Comparing the HS-SPME, Vac-HS-SPME, and HiSorb Techniques

Researchers compared three headspace-based extraction techniques for analyzing volatile organic compounds (VOCs) in virgin olive oil: headspace solid-phase microextraction (HS-SPME), vacuum-assisted HS-SPME (Vac-HS-SPME), and HiSorb (a high-capacity sorptive extraction probe). Volatile compounds are central to olive oil's aroma and quality classification, with LOX-pathway-derived C5/C6 compounds contributing desirable green and fruity notes, while C7-C11 compounds are generally associated to off-flavors from oxidation or microbial activity.

While HS-SPME has been the standard method since the 1990s due to its solvent-free, sensitive extraction, it has limitations including reduced sensitivity for certain compounds and competitive adsorption effects. Vac-HS-SPME and HiSorb have been proposed to help overcome these shortcomings by improving extraction efficiency, but neither has been well-studied in olive oil matrices, and no prior work has directly compared all three techniques together.

The authors of the paper resulting from this study (which was published in Microchemical Journal¹) stated that their research provides the first systematic, controlled comparison of the three sampling methods on a representative sample, evaluating 34 target compounds for extraction coverage, signal intensity, and selectivity to better understand how each technique shapes the resulting volatile profile.

LCGC International spoke to Irene Digiglio andGiorgia Purcaro, lead and corresponding authors of that paper, about this work.

Why is comprehensive two-dimensional gas chromatography coupled with mass spectrometry (GC×GC-MS) particularly advantageous for analyzing volatile compounds in virgin olive oil compared to one-dimensional GC-MS?

Compared to conventional one-dimensional GC, the GC×GC-MS significantly advances virgin olive oil volatilomics by coupling two orthogonal stationary phases via a modulator. This approach drastically increases peak capacity and minimizes the co-elution of structurally related isomers, providing cleaner mass spectra for reliable identification. Additionally, the modulation process focuses the eluate into extremely narrow pulses, enhancing the signal-to-noise ratio (S/N) and boosting sensitivity for key trace compounds related to sensory profiles or geographical authenticity. Finally, by separating compounds across a two-dimensional space rather than a single time axis, GC×GC generates structured, chemically ordered retention patterns that simplify data interpretation and marker screening.^1,2

How do the extraction principles of headspace solid-phase microextraction (HS-SPME), vacuum-assisted (Vac-HS-SPME), and high-capacity sorptive extraction (HiSorb) differ, and how might these differences affect the chromatographic profile obtained for olive oil volatile organic compounds (VOCs)?

HS-SPME is a conventional equilibrium-based technique governed by the partition coefficients between the oil matrix, the headspace, and the fiber coating. Highly volatile, low-molecular-weight compounds are readily extracted as they easily partition into the gas phase. Conversely, semi-volatile constituents or those with higher thermodynamic affinity for the lipid matrix tend to remain preferentially retained in the liquid phase. Chromatographically, this typically results in a profile in which major, highly volatile green-note compounds are well-represented, while heavier volatile fractions may appear less prominent.

Vac-HS-SPME accelerates these mass transfer kinetics by reducing the internal pressure of the vial, lowering the transport resistance at the liquid-gas interface. This approach enhances the recovery of semi-volatile and heavier compounds without requiring excessive heating, which could otherwise induce thermal degradation artifacts in the oil. Consequently, it yields a chromatogram with a significantly enriched late-elution region.^3,4

HiSorb, on the other hand, shifts the thermodynamic equilibrium by using a high-capacity probe with a larger adsorbent phase volume compared to a standard fiber. This configuration maximizes absolute sensitivity and reduces competitive displacement effects across the entire volatile fraction.⁵

In summary, while standard HS-SPME provides a profile dominated by the major highly volatile compounds, Vac-HS-SPME broadens the analytical window toward fewer volatile constituents by accelerating their kinetic release, and HiSorb enhances overall peak areas, making it particularly suitable for comprehensive untargeted screening.

What factors should be considered when selecting an SPME fiber coating for the analysis of volatile compounds in olive oil?

When selecting an SPME fiber coating for olive oil volatile analysis, several critical factors must be considered, starting with the nature of the sample matrix and the specific analytical objective. Due to the complexity of the olive oil volatilome, which spans a wide range of volatilities, polarities, and molecular weights, the choice of the coating must align with the physicochemical properties of the target analytes.

A second key factor is the extraction mechanism, as SPME coatings can operate via absorption (liquid phases like PDMS), adsorption (porous solid phases like DVB and CAR), or a combination of both. The affinity between the analytes and these materials determines the extraction selectivity and the relative abundance of the recovered compounds. Particular attention to the extraction time is needed when the adsorption mechanism is exploited, as it can be subject to the displacement effect, thereby reducing the extraction yield of less-affine, and usually less abundant, compounds. Furthermore, coating thickness must be evaluated: while thicker coatings generally enhance thermodynamic capacity for semi-volatile constituents, they may influence equilibration times, whereas thinner coatings can facilitate the thermal desorption of higher-boiling compounds. Therefore, coating thickness represents a compromise between extraction efficiency, time, and analytical robustness.⁶

To overcome the limitations of individual phases and obtain a comprehensive volatilome profile, a mixed-trivalent coating — specifically DVB/CAR/PDMS — is generally preferred for olive oil analysis. The complementary nature of its three phases allows the system to simultaneously target compounds across diverse chemical classes by balancing absorption and adsorption mechanisms for low-, medium-, and high-molecular-weight volatiles.

