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

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PeerJ Analytical Chemistry


The availability of water resources in the world is gaining increasing attention. It is frequently associated to climatic factors (drought, climate changes), as well as by the increasing demand for clean water (due to the intense population growth) and decreasing quality. The latter is a direct consequence of the pollution promoted by domestic, rural and industrial activities. Approximately half of the world’s population currently suffers from moderate and 10% from extreme water scarcity (Banjac et al., 2015; Estrada-Arriaga et al., 2016; Johnson et al., 2016; Cunha, Araujo & Marques, 2017; De Araujo, Bauerfeldt & Cid, 2017; De Araujo et al., 2019).

Concerns about exposure to the endocrine disruptors (EDs) micropollutants have been increasing over the years due to the possible damage that can be caused to exposed organisms. (Meyer, Sarcinelli & Moreira, 1999; Bila & Dezotti, 2007; Cirja et al., 2008; Vela-Soria et al., 2014; Rodríguez-Gómez et al., 2015; Caldas et al., 2016;Vela-Soria et al., 2014; Starling, Amorim & Leão, 2019). According to the United States Environmental Protection Agency (USEPA), an ED is “an exogenous agent that interferes with the synthesis, secretion, transport, metabolism, binding or elimination of the body’s natural hormones, which are responsible for homeostasis, reproduction, development and/or behavior” (Bila et al., 2007; Camilleri et al., 2015; Kabir, Rahman & Rahman, 2015).

The disruption of endocrine functions may be associated with interference in the synthesis, secretion, transport, binding, action or elimination of the natural hormones of organisms, thus triggering a new hormonal response. By mimicking the action of an endocrine hormone, a substance exaggerates or improperly triggers a false stimulus, causing a damage to the exposed organisms even at low concentrations (Bila & Dezotti, 2007; Castro-Correia & Fontoura, 2015, Rodríguez-Gómez et al., 2015).

The main sources of EDs in water bodies are punctual (such as domestic and industrial effluents) or diffuse (such as pesticides from agricultural areas). Several investigations report the recalcitrance of many of these compounds to degradation in wastewater treatment plants and in treatability studies. Due to the ineffectiveness of most wastewater treatment systems, micropollutants in the effluents are discharged into the receiving water bodies, in concentration levels which are high enough to cause chronic toxicity or to trigger an endocrine reactions (Funasa, 2014; Camilleri et al., 2015; Castro-Correia & Fontoura, 2015; Dias et al., 2015). Some EDs, such as bisphenol A (BPA), 4-nonylphenol (4NP), benzophenone (BP) and phthalates, such as diethylphthalate (DEP), have attracted the attention of the scientific community due to the frequent detection and quantification of these analytes in water samples, for example in raw sewers or treated waters. 4NP is a byproduct from the biological degradation of alkylphenol polyethoxylates, widely used as nonionic surfactants in household cleaning products (Moreira et al., 2011; De Araujo, Bauerfeldt & Cid, 2017). DEP is a synthetic substance used as an ingredient in cosmetic formulation and to increase the flexibility of plastic materials used in the manufacture of toys, household items, auto parts and others (Viecelli et al., 2011; Farajzadeh & Mogaddam, 2012a; Farajzadeh & Mogaddam, 2012b). BPA is a degradation product of some polymers, such as polycarbonates and epoxide resins, and is used as an antioxidant in polyvinyl chloride (PVC) plastics (Chen et al., 2011; Doerge et al., 2011; Bomfim et al., 2015). Finally, BP is commonly found in UV filter formulations despite its well-known disruptive endocrine activity proved by in vivo and in vitro assays assays (Vela-Soria et al., 2014; Wang et al., 2017).

