Cryogenic-zone-compression gas chromatography-mass spectrometry for the determination of 16 polycyclic aromatic hydrocarbons in extra virgin olive oil

The goal of this study was to develop a straightforward and green analytical method for the semi-quantitative determination of 16 polycyclic aromatic hydrocarbons (PAHs) in extra virgin olive oil. The method uses cryogenic-zone-compression gas chromatography coupled with single quadrupole mass spectrometry (CZC GC-QMS), allowing for a significant enhancement in signal-to-noise ratio and simplifying the sample preparation to a single extraction step using only 500 µL of acetonitrile.

This approach aligns with green analytical chemistry principles, offering improved sensitivity—an average 14-fold increase in s/n ratio—while maintaining satisfactory limits of quantification, accuracy, and precision. The method was applied to ten EVOO samples, where trace levels of PAHs were detected in six of them.

The original article

Cryogenic-zone-compression gas chromatography-mass spectrometry for the determination of 16 polycyclic aromatic hydrocarbons in extra virgin olive oil

Alessia Arena, Mariosimone Zoccali, Peter Q. Tranchida, Luigi Mondello 

Journal of Chromatography A, Volume 1732, 13 September 2024, 465248

https://doi.org/10.1016/j.chroma.2024.465248

licensed under CC-BY 4.0

Selected sections from the article follow. Formats and hyperlinks were adapted from the original.

Humans are exposed to a wide range of chemicals throughout their lifetimes, such as polycyclic aromatic hydrocarbons (PAHs) which require particular attention due to their toxicity [1]. PAHs constitute a class of organic compounds composed of two or more aromatic rings, primarily formed through incomplete combustion or pyrolysis of organic matter [1]. One of the main routes of exposure is through food, particularly fatty foods, given the lipophilic nature of these contaminants [2]. Sixteen PAHs have been highlighted by the International Programme on Chemical Safety, the Scientific Committee on Food, and the Joint FAO/WHO Expert Committee on Food Additives as mutagenic and genotoxic in somatic cells, and have also shown clear carcinogenic effects [1]. According to the European (EU) Regulation 835/2011, a maximum level (ML) of 2.0 μg kg⁻¹ for benzo[a]pyrene (BaP) and of 10.0 μg kg⁻¹ for the sum of BaP, benz[a]anthracene (BaA), chrysene (CHR), and benzo[b]fluoranthene (BbFA) has been established for fats and oils [3]. At the same time, following the Green Analytical Chemistry guidelines, it is crucial to develop environmental-friendly analytical methods that do not trigger further damage towards the environment and humans [4].

Sample preparation has a strong impact on the environment, and many efforts have been made to reduce such an effect. In particular, in recent years the present Authors have focused attention toward a reduction of solvent use during analyte extraction, accompanied by the elimination of purification and final concentration steps [[5], [6], [7], [8]]. In such a respect, an additional approach that can enable the avoidance of purification/concentration steps, along with a reduction of sample solution volumes injected (leading to a reduced need for instrumental maintenance), consists in "cryogenic solute trapping", proposed by Marriott and Kinghorn in 1996 [9]. The approach allowed for an increase in signal-to-noise ratio (s/n) values prior to detection, by using a cryogenic trap positioned at the end of the capillary column. Analyte release was enabled by thermal desorption from the GC oven heat. The same concept was applied in 2011 by Patterson et al. for the measurement of dioxins in human serum [10], and in 2016 by L'Homme and Focant for the analysis of polychlorinated biphenyls and dichlorodiphenyldichloroethylene in serum samples [11]. In both cases, a liquid nitrogen jet-cooled thermal modulator used for comprehensive two-dimensional gas chromatography (GC×GC) was exploited, and this approach was defined as "cryogenic-zone-compression" (CZC). It is noteworthy that, prior to the invention of GC×GC itself [12], Phillips and co-workers used thermal desorption modulation for the scope of analyte concentration and release [13].

The objective of the present study was to develop a CZC gas chromatography-single quadrupole mass spectrometry (GC-QMS) method for the determination of 16 PAHs in extra virgin olive oil (EVOO). A total of 10 commercial EVOOs were subjected to investigation. The liquid-liquid extraction process involved the use of 500 µL of acetonitrile. The quali/semi-quantitative PAH determination was performed in the selected-ion-monitoring mode (SIM). The developed method, which can be considered as a rapid green or red light one, was characterized by sufficient analyte detectability in relation to regulated PAH concentration limits.

2. Materials and methods

2.2. CZC GC-QMS conditions

The CZC GC-QMS system was composed of a GC oven (GC-2010, Shimadzu Corporation, Kyoto, Japan), a single quadrupole mass spectrometer (QP2020, Shimadzu). The GC-QMS system was equipped with an AOC-20i autosampler, and a Zoex II (liquid nitrogen-free modulator) cryogenic modulator. The split/splitless injector (350 °C) contained an empty liner (volume: 860 μL). The injection volume was 3 μL, in the splitless mode, using the “high-pressure injection mode” function (400 kPa for 0.5 min).

