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Article

Simultaneous Determination of Polycyclic Aromatic Hydrocarbons and Anthraquinone in Yerba Mate by Modified MSPD Method and GC-MS

by
Dylan M. Hoffmann
,
José D. da Silva
,
Igor F. de Souza
,
Gabriel A. B. Prates
,
Vagner A. Dutra
,
Osmar D. Prestes
and
Renato Zanella
*
Chemistry Department, Federal University of Santa Maria (UFSM), Santa Maria 97105-900, RS, Brazil
*
Author to whom correspondence should be addressed.
Separations 2025, 12(9), 240; https://doi.org/10.3390/separations12090240
Submission received: 3 August 2025 / Revised: 28 August 2025 / Accepted: 2 September 2025 / Published: 4 September 2025
(This article belongs to the Topic Advances in Analysis of Food and Beverages, 2nd Edition)
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Abstract

Yerba mate (Ilex paraguariensis) is widely consumed in South America and is valued for its bioactive compounds, such as polyphenols and methylxanthines. However, during traditional processing, mainly in the fire-based scorch and drying steps, polycyclic aromatic hydrocarbons (PAHs) and anthraquinone (AQ), substances with carcinogenic potential, may be formed. This study aimed to develop and validate an analytical method based on the balls-in-tube matrix solid-phase dispersion technique (BiT-MSPD) and analysis by gas chromatography with mass spectrometry (GC-MS) for the simultaneous determination of 16 priority PAHs and AQ in yerba mate. Parameters such as sorbent type, solvent, sample-to-sorbent ratio, and extraction time were optimized. The method showed good linearity (r2 > 0.99), detection limits between 1.8 and 3.6 µg·kg−1, recoveries ranging from 70 to 120%, and acceptable precision (RSD ≤ 20%). The method was applied to 31 yerba mate samples, including 20 commercial samples and 11 collected at different stages of processing. Most commercial samples showed detectable levels of PAHs, with some exceeding the limits established by the European Union. AQ was detected in 40% of the samples, with some values above the permitted limit of 20 µg·kg−1. The results confirm that scorch (sapeco) and drying contribute to contaminant formation, highlighting the need to modernize industrial processing practices. The proposed method proved to be effective, rapid, and sustainable, representing a promising tool for the quality control and food safety monitoring of yerba mate.

