Identification of minor nitrosation products of the alkaloid gramine by mass spectrometry.

The nitrosation of gramine, a tertiary amine alkaloid present in barley malt, was carried out by reaction with sodium nitrite in buffered acetic acid (pH 3.4) for 1 hr at room temperature. Following the previous isolation and identification of the major nitrosation products (Ahmad et al. Fd Chem. Toxic. 1985, 23, 841), two minor products were isolated by HPLC and identified as indolin-3-one oxime and indole-3-aldehyde. Identification was based on mass spectrometry. The results give strong support to the hypothesis that gramine does not undergo nitrosation by nitrosative dealkylation.

Synthesis of N1-nitroso-3-nitromethylindole: a nonvolatile N-nitroso compound isolated from the nitrosation of the alkaloid gramine.

An approach to the synthesis of N1-nitroso-3-nitromethylindole, a nitrosation product of the alkaloid gramine, is described. The method involves a few reaction steps starting from indole-3-carbinol.

Isolation and characterization of products from the nitrosation of the alkaloid gramine.

The nitrosation of gramine, a tertiary amine alkaloid present in barley malt, was carried out by reaction with sodium nitrite in buffered acetic acid (pH 3.4) for 1 hr at room temperature. Two major non-volatile products of the nitrosation reaction were isolated by preparative HPLC and characterized as indole-3-carboxylic acid and N1-nitroso-3-nitromethylindole. This interpretation was supported by spectral data. The nature of these products indicated that gramine did not undergo nitrosation by the expected mechanism of nitrosative dealkylation. A mechanism is offered to explain the labile nature of the dimethylamino group found in gramine.

Identification and mutagenicity of aflatoxicol-M1 produced by metabolism of aflatoxin B1 and aflatoxicol by liver fractions from rainbow trout (Salmo gairdneri) fed beta-naphthoflavone.

beta-Naphthoflavone (beta NF) fed to rainbow trout (Salmo gairdneri) at 50 or 500 ppm in the diet, modified the in vitro metabolism of aflatoxin B1 (AFB1) by the postmitochondrial fraction (PMF) of the liver. Production of aflatoxicol (AFL) was significantly less in the 500 ppm beta NF-fed group (33.9 ng/mg protein) than in the control group (45.7 ng/mg protein), aflatoxin M1 production was dependent on the dose of beta NF, being greatest in the 500 ppm beta NF-fed group (48.9 ng/mg protein), intermediate in the 50 ppm beta NF-fed group (3.7 ng/mg protein), and was not detected in controls. A new trout metabolite, 4-hydroxyaflatoxicol (aflatoxicol M1, AFLM1) was also detected in small amounts from in vitro metabolism by liver PMF from beta NF-fed trout. Sufficient quantities of AFLM1 for confirmation of identity by ultraviolet spectra, mass spectra and nuclear magnetic resonance spectra were prepared by biotransformation of AFL using liver microsomes and isolation by HPLC. In a modified Ames mutagen assay with Salmonella typhimurium TA98, ALFM1 was 4.1% as mutagenic as AFB1 in a previous determination. The carcinogenicity of AFLM1 to rainbow trout is expected to be considerably less than that of AFB1.

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.

Nitrosation of N-methyltyramine and N-methyl-3-aminomethylindole, two barley malt alkaloids.

The secondary amine barley malt alkaloids N-methyltyramine and N-methyl-3-aminomethylindole were synthesized, nitrosated in dilute acetic acid and the products characterized by mass spectrometry and NMR spectroscopy. The nitrosation products of N-methyltyramine were p-hydroxy-m-nitro-N-nitroso-N-methyl-2-phenylethylamine and p-hydroxy-N-nitroso-N-methyl-2-phenylethylamine. Upon nitrosation, N-methyl-3-aminomethylindole formed N-nitroso-N-methyl-3-aminomethylindole and the dinitrosated product N1-nitroso-N-nitroso-N-methyl-3-aminomethylindole.

Frontalin in the male mountain pine beetle.

Frontalin andexo-brevicomin were identified by GC-MS in air drawn over maleDendroclonus ponderosae Hopk. (MPB) from Oregon that had joined females for 1-2 days in the bark of lodgepole and ponderosa pine logs. Unfed males releasedexo- andendo-brevicomin but not frontalin. These three compounds were not detected in either unfed or fed females. Arrestment of males bytrans-verbenol and terpenes in an olfactory walkway was reduced by the addition of racemic frontalin; production of attractant chirps also diminished. Racemic frontalin also strongly reduced the aggregation of MPB in lodgepole and ponderosa pine stands to sticky traps baited with the aggregation pheromonetrans-verbenol and host terpenes; however, the function of the natural enantiomer of frontalin in MPB is unknown.

Combination of a GC-TEA and a GC-MS-data system for the microgram/kg estimation and confirmation of volatile N-nitrosamines in foods.

