Simple Determination of Haloacetic Acids (HAA) by IC and MS

Simple Determination of Haloacetic Acids (HAAs) with Ion Chromatography and Mass Spectrometry

Introduction

Haloacetic acids (HAAs) typically emerge as disinfection by-products in water treatment processes. Some HAAs are monitored under regulatory frameworks and classified as potential carcinogens. Traditionally, HAAs are analyzed using gas chromatography (GC), a technique requiring time-consuming sample extraction and derivatization, resulting in higher cost per analysis. Mass spectrometry (for example, single or triple quadrupole MS systems) combined with Ion Chromatography (IC) is a powerful tool capable of performing many challenging analytical tasks, such as detecting HAAs at μg/L levels in drinking water samples. After compounds in a sample are separated via IC, mass selective detection guarantees identification and quantitation of analytes, including low concentration limits. By incorporating automated Metrohm Inline Sample Preparation (MISP) techniques into IC systems, various sample types can be easily analyzed without the need for extensive and manual laboratory work. Automation of both sample preparation steps and analysis enables obtaining more reliable and reproducible data. This research article aims to explain the intricacies of IC-MS technique and the benefits of this hybrid method used for accurate and precise measurement of HAAs in water samples.

Overview of IC Combined with MS Technique

IC typically overcomes challenging separation problems by using interactions between ionic species in liquid samples and a fixed separation column, with conductivity detection. Mass detection performed with a mass spectrometer (for example, MS or MS/MS) as a secondary independent detector for IC records the mass-to-charge ratios (m/z) of selected relevant analytes. Thus, mass detection ensures obtaining results with certainty by identifying compounds present in challenging matrices. Thanks to IC-MS technique, quantitation of simultaneously separated compounds can also be performed and detection limits can be significantly improved. High-performance liquid chromatography mass spectrometry (HPLC-MS) is a well-established analytical technique. However, separation of ionic compounds using this conventional HPLC-MS technique is difficult. When the IC-MS combination is used, this difficulty can be easily overcome. For these analytes, IC-MS technique can be applied as a successful method with the help of special columns optimized for ion separation. Suppressor units within the IC system minimize the ion load from the eluent to optimize signal-to-noise ratios for both conductivity and MS detection. Sequential suppressor systems (chemical and carbon dioxide suppression) are used in Metrohm IC systems, positioned after the separation column and before the detector, with unconditional 10-year warranty offered especially for anion chemical suppressor units. IC-MS is a robust and easy-to-use technique for determination of analytes such as inorganic anions, organic acids, HAAs, oxyhalides, alkali and alkaline earth metals. By adding Metrohm Inline Sample Preparation (MISP) techniques to the system configuration, it becomes possible to easily analyze not only water samples, but also chemicals, organic solvents, or post-blast residues.  

Mass Detection for Small Ionic Compounds

While many analytes can be detected with IC through conductivity technique, other common detectors such as UV/VIS or amperometric detection can also be used. Analyte identification is performed by comparing the retention times of peaks detected in the chromatogram with peaks in prepared standard solutions. For multicomponent samples or samples with extreme concentration differences between the analyte and sample matrix, this comparison can be difficult. A mass detector connected in series with a conductivity detector matches the conductivity signal with the corresponding mass-to-charge signal, ensuring low detection limits and accurate peak identification. Combining IC with a mass detector significantly expands the application areas for this hybrid system. Molecular ions are typically analyzed using single quadrupole mass spectrometers (Figure 1). Higher-level mass spectrometers such as triple quadrupole have greater sensitivity and can elucidate structural details through fragmentation studies. [caption id="attachment_145334" align="aligncenter"] Figure 1. Schematic representation of the working principle of single quadrupole MS.[/caption]

