Gas chromatographic method for analysis of conjugated linoleic acids isomers (c9t11, t10c12, and t9t11) in broth media as application in probiotic studies

A gas chromatography (GC) procedure is assayed for analysis of conjugated linoleic acid (CLA) isomers cis-9, trans-11-octadecadienoic (c9t11); trans-10, 12 cis-octadecadienoic (t10c12); and trans-9, trans-11-octadecadienoic (t9t11) in culture broth by GC using NaOH-BF3 in methanol for methylating and a long capillary (100 m) high-polarity column. Repeatability of the method is assessed; the coefficient of variation for CLA isomers ranges from 4.62 for c9t11 to 8.19 for t9t11. Recovery ranges between 88.01 and 89.76, with a mean value of 89.06 for all CLA isomers studied. This method may be considered advantageous for analysis of CLA isomers in probiotics cultures samples.     

Improved HRGC separation of cis, trans CLA isomers as Diels-Alder adducts of alkyl esters

This paper reports the separation of four isomers of conjugated linoleic acid (CLA), c,t/t,c-8,10; c,t/t,c-9,11; c,t/t,c-10,12; c,t/t,c-11,13, after reaction of esterification with aliphatic alcohols of different chain length and adduct formation with 4-methyl-1,2,4-triazoline-3,5-dione (MTAD). The high resolution gas chromatographic analyses were carried out using a simple 50-m cyanopropyl polysiloxane capillary column both with a flame ionization detector and a mass spectrometer. The resolution between the two pair of isomers: c,t/t,c-9,11 and c,t/t,c-10,12 and between c,t/t,c-10,12 and c,t/t,c-11,13 isomers were good for all the investigated alkyl esters and increased with the chain length of alcohol esterified to carboxylic moiety of CLA isomers. The most interesting result was relative to the c,t/t,c-8,10 and c,t/t,c-9,11 isomers, critical pair of isomers also when analyzed with a 120-m cyanopropyl polysiloxane capillary column; their resolution also increased from methyl to hexyl esters of CLA isomers and reached an acceptable value (0.8) in the case of hexyl esters. The best resolutions of the four considered CLA isomers were obtained with the hexyl esters of MTAD adducts of the isomers, without excessive analysis time. This method was useful and simple to evaluate the profile of the four main c,t isomers in commercial CLA samples.     

Improved identification of conjugated linoleic acid isomers using silver-ion HPLC separations

Silver-ion high-performance liquid chromatography (Ag+-HPLC) has been shown to be effective in the resolution of most of the isomers of conjugated octadecadienoic acids (18:2), also known as conjugated linoleic acid (CLA). The CLA isomers identified in natural fats from ruminants are a mixture of numerous positional and geometric isomers from 7,9- to 12,14-18:2. Ag+-HPLC separates both geometric (trans,trans < cis/trans < cis,cis) and positional CLA isomers using the mobile phase hexane/acetonitrile (99.9:0.1). The elution volumes for the CLA isomers were not only affected by the concentration of acetonitrile (in the prepared mobile phase) but also with successive runs during the day using a prepared mobile phase batch, due to the partial solubility of acetonitrile in hexane. However, this drift does not affect the relative resolution of the CLA isomers. The addition of diethyl ether to the mobile phase partly stabilizes the solvent mixture. In order to facilitate the interpretation of Ag-+HPLC chromatograms, the relative retention volumes (RRV) were calculated for each CLA isomer. Toluene was added to all the test portions and served as an estimator of dead volume, whereas the elution of the ubiquitous 9c,11t-CLA isomer was chosen as unity (1.00). Expressing the elution of all the CLA isomers as their RRV greatly helped to standardize each CLA isomer, resulting in relatively small coefficients of variation (% CV) for the trans,trans (<1.5%) and cis/trans (<0.5%) CLA isomers. The identification of the CLA isomers was further facilitated by synthesis of authentic CLA isomers. All the geometric CLA fatty acid methyl esters (FAME) from positions 6,8- to 13,15-CLA were commercially available or synthesized by a combination of partial hydrazine reduction of known polyunsaturated fatty acids followed by alkali isomerization, isolation of products, and further iodine-catalyzed geometric isomerization. Based on expressing the elution volume as RRV and the availability of the synthetic CLA isomers, a unique reversal of the elution order of the c/t CLA isomers was found. It is also proposed that the retention times of CLA isomers by gas chromatography (GC) should be expressed as their relative retention times (RRT) relative to methyl gamma-linoleneate. The availability of CLA reference materials and the application of RRV and RRT to Ag+-HPLC and GC separations, respectively, will greatly improve in the identifications of CLA isomers.     

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.     

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.     

