Determination of haloacetic acid in water for human consumption by Paula S. Rosa, Margarida S. Romão, Georgina S. Felisberto Laboratório de Análises do Instituto Superior Técnico, Universidade de Lisboa, Portugal December, 2015 Abstract The objective of this work is the implementation and validation of an analytical method for the analysis of haloacetic acids (HAAs) in drinking water. In terms of legislation, the USEPA requires that the limit for the concentration of HAAs5 (MCAA, DCAA, TCAA, MBAA and DBAA) should not exceed 60 μg/l. WHO imposes to MCAA, DCAA and TCAA the limits of 20 μg/l, 50 μg/l and 200 μg/l, respectively. The ERSAR imposes that the limit for the concentration of HAAs3 (MCAA, DCAA, TCAA) should not exceed 100 μg/l. For the determination of these compounds a method that combines the efficiency of ultra-performance liquid chromatography with mass spectroscopy () was developed. First the operating conditions of the mass spectrometer were optimized by varying the parameters (cone voltage and collision energy) related to the formation of precursor ions and product ions of each HAAs. Then the chromatographic conditions were optimized so as to obtain a higher sensitivity and resolution for each compound in the shortest possible time. After the implementation of the analytical method it was carried out its validation. Limits of quantification for the HAAs study were below the reference values imposed by USEPA, OMS and ERSAR entities. Drinking water samples from the area of Lisbon and Algarve were analysed in order to examine the presence of the halogenated compounds, and the highest concentration value was 22 μg/l of TCAA in a sample. In none of the samples analysed, the reference values of the entities USEPA, WHO and ERSAR have been exceeded. Keywords: haloacetic acids, drinking water, 1. Introduction Water supply for human consumption and the monitoring of its quality, is an essential public service to the welfare of citizens and public health. It can be said that Portugal is advanced in this obligation, covering practically the entire population with an adequate service supply. Nationally drinking water has excellent quality, with 98.4% of safe water (ERSAR - 2014), placing Portugal at the level of the most advanced countries of the European Union [1]. The quality required is based not only on the analysis of physical-chemical parameters but also in the compliance with the parametric values according to the law decree 306/2007, forcing the water to be free of pathogenic micro-organisms. This is because drinking water is a major source of microbial pathogens [2]. The treatment to which water intended for human consumption is subjected, is composed by various processes, one of them being the disinfection. The chemicals used as disinfectants such as chlorine and sodium hypochlorite, react with natural organic matter present in the water, forming the so-called disinfection by-products. There is a wide variety of disinfection by-products that involve serious risks to public health, being trihalomethanes (THMs) and haloacetic acids the (HAAs), the principals ones [3]. The compounds under study in this work are the HAAs, representing the main group of halogenated and non-volatile disinfection by-products. There are nine different haloacetic acids: monochloroacetic acid (MCAA), dichloroacetic acid (DCAA), trichloroacetic acid (TCAA), monobromoacetic acid (MBAA), dibromoacetic acid (DBAA), tribromoacetic acid (TBAA) bromochloroacetic acid (BCAA), bromodichloroacetic acid (BDCAA) and dibromochloroacetic acid (DBCAA). They are substituted carboxylic acids, by halogen groups conferring to HAAs higher acidity and are polar compounds with relatively high boiling points and high water solubility. In terms of current legislation, USEPA has as reference value a limit of 60 μg/l for the sum of HAAs5 (MCAA, DCAA, TCAA, MBAA and DBAA) [4]. 1
