Quantitative Method for the Gas Chromatographic Analysis of Short-Chain Monocarboxylic and Dicarboxylic Acids in Fermentation Media

A method for the preparation and gas chromatographic analysis of the butyl esters of volatile (C-1-C-7) and nonvolatile (lactic, succinic, and fumaric) acids in microbial fermentation media is presented. Butyl esters were prepared from the dry salts of the acids. The esters were separated by temperature programming on a column of Chromosorb W coated with Dexsil 300 GC liquid phase and analyzed with a flame ionization detector. Apparent recoveries with butanol-HCl or butanol-H2SO4 as butylating agents were 80 to 90% for most acids. Chromatographic profiles of the butyl esters demonstrated that both volatile and nonvolatile acids can be detected and separated in 24 min on a single column. Standard calibration curves (peak area versus concentration) of the butyl esters were linear in the range of 5 to 40 mumol of acid per ml. The advantages of using an internal standard (heptanoic acid) for quantitating fatty acids in a mixture are given. Chromatograms of butylated fermentation media in which rumen anaerobic bacteria were grown illustrated that this method is useful for determining short-chain volatile and nonvolatile acids of toxonomic significance.     

Definition of total biosynthesis pathway of taurolipids in Tetrahymena cells

Taurine-combined fatty acids were found in the lipotaurine fraction of cells of Tetrahymena thermophila. Taurine and fatty acid moieties of the compounds were identified by nuclear magnetic resonance and gas chromatography-mass spectrometry, respectively. The molar ratio of fatty acid methyl esters and taurine in the hydrolysate of the lipotaurine fraction by methanolic hydrochloric acid hydrolysis, was 1.06:1.00. From the results, the structures of six taurine-combined fatty acids including lipotaurine in the fraction were identified. These structures suggest that the compounds are precursors of lipotaurine as an intermediate of taurolipids biosynthesis, and lipotaurine is biosynthesized via 2-(octadecanoylamino)ethanesulfonic acid and 2-(7-hydroxy-13-octadecenoylamino)ethanesulfonic acid. From the results of the present study and our previous studies, the total biosynthesis pathway of taurolipids is defined.     

A gas-liquid-chromatographic procedure for separating a wide range of metabolites occuring in urine or tissue extracts

1. A gas–liquid-chromatographic procedure is described which permits separation and identification on the same chromatogram of a wide range of substances occurring in urine or tissue extracts. The method uses hydrogen flame ionization, which detects organic compounds whether free or conjugated with no requirement for specific reactive groups. 2. For chromatography, carboxyl groups are quantitatively converted into methyl esters or trimethylsilyl esters. Phenolic, alcoholic and potential enolic groups are converted into trimethylsilyl ethers. Separations are carried out on a 6ft. column of either 10% F-60 (a polysiloxane) or 1% F-60, temperature programming at 2°/min. being used over such part of the temperature range 30°–260° as is required. Propionyl derivatives of hydroxy compounds can also be used, but only on a non-quantitative basis. Derivatives and columns have been selected for optimum range of usefulness when large numbers of samples are examined by using automated gas chromatography. 3. The method is applicable to: fatty acids above butyric acid; di- and tri-carboxylic acids; hydroxy acids and keto acids; polyhydroxy and alicyclic compounds such as glycerol, inositol, quinic acid, shikimic acid, ascorbic acid and sugar alcohols; aromatic hydroxy and acidic compounds, both benzenoid and indolic; sesquiterpenes; steroids; glycine conjugates; mercapturic acids; glucuronides. It is not satisfactory for sulphate conjugates, iminazoles or polypeptides. 4. Methylene units provide an accurate and reproducible parameter for characterizing peak position. Methylene unit values are reported for a large variety of substances occurring in, or related to those occurring in, urine and tissue extracts. 5. The nature of derivatives was confirmed by combining gas chromatography with mass spectrometry. Combined gas chromatography–mass spectrometry gives a diagnostic tool of great power in the evaluation of metabolic patterns, and various uses are discussed.     

