Open access peer-reviewed chapter
Abstract
Gas chromatography-mass spectrometry is a powerful tool to analyze flavor and fragrance from raw materials to the final commercial products. During the development of new technologies, most focuses have been given to novel columns, advanced detectors, and automation designs to leverage the instrument capabilities. The fundamental factors including polarity impact on sample homogenization and chemical interaction between analytes and extraction solvents are not equally emphasized during the sampling procedures. The current project focused on the liquid extraction procedures prior to GCMS analysis. Significant nucleophilic reactions were found to take place when a water-ethanol solvent was tried to extract flavor and fragrance ingredients. The isooctane in water-isooctane extraction system is friendly with GC columns and effective to extract the volatiles. However, the surfactants, humectants, and polymers in consumer cleaning products have significant impact on analyte distribution between water and isooctane solvents. The enhanced solubility of certain ingredients in water phase will change their profiling information in isooctane. During such extractions, hydrophilic volatile ingredients can be missed and the results become unreliable. For this reason, a newly designed water-n-propanol-isooctane extraction system was compared. This one-phase sample solution follows the homogenization rule in analytical chemistry and be more representative to the original samples.
Keywords
- liquid extraction
- gas chromatography
- mass spectrometry
- flavor
- fragrance
- volatiles
- consumer products
1. Introduction
Personal and oral care products that serve subtle human needs are tied inextricably to sensory values of taste, odor, and texture. Moreover, failure to meet flavor and fragrance expectations may often signal poor quality and could even be related to inherently subjective influence. For these commercial reasons, product manufacturers, distributors, buyers, wholesale and retail sellers, and especially consumers need reliable ways to assess product flavor and fragrance quality. From the perspective of a consumer product company, “a reliable assessment” of a chemical mixture—whether it is raw materials or final products—calls for the need to make a correct analytical measurement. A “reliable assessment” should be objective; even the term “flavor and fragrance” could often be inherently subjective [1].
Flavor and fragrance are typically consistent with volatile ingredients. The determination of volatile components in a mixture is a process widely used in many disciplines, such as flavor, fragrance, environmental, food, forensic, oil, pharmaceutical, and consumer product analysis [2, 3]. The method of choice for many of these analyses can be simply described as a sampling procedure plus instrumental analysis such as GCMS. Solvent extraction is the most common sampling step before the GCMS analysis is performed. During extraction, all volatile ingredients should be transferred and dissolved into extraction solvents. Qualitative and quantitative studies of chemical compounds from different matrices, such as plant materials, drugs, and consumer products, rely mostly on the selection of appropriate extraction methods [4, 5]. Extraction plays a significant and crucial role for the final outcome. Extraction methods are sometimes referred to as “sample preparation techniques.” Most of the time, this part of the study is neglected and done by non-trained research personnel [6, 7], despite two-thirds of the analytical chemist workforce accounting for sample preparation techniques. Most researchers believe in the importance of sample preparation during any analytical studies because the analytes could be missed or alternated without a suitable sampling procedure [8]. Even with very selective mass detectors such as Thermo orbitrap or time of flight, sample cleanup procedure is still a critical stage for the final analytical results [9]. It is true that the development of modern chromatographic and spectrometric techniques makes chemical compound analysis easier than before, but a successful method still heavily depends on sampling procedures, input parameters, and exact nature of materials [10]. High-resolution mass detectors can filter out a large part of sample matrix noise due to its mass selectivity and ensure more precise results. However, the detector cannot guarantee the high accuracy if certain ingredients are not fully extracted and transferred into the instrument
Common factors affecting extraction processes are sample matrix property, solvent selection, operation temperature, pressure, and time [11]. Those common factors could cause the chemical interaction between extraction solvents and chemical composition to be analyzed. The chemical interaction could not only cause chemical structure changes such as chemical reaction but also alternate a substance solubility, precipitation, solvation, complexation, and dissociation [12]. For cleaning consumer products such as toothpaste, the matrix usually contains a significant amount of silica base, humectants such as glycerin or sorbitol and polymers such as polyethylene glycol (PEG) and xanthan gum, and certain amount of surfactants. Silica usually has poor solvent solubility, and polymers usually contain both hydrophobic and hydrophilic segments inside their structures. That is why these products cannot be evenly suspended with organic solvents or even very polar alcohols such as methanol or ethanol. Water is necessary to suspend the toothpaste matrix and provide a homogenized sampling procedure. For this reason, we often use common extraction solvents such as methanol or ethanol with water to directly extract analytes from oral and personal care products for GCMS analysis [13]. In addition, highly hydrophobic organic solvents such as isooctane (2,2,4-trimethylpentane) were used to extract flavors and fragrance from the water phase with a fractionation operation. Those two methods have been alternatively used for the quantification of flavor and fragrance based on their polarity difference. However, those two methods faced the challenges during flavor and fragrance profiling projects for quality evaluation. Some existing ingredients were missing in GCMS chromatograms due to two extraction phases and ingredient distribution impacted by the sample matrix. The chemical interactions between flavor and fragrance ingredients and extraction solvents can change flavor and fragrance profiles during their chemical analysis. To overcome those challenges, a water organic miscible solvent system was developed and used for flavor and fragrance extraction in cleaning products such as toothpaste. In this study, three extraction procedures were explored and compared. Their positive and negative attributes were discussed. A general extraction summary is provided at the end.
