Presence Of Trimethylsulfonium Hydroxide Research Articles

Rapid Analysis of Fatty Acid Composition in Polysorbate 80 by Gas Chromatography with On-line Pyrolytic Methylation Technique

A novel method of on-line pyrolytic methylation–gas chromatography was developed for the rapid analysis of fatty acid composition in Polysorbate 80 without any tedious pre-treatment steps. Fatty acids in Polysorbate 80 were converted into their corresponding fatty acid methyl esters in the presence of trimethylsulfonium hydroxide with a vertical microfurnace pyrolyzer at 300 °C. The premixing procedure of sample and organic alkali reagent was necessary before the on-line pyrolytic methylation to improve the repeatability. The relative standard deviations for peak areas of fatty acids in Polysorbate 80 were over the range of 0.3–9.1% (n = 5). Six Polysorbate 80 samples, consisting of three samples of pharmaceutical grade and three samples of non-pharmaceutical grade, were analyzed to evaluate the feasibility of the proposed method. The relative percentages (%) of fatty acids for samples of pharmaceutical grade meet the Chinese Pharmacopoeia requirements with the amount of oleic acid varying from 78.4 to 89.3%. On the other hand, the relative percentages (%) for palmitic acid and stearic acid in samples of non-pharmaceutical grade were out of the specification limits, with the amount of oleic acid varying from 62.0 to 63.5%. The quantitative results determined by on-line pyrolytic methylation were in agreement with those obtained by off-line methylation. The result proved that gas chromatography with on-line pyrolytic methylation technique is of great value for rapid screening analysis of Polysorbate 80 samples in bulks.

Analysis of fatty acid composition in cottonseed by gas chromatography with on-line pyrolytic methylation

A method of on-line pyrolytic methylation-gas chromatography was developed for the analysis of fatty acid composition in cottonseed. Fatty acids in cottonseeds were converted to their corresponding fatty acid methyl esters in the presence of trimethylsulfonium hydroxide at 300℃. The major fatty acids were linoleic acid (C18:2), oleic acid (C18:1), palmitic acid (C16:0), stearic acid (C18:0), myristic acid (C14:0), palmitoleic acid (C16:1), arachidic acid (C20:0) and docosanoic acid (C22:0). The unsaturated fatty acid content varied from 66.30% to 72.54%, and linoleic acid content varied from 43.20% to 53.61%. The RSDs of the peak areas of the fatty acids were less than 10% (n=5). The fatty acid compositions in the cottonseed samples, obtained from different growing places and different edible oil samples were compared by statistical analysis. The results showed that fatty acid compositions in cottonseed samples from different regions were similar. The fatty acid composition in cottonseed samples was closest to that of corn oil, and the similarity varied from 0.960 to 0.992. The method is simple, rapid, accurate, and is suitable for the analysis of fatty acid composition in cottonseed.

Determination of glyceride and free fatty acid residuals in biodiesel by thin layer chromatography combined with on-line pyrolytic methylation gas chromatography

A novel method on basis of thin layer chromatography (TLC) combined with on-line pyrolytic methylation gas chromatography (OPM–GC) in the presence of trimethylsulfonium hydroxide (TMSH) was developed and applied to the determination of glyceride and free fatty acid residuals in biodiesel. Both glycerides and free fatty acids in biodiesel were separated by TLC alumina sheets, and then the cut spots were analyzed directly by pyrolytic methylation gas chromatography method in a pyrolyzer at 350°C without complex pretreatment. In this process, glycerides and free fatty acids were converted into their corresponding fatty acid methyl esters, and quantified well by the total peak areas of formed esters. Thus obtained calibration curves of monostearin, distearin, tristearin and oleic acid exhibited good linearity with the regression coefficients from 0.9766 to 0.9950 at the concentrations ranged from 300 to 5000mgL−1. The limits of detection were in the range of 100–200mgL−1, and the RSDs (n=3) ranged from 3.7% to 12.5%, calculated for monostearin, distearin, tristearin and oleic acid at the concentration level of 2000mgL−1. The potential of the proposed method was assessed by the determination of glycerides and free fatty acids in real biodiesel samples. The results prove that this TLC–OPM–GC technique is a simple, accurate and low solvent consuming method for the simultaneous determination of glyceride and free fatty acid residuals in biodiesel.

