Development and validation of an analytical method for detection and quantification of benzophenone, bisphenol A, diethyl phthalate and 4-nonylphenol by UPLC-MS/MS in surface water

Chemical and reagents

BP, BPA, DEP and 4NP, with 99% purity, were purchased from Sigma-Aldrich (St. Louis, USA). Methanol, acetonitrile (LC–MS grade) and ammonium hydroxide (28.0–30.0%) were purchased from J.T. Baker^® (Phillipsburg, USA). Ultrapure water was obtained from a Milli-Q Direct 8 (Millipore^®). SPE cartridges were used to promote the clean-up and pre-concentration of the analytes in the preparation of real samples. The SPE cartridges Bond Elut C18^® (500 mg/3 mL) were purchase from Las do Brasil (Brazil).

Instrumentation and software

Analyses were performed using an ultra-performance liquid chromatography (UPLC-MS/MS) (Waters Milford, MA, USA) equipped with an Acquity UPLC binary pump liquid chromatograph with a Xevo TQD MS/MS triple quadrupole detector, autosampler and column temperature controller. Details on the chromatographic method are described elsewhere (De Araujo et al., 2019).

Electrospray ionizations in positive and negative modes (ESI+ and ESI- respectively) were used as ionization source. The drying gas, as well as the nebulizing gas, was nitrogen generated by pressurized air in a Nitrogen Generator Genious NM32LA (Peak, USA). The nebulizing gas flow was 50 L h⁻¹ whereas the desolvation gas flow was 1100 L h⁻¹.

To operate in the MS/MS mode, the collision gas was argon 99.99% (Air Products, Brazil) in the collision cell. The optimized values were as follows: capillary voltage 3.2 kV; source temperature 150 °C; desolvation temperature 600 °C. For quantification and identification, the best collision energies were optimized to promote more intense signals used in multiple reaction monitoring (MRM) mode. Thus, two transitions were selected: one transition with the highest signal intensity was selected for quantification and another transition, with the lower signal intensity, for identification. Table 1 shows the optimized MRM transitions for the EDs with their respective retention times (Tret). The software MassLynx (Waters, USA), version 4.1, performed analytical instrument control, data acquisition and treatment.

Preparation of the laboratory matrix control sample

A composite sample (control sample) was prepared in order to gather all possible interferents and matrix effect that we should find in real samples collected. Surface water samples were collected at four different locations along the Guandu River: Paracambi (PBI; −22.663144; −43.742502), Seropédica (SER; −22.806417; −43.626079), Nova Iguaçu (NIG; −22.817486; −43.624333) and Rio de Janeiro (RIO; −22.897108; −43.734804) in order to gather all possible interferents and matrix effect that we should find in real samples collected. The sites are characterized by rural, industrial and high population density areas. This composite control sample was prepared by filtration on glass fiber filter (1.00 µm pores size) followed by the SPE procedure. The target analytes were not detected, in comparison to the non-spiked matrix (sample control) and spiked matrix (sample control spiked with limit of quantification (LOQ) values for each analyte). Thus, the composite sample has been adopted as the matrix control for the validation and sample preparation tests.

Sample preparation

The sample preparation procedure, previously developed in our laboratory (de (De Araujo, Bauerfeldt & Cid, 2018), was applied aiming at the extraction, cleaning and concentration of EDs and optimized in order to achieve its best performance concerning the recovery of BP, BPA and DEP and also the necessity of promoting greater concentration factor thus, obtaining lower limits of quantification and detection (at the ng L⁻¹ level, for environmental monitoring purposes). Different sample volumes (250, 500 and 1,000 mL) were evaluated. During these tests, the maximum load the cartridge was capable of adsorbing was evaluated. Each test was carried out in triplicate. Three solutions were prepared by spiking of the BP, BPA, DEP and 4NP standards to the matrix control sample at the same concentration level of 25.0 µg L⁻¹ but with different volumes. Thus, each solution percolated by the cartridge had a different analyte mass (6.25 µg, 12.5 µg and 25.0 µg). After percolation, an aliquot of the eluate was taken and injected into the UPLC-MS/MS and the presence or absence of signals was verified, allowing to conclude whether the analytes were completely adsorbed by cartridge. Tests for other elution volumes and two-step elution with the same final volume also performed. Each test was carried out in triplicate. In all tests the cartridge was conditioned with 5.00 mL of methanol followed by 5.00 mL of ultrapure water.

Method validation

All validation experiments were performed according to the USEPA 8000D guidelines (Usepa, 2018). The parameters selectivity, precision (relative standard deviation - % RSD), accuracy, LOQ, limit of detection (LOD), linearity, matrix effect and robustness were evaluated as previously described (De Araujo et al., 2019).

Spiked matrix control samples were prepared, using of the filtered composite control sample, at concentration levels of 50.0 ng L⁻¹ (low), 250 ng L⁻¹ (medium) and 500 ng L⁻¹ (high) in two days (intra-day 1 and 2) and submitted to SPE procedure. The precision and accuracy of the analytical method were determined by analyzing sets of 5 replicates of the spiked matrix control samples. The extracts were injected in triplicate in the UPLC-MS/MS. The precision of the method was evaluated in terms of the % RSD. Accuracy was calculated by comparing the measured concentration with the nominal concentration as the mean recovery percent (%).

The LOQ is defined for each analyte as the lowest concentration level that can be quantified with RSD <20% and accuracy found within the range from 70–130%. The LOD for the 4NP, was calculated according to (Shrivastava & Gupta, 2011), while for the BPA, BP and DEP, the LOD were calculated based on the standard deviation of the blanks replicates, according to (Usepa, 2014).