2. Materials and methods
2.1 Chemical reagents and materials
Isooctane (2, 2, 4-Trimethylpentane, C8H18) was purchased from VWR (Radnor, PA, USA). Anhydrous sodium sulfate (Na2SO4) was purchased from Sigma-Aldrich (MilliporeSigma, St Louis, MO, USA). n-propanol (n-propyl alcohol, C3H8O) was purchased from Honeywell (Charlotte, NC, USA). Wintergreen oil (CAS#: 90045–28-6), lemongrass oil (CAS#: 8007-02-1), and peppermint oil (CAS#: 8006-90-4) were purchased from MilliPoreSigma). PTFE membrane filter was obtained from VWR.
2.2 Instrumentation
A Genie 2 vortex mixer from MilliPoreSigma was used to assist sample dispersion in a minimal amount of time. The Eppendorf centrifuge 5810R from MilliPoreSigma was used to centrifuge the toothpaste sample for 10 min at 5000 g before the GCMS analysis.
A gas chromatography system 6890 N combined with 5975 mass spectrometry detector (MSD) from Agilent Technology (Santa Clara, CA, USA) plus multiple-Purpose-Sampler (MPS2) from Gerstel (Linthicum, MD, USA) was used for flavor analysis. Separation was accomplished using the GC column HP-5MS (30 m x 0.25 mm x 0.25 μm, length x inside diameter x film thickness, Agilent Technologies). A 1 μL sample was injected in the splitless mode. The oven temperature was initially held at 80°C for 1 min. Thereafter, the temperature was raised at 4°C/min until 150°C and held for 1.5 min. Total running time is 20 min. Helium was used as the carrier gas and delivered at 1 mL/min constant flow rate. The gas pressure and velocity were at 8.2 psi and 37 cm/sec, respectively. The injector temperature was set at 250°C, and the interface temperature between GC oven and MS detector chamber was 250°C. The MS detectors were tuned with the standard spectrum auto-tune, and the MS data for total ion chromatogram (TIC) were acquired in the full scan mode (m/z of 29–450 at a scan rate of 3 scan/sec using electron ionization (EI) with electron energy of 70 eV. The MS source and quat temperatures were 230°C and 150°C, respectively.
2.3 Sampling procedure with ethanol-water system
2.3.1 Standard preparation using ethanol-water solvents
About 100 mg of flavor oil was added into 49.9 grams of a 1:8 water-ethanol solution. Total weight of the solution was 50.0 grams. Three flavor stock solutions (2000 ppm) from wintergreen, lemongrass, and peppermint oils were used as reference standards in this project. The stock solution was then diluted 20-fold with the 1:8 water-ethanol solution, reaching target concentrations of 100 ppm. This standard solution was transferred into a 2-mL autosampler vials for GCMS analysis.
