Equivalent Chain Length Values Research Articles

Identification and Total Synthesis of Two Previously Unreported Odd-Chain Bis-Methylene-Interrupted Fatty Acids with a Terminal Olefin that Activate Protein Phosphatase, Mg2+/Mn2+-Dependent 1A (PPM1A) in Ovaries of the Limpet Cellana toreuma.

Diverse non-methylene-interrupted (NMI) fatty acids (FAs) with odd-chain lengths have been recognized in triacylglycerols and polar lipids from the ovaries of the limpet Cellana toreuma, however their biological properties remain unclear. In this study, two previously unreported odd-chain NMI FAs, (12Z)-12,16-heptadecadienoic (1) and (14Z)-14,18-nonadecadienoic (2) acids, from the ovary lipids of C. toreuma were identified by a combination of equivalent chain length (ECL) values of their methyl esters and capillary gas chromatography-mass spectrometry (GC-MS) of their 3-pyridylcarbinol derivatives. On the basis of the experimental results, both 1 and 2 were synthesized to prove their structural assignments and to test their biological activity. The ECL values and electron impact-mass (EI-MS) spectra of naturally occurring 1 and 2 were in agreement with those of the synthesized 1 and 2. In an in vitro assay, both 1 and 2 activated protein phosphatase, Mg2+/Mn2+-dependent 1A (PPM1A) up to 100 μM in a dose-dependent manner.

English

Fatty acid profile of Manketti oil extracted by screw press (SPE), supercritical fluid, Soxhlet (SE) and mechanical shaking (MS) extractions determined by chromatography (GC). Eight fatty acids were identified using authentic standards; two were calculated by equivalent chain lengths (ECL) values; α-eleostearic acid was identified by GC and spectroscopic techniques while three could not be identified. The results indicate that, extractions using hexane, SE and MS, had higher concentrations of total saturated fats of 15.71 and 16.27%, respectively, compared to SPE and SFE with 15.51 and 15.13%, respectively. On the other hand, SPE and SFE respectively had concentrations of unsaturated fats of 82.55 and 82.90%, compared to SE and MS, which had 82.08 and 81.86%, respectively. SPE and SFE had significantly higher (P < 0.05) concentrations of α-eleostearic acid of 27.01 and 26.86% compared to SE and MS with 26.28 and 26.11%. Hence, Manketti oil that is relatively rich in unsaturated fats can be obtained by SPE and SFE. Additionally, SPE and SFE oil is free of organic solvent residues, hence is suitable for edible purposes. The existence of α-eleostearic acid in this Manketti sample has been proven by GC, Gas chromatography-mass spectrometry (GC-MS), infrared (IR), ultraviolet (UV) and nuclear magnetic resonance (NMR) techniques. The results strongly agree with literature. The result is important since α-eleostearic acid is known to have the ability to lower antitumor and hepatic cholesterol; so Manketti oil could be used for neutraceutical purposes. Key words: Manketti oil, fatty acids, screw-press, supercritical fluid, solvent extraction, α eleostearic acid.

Characterisation of capillary ionic liquid columns for gas chromatography–mass spectrometry analysis of fatty acid methyl esters

Due to their distinct chemical properties, the application of ionic liquid (IL) compounds as gas chromatography (GC) stationary phases offer unique GC separation especially in the analysis of geometric and positional fatty acid methyl ester (FAME) isomers. Elution behaviour of FAME on several commercialised IL capillary columns including phosphonium based SLB-IL59, SLB-IL60, SLB-IL61 and SLB-IL76 and imidazolium based SLB-IL82, SLB-IL100, and SLB-IL111 as well as a general purpose column SLB-5ms, were evaluated in gas chromatography–mass spectrometry (GC–MS) analysis. The phases were further characterised by using a linear solvation energy relationship (LSER) approach according to the equivalent chain length (ECL) index of FAME. Among all tested IL columns, elution temperatures of saturated FAME increased as their McReynolds’ polarity value decreased, except for IL60. ECL values increased markedly as the stationary phase polarity increased, particularly for the polyunsaturated FAME. The LSER study indicated a lowest l/e value at 0.864 for IL111, displaying phase selectivity towards unsaturated FAME, with higher peak capacity within a carbon number isomer group. s and e descriptors calculated from LSER were validated by excellent correlation with dipole moments and lowest unoccupied molecular orbital (LUMO) energies, with R2 values of 0.99 and 0.92 respectively, calculated using GAUSSIAN.