Six-point analytical curves were obtained by analysis of spiked matrix control samples at concentration levels between 50.0 to 500 ng L⁻¹ for BPA and DEP and 10.0 to 500 ng L ⁻¹ for BP and 4NP. All spiked matrix control samples were subject to extraction and clean-up procedures. Similar procedure was performed used ultrapure water for the preparation of the standards for comparision. F-test was applied in order to evaluate the matrix effect, based on the calibration factors, with a confidence limit of 95% and 24 degrees of freedom.

The robustness of an analytical method is the ability of the method be not affected by small variations in method execution parameters. Robustness provides an indication of method reliability during routine application (Eurochem, 2014). To evaluate the robustness of the method, change of the flow of the mobile phase was chosen. Samples previously used in the linearity assay were taken and the mobile phase flow was changed from 0.40 mL min⁻¹ to 0.30 mL min⁻¹. Thus, two analytical curves with different flows were obtained. Based on the same concept of the calibration factor described in the matrix effect, it was possible to evaluate, through the F test, if the analytical curves presented any significant difference, assuming a confidence limit of 95% and 24 degrees of freedom.

Application of the developed method to real surface water samples

Determination by UPLC-MS/MS

The MS/MS operation mode was described. As above mentioned, the two most intense transitions were selected for the identification and further quantification of the target analytes. The highest intense transition was used for quantification and the second, for identification. The retention time, class, log K_ow, MRM transitions and collision energy for each compound analyzed are described in Table 1.

Figure 1 presents the UPLC-MS /MS total ion chromatograms (TIC) of the four EDs analyzed in this work and a sample where the BP was quantified in April 2018. The total running time was 8.0 min.

Optimization of the SPE sample preparation step

The optimization of the SPE procedure was performed in order to guarantee the conditions for the best recovery of the analytes under study. Figure 2 illustrates the maximum load the cartridge is able to adsorb.

As can be seen from Fig. 2, the maximum retention of the cartridge is 12.5 µg. Thus, the sample volume of 500 mL was selected, since the expected concentration of micropollutants in surface water samples is less than the saturation capacity of the cartridge.

In order to achieve 70–130% recovery as recommended by the USEPA 8000D Guide (Usepa, 2018), two volume of solvent were tested and elution in one and two steps was also tested (Fig. 3).

As it can be seen from Fig. 3, the adoption of a two-step elution using the four mL (2 × two mL) elution volume led to a gain in the recovery of the analytes, which are, however, still below the recommended range (70–130%), as recommended by the USEPA 8000D Guide (Usepa, 2018). Alternatively, the elution volume was increased to five mL and an increase in the recovery of all analytes was observed. In order to promote better recovery values, the 2.5 mL two-step elution was tested and, as shown in Fig. 4, an increase in the recovery rate of all analytes was noted, being all analytes within the recommended recovery range (Usepa, 2018).

The increased recovery is achieved with two-step elution since this is similar to solid–liquid extraction. Efficient extraction of analytes from solid to extracting liquid is guaranteed by repeated extractions. Neutral compounds can have substantial distribution coefficient (K_D) values, making extraction easier. On the other hand, organic compounds that form hydrogen bonds with water, are partially soluble in water or are ionogenic (weak acids or bases), may have lower K_D, thus hindering their complete extraction, which requires numerous extractions. Additionally, the sample matrix may contain influence on the value of K_D (Mitra, 2003).

Method validation

Validation of the analytical method was performed by analyzing spiked matrix control samples and was assessed in agreement to the recommendation in the USEPA 8000D guide (Usepa, 2018).

Application in real samples

Table 6 shows the physicochemical parameters, obtained from analysis of the samples at collection sites, in April, May, June, July, and August 2018.

A chromatogram of the environmental samples is shown in Fig. 5, in which the quantification of the four EDs in the collected surface water sample can be seen. The results are presented in Table 7.

BP was detected (below LOQ) in one sample (July) and quantified in the other four samples (April, May, June, August); 4NP was detected in all samples but quantified in only one (July); BPA was quantified in four samples and DEP was quantified in all five samples.

In Guandu river, the range (min–max) found for DEP was 259.40–2. 56 ×10³ ng L⁻¹ (the maximum value being 10 times higher than the threshold value for drinking water according to the USEPA 816-F-09-004 regulation (Usepa, 2009). For proper quantification of DEP in the samples, it was necessary to dilute the samples prior to the SPE procedure, in order to guarantee that the concentration of DEP could be found within the linear range. With regard to the European regulations, the European Union has included 4NP, octylphenol (OP) and DEHP in the list of the 33 priority substances in environmental waters and has established maximum concentrations levels (based on environmental quality standards) at 300 ng L⁻¹ for 4NP, at 100 ng L⁻¹ for OP and at 1.30 ×10³ ng L⁻¹ for DEHP (Dévier et al., 2013).

DEP is often found in formulations of medicines, perfumes, nail polishes, shampoos, toys and other consuming goods (Gómez-Hens & Aguilar-Caballos, 2003; Viecelli et al., 2011). Phthalates are of great concern today due to their intensive utilization, especially for the purpose of increasing the flexibility and strength of plastic packaging; however phthalates are not chemically bound to the packaging plastic, which facilitates the release from the plastic into aqueous matrices (Farajzadeh & Mogaddam, 2012a; Farajzadeh & Mogaddam, 2012b). The main source of DEPs found in surface water is the untreated or insufficiently treated sewage and industrial effluents discharged into water bodies. Another source of environmental contamination is the leaching from plastic waste disposed in open dumps or uncontrolled landfills, when the environmental conditions (pH, temperature, contact time among other conditions) are in favor of the leaching of phthalates once the plastics are in contact with water (Bošnir et al., 2007; Souza et al., 2012).