Gas Chromatography-mass Spectrum Research Articles

Structure of Saccharomyces cerevisiae mating hormone a-factor. Identification of S-farnesyl cysteine as a structural component.

Mating type a cells of the yeast Saccharomyces cerevisiae produce a mating hormone, the a-factor, that we have previously characterized as a very hydrophobic, modified dodecapeptide (Betz, R., Crabb, J. W., Meyer, H. E., Wittig, R., and Duntze, W. (1987) J. Biol. Chem. 262, 546-548). We have investigated the molecular structure in detail using mass spectrometry and proton NMR spectrometry of the intact hormone and authentic component molecules. Tandem mass spectrometry confirms the previously determined peptide sequence of the hormone and shows that it contains additional structural components with masses of 205 and 15 daltons. These were identified by proton NMR and mass spectrometry as a farnesyl (C15H25) residue and a terminal methyl ester group. The farnesyl moiety is attached to the sulfur atom of the carboxyl-terminal cysteine residue, as revealed by NMR of synthetic S-farnesyl cysteine methyl ester. The stereochemical configuration of the farnesyl moiety was determined to be trans,trans by comparison of gas chromatography retention times, mass spectra, and NMR spectra with those of standards. These results define the structure of a-factor as: (Sequence: see text). Replacement of the farnesyl by a methyl group leads to a partial reduction in specific biological activity of the a-factor, whereas hydrolysis of the carboxyl-terminal methyl ester causes a complete loss of activity.

Determination of Rosin Ester Gum Emulsifiers in Fruit Juices by Gas Chromatography

A method for detection and determination of rosin ester gum in fruit juice was established as follows. Rosin ester as a component of ester gum was extracted with benzene from the sample, and saponified with N/2 KOH-ethanol solution. The rosin acids were extracted with diethyl ether in acidic condition, derivatized with TMS reagent and determined by gas chromatography (GC). Seven kinds of ester gum standard were analyzed by the proposed method, and it was found that contents of dihydroabietic acid and abietic acid in the ester gums ranged between 33.8–75.1% and 0–36.7%, respectively, but the total contents of them were 66.7–75.1% (average, 70.9 ± 3.0%). Dihydroabietic acid and abietic acid derived from ester gum in 6 kinds of imported fruit juice were identified by GC-mass spectra and quantitated by GC. The contents of ester gum in samples estimated from the two peaks were 15.2–33.9 ppm. Recoveries of ester gum added to sample at 50 and 500 ppm were more than 92.7%, and the detection limit of ester gum was 0.5 μg (2 ppm in sample) by the proposed method.

A21978C, a complex of new acidic peptide antibiotics: isolation, chemistry, and mass spectral structure elucidation.

A21978C, produced by Streptomyces roseosporus, NRRL 11379, is a complex of new acidic lipopeptolide antibiotics which inhibits Gram-positive bacteria. HPLC separation of the various components from the purified complex resulted in the isolation of A21978C1, -C2 and -C3 (major components) and -C4, -C5, and -C0 (minor components). Each of these components was fermented with cultures of Actinoplanes utahensis (NRRL 12052) to give the identical inactive peptide ("A21978C nucleus") by removal of the fatty acid acyl groups from the N-terminus. This peptide was composed of 13 amino acids: L-kynurenine, L-threo-3-methylglutamic acid, L-asparagine, L-aspartic acid (3 residues), glycine (2 residues), L-tryptophan, L-ornithine, D-alanine, D-serine and L-threonine. The amino acid sequence was determined using a combination of the Edman degradation and gas chromatography mass spectrum (GC-MS) analysis of appropriately derivatized peptides obtained from partial hydrolysis. Each major component was shown to be acylated with a branched chain fatty acid at the N-terminus and the structure of this fatty acid was determined by 1H NMR and mass spectral methods. A structure for A21978C was assigned on the basis of this degradative and physico-chemical information.

Degradation of bromazepam by the intestinal microflora.