2.3.2 Sample preparation using ethanol-water solvents
About 0.5 gram of toothpaste sample was weighed into a 50-mL polypropylene conical tube. The weight of 3.2 gram of water was added into the tube and mixed with toothpaste by using a Genie 2 vortex mixer into slurry. Next, a weight of 21.3 gram of ethanol was added to the tube. The solution was then mixed thoroughly with a Genie 2 vortex mixer, and the mixture was centrifuged by using Eppendorf centrifuge 5810R for 10 min at 5000 g. The top layer of solution was filtrated with a 0.2 μm PTFE membrane and transferred into a 2-mL autosampler vial for GCMS analysis.
2.4 Sampling procedure using isooctane with saturated sodium sulfate water solution
2.4.1 Preparation of a saturated sodium sulfate solution
About 40.0 gram of anhydrous sodium sulfate salt was transferred into a 250-mL Erlenmeyer flask with 100 mL of distilled water and mixed thoroughly with a magnetic stirring bar. After excess sodium sulfate started to rest at the bottom of the container, the upper clear saturated solution was carefully transferred to another clean glass jar for use.
2.4.2 Standard preparation using isooctane solvent
About 100 mg of flavor oil was added into 49.9 gram of isooctane. The total weight of the solution was 50.0 gram. Three flavor stock solutions (2000 ppm) from wintergreen, lemongrass, and peppermint oils were used as reference standards. Then, the stock solution was diluted 20-fold with isooctane solvent, reaching target concentrations of 100 ppm. This standard solution was transferred into 2-mL autosampler vials for GCMS analysis.
2.4.3 Sample preparation using isooctane with saturated sodium sulfate water solvents
About 0.5 gram of toothpaste sample was weighed into a 50 mL polypropylene conical tube. In total, 24.5 gram of saturated sodium sulfate water solution was added into the tube and toothpaste was mixed and suspended with a Genie 2 vortex mixer into the slurry. About 12.5 gram such aqueous toothpaste solution was mixed with 12.5 gram of isooctane solvent. This combination was then mixed thoroughly with the Genie 2 vortex mixer. The mixture was centrifuged by using Eppendorf centrifuge 5810R for 10 min at 5000 g. The top clear solution of isooctane was transferred into a 2-mL autosampler vial for GCMS analysis.
2.5 Sampling procedure with water-n-propanol-isooctane (1:8.5:15) solvent system
2.5.1 Standard preparation using water-n-propanol-isooctane (1:8.5:15) as solvent
About 100 mg of flavor oil was added into 49.9 gram of water-n-propanol-isooctane solvents. Total weight of the solution was 50 gram. Three flavor stock solutions (2000 ppm) from wintergreen, lemongrass, and peppermint oils were used as reference standards. The stock solution was then diluted 20-fold with water-n-propanol-isooctane solvents, reaching target concentrations of 100 ppm. This standard solution was transferred into 2-mL autosampler vials for GCMS analysis.
2.5.2 Sample preparation using water-n-propanol-isooctane (1:8.5:15) as solvent
About 0.5 gram of toothpaste sample was weighed into a 50-mL polypropylene conical tube. About 1.0 gram of water was added into the tube and toothpaste was mixed and suspended with the Genie 2 vortex mixer into the slurry. Next, 8.5 gram of n-propanol solvent was added. The mixture was then mixed thoroughly with the Genie 2 vortex mixer again. Lastly, 15.0 gram of isooctane was added. The combination was then blended thoroughly with Genie 2 vortex, and the mixture was centrifuged by using the Eppendorf centrifuge 5810R for 10 min at 5000 g. The top solution was filtrated with a 0.2 μm PTFE membrane and transferred into a 2-mL autosampler vial for GCMS analysis.
