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.     

Development of a capillary electrophoretic method for the separation of diastereoisomers of a new human immunodeficiency virus protease inhibitor

A capillary electrophoretic (CE) method was developed for the separation of diastereoisomers of a new human immunodeficiency virus (HIV) protease inhibitor TMC114. In total 16 isomers of this drug have been synthesized (eight pairs of enantiomers). We succeeded in the separation of the eight diastereoisomers, but no enantiomers could be separated. Because of the high similarity and water-insolubility of these isomers, the separation is a real challenge. Different CE modes were tried out: capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), micellar electrokinetic capillary chromatography (MEKC), and microemulsion electrokinetic capillary chromatography (MEEKC). Only MEEKC offered resolution of these compounds.     

Comparative study of capillary zone electrophoresis and micellar electrokinetic chromatography for the separation of twelve aromatic sulphonate compounds

Summary Two modes of capillary electrophoresis (CE), capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC), were investigated for the separation of 12 aromatic sulphonate compounds. In CZE, although the voltage applied, the buffer concentration and the pH were optimized for effective separation of the compounds studied, under the best conditions four of the five amino compounds coeluted, as did naphthalene-1-sulphonic acid and naphthalene-2-sulphonic acid. In MEKC, sodium dodecyl sulphate (SDS) and Brij 35 were chosen as the anionic and nonionic surfactants and the effect of the concentration of micelles was examined. The effect of adding methanol as the organic modifier was also investigated with each of these micellar systems. All the analytes, including the isomers, were completely separated by use of MEKC with Brij 35 but when SDS was used only 11 compounds were separated because two amino compounds coeluted.     

Highly efficient separation of isomeric epoxy fatty acids by micellar electrokinetic chromatography

A capillary electrophoresis (CE) method has been developed for simple and direct separation of cis- and trans-12,13-epoxy-9(Z)-octadecenoic acid and 9,10-epoxy-12(Z)-octadecenoic acid isomers. Separation was performed in micellar electrokinetic capillary chromatography (MEKC) using a buffer consisting of 25 mM borate (pH 9.20), 10 mM sodium dodecyl sulfate (SDS) and 10% v/v acetonitrile. The key variables, concentrations of SDS and organic modifier, were optimized by the application of a factorial experimental design. The use of a low micellar concentration, just above critical micelle concentration (CMC), in a background electrolyte containing an organic modifier not only made it possible to dissolve and separate highly hydrophobic fatty acid isomers, but also resulted in improved separation efficiency and selectivity. Separation efficiency up to 4 x 10(5) theoretical plates/m was achieved under an optimized condition. Also investigated were the influence of temperature on separation and the effect of organic modifier concentration on the dynamic exchange of the analytes between micelles and the bulk of the buffer solution. Direct UV was applied for detection of the fatty acids.     

Investigation of solvent effects in capillary electrophoresis for the separation of biological porphyrin methyl esters

The effects of organic solvents on the capillary electrophoresis (CE) separation of a number of important biological porphyrin methyl esters - six weakly basic, hydrophobic cyclic tetrapyrroles possessing two and four to eight methyl ester groups around the periphery of the porphyrin ring - were investigated in the mode of micellar electrokinetic chromatography (MEKC), microemulsion electrokinetic chromatography (MEEKC), and nonaqueous CE. In aqueous MEKC, partial separation of the six neutral porphyrin methyl esters was obtained with an organic modifier (acetonitrile) in the concentration range between 20 and 40%, in which sodium dodecyl sulfate (SDS) molecules might be present in the form of SDS micelles and/or SDS micelle-like aggregates. Relatively stable SDS micelles can be formed in nonaqueous MEKC using formamide as the separation medium, but the separation of the target analytes remained unsatisfactory. Improved resolution of all six porphyrin methyl esters was obtained using MEEKC with the running buffer consisting of 0.8% w/w n-heptane (oil phase), 2.25% w/w SDS and 1.0% w/w Brij 35 (mixed surfactant), 6.6% w/w 1-butanol (cosurfactant), and 30% v/v 2-propanol (second cosurfactant), but reproducibility in terms of peak areas for certain porphyrins (especially uroporphyrin I octamethyl ester) was found to be very poor. Best separation performances were achieved with nonaqueous CE separations in which the weakly basic porphyrin methyl esters were protonated under strongly acidic conditions (e.g., using 10 mM perchloric acid) in mixed organic solvents. For example, using a 50:50 mixture of methanol and acetonitrile as the separation medium, baseline separation of all six (positively charged) porphyrin methyl esters can be obtained within 3 min and the average precision (RSD, N = 13) in terms of migration time and peak area were 0.55 and 2.16%, respectively.     

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.     

