Silver Ion High-performance Liquid Research Articles

Impact of column temperature on triacylglycerol regioisomers separation in silver ion liquid chromatography using heptane-based mobile phases

This work presents the investigation of the use of heptane as an alternative and less toxic mobile phase to the most used hexane for triacylglycerols (TAGs) analysis in silver ion high-performance liquid chromatography (Ag+-HPLC). The impact of column temperature (in the 5 °C-35 °C range) on the retention and resolution of five pairs of regioisomers relevant for the confectionery industry was investigated using a heptane-based mobile phase modified with acetonitrile (ACN). The retention behaviour was compared for a standard TAG mixture and an interesterified cocoa butter. The temperature effect previously observed with hexane-based mobile phases was confirmed for this new system, and it was also observed that the ACN concentration had an important impact on the strength of the temperature effect, with a higher ACN concentration leading to a lesser impact of temperature on the TAGs’ elution behaviour. In general, the study allowed to conclude on the equivalence of hexane and heptane for TAGs regioisomers separation in Ag+-HPLC, independently of the used temperature or the ACN concentration. In addition, the applicability of heptane-based mobile phases for the separation of TAGs regioisomers was demonstrated on three other confectionary fat samples, namely palm olein, interesterified palm olein, and interesterified shea olein.

Silver Ion High-Performance Liquid Chromatography-Atmospheric Pressure Chemical Ionization Mass Spectrometry: A Tool for Analyzing Cuticular Hydrocarbons.

Aliphatic hydrocarbons (HCs) are usually analyzed by gas chromatography (GC) or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. However, analyzing long-chain HCs by GC is difficult because of their low volatility and the risk of decomposition at high temperatures. MALDI cannot distinguish between isomeric HCs. An alternative approach based on silver ion high-performance liquid chromatography (Ag-HPLC) is shown here. The separation of HC standards and cuticular HCs was accomplished using two ChromSpher Lipids columns connected in series. A gradient elution of the analytes was optimized using mobile phases prepared from hexane (or isooctane) and acetonitrile, 2-propanol, or toluene. HCs were detected by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Good separation of the analytes according to the number of double bonds, cis/trans geometry, and position of double bonds was achieved. The retention times increased with the number of double bonds, and trans isomers eluted ahead of cis isomers. The mobile phase significantly affected the mass spectra of HCs. Depending on the mobile phase composition, deprotonated molecules, molecular ions, protonated molecules, and various solvent-related adducts of HCs were observed. The optimized Ag-HPLC/APCI-MS was applied for characterizing cuticular HCs from a flesh fly, Neobellieria bullata, and cockroach, Periplaneta americana. The method made it possible to detect a significantly higher number of HCs than previously reported for GC or MALDI-MS. Unsaturated HCs were frequently detected as isomers differing by double-bond position(s). Minor HCs with trans double bonds were found beside the prevailing cis isomers. Ag-HPLC/APCI-MS has great potential to become a new tool in chemical ecology for studying cuticular HCs.

Separation and quantification of olefins and diolefins in cracked petroleum fractions using silver-ion high performance liquid chromatography

A method using normal phase high performance liquid chromatography for the analysis of olefins and diolefins in cracked petroleum samples is proposed and validated in this work. A silver-impregnated silica column is used to separate olefin and diolefin species, resulting in accurate quantification of both group-types. Separations are completed using an isocratic hexane mobile phase, with no sample clean up or backflushing required. Results from the analysis of 11 model compounds are shown, as well as quantification data for four representative samples; figures of merit are also described.

More than 170 polyunsaturated tocopherol-related compounds in a vitamin E capsule: Countercurrent chromatographic enrichment, gas chromatography/mass spectrometry analysis and preliminary identification of the potential artefacts

Tocopherols and tocotrienols (usually summed up as vitamin E) are a class of structurally related natural antioxidants. Commonly, only some of the eight classic representatives (four tocopherols and four tocotrienols) are found with varied composition in food. In this study we fractionated 230mg oil from commercial vitamin E supplement capsules by countercurrent chromatography (CCC) and subsequent analysis by gas chromatography with mass spectrometry (GC/MS) of silylated CCC fractions showed that these eight isomers represented only about 70% of total tocopherol compounds. Detailed analysis enabled the detection of 161T3 isomers (α-, γ- and δ-T3) along with 18 tetra- and several penta-unsaturated isomers (tocools), two tocomonoenol isomers, and several degradation products with shorter isoprenoid side chain (apo-tocools). Altogether, over 170 tocool compounds, most likely artefacts which originated from an inappropriate oil refining process were described in this study. Silver ion high performance liquid chromatography (Ag+-HPLC) was used to separate one fraction rich in γ-T3 into four peaks each consisting of at least five peaks according to GC/MS. About ten γ-T3 isomers were also detected in rice bran oils from one producer bought retail in Germany.

