparations for CLA enrichment of animal products. This is particularly important because absorption and metabolism will alter the ingested-CLA profile in the animal fed.     

Regiospecific characterisation of the triacylglycerols in animal fats using high performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry

High performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry (HPLC-APCI MS) was applied to the characterisation of triacylglycerols (TAGs) in animal fats. The major TAGs in four fats (beef, chicken, lamb and pork) were identified and positional isomers assigned according to their APCI mass spectra. Beef and lamb fat TAGs were confirmed as containing higher proportions of saturated fatty acids compared with those of chicken and pork. HPLC-APCI MS was also shown to be of value in providing regiospecific information for the fatty acids in individual TAG species. For example, beef and lamb fat were shown to contain both cis- and trans-isomers of the 18:1 fatty acid, whilst chicken and pork contained only the cis-isomer. When the position of fatty acid substitution was determined from the APCI spectra, whilst the cis- 18:1 was predominantly found in the 2-position of the TAG, the trans-18:1 showed a preference for the 1/3-position. Similarly, it was confirmed that although the 2-position of beef, chicken and lamb fat TAGs was dominated by unsaturated fatty acids, in pork fat, a characteristically high proportion of palmitic acid was seen in this position. The TAGs identified compared well with those reported previously. The distributions of 2-position fatty acids seen in lamb and pork fat compared favourably with those obtained by the more traditional method of lipase degradation. Although the distributions for chicken and beef showed some discrepancies, these can be attributed to weaknesses in the quantification procedure or the specificity of the lipase. Overall, the technique of HPLC-APCI MS has been shown to be very powerful for the regiospecific analysis of animal fats.     

Utilisation of reversed-phase high-performance liquid chromatography as an alternative to silver-ion chromatography for the separation of cis- and trans-C18:1 fatty acid isomers

Silver ion-high-performance liquid chromatography (HPLC) has been commonly used for the separation and the analysis of trans-18:1 isomers in partially hydrogenated oils and milk fat. This paper describes an easy HPLC method using two reversed-phase columns. The cis- and trans-18:1 fatty acids isomers as methyl esters were eluted as two separate fractions. The collected fractions were analysed by gas chromatography (GC). The purity of the two fractions were tested by GC-MS and GC-Fourier transform IR.     

Enhanced Resolution of Conjugated Linoleic Acid Isomers by Tandem-Column Silver-Ion High Performance Liquid Chromatography

A commercial mixture of conjugated linoleic acid (CLA) isomers, reportedly consisting of six components, was recently resolved into 12 peaks attributed to CLA isomers using silver-ion high performance liquid chromatography (Ag⁺-HPLC). In this study, the coupling of two analytical silver-ion high performance liquid chromatography columns (tandem-column Ag⁺-HPLC) in series led to the enhanced resolution of CLA isomers. Many CLA isomers were baseline resolved and the pair 18 : 2 8,10 c/t and 18 : 2 7,9 c/t found in cheese products, was resolved for the first time. In this work, a similar commercial CLA mixture was separated into 16 peaks, while CLA isomers from cheese also gave rise to 16 peaks. As expected, the CLA isomers were separated into three geometric groups in the order trans,trans, cis/trans, and cis,cis. Semi-preparative Ag⁺-HPLC, followed by gas chromatography–mass spectroscopy of the dimethyloxazoline derivatives, was used to confirm the identity of the newly resolved positional CLA isomers. The double bond configuration of CLA isomers was established by gas chromatography–Fourier transform infrared spectroscopy. Two minor t,t CLA isomers found in cheese, presumably 18 : 2 t6t8 and 18 : 2 t13t15, were also separated. The CLA isomeric composition of 16 commercial cheese products was determined.     

