Persistence assessment of cyclohexyl- and norbornyl-derived ketones and their degradation products in different OECD screening tests.

The persistence of synthetic cyclohexyl- and norbornyl-derived ketones was assessed by using OECD 301F and 301D biodegradation tests. While cyclohexyl-derived ketones either reached or came close to the pass level (60%) after 60 d, the corresponding norbornyl derivatives yielded significantly less biodegradation (<40%). By analyzing extracts at 60 d, the key degradation products of four norbornyl derivatives were identified. Consistently, 2-bicyclo[2.2.1]heptane carboxylic acid was found as a principal degradation product with minor quantities of bicyclo[2.2.1]heptan-2-one and 2-bicyclo[2.2.1]heptane acetic acid. When the three degradation products were re-synthesized and tested individually for biodegradability, the former two were found to be ultimately biodegradable after 60 d in OECD 301D tests, thus proving non-persistence. Similarly, 2-bicyclo[2.2.1]heptane acetic acid was found to be degraded significantly, albeit with long lag phases exceeding 60 d in the case of freshwater inoculum, then ultimately reaching the pass level. On the other hand, norbornyl ketones were still only partially biodegradable in the same test. We conclude that despite the potential for ultimate biodegradation of norbornyl-derived ketones, current screening tests yield an incomplete picture of their biodegradability, particularly when applying strict OECD criteria. The appearance of long lag phases when re-testing norbornyl ketone degradation products underlines the importance of extending tests to well beyond 28 and even 60 d in the case of freshwater inocula.

Elucidation of the upper pathway of alicyclic musk Romandolide degradation in OECD screening tests with activated sludge.

The degradation of Romandolide ([1-(3',3'-dimethyl-1'-cyclohexyl)ethoxycarbonyl] methyl propanoate), a synthetic alicyclic musk, by activated sludge inocula was investigated using both the manometric respirometry test OECD 301F and the CO2 evolution test. In addition to measuring its biodegradability, key steps of the upper part of the metabolic pathway responsible for Romandolide degradation were identified using extracts at different time points of incubation. Early metabolism of Romandolide yielded ester hydrolysis products, including Cyclademol (1-(3,3-dimethylcyclohexyl)ethanol). The principal metabolites after 31 days were identified as 3,3-dimethyl cyclohexanone and 3,3-dimethyl cyclohexyl acetate. Formation of 3,3-dimethyl cyclohexanone from Cyclademol by sludge was confirmed in subsequent experiments using Cyclademol as a substrate, indicating the involvement of an oxygen insertion reminiscent of a Baeyer-Villiger oxidation. Further mineralization of 3,3-dimethyl cyclohexanone was also confirmed in subsequent studies. Three steps were thus required for complete biodegradation of the alicyclic musk: (1) successive ester hydrolyses leading to the formation of Cyclademol with concomitant degradation of the resulting acids, (2) conversion of Cyclademol into 3,3-dimethyl cyclohexanone, and (3) further mineralization via ring cleavage.

Strategy for the identification of key odorants: application to shrimp aroma.

The GC-SNIF technique was used to obtain the olfactograms of shrimps, and their impact odorants were identified by MS hyphenated to the GC/olfactometric system and by comprehensive two-dimensional GC hyphenated to a time-of-flight MS. Confirming these identifications by their linear retention indices required application of a new strategy to compare retention indices between both instruments and with the in-house database. The aldehydes were confirmed by using their pentafluorophenylhydrazone derivatives, and 2-ethyl-3,5-dimethyl pyrazine had to be resolved from co-eluting compounds and then identified by multidimensional GC hyphenated to an MS and a sniff port. In both shrimp products, the most important odorants were trimethylamine, 2-acetyl-1-pyrroline, and 2-ethyl-3,5-dimethyl pyrazine, together with common carbonyl compounds.

Determination of air-to-water partition coefficients using automated multiple headspace extractions.