Application of Silver Ion High-Performance Liquid Chromatography for Quantitative Analysis of Selected n-3 and n-6 PUFA in Oil Supplements.

The aim of this study was to develop a simple method for simultaneous determination of selected cis/cis PUFA-LNA (18:2), ALA (18:3), GLA (18:3), EPA (20:5), and DHA (22:6) by silver ion high-performance liquid chromatography coupled to a diode array detector (Ag-HPLC-DAD). The separation was performed on three Luna SCX Silver Loaded columns connected in series maintained at 10 °C with isocratic elution by 1% acetonitrile in n-hexane. The applied chromatographic system allowed a baseline separation of standard mixture of n-3 and n-6 fatty acid methyl esters containing LNA, DHA, and EPA and partial separation of ALA and GLA positional isomers. The method was validated by means of linearity, precision, stability, and recovery. Limits of detection (LOD) for considered PUFA standard solutions ranged from 0.27 to 0.43 mg L(-1). The developed method was used to evaluate of n-3 and n-6 fatty acids contents in plant and fish softgel oil capsules, results were compared with reference GC-FID based method.

Analysis of Trans Fat in Edible Oils with Cooking Process

Trans fat is a unsaturated fatty acid with trans configuration and separated double bonds. Analytical methods have been introduced to analyze trans fat content in foods including infrared (IR) spectroscopy, gas chromatography (GC), Fourier transform-infrared (FT-IR) spectroscopy, reverses-phase silver ion high performance liquid chromatography, and silver nitrate thin layer chromatography. Currently, FT-IR spectroscopy and GC are mostly used methods. Trans fat content in 6 vegetable oils were analyzed and processing effects including baking, stir-frying, pan-frying, and frying on the formation of trans fat in corn oil was evaluated by GC. Among tested vegetable oils, corn oil has 0.25 g trans fat/100 g, whereas other oils including rapeseed, soybean, olive, perilla, and sesame oils did not have detectable amount of trans fat content. Among cooking methods, stir-frying increased trans fat in corn oil whereas baking, pan-frying, and frying procedures did not make changes in trans fat content compared to untreated corn oils. However, the trans fat content was so low and food label can be declared as ‘0’ trans based on the regulation of Ministry of Food ad Drug Safety (MFDS) (< 2 g/100 g edible oil).

Analysis of wax esters by silver-ion high-performance liquid chromatography–tandem mass spectrometry

Wax esters (WEs), esters of long-chain fatty acids and long-chain alcohols, were analysed by Ag-HPLC/APCI-MS/MS. Two ChromSpher Lipids columns connected in series (a total length of 50cm) and hexane–2-propanol–acetonitrile mobile phases were used to achieve good separation of the molecular species. The chromatographic behaviour of WEs was studied under optimised conditions: retention increased with the number of double bonds and with the temperature (15–35°C); retention times were affected by the double-bond position, trans isomers eluted earlier than cis isomers, and the WEs were partially separated depending on the aliphatic-chain length. The WEs provided simple APCI spectra with [M+H]+ ions, the MS/MS spectra showed fragments, which allowed their identification. The method was applied for an analysis of the WE mixtures from jojoba oil and human hair and the results were compared with analogous data from an optimised RP-HPLC system.