Degradation of four benzodiazepines, i.e. bromazepam, diazepam, desmethyldiazepam, and 7-bromo-1, 3-dihydro-5-phenyl-2H-1, 4-benzodiazepin-2 (1H) -one, in a broth containing human feces was studied. Degradation of bromazepam was also investigated in a fecal medium, a homogenate of the liver and a homogenate of intestinal epithelial cells of rat. More than 80% of bromazepam, which contains a pyridine ring in its structure, was degraded in the broth containing 8% human feces under both anaerobic and aerobic conditions. The major degradation product of bromazepam was identified as 2- (2-amino-5-bromobenzoyl) pyridine by means of thin layer chromatography and gas chromatography-mass spectra analyses. About 70, 90, and 90% of bromazepam remained in the fecal medium, homogenate of the liver and homogenate of intestinal epithelial cells of rat, respectively, after incubation for 5 h at 37 °C.Therefore, degradation of bromazepam by the intestinal microflora may be considered to be one of the major factors influencing the bioavailability after oral administration to man.

Calcium ionophore and chemotactic peptide stimulation of peptidoleukotriene synthesis in DMSO-differentiated HL60 cells.

The human promyelocytic leukemia cell line HL60 can be differentiated to mature granulocytes upon exposure to DMSO (1.3%, 6 days). The ability of these cells to metabolize arachidonic acid via the 5-lipoxygenase pathway to form 5-HETE, LTB4, and 5,12-diHETEs, has been previously documented. However, the production of peptidoleukotrienes by DMSO-differentiated HL60 cells has not been previously reported. Arachidonic acid metabolites produced via 5-lipoxygenase were identified by reverse-phase, high-performance liquid chromatography, immunoreactivity specific for peptidoleukotriene, glutamyl transpeptidase transformation, characteristic UV spectra, and GC mass spectra. Leukotriene synthesis in the DMSO-differentiated HL60 cell is maximal at 5 min when stimulated with the calcium ioniphore, A23187 (1 microM), in the presence of calcium. These cells produce 12.94 +/- 1.8 ng/10(6) cells of LTC4 and 3.8 +/- 0.4 ng/10(6) cells of LTB4. LTC4 and LTB4 are also synthesized in the undifferentiated cell when stimulated with 1 microM A23187 and 1 mM Ca2+, but in much smaller quantities, i.e., 1.91 +/- 0.42 ng/10(6) cells of LTC4 and 0.41 ng +/- 0.06/10(6) cells of LTB4. The synthetic chemotactic peptide, f-Met-Leu-Phe, also elicits formation of LTC4 and LTB4 in a dose-dependent manner in the presence of exogenously added calcium. Maximal stimulation of DMSO-differentiated cells with f-Met-Leu-Phe produces 2.5 +/- 0.2 ng of LTC4 and 1.45 +/- 0.2 ng of LTB4 per 10(6) cells. The observation that DMSO-differentiated HL60 cells produce LTC4, as well as other 5-lipoxygenase products, increases the utility of this cell line for unraveling the regulation of leukotriene biosynthesis by granulocytes.

Chemical nature of a desmutagenic factor from burdock (Arctium lappa LINNE).

Studies were carried out to elucidate the chemical nature of the desmutagenic factor of Burdock (Arctium lappa Linne), Which is shown to be a complex polymer having a molecular weight of about 300,000. Its desmutagenic effects are demonstrated on indirect mutagens such as Trp-P-1 and Trp-P-2, as well as on direct mutagens such as 2-nitro-1,4-diamino benzene and 4-nitro-1,2-diamino benzene. The present findings suggest that this factor is not a protein because of the low content of nitrogen and the non-color reaction of its hydrolysate with ninhydrin. The desmutagenic factor probably possesses carboxyl groups as indicated by a 13C-NMR spectrum, potentiometric titration and analysis of its pyrolysates. This analysis of the factor’s pyrolysates and its reductive degradation substances on the GC-mass spectrum provided evidence that the factor also has an aromatic ring as a basic structure. The factor may be a lignin-like compound having a 10% sugar content.