Graphical Abstract

1. Introduction

Yerba mate (Ilex paraguariensis), a plant native to South America, plays an important role in the culture and economy of countries such as Brazil, Argentina, Paraguay, and Uruguay, and is traditionally consumed in the form of chimarrão, tereré, and mate tea [1,2]. In addition to its popular consumption, yerba mate has been widely used in the food, cosmetic, pharmaceutical, and agricultural industries due to its rich phytochemical composition and functional properties [3,4,5].
Its chemical composition includes phenolic compounds, saponins, methylxanthines, and minerals, and it is noted for its antioxidant, anti-inflammatory, hypoglycemic, and stimulant potential [2,6]. Polyphenols, such as chlorogenic acids, and methylxanthines, such as caffeine and theobromine, are primarily responsible for these effects. The concentration of these compounds varies significantly depending on the cultivation system, soil and climate conditions, processing methods, and harvest season [7,8,9].
Despite its health benefits, recent studies warn of the contamination of yerba mate by toxic compounds, such as polycyclic aromatic hydrocarbons (PAHs) and anthraquinone (AQ), especially as a result of the scorch (sapeco) and fire drying stages, commonly carried out with wood burning [10,11]. PAHs are persistent organic compounds formed by the incomplete combustion of organic matter and are associated with carcinogenicity, mutagenicity, and bioaccumulation [12,13]. Exposure to these contaminants can occur through ingestion, inhalation, or skin contact, with smoked and dried foods being the main food vehicles [12,14].
Among the more than 100 existing PAHs, 16 compounds (Table 1) have been classified by the European Food Safety Authority (EFSA) as priorities for monitoring in food due to their toxicity and frequent occurrence, with emphasis on benzo[a]pyrene (BaP), considered a risk marker due to its proven carcinogenicity (Group 1 by IARC) [11,15]. Initially, the regulatory limit in the European Union considered only BaP, but since 2011, Regulation (EU) No. 835/2011 has established that the sum of four PAHs (BaP, benzo[a]anthracene, chrysene, and benzo[b]fluoranthene) should not exceed 50 µg·kg−1 in dried herbs, while the individual limit for BaP remains at 10 µg·kg−1. This change was motivated by the observation that the presence of other toxic PAHs was common even in the absence of BaP [12,16]. Such regulations aim to reduce the chronic exposure of the population to these compounds and are indicative of the importance of sensitive analytical methods for their monitoring.
Anthraquinone, in turn, is an oxygenated polycyclic aromatic hydrocarbon used in various industries, such as paper, dyes, and agriculture. Despite its widespread application, AQ is classified as possibly carcinogenic (IARC Group 2B) (Table 1) and its maximum residue limit (MRL) has been set at 0.02 mg·kg−1 in teas by the European Union, based on the minimum analytical determination limit [11,16,17].
Regular consumption of yerba mate infusions, often at high temperatures, increases the risk of adverse effects, not only due to the presence of contaminants, but also due to thermal damage to the esophageal mucosa, which can promote the action of carcinogenic compounds. Epidemiological studies conducted in Brazil, Paraguay, Argentina, and Uruguay point to an association between yerba mate consumption and an increase in the incidence of cancer of the esophagus, mouth, and pharynx [2,18].
In this context, monitoring and controlling the presence of PAHs and AQ in yerba mate products is essential. However, the complexity of the plant matrix and the low concentration of analytes require sensitive and selective analytical methods. Sample preparation is a critical step in this process, as it should promote the efficient extraction of target compounds and the removal of matrix interferents, such as chlorophylls and phenolic compounds, which can compromise the selectivity and detectability of the method [19,20,21]. Soxhlet extraction, although efficient, requires long analysis times (up to 24 h) and large volumes of solvents [22]. Alternative methods, such as ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE), have been explored for their speed and lower solvent consumption [23]. Liquid–liquid extraction (LLE) is still used, but it has limitations in terms of selectivity and the need for additional purification steps [24]. More modern techniques, such as the QuEChERS method, have shown promise, especially when combined with d-SPE, using sorbents such as PSA, C18, Florisil, or diatomaceous earth [25,26]. However, these approaches may still have limitations in the extraction of lipophilic and heat-resistant compounds such as PAHs. Several techniques have been used in the literature [27,28,29,30,31,32,33,34,35,36,37,38,39,40] to determine PAHs and AQ in food and plants, as highlighted in Table S1.
The matrix solid-phase dispersion (MSPD) technique, introduced by Barker et al. [41] in the 1980s, has established itself as an effective and versatile alternative for the preparation of complex samples, especially those of plant, animal, or food origin. Unlike conventional methods, MSPD allows for the simultaneous combination of extraction, homogenization, and purification of analytes in a single step. This is possible by directly mixing the solid sample with an appropriate sorbent (such as C18, PSA, Florisil, or diatomaceous earth), forming a dispersed matrix accessible to the extracting solvent. The choice of sorbent directly influences the purification capacity of the matrix [42,43,44]. Among the main advantages of MSPD are low consumption of organic solvents, reduced preparation time, minimization of successive purification steps, and compatibility with modern chromatographic systems, such as GC-MS and LC-MS. In addition, the technique is easily adaptable to different analytical scales, making it particularly attractive for laboratories that handle a high number of samples and seek more sustainable, selective, and economically viable methods [45,46,47].
In this work, we propose the development and validation of an analytical method based on a modification of the MSPD technique, called BiT-MSPD (balls-in-tube matrix solid-phase dispersion) [43], associated with gas chromatography coupled with mass spectrometry (GC-MS), for the simultaneous determination of 16 priority PAHs and anthraquinone in yerba mate samples. The establishment of a comprehensive method is important because it will reduce the time and cost of analyzing these compounds in yerba mate. The proposed method seeks to combine analytical efficiency with sustainability, with a view to application in quality control and food safety programs.

2. Materials and Methods

2.1. Reagents and Materials

The reagents, solvents, and materials used in this study were as follows: HPLC-grade acetonitrile (J.T. Baker, Ecatepec, Mexico); ethyl acetate (Scharlab, Sentmenat, Spain); HPLC-grade acetone (J.T. Baker, Ecatepec, Mexico); hexane (95% n-hexane, J.T. Baker, Ecatepec, Mexico); ultrapure water obtained by a Milli-Q Direct 3UV® system (Millipore, Molsheim, France) with a resistivity of 18.2 MΩ·cm−1; Bondesil C18 sorbent, with a particle diameter of 40 μm (Sigma-Aldrich, San Luis, CA, USA); Bondesil PSA sorbent, with a particle diameter of 40 μm (Agilent Technologies, Santa Clara, CA, USA); Florisil® sorbent (Agilent Technologies, Santa Clara, CA, USA); diatomaceous earth sorbent and calcium chloride (Sigma-Aldrich, San Luis, CA, USA); polypropylene tubes with capacities of 15 and 50 mL (Sarstedt, Sarstedt, Germany); ceramic homogenizing rods (Agilent Technologies, Santa Clara, CA, USA); 2 and 5 mL Eppendorf microtubes (Eppendorf, Hamburg, Germany); 2 mL glass vials (Agilent Technologies, Santa Clara, CA, USA); Extran® neutral and alkaline cleaning agents (Merck, Rio de Janeiro, Brazil); analytical standards of the compounds under study (Table S2); and general laboratory glassware.

