Determination of Organophosphate Esters in Water Samples Using Gas Chromatography– Mass Spectrometry and Magnetic Solid-Phase Extraction Based on Multi-Walled Carbon Nanotubes

A method based on gas chromatography–mass spectrometry (GC–MS) coupled with magnetic solid-phase extraction (SPE) with multi-walled carbon-nanotube-coated Fe₃O₄ as adsorbent was developed for the analysis of four organophosphate esters (OPEs) in ambient water samples. The magnetic multi-walled carbon nanotube composites were prepared by hydrothermal synthesis and characterized with scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FT-IR), and superconducting quantum interference device (SQUID) magnetometry. The extraction and desorption conditions, such as adsorbent dosage, adsorption time, eluent type, and eluent volume, were studied. The adsorbent was used to extract analytes within 50 min. The limit of detection was in the range of 0.038–1 μg/L and the limit of quantitation was between 0.10 and 3.59 μg/L. The method was applied to analyze organophosphate esters in environmental water samples. A 72.5–89.1% recovery was obtained by analyzing spiked samples with low-, medium-, and high-concentration analytes. The relative standard deviations were less than 10%. The method has comparable sensitivity and accuracy and can be successfully used to detect organophosphate esters in environmental water samples.

Organophosphate esters (OPEs) are common flame retardants and plasticizers and are used in the production of plastic foam, electronics, furniture, textiles, and construction materials (1,2). It has been reported that the widespread application of OPEs has led to their becoming an emerging pollutant. Most OPEs are primarily combined with the product through physical action. Therefore, they are easily released into the surrounding environmental media using volatilization, abrasion, and even dissolution. They have been detected in surface water (3,4), groundwater (5), wastewater (6,7), atmospheric particulate matter (8, 9), and dust (10). The previous studies found that some commonly used OPEs have carcinogenicity, neurotoxicity, and hepatoxicity, which could threaten human health and ecosystems (11,12). Exposure to triphenyl phosphate (TPhP) disturbed hormone levels and sperm quality in fish (13). Tributyl phosphate (TnBP)
and Tris (2-ethylhexyl) phosphate (TEHP) showed strong irritation to the skin (1).
In recent years, the United States, the European Union, and other countries around the world have set up relevant laws and regulations to limit the use of OPEs in domestic products. Organizations and institutions worldwide have also begun to pay close attention to the issue of environmental pollution from OPEs, bringing about much research on the subject.

There are many kinds of pretreatment methods for OPE extraction in water samples, such as liquid–liquid extraction (LLE) (14), membrane-assisted solvent extraction (MASE) (6), solid-phase extraction (SPE) (3,15), and solid-phase microextraction (SPME) (16,17). LLE and SPE are the most commonly used pretreatment methods. However, these methods are cumbersome and require a large amount of organic solvent for pretreatment of the extraction column. Moreover, LLE and SPE methods have poor recoveries for polar and volatile (for example, trimethyl phosphate [TMP], Tri(2-chloroethyl) phosphate [TCEP]) or hydrophobic (for example, TEHP) compounds (15,18,19). Magnetic solid-phase extraction (MSPE) has been developed as a new type of sample preparation technique and has received considerable attention. MSPE uses magnetic or magnetizable material as the sorbent for the extraction or preconcentration of the target analytes (20). The method also overcomes the column plugging problem that often occurrs in SPE, greatly simplifying the SPE process and enhancing the extraction efficiency. Currently, MSPE is widely used for qualitative and quantitative analysis of various organic pollutants and inorganic metal ion contaminants in an environmental sample when combined with high performance liquid chromatography (HPLC) or gas chromatography (GC) (21–23). This makes the method ideal for application in the field of biological separation and environmental analysis. However, MSPE is currently rarely used for OPEs analysis.

Instrumental analysis of OPEs is mainly conducted using the GC and nitrogen–phosphorous detector method (GC–NPD) (4), GC–mass spectrometry (GC–MS) (17,23), and liquid chromatography–tandem mass spectrometry (LC–MS/MS) (14,24). Among them, GC–NPD offers poor stability, requiring regular replacement parts, while the sample matrix can affect the electrospray ionization source of LC–MS/MS. In this article, a method based on MSPE–GC–MS for the analysis of OPEs in river water samples was developed. The MSPE and GC–MS detection conditions were investigated. The method offers a wide linear range and good sensitivity and recovery for both polar and nonpolar OPEs. It can be successfully used to detect OPEs in environmental water samples.