Lipase-catalyzed synthesis and characterization of flaxseed oil-based structured lipids

The biosynthesis of structured lipids (SLs) was carried out by the interesterification of flaxseed oil (FO) and tricaprylin (TC) in an organic solvent medium (OSM), using selected commercial lipases, including Amano DF, Novozym 435, Lipozyme TL-IM and Lipozyme RM-IM. The fatty acyl chains of the synthesized triacylglycerols (TAGs) were identified by atmospheric pressure chemical ionization/mass spectrometric (APCI/MS) analysis, while the fatty acid positional distribution of the MLM- and MML-SLs (M-medium and L-long chain fatty acids) was determined by silver-ion high-performance liquid chromatographic (Ag+/HPLC) analysis. The effects of reaction temperature (Tr, 30–50°C), enzyme concentration (Ec, 0.5–4%, w/v), initial water activity (aw, 0.05–0.43) and reaction time (Rt, 0–72h) on the efficiency of the enzymes, were studied. The bioconversion yield (%) of the synthesized MLM- and MML-SLs was monitored under the established reaction parameters for each lipase. The maximum yield of MLM-SLs was obtained in the order, of Novozym 435>Lipozyme TL-IM>Lipozyme RM-IM>Amano DF. Moreover, considering the ratio of the MLM- to MML-SLs produced by each enzyme, Novozym 435 and Lipozyme TL-IM were selected as the most effective enzymes for interesterification of FO and TC.

Oxidative modifications of conjugated and unconjugated linoleic acid during heating

The oxidative stability of conjugated linoleic (CLA) and linoleic (LA) acids in different chemical forms (free acids, methyl esters and homogeneous triacylglycerols) was compared. All model systems were heated at 180°C for different times (15, 30, 45 and 60min). The primary oxidation products were evaluated by spectrophometric analysis, while the volatile compounds were determined by solid phase micro-extraction (SPME), coupled with gas chromatography–mass spectrometry (HRGC–MS). The isomer profile modifications were investigated by silver-ion high performance liquid chromatography (Ag+-HPLC) equipped with an UV detector. Generally, peroxide values decreased during the heating time. Among the volatiles, saturated aldehydes were the most represented compounds. Isomerization of cis,trans and trans,cis CLA to trans,trans isomers was observed mainly for the methyl form of CLA. The three different chemical forms of LA never showed isomerization phenomena.

Triacylglyceride profiling in serum by silver ion high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

A simple and effective method was developed to investigate triacylglyceride (TAG) compounds in biological matrices using silver ion high performance liquid chromatography-mass spectrometry (Ag+ -HPLC-MS). The TAG compounds from mouse serum were extracted by a modified Folch method using the classical CHC13-MeOH solvent system. The extract was separated on a Varian ChromSpher 5 Lipids column with isocratic elution using acetonitrile-hexane (1: 99, v/v) as mobile phase at a flow rate of 0.75 mL/min, and detected by a mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) source in positive ion mode to acquire sufficient MS information of TAG compounds by enhanced mass spectrometry (EMS), enhanced product ion (EPI) and neutral loss (NL) scans. The identification of TAG compounds was based on their chromatographic behaviors and MS data. Besides, the NL scans of thirty fatty acids were performed to further validate the results. Finally, 66 TAG compounds as well as 5 cholesteryl ether (CE) compounds were obtained from the mouse serum extract. This method is simple, reproducible and also suitable for the analysis of TAG compounds in other samples.

Predicting conjugated linoleic acid (CLA) composition in Brazilian dairy products by multiple regression analysis based models

The analysis of conjugated linoleic acids (CLA) in dairy products is of concern for human health, but requires a combination of different analytical methods that are laborious and time consuming. The aims of the present study were to develop mathematical models based on data of the CLA and fatty acid composition of Brazilian dairy products and to apply the developed models for the calculation of CLA based on a basic analysis of fatty acid composition. The fatty acid composition of five types of dairy products was determined by gas chromatography (GC); analysis of CLA was achieved by silver-ion high-performance liquid chromatography and GC-mass spectrometry. Stepwise multiple regression analysis was performed to model the composition of CLA. All the six significant models of CLA composition (total CLA; total cis,trans, trans,cis CLA; cis-9,trans-11 CLA; trans-7,cis-9 CLA; trans-11,cis-13 CLA; and total trans,trans CLA) were physiologically consistent, had appropriate goodness of fit, and were fairly robust. According to the results of Pearson correlation analysis between estimates and experimental data (r = 0.78–0.91 and 0.0000 < P < 0.0028), the models could be used to rank dairy products according to the estimates of total CLA, trans-7,cis-9 CLA; trans-11,cis-13 CLA; total cis,trans, trans,cis CLA; and total trans,trans CLA, but not for cis-9,trans-11. The adjusted regression models allowed the estimation of CLA composition in a variety of Brazilian dairy products.