Complete separation of the geometical isomers of linolenic acid by high performance liquid chromatography with a silver ion column

A method has been developed for separation of linolenic acid and its seven isomers by HPLC on a silver-ion-loaded column. The standard 18:3 isomers, isolated from a heated linseed oil or prepared by isomerization of linolenic acid, were converted into phenacyl esters and detected by UV at 238 nm. The use of low temperature (10 °C) combined with a gradient of dichloromethane and methanol enabled separation of all the cis/trans isomers. The peaks were identified by comparison of ECL values with those of a standard mixture, by chromatographing collected HPLC fractions on a polar GC column. HPLC quantification was compared with GC analysis. There was satisfactory agreement between the tow methods. This method could be used for seperation, collection and quantification of 18:3 fatty acids with trans double bonds in different oils and foods.     

Optimization of the selectivity of a cyanopropyl stationary phase for the gas chromatographic analysis of trans fatty acids

The quantification of monounsaturated and polyunsaturated trans fatty acids in partially hydrogenated fats by gas-liquid chromatography on a CP-Select CB-FAME capillary column was optimized using equivalent chain length values of fatty acids methyl esters that could coelute in the temperature range from 155 to 200 degrees C. The most appropriate temperature for the simultaneous determination of these trans isomers is around 197 degrees C. It is proposed a system to discriminate trans from cis octadecenoic fatty acid methyl esters based on the angular coefficient values of the equivalent chain length curves. The limits of detection and quantification were 0.28 and 0.93 mg g(-1). Quantification was performed in the range from 0.93 to 280 mg g(-1). Quantification accuracy was estimated by spike recovery of elaidic and trans-13-octadecenoic acids in hydrogenated fat samples. The obtained levels were from 97.60 to 103.28% for elaidic acid and from 98.12 to 99.27% for trans-13-octadecenoic acid. These results indicate that the accuracy of the methodology proposed for the quantification of monounsaturated and polyunsaturated trans fatty acids in hydrogenated fats is adequate.     

Fatty acid profile of Canadian dairy products with special attention to the trans-octadecenoic acid and conjugated linoleic acid isomers

Current scientific evidence indicates that consumption of industrial trans fatty acids (TFA) produced via partial hydrogenation of vegetable oils increases the risk of coronary heart disease. However, some studies have suggested that ruminant TFA, especially vaccenic acid (VA or 11t-18:1) and rumenic acid (RA or 9c,11t-18:2), which is a conjugated linoleic acid (CLA) isomer, may have potential beneficial health effects for humans. To date, no concerted effort has been made to provide detailed isomer composition of ruminant TFA and CLA of Canadian dairy products, information that is required to properly assess their nutritional impacts. To this end, we analyzed the fatty acid profile of popular brands of commercial cheese (n = 17), butter (n = 12), milk (n = 8), and cream (n = 4) sold in retail stores in Ottawa, Canada, in 2006-2007 by silver nitrate thin-layer chromatography and gas liquid chromatography. The average total TFA content of cheese, butter, milk, and cream samples were 5.6, 5.8, 5.8, and 5.5% of total fatty acids, respectively. VA was the major trans-octadecenoic acid (18:1) isomer in all the Canadian dairy samples with average levels of (as % total trans-18:1) 33.9% in cheese, 35.6% in butter, 31.0% milk, and 30.1% in cream. The different dairy products contained very similar levels of CLA, which ranged from 0.5 to 0.9% of total fat. RA was the major CLA isomer of all the dairy products, accounting for 82.4-83.2% of total CLA. There were no significant differences (P > 0.05) in the fatty acid profile between the 4 different dairy groups, which suggests lack of processing effects on the fatty acid profile of dairy fat.     

