[Influence of ethanol content on the detection of volatile components in Huangjiu].

Huangjiu (Chinese rice wine) is a traditional Chinese fermented wine with a unique flavor. The components of this wine are complex, and the ethanol content of different Huangjiu preparations varies greatly. In this study, changes in the chromatographic peak areas of the volatile components of Huangjiu samples with different ethanol contents were measured using headspace-gas chromatography (HS-GC). The influence of ethanol on the quantitative detection of different volatile components of Huangjiu at gas-liquid equilibrium was also analyzed. When the ethanol content of Huangjiu was in the range of 10%-19% vol, the peak areas of 16 volatile components (i. e., sec-butanol, n-propanol, isobutanol, n-butanol, isoamyl alcohol, ß-phenyl-ethanol, acetaldehyde, isovaleraldehyde, benzaldehyde, ethyl formate, ethyl acetate, isobutyl acetate, isoamyl acetate, ethyl hexanoate, ethyl lactate, and diethyl succinate) were negatively correlated with the ethanol content. Increases in the ethanol content of the liquor changed the gas-liquid equilibrium of most other trace volatile components. In addition, only the peak area of acetal was positively correlated with ethanol content. The content of acetal in Huangjiu was affected by the alcohol content, and its decomposition reaction occurred along with the dilution process. The influence coefficient of ethanol content on the peak area of the above compounds ranged from -12.4% to 4.9%. The vapor pressure of most volatile components decreased with increasing ethanol content, and different components were affected in different ways. Compared with those of other components, the peak areas of methanol, furfural, and acetic acid were less affected by the ethanol content. These components were also affected by other factors, such as ionization and chemical reactions occurring during the dilution process. When different wine samples were adjusted to the same ethanol content, the concentration of volatile components in these samples became proportional to the total chromatographic peak area and the influence of the matrix effect of ethanol on the quantitative analysis was effectively eliminated. Thus, when researchers use pretreatment methods based on the principle of gas-liquid balance to carry out the quantitative detection of flavor components, they should adjust different rice wine samples to the same alcohol content to effectively control the matrix effect caused by differences in ethanol content and achieve accurate quantitative analysis.

Identification and quantification of dimethyl acetals from plasmalogenic lipids in lamb intramuscular fat under different derivatization procedures.

Meat lipids are mostly comprised by triacylglycerols, but small amounts of plasmalogens are also present in intramuscular fat. The purpose of this study was to evaluate the effect of lipid derivatization on the presence of dimethyl acetal (DMA) molecules from plasmalogenic lipids in intramuscular fat samples. Three different methods of methylation were assayed. Acid-catalyzed methanolysis using HCl, the traditional procedure to derivatize meat lipids, was compared to two base-catalyzed methanolysis based on the ISO International standard procedure using either KOH and/or NaOCH3 which, apparently, are only able to methylate fatty acids from triacylglycerols. DMA compounds were isolated by thin layer chromatography and then identified by gas chromatography-mass spectrometry. The most prominent DMA molecules detected were 16:0 and 18:0, but also minor amounts of monounsaturated and branched-chain DMA were quantified. Acid methylation yielded the highest amounts of DMA. However, the present article demonstrates that ISO standard based methylation procedures could also generate DMA derivatives in considerable quantities, which is not usually considered and may interfere with the determination of fatty acid methyl esters (FAME) from triacylglycerides. The current research warns scientist about possible FAME misidentifying and overestimations in intramuscular fat analysis using basic methylation and the need to consider the presence of DMA in samples that contain plasmalogens.

Acetaldehyde reactions during wine bottle storage.

Acetaldehyde is a major wine oxidation product. Here, three Cabernet Sauvignon wines, containing different levels of acetaldehyde from different micro-oxygenation (mOx) regimes, including yeast-mediated treatments, were aged under closures differing in oxygen ingress. Oxygen, phenolics, carbonyls and heterocyclic acetals were measured. Acetaldehyde levels at bottling was a significant factor in the phenolic compound profile after one year, with anthocyanins most affected, then flavonols, flavonoids and hydroxycinnamic acids, but there were negligible effects on benzoic acids. The effect of bottle closures with increased oxygen ingress had a similar trend. Increased acetaldehyde levels and oxygen ingress also yielded higher levels of the heterocyclic acetals from glycerol. These changes reflect aging, and suggest that managing mOx during production could be used to reduce the time needed to achieve some aged wine characteristics.