Characterization of the disappearance and formation of biohydrogenation intermediates during incubations of linoleic acid with rumen fluid in vitro1

Dietary unsaturated fatty acids are extensively hydrogenated in the rumen, resulting in the formation of numerous intermediates that may exert physiological effects and alter the fat composition of ruminant-derived foods. A batch culture method was used to characterize the hydrogenation of linoleic acid (LeA) by strained rumen fluid in vitro. Incubations (n=5) were performed in 100-mL flasks maintained at 39°C containing 400mg of grass hay, 50mL of buffered rumen fluid, and incremental amounts of LeA (0, 1.0, 2.5, 5.0, or 10.0mg) for 0, 1.5, 3.0, 4.5, 6.0, and 9.0h. The fatty acid composition of flask contents was determined using complimentary silver-ion thin-layer chromatography, gas chromatography mass-spectrometry, and silver-ion high-performance liquid chromatography. Linoleic acid was extensively (98.1, 97.6, 98.0, and 89.8% for additions of 1.0, 2.5, 5.0, and 10.0mg of LeA, respectively) hydrogenated over time. Complete reduction of LeA to 18:0 was inhibited in direct relation to the amount of added substrate, the extent of which was greatest for the highest amount of LeA addition. Recoveries of 1.0, 2.5, 5.0, and 10.0mg of added LeA as 18:0 averaged 73.6, 65.0, 57.3, and 10.7%, respectively. Incubation of incremental amounts of LeA resulted in a time-dependent accumulation of geometric isomers of 9,11 and 10,12 conjugated linoleic acid, several nonconjugated 18:2 isomers, and a wide range of cis 18:1 and trans 18:1 intermediates. Several unusual intermediates including cis-6,cis-12 18:2; cis-7,cis-12 18:2; and cis-8,cis-12 18:2, were found to accumulate in direct relation to the amount of added LeA, providing the first indications that hydrogenation of LeA by ruminal bacteria may also involve mechanisms other than hydrogen abstraction or isomerization of the cis-12 double bond. Fitting of single-pool, first-order kinetic models to experimental data indicated that the rate of LeA disappearance decreased with increases in substrate availability. Reduction of 18:1 and 18:2 intermediates occurred at much lower rates compared with conjugated linoleic acid and nonconjugated 18:2 isomer formation. In conclusion, the extent of LeA biohydrogenation in vitro was shown to be time- and dose-dependent with evidence that LeA is hydrogenated by ruminal bacteria via several distinct metabolic pathways. The accumulation of several unusual 18:2 isomers indicates that biohydrogenation of LeA also proceeds via mechanisms other than isomerization of the cis-12 double bond.

Short Communication: Rapid separation of cis9, trans11- and trans7,cis9-18:2 (CLA) isomers from ruminant tissue using a 30 m SLB-IL111 ionic column

Turner, T., Rolland, D. C., Aldai, N. and Dugan, M. E. R. 2011. Short Communication: Rapid separation of cis 9, trans 11- and trans 7,c is 9-18:2 (CLA) isomers from ruminant tissue using a 30 m SLB-IL111 ionic column. Can. J. Anim. Sci. 91: 711–713. Rumenic acid (cis9,trans11-18:2) is the main natural isomer of conjugated linoleic acid (CLA). Rumenic acid has many purported health benefits, but effects of most other CLA isomers are unknown. Typically trans7,cis9-18:2 is the second most abundant CLA isomer, but it co-elutes with rumenic acid on conventional polar gas chromatography (GC) columns, requiring complimentary analysis with silver-ion high performance liquid chromatography (Ag+-HPLC). Herein we report a rapid method for analyzing rumenic acid and trans7,cis9-18:2 using a 30 m ionic-liquid GC column. Optimal resolution of the two CLA isomers was at 145°C and analysis of backfat from barley-fed cattle compared well with GC/Ag+-HPLC (y=0.978x – 0.031, r=0.985, P&lt;0.001).