Alkyl esters of hydroxycinnamic acids with improved antioxidant activity and lipophilicity protect PC12 cells against oxidative stress

Hydroxycinnamic acids (HCAs) are phenolic compounds present in dietary plants, which possess considerable antioxidant activity. In order to increase the lipophilicity of HCAs, with the aim of improving their cellular absorption and expansion of their use in lipophilic media, methyl, ethyl, propyl and butyl esters of caffeic acid and ferulic acid have been synthesized. All caffeate esters had a slightly lower DPPH IC(50) (13.5-14.5 μM) and higher ferric reducing antioxidant power (FRAP) values (1490-1588 mM quercetin/mole [mMQ/mole]) compared to caffeic acid (16.6 μM and 1398 mMQ/mole, respectively) in antioxidant assays. In contrast, ferulate esters were less active in DPPH (56.3-74.7 μM) and FRAP assays (193-262 mMQ/mole) compared to ferulic acid (44.6 μM and 324 mMQ/mole, respectively). Redox properties of HCAs were in line with their antioxidant capacities, so that compounds with higher antioxidant activities had lower oxidation potentials. Measurement of partition coefficients disclosed the higher lipophilicity of the esters compared to parent compounds. All esters of caffeic acid significantly inhibited hydrogen peroxide-induced neuronal PC12 cell death assessed by MTT assay at 5 and 25 μM. However, caffeic acid, ferulic acid and ferulate esters were not able to protect the cells. In conclusion, these findings suggest that alkyl esterification of some HCAs augments their antioxidant properties as well as their lipophilicity and as a consequence, improves their cell protective activity against oxidative stress. These compounds could have useful applications in conditions where oxidative stress plays a pathogenic role.     

Stimulation of nitrogen removal in the rhizosphere of aquatic duckweed by root exudate components

Plants can stimulate bacterial nitrogen (N) removal by secretion of root exudates that may serve as carbon sources as well as non-nutrient signals for denitrification. However, there is a lack of knowledge about the specific non-nutrient compounds involved in this stimulation. Here, we use a continuous root exudate-trapping system in two common aquatic duckweed species, Spirodela polyrrhiza (HZ1) and Lemna minor (WX3), under natural and aseptic conditions. An activity-guided bioassay using denitrifying bacterium Pseudomonas fluorescens showed that crude root exudates of the two species strongly enhanced the nitrogen-removal efficiency (NRE) of P. fluorescens (P < 0.05) under both conditions. Water-insoluble fractions (F) obtained under natural conditions stimulated NRE to a significant extent, promoting rates by about 30 %. Among acidic, neutral and basic fractions, a pronounced stimulatory effect was also observed for the neutral fractions from HZ1 and WX3 under both conditions, whereas the acidic fractions from WX3 displayed an inhibitory effect. Analysis of the active fractions using gas chromatography/mass spectrometry (GC/MS) revealed that duckweed released fatty acid methyl esters and fatty acid amides, specifically: methyl hexadecanoate, methyl (Z)-7-hexadecenoate, methyl dodecanoate, methyl-12-hydroxystearate, oleamide, and erucamide. Methyl (Z)-7-hexadecenoate and erucamide emerged as the effective N-removal stimulants (maximum stimulation of 25.9 and 33.4 %, respectively), while none of the other tested compounds showed stimulatory effects. These findings provide the first evidence for a function of fatty acid methyl esters and fatty acid amides in stimulating N removal of denitrifying bacteria, affording insight into the “crosstalk” between aquatic plants and bacteria in the rhizosphere.     

Synthetic transformations of higher terpenoids. XXVI. 16-acetylaminomethyllabdanoids and their cytotoxicity

Coupling of methyl 16-aminomethyllambertianate with N-Boc-protected ω-amino acids resulted in 16-(N-Boc-aminononan)- and 16-(N-Boc-aminoundecan)amidomethyllabdanoids. Interaction of methyl aminomethyllambertianate with bicyclo[2.2.1]hept-5-en-2,3-dicarboxylic acid anhydride led to the amide of bicyclo[2.2.1]heptan-1,2-dicarboxylic acid with a labdanoid substituent. Reaction of methyl 16-aminomethyllambertianate with chloroacetyl chloride resulted in methyl 16-(chloroacetylaminomethyl)lambertianate; coupling of the latter with methyl esters of amino acids gave the corresponding amides of methyl lambertianate. The compounds obtained were more cytotoxic toward CEM-13, MT-4, and U-937 tumor cell lines as compared with lambertianic acid; the dose inhibiting tumor cell viability by 50% (CCID₅₀) of the more active compounds was 3.9–9.9 μM.     