Quantitation, Organoleptic Contribution, and Potential Origin of Diethyl Acetals Formed from Various Aldehydes in Cognac.

Cognac wine distillate (WD), especially that produced during aging, is marked by complex and elegant aroma. This work aimed at expanding the knowledge on the Cognac WD aroma by a sensory-guided approach, involving a fractional-distillation technique and gas chromatography coupled to olfactometry and mass spectrometry (GC-O-MS). In doing so, a fruity-odor zone was highlighted in WD extracts that was attributed to the diethyl acetal family. Ten additional diethyl acetals were detected by GC-MS. Next, an assay method was developed and validated for seven of these diethyl acetals. Their detection thresholds were evaluated in a model solution of water/ethanol (60:40, v/v). 1,1-Diethoxy-3-methylbutane was shown to present a significant organoleptic impact because its olfactory-detection threshold (323 µg/L) is lower than its range of concentrations in WD (461 to 3337 µg/L). Given that diethyl acetals result from reactions between ethanol and aldehydes, quantitative correlations between diethyl acetals and corresponding aldehydes were considered.

Incorporation of Black Soldier Fly (Hermetia illucens L.) larvae fat or extruded linseed in diets of growing rabbits and their effects on meat quality traits including detailed fatty acid composition.

The inclusion of Black Soldier Fly (BSF) fat or extruded linseed (LIN) in diets for growing rabbits on meat fatty acids (FA), dimethyl acetals (DMA), oxidative stability and color was evaluated. Forty-eight rabbits with 35 days of age were individually housed, fed one of 4 diets (LIN-Low, 30 g/kg of fat from LIN; LIN-High, 60 g/kg of fat from LIN; BSF-Low, 30 g/kg of BSF fat; BSF-High, 60 g/kg of BSF fat) and slaughtered after 5 weeks. Diets with BSF reduced the intramuscular FA but increased the 12:0 and 14:0 in meat compared to LIN, whereas LIN diets increased the 18:3n-3 deposition in meat. Regressions between FA intake and FA meat concentration indicate that the deposition of 12:0 and 14:0 in the meat of BSF-fed rabbits was lower than the deposition of n-3 PUFA in the meat of LIN-fed rabbits. Overall, lipid profiles of meat from BSF-fed rabbits were less healthy, but meat from LIN-fed rabbits was the more susceptible to oxidation.

Tandem Mass Spectrometry Imaging and in Situ Characterization of Bioactive Wood Metabolites in Amazonian Tree Species Sextonia rubra.

Driven by a necessity for confident molecular identification at high spatial resolution, a new time-of-flight secondary ion mass spectrometry (TOF-SIMS) tandem mass spectrometry (tandem MS) imaging instrument has been recently developed. In this paper, the superior MS/MS spectrometry and imaging capability of this new tool is shown for natural product study. For the first time, via in situ analysis of the bioactive metabolites rubrynolide and rubrenolide in Amazonian tree species Sextonia rubra (Lauraceae), we were able both to analyze and to image by tandem MS the molecular products of natural biosynthesis. Despite the low abundance of the metabolites in the wood sample(s), efficient MS/MS analysis of these γ-lactone compounds was achieved, providing high confidence in the identification and localization. In addition, tandem MS imaging minimized the mass interferences and revealed specific localization of these metabolites primarily in the ray parenchyma cells but also in certain oil cells and, further, revealed the presence of previously unidentified γ-lactone, paving the way for future studies in biosynthesis.

Effect of different types of olive oil pomace dietary supplementation on the rumen microbial community profile in Comisana ewes.

Olive oil pomace (OOP) is a bio-waste rich in highly soluble polyphenols. OOP has been proposed as an additive in ruminant feeding to modulate rumen fermentations. Three groups of ewes were fed the following different diets: a control diet and two diets supplemented with OOP, obtained with a two-phase (OOP2) or three-phase (OOP3) olive milling process. Rumen liquor (RL) showed a higher content of 18:3 cis9 cis12 cis15 (α-linolenic acid, α-LNA) with OOP2 inclusion, and of 18:2 cis9 trans11 (rumenic acid, RA) with OOP3 inclusion. The overall composition of the RL microbiota did not differ among treatments. Significant differences, between control and treated groups, were found for six bacterial taxa. In particular, RL microbiota from animals fed OOPs showed a reduction in Anaerovibrio, a lipase-producing bacterium. The decrease in the Anaerovibrio genus may lead to a reduction in lipolysis, thus lowering the amount of polyunsaturated fatty acids available for biohydrogenation. Milk from animals fed OOP showed a higher content of 18:1 cis9 (oleic acid, OA) but the α-LNA concentration was increased in milk from animals treated with OOP2 only. Therefore, inclusion of OOP in ruminant diets may be a tool to ameliorate the nutritional characteristics of milk.

