Here, we present a protocol for the sequential targeted quantification and non-targeted analysis of fluorinated compounds in water by mass spectrometry. This methodology provides quantitative levels of known fluorochemical compounds and identifies unknown chemicals in related samples with semi-quantitative estimates of their abundance.
This approach allows for the quantitation of known PFAS and the discovery of new substances in the same samples, compared to targeted approaches alone that miss emerging compounds of concern. This protocol targets a wider variety of PFAS compounds, which are representative of the newest emerging chemicals. If the concentration step is adapted for the compounds, the measurement methods used here could be applied to any class of chemicals.
To collect a sample from the field, wear nitrile gloves and collect 500 to 1, 000 milliliters of water from the field location in a clean, high-density polyethylene, HDPE, or polypropylene bottle. Add five milliliters of 35%nitric acid preservative to the sample bottle and to a field blank, and transport the samples back to the lab. Pour each sample into individual, pre-cleaned, one-liter, high-density polypropylene graduated cylinders, and record the exact volume of the samples.
Add 10 milliliters of methanol to each emptied sample bottle before capping and shaking vigorously to rinse absorbed per-and polyfluoroalkyl substances, or PFAS, from the bottle interiors. Then, return the measured water sample to the rinsed bottle with the methanolic rinse. To obtain a standard curve for quantitation, fill eight empty, one-liter sample bottles with PFAS-free deionized water, and label the bottles with eight evenly spaced concentrations covering the desired quantitation range.
Next, add a quantity of native PFAS mix to each bottle to yield the appropriate PFAS concentrations, and add the internal standard PFAS mixture to each sample at a concentration approximating the midpoint of the calibration curve. Then, filter the samples through glass fiber A filters under gentle vacuum into individual, pre-cleaned, one-liter high-density polypropylene vacuum flasks. If particulate matter remains within the bottle, rinse the additional deionized water, and filter the wash into the flask.
For the solid-phase extraction, return the filtered water to the sample bottle, and condition a weak anion exchange, or WAX, cartridge with 25 milliliters of methanol and an additional 25 milliliters of deionized water. Position the pump draw tubing within the filtered sample bottles, and label solid-phase extraction, or SPE, cartridges with the appropriate sample names. Pump 500 milliliters of each sample through each cartridge at a steady flow rate of 10 milliliters per minute, discarding the flow-through.
Remove the WAX SPE cartridges from the piston pumps, and transfer the SPE cartridge to a vacuum manifold, equipped with external glass reservoirs. Flush each SPE cartridge with four milliliters of 25-millimolar, pH four sodium acetate buffer under gentle vacuum, followed by a four-milliliter neutral methanol wash. At the end of the wash, place a 15-milliliter polypropylene centrifuge tube beneath each SPE cartridge to collect the eluent, and elute the samples with four milliliters of 0.1%ammonium hydroxide in methanol.
Then, remove the elution tubes, and reduce the eluate volume to 500 to 1, 000 microliters by evaporation under dry nitrogen stream in a water bath at 40 degrees Celsius. For targeted liquid chromatography with tandem mass spectrometry, dilute 100 microliters of sample extract with 300 microliters of two-millimolar ammonium acetate buffer in a high-pressure liquid chromatography sample vial, and prepare an analytical worklist consisting of the standard curve, samples, and an additional replicate of the standard curve to assess instrumental drift across the run. Analyze the samples using standard liquid chromatography and mass spectrometry methods established for the targeted compounds of interest.
At the end of the analysis, generate a standard curve from the standard samples using the peak area ratio of the analyte to the internal standard versus the concentration of analyte, and generate a quadratic regression formula with one over x weighting for concentration prediction. Quantitate the targeted analytes in each sample using the prepared standard curves and area ratio for each measurement. If the concentration exceeds the calibration range, dilute the original sample with deionized water spiked with the appropriate internal standard concentration, and re-extract to bring the concentration into the appropriate range.
For non-targeted liquid chromatography with tandem mass spectrometry, dilute 100 microliters of sample extract with 300 microliters of two-millimolar ammonium acetate buffer into a high-pressure liquid chromatography sample vial. After setting a worklist as demonstrated, use the instrument software to collect liquid chromatography-mass spectrometry data with a wide scan in data-dependent mode. For non-targeted data processing, open the appropriate molecular feature extraction software package, and select Add/Remove Sample Files and Add Files, and select the raw data from the non-targeted experiment.
Click OK, and select Batch Recursive Feature Extraction and Open Method to load a pre-established method or to manually edit the software settings. For each feature remaining after filtering, generate predicted chemical formulas from the exact mass and composite mass spectrum, and open the Environmental Protection Agency CompTox Chemicals Dashboard Batch Search tool. To search the predicted chemical formulas or neutral masses against, to return potential chemical structures, select the identifier type, and paste the list of identifiers into the identifier box.
Select Download Chemical Data and any physical, chemical, or toxicology data desired for potential matches from the drop-down menu. Then, confirm the structures using the available standards and/or targeted high-resolution tandem mass spectrometry matching of the fragments against the spectra from the databases, in silico theoretical spectra, or manual curation. Quantitative liquid chromatography with tandem mass spectrometry results are presented in the form of ion chromatograms for the total ion chromatogram and the extracted ion chromatograms of specific chemical transitions for measured chemicals.
The integrated peak area of a chemical transition is related to the compound abundance and can be used to calculate the exact concentration using a calibration curve normalized to an internal standard. Non-targeted analysis using a full MS scan yields a total ion chromatogram for samples, which allows ad hoc generation of extracted ion chromatograms for individual ions. PFAS compounds have negative mass defects due to their preponderance of fluorine atoms, and polyfluorinated compounds have positive but substantially smaller mass defects than homologous organic materials.
A second method filtering step is to identify homologous series containing repeating units common to PFAS species using an appropriate software package. From high-resolution MS data, one or more putative chemical formulas can be matched against the isotopic fingerprint of the mass spectrum and scored. Chemical formulas can be further confirmed, and some structural information can be garnered from tandem mass spectrometry data.
Further mass spectrometry experiments can be used to confirm the identity of new compounds, and sample comparisons can give information about the prevalence and relative quantity of chemicals. It is very important to have appropriate blanks and quality control samples for the matrix to validate the quantitative and non-targeted measurements. The strategies shown have become the new approach to environmental screening for PFAS by allowing the discovery of unknowns and the prediction of their identities.
