A joint study between the University of Tasmania (Hobart, Australia) and Trajan Scientific and Medical (Ringwood, Australia) coupled a compact capillary liquid chromatography (capLC) system with two small-footprint and portable single quadrupole mass spectrometers for the analysis of haloacetic acids (HAAs) in water samples. The resulting system is compatible with the on-site analysis within water treatment plants and provides a cost-effective and green solution for the quantification of the nine haloacetic acids recommended for monitoring by United States Environmental Protection Agency (USEPA). LCGC International spoke to Ibraam Mikhail of the University of Tasmania, lead author of the paper resulting from this work (1).
What are disinfection by-products (DBPs), and why do they form during water treatment?
Key Points
- ·Disinfection by-products (DBPs), including haloacetic acids (HAAs), form when disinfectants like chlorine react with natural organic matter. HAAs are persistent, prevalent, and linked to health risks such as cancer, making their monitoring in drinking and swimming pool water crucial.
- ·Portable capillary liquid chromatography (capLC) coupled with single quadrupole electrospray ionization-mass spectrometry (ESI-MS) enables sensitive, on-site HAA detection without ion-pairing reagents, overcoming challenges of retention and ion suppression common in conventional methods.
- ·High chromatographic resolution and method validation are essential for accurate, regulatory-compliant HAA quantification, with ongoing research focusing on field deployment and expanding contaminant monitoring capabilities.
Disinfection by-products (DBPs) are chemicals formed when disinfectants like chlorine react with natural organic matter (NOM) in water. This process, while essential for safe drinking water, creates various halogenated compounds from reactions with natural organic substances, such as fragments of humic or fulvic acids, small organic acids, or amino acids. This highlights a key trade-off in water treatment: ensuring microbial safety while managing chemical risks.
Explain why haloacetic acids (HAAs) are considered a significant class of DBPs.
HAAs are a significant class of disinfection by-products due to their prevalence, persistence, and health implications. They are the second largest group of halogenated DBPs found in water, after trihalomethanes. Unlike volatile trihalomethanes, HAAs are highly soluble and minimally volatile, meaning they persist in the water system longer, and so if present in treated potable waters are very likely to be ingested by consumers.
Describe the health concerns associated with exposure to HAAs in drinking water.
Due to their high solubility, HAAs are readily absorbed into the bloodstream from the gastrointestinal tract, making them highly bioavailable. Exposure to HAAs in drinking water has been linked to several health concerns, including an increased risk of colorectal and bladder cancers, as well as a reduction in sperm count and motility. In particular, the United States Environmental Protection Agency (USEPA) classifies dichloroacetic acid (DCAA) as a likely human carcinogen and recognizes trichloroacetic acid (TCAA) as potentially carcinogenic based on suggestive evidence.
Why are HAAs particularly relevant in swimming pool water analysis compared to drinking water?
HAAs are especially relevant in swimming pool water because of continuous disinfectant dosing and the presence of high levels of anthropogenic organic matter (for example, as delivered through sweat or personal care products) which react with chlorine to form elevated levels of DBPs. Given their low volatility, HAAs can accumulate over time, with DCAA and TCAA concentrations reported up to 2,400 and 2,600 µg/L, respectively in Australia (2).
What role does ion-pairing reagents play in LC analysis of HAAs, and what are the downsides of using them in ESI-MS applications?
Ion-pairing reagents like tributylamine play a key role in LC analysis of HAAs by improving the retention of these highly polar compounds on reversed-phase columns. HAAs, being small and hydrophilic carboxylic acids, typically exhibit poor retention and/or selectivity on conventional C18 functionalized phases. The ion-pairing reagent forms more hydrophobic complexes with the negatively charged HAA molecules, allowing for stronger interaction with the stationary phase and thus improved retention. However, when coupled with electrospray ionization-mass spectrometry (ESI-MS), these reagents are notorious for causing ion suppression and system contamination, complicating routine HAAs analysis.
Explain why achieving high chromatographic resolution is essential when using single quadrupole MS detection for HAA analysis.
Achieving high chromatographic resolution is essential when HAAs with a single quadrupole mass spectrometer due to the MS's inherent limitations in selectivity and resolving power. Single quadrupole MS struggles to differentiate between ions with identical or very similar mass-to-charge ratios (m/z). Further complicating this is the fact that each HAA can form various ionic species during electrospray ionization, such as pseudomolecular ions, decarboxylated ions, and formate adducts. This means a single HAA can produce signals at several m/z values, and, critically, different HAAs might yield ions at the same m/z through different ionization pathways. Therefore, sufficient chromatographic resolution is indispensable to separate these compounds based on their unique retention times, allowing for accurate identification and quantification.
What are the benefits of using a small-footprint capillary LC (capLC) system for environmental monitoring?
Small-footprint capLC systems offer substantial benefits for environmental monitoring, especially for field-based and on-site analysis. Their enhanced portability, exemplified by "briefcase-sized" chromatograph, allows for direct environmental deployment, thereby eliminating the need to ship samples to centralized laboratories. Additionally, the capillary format limits any transport of large volumes of solvent, which otherwise presents considerable safety issues and limits mobility. This capability enables real-time decision-making, rapid response to contamination events, and more frequent monitoring for regulatory compliance. Ultimately, capLC systems facilitate immediate verification of treatment effectiveness and prompt detection of excursions above regulatory limits, allowing for swift corrective action.
Discuss the trade-offs between selectivity, sensitivity, and throughput when developing a chromatographic method for HAA monitoring.
