Plant metabolomics requires that as many metabolites as possible are extracted to obtain a reliable picture of the sample under study. Therefore, sample preparation plays a crucial role. In recent years, several efforts have been made to improve the environmental friendliness of sample preparation, including in the plant sector. However, the environmental friendliness of a method cannot be evaluated without also considering its productivity and, more importantly, its analytical performance to ensure not only environmental friendliness but also the “sustainability” of sample preparation approaches.

Plants and natural products are rich sources of a variety of bioactive metabolites that can be used for several applications, such as food and health. Their analyses need to cover a wide range of topics, from metabolomics studies to quality and safety controls. In particular, metabolomics studies for medicinal plants are growing rapidly and aim, for example, to identify new bioactive compounds, determine balsam time, monitor plant quality, and correlate the chemical composition of a natural product with its quality. Despite the remarkable technological advances made in recent years, the chemical diversity of primary and secondary metabolites of complex natural products still poses challenges for analysis. They affect all analytical steps from sample collection and sample preparation to analysis and data processing (1).

The main challenges in the analysis of natural products are related to the complexity of the plant metabolome, which, in addition to the abundant primary metabolites, is characterized by the presence of hundreds of specialized (secondary) metabolites belonging to different chemical classes, which are often present in very different amounts and, in some cases, are susceptible to degradation. Some of these compounds (such as chlorophylls and other pigments in photosynthetic tissues) could also interfere with extraction or analysis of target compounds. It is also important to remember that the samples are mainly solids and that plant cells are protected by thick and robust lignocellulosic walls that should be disrupted if efficient extraction of intracellular metabolites is to be achieved. All these factors have led to the analysis of natural products being quite conservative, especially in terms of sample preparation, which still mainly uses traditional extraction techniques, such as liquid-liquid extraction (LLE) and Soxhlet extraction, and involves high consumption of toxic and volatile organic solvents.

The use of microextraction techniques and new classes of more sustainable extraction phases are gaining importance as they meet the criteria of green analytical chemistry (GAC) (2), particularly green sample preparation (GSP) (3,4), and are thusly applied in the plant field (5). In this sense, several metric tools have been developed in recent years to assess the greenness of a method and its compliance with GAC principles. Of fundamental importance was the development of the AGREEprep metric tool, which focuses on sample preparation and is based on the ten principles of GSP (6). At the same time, the need has arisen in recent years to conduct a holistic and comprehensive evaluation of an analytical methodology and to balance the environmental friendliness of a method with its analytical performance and practical efficiency (7,8). A global assessment of analytical methods would be beneficial to industry and quality control laboratories because, in addition to increasing interest in improving the environmental impact of analyses, industry and official laboratories are dealing with a multiplication of norms and quality standards that require accurate and reliable measurements, as well as practical considerations such as productivity, cost, and simplicity of methods. In addition, assessing the environmental impact of a method (which provides an ecological outcome) along with assessing its analytical performance (which ensures the quality of the results and thus the social impact) and its productivity (which measures the economic impact) would help to measure the degree of “sustainability” of a given method (8,9). Some tools have also been developed to critically and globally evaluate analytical methods by balancing considerations of a method’s environmental friendliness with its analytical efficiency and practical efficiency (7,10), but their application is rather scarce, particularly in the assessment of sample preparation methods. However, in plant-based metabolomics studies, reliable sample preparation is essential because, as Mushtaq and coauthors noted, “the value of the information obtained from a metabolomic study depends on how much of the metabolome is present in analyzed samples. Thus, only a comprehensive and reproducible extraction method will provide reliable data, because the metabolites that will be measured are those that were extracted and all conclusions will be built around this information” (11).

 will be presented here to demonstrate the importance of evaluating the overall performance of sample preparation methods, especially in the plant field.

 is a fascinating plant that has been used for recreational, medicinal, textile, and food purposes since ancient times. From a chemical point of view, it is a very complex matrix, as it contains several classes of specialized metabolites, including more than 100 cannabinoids, 120 terpenoids (monoterpenes, sesquiterpenoids, and triterpenoids) and several flavonoids among the more than 500 compounds identified (12). The plant metabolome is highly variable depending on the part of the plant considered (Figure 1). Cannabinoids and terpenoids are produced and stored in the secretory cells of the glandular trichomes, which are located in the aerial parts of the cannabis plant and especially on the upper surfaces of the seedless female flowers. Therefore, they are mainly found in the inflorescences of the plant, while their content in the leaves decreases sharply, and they are almost absent in the barks of the stems and in the roots. In contrast, the content of flavonoids is highest in the leaves, while they are less present in the inflorescences and almost absent in the roots and stem barks (12).

Classification and taxonomy of this plant is often difficult and ambiguous due to the high variability within the genus. Usually, a distinction is made between drug (cannabis) and fiber (hemp) types, based on the higher content of tetrahydrocannabinol (THC) and cannabidiol (CBD), respectively. Although attention has focused mainly on these two main markers, there is growing interest in exploring the bioactivity of 

 in terms of potential synergistic effects, the so-called entourage effect, which could contribute to or modulate the therapeutic properties of cannabis or hemp extracts. Synergistic effects have already been demonstrated in research on combinations of phytocannabinoids and phytocomplexes of cannabinoids and terpenoids. In addition, phenolic compounds are also known for their broad biological activity (13), and it is therefore important to determine the entire specialized metabolome to obtain an accurate characterization of the plant. In this sense, the extraction step is of fundamental importance, since significant differences in bioactive chemical profiles are observed in the extracts obtained with the different protocols (14).

As mentioned earlier, the inflorescences of cannabis are mainly characterized by the more volatile terpenoids and the semi-volatile cannabinoids. The most common method for extraction of cannabinoids is solid-liquid extraction (SLE) using organic solvents (such as ethanol, methanol, or acetone) coupled to high performance liquid chromatography (HPLC) or gas chromatography (GC) combined with mass spectrometry (MS). Organic solvents can also be used for the extraction of terpenoids (which are subsequently analyzed by GC), so that the two classes of compounds can be determined simultaneously by methods that can have strong environmental implications (15). Figure 2 shows the AGREEprep results for the method developed by Jin and coauthors to isolate terpenoids and cannabinoids using methanol as the extraction solvent (12). A final score of 0.27 is obtained, with several parameters showing very critical values.

Thanks to their volatile nature, the isolation of terpenes, especially mono- and sesquiterpenes, from plant raw materials can be performed by headspace solid-phase microextraction (HS-SPME) online combined with GC–MS analysis. Recovery of the semi-volatile cannabinoids from solid matrices by HS-SPME is also possible, but requires long sampling times due to their low volatility and low tendency to escape into the headspace. Indeed, poor recovery of cannabinoids is observed when using conventional sampling conditions (90 °C and 30 min) (Figure 3a) (16).

When investigating the possibility of sampling at higher temperatures, it is possible to observe that these conditions significantly discriminate against the recovery of more volatile markers (such as terpenoids) due to a reduction in the partition coefficient between the fiber and the headspace and an enhancement of competitive adsorption and displacement of low molecular weight analytes, given the extremely high amount of cannabinoids extracted (Figure 3b). Moreover, high temperature during sampling, especially when combined with relatively long extraction times, can lead to decomposition of cannabinoids and the formation of other components or artifacts (17). In fact, by submitting a CBD standard HS-SPME for only 5 min at 150 °C, it is possible to observe that the compound is degraded forming cannabinoids, including cannabichromene (CBC), Δ

