Addressing challenges when implementing PFAS LC MS analysis methods for water samples

Introduction to PASAs and LCMS Analysis

Overview of the Webinar

• The webinar focuses on challenges in implementing PASA's LCMS analysis methods for water samples.

• Matt James, a senior research scientist at Avantor Sciences, presents his insights and experiences.

Background of the Presenter

• Matt has a PhD from the University of Bristol and extensive experience in pharmaceutical applications and method development.

• His recent work includes HPLC and UHPLC method transfer, solid core phases, and analyzing nitramines by LCMSMS.

Understanding PASAs

Definition and Classification

• PASAs (Per- and Polyfluoroalkyl Substances) are synthetic fluorinated chemicals used since the 1950s, classified as polymeric or non-polymeric. Focus is on non-polymeric types like PFOA and PFOS.

• The OECD defines PASAs broadly; any chemical with a perfluorinated methyl or methylene group qualifies as a PASA. This definition impacts regulatory measures significantly.

Environmental Presence

• Over 4,500 known or likely PASAs have been identified in recent years; databases show over 12,000 compounds in the US alone. This indicates a vast number of potential target analytes for analysis.

• Their widespread use leads to environmental persistence due to strong carbon-fluorine bonds that resist degradation, raising concerns about bioaccumulation and toxicity linked to long-term exposure.

Health Concerns Related to PASAs

Toxicity Issues

• Many studied PASAs are moderately to highly toxic; long-term exposure is associated with various health issues costing Europe over €50 billion annually in health-related expenses.

Regulatory Landscape

• Regulations dictate sensitivity requirements for analytical methods; drinking water analysis must detect levels down to parts per trillion (ppt). Current US regulations set limits for specific compounds at four ppt with goals for zero contamination levels.

Analytical Approaches for PASA Detection

Methodologies Employed

• Total fluorine measurements provide rapid but non-specific results; mass spectrometry coupled with liquid chromatography offers high specificity needed for low-level detection of individual components.

Techniques Discussed:

1. Targeted Mass Spectrometry: Utilizes triple quadrupole instruments focusing on known compounds but requires available analytical standards which can be limited.

2. High Resolution Mass Spectrometry: Allows broader compound identification but is more common in research settings.

3. GC-MS: Useful for volatile compounds but less sensitive than LC-MS techniques.

Setting Up LCMS Methods for Analysis

Key Considerations

• Triple quadrupole mass spectrometers using electrospray ionization (negative mode) allow quantitative analysis with high specificity down to parts per trillion levels through solid phase extraction (SPE).

Sample Preparation Process:

1. Load water sample onto SPE cartridge.

2. Elute with organic solvent.

3. Concentrate sample before injection into LCMS system.

Developing Effective LC Methods

Chemical Properties Impacting Analysis

• Understanding physicochemical properties such as pKa values helps define analytical procedures; many PASAs exist in negatively charged forms under practical mobile phase conditions aiding detection efficiency.

Structural Diversity Challenges:

1. Short-chain vs long-chain differences affect hydrophobicity requiring gradient modes during reverse-phase liquid chromatography.

2. Sticky nature of longer chain compounds necessitates careful handling during sample preparation.

Addressing Background Contamination

Managing System Contamination

• Ubiquitous nature of PASAs means solvents may contain trace amounts leading to background peaks interfering with quantification.

Solutions Proposed:

1. Replace certain system components with non-PASA containing equivalents.

2. Use delay columns specifically designed to trap background contaminants before sample injection ensuring accurate quantitation.

Injection Volume Considerations

Optimizing Sensitivity

• Increasing injection volume can enhance sensitivity but risks distorting peak shapes due to high organic solvent percentages required in samples.

Recommendations:

1 . Maintain optimal injection volumes while considering solvent composition carefully.

2 . Ensure calibration standards reach room temperature before use.

This structured summary provides an organized overview of key points discussed throughout the webinar regarding the challenges faced when implementing LCMS methods for analyzing PASAs in water samples while highlighting critical insights into methodology development, regulatory implications, health concerns related to these substances, and effective strategies employed within laboratory settings.

EPA Method 533 for Drinking Water Analysis

Overview of Methodology

• The method focuses on achieving low parts per trillion (ppt) levels in drinking water analysis using a 10 cm C18 column and ammonium acetate with methanol as the organic modifier.

• A sample diluent of methanol and water (80:20) is used, allowing for a 5 microliter injection volume.

Standard Solutions and Preconcentration

• Standard solutions range from 100 to 2500 nanograms per liter, with EPA Method 533 incorporating a 250-fold sample preconcentration step. This results in actual concentrations of about 4 to 10 nanograms per liter in samples.

• The method has a long gradient program lasting approximately 25 minutes, leading to total cycle times of around 35 to 40 minutes due to necessary column re-equilibration.