2.2. Yerba Mate Samples and Application of the Proposed Method

The analytical method evaluation and validation tests were performed with “blank” yerba mate samples. For processing, 1 kg of each sample sold in the city of Santa Maria-RS, Brazil, from different brands, was purchased. The samples were only sieved to remove the yerba mate stems, except for the pure leaf type herbs, where no process was performed, and then placed in frasks with a capacity of 200 g and kept at a temperature of −10 °C.
The method developed was validated and applied to determine PAHs and anthraquinone in 31 samples of yerba mate, including 20 commercial samples from nine different cities (identified as EV01 to EV20), three of which were pure leaf type and the rest traditional type, and 11 samples obtained at different stages of yerba mate processing (identified as YM01 to YM11). All samples were purchased in July 2025 at different establishments in the state of Rio Grande do Sul, Brazil, with different brands and batches.

2.3. Instrumentation

The analyses and experimental procedures were performed using the following equipment: Milli-Q Direct 3UV® water purification system (Millipore, L’Isle D’Abeau Chesnes, France); refrigerated centrifuge for 15 and 50 mL tubes (NT 825, Novatécnica, São Paulo, Brazil); vortex mixer model VX-38 (IONLAB, Araucária, Brazil); multi-turbo vortex mixer (Fisher Scientific, Waltham, MA, USA); UX-420H and APX-200 analytical balances (Shimadzu, Kyoto, Japan); automatic micropipettes with variable volumes (Brand, Brand, Germany; and Eppendorf, Hamburg, Germany); Polytron PT 3100 homogenizer (Kinematica, Malters, Switzerland); and gas chromatography coupled with mass spectrometry (GC-MS) system model QP2010 SE from Shimadzu (Kyoto, Japan).

2.4. GC-MS Analysis

The chromatographic conditions for the analysis of polycyclic aromatic hydrocarbons (PAHs) and anthraquinone (AQ) were established based on the method described by Smith and Lynam [48], with some specific modifications to adapt to the yerba mate matrix. Table S2 shows the retention time of the compounds with their monitored ions and Figure S2 shows a GC-MS chromatogram in SIM mode, from an analytical solution extracted from the blank matrix spiked at 240 µg kg−1. The compounds were separated by GC and identified and quantified by mass spectrometry (MS). It can be seen that compounds 15 and 16 appear overlapping on the chromatogram, but the overlapping compounds have ions with different mass/charge ratios (m/z), making it possible to detect and quantify both using GC-MS without interference. The analyses were performed using gas chromatography coupled with mass spectrometry (GC-MS) using an SH-Rtx-5MS capillary column (30 m × 0.25 mm × 0.25 µm). The oven temperature program started at 50 °C (maintained for 0.4 min), with an increase of 25 °C·min−1 to 195 °C, followed by an increase of 10 °C·min−1 to 265 °C (maintained for 1 min), and then an increase of 20 °C·min−1 to 305 °C, which was maintained for 7.8 min. The total analysis time was 24 min. The injector temperature was set to 320 °C, with 1 µL injection in splitless mode. The carrier gas used was helium (grade 6.0), with a constant flow rate of 1.7 mL·min−1. The ionization source temperature was 260 °C and the interface temperature was 330 °C.

2.5. Sample Preparation Evaluation

The sample preparation method proposed in this study was developed based on the technique named BiT-MSPD described for the multiresidue determination of pesticides in food [43]. However, due to the specific characteristics of yerba mate and the compounds analyzed, some modifications to the method were necessary to optimize extraction efficiency. The tests included the type of extraction solvent, the type of sorbent used as a dispersing agent, the saponification of the final extract for pigment removal, and the dilution ratio of the final extract. All tests were conducted in triplicate with a blank sample spiked at 60 µg kg−1, where the recovery percentage (70–120%) was evaluated with RSD values ≤ 20%, as established by INMETRO [19] and SANTE [20].

2.6. Established Sample Preparation Procedure

Figure 1 shows a diagram of the modified MSPD method validated in this study. The procedure consists of weighing 0.5 g of sample in a 50 mL polypropylene Falcon tube, adding two ceramic homogenizing sticks, and shaking manually for 1 min. After that, 0.5 g of PSA sorbent is added and shaken manually for 1 min, and then 3 mL of ethyl acetate is added and shaken for 1 min. Afterwards, the tube is centrifuged at 1559× g for 8 min. The extract is diluted with the extraction solvent itself (1:1) and filtered through a 13 mm PTFE syringe filter with a porosity of 0.22 µm.