Table 1 - Optimized instrumental and MRM conditions of HAAs. Compound Retention Time (min) Dwell Time (s) Cone Voltage (V) Precursor Ion MRM1 Collision Energy (ev) Product Ion Cone Voltage (V) Precursor Ion MRM2 Collision Energy (ev) Product Ion MCAA 1.61 0.035 20 92.90 6 34.68 20 94.90 10 37.00 DCAA 1.70 0.035 19 127.78 6 82.75 19 127.78 16 34.87 MBAA 1.83 0.035 17 138.90 12 80.80 17 136.85 17 78.87 BCAA 1.84 0.035 18 170.80 10 126.82 18 170.80 10 78.80 DBAA 2.03 0.035 20 216.70 15 172.80 20 216.70 18 78.80 TCAA 2.80 0.151 20 162.90 9 118.90 12 160.90 10 116.85 MCAA 1.61 0.035 20 92.90 6 34.68 20 94.90 10 37.00 WHO indicates only three reference values with the limits of 20 μg/l, 50 μg/l and 200 μg/l for MCAA, DCAA and TCAA, respectively [3]. In Portugal, the ERSAR has as reference value the limit for the sum of the concentrations of MCAA, DCAA and TCAA, 100 μg/l [5]. The growing interest in these compounds is based on the fact that they have some toxicological effects (according to the USEPA, some are probable carcinogenic), which is why the quantitative control of them should be regulated [6]. From the literature research done, it was found that the methods with most applicability in the analysis of HAAs are gas chromatography with electron capture detection () and ion chromatography with detection by mass spectrometer (IC-MS). These are very time consuming methods, mainly due to the process of sample preparation, and high solvent costs, so it makes sense to implement a new methodology for the analysis of HAAs in human drinking water, more efficiently. This study aims to validate an analytical method by ultra-performance liquid chromatography with detection by tandem mass spectrometry (MS / MS), which allows the analysis of haloacetic acids in drinking water by direct sample injection. The HAAs under study were MCAA, DCAA, TCAA, MBAA, DBAA, TBAA and BCAA. The MS parameters were optimized through direct infusions of the individuals HAAs. Different conditions (columns and mobile phases) were tested in order to obtain an efficient chromatographic separation. This method was then applied to water samples from Algarve and Lisbon public water supplies, Portugal. In LAIST, where this study was carried out, the analysis of haloacetic acids is currently done by GC. Therefore, a comparison is made between this technique and the technique developed in this work by UPLC. 2. Experimental 2.1. Chemicals and reagents The HAAs (purity >99%), MCAA, DCAA, TCAA, MBAA, DBAA, BCAA used were from Riedel Haën. Methanol and acetonitrile HPLC grade were obtained from CARLOERBA. MTBE obtained from Acros Organics. HPLC grade acetic acid and ascorbic acid were purchased from ABSOLVE. Ultrapure water was prepared by a Milli-Q Synthesis water purification system (Millipore). Stock solutions (1 g/l in MTBE) for all single standard substances were prepared, and then 1 mg/l mixture of the HAAs was made in methanol. More diluted solutions were prepared by suitable dilution with the samples. All solutions were stored at 4ᵒC. 2.2. Sample preparation Different water samples from Lisbon and Algarve public water supplies were collected. To all samples ascorbic acid (final concentration of 0.03 g/l) for removing residual chlorine and acetic acid (final concentration of 0.1%) were added and the solutions were stored at 4ᵒC. 2.3. Liquid chromatography and mass spectrometry The LC apparatus was an ACQUITY UPLC system by 2
Waters. Separation of HAAs was achieved using a Atlantis dc18 column (5μm; 2.1mm 150 mm). The column was maintained at 40ᵒC, the flow rate was 0.4 ml/min and the injection volume was 15 μl. Methanol (A) and ultrapure water containing 0.2% (v/v) acetic acid (B) were used as mobile phases. The gradient was increased from initial 5% to 40% of solvent A linearly within 2 minutes. Then the mobile phase A was increased to 100% in another 1 minute and kept for 4 minutes. Finally, the gradient was returned to the initial conditions of 5% A for a 2 minutes re-equilibrium before the next injection. The total run time was 9 minutes. Mass spectrometry was performed using a Waters Xevo TQD (triple quadrupole) detector equipped with an electrospray ionization source (API - ESI) in the negative ion mode. The optimized MS parameters were as follows: source temperature, 150ᵒC; desolvation