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.

Gas Chromatographic Separation of Docosenoic Acid Positional Isomers on an SLB-IL100 Ionic Liquid Column

Gas chromatography (GC) of docosenoic acid (22:1) has been performed for the separation of positional isomers on the novel SLB-IL100 column with a highly polar ionic liquid stationary phase. A test mixture of 22:1 methyl esters prepared from total lipids of flounder was subjected to GC on a 60 m×0.32 mm i.d. column at an isothermal temperature of 150°C-180°C. On this column, all five positional isomers separated in the elution order of 22:1n-15, 22:1n-13, 22:1n-11, 22:1n-9, and 22:1n-7. The positional isomers, 22:1n-15, 22:1n-13, and 22:1n-11, unresolvable on conventional polar polymer phase columns, were almost completely separated from each other within 24 min at 170°C. The equivalent chain length values of 22:1n-11 to 22:1n-7 were parallel to those on polyethylene glycol and cyanopropyl polysiloxane columns, whereas 22:1n-15 and 22:1n-13 were relatively lower and closer to saturated 22:0 acid. Similar findings were also obtained for co-injected 20:1n-15 to 20:1n-11 isomers. Analysis of fish 22:1 revealed that 22:1n-13 is not always a minor isomer, as previously reported for several samples. The results of this study confirm the view that SLB-IL100 is a powerful tool for GC analysis of monounsaturated fatty acids.

Establishment of reliable mass spectra and retention indices library: Identification of fatty acids in human plasma without authentic standards

Gas chromatography mass spectrometry (GC–MS) is routinely employed to analyze small molecules in various samples. The more challenge of GC–MS data processing is to identify the unknown compounds in samples. Mass spectra and retention indices library searching are commonly used method. However, the current libraries are often built through collecting data from different groups. To unknown compounds with similar mass spectra and retention indices (e.g. geometric (cis/trans) isomers), the inaccurate results sometime are supplied. In this case, the costly standard compounds have to be used in every analysis. In this report, taking identification of fatty acids as an example, we proposed a strategy of establishment of special database constructed by equivalent chain length (ECL) values in uniform conditions and mass spectra of fatty acid methyl esters (FAMEs). The mass spectral characteristics were firstly used to identify all expected straight saturated fatty acids, and subsequently calculate the ECL for fatty acids in the sample. Finally, the ECL values of fatty acids in the sample were compared with those of fatty acids in the customized database to identify their structures. The results showed that the method developed in this report could effectively identify similar unknown compounds (FAMEs in the human plasma) after validated by the authentic standards.

GC Separation of cis‐Eicosenoic Acid Positional Isomers on an Ionic Liquid SLB‐IL100 Stationary Phase

AbstractGas chromatography (GC) of cis‐eicosenoic acid (20:1) positional isomers has been investigated on a capillary column of ionic liquid 1,9‐di(3‐vinyl‐imidazolium)nonane bis(trifluoromethyl)sulfonylimidate stationary phase (SLB‐IL100). A test mixture of isomeric 20:1 methyl esters was prepared from flathead flounder flesh lipids. On a 60‐m column operated at 150–180 °C, six peaks appeared in the elution order of 20:ln‐15 → 20:ln‐13 → 20:ln‐11 → 20:ln‐9 → 20:ln‐7 → 20:ln‐5. These peaks were baseline resolved within 20 min at 180 °C. The 20:ln‐13 and 20:ln‐11 isomers, poorly resolved on conventional polar polysiloxane stationary phases, were completely separated from each other with separation factor α = 1.02 and peak resolution (Rs) ≥ 1.57. When equivalent chain length (ECL) values were compared between the SLB‐IL100 and CP‐Sil 88 (biscyanopropyl polysiloxane), those of 20:ln‐15 and 20:ln‐13 exceptionally tended to be lower on the SLB‐IL100. The excellent separation of 20:1 isomers seems due to less retention of 20:ln‐15 and 20:ln‐13 on SLB‐IL100 rather than simply due to its high polarity. Analysis of herring oil 20:1 revealed the occurrence of 20:ln‐13 in the Pacific herring but not in the Atlantic herring. The ionic liquid stationary phase, SLB‐IL100, is effective for analyzing 20:1 isomers occurring in fish and other natural oils.