Formation of Catechols via Removal of Acid Side Chains from Ibuprofen and Related Aromatic Acids

Although ibuprofen [2-(4-isobutylphenyl)-propionic acid] is one of the most widely consumed drugs in the world, little is known regarding its degradation by environmental bacteria. Sphingomonas sp. strain Ibu-2 was isolated from a wastewater treatment plant based on its ability to use ibuprofen as a sole carbon and energy source. A slight preference toward the R enantiomer was observed, though both ibuprofen enantiomers were metabolized. A yellow color, indicative of meta-cleavage, accumulated transiently in the culture supernatant when Ibu-2 was grown on ibuprofen. When and only when 3-flurocatechol was used to poison the meta-cleavage system, isobutylcatechol was identified in the culture supernatant via gas chromatography-mass spectrometry analysis. Ibuprofen-induced washed-cell suspensions also metabolized phenylacetic acid and 2-phenylpropionic acid to catechol, while 3- and 4-tolylacetic acids and 2-(4-tolyl)-propionic acid were metabolized to the corresponding methyl catechols before ring cleavage. These data suggest that, in contrast to the widely distributed coenzyme A ligase, homogentisate, or homoprotocatechuate pathway for metabolism of phenylacetic acid and similar compounds, Ibu-2 removes the acidic side chain of ibuprofen and related compounds prior to ring cleavage.     

Synthesis and pharmacological activity of amides and ozonolysis product of maleopimaric acid

The synthesis of a new group of maleopimaric acid amides containing fragments of the methyl esters of amino acids, aliphatic amines, imidazole and N-methylpiperazine was carried out. Ozonolysis of methyl maleopimarate flows through the cleavage of double bond C18(19) and the disclosure of anhydrous cycle with formation of secotriacid. As a result of screening of anti-inflammatory and antiulcer activity of maleopimaric acid derivatives new effective compounds such as methyl esters of maleopimaric acid and product of ozonolysis - diterpenic secotriacid, maleopimaric acid amide with L-leucine were revealed. An important advantage of the compounds studied is the low toxicity and the presence of bidirectional activity in the absence of adverse effects on the animal.     

Steric analysis of 3-, ω4-, ω3- and ω2-hydroxy acids and various alkanols by gas-liquid chromatography

D-Phenylpropionate (PP) derivatives of racemic hydroxy acid methyl esters and alkanols were prepared and the gas-liquid chromatographic separation of diastereoisomers was investigated using QF-1 as stationary phase. Good separations were obtained for the diasteroisomeric PP-derivatives of methyl 3-, 15-, 16-, and 17-hydroxyoctadecanoates. Less separation was observed for methyl 2- and 14-hydroxyoctadecanoates as PP-derivatives and there was no visible separation for PP-derivatives of 4-, 7-, or 13-hydroxyoctadecanoic acid methyl esters. The use of optically active 15-, 16- and 17-hydroxyoctadecanoic acids showed, that in these cases, the diastereoisomers containing L-hydroxy acids had shorter retention times than the ones containing D-hydroxy acids. On the other hand, the D-phenylpropionate derivative of methyl 3D-hydroxydecanoate had shorter retention time than the derivative of its L-enantiomer. PP-derivatives of 3-hexanol, 3-heptanol, 3-octanol, 2-octanol and 2-eicosanol could be resolved by gas-liquid chromatography.     