Evidence that Cerambycid Beetles Mimic Vespid Wasps in Odor as well as Appearance.

We present evidence that cerambycid species that are supposed mimics of vespid wasps also mimic their model's odor by producing spiroacetals, common constituents of vespid alarm pheromones. Adults of the North American cerambycids Megacyllene caryae (Gahan) and Megacyllene robiniae (Forster) are conspicuously patterned yellow and black, and are believed to be mimics of aculeate Hymenoptera, such as species of Vespula and Polistes. Adult males of M. caryae produce an aggregation-sex pheromone, but both sexes produce a pungent odor when handled, which has been assumed to be a defensive response. Headspace aerations of agitated females of M. caryae contained 16 compounds with mass spectra characteristic of spiroacetals of eight distinct chemical structures, with the dominant compound being (7E,2E)-7-ethyl-2-methyl-1,6-dioxaspiro[4.5]decane. Headspace samples of agitated males of M. caryae contained five of the same components, with the same dominant compound. Females of M. robiniae produced six different spiroacetals, one of which was not produced by M. caryae, (2E,7E)-2-ethyl-7-methyl-1,6-dioxaspiro[4.5]decane, and five that were shared with M. caryae, including the dominant (2E,8E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane. The latter compound is the sole spiroacetal produced by both males and females of a South American cerambycid species, Callisphyris apicicornis (Fairmaire & Germain), which is also thought to be a wasp mimic. Preliminary work also identified spiroacetals of similar or identical structure released by vespid wasps that co-occur with the Megacyllene species.

Flower Visitors of Campanula: Are Oligoleges More Sensitive to Host-Specific Floral Scents Than Polyleges?

The pollen diet provided by adult bees to their offspring varies immensely. While some species collect pollen on several plants irrespective of their phylogenetic relatedness (polyleges), others collect only on plants within a genus or family (oligoleges). Floral scents play a central role in bee-plant interactions. To locate flowers, polyleges are assumed to rely on compounds commonly found as floral scent constituents, whereas oligoleges rely on unusual compounds to recognize host flowers unambiguously. Campanula flowers are visited by both polylectic and oligolectic species, and their scent bouquets consist of common and unusual (e.g., spiroacetals) volatiles. In a comparative approach, we performed electroantennographic analyses to investigate the antennal responses of three polyleges and three oligoleges to three common volatiles and four spiroacetals. We hypothesized that: 1) oligoleges and polyleges should respond similarly to common flower volatiles, and 2) Campanula oligoleges should be more sensitive to spiroacetals than are polyleges. In corroboration, we found that antennal sensitivity to common volatiles was similar among bees irrespective of pollen diet, whereas oligoleges of Campanula were more sensitive to spiroacetals than polyleges. Newly emerged bees of the Campanula oligolege Chelostoma rapunculi rely on spiroacetals for recognizing host-flowers, and our results suggest that this might also be true for other Campanula oligoleges, since Chelostoma campanularum and Hoplitis mitis also were able to perceive these specific volatiles at very low concentrations. Together, our results provide interesting insights into the significance of olfactory adaptations in oligolectic and polylectic bee species.

New 2-methyl-13-icosenoic acid from the temperate calcisponge Leuconia johnstoni.

The fatty acid composition of the temperate calcareous marine sponge Leuconia johnstoni Carter 1871 (Calcaronea, Calcarea) was characterized for the first time in specimens collected off the Brittany coast of France over four years from October 2005 to September 2008. Forty-one fatty acids (FA) with chain lengths ranging from C14 to C22 were identified as fatty methyl esters (FAME) and N-acyl pyrrolidide (NAP) derivatives by gas chromatography-mass spectrometry (GC-MS). Twenty-two saturated fatty acids (SFA) were identified accounting for 52.1-59.0% of the total FA and dimethylacetals (DMA). In addition, among the SFA, we noticed the presence of numerous methyl-branched iso and anteiso FA, suggesting a large number of associated bacteria within L. johnstoni. Thirteen monounsaturated fatty acids (MUFA, 28.0-36.0% of total FA + DMA) were also identified as well as six polyunsaturated fatty acids (PUFA, 4.0-8.2%). A noticeable DMA was detected at a high level, particularly in September 2008 (11.8%), indicating the presence of plasmalogens in this sponge species. This calcareous sponge lacked the non-methylene-interrupted FA (NMI FA) with a Δ5,9 system typical of siliceous Demosponges and Hexactinellids. The occurrence of the unusual 8,13-octadecadienoic acid was reported for the first time as a minor PUFA in a calcareous sponge. The major FA, representing 20-25% of this sponge FA, was identified as the new 2-methyl-13-icosenoic acid from mass spectra of its methyl ester and its corresponding N-acyl pyrrolidide derivatives as well as a dimethyl disulfide adduct.

