Disposable Pipette Extraction Using Ionic Liquid-Intercalated Montmorillonite for Polycyclic Aromatic Hydrocarbons Determination in Seawater

Abstract

A disposable pipette extraction method using ionic liquid-intercalated montmorillonite as sorbent was developed and optimized for the determination of low-molecular-weight polycyclic aromatic hydrocarbons in seawater by gas chromatography-mass spectrometry. Multivariate experimental designs were employed to optimize the extraction parameters, enabling a miniaturized procedure that required small amounts of sorbent, sample, and solvent. The optimized conditions included 15 mg of sorbent, 2.6 mL of sample, 13 extraction cycles, 10 desorption cycles, and 250 µL of hexane as desorption solvent. The method exhibited good linearity over the range 0.1-3.0 µg L-¹ (determination coefficient (R²) > 0.99), with limits of detection and quantification of 0.03 and 0.10 µg L-¹, respectively. Recovery values ranged from 71 to 123%, with deviations attributed to normal experimental variability rather than systematic bias. The optimized protocol provided efficient extraction using a simplified workflow and reduced reagent consumption, in agreement with green analytical chemistry principles. Application to a seawater sample confirmed the occurrence of polycyclic aromatic hydrocarbons and enabled reliable quantification at trace levels (ng-µg L-¹), demonstrating the suitability of the method for routine environmental monitoring.

Keywords:
green analytical chemistry; microextraction; sorbent-based extraction; marine contamination; method validation; trace organic pollutants

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are a class of hydrophobic organic contaminants widely distributed in aquatic environments, mainly as a result of incomplete combustion processes and petroleum-related activities. Due to their toxic, mutagenic, and carcinogenic properties, PAHs pose significant risks to ecosystems and human health. Large-scale environmental contamination events, such as the oil spill that affected the Brazilian coastline in 2019, highlight the relevance of monitoring these compounds in marine environments. This incident impacted more than 11 states and over 1,000 coastal locations, causing severe damage to fauna and highly sensitive ecosystems, including mangroves and coral reefs.¹,²

Although the determination of PAHs in water matrices is well established, the occurrence of these compounds at trace concentrations and their wide range of physicochemical properties still requires efficient and selective sample preparation strategies. Consequently, efficient sample preparation and preconcentration steps are essential prior to instrumental analysis.³,⁴ Recent reviews⁵ have reinforced the importance of advanced and greener extraction strategies for trace hydrophobic contaminants in complex aquatic matrices, emphasizing the need for miniaturized and selective approaches.

In addition to analytical challenges, the environmental and regulatory relevance of PAHs further reinforces the need for sensitive methodologies. Polycyclic aromatic hydrocarbons are classified as priority pollutants due to their persistence and carcinogenic potential. The United States Environmental Protection Agency (EPA) has established a maximum contaminant level (MCL) of 0.2 μg L^-1 for benzo[a]pyrene in drinking water and recommends non detectable levels for carcinogenic PAHs in ambient waters.⁶ These stringent regulatory limits, often at trace levels, highlight the importance of developing sensitive, reliable, and environmentally sustainable analytical methods for PAH determination in aquatic systems. In this context, the limit of quantification achieved in this study (0.1 μg L^-1) falls within the range of internationally relevant regulatory thresholds, supporting the applicability of the proposed method for environmental monitoring.

Traditionally, liquid-liquid extraction (LLE) has been used for PAHs extraction from aqueous samples; however, this technique has progressively fallen into disuse due to its high solvent consumption, large sample volume requirements, and labor-intensive procedures.³,⁷,⁸ Solid-phase extraction (SPE) has emerged as an alternative, providing high recovery efficiencies, but it still involves multiple operational steps and significant consumption of sorbents and solvents.³,⁷,⁸ Sorbent based microextraction techniques, such as SPE, solid-phase microextraction (SPME), and disposable pipette extraction (DPX), have emerged as promising approaches for the extraction of PAHs, offering improved selectivity, reduced solvent consumption, and suitability for complex environmental matrices.⁹

In response to these limitations, there has been growing interest in the development of miniaturized and environmentally friendly sample preparation techniques that reduce solvent usage, sample volume, and analysis time, while maintaining analytical performance.³ DPX has gained attention in recent years as a simple, cost-effective, and versatile technique for the extraction of a wide range of analytes from different matrices.⁷,¹⁰ DPX devices consist of a pipette tip containing a small amount of sorbent retained between two filters, allowing rapid interaction between the analyte and the extraction phase. The technique enables easy optimization of critical parameters such as sorbent type, extraction and desorption cycles, solvent selection, sample volume, pH, salinity, and equilibrium time.¹⁰

Although DPX has been successfully applied to the determination of PAHs in aqueous samples, its application to seawater matrices remains scarcely explored.³,⁷,⁸ At the same time, recent contributions have highlighted the growing interest in combining DPX with innovative sorbent materials to enhance extraction efficiency and environmental compatibility.¹¹

The selection of an appropriate sorbent is a key factor in achieving adequate selectivity and extraction efficiency in DPX-based methods. In this context, montmorillonite is a layered clay mineral capable of intercalating various compounds, while ionic liquids are organic salts that exhibit low volatility, high thermal stability, and strong affinity for organic molecules. The intercalation of ionic liquids into the montmorillonite structure results in a hybrid material that combines the advantages of both components.¹²,¹³