3. Results and discussion
3.1 Sampling procedure with ethanol-water system
Due to the specific properties of cleaning products we described in the introduction section, aqueous methanol and ethanol are very common liquid extraction solvent systems used for toothpaste sampling procedures [13, 14]. Water can easily suspend toothpaste into the slurry and organic solvents can effectively extract most analytes. The ratio between organic solvent and water can further determine the polarity of the analyte to be extracted. Because most flavor ingredients are hydrophobic, we used a high ratio of organic solvent at 1:8 ratio of water:ethanol to extract toothpaste flavors. The extraction efficiency and recovery were close to 100% as predicted because ethanol not only has good solubility for most flavor ingredients but also possesses penetration capabilities into toothpaste matrix materials such as silica and polymers. However, the hydroxyl group in ethanol structure has a very strong nucleophilic attaching capability, which can easily react with the compounds having carboxyl groups including esters. When quantification of methyl salicylate in wintergreen flavor and related products was tried with this extraction system, ethyl salicylate was identified within 30 min after sampling shown in Figure 1. From 30 min, 6 hours to overnight time periods, methyl salicylate continuously decreased and ethyl salicylate correspondingly increased, which is demonstrated well in Figure 1. In addition, when quantification of lemongrass flavor oil was tried with this same procedure, a new citral diethyl acetal peak at retention time of 15 min was observed and identified 60 min after sampling shown in Figure 2. Those reacted products indicated that the quantity of original flavor ingredients can be changed due to the reaction with extraction solvents. The final results on the GCMS instruments then become unreliable. Specifically, if an integrated flavor profile is needed to evaluate the flavor quality, the aqueous methanol and ethanol systems could become challenging.
Figure 1.
The GCMS chromatogram of wintergreen flavor extracted by using a water-ethanol solvent system. The peaks of menthol and anethole at the retention times around 10.87 and 14.25 min displayed similar ion count intensity. The methyl salicylate peak intensity at 11.54 min was continuously reduced from brown, green and purple to black after sample extraction, and ethyl salicylate peak intensity at 13.82 min was continuously increased from brown, green, and purple to black. The structure change is demonstrated inside figure.
Figure 2.
The GCMS chromatogram of lemongrass flavor extracted by using water-ethanol solvent system. The citral peak intensity at 11.37 min was reduced from green to red after extraction. A new peak at 15.01 min was observed and identified as citral diethyl acetal 60 min after extraction. Both citral and citral diethyl acetal are shown in figure.
3.2 Sampling procedure using isooctane with sodium sulfate saturated aqueous solution fractionation
Most volatile ingredients are hydrophobic or with a middle range of polarity. Usually, they can be well dissolved into hydrophobic solvents such as isooctane. To enhance the extraction efficacy, dispersive liquid-liquid microextraction methods have been reviewed [15]. The emulsion formed between the organic solvent and water can increase the contact surface and enhance the extraction efficiency. Many reports [16, 17, 18, 19, 20] have found that the addition of a water-soluble inorganic salt can aid in sufficient dispersion of extraction solvent at microliter level into aqueous phase. Specifically, Na2SO4 could enhance the formation of the emulsion between aqueous sample solution and organic solvent [17]. Many studies discovered that a vortex-assisted mixing method helped disperse emulsion and improved extraction efficiency [21, 22, 23]. Therefore, an isooctane-water system is commonly used to extract flavor from the toothpaste. Water with Na2SO4 was used to disperse and suspend toothpaste matrix into loss slurry at first, and then, isooctane was applied to extract the flavor ingredients. After vortex-assisted mixing, centrifuging at 5000 rpm for 10 min was performed to collect the top isooctane phase for GCMS analysis of flavor ingredients. Most flavor ingredients can be successfully extracted out in this manner, and this method was successful in quantifying menthol and methyl salicylate in the corresponding flavor oils and related products with good recovery and reproducibility (data are not shown here). The advantage of this extraction system is there is no water in the injection solvent, which should improve column life based on the traditional GC column stability consideration. Most modern GC columns except wax and free fatty acid phase (FFAP) columns have significantly improved tolerance to water except when strong acid or base is present in injection liquid. But the presence of water in the injected solvents still exhibited more column bleeding at elevated temperatures. One big disadvantage of this method is that two liquid phases were involved in the sampling procedures, which could be against the sample homogenization rule in analytical chemistry. The situation is even worse when a full integrated flavor profile is required to evaluate the flavor quality. The flavor ingredients could be unevenly partitioned between those two phases. If only the organic phase is used for injection, this uneven distribution in the two separated liquid phases could twist and change the final analytical results. This phenomenon could be very significant for cleaning products such as toothpastes. Common toothpastes contain about 30% of either glycerol or sorbitol as humectant. Those polyalcohols will mainly stay with water, and they can dramatically enhance the flavor solubility in the water phase and significantly change the flavor ingredient distribution between water and isooctane phases [24]. Polyalcohol impact on flavor distribution during the two-phase fractionation may not be obvious for nonpolar flavor ingredients because they can be well dissolved in hydrophobic isooctane solvent. That is why the quantitation of menthol and methyl salicylate can exhibit a good accuracy and precision result after flavor ingredients were formulated with toothpaste matrix. However, if a full flavor ingredient profile needs to be analyzed to evaluate the flavor quality, this extract system could bring some errors for the final results. Here is an example. When the current procedure was compared with the method described in the Section 3.3 below, the results of toothpaste flavor are shown in Figure 3. The two peaks at the retention times of 3.43 and 5.59 min are missing when the current two-phase, water-isooctane, extraction method was applied. Those two peaks were identified as acetic and propanoic acids by using the NIST library and chemical standard. They are the flavors corresponding to sour and cheese characters and commonly used in toothpaste [25]. Those two flavor ingredients are very critical to flavor quality evaluation. Due to their polarity and high content of humectants such as glycerol or sorbitol in the water phase, they will stay in the water phase and cannot be injected into the GCMS instrument.