Synthesis of C (6)-epimer derivatives of diacetoxy acetal derivative of santonin and their inducing effects on HL-60 leukemia cell differentiation.

Induction of differentiation is a new and promising approach to leukemia therapy, well illustrated by the treatment of acute promyelocytic leukemia with 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] or all-trans retinoic acid (ATRA). Using combination of either 1,25-(OH)(2)D(3) or ATRA and chemotherapy, adverse effects 1,25-(OH)(2)D(3) or ATRA such as hypercalcemic effects have decreased, and long-term survival has improved. In a previous study, we demonstrated that santonin could be chemically modified into a diacetoxy acetal derivative of santonin with strong differentiation-inducing activity. In this study, we further synthesized C(6)-epimer derivatives of diacetoxy acetal derivative of santonin and tested their effects on HL-60 cell differentiation. Some of the C(6)-epimer derivatives themselves induced increases in cell differentiation. Especially, (11S)-3,3-(ethylenedioxy) eudesmano-13-ol-6ß-acetate (7) was demonstrated to induce differentiation with larger than 80% of the cells attaining a differentiated phenotype. Importantly, 7 strongly enhanced differentiation of HL-60 cells in a dose-dependent manner when combined with either low doses of 1,25-(OH)(2)D(3) or ATRA. The ability to enhance the differentiation potential of 1,25-(OH)(2)D(3) or ATRA by 7 may improve outcomes in the therapy of acute promyelocytic leukemia.

In-depth search focused on furans, lactones, volatile phenols, and acetals as potential age markers of Madeira wines by comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry combined with solid phase microextraction.

The establishment of potential age markers of Madeira wine is of paramount significance as it may contribute to detect frauds and to ensure the authenticity of wine. Considering the chemical groups of furans, lactones, volatile phenols, and acetals, 103 volatile compounds were tentatively identified; among these, 71 have been reported for the first time in Madeira wines. The chemical groups that could be used as potential age markers were predominantly acetals, namely, diethoxymethane, 1,1-diethoxyethane, 1,1-diethoxy-2-methyl-propane, 1-(1-ethoxyethoxy)-pentane, trans-dioxane and 2-propyl-1,3-dioxolane, and from the other chemical groups, 5-methylfurfural and cis-oak-lactone, independently of the variety and the type of wine. GC × GC-ToFMS system offers a more useful approach to identify these compounds compared to previous studies using GC-qMS, due to the orthogonal systems, that reduce coelution, increase peak capacity and mass selectivity, contributing to the establishment of new potential Madeira wine age markers. Remarkable results were also obtained in terms of compound identification based on the organized structure of the peaks of structurally related compounds in the GC × GC peak apex plots. This information represents a valuable approach for future studies, as the ordered-structure principle can considerably help the establishment of the composition of samples. This new approach provides data that can be extended to determine age markers of other types of wines.

Desulfotignum toluenicum sp. nov., a novel toluene-degrading, sulphate-reducing bacterium isolated from an oil-reservoir model column.

A Gram-negative, sulphate-reducing bacterium (strain H3(T)) was isolated from an oil-reservoir model column. The new isolate was able to oxidize toluene coupled to hydrogen sulphide production. For growth, the optimum salt concentration was 1.5 % (w/v), the optimum pH was 7.2 and the optimum temperature was 34 degrees C. The cells were straight to slightly curved rods, 0.6-1.0 microm in diameter and 1.4-2.5 microm in length. The predominant fatty acids were C(16 : 0), C(16 : 1)omega7c and C(17 : 0) cyclo, and the cells also contained dimethylacetals. Cloning and sequencing of a 1505 bp long fragment of the 16S rRNA gene showed that strain H3(T) is a member of the Deltaproteobacteria and is related closely to Desulfotignum balticum DSM 7044(T). The G+C content of the DNA was 52.0 mol% and the DNA-DNA similarity to D. balticum DSM 7044(T) was 56.1 %. Based on differences in DNA sequence and the unique property of toluene degradation, it is proposed that strain H3(T) should be designated a member of a novel species within the genus Desulfotignum, for which the name Desulfotignum toluenicum sp. nov. is proposed. The type strain is H3(T) (=DSM 18732(T)=ATCC BAA-1460(T)).