2.7. Method Validation

To ensure suitability for use, all steps of the analytical method must be validated. In Brazil, ANVISA and INMETRO provide guidelines for analytical validation, such as Resolution RE No. 27/2012 and DOQ-CGCRE-008/2020 [19], respectively. International bodies such as SANTE [20] also provide guidelines aimed at ensuring the reliability and comparability of the analytical results of the developed method.
Selectivity is one of the main parameters evaluated, and this was achieved by comparing the chromatograms of the spiked blank sample. Linearity was evaluated using an analytical curve obtained from the extraction of the spiked blank matrix at concentration levels corresponding to 0.5, 1, 2, 5, 10, 20, and 50 µg L−1, considered adequate for compounds that presented a determination coefficient (r2) ≥ 0.99. The matrix effect was evaluated by comparing the slopes of the calibration curves in solvent (ethyl acetate) and from extracted spiked blank matrix (n = 3). Extraction efficiency was evaluated by average recovery, and precision in terms of repeatability (RSDr, within the same day) and intermediate precision (RSDpi, at different days) were evaluated, based on the injection of the analytical curve and spiked blank samples at 12, 60, and 240 µg kg−1 (n = 6). The limit of quantification was established as the lowest point on the calibration curve with a signal-to-noise ratio (S/N) > 10, and the limit of detection (LOD) was determined by dividing the LOQ by 3.333 [19]. The parameters evaluated for method validation are listed in Table 2. All analyses were performed in a laboratory accredited by ISO/IEC 17025/2017, following quality criteria.

2.8. Statistical Analysis

The data from the spectrophotometric and recovery tests were submitted to analysis of variance (ANOVA) and Tukey’s test for comparison of means. Statistical analyses were conducted using Minitab® with a 5% significance level.

3. Results and Discussion

3.1. Sample Preparation Method

Considering the complexity of the yerba mate plant matrix and the low concentrations of the target compounds (PAHs and AQ), it was necessary to develop an effective sample preparation strategy based on the solid-phase matrix dispersion (MSPD) technique. The methodology was optimized based on the evaluation of different critical variables, such as the type of extraction solvent, the sorbent used for matrix dispersion, and the final extract dilution step.
A modification of the MSPD method developed in comparison to the method of Kemmerich et al. [43] was the replacement of metal beads with ceramic homogenizing rods, which are 2.5 cm long, 1 cm thick, and weighing 4 g, similar in mass to the metal balls, to provide effective homogenization and maceration of the sample with the sorbent.
Acetonitrile, hexane, ethyl acetate, and acetone were tested in the evaluation of extraction solvents. Ethyl acetate was selected as the ideal solvent because it presented high recovery rates (70–120%) (Figure 2) with an RSD ≤ 20% for all compounds and, at the same time, reduced the co-extraction of matrix interferents, such as phenolic compounds and chlorophylls. The choice was corroborated by spectrophotometric tests and statistical analysis (Tukey test, p < 0.05) (Table S3), which indicated the superior performance of ethyl acetate compared to acetone, although the latter also proved to be efficient in the extraction of analytes.
Among the sorbents evaluated for matrix dispersion (C18, PSA, Florisil, silica, diatomaceous earth, calcined diatomaceous earth, and calcium chloride), PSA showed the best results in extract cleaning (Figure S1), with a significant reduction in plant interferents, as demonstrated by the low absorbances in the characteristic bands of chlorophylls a and b and in the total phenolics assay. The statistical data indicated significant differences (p < 0.05) between PSA and the other sorbents for all parameters evaluated and are shown in Table 3. Due to its high cost, the PSA mass used was optimized to 0.5 g, in a 1:1 ratio with the sample, maintaining the efficiency in the purification of the extracts.
Alkaline hydrolysis was tested as an additional purification step, using 6 mol L−1 NaOH followed by liquid–liquid extraction with hexane. The tests performed are listed in Table S4. Although it promoted a reduction in chlorophyll and phenolic content, this step resulted in an increase in chromatographic interferents in the hydrolyzed extracts, especially in compounds such as fluoranthene and pyrene (Figure 3), which compromised the selectivity and detectability of the method. Due to the limitation of the single quadrupole GC-MS system, which can suffer interference in the ionization source, this step was discarded.
Finally, the dilution of the final extract was evaluated (undiluted, 1:1, and 1:4, extract/solvent). The 1:1 dilution was the most suitable, as it reduced interferents without significantly impairing the recovery of analytes, as shown in Figure 3, ensuring greater reproducibility and analytical sensitivity. Based on these results, the final conditions adopted for the sample preparation stage involved the use of 0.5 g of PSA as a sorbent, ethyl acetate as an extractant solvent, and a 1:1 dilution of the final extract, providing a robust, selective, and economically viable method for the analysis of PAHs and AQ in yerba mate.

