B-265 Evaluation of LC-MS/MS Methodologies for 37 Per- and Poly-fluoroalkyl Substances (PFAS) in Serum
Authors: Cho K , Hur J , Cho S , Lee J , Kim G , Han J , Lee S 
Published: October 2024

Abstract

Background
PFOS and PFOA, extensively used in industrial products like firefighting foams, food packaging, textiles, and various industrial application over the past decades, have been regulated due to their strong persistence, bioaccumulative, and toxicity. In response, industries are developing more degradable and less bioaccumulative PFAS replacements, including shorter chain PFAS and fluoroether alternatives such as perfluorobutanoic acid (PFBA) and hexafluoropropylene oxide dimer acid (HFPO-DA, GenX). However, the health effect of these replacements remains unclear. Despite increasing interest in diagnosing their presence in serum sample, the large number of compound and physicochemical diversity make it difficult to establish appropriate methods, including sample preparation and LC-MS analytical parameter. In this study, we propose appropriate sample preparation procedure based on chemical properties by comparing several PFAS extraction method. Additionally, we demonstrate the significant impact of MS parameters during analysis.

Methods
Serum samples fortified with 37 native PFAS standards adjusted to 1 ppb, including regulated substances (e.g., PFOS and PFOA) and replacements were prepared. PFAS extraction from fortified serum samples was conducted using weak anion exchange (WAX) cartridge, enhanced matrix removal cartridge (EMR), and liquid-liquid extraction (LLE), Eluates were evaporated and reconstituted in the 50 % acetonitrile solution fortified with isotope-labeled PFAS standards. Signals of 37 of the PFAS compounds were compared while adjusting the curtain gas, ion source gas1, and ion source gas2 flow and source temperatures, respectively. LC-MS/MS analysis was performed using SCIEX 6500 (Sciex, Framingham, MA, USA) combined with ExionLC (Sciex, Framingham, MA, USA, flow rate: 0.3 mL/min).

Results
The number of carbons in PFAS compounds affected the extraction efficiency in both the LLE and EMR extraction procedure. It was observed that extraction efficiency decreased as the number of carbons increased in both methods. On the other hand, the extraction efficiency of the WAX extraction procedure was affected by the pKa value of acidic group rather than the number of carbons. Specifically, PFAS compounds with carboxylic acid residue exhibited lower extraction efficiency in WAX method compared to sulfonic acid PFAS compounds, and this efficiency was also lower than that of the LLE and EMR methods. In the comparison of MS parameters, it was found that PFAS compounds with phosphate ester structure (6:2diPAP and 8:2diPAP) exhibited a significant decrease in signal when the curtain gas flow was increased. Conversely, very long fluoroalkyl PFAS compounds (such as PFHxDA=C16, PFODA=C18) showed a higher signal at higher curtain gas flow, although partial in-source fragmentation occurred in this case. The ion-source gas and source cone flow did not significantly affect the PFAS signal.

Conclusions
As the usage of PFAS replacements increases, so does the need for their monitoring. This study underscores that physicochemical diversity of PFAS compounds can significantly affect PFAS monitoring, depending on the sample preparation and MS parameters. We believe that this study will aid in selecting appropriate pretreatment methods and parameters for analyzing specific types of PFAS.