The analytical column was an SLB-PAHms [silphenylene polymer, virtually equivalent in polarity to poly (50 % diphenyl/50 %methylsiloxane)] of dimensions 30 m × 0.25 mm I.D. × 0.10 μm d_f (Merck Life Science). The GC oven temperature was programmed as follows: 110 °C (0.5 min) to 200 °C at 30 °C min⁻¹, increased to 350 °C at 5 °C min⁻¹ (for 10 min), with a total run time of 43.5 min.

To achieve the CZC approach, the end of the analytical column was looped once using the metal support of the cryogenic modulator (Figure S1). A “compression” period of 18 s was applied, with a hot pulse (400 °C) of 350 ms. The cold jet flow was 9 L min⁻¹ (−90 °C).

Helium was used as carrier gas; after the high-pressure injection step, the initial pressure was 136.6 kPa (constant linear velocity mode: 50 cm s⁻¹).

Electron ionization was performed at 70 eV. The interface and ion source temperatures were 350 and 280 °C, respectively. The target analyses were carried out in the SIM acquisition mode, with an acquisition frequency of 10 and 100 Hz for the conventional and CZC GC-QMS analysis, respectively. The GCMS Solution v.4.45 software (Shimadzu Corporation) was used for data collection and processing.

3. Results and discussion

3.1. Development of the CZC GC-QMS method

For the development of the CZC GC-QMS method, a cryogenic modulator (loop-type) designed for GC×GC was utilized. Unlike GC×GC, where continuous and sequential modulation is carried out [14], with CZC the aim is to entrap/compress each compound in a single cooling period and rapidly release it with a hot pulse. In such a manner, band broadening is practically nullified allowing for a great increase in s/n values (and reduction of detection limits).

Considering that the modulator used does not allow for programmed variation in the cold and hot jet flows during the analysis, the initial step of the research was focused on fine tuning of the cold jet flow (ranging from 5 to 16 L min⁻¹) and on entrapment time duration; the temperature (ranging from 300 to 450 °C) and duration (ranging from 100 to 500 ms) of the hot jet were also finely optimized. This, to efficiently entrap and release each compound within a single cold-hot cycle. The efficient entrapment of the lowest boiling-point PAH (BcFL) was achieved using a cold jet flow of 9 L min⁻¹. On the other hand, the release of the highest boiling-point PAH was performed by using a hot jet pulse of 350 ms and a temperature of 400 °C.

The duration of the compression period of 18 s was experimentally optimized, considering both the widest peak base width (10.2 s considering the partially co-eluting compounds IP and DBahA) and potential retention time shifts. The final outcome consisted of PAH peaks with a base width in the 120–420 ms range, with an average value of 217 ms. Instead, without using CZC, the peak base widths were in the 4.8–8.4 s range, with an average value of 6.5 s (without considering the sum of IP and DBahA). Therefore, considering the average values for both the approaches, a compression factor of about 30 times was obtained.

The use of the SIM mode (used in all stages of this research) enabled both an enhancement of analyte detectability and the use of an acquisition frequency (100 Hz) sufficient to generate an adequate number of data points (a minimum of 12 were attained) for reliable quantification. It should be noted that for isobaric compounds, the SIM mode may introduce errors in quantification if the components of interest are not baseline resolved. For this reason, considering the partial co-elution between IP and DBahA, the two compounds were trapped together, released in a single pulse, and then quantified as a sum. However, the current EU legislation does not report a specific ML, for such compounds, therefore not affecting the validity of the proposed method [3].

Fig. 1 reports the chromatograms obtained by using the CZC GC-QMS approach (lower signals) in comparison with the one acquired without CZC (upper signals), analyzing a blank EVOO sample spiked at the 10 µg kg⁻¹ concentration level. Fig. 2 reports four zoomed-in segments of the two chromatograms shown in Fig. 1, involving BaA, CPP, CHR and the IS, along with BaP. The CZC GC-QMS and GC-QMS chromatogram segments are characterized by a different y-axis maximum intensity, to enable an easier visual comparison between the two methods.

Journal of Chromatography A, Volume 1732, 2024, 465248: Fig. 1. Comparison of the two investigated approaches-conventional GC-QMS

Journal of Chromatography A, Volume 1732, 2024, 465248: Fig. 2. Conventional GC-QMS and CZC GC-QMS zoomed-in segments from Fig. 1 (abbreviations are defined in Section 2).

4. Conclusions

The CZC-GC-QMS method herein proposed has a demonstrated effectiveness for the determination of PAH contamination in EVOO, enabling a notable reduction in the sample preparation process and solvent usage. An analyte detectability comparison between conventional GC-QMS and CZC-GC-QMS revealed the superior performance of the latter approach. Notwithstanding the high number of analysis carried, no degradation of the column and MS performances were observed. In future studies, the CZC-GC-QMS approach will be exploited to target analyze various other types of food and biological samples.