3.2. Validation of the Method

The selectivity of the method was confirmed by the absence of interferents in the chromatograms of the blank samples, even after spiking with the analytes, indicating that the matrix does not interfere with detection. The linearity was satisfactory for all compounds, with a coefficient of determination (r2) ≥ 0.99 in analytical curves extracted from spiked blank matrix, covering the range of 0.5 to 50 µg·L−1, except for anthraquinone, whose linear range started at 1 µg·L−1.
Due to the strong influence of the matrix on the response of the analytes, it was not possible to construct curves in solvent, so the curve extracted from the matrix was used. The limits of detection (LOD) and quantification (LOQ) were 1.8 µg·kg−1 and 6 µg·kg−1, respectively, for 16 compounds; for anthraquinone, the values were 3.6 and 12 µg·kg−1. These limits meet the European Commission [16] criteria for PAHs in dried herbs.
Accuracy and precision tests (using spiked blank sample at 12, 60, and 240 µg·kg−1, over 5 days) showed recoveries between 70% and 120% with RSD ≤ 20% for almost all compounds. The exception was anthraquinone at the lowest spiked level, which had insufficient precision, possibly due to the lower sensitivity of AQ in the analysis, which may be related to the high boiling point of the compound. Compared to methods such as QuEChERS with GC-MS/MS [26], the validated method obtained compatible recoveries and limits, even using a single quadrupole GC-MS, demonstrating good robustness and applicability. By applying the proposed sample preparation method in conjunction with the GC-MS/MS technique, it is expected that even lower LOQs than those obtained by GC-MS will be achieved, which would make it possible to verify the occurrence of analytes at concentration levels more suitable for quality control.

3.3. Application of the Validated Method

The modified MSPD method was applied to 31 yerba mate samples, 20 of which were commercial, originating from nine cities (three pure leaf and 17 traditional), and 11 samples collected at different stages of processing. The samples were coded as EV01 to EV20 (commercial) and YM01 to YM11 (processing), with only YM01 and YM02 having no contact with smoke. The results for commercial samples and from different stages of processing are presented in Table 4 and Table 5, respectively.
Of the commercial samples, nine showed quantifiable contamination by PAHs, notably phenanthrene, present in all (11 to 81 µg·kg−1), followed by fluoranthene (seven samples), pyrene (five), acenaphthylene (four), benzo[b]fluoranthene and anthracene (three), fluorene (two), and naphthalene, benzo[a]pyrene (BaP), and benzo[g,h,i]perylene (one each).
Samples EV08, EV10, and EV12 were the most contaminated (seven PAHs detected), while EV01, EV11, EV13, EV15, and EV17 had one or no compounds above the LOQ. No sample exceeded the maximum limits allowed by the European Union: BaP (10 µg·kg−1) or the sum of the four priority PAHs (BaP, BbF, Chr e BaA, 50 µg·kg−1). Only sample EV07 had BaP above the LOQ.
The results obtained are consistent with those of Górka et al. [26], who applied the QuEChERS method to yerba mate samples from three countries, observing phenanthrene in all Brazilian samples (46 to 126 µg·kg−1), in addition to the detection of pyrene and anthracene, compounds also found in this study.
In the samples collected during the different processing stages, it was observed that only YM01 and YM02, both without exposure to smoke, did not present compounds detected above the limit of quantification (LOQ). In contrast, the other samples, which differed only in drying and grinding methods, had contact with smoke during the sapeco process and showed contamination of three or more quantified HPAs, consistently showing that contamination occurs predominantly during the sapeco and/or drying stages, when there is direct contact with combustion products. Among the compounds monitored, benzo[a]pyrene (BaP) was the most frequently detected, with eight samples exceeding the regulatory limit of 10 µg kg−1, reaching concentrations between 10 and 50 µg kg−1. The other three HPAs regulated by the European Commission were also identified, with sample YM09 showing a sum of these four compounds of 116 µg kg−1, which is above the established limit. In addition, phenanthrene (Phe), fluoranthene (Fa), and pyrene (Pyr) compounds were widely detected, with seven samples showing concentrations above the LOQ, ranging from 12 to 113, 33 to 70, and 44 to 99 µg kg−1, respectively. On the other hand, the compounds dibenzo[a,h]anthracene (DBahA), acenaphthene (Acp), acenaphthylene (Acy), and fluorene (Flr) were not detected above the LOQ. Sample YM09, presenting the greatest diversity of contaminants, with the detection of ten HPAs above the LOQ. These results obtained using the modified MSPD method confirm the contamination of yerba mate by HPAs and anthraquinone, particularly due to exposure to smoke during processing. These findings are consistent with the literature, as demonstrated by Vieira et al. [49], who also observed higher concentrations of HPAs in the processing stages where direct contact with smoke occurs.