Combination of IC and MS Equipment

Metrohm IC systems can work together with different MS devices (Figure 2) from any supplier. Synchronization of both devices is performed using a remote box and appropriate cable connections, so that after sample injection, the MS detector is notified to begin recording through the connection box. Regarding hardware connection, a simple capillary tube connection between the conductivity detector and the MS detector is sufficient. The conductivity detector is a non-destructive detector with low dead volume (>1 μL) and the outlet capillary tube (a PEEK capillary with 0.25 mm internal diameter) is typically connected to the MS injection valve or directly to the ion source. [caption id="attachment_145335" align="aligncenter"] Figure 2. Synchronization of Metrohm IC with an MS device from any supplier is possible.[/caption] Additional components that can be added to the system allow for improved applications. A splitter can optimize the sample flow rate reaching the MS for an ideal signal-to-noise ratio. A switch valve can also be used to direct flow to the MS only when elution of the analytes of interest is expected. To minimize MS contamination, matrix peaks can be separated, system conditioning, and automatic sample preparation operations can be directed to waste flow. Some applications may require post-column derivatization (PCR) with the addition of reagents (for example, organic solvent) before MS analysis, or may require an additional suppressor module to adjust pH or remove matrix ions to prevent signal suppression.  

General Approach for All MS Types

If there is no common software to run the systems, both devices are operated through their own software. In such cases, sample tables must be copied to both software applications and data evaluation is typically performed in the respective device software (for example, processing of conductivity signals in IC software and m/z signals in MS software). This approach is generally used for high-level MS analysis with specialized MS software. For example, while Metrohm IC systems are operated with MagIC Net software, the MS detector can be controlled by software from manufacturers such as Agilent (MassHunter™), Waters (MassLynx™), or AB Sciex (Analyst®). These flexible and high-performance configurations can reveal their true potential in development laboratories and research institutions. On the other hand, operating these configurations requires trained personnel. Otherwise, for IC-MS configurations controlled by two independent software applications, working under full compatibility can be difficult for analysts.  

Metrohm IC Drivers for Single Common Software

Native IC drivers enable IC systems, including high-precision burettes such as auto-samplers and Metrohm Dosino technology, to be controlled and operated with MS detectors under the same software. Single software for IC and MS systems facilitates user-friendly applications and robust operation. Since progression is through a single software platform, training processes become faster and easier, while data integrity and full compatibility are ensured with these drivers. Metrohm can provide integrated solutions through IC drivers for Waters Empower™ 3 and Agilent OpenLab CDS software.  

US EPA 557 Compliant IC-MS Detection in Water Samples

Analysis of Haloacetic Acids with Technique Chlorine in various forms is used as the primary disinfection method in municipal water systems and can produce disinfection by-products, including HAAs, in trace amounts (at μg/L levels). While definitive evidence has not yet been established, continuous exposure to high concentrations of these HAAs is thought to carry potential cancer risk. Various global organizations, including the U.S. Environmental Protection Agency (EPA), have defined a threshold value for maximum HAA content in municipal water supplies as a health precaution. The European Parliament recently revised the "Drinking Water Directive" by defining the total limit of nine HAAs as 60 μg/L. For this reason, interest in measuring HAA concentrations in trace amounts in municipal water supply sources has increased significantly.  

Achieve Lowest Detection Limits with IC and MS

To address the need to measure various HAAs at μg/L levels in municipal water supply samples, a robust method was developed using IC-MS/MS detection technique compliant with US EPA Method 557. Within this method, the greatest advantage of using MS/MS with a triple quadrupole mass spectrometer compared to using MS with a single quadrupole mass spectrometer is the ability to prevent potential interferences while achieving lower detection limits and improved selectivity. Other analytical methods require complex and labor-intensive sample derivatization procedures to enable detection of analytes. In the developed IC-MS/MS method, internal standards are added to water samples in known amounts and samples are analyzed directly without requiring any derivatization, cleaning, or pre-concentration steps. This method saves significant time per sample, improves analytical efficiency, and reduces operational costs. Another advantage of using a Metrohm IC system for this application is that direct addition of organic solvent to the eluent not only increases dissolution of analytes at the electrospray ionization (ESI) interface but also improves chromatographic separation. Within the method followed in this study, a Metrohm IC connected to an Agilent 6470 triple quadrupole MS was used to analyze nine HAAs, dalapon, and bromate ions via the IC-MS/MS system (Table 1). These compounds were separated on a Metrosep A Supp 7 - 250/4.0 separation column using a dual-channel gradient system. The analytes examined within the method and technical parameters are shown in Table 2. Following suppression, analytes are transferred to the MS and selected MRM (multiple reaction monitoring) transitions are used for quantitation (Figure 3). [caption id="attachment_145336" align="aligncenter"] Table 1. IC-MS system components[/caption] [caption id="attachment_145337" align="aligncenter"] Table 2. Retention times and MS/MS acquisition parameters for nine HAAs, dalapon, and bromate ions.[/caption] [caption id="attachment_145338" align="aligncenter"] Figure 3. Analytes in mixture, each at 50 μg/L, and internal standards at 100 μg/L, separated using Metrosep A Supp 7 - 250/4.0 column. Analytes are shown in black, internal standards in blue.[/caption] Calibration performed for this study covered a concentration range of 1–500 μg/L with linear regression. MagIC Net was used as chromatographic software and Agilent Mass Hunter™ software was used to operate the MS. Reagents used in the analyses are listed in Table 3, while IC and MS parameters are listed in Tables 4-1 and 4-2.  