The incorporation of ionic liquids into montmorillonite has been shown to enhance surface area, porosity, and affinity toward hydrophobic organic compounds, including PAHs.¹² This behavior is attributed to the unique molecular structure and charge distribution of ionic liquids, which promote strong interactions with aromatic compounds.¹²,¹³ When used as a sorbent in DPX, ionic liquid-intercalated montmorillonite (IL-MMT) offers additional driving forces for PAHs extraction, improving both efficiency and selectivity.⁶,¹²,¹³ Furthermore, the miniaturized configuration of DPX enables rapid extraction using reduced amounts of sorbent, solvent, and sample, aligning with green analytical chemistry principles.¹⁰,¹⁴

Despite the growing interest in DPX and the promising properties of IL-MMT, no studies have reported the application of IL-MMT as a sorbent phase in DPX for the extraction of PAHs from seawater. Therefore, the objective of this work is to develop and optimize a DPX based method employing IL-MMT as an eco-friendly sorbent for the extraction and determination of PAHs in seawater samples, followed by gas chromatography-mass spectrometry (GC-MS) analysis.

Experimental

Analytical standards, solvents, and reagents

Higher priority polycyclic aromatic hydrocarbons according to the European Community were used in this study: naphathlene (Naf); acenaphthylene (Acy); acenaphthene (Ace); fluorene (Flu); phenantrene (Phe); anthracene (Ant); fluoranthene (Fla); pyrene (Pyr); benzo[a]anthracene (BaA); crysene (Crys); benzo[b]fluoranthene (BbF); benzo[k]fluoranthene (BkF); benzo[a]pyrene (BaP); indeno[1,2,3-cd]pyrene (Ind); dibenzo[a,h]anthracene (DahA); benzo[g,h,i]perylene (BghiP); five surrogates deuteraded standards naphtalene (Nap[²H₈]); acenaphthene (Ace[²H₁₀]); phenanantrene (Phe[²H₁₀]); crysene (Cris[²H₁₂]) and perylene (Per[²H₁₂]) and a deuterated internal standard p-terphenyl (p-Ter[²H₁₄]) all from Merck (Darmstadt, Germany).

All solvents (acetone, hexane, and acetonitrile) used for standards and sample preparation are from the chromatographic purity from J.T. Baker (Mexico City, Mexico). High purity water (resistivity of 18.2 mΩ cm) used for solutions elaboration was obtained in permution reverse osmosis system (Curitiba, Brazil) followed by a purification in a Millipore UV Simplicity system (Molsheim, France).

The stock mixture solutions of PAHs standards were prepared in hexane at concentrations of 80.0 mg L^-1. These were kept in the freezer (-18 ºC) until the moment of the preparation of the intermediate solutions. The working solutions were made daily using the same solvent used in stock solution at a concentration of 5.00 mg L^-1.

The IL-MMT was previously modified according to the procedure described by Fiscal-Ladino et al.¹² Detailed physicochemical characterization of the IL-MMT material, including structural and surface properties, is fully reported in that study and will not be repeated here, as the present work focuses on the analytical application of the sorbent. Briefly, one gram of MMT was dispersed in 3.0 g of a 1-hexadecyl-3-methylimidazolium bromide (HDMIM-Br) solution in methanol (13% v/v), and the dispersion was vigorously stirred for 1 h at room temperature. The mixture was filtered, and the solid material was washed three times with methanol (20 mL each) to remove excess ionic liquid, followed by three washes with distilled water. The solid was then dried in an oven at 105 °C for 24 h.

Materials decontamination

Before use, all glassware, and other materials were cleaned by sonication in a Unique ultrasonic bath (São Paulo, Brazil) using ultra-pure water three times for 10 min each. Afterwards, the non-volumetric glass materials were oven-dried at 105 °C for 24 h and then calcined in a muffle for 4 h at 400 °C. The volumetric items were dried in a laminar flow cabinet for 24 h and flushed with acetone before use.

Instrumentation and chromatographic parameters

GC-MS was used for PAHs separation, identification and quantification. A gas chromatograph Shimadzu (Kyoto, Japan) model QP2010-TQ8040 equipped with an analytical capillary column Phenomenex ZB-5ms (Torrence, USA) 30 m × 0.25 mm × 0.25 µm coupled to mass spectrometer and an autosampler Palm Shimadzu model AOC-5000 plus was used. The equipment was operated with high-purity analytical helium 99.999% as carrier gas (constant flow), supplied by White Martins (Paraná, Brazil).

The chromatographic conditions employed were determined through adaptations of past work conducted by our group.¹⁵,¹⁶ Analytical standards and samples were injected at 1 µL volume, splitless mode, with injector at 270 ºC, without pressure pulse and 4 min sampling time. The chromatographic column was initially kept at 40 °C for 2 min, followed by heating at 50 °C min^-1 to 80 °C, gradient 10 °C min^-1 to 240 °C, held for 2 min, heating at 2 ºC min^-1 to 260 ºC, held for 5 min and finally ramp at 20 °C min^-1 to 300 °C, held for 5 min. The transfer line was operated at 280 ºC, and the ion source at 230 ºC. The GC MS system was operated using high carrier gas at a flow rate of 1.56 mL min^-1. Aiming to increase the selectivity and detectability the analytes were quantified using selective ion monitoring (SIM), shown in Table 1.