4. Conclusions

An efficient analytical method was developed and validated in this study for determining polycyclic aromatic hydrocarbons (PAHs) and anthraquinone (AQ) in yerba mate, using the modified MSPD method coupled with GC-MS. The method integrated extraction and cleanup in a single step, with good results in terms of selectivity, precision, recovery, and detection limits compatible with European legislation.
Application to commercial samples from Rio Grande do Sul revealed contamination by PAHs, especially in products exposed to smoke during processing. These findings reinforce the health risks associated with frequent consumption of the beverage and ingestion at high temperatures, pointing to the need for improvements in drying methods and greater sanitary control.
The BiT-MSPD method showed promise for the analysis of contaminants in complex plant matrices and can be adapted for other substances such as pesticides. Further studies should focus on optimizing the purification of extracts and on processing alternatives that reduce the formation of contaminants, contributing to the safety and value of yerba mate in the market.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/separations12090240/s1: Figure S1: Coloration of the extracts obtained with different sorbents: (A) PSA, (B) Florisil, (C) calcium chloride, (D) diatomaceous earth, (E) calcined diatomaceous earth, (F) silica, and (G) C18; Figure S2: Chromatogram obtained by GC-MS in SIM mode from the blank sample spiked at 240 µg kg−1. (1) Naphthalene; (2) Acenaphthylene; (3) Acenaphthene; (4) Fluorene; (5) Phenanthrene; (6) Anthracene; (7) Anthraquinone; (8) Fluoranthene; (9) Pyrene; (10) Benzo[a]anthracene; (11) Chrysene; (12) Benzo[b]fluoranthene; (13) Benzo[k]fluoranthene; (14) Benzo[a]pyrene; (15) Indeno[1,2,3-c,d]pyrene; (16) Dibenzo[a,h]anthracene; (17) Benzo[g,h,i]perylene.; Table S1: Review of sample preparation methods for the determination of PAHs in plant matrices and chromatographic conditions; Table S2: Compounds analyzed by GC-MS with their respective retention times (tR) and ions monitored; Table S3: Data from the evaluation of the absorbance of chlorophyll a and b and total phenolic compounds of the extract obtained with each solvent evaluated (n = 3); Table S4. Tests carried out to analyze alkaline hydrolysis (n = 3).

Author Contributions

Conceptualization, R.Z., O.D.P. and D.M.H.; methodology, D.M.H., G.A.B.P., I.F.d.S. and V.A.D.; validation, D.M.H., J.D.d.S., G.A.B.P. and I.F.d.S.; formal analysis, D.M.H., J.D.d.S. and V.A.D.; writing—original draft preparation, D.M.H., J.D.d.S., G.A.B.P. and I.F.d.S.; writing—review and editing, R.Z.; visualization, G.A.B.P. and I.F.d.S.; supervision, O.D.P.; project administration, R.Z.; funding acquisition, R.Z. and O.D.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)/INCT-ALIM number 406760/2022-5.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors gratefully acknowledge the support of the Brazilian agencies CNPq and CAPES.