Figure 3.
The GCMS chromatographic flavor profiles from a commercial toothpaste product. The compounds were identified by mass spectra, NIST library, and reference standards. The green chromatogram is extracted by using one phase of water-n-propanol-isooctane (1:8.5:15) solvent system, and the red one is extracted by using two phases of water-isooctane solvent system with fractionation separation.
3.3 Sampling procedure with water-n-propanol-isooctane (1:8.5:15) solvent system
To avoid the disadvantage of water-isooctane two-phase application, a new extraction system composed of water-n-propanol-isooctane (1:8.5:15) was explored for flavor ingredient extraction from toothpaste matrix. This single-phase system mimics toothpaste polarity and allows flavor ingredients to be equitably transferred from high viscous paste to light clear extraction solvents. A minimum amount of water was applied to disperse and suspend the toothpaste matrix, and n-propanol was used not only for further dispersion of toothpaste matrix but also to enhance the water solubility in isooctane to avoid phase separation and constitute a one-phase extraction solvent system. Isooctane is a major extraction solvent to get flavor ingredients from the toothpaste matrix. This system was well aligned with the homogenization rule during sampling procedures, which enabled high confidence in detecting all the flavors on the GCMS instrument analysis. By using this ratio of three solvent compositions to mix different commercial toothpaste products, no phase separation was observed and sampling homogenization was achieved during vortex-assisted mixing. This system can dissolve and extract not only hydrophobic flavor ingredients but also hydrophilic flavor ingredients including acetic and propanoic acids, which is demonstrated in Figure 3 and described in Section 3.2. For a different consumer cleaning product, the ratio of three solvents would need to be slightly adjusted to make sure no phase separation and dominant isooctane is present.
4. Conclusion
This study explored three liquid extraction systems for sampling flavor ingredients from toothpaste products. They are representative liquid sampling procedures to most consumer cleaning products. The first extraction system is the simplest and most eco-friendly13. Due to high water activity and nucleophilic attacking capability from the hydroxyl group in ethanol, nucleophilic substitution reaction of ethoxyl from methoxyl group in methyl salicylate compounds and nucleophilic addition reaction from citral to citral diethyl acetal were observed after extraction procedures. In the second system, water-isooctane solvent mixture with two-phase fraction provided a cleaner extraction solution for the GCMS instrumental analysis, especially for hydrophobic flavor ingredient quantitation. However, the ingredient distribution in two phases could be against the homogenization rule to present the holistic sample profiles. Specifically, the high contents of polyalchols such as glycerol and sorbitol present in toothpaste products could prevent polar flavor ingredients such as acetic and propanoic acids from isooctane phase. Therefore, this solvent extraction system is not ideal for flavor profile analysis, and the results could be misleading for flavor quality evaluation. The third liquid extraction system with small amounts of water, suitable amount of n-propanol and large amounts of isooctane provided a one-phase extraction media after mixing with the toothpaste products, which can better satisfy the sampling homogenization rule in the analytical chemistry. This system is not only suitable for flavor ingredient quantification but also can be used for toothpaste product flavor profiling analysis.
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