Monitoring cellular responses upon fatty acid exposure by Fourier transform infrared spectroscopy and Raman spectroscopy

We investigated the applicability of FTIR-spectroscopy as a high throughput screening method for detection of biochemical changes in intact liver cells in bulk upon fatty acid exposure. HepG2 cells adapted to serum free (HepG2-SF) growth were exposed to four different fatty acids, three octadecenoic acids, differing in cis/trans-configuration or double bond position (oleic acid, elaidic acid and vaccenic acid) as well as palmitic acid in three days. High throughput FTIR spectroscopic measurements on dried films of intact cells showed spectra with high signal-to-noise ratio and great reproducibility. When applying principal component analysis (PCA) a clear discrimination between fatty acid exposures was observed. Higher levels of triacylglycerides were accumulated in cells exposed to elaidic acid than when exposed to the other fatty acids; the least accumulation appeared to be in cells exposed to palmitic acid. An increased absorption at ~966 cm(-1) corresponding to trans-double bond was detected upon elaidic acid exposure but not upon vaccenic acid exposure. Instead, upon vaccenic acid exposure two new absorption bands were observed at 981 and 946 cm(-1) due to the presence of double bond conjugation. Raman spectroscopy on single cells, with and without treatment by vaccenic acid, confirmed the presence of conjugation. By fatty acid composition analysis, the conjugation was further specified to be conjugated linoleic acid (CLA) isomers. Thus, instead of being preserved as a monounsaturated fatty acid, vaccenic acid was converted into CLA in HepG2 cells. The results demonstrate the applicability of high-throughput FTIR spectroscopy as an explorative method in in vitro systems from which biologically relevant hypotheses can be generated and further investigated.     

毛细管气相色谱法测定乳脂中的cis-9,trans-11-共轭亚油酸

建立了测定乳脂中cis-9, trans-11-共轭亚油酸(CLA)的毛细管气相色谱方法。样品经正己烷-异丙醇提取、甲醇-甲醇钠甲酯化后,进行气相色谱分析;采用程序升温,以保留时间定性,外标法定量。采用该方法测得共轭亚油酸的回收率为100.26%,相对标准偏差（RSD）为1.9%(n=6),检测限为1 mg/L。该方法样品用量少,前处理简单,建立的实验条件准确可靠,不仅可以用来测定cis-9,trans-11-CLA的含量,而且对于乳制品中所含的其他脂肪酸的分析测定也具有指导意义。     

Effect of conjugated linoleic acid immobilization on the hemocompatibility of cellulose acetate membrane

Conjugated linoleic acid (CLA) was covalently immobilized onto cellulose acetate (CA) membranes. The effects of CLA immobilization on the blood coagulation, platelet aggregation, and oxidative stress were evaluated using human blood. The resulting CLA grafting CA membranes were characterized with X-ray photoelectronic spectroscopy (XPS). The complete blood count (CBC) and coagulation time (CT) was evaluated in vitro for the hemocompatibility. Human serum albumin (HSA) and human plasma fibrinogen (HPF) was evaluated for the protein affinity. The production of reactive oxygen species (ROS) was measured by chemiluminescence (CL) method to evaluate the oxidative stress. The results showed that the CLA-immobilizing CA membrane could keep the CBC values more stable than unmodified CA membrane. The CLA-immobilized CA membranes also showed longer CT and less adsorption of plasma proteins. CLA-immobilized CA membrane could keep the CL counts of hydrogen peroxide and superoxide values more stable than unmodified CA membrane. These results suggest that a CLA-immobilized CA membrane could offer protection for patients against oxidative stress and would be helpful for reducing the dosage of anticoagulant during hemodialysis.     

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.     

Chemometric deconvolution of gas chromatographic unresolved conjugated linoleic acid isomers triplet in milk samples

A generally known problem of GC separation of trans-7;cis-9; cis-9,trans-11; and trans-8,cis-10 CLA (conjugated linoleic acid) isomers was studied by GC–MS on 100 m capillary column coated with cyanopropyl silicone phase at isothermal column temperatures in a range of 140–170 °C. The resolution of these CLA isomers obtained at given conditions was not high enough for direct quantitative analysis, but it was, however, sufficient for the determination of their peak areas by commercial deconvolution software. Resolution factors of overlapped CLA isomers determined by the separation of a model CLA mixture prepared by mixing of a commercial CLA mixture and CLA isomer fraction obtained by the HPLC semi-preparative separation of milk fatty acids methyl esters were used to validate the deconvolution procedure. Developed deconvolution procedure allowed the determination of the content of studied CLA isomers in ewes’ and cows’ milk samples, where dominant isomer cis-9,trans-11 is eluted between two small isomers trans-7,cis-9 and trans-8,cis-10 (in the ratio up to 1:100).     