temperature, 500ᵒC; capillary voltage, 0,5 kv; desolvation gas flow, 1000 L/h; and cone gas flow, 50 L/h. Finally, the data acquisition was performed in the multiple reaction monitoring mode (MRM) to maximize sensitivity of detection. Infusions were performed in order to determine the best precursor and products ions, and to optimize the cone voltage and collision energy for each analyte. The precursor ion for TBAA was [M COOH], and [M H] were selected as the precursor ions for the other six HAAs. In Table 1 are summarised the optimized conditions for each HAA. In the particular case of TBAA, it was not possible to find a precursor ion with good intensity. Therefore this compound was no longer studied. 2.4. Quantification Identification of the six HAAs in drinking water was accomplished by comparing the retention time and mass ratio between product ions for each haloacetic acid. Six point calibration curves were constructed from the standard solutions in a concentration range between 5 and 50 μg/l, depending on the individual compound. To avoid sample contamination, all equipment rinses were done with methanol, and laboratory blanks were analysed to assess potential sample contamination. Recoveries were evaluated by spiking standard solutions to a drinking water sample at three concentration levels for each HAA in replicates of two, and the original concentration was determined prior to the fortification experiment. Because no sample extraction steps were included in this method, the recovery data reflected the ion suppression. Because observed recoveries were below the acceptable values ( 80%) for some HAAs (as discussed below) quantification was done using the standard addition method. Data was analysed using Waters MassLynx V4.1 and Microsoft Excel 2013. The limits of detection (LODs) and limits of quantitation (LOQs) were defined as signal-tonoise (S/N) ratios at 3 and 10, respectively. 3. Results and discussion 3.1. Optimizing analytical conditions Because liquid chromatographic conditions, especially the solvent conditions can greatly influence the separation of the desired compounds and ESI sensitivity, the effects of mobile phase composition on the sensitivity and separation were investigated. In previous studies, water containing acids was the commonly used aqueous mobile phase in HAAs analysis in order to reduce the dissociation of HAAs and to improve their retention and separation on liquid chromatography columns [7]. In this study, methanol/water containing acetic acid at 0.1% and 0.2% were investigated and the latter was used as the aqueous mobile phase due to the relatively high sensitivity and good separation for most Haas. One of the most important tasks in developing a LC- method is the selection of a proper chromatography column. Conventional RP C18 column was reported to have difficulty to retain HAAs due to the non-polar stationary phase, so great efforts have been made to select a suitable column [6]. In this study, five types of RP columns were investigated to analyse HAAs: Acquity BEH C8 (1.7 μm; 2.1 mm 100 mm), Acquity BEH C18 (1.7 μm; 2.1 mm 100 mm), CORTECS C18 (1.6 μm; 2.1 mm 100 mm), CORTECS HILIC (1.6 μm; 2.1 mm 100 mm) and Atlantis dc18 (5 μm; 2.1 mm 150 mm). HILIC UPLC column failed to separate the six HAAs. Acquity BEH C18 and CORTECS C18 columns would theoretically provide a better retention of HAAs than a BEH C8 column, because longer chain lengths may be more appropriate for retention of small hydrophilic 3
2.03;8844;103901 1.84;8343;99866 2.80;1631;15902 1.83;1702;19449 1.70;103;1488 1.60;597;6924 Fig. 1 Total ion chromatogram for each HAA, to a concentration of 50 μg/l in Atlantis dc18 column. molecules [6]. It was found that Acquity BEH C8 and Atlantis dc18 columns provided better retention and separation than all the others. The BEH C8 column was actually better for capturing HAAs than all C18, which would be due to the fact that the longer C18 chains laydown during the early aqueous period of the gradient and therefore the hydrophilic HAAs were not captured. Although the Acquity BEH C8 column was the column were