On-line identification of trans- and cis-resveratrol by nonaqueous capillary electrophoresis/fluorescence spectroscopy at 77 K

This work presents a novel method for the accurate determining trans- and cis-resveratrol (3,5,4'-trihydroxystilbene) by nonaqueous capillary electrophoresis/fluorescence spectroscopy at 77 K. The proposed method permits not only the separation of resveratrol isomers, but also ensures that on-line spectra are readily distinguishable and unambiguously assigned. The experimental results also indicate that the effect of nonaqueous capillary electrophoresis buffer and low-temperature technique increase the detection limit by more than 150-fold.     

Enzymatic activity measurement of phospholipid hydroperoxide glutathione peroxidase by capillary electrophoresis

Based on the separation of 1-palmitoyl-2-(13-hydroperoxy-cis-9,trans-11-octadecadienoyl)-L-3- phosphatidylcholine (PC-OOH) and 1-palmitoyl-2-(13-hydroxy-cis-9,trans-11-octadecadienoyl)-L-3- phosphatidylcholine (PC-OH) and the quantitative determination of PC-OH, the enzymatic activity of phospholipid hydroperoxide glutathione peroxidase (PHGPx) can be measured by capillary electrophoresis. The separation was carried out in a fused-silica capillary (30 cm x 100 microns id) at 15 kV positive voltage. Sodium borate (100 mM; pH = 8.4) was used as the running buffer, and the photodiode array detector wavelength was 232 nm. The determination can be completed in 5 min. The detection limit was 5 pmol; and the relative standard deviation (RSD) of the peak area was less than 1% with an average recovery of 98.6%. Compared with traditional methods such as HPLC and spectrophotometry, it is faster and more convenient. Using capillary electrophoresis, the enzymatic activities of PHGPx expressed by the rice PHGPx gene in E. coli. M15 was determined as 1.25 x 10(-5) mumol min-1, and the specific activity of partially purified trans-gene PHGPx was 3.1 x 10(-2) mumol min-1 per mg. The stability of the trans-gene PHGPx was also studied.     

Separation and determination of the anthraquinones in Xanthophytum attopvensis pierre by nonaqueous capillary electrophoresis

A nonaqueous capillary electrophoresis (NACE) method with direct on-column UV detection has been developed for the separation of the pharmaceutically important anthraquinones from the total grass of Xanthophytum attopvensis pierre extract. The separation of three main anthraquinones (1-hydroxy-2-methoxy-3-hydroxymethyl-9, 10-anthraquinone-1-O-β-d-glucoside (1), rubiadin- 1-methylether (2) and 1-methoxy-2-formyl-3-hydroxy-9, 10-anthraquinone (3)) was optimized with respect to concentration of sodium cholate (SC) and acetic acid, addition of acetonitrile (ACN), and applied voltage. Baseline separation was obtained for the three analytes within 5 min using a running buffer containing 50 mM sodium cholate (SC), 1.0% acetic acid and 40% ACN in methanol. The method of NACE for the separation and determination of bioactive ingredient in traditional Chinese medicines was discussed.     

Determination of polyphenol components in herbal medicines by micellar electrokinetic capillary chromatography with Tween 20

A simple and convenient method of micellar electrokinetic capillary chromatography (MEKC) using polyoxyethylene sorbitan monolaurate (Tween 20) to form single micelle and methanol as a buffer additive was introduced for the simultaneous determination of five polyphenols, including scopoletin, rutin, esculetin, chlorogenic acid and caffeic acid. A running buffer solution of pH 9.3, 20 mmol/L sodium tetraborate containing 64 mmol/L Tween 20 and 9% (v/v) methanol was adopted in the separation. Because rutin and esculetin were difficult to be separated by capillary zone electrophoresis (CZE) and SDS-based MEKC, Tween 20-based MEKC was adopted and the polyphenols were separated satisfactorily. The proposed method was used to determine the polyphenol components in the herbal medicine of Cortex fraxini. The separation mechanism of Tween 20-based MEKC for the polyphenols was discussed preliminarily.     