Materials and Method

Materials: Organophosphate esters (TnBP, TCEP, TPhP, and TEHP) were purchased from AccuStandard (AccuStandard Inc.). Their full names, abbreviations, chemical structures, and octanol-water partition coefficient (K_ow) are shown in Table 1. Ethyl acetate and multi-walled carbon nanotubes were obtained from the Aladdin Reagent Company. Tetraethyl orthosilicate (TEOS), polyethylene glycol, FeCl₃•6H₂O, aqueous ammonia (25 wt% aqueous NH₃), and ethylene glycol were the products of the Tianjin Damao Chemical Reagent Factory.

The OPEs stock solution with a concentration of 10 mg/mL was prepared with ethyl acetate and was stored in a refrigerator at 4 °C. According to experimental requirements, other solutions were diluted by using the stock solution. Unless specifically mentioned, ultrapure water was used in all experiments.

Instruments: The analysis was performed using an Agilent 7890 gas chromatograph coupled to an M7-300EI single quadrupole mass spectrometer (GC–MS) system. The morphology of the prepared material was observed by using a Tecnai-G20 transmission electron microscope (FEI) and a JSM-7500F scanning electron microscope (Japan Electronics). Magnetic measurements were performed by using a superconducting quantum interference device (SQUID) and a magnetometer (VSM, Quantum Design). The functional groups of the synthesized adsorbent were measured using a Fourier-transform infrared spectroscopy (FT-IR) spectrometer (920, Tuopu). Water was purified through a Milli-Q system (Millipore).

Preparation of MSPE Sorbent:

Synthesis of Fe₃O₄: Fe₃O₄ magnetic nanoparticles were synthesized by using a solvothermal method (25). First, 1.35 g of FeCl₃•6H₂O was added to a beaker containing 40 mL of ethylene glycol. The solution was then stirred for 15 min. Second, 3.6 g of anhydrous sodium acetate and 1.0 g of polyethylene glycol were added to the mixed solution. Stirring was continued even after the formation of a uniform yellow thick liquid. The sample was then transferred to a 50-mL polytetrafluoroethylene autoclave, which was sealed and placed in an oven at 190 °C for 8 h. The new-solid sample was then cooled to room temperature and the Fe3O4 nanoparticles were separated with the magnet. After washing three times with ultrapurewater and anhydrous ethanol, the sample was vacuum dried at 60 °C for 2 h. The Fe₃O₄ black powder was obtained.

Synthesis of Fe₃O₄@SiO₂ Microspheres: SiO₂-coated Fe₃O₄ microspheres were prepared via a modified Stöber synthesis (26). First, 0.1 g Fe3O4 nanoparticles were added to 50 mL HCl (0.1 mol/L) and the solution underwent ultrasound treatment for 10 min. After washing with distilled water, the Fe₃O₄ was added to a three-necked round bottom flask containing 80 mL of absolute ethanol, 20 mL of water, and 1 mL of aqueous ammonia. The solution was mixed well through mechanical agitation. A 0.03 g measure of tetraethyl orthosilicate (TEOS) was added dropwise and stirred at room temperature for 6 h. Finally, the product was separated with a magnet, washed three times with distilled water and absolute ethanol, respectively, and vacuum dried at 60 °C for 2 h. The Fe₃O₄@SiO₂ microspheres were obtained.

Synthesis of Fe₃O₄@SiO₂ Multi-Walled Carbon Nanotubes: Fe₃O₄@SiO₂ (0.004 g) and multi-walled carbon nanotubes (MWCNTs) (0.016 g) were added to deionized water. The mixture was sonicated for 15 min to ensure complete self-assembly of MWCNTs onto Fe₃O₄@SiO₂ nanoparticles. A black homogeneous liquid was formed.

Sample Preparation by MSPE: Several elements of extraction, such as adsorbent dosage, adsorption time, elution solvent, and elution time, were studied. During MSPE, 20 mg Fe₃O₄@SiO₂ MWCNTs were dispersed to 20-mL pure water solution spiked with four OPEs (10 μg/L), which was subjected to a constant-temperature 25 °C water bath with vibration mixing for 50 min. After the oscillation extraction, Fe₃O₄@SiO₂ MWCNTs that had absorbed four OPEs were separated by a magnet. The supernatant was discarded. A 1400 μL measure of the ethyl acetate was then used to desorb the OPEs by ultrasonic treatment for 20 min. The obtained elution solution containing OPEs was aspirated off with nitrogen. The residual sample was dissolved with 200 μL ethyl acetate and then analyzed by GC–MS.

GC–MS Analysis: The GC conditions were as follows. A 30 m × 0.32 mm, 0.25‑µm HP-5 column (Agilent, J&W) was used for separation of the target analytes. The carrier gas was helium (purity ≥ 99.999%) and the flow rate was 1.2 mL/min. For the heating program, the initial temperature was 70 °C, heating at 15 °C/min to 200 °C, then at 10 °C/min to 280 °C (where the temperature was held for 5 min). The inlet temperature was 280 °C and the interface temperature was 280 °C. The splitless mode was adopted and the injection volume was 1 μL.

