Shun-Hsin Liang & Mike Chang detail simultaneous analysis of ultrashort-chain, alternative and legacy PFAS
LC-MS/MS methods for the analysis of legacy short-chain(C4, C5) and long-chain (>C5) per- and polyfluoroalkyl substances (PFAS) based on reversed-phase (RP) chromatography are well established. With proper modification, these methods often can also be used for LC-MS/MS analysis of alternative PFAS, such as HFPO-DA (GenX) and ADONA, which are perfluoroalkyl ether carboxylic acids used as PFOA substitutes.
However, current LC methods may not be suitable for the analysis of newly trending ultrashort-chain(C2, C3) PFAS environmental contaminants, mainly due to their insufficient retention on typical RP columns.
Currently, methods that allow the desired combined analysis of ultrashort-chain PFAS with alternative and legacy PFAS are very rare.
To fill this gap, Restek has developed a procedure for the simultaneous quantification of a wide range of chain lengths and structures including C3, C4, C8, and alternative PFAS in a variety of water samples.
What were the experimental conditions?
Samples of tap water collected from the Restek facility and three diverse water types (Chicago river water, groundwater and publicly owned treatment works [POTW] effluent water) supplied by the U.S. EPA were included in this study.
Each water type was fortified at 10 (20 ppt for PFPrA) and 80 ppt in duplicate per batch with a total of three batches being prepared and analysed on different days. To prepare water samples, 250 µL aliquots were mixed with 250 µL of 40:60 reagent water:methanol and 5 µL of internal standard solution(5 ng/mL of 13C2-PFHxA, 13C2-PFOA, 13C4-PFOS in methanol) in polypropylene vials sealed with polyethylene caps.
Calibration standards were prepared by fortifying reagent water (Optima LC-MS water) with ten PFAS analytes across a range of 5–400 ng/L. The calibration standard solutions were then processed following the sample preparation procedure detailed above.
How the PFAS analysis was conducted
LC-MS/MS analysis was performed using a Raptor C18 analytical column (2.7 µm; 100 mm x 3.0 mm) and a Shimadzu Nexera X2 HPLC coupled to a Sciex 4500 MS/MS. A PFAS delay column (cat.# 27854) was installed between the pump mixer and the injector in order to prevent coelution of any instrument-related PFAS with target analytes in the sample. Instrument conditions were as follows and analyte transitions are provided in Table 1.
Mobile phase A: 5 mM ammonium acetate in water
Mobile phase B: Methanol
Gradient Time (min) %B
Flow rate: 0.25 mL/min
Run time: 11 minInjection volume: 10 µL
Column temp.: 40°C
Ion mode: Negative ESI
Ion spray voltage:-2,000
Source temp.: 450°C
PFAS analysis results and discussion
The calibration range was demonstrated to be 10-400 ppt for PFPrA and 5-400 ppt for all other analytes. All compounds showed acceptable linearity with r values ≥ 0.999 and deviations of <20%. 11Cl-PF3OUdS is the only analyte that was quantified using a quadratic regression (1/x weighted) standard curve. All other analytes were quantified using a 1/x weighted linear regression.
Analyte peak shapes, retention, and intensity were similar across the reagent water and field water samples. There was a higher baseline for the PFPrA signal in all field water samples (Fig. 1), but this did not have a negative impact on peak integration and quantification of PFPrA. No matrix interference was observed for all water samples prepared by two-fold dilution.
The unfortified water samples showed various levels of C3, C4, and C8 PFAS with no detectable PFPrS, ADONA, HFPO-DA, 9Cl-PF3ONS and 11Cl-PF3OUdS (Table 2). For accuracy (%recovery) calculation, the measured amount of analyte in the fortified sample was adjusted based on the unfortified concentration. Table 3 shows the accuracy and precision results calculated across all three batches of data. Method accuracy was demonstrated by recovery values that were within 20% of the nominal concentration for both fortified sample levels, as well as for the LLOQ concentration standard prepared in reagent water. The %RSD was <15%, indicating acceptable method precision.
PFAS analysis conclusion
The LC-MS/MS method presented here provides a robust procedure for quantitating diverse PFAS of various chain lengths and structures in a wide range of water sample types. Based on a simple direct injection procedure using a Raptor C18 column with a PFAS delay column, the analytical method was demonstrated to be fast, rugged, and sensitive with acceptable accuracy and precision.
This approach is suitable for labs needing to report an extended list of PFAS compounds, including the analysis of ultrashort-chain PFAS, for potable and non-potable water testing.
Shun-Hsin Liang & Mike Chang are with Restek