An analytical procedure has been outlined in which volatile N-nitrosamines in foods are isolated by vacuum distillation, cleaned up, identified and quantified by GC-TEA and qualitatively confirmed by GC-MS, coupled with a data system. Spectra are presented which demonstrate that this technique is capable of confirming NDMA and NPYR in a variety of foods at levels of 1 to 10 micrograms/kg. Unequivocal confirmation is based on three types of evidence: (1) the compound is TEA positive; (2) the compound has the correct retention time on a packed and on a capillary column; (3) the complete mass spectrum closely matches standard spectra. These procedures have been used to confirm the presence of trace levels of NDMA in domestic beer and in non-fat dry milk.

Confirmation of low microgram/kg amounts of volatile N-nitrosamines in foods by low resolution mass spectrometry.

Volatile N-nitrosamines were confirmed in foods at concentrations of less than 10 micrograms/kg by full scan low resolution mass spectrometry. Ground samples were vacuum-distilled from mineral oil and condensed in liquid nitrogen-cooled vapor traps. The thawed distillate was extracted, the extract was cleaned up and concentrated, and the N-nitrosamine content was determined by combined gas chromatograph-thermal energy analysis. Positive samples were further cleaned up, trapped from a gas chromatographic column, and purged into a gas chromatograph-mass spectrometer for qualitative confirmation by full scan low resolution mass spectrometry. This procedure was applied to foods spiked at 1 microgram/kg and to fried commercial bacons with volatile N-nitrosamine contents of 2 to 5 micrograms/kg.

Aflatoxin B1 and aflatoxicol metabolism in rainbow trout (Salmo gairdneri) and the effects of dietary cyclopropene.

Aflatoxin M1 was identified as a minor metabolite formed by the in vitro incubation of aflatoxin B1 with the postmitochondrial fraction or microsomes from rainbow trout (Salmo gairdneri) liver. Fresh, whole trout liver converted perfused aflatoxin B1 to aflatoxicol and aflatoxin M1, and converted perfused aflatoxicol to aflatoxin B1 and aflatoxin M1. AFB1 reductive activity was found in the 105,000 xg supernatant, while activity converting AFL to AFB1 was distributed about equally in the 105,000 xg supernatant and in the microsomal pellet. Transformation of AFL to AFB1 was not inhibited by CO, but formation of AFM1 was completely blocked. Cyclopropene fatty acids (CPFA)-fed fish produced only one-half to one-third as much AFL from AFB1 as controls, and they produced no detectable AFM1. Most of the unreacted AFB1 was recovered by extraction of the incubation medium by organic solvent whereas, in controls, much of it remained bound to the protein. There was no difference in conversion of AFL to AFB1 when results were expressed in terms of postmitochondrial protein levels. CPFA-fed fish had lower microsomal protein and cytochrome P-450 levels and NADPH-cytochrome c reductase and aldrin epoxidation activities than did controls.

N-Nitroso-3-hydroxypyrrolidine in fried bacon and fried out fat.

N-Nitroso-3-hydroxypyrrolidine was extracted from fried bacon and fried-out fat with water:methanol (3:2). After elimination of residual nitrite with ammonium sulfamate, the nitrosamine was removed from the water:methanol by continuous extraction with dichloromethane. Identification was accomplished by conversion of N-nitroso-3-hydroxypyrrolidine to the trimethylsilyl derivative followed by separation on a capillary column and characterization by gas chromatography-mass spectrometry. Quantification and recovery studies were conducted without derivatization. Concentrated dichloromethane extracts were analyzed using a gas chromatographic-thermal energy analyzer system. Recovery of N-nitroso-3-hydroxypyrrolidine from fried bacon and fried-out fat was 62 and 63%, respectively. Five commercial samples of fried bacon contained 0.4-3.9 ppb, while the fried-out fat contained 0.3-2.2 ppb N-nitroso-3-hydroxypyrrolidine.

Rat urinary metabolites of [9,10-methylene-14C] sterculic acid.

1. The metabolism of [9,10-methylene-14C] sterculic acid was studied in corn oil and Stercula foetida oil fed rats. The majority of the radioactivity was excreted into the urine as short chain dicarboxylic acids. The main urinary metabolites were cis-3,4-methylene adipic acid, cis-3,4-methylene suberic acid, trans-3,4-methylene adipic acid, cis-3,4-methylene pimelic acid, and cis-3,4-methylene azelic acid. 2. Formation of these urinary metabolites requires alpha-, beta-, and omega-oxidation plus reduction of the cyclopropene ring to a cyclopropane ring. Sterculic acid must be transported through both mitochondrial and microsomal systems. 3. Other non-radioactive urinary compounds were also identified. A proposed pathway for the metabolism of sterculic acid and possible detrimental effects caused by these metabolites is discussed.