UPLC-ESI/MS analysis of disinfection by-products (perchlorate, bromate, nitrate, nitrite and sulfite) in micro-filtered drinking water obtained from spring, well and tap water (desalinated) sources

2.1 Reagents and chemicals

Solvents and reagents used were of LC and analytical-reagent, respectively. Stock (ClO₄⁻, BrO₃⁻, NO₃⁻ and NO₂⁻) solutions (1000 µg/mL) of TraceCERT® grade were achieved from Merck. SO₃^2–, ACS reagent, ≥98.0% supplied from Sigma-Aldrich (Darmstadt, Germany). Acetonitrile, methanol and formic acid were purchased from Sigma-Aldrich (Darmstadt, Germany). Ultra-pure water (Milli–Q) was used for standard, sampling and mobile phase purposes, achieved from a water purification system of model Advantage (A10) from Millipore Corporation (Bedford, USA).

2.2 Water samples

A total of seventy-five water samples including spring bottled water (11 samples, international brand) and well bottled water (34 samples, national brand) were purchased from the local super markets based in the Saudi Arabia. Tap water (30 samples, desalinated) were achieved from five locations/city, Saudi Arabia (Tables 1 and 2). Samples were filtered by means of polytetrafluoroethylene (Chromafil Xtra, PTFE) syringe filter (0.20 μm) before being injection into the UPLC-ESI/MS system. Solutions were freshly prepared to evade any dreadful conditions of the studied compounds.

2.3 Analysis of water samples

For the purpose of system calibration curve and standard addition procedures, the individual stock solution was diluted and mixed all together at needed concentrations. The calibration solutions were prepared ranging from 5 to 100 µg/L (ClO₄⁻), 5–50000 µg/L (BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2–). The standard addition solutions were prepared at concentrations comprising two non-spiked and three spiked levels. Solutions including water samples were filtered using polytetrafluoroethylene (Chromafil Xtra, PTFE) syringe filter (0.20 μm) (Macherey-Nagel GmbH, Duren, Germany). To evade any microbial contamination in solutions and water samples, they were refrigerated until UPLC-ESI/MS analysis. Ultra-pure water (Milli–Q) was screened out for the presence of oxyhalides and inorganic ions, found free from these compounds. Subsequently, the Milli–Q water was used for the preparation of standards and mobile phase.

To assess the efficacy of the optimized UPLC-ESI/MS technique and matrix influence on ³⁵ClO₄⁻, ⁸¹BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2– retention time, peaks symmetry and their intensities, determination was performed by standard addition quantification method which contain two non-spiked samples at zero levels and spiked samples at three levels (50%, 200% and 600%). The levels demonstrating that the amounts of ³⁷ClO₄⁻, ³⁵ClO₄⁻, ⁸¹BrO₃⁻, ⁷⁹BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2– increase after spiking in the studied samples. All samples were analyzed in triplicates (n = 3), three individual analyses of each sample. Estimation procedures were carried out using a statistical models based on analysis of variance (ANOVA). The recovery of the studied compounds was calculated from the slope achieved between the spiked and found concentrations of the ³⁵ClO₄⁻, ⁸¹BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2– in analyzed samples.

2.4 UPLC

For the efficient separation of ³⁵ClO₄⁻, ⁸¹BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2– in drinking water, we used an ultra-performance liquid chromatography (UPLC) from Waters (Milford, USA). All compounds were separated using an analytical column based on BEH C₁₈ with dimension 50 mm × 2.1 mm i.d. and particle size of 1.7 µm (Waters, Milford, USA). To save the column during sample analysis, a pre-column based on VanGuard™ BEH C₁₈ with particle size 1.7 µm was used. The best chromatographic parameters for the determination of ³⁵ClO₄⁻, ⁸¹BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2– in water samples was attained by isocratic mobile phase elution mode, comprising methanol (75%) and MilliQ water (24.99%, HCOOH 0.01%) mobile phase at a flow speed of 0.2 mL/min. The column temperature was maintained at 25 °C during the sample analysis. The sample was injected with a volume of 5 µL.

