Mass Spectrometry: An Essential Tool for the Flavor and Fragrance Industry.

Over the past decades, mass spectrometers have become standard instruments in the analytical laboratories of the flavor and fragrance (F&F) industry. As the expansion of the fields of use of flavors and fragrances and the launch of numerous new technologies designed to enhance their performance have led to a diversification and increased complexity of the questions asked to the analytical chemists, mass spectrometry remains an essential tool to address them. The capabilities of the latest generation of  instruments combined with advanced molecular separation and data processing tools allow to tackle many of these challenges. Through different examples, and focusing on the period that followed our 2014 review [1], this article illustrates different ways MS is contributing to the discovery development and commercialization of F&F ingredients and technologies, for both volatile and non-volatile molecules.

Quantification of Residual Perfume by Py-GC-MS in Fragrance Encapsulate Polymeric Materials Intended for Biodegradation Tests.

Perfume encapsulates are widely used in commercial products to control the kinetic release of odorant molecules, increase storage stability and/or improve deposition on different substrates. In most of the cases, they consist of core-shell polymeric microcapsules that contain fragrance molecules. A current challenge is to design and produce polymeric materials for encapsulation that are both resistant and non-persistent. The selection of such eco-friendly formulations is linked to a deep understanding of the polymeric material used for encapsulation and its biodegradation profile. To collect this information, pure samples of capsule shells are needed. In this article we present an innovative quantification method for residual volatiles based on pyrolysis-GC-MS to enable validation of sample quality prior to further testing. The presented analytical method also led to the development of a robust and comprehensive purification protocol for polymers from commercial samples. Standard techniques are not suited for this kind of measurement due to the non-covalent embedding of volatiles in the 3D structure of the polymers. We demonstrated the confounding impact of residual volatiles on the estimated biodegradability of fragrance encapsulates.

Intestinal Fish Cell Barrier Model to Assess Transfer of Organic Chemicals in Vitro: An Experimental and Computational Study.

We studied the role of the fish intestine as a barrier for organic chemicals using the epithelial barrier model built on the rainbow trout (Oncorhynchus mykiss) intestinal cell line, RTgutGC and the newly developed exposure chamber, TransFEr, specifically designed to work with hydrophobic and volatile chemicals. Testing 11 chemicals with a range of physicochemical properties (logKOW: 2.2 to 6.3, logHLC: 6.1 to 2.3) and combining the data with a mechanistic kinetic model enabled the determination of dominant processes underlying the transfer experiments and the derivation of robust transfer rates. Against the current assumption in chemical uptake modeling, chemical transfer did not strictly depend on the logKOW but resulted from chemical-specific intracellular accumulation and biotransformation combined with paracellular and active transport. Modeling also identified that conducting elaborate measurements of the plastic parts, including the polystyrene insert and the PET filter, is unnecessary and that stirring in the TransFEr chamber reduced the stagnant water layers compared to theoretical predictions. Aside from providing insights into chemical uptake via the intestinal epithelium, this system can easily be transferred to other cell-based barrier systems, such as the fish gill or mammalian intestinal models and may improve in vitro-in vivo extrapolation and prediction of chemical bioaccumulation into organisms.

Country-Specific Environmental Risks of Fragrance Encapsulates Used in Laundry Care Products.

Fragrance encapsulates (FEs) are designed to deliver fragrance components, notably in laundry care products. They are made of thermoset polymeric shells surrounding the fragrance content. These materials enter the environment mainly during laundry washing, but little is known about their distribution in and impact on the environment. The aim of the present study was to estimate the environmental concentrations of FE shells in freshwater, sediment, and soil compartments for 34 selected countries and to compare them with ecotoxicological effects. Probabilistic material flow analysis was used to estimate worst-case predicted environmental concentrations (PECs). The lowest freshwater PEC was predicted for Finland (0.00011 µg/L) and the highest for Belgium (0.13 µg/L). Accumulation of FE shells between 2010 and 2019 was considered for sediments and sludge-treated soils. The PECs in sediments ranged from 3.0 µg/kg (Finland) to 3400 µg/kg (Belgium). For sludge-treated soil, the concentration was estimated to be between 0 (Malta and Switzerland) and 3600 µg/kg (Vietnam). Ecotoxicological tests showed no effects for FE shells at any tested concentration (up to 2700 µg/L freshwater, 5400 µg/kg sediment, and 9100 µg/kg soil), thus not allowing derivation of a predicted-no-effect concentration (PNEC). Therefore, to characterize the environmental risks, the PEC values were compared with highest-observed-no-effect concentrations (HONECs) derived from ecotoxicological tests. The PEC/HONEC ratios were 9.3 × 10-6 , 0.13, and 0.04 for surface waters, sediments, and sludge-treated soils, respectively, which are much below 1, suggesting no environmental risk. Because the PEC values constitute an upper boundary (no fate considered) and the HONEC values represent a lower boundary (actual PNEC values based on NOECs will be higher), the current risk estimation can be considered a precautionary worst-case assessment. Environ Toxicol Chem 2022;41:905-916. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Investigations On the Fish Acute Toxicity of Fragrance Ingredients Involving Chinese Fish Species and Zebrafish Embryos.