Validation of Thermally Assisted Hydrolysis and Methylation–Gas Chromatography Using a Vertical Microfurnace Pyrolyzer for the Compositional Analysis of Fatty Acid Components in Microalgae

Thermally assisted hydrolysis and methylation-gas chromatography (THM-GC) in the presence of trimethylsulfonium hydroxide, using a vertical microfurnace pyrolyzer, was validated for the compositional analysis of fatty acid components in microalgae. The chromatograms of a microalga, Pavlova lutheri , obtained under optimized THM conditions clearly showed a series of fatty acid methyl esters including thermally labile polyunsaturated fatty acid components. On the basis of these peak areas, their chemical compositions were rapidly determined without using any tedious sample pretreatment with a precision of <8% relative standard deviation. Moreover, the compositions thus obtained were in good agreement with those obtained by the conventional technique involving solvent extraction. Finally, the THM-GC technique was applied for the compositional analysis of fatty acid components in a newly found microalga, Coccomyxa gloeobotrydiformis . The obtained data showed a high abundance (24 mol %) of α-linolenic acid components, suggesting its potential usefulness as feed sources and/or functional foods.

Characterization of Alkylphenol Components in Ginkgo biloba Sarcotesta by Thermochemolysis–Gas Chromatography/Mass Spectrometry in the Presence of Trimethylsulfonium Hydroxide

A method to determine alkylphenols in Ginkgo biloba sarcotesta, especially focusing on polyunsaturated ginkgolic acid components, was developed on the basis of thermochemolysis–gas chromatography/mass spectrometry with a DB-23 column in the presence of trimethylsulfonium hydroxide [(CH3)3SOH]. By using this technique, powder sample of Ginkgo biloba sarcotesta can be directly analyzed without any tedious and time-consuming pretreatment. On the resulting chromatograms, seven kinds of ginkgolic acids including polyunsaturated ginkgolic acid GA17:2 and two kinds of ginkgols were clearly observed as their methyl derivative. Based on the peak areas, the ginkgolic acid compositions are in good agreement with these obtained by a high-performance liquid chromatography method dedicated to analysis of ginkgolic acid.

Rapid screening of hardwood and softwood lignin using low temperature thermochemolysis with a GC injection port

A low temperature thermochemolysis method was developed to rapidly differentiate softwood and hardwood lignin. The technique uses heat at 350 °C in the presence of trimethylsulfonium hydroxide (TMSH), whereby the sample is introduced via a fabricated solids injector into the injection port of a gas chromatography–mass spectrometry (GC–MS) instrument. No major modification or external devices are required for instrument conversion and the switchover to normal use is complete within minutes. Three different softwood lignin and two hardwood lignin samples were examined. All the thermochemolysis results revealed the presence of either yangambin, a hardwood dimeric marker containing syringyl groups, or sylvatesmin, a softwood dimeric marker containing guaiacyl groups. The results were confirmed by way of thermochemolysis using a traditional micro-furnace pyrolyzer. The study also revealed the presence of 3,3′,4,4′-tetramethoxystilbene, a marker for both types of lignin. It appears that the thermochemolysis-TMSH technique used with a 350 °C injection port produces larger methylated units of the lignin substructures than those obtained using thermochemolysis in the presence of the more common methylating agent, tetramethylammonium hydroxide (TMAH), which is often used at much higher temperature. In addition to lignin markers, other compounds such as resin acid methyl esters were liberated, but only as a result of thermochemolysis. These extract-like materials were not observed using solvent extraction–methylation, which indicates that these resin acid units may be covalently bound to the softwood lignin macrostructure.

Analysis of catechins and condensed tannins by thermally assisted hydrolysis/methylation-GC/MS and by a novel two step methylation

The objective of this study is to observe high molecular weight markers of catechins and condensed tannins by thermally assisted hydrolysis/methylation (THM)-GC/MS. Techniques for formation of intact methylated flavanols of catechins using THM in the presence of trimethylsulfonium hydroxide (TMSH), and, of a dimer marker of condensed tannins using a novel two step methylation technique, are presented. The two step methylation procedure involves pre-methylation of the sample with trimethylsilyl diazomethane (TMS-diazomethane) followed by THM. The dimer marker, a methylated product containing the C–C linkage between adjacent flavanol units, has a molecular weight of 540. Intact methylated flavanols of catechins were also successfully observed as high molecular weight compounds including partially methylated catechin and epicatechin (3-flavanol, 3′,4′,5,7-tetramethoxy, cis/trans; m/ z = 346), epigallocatechin and gallocatechin (3-flavanol, 3′,4′,5,5′,7-pentamethoxy, cis/trans; m/ z = 376). These techniques were successfully applied to the analysis of series of condensed tannins isolated from plants, and catechins and other phenolics present in (hot water) extracts of tea leaves. In green tea the major catechins were identified as epicatechin and epigallocatechin along with flavonols and tannin dimers.