2.5 ESI/MS

The proficient identification of ³⁵ClO₄⁻, ⁸¹BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2– in drinking water, a triple quadrupole mass spectrometer (Quattro Premier™, Micromass, Milford, USA) comprising an electrospray ionization source (ESI, Z–spray) attached with UPLC system. The apparatus was functioned in negative electrospray ionization mode. To choose the utmost abundant ions from individual compounds, data acquisition was carried out in full scan mode with mass-to-charge ratio values (m/z, 0–150). The selected ion recording (SIR) scanning mode was used to record the ions having greater intensities. Features affecting the target compounds ion transmission were optimized through infusing a ClO₄⁻, BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2– standard mixture solution at concentration 5 µg/mL. Optimized ESI/MS operational conditions were monitors for instance capillary voltage (3.5 kV), desolvation temperature (350 °C), source temperature (120 °C), cone gas flow speed (60 L/h), desolvation gas flow speed (600 L/h), and cone voltage (V): ³⁷ClO₄⁻ (50 V, m/z 101.14), ³⁵ClO₄⁻ (46 V, m/z 99.13), ⁸¹BrO₃⁻ (36 V, m/z 129.06), ⁷⁹BrO₃⁻ (50 V, m/z 127.09), NO₃⁻ (42 V, m/z 62.17), NO₂⁻ (72 V, m/z 46.25) and SO₃^2– (60 V, m/z 80.09). For cone gas, nitrogen of high purity was obtained from nitrogen generator of NM30LA model (Peak Scientific Inchinann, United Kingdom). For collision gas, argon of high purity was obtained from Speciality Gas Centre (Jeddah, Saudi Arabia). The mass spectrometer primary vacuum was produced from Oerlikon rotary vacuum pump of SOGEVAC SV40 BI model (Paris, France). The instrument handling and data acquisition were carried out by means of MassLynx V4.1 software (Waters, Milford, USA). The MS SIR parameters have been presented in Table 3.

2.6 Method validation

The performance of the optimized UPLC-ESI/MS method was evaluated in terms of linearity, limits of detection (LOD, signal-to-noise ratio of 3), limits of quantification (LOQ, signal-to-noise ratio of 10), and precision (run–to–run, five replicate analysis of a standard mixture solution in the same day) and (day–to–day, five replicate analysis of the same standard mixture solution over three successive days.

3.1 Method optimization (UPLC-ESI/MS)

The studied disinfection by-products oxyhalides (³⁵ClO₄⁻ and ⁸¹BrO₃⁻) and inorganic anions (NO₃⁻, NO₂⁻ and SO₃^2–) in drinking water were fully resolved by the UPLC-ESI/MS method, the chromatograms with high peak symmetry has been demonstrated in Fig. 1. The ESI mass spectra were primarily studied via infusing solution mixture of ClO₄⁻, BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2– standards at concentration of 5 µg/mL. The mass spectra achieved during ESI/MS optimization has been shown in Fig. 2. The mass spectra of these oxyhalides and inorganic ions are illustrating high intensities with higher ion counts. The ClO₄⁻ and BrO₃⁻ have resulted two isotopes, ³⁷ClO₄⁻ (m/z, 101.14) and ³⁵ClO₄⁻ (m/z, 99.13), and ⁸¹BrO₃⁻ (m/z, 129.06) and ⁷⁹BrO₃⁻ (m/z, 127.09). The oxyhalides demonstrate the identical mass transitions. The NO₃⁻, NO₂⁻ and SO₃^2– have resulted single ion with higher intensities and ion counts i.e. NO₃⁻ (m/z, 62.17), NO₂⁻ (m/z, 46.25) and SO₃^2– (m/z, 80.09) (Fig. 2). The oxyhalides isotopic transitions ³⁷ClO₄⁻ and ⁸¹BrO₃⁻, and individual inorganic anions transitions used for quantification purposes. Current result specified that in the negative ESI method, the studied compounds could form the lesser mass ions by deprotonation (removal of a proton, H⁺) (Gioumouxouzis et al., 2015). The selected on recording (SIR) conditions acquired with the ESI/MS system has been illustrated in Table 3. The NO₃⁻, NO₂⁻ and SO₃^2– ion transitions do not display an uninterrupted interference with ClO₄⁻ and BrO₃⁻ ion transitions. Nevertheless, in most of the commercial or municipal drinking waters, NO₃⁻, NO₂⁻ and SO₃^2– existing at amounts much greater than those of the studied ClO₄⁻ and BrO₃⁻ ions (Alsohaimi et al., 2012; Khan et al., 2013, 2014, 2016, 2020a, 2020b). To authenticate the occurrence and influence of NO₃⁻, NO₂⁻ and SO₃^2– on ClO₄⁻ and BrO₃⁻ ion transitions, the ion transitions of NO₃⁻, NO₂⁻ and SO₃^2– were tested by means of individual standards and subsequently examined along with the ClO₄⁻ and BrO₃⁻ ion transitions, and found no ions interference that could effect on the performance of the method. Based on these outcomes, the water samples were directly analyzed after micro filtration, found no matrix interferences and offered more consistent quantification of the targeted compounds (Alsohaimi et al., 2012; Khan et al., 2013, 2016, 2020b).