While zebrafish (Danio rerio) have been accepted worldwide for evaluating chemical hazards to aquatic vertebrates, and in some countries it is mandated to generate fish toxicity data using native species, such as Chinese rare minnow (Gobiocypris rarus) in China. This represents an additional regulatory constraint that may cause redundant tests, additional animal uses, and higher costs. Previous studies showed that juvenile G. rarus was more sensitive than zebrafish juveniles and embryos to metals. To better understand the sensitivity of G. rarus to organic chemicals, we selected 29 fragrance ingredients belonging to various chemical classes and with differing physicochemical properties, for which good quality zebrafish acute toxicity data were available and tested them with juvenile G. rarus and embryo D. rerio using the Organisation of Economic Co-operation and Development test guidelines. Chemical toxicity distribution (CTD) and chemical ratio distribution (CRD) models were established to systematically compare the sensitivity between juveniles of G. rarus and D. rerio, as well as between D. rerio embryos and juveniles. The results of the CTD models showed that for tested chemicals, the sensitivity of juvenile G. rarus was similar to that of D. rerio juveniles and embryos. The CRD comparisons revealed that juvenile G. rarus was slightly less sensitive by a factor of ~2 than juvenile D. rerio to ingredients belonging to Verhaar class 3 and Ecological Structure Activity Relationship ester class, while comparable to other chemicals. These comparative experiments demonstrated that fish toxicity data with G. rarus can be submitted for use in chemical registrations outside China, which would avoid repeating animal tests using D. rerio. Meanwhile, the similar sensitivity of zebrafish juveniles and embryos to fragrance ingredients confirmed the suitability of replacing juveniles by zebrafish embryos. Environ Toxicol Chem 2022;41:2305-2317. © 2022 SETAC.

Extraction, thermal cleanup, and GC/MS quantification with disposable solid extractants: application to hydrophobic analytes in aqueous surfactant solutions.

Fragranced consumer products are generally formulated together with surfactants. In application, these products are often highly diluted with water. Analyzing trace amounts of fragrance ingredients in such mixtures is challenging and usually requires either time-consuming sample cleanup or extensive cleaning of the trapping device to avoid memory effect and cross-contamination between samples. To overcome these limitations, a new disposable extraction device has been developed to be used in combination with a thermodesorption-GC-MS unit. Made of PDMS foam cylinders, it efficiently extracts trace amounts of hydrophobic compounds from complex aqueous solutions and provides an online sample cleanup, thanks to the controlled desorption temperature, which allows retaining the low volatile constituents of the matrix within the absorptive foam. Combined with a stable isotope dilution assay, accurate quantifications of Cetalox(®), Muscenone™, Helvetolide(®), Polysantol(®), Dartanol, and Myrrhone(®) from aqueous solution containing surfactant and from water from the aeration tank of a sewage plant were successfully conducted. LOQ varied between 1 and 25 ppb (20% confidence interval, α = 0.1).

In Vitro Biotransformation Assays Using Liver S9 Fractions and Hepatocytes from Rainbow Trout (Oncorhynchus mykiss): Overcoming Challenges with Difficult to Test Fragrance Chemicals.

In vitro metabolic stability assays using rainbow trout (Oncorhynchus mykiss) isolated hepatocytes (RT-HEP) or hepatic S9 fractions (RT-S9) were introduced to provide biotransformation rate data for the assessment of chemical bioaccumulation in fish. The present study explored the suitability of the RT-HEP and RT-S9 assays for difficult test chemicals, and the in vitro-based predictions were compared to in silico-based predictions and in vivo-measured bioconcentration factors (BCFs). The results show that volatile or reactive chemicals can be tested with minor modifications of the in vitro protocols. For hydrophobic chemicals, a passive dosing technique was developed. Finally, a design-of-experiment approach was used to identify optimal in vitro assay conditions. The modified assay protocols were applied to 10 fragrances with diverse physicochemical properties. The in vitro intrinsic clearance rates were higher in the S9 than in the hepatocyte assay, but the in vitro-in vivo (IVIV) predictions were comparable between the 2 assays. The IVIV predictions classified the test chemicals as nonbioaccumulative (BCF < 2000), which was in agreement with the in vivo data but in contrast to the in silico-based predictions. The findings from the present study provide strong evidence that the RT-HEP and RT-S9 assays can provide reliable estimates of in vivo biotransformation rates for test chemicals with difficult physicochemical properties. Environ Toxicol Chem 2020;39:2396-2408. © 2020 SETAC.

