Introduction of a small volume ethyl acetate based liquid-liquid extraction procedure for analysis of polycyclic aromatic hydrocarbons in wastewater by atmospheric pressure gas chromatography-mass spectrometry and evaluation of method greenness

• Eco-friendly method meets sensitivity requirement without evaporative concentration.
• Enhanced selectivity due to use of PAHs specific column and soft ionization source.
• Method accuracy proved with validation, MU calculation and PT participation.
• Simple alternative to complex conventional methods for PAHs testing in water.
• High-throughput method ranked excellent in greenness evaluation by AES and GAPI.

Abstract

The Small Volume Ethyl Acetate based Liquid-Liquid Extraction (SVEA-LLE) method, using Atmospheric Pressure Gas Chromatography-Mass Spectrometry (APGC-MS/MS) with Dual Head Robotic Tool Change (DH-RTC) Prep and Load (PAL) autosampler, was evaluated for the analysis of 17 PAHs in water and wastewater. The extraction method was first evaluated for extraction efficiency with respect to time using a one-way Analysis of Variance (ANOVA) and the effect of acidification. Method validation was performed as per SANTE/11312/2021 v2 2024 for analysis of PAHs in wastewater. Linearity was determined over a range of 5 to 80 ng/ml, meeting the acceptable coefficient of determination (r2) and the values of % deviation from back-calculated concentration. The method was observed to be sensitive enough, as the estimated Limits of Detection (LOD) and Quantification (LOQ) were observed to be lower than or close to the Limits specified in EPA 625.1/2016 method, without the need for evaporative concentration. During the method validation trials, it was observed that 82.71 % of the observations were below 10 % RSD, 14.19 % were in the range of 10 % to 15 % RSD, and 3.08 % were between 15 % and 20 % RSD (n = 6). Similarly, in the case of mean % recovery, 92.59 % of the observations were between 80 to 120 % recovery, 3.70 % of the observations were between 70 to 80 % recovery, 3.08 % of the observations were 120 to 130 % recovery (n = 6). It was observed that mean % recovery of robustness study (n = 18) ranged from 90.21 to 117.82 (observed for phenanthrene and benzo (a) pyrene respectively). Further, the % RSD from robustness study ranged between 5.11 to 18.98 (observed for acenaphthylene and pyrene respectively). Measurement Uncertainty of all the analytes ranged from 3.73 to 8.92 % (observed for fluorene and pyrene, respectively). The method described has been observed to be fit for purpose, as it meets the method validation requirements with acceptable results in PT participation. The greenness assessment of the method using tools like the Analytical Eco-Scale (AES) and the Green Analytical Procedure Index (GAPI) indicated that method ranks excellent in greenness evaluation and is eco-friendly. The optimized sample preparation method is short, safe, and easy with an extract suitable for direct injection onto GC column, eliminating the concentration and reconstitution steps for high-throughput analysis of PAHs in water and wastewater.

1. Introduction

Water is essential in all aspects of life in adequate quantities and quality. However, water in nature picks up some amounts of chemical impurities, most of which are anthropogenic and these significantly impact its quality [1]. Chemical pollutants have been released into the environment since the industrial revolution, but their release and dispersal have accelerated markedly in the last half-century [2]. Polycyclic Aromatic Hydrocarbons (PAHs) are a major group of chemicals that pose serious threats to human health and the environment. PAHs are compounds with two to seven fused aromatic rings, that can have substituted groups attached, ranging from naphthalene (C₁₀H₈, two rings) to coronene (C₂₄H₁₂, seven rings) with molecular masses from 128 to 300 Da [3]. More than 80 % of the total PAHs contribution to environmental and health concerns can be attributed to the first 16 EPA priority PAHs shown in Table 1.