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Fig. 1

Optimized UPLC-ESI/MS chromatograms of oxyhalides isotopes ³⁷ClO₄⁻ (m/z, 101.14) and ³⁵ClO₄⁻ (m/z, 99.13), and ⁸¹BrO₃⁻ (m/z, 129.06) and ⁷⁹BrO₃⁻ (m/z, 127.09), and inorganic anions NO₃⁻ (m/z, 62.17), NO₂⁻ (m/z, 46.25) and SO₃^2– (m/z, 80.09) (1 µg/mL).

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Fig. 2

Mass spectra (ion signal as a function of mass-to-charge ratio) of ³⁷ClO₄⁻ (m/z, 101.14) and ³⁵ClO₄⁻ (m/z, 99.13), and ⁸¹BrO₃⁻ (m/z, 129.06) and ⁷⁹BrO₃⁻ (m/z, 127.09), and NO₃⁻ (m/z, 62.17), NO₂⁻ (m/z, 46.25) and SO₃^2– (m/z, 80.09) in some of the analyzed water sample.

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The best separation behavior of ClO₄⁻, BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2– was achieved using UPLC. Primarily, various analytical columns based on hydrophilic interaction liquid chromatography (HILIC) and UPLC™ Ethylene Bridged Hybrid (BEH) C₈ and C₁₈ columns of 1.7 µm particle were studied. The columns characteristically offer great retention of oxyhalides and inorganic ions (Khan et al., 2016, 2020b). Individual columns were optimized by means of a standard mixture solution (1 µL/mL) of ClO₄⁻, BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2– at different mobile phase flow speed ranged from 100 to 500 µL/min. Mobile phases for instance acetonitrile, methanol and water were optimized either separately or in diverse proportions in isocratic elution mode. The sample injection volume was fixed (5 µL) in all cases. Nevertheless, it did not effect on analysis as reported in the previous studies (Khan et al., 2016, 2020a, 2020b). The aim was to achieve the excellent symmetric peaks of the analyzed compounds by applying these conditions. Initially, the columns were studied using separate mobile phase at various flow rates, the outcomes show the weaker retention characteristics and formed asymmetrical peaks with complex base peak noise and the elution time of the targeted compounds. To maintain the symmetrical peak shape, the organic modifier (formic acid) was also added in the separate mobile phase nonetheless the symmetry of the respective peaks was enhanced, but formed peaks with diminutive complex base noise and the elution time. Subsequently, the columns were investigated by means of numerous mobile phase proportions comprising HCOOH as organic modifier at certain levels. The column BEH C₁₈ and isocratic binary mobile phase methanol (75%) and water (24.99%, HCOOH 0.01%) at a flow rate 0.2 mL/min offered high intense peaks with tremendous symmetry of the studied compounds. The column dead volume was studied and found to be 0.48 min, and the ClO₄⁻, BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2– eluted between 0.75 min and 0.78 min, depending on the types of compounds (Fig. 1). The higher mobile phase flow speed may offer the elution of targeted compounds at lower retention time, but the response of the peak intensity may have decreased owing to the presence of additional ions arise from applied mobile phases into the ESI/MS (Khan et al., 2020a), consequently low ionization of the targeted compounds occur and led to lower the intensities of the compounds (Khan et al., 2020a). So, the BEH C₁₈ column and mobile phase methanol (75%) and water (24.99%, HCOOH 0.01%) at a flow rate 0.2 mL/min were preferred for the quantitative identification of ClO₄⁻, BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2– in bottled and tap water samples. To avoid any sample carryover, initially the Milli-Q water was analyzed and found free from ClO₄⁻, BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2–. Subsequently, the Milli-Q water was injected after each analyzed samples and found no any unwanted compounds or sample matrices remain present throughout the analysis. To our knowledge, the current optimized UPLC-ESI/MS method is as one of the highly sensitive and rapid method, which offered analysis times lower than one minute without need of sample pretreatment and column preconditioning during the analysis.

3.2 Performance of the method

The method validation in terms of coefficient of determination (R²), limits of detection (LOD), limits of detection (LOQ) and precision was evaluated. The R² was measured at concentrations ranged from 5 to 100 µg/L (ClO₄⁻) and 5 to 50000 µg/L (BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2–), and R² values found > 0.995–0.999 depending upon the type of compounds. The LOD and LOQ were established at signal-to-noise ratio of 3 and 10, respectively. The LOD (0.016–0.043 µg/L) and LOQ (0.051–0.104 µg/L) were achieved. The precision repeatability was assessed from five replicate analysis of a standard mixture solution of ClO₄⁻, BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2– at 0.03 µg/mL in the similar day, and reproducibility was determined from five replicate analysis of the identical standard mixture solution over three successive days. The precision repeatability (1.54–2.28%) and reproducibility (3.13–4.08%) in terms of RSD% were achieved. The obtained R², LOD, LOQ, repeatability and reproducibility values of the optimized UPLC-ESI/MS method have been presented in Table 4. The system has offered the excellent quality parameters, hence the UPLC-ESI/MS method could be proposed for the reliable analysis of oxyhalides and inorganic ions in drinking water.

