Gas Chromatography-Ion Mobility Spectrometry for Characterizing Different Species of Origanum and Detecting Blends of Its Products

Gas chromatography-ion mobility spectrometry (GC-IMS) has emerged as a complementary technique to GC-MS for food-aroma analysis and offers several advantages for aroma profile characterization. Applications include the authentication of olive oils, (14) monofloral honeys, (15) and many more. (16)

Many GC-IMS approaches in food analysis use HS sampling. (16) It eliminates the need for sample preparation and is optimal for transferring the compounds of interest into the GC system. After GC separation, IMS provides an additional dimension via drift time. Furthermore, it is a very sensitive, less costly method compared to a conventional quadrupole MS. Unfortunately, the capabilities for identifying compounds in IMS are limited, as it lacks the powerful databases that MS have. Therefore, in the analytical setup used, a quadrupole MS is installed on a separate flow line, as described by Budzyńska et al. (17) and Augustini et al. (18) Coupled with the NIST MS database and the peak-assignment approach of Augustini et al., (18) this method enables reliable identification of most GC-IMS signals. Using this HS-GC-MS/-IMS configuration, it is demonstrated how GC-IMS can characterize Origanum volatilomes by analyzing five (sub)species, including one species represented by material from two geographic origins. To minimize environmental effects on volatile expression, reference seeds were grown under identical conditions in an indoor greenhouse prior to analysis. This approach helps to compare chemotypic differences among taxa and origins under controlled growth conditions.

Beyond chemotaxonomy and quality control, a further motivation for robust analytical fingerprinting of Origanum is the widespread issue of oregano adulteration. According to the European Spice Association, only the leaf product derived from Origanum vulgare ssp. hirtum and Origanum onites may be called oregano, while the International Organization for Standardization defines oregano in ISO 7925:1999–10 as leaves from the genus Origanum except Origanum majorana. ISO also specifies that the proportion of stalks and other plant parts shall not exceed 3% and that extraneous matter shall not exceed 1%, a requirement often violated in commercial oregano for economic reasons. Maquet et al. (19) reported that 48% of oregano samples collected across the supply chain in the European Union were adulterated. Common adulterants include leaves from Cistus spp. for a dark green color, from olive (Olea europaea), and myrtle (Myrtus communis) for financial reasons. Another possibility could be an accidental admixture with other herbs, such as marjoram (O. majorana) or thyme (Thymus spp.). (20) These types of food fraud are challenging for consumers and regulatory authorities to identify. One possibility is to analyze leaf material macroscopically or microscopically for foreign matter. (21) However, a variety of analytical approaches have been developed to detect oregano adulteration. Fourier-transform infrared (FT-IR) spectroscopy has been used to screen for olive, myrtle, sumac, hoary rock-rose, and hazelnut leaf adulterants. (22) Flügge et al. (23) combined next-generation sequencing (NGS) with nuclear magnetic resonance (NMR) spectroscopy to detect olive, myrtle, hazelnut, and pink rock-rose (Cistus) leaves. Liquid chromatography–mass spectrometry (LC-MS) targeting oleuropein has been developed specifically to identify olive leaf adulteration (24) and is also used by Maquet et al. (19) as an analytical method for an EU-wide control of adulteration. Rocomora-Rivera et al. (25) were the first to apply GC-IMS for the detection of oregano adulteration. Using orthogonal partial least-squares discriminant analysis (OPLS-DA), they identified admixtures containing olive leaves, but they did not test other potential adulterants. They also applied the method to commercially available oregano samples. However, their results were not verified by other analytical techniques. In the presented study, the feasibility of GC-IMS for detecting common adulterants in oregano, namely the leaves of hoary rock-rose, olive, myrtle, thyme, and marjoram, is tested by identifying marker compounds. Because only one authentic oregano type and no commercially adulterated samples were available, blends were prepared by combining oregano with candidate adulterant leaf material to simulate adulteration scenarios.

The objectives of this study are to evaluate HS-GC-IMS for volatilome profiling of five Origanum (sub)species grown under controlled conditions and assess the technique’s ability to detect common leaf adulterants in oregano blends.

An AOC5000 (Shimadzu Corporation, Kyoto, Japan) HS sampler incubating for 30 min at 120 °C was used for the static HS analysis of the samples. 500 μL of the HS was injected into a GC 2010-Plus (Shimadzu Corporation), equipped with two split injectors with a split of 1:20 and two identical analytical columns (HP-5 MS UI 30 m × 0.25 mm × 0.5 μm; Agilent, Santa Clara, CA, USA), each leading to one spectrometer (IMS or MS). A constant flow rate (35 cm/s) of helium 5.0 (Messer Industriegase, Siegen, Germany) was used. The GC temperature program started at 40 °C, held for 4 min, then heated with 3 °C/min to 150 °C, then heated 10 °C/min to 220 °C, held for 12.33 min, for a total program time of 60 min.

One analytical column was connected to the quadrupole MS QP-2010 Ultra (Shimadzu Corporation) in scan mode (m/z 32–300 at 3.3 Hz) with electron ionization (70 eV) and the other to the ion mobility spectrometer (G.A.S. Gesellschaft für analytische Sensorsysteme mbH, Dortmund, Germany). The drift tube (dimensions: 15.2 mm × 53 mm; temperature: 80 °C) was flushed with 75 mL/min drift gas (nitrogen 5.0, produced by an in-house generator), and ionization was performed by atmospheric-pressure chemical ionization with a ³H source. The field strength was 500 V/cm.

3. Results and Discussion

3.1. Identification of Volatiles in Origanum

3.4. Comparison of the Species

The first main topic of this study is to investigate species-specific variations in the volatilome. The HS-GC-IMS chromatograms for each species are illustrated in Figure 6, where all Origanum species can be compared visually. Distinct differences in peak pattern and intensity between the samples are evident.

ACS Food Sci. Technol. (2026): Figure 6. Headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS) chromatograms of five different species, one from two origins, Germany (DE) and Greece (GR), with the drift time on a reactant ion peak (RIP) relative scale (RIP position: ∼4.9 ms), the retention time in seconds on the y-axis, and the intensity in V on a color scale.

It can be seen that the species O. vulgare ssp. hirtum contains more monoterpenes/monoterpenoids, especially carvacrol, than O. vulgare ssp. vulgare, which shows higher amounts of sesquiterpenes. This comparison provides insight into the profile of each species, as even two different plants of the same species can exhibit distinct profiles.

3.5. Detection of Blends in Oregano

After investigating the volatilomes of different Origanum species, the potential of this technique to detect food fraud can be demonstrated by comparing the volatilomes of oregano and its adulterants. The capabilities and limitations of GC-IMS for identifying oregano adulteration were assessed within this feasibility study by using self-made blends, as no intentionally fraudulent samples were available. For this purpose, common adulterants for oregano, namely leaves from olive, thyme, marjoram, myrtle, and hoary rock-rose, were added in different mass ratios (10%, 30%, and 50%) to ground oregano known to be pure and from O. vulgare ssp. hirtum. The sample material is shown in Figure 9 as a photograph.

ACS Food Sci. Technol. (2026): Figure 9. Photograph of the sample material: Oregano (O. vulgare ssp. hirtum), olive (O. europaea L.), thyme (Thymus vulgaris L.), marjoram (O. majorana L.), myrtle (M. communis L.), hoary rock-rose (C. incanus L.).