Separation of isomers of dienoic acids by electromigration techniques

Different capillary electromigration techniques were employed to resolve geometrical isomers of sorbic acid, decadienoic acid, and ethyl sorbate. Since these substances differ in their polarity, shape, and size, various electromigration approaches were investigated to separate the four geometrical isomers of each compound. With capillary electrophoresis (CE) modified with a cyclodextrin (β‐CD) the four isomers of sorbic acid were separated using a buffer that consists of 60 mM tetraborate and 8 mg/mL β‐CD. The separation of decadienoic acid geometrical isomers was not possible, even at elevated tetraborate and cyclodextrin concentrations. The four isomers of decadienoic acid were successfully separated using micellar electrokinetic chromatography (MEKC) with a buffer consisting of 30 mM tetraborate and 100 mM SDS and microemulsion electrokinetic chromatography (MEEKC). Ethyl sorbate is the least polar of all the studied substances and its isomers could not be separated by MEKC or MEEKC. The resolution was improved and isomers were fully separated using capillary electrochromatography (CEC) with ODS stationary phase and a mobile phase consisting of 10 mM boric acid in 50% acetonitrile. Minor differences in the polarity and the shape of isomers and high resolving power of the applied techniques were sufficient for separation of very similar compounds. We have shown that versatile electromigration techniques can be applied for separation of geometrical isomers of dienoic acids and their esters.     

Profiling and screening analysis of 27 aromatic amino acids by capillary electrophoresis in dual modes

An efficient capillary electrophoretic (CE) profiling and screening system based on dual modes of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) was developed for the simultaneous determination of 23 nonprotein amino acids (NPAAs) and 4 protein amino acids with aromatic moiety. It involves separation by an uncoated fused-silica capillary under phosphoric acid buffer in CZE mode and by another uncoated fused-silica capillary under neutral sodium dihydrogen phosphate buffer containing sodium dodecyl sulfate in MEKC mode. Migration orders of the amino acids studied on the two separation modes under each optimum condition were very different. The repeatability of migration times measured by the CZE and MEKC was found to be better than 4.8 and 3.4%, respectively, thereby enabling to cross-check the identification of each amino acid. The method linearity and limit of detection of the CZE for each amino acid were found to be adequate for the assay of aromatic amino acids. When the present CE profiling and screening analysis in dual modes was applied to plant seeds, NPAAs such as mimosine from Mimosa pudica Linné, and 2-phenylglycine from Lindera erythrocarpa Makino were positively detected along with tryptophan, phenylalanine and tyrosine.     

Rapid reversed-phase high-performance liquid chromatographic determination of the regiospecificity of lipoxygenase products on linoleic acid

A new reversed-phase high-performance liquid chromatography method for the separation of regioisomeric products from lipoxygenase acting on linoleic acid was studied. The addition of salts to the mobile phase improved the retention and separation behaviour of 13-hydroperoxy-9,11-octadecadienoic acid and 9-hydroperoxy-10,12-octadecadienoic acid with respect to the results obtained with other mobile phases reported in the literature. The effect of the pH and ionic strength of the buffer on the retention times, capacity factor and separation factor of these lipoxygenase products were also studied. The pH optimum coincided with the pKa of linoleic acid (close to 7 depending on the fatty acid concentration). Phosphate concentrations close to 100 mM considerably reduced the retention times and led to better separation of the mixture of both products. Finally, this method was applied to the identification and separation of two linoleic acid hydroxides (13-hydroxy-9,11-octadecadienoic acid and 9-hydroxy-10,12-octadecadienoic acid) obtained by the reduction of their corresponding hydroperoxides.     

Enantiomeric and isomeric separation of herbicides using cyclodextrin-modified capillary zone electrophoresis

Cyclodextrin-modified capillary zone electrophoresis (CD-CZE) was applied successfully to the enantiomeric and isomeric separation of three herbicides (imazaquin, diclofop and imazamethabenz). Commercially available cyclodextrins were evaluated for separation of the enantiomers and isomers of the three herbicides having varied molecular structures. The enantiomers of imazaquin and diclofop, and the isomers of imazamethabenz could be resolved with a resolution of ≥1.5. The resolution was found to depend on pH of the run buffer, cyclodextrin type and cyclodextrin concentration. By employing mixed cyclodextrins in the running buffer, the three herbicides were simultaneously separated in a single run. In addition, rapid (less than 3 min) enantiomeric separation is demonstrated using imazaquin as a model herbicide. The reported capillary electrophoresis (CE) methods are simple, rapid, efficient and reproducible and our results demonstrate that CE provides a powerful analytical tool for enantiomeric and isomeric separation of herbicides.     

Separation of ritalin racemate and its by-product racemates by capillary electrophoresis

Ritalin, [(+)-threo]methylphenidate hydrochloride, is a chiral drug substance with two chiral centers. The drug substance may contain three pairs of enantiomers, [(+)-threo], [(-)-threo], [(+)-erythro] and [(-)-erythro] isomers, and its degradation products, threoritalinic acid racemate. Determination of the optical purity of ritalin drug substance and the amount of its by-product isomers is a critical step in the single-isomer drug development. In order to efficiently recognize the three pairs of enantiomers by one method, capillary electrophoresis (CE) was employed for the separation. The three pairs of enantiomers in CE showed different enantioselectivities with eight different types of CDs. Only 2,6-di-o-methyl-beta-cyclodextrin (DM-beta-CD) and carboxymethyl-beta-cyclodextrin (CM-beta-CD) showed enantioselectivity to all these pairs of enantiomers. With respect to separation resolution and efficiency, DM-beta-CD was chosen as the chiral selector. For optimization of the separation conditions, the concentration of DM-beta-CD, pH of the buffer solution, and temperature of the capillary were further studied.     