Squid Oil and Fat Production from Squid Wastes Using Subcritical Water Hydrolysis: Free Fatty Acids and Transesterification

Application of subcritical water (sub-CW) treatment on the yield of oil and fat produced from squid waste entrails was studied experimentally in the temperature range of 443−653 K with a reaction time of 1−40 min. This process showed that the oil and the fat phases contain nine free fatty acids which were produced during hydrolysis of the triglyceride molecules and showed a maximum total yield at 513 K and 40 min (0.44 kg/kg oil). The order of the yields for the nine fatty acids, including omega-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), was found to be DHA > palmitic > oleic > EPA > others. On the other hand, transesterification of the oil and the fat phases in the presence of trimethylsulfonium hydroxide (TMSH) which converted the free fatty acids to the corresponding fatty acid methyl esters (FAMEs) was studied. The total yield of FAMEs in the oil and the fat phases illustrated a peak at 473 and 493 K, respectively.

Fatty acid profiling of lipid classes by silica rod TLC-thermally assisted hydrolysis and methylation–GC/MS

Advances towards the coupling of thin layer chromatography (TLC) Chromarods to gas chromatography/mass spectrometric (GC/MS) analysis were accomplished by introducing an intact TLC silica-coated rod (15 cm) into a vertical furnace pyrolyzer coupled to a GC/MS instrument. Using this technique, a fatty acid profile corresponding to the neutral and polar fraction of a lipid extract previously separated by TLC was obtained. The fatty acids were analyzed directly off the TLC rod, as their methyl derivatives were obtained following thermally assisted hydrolysis methylation (THM) in the presence of trimethylsulfonium hydroxide (TMSH). The results show that the THM reaction is successful on lipids that are adsorbed on the silica surface. Using lipid standards, differences in the fatty acid yield for the THM reaction of phospholipids (PL), triacylglycerides (TAG) and free fatty acids (FFA) were observed. Compared to conventional TMSH/lipid solution analysis, there was a significant decrease in the degree of isomerization for polyunsaturated fatty acids (PUFA) using the TMSH/silica-supported reaction, when a relatively high TMSH/lipid molar ratio (>2500) is used. The fatty acid profile for a marine (cold water) lipid sample using silica-supported THM compared well with results obtained by conventional lipid extraction followed by BF 3–methanol esterification. This study's success of in situ analysis of intact chromatographic bands illustrates the strong potential for TLC–GC/MS.

Highly sensitive determination of lipid components including polyunsaturated fatty acids in individual zooplankters by one-step thermally assisted hydrolysis and methylation-gas chromatography in the presence of trimethylsulfonium hydroxide

Thermally assisted hydrolysis and methylation-gas chromatography (THM-GC) in the presence of trimethylsulfonium hydroxide (TMSH) was applied to the analysis of the lipid contents and their fatty acid compositions in individual zooplankters, especially focusing on polyunsaturated fatty acid (PUFA) components. On the resulting chromatograms, fatty acid components including PUFAs such as eicosapentaenoic acid (EPA; C20:5) and docosahexaenoic acid (DHA; C22:6) in single zooplankters were clearly observed as their methyl esters without doing any tedious and time-consuming pretreatment. By using this technique, the lipid contents and fatty acid compositions in a powdered plankter sample prepared by cryomilling homogenization were estimated on the basis of the observed peak intensities with relative standard deviations of less than 6%. The fatty acid compositions thus determined were in good agreement with those obtained by the conventional technique involving solvent extraction of the samples for every fatty acid component including PUFAs. The differences among individual zooplankter samples were then evaluated by THM-GC method. As a result, the chemical compositions of EPA and DHA showed relatively large inter-individual fluctuations even for the samples cultured under the same conditions. This suggests that lipid biosynthesis of the highly unsaturated fatty acid components and their consumption proceeded differently depending on the life history for each individual zooplankter.