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 °C. The most appropriate temperature for the simultaneous determination of these trans isomers is around 197 °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.

Unusual Fatty Acids in the Fat Body of the Early Nesting Bumblebee, Bombus pratorum

Unusual fatty acids with 24, 26, and 28 carbon atoms were found in triacylglycerols (TAGs) isolated from fat body tissue of bumblebee Bombus pratorum. The most abundant one was (Z,Z)-9,19-hexacosadienoic acid. Its structure was determined by mass spectrometry after derivatization with dimethyl disulfide and by infrared spectroscopy. ECL (equivalent chain length) values of its methyl ester were determined on both DB-1 and DB-WAX capillary columns. (Z,Z)-9,19-Hexacosadienoic acid is quite rare in nature. So far it has been identified only in marine sponges, and this work is the first evidence of its occurrence in a terrestrial organism. HPLC/MS analysis of the bumblebee TAGs showed that (Z,Z)-9,19-hexacosadienoic acid is present in one third of all TAG molecular species. As it was found in all sn-TAG positions, it is likely that (Z,Z)-9,19-hexacosadienoic acid is transported to tissues. Interestingly, labial gland secretion of B. pratorum was found to contain (Z,Z)-7,17-pentacosadiene, a hydrocarbon with markedly similar double bond positions and geometry. Possible biosynthetic relationships between these two compounds are discussed.

Rapid identification of fatty acid methyl esters using a multidimensional gas chromatography–mass spectrometry database

A multidimensional approach for the identification of fatty acid methyl esters (FAME) based on GC/MS analysis is described. Mass spectra and retention data of more than 130 FAME from various sources (chain lengths in the range from 4 to 24 carbon atoms) were collected in a database. Hints for the interpretation of FAME mass spectra are given and relevant diagnostic marker ions are deduced indicating specific groups of fatty acids. To verify the identity of single species and to ensure an optimized chromatographic resolution, the database was compiled with retention data libraries acquired on columns of different polarity (HP-5, DB-23, and HP-88). For a combined use of mass spectra and retention data standardized methods of measurement for each of these columns are required. Such master methods were developed and always applied under the conditions of retention time locking (RTL) which allowed an excellent reproducibility and comparability of absolute retention times. Moreover, as a relative retention index system, equivalent chain lengths (ECL) of FAME were determined by linear interpolation. To compare and to predict ECL values by means of structural features, fractional chain lengths (FCL) were calculated and fitted as well. As shown in an example, the use of retention data and mass spectral information together in a database search leads to an improved and reliable identification of FAME (including positional and geometrical isomers) without further derivatizations.

Prediction of equivalent chain lengths from two-dimensional fatty acid retention indices

A recently introduced two-dimensional fatty acid retention index system (2D-FARI) was used as basis for prediction of equivalent chain lengths (ECL) of fatty acid methyl esters (FAME) on a BPX-70 stationary phase. Models for the relationship between 2D-FARI data and ECL values of a calibration sample with 30 common fatty acids were established by a simple multivariate regression. The models were thereafter applied on 2D-FARI data for other FAMEs and used to predict the ECLs for these compounds. The 2D-FARI values for the fatty acids in the calibration sample are given by definition. Thus, the only information necessary to calculate the ECL value for a compound run under identical conditions as the calibration sample is the 2D-FARI values for the compound, which can be acquired from literature data. The method was validated with test sets analysed with different temperature and flow programs. ECLs of various marine FAME and trans isomers of Eicosapentaenoic and Docosahexaenoic acid were predicted with root mean squared error of prediction from 0.002 to 0.012 ECL units.

