Dwight W. Miller

Sample preparation for the analysis of flavors and off-flavors in foods

Off-flavors in foods may originate from environmental pollutants, the growth of microorganisms, oxidation of lipids, or endogenous enzymatic decomposition in the foods. The chromatographic analysis of flavors and off-flavors in foods usually requires that the samples first be processed to remove as many interfering compounds as possible. For analysis of foods by gas chromatography (GC), sample preparation may include mincing, homogenation, centrifugation, distillation, simple solvent extraction, supercritical fluid extraction, pressurized-fluid extraction, microwave-assisted extraction, Soxhlet extraction, or methylation. For high-performance liquid chromatography of amines in fish, cheese, sausage and olive oil or aldehydes in fruit juice, sample preparation may include solvent extraction and derivatization. Headspace GC analysis of orange juice, fish, dehydrated potatoes, and milk requires almost no sample preparation. Purge-and-trap GC analysis of dairy products, seafoods, and garlic may require heating, microwave-mediated distillation, purging the sample with inert gases and trapping the analytes with Tenax or C18, thermal desorption, cryofocusing, or elution with ethyl acetate. Solid-phase microextraction GC analysis of spices, milk and fish can involve microwave-mediated distillation, and usually requires adsorption on poly(dimethyl)siloxane or electrodeposition on fibers followed by thermal desorption. For short-path thermal desorption GC analysis of spices, herbs, coffee, peanuts, candy, mushrooms, beverages, olive oil, honey, and milk, samples are placed in a glass-lined stainless steel thermal desorption tube, which is purged with helium and then heated gradually to desorb the volatiles for analysis. Few of the methods that are available for analysis of food flavors and off-flavors can be described simultaneously as cheap, easy and good.

Microbial metabolism of pyrene

The isolation and identification of pyrene metabolites formed from pyrene by the fungus Cunninghamella elegans is described. C. elegans was incubated with pyrene for 24 h. Six metabolites were isolated by reversed-phase high-performance liquid (HPLC) and thin-layer chromatography (TLC) and characterized by the application of UV absorption, 1H-NMR and mass spectral techniques. C. elegans hydroxylated pyrene predominantly at the 1,6- and 1,8-positions with subsequent glucosylation to form glucoside conjugates of 1-hydroxypyrene, 1,6- and 1,8-dihydroxypyrene. In addition, 1,6- and 1,8-pyrenequinones and 1-hydroxypyrene were identified as metabolites. Experiments with [4-14C]pyrene indicated that over a 24-h period, 41% of pyrene was metabolized to ethyl acetate-soluble metabolites. The glucoside conjugates of 1-hydroxypyrene, 1,6- and 1,8-dihydroxypyrene accounted for 26%, 7% and 14% of the pyrene metabolized, respectively. Pyrenequinones accounted for 22%. The results indicate that the fungus C. elegans metabolized pyrene to non-toxic metabolites (glucoside conjugates) as well as to compounds (pyrenequinones) which have been suggested to be biologically active in higher organisms. In addition, there was no metabolism at the K-region of the molecule which is a major site of enzymatic attack in mammalian systems.

Stereoselective formation of a K-region dihydrodiol from phenanthrene by Streptomyces flavovirens

The metabolism of phenanthrene, a polycyclic aromatic hydrocarbon (PAH), by Streptomyces flavovirens was investigated. When grown for 72 h in tryptone yeast extract broth saturated with phenanthrene, the actinomycete oxidized 21.3% of the hydrocarbon at the K-region to form trans-9,10-dihydroxy-9,10-dihydrophenanthrene (phenanthrene trans-9,10-dihydrodiol). A trace of 9-phenanthrol was also detected. Metabolites isolated by thin-layer and high performance liquid chromatography were identified by comparing chromatographic, mass spectral, and nuclear magnetic resonance properties with those of authentic compounds. Experiments using [9-14C]phenanthrene showed that the trans-9,10-dihydrodiol had 62.8% of the radioactivity found in the metabolites. Circular dichroism spectra of the phenanthrene trans-9,10-dihydrodiol indicated that the absolute configuration of the predominant enantiomer was (−)-9S,10S, the same as that of the principal enantiomer produced by mammalian enzymes. Incubation of S. flavovirens with phenanthrene is an atmosphere of 18O2, followed by gas chromatographic/mass spectral analysis of the metabolites, indicated that one atom from molecular oxygen was incorporated into each molecule of the phenanthrene trans-9,10-dihydrodiol. Cytochrome P-450 was detected in 105,000×g supernatants prepared from cell extracts of S. flavovirens. The results show that the oxidation of phenanthrene by S. flavovirens was both regio- and stereospecific.

Microwave mediated distillation with solid-phase microextraction: determination of off-flavors, geosmin and methylisoborneol, in catfish tissue

Abstract Presented is a rapid distillation device for use with solid-phase microextraction (SPME). We apply this device specifically for determining two semivolatile off-flavor compounds, methylisoborneol and geosmin, in channel catfish. The presence of these two compounds in channel catfish filets results in unwelcome tastes. In the presented procedure, a catfish tissue sample is placed within a sample container located inside the microwave device. Microwave radiation is applied and distillates formed migrate through a condenser via a purge gas and are collected in a sample vial. A SPME fiber is placed within the stirred collected distillate and methylisoborneol and geosmin are extracted. Qualitative and quantitative results of these extractions are obtained using a gas chromatograph-ion trap mass spectrometer. This solventless technique results in detection limits far below the human threshold for these off-flavor compounds in channel catfish.

Use of 13C NMR spectrometric data to produce a predictive model of estrogen receptor binding activity.