Extending the concept of predicting fish acute toxicity in vitro to the intestinal cell line RTgutGC.

Testing chemicals for fish acute toxicity is a legal requirement in many countries as part of environmental risk assessment. To reduce the numbers of fish used, substantial efforts have been focussed on alternative approaches. Prominently, the cell viability assay with the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, has been recognized, owing to its high predictive power and robustness. Like gills, the intestine is considered a major site of chemical uptake and biotransformation but, in contrast to gills, is expected to be exposed to rather hydrophobic chemicals, which enter the fish via food. In the present study, we therefore aimed to extend the cell bioassay to the rainbow trout epithelial cell line from intestine, RTgutGC. Using 16 hydrophobic and volatile chemicals from the fragrance palette, we showed that also the RTgutGC cell line can be used to predict fish acute toxicity of chemicals and yields intra-laboratory variability in line with other bioassays. By comparing the RTgutGC toxicity to a study employing the RTgill-W1 assay on the same group of chemicals, a fragrance specific relationship was established which reflects an almost perfect 1:1 relationship between in vitro and in vivo toxicity results. Thus, both cell lines can be used to predict fish acute toxicity, either by using the obtained in vivo-in vitro relationship or by taking the in vitro results at face value. We moreover demonstrate the derivation of non-toxic concentrations for downstream applications which rely on a healthy cell state, such as the assessment of biotransformation or chemical transfer.

Effects of variation in modulator temperature during cryogenic modulation in comprehensive two-dimensional gas chromatography.

Many modulation systems in comprehensive 2D GC (GC x GC) are based on cryogenic methods. High trapping temperatures in these systems can result in ineffective trapping of the more volatile compounds, whilst temperatures that are too low can prevent efficient remobilisation of some compounds. To better understand the trapping and release of compounds over a wide range of volatilities, we have investigated a number of different constant temperature modulator settings, and have also examined a constant temperature differential between the cryo-trap and the chromatographic oven. These investigations have led us to modify the temperature regulation capabilities of the longitudinally modulated cryogenic system (LMCS). In contrast to the current system, where the user sets a constant temperature for the cooling chamber, the user now sets the temperature difference between the cryo-trap and the chromatographic oven. In this configuration, the cooling chamber temperature increases during the chromatographic run, tracking the oven temperature ramp. This produces more efficient, volatility-dependent modulation, and increases the range of volatile compounds that can be analysed under optimal trap-and-release conditions within a single analytical run. This system also reduces cryogenic fluid consumption.

From the Representativeness of Sampled Odors to an Olfactive Camera.

Although representativeness is often a prerequisite when sampling odors, the methods used have never been assessed from the analytical and sensory perspective simultaneously. We validate several critical innovations in the methods used to sample odors, starting with a previously developed static-and-trapped headspace (S&T-HS) cell, to minimize sorptive biases and allow for thermodesorption of trapped odors. The addition of a desorption oven allows for restoration and testing of odors sampled by not only S&T-HS but also other techniques (solid-phase microextraction, headspace sorptive extraction, purge-and-trap headspace). The S&T-HS cell exhibits satisfactory representativeness, much higher than the three other techniques. This allows, for the first time, a proposal to use this technology as an olfactive camera to capture and restore an odor. The method was tested on a sample of a complex fresh ashtray odor.

Chiral multidimensional gas chromatography (MDGC) and chiral GC-olfactometry with a double-cool-strand interface: application to malodors.

Volatile sulfur compounds such as 3-methyl-3-sulfanylhexan-1-ol (1) are largely responsible for axillary-sweat malodors. In this work, we describe the determination of the enantiomer ratio of the trace constituent 1 and the odor description of its antipodes (R)- and (S)-1 by means of multidimensional gas chromatography (MDGC) in combination with chiral gas chromatography-olfactometry (GC-O). This technique allowed the on-line evaluation of the sensory character of both enantiomers via a sniffing port, and is based on a novel double-cool-strand interface (DCSI). First, the system's inertness was tested towards the labile compound 2-methylfuran-3-thiol (MFT; 2). Then, the DCSI was used in a new configuration to achieve olfactive characterization by means of chiral GC-O. In contrast to direct smelling after the chiral column, our technique allows, for the first time, to significantly delay the perception of the second-eluting enantiomer after the first one. This lowers the risk of sensory saturation, as the panelist can recover from the first stimulus, before evaluating the second one. To help programming the DCSI, a dedicated program was set up. The enantiomer ratio of the sweat malodor 1 was determined as (S)/(R) 3 : 1, and the dominating (S)-isomer was shown to largely impart its specific character to the overall odor of the sweat extract.

Good quantification practices of flavours and fragrances by mass spectrometry.