-THC (Figure 3c) (16). Thus, the HS-SPME method is certainly greener (Figure 2), but it can provide unreliable and misleading results. To maintain the optimal greenness of HS-SPME and avoid the risk of artifact formation, a very interesting option is the possibility of sampling at reduced pressure. As described in detail by Psillakis (18), vacuum is a powerful experimental parameter to consider to increase the extraction kinetic of semi-volatile compounds during the HS-SPME process. This is because, in the case of semi- volatiles and under non-equilibrium conditions, reduced pressure in the sample container decreases the resistance to mass transfer in the gas zone at the interface between the solid and the headspace. As a result, higher extraction efficiencies for semi-volatile compounds can be achieved in shorter sampling times and at milder extraction temperatures. Indeed, it can be observed that when sampling at a mild temperature (90 °C), regardless of the sampling time, the amount of CBD extracted with vacuum is several times higher than the amount obtained under regular conditions. In only 5 min, a sufficient amount of CBD could be extracted with vacuum to achieve an acceptable instrument sensitivity and to obtain a good picture of CBD abundance in inflorescences (Figures 3a and 3b). Moreover, no degradation of CBD can be observed at this temperature (Figure 3c) (16). This approach somewhat reduces the environmental friendliness of the method (Figure 2), since an additional step is introduced, but it also allows reliable results to be obtained. The three methods can be evaluated in terms of their overall performance. Among the available tools, the RGB model was chosen because it is flexible in the selection of parameters to be evaluated and the assignment of their relative weights, making it easier to adapt to the scope of the analysis and the objectives to be achieved. The name RGB is derived from the three primary colors that correspond to the three main parameters of each analytical method. The red color represents the analytical performance of the method, the green color represents its safety and greenness, and the blue color represents its productivity and practical effectiveness. An overall method score (called method brilliance) is calculated by combining the results of the three attributes, also taking into account the relative importance that the user attributes to each of them. The comparison of the RGB results is shown in Figure 4 and highlights that the analytical performance of the Vac- HS-SPME method is comparable to the conventional method, while strong improvements are obtained in terms of greenness and productivity, indicating that the method is the most reliable for metabolomics characterization of 

As already mentioned, the other parts of the plant are characterized by a different phytocomplex (12). In particular, studies have shown that the aerial parts of hemp (stems and leaves) are mainly characterized by the presence of flavonoids and non-psychotomimetic cannabinoids, which can be simultaneously extracted by ultrasound-assisted methanol solid-liquid extraction (13). The method is reliable, but again, evaluation of the AGREEprep score shows that the method is quite impactful on the environment (Figure 5a).

Deep eutectic solvents (DES) are a more environmentally friendly alternative to conventional solvents thanks to their ease of preparation and low raw material costs. They consist of two or more components that form a hydrogen bonding network, which is key to the formation of the DES. Various natural compounds have been used as hydrogen bond donor (HBD) or acceptor (HBA) to produce both hydrophilic and hydrophobic natural DES (NADES). Hydrophobic natural terpenoids and phenolic compounds (carvacrol, eugenol, linalool, menthol, terpinen-4-ol, and thymol) were used in a dispersive solid-liquid microextraction (DSLME) to isolate non-volatiles from hemp leaves (Figure 6) (19).

The method is certainly faster, more user-friendly, and greener than the conventional method (Figure 5a). Indeed, it only requires 100 mg of the hemp plant, 2 mL of water as cosolvent and 100 μL of eutectic solvent, which is dispersed in the sample by a vortex step, followed by 10 min of ultrasound to help release the target compounds from the plant. The NADES phase is then separated by centrifugation before analysis by HPLC. NADES are very effective in extracting the more hydrophobic cannabinoids (Figure 6b), but are less effective than the conventional method in isolating the more polar flavonoid glycosides. For this reason, a new class of hydrophobic compounds has been developed. The structure of polar choline was used as a model for the development of a new HBA. The hydroxyl functional group of choline was retained to improve the polarity of the new compounds and their ability to form hydrogen bonds, but the length of the alkyl chain substituents appended to the ammonium head group was increased to improve hydrophobicity and broaden the range of application. The developed [Ch

]-based salts have been mixed with thymol to form DESs, which showed better extraction of hydrophilic compounds (such as flavonoids) compared to the NADES, while maintaining the same good enrichment for cannabinoids (see Figure 6b) (20). The environmental impact assessment of the method shows a reduction in performance due to the need to synthesize the [Ch

]-based salt (Figure 5a). However, the RGB results presented in Figure 5b show better overall performance of this latter method, which is therefore more suitable for reliable metabolomics characterization of the nonvolatile fraction of 

Scientists working in analytical chemistry and, in particular, in the field of sample preparation have made great efforts to improve the environmental performance of their methods, thanks in part to the increasing attention paid to environmental sustainability by government agencies and the public. This improvement is essential as the world is committed to addressing climate and environmental challenges. However, we have shown here that, especially for complex samples, the environmental friendliness of a method should be evaluated along with its productivity and, more importantly, its analytical performance to ensure not only environmental friendliness but also “sustainability” of the results. New metric tools that also take these aspects into account and give appropriate importance to the sample preparation step are therefore desirable for the future.

(1) Waris, M.; Koçak, E.; Gonulalan, E. M.; Demirezer, L. O.; Kır, S.; Nemutlu, E. 

Metabolomics Analysis Insight into Medicinal Plant Science

(2) Gałuszka, A.; Migaszewski, Z.; Namieśnik, J. The 12 Principles of Green Analytical Chemistry and the SIGNIFICANCE Mnemonic of Green Analytical Practices. 

(3) López-Lorente, Á.I.;Pena-Pereira, F.; Pedersen-Bjergaard, S.; Zuin, V. G.; Ozkan, S. A.; Psillakis, E. The Ten Principles of Green Sample Preparation. 

(4) Kokosa, J. M.; Przyjazny, A. Green Microextraction Methodologies for Sample Preparations. 

(5) Mastellone, G.; Marengo, A.; Sgorbini, B.; Rubiolo, P.; Cagliero, C. New Phases for Analytical Scale Extraction from Plants: Current and Future Trends. 

(6) Wojnowski, W.; Tobiszewski, M.; Pena-Pereira, F.; Psillakis, E. AGREEprep – Analytical Greenness Metric for Sample Preparation. 

(7) Nowak, P. M.; Kościelniak, P.; Tobiszewski, M.; Ballester-Caudet, A.; Campíns-Falcó, P. Overview of the Three Multicriteria Approaches Applied to a Global Assessment of Analytical Methods. 

(8) Nowak, P. M.; Wietecha-Posłuszny, R.; Pawliszyn, J. White Analytical Chemistry: An Approach to Reconcile the Principles of Green Analytical Chemistry and Functionality. 

(9) Sheldon, R. A. Metrics of Green Chemistry and Sustainability: Past, Present, and Future. 

32-48. DOI: 10.1021/acssuschemeng.7b03505

(10) Bechis, G.; Raccary, B.; Sarrazin, E.; Corbi, E.; Peres, C.; David, N.; Bicchi, C.; Cagliero, C. Assessing the Environmental and Overall Performance of Gas Chromatographic Analyses. Development of a Comprehensive Evaluation Framework and Application to Routine Chiral Analyses of Fragrances as a Case Study. 

(11) Mushtaq, M. Y.; Choi, Y. H.; Verpoorte, R.; Wilson, E. G. Extraction for Metabolomics: Access to the Metabolome. 

(12) Jin, D.; Dai, K.; Xie, Z.; Chen, J. Secondary Metabolites Profiled in Cannabis Inflorescences, Leaves, Stem Barks, and Roots for Medicinal Purposes. 

(13) Mastellone, G.; Marengo, A.; Sgorbini, B.; Scaglia, F.; Capetti, F.; Gai, F.; Peiretti, P. G.; Rubiolo, P.; Cagliero, C. Characterization and Biological Activity of Fiber-Type 

 L. Aerial Parts at Different Growth Stages. 

(14) Bowen, J. K.; Chaparro, J. M.; McCorkle, A. M.; Palumbo, E.; Prenni, J. E. The Impact of Extraction Protocol on the Chemical Profile of Cannabis Extracts from a Single Cultivar. 

(15) Micalizzi, G.; Vento, F.; Alibrando, F.; Donnarumma, D.; Dugo, P.; Mondello, L. 

.: A Comprehensive Review on the Analytical Methodologies for Cannabinoids and Terpenes Characterization. 

 461864. DOI: 10.1016/j.chroma.2020.461864

(16) Capetti, F.; Rubiolo, P.; Mastellone, G.; Marengo, A.; Sgorbini, B.; Cagliero, C. A Sustainable Approach for the Reliable and Simultaneous Determination of Terpenoids and Cannabinoids in Hemp Inflorescences by Vacuum Assisted Headspace Solid-Phase Microextraction. 