Characterization of fatty acid and triacylglycerol composition in animal fats using silver-ion and non-aqueous reversed-phase high-performance liquid chromatography/mass spectrometry and gas chromatography/flame ionization detection

Fatty acid (FA) and triacylglycerol (TG) composition of natural oils and fats intake in the diet has a strong influence on the human health and chronic diseases. In this work, non-aqueous reversed-phase (NARP) and silver-ion high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometry detection and gas chromatography with flame-ionization detection (GC/FID) and mass spectrometry detection are used for the characterization of FA and TG composition in complex samples of animal fats from fallow deer, red deer, sheep, moufflon, wild boar, cock, duck and rabbit. The FA composition of samples is determined based on the GC/FID analysis of FA methyl esters. In total, 81 FAs of different acyl chain length, double bond (DB) number, branched/linear, cis-/ trans- and DB positional isomers are identified. TGs in animal fats contain mainly monounsaturated and saturated FAs. High amounts of branched and trans-FAs are observed in the samples of ruminants. In NARP mode, individual TG species are separated including the separation of trans- and branched TGs. Silver-ion mode provides the separation of TG regioisomers, which enables the determination of their ratios. Great differences in the preference of unsaturated and saturated FAs in the sn-2 position on the glycerol skeleton are observed among individual animal fats. Unsaturated FAs are preferentially occupied in the sn-2 position in all animal samples except for wild boar with the strong preference of saturated FAs in the sn-2 position.

Quantitative determination of conjugated linoleic acid isomers by silver ion HPLC in ewe milk fat

The aim of this research was to validate a method for the quantification of conjugated linoleic acid (CLA) isomers in the fat of ewe's milk. The isomers in the form of methyl ester (FAME) are separated by silver ion high performance liquid chromatography (Ag+HPLC) and the quantification was based on the measurement of the integrated area under the 231nm peaks, using external CLA reference standards. The response linearity is maintained in a wide range of concentrations and the quantification limit estimated was of 0.005μg/injection for the cis-9,trans-11 and trans-10,cis-12 isomers. Referred to the samples this limit would be 0.008mg/g of fat in the usual working conditions. In the precision assay an RSDr of 2.14% for the isomer cis-9,trans-11 was calculated and for the rest of the CLA isomers varied between 2.08 and 7.5%. The recovery assays were performed adding CLA cis-9,trans-11 triglyceride and the mean recovery was 96%. The method proposed allows the determination of the individual content of different CLA isomers using one single technique, HPLC and supposes saving time and resources.

Regioisomeric analysis of triacylglycerols using silver-ion liquid chromatography–atmospheric pressure chemical ionization mass spectrometry: Comparison of five different mass analyzers

Silver-ion high-performance liquid chromatography (HPLC) coupled to atmospheric pressure chemical ionization mass spectrometry (APCI-MS) is used for the regioisomeric analysis of triacylglycerols (TGs). Standard mixtures of TG regioisomers are prepared by the randomization reaction from 8 mono-acid TG standards (tripalmitin, tristearin, triarachidin, triolein, trielaidin, trilinolein, trilinolenin and tri- gamma-linolenin). In total, 32 different regioisomeric doublets and 11 triplets are synthesized, separated by silver-ion HPLC using three serial coupled chromatographic columns giving a total length of 75 cm. The retention of TGs increases strongly with the double bond (DB) number and slightly for regioisomers having more DBs in sn-1/3 positions. DB positional isomers (linolenic vs. γ-linolenic acids) are also separated and their reverse retention order in two different mobile phases is demonstrated. APCI mass spectra of all separated regioisomers are measured on five different mass spectrometers: single quadrupole LC/MSD (Agilent Technologies), triple quadrupole API 3000 (AB SCIEX), ion trap Esquire 3000 (Bruker Daltonics), quadrupole time-of-flight micrOTOF-Q (Bruker Daltonics) and LTQ Orbitrap XL (Thermo Fisher Scientific). The effect of different types of mass analyzer on the ratio of [M+H−R i COOH] + fragment ions in APCI mass spectra is lower compared to the effect of the number of DBs, their position on the acyl chain and the regiospecific distribution of acyl chains on the glycerol skeleton. Presented data on [M+H−R i COOH] + ratios measured on five different mass analyzers can be used for the direct regioisomeric determination in natural and biological samples.