Fast analysis by gas-liquid chromatography. Perspective on the resolution of complex fatty acid compositions

Separation of fatty acids as methyl ester (FAME) derivatives has been carried out using short and highly polar capillary column developed for fast gas-liquid chromatography (GLC) applications. The GLC parameters have been optimized in order to achieve separation of FAME ranging from 4:0 (butyric acid) to 24:1 in less than 5 min. Milk fat that has by far the most complex fatty acid composition among edible fats and oils has been used to optimize the method. The volume of the oven has been reduced in order to allow for a heating rate of 120 degrees C/min and to rapidly cool-down to the initial temperature (50 degrees C) of the GLC program. The GLC conditions developed are not suitable to achieve separation of positional and geometrical isomers of octadecenoic acid but are useful to perform separation of major fatty acids in milk fat. The conditions developed could be used to analyze edible fats and oils or biological samples such as plasma or red blood cell lipids. The results confirmed that short and highly polar fast columns operating under optimal conditions could be used to separate the fatty acids in various matrices.     

Analysis of triacylglycerol and fatty acid isomers by low-temperature silver-ion high performance liquid chromatography with acetonitrile in hexane as solvent: limitations of the methodology

Silver ion HPLC (Ag-HPLC), utilizing columns containing silver ions bonded to a silica substrate and acetonitrile in hexane as solvent, has proven to be a powerful technology for the analysis of geometric (cis or trans) or positional fatty acids, fatty acid ester (primarily methyl ester; FAME), or triacylglycerol (TAG) isomers. Previous studies had demonstrated that, unlike gas chromatography, samples eluted more rapidly at lower temperatures (at 20 degrees C versus 40 degrees C, for example). A low-temperature bath [dual-column Ag-HPLC; isocratic solvent systems of 0.3 to 0.7% acetonitrile (ACN) in hexane] was utilized to study the application of this system at low (below 0 degrees C) temperatures for analysis of FAME (zero to six double bonds) and TAG [SSS, OOO and LLL, where S=stearic acid (18:0), O=oleic acid (9c-18:1), and L=linoleic acid (9c, 12c-18:2)] standards. While FAME elution times continued to decrease from 0 degrees C to -10 degrees C, they began to increase at -20 degrees C. A similar situation was noted for the TAG isomers, except that retention times began to increase below 0 degrees C. The lower temperature limit of the Ag-HPLC/ACN in hexane system is thus ca. -25 degrees C. Increasing sample elution times and pump head pressures upon sample injection were noted at temperatures of -25 degrees C to -40 degrees C. Equilibration times at each temperature could be reduced to ca. 15 min without loss of resolution and with retention times of +/-2%. Temperature, rather than solvent composition, can therefore be utilized with the Ag-HPLC/ACN in hexane solvent system to optimize elution times and resolution(s) of FAME and TAG isomers.     

Separation of geometric isomers of native lutein diesters in marigold (Tagetes erecta L.) by high-performance liquid chromatography-mass spectrometry

Lutein is found in many foods; the richest and purest plant source is marigold flower (Tagetes erecta L.). In this plant, lutein is in the form of saturated fatty acid diesters. By using a binary mobile phase consisting of ethyl acetate and acetonitrile-methanol (9:1), improved separation was achieved on a C18-bonded phase. The unique absorption of lutein cis isomers at 330nm was used in combination with MS to identify the novel cis-lutein isomeric dimyristate, myristate-palmitate, dipalmitate, and palmitate-stearate diesters, as well as the rare combinations of both trans- and cis-lutein laurate-palmitate and trans- and cis-lutein myristate-stearate. The presence of the all-trans-lutein laurate-myristate, dimyristate, myristate-palmitate, palmitate-stearate, and distearate diesters, reported by others, was also confirmed.     

Gas chromatography and silver-ion high-performance liquid chromatography analysis of conjugated linoleic acid isomers in free fatty acid form using sulphuric acid in methanol as catalyst

This study used GC and silver-ion HPLC to examine the effects of temperature and time on methylation of individual and mixtures of conjugated linoleic acid (CLA) isomers in free fatty acid form using sulphuric acid as catalyst. In the conditions tested (temperatures between 20 and 50 degrees C and times between 10 and 60min) methylation was complete while avoiding isomerization of conjugated dienes and the formation of artefacts that could interfere with chromatographic determinations. An analytical method using solvent extraction of the lipids followed by selective elution of the free fatty acids from aminopropyl bonded phase columns and methylation with H(2)SO(4) in mild conditions was then applied to determine the CLA isomers in free fatty acid form in rumen fluid, and the results were evaluated.     