(17) Czégény, Z.; Nagy, G.; Babinszki, B.; Bajtel, Á.; Sebestyén, Z.; Kiss, T.; Csupor-Löffler, B.; Tóth, B.; D. Csupor, D. CBD, a Precursor of THC in E-Cigarettes. 

(18) Psillakis, E. Vacuum-Assisted Headspace Solid-Phase Microextraction: A Tutorial Review. 

(19) Mastellone, G.; Marengo, A.; Sgorbini, B.; Rubiolo, P.; Cagliero, C. Development of a Dispersive Solid-Liquid Microextraction Method Using Natural Eutectic Solvents for a Greener Extraction of Phytochemicals from Fiber-Type 

(20) Mastellone, G.; Abbasi, N. M.; Cagliero, C.; Anderson, J. L. New Class of Tunable Choline Bromide-Based Hydrophobic Deep Eutectic Solvents for the Extraction of Bioactive Compounds of Varying Polarity from a Plant Matrix. 

6665–6675. DOI: 10.1021/acssuschemeng.3c00185

 is an Associate Professor with the Department of Pharmaceutical Science and Technology at the University of Torino, in Torino, Italy. Direct correspondence to: 

Events

To measure a method’s environmental friendliness and its sustainability, one must consider its productivity and analytical performance.

From “Green” to “Sustainable” Sample Preparation in Omics Studies in the Natural Product Field: Case Studies Dealing with Cannabis sativa L.

Environmental compartments are characterized by their large size and the heterogeneous distribution of the target analytes. Onsite extraction procedures are especially useful in this scenario, allowing the development of ambitious sampling campaigns (including a larger number of locations and periods). This article outlines the relevance of extraction techniques, including exhaustive and non-exhaustive ones, in onsite strategies. However, only stirred units are discussed and described in detail. The discussion of the analytical performance (for example, sensitivity and precision) is intentionally avoided to focus the attention on the devices that can be applied (selecting the sorptive phase) to almost any analytical problem. The impact of open technologies (microprocessors and 3D printing) in the design of these units is also presented.

Onsite environmental analysis is a simplified procedure that measures the target analytes at the sampling point. This strategy reduces errors by eliminating unnecessary steps that add to the total variance of the results (1). Onsite analysis also maintains the sample’s representativeness by avoiding storage and transportation, which can alter the sample composition (in ways such as, but not limited to, analyte losses by adsorption in the vessel walls). However, onsite analysis often depends on portable instruments with lower performance than benchtop facilities. The sensitivity and selectivity can be improved by applying an onsite sample preparation step. This combined approach is practical for those problems where the sample matrices are not very complex, and the concentration thresholds are not very strict. If these conditions are not fulfilled, benchtop instruments cannot be avoided. In this case, onsite extraction can be developed in a different way. The analytes are in situ isolated and retained in a sorptive phase, which can be liquid or solid. Once retained, the analytes are less prone to be lost by evaporation or chemical degradation (if the phase is appropriately dried and stored). The sorptive phase is transported to the laboratory, where it can be stored for the final analysis. The portability and affordability of the sample preparation devices are essential in making the last approach practical and useful.

A sample preparation procedure should be as simple and miniaturized as possible to be portable. A closer look at these characteristics reveals some sustainability-related connotations. In fact, simplicity and miniaturization result in procedures with a lower requirement of consumables (solvents, reagents) and energy, thus reducing the environmental impact. It is not surprising that López-Lorente and associates selected portability as the first of the ten principles of green sample preparation (GSP) (2).

The distribution of target analytes in a given environmental compartment is non-uniform, varying both spatially and temporally. Therefore, to have a complete understanding of this distribution, it is necessary to take multiple samples from different locations and at different times. The cost of this multisampling approach dramatically depends on the price of the extraction units. Therefore, affordable devices permit the definition of more ambitious sampling campaigns.

Thanks to their inherent characteristics, microextraction techniques are ideal tools for onsite sample preparation. Solid-phase microextraction (SPME) and related techniques are paradigmatic in this sense. Their miniaturized nature also allows the design of special sampling devices capable of extracting the analytes at difficult-to-access locations. For example, Grandy and co-authors integrated a thin film microextraction device in a floating aerial drone for onsite water extraction at remote locations (3). In the same way, SPME devices have been implemented in aerial drones for environmental air sampling and extraction (4). Readers interested in deepening onsite environmental sample preparation can find further information in recent literature where the topic has been reviewed (5,6). Instead, this article focuses on our experience developing onsite environmental extraction based on stirred devices.

Exhaustive and Non-Exhaustive Extraction Techniques

Green analytical chemistry (GAC) (7) and GSP (2) principles recommend minimizing the sample volume, as this parameter directly influences the resources needed for the analytical procedure. Environmental analysis is particular since the large size of the studied compartments requires a relatively high sample volume to assess its representativeness. Onsite processing of these volumes (typically in the range of hundreds of mL to a few of L) is challenging since close contact of the sample with the sorptive phase is required to isolate the analytes efficiently. Additionally, the sample volume defines the amount of analyte that is extracted. This effect, which influences the sensitivity of the determination, is typically more marked in exhaustive extractions where the sample volume must be precisely measured. The need to accurately measure the sample volume when applying a non-exhaustive technique depends on the experimental conditions under which the extraction is carried out. In fact, in SPME, when the sample is much larger than the sorptive phase volume, the amount of analyte extracted becomes independent of the sample volume. Rather than being a drawback, this fact allows the development of in situ extraction, where the sorptive phase is directly introduced in the environmental compartment without an apparent (although it is implicit) sampling step. The effect of the sample volume must be understood before designing an onsite extraction.

In solid-phase extraction (SPE), the sample is forced to flow through the sorbent bed, thus increasing the efficiency. In this case, Schulze and associates reported a device capable of onsite processing up to 50 L of sample (8). The device consisted of a customizable SPE cartridge that can be filled with the adequate sorbent, or mixture of sorbents, ad-hoc selected depending on the target analytes. A prefiltration element is incorporated to avoid clogging the cartridge with the suspended particulate matter. A pressurized system is implemented to flow the sample through the cartridge. The device has a power demand of 12 V that can be supplied using a car battery.

In nonexhaustive extraction approaches, like SPME, the extraction significantly depends on the diffusion of the analytes from the bulk sample to the sorptive phase. The sample agitation typically improves this diffusion. In 2018, Piri-Moghadam and associates described a bottle-based apparatus for onsite extraction (9). In this approach, 1 L of the sample is initially taken in a glass bottle and closed with a polytetrafluoroethylene (PTFE) stopper. The thin film microextraction (TFME) phase hangs from the stopper, held by a polymeric thread. A sinker, placed at the end of the thread, guarantees the immersion of the sorptive phase into the sample. An orbital agitator is used to enhance the diffusion of the analytes. Liu and co-authors recently reported an onsite extraction technique consisting of a glass bottle whose inner surface were coated with a polydimethylsiloxane/divinylbenzene film (10). The sample is introduced into the bottle and stirred to retain the analytes (volatile hydrocarbons in this particular example) in the coated walls of the bottle. After the extraction, the sample is discharged, and the vial can be transported to the lab or analyzed in a portable chromatograph.

Drills are cheaper and more portable than orbital shakers and magnetic stirrers and can be operated with batteries. Moreover, they open the possibility of integrating the sorbent phase into the stirring element, which is an additional advantage for the diffusion of the analytes. In 2009, Qin and coauthors proposed using planar sorptive phases coupled to a portable drill as a miniaturized extraction system in environmental analysis (11). Mao and associates have also proposed the combination of stir-bar sorptive extraction (SBSE) with a portable electric drill (12). In this case, the stir-bar is magnetically attached to the drill that is used for sample agitation.

To improve the versatility of the sorptive phases available, our research group described the synthesis of borosilicate disks modified with carbon nanohorns as a sorbent phase for the extraction of an endocrine disruptor, benzophenone-3, from water samples (13). As shown in Figure 1, the disks were easily attached to a portable drill that allowed them to be agitated inside the sample. This agitation improved the diffusion of the analytes, allowing their quick isolation, thus facilitating sampling at different locations in a single working day.

