Integrated LC-MS/MS Workflow for Simultaneous PFAS and Cyanotoxin Analysis: Evaluating Mobile Phase Acid Effects to Improve Accuracy and Robustness

Significance of the topic

The combined analysis of per- and polyfluoroalkyl substances (PFAS) and cyanotoxins addresses two priority classes of drinking-water contaminants regulated or monitored by the U.S. EPA. PFAS originate from industrial and consumer product sources and include short- and long-chain analytes with differing chromatographic and ionization behavior. Cyanotoxins arise from harmful algal blooms and include diverse peptides and small alkaloids. Integrating these analyses on a single LC–MS/MS platform can reduce instrument demand, improve laboratory throughput, and provide a practical workflow for routine monitoring if method switching and cross-contamination are controlled.

Objectives and overview of the study

This work evaluated an integrated LC–MS/MS workflow that runs EPA Methods 533 (PFAS), 544 and 545 (cyanotoxins) on a single Shimadzu LCMS-8060RX system with automatic method switching. Key goals were to demonstrate analytical performance across methods, assess interchange-related contamination risks, test a short automated rinse for method transitions, and investigate the influence of mobile phase acid (formic vs acetic acid) on PFAS signal integrity and robustness.

Methodology and instrumentation

The study combined chromatographic and MS conditions tailored to each EPA method while using one triple-quadrupole MS with programmable flow-path switching. Principal instrumentation and LC conditions documented in the study included:

• LC–MS system: Shimadzu LCMS-8060RX with automated method-switching capability.
• Analytical columns: Shim-pack GIST C18 (3 μm, 2.1 × 50 mm), Shim-pack GIST C18 (2 μm, 2.1 × 100 mm), and Shim-pack Velox SPC18 (2.7 μm, 2.1 × 100 mm) employed depending on method.
• Delay column for PFAS: Shim-pack GIST C18 (5 μm, 3.0 × 50 mm) used in the PFAS flow path to reduce background PFAS contamination; bypassed for cyanotoxin methods.
• Injection volumes and oven temperatures: method-specific (examples reported: 2 μL to 20 μL; oven temps around 40–45 °C).
• Mobile phases: EPA 533 used 5 mM ammonium acetate in LC–MS grade water (A) and methanol (B); EPA 544/545 used 0.2% acetic acid in water (A) and 0.2% acetic acid in methanol or acetonitrile (B).
• Flow rates and run times: PFAS method (0.25 mL/min, 18 min); cyanotoxin methods (0.3 mL/min, ~8 min).
• MS ionization: ESI negative for PFAS; ESI positive for cyanotoxins.
• Batch design: a 294-injection sequence over 54 hours including nulls, solvent blanks, rinses, and standards to evaluate robustness and carryover across method switching.

Main results and discussion

Performance and calibration:

• Calibration for all targeted analytes followed the respective EPA methods and produced linear responses with R² > 0.99 across the calibrated ranges.
• Accuracy of injections remained in the 80–120% range and precision (%RSD) was <15% across LLOQ, mid, and HLOQ levels in continuing calibration checks.

Method switching and rinsing:

• Automatic method switching with a programmed five-minute rinse between PFAS and cyanotoxin methods effectively controlled cross-contamination; continuing calibration checks after triplicate injections showed analyte recoveries within acceptance criteria.
• Batch testing (294 injections over 54 hours) confirmed stable performance and reliable transitions when the rinse protocol was applied.

Mobile-phase acid effects:

• Comparative experiments showed that prolonged exposure to formic acid (>30 hours) or a single overnight contamination event led to substantial ion suppression for several PFAS (example chromatograms reported for PFBA and PFNA at 5 ng/mL).
• Acetic acid did not produce observable ion suppression of PFAS regardless of exposure time, even when formic acid passed only through LC lines (without contacting the column) produced signal loss of FOSE-class compounds in separate tests (EPA Method 1633 context).
• The data support replacing formic acid with acetic acid in cyanotoxin mobile phases when PFAS analysis follows cyanotoxin runs, reducing the risk of PFAS signal suppression caused by residual formic acid contamination.

Benefits and practical applications of the method

• Consolidation of PFAS and cyanotoxin analyses on a single triple-quadrupole MS reduces capital and maintenance costs by minimizing redundant instrumentation.
• Automated method switching and a short, five-minute rinse minimize manual handling, reduce downtime between methods and enhance sample throughput for routine monitoring laboratories.
• Using a delay column in the PFAS flow path effectively lowers laboratory background levels of PFAS, improving method sensitivity and reliability for low-level targets.
• Selecting acetic acid for cyanotoxin mobile phases when integrated with PFAS analysis provides a practical mitigation against ion-suppression artifacts that would otherwise compromise PFAS quantitation.

Future trends and applications

Potential developments and broader applications stemming from this work include:

• Expanded integration of additional targeted methods (e.g., other emerging contaminants) on single platforms using programmable flow-paths and automated rinsing.
• Systematic evaluation of alternative buffers, organic modifiers, and detergent-free cleaning agents to further reduce cross-method contamination and extend column life.
• Automation of conditional rinse and cleaning protocols based on real-time QC metrics or method sequence to optimize runtime and reduce unnecessary rinses.
• Material-compatibility and surface-adsorption studies for tubing, valves, and fittings to identify components that minimize PFAS carryover.
• Broader interlaboratory validation and robustness testing across instrument makes and column chemistries to confirm transferability of the acetic-vs-formic acid observation for PFAS classes including FOSE and FOS-related compounds.

Conclusion

The study demonstrates that an integrated LC–MS/MS workflow running EPA Methods 533, 544 and 545 on a single Shimadzu LCMS-8060RX is feasible, robust, and throughput-efficient when automatic method switching and a short rinse protocol are employed. Calibration and QC performance met method acceptance criteria across hundreds of injections. Crucially, replacing formic acid with acetic acid in cyanotoxin mobile phases prevents PFAS ion suppression observed with formic acid exposure, improving accuracy when PFAS analyses are performed on a shared system. Overall, this approach offers a practical path to consolidate routine monitoring of PFAS and cyanotoxins while maintaining analytical integrity.

Instrumentatio n used

Key instruments and consumables reported:

• Shimadzu LCMS-8060RX triple-quadrupole mass spectrometer with method-switching plumbing.
• Shim-pack GIST C18 analytical columns (various dimensions and particle sizes) and Shim-pack Velox SPC18 column.
• Shim-pack GIST C18 delay column (3.0 × 50 mm, 5 μm) for PFAS background removal.

Reference

Selected standards and method documents referenced in the work:

• U.S. Environmental Protection Agency, Method 533, Determination of PFAS in drinking water.
• U.S. Environmental Protection Agency, Method 544, Determination of cyanotoxins in drinking water.
• U.S. Environmental Protection Agency, Method 545, Determination of cyanotoxins in drinking water.
• U.S. Environmental Protection Agency, Method 1633, PFAS in solids and related analyses (discussed with respect to FOSE compounds).
• Shimadzu Scientific Instruments, LCMS-8060RX system and Shim-pack column product lines (manufacturer application data as reported by the authors).

Note: Study authors and funding are affiliated with Shimadzu Corporation. The work is presented for research use and is not intended for diagnostic procedures.