Melatonin can be differentially metabolized in the rat to produce N-acetylserotonin in addition to 6-hydroxy-melatonin.

It has been generally agreed that the metabolism of the pineal hormone melatonin (aMT) consists of 6-hydroxylation followed by sulfate or glucuronide conjugation. The urinary assay of 6-hydroxy-melatonin (6-HaMT) is valued as a means of providing integrated information on aMT production. However, we show, in this study, that aMT has two principal urinary metabolites, N-acetylserotonin (NAS) as well as 6-HaMT. Rats were administered varying doses of aMT and their urines were collected and analyzed by thin layer chromatography and gas chromatography-mass spectrometry (GCMS). Thin layer chromatography of the urinary metabolites showed the expected pattern, a major spot at Rf 46%, the position of 6-sulfatoxy-melatonin, a less intense spot at Rf 32%, the position of 6-glucuronide-melatonin and a weak spot at Rf 78%, the unconjugated metabolite. However, after deconjugation and derivitization, GCMS analysis of the urines, or of the spot at Rf 46%, showed two products, one of which had the same GC retention time and mass spectrum as 6-HaMT, whereas the other had the GC retention time and mass spectrum of NAS. When deuterated aMT was administered, GCMS analysis showed the presence of deuterated 6-HaMT and deuterated NAS, proving that NAS was metabolized directly from aMT and not produced somewhere else in the body in response to aMT. Finally, GCMS analysis of urines after the administration of 6-HaMT or of NAS showed only one metabolic product in each case, i.e. 6-HaMT and NAS, respectively. This suggested that the conversion of aMT to 6-HaMT and NAS resulted from two independent metabolic pathways. It is understandable that research workers who relied entirely on chromatography should have failed to distinguish NAS and its conjugates from 6-HaMT and its conjugates since the chromatographic and staining properties of the two indoles are almost indistinguishable.

Purification and identification of formyl-methionyl-leucyl-phenylalanine as the major peptide neutrophil chemotactic factor produced by Escherichia coli.

Chemotactic factor-enriched butanol extracts from Escherichia coli culture filtrates were fractionated and purified by high pressure liquid chromatography. The yield from individual fractions of biological activity (lysosomal enzyme secretion) and antigenic activity (competition with [3H]fMet-Leu-Phe for binding to rabbit anti-fMet-Leu-Phe) revealed an average 50% recovery of original material. Five peaks of biological activity were separated as demonstrated by enzyme-releasing activity. Three of these peaks coincided exactly with peaks of antigenic activity, suggesting that at least 3 and as many as 5 distinct formyl-methionyl peptides had been separated. The majority of recovered activity appeared in peak 3 and represented 70% of the total biological and antigenic activities recovered. The five peak fractions were subsequently analyzed by dipeptidyl carboxypeptidase gas chromatography-mass spectrometry (DCP/GC-MS) to determine amino acid sequences. After digestion, the formyl-Met peptide was demonstrated in only one of the five peak fractions (peak 3). Furthermore, both the GC retention times and mass spectra indicated that peak 3 contained formyl-methionyl-leucyl-phenylalanine. The DCP/GC and MS data were confirmed with tests made on authentic fMet-Leu-Phe. Butanol extracts from E. coli filtrates to which were added synthetic fMet-Leu-Phe resulted in increased biological and antigenic activity in the precise high pressure liquid chromatography fractions of peak 3 where the fMet-Leu-Phe produced by E. coli was found. Finally, the analysis of recovered biological and antigenic activities indicated that the formyl peptides were found in nanomolar concentrations in culture filtrates. These results demonstrate that the NH2-terminal formyl peptides produced by E. coli, of which formyl-methionyl-leucyl-phenylalanine appears to be the major component, are the peptide mediators responsible for leukocyte chemotactic activity in the bacterial culture extracts.