Carbon disks provided outstanding performance. However, they were not commercially available, thus restricting their potential application in routine analysis. To overcome this limitation, commercial polymeric membranes were proposed as sorbent phases in 2019 (14). The wide diversity of commercial membranes significantly increased the technique’s applicability for extracting different contaminants. The robust coupling of these membranes with portable drills posed a challenge. Magnets were proposed as extraction supports to achieve this integration. The polymeric membranes were placed on top of the magnet to which they were attached using a metal washer. Blades were attached to the unit to improve the stirring capability of the devices. The extraction unit is represented in Figure 2a. Although the initial results were promising, the metal washer seemed to reduce the extraction kinetics by creating a reduced diffusion layer over the sorptive phase. To solve the latter limitation, magnetic membranes were proposed as sorptive phases in 2021 (15). The magnetic membranes (Figure 2b), prepared by the control coating of a paper with a polymeric magnetic nanocomposite, could be attached to the magnet directly. Avoiding the metallic washer permits the direct interaction of the sorptive phase with the sample.

Simplicity is an added value in onsite extraction techniques as it directly impacts the price of the designed units. In the previous investigations, portable drills were used to agitate the sorbent phases. The price of these drills, around 50 € ($53.00), and the need to recharge their batteries relatively frequently are significant handicaps in designing ambitious sampling campaigns. In 2022, we evaluated the replacement of these drills with electric mini-motors (16). The reduced cost of these motors (about 0.4 € [$0.42] per unit), opens the possibility of manufacturing many sampling devices, thus increasing the number of locations that can be sampled. In addition, these motors operate efficiently with portable 5V batteries, giving them greater autonomy. Figure 3a shows the designed extraction unit. The electric motor is integrated into the stopper of a glass bottle (Figure 3b). The extraction element (a magnet on which the sorbent phase is fixed) is located at the bottom of the unit, where a blade is also placed to improve the diffusion of the analytes (Figure 3c). Both elements, motor and magnet, are connected by a metal rod that transmits the movement. The extraction procedure involves several well-defined sequential steps. Initially, a sample volume in the range of 2.5 L is taken and placed in the glass bottle (Figure 3d). The extraction unit is introduced into the sample by simply closing the stopper, and then 5 V is applied to the motor to agitate the sorptive phase inside the sample. After the extraction, the sorptive phase is recovered and transported to the laboratory for final analysis. A new sorptive phase was designed to boost the extraction of the target analytes. Hydrophilic lipophilic balance particles, which are a common sorbent in environmental analysis, were immobilized using adhesive tape as the binder in a magnetic tape. The microparticles were fixed on the surface of the tape, having a high superficial area to contact with the analytes.

Open technologies can play a fundamental role in lowering the fabrication costs of these devices. Recently, the research group has developed a device based on Arduino technology that also integrates sensors for online monitoring of various parameters (17). A temperature sensor and conductivity sensor are incorporated into the stopper, allowing the measurement of these parameters during the onsite extraction. The motor is operated by the Arduino microprocessor that controls the extraction time. The onsite extraction procedure is schematically presented in Figure 4, consisting of well-defined steps. Initially, a defined sample volume is taken, and the internal standard is added (Figure 4.1). The extraction is performed while the temperature and conductivity values are monitored (Figure 4.2–3). After the extraction, the sorptive phase is dried (Figure 4.4), and a QR code label containing the information of the sample (GPS coordinates of the sampling point, temperature, conductivity, and other data) is printed in a portable printer (Figure 4.5). The QR label is attached to a plastic bag (Figure 4.6) where the sorptive phase is stored for its transportation to the lab for final analysis (Figure 4.7).

3D printing is a disruptive technology with broad applications in analytical chemistry, and it has been recently used to fabricate stirred extraction devices to be deployed onsite (18). Vargas-Muñoz and associates designed a stirrer whose paddles were 3D printed and coated with a metal-organic framework to extract phenols. After the extraction, the paddles can be removed from the stirrer and chemically eluted before the analysis.

Onsite sample preparation is a useful strategy in environmental analysis with positive effects on the metrological quality (representativeness) and the affordability (easier sampling logistics) of the analytical procedures. Extraction techniques, both exhaustive and non-exhaustive ones, have demonstrated potential in this field. However, due to their miniaturized and simplified character, microextraction technique seems to be a better option. This article outlined the role that stirred-based techniques can play in onsite extraction, presenting our perspective on the topic. The introduction of open technologies (microprocessors and 3D printing) in the design of these devices improves their affordability, which is critical to extending their use. There is room for improvement in this field. Directly coupling these onsite devices with instrumental techniques (spectroscopic and spectrometric ones) can simplify the analytical procedures even more.

Financial support from the Andalusian counseling of “Transformación Económica, Industria, Conocimiento y Universidades” (PY20_00461) is gratefully acknowledged. This article is based upon work from the Sample Preparation Study group and Network, supported by the Division of Analytical Chemistry of the European Chemical Society.

(1) Pawliszyn, J. Why Move Analysis from Laboratory to On-Site? 

(2) López-Lorente Á. I. et al. The Ten Principles of Green Sample Preparation. 

(3) Grandy, J. J.; Galpin, V.; Singh, V.; Pawliszyn, J. Development of a Drone-Based Thin-Film Solid-Phase Microextraction Water Sampler to Facilitate On-Site Screening of Environmental Pollutants. 

(19), 12917–12924. DOI: 10.1021/acs.analchem.0c01490

Aerial Drone as a Carrier for Miniaturized Air Sampling Systems. 

(5) Sajid, M. Advances in On-Site Analytical Sample Preparation for Analysis of Environmental Waters: A Review. 

(6) Casado-Carmona, F. A.; Lasarte-Aragonés, G.; Lucena, R.; Cárdenas, S. "Green Sample Preparation Techniques in Environmental Analysis" in 

(7) Gałuszka, A.; Migaszewski, Z.; Namieśnik, J.The 12 Principles of Green Analytical Chemistry and the SIGNIFICANCE Mnemonic of Green Analytical Practices. 

(8) Schulze, T. et al. Assessment of a Novel Device for Onsite Integrative Large-Volume Solid Phase Extraction of Water Samples to Enable a Comprehensive Chemical and Effect-Based Analysis. 

(9) Piri-Moghadam, H.; Gionfriddo, E.; Grandy, J. J.; Alam, Md. N.; Pawliszyn, J. Development and Validation of Eco-Friendly Strategies Based on Thin Film Microextraction for Water Analysis. 

(10) Liu, X. et al. Sample Bottle Coated with Sorbent as a Novel Solid-Phase Extraction Device for Rapid On-Site Detection of BTEX in Water. 

(11) Qin, Z.; Bragg, L.; Ouyang, G.; Niri, V. H.; Pawliszyn, J. Solid-Phase Microextraction Under Controlled Agitation Conditions for Rapid On-Site Sampling of Organic Pollutants in Water. 

(12) Mao, X.; Hu, B.; He, M.; Fan, W. Stir Bar Sorptive Extraction Approaches with a Home-Made Portable Electric Stirrer for the Analysis of Polycyclic Aromatic Hydrocarbon Compounds in Environmental Water. 

 16–24. DOI: 10.1016/j.chroma.2012.08.062

(13) Roldán-Pijuán, M.; Lucena, R.; Cárdenas, S.; Valcárcel, M. Micro-Solid Phase Extraction Based on Oxidized Single-Walled Carbon Nanohorns Immobilized on a Stir Borosilicate Disk: Application to the Preconcentration of the Endocrine Disruptor Benzophenone-3. 

87–94. DOI: 10.1016/j.microc.2014.02.015

(14) Casado-Carmona, F. A.; del C. Alcudia-León,M.; Lucena, R.; Cárdenas, S. Portable Stir Membrane Device for On-Site Environmental Sampling and Extraction. 

 360359. DOI: 10.1016/j.chroma.2019.07.013

(15) Casado-Carmona, F. A.; Lucena, R.; Cárdenas, S. Magnetic Paper-Based Sorptive Phase for Enhanced Mass Transference in Stir Membrane Environmental Samplers. 