Conflicts of Interest

The authors declare no conflicts of interest. The funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Separations 12 00240 g001
Figure 1. Flowchart of the proposed MSPD method.
Figure 1. Flowchart of the proposed MSPD method.
Separations 12 00240 g001
Separations 12 00240 g002
Figure 2. Recovery of compounds for each solvent evaluated at the spike level of 60 µg kg−1 (n = 3).
Figure 2. Recovery of compounds for each solvent evaluated at the spike level of 60 µg kg−1 (n = 3).
Separations 12 00240 g002
Separations 12 00240 g003
Figure 3. Chromatogram of the extract with hydrolysis of the spiked blank sample at 240 µg kg−1 of fluoranthene and pyrene. (A) with hydrolysis; (B) without hydrolysis.
Figure 3. Chromatogram of the extract with hydrolysis of the spiked blank sample at 240 µg kg−1 of fluoranthene and pyrene. (A) with hydrolysis; (B) without hydrolysis.
Separations 12 00240 g003
Table 1. List of priority PAHs for monitoring by EFSA and their physical–chemical characteristics and carcinogenicity [11].
Table 1. List of priority PAHs for monitoring by EFSA and their physical–chemical characteristics and carcinogenicity [11].
CompoundsAbbreviationCASBoiling Point (°C)log KowMolecular Mass (Da)Carcinogenicity
AcenaphtheneAcp83-32-9963.98154.23
AcenaphthyleneAcy208-96-82754.07152.23
AnthraceneAnt0120-12-73424.45178.23
AnthraquinoneAQ84-65-13793.39208.22B
Benz[a]anthraceneBaA56-55-34385.61228.32B
Benzo[a]pyreneBaP50-32-84956.06252.31
Benzo[b]fluorantheneBbF205-99-24816.04252.32B
Benzo[g,h,i]peryleneBghiP191-24-25506.5276.33
Benzo[k]fluorantheneBkF207-08-94806.06252.32B
ChryseneChr0218-01-094485.9228.32B
Dibenzo[a,h]anthraceneDBahA53-70-35246.84278.32A
FluorantheneFa206-44-03754.9202.33
FluoreneFlr86-73-72954.18166.23
Indeno[1,2,3-c,d]pyreneInd193-39-55366.58276.32B
NaphthaleneNph91-20-32183.29128.22B
PhenanthrenePhe85-01-83404.45178.23
PyrenePyr129-00-03934.88202.33
Caption: CAS: Chemical Abstracts Service; Group 1: Carcinogenic to humans; Group 2A: Probably carcinogenic to humans; Group 2B: Possibly carcinogenic to humans; and Group 3: Not classifiable as carcinogenic to humans.
Table 2. Method validation results for linearity (r2), linear range, limit of quantification (LOQ), limit of detection (LOD), accuracy (recovery, %) and precision (repeatability and intermediate precision, RSD %) (n = 6).
Table 2. Method validation results for linearity (r2), linear range, limit of quantification (LOQ), limit of detection (LOD), accuracy (recovery, %) and precision (repeatability and intermediate precision, RSD %) (n = 6).
Compoundsr2Linear Range
(µg L−1)		Method LOD
(µg kg−1)		Method LOQ
(µg kg−1)		Repeatability
Recovery (RSDr), %		Intermediate Precision
Recovery (RSDpi), %
Spike Level (µg kg−1)Spike Level (µg kg−1)
12602401260240
Acp0.99980.5–501.8693 (13)94 (4)101 (3)85 (19)82 (10)87 (1)
Acy0.99990.5–501.86110 (9)97 (2)100 (3)77 (14)83 (3)89 (2)
Ant0.99940.5–501.8689 (19)98 (4)104 (2)79 (18)77 (5)77 (2)
AQ0.99951–503.61295 (25)120 (7)107 (8)70 (59)74 (19)82 (10)
BaA0.99980.5–501.86106 (20)96 (6)108 (4)97 (10)88 (5)88 (5)
BaP0.99910.5–501.86105 (9)89 (9)105 (6)119 (8)84 (9)91 (6)
BbF0.99910.5–501.86114 (10)98 (4)103 (4)120 (2)93 (2)90 (5)
BghiP0.99980.5–501.8680 (16)89 (2)98 (3)116 (9)86 (3)94 (3)
BkF0.99960.5–501.86116 (6)99 (4)108 (4)78 (17)87 (4)96 (8)
Chr0.99950.5–501.86108 (14)85 (11)102 (3)95 (14)76 (3)77 (4)
DBahA0.99880.5–501.86110 (12)89 (8)98 (3)117 (9)106 (10)111 (9)
Fa0.99980.5–501.86110 (17)95 (5)97 (4)94 (16)80 (4)85 (5)
Flr0.99930.5–501.86119 (1)96 (3)101 (4)78 (16)77 (6)84 (3)
Ind0.99960.5–501.86118 (14)98 (6)105 (3)99 (20)94 (6)106 (6)
Nph0.99990.5–501.8678 (12)97 (4)107 (2)103 (12)78 (7)82 (3)
Phe0.99980.5–501.86101 (18)104 (5)107 (3)79 (16)70 (3)76 (3)
Pyr0.99970.5–501.86120 (19)97 (3)101 (1)98 (16)74 (8)84 (2)
Table 3. Data from the evaluation of the absorbance of chlorophyll a and b and total phenolic compounds of the extracts obtained with each solvent evaluated (n = 3).
Table 3. Data from the evaluation of the absorbance of chlorophyll a and b and total phenolic compounds of the extracts obtained with each solvent evaluated (n = 3).
Sorbents or SaltsChlorophyll a
(664 nm)		Chlorophyll b
(647 nm)		Total Phenols
(765 nm)
C180.500 ± 0.013 b0.223 ± 0.008 a0.629 ± 0.037 ab
CaCl20.493 ± 0.017 b0.261 ± 0.032 a0.667 ± 0.123 ab
Florisil0.530 ± 0.016 ab0.251 ± 0.020 a0.736 ± 0.073 ab
PSA0.149 ± 0.024 c0.107 ± 0.023 b0.405 ± 0.060 b