The mass spectrometry conditions were as follows: EI ion source, energy = 70 eV, temperature = 230 °C, quadrupole temperature = 150 °C, and the data acquisition mode was a selected ion monitoring (SIM) scan. The SIM ions (m/z) of TnBP were 99, 155; TCEP were 63, 249; TPhP were 326, 77; TEHP were 99, 113.

Results and Discussion

Characterization of Fe₃O₄@SiO₂ MWCNTs:The preparation procedure of Fe₃O₄@SiO₂ MWCNTs is shown in Figure 1. The structure and composition were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and FT-IR. Figure 2(a) displays the SEM image of the pristine MWCNTs. It can be seen that the MWCNTs are long with a rather smooth surface and curly shape. With the help of references 27 and 28, and the SEM and TEM images of Fe₃O₄@SiO₂ MWCNT nanocomposites shown in Figure 2(b)–2(d), it was found that the material is nearly in a core–shell structure, with black solid spheres showing Fe₃O₄ and a surface coating of a thin layer of SiO₂ (Figure 2[c] and 2[d]). The SiO₂ shell modified by the Stöber method not only protected the Fe3O4 magnetic shell from O₂ oxidation in the air but also prevented Fe3O4 agglomeration. Almost all the Fe₃O₄@SiO₂ nanoparticles were preferentially adhered to the surfaces of MWCNTs, indicating that the MWCNTs had been successfully modified (Figure 2[b]).

The FT-IR spectra of the Fe₃O₄@SiO₂ MWCNT composite are shown in Figure 3(a). The characteristic absorption peak of Fe–O in Fe₃O₄ is near 631 cm-1 (29). In the vicinity of 1045 cm-1, the stretching vibration characteristic absorption peak of Si–O–Si can be observed, indicating that SiO2 was successfully modified on the surface of Fe₃O₄ (30). The bands around 1630 cm-1 can be assigned to the stretching vibration peak of C = C in MWCNTs (31). These results confirmed that the Fe₃O₄@SiO₂ MWCNT magnetic composites were successfully prepared.

A magnetometer was used to study the magnetic properties of the adsorbent. Figure 3(b) shows the magnetization curve (hysteresis loop) of Fe₃O₄@SiO₂ MWCNT nanoparticles. The saturation magnetization was 7.68 emu/g. The curve is smooth and symmetrical at the origin, and no remanence (residual magnetism) was found, indicating superior magnetic character.

Effect of the Amount of Magnetic Adsorbent: A different mass of magnetic adsorbent (5–30 mg) was added to a 20 mL spiked solution to study OPE extraction efficiency. The results are shown in Figure 4(a). The peaks of OPEs significantly increased with the increase of adsorbent amount within the range of 5–20 mg. When the adsorbent added was greater than 20 mg, the OPE extraction efficiency decreased. It might be that excessive adsorbent enhanced the adsorption capacity between the adsorbent and the analytes and resulted in incomplete elution of the analytes. Therefore, the amount of magnetic adsorbent added was set at 20 mg.

Effect of Extraction Time:Extraction time is one of the most important parameters in MSPE. The effect of extraction time on the adsorption of OPEs was determined by testing extraction times of 20–80 min. The results are shown in Figure 4(b). With the extension of time, the extraction efficiency gradually increased but then decreased after 50 min. It was speculated that volatilization of eluent caused sample loss and decreased the extraction efficiency. Based on the above experimental results, 50 min was chosen as the best extraction time.

Effect of Eluent: The eluent is also an important factor affecting extraction efficiency. The effects of methanol, acetonitrile, 1:1 methanol–acetonitrile (v/v), and ethyl acetate on the OPE extraction efficiency were investigated, respectively. The results are shown in Figure 5(a). The ethyl acetate solution was the most efficient eluent. This result is consistent with previous literature (32–34) and so the ethyl acetate was chosen as the eluent.

Elution Volume and Elution Time: The influence of the eluent volume on the extraction efficiency of OPEs was also investigated. An eluent volume of 1400 μL was sufficient to obtain better extraction efficiency. More elution had almost no effect on the extraction results but extended the concentration time. Therefore, a volume of 1400 μL ethyl acetate was used to elute the sample.

Additionally, the influence of the elution time on the extraction efficiency of OPEs was also investigated. With ultrasonic assistance, OPEs were eluted quickly and efficiently from the MSPE adsorbent surface. As is shown in Figure 5(b), when elution time was extended over 20 min, extraction efficiency decreased, so 20 min was chosen as the elution time.