For a quantitative assessment of the quality of surface water concerning the concentration levels of these EDs, accurate analytical methods are required. (Giesy et al., 2002; Silveira et al., 2013;Arbeláez et al., 2015; Caldas et al., 2016; Starling, Amorim & Leão, 2019). Preparation of water surface water samples, prior to the analytical determination of these four EDs, is required and solid phase extraction (SPE) is the most applied method. However, liquid–liquid Extraction (LLE), solid phase micro extraction (SPME), dispersive liquid–liquid microextraction (DLLME), SPE online, stir bar sorptive extraction (SBSE) are often reported (Farajzadeh & Mogaddam, 2012a; Farajzadeh & Mogaddam, 2012b; Zaater, Tahboub & Sayyed, 2014; Selvaraj et al., 2014; Wu et al., 2015; Caballero-Casero, Lunar & Rubio, 2016; Olatunji et al., 2017; Rozaini et al., 2017; De Araujo, Bauerfeldt & Cid, 2018; Barreca et al., 2019; He & Aga, 2019; Król & Dudziak, 2019). The quantitative analysis is frequently performed by the liquid chromatography coupled to mass spectrometry (LC-MS/MS) and gas chromatography coupled to mass spectrometry (CG-MS) techniques, although other detectors of other kinds coupled to the liquid chromatography are used, such as liquid chromatography with ultraviolet detection (LC-UV), liquid chromatography with diode array detection (LC-DAD) and liquid chromatography with fluorescence detector (LC-FLD) (Zgoła-Grześkowiak et al., 2009; Chen et al., 2010; Lou et al., 2012; Ciofi et al., 2014; Padhye et al., 2014; Camilleri et al., 2015; Terzopoulou, Voutsa & Kaklamanos, 2015;, Asati, Satyanarayana & Patel, 2017; Comtois-Marotte et al., 2017; De Araujo, Bauerfeldt & Cid, 2018. The realization that conventional water and sewage treatment systems do not completely remove most of these micropollutants opens a discussion of a possible worrisome public health problem, since micropollutants may be present in the water supply (Matsuo et al., 2011; Moreira et al., 2011; Silveira et al., 2013; Tran, Hu & Ong, 2013; Dai et al., 2014; Mann et al., 2016; Valls-Cantenys et al., 2016; Tröger et al., 2018; Starling, Amorim & Leão, 2019).

The Guandu river basin is the key source of water supply for approximately 9 million inhabitants in the metropolitan region of Rio de Janeiro State (Cetesb, 2011). In the basin, Guandu river is the most important component. Rio dos Poços, Queimados, Macaco, Ribeirão das Lajes rivers, among others, are also part of the Guandu basin. Pollution of these rivers is potentialized by the discharge of untreated (or only partially treated) urban and industrial effluents. The runoff from agricultural areas also contributes to pollution of the Guandu river basin. A large part of the sewage discharged into the tributaries of the basin has the water damming area for collection and subsequent treatment and distribution. Thus, the studied site (dam to collect water for supply), is characterized by being a region with a complex matrix, due to the amount of untreated sewage that is discharged in the region.

Previous investigations concerning the Guandu basin waters revealed the presence of three psychoactive drugs (bromazepam, clonazepam and diazepam) in most samples (De Araujo et al., 2019) and the presence of 4NP in relatively high concentrations (De Araujo, Bauerfeldt & Cid, 2018). The need for the development of a new analytical methodology arises due to the complexity of the matrix as described above and also by the scarcity of previously published works concerning this site. Another investigation (Dias et al., 2015) revealed that 20–50% of the Guandu river water samples presented estrogenic activity expressed as 17β-estradiol (E2) equivalents or E2-EQ. Additionally, after treating the water in a conventional water treatment plant, estrogenic activity was still found in 8% of the water samples in levels higher than the reference established by the authors as 1 ng L−1 E2-EQ. These findings strongly indicate the importance of developing proper analytical methods for the determination of EDs, particularly for monitoring of the Guandu river, which is of high relevance in terms of water supply and receives heavy discharge of treated and untreated urban sewage and industrial effluents.

Therefore, the objective of the present study is to develop and validate an analytical method based on LC-MS/MS to determine four EDs: BP, BPA, DEP and 4NP in the Guandu river basin, Rio de Janeiro State, Brazil.

Materials & Methods

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.

Table 1:
Endocrine disruptor class, log Kow and UPLC-MS/MS parameters.
Analyte Class Log Kowa Tret (min) Ionization Transitions MRM, m/z (CEb, eV)
Quantification Identification
BP PCPc 3.18 4.01 Positive 316 > 105 (15) 316 > 77 (30)
BPA Plasticizer 3.22 3.77 Negative 227 > 212 (18) 227 > 133 (25)
DEP Plasticizer 2.42 3.81 Negative 223 > 149 (20) 223 > 177 (10)
4NP Surfactants 5.76 5.05 Negative 219 > 106 (21) 219 > 119 (34)
DOI: 10.7717/peerjachem.7/table-1


CE, collision energy.
Personal care product.

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 L1 (low), 250 ng L1 (medium) and 500 ng L1 (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

Sampling location and procedure

Five composite surface water samples of 4.0 L each were collected monthly from a selected sampling point at Guandu river, NIG, Rio de Janeiro State for five months (April to August 2018). The sampling point was in the dammed area used as a source for water abstraction for treatment and distribution. The sampling procedure, preservation and transport conditions followed the instructions of the Brazilian National Guide for Collection and Preservation of Environmental Samples (Ceteb, 2011). Samples were collected in a 4.0 L amber vial, with enough volume to perform the determination assays and to store for further testing, if necessary. At the time of collection, with the help of a HORIBA U-52 multiparameter probe, temperature, pH, Oxidation Reduction Potential (ORP), conductivity, turbidity, dissolved oxygen and dissolved total solids were measured.

