Olivier P. Haefliger

Real-Time Monitoring of Fragrance Release from Cotton Towels by Low Thermal Mass Gas Chromatography Using a Longitudinally Modulating Cryogenic System for Headspace Sampling and Injection

An innovative headspace sampling and injection system for gas chromatography was designed using a longitudinally modulating cryogenic system mounted around the sampling loop of a two-position loop injector. The setup was hyphenated to a fast low thermal mass gas chromatograph, allowing transient concentrations of semivolatile analytes to be monitored in real time with a time resolution of 4.5 min. The performance of the instrument, and in particular its cryotrapping efficiency, was characterized using a mixture of long-chain alkanes, methyl esters, ethyl esters, and alcohols of different volatilities. The device was found to be ideally suited to the analysis of semivolatile compounds with boiling points ranging between 190 and 320 degrees C, which are typical for a majority of perfumery raw materials. The new instrument was successfully used to monitor the release of eight odorant compounds from cotton towels to which fabric softener had been applied that alternatively contained the fragrance in free form or in microencapsulated form. The analytical results, unprecedented in their level of precision and time resolution for such an application, evidenced the major impact of microencapsulation technology on the kinetics of fragrance release during the drying of the towels and on the triggering of additional fragrance release by applying mechanical stress to the fabric to rupture the microcapsule walls.

Complexation/encapsulation of green tea polyphenols in mixed calcium carbonate and phosphate micro-particles

We used a double-jet mixer to encapsulate water-soluble polyphenols, green tea extract (GTE), with calcium-based inorganic materials. The device mixed calcium chloride solutions with a solution of carbonate and phosphate in the presence of a GTE solution, and formed micro-particles which capture the GTE molecules. The micro-particles were analysed by liquid chromatography coupled to tandem mass spectroscopy to determine the encapsulation yield and loading of the different GTE components. We established correlations between (1) the efficiency of the GTE encapsulation and the composition of the mixed anion solutions and (2) the protonation degree of the ions and the molar ratio of calcium cations and carbonate/phosphate anions. An optimal and reproducible GTE loading of about 40% with an encapsulation yield of 65% was observed for a carbonate/phosphate molar composition of 4 : 1. In addition, our experimental results showed that the process is selective and favours the encapsulation of gallated species which form stronger complexes with calcium cations.

Engineering cytochrome P450 BM3 of Bacillus megaterium for terminal oxidation of palmitic acid.

Directed evolution via iterative cycles of random and targeted mutagenesis was applied to the P450 domain of the subterminal fatty acid hydroxylase CYP102A1 of Bacillus megaterium to shift its regioselectivity towards the terminal position of palmitic acid. A powerful and versatile high throughput assay based on LC-MS allowed the simultaneous detection of primary and secondary oxidation products, which was instrumental for identifying variants with a strong preference for the terminal oxidation of palmitic acid. The best variants identified acquired up to 11 amino acid alterations. Substitutions at F87, I263, and A328, relatively close to the bound substrate based on available crystallographic information contributed significantly to the altered regioselectivity. However, non-obvious residues much more distant from the bound substrate showed surprising strong contributions to the increased selectivity for the terminal position of palmitic acid.

Release kinetics of actives from chewing gums into saliva monitored by direct analysis in real time mass spectrometry.

Direct analysis in real time mass spectrometry (DART-MS) was used to monitor the release kinetics of a taste-refreshing compound from chewing gums into the saliva of subjects. A new DART-MS sample probe was designed which was about four times more sensitive than the current benchmark probe. This decreased the impact of the dilution of the saliva samples that was required to minimize ion suppression effects and make quantitative analyses without an internal standard possible. The new probe was also about three times more reproducible, which allowed quantitative measurements to be conducted manually without requiring the enhanced precision provided by an automatic sample positioner. The accuracy of analyses performed by DART-MS was verified by comparing the results obtained from saliva samples analyzed both by DART-MS and by a more classical liquid chromatography/mass spectrometry (LC/MS) method. This investigation showed good agreement between the two techniques. DART-MS could then be used to objectively demonstrate the efficiency of a granular carbohydrate-based delivery system to boost for a few minutes the release of a lipophilic flavor raw material with a high octanol/water partition coefficient, cyclohexanecarboxamide, N-ethyl-5-methyl-2-(1-methylethyl) (WS-3), from chewing gum into saliva.