PAHs are generated from both natural and anthropogenic sources and are ubiquitous in nature [3]. PAHs enter shallow coastal, estuarine, lake, and riverine environments through petroleum spills, treated industrial and municipal wastewater discharges, urban and suburban stormwater runoff, chemical refineries, recreational and commercial boats, volcanoes, and atmospheric fallout from vehicle exhaust [4,5]. PAHs in water bodies are subject to distribution and accumulation among the water column, suspended particulate matter, bottom sediments, and biota [6], due to their low vapor pressure, non-polarity, lipophilicity, and high hydrophobicity [7]. The contamination of sediments from aquatic bodies by PAHs was evaluated by comparing the PAH concentration in sediment samples with the effects-based guideline values, like the Effects Range Low (ERL) and Effects Range Median (ERM) established by the United States National Oceanic and Atmospheric Administration (USNOAA) [8,9]. Table 1 presents the ERL and ERM concentrations for the 16 priority PAHs. The ERL values suggest a likelihood of serious biological effects on aquatic organisms, while ERM values indicate a greater risk of damaging biological effects on aquatic life [9].

PAHs are generally carcinogenic and mutagenic and may induce lung, bladder, and skin cancer [13]. The carcinogenic nature of these compounds is a major concern and are classified into five groups based on their carcinogenic potential [3]. Group 1 consists of substances with carcinogenic potential for humans, and benzo (a) pyrene with sufficient toxicological data, is the only member of this group [10,11]. Group 2A PAHs are those that are probably carcinogenic to humans, with dibenz (a,h) anthracene being a major example among the EPA priority contaminants in this group. Eight of the PAHs from groups 1, 2A, and 2B have been identified by the United States Environmental Protection Agency (USEPA) and the International Agency for Research on Cancer (IARC) as being of high risk to humans and are therefore used to assess the level of pollution in the environment (Table 1). Aside from their confirmed carcinogenicity, all eight PAHs were also found to be genotoxic alongside benzo (g,h,i) perylene which was not classified as being carcinogenic to humans [12]. In addition, exposure to high levels of PAHs has been shown to produce immunosuppressive effects and cause oxidative stress during their metabolism [14]. Bioconcentration and bioaccumulation of PAHs in organisms occur through various routes, including ingestion, inhalation, or dermal contact pathways [3]. The USEPA has established Toxicity Equivalency Factors (TEFs) to quantify their level of toxicity. The highest TEF of 1 was assigned to benzo (a) pyrene, while lower values as shown in Table 1, were assigned to the other six PAHs. Toxicity Equivalent Concentration (TEC) of an individual PAH can be calculated by multiplying the concentration of the congener in the environmental sample with the respective TEF [3].

Traditionally, PAHs are analyzed in different types of water and other environmental matrices using standard reference methods as detailed in Table 2, with Liquid-Liquid Extraction (LLE) or Solid Phase Extraction (SPE) and detection using Electron Impact (EI) ionisation and Gas Chromatography with Mass Spectrometry or Liquid Chromatography with specific detectors. These conventional extraction methods generally involve several steps including repeated extraction of large volume of sample using large quantities of carcinogenic solvent such as dichloromethane (DCM), followed by filtration, clean-up steps (including GPC if required), column chromatography or Solid Phase Extraction (SPE), evaporative concentration of large volume of extract, and reconstitution in a suitable solvent for GC or LC analysis. These steps as detailed in Table 2, make conventional methods laborious and time-consuming. Environmental restrictions should also be considered as DCM is carcinogenic and is difficult to use in some of the countries due to its legal restrictions. The U.S. Environmental Protection Agency finalized a ban on most uses of DCM on April 30, 2024, citing it as a dangerous chemical.

Typical solvents used for extraction in analytical methods are acetone [[30], [31], [32], [33]], ethyl acetate [[34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55]], and acetonitrile [[56], [57], [58], [59], [60]]. Ethyl acetate has been shown to be equivalent to water-miscible solvents in terms of extraction efficiency, for both polar and non-polar pesticides in vegetables, fruits and dry products [35,36,55,[32], [61]]. Ethyl acetate has good wettability in GC columns, which is beneficial for solvent trapping of the most volatile analytes, which is required for refocusing after injection. Its vapor pressure and expansion volume during evaporation also favor large-volume injection. The ethyl acetate extract can also be used for LC analysis after reconstitution into methanol [40,44,47,55], as is done in acetone-based methods [62]. Ethyl acetate is an equally acceptable as extraction solvent for different products and is especially suitable for extracting high-sugar commodities due to limited solubility of sugar in ethyl acetate [35,44,48,55,63,64]. Ethyl acetate is approved by the U.S. Food and Drug Administration (FDA) for use as a food additive and flavoring agent, indicating its relative safety in small, regulated amounts. Also, it does not fall under the hazardous category according to the EPA's list of Toxic Release Inventory (TRI), nor is it classified as PBT (Persistent, Bio-accumulative, and Toxic).