IC Reagents

[caption id="attachment_145339" align="alignleft"] Table 3. Reagents used for HAA analyses.[/caption]        

IC Parameters

[caption id="attachment_145340" align="alignleft"] Table 4-1. IC system parameters[/caption]      

MS/MS (Triple Quadrupole) Parameters

[caption id="attachment_145341" align="alignleft"] Table 4-2. MS system parameters[/caption]             An additional Dosino unit is used to set and optimize the MS, providing a constant analyte solution flow (0.2 mg/L) at 0.5 mL/min directly to the ESI source. Thus, MS source settings are automatically optimized for each analyte without chromatographic separation. During the first 7 minutes following injection and between 27 - 28.5 minutes, eluent flow is directed to waste. This diversion prevents detection of non-retained or off-target compounds and also minimizes contamination of the MS source, reducing the need for frequent cleaning. The performance of the developed method was demonstrated by adding known amounts of analytes to water samples and measuring their recoveries (Table 5). [caption id="attachment_145342" align="aligncenter"] Table 5. Results of five replicate analyses of municipal water samples with 25 μg/L of each analyte added.[/caption] In addition to these studies, municipal water samples from the Tampa Bay region (Florida, USA) were also analyzed for HAA content. These samples contained total HAAs in amounts less than 60 μg/L, the critical limit according to US EPA guidelines (Table 6). [caption id="attachment_145343" align="aligncenter"] Table 6. HAA results in two different municipal water samples.[/caption] Analysis of HAAs in Water Samples with IC and Different Brand MS Detectors The same application study was also performed with a Metrohm IC and AB Sciex QTRAP 6500+ combination. Chromatographic separation was provided under the same conditions as the previous application (Figure 3) and MS detection analysis parameters were adapted for this device. Bromate, dalapon, and ten HAA compounds were detected in the range of 0.2 μg/L and, depending on the relevant analyte, even 0.02 μg/L - 200 μg/L. With this configuration, chromatographic separation was performed using AB Sciex Analyst® software (Figure 4). Monofluoroacetic acid (MFA), difluoroacetic acid (DFA), monoiodoacetic acid (MIA), and trifluoroacetic acid (TFA) were also additionally investigated in water samples, but their amounts could not be determined. Spike tests on tap water (Figure 5) and bottled water (Figure 6) samples proved the accuracy of this method and its robustness against matrix effects. [caption id="attachment_145344" align="aligncenter"] Figure 4. Ten HAA compounds (including TFA and TBA), bromate and dalapon ions (200 μg/L of each analyte, 100 μL injection volume), separated using Metrosep A Supp 7 - 250/4.0 column.[/caption] [caption id="attachment_145345" align="aligncenter"] Figure 5. Sample chromatograms for TCA (A) and BDCA (B) spiked at 2 μg/L in tap water samples.[/caption] [caption id="attachment_145346" align="aligncenter"] Figure 6. Sample chromatograms for tribromo acetic acid (TBA) spiked at 2 μg/L in bottled water samples.[/caption] As a result of the applications, it was demonstrated that the Metrohm IC system can be connected to MS detectors from any supplier, enabling sensitive and accurate analysis in different water samples.