1-Methylcyclohex-2-en-1-ol as an aggregation pheromone ofDendroctonus pseudotsugae

1-Methylcyclohex-2-en-1-ol (1,2-MCH-ol) was synthesized, identified as a compound found in volatiles of the female Douglas-fir beetle, and shown by bioassays to be an aggregation pheromone. 1,2-MCH-ol matches in both GC retention index and mass spectrum a compound released by the female after feeding. 3,3-MCH-ol was also synthesized as a candidate compound; its mass spectrum is presented because published mass spectra are incorrect for this compound. Synthetic 1,2-MCH-ol increased arrestment and stridulation of males in olfactory walkways and increased trap catches of flying beetles. Males were more responsive to 1,2-MCH-ol than females.

High Performance Liquid Chromatography (HPLC) determination of endogenous serotonin released from aggregating platelets

High performance liquid chromatography with electrochemical detection was developed for the determination of serotonin (5-hydroxytryptamine) in rabbit platelet and plasma. A peak from tissue extract (peak a) was identified as serotonin based on retention time on the chromatogram, using a potential that gave limiting current on both a current-potential curve and gas chromatography-mass spectrum, as compared with that of authentic serotonin. For the assay of serotonin, 5-hydroxy- N-methyltryptamine was used as an internal standard and the extraction recovery of the serotonin added to the platelet-rich plasma was almost total. Furthermore, using this highly sensitive method, it has been demonstrated that the release of endogenous serotonin from rabbit platelets occurs during collagen-induced platelet aggregation but not during adenosine diphosphate-induced platelet aggregation. The procedures described should prove to be simple and useful for pharmacological and physiological investigations on the relationship between platelet aggregation and release reaction.

カボチャの花の成分研究

The methanol extract of the male flowers of Cucurbita pepo was shown to contain glutinol, lupeol, α-spinasterol, stigmast-7-enol, α-spinasteryl-β-D-glucoside, stigmast-7-enyl-β-D-glucoside, adenine, adenosine, eleven fatty acids and twenty-three amino acids. Four paraffins were also identified with gas chromatography-mass spectrum from the essential oil. And sex differentiation of the constituents of male and female flowers was studied.

Amino acids of the Murchison meteorite: I. Six carbon acyclic primary alpha-amino alkanoic acids.

Six of the seven chain isomers of six-carbon acyclic primary alpha-amino alkanoic acids (leucine isomers) have been either identified or confirmed in hot-water extracts of the Murchison meteorite using combined gas chromatography-mass spectrometry (GC-MS) and ion exchange chromatography. 2-Amino-2-ethylbutyric acid, 2-amino-2,3-dimethylbutyric acid, pseudoleucine, and 2-methylnorvaline were positively identified by GC-MS. These amino acids have not been previously reported to occur in natural materials and may be uniquely meteoritic in origin. The presence of leucine and isoleucine (including the diastereoisomer, alloisoleucine) was confirmed. Peaks corresponding to norleucine were seen by ion-exchange and gas chromatography but characteristic mass spectra were not obtained. The alpha-branched chain isomers in this series are quantitatively the most significant. These results are compared with literature data on amino acid synthesis by electrical discharge and Fischer-Tropsch-type catalysis. Neither model system produces an amino acids suite that is completely comparable to that found in the Murchison meteorite.

3-Trifluoromethyl-3-phenyldiazirine. A new carbene generating group for photolabeling reagents.

The synthesis of 3-trifluoromethyl-3-phenyldiazirine (TPD) is reported in an overall yield of 60% based on 2,2,2-trifluoroacetophenone as starting material. TPD is rapidly photolyzed on irradiation near 350 nm to yield 35% of the diazoisomer and 65% of the corresponding carbene. No internal rearrangement of the latter compound by fluorine migration was detected. Photolysis in methanol yielded the product of a formal OH insertion in greater than 95% yield. Photolysis in cyclohexane gave at least a 50% yield of the CH insertion product at a diazirine concentration of 15 mM. The products were identified by gas chromatographic mass spectra and by 19F NMR spectra. In the dark the diazirine is stable in 1 M acid or base and at temperatures as high as 75 degrees C for at least 30 min. The diazoisomer is much less photolabile and is stable in 0.1 M acetic acid for at least 12 h. The synthesis of a derivative of TPD containing the 2-hydroxyethyl[O-tosylate] group on the para position of the phenyl ring is described. This compound permits the easy attachment of the TPD function to other molecules. It is suggested that the ease of synthesis, dark stability, rapid photolysis, and reactivity of the carbene may make this group useful in the preparation of various photochemical probes.

