Overcoming Limitations in Food Analysis with Cryogen-free Trap Focusing: Enhancing Sensitivity, Selectivity, and Throughput

In the fields of food safety and product development, the ability to reliably detect and quantify trace‑level volatile and semi-volatile organic compounds (VOCs and SVOCs) is essential. Whether the aim is to ensure regulatory compliance by monitoring for toxic residues or to maintain consistent sensory profiles in consumer products, laboratories must deliver both analytical sensitivity and specificity, often across a wide concentration range.

Two techniques frequently used in these contexts are solid-phase microextraction (SPME) (1) and static headspace sampling (HS), both coupled to gas chromatography–mass spectrometry (GC–MS or GC–MS/MS). While these approaches are well established, they are not without limitations. When analytes are desorbed directly into the GC inlet—as is typically the case—volatile compounds may not refocus effectively at the head of the column, resulting in peak broadening, coelution, and loss of resolution. Moreover, in high-throughput laboratories, the need for labor-intensive methods such as QuEChERS, as recommended by EU regulations, can present logistical and cost challenges.

Consumer demand for traceability and minimal processing is also driving the adoption of techniques that avoid solvents, minimize thermal degradation, and reduce the risk of contamination. As the number of analytes under regulatory scrutiny increases, workflows must adapt to deliver higher sensitivity and better selectivity while maintaining throughput. These limitations have led researchers to explore improved focusing and enrichment techniques to boost sensitivity in headspace analysis (2).

One approach that addresses many of these issues is the use of multi-step enrichment on a cryogen-free focusing trap. This article will illustrate the benefits of integrating such focusing traps into HS and SPME workflows, drawing on case studies involving both aroma profiling and contaminant detection of cola and garlic samples.

Principles of Cryogen-free Trap Focusing

Trap-based enrichment is widely used in thermal desorption (TD) and dynamic headspace techniques, but has not traditionally been employed with SPME
or syringe-based HS sampling. However, by integrating a cryogen-free focusing trap into these workflows, analysts can combine the benefits of sorptive extraction with enhanced sensitivity and selectivity.

The trap is typically a short, narrow tube packed with a suitable sorbent material. Sorbent selection is dictated by the volatility range and polarity of the target analytes; multi-bed traps may be used to extend performance across diverse compound classes. Volatile analytes are transferred onto the trap during desorption of the SPME fiber or sampling from the vial headspace. A purge step follows to eliminate residual water or oxygen, which can interfere with GC–MS analysis.

The key advantage of the trap is its ability to retain analytes at a low temperature and then release them in a sharp thermal pulse. This is achieved by rapidly heating the trap, often at up to 100 °C/s, while maintaining a high carrier gas linear velocity. The resulting injection band is much narrower than that produced by direct desorption into the GC inlet, reducing band broadening and improving peak shape. This effect is particularly valuable for early-eluting compounds, which otherwise risk coelution or detection below the noise threshold.

Cryogen-free traps operate without the need for liquid cryogens. Instead, they use Peltier or mechanical cooling to reach sub-ambient temperatures. This eliminates safety risks, consumable costs, and the logistical burden of cryogen handling. When combined with automated sample introduction platforms, these traps enable workflows such as multi-step enrichment (MSE) and split-flow re-collection, which further enhance method sensitivity and dynamic range.

Aroma Profiling in Complex Matrices: Cola and Garlic

The first application explored the benefits of cryogen-free trap focusing for flavor and aroma profiling, using a carbonated cola drink and a commercial garlic powder. Both are challenging matrices: cola contains both volatile flavour compounds and interfering sugars, while garlic is rich in reactive sulphur species.

Each sample was prepared in 20 mL headspace vials: 5 mL of cola with 1 g of NaCl, and 1 g of garlic powder, which was weighed directly into the vial. Both were analyzed using SPME Arrow extraction with a polydimethylsiloxane/carbowax resin/divinybenzene (PDMS/CWR/DVB) sorbent phase. Vial equilibration and extraction were performed at 40 °C with agitation. The SPME Arrow was then either desorbed directly into the GC inlet (260 °C, 5 min, split 10:1) (“direct Arrow”), or via a focusing trap held at 20 °C and subsequently desorbed at 300 °C (“Arrow-trap”).

In cola, the direct desorption of the SPME Arrow to the GC (“direct Arrow”) method identified 58 volatile compounds. The focusing trap (“Arrow-trap”) method identified 89. This improvement was most evident among early-eluting compounds, which often exhibit band broadening without refocusing. One example, 3-furaldehyde, exhibited a signal-to-noise (S/N) ratio of 6.7 with Arrow-trap, compared to just 3.2 with direct Arrow. The improvement in peak shape also led to a better spectral match factor, reaching the threshold for confident identification (Figure 1).

A similar effect was seen in garlic. Direct Arrow desorption yielded 35 identifiable compounds; Arrow–trap increased this to 45. Sulfur-containing compounds, which are often labile and elute early, benefited from enhanced resolution. Acetic acid and allyl alcohol could only be baseline‑resolved when both peaks were sharpened by refocusing on the trap.