Table 1
Compounds, abbreviations, monitoring window and m/z target ions

Compound	Abbreviation	Acquisition time window / min	Target iona / (m/z)
bNaphthalene[2H8]	Nap[2H8]	6.50-10.78	136, 137, 108
Naphthalene	Nap	6.50-10.78	128, 127, 129
Acenaphthylene	Acy	10.78-13.57	152, 151,150
bAcenaphthene[2H10]	Ace[2H10]	10.78-13.57	162, 164,160
Acenaphthene	Ace	10.78-13.57	153, 154, 152
Fluorene	Flu	13.57-15.29	166, 165, 164
bPhenanthrene[2H10]	Phe[2H10]	15.29-17.89	188, 187, 184
Phenanthrene	Phe	15.29-17.89	178, 176, 152
Anthracene	Ant	15.29-17.89	178, 176, 179
Fluoranthene	Fla	17.98-22.52	202, 200, 203
Pyrene	Pyr	17.98-22.52	202, 200, 201
cp-Terphenyl[2H14]	p-Ter[2H14]	17.98-22.52	244, 243, 245
bChrysene[2H12]	Chr[2H12]	22.52-27.52	240, 236, 241
bPerylene[2H12]	Per[2H12]	30.93-33.95	264, 260, 265

• ^a
  Target ions in italic; ^bsurrogate standards; ^cinternal standard.

Although a broader set of PAHs was included in the standards mixture, the validation was restricted to eight low-molecular-weight compounds, selected based on their distinct physicochemical characteristics. Their higher volatility and lower hydrophobicity lead to reduced affinity for the sorbent phase and increased susceptibility to experimental losses, making them more challenging to extract, particularly in miniaturized systems such as DPX. Therefore, these analytes were considered suitable probes for critically assessing the efficiency, robustness, and analytical performance of the proposed method.

Preparation of DPX device

The extraction device was prepared using two pipette tips (1250 and 200 µL), calcined glass wool, and IL-MMT (Figure 1). Initially, approximately 2 cm was removed from the bottom of the 1250 µL pipette tip. A 0.5 cm layer of glass wool was then placed at the bottom to serve as a support, followed by the addition of the IL-MMT sorbent. Another 0.5 cm layer of glass wool was placed above the sorbent to prevent its loss and to ensure proper solvent flow during the extraction and elution steps. Finally, the 200 µL pipette tip was fitted to the lower end of the modified 1250 µL tip to complete the extraction device.

Figure 1
Schematic representation of the miniaturized DPX device containing IL-MMT sorbent and glass wool packed into a 1250 µL pipette tip, coupled to a 200 µL micropipette tip for extraction of low-molecular-weight PAHs from aqueous samples.

DPX procedure

For the extraction, the DPX device was activated by 1 mL hexane and 1 mL deionized water, successively. Then, the sample was loaded manually into the conditioned tip and dispensed back into the same sample tube, which was referred to as one aspirating/dispensing cycle. The sample was repeatedly performed with aspirating/dispensing cycles, allowing the analytes to be adsorbed on the sorbent sufficiently. After, the eluate was discarded. Finally, the sorbed analytes on the IL-MMT were repeated with the solvent by aspirating/dispensing cycles. Subsequently, the eluate was ready for GC-MS analysis. The schematic diagram of the DPX procedure is presented in Figure 2.

Figure 2
Schematic representation of the DPX extraction procedure using IL-MMT for extraction of low-molecular-weight PAHs from aqueous samples.

Optimization of the DPX procedure

For the development of the analytical protocol, the following six extraction parameters were optimized: sorbent mass, sample volume, extraction cycles, desorption cycles and desorption solvent. A fractional factorial design was used to determine the most significant factors, followed by a central composite orthogonal design to optimize the DPX procedure, with the statistical significance of the effects evaluated by analysis of variance (ANOVA) based on the estimated model coefficients and their confidence intervals.

To perform the optimization, an aqueous solution of concentration 1 µg L^-1 was used. Initially, a 2^6-2 fractional factorial design (levels +1 and -1) was performed. The variables and evaluated levels are present in Table 2.

Table 2
Variables and levels of the 2^6-2 fractional factorial design

Variable	Level
	-1	+1
Mass of sorbent / mg	15	20
Sample volume / mL	2.5	5.0
Extraction cycles	10	15
Desorption cycles	10	15
Solvent	acetone	hexane
Solvent volume / µL	250	500

Based on the results obtained from the fractional factorial design, the experimental domain was narrowed to refine the optimization process. With the two most significant variables, a two-factor central composite design, with five levels (-α, -1, 0, +1, +α) and a central point in triplicate, was performed (Table 3). The analytical response in all experiments was the peak area of the studied PAHs. A desirability function was calculated to obtain one response vector in both experimental designs. The design matrix, desirability function, factor evaluation and optimization were performed using MODDE pro software (version 13.1, Sartorius Stedim Biotech, Sweden).