Gas-liquid chromatographic method for analysing complex mixtures of fatty acids including conjugated linoleic acids (cis9trans11 and trans10cis12 isomers) and long-chain (n-3 or n-6) polyunsaturated fatty acids. Application to the intramuscular fat of beef meat

The optimisation and validation of a gas-liquid chromatographic (GLC) method using direct saponification with KOH/methanol followed by a derivatization with (trimethylsilyl)diazomethane was carried out trying to overcome all the difficulties posed by the analysis of complex mixtures of fatty acids (FAs) in animal fat tissues. The presented method allowed sensitive, selective and simultaneous determination of a wide range of different FAs, including short-chain FAs, branched-chain FAs and conjugated linoleic acid isomers in the same GLC run along with other well known saturated, monounsaturated and polyunsaturated FAs. To demonstrate the feasibility of the procedure, the total FA profile of beef meat was characterised.     

Characterization and quantification of triacylglycerols in peanut oil by off‐line comprehensive two‐dimensional liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry

The complexity of natural triacylglycerols (TAGs) in various edible oils is prodigious due to the hundreds of set is of TAG compositions, which makes the identification of TAGs quite difficult. In this investigation, the off‐line 2D system coupling of nonaqueous RP and silver‐ion HPLC with atmospheric pressure chemical ionization MS detection has been applied to the identification and quantification of TAGs in peanut oil. The method was successful in the separation of a high number of TAG solutes, and the TAG structures were evaluated by analyzing their atmospheric pressure chemical ionization mass spectra information. HPLC and MS conditions have been optimized and the fragmentation mechanisms of isomers have been validated. In addition, an internal standard approach has been developed for TAG quantification. Then this system was applied in peanut oil samples and there was a total of 48 TAGs including regioisomers that have been determined and quantified.     

Syn/anti isomerization of 2,4-dinitrophenylhydrazones in the determination of airborne unsymmetrical aldehydes and ketones using 2,4-dinitrophenylhydrazine derivation

Aldehydes and ketones readily react with 2,4-dinitrophenylhydrazine (2,4-DNPH) to form the corresponding hydrazones. This reaction has been frequently used for the quantification of airborne carbonyl compounds. Since unsymmetrical aldehydes and ketones are known to form isomeric 2,4-dinitrophenylhydrazones (syn/ anti-isomers), the influence of isomerization on the practicability and accuracy of the 2,4-DNPH-method using 2,4-dinitrophenylhydrazine-coated solid sorbent samplers has been studied with three ketones (methyl ethyl ketone (MEK), methyl isopropyl ketone (MIPK), and methyl isobutyl ketone (MIBK)). With all three ketones the reaction with 2,4-DNPH resulted in mixtures of the isomeric hydrazones which were separated by HPLC and GC and identified by mass spectroscopy and ¹H nuclear magnetic resonance spectroscopy. The isomers show similar chromatographic behaviour in HPLC as well as in GC, thus leading to problems in quantification and interpretation of chromatographic results.     

脂肪酸的色谱保留时间规律与质谱特征研究及其在食品分析中的应用

用石油醚提取食品中的脂肪,经甲酯化反应后,采用HP-88(100m×0.25mm,0.33μm)弹性石英毛细管柱分离脂肪酸甲酯的同系物及异构体,GC/MS法测定。研究了不同链长脂肪酸的同系物及异构体的气相色谱出峰顺序,得到其保留时间规律;研究了不同脂肪酸的质谱断裂规律,选择3个特征离子来鉴定脂肪酸成分。建立了3个特征离子确定脂肪酸碳数及双键数目,色谱保留时间规律确定脂肪酸顺反异构体及双键位置异构体的方法。本法无需标准品即可快速测定脂肪酸同系物及异构体的含量,适用于脂肪酸组成的研究;及油脂、食品中脂肪酸,特别是反式脂肪酸的测定。     