The importance of sample preparation in developing greener analytical methods continues to be the focus of many studies. Major aspects of the eco-friendly and green sample preparation include the use of safe solvents, reagents, and materials that are renewable, recyclable and reusable, minimizing waste generation and energy demand; and enabling high-throughput, miniaturization, procedure simplification, automation, and operator safety. Studies have suggested applying direct analytical techniques to avoid sample preparation for greener analytical methods. However, any ‘green’ actions taken during the sample preparation steps negatively impacted the accuracy, precision, selectivity, sensitivity, and detectability of the analytical process [75]. Therefore, this study emphasised developing an analytical method that is simple, high-throughput, resource efficient, safe for both human health and the environment, while ensuring that accuracy, precision, specificity, sensitivity are not compromised.

In GC–MS/MS, the most widely used technique of ionization is Electron Impact (EI) ionisation due to its ability to ionize broad range of organic compounds. It is well known that the EI source produces high fragmentation of molecules, often leading to absence of molecular ions in most cases in EI spectra. Due to the absence of quasi-molecular ions in MS spectra sensitivity and specificity of the method gets significantly affected. Chemical ionization (CI) or high-resolution mass spectrometry, if available, can provide additional information needed for exhaustive elucidation [[65], [66], [67]]. It has been observed in the literature that, in addition to the selectivity advantage arising from the ability to use the quasi-molecular ion, sensitivity is also substantially improved with the use of APGC-MS/MS [68].

The objective of the current study is to develop a simple, safe, and high-throughput alternative procedure to the complex conventional methods for the analysis of PAHs in water. The proposed method was subjected to optimization, verification, validation, Measurement Uncertainty (MU) calculation, Proficiency Testing (PT), and greenness evaluation using tools such as the Analytical Eco-Scale (AES) and Green Analytical Procedure Index (GAPI).

2. Experimental

2.2. Sample preparation

The Small Volume Ethyl Acetate-based Liquid-Liquid Extraction (SVEA-LLE) method was used for sample preparation. A 10 mL wastewater sample prepared by homogeneously mixing of pre-screened 100 control samples collected from different locations was aliquoted into 50 mL polypropylene centrifuge tubes for sample control, calibration curve (CC), and quality control (QC). All CC and QC samples were spiked with 17 PAHs at the selected concentration level, and 11 internal standards were spiked at a fixed concentration at the midpoint of the calibration curve. A 10 mL of ethyl acetate were added to the aliquoted test samples in centrifuge tubes. The contents were mixed for 30 seconds, and the tubes were set aside for 15 minutes. Then, 4 g of anhydrous sodium sulfate was added followed by vigorous manual shaking for 30 seconds, vortex mixing for five minutes and centrifugation of the tubes at 4000 rpm for 20 minutes in a refrigerated (10 °C) centrifuge. Finally, 1 mL of the supernatant was transferred into 2 ml GC vials for further injection of 1 µL sample volume into the GC injector port.