Table 3
Variables and work range of central composite design

Variable	Work range
Samples volume / mL	2.5-7.5
Extraction cycles	12-18

Salinity effect

To evaluate the effect of salinity on the extraction efficiency, aqueous solutions (1 µg L^-1) containing the target PAHs and five deuterated PAHs were prepared. The salinity was adjusted by adding sodium chloride (NaCl) at concentrations of 0, 0.5, 2.0, and 3.5% (m/v), with the highest value selected to approximate typical seawater salinity.¹⁷ The extraction was performed under the optimized conditions established by the factorial design.

These conditions were defined to simulate seawater matrices and to assess the influence of ionic strength on the extraction process. In addition, the applicability of the method was evaluated using a seawater sample collected from a coastal environment (Praia de Leste, Paraná, Brazil). The sample was transported in a glass container and stored in a thermal box with ice prior to analysis. Before extraction, it was filtered through a 0.45 µm membrane to remove suspended particulate matter and ensure compatibility with the DPX procedure.

Validation parameters

The proposed method was validated in terms of linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ). Calibration curves were constructed using standard solutions prepared in hexane by plotting the peak area ratio of each analyte to the internal standard as a function of analyte concentration. The LOQ was operationally defined as the lowest calibration level at which adequate chromatographic resolution and repeatability were achieved, based on five independent replicates (n = 5). The LOQ was established at 0.10 µg L^-1 for all evaluated PAHs, while the LOD was derived from the LOQ using the relationship LOD = LOQ / 3.3, in accordance with widely adopted analytical validation practices and Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMETRO) guidelines.¹⁸

Precision and accuracy were evaluated through recovery experiments using spiked seawater samples at concentrations of 0.6, 1.4, and 2.75 µg L^-1. Precision was expressed as relative standard deviation (RSD), based on three independents manual DPX extractions (n = 3), each comprising multiple aspirating/dispensing cycles under optimized conditions, while accuracy was assessed from the recovery percentages obtained for each PAH. Recoveries were calculated by comparing the peak area ratios (analyte/internal standard) obtained for spiked samples after extraction with those from calibration standards prepared in hexane at equivalent concentrations. The reported values correspond to apparent recoveries, reflecting both extraction efficiency and potential matrix effects. Although matrix effects were not evaluated separately, the use of deuterated internal standards minimized signal variations and improved analytical accuracy.

The statistical evaluation of recoveries was performed using the t-test of student, comparing the experimental mean recovery of each PAH with the theoretical value of 100% (H₀: μ = 100%). Values of p higher than the adopted significance level (α = 0.05) were considered indicative of no significant difference. Deviations from the theoretical value were expressed as recovery deviation (∆r, %), providing a complementary measure of method performance. Statistical analyses were carried out using the Python programming environment (Python 3.x) with the SciPy library.¹⁹

It is important to note that the LOQ was defined based on calibration criteria, and recovery experiments were performed at concentration levels above the LOQ to ensure a robust evaluation of method accuracy and precision under conditions of reliable quantification.

Results and Discussion

Optimization of DPX procedure

The multivariate approach enabled the identification of the combined contribution of these variables using a reduced number of experiments, thereby facilitating a more efficient interpretation of system behavior.²⁰,²¹ In this context, the effects of the main experimental variables - namely sorbent mass (SorMas), solvent type (Sol), solvent volume (SolVol), number of extraction cycles (ExtCyc), sample volume (SamVol), and number of desorption cycles (DesCyc) - could be evaluated in an integrated manner with respect to extraction efficiency, allowing a consistent assessment of the overall method performance.

Fractional factorial design

The simultaneous evaluation of the six extraction parameters using a fractional factorial design enabled the integrated identification of the variables with the greatest influence on extraction performance (Figure 3). The obtained responses exhibited high variability, which is associated with the extraction of analytes with distinct physicochemical properties.

Figure 3
Coefficients obtained from the fractional factorial design for the global desirability function, which integrates the responses of all evaluated PAHs. SorMas: sorbent mass; Sol: solvent; VolS: solvent volume; CicD: number of desorption cycles; VolA: sample volume; CicE: number of extraction cycles; N: number of experiments; DF: degrees of freedom; Cond.: condition; Q²: predictive coefficient.

Under these conditions, optimization based on individual responses proved to be limited, as the optimal condition for one analyte does not necessarily correspond to that for the others. To overcome this limitation, the experimental responses were integrated into a single global parameter, allowing direct comparison among the evaluated conditions and identification of the most relevant variables.²⁰

The desirability function enabled the integration of multiple responses into a single dimensionless value, in which values close to zero indicate unsatisfactory performance and values close to one represent near-optimal conditions, thereby allowing evaluation of the combined effect of the variables on overall extraction efficiency.²⁰

Figure 3 depicts the coefficient values and confidence intervals of each factor, allowing the identification of statistically relevant effects based on the magnitude of the estimated coefficients and whether their confidence intervals crossed zero. It was possible to determine which variables would be investigated in the method optimization procedure. Although only the SorMas was determined as non-significant for the extraction procedure, the factors SamVol and number of ExtCyc presented a higher impact on the global desirability when compared to the remaining factors. Therefore, only the two most significant factors were selected to perform a central composite design and optimize the extraction condition. The variables SolVol, DesCyc and SorMas were held constant in the subsequent stage at the lower level (-1), corresponding to 250 µL for SolVol, 10 cycles for DesCyc and 15 mg for SorMas, in order to minimize residuals at the end of the process and improve analytical frequency. Hexane was determined as the desorption solvent once a higher global desirability was achieved compared to the experiments using acetone.