Analysis of conjugated linoleic acids as 9-anthrylmethyl esters by reversed-phase high-performance liquid chromatography with fluorescence detection

A simple and highly sensitive method for determining the fatty acid composition of food lipids containing conjugated linoleic acid (CLA) is described. The method is based on the separation of the 9-anthrylmethyl ester derivatives of saturated and unsaturated (conjugated and non-conjugated) fatty acids by reversed-phase high-performance liquid chromatography with fluorescence detection. Just like the other fatty acids, CLA reacts readily with 9-anthryldiazomethane at room temperature to produce 9-anthrylmethyl esters without isomerization and decomposition of the conjugated double bonds. Clear resolution of the individual fatty acids as their 9-anthrylmethyl esters is achieved on a highly efficient octadecylsilylated silica column (150- x 3-mm i.d., 3-microm particle size) using a stepwise gradient elution with methanol-water. The method is standardized with commercially available CLA isomers (cis-9, trans-11 and trans-10, cis-12-octadecadienoic acids, and their cis,cis and trans,trans isomers) and applied for determination of the fatty acid compositions of milk and sdairy products.     

Conjugated linoleic acid determination in human milk by fast-gas chromatography

An efficient direct method for measuring c9,t11- and t10,c12-conjugated linoleic acid (CLA) isomer content in human and rat milk was developed and validated using an RTX-2330 capillary column (40 m × 0.18 mm × 0.1 μm). In comparison with the commonly used 100 m × 0.25 mm × 0.20 μm columns, this new type of fast column allowed the separation of FAMEs with the same resolution but in much less time. An additional advantage for biological samples was that only a small volume of sample was needed. Two different procedures were tested in order to select the best methylation of CLA isomers, and the alkali plus acid-catalyzed procedure was selected. The precision results showed relative standard deviations (R.S.D.) of repeatability and reproducibility ranging between 0.10 and 8.71%. The application of this method to human and rat milk samples showed that it was a rapid, simple and reliable method for the analysis of biological samples.     

Effective Assignment of α2,3/α2,6‐Sialic Acid Isomers by LC‐MS/MS‐Based Glycoproteomics

Distinct structural changes of the α2,3/α2,6‐sialic acid glycosidic linkages on glycoproteins are of importance in cancer biology, inflammatory diseases, and virus tropism. Current glycoproteomic methodologies are, however, not amenable toward high‐throughput characterization of sialic acid isomers. To enable such assignments, a mass spectrometry method utilizing synthetic model glycopeptides for the analysis of oxonium ion intensity ratios was developed. This method was successfully applied in large‐scale glycoproteomics, thus allowing the site‐specific structural characterization of sialic acid isomers.     

Cancer Influences the Elemental Composition of the Myocardium More Strongly than Conjugated Linoleic Acids-Chemometric Approach to Cardio-Oncological Studies

The aim of the study was to verify in a cardio-oncological model experiment if conjugated linoleic acids (CLA) fed to rats with mammary tumors affect the content of selected macro- and microelements in their myocardium. The diet of Sprague–Dawley females was supplemented either with CLA isomers or with safflower oil. In hearts of rats suffering from breast cancer, selected elements were analyzed with a quadrupole mass spectrometer with inductively coupled plasma ionization (ICP-MS). In order to better understand the data trends, cluster analysis, principal component analysis and linear discriminant analysis were applied. Mammary tumors influenced macro- and microelements content in the myocardium to a greater extent than applied diet supplementation. Significant influences of diet (p = 0.0192), mammary tumors (p = 0.0200) and interactions of both factors (p = 0.0151) were documented in terms of Fe content. CLA significantly decreased the contents of Cu and Mn (p = 0.0158 and p = 0.0265, respectively). The level of Ni was significantly higher (p = 0.0073), which was more pronounced in groups supplemented with CLA. The obtained results confirmed antioxidant properties of CLA and the relationship with Se deposition. Chemometric techniques distinctly showed that the coexisting pathological process induced differences to the greater extent than diet supplementation in the elemental content in the myocardium, which may impinge on cardiac tissue’s susceptibility to injuries.     

提高维甲酸毛细管电泳分析灵敏度的微柱固相萃取法研究

本文发展了一种适于复杂样品预处理的微柱固相萃取装置。该装置与毛细管电泳结合用于13-顺-维甲酸和全反式-维甲酸异构体的分析,灵敏度可提高100倍以上。维甲酸异构体的微柱固相萃取过程包括柱老化、上样、洗涤、洗脱以及柱再生等步骤。将该法用于加标血浆样品中的维甲酸分析,得到满意的结果。     

Development of HPLC with fluorescent detection using NBD‐F for the quantification of colistin sulfate in rat plasma and its pharmacokinetic applications