Vac-HS-SPME reduces headspace pressure during extraction. How does this affect mass transfer kinetics, and which classes of volatile compounds would be expected to benefit most from this approach?

The effects of Vac-HS-SPME on mass transfer kinetics can be explained using the two-film theory, in which the extraction rate is governed by the boundary layer resistances at the sample-headspace interface. Reducing the internal pressure of the vial does not have a uniform effect on all volatile compounds; rather, it selectively accelerates the mass transfer of compounds that inherently have lower diffusion coefficients in the gas phase. Since diffusion coefficients in the gas phase are inversely proportional to pressure, lowering the total pressure dramatically increases molecular diffusivity in the headspace for these heavier or more polar analytes, thereby decreasing their gas-film resistance. In olive oil analysis, this kinetic advantage primarily benefits semivolatile compounds and high-boiling-point constituents, which are traditionally retained within the viscous lipid matrix due to their lower volatility and thermodynamic affinity for oil. In viscous matrices, such as olive oil, a beneficial combination is reduced pressure and milder temperature, which reduce matrix viscosity and thus mass transfer within the sample, accelerating the replenishment of the headspace. Chemical classes that typically benefit most from Vac-HS-SPME include sesquiterpenes (such as copaene, α-muurolene, and β-caryophyllene), along with aliphatic aldehydes, alcohols, and high molecular weight esters.^1,3,4

HiSorb employs a sorbent volume approximately 100 times larger than conventional SPME fibers. How might this increased extraction capacity influence chromatographic sensitivity, peak shape, and potential matrix effects?

The 100-fold increase in sorbent volume significantly alters the phase ratio, potentially shifting the thermodynamic equilibrium toward a much higher extraction capacity. This is expected to dramatically improve analyte mass accumulation, thereby increasing chromatographic sensitivity and facilitating the detection of trace or semivolatile compounds. Furthermore, the increased specific surface area could mitigate competitive displacement effects, potentially ensuring a more representative extraction of the entire volatile fraction.

From a chromatographic perspective, introducing a greater sorbent mass simply requires targeted optimization of thermal desorption and focusing parameters, such as using the system's dedicated trap, to ensure rapid transfer to the column and maintain sharp, narrow peaks.⁷

When analyzing complex food matrices such as virgin olive oil, what chromatographic challenges arise from the presence of hundreds of volatile compounds spanning a wide concentration range?

When analyzing complex matrices such as extra virgin olive oil, the presence of hundreds of volatile compounds with similar structures, boiling points, or functional groups often leads to co-elution and poorly resolved peaks, potentially compromising method specificity and quantification accuracy. Furthermore, the wide concentration range within the volatile mass introduces a significant challenge in terms of linear dynamic range. Optimizing a single injection to detect trace components often causes the chromatographic column to be overloaded by highly concentrated compounds and the mass spectrometer detector to saturate. Conversely, sample dilution to maintain major peaks within the linear range can push crucial trace markers below the signal-to-noise detection threshold.

Addressing these competing analytical requirements emphasizes the need for careful method optimization and the implementation of advanced instrument configurations. Therefore, the use of full two-dimensional gas chromatography (GC×GC) could lead to a significant improvement in separation power, dramatically increasing resolution and allowing the isolation of trace components that would otherwise remain hidden under more dominant signals.^1,2,8

Competitive adsorption is a known limitation of HS-SPME. How can chromatographic data be used to identify and evaluate competitive adsorption effects during method development?

The simplest approach to monitoring the possible presence of the competitive effect is to profile the extraction kinetics across multiple time points. Highly volatile compounds often occupy the fiber sites first; over time, analytes with higher specific affinity for the coating can competitively replace them. This results in a paradoxical decrease in the chromatographic peak areas of these compounds at longer extraction times.

Many key olive oil aroma compounds are present at trace levels (ng/kg). What chromatographic and sample preparation strategies would you use to maximize their detection and quantification?