A Method for Detecting Volatile Amines in Grapes and Wines

A method for determining amines is given. The method involves double vacuum distillation, derivatizing with trifluoroacetic anhydride, extraction with ether after sodium bicarbonate wash, and gas chromatography of the sample in a gas chromatograph (GC) equipped with a nitrogen detector. Recovery and reproducibility studies are shown. Examples of gas chromatographic-mass spectra (GC-MS) and data of retention times are given. The sensitivity of the method is demonstrated by chromatograms of Cabernet Sauvignon, Pinot noir, White Riesling, and Carmine that show the presence of some amines positively identified, some tentatively identified, and some as yet unknown.

Aromatic hydroxylation of beta-adrenergic antagonists. Formation of 4'- and 5'-hydroxy-1-(isopropylamino)-3-[2'-allyloxy)phenoxy]-2-propanol from oxprenolol.

The metabolic aromatic hydroxylation of oxprenolol [1-(isopropylamino)-3-[2'-(allyloxy)phenoxy]-2-propanol] in rats was examined. Synthesis of the isomeric ring methoxyoxprenolols (3b-6b) was accomplished from the isomeric methoxysalicylaldehydes by O-allylation, followed by Baeyer-Villiger oxidation. The propanolamine side chain was elaborated by O-alkylation of the Bayer-Villiger product with epichlorohydrin and subsequent oxirane opening with isopropylamine. Gas chromatography-mass spectra of the trifluoroacetyl derivatives of these standards was compared with urinary metabolites obtained from the rat, after methylation with diazomethane and derivatization with trifluoroacetic anhydride. Both 4'- and 5'-hydroxyoxprenolol (4a and 5a) were present in an approximate 4:1 ratio. No 3'- or 6'-hydroxyoxprenolol (3a and 6a) was detected. The metabolites obtained from a human urine treated in the same manner gave similar results with both 4a and 5a present.

Identification of Lower Terpenoids from Gas-Chromatography-Mass Spectral Data by On-Line Computer Method

It was found that the less intense ion peaks of the mass spectra of lower terpenes can play an important role in distinguishing compounds that have similar mass spectra. Two computer programs were developed that generate, from combined gas chromatographic—mass spectra data, F-1 weights (ANOVA) of mass ion peaks between 41 and 300. The library program FIXLIB contains mass spectral and retention data from known mono- and sesquiterpenes, as well as some phenyl propanoid ethers, which are then used for identification of unknowns. The identification program GETID allows rapid comparison of mass spectra and retentions of compounds that fall within specified retention windows. Weighted similarity ratios are calculated which establish the relative similarities of an unknown with the reference compounds falling into a given retention window. An absolute similarity is then calculated for the unknown and that reference compound which has the closest fit, which provides the measure of confidence for the identification. The method was applied to the identification of lower terpenes and phenylpropanoid ethers found in the volatile leaf oils of three true fir and three juniper species, as well as two Douglas-fir varieties. All compounds of previously known identity were correctly identified by the GETID program.