Method Evaluation: Under these optimal conditions, the detection limit (LOD), the limit of quantitation (LOQ), recovery, and precision were used to evaluate the developed MSPE–GC–MS method. The instrumental linearity was performed by detecting OPE standard solutions with 0.1, 0.5, 1, 2, 5, and 10 μg/mL. Good linearity with coefficient R2 = 0.9914–0.9990 was obtained in this range (Table 2). The LOD and LOQ—defined as three times and ten times signal-to-noise—was achieved by analyzing four spiked OPEs in a pure water sample. The LODs of TnBP, TCEP, TPhP, and TEHP were 0.45, 0.56, 1, and 0.038 μg/L, respectively. The LOQs of TnBP, TCEP, TPhP, and TEHP were 1.51, 1.35, 3.59, and 0.1 μg/L, respectively. In pure spiked water solutions, the average recoveries of TnBP, TCEP, TPhP, and TEHP were 84.3%, 90.7%, 83.8%, and 79.4%, respectively. According to previously reported results (35), the high extraction efficiency can be attributed to the fact that the OPEs can interact with the Fe₃O₄@SiO₂ MWCNT adsorbents via a Brønsted acid-base interaction, van der Waals forces, and a π-π electron donor-acceptor (EDA) interaction between the OPEs and the functionalized groups of the adsorbents.

Actual Water Sample Detection: To determine the applicability of this method, water from the Songhua River (Jilin Province, China) was selected for testing. First, water samples were centrifuged at 10,000 rpm for 10 min to remove sediment, and the supernatant was passed through a 0.45 μm filter. Then the water sample was processed as per section named “Sample Preparation by MSPE” and extracted OPEs were analyzed with GC–MS. No OPEs were detected. OPEs were then added to Songhua River water samples to make the concentrations of 2.5, 10, and 25 μg/L. The recoveries of OPEs spiked at the low, middle, and high concentrations were 72.5–89.1%, with mean relative standard deviations (RSDs) ranging from 3.4% to 9.7% (Table 3). There were inorganic salts, organic matter, microorganisms, and some impurities found in the Songhua River water. The extraction efficiency of the adsorbent therefore had a certain impact. However, recoveries of four OPEs in environmental water were acceptable.

Compared With Other Methods: The MSPE–GC–MS method established in this paper was compared to other methods (Table 4). It was shown that MSPE–GC–MS had preferable recoveries for both polar and hydrophobic OPEs (TCEP and TEHP), and required less organic solvent compared to LLE and SPE methods (18, 36). However, the LOD and LOQ of this method were higher than previous studies. This could possibly be attributed to the smaller sample volume used in this study. The SPME method was a solvent-free sample preparation method, which also had a lower LOD (4). The recovery of polar TCEP in MASE was only 5%. Moreover, the MASE method was time-consuming, and the cost of membrane is higher (6).

Conclusions

In this study, Fe₃O₄@SiO₂ MWCNT magnetic composite was prepared and used to analyze organophosphate esters in water samples in combination with GC–MS for the first time. This method requires only 20 mg of magnetic adsorbent, isothermally oscillated for 50 min, 1400 μL of eluting solvent, and 20 min of elution time. The method has the advantages of good recoveries, a wide linear range, low consumption of organic solvents, and low preparation cost of magnetic composite materials. This method could be successfully used to extract and analyze organophosphates (TnBP, TCEP, TPhP, and TEHP) in environmental water samples.

Acknowledgements

This work was supported by the Project of Science and Technology Development of Jilin Province (No. 20190303116SF and 202002008JC), the Research and Development Project for Industrial Technology of Jilin Province (No. 2020C028-1), the Talents Project for Innovation and Entrepreneurship of Jilin province (No. 2020030), and the Project of Science and Technology of the Education Department of Jilin Province (No. JJKH20210242KJ). The financial support from the Key Laboratory of Fine Chemicals of Jilin Province is also acknowledged.

Conflict of Interest

There are no conflicts of interest to declare.

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Xiyue Wang is a lecturer at the Jilin Institute of Chemical Technology.

Yuanyuan Tian is a student at the Jilin Institute of Chemical Technology.

Lili Lian is a professor at the Jilin Institute of Chemical Technology.

Hao Zhang is a lecturer at the Jilin Institute of Chemical Technology.

Bo Zhu is an associate professor at the Jilin Institute of Chemical Technology.

Wenxiu Gao is an associate professor at the Jilin Institute of Chemical Technology.

Dawei Lou is a professor at the Jilin Institute of Chemical Technology.