2.3. Instrumentation

Data were acquired using a GC system (Agilent 8890B, Palo Alto, CA, USA) coupled to a triple quadrupole (QqQ) mass spectrometer (Xevo TQ-XS, Waters Corporation, Manchester, UK), with the source operating in atmospheric pressure that is APGC mode and equipped with Dual Head Robotic Tool Change (DH-RTC) Prep and Load (PAL) autosampler (CTC Analytics AG, Zwingen, Switzerland). A fused silica Select PAH GC capillary column (Length 30 m x I.D. 0.25 mm x film 0.15 µm) (Agilent part no. CP7462 J&W Scientific, Folsom, CA, USA) was used for GC separation. A deactivated capillary (column without stationary phase) guard column (0.75 meter) and retention gap (0.75 meter) were connected to the front and back end of the main column, respectively, using multi union kit. The injector was operated in pulsed spitless mode, injecting 1 µL at 280°C. The oven temperature was programmed as follows: 50°C (hold for 1.0 min), ramped at 10°C/min to 120°C (no hold), and ramped at 20°C/min to 320°C (hold for 10.0 min). Helium was used as a carrier gas in constant flow mode (2 mL/min). A pulsed spitless injection was carried out using an initial pressure of 32 psi, maintained for 1.2 minutes, and then switched to a constant flow mode (2 mL/min), which corresponded to a linear velocity of 41.087 cm/s. In the MRM method, automatic dwell time was applied to obtain 12 to 15 points per peak. The interface temperature was set to 330°C, with N₂ as heated transfer line gas at 280 mL/min. Source temperature was set at 150°C, and auxiliary and cone gases were set at 200 and 260 L/h respectively. The corona discharge pin in the source was operated at 2 µA, and dry source conditions were maintained for charge transfer-based ionization. MS was operated in MRM mode for acquisition with MS method events: corona current of 20 µA for the first 3 min and after 3 min it was set to 2 µA and cone gas set to 50 mL/min for first 3 min and after 3 min it was set to 260 mL/min. The autosampler (DH-RTC PAL) used was equipped with tools for liquid injection, SPME arrow extraction an agitator and stirrer, a vortex, a conditioning station and syringe washing station. During this work, the DH-RTC PAL with liquid injection was used for injection of 1 µL using 10 µL syringe with injection and fill speeds of 100 and 20 µL/sec respectively. The injection was performed with 6 pre-cleaning cycles of ethyl acetate and 6 post-cleaning cycles of acetonitrile to avoid carryover. Chronos software was used to control, create, and load batch sequences using the DH-RTC PAL autosampler. Optimized MRM transitions, with Retention Time (RT), Quantifier (Q1), Qualifier (Q2), cone voltages, and collision energy are as shown in Table 3.

2.4. Outlines of method optimisation and validation

Small Volume Ethyl Acetate based Liquid-Liquid Extraction (SVEA-LLE) was evaluated for its fitness for purpose. The optimized instrument method parameters of APGC-MS/MS with DH-RTC-PAL were evaluated for consistency in RT, Ion Ratio, area of the qualifier and quantifier ion, calculated concentration, peak asymmetry factor and chromatographic resolution of the isomeric peaks. The ethyl acetate-based extraction method was verified for its performance in MilliQ water and wastewater to assess the linearity, recovery, and repeatability. The extraction efficiency of the method was evaluated by acidification and an increase in extraction time. Furthermore, the method was fully validated in wastewater for method performance criteria outlined in the method validation guideline that is SANTE/11312/2021 v2 2024 [69]. Measurement Uncertainty (MU) of the method was calculated at the targeted Limit of Quantification (LOQ), by referring to the international guidelines that is JCGM 100:2008 and EURACHEM/CITAC Guide CG 4 [70,71]. The validated method was further validated for its accuracy by participating in Proficiency Testing (PT) program conducted in accordance with ISO/IEC 17043:2023 [72] by Phenova for water pollution for program code of WP0723.

3. Results and discussion

3.1. Method optimization and evaluation

3.1.1. Instrument method optimization

Optimized MRM transitions with cone voltages and collision energy as shown in Table 3 were verified using PAHs standard solutions in ethyl acetate, with the mass spectrometer operating in MRM mode to check the response and selection of quantifier and qualifier. During the initial method development trials, it was observed that in a sequence of continuous injections chromatographic peak shapes of high boiling PAHs were getting affected. PAHs with high molecular weight are always difficult to analyse with GC due to their low volatilities, tendency to decompose when subjected to high temperature, and possibility of adsorbing onto the GC inlet and column [73]. The loss of peak shape in continuous operation was attributed to a relatively long length of transfer line and limitation in heating it due to the maximum temperature of the column (325°C). To ensure stability of peak shape in long run, deactivated capillary column of 0.75 meters without stationary phase was used as a guard column, and retention gap was connected to main column with multi union kit. It has been observed that connecting guard column and retention gap as well as maintaining the transfer line temperature at 330°C helped in maintaining symmetrical peak shape during continuous instrument operation without the need for routine maintenance. This improvement in continuous operation for high-boiling PAHs was attributed to the readily volatile compounds entering the main column from guard column. Elevated transfer line temperature prevents the formation of cold spots in transfer line and helps to ensure complete removal of high-boiling matrix compounds from column.