Central composite orthogonal design

The two most significant variables from the fractional factorial design - sample volume and number of extraction cycles - were selected for a new experimental design aimed at optimizing the extraction conditions. The design chosen for this stage was a central composite orthogonal design with five levels, including a triplicate central point, resulting in a total of 11 experiments.

The number of ExtCyc exhibited a positive coefficient in the fractional factorial design; therefore, the upper level of ExtCyc (15 cycles) was set as the central point in the new experimental design. Although the SamVol presented a negative coefficient (Figure 3), lower sample volumes hindered effective contact between the sample and the sorbent material. Consequently, a volume of 2.5 mL was defined as the minimum level in the central composite design (-α).

For model evaluation, the mean, standard deviation, and RSD (%) of the peak areas obtained from the triplicate central point are presented in Table 4.

Table 4
Mean, estimated standard deviation, and RSD for the peak area of the central point in the central composite orthogonal design

PAH	Mean	Standard deviation	RSD / %
Naphthalene	4839439	484729	10.2
Acenaphthylene	3033096	43691	1.4
Acenaphthene	2027931	14934	1.1
Fluorene	2001082	17527	1.2
Phenanthrene	3188071	252978	8.6
Anthracene	2355434	197888	8.9
Fluoranthene	2875299	507504	18.8
Pyrene	3391653	574732	17.2

RSD values ranged from 1.1 to 18.8% (Table 4), all within the generally accepted limit of 20% for the studied concentration of 1 µg L^-1, as recommended by Association of Official Analytical Chemists (AOAC) International.²² Considering the homoscedastic behavior of the central composite design, it can be stated that both the central point and the remaining optimization conditions exhibited satisfactory RSD values.

In addition, the desirability function was calculated for each experiment based on the results obtained from the new experimental design, with the corresponding values presented in Figure 4.

Figure 4
Desirability values obtained for the central composite orthogonal design applied to the optimization of DPX extraction of low-molecular-weight PAHs using IL-MMT as sorbent. N: number of experiments; DF: degrees of freedom; Cond.: condition; RSD: relative standard deviation; Q²: predictive coefficient.

Desirability values ranged from 0.80 to 1.0 (Figure 4). The lowest desirability value, approximately 0.80, was observed for experiment 3, whereas experiments 2 and 8 exhibited values between 0.80 and 0.90. In contrast, the triplicate central point (experiments 9-11), as well as experiments 4 and 6, showed desirability values above 0.90. For the remaining experiments (1, 5, and 7), the values were close to 1. As previously discussed, desirability values close to 1 are the most desirable for this experiment.²⁰

Based on the obtained desirability values, a response surface plot was generated (Figure 5) to evaluate the optimal conditions for the extraction process.

Figure 5
Response surface plots obtained from the central composite orthogonal design for optimization of DPX extraction of low-molecular-weight PAHs using IL-MMT sorbent.

Through the response surface analysis (Figure 5), the optimal condition for extraction can be determined. It is observed that the desirability values varied between 0.82 and 1.0, with values close to 1 being the most desirable for the extraction process. Desirability values close to 1 correspond to 13 extraction cycles and samples volumes of 2.6 mL.

Lower sample volumes and higher numbers of extraction cycles favor the extraction process, likely due to a higher percentage of the samples in contact with the sorbent material. The behavior has been previously reported in earlier studies.⁷,²³

Optimal condition for the extraction

The application of the factorial design allowed the optimization of the DPX extraction procedure, clearly demonstrating the influence of the evaluated variables on PAH recovery when IL-MMT was employed as the sorbent. The best experimental condition resulted in improved extraction efficiency, and the optimized parameter values are presented in Table 5.

Table 5
Optimal condition for the extraction based on the combination of factorial designs

Variable	Optimal condition
Mass of sorbent	15 mg
Solvent	hexane
Solvent volume	250 µL
Extraction cycles	13
Desorption cycles	10
Sample volume	2.6 mL

Overall, the optimized conditions showed trends consistent with the DPX extraction mechanism and the hydrophobic nature of PAHs. The lower sorbent mass and the use of hexane tended to favor analyte desorption, while the reduced solvent volume may have contributed to a preconcentration effect.²⁴,²⁵ In addition, a higher number of extraction cycles likely increased the contact between the sample and the IL-MMT sorbent, enhancing mass transfer, whereas multiple desorption cycles tended to improve analyte recovery. Together, these factors help explain the improved extraction performance observed under the optimized conditions.²⁴

Salinity effect

Figure 6 suggests that increasing solution salinity tends to be associated with higher PAH/internal standard ratios. This behavior can be qualitatively attributed to the salting-out effect, in which an increase in ionic strength may reduce the solubility of organic compounds in water and influence their octanol-water partitioning. Under higher salt concentrations, PAHs may become less soluble in the aqueous phase, favoring their migration toward the sorbent material.²⁶-²⁸

Figure 6
Effect of salinity on PAHs extraction using IL-MMT for DPX. Extraction conditions: mass of sorbent: 15 mg; samples volume: 2.6 mL; extraction cycles: 10; solvent: hexane; solvent volume: 250 µL and desorption cycles: 13; Naf: naphathlene; Acy: acenaphthylene; Ace: acenaphthene; Flu: fluorene; Phe: phenantrene; Ant: anthracene; Fla: fluoranthene; Pyr: pyrene.