To analyze olive oil aroma compounds at trace levels (ng/kg), preconcentration techniques are generally used. The most widely used is headspace solid-phase microextraction (HS-SPME). A trivalent DVB/CAR/PDMS mixed coating is typically chosen to cover a wide range of volatilities and polarities.⁸ However, extraction time and temperature require a careful trade-off: insufficient thermal conditions or timing can limit analyte release from the viscous lipid matrix, while excessive temperatures or prolonged times risk inducing artifacts or promoting competitive adsorption at the expense of minor components. From a separation perspective, comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC-MS) offers decisive advantages over conventional one-dimensional GC, drastically reducing co-elutions and increasing peak capacity.^1,3 The modulation process plays a crucial role in enhancing analytical sensitivity. Specifically, cryogenic modulation provides significant compression of the chromatographic band , which leads to signal-to-noise (S/N) ratio increase. While this beneficial effect is typically less pronounced with flow modulation, a noticeable improvement in sensitivity is still achieved, primarily driven by the enhanced overall separation efficiency. Finally, for reliable quantification, relying on a single internal standard is often insufficient because of variability in extraction efficiencies and detector responses. An effective strategy involves using a mixture ofinternal standards to carefully compensate for variations across chemical classes.⁸

If you were tasked with validating a GC×GC-MS method for olive oil volatilomics, what chromatographic performance parameters would you evaluate?

Once properly optimized, the validation protocol for a GC×GC method is not substantially different from that required for conventional one-dimensional GC-MS. The main exception is that two chromatographic separations must be considered instead of one; therefore, resolution must be systematically monitored in both the first and second dimensions.

Suppose Vac-HS-SPME and HiSorb produce substantially different chromatographic fingerprints for the same olive oil sample. How would you determine whether these differences arise from extraction selectivity, extraction capacity, chromatographic separation, or detector response?

To determine whether fingerprint discrepancies between Vac-HS-SPME and HiSorb arise from extraction selectivity, capacity, chromatographic separation, or detector response, a systematic diagnostic workflow can be applied. First, comparing the relative abundances of distinct chemical classes or compounds reveals extraction selectivity, where preferential enrichment of specific classes implies underlying thermodynamic or kinetic shifts.^1,3 Conversely, extraction capacity can be isolated by evaluating absolute peak areas; a uniform and proportional increase in signal across the entire volatilome indicates that the variations are driven primarily by the larger phase volume of HiSorb, while non-proportional shifts between major and minor constituents indicate competitive displacement or vacuum-accelerated mass transfer. From an instrumental (i.e. chromatographic separation) point of view, unless column capacity overload occurs, which is usually controlled during optimization by adjusting the split ratio, systematically running a quality control sample ensures that the observed difference results exclusively from the unique characteristics of the sample preparation technique used.

References

1. Digiglio, I.;Damien Eggermont, D.; Natasha Damiana Spadafora, N. et al. Comparison of Headspace Pre-Concentration Techniques for Volatile Profiling of Virgin Olive Oil by Comprehensive Two-Dimensional Chromatography. Microchem. J. 2026, 226, 118339. DOI: 10.1016/j.microc.2026.118339
2. Chen, E.; Ma, Z.; Geng, X. et al. Comprehensive Two-Dimensional Gas Chromatography Technique and Its Applications in Fermented Food Aroma Analysis: A Review. Food Biosci. 2024, 62, 105473.DOI:10.1016/j.fbio.2024.105473
3. Mascrez, S. et al. Vacuum-Assisted and Multi-Cumulative Trapping in Headspace Solid-Phase Microextraction Combined with Comprehensive Multidimensional Chromatography-Mass Spectrometry for Profiling Virgin Olive Oil Aroma.” Food Chem. 2024, 442, 138409. DOI:10.1016/j.foodchem.2024.138409
4. Psillakis, E. Vacuum-Assisted Headspace Solid-Phase Microextraction: A Tutorial Review. Anal. Chim. Acta 2017, 986, 12–24, DOI: 10.1016/j. aca.2017.06.033
5. Hearn, L.; Cole, R.; Spadafora, N. D. et al. Volatile and Semi-Volatile Compounds in Flavoured Hard Seltzer Beverages: Comparison of High-Capacity Sorptive Extraction (HiSorb) Methods, Adv. Sample Prep. 2022, 3, 100032. DOI:10.1016/j.sampre.2022.100032.
6. Shirey, R. E. SPME Commercial Devices and Fibre Coatings in Handbook of Solid Phase Microextraction; J. Pawliszyn, Ed. Elsevier, 2012. DOI: 10.1016/B978-0-12-416017-0.00004-8
7. Paiva, A. C., Crucello, J., de Aguiar Porto, N. et al. Fundamentals of and Recent Advances in Sorbent-Based Headspace Extractions. Trends Anal. Chem. 2021,139, 116252. DOI: 10.1016/j.trac.2021.116252
8. Cecchi, L.; Migliorini, M.; Mulinacci, N. Virgin olive Oil Volatile Compounds: Composition, Sensory Characteristics, Analytical Approaches, Quality Control, and Authentication. J. Agric. Food Chem. 2021, 69 (7), 2013–2040. DOI: https://doi.org/10.1021/acs.jafc.0c07744.
9. Stilo, F.; Segura Borrego, M. D. P.; Bicchi, C. et al. Delineating the Extra-Virgin Olive Oil Aroma Blueprint by Multiple Headspace Solid Phase Microextraction and Differential-Flow Modulated Comprehensive Two-Dimensional Gas Chromatography. J Chromatogr A.2021, 1650, 462232. DOI:10.1016/j.chroma.2021.462232