Achieving high selectivity often sacrifices throughput, as complex gradient elution programs and column equilibration steps prolong the overall analysis time. For example, a 10-minute gradient for HAA separation, coupled with equilibration, extends the total cycle time. Sensitivity optimization also presents trade-offs. While extended sample preparation such as solid-phase extraction can significantly enhance sensitivity, it simultaneously increases analysis time, negatively impacting throughput. Conversely, direct injection approaches maximize throughput and simplify protocols, but a limited to simpler matrices like tap water. However, for more complex matrices like swimming pool water, a 10-fold dilution might be necessary to minimize matrix effects and maintain selectivity, though this directly compromises sensitivity. Despite this compromise, the method we developed still allows for detection of HAAs below the acceptable limits.
What challenges might arise in using liquid chromatography-electrospray ionization-mass spectrometry (LC–ESI-MS) for HAA analysis without ion-pairing reagents?
When using LC-ESI-MS for HAAs analysis without ion-pairing reagents, the primary challenge is the poor retention of these highly polar carboxylic acids on conventional reversed-phase chromatographic columns. As mentioned above, HAAs exhibit minimal interaction with traditional C18 stationary phases, causing them to elute near the void volume and resulting in inadequate chromatographic resolution. This lack of selectivity hinders the reporting of individual HAA levels, particularly for compounds sharing similar m/z values in MS detection. To overcome this, specialized columns, such as polar-modified C18 columns, are necessary to enhance the retention of these hydrophilic analytes.
Why might capLC be preferable to conventional LC for HAAs analysis in certain field applications?
CapLC offers distinct advantages over conventional LC for HAA analysis in field applications primarily due to significantly reduced solvent consumption and enhanced mass sensitivity. CapLC systems consume only a few liters of mobile phase annually, drastically simplifying logistics in remote locations by minimizing solvent transport, storage, and waste disposal. Furthermore, their enhanced mass sensitivity, stemming from lower flow rates and smaller column dimensions, enables the detection of trace-level HAAs at regulatory limits without the need for time-consuming sample preconcentration. This not only simplifies field protocols and reduces analysis time but also minimizes sample volume requirements, which is crucial when sample availability is limited.
How does the formation of formate adducts or decarboxylation products complicate LC–MS data interpretation, and how can chromatographic separation help mitigate these issues?
In LC–MS analysis of HAAs, formate adducts and decarboxylation products generate multiple ion species for a single compound, complicating peak identification, especially with single quadrupole MS. Chromatographic separation helps resolve this issue by separating compounds based on retention time, allowing accurate identification and quantification even when mass signals overlap.
Describe how method validation (linearity, precision, detection limits) is influenced by chromatographic performance.
Method validation parameters are profoundly influenced by chromatographic performance. Good peak shape and resolution improve linearity by ensuring accurate and consistent integration. Moreover, the method's precision relies heavily on the stability of retention times, the repeatability of peak areas, and consistent MS response. These factors collectively guarantee reliable results across all your analyses. Finally, detection limits (LODs) and quantitation limits (LOQs) are significantly impacted by chromatographic performance. Chromatographic resolution is crucial for effectively isolating the analyte signal from co-eluting matrix interferences and other compounds. This improved separation leads to a higher signal-to-noise ratio, which directly translates to lower LODs and LOQs, enabling more sensitive detection and accurate quantification of HAAs at lower concentrations.
Given the different guidelines (for example, those of the USEPA and World Health Organization [WHO}), how would you ensure the chromatographic method meets regulatory requirements for HAA monitoring?
To ensure a chromatographic method meets regulatory requirements for HAA monitoring, such as those from the USEPA and WHO, we would focus on three critical aspects, chromatographic selectivity, sensitivity, and validation. The method must demonstrate sufficient selectivity to distinguish and resolve all nine HAAs (HAA9) as regulatory limits often apply to individual HAA compounds rather than total concentrations. The method's LODs must be consistently below the maximum contamination levels (MCLs) set by regulatory bodies. This includes the USEPA's limit of 60 µg/L for HAA5, chloroacetic acid (CAA), dichloroacetic acid (DCA), trichloroacetic acid (TCA), bromoacetic acid (BAA), and dibromoacetic acid (DBAA), in drinking water, as well as being capable of detecting HAAs below WHO's suggested guideline levels for specific HAAs (e.g., CAA < 20 µg/L, DCAA < 50 µg/L, and TCAA < 200 µg/L). The entire method must undergo comprehensive validation following established protocols recognized by regulatory agencies. This includes a thorough assessment of linearity, precision, accuracy, LODs, and LOQs, using standardized procedures to ensure its acceptance and reliability for regulatory compliance.
What are your next steps concerning this research?
The next phase of this research focuses on three main areas: (1) deploying the capLC–ESI-MS platform in water treatment plants and distribution systems for at-the-spot HAAs monitoring; (2) expanding the method to include other environmental contaminants building on our current work with HAAs and per- and polyfluoroalkyl substances (PFAS); and (3) developing automated, portable sample preparation protocols to enhance sensitivity and streamline field operation. These potential advancements aim to extend the system’s utility for decentralized, high-impact environmental monitoring.
References
- Mikhail, I. E.; Lam, S. C.; Coates, L. J. et al. Determination of Haloacetic Acids in Municipal Tap Water and Swimming Pool Water Using Portable Capillary Liquid Chromatography-Mass Spectrometry. J. Chromatogr. A 2025, 1751, 465941 DOI: 10.1016/j.chroma.2025.465941
- Yeh, R. Y.; Farré, M. J.; Stalter, D. et al. Bioanalytical and Chemical Evaluation of Disinfection By-Products in Swimming Pool Water. Water Res. 2014, 59, 172-184. DOI: 10.1016/j.watres.2014.04.002