Some new perfluoroethers and copolymers from ‘Dewar’ hexafluorobenzene

The thermal and photolytic reactions of hexafluorobicyclo[2.2.0]hexa-2,5-diene and bis(fluoroxy)difluoromethane yielded four-membered fused-ring-containing perfluoroethers and copolymers. The reaction products were analyzed by gas chromatography, gas chromatography-infrared, gas chromatography-mass spectra, field ionization mass spectra and 19FNMR. The elucidation of the polymer microstructures was assisted by the 19FNMR spectroscopy of the related compounds, hexafluorobicyclo[2.2.0]-hexanes and -hexene in the volatile fraction. An increase of the concentration of ‘Dewe’ C6F6 in hexafluorobenzene for copolymerization with CF2(OF)2 decreased the six-membered ring moieties. At about 90% level of the para-bonded C6F6, no six-membered ring-containing perfluoroethers were found in the volatile fraction and only a low concentration of the six-membered ring moieties were in the polymeric fractions. The molecular weights of the copolymers ranged from 1000 to over 10,000. The microstructures of the thermal and photopolymers about 1000 range were suggested.

Electron capture gas chromatography detection and mass spectrum identification of 3-(2'-ketohexyl)indoline in spinal fluids of patients with tuberculous meningitis.

A basic, extractable, indolic type of compound, which was derivatized with heptafluorobutyric anhydride and pyridine, was obtained from the cerebrospinal fluids of patients with acute tuberculous meningitis. The compound was detected by frequency-pulsed, modulated electron capture gas-liquid chromatography, and it was tentatively identified by mass spectrometry as 3-(2'-ketohexyl)indoline. The compound was found to be valuable for differentiating between tuberculous, cryptococcal, and aseptic meningitides.

Structure determination of N-acetyl amino sugar derivatives and disaccharides by gas chromatography and mass spectroscopy

Gas chromatography retention times and mass spectra fragmentation patterns were obtained for the trimethylsilyl ethers of 2-acetamindo-2-deoxy- d-glucose, -galactose, and -mannose, (GlcNAc, GalNAc, and ManNAc, respectively), β-1,3-dimethoxy-GlcNAc, the α- and β-methoxy glycosides of GlcNAc and GalNAc, the α- and β-methoxy furanosides of GalNAc, and β-1-4 and β-1-6 linked 2,2′-diacetamido-2,2-dideoxy-glucosylglucoses, and a glycopeptide linkage compound, 2-acetamido-1- N-β- l-aspartyl-2-deoxy-β- d-glycopyranosylamine (GlcNAc-Asn). The mass spectra of these derivatives are discussed in detail. From the mass spectra of the trimethylsilyl derivatives of the free and partially methylated monosaccharides it is possible to determine which and how many positions are methylated and whether the ring is in the pyranose or furanose form. Fragmentation patterns of trimethylsilyl disaccharide derivatives show that it is possible to determine linkage position. Anomeric configuration and other steriochemical features such as axial or equatorial configuration cannot be easily determined from fragmentation patterns but can be determined from gas chromatography retention times, optical rotatory dispersion, and circular dichroism (3). A combination of these methods provides a means of solving the structural and stereochemical problems of carbohydrates.

Separation and identification of fatty acids as anti-inflammatory principle from mycelium of Streptomyces sp. (author's transl)

Erythrocyte membrane stabilizing fraction (EMSF) was purified from the acetone extract of the mycelium of Streptomyces sp. F4818 by the combination of chromatography on silica gel and preparative thin-layer chromatography on silica gel. 1) Heat-induced hemolysis was significantly inhibited by EMSF at a concentration of 2 μg/ml and the hypotonic hemolysis was also inhibited by about 50% at a concentration of 12 μg/ml of EMSF. 2) EMSF showed high anti-inflammatory activity for carrageenin-induced edema in the hind paw of rats at the oral dose of 50 mg/kg. 3) It was proved that EMSF was a fatty acid mixture, consisting of C14 : 0, iso- or anteiso-C14 : 0, C15 : 0, iso C15 : 0, C16 : 0, iso-C16 : 0, iso- or anteiso-C17 : 0, C18 : 0, and C18 : 1 from the result of gas chromatography-mass spectra (GC-MS) analysis on their methyl esters. 4) Palmitic acid, the main fatty acid component in EMSF, markedly suppressed the heat-induced, hypotonic hemolysis in vitro and carrageenin-induced edema in vivo.