better chromatograms were obtained, due to fortuitous reasons the work could not proceeded with that column. The column chosen was Atlantis dc18 column, which also provide good retention and separation of all HAAs. HAAs separation can be seen in Fig. 1. The column temperature is an important parameter in chromatographic separation because it has a direct influence on mass transfer. Since the higher the temperature, the lower the viscosity of the mobile phase, mass transfer process increases giving greater sensitivity in the chromatographic separation. On the other hand, at lower temperatures compounds are retained on the column due to the increased viscosity of the mobile phase. In this work three different operating temperatures of the column were tested: 30, 40 and 45 ᵒC. Since the maximum temperature supported by dc18 column is 45 ᵒC, this temperature was immediately excluded. It was necessary to reach a compromise between the two remaining temperatures. Considering that there were not appreciable difference between the retention times obtained for 30 and 40 ᵒC, it was established as optimal column temperature, 40 ᵒC. In these conditions good resolution was obtained in a total run time of 9 minutes. High injection volume implies higher sensitivity for the compounds. But, higher volumes lead to peaks enlargement which results from the column saturation. The chromatograms that present better peaks are the ones that correspond to an injection volume of 30 μl (injection volumes studied: 10, 20 and 30 μl). 3.2. Quantification and method validation The methods of standard addition and external calibration were applied for quantification of the HAAs. Calibration curves were constructed from 5 to 50 μg/l (the standard concentration levels for all HAAs were at 5, 10, 20, 30, 40 and 50 μg/l, except for MCAA, which were 10, 20, 30, 40 and 50 μg/l). Calibration graphics were linear with good correlation coefficients (R) all being greater than 0.995. The LODs (n = 6) of MCAA, DCAA, TCAA, MBAA, DBAA, BCAA were 1.5, 1.6, 0.62, 1.3, 0.90, and 0.33 μg/l, respectively (RSDs were 2.7 8.7%), and their LOQs (n = 6) were 4.6, 4.7, 1.9, 3.8, 2.7,and 1.0 μg/l, respectively (RSDs 4
Area Area Table 2 - Conductivity (μs/cm) and ph values of the different types of w ater used in this test. Tipo de Água Conductivity (μs/cm) ph Ultrapure 0,055 5,4±0,1 Luso 50 [8] 5,7±0,1 Penacova 49 [9] 5,3±0,4 Tap Water 1 250 7-8 Tap Water 2 250 7-8 were 2.0 8.7%). The above description indicates that without pre-enrichment steps, our method was sensitive enough to directly analyse HAAs in drinking water, according to the values referred by USEPA, OMS and ERSAR entities. The intra-day and inter-day precision were calculated by the relative standard deviations (RSDs) at two concentration levels for each HAA within the linear ranges. The intra-day RSDs (n = 6) were below 8.7% as were inter-day RSDs results. The mean recoveries (n > 40) of four HAAs in the spiked water samples were above 80% to DCAA, TCAA, DBAA and BCAA, suggesting no apparent signal suppression in this compounds. On the other hand the mean recoveries (n > 40) of MCAA and MBAA in the spiked water samples were 57 79% suggesting signal suppression in this compounds. The matrix effects were overcame by using the standard addition calibration method. In order to verify the efficiency of standard addition calibration method, a matrix simulation was carried out. It was used a bottle water which was known to be HAAs free. This matrix was spiked with a concentration of 20 μg/l from each HAA. Then the standard addition calibration method was carried, and the results obtain were very satisfying, because the recuperation values were 80% for each HAA. The ratio between the two transitions of a compound (MRM1 and MRM2) was used to confirm its identity. Along the working range this ratio was studied for each compound to confirm its stability and respective RSD was between 6 to 16 %. In order to highlight clearly the effects of matrix during analyses, two different tests were made. Test 1 consisted on identifying the differences in slopes of the calibration curves when using different matrices. There were used five different matrices, with characteristics show in 1000 800 600 400 200 0 Table 2. In Fig. 2 the calibration curves obtained for the MCAA (the most influenced compound by matrix composition) are shown highlighting the matrix effect. Test 2 consists in varying the percentage of ultrapure water against the percentage of the sample matrix. Different solutions were prepared with the same concentration of HAA (30 μg/l) at 0, 10, 30, 60 and 100% of tap water and the results for MCAA are shown in Fig. 3. 