3.3 Application of the real samples

The optimized method (UPLC–ESI/MS) was practically applied for the analysis of ClO₄⁻, BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2– in drinking water obtained from spring, well and tap water sources. A total of seventy-five samples were analyzed, the outcomes (amounts and recovery) have been presented in Table 1 and 2. It is noticeable that all samples contain both oxyhalides and inorganic ions at higher concentrations. In spring water, the amounts of ClO₄⁻ (5.33–22.64 µg/L), BrO₃⁻ (7.52–16.73 µg/L), NO₃⁻ (5.42–12.14 mg/L), NO₂⁻ (1.08–4.37 mg/L) and SO₃^2– (6.84–32.45 mg/L) were found, whereas well and tap water contained ClO₄⁻ (4.20–21.33 µg/L), BrO₃⁻ (8.05–15.13 µg/L), NO₃⁻ (1.27–16.11 mg/L), NO₂⁻ (0.43–10.77 mg/L) and SO₃^2– (7.14–36.10 mg/L), and ClO₄⁻ (0.89–7.37 µg/L), BrO₃⁻ (5.15–14.68 µg/L), NO₃⁻ (1.12–6.33 mg/L), NO₂⁻ (0.67–3.95 mg/L) and SO₃^2– (5.40–23.26 mg/L), respectively. The recovery values of the study compounds were found between 96% and 99% in spring and well water samples, however, the tap water produced 94% and 99%. The reason might be due to tap water contained higher amounts of salt and other matrices than spring and well water. The high recovery values specify that compounds loss in the filtration phase and ion suppression in the MS source (ESI) were negligible. In Fig. 3, the UPLC–ESI/MS chromatograms of ClO₄⁻, BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2– in spring water sample (SW1) have been presented. The method offered chromatographic peaks with excellent symmetry and no other interfering compounds eluting at the same retention time of the targeted compounds. The investigation about the occurrence of ClO₄⁻, BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2– in drinking water are essentially unexplored, many earlier studies reported either single or few of them were determined in drinking water (Liu et al., 2002; Alsohaimi et al., 2012; Khan et al., 2016, 2020a, 2020b; Liu and Croué, 2016; Djam et al., 2020). The oxyhalides for instance chlorite, chlorate and BrO₃⁻ were measured by IC in 168 bottled water (mineral and drinking water) samples commercialized in Iran, and the obtained amounts ranged from 37.04 and 33.58 μg/L (Djam et al., 2020). Liu et al., have analyzed Beijing’s drinking water for the determination of trace level BrO₃⁻ and ClO₄⁻ by IC with an evaporative preconcentration method, and achieved 0.58 μg/L and 0.79 μg/L, respectively (Liu et al., 2002). Khan et al., have also determined either single or in combination of three ions in drinking water and non-alcoholic beer by UPLC-MS method (Alsohaimi et al., 2012; Khan et al., 2013, 2014, 2016, 2020a, 2020b). The amounts of these ions in various matrices were found in good agreement with those achieved in the current study. Nonetheless, the increasing demand of uncontaminated drinking water either in the form of tap, spring or bottled water, and guidelines established by various national and international regulatory agencies (Food and Administration, 1986; EPA, 2018), it was highly essential to optimized a reliable method (UPLC-ESI/MS) that could analyze such type of compounds in drinking water. In this study, the amounts of ClO₄⁻, BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2– were found beyond the MCL and DWEL regulated by the EPA (EPA, 2018) and FDA (Food and Administration, 1986), respectively. The outcomes also revealed that the tap water produced lower levels of these contaminants especially in cities with low population densities (Fig. 4). The ClO₄⁻, BrO₃⁻, NO₃⁻, NO₂⁻ and SO₃^2– were remain present in Saudi Arabian drinking water. Therefore, the identifying and reducing the level of these compounds in water samples are highly required additional fears to meet better essentials of policies for end users.

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Fig. 3

UPLC-ESI/MS chromatograms of ³⁷ClO₄⁻ (m/z, 101.14), ⁸¹BrO₃⁻ (m/z, 129.06), NO₃⁻ (m/z, 62.17), NO₂⁻ (m/z, 46.25) and SO₃^2– (m/z, 80.09) obtained in spring water sample (SW1).

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Fig. 4

Variation of oxyhalides and inorganic anions concentrations vs population density.

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