Results and Discussion

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 Kow, 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.

UPLC-MS/MS total ion chromatograms (TIC) of each endocrine disruptor under studyat10.0 µg L-1. (A) BPA; (B) 4NP; (C) DEP; (D) BP; (E) sample of april/2018.

Figure 1: UPLC-MS/MS total ion chromatograms (TIC) of each endocrine disruptor under studyat10.0 µg L-1. (A) BPA; (B) 4NP; (C) DEP; (D) BP; (E) sample of april/2018.

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.

Maximum load (in mass) a cartridge is able to adsorb.

Figure 2: Maximum load (in mass) a 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).

Effect of the solvent volume and sequential elution on the recovery.

Figure 3: Effect of the solvent volume and sequential elution on the recovery.

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).

UPLC-MS/MS chromatograms of each ED from a positive sample of surface water with the corresponding LOQ and a non-spiked matrix control sample (blank matrix).

Figure 4: UPLC-MS/MS chromatograms of each ED from a positive sample of surface water with the corresponding LOQ and a non-spiked matrix control sample (blank matrix).

(A) BP; (B) BPA; (C) DEP; (D) 4NP.

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 (KD) 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 KD, thus hindering their complete extraction, which requires numerous extractions. Additionally, the sample matrix may contain influence on the value of KD (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).


The selectivity of the method was established by analysis of non-spiked (Blank) and spiked matrix control sample at the LOQ values (50.0 ng L−1 for BPA and DEP and 10.0 ng L−1 for BP and 4NP). The chromatograms (Fig. 4) were evaluated and the existence of a signal was verified in the chromatograms of the non-spiked matrix control sample (Blank) with intensity lower than the signal intensities of the respective LOQs.

Thus, the selectivity of the method was considered satisfactory, since the non-spiked matrix control sample (Blank) showed signals with lower intensities than the analyte signals, at the LOQ level, for each analyte, causing no interference in the quantification.

Precision and accuracy

Intra-day and inter-day precision and accuracy were established by analyzing spiked matrix control sample (n = 5) at three different concentrations: 50.0 ng L−1 (low), 250 ng L−1 (medium) and 500 ng L−1 (high), in two consecutive days (intra-days 1 and 2.) Results for the inter-day precision were found between 1.46 and 10.32% for 4NP and BP respectively. Regarding the accuracy, results were obtained between 80.43 and 104,32% for DEP and BP respectively (Table 2). Results were satisfactory and within the recommended values (RSD < 20%; accuracy between 70 and 130%) (Usepa, 2018). The precision and accuracy values of the method are quite consistent with other reports (Chen et al., 2010; Li et al., 2012; Selvaraj et al., 2014; De Araujo, Bauerfeldt & Cid, 2017; De Araujo, Bauerfeldt & Cid, 2018).

Table 2:
Accuracy and precision of target compounds in surface water.
Intra day Inter day
day 1 day 2
Comp Spiked level (ng L−1) Found (ng L−1) RSD (%) Rec (%) Spiked level (ng L−1) Found (ng L−1) RSD (%) Rec (%) Spiked level (ng L−1) Found (ng L−1) RSD (%) Rec (%)
BP 50 46.71 4.74 93.42 50 57.61 1.85 115.22 50 52.16 1.85 104.32
250 266.56 5.94 106.62 250 208.73 11.03 83.49 250 245.78 10.32 98.31
500 435.52 3.87 87.71 500 419.47 4.10 83.89 500 429.01 4.92 85.80
BPA 50 49.86 3.55 99.71 50 48.26 4.83 96.52 50 49.06 2.16 98.11
250 210.36 3.97 84.15 250 216.53 1.64 86.61 250 213.45 1.64 85.38
500 419.65 6.70 83.93 500 405.08 5.00 81.02 500 412.36 5.00 82.47
DEP 50 53.68 4.93 107.37 50 47.01 4.70 94.01 50 50.35 3.43 100.69
250 213.02 4.66 85.21 250 219.85 2.31 87.94 250 201.08 2.29 80.43
500 490.30 1.38 98.06 500 469.95 2.73 93.99 500 480.12 2.73 96.02
4NP 50 46.44 0.89 92.87 50 46.42 1.46 92.84 50 46.43 1.46 92.86
250 239.23 2.08 95.69 250 207.63 5.55 83.05 250 223.43 5.55 89.37
500 412.49 4.62 82.51 500 493.95 4.92 98.79 500 453.22 4.92 90.64
DOI: 10.7717/peerjachem.7/table-2


Mean value from quintuplicate samples injected in triplicates (in total, 30 measurements).