400 300 200 100 0 0 10 30 60 100 % Tap Water Fig. 3 Graphical representation of the area variation, depending on the tap w ater percentage, for the MCAA. The results show that with 100% tap water, the obtained value is half of what it is obtained with 100% ultrapure water. The expanded uncertainties estimated were based in precision and accuracy, then 20%. Test 1 0 10 20 30 40 50 Concentration (μg/l) Ultrapure Water Luso Water Penacova Water Tap Water 1 Tap Water 2 Fig. 2 Graphical representation of the calibration curves obtained for the MCAA, in different matrices. Test 2 3.3. Environmental application presenting values lower This method was applied to determine six HAAs in tap water samples collected from different regions in Algarve and Lisbon, Portugal and the results are presented in Table 3. The presence of HAAs was not detected in most situations. Taken into account the LQs of the method this means that even if present their concentrations are below the values required by the USEPA, WHO and ERSAR authorities. The water samples from Lisbon, presented a 5
Table 3 Concentrations (μg/l) of HAAs in the w ater samples from different regions, where LQ values are 10 μg/l for MCAA and 5 μg/l for the remaining HAAs. Samples MCAA DCAA TCAA MBAA DBAA BCAA A <10 <5 <5 <5 <5 6±1 B <10 <5 <5 <5 <5 <5 C <10 <5 <5 <5 <5 <5 Algarve D <10 <5 <5 <5 5±1 <5 E <10 <5 <5 <5 6±1 <5 F <10 <5 <5 <5 <5 <5 G <10 <5 <5 <5 6±1 6±1 H <10 7±1 <5 <5 <5 <5 I <10 <5 19 ± 2 <5 <5 <5 J <10 <5 22 ± 2 <5 <5 <5 K <10 <5 9 ± 1 <5 <5 <5 Lisbon L <10 <5 14 ± 1 <5 <5 <5 M <10 <5 13 ± 1 <5 <5 <5 N <10 <5 9 ± 1 <5 <5 <5 O <10 <5 15 ± 1 <5 <5 <5 P <10 <5 11 ± 1 <5 <5 <5 TCAA concentration always greater than 9 μg/l that however is lower than the suggested limits. Method Comparison Analyses were performed simultaneously with method in real samples. The results are shown in Table 4. The comparison between method and method allowed to conclude that although method offers lower LQ values to DCAA, TCAA, MBAA, DBAA and BCAA (LQ = 1 μg/l), there are significant advantages of using, such as less time consuming in sample preparation and analysis together with much less solvents consuming. Also because manual injection is needed in method, method provides a safer environment to the analyst. And at last, because with method there is no need to sample preparation, the response time to the client is lower than with method. Table 4 - Results obtained in HAAs analysis by HPLC-MS / MS and methods, in 5 real samples. Samples A B C H X Method MCAA < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 DCAA < 5 5±1 < 5 4±1 < 5 3±1 7±1 5±1 < 5 6±1 TCAA < 5 1.0±0.2 < 5 2.0±0.4 < 5 < 1 < 5 1.0±0.2 11±2 12±2 MBAA < 5 - < 5 - < 5 - < 5 - < 5 - DBAA < 5 5±1 < 5 4±1 < 5 4±1 < 5 5±1 < 5 1.0±0.2 BCAA 6±1 - < 5 - < 5 - < 5 - < 5 - Note: The uncertainty associated with each HAA by method presents a value of 20%. 6
4. Conclusions An efficient, sensitive and simple method was developed for the direct analysis of six HAAs (MCAA, DCAA, TCAA, MBAA, DBAA and BCAA) in drinking water samples using chromatographic separation by ultraperformance liquid chromatography with electrospray ionization and detection by mass spectrometry (ESI-MS / MS). The mass ratio and the retention times allowed the identification of each of haloacetic acids. The LQ values determined for all six HAA are lower than the reference values imposed by USEPA, WHO and ERSAR entities. However and in a future perspective, it would be interesting to lower these values. The method was