An alternative approach for the estimation of equivalent temperature in gas chromatography.

The temperature at which the isothermal retention time (tRiso) is equal to the temperature-programmed retention time (tRTPGC) or the isothermal retention index (Iiso) is equal to the temperature-programmed retention index (ITPGC) is defined as the equivalent temperature (Teq). The Teq of one-, two-, three-, and four-step temperature-programmed gas chromatography (TPGC) of unsaturated fatty acid methyl esters (FAMEs) from Chinese mustard seed oil is calculated. All of the tR values obtained at the Teq (tRTeq) are very close to the tRTPGC. The highest difference for each chromatogram is less than 0.5%. The slight deviation may partly arrive from the difficulty in setting the desired carrier gas flow rate. Also, good agreement among equivalent chain length values determined by different methods is observed, including the graphical method at the Teq. Therefore, it is speculated that the proposed method may facilitate the GC identification of FAMEs as well as other organic compounds in TPGC by using the available isothermal retention index database.

Identification of fatty acids in gas chromatography by application of different temperature and pressure programs on a single capillary column

A method for the identification of fatty acid methyl esters (FAME) based on the analysis of shifts in equivalent chain lengths (ECL) is described. The method is based on two-dimensional retention data achieved on one capillary column. Various temperature and pressure programs are applied on the same cyanopropyl column and the shifts in the ECL values are analysed by multivariate methods. The chain length, number of double bonds, and the double bond positions can be determined with high accuracy. The same procedure is suitable for determination of the number of trans and cis double bonds in trans fatty acids, and for detection of artefacts in fatty acid methyl ester chromatograms.

Determination of fatty acids in vegetable oil by reversed-phase liquid chromatography with fluorescence detection

The effect of temperature and organic solvent composition (acetonitrile and methanol) on the reversed-phase separation of coumarin-derivatized fatty acids according to their carbon number (C14 to C22), the degree of unsaturation, as well as cis/ trans (C18:1 c/t, C18:2 cc/tt, C18:3 ccc/ttt) configuration was investigated to find out the effective separation condition. Based on the linear plots of the logarithm of the capacity factor of saturated fatty acids versus their carbon number, the equivalent chain length (ECL) of unsaturated fatty acids was calculated. The ECL values were found to be significantly altered and the differentiation between cis and trans fatty acids was increased when either the temperature or organic solvent composition was decreased. These results generally led to a better resolution at the expense of separation time. A ternary gradient composed of water, acetonitrile, and methanol was then developed to elute the solutes at 55°C within a separation time of 40 min with a minimum resolution of 1.0 for the worst pair. This method was demonstrated to resolve the fatty acids in a vegetable shortening.

Simplifying Fatty Acid Analyses Using a Standard Set of Gas--Liquid Chromatographic Conditions: II. Equivalent Chain Length Values for cis- and trans- Isomers of Monoethylenic C18 Fatty Acid Methyl Esters for Carbowax-20M Liquid Phase

The Carbowax-20M liquid phase is becoming the standard in gas chromatography for general fatty acid analyses because it simplifies the work and reduces the chance for errors in identification of fatty acids. Isothermal analysis using the Carbowax-20M column, as opposed to temperature-programming, provides further simplification and ruggedness to the analysis. An extensive compilation of characteristic separation factors for 59 different fatty acid methyl esters has appeared, but it included gas-liquid chromatography elution information on only four of the approximately 20-24 isomeric octadecenoates that occur in foods containing ruminate fats and partially-hydrogenated vegetable oils. This contribution serves to fill that void by providing the equivalent chain length elution factors for 25 additional trans- and cis-monoethylenic fatty acid methyl esters.

An Alternative Method for the Calculation of Equivalent Chain Length or Carbon Number of Fatty Acid Methyl Esters in Gas Chromatography

The carbon number or equivalent carbon number (ECN) of fatty acid methyl esters (FAMEs) separated on a 30-m × 0.32-mm-i.d. Omegawax 320 capillary column is calculated directly from the retention data. When the described equation is used to identify FAMEs in a mixture of FAMEs from rambutan and para-rubber seed oils, the calculated ECN values are very close to the widely accepted equivalent chain length values described in the literature. Hence, the equation can probably be used as an identification tool for FAMEs.