3.1.2. Column chromatography

A major challenge in PAHs analysis is chromatographic resolution of the isomeric peaks. Out of targeted 17 PAHs there are 5 pairs (phenanthrene and anthracene, fluoranthene and pyrene, Benzo (a) anthracene and chrysene, benzo (b) and (k) fluoranthene, and dibenz (ah) anthracene and indeno (1,2,3-cd) pyrene) of isomeric or closely eluting PAHs. These pairs exhibit same molecular mass (Table 1) and/or close elution. These isomeric PAHs are challenging to resolve chromatographically and maintain peak shape, which makes their accurate quantification difficult in routine testing. The chromatography of five isomeric pairs of PAHs was evaluated on a Select PAH GC column which offers enhanced selectivity towards PAHs. It was observed that these four challenging isomeric pairs were resolved base-to-base on the Select PAH GC column as shown in Fig. 1. The most challenging pair of the isomer that is benzo (b) and (k) fluoranthene was well separated chromatographically, as base line valley between the two peaks was less than 25 % as showed in Fig. 1. It was observed that the baseline valley between the two peaks of benzo (b) and (k) fluoranthene increased to 50 % upon continuous operation for two weeks without maintenance. This may be because of the high boiling point (Table 1) of these isomers which is higher than the maximum inlet temperature of the GC, causing incomplete evaporation and deposition of high boiling compounds on the inlet and front end of the GC column. Therefore, the approach of reporting the results as sum of benzo b and benzo k fluoranthene is recommended. However, in the case of single isomer presence in the unknown sample, individual peak integration and reporting are recommended.

Journal of Chromatography A, Volume 1740: Fig. 1. Chromatographic separation of the 5 pairs of isomeric PAHs.

3.1.3. System suitability Test for optimised instrument method

As the APGC-MS/MS system was configured with DH-RTC PAL autosampler, which is a relatively a new instrument configuration, therefore the system suitability was evaluated by subjecting the optimised instrument method to continuous injections (n = 10) @ 50 ng/ml concentration. During this study, the consistency of Retention Time (RT), ion ratio, peak shape, area (Q1 and Q2), and calculated concentration were evaluated for 17 PAHs and 11 Internal Standards (IS). The results for RT were consistent for all the compounds as they did not deviate by more than the acceptable tolerance of ± 0.1 minutes. It was observed that the ion ratio (Q2/Q1) for all compounds was consistent as % relative standard deviation for the ion ratio was less than acceptable tolerance of 30 %. Average peak asymmetry factor for 23 compounds was less than 1.5, with acceptable repeatability and for 6 compounds, it ranged from 1.5 to 1.8. It was observed that the % RSD of the response in terms of area for both Q1 and Q2 was less than 15 % for all compounds, except for the Q2 ion of benzo (b) fluoranthene, which was 15.96 %. With the use of Internal Standards (IS) for quantification, the % RSD for calculated concentrations was less than 10 % for all 17 targeted PAHs and 11 IS. The results from the trials as shown in Table 3 indicates that the instrument method is well optimised and ready for further method verification in MilliQ water and wastewater using SVEA-LLE protocol.