~1H-NMR studies on cis-trans isomers of spheroidenes

All-trans spheroidene was extracted from the cells of Rhodobacer sphaeroides 2.4.1 and a set of cis-trans isomers were isolated from an isomeric mixture obtained by iodine-sensitized photo-isomerization of the all-trans isomer by high-pressure liquid chromatography (HPLC). On the basis of assignment of the 1H-NMR spectra (COSY, long-range COSY, rekyed COSY, ROESY and NOESY) of the all-trans isomer, the configurations of cis isomers were determined by the isomerization shifts of the olefinic 1H signals. The peaks of HPLC could be assigned as follows: A: 9,13'-cis, B1: 5,13-cis, B2: 13,9'-cis, C: 13-cis, E: 9-cis, F: 13'-cis, G: 5,9'-cis, H: 9'-cis, I: all-trans.     

A reversed-phase high-performance liquid chromatographic method to analyze retinal isomers

A high-performance liquid chromatographic (HPLC) procedure was developed to separate all-trans-, 13-cis-, 11-cis- and 9-cis-retinal isomers. Two reversed-phase Vydac C18 columns in series were used with an isocratic solvent system of 0.1 M ammonium acetate-acetonitrile (40:60, v/v) as mobile phase and all-trans-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-no natetraene-1-ol (TMMP) as internal standard. Prior to HPLC, the retinal isomers were efficiently extracted in their original isomeric conformation using dichloromethane-n-hexane in the presence of formaldehyde. This technique is suitable for the assay of 11-cis- and all-trans-retinal isomers in retina.     

Oxygenated acids of the reserve lipids of Rumex paulsenianus

The composition of the fatty mono- and dihydroxy acids of the fruit ofRumex paulsenianus Rech. fil. (familyPolygonaceae) has been determined by a combination of IRS, UVS, GLC, and mass-spectrometric methods. (22) Monohydroxy acids of the C₁₄–C₂₀ series, including isomers and isologues, and four dihydroxy acids from C₁₈ to C₂₀, including a new isomeric 9-OH-10, 12–17:2 acid have been detected.     

Use of Isopropanol as a Modifier in a Hexane-Acetonitrile Based Mobile Phase for the Silver Ion HPLC Separation of Positional Isomers of Triacylglycerols Containing Long Chain Polyunsaturated Fatty Acids

A high resolution approach to silver ion HPLC was studied for the separation of positional isomers of triacylglycerols (TAGs) containing long chain polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA) in enzymatically synthesized structured TAGs. Isopropanol was used as a novel modifier in a hexane-acetonitrile based mobile phase for silver ion HPLC. Peak identification was based on HPLC-mass spectroscopy and selectivities of lipases. Positional isomers of TAGs containing one molecule of EPA, DHA, or DPA with saturated fatty acids (FAs) such as caprylic acid and palmitic acid were separated within 13 min using a gradient of hexane-isopropanol-acetonitrile as mobile phase. TAGs containing two or more EPA, DHA, or DPA were also separated from each other within 25 min, but their positional isomers were unresolved. The retention characteristics of the TAG were found to be related to the number of carbon atoms in the FAs present in addition to the number of double bonds and their isomeric configuration. One isomer with an unsaturated FA in the sn-2 position eluted faster than the other with the unsaturated FA in the sn-1 or 3 position. Species with longer chain FAs attached to TAGs with the same degree of unsaturation eluted faster than those that have shorter chain FAs. For example, docosapentaenoylhexadecanoyloctanoin (DPA/C16/C8) was eluted faster than dioctanoyldocosapentaenoin (DPA/C8/C8).