Stereospecific analysis of phospholipid classes in skeletal muscle from rats fed different fat sources.

The fatty acid (FA) and dimethylacetal profiles of the sn-1 and sn-2 positions of different phospholipid (PL) classes from skeletal muscle of rats as affected by dietary FA profiles were studied. Rats were fed either a control diet, an olive oil-enriched diet, or a sunflower oil-enriched diet. The FA composition of both positions of the studied PL classes was affected by diet to different extents. The FA composition of the sn-2 position of phosphatidylserine was the most influenced by diet, while phosphatidylinositol was less affected by dietary modification. The FA profile of phosphatidylcholine reflected consumed FA better than any other studied PL. Thus, olive oil rats showed higher oleic acid (C18:1 n-9) contents in both positions of phosphatidylcholine, and sunflower oil rats had higher proportions of arachidonic acid (C20:4 n-6) in the sn-1 position of this PL class. Dimethylacetals were scarcely affected by diet, and only the dimethylacetal composition of phosphatidylethanolamine showed significant modifications.

Characterization of aroma compounds of chinese "Wuliangye" and "Jiannanchun" liquors by aroma extract dilution analysis.

Aroma compounds in Chinese "Wuliangye" liquor were identified by gas chromatography-olfactometry (GC-O) after fractionation. A total of 132 odorants were detected by GC-O in Wuliangye liquor on DB-wax and DB-5 columns. Of these, 126 aromas were identified by GC-mass spectrometry (MS). Aroma extract dilution analysis (AEDA) was further employed to identify the most important aroma compounds in "Wuliangye" and "Jiannanchun" liquors. The results showed that esters could be the most important class, especially ethyl esters. Various alcohols, aldehydes, acetals, alkylpyrazines, furan derivatives, lactones, and sulfur-containing and phenolic compounds were also found to be important. On the basis of flavor dilution (FD) values, the most important aroma compounds in Wuliangye and Jiannanchun liquors could be ethyl butanoate, ethyl pentanoate, ethyl hexanoate, ethyl octanoate, butyl hexanoate, ethyl 3-methylbutanoate, hexanoic acid, and 1,1-diethoxy-3-methylbutane (FD > or = 1024). These compounds contributed to fruity, floral, and apple- and pineapple-like aromas with the exception of hexanoic acid, which imparts a sweaty note. Several pyrazines, including 2,5-dimethyl-3-ethylpyrazine, 2-ethyl-6-methylpyrazine, 2,6-dimethylpyrazine, 2,3,5-trimethylpyrazine, and 3,5-dimethyl-2-pentylpyrazine, were identified in these two liquors. Although further quantitative analysis is required, it seems that most of these pyrazine compounds had higher FD values in Wuliangye than in Jiannanchun liquor, thus imparting stronger nutty, baked, and roasted notes in Wuliangye liquor.

Cyclization reactions of acylium and thioacylium ions with isocyanates and isothiocyanates: gas phase synthesis of 3,4-dihydro-2,4-dioxo-2H-1,3,5-oxadiazinium ions.

Gas-phase reactions of several acylium and thioacylium ions, that is H2C=N-C+=O, H2C=N-C+=S, O=C=N-C+=O, S=C=N-C+=O, H3C-C+=O, and (CH3)2N-C+=O, with both a model isocyanate and isothiocyanate, that is, C2H5-N=C=O and C2H5-N=C=S, were investigated using tandem-in-space pentaquadrupole mass spectrometry. In these reactions, the formation of mono- and double-addition products is observed concurrently with proton transfer products. The double-addition products are far more favored in reactions with ethyl isocyanate, whereas the reactions with ethyl isothiocyanate form, preferentially, either the mono-addition product or proton transfer products, or both. Retro-addition dominates the low-energy collision-induced dissociation of the mono- and double-addition products with reformation of the corresponding reactant ions. Ab initio calculations at Becke3LYP//6-311 + G(d,p) level indicate that cyclization is favored for the double-addition products and that products equivalent to those synthesized in solution, that is, of 3,4-dihydro-2,4-dioxo-2H-1,3,5-oxadiazinium ions and sulfur analogs, are formed.