In this context, the results indicate that salinity does not appear to impair extraction efficiency and may contribute positively to the process up to a certain extent. However, a tendency toward a plateau in extraction efficiency is observed at higher salinity levels, suggesting that further increases in ionic strength may not significantly enhance the salting-out effect under the studied conditions.

Nevertheless, the complexity of natural seawater matrices, including dissolved organic matter and competing species, may influence analyte recovery, and thus the method performance should be confirmed using real samples.

Validation parameters

The analytical performance of the proposed method is discussed below, considering linearity, LOD, LOQ, accuracy, and precision.

Linearity, LOD and LOQ

Linearity, as well as LOD and LOQ, showed adequate performance for the proposed method. The linear range of the analytical curve extended from 0.1 to 3.0 µg L^-1, with five replicates at each concentration level. The analytical parameters, including slope, intercept, coefficient of determination (R²), RSD, LOD, and LOQ, are presented in Table 6, while the chromatogram corresponding to the LOQ is shown in Figure 7.

Table 6
Parameters evaluated in linearity, LOD and LOQ

PAH	Slope	Intercept	R2	RSD / %	LOD / (µg L-1)	LOQ / (µg L-1)
Naphthalene	0.0227	-0.0057	0.9911	8.99	0.03	0.10
Acenaphthylene	0.0283	-0.0018	0.9923	7.54	0.03	0.10
Acenaphthene	0.0175	-0.0006	0.9955	10.00	0.03	0.10
Fluorene	0.0209	-0.0011	0.9978	10.90	0.03	0.10
Phenanthrene	0.0321	0.00007	0.9904	11.10	0.03	0.10
Antrhracene	0.0279	-0.0021	0.9912	14.70	0.03	0.10
Fluoranthene	0.0347	-0.0017	0.9969	11.51	0.03	0.10
Pyrene	0.0378	-0.0021	0.9967	8.71	0.03	0.10

Figure 7
GC-MS chromatogram obtained for low-molecular-weight PAHs at a concentration of 1.0 µg L-¹ after the DPX procedure using IL-MMT sorbent. The y-axis represents the GC-MS signal intensity (arbitrary units, a.u.). Naf: naphathlene; Acy: acenaphthylene; Ace: acenaphthene; Flu: fluorene; Phe: phenantrene; Ant: anthracene; Fla: fluoranthene; Pyr: pyrene.

Additional chromatographic data are provided in the Supplementary Information (SI) section, including a representative chromatogram showing all monitored compounds - target PAHs, deuterated surrogate standards, and the internal standard (Figure S1) - as well as chromatograms illustrating the separation of PAHs together with surrogate standards and the internal standard at different concentration levels (Figure S2).

The R², ranging from 0.9904 to 0.9978, indicate adequate linear behavior across the entire concentration range evaluated for all PAHs. The RSD values, mostly below 15%, demonstrate good repeatability of the method at µg L^-1 levels. In addition, the low LOD (0.03 µg L^-1) and LOQ (0.10 µg L^-1) indicate sensitivity compatible with the determination of PAHs at trace concentrations typically found in natural waters. These figures are consistent with the performance reported for recent analytical methods applied to environmental samples, particularly those based on solid-phase extraction and GC-MS, which commonly achieve low detection limits and satisfactory precision for PAH monitoring.⁴ Collectively, these results confirm the reliability and suitability of the optimized DPX method for quantitative environmental analysis.

Accuracy and precision

Considering the linear range evaluated for PAHs (0.1-3.0 µg L^-1), the recovery results obtained for samples fortified at 0.60, 1.40 and 2.75 µg L^-1 demonstrated satisfactory method performance across the entire working range (Table 7). The mean recoveries (n = 3) were, in most cases, close to the theoretical recovery of 100%, with moderate data dispersion, as expressed by the RSD (%) values (Table 7). Statistical evaluation using the Student’s t-test indicated, for the majority of PAHs and concentration levels, no statistically significant difference relative to the theoretical recovery (Table 7). Deviations observed above or below 100% were predominantly attributed to experimental variability and do not indicate systematic bias of the method.

Table 7
Mean recovery (n = 3) and statistical evaluation by Student’s t-test (α = 0.05) of PAH extraction using IL-MMT in DPX