Gas-liquid chromatographic properties of positional isomers of methyl thia, selena, and tellura laurate analogs.

Gas-liquid chromatographic analyses of three complete series of synthetic positional isomers of methyl thia, selena, and tellura laurate analogs were carried on a nonpolar (SE-30) and a polar (SP-2330) stationary phase. The average ECL (equivalent chain length) values of the thia, selena, and tellura laurate on SE-30 stationary phase were 13.8, 14.8, and 15.7, respectively, while on SP-2330 the average values for the same series were 17.1, 19.0, and 19.1, respectively. Positional isomers with the heteroatom at the 2-position exhibited the lowest ECL values, while those with the heteroatom at the omega-1 position gave the highest ECL values and were readily separated from the other positional isomers of the same series of analogs by this technique.

Standard equivalent chain length values of monoenic and polyenic (methylene interrupted) fatty acids

AbstractThe equivalent chain length (ECL) values of the methyl esters of 83 defined fatty acids (FAs) have been determined by gas chromatog‐raphy (GC) on three fused silica DB‐WAX and three DB‐1 columns. ECL values of further 46 FAs were calculated by different methods. Conditions of chromatography, methods of ECL values calculation and differences between ECL values on individual columns and between trans‐ and cis‐isomers of corresponding FAs are also discussed.

Simplifying Fatty Acid Analyses in Multicomponent Foods with a Standard Set of Isothermal GLC Conditions Coupled with ECL Determinations

It is common to use some sort of temperature program in the analysis of fatty acid methyl esters. For samples that contain only a few components or in situations where all samples are basically the same, temperature programming can offer certain advantages. Unfortunately, samples such as these are certainly not the norm in routine food fatty acid analysis. A gas-liquid chromatographic column, the Carbowax-20M fused-silica phase (cross-linked) recommended by Ackman as a standard for fatty acid analysis (it simplifies the work and reduces the chance for errors), is used to chromatograph mixtures of methyl esters at column temperature settings that range from 170°C to 250°C. It is determined that three specific temperatures (183°C, 212°C, and 247°C) permit complete resolution of a wide array of acids under strictly isothermal conditions. Equivalent chain length values have been compiled for use in assigning identities of fatty acid methyl esters. Also, the specific responses of individual methyl esters to alterations in column temperature can be of use in the identification of peaks in complex mixtures.

Gas chromatographic analysis of fatty acid methyl esters.

The full process of fatty acid methyl ester (FAME) analysis consists of esterification of lipids, and of injection, separation, identification and quantitation of the FAMEs. In order for the required accuracy and precision to be attained, each of these steps has to be optimized. Esterification of lipids can be carried out with several reagents based on acid-catalysed or base-catalysed reactions. The advantages and disadvantages of these reagents are discussed. The most critical step in the gas chromatographic analysis of FAMEs is sample introduction. The classical split injection technique, which is the most widely used technique in the analysis of FAMEs, has the potential disadvantage of boiling-point-dependent sample discrimination. Cold injection of the sample, either on-column or by programmed-temperature vaporization, does not present this problem and should therefore be preferred. Modern, commercially available fused-silica capillary columns offer excellent separation of FAMEs from biological samples. Very polar stationary phases give excellent separation of all FAMEs but have relatively low thermal stability, resulting in long retention times. Non-polar phases have a much greater thermal stability but inferior selectivity. For many analyses, phases of intermediate polarity, which combine the advantages of a relatively high resolution capability with relatively high thermal stability, are the most suitable. FAMEs can be identified by comparison of their retention times with those of individual purified standards or secondary standards based on lipids that have been well characterized in literature. Relative retention times and equivalent chain-length values also provide useful information. FAMEs can be quantitated by peak areas via calibration factors, and absolute concentrations can be determined by adding an internal standard. Among numerous applications in biomedical research, the analysis of fatty acids from body tissues may contribute to the understanding of the link between the dietary intake of fatty acids and the diseases with which these acids are associated.