Few bicyclic acetals at reducing end of low-molecular-weight heparins: might they restrict specification of pharmacopoeia?

The alkaline hydrolysis of heparin benzyl ester originates enoxaparin. The depolymerization by beta-elimination is the primary effect of reaction; but side reactions can happen and the bicyclic acetal at the reducing end of glucosamine N,6-disulphate, called 1,6-anhydro ring, is a product of a side reaction. The amount of this predictable moiety of enoxaparin can be controlled to a lowest extent (6%) and to extent higher than 40% by modulating the alkalinity and duration of the reaction of hydrolysis. With the exclusion of the beta-elimination effects and of these non significative side reactions, the chemical structure of the parent heparin is entirely maintained in enoxaparin as it results by the same profiles of constituent disaccharides. The content of 1,6-anhydro rings is assessed by a not yet validated NMR method. The chains of enoxaparin bearing, at their reducing end, 1,6-anhydro rings could be regarded as Related Substances of enoxaparin. If present, even in an amount less than, or equal to, 30% of chains, these "related substances" affect neither activities nor safety of enoxaparin.

GIAO/DFT evaluation of 13C NMR chemical shifts of selected acetals based on DFT optimized geometries.

DFT/B3LYP calculations of the ground-state conformation of eight cyclic and acyclic acetals are presented and compared with experimental data. Results of single-point GIAO/DFT calculations at five different levels of theory show that isotropic shieldings need to be empirically scaled to achieve agreement with experimental chemical shifts. Statistical evaluation of data indicates that the most accurate prediction of 13C chemical shifts is achieved at the MPW1PW91/6-311G** level of theory. An empirical equation describing the relationship between delta values and shielding constants is postulated. This equation has been applied to the non-chair ground-state conformation of the six-membered acetonide and to the conformationally flexible benzodioxonine derivative. The agreement observed between the experimental and predicted chemical shifts shows that calculations at the MPW1PW91/6-311G** level of theory are adequate for addressing questions of conformation.

Concerning the stability of benzyl alcohol: formation of benzaldehyde dibenzyl acetal under aerobic conditions.

An impurity in benzyl alcohol was identified as benzaldehyde dibenzyl acetal (BDBA). The component BDBA is reversibly formed by the reaction of benzyl alcohol with benzaldehyde, an oxidative degradation product of benzyl alcohol. Whereas, BDBA is a known chemical entity, it is not typically controlled in commercial benzyl alcohol since it cannot be formed in the absence of benzaldehyde, which is itself generally controlled. However, once commercial benzyl alcohol is exposed to the atmosphere, formation of BDBA is possible. The synthesis and characterization of BDBA is reported. The ability of BDBA to react with alcohols to form other types of acetals, and the impact of low levels of BDBA on the quantitative analysis of pharmaceutical products, are considered.

Direct electrospray tandem mass spectrometry of the unstable hydroperoxy bishemiacetal product derived from cholesterol ozonolysis.

Cholesterol is the most abundant neutral lipid in the epithelial lining fluid of the lower airways of the lung also known as pulmonary surfactant and a potential target for reaction with ambient ozone when inspired into the human lung. The isolated double bond of cholesterol has been shown to be susceptible to attack by ozone, but the major reaction product from cholesterol ozonolysis had been remarkably difficult to structurally characterize. Recently, NMR and X-ray crystallography have been used to suggest the formation of a hydroperoxy, hydroxy hemiacetal product, using various derivatives and models of cholesterol to stabilize this chemically reactive product. Electrospray ionization mass spectrometry was used to study the somewhat unstable ozonolysis product of cholesterol which was found to display unique ionization and fragmentation properties when collisionally activated. The electron-deficient carbon atoms of this highly oxygenated product permitted covalent attachment of an acetate anion during negative ion electrospray ionization, leading to the formation of abundant adduct ions at m/z 511. Surprisingly, positive ions were not readily formed. Collision induced dissociation of the adduct anion yielded a major ion at m/z 477, corresponding to the loss of hydrogen peroxide. The most abundant fragment ion following collisional activation was observed at m/z 93, resulting from a complex rearrangement subsequent to the attack of the hydroperoxide anion on the carbon center of the acetate adduct. Based on the interpretation of the tandem mass spectral data, the major cholesterol ozonization product was characterized as a hydroperoxy, hydroxy hemiacetal derivative, which was consistent with the NMR and X-ray crystallographic studies which were carried out on the more stable methyl ether derivative.