validated. In terms of precision the intermediate precision and repeatability have variances below 10%. With regard to accuracy, the average recoveries for DCAA, TCAA, DBAA and BCAA were above 80% while the average of monohalogenados acid (MCAA and MBAA) were less than 80% duo to matrix effects. This difficulty was overcome using the standard addition method. The expanded uncertainties estimated, for each of haloacetic acids have acceptable values below 20%. The developed method () has several advantages over method, such as no preparation time, less analysis time, less response time to the customer, and less solvent exposure for the analyst, being therefore a more secure method. The developed methodology was applied in the determination of HAAs in real samples of drinking water from different points of collection (public water supply of Lisbon and Algarve). The results obtained show that the presence of HAAs is vestigial, for most analytes under study, with existing concentrations below the values required by the USEPA, WHO and ERSAR entities. The water from Lisbon, which was tested in various sessions, presented a TCAA concentration always greater than 9 μg/l, which however never exceeded the reference values of the aforementioned entities. Since water of swimming pools suffer a larger chlorination process, it would be interesting to analyse those in the future, because according to the literature, concentrations that exceed the limits of the USEPA were found in some situations [10] [11]. As future prospects, it is suggested to attempt to implement and validate the method in the analysis of TBAA and also a depth study of the matrix effects, as evidenced by tests performed which showed its influence in the determination of haloacetic acids in drinking water. Acknowledgements Thanks are due to Professor Margarida Romão, Engineer Georgina Sarmento, Ana Cruz, Hélder Gajeiro and all members from LAIST for the opportunity and all the help. References [1] ERSAR, "A qualidade da água - O consumidor e os serviços de águas e resíduos", 2013. [2] N. J. Ashbolt, Microbial contamination of drinking water and disease outcomes in developing regions, Toxicology, vol. 198, p. 360, 2004. [3] WHO - World Health Organization, Guidelines for Drinking-water Quality, 2011. [Online]. Available: http://whqlibdoc.who.int/publications/2011/978924 1548151_eng.pdf. [4] EPA United States Environmental Protection Agency, Water: Basic Information about Regulated Drinking Water Contaminants, December 2013. [Online]. Available: http://water.epa.gov/drink/contaminants/basicinfor mation/disinfectionbyproducts.cfm#what disinfection byproducts does EPA regulate, how are they formed, and what are their health effects in drinking water at levels above the maximum contaminant level?. [Accessed March 2015]. [5] ERSAR, Recomendação ERSAR n.º 02/2011 - ESPECIFICAÇÃO TÉCNICA PARA A CERTIFICAÇÃO DO PRODUTO ÁGUA PARA CONSUMO HUMANO, 2011. [6] L. Meng, S. Wu, F. Ma, A. Jia and J. Hu, Trace determination of nine haloacetic acids in drinking water by liquid chromatography-electrospray tandem mass spectrometry, Journal of Chromatography A, vol. 1426, pp. 1-252, 2010. 7
[7] C. Y. Chen, S. N. Chang and G. S. Wang, Determination of Ten Haloacetic Acids in Drinking Water Using High Performance and Ultra Performance Liquid Chromatography - tandem Mass Spectrometry, J Chromatogr Sci., vol. 47, no. 1, pp. 67-74, 2009. [8] CESAB, Cronograma relativo à Zona de Abastecimento: Luso, 2012. [Online]. Available: http://www.cmmealhada.pt/ficheiros/ambiente/aguassaneament o/za_luso4tri11.pdf. [Accessed 2015]. [9] CALDAS DE PENACOVA, SOP Business Centers, 2015. [Online]. Available: http://sopbusiness-centers.webnode.pt/products/aguacaldas-de-penacova-0-33-lt/. [Accessed 2015]. [10] G. M. Cardador MJ, Determination of haloacetic acids in human urine by headspace gas chromatography mass spectrometry, J Chromatogr B, vol. 878, p. 21, 2010. [11] X. W. e. all, Haloacetic acids in swimming pool and spa water in the United States and China, Frontiers of Environmental Science & Engineering, vol. 8, no. 6, pp. 820-824, 2014. 8