Studies of Aedes aegypti (Diptera: Culicidae) ovipositional responses to newly identified semiochemicals from conspecific eggs

Abstract  The chemical factors influencing the selection of oviposition site by gravid females of various mosquito species have been the subject of numerous investigations. Recent studies have revealed this behaviour to be controlled by semiochemicals. Here we report studies on semiochemicals of egg origin and their effect on the ovipositional behaviour of Aedes aegypti. The compounds present in egg extracts of Ae. aegypti mosquitoes were isolated and identified by gas chromatography-mass spectrometry. They were then evaluated for their effect on ovipositional behaviour against gravid females of Ae. aegypti mosquitoes at different concentrations. Gravid female Ae. aegypti were found to be sensitive to all the identified compounds: 6-hexanolactone, methyl dodecanoate, dodecanoic acid, methyl tetradecanoate, tetradecanoic acid, methyl (Z)-9-hexadecenoate, methyl hexadecanoate (Z)-9-hexadecenoic acid, hexadecanoic acid, methyl (Z)-9-octadecenoate, methyl octadecanoate (Z)-9-octadecenoic acid and octadecanoic acid. Among them, dodecanoic and (Z)-9-hexadecenoic acids showed significant positive ovipositional response at different concentrations whereas all the esters showed deterrent/repellent ovipositional effect.     

Gas chromatography-mass spectrometry determination of metabolites of conjugated cis-9,trans-11,cis-15 18:3 fatty acid

Structural determination of polyunsaturated fatty acids by gas chromatography-mass spectrometry (GC-MS) requires currently the use of nitrogen containing derivatives such as picolinyl esters, 4,4-dimethyloxazoline or pyrrolidides derivatives. The derivatization is required in most cases to obtain low energy fragmentation that allows accurate location of the double bonds. In the present work, the following metabolites of rumelenic (cis-9,trans-11,cis-15 18:3) acid, from rat livers, were identified: cis-8,cis-11,trans-13,cis-17 20:4, cis-5,cis-8,cis-11,trans-13,cis-17 20:5, cis-7,cis-10,cis-13,trans-15,cis-19 22:5, and cis-4,cis-7,cis-10,cis-13,trans-15,cis-19 22:6 acids by GC-MS as their 4,4-dimethyloxazoline and methyl esters derivatives. Specific fragmentation of the methyl ester derivatives revealed some similarity with their corresponding DMOX derivatives. Indeed, intense ion fragments at m/z=M+-69, corresponding to a cleavage at the center of a bis-methylene interrupted double bond system were observed for all identified metabolites. Moreover, intense ion fragments at m/z=M+-136, corresponding to allylic cleavage of the n-12 double bonds were observed for the C20:5, C22:5, C22:6 acid metabolites. For the long chain polyunsaturated fatty acids from the rumelenic metabolism, we showed that single methyl esters derivatives might be used for both usual quantification by GC-FID and identification by GC-MS.     

Comparison of Volatile Compounds from Chungkuk-Jang and Itohiki-Natto

Volatile compounds from two South-East Asian fermented soybean foods, Chungkuk-jang (CKJ) and Itohiki-natto (natto), were analysed by gas chromatography-mass spectrometry (GC-MS), gas chromatography (GC), and GC-sniffing. A total of 112 compounds were identified. A large amount of ethanol was detected from CKJ, while acetone and methyl isobutyrate were major components of natto. The characteristic odor compounds of CKJ were some ethyl esters of short chain fatty acids, diallyl disulfide, and several natto-like odor compounds were identified as ammonia, 2,5-dimethylpyrazine, and 2-methylbutanoic acid.     

Quantitative analysis of fatty acid precursors in marine samples: direct conversion of wax ester alcohols and dimethylacetals to FAMEs

To apply fatty acid analyses to the study of foraging ecology and diet determination, all compounds that may be deposited as fatty acids in a predator must be quantified in the prey. These compounds include the usual fatty acids in acyl lipids, but also the alcohols of wax esters and the vinyl ethers of plasmalogens. In routine fatty acid analysis, samples are extracted and transesterified (methylated), resulting in the formation of fatty acid methyl esters (FAMEs); however, fatty alcohols and dimethylacetals (DMAs) are also generated if wax esters or plasmalogens are present. Here, we present a new method using a modified Jones' reagent to oxidize these alcohols and DMAs to free fatty acids (FFAs). These FFAs are then easily methylated and quantitatively recombined with FAMEs from the same sample. This generates a fatty acid signature of prey that is equivalent to that which the predator has available for deposition upon digestion of that prey. This method is validated with alcohol and DMA standards. Its application to typical marine samples is also presented, demonstrating the change in effective fatty acid signature after inclusion of fatty acids derived from wax esters and plasmalogens.     