Air-to-water partition coefficients are experimentally determined using a multiple headspace extraction procedure and an automated headspace cell. The approach is first validated with 2-butanone and then applied to a homologous series of methyl ketones. As adsorptions of the most hydrophobic compounds occurred in the sampling cell, technical improvements have been tested. This study represents the first attempt to overcome analyte adsorptions by studying and minimizing the effect of the cell's adsorption of hydrophobic analytes on the determination of their partition coefficients. The present method allows the measurement of several analytes at the same time, in the ppm range, without calibration, and with a limited manpower.

Automated multiple headspace extraction procedure: adsorption modeling and determination of air-to-water partition coefficients.

The present work makes the first attempt to take into account adsorptions in the determination of partition coefficients by modeling the multiple headspace extraction (MHE) process. Modeling a six-step MHE procedure of a homologous series of methyl ketones revealed that their adsorption-desorption on the walls was the rate-limiting step. Moreover, a comparison between experimental and predicted MHE plots shows that only the last MHE points were affected by adsorption phenomena. Using cell materials with the lowest sorptive properties, partition coefficients were then accurately calculated from the first four MHE steps. This kinetic approach supports previous work in which adsorptions were lowered owing to the choice of sampling cell materials. It also justifies some reproducibility limitations of the MHE quantification procedure mentioned in the literature.

Hyphenation of quadrupole MS to GC and comprehensive two-dimensional GC for the analysis of suspected allergens: review and improvement.

This survey reviews papers that have been previously published on the quantitative analysis of suspected allergens. The routine gas chromatography (GC)-mass spectrometry (MS) method allows their evaluation in most fragrances, but the application of an automated data treatment sometimes leads to over- or underestimations when target compounds are coeluted or shifted because of the presence of other fragrance ingredients. In such cases, an appropriate retreatment of data generated by the routine analysis is proposed to better estimate these shifted or coeluted peaks. A second and more sophisticated approach, based on comprehensive bidimensional GC hyphenated to quadrupole MS, overcomes coelution problems. However, its use is still time consuming because of the lack of a commercial program. In this work, a software prototype is tested to reprocess the data. It dramatically shortens the data treatment and offers good quantitative results.

Improved hardware and software for quick gas chromatography-olfactometry using CHARM and GC-"SNIF" analysis.

A multisniffing system has been developed to allow three panelists to simultaneously participate in a GC-olfactometric analysis. This device, associated with a computerized data treatment, allows shortening CHARM and GC-"SNIF' analyses to less than 1 week and less than 1 day, respectively. The program was developed as an extension of an existing commercial chromatography data system, as usual GC processing functions are suited to the treatment of olfactograms (plots of odor response versus GC elution time). Because of the improved algorithm, the consequences of gaps in coincident responses were minimized, and the systematic use of a panel improved the repeatability of CHARM olfactograms. Comparing both methods, GC-SNIF repeatability appears to be higher than that of CHARM, as the former method uses a larger panel, but in a shorter lapse of time.

Origin of acetaldehyde during milk fermentation using (13)C-labeled precursors.

Acetaldehyde formation by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus during fermentation of cow's milk was investigated using (13)C-labeled glucose, L-threonine, and pyruvate with a recent static-and-trapped-headspace technique that does not require derivatization of acetaldehyde prior to gas chromatography-mass spectrometry. Over 90% and almost 100% of acetaldehyde originated from glucose during fermentation by L. delbrueckii subsp. bulgaricus and S. thermophilus, respectively, taking into account both singly and doubly labeled acetaldehyde. As both microorganisms showed threonine aldolase activity and formed labeled acetaldehyde from (13)C-labeled threonine during the fermentation of milk, this amino acid should also contribute to the acetaldehyde produced.

Vicinal diketone formation in yogurt: (13)C precursors and effect of branched-chain amino acids.

