Simultaneous Determination of Four Per- and Polyfluoroalkyl Substances (PFAS) in DHA Products using LC-MS/MS for Quality Control and Risk Assessment

BACKGROUND: Docosahexaenoic acid (DHA) is a critical dietary supplement for vulnerable populations (e.g., infants, pregnant women), but contamination by per- and polyfluoroalkyl substances (PFAS)-persistent "forever chemicals"-poses potential health risks. Currently, no standardized methods exist for PFAS detection in DHA products, hindering quality control and risk assessment. OBJECTIVE: To develop and validate a sensitive LC-MS/MS method for simultaneous determination of four PFAS (PFOA, PFODA, PFOS, and PFBS) in DHA matrices (algal oil and fish oil) and support product safety evaluation. METHODS: Samples were extracted with 50% methanol/acetonitrile (v/v) via ultrasonication (40 °C, 20 min), followed by centrifugation and filtration. Chromatographic separation was achieved on a Phenomenex Kinetex F5 column (100 × 3.0 mm, 2.6 μm) with gradient elution (mobile phase: 2 mmol/L ammonium formate aqueous solution-methanol) in 12 min. Detection was performed via negative electrospray ionization (ESI-) in multiple reaction monitoring (MRM) mode. Method validation included linearity, limits of detection (LODs)/quantitation (LOQs), accuracy, precision, matrix effects, and stability. Thirteen commercial DHA products (11 algal oil, 2 fish oil) were analyzed. RESULTS: Linearity (r ≥ 0.99) were achieved for PFAS; LODs/LOQs: 0.02/0.04 ng/mL. Recoveries at three spiking levels (0.1, 0.5, 1.0, 1.5 ng/mL) were ranged from 92.1%-108.4%, with RSDs of 2.8%-5.6%. Matrix effects were effectively corrected via matrix-matched calibration. Trace PFAS were detected in 3/13 samples, all below regulatory limits. CONCLUSIONS: The developed method is reliable for routine PFAS detection in DHA matrixes, providing practical technical support for quality control and risk assessment for DHA supplements for vulnerable populations. HIGHLIGHTS: Rapid (12 min), sensitive, and applicable to both algal oil and fish oil matrixes.