EPA Method 1633, finalized in 2024, analyzes 40 per- and polyfluoroalkyl substances (PFAS) across multiple matrices using LC–MS/MS. The method is performance-based, allowing labs to adjust extraction and sample preparation steps if they meet quality control criteria. In water testing, many workflows concentrate samples 100-fold to achieve parts-per-trillion detection, but that process also amplifies background contamination and procedural drift, leading to quantitation problems.

This article identifies the three steps that most affect PFAS quantitation in EPA 1633 water testing and the practices labs use to control them.

PFAS Contamination Sources That Raise Blanks

EPA Method 1633 analyzes PFAS through solid-phase extraction (SPE) followed by LC–MS/MS. Samples are fortified with isotopically labeled internal standards before extraction. Quantitation uses isotope dilution to track recovery and correct for matrix suppression.

Any part of this process—from solvents and samplers to sample-contacting surfaces—can introduce PFAS contamination. EPA Method 1633 requires field, method, and equipment blanks to isolate contamination sources.

Water testing labs rely on several practical steps to reduce system contamination:

• Avoid polytetrafluoroethylene (PTFE) in tubing, valve seals, caps, and vials. PEEK, polypropylene, or stainless steel alternatives reduce background contamination.
• Install a delay column between the LC pump and autosampler to divert system-generated PFAS to waste before sample introduction, lowering background interference.
• Use LC–MS grade solvents screened for PFAS to maintain quantitation at ng/L levels.

These material choices reduce blank interference, but sample preparation still introduces its own variability.

Solid-Phase Extraction Steps That Influence PFAS Recovery

SPE is where most PFAS recovery issues begin in water samples. Short-chain PFAS interact weakly with the sorbent and shift when loading rates, wash strength, or concentration steps change. Stable extraction depends on repeatable execution.

Specific handling points shape whether extraction stays consistent:

• Cartridge conditioning: Weak anion-exchange (WAX) sorbents are the preferred choice for PFAS extraction because they retain anionic compounds across varying chain lengths. However, WAX cartridges perform best with proper conditioning: alkaline methanol primes the sorbent surface, and alkaline elution releases both short- and long-chain compounds in a single fraction without leaving residues.
• Sample loading: Moderate, consistent loading is essential for preserving contact time between sample and sorbent. Rapid loading moves polar PFAS off the cartridge early, introducing scatter.
• Washing and elution: Balancing wash and elution strength is critical for keeping all target compounds in the same fraction. However, the margin is narrow: wash conditions that are too strong strip short-chain PFAS prematurely, while elution that is too mild leaves long-chain PFAS bound to the sorbent.
• Concentration: Certain PFAS precursors are sensitive to evaporation conditions. Leaving a small residual solvent volume protects volatile species before LC–MS/MS analysis.

More SPE failures trace to technique variation than to cartridge choice.

LC–MS/MS Conditions That Support Stable Quantitation

LC–MS/MS exposes instabilities that upstream steps mask. Background peaks, retention shifts, and carryover effects emerge even when calibration files look clean. Each symptom traces to a specific LC system component.

Steps that maintain LC–MS/MS stability:

• Columns: Columns engineered to limit PFAS bleed keep early retention windows clean, reducing ghost peaks that overlap with short-chain PFAS.
• Mobile phases: PFAS-free solvents buffered with ammonium acetate maintain steady retention across runs. Fresh buffers and mobile-phase filtration reduce retention drift and ion suppression.
• Carryover control: PFAS can linger in the autosampler needle, valve, and transfer line, reappearing in subsequent injections. Needle rinses with strong polar organic solvents (methanol or acetonitrile) and waste diversion of early run segments clear residual PFAS before quantitation.
• Acquisition settings: Adequate dwell time on key MRM transitions preserves signal quality when PFAS co-elute in tight windows. Periodic tuning of transitions and source parameters maintains sensitivity across long sequences.

LC–MS/MS stability depends as much on upstream sample prep as on instrument tuning.

Conclusion

EPA 1633 PFAS failures often appear as instrument problems but originate in sample preparation. Labs that address materials, extraction discipline, and LC system stability avoid chasing false readings.

Download Merck's application note for the complete EPA 1633 workflow, including SPE and cleanup protocols with recovery data for all 40 PFAS in water.