Addition of branched-chain amino acids (BCAA) or an inhibitor of the BCAA biochemical pathways during fermentation of milk with a lac(-) mutant of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strongly influenced the formation of two aroma-impact compounds, 2,3-butanedione and 2,3-pentanedione, as well as their direct precursors 2-acetolactate and 2-acetohydroxybutyrate. This suggests a connection between vicinal diketone formation and BCAA biosynthesis in yogurt bacteria. A recently developed static-and-trapped headspace technique combined with gas chromatography-mass spectrometry demonstrated incorporation of (13)C from [U-(13)C(6)]-D-glucose and [U-(13)C(4)]-L-threonine into both vicinal diketones. For 2,3-butanedione, glucose is the major precursor via pyruvate and activated acetaldehyde. For 2, 3-pentanedione, L-threonine is a precursor via 2-ketobutyrate, but glucose is the major contributor via activated acetaldehyde and, possibly, also via 2-ketobutyrate, which is a degradation product of 3-methylaspartate, an intermediate in glutamate synthesis.

Sensory investigation of yogurt flavor perception: mutual influence of volatiles and acidity.

The sensory properties of traditional acidic and mild, less acidic yogurts were characterized by a trained panel using a descriptive approach. Many of the descriptive attributes varied almost linearly with pH, showing either a positive or negative correlation with increasing acidity. The panel was very sensitive to acidity differences, as demonstrated by the linear relationship between acidity perception and pH. Important flavor differences were found between the two classes of yogurt. They were mainly due to differences in acidity and not to different concentrations of the three impact aroma compounds, acetaldehyde, 2,3-butanedione, and 2, 3-pentanedione. This emphasizes the importance of acidity in yogurt flavor. Deodorization and impact aroma compound addition had much less influence on yogurt flavor than pH variations.

First attempt of odorant quantitation using gas chromatography-olfactometry.

An aroma compound was quantitated for the first time by GC-olfactometry (GC-O) on the basis of the detection frequency of odorants by a panel of 8-12 persons. The method was previously optimized regarding the coincidence of olfactometric peak apexes and the repeatability of peak height and area over 4 months. The number of required calibration points and the confidence interval of the curve were investigated. This technique was then tested by quantifying a model solution of 1-octen-3-one. The standard addition method was found to be unsuitable in this context, but external calibration gave excellent results in the ppt range. GC-O was then challenged using one of the most sensitive and selective methods, GC/MS, to quantitate 1-octen-3-one in coffee, a complex aroma. Results showed performances comparable to GC/MS/MS for this odorant, or even better as the latter required 75-500 times more sample to perform the quantitation. However, at such a low concentration, overestimation cannot be excluded with either technique because of possible coelution of odorants or isobaric ions, respectively. These results show that GC-olfactometry can compete with the most sensitive and selective techniques, such as MS, for determination of extremely intense odorants, because little sample preparation is required and there is no need for the synthesis of labeled compounds.

Aroma comparisons of traditional and mild yogurts: headspace gas chromatography quantification of volatiles and origin of alpha-diketones.

A quick headspace GC method for quantification of volatiles was developed, involving only minor sample preparation. Yogurt flavor compounds could be quantified in the micrograms per kilogram to milligrams per kilogram range without any difficulty, despite the complex matrix. Volatiles of traditional acidic and mild, less acidic yogurts were compared, and important differences were found for acetaldehyde, 2,3-butanedione, and 2,3-pentanedione. Concentrations of 2,3-butanedione and 2,3-pentanedione increased 2-3-fold in mild, less acidic yogurts compared to traditional acidic ones. This is due to accumulation of the precursors of the diketones, 2-acetolactate and 2-acetohydroxybutyrate, during fermentation in mild, less acidic yogurt. These precursors are subsequently converted to the corresponding diketones during storage. On the contrary, acetaldehyde formation was reduced in the mild yogurt, due to growth differences between the lactic acid bacteria used for fermentation of the milk. The quantitative results presented in this study validate previous GC sniffing conclusions (Ott et al. J. Agric. Food Chem. 1997, 45, 850-858), showing that yogurt aroma is the superposition of impact flavor compounds generated by fermentation on milk compounds.