Nonaqueous capillary electrophoresis with laser-induced fluorescence detection: a case study of comparison with aqueous media

A novel method based on separation by nonaqueous capillary electrophoresis (NACE) combined with laser-induced fluorescence (LIF) detection was developed and compared with classic aqueous modes of electrophoresis in terms of resolution of solutes of interest and sensitivity of the fluorescence detection. Catecholamines derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) were chosen as test analytes for their subtle fluorescence properties. In aqueous systems, capillary zone electrophoresis (CZE) was not suitable for the analysis of test analytes due to complete fluorescence quenching of NBD-labeled catecholamines in neat aqueous buffer. The addition of micelles or microemulsion droplets into aqueous running buffer can dramatically improve the fluorescence response, and the enhancement seems to be comparable for micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC). As another alternative, NACE separation was advantageous when performing the analysis under the optimum separation condition of 20 mM sodium tetraborate, 20 mM sodium dodecyl sulfate (SDS), 0.1% (v/v) glacial acetic acid, 20% (v/v) acetonitrile (ACN) in methanol medium after derivatization in ACN/dimethyl sulfoxide (DMSO) (3:2, v/v) mixed aprotic solvents containing 20 mM ammonium acetate. Compared with derivatization and separation in aqueous media, NACE-LIF procedure was proved to be superior, providing high sensitivity and short migration time. Under respective optimum conditions, the NACE procedure offered the best fluorescence response with 5-24 folds enhancement for catecholamines compared to aqueous procedures. In addition, the mechanisms of derivatization and separation in nonaqueous media were elucidated in detail.     

Novel approach to the analysis and use of fullerenes in capillary electrophoresis

The analysis and use of fullerenes in capillary electrophoresis (CE) was investigated. Sodium dodecyl sulfate (SDS) was used to solubilize fullerenes C60, C70, and a mixture of C60 and C70 in water. The behavior of the solutions of the C60- and C70-SDS complexes was examined by CE with on-line UV-Vis diode array detection. This study included the use of a C60-SDS complex as a new method of micellar electrokinetic chromatography (MEKC) for the separation of polycyclic aromatic hydrocarbons (PAHs) using CE with uniwavelength detection. Since SDS micelles act as a pseudostationary phase in which the PAH compounds partition with their hydrophobic interior, the addition of C60 within the micelles enhanced separation of the PAHs. The preliminary results using C60-MEKC with SDS were compared to those obtained with MEKC with SDS. The capillary electrophoretic separations were performed in 10 mM borate-phosphate buffer with 100 mM SDS at pH 9.5.     

Nonaqueous capillary electrophoresis in coated capillaries: an interesting alternative for proteomic applications

This work brings together some contributions for the use of nonaqueous media for proteomic analysis, for both capillary electrophoresis (CE) separation and the preparation of tryptic digests. First, a ternary nonaqueous buffer consisting of 60/30/10 v/v methanol/acetonitrile/acetic acid with 12.5 mmol/L ammonium acetate was optimized for CE separation of the tryptic digest of lysozyme. Lysozyme was chosen as a model system for the protein digestion, which has also been prepared in an organic-rich medium with methanol/50 mmol/L NH(4)HCO(3), pH 8.0 (60/40 v/v). The separation results were compared to in silico (PeptideCutter program) digestion conditions, and high-efficiency peak separation (18 peaks) was obtained in 20 min with an electric field of 350 V/cm. In addition, we have evaluated the stability of a coated capillary with poly-N,N-dimethylacrylamide (60/30 cm total/effective length and 75 microm ID) for over 100 runs of tryptic digest with the nonaqueous background electrolyte solvent system. The migration times for ten selected peptide peaks presented 3-7% relative standard deviation.     

Efficiency studies in nonaqueous capillary electrophoresis

Nonaqueous capillary electrophoresis (NACE) is a relatively new area with several advantages that include enhanced efficiency and improved detection sensitivity. The goal of this study was to investigate the influence of NACE compared to aqueous CE on the separation efficiency of oligosaccharides. The applied voltage and buffer concentration were optimized for the aqueous and nonaqueous buffer media to minimize the band broadening effects of Joule heating and electrophoretic dispersion. At the optimized conditions a 1.5-fold enhancement in efficiency was obtained with the nonaqueous buffer medium.     

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。