Silica0.610 ± 0.023 a0.311 ± 0.018 a1.041 ± 0.166 a
Diatomaceous earth0.481 ± 0.014 b0.225 ± 0.019 a0.467 ± 0.037 b
Calcined diatomaceous earth0.502 ± 0.007 b0.243 ± 0.016 a0.394 ± 0.029 b
Values are presented as mean ± standard error of the mean. Different lowercase letters in the same row indicate statistically significant differences (p < 0.05) between sorbents, according to Tukey’s test.
Table 4. Positive results (µg kg−1) of the applications of the proposed method for the determination of PAHs and anthraquinone in commercial yerba mate samples.
Table 4. Positive results (µg kg−1) of the applications of the proposed method for the determination of PAHs and anthraquinone in commercial yerba mate samples.
CompoundsConcentration in Commercial Yerba Mate Samples (µg kg−1)
EV02EV03EV04EV05EV06EV07EV08EV09EV10EV12EV14EV16EV18EV20
Acyn.d.n.d.<LOQ<LOQ<LOQ<LOQ23.64n.d.38.3316.81n.d.<LOQn.d.16.17
Antn.d.n.d.<LOQn.d.n.d.n.d.13.73n.d.12.47<LOQn.d.n.d.<LOQ9.99
AQn.d.34.54<LOQ<LOQn.d.n.d.21.76n.d.<LOQn.d.n.d.n.d.36.6121.42
BaAn.d.<LOQ<LOQn.d.n.d.n.d.<LOQn.d.<LOQn.d.n.d.n.d.<LOQ<LOQ
BaP<LOQ<LOQ<LOQ<LOQn.d.7.02<LOQ<LOQ<LOQ<LOQ<LOQn.d.<LOQ<LOQ
BbF<LOQ12.966.96<LOQ<LOQ7.36<LOQ<LOQ<LOQ<LOQ<LOQ<LOQ<LOQ<LOQ
BghiPn.d.n.d.n.d.n.d.n.d.11.42<LOQn.d.<LOQn.d.n.d.n.d.n.d.n.d.
BkFn.d.<LOQn.d.n.d.n.d.<LOQn.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.
Chrn.d.n.d.<LOQn.d.n.d.<LOQ<LOQn.d.n.d.n.d.n.d.n.d.<LOQ<LOQ
Fan.d.8.30<LOQ11.54n.d.32.0630.75n.d.29.4610.11n.d.n.d.<LOQ16.82
Flrn.d.n.d.n.d.n.d.n.d.31.36n.d.n.d.14.48n.d.<LOQn.d.n.d.n.d.
Nphn.d.n.d.n.d.n.d.n.d.n.d.8.61n.d.<LOQn.d.n.d.n.d.n.d.<LOQ
Phen.d.30.9018.4211.74n.d.18.7981.31n.d.53.6019.79n.d.n.d.17.4763.79
Pyrn.d.<LOQ<LOQ12.71n.d.36.6623.44n.d.21.94<LOQn.d.n.d.n.d.9.28
∑PAHs086.7025.3735.990144.67203.240170.2846.700054.08137.48
n.d.: not detected.
Table 5. Positive results (µg kg−1) of the applications of the modified MSPD method in this study for the determination of PAHs and anthraquinone in yerba mate samples at different stages of processing.
Table 5. Positive results (µg kg−1) of the applications of the modified MSPD method in this study for the determination of PAHs and anthraquinone in yerba mate samples at different stages of processing.
CompoundsConcentration Found in Yerba Mate Samples at Different Stages of Processing (µg kg−1)
YM03YM04YM05YM06YM07YM08YM09YM10YM11
Antn.d.n.d.<LOQn.d.n.d.8.356.68n.d.n.d.
AQ28.95n.d.n.d.n.d.n.d.20.1318.56n.d.n.d.
BaA<LOQn.d.3.42n.d.n.d.<LOQ6.719.07n.d.
BaP14.9811.9911.23<LOQ10.239.8214.1049.9413.51
BbF7.26<LOQ11.20n.d.n.d.<LOQ11.5041.928.96
BghiPn.d.2.57n.d.n.d.1.15n.d.n.d.31.640.49
BkF16.23<LOQ10.28<LOQ<LOQ<LOQ12.6646.5013.95
Chr<LOQn.d.11.27n.d.n.d.<LOQ16.8715.21n.d.
Fa40.1052.8749.4667.2770.0833.5957.88n.d.n.d.
Ind<LOQn.d.n.d.n.d.n.d.n.d.n.d.23.35n.d.
Nphn.d.n.d.<LOQn.d.n.d.n.d.<LOQ<LOQ7.09
Phe26.8912.7691.1449.4747.2476.53113.16n.d.n.d.
Pyr48.2479.3759.5892.5399.6644.2165.25n.d.n.d.
∑PAHs182.64159.56247.57209.27228.36192.64323.36217.6344.00
∑4PAHs22.2411.9937.120.0010.239.8249.18116.1422.47
n.d.: not detected.
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Hoffmann, D.M.; da Silva, J.D.; de Souza, I.F.; Prates, G.A.B.; Dutra, V.A.; Prestes, O.D.; Zanella, R. Simultaneous Determination of Polycyclic Aromatic Hydrocarbons and Anthraquinone in Yerba Mate by Modified MSPD Method and GC-MS. Separations 2025, 12, 240. https://doi.org/10.3390/separations12090240

AMA Style

Hoffmann DM, da Silva JD, de Souza IF, Prates GAB, Dutra VA, Prestes OD, Zanella R. Simultaneous Determination of Polycyclic Aromatic Hydrocarbons and Anthraquinone in Yerba Mate by Modified MSPD Method and GC-MS. Separations. 2025; 12(9):240. https://doi.org/10.3390/separations12090240

Chicago/Turabian Style

Hoffmann, Dylan M., José D. da Silva, Igor F. de Souza, Gabriel A. B. Prates, Vagner A. Dutra, Osmar D. Prestes, and Renato Zanella. 2025. "Simultaneous Determination of Polycyclic Aromatic Hydrocarbons and Anthraquinone in Yerba Mate by Modified MSPD Method and GC-MS" Separations 12, no. 9: 240. https://doi.org/10.3390/separations12090240

APA Style

Hoffmann, D. M., da Silva, J. D., de Souza, I. F., Prates, G. A. B., Dutra, V. A., Prestes, O. D., & Zanella, R. (2025). Simultaneous Determination of Polycyclic Aromatic Hydrocarbons and Anthraquinone in Yerba Mate by Modified MSPD Method and GC-MS. Separations, 12(9), 240. https://doi.org/10.3390/separations12090240

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