To further improve coverage, multi‑step enrichment (MSE) was employed for garlic. Instead of a single 10-min extraction, three 3.3-min extractions were performed from the same vial. Between each extraction, the Arrow was desorbed and its analytes collected on the trap. The focusing trap was held at a low temperature throughout and only desorbed once after the final Arrow desorption, so that analytes from all three extractions were injected into the GC system together. This process avoids saturation of the SPME phase and allows a greater total analyte load. As a result, the number of identifiable compounds increased from 45 to 55, including important aroma‑active species such as diallyl disulphide and 4-methylpyridine (Figure 2).

Trace-level Fumigant Analysis: EtO and ECH in Spices

Ethylene oxide (EtO) and epichlorohydrin (ECH) are fumigants used to sterilize spices and dried goods. However, their toxicity has led to strict regulations, with EU maximum residue limits set at 0.05 mg/kg for EtO in many commodities. Following a global product recall of contaminated sesame seeds, laboratories are seeking more sensitive, robust, and automatable methods to meet current EU regulations (3).

To address this, a headspace–trap method using cryogen-free focusing was tested on chilli, groundnut, and turmeric samples. Each 20-mL vial contained 2 g of finely ground sample, fortified with EtO or ECH across a range from 0.005–0.125 mg/kg. Extraction was performed by static headspace at 70 °C for 10 min, followed by three 5 mL HS withdrawals with a 3 min delay between each. The analytes were trapped on an ETO-specific focusing trap, purged, then desorbed at 250 °C for 3 min into the GC–MS/MS system.

Detection was carried out in multiple reaction monitoring (MRM) mode. EtO was monitored using transitions 44 > 29 and 44 > 15; ECH used 80 > 44 and 80 > 31. Excellent linearity was achieved across the calibration range (R² > 0.99), with reproducibility (RSD) under 20% and recoveries between 77–103%. Crucially, the method achieved quantitation at 0.005 mg/kg—10 times below the typical maximum residue limit (MRL) (Figure 3).

Compared to conventional QuEChERS workflows, which require solvent extraction, clean-up, and centrifugation, the HS–trap approach is faster, cleaner, and less labor-intensive. It also removes the risk of solvent suppression in electron ionization (EI) GC–MS and improves reproducibility through full automation.

Extending Concentration Range with High/Low Analysis

Another common challenge in food VOC analysis is the wide dynamic range of compound abundances. In cola, for instance, certain aroma compounds are highly abundant and risk overloading the GC column, while others are present at trace levels.

To overcome this, the split outlet during the initial Arrow–trap analysis (50:1 split ratio) was connected to a sorbent-packed re-collection tube. This tube was then analyzed using a lower split (5:1), allowing trace-level compounds to be visualized without repeating the SPME extraction (Figure 4).

This “High/Low” workflow provides an effective way to extend the dynamic range of analysis from a single vial. In practice, the initial chromatogram reveals major peaks with good symmetry and no overloading, while the re‑collection run captures minor peaks with enhanced sensitivity. This technique is particularly valuable when sample availability is limited or when instrument throughput is a priority.

Conclusion

Cryogen-free trap focusing provides a robust, flexible enhancement to both headspace and SPME–GC workflows. It improves chromatographic quality, enhances sensitivity, and facilitates detection across a wide concentration range. Through examples from both flavor profiling and contaminant monitoring, we have shown how integrating a focusing trap can resolve analytical challenges such as poor peak shape, coelution, phase saturation, and low analyte recoveries.

This approach also lends itself well to automation, cryogen-free operation, and advanced workflows such as multi‑step enrichment and high/low re-analysis. Together, these capabilities enable modern laboratories to meet the increasing demands of food analysis to deliver reliable, reproducible data with minimal
manual intervention. As regulations evolve and sample complexity
increases, methods that combine analytical rigor with operational
simplicity will be key to future-proofing laboratory workflows.

References

(1) Majors, R. E. Solid-Phase Microextraction: A Practical Guide. LCGC North Am. 2003, 21 (5), 424–431.

(2) Snow, N. H.; Bullock, J. M. Novel Approaches for Improving Sensitivity in Headspace Analysis. J. Chromatogr. A 2009, 1217 (16), 2726–2735. DOI: 10.1016/j.chroma.2009.01.015

(3) European Commission. SANTE/11312/2021: Analytical Quality Control and Method Validation Procedures for Pesticide Residues Analysis in Food and Feed; European Commission: Brussels, 2021. https://food.ec.europa.eu/system/files/2022-01/pesticides_mrl_guidelines_wrkdoc_2021-11312.pdf

Lucy Hearn received an MSc in green chemistry and sustainable industrial technologies from the University of York, UK, where her final-year project focused on the extraction and isolation of cannabinoids from hemp using supercritical CO₂. She later took up the role of application specialist at Markes, providing technical and application support to users around the world, and developing applications using extraction and enrichment techniques for GC–MS.

Rebecca Cole received a BSc in genetics from the University of Cardiff, UK, and subsequently undertook research for a PhD at the University of Bristol on the population genetics of parasitic nematodes. Subsequently, Rebecca joined Markes International as an application specialist and is involved in applications using extraction and enrichment techniques for GC–MS.