A study of the sulphur amino acids of rat tissues

1. In a study of the metabolism of l-[(35)S]methionine in vivo, the labelled sulphur compounds of rat liver and brain were separated first by ion-exchange chromatography into two fractions containing (i) free sulphur amino acids such as methionine, cystathionine, cyst(e)ine and homocyst(e)ine and (ii) glutathione. 2. Two-dimensional paper chromatography with butan-1-ol-acetic acid or propionic acid-water in the first direction and 80% acetone or acetone-ethyl methyl ketone-water in the second direction was found superior to other solvent systems for separating the sulphur amino acids. 3. At 10min. after injection of [(35)S]methionine only a small part of the (35)S was found combined in free methionine or other free sulphur amino acids. 4. Evidence was obtained of the presence of adenosyl[(35)S]methionine and adenosyl[(35)S]homocysteine in perchloric acid extracts of rat liver and brain. 5. The trans-sulphuration pathway was active in brain as well as in liver.     

Location of double bonds in two unsaturated forms of phytanic acid from Refsum disease as determined by mass spectrometry

A monounsaturated and a triunsaturated form of phytanic acid (3,7,11,15-tetramethylhexacosanoate) were isolated from plasma lipids of a patient with Refsum disease. Both were converted to their methyl esters, oxidized to polyhydroxy acids by treatment with OsO4 and converted to their vicinal trimethylsilyl ethers. These derivatives were analyzed by gas chromatography-mass spectrometry using both electron impact ionization (at 21 and 70 eV) and chemical ionization conditions to obtain clear evidence to establish the structure of the monounsaturated form of phytanic acid as 3,7,11,15-tetramethylhexadec-15-monoenoic acid and that of the triunsaturated form of phytanic acid as 3,7,11,15-tetramethylhexadec-6,10,14-trienoic acid. The possible metabolic and dietary sources for these novel fatty acids are discussed.     

Formation of catechols via removal of acid side chains from ibuprofen and related aromatic acids

Although ibuprofen [2-(4-isobutylphenyl)-propionic acid] is one of the most widely consumed drugs in the world, little is known regarding its degradation by environmental bacteria. Sphingomonas sp. strain Ibu-2 was isolated from a wastewater treatment plant based on its ability to use ibuprofen as a sole carbon and energy source. A slight preference toward the R enantiomer was observed, though both ibuprofen enantiomers were metabolized. A yellow color, indicative of meta-cleavage, accumulated transiently in the culture supernatant when Ibu-2 was grown on ibuprofen. When and only when 3-flurocatechol was used to poison the meta-cleavage system, isobutylcatechol was identified in the culture supernatant via gas chromatography-mass spectrometry analysis. Ibuprofen-induced washed-cell suspensions also metabolized phenylacetic acid and 2-phenylpropionic acid to catechol, while 3- and 4-tolylacetic acids and 2-(4-tolyl)-propionic acid were metabolized to the corresponding methyl catechols before ring cleavage. These data suggest that, in contrast to the widely distributed coenzyme A ligase, homogentisate, or homoprotocatechuate pathway for metabolism of phenylacetic acid and similar compounds, Ibu-2 removes the acidic side chain of ibuprofen and related compounds prior to ring cleavage.     

Detection of bacterial contamination in cultures of eucaryotic cells by gas chromatography-mass spectrometry