PAH	Concentration / (µg L-1)	Recovery / %	RSD / %	∆r / %	p-value	F value
Naphthalene	0.60	78	14	-22	0.0730	12.221
Acenaphthylene	0.60	91	31	-9	0.6359	0.306
Acenaphthene	0.60	86	25	-14	0.3765	1.272
Fluorene	0.60	100	21	0	1.0000	0.000
Phenanthrene	0.60	114	24	+14	0.4695	0.783
Anthracene	0.60	82	14	-18	0.1134	7.350
Fluoranthene	0.60	97	24	-3	0.8442	0.050
Pyrene	0.60	108	13	+8	0.4266	0.980
Naphthalene	1.40	123	3	+23	0.0085	115.924
Acenaphthylene	1.40	103	18	+3	0.8052	0.079
Acenaphthene	1.40	95	22	-5	0.7188	0.172
Fluorene	1.40	110	26	+10	0.6063	0.367
Phenanthrene	1.40	95	21	-5	0.7072	0.187
Anthracene	1.40	114	22	+14	0.4359	0.933
Fluoranthene	1.40	93	20	-7	0.5814	0.425
Pyrene	1.40	85	24	-15	0.3308	1.622
Naphthalene	2.75	101	18	+1	0.9329	0.009
Acenaphthylene	2.75	91	9	-9	0.1977	3.614
Acenaphthene	2.75	92	13	-8	0.3675	1.333
Fluorene	2.75	95	13	-5	0.5572	0.488
Phenanthrene	2.75	78	8	-22	0.0255	37.773
Anthracene	2.75	97	14	-3	0.7392	0.146
Fluoranthene	2.75	76	4	-24	0.0052	192.000
Pyrene	2.75	71	8	-29	0.0126	77.655
RSD: relative standard deviation; ∆r: deviation from the theoretical recovery (100%); F: Fisher’s F statistic; Fcritical = 18.51 at α = 0.05, with df1 = 1 and df2 = 2.

Among the evaluated PAHs, fluorene, phenanthrene and anthracene generally exhibited ∆r values closer to zero across the studied concentration range, indicating good agreement with the theoretical recovery of 100% (Table 7). In contrast, phenanthrene, pyrene, fluoranthene and naphthalene showed statistically significant differences relative to the theoretical value at least one of the evaluated concentration levels, as indicated by p-values lower than 0.05 (Table 7). These deviations do not suggest systematic bias of the method and are mainly attributed to experimental variability associated with differences in the physicochemical properties of the analytes, such as volatility, hydrophobicity and affinity for the sorbent, as well as more pronounced matrix effects for specific compounds. In particular, higher molecular weight PAHs tended to exhibit larger deviations from 100%, while more volatile compounds, such as naphthalene, may be more susceptible to experimental losses, resulting in occasional variations in recovery values.

The recovery values obtained for the evaluated PAHs ranged from 71 to 123% (Table 7). Most results were within the acceptance criteria commonly adopted for methods applied to concentrations below 10 µg L^-1, according to INMETRO¹⁸ guidelines. Recovery values slightly above this range were observed in specific cases, notably for naphthalene at one concentration level, and are attributed to experimental variability associated with its higher volatility rather than to systematic method bias, as supported by the statistical evaluation. The associated uncertainties, expressed as RSD (%), ranged from 3 to 31%, with higher values observed at lower concentration levels.

Considering the evaluated parameters, the method can be regarded as suitable for its intended purpose, providing adequate and consistent extraction of PAHs across the studied concentration range.

Comparison with previous works

A comparison with previously reported methods (Table 8) highlights the advantages of the proposed procedure in terms of miniaturization, sustainability, and operational simplicity. In contrast to conventional extraction techniques such as solid-phase extraction (SPE) and liquid-liquid extraction (LLE), which generally require larger sample volumes, higher solvent consumption, and multiple preparation steps,³,⁷,⁸ the proposed method enabled the determination of eight low-molecular-weight PAHs in water samples using an environmentally friendly sorbent in a small amount (15 mg), together with reduced solvent (250 µL) and sample (2.6 mL) volumes, significantly decreasing reagent consumption and waste generation.

Table 8
Comparison between the developed method and those reported in the literature

Analyte	Sample (type / volume)	Extraction solvent (type / volume)	Sorbent (type / mg)	Technique	Recovery / %	LOQ / (µg L-1)	Reference
8 PAHs	seawater / 2.5 mL	hexane / 250 µL	IL‑MMT / 15	GC-MS	71-123	0.10	this work
12 PAHs + 2 hormones	river water / 700 µL	ACN:MeOH 1:1 / 200 µL	polypyrrole / 5	LC-DAD	41-139	5.0-20	30
4 PAHs	lotus root / 5 mL	qcetone:qcetic acid:water 8:1:1 / 1 mL	PUP polymer / 3	LC-FLD	84.6-114.3	0.29-0.93	31
2 PAHs	microalgae cultures / 1 mL	ACN / 500 µL	C18 / 50	LC-UV	83-93	2.3-2.9	32
5 PASHs	water & petrochemical residues / 700 µL	MeOH / 200 µL	8‑eHQ‑Pd silica / 20	GC-MS	72.7-131.2	0.2-2.7	33

The extraction protocol comprises only two steps -extraction and desorption - eliminating additional operations such as agitation, centrifugation, or solvent evaporation,³,⁷,⁸ thereby simplifying sample preparation, shortening analysis time, and lowering energy demand. These features align the method with the principles of green chemistry, particularly waste prevention, minimization of auxiliary solvent use, and improved energy efficiency.²⁹

Despite this streamlined approach, the limits of quantification and recoveries remained satisfactory, demonstrating analytical performance comparable to that of conventional methods reported in the literature. Overall, the proposed method represents an efficient, sustainable, and competitive alternative for the determination of PAHs in aqueous matrices.