3.1.4. Effect of acidification on efficiency of extraction

Sample pH is an important parameter that can affect the distribution constant, hydrolysis, and form of analytes. The pH value influences the ionization status and solubility of the analytes. For the efficient extraction of ionizable and relatively polar compounds, pH of the sample solution plays a decisive role. The sample solution pH should be lower than the pKa of the analytes to obtain the target analytes in their unionized forms, so that they have a higher tendency to partition into the organic phase. In standard reference methods from the EPA and the APHA extraction efficiency was evaluated under acidic and neutral condition for all targeted analytes. Therefore, wastewater samples spiked at 10.00 ng/ml were analysed for targeted PAHs by lowering their pH down to 2 with formic acid to evaluate the extraction efficiency of PAHs in acidic environment. The average area of the targeted PAHs and IS was compared and the Relative Percent Deviation (RPD) of average area is calculated in acidified and without acidified (base-neutral) samples (n = 6). There was no significant improvement in the average response upon acidification, as RPD between the average areas with and without acid extraction was less than 5 for all targeted PAHs, as showed in Fig. 2. For targeted PAHs, the RPD ranged from a minimum of 0.64 to 4.83 for anthracene and benzo (ghi) perylene respectively. This observation is in line with APHA 6410B, EPA 625.1, and 8270D, as PAHs are categorised as base-neutral extractables in these methods.

Journal of Chromatography A, Volume 1740: Fig. 2. Study of effect of acidification.

3.1.5. Effect of extraction time on efficiency of extraction

It was observed during the trials conducted to evaluate extraction efficiency with respect to time (2, 5, and 10 minutes) that extracting samples for 5 minutes produced the best results in terms of area and % RSD. Analysis of variance (ANOVA) shows that absolute area increased significantly (p<0.05) at the 5-minute extraction compared to the 2-minute extraction for all targeted PAHs, except for anthracene. It was observed that for 8 high-boiling and late-eluting PAHs (benzo (a) anthracene, chrysene, benzo (b) fluoranthene, benzo (k) fluoranthene, benzo (a) pyrene, indeno (1,2,3-cd) pyrene, dibenz (ah) anthracene and benzo (ghi) perylene) the area increased from 1.5 to 2.4 times upon 5 minutes extraction compared to the 2-minute extraction. Furthermore, there was no additional improvement when the results for extraction times of 5 and 10 minutes were compared as shown in Table 4. The percentage relative standard deviation (% RSD) of the calculated concentration using internal standards at a 5-minute extraction time for all targeted PAHs was observed to range from 0.59 to 8.15, for benzo (b) fluoranthene and anthracene respectively. Therefore, an extraction time of 5 minutes without acidification of the samples was chosen for sample extraction in the subsequent method verification and validation trials.

3.1.6. Initial SVEA-LLE method verification and applicability study in types of water matrices

The optimized SVEA-LLE method using APGC-MS/MS with a DH-RTC PAL autosampler was verified and assessed for its applicability in MilliQ water (MQ) and wastewater (WW). A five-point procedural calibration curve was prepared in (MQ) water and used to assess precision and recovery (n = 6) at concentration of 5 and 10 ng/ml (MQ water) and at 20 ng/ml (WW). Method verification parameters such as consistency of retention time (RT), ion ratio, linearity, % recovery, and % RSD were verified before proceeding with the final validation of the method in wastewater. Retention Time (RT) and Ion ratio for the targeted 17 PAHs were observed to be consistent in both types of water. Ion ratios were observed to be consistent in both MilliQ water and wastewater matrices and with deviations not exceeding 10.09 %, as observed in the case of 2 chloronaphthalene. The response for all 17 PAHs was observed to be linear, and coefficient of determination (r²) was observed to be ranging between 0.9914 to 0.9998 for naphthalene and phenanthrene respectively. The average % recoveries in MilliQ water at both the levels ranged from a minimum of 77.52 to maximum of 106.35 % for fluoranthene and dibenz (ah) anthracene respectively. The % RSDs ranged from a minimum of 3.81 to a maximum of 11.55 % for 2-chloronaphthalene and acenaphthylene respectively. Recoveries in wastewater at 20 ng/ml were observed to range from a minimum of 88.98 to a maximum of 103.02 % for benzo (k) fluoranthene and phenanthrene respectively. The % RSDs ranged from a minimum of 3.30 % to a maximum of 11.81 % for dibenz (ah) anthracene and phenanthrene respectively. The results shown in Fig. 3, for % recovery and % RSD are acceptable in both MilliQ water and wastewater using the SVEA-LLE protocol. Further validation of the method was conducted in wastewater considering it the most challenging type of water matrix.

Journal of Chromatography A, Volume 1740: Fig. 3. SVEA-LLE Method Verification Results.