The use of gas chromatography-mass spectrometry for early detection of bacterial contaminations in cultures of baker's yeast, Penicillium chrysogenum, and an animal cell line was evaluated; muramic acid and characteristic cellular fatty acids were used as analytes. By analyzing branched-chain and cyclopropane-substituted fatty acids as methyl esters, Staphylococcus epidermidis, Bacillus subtilis, Lactobacillus reuteri, Enterobacter cloacae, and Pseudomonas fluorescens were detected in a 500-fold excess (w/w) of baker's yeast; the amounts injected corresponded to 300 ng (dry mass) of the bacteria. Contamination with Bacillus was detected in cultures of Penicillium chrysogenum and animal cells by analyzing muramic acid, both as its alditol acetate derivative, using electron impact ionization, and its trifluoroacetyl methyl glycoside derivative, using negative ion-chemical ionization. The trifluoroacetylated derivative was detected in injected amounts corresponding to 1 x 10(3) bacterial cells in the contaminated animal cell line, whereas amounts corresponding to 1 x 10(5) bacteria were required for detection of the alditol acetate derivative; the amounts in the original samples were 5 x 10(5) and 5 x 10(6), respectively. However, the alditol acetate method exhibited lower chemical interferences than the trifluoroacetyl methyl glycoside procedure. The results show the potential of using gas chromatographic-mass spectrometric analysis of cellular constituents for the detection of bacterial contaminations in eucaryotic cultures as an alternative to conventional microbiological methods. (c) 1993 John Wiley & Sons, Inc.     

Synthesis and biological activity of some new 3,6-dinitro-1:8-naphthaloyl- and 3,6-diamino-1:8-naphthaloylamino acids and dipeptide derivatives

Synthesis of a series of 3,6-dinitro-1:8-naphthaloylamino acids (II-IX) and some of their corresponding methyl esters (X-XVI) and 3,6-diamino-1:8-naphthaloylamino acid derivatives (XXIX-XXXVI) is described. Coupling of 3,6-dinitro-1:8-naphthaloylamino acids with amino acid methyl ester hydrochlorides in dioxane-DMF-Et3N medium using DCC method furnishes the desired 3,6-dinitro-1:8-naphthaloyldipeptide methyl esters (XVII-XXVIII). Most of the synthesized 3,6-dinitro-1:8-naphthaloylamino acids, esters and dipeptide derivatives (compounds III-VI, XI-XV, XVII, XIX-XXI, XXIII and XXV) and 3,6-diamino-1:8-naphthaloylamino acid derivatives (XXIX-XXXV) were found to be active against a number of microorganisms.     

High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids.

A sensitive negative chemical ionization (NCI) gas chromatography-mass spectrometry (GC-MS) method for the detection of pentafluorobenzyl (PFB) esters of deuterated fatty acids is described. Deuterated linoleic [18:2n-6 2H4-9,10,12,13] and linolenic [18:3n-3 2H5-17,17,18,18,18] acids were converted to chain-elongated and desaturated products during incubations with homogenates prepared from rat liver. The extracted fatty acids were derivatized with pentafluorobenzyl bromide and analyzed in the negative ion mode by GC-MS. The detection limit of the PFB esters in NCI using selected ion monitoring was below 10 femtograms. In general, detection of the PFB derivatives using the negative ion mode was more than three orders of magnitude more sensitive than using a positive chemical ionization (PCI) method with methyl ester derivatives. The PFB esters of the 2H4-18:2n-6 metabolites eluted with their unlabeled analogues, whereas the PFB esters of the 2H5-18:3n-3 metabolites were resolved from the unlabeled compounds on polar capillary FFAP columns. Isotope ratios of the 2H4-18:2n-6 metabolites were used to quantify the deuterated compounds from standard dilution curves generated from the ion abundances of the unlabeled fatty acids. The 2H5-18:3n-3 metabolites were quantified similarly using 18:3n-3. This method is feasible for the study of the in vivo metabolism of deuterated essential fatty acids in whole animals.     

Gas chromatographic-mass spectrometric detection of 2- and 3-hydroxy fatty acids as methyl esters from soil,sediment and biofilm

Hydroxy fatty acids (OH-FAs) can be used in the characterization of microbial communities, especially Gram-negative bacteria. We prepared methyl esters of 2- and 3-OH-FAs from the lipid extraction residue of soil, sediment, and biofilm samples without further purification or derivatization of hydroxyl groups. OH-FA methyl esters were analyzed using a gas chromatograph equipped with a mass selective detector (GC-MS). The ions followed in MS were m/z 103 for 3-OH-FAs and m/z 90 and M-59 for 2-OH-FAs. The rapid determination of 3- and 2-OH-FAs concomitantly with phospholipid fatty acids provided more detailed information on the microbial communities present in soil, sediment, and drinking water biofilm.