(16) Casado-Carmona, F. A.; Jiménez-Soto, J. M.; Lucena, R.; Cárdenas, S. Portable Stirring Device for the On-Site Extraction of Environmental Waters Using Magnetic Hydrophilic-Lipophilic Balance Tape. 

(17) Casado-Carmona, F. A.; Lucena, R.; Cárdenas, S. Lab in a Bottle, Open-Source Technologies for the Design of Affordable Environmental Samplers Integrating On-Site Extraction, 

 (3), 109713, DOI: 10.1016/j.jece.2023.10971318.

(18) Vargas-Muñoz, M. A.; Palomino, C.; Turnes, G.; Palacio, E. A Sampling Platform Incorporating 3D-Printed Paddles-Stirrers Coated with Metal-Organic Framework MIL-100(Fe) for in-situ Extraction of Phenolic Pollutants in Biodigester Supernatant and Wastewater Effluent Samples. 

Francisco Antonio Casado-Carmona, Rafael Lucena, and Soledad Cárdenas are with the Chemical Institute for Energy and Environment (IQUEMA), part of the Department of Analytical Chemistry, at the University of Córdoba in Córdoba, Spain. Direct correspondence to: 

This article outlines the relevance of extraction techniques, including exhaustive and non-exhaustive ones, in onsite strategies.

Onsite Environmental Extraction Based on Portable and Affordable Stirred Devices

Headspace-solid-phase microextraction (HS-SPME) is a widely used technique for the analysis of volatile compounds from different kinds of samples and with multiple fields of applications. Despite of its applicability, it can require a long extraction time and it is limited by the volatility of the analytes of interest and the need for equilibrium conditions between the sample and the headspace. Recently, two additional approaches have been proposed to reduce the extraction time and broaden the coverage to less volatile compounds, namely vacuum-assisted HS-SPME and multi-cumulative trapping HS-SPME. This paper illustrates the principle of the two techniques and their potential for extending the applicability of HS-SPME, using them as alternatives or combined to characterize olive oil samples in terms of quality.

Solid-phase microextraction (SPME), was introduced in the 1990s by Pawliszyn and collaborators (1,2), and represents one of the most widely applied techniques for volatile and semi-volatile analysis. When SPME is applied to the headspace (HS), it involves the equilibrium between three phases, and thus, two equilibrium processes—namely, the sample and the HS (characterized by 

, distribution constant HS-sample) and the HS and the fiber (characterized by 

). Reaching equilibrium conditions guarantees to maximize the sensitivity. However, when dealing with complex samples and untargeted analysis, the equilibrium condition may not be reached for all the compounds within an acceptable time, and even some analytes may not be properly extracted at equilibrium conditions. Moreover, longer extraction times may promote the occurrence of a competition phenomenon (for example, displacement effect) when solid sorbents are used (3,4). Nevertheless, the direct correlation between the amount extracted (

) is maintained before and at the equilibrium (equation [1]) (5):

 represent the volume of the sample, the HS, and the fiber coating, respectively.

This proportionality can also be expressed as:

where A is the chromatographic area, and ß the phase ratio (ß = 

). This relationship is valid as long as HS saturation is avoided (6). The HS linearity range depends on the actual concentration in the sample, but also the sampling temperature, time, and ß. The linearity condition of the total sample is a compromise between trace and major components. The same compromise is necessary in terms of extraction time. To accelerate the kinetics of extraction, stirring of the sample and increased temperature are usually applied. A powerful alternative that is gaining attention is the use of vacuum-assisted HS extraction (Vac-HS). It was proposed for the first time in the early 2000s (7,8), but it has gained attention starting from 2012, thanks to the work of Psillakis and associates (9–13). Another alternative to improve the sensitivity of trace compounds using HS-SPME is to perform multiple-cumulative trapping (MCT) extractions from the same samples, thus depleting the HS of the most volatile compounds and facilitate the extraction of the less volatile compounds (14,15).

In this paper, the two aforementioned approaches, namely Vac-HS-SPME and MCT-SPME, are discussed in the context of virgin olive oil profiling.

The fundamental theory of Vac-HS-SPME in water-based samples was nicely described in a tutorial paper published in 2017 by Psillakis and collaborators (16), and lately extended to oil based samples (17). In both cases, the use of reduced pressure does not impact the thermodynamics of the extraction but only the kinetics. It plays a fundamental role in improving the mass transfer from the sample (liquid or solid) to the HS (16,18).

To explain the limitation of the mass transfer at the interface, the two-film theory is used. This theory assumes a homogenous distribution within the bulk of each phase, while a concentration gradient is present in the stagnant thin layers at the interfaces. This theory successfully explained the behavior of volatile compounds from water-based samples when the pressure is lowered (19–21). According to this theory, the overall mass transfer coefficient, 

, at the interface can be modeled as follows:

 are the mass transfer coefficients for the gas and liquid boundary layers, and 

 is the gas-liquid phase partition coefficient. Once in the HS, the mass transfer of the analytes to the fiber can be neglected, although it is also improved in Vac-HS-SPME (9,18,22), as shown for other high-capacity sorbents (such as, the stir bar and liquid microdrop) (23,24). Therefore, the overall resistance to transfer from liquid to the gas phase (1/

) can be considered as two diffusional resistances in series.

The diffusion coefficient in the gas-phase, 

, has an inverse proportionality to the total pressure in the system (9). On the other hand, the liquid phase resistance is highly dependent from the viscosity of the medium (η) and the temperature (T). The Wilke-Chang (25) formula (Equation [4]) describes the diffusion coefficient in the liquid phase (

 is the molecular weight of the solvent, and 

 is the molar volume of the solute. Therefore, when vacuum is applied to HS-SPME, 

, reducing the gas-phase resistance (see the term 1/(

) in equation 3) (9,16). However, the positive impact in extraction efficiency will be observed for analytes for which 

 values for solutes in olive oil as a solvent are substantially different from those with water as a solvent, due to the differences in solute-solvent and solvent-solvent molecular interactions (26). Therefore, the use of Vac-HS- SPME for water- or oil-based samples mainly differs due to the viscosity of the two media.

Accordingly, lowering the total pressure will improve the overall mass transfer coefficient for analytes where gas-phase resistance controls their volatilization rate. It results in faster HS-SPME extraction kinetics and shorter equilibration times. However, for analytes and samples where the liquid diffusion is the limiting step (as in highly viscous medium, such as oils), the impact of reducing pressure on recoveries is less important. It plays a synergic effect with temperature, which reduces the viscosity, and thus increases DL. Figure 1 shows an example of the combined impact of reduced pressure and temperature on a series of compounds extracted from the HS of an extra virgin olive oil. In particular, semi-volatile compounds, such as for instance 2-decenal and α-farnesene, benefit significantly from the temperature increase (Figure 1). In fact, at higher temperature the increase in DL allows the analytes to rapidly reach the liquid-gas interface, and quickly replenish the HS by the facilitated mass transfer due to the reduced pressure.

In the case of water samples, a clear criterion for predicting the effect of vacuum on the extraction of the compounds of interest have been established. However, due to the limited availability of air-olive oil partitioning data (such as K

 values), and the complex interactions of solutes with olive oil, it is not possible to establish a similar criterion for oily samples.

Multiple-Cumulative Trapping HS-SPME in Fatty Samples

As previously mentioned, equilibrium may require a long extraction time to be reached. The longer the extraction time, the higher the risk of displacement effects when using a porous fiber, such as DVB/CAR/PDMS (3,27). Therefore, shorter extraction times are often preferred (also improving the throughput), but this comes at the expense of sensitivity. The payback was calculated for 49 compounds extracted from extra virgin olive oil, comparing the ratio of the responses obtained when extracting for 30 and 10 minutes (28). It was shown that the increment ranged between 0.6 to 2.6 folds, with a clear decreasing trend in uptake gain at 30 min for the less volatile analytes, which showed a ratio of about 1 (Figure 2a).

The use of MCT-HS-SPME was applied to minimize the displacement and enhance the uptake of less volatile compounds. Indeed, as shown in Figure 2b, there is a clear difference on how much better the semi-volatile compounds are extracted when performing MCT-HS-SPME with three extractions of 10 min, compared to a single extraction of 30 min.