Although no formal assessment using green analytical chemistry metrics such as AGREE or AGREEprep was performed - tools designed to quantitatively evaluate the environmental impact and sustainability of analytical procedures - the proposed method presents key features associated with sustainable analytical approaches, including reduced consumption of sorbent, solvent, and sample, as well as a simplified and low-energy workflow. These characteristics suggest that the method is aligned with green analytical chemistry principles and would likely achieve favorable performance in such metrics, although a quantitative evaluation was not within the scope of this study.

Application of the analytical method in aqueous samples

The validated method was applied to a seawater sample collected in Praia de Leste, Pontal do Paraná-PR, located between the Paranaguá and Guaratuba bays, a coastal region influenced by intense maritime traffic due to its proximity to the port of Paranaguá. This context makes the area particularly relevant for assessing the presence of PAHs in coastal waters associated with port activities and maritime transportation. The detected PAHs are presented in Table 9.

Table 9
Concentration of PAHs in seawater samples analyzed

PAH	Concentration / <sc>(</sc>µg L-1)
Naphthalene	0.31 ± 0.04
Acenaphthene	< 0.10
Fluorene	0.14 ± 0.03
Anthracene	0.13 ± 0.01
Fluoranthene	0.14 ± 0.02
Pyrene	0.16 ± 0.01

A representative GC-MS chromatogram of a simulated seawater sample fortified with PAHs and the internal standard is provided in the SI section (Figure S3).

As summarized in Table 9, the analyzed seawater sample confirmed the occurrence of PAHs, demonstrating the applicability of the proposed method to a real coastal matrix. Acenaphthene was detected below the limit of quantification of the method, whereas naphthalene, fluorene, anthracene, fluoranthene, and pyrene were reliably quantified at concentrations between 0.13 and 0.31 µg L^-1 (Table 9). The results agree with the recovery performance and statistical evaluation presented in Table 7, with fluorene and fluoranthene exhibiting more stable analytical behavior, while naphthalene, anthracene, and pyrene showed slightly higher variability in ∆r. Importantly, all compounds met the established recovery acceptance criteria, with no evidence of systematic bias, indicating that the performance observed in the real sample is consistent with the characteristics verified during method validation.

Dissolved PAH concentrations in marine environments are known to vary widely, typically spanning from ng L^-1 levels in offshore or open-ocean waters to µg L^-1 levels in more impacted coastal regions, depending on local anthropogenic inputs such as shipping activities, wastewater discharge, and atmospheric deposition.³⁴-³⁷ Within this framework, the satisfactory quantification achieved here highlights the suitability of the proposed procedure for PAH determination in seawater and supports its application in routine monitoring of coastal environments.

Dissolved PAH concentrations in marine environments are known to vary widely, typically spanning from ng L^-1 levels in offshore or open-ocean waters to µg L^-1 levels in more impacted coastal regions, depending on local anthropogenic inputs such as shipping activities, wastewater discharge, and atmospheric deposition.³⁴-³⁷ In this context, elevated PAH concentrations have been reported in impacted coastal environments, in some cases approaching the µg L^-1 range, supporting the applicability of the LOQ obtained in this study for environmental monitoring purposes.

Although the LOQ obtained in this study (0.10 µg L^-1) is suitable for the determination of PAHs in coastal and impacted environments, it may not be sufficient for the quantification of ultra-trace levels typically found in remote or open-ocean waters without additional preconcentration steps.

To confirm the extraction efficiency, a known concentration (1.50 µg L^-1) of deuterated PAHs was added. The recovery for these compounds ranged from 89.7 to 119.7%, demonstrating acceptable values.

Conclusions

The optimization of the DPX extraction procedure using IL-MMT as sorbent provided satisfactory analytical performance for the determination of low-molecular-weight PAHs in aqueous samples. The method exhibited adequate linearity (R² > 0.99), limits of quantification compatible with trace-level analysis, and recovery values ranging from 71 to 123%, with deviations attributable to normal experimental variability rather than systematic bias.

The protocol required small amounts of sorbent and reduced sample and solvent volumes, while maintaining a simplified and efficient extraction workflow, thereby contributing to lower reagent consumption and improved operational practicality.

Salinity simulation at levels comparable to those found in seawater indicated the potential applicability of the method to saline environmental matrices, enabling the determination of PAHs at ng-µg L^-1 levels. The satisfactory recoveries of deuterated standards indicated adequate extraction efficiency and robustness under the tested conditions. Nevertheless, analyses using real samples are recommended to further confirm the method performance in more complex matrices.

Overall, the proposed approach represents a simple, reliable, and environmentally favorable alternative for routine monitoring of PAHs in coastal and aquatic environments.

Supplementary Information

Supplementary information is available free of charge at http://jbcs.sbq.org.br as PDF file.

Supplementary PDF

Supplementary PDF

Acknowledgments

The authors are grateful to the CNPq for the financial support which made this research possible. ChatGPT version 5.2 (OpenAI) was used to improve the fluency of the English language and to assist in checking data during the statistical analysis.

Data Availability Statement

All data are available in the text.

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Edited by

• Editor handled this article:
  César Ricardo Teixeira Tarley (Associate)

Publication Dates

• Publication in this collection
  24 Aug 2026
• Date of issue
  2026

History

• Received
  12 Mar 2026
• Accepted
  06 July 2026
• Published
  07 Aug 2026