Optimizing Analysis Time

Flexibility in Chromatography

• EPA methods allow flexibility; by increasing flow rates and simplifying gradients, analysis time can be reduced significantly—down to about 17 minutes after re-equilibration.

• The focus is shifting towards analyzing a broader range of perfluoroalkyl substances (PFAS), including shorter chain compounds that are challenging to retain using reverse phase chromatography.

Adjustments for Sample Dilution

• A higher concentration of methanol (96%) is used in the sample diluent, reducing injection volumes slightly to avoid peak shape issues while maintaining effective separation within approximately ten minutes using acetonitrile as an organic modifier.

Mass Spectrometry Optimization

Tuning Parameters for Sensitivity

• Mass spectrometry source parameters should be optimized specifically for each application rather than relying solely on generic application notes, especially when targeting low sensitivity levels.

Method Validation Examples

Achieving Regulatory Standards

• An example shows the separation of PFAS compounds under EPA Method 5371 with tight criteria for peak shapes; lower injection volumes help meet regulatory requirements effectively down to four parts per trillion concentrations.

Rapid Screening Techniques

Quick Analysis Methods

• A rapid analysis method utilizing a short column allows screening water samples quickly (in just over one minute) while still retaining good peak shapes despite high injection volumes due to the use of a diluted sample matrix (50/50 methanol-water).

Controlling Background Contamination

Sources of PFAS Contamination

• Background contamination from lab consumables such as vials, solvents, reagents, and even clothing can significantly affect analytical results; identifying these sources is crucial for accurate analysis.

Recommendations for Reducing Contamination:

• Avoid PTFE materials and fluoropolymers in workflows.

• Use dedicated glassware made from polypropylene instead of communal equipment.

• Regularly test new batches of consumables and solvents before use to ensure they are free from detectable PFAS contaminants.

Testing Lab Materials

Procedures for Identifying Contaminants

• Testing involves placing materials into clean centrifuge tubes with solvent and vortexing them before analyzing via LC-MS; this helps identify potential sources like sticky tape or latex gloves that may contain detectable PFAS levels.

Notable Findings:

• Common lab items such as tissues made from recycled paper were found to contain extractable PFAS compounds due to varying content within the recycled material itself.

Sample Preparation Challenges

Importance of Clean Sample Prep Equipment

• Sample preparation steps pose significant risks for contamination; high-quality filters should be used without PTFE components, which have been shown to introduce background contamination during analyses.

Blowdown Step Issues:

• Nitrogen blowdown equipment was identified as a source of consistent contamination across various setups; switching nitrogen sources helped mitigate this issue effectively without compromising sample integrity during analysis procedures.

Conclusion on Analytical Practices

Summary Insights

• Achieving ppt level sensitivity requires careful consideration of analyte properties when developing LC-MS methods; employing appropriate protocols ensures compliance with regulatory standards while minimizing contamination risks throughout the analytical workflow.

Key Considerations for PASAS Analysis in LCMS

Importance of Delay Columns

• The use of a delay column is essential for any form of PASAS (Passive Sampling and Analysis System) analysis by LCMS (Liquid Chromatography-Mass Spectrometry).

Addressing Sample Contamination

• Discussed the serious concern regarding potential contamination of samples during background laboratory PASAS.

• Suggested several key approaches to mitigate contamination risks, including:

• Eliminating past-containing materials.

• Selecting appropriate consumables.

• Continual testing of consumables.

• Maintaining good work practices.

• Using high-grade solvents and reagents.

Available Resources

• Mentioned resources available for further assistance, including:

• A workflow solutions guide for PASAS analysis accessible via a QR code.

• Avantor's Knowledge Zone with technical resources like knowledge notes and white papers.

• A chromatography application library containing application notes discussed during the presentation.

Q&A Session Insights

Questions on Ammonium Acetate Usage

• Responded to a question about not using ammonium acetate in the mobile phase B line, explaining that it’s typically suggested to maintain ionic strength by using the same buffer in both lines.

• Clarified that following EPA methods led to using pure organic solvents without issues; however, ammonium acetate could be added if retention drift occurs.

Acetic Acid Application

• Addressed inquiries about adding acetic acid to samples or mobile phases. Notably:

• It can neutralize ammonium hydroxide from certain EPA methods (e.g., EPA 1633).

• Adding acetic acid may increase retention but could also affect later eluting compounds due to pKa considerations.

System Maintenance Recommendations

Cleaning Protocol Before Analysis

• Emphasized the importance of cleaning the system daily before starting any PASAS LCMS analysis. Recommended steps include:

• Flushing the entire system with organic solvent to remove accumulated past residues.

• Directing waste initially away from the mass spectrometer and then into it for final cleaning.

Daily Maintenance Practices

• Suggested cleaning the mass effect source daily according to manufacturer guidelines as an additional precautionary measure before beginning analyses.