Therefore, the use of MCT-HS-SPME proved to be advantageous in further enriching the overall information obtained from HS profiling, while maintaining the key benefit of not affecting sample throughput. This was then extended to the optimization of an additional variable: the number of repeated extractions needed to improve the information extracted (in terms of discrimination capability), but avoiding to increase the noise response, and do not modify the real perceived volatile profile. This is especially important when the intention is to determine the correlation between the volatile profile and aroma, as in the present case of olive oil analysis. It was found that repeating the extraction six times was, in fact, detrimental to differentiating between different olive oil qualities (extra virgin, virgin, and lampante) (28).

Vacuum-Assisted and Multiple-Cumulative Trapping HS-SPME in Fatty Samples 

Following the previously discussed advantages of Vac- and MCT-HS- SPME, both should provide similar benefits towards the less volatile compounds. Indeed, when comparing their extraction efficiencies, similar or slightly higher responses can be observed for Vac-HS-SPME (orange bars in Figure 3, a ratio of 1 means equal response). It was decided to evaluate the combination of the two sampling approaches to evaluate the potential of further increasing the responses of the less-volatile compounds in olive oil analysis.

Preliminary tests were carried out to verify that the reduced pressure could be maintained after repeated piercing of the septum, and it proved the MCT-Vac-HS-SPME approach was feasible. Therefore 0.1 g of olive oil was extracted three times for 10 min under regular and reduced pressure conditions. For the four targeted compounds reported in Figure 3, a synergic effect can be easily observed. This is particularly true for the less volatile ones, such as 2-decenal and α-farnesene, where an increment of 4.5-5 times can be observed compared to 3-times 10 min regular pressure MCT-HS-SPME.

Cross-Sample Evaluation Using Enhanced HS-SPME

Physically extracted olive oil is classified into different commercial categories (such as extra virgin oil [EVO], virgin oil [VO], and lampante oil [LO]) based on physicochemical and sensory parameters, according to European Regulation No 2568/1991 and following modifications (29). Although the sensory evaluation is highly standardized (29,30), it presents a problem due to the inherent low robustness and reproducibility of panel evaluations. Therefore, it is clear the necessity for a robust and objective analytical method to support this classification. Moreover, this application is further complicated by the many variables affecting the aroma profile such as cultivar, geographical origin, fruit ripeness, processing practices, and storage conditions.

Although there is a clear enhanced response of the less volatile compounds by the implementation of reduced pressure conditions (in particular for MCT), this required further evaluation to determine whether it reflects in a gain of overall information obtained from the volatile profile, especially for olive oil. This was preliminary tested in a study consisting of about 70 samples of olive oil of different quality and geographical origins (Italy, Spain-Portugal, Spain, Spain-Tunisia, and Tunisia) using MCT-HS-SPME-GC–MS (31). As it can be observed in Figure 4, the higher quality EVO (blue dots on the left side of Figure 4) were clearly differentiated from the samples that presented some sensory defects (VO and LO, reddish dot on the left side of the top principal component analysis [PCA]). At first glance, it may be seen as the proposed approach does not allow for discrimination between the VO and LO. However, when adding PC3 (here emphasized with a hierarchical cluster analysis) there was a satisfactory discrimination between VO and LO. Although some misclassifications occurred, it has to be kept in mind that the classification into LO can also be related to chemical parameters, such as the acidity or the peroxide value. Thus, the sensory defects may not be the only thing responsible for the classification.

Moreover, EVO samples could be further classified based on the geographical origin of the olives. Italian and Spanish oils were very clearly discriminated, as well as Tunisian ones, although only very few samples were available. Unfortunately, no pure Portugal samples were available, and the Tunisian ones were too few to provide enough statistical power to differentiate the mixtures. Nevertheless, their content in the mixtures was far less than 10% of these different origins, thus justifying their clustering with the Spanish EVO.

The implementation of combined MCT-Vac-HS-SPME was also extended to the use of a multidimensional comprehensive GC (GC×GC)–MS separation. The combination of the more powerful separation technique (GC×GC), and the enrichment during sampling, provided a very satisfactory separation of the three groups of olive oil (EVO, VO, and LO). This very good classification obtained with MCT-Vac-HS-SPME is shown in Figure 5.

Vac-HS-SPME and MCT-HS-SPME are two useful alternative techniques that can be used on their own or combined to enhance the profiling of volatile and semi-volatile compounds. The two techniques can be easily implemented and have both shown a significant improvement in the recovery of the semi-volatile compounds. Moreover, if coupled to GC×GC, the enriched extraction profiles can be more comprehensively separated, allowing for a substantial improvement of the discrimination ability among olive oil samples of different quality (that is, EVO, VO and LO) based on the information rich chromatographic fingerprints obtained.

This article is based upon work from the Sample Preparation Study Group and Network, supported by the Division of Analytical Chemistry of the European Chemical Society.

The authors declare no competing financial interest.

(1) Zhang Z.; Pawliszyn, J. Headspace Solid-Phase Microextraction. 

(2) Pawliszyn, J. Theory of Solid-Phase Microextraction. 

(3) Górecki, T.; Yu, X.; Pawliszyn, J. Theory of Analyte Extraction by Selected Porous Polymer SPME Fibres. 

(4) Gionfriddo, E.; Souza-Silva, É. A.; Pawliszyn, J. Headspace Versus Direct Immersion Solid Phase Microextraction in Complex Matrixes: Investigation of Analyte Behavior in Multicomponent Mixtures. 

Static Headspace-Gas Chromatography: Theory and Practice

(7) Brunton, N. P.; Cronin, D. A.; Monahan, F. J. The Effects of Temperature and Pressure on the Performance of Carboxen/PDMS Fibres During Solid Phase Microextraction (SPME) of Headspace Volatiles from Cooked and Raw Turkey Breast. 

(8) Darrouzès, J.; Bueno, M.; Pécheyran, C.; Holeman, M.; Potin-Gautier, M. New Approach of Solid-Phase Microextraction Improving the Extraction Yield of Butyl and Phenyltin Compounds by Combining the Effects of Pressure and Type of Agitation. 

(9) Psillakis, E.; Yiantzi, E.; Sanchez-Prado, L.; Kalogerakis, N. Vacuum-Assisted Headspace Solid Phase Microextraction: Improved Extraction of Semivolatiles by Non-Equilibrium Headspace Sampling Under Reduced Pressure Conditions. 

(10) Psillakis, E.; Mousouraki, A.; Yiantzi, E.; Kalogerakis, N. Effect of Henry's Law Constant and Operating Parameters on Vacuum-Assisted Headspace Solid Phase Microextraction. 

(11) Psillakis, E.; Yiantzi, E.; Kalogerakis, N. Downsizing Vacuum-Assisted Headspace Solid Phase Microextraction. 

(12) Yiantzi, E.; N. Kalogerakis, N.; Psillakis, E. Design and Testing of a New Sampler for Simplified Vacuum-Assisted Headspace Solid-Phase Microextraction. 

(13) Trujillo-Rodríguez, M. J. et al., Vacuum-Assisted Headspace-Solid Phase Microextraction for Determining Volatile Free Fatty Acids and Phenols. Investigations on the Effect of Pressure on Competitive Adsorption Phenomena in a Multicomponent System. 

(14) Oliver-Pozo, C.; Trypidis, D.; Aparicio, R.; Garciá-González, D. L.; Aparicio-Ruiz, R.Implementing Dynamic Headspace with SPME Sampling of Virgin Olive Oil Volatiles: Optimization, Quality Analytical Study, and Performance Testing. 

(15) Chin, S. T.; Eyres, G. T.; Marriott, P. J. Cumulative Solid Phase Microextraction Sampling for Gas Chromatography-Olfactometry of Shiraz Wine. 

(16) Psillakis, E. Vacuum-Assisted Headspace Solid-Phase Microextraction: A Tutorial Review. 

(17) Mascrez, S.; Psillakis, E.; Purcaro, G. A Multifaceted Investigation of the Effect of Reduced Pressure Condition on the Headspace-Solid Phase Microextraction of Extra-Virgin Olive Oil

(18) Górecki, T. Effect of Sample Volume on Quantitative Analysis by Solid-phase Microextraction Part 1. Theoretical Considerations. 