Colistin sulfate, composed of a mixture of colistin A sulfate (CLA) and colistin B sulfate (CLB), is available for treating life‐threatening infections caused by multidrug‐resistant Gram‐negative bacteria. In this study, the CLA and CLB were quantified separately. Colistin sulfate was extracted from rat plasma with a solid‐phase extraction C₁₈ cartridge and reacted with 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F), and the fluorescent derivatives were subjected to reversed‐phase high‐performance liquid chromatography analysis and used to investigate the pharmacokinetics of CLA and CLB in rat plasma. The recovery rates of CLA and CLB were 41.2 ± 4.4 and 45.5 ± 3.1%, respectively. The recovery rate calculated from the total area of CLA and CLB was 43.9 ± 3.6%. When 2 mm NBD‐F and 10 mm boric acid buffer (pH 9.5) were added to colistin sulfate, the highest recovery rate was obtained. The best heating time was 5 min at 60°C. The lower limits of quantification for CLA, CLB and the total area of CLA and CLB were 0.05, 0.05 and 0.1 μg/mL; the coefficients of variations were 13.5, 14.5 and 14.1%, respectively. This method was found to have acceptable linearity, precision and accuracy, and has been successfully applied to a pharmacokinetic study in rat plasma.     

Analysis of conjugated linoleic acid-enriched triacylglycerol mixtures by isocratic silver-ion high-performance liquid chromatography

Silver-ion HPLC (Ag-HPLC) was applied to the fractionation of a triacylglycerol (TAG) sample enriched (>80%) with conjugated linoleic acid (CLA). After conversion of the TAGs to fatty acid methyl esters using sodium methoxide in methanol, Ag-HPLC (dual-column; isocratic solvent system of 0.1% acetonitrile in hexane; UV detection at 233 nm) was used to determine the CLA isomer distribution (50:50 mixture of 9c 11t- and 10t,12c-18:2). Three or four Ag-HPLC columns connected in series (0.6-1.0% acetonitrile in hexane as solvent; UV detection at 206 nm) were used to analyze the sample in TAG form. Elution times for CLA-enriched TAGs averaged 30 min or less. Isocratic solvent conditions were used to eliminate the solvent equilibration times (often 30 min or more) required between sample injections when solvent programming is used. The ratio of TAGs containing three vs. only two CLA molecules was found to be approximately 3 to 1. Ag-HPLC has thus been shown to be a useful method for rapidly analyzing not only CLA isomers as esters, but also in the TAG form.     

Capillary electrophoretic analysis of the derivatives and isomers of benzoate and phthalate

A capillary electrophoretic method for the analysis of 12 commonly found derivatives and isomers of benzoate and phthalate, including p-toluic acid, p-acetamido and p-hydroxy derivatives of benzoic acid, salicylic acid and its acetyl ester, 2- and 4-isomers of carboxybenzaldehyde, meta-, para-, and ortho-isomers of phthalic acid, and monomethyl terephthalic acid was developed. Capillary electrophoresis (CE) was performed in the free zone electrophoresis mode. Performing CE in 10 mM phosphate buffer, pH 7.0 could separate most of the benzoic acid derivatives except the structural or positional isomers. The positional isomers of phthalic acids could be completely separated with co-addition of alpha- and beta-cyclodextrins. Addition of poly(ethylene glycol) 600 (4%) could further resolve some structural isomers. The CE method developed here is rapid, i.e. complete separation could be achieved in less than 8 min for the nine monoanionic benzoate derivatives and in less than 14 min for the three dianionic phthalate isomers. The new method has good precision and linearity and can be readily applied to real samples for quantitative analysis. It is sensitive and can detect sub-ppm (w/w) level of impurity in real terephthalic samples.     

Determination of dopamine-3- and 4-O-sulphate in human plasma and urine by anion-exchange high-performance liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic (HPLC) method is reported for the determination of dopamine-3- and -4-O-sulphate isomers in human plasma and urine using an anion exchanger coupled with post-column hydrolysis and fluorimetric detection. Samples of plasma or urine are partially purified on Dowex 1 and Dowex 50 columns and separated using HPLC. These compounds are then hydrolysed and determined automatically by the p-aminobenzoic acid method in a continuous-flow reaction system. As the p-aminobenzoic acid method is very specific for dopamine, it is also possible to determine the isomers by injecting 5-20 microliter of urine or 100-200 microliter of deproteinized plasma directly into the HPLC system without clean-up. The detection limit of the method for both isomers is 0.3 pmol. In normal subjects, the plasma levels of dopamine-3- and -4-O-sulphate are 26.5 (S.D. 11.1) and 2.68 (S.D. 0.34) pmol/ml, and their urinary excretion rates are 1.73 (S.D. 0.56) and 0.27 (S.D. 0.04) nmol/min, respectively. Thus the two isomers are present in both plasma and urine and their urinary excretions reflect directly their plasma levels.