Analytical curves were prepared with both matrix control sample and ultrapure water, containing analytes in concentrations levels ranging from 50.0 to 500 ng L−1 for BPA and DEP and 10.0 to 500 ng L−1 for BP and 4NP. Results are shown in Table 3. The R2 coefficients range from 0.992 to 0.999 for BP and DEP respectively, indicating excellent linearity for all analytes.

Table 3:
LOQ, LOD, slope, intercept, determination coefficients (R2) for linearity tests.
Comp LOQ LOD intra day inter day
day 1 day 2
ng L−1 ng L−1 slope intercept R2 slope intercept R2 slope intercept R2
BP 10.00 5.72 658.40 2,413.01 0.993 654.63 2,453.46 0.992 651.01 2,634.58 0.992
BPA 50.00 5.61 30.43 −66.06 0.998 30.50 −59.79 0.997 30.47 −63.10 0.997
DEP 50.00 2.71 2,037.53 10,208.03 0.996 2,075.22 9,314.53 0.999 2,053.88 9,848.56 0.996
4NP 10.00 0.87 66.91 −27.53 0.999 62.28 17.99 0.998 64.55 −3.43 0.996
DOI: 10.7717/peerjachem.7/table-3

Matrix effects must be investigated in quantitative LC-MS/MS determinations (Postigo, De & Barceló, 2008; Gros & Petrovic, 2009; Gros, Rodríguez-mozaz & Barceló, 2012). As above mentioned, matrix effect was evaluated from F values (Fcalc) resulting from the comparison of the calibration factors (Fc) calculated for each analyte from the analytical curves obtained from standards prepared with ultrapure water and matrix control samples (see Table 4).

Table 4:
Evaluation of matrix effect.
Comp Ultrapure water Matrix (surface water) Fcalca
Slope Intercept R2 Slope Intercept R2
BP 368.65 2,248.66 0.990 798.01 1,433.27 0.992 50.46
BPA 13.76 40.48 0.992 32.74 −98.94 0.992 1.00
DEP 651.86 5,357.51 0.993 2,201.35 9,873.76 0.993 12.14
4NP 18.68 21.57 0.990 65.83 4.63 0.996 6.22
DOI: 10.7717/peerjachem.7/table-4


Fcritical = 1.98. (interval of confidence: 95%).

Calculated F values for BP, DEP and 4NP are greater than the critical F value, suggesting that the analytical curves are not statistically equal. Thus, the matrix must be showing significant influence on the determination of these analytes. Thus, it is strongly suggested that an analytical curve prepared in a spiked matrix is adopted for the determination of BP, DEP and 4NP. An alternative should be found on the adoption of the standard addition method. For BPA, the calculated F value is lower than the critical F value, suggesting that the analytical curves in ultra-pure water and spike matrix are equivalent. Even though, quantification of all analytes was performed by comparison with the spiked matrix analytical curves.

Limit of quantification and limit of detection

LOQ were determined for each analyte, according to the validation criteria. LOQ values are 10.0 ng L−1 for BP and 4NP and 50.0 ng L−1 for BPA and DEP. LOD, defined according to the literature (Shrivastava & Gupta, 2011; Eurochem, 2014), varied between 0.87 and 5.72 ng L−1 for 4NP and BP respectively. LOQ and LOD values are shown in Table 3.

Current legislation recommends maximum permitted levels for some EDs in water. According to the USEPA 816-F-09-004 (Usepa, 2009), the threshold concentration level for di(2-ethylhexil) phthalate (DEHP), an ED that can cause reproductive difficulties, liver problems and increased risk of cancer, is 0.60 µg L−1. The threshold value for 4NP in freshwater samples is 28.00 µg L−1 and in saltwater it is 7.00 µg L−1 according to USEPA 822-R-05-005 (Usepa, 2005). An analytical method for the determination of 4NP in the same basin has been previously reported (De Araujo, Bauerfeldt & Cid, 2018). However, comparisons between LOQ and LOD are not possible due to the difference between hyphenated techniques (LC-DAD and UPLC-MS/MS). Nevertheless, the reported LOQ and LOD values, obtained from SPE-LC-MS/MS technique, are similar to some previously reported limits, in investigations using similar hyphenated techniques (Li et al., 2012; Camilleri et al., 2015; Wooding, Rohwer & Naudé, 2017; Chang et al., 2018).