A Novel Method for Preservation of Labile Lipid Samples at Ambient Temperature with Oxygen Absorber

A novel method for preserving labile lipid samples at ambient temperature was developed with a commercially available oxygen absorber. In order to optimize the preservation conditions, the changes in the fatty acid composition of a triacylglycerol mixture during storage were determined by thermally assisted hydrolysis and methylation gas chromatography in the presence of trimethylsulfonium hydroxide. The polyunsaturated fatty acid (PUFA) components of the sample proved to degrade during preservation at room temperature through autoxidation induced by the trace amount of residual oxygen in a plastic bag for storage even coexisting with the oxygen absorber. On the other hand, once the lipid sample in the plastic bag with the oxygen absorber was subjected to 7 days “pretreatment” in a refrigerator at about 2°C, the PUFA components of the lipid sample in the bag were kept almost completely unchanged during further preservation at room temperature for at least four weeks. The observed results indicated that the residual oxygen in the plastic bag was sufficiently eliminated by the oxygen absorber during the “pretreatment” at about 2°C. Therefore, the labile lipid samples can be preserved at room temperature for a long time without deterioration by using oxygen absorber combined with the appropriate “pretreatment” storage in chilled conditions for a certain period. Moreover, reliable transportation of various biomaterial samples to a distant laboratory is able to be carried out conveniently at ambient temperature by this novel storing method without utilizing any special freezing system.

Compositional analysis of polyunsaturated fatty acid oil by one-step thermally assisted hydrolysis and methylation in the presence of trimethylsulfonium hydroxide

Thermally assisted hydrolysis and methylation-gas chromatography (THM-GC) in the presence of trimethylsulfonium hydroxide (TMSH) was applied to the determination of fatty acid composition of lipids containing polyunsaturated fatty acid residues. By using this method, the polyunsaturated fatty acid (PUFA) components as well as saturated and monounsaturated fatty acid residues were observed almost quantitatively as their methyl esters on the resulting chromatogram without causing any appreciable isomerization and/or degradation. On the basis of these peak intensities, the fatty acid compositions in soybean oil and sardine oil samples were rapidly and precisely estimated with less than 6% of the relative standard deviations even for PUFA components such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

S-methyl derivatives from thiol compounds by the pyrolytic reaction with trimethylsulfonium hydroxide.

Base-catalyzed transesterification of acyl lipids with methanol in the presence of trimethylsulfonium hydroxide (TMSH) is an easy and convenient method for the preparation of fatty acid methyl esters for gas chromatography (GC) analyses. Free fatty acids are converted to fatty acid methyl esters by the pyrolytic reaction with TMSH as well. We have found that lipids and other compounds containing thiol groups are also converted easily to the corresponding methyl sulfides (methyl thioethers) by the pyrolytic reaction with TMSH occurring in the injector of the gas chromatograph. For example, alkane thiols such as dodecane thiol and octadecane thiol are converted to the corresponding alkyl methyl sulfides, whereas bis(methylthio) derivatives are formed from alpha,omega-alkane dithiols, e.g., 1,8-octanedithiol, and from 2,3-dimercaptopropan-1-ol (dimercaprol). Furthermore, 3beta-mercaptocholest-5-ene (thiocholesterol) is converted to 3-methylthiocholest-5-ene by the same reaction. The S-methylation reactions which finally lead to the corresponding methylthio derivatives of lipids and other compounds with thiol groups may be of diagnostic value for the structural analysis of such compounds by GC and GC/mass spectrometry.

Formation of N- and O-methyl derivatives of lipids containing amino, amide or hydroxy groups by the pyrolytic reaction with trimethylsulfonium hydroxide

Base-catalyzed transesterification of acyl lipids with methanol in the presence of trimethylsulfonium hydroxide (TMSH) is an easy and convenient method for the preparation of fatty acid methyl esters for GC analyses. However, lipids containing functional groups such as amino, amide and hydroxy groups are converted in varying degrees to the corresponding N- and O-methyl derivatives by the pyrolytic reaction of TMSH occurring in the injector of the gas chromatograph. For example, lipids containing amino or amide groups are converted into the corresponding N-methyl and N,N-dimethyl derivatives, fatty acid ethanolamides to the corresponding N-methyl, O-methyl and N,O-dimethyl derivatives, whereas alkyl methyl ethers are formed from long-chain alcohols. Furthermore. 2-O- and 3-O-monomethyl ethers as well as 2,3-di-O-methyl ethers are formed from 1-O-alkylglycerols, methoxy fatty acid methyl esters from hydroxy fatty acids as well as steryl 3β-O-methyl ethers from cholesterol and other sterols. Since some of the mentioned artefacts may interfere with fatty acid methyl esters in GC separations the TMSH derivatization method is recommended only with caution for lipids containing amino, amide and hydroxy groups. The methylation reactions, which finally lead to the corresponding N-methyl, O-methyl, N,N-dimethyl or N,O-dimethyl derivatives of fatty acids and lipids may, however, be of some diagnostic value for the structural analysis of such lipids by GC/MS.