IC and Single Quadrupole MS Detector Combination: Robust and Easy to Use

The current ISO standard (ISO 23631:2006) recommends the use of GC-MS technique to measure six haloacetic acids in water samples. A soon-to-be released drinking water directive draft suggests that nine HAAs can be detected by other analytical techniques (for example, IC-MS or IC-MS/MS) such that individual concentrations do not exceed 5 μg/L and total concentration does not exceed 60 μg/L. In an inter-laboratory study conducted for method development purposes, it was demonstrated that the combination of Metrohm IC with a single quadrupole MS (Waters SQ Detector 2) is a suitable system configuration for HAA detection in water samples (Table 7). The ability to control the entire system with Empower™ 3 software makes the application compatible and easy to use for operators at all levels. Nine HAAs detected in different water matrices were analyzed within 35 minutes using Metrosep A Supp 5 - 250/2.0 column and Metrosep A Supp 10/4.0 guard column. A 2 mm microbore separation column is particularly suitable for MS detection due to its lower required flow rates. The ideal eluent flow rate for this separation column is 0.2 mL/min, which is excellent for MS detection. The additional guard column also improves the separation between MCA, MBA, and chloride. Furthermore, reducing the column oven temperature to 10°C further enhances the separation between analytes. Following suppression, conductivity signals and selected ion registrations were collected. Corresponding 13C standards were used to avoid matrix effects and ensure accurate quantitation. With this configuration, it is possible to detect HAAs at concentrations of 5 μg/L or less in all tested sample matrices (Figures 7 and 8). [caption id="attachment_145347" align="aligncenter"] Table 7. Calculated limit of quantitation (LOQ) for HAAs in ultrapure water (S/N = 10 extrapolation).[/caption] [caption id="attachment_145348" align="aligncenter"] Figure 7. Sample chromatograms in drinking water sample for MCA ion (m/z 93; spike concentrations 5, 10, 20, 30, 50, and 100 μg/L) detected with Metrosep A Supp 5 - 250/2.0 separation column.[/caption] A similar approach was also carried out using an Agilent single quadrupole MS detector. In this case, LCMRL (lowest concentration minimum reporting level) values below 0.4 μg/L were achieved for six HAAs. During the period this work was conducted, two different software applications were used, one for chromatography and one for MS. Today, however, a single software solution based on Agilent OpenLab CDS for Metrohm IC and MS (along with Metrohm IC driver) ensures ease of use and full traceability for instruments.  

General Conclusion

Ion chromatography is a powerful analytical tool particularly suitable for use in laboratories with challenging sample matrices containing multiple analytes at various concentrations. For this reason, the unique combination of IC with MS for applications involving ionic and polar molecules is becoming increasingly popular. Determination of HAAs in drinking water samples is an important parameter for securing water sources and protecting public health in this context. When it comes to analyzing HAAs at low concentrations, Metrohm IC is the ideal analytical solution for separating these compounds. Thanks to the ability to be combined with any MS detector, various separation column options, and many automated Metrohm Inline Sample Preparation (MISP) techniques, trace-level analysis has never been easier with our robust ion chromatography systems.   References 1. Metrohm White Paper WP-066EN 4868833 4886948 "An introduction to ion chromatography mass spectrometry (IC-MS)" 2. Metrohm White Paper WP-075EN 5034328 5034328 "Simple determination of haloacetic acids (HAAs) in potable water with ion chromatography hyphenated to mass spectrometry" 3. Metrohm Application Work AWUS6-0255-052019 "Trace Level Haloacetic Acid (HAA), Dalapon, and Bromate Analysis in Water Using a Metrohm Ion Chromatograph and an Agilent Triple Quadrupole Mass Spectrometer (IC-MS/MS) Conforming to USEPA Method 557" 4. Metrohm Application Note AN-M-15 "Trace haloacetic acids, dalapon, and bromate in water" 5. Metrohm Application Note AN-M-16 "Resolving haloacetic acids in water" 6. Triple Quadrupole Mass Spectrometer (IC-MS/MS) Conforming to USEPA Method 557"   Elif Metin Kulaksız Senior Ion Chromatography Application Specialist Metrohm Turkey