We have developed a spectroscopic data-activity relationship (SDAR) model based on 13C NMR spectral data for 30 estrogenic chemicals whose relative binding affinities (RBA) are available for the alpha (ERalpha) and beta (ERbeta) estrogen receptors. The SDAR models segregated the 30 compounds into strong and medium binding affinities. The SDAR model gave a leave-one-out (LOO) cross-validation of 90%. Two compounds that were classified incorrectly in the SDAR model were in the transition zone between classifications. Real and predicted 13C NMR chemical shifts were used with test compounds to evaluate the predictive behavior of the SDAR model. The 13C NMR SDAR model using predicted 13C NMR data for the test compounds provides a rapid, reliable, and simple way to screen whether a compound binds to the estrogen receptors.

Identification of xyloside conjugates formed from anthracene by Rhizoctonia solani

Biotransformation experiments showed that a strain of Rhizoctonia solani was able to metabolize anthracene, a tricyclic aromatic hydrocarbon. The fungus was grown in a complex liquid medium containing [9-14C]anthracene; after 6 d, 98·8% of the anthracene had been converted to ethyl acetate-extractable metabolites. These compounds were separated by high-performance liquid chromatography (hplc) and detected by ultraviolet (uv) absorbance and liquid scintillation counting. The major metabolites were identified by their uv, mass, and nuclear magnetic resonance spectra. One of the principal metabolites was identified as trans-1,2-dihydroxy-1,2-dihydroanthracene (anthracene trans-1,2-dihydrodiol), which was shown by circular dichroism spectroscopy to be a mixture of two enantiomers. Chiral stationary phase hplc was used to resolve the trans-1,2-dihydrodiol into a (−)-1S,2S enantiomer (60%) and a (+)-1R,2R enantiomer (40%). The other principal metabolites were novel xyloside conjugates of anthracene: 1-O-(2-hydroxy-trans-1,2-dihydroanthryl)-β- d -xylopyranoside, 2-O-(1-hydroxy-trans-1,2-dihydroanthryl)-β- d -xylopyranoside, and 1-O-anthryl-β- d -xylopyranoside. Anthraquinone was found in the culture media but was also present in non-inoculated controls.

Fungal transformations of antihistamines: metabolism of methapyrilene, thenyldiamine and tripelennamine to N-oxide and N-demethylated derivatives

1. Strains of the fungus Cunninghamella elegans ATCC 9245 and 36112 were tested for their ability to transform the antihistamines methapyrilene (I), thenyldiamine (II) and tripelennamine (III). 2. Antihistamine metabolites were isolated by h.p.l.c., and identified by their 1H-n.m.r. and mass spectral properties. 3. All three drugs were transformed by both C. elegans strains to N-oxidized and N-demethylated derivatives. Metabolism during 96 h of incubation amounted to 85% for (I), 64% for (II), and 83% for (III). Metabolites soluble in organic solvents amounted to 62% to 86% of the total metabolism; approximately 88% to 95% of the organic-soluble metabolites were N-oxide derivatives of each antihistamine.

Structure analysis of proximadiol (cryptomeridiol) by 13C NMR spectroscopy

Abstract The sesquiterpene diol with antispasmodic properties, earlier isolated from Cymbopogon proximus, is shown to be identical with cryptomeridiol.

Biodegradation of 1-nitropyrene

The metabolism of 14C-labeled 1-nitropyrene in microcosms containing nonsterile estuarine sediments, and in cultures of a Mycobacterium sp. previously isolated from oil-contaminated sediments was investigated. Although mineralization of 1-nitropyrene by pure cultures of the Mycobacterium sp. totaled only 12.3% after 10 days of incubation, over 80% of the ethyl acetate extractable 14C-labeled compounds consisted of 1-nitropyrene metabolites. High pressure liquid chromatographic analysis of 1-nitropyrene degradation products indicated that two major metabolites were formed. They were identified as 1-nitropyrene cis-9,10-and 4,5-dihydrodiols, based on their UV-visible, mass and NMR spectra. Time course studies in microcosms showed that 1-nitropyrene was degraded slowly under aerobic and anaerobic conditions in estuarine sediments. Less than 1% had been converted to 14CO2 after 8 weeks of aerobic incubation. The addition of 1-nitropyrene to anaerobic sediments resulted in no 14CO2 evolution; however, the nitro group of 1-nitropyrene was reduced to form 1-aminopyrene. Although the mineralization of 1-nitropyrene in sediments was slow, the Mycobacterium sp. metabolized 1-nitropyrene in pure culture. This bacterium appears promising for the bioremediation of this ubiquitous pollutant in contaminated waste.

Determination of methylisoborneol in channel catfish pond water by solid phase extraction followed by gas chromatography-mass spectrometry

Abstract Several compounds exist which are responsible for unwanted taste and odor characteristics that have been found in the edible tissue of the channel catfish (Ictalurus punctatus). Methylisoborneol (MIB), one compound responsible for these off-flavors, is produced through the metabolism of cyanobacteria in aqueous systems such as reservoirs and ponds. The channel catfish accumulates MIB in its tissues from the intake of its culture-pond water. Current methods of analysis for MIB, such as closed- and open-loop stripping, and liquid-liquid extraction, are time-consuming and labor-intensive. A method for the analysis of MIB in catfish culture-pond water is described which, unlike other methods, is rapid, inexpensive, and does not require specialized sample preparation equipment. This method utilizes C18 solid-phase extraction followed by capillary gas chromatography with detection by mass spectrometry. Standard MIB and the internal standard, butylisoborneol (BIB), were prepared from the reaction of d -camphor with methylmagnesium chloride and n-butyllithium, respectively. Extraction efficiencies for MIB in channel catfish pond water averaged 89% at 101 parts per trillion (ppt) and 84% at 202 parts per billion (ppb). The detection limit of the method was calculated to be 11.5 ppt.