 (10), 1079–1086. DOI: 10.1039/A701303E

(19) Chao, H.-P. Volatilization Characteristics of Organic Solutes in Stirred Solution. 

(20) Mackay, D.; Wolkoff, A. W. Rate of Evaporation of Low-Solubility Contaminants from Water Bodies to Atmosphere. 

(21) Mackay, D.; Yuen, T. K.Volatilization Rates of Organic Contaminants from Rivers. 

(22) Koziel, J.; Jia, M.; Pawliszyn, Air Sampling with Porous Solid-Phase Microextraction Fibers. J. 

(23) Solomou, N.; Bicchi, C.; Sgorbini, B.; Psillakis, E. Vacuum-Assisted Headspace Sorptive Extraction: Theoretical Considerations and Proof-of-Concept Extraction of Polycyclic Aromatic Hydrocarbons from Water Samples. 

(24) Psillakis, E.; Koutela, N.; Colussi, A. J. Vacuum-Assisted Headspace Single-Drop Microextraction: Eliminating Interfacial Gas-Phase Limitations. 

(26) Schwarzenbach, R. P.; Gschwend, P. M.; Imboden, D. M. 

Environmental Organic Chemistry, Second Edition

(27) Trujillo-Rodríguez, M. J. et al. Vacuum-Assisted Headspace-Solid Phase Microextraction for Determining Volatile Free Fatty Acids and Phenols. Investigations on the Effect of Pressure on Competitive Adsorption Phenomena in a Multicomponent System. 

(28) Mascrez, S.; Purcaro, G. Enhancement of Volatile Profiling Using Multiple-Cumulative Trapping Solid-Phase Microextraction. Consideration on Sample Volume. 

(29) European Commission Regulation No 2568/1991, Commission Regulation (EEC) No 2568/91 of 11 July 1991 on the Characteristics of Olive Oil and Olive-Residue Oil and on the Relevant Methods of Analysis. 

https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:01991R2568-20161204

https://www.internationaloliveoil.org/wp-content/uploads/2019/11/COI-T20-Doc.-15-REV-10-2018-Eng.pdf

(31) Spadafora, N. D.; Mascrez, S.; McGregor, L.; Purcaro, G. Exploring Multiple-Cumulative Trapping Solid-Phase Microextraction Coupled to Gas Chromatography–Mass Spectrometry for Quality and Authenticity Assessment of Olive Oil. 

 are with Gembloux Agro-Bio Tech, at the University of Liège, in Gembloux, Belgium. 

is with the Laboratorio de Investigación y Desarrollo de Métodos Analíticos at the Universidad Nacional de La Plata, in La Plata, Argentina. Direct correspondence to: 

Headspace-solid-phase microextraction (HS-SPME) is widely used for the analysis of volatile compounds from different kinds of samples.

Enhance the Performance of Solid-Phase Microextraction by Exploiting Vacuum-Assisted Headspace and Multicumulative Trapping  for Olive Oil Characterization

Passive samplers have been developed in many different forms and used in different fields of study because of their unique capabilities. One part of recent reports has focused on benefiting from the advantages of passive sampling in areas such as wastewater-based epidemiology and non-targeted analysis. The other part mainly deals with novel approaches to improve the reliability and efficiency of the sampling process. This paper reviews major advances and new applications of this sampling strategy based on recently published scientific publications.

Among the common sampling strategies, that is grab, composite, and passive sampling, the latter has some unique features that have made it a useful tool, especially for environmental studies (1). Passive sampling can provide reliable data on the time-averaged concentration of contaminants. Temporary contamination events, such as provisional discharges and accidental spills which are not normally captured by other sampling strategies, could be recorded (chemically) by the passive sampler. In addition, this approach can enrich the trace contaminants over a rather long exposure and also provide toxicologically relevant data on bioavailable concentrations of the contaminants (2–4). Passive sampling is also possible and useful for the study of atmospheric organic contaminants, in similar approaches (of course taking into account the differences in receiving phases, sampler design, and analytes) (5). Despite its advantages, passive sampling needs a more complicated calibration procedure in order to produce valid quantitative data. This could be done using theoretical estimation of the partition coefficients of the analytes between the sorbents and the sampled media, considering its physicochemical properties (6). Another strategy (considered to be more reliable) is to calibrate the passive sampler in the real environment by comparing its analytical results with those of active sampling (7). In the following section, the most recent applications of various passive sampling devices will be briefly reviewed (Figure 1).

Applications and Developments of Passive Sampling 

Since the first reports on quantitative applications of passive sampling for the determination of sulfur dioxide in air in 1973, diverse sorption phases and sampler designs have been reported for studying water and air contamination. Among them, semi-permeable membrane devices (SPMDs) (8), low-density polymer devices (LDPE) (9) providing higher surface areas and reduced consumable costs (10), silicon rubber (SR), Chemcatcher (11), the polar organic chemical integrative sampler (POCIS) (12), and solid-phase microextraction (SPME) (13) have been implemented for water sampling purposes. Regarding atmospheric contamination, SPMDs, similar to what is being used for water sampling, with various sorbing phases (14–16) have been utilized. Furthermore, devices based on porous materials have been developed for different semi-volatile compounds in the atmosphere, using for example polyurethane foam (PUF) (17), naphthylisothiocyanate (XAD) (18), and activated carbon (19) as sorbents.

Wastewater-based epidemiology (WBE) is one of the most recent application fields of passive sampling. In this field of study, untreated wastewater is collected and analyzed for the content and variations of health-related compounds (biomarkers), and the data are compared with the public health situation of the catchment (20). Obviously, grab sampling could only provide a snapshot of the wastewater under study, while the composition of the media is continuously changing due to the population routines as well as the social and climatic conditions, such as working days and holidays, vacation periods, seasonality, and atmospheric precipitations. Composite sampling, as it is conventionally used, could resolve such problems, but its implementation requires higher costs of operation and maintenance and also could produce large volumes of samples to be handled. Alternatively, passive sampling could significantly reduce cost and labor and at the same time eliminate or simplify many sample pretreatment steps, such as preservation, storage, filtration, extraction, and preconcentration.

One of the most recent applications of passive sampling in WBE studies is the investigation of the spread and dynamics of the SARS-CoV-2 virus (21). In this work, authors deployed polyethylene plastic strips, as simple forms of the sorbing phase, cotton cloth sheets, and unraveled polypropylene plastic ropes to sample the wastewater. The samplers then were transferred to the laboratory to extract and analyze SARS-CoV-2 RNA. The results suggested that considering the enrichment of the target analytes on the sampler, the sensitivity of the passive sampling was better than conventional composite sampling, and it also could provide a better estimation of the infected population rather than the number of incidents. In a similar study, it has been shown that passive sampling using torpedo sampling units (containing a combination of readily available electronegative membranes, cotton buds, and medical gauze) not only makes detection of various viruses in municipal wastewater possible but also could be implemented in much smaller scales, such as an individual building, to produce point-specific data (22).

Another interesting development in the application of passive sampling in WBE is the study of illicit drugs and their metabolites and hence, the assessment of the consumption rates of such chemicals (23). Contrary to the abovementioned examples, efficient uptake of the chemicals from the complex matrix of untreated wastewater needs strong chemical sorbents and in this case a POCIS consisting of Oasis HLB (a copolymer made from hydrophilic N-vinylpyrrolidone and lipophilic divinylbenzene monomers) sandwiched between two polyether sulphone membranes was deployed. The passive sampling method was calibrated in situ for drugs such as cocaine, amphetamine, methamphetamine, and morphine. A few other biomarkers, such as the antihistamine cetirizine and metoprol acid (a metabolite of metoprolol and atenolol), which are more regularly consumed and released into wastewater, were simultaneously analyzed to produce a better estimation of the population, despite its temporal variations.