Fatty acid and CLA composition of Brazilian dairy products, and contribution to daily intake of CLA

In the present study, the conjugated linoleic acid (CLA) contents of dairy products most consumed by the population living in the southeast region of Brazil (whole milk, “Prato” cheese and butter) were determined. In addition, the contribution of these dairy foods to the daily intake of CLA isomers was estimated. Fatty acid composition was determined by gas chromatography. Quantitative analysis of CLA isomers was done by silver-ion high-performance liquid chromatography (HPLC) and their identification was achieved by gas chromatography–mass spectrometry (GC–MS) of dimethyloxazoline (DMOX) derivatives. Eleven CLA isomers were identified and the predominant were cis-9, trans-11 and trans-7, cis-9. The total CLA contents in whole milk, “Prato” cheese and butter ranged from 4.0 to 8.4 mg/g fat and were similar to those reported for dairy products from other countries. Whole milk samples had significantly higher contents of total CLA, when compared to “Prato” cheese and butter, and the total CLA contents were significantly different between the commercial brands of each dairy product. The estimated average daily intake of total CLA in southeastern Brazil was 36 mg/day, which was lower than those of European and North American countries, and milk was the main food source of CLA in the diet.

Separation of isomeric octadecenoic fatty acids in partially hydrogenated vegetable oils as p-methoxyphenacyl esters using a single-column silver ion high-performance liquid chromatography (Ag-HPLC)

A protocol for separation of positionally isomeric trans- and cis-octadecenoic fatty acids as their p-methoxyphenacyl esters, using a single column silver ion high-performance liquid chromatography (Ag-HPLC) with UV detection, is described. The procedure involves hydrolysis of oil to free fatty acids, derivatization to p-methoxyphenacyl esters, purification and separation by Ag-HPLC. The procedure is robust and relatively rapid. In total, it takes about 16 h or overnight and a further 5 h to carry out. Its feature and value consist in the application of p-methoxyphenacyl esters in Ag-HPLC, thus ensuring excellent simultaneous separation of both configurational and positional fatty acid isomers on a single column by a single run. Quantification is not affected by the fatty acid structure, and the method is highly sensitive. As fatty acid isomerization is a strong evidence for adulteration of lipid-containing food products with chemically modified oils and fats, the procedure is especially appropriate for detection and authenticity analysis in many areas of food manufacturing and quality control.

Two methods for the separation of monounsaturated octadecenoic acid isomers

The identification and quantification of complex mixtures of cis and trans octadecenoic (18:1) fatty acid isomers presents a major challenge for conventional one-dimensional GC/FID analysis of their methyl esters. We have compared the use of two methods to achieve optimized separations of positional and geometrical octadecenoic fatty acid isomers—comprehensive two-dimensional gas chromatography (GC × GC), and silver ion high performance liquid chromatography interfaced to atmospheric pressure photoionization (APPI) mass spectrometry. Nine isomers of octadecenoic acid methyl ester were well separated on a single silver ion column with a mobile phase of 0.018% acetonitrile and 0.18% isopropanol in hexane. Reproducible retention times were obtained with relative standard deviations of around 1% over 5 injections. The extra selectivity and reproducibility afforded by APPI-MS, together with the wide separation of cis and trans isomers by silver ion chromatography, resulted in a promising method for measurement of octadecenoic acid FAME. The GC × GC separation was performed using various column combinations, and optimal separation was obtained by coupling an ionic liquid column (Supelco SLB-IL100 [1,9-di(3-vinyl-imidazolium) nonane bis(trifluoromethyl) sulfonyl imidate]) in the first dimension with a SGE BPX50 (50% phenyl polysilphenylene-siloxane) in the second dimension. These methods have been applied to the analysis of octadecenoic acid in milk and beef fat.

2-Propanol in the mobile phase reduces the time of analysis of CLA isomers by silver ion-HPLC

Individual isomers of octadecadienoic acid (C18:2) with conjugated double bonds (conjugated linoleic acids; CLA) exert different biological activities. Their distribution in food and tissues differs. Therefore, the separation of the various positional and geometric isomers is important. The time of analysis using silver ion-high performance liquid chromatography can extend up to 90 min. The aim of this study was to reduce this time. The time of analysis reduced from ca. 90 min onto 45 to 35 min, respectively, by the addition of 0.05% or 0.1% (v/v) 2-propanol to the mobile phase [acetonitrile (0.1%; v/v) and diethyl ether (0.5%; v/v) in n-hexane]. There was no effect on resolution of the 17 individual CLA isomers of the CLA mixture. Regarding the lowest coefficient of variation and an adequate baseline separation the use of 0.05% 2-propanol in the mobile phase is recommended, without any disadvantages and adverse effects on the service life of columns. In conclusion, adding 0.05% or 0.1% 2-propanol to the mobile phase shortens the time of analysis of CLA isomers, saves solvents and reduces costs.