New sample preparation for quantitative laser desorption mass spectrometry and optical spectroscopy

Several analytical mass spectrometric and optical spectroscopic methods require a step during which a nonvolatile substance is desorbed by a laser pulse. It is, however, very difficult to use these methods for quantitative measurements because an accurate control over the amount desorbed by the laser pulse is generally not possible, especially when mixtures of several substances are used. We report a new fast and convenient sample preparation procedure that solves these problems. A solution of the analytes is mixed with a solution of poly(vinyl chloride) to obtain a homogeneous and vacuum-stable thin polymer membrane after the solvent has evaporated. Laser ablation is then performed directly from this membrane, allowing an accurate control of the amount of ablated analytes and excellent reproducibility. Quantitative laser desorption mass spectrometry over three orders of magnitude as well as optical spectroscopic measurements using this sample preparation method are demonstrated for polycyclic aromatic hy...

Selective Coprecipitation of Polyphenols in Bioactive/Inorganic Complexes

We investigate the formation of bioactive/inorganic coprecipitates of polyphenols catechins with calcium carbonate and calcium phosphate. Extracted from the leaves of Camellia sinensis, green tea catechins are efficient free radical scavengers, but their purported benefits from the perspective of prevention, health, and nutritional physiology are accompanied by unpleasant organoleptic characteristics: they are notoriously bitter. Selective complexation of polyphenols with metal salts is a possibility to mask or inactivate bitterness and/or off-flavors. We produce such complexes using a continuous coprecipitation process. With excess calcium chloride present in the matrix we observe a correlation of the carbonate to total anions molar ratio with the catechin load. To characterize the composition and structure of the coprecipitates we combine elemental analysis, scanning electron microscopy, X-ray powder diffraction, and liquid chromatography coupled to tandem mass spectroscopy (LC/MS-MS). We quantify the release kinetics in different model environments to predict the behavior of the catechins from the coprecipitates in model media simulating the conditions during oral ingestion and storage. The dissolution data suggest that the release profile of these delivery systems can be influenced and fine-tuned via the anion composition of the mineral carrier.

Stripping of aroma compounds during beer fermentation monitored in real-time using an automatic cryotrapping sampling system and fast gas chromatography/mass spectrometry

Improvements are reported for a recently described instrument made up of an innovative headspace sampling and injection system built using a longitudinally modulating cryogenic system mounted around the sampling loop of a two-position loop injector, which had been hyphenated to a low thermal mass gas chromatograph. Replacing the bare capillary used previously for cryotrapping by a coated capillary opened additional potential fields of application for the instrument since more volatile analytes, with boiling points above 140 °C instead of 190 °C, could now also be quantitatively cryotrapped. Detection using a mass spectrometer instead of a flame ionization detector enabled the investigation of dynamic processes that release complex mixtures of volatile compounds in widely varying amounts. The performance of the improved system was illustrated by monitoring in real time, with 5 min resolution, of the stripping of volatile aroma compounds by fermentative carbon dioxide during beer fermentation. Compounds derived from hops, including monoterpenes, sesquiterpenes and some esters, which are present in the initial wort but are not formed subsequently, were observed to be stripped off early in the fermentation process. Fusel alcohols are the earliest volatile compounds derived from yeast metabolism, which are found to be released in significant amounts from the wort. More than ten fermentation-derived esters were observed to be stripped from the wort later into the fermentation. Although similar, their release kinetics exhibited clear differences which were the consequences of their different biosynthetic production rates and physical–chemical properties. The release curves of further compounds and how they can lead to the elucidation of their origin are also discussed.