Over the past 15 years, chromatographic techniques with mass spectrometric detection have been increasingly used to monitor the rapidly expanded list of regulated flavour and fragrance ingredients. This trend entails a need for good quantification practices suitable for complex media, especially for multi-analytes. In this article, we present experimental precautions needed to perform the analyses and ways to process the data according to the most recent approaches. This notably includes the identification of analytes during their quantification and method validation, when applied to real matrices, based on accuracy profiles. A brief survey of application studies based on such practices is given.This article is part of the themed issue 'Quantitative mass spectrometry'.

Limits of rapid log P determination methods for highly lipophilic and flexible compounds.

Lipophilicity is of crucial importance in many fields including pharmaceutical, environmental, cosmetic and food industries. Whereas different experimental strategies have been developed for rapid lipophilicity determination of new chemical entities, log P determination of highly lipophilic compounds is always challenging. In this study, three published chromatographic methods have been compared on a series of phenylalkanoic acids including the pro-perfume HaloscentD (HD-C12). Different log P values were obtained depending on the chromatographic method used for log P estimation. Molecular modelling suggested that log P variations may be due to the chromatographic conditions applied (isocratic or gradient mode, ratio methanol/water in the mobile phase), responsible of specific conformations of the molecule in solution. Thus, for flexible compounds, published methods have to be used with caution and considered as a good tool to estimate a log P range, depending on the molecular conformational state.

Multidimensional gas chromatography using a double cool-strand interface.

Commercial interfaces for multidimensional gas chromatography (MDGC) are based either on a valve or a pneumatic switching between columns. Both exhibit significant drawbacks and only few suppliers exist. An extremely simple interface has been set up to overcome these limitations without requiring any pneumatic control or valves switching. This new MDGC design is based on the cryo-control of the analyte transfer from the first to the second column through two cool strands of a capillary. This technique is simple to implement and does not require any special column connections. Applications involve non-polar/polar phase combinations, as well as chiral analysis, hyphenation to a conventional mass spectrometer, and olfactometric detection. In contrast to conventional MDGC configuration, the present configuration allows the use of a single oven to operate both columns at different temperatures.

Neuronal pattern separation in the olfactory bulb improves odor discrimination learning.

Neuronal pattern separation is thought to enable the brain to disambiguate sensory stimuli with overlapping features, thereby extracting valuable information. In the olfactory system, it remains unknown whether pattern separation acts as a driving force for sensory discrimination and the learning thereof. We found that overlapping odor-evoked input patterns to the mouse olfactory bulb (OB) were dynamically reformatted in the network on the timescale of a single breath, giving rise to separated patterns of activity in an ensemble of output neurons, mitral/tufted (M/T) cells. Notably, the extent of pattern separation in M/T assemblies predicted behavioral discrimination performance during the learning phase. Furthermore, exciting or inhibiting GABAergic OB interneurons, using optogenetics or pharmacogenetics, altered pattern separation and thereby odor discrimination learning in a bidirectional way. In conclusion, we propose that the OB network can act as a pattern separator facilitating olfactory stimulus distinction, a process that is sculpted by synaptic inhibition.

Analysis of flavor and perfume using an internally cooled coated fiber device.

A miniaturized internally cooled coated fiber device was applied for the analysis of flavors and fragrances from various matrices. Its integration with a CTC CombiPAL autosampler enabled high throughput for the analysis of analytes in complex matrices that required simultaneous heating of the matrices and cooling of the fiber coating to achieve high extraction efficiency. It was found that up to ten times increase of extraction efficiencies was observed when the device was used to extract flavor compounds in water, even when limited sample temperatures were used to preserve the integrity of target compounds. The extraction of the flavor compounds in water with the device was reproducible, with RSD not larger than 15%. The lower limits of the linear ranges were in the low ppb range, which was about one order of magnitude smaller than those obtained with the commercialized 100 microm PDMS fibers. Exhaustive extraction of some perfume ingredients from a complex matrix (shampoo) was realized. All achieved recoveries were not less than 80%. The repeatability of the extraction of the perfume compounds from shampoo was better than 10%. The linear ranges were about 1-3000 microg/g, and the LOD was about 0.2-1 microg/g. The automated internally cooled coated fiber device was demonstrated to be a powerful sample preparation tool in flavor and fragrance analysis.

Standard gas addition: a calibration method for handling temporal drifts of mass spectrometry-based sensors.

This work describes a new method for the correction of signal drift of a MS-based sensor by standard gas addition (SGA). It consists of introducing a gaseous He-Xe mixture continuously and independently of the carrier gas into the mass spectrometer source. To mimic the disturbances generated by periodical tuning of the mass detector, three series of adjustments were made to the main acquisition parameters. The influence of the SGA signal correction on the discriminating power of the data was evaluated from the analysis of three batches of cheeses by dynamic headspace coupled to mass spectrometry. SGA afforded a good correction of the main types of drift classically observed in mass spectrometry.