3.1.7. Evaluation of the greenness of the method using AES and GAPI

The Analytical Eco-Scale (AES) was used to evaluate the method's greenness, and it was calculated by allotting Penalty Points (PPs) to factors in the analytical procedure that do not align with the principles of a perfect green analysis. A green analysis is considered ideal if it has an Eco-Scale value of 100, excellent if greater than 75, acceptable if greater than 50, and inadequate if less than 50. Penalty Points (PPs) were assigned to each of the four main parameters of the analytical procedure that deviate from an ideal green analysis: amount and hazardousness of reagents, the energy consumption of the equipment used, and generation of waste. In the proposed method, ethyl acetate was used, which does not fall under the hazardous category according to the EPA's list of Toxic Release Inventory (TRI), nor is it classified as PBT (Persistent, Bio-accumulative, and Toxic) substance. However, due to the high flammability of ethyl acetate when exposed to heat, hot surfaces, sparks, open flames and other ignition sources, a hazard penalty points of 1 was allotted for less severe hazards, as it is stable under recommended storage conditions. A penalty points of 2 is allotted because the amount is 10 ml. Other penalty points are assigned based on the instruments used, energy consumption per sample, and the amount and treatment of waste. The proposed method achieved a score of 89 (Table 5, after subtracting 11 Penalty Points from 100), indicating its excellence as a green methodology.

The Green Analytical Procedure Index (GAPI) is a semi-quantitative tool consisting of five pentagrams that represents different stages of analytical process: sampling, sample preparation, reagents and chemicals, instrumentation, and other general aspects of method. It provides sufficient data to assess and measure the environmental impact associated with each step of the analytical approach, from sampling to the final instrumental analysis. The three main colors of the symbol- green, yellow, and red- represent low, medium, and high impact, respectively. More number of green colours in the pentagram shape of GAPI means a safe method. The application of GAPI for the proposed method, as shown in Table 6, and the pentagrams demonstrate that the method satisfies most of the criteria and confirms the proposed method is eco-friendly.

4. Conclusion

An innovative Small Volume Ethyl Acetate based Liquid-Liquid Extraction (SVEA-LLE) method for the determination of Polycyclic Aromatic Hydrocarbons (PAHs) in water and wastewater matrices was developed with satisfactory results using APGC-MS/MS with DH-RTC PAL autosampler. Ethyl acetate-based extraction offers benefits of low toxicity, low cost, suitability for direct injection into GC, reducing reconstitution step and increasing throughput of commercial sample analysis. The soft ionization allowed the use of quasi-molecular ions as a parent ion for targeted PAHs which helped to improve the selectivity of the method. The method was successfully verified in MilliQ water and wastewater and further fully validated in wastewater, making this approach suitable for analysis of 17 PAHs in different types of water samples. The presented method offers a satisfactory quantification limit with a 10 mL sample volume and extraction solvent due to the highly sensitive mass spectrometer, without the need for evaporative concentration, when compared to conventional LLE methods (EPA 625.1, APHA 6410B, EPA 3510C, EPA 8100, EPA 8410, EPA 8270D, EPA 610, EPA 8310, EPA 550.1, EPA 525.3, and EPA 1625), which typically require a 1-liter sample volume. Results obtained during the validation study indicate that the proposed method provides adequate sensitivity, specificity, linearity, trueness, precision, robustness, and overall accuracy, with the capacity for high-throughput analysis and monitoring of PAHs in different types of water matrices. The accuracy of the method was further demonstrated by the successful participation in the international Proficiency Testing (PT) program for targeted PAHs. Calculated Measurement Uncertainty (MU) was found to be acceptable for the quantitative analysis of the PAHs, demonstrating that the method is fit for its intended purpose and provides an efficient alternative to the time consuming and laborious conventional LLE methods for PAH analysis in different types of water matrices. The greenness assessment of the method using tools like the Analytical Eco-Scale (AES) and the Green Analytical Procedure Index (GAPI) indicated that method ranks excellent in greenness evaluation and is more eco-friendly. This high-throughput, simple, accurate, and eco-friendly method (SVEA-LLE) can be applied for the routine analysis of PAHs by different pollution control and water testing laboratories for large numbers of water samples, following in-house method verification and validation.