The highly variable flow of the surface water, especially rivers, could be a serious challenge since it has a dramatic effect on the sampling rate and hence the accuracy and precision of quantitative data. A helpful technique to compensate for the environmental condition changes is to use performance and reference compounds (PRCs). These compounds are selected so that they do not naturally exist in the studied water and are spiked to the sorbent prior to the deployment of the sampler. Then, the dissipation of these compounds during the sampling period is used to estimate and correct the uptake rate of the target analytes. It has been shown that this technique is suitable for hydrophobic (nonpolar) sorbents (24) but the results are not as reliable for hydrophilic samplers. As a solution, a novel approach has been introduced in which a PRC-spiked nonpolar silicon disk has been implemented in parallel with the polar sampler so that the latter was implemented for collecting and quantitating the polar target compounds (of diverse classes) and the former was used to estimate the changes in the sampling rates and to correct the quantitative results (25).

Passive sampling has also been deployed for groundwater monitoring. Regarding the significantly slower movement of the water body, the target analytes could reach an approximate equilibrium between water and the sampler sorbent, a fact that facilitates the calibration of the passive sampler. In addition, spatial (vertical) distribution of the contaminants, as an important feature of groundwater contamination studies, could be determined by deploying so-called diffusive equilibrium high-resolution passive samplers. This type of passive sampler consists of a series of sorbent cells installed inside a stainless steel rod which could be inserted into the groundwater, and the sampling process takes place at different depths. The approach has been reported to be a versatile means for studying groundwater contamination with per- and polyfluoroalkyl substances (PFAS) (26).

Study of sediments is another field in which passive samplers could play an important role. As an example, a passive sampling strategy based on POCIS has been developed for the determination of PFAS in sediments (as well as water) and the results have been used to estimate the bioaccumulation potential of these compounds (27). Beside advantages such as preconcentration of the analytes and time-weighted average concentration values, passive samplers could imitate the uptake behavior of the living organisms and therefore, provide helpful information on the impacts of the contaminants.

Passive samplers have been used in the determination of diverse air contaminants over the past five decades. There are numerous examples in this field, however, studying the transport and exchange of contaminants between different environmental media, using passive samplers, is attracting increasing focus. For example, diffusive exchange flux between the atmospheric gas phase and the freely dissolved water phase of a large set of hydrophobic organic compounds has been studied using the data extracted from passive sampling of water (LPDE and SR) and air (PUF) in high-mountain lakes (28). Such studies could reveal the transport of atmospheric contaminants and also the role of the local anthropogenic pollution sources in the contamination of remote ecosystems.

Regarding the long deployment time of passive samplers which enables collection of both long-term and temporary contamination events in the medium, as well as their preconcentration capability, the number of the collected chemical compounds and also their concentrations could be much higher compared with methods that use grab sampling. Therefore, passive sampling can be of greatest importance in non-targeted analysis, as a recent and growing field of environmental analytical chemistry (29). It has also been shown that when passive sampling is followed by sensitive analytical instrumentation, such as liquid chromatography–high resolution mass spectrometry (LC– HRMS), the number of detected compounds is increased in such a way that implementing advanced data processing methods could become inevitable (30).

It is clear now that despite the difficulty of calibrating passive samplers for quantitative analyses, they benefit from significant advantages such as time-weighted average data, greener analytical methods, and more time, effort, and cost-effective procedures that rationalize their implementation. That is the reason behind the spreading use of this sampling strategy, as can be seen in its progressive novel application fields. Regarding the general modality of this short review, the theoretical aspects and the entire range of the diverse application fields of passive sampling have not been covered and the interested reader is encouraged to refer to more comprehensive and specialized review articles (6).

(1) Godlewska, K.; Stepnowski, P.; Paszkiewicz, M. Pollutant Analysis Using Passive Samplers: Principles, Sorbents, Calibration and Applications. A Review. 

 (1), 465-520. DOI: 10.1007/s10311-020-01079-6

(2) Silvani, L.; Riccardi, C.; Eek, E.; Paini, M. P.; Morin, N. A. O.; Cornelissen, G.; Oen, A. M. P.; Hale, S. E. Monitoring Alkylphenols in Water Using the Polar Organic Chemical Integrative Sampler (POCIS): Determining Sampling Rates via the Extraction of PES Membranes and Oasis Beads. 

(3) Tapie, N.; Devier, M. H.; Soulier, C.; Creusot, N.; Le Menach, K.; Aït-Aïssa, S.; Vrana, B.; Budzinski, H. Passive Samplers for Chemical Substance Monitoring and Associated Toxicity Assessment in Water. 

 (10), 2418–2426. DOI: 10.2166/wst.2011.129 (acccessed 8/20/2022).

(4) Thomatou, Α.-Α.; Zacharias, I.; Hela, D.; Konstantinou, I. Passive Sampling of Selected Pesticides in Aquatic Environment Using Polar Organic Chemical Integrative Samplers. 

 (7), 1222-1233. DOI: 10.1007/s11356-010-0436-6

(5) Bartkow, M. E.; Jones, K. C.; Kennedy, K. E.; Holling, N.; Hawker, D. W.; Müller, J. F. Evaluation of Performance Reference Compounds in Polyethylene-Based Passive Air Samplers. 

(6) Salim, F.; Górecki, T. Theory and Modelling Approaches to Passive Sampling. 

 (10), 1618-1641. DOI: 10.1039/C9EM00215D

(7) Allinson, M.; Cassidy, M.; Kadokami, K.; Besley, C. H. In situ Calibration of Passive Sampling Methods for Urban Micropollutants Using Targeted Multiresidue GC and LC Screening Systems. 

(8) Huckins, J. N.; Tubergen, M. W.; Manuweera, G. K. Semipermeable Membrane Devices Containing Model Lipid: A New Approach to Monitoring the Bioavaiiability of Lipophilic Contaminants and Estimating Their Bioconcentration Potential. 

(9) Booij, K.; Sleiderink, H. M.; Smedes, F. Calibrating the Uptake Kinetics of Semipermeable Membrane Devices Using Exposure Sandards. 

(10) Lohmann, R. Critical Review of Low-Density Polyethylene’s Partitioning and Diffusion Coefficients for Trace Organic Contaminants and Implications for Its Use As a Passive Sampler. 

(11) Kingston, J. K.; Greenwood, R.; Mills, G. A.; Morrison, G. M.; Björklund Persson, L. Development of a Novel Passive Sampling System for the Time-Averaged Measurement of a Range of Organic Pollutants in Aquatic Environments. 

(12) Alvarez, D. A.; Stackelberg, P. E.; Petty, J. D.; Huckins, J. N.; Furlong, E. T.; Zaugg, S. D.; Meyer, M. T. Comparison of a Novel Passive Sampler to Standard Water-Column Sampling for Organic Contaminants Associated with Wastewater Effluents Entering a New Jersey Stream. 

(13) Chen, Y.; Pawliszyn, J. Kinetics and the On-Site Application of Standards in A Solid-Phase Microextration Fiber. 

(14) Schuster, J. K.; Gioia, R.; Breivik, K.; Steinnes, E.; Scheringer, M.; Jones, K. C. Trends in European Background Air Reflect Reductions in Primary Emissions of PCBs and PBDEs. 

(15) Paschke, H.; Popp, P. New Passive Samplers for Chlorinated Semivolatile Organic Pollutants in Ambient Air. 

(16) Esteve-Turrillas, F. A.; Ly-Verdú, S.; Pastor, A.; de la Guardia, M. Development of a Versatile, Easy and Rapid Atmospheric Monitor for Benzene, Toluene, Ethylbenzene and Xylenes Determination in Air. 

Passive samplers come in many forms and can be used in various fields. Here, we review multiple advances made to this sampling strategy.

Advances in Sample Preparation | LCGC Supplements

From “Green” to “Sustainable” Sample Preparation in Omics Studies in the Natural Product Field: Case Studies Dealing with Cannabis sativa L.

Onsite Environmental Extraction Based on Portable and Affordable Stirred Devices

Enhance the Performance of Solid-Phase Microextraction by Exploiting Vacuum-Assisted Headspace and Multicumulative Trapping for Olive Oil Characterization

Recent Advances and Applications of Passive Sampling Devices

Vacuum-Assisted Headspace Solid-Phase Microextraction Sampling Method for the Extraction of Semi-Volatile Compounds: An Overview