The same standards used for the linearity test were analyzed under two different mobile phase flow conditions (0.40 and 0.30 mL min−1), in order to investigate the robustness of the method. F test was applied showing no significant difference for the calibration factors. Table 5 illustrates the results for the robustness of the method. As can be seen from Table 5, the developed method proved to be robust for all analytes.

Table 5:
Evaluation of matrix effect.
Control Flow Fcalca
Comp Slope Intercept R2 Slope Intercept R2
BP 798.02 1,433.27 0.992 766.14 1,442.23 0.995 1.08
BPA 32.74 −98.94 0.992 28.87 −47.91 0.994 1.01
DEP 2,201.35 9,873.75 0.993 2,221.35 6,678.87 0.990 1.03
4NP 65.83 4.63 0.997 61.14 2.35 0.995 1.17
DOI: 10.7717/peerjachem.7/table-5


Fcritical = 1.98. (interval of confidence: 95%).

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.

Table 6:
Physicochemical characteristics of the samples measured on site during sampling.
Parameter/Date April 13 May 11 June 08 Min–Max
Time 9:47 AM 10:57 AM 10:08 AM 9:47–10:57
Temperature (°C) 26.11 24.54 23.41 23.4–26.1
pH 7.25 7.05 7.49 7.05–7.49
ORPa (V) 0.16 0.18 0.17 0.16–0.18
Condutivity (mS cm−1) 0.12 0.13 0.12 0.12–0.13
Turbidity (NTU) 16.47 8.54 5.41 5.41–16.5
DOb (mg L−1) 6.81 7.47 7.96 6.81–7.96
TDSc (g L−1) 0.021 0.063 0.081 0.021–0.081
DOI: 10.7717/peerjachem.7/table-6


Obs: July and August: probe under maintenance.

Oxidation Reduction Potential.
Dissolved Oxygen.
Total dissolved solid.

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.

Chromatograms of each ED for samples collected between April and August 2018.

Figure 5: Chromatograms of each ED for samples collected between April and August 2018.

(A) BP; (B) BPA; (C) DEP; (D) 4NP.

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).

Table 7:
Determination of BP, BPA, DEP and 4NP in surface water (ng L−1).
Compound April May June July August Mean Max Min
BP 286.20 42.36 41.66 da da 123.41 286.20 41.66
BPA 182.04 76.25 65.14 57.32 da 95.18 182.04 57.32
DEP 1.30 × 103 456.77 259.40 2.56 × 103 545.19 1.02 × 103 2.05 × 103 259.40
4NP da da da 13.48 da
DOI: 10.7717/peerjachem.7/table-7


detected (value < LOQ).


The present study describes the optimization and application of an analytical method for the determination of four EDs in surface water samples using SPE for sample cleaning, extraction and pre-concentration and subsequent analysis by UPLC-MS/MS. There was an optimization of the sample volume percolated by the SPE cartridge. The elution volume was also optimized considering sequential elutions. Bond Elut C18 cartridge was used for analyte extraction. The sample preparation was efficient (recovery >90% and RSD <11.03%) for extraction, pre-concentration and clean-up of BP, BPA, DEP and 4NP from surface water samples. UPLC-MS/MS analysis allowed the simultaneous determination of the four EDs in a fast-chromatographic run (8 min). In addition, the method was selective, robust and sensitive, with relatively low LOQ values, found within the ranges commonly reported in the literature. Analytical curves were developed with coefficient of determination greater than 0.99. Matrix effect was verified for three out of four analytes: BP, DEP and 4NP. For these EDs, the adoption of the standard addition method is highly recommended.

The method was applied for the determination of the EDs in surface water samples from a very important water supply source in the Rio de Janeiro State. The maximum concentrations of BP, BPA, DEP and 4NP in five samples collected monthly during a five-month period in 2018 were 286.20, 182.04, 2. 56 ×103 and 13.48 ng L−1.

Finally, based on the results and the lack of regulation in Brazil and many other countries in the world regarding threshold values for these EDs, monitoring of other relevant water bodies must be done, followed by ecological and human health risk assessment. Moreover, the concentration levels found in the water samples are high enough to justify future investigations on the presence of these micropollutants in drinking water as well as to extend the monitoring for the search of similar pollutants and their metabolites.

Supplemental Information

The results for the optimization of the extraction methodology

DOI: 10.7717/peerj-achem.7/supp-1

Data processing for the validation of the methodology

DOI: 10.7717/peerj-achem.7/supp-2

Data monitoring of the four sampling points

Demonstrates that the methodology has been validated and useful for the purpose of determining the DEs in surface water samples.

DOI: 10.7717/peerj-achem.7/supp-3
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