Manual extraction of PFAS in drinking water in compliance with EPA method 537.1

Introduction

Per- and polyfluorinated alkyl substances (PFAS) have been used abundantly since their inception in the twentieth century and have become a closely monitored class of compounds within environmental testing. This application note outlines a procedure for those seeking to become compliant with EPA Method 537.1. The data presented was generated using a Biotage® VacMaster™ vacuum manifold with a Large Volume Extraction (LVE) kit designed for PFAS work in conjunction with ISOLUTE® 101 SPE cartridges and a TurboVap® LV system. This note will demonstrate that the solution outlined is capable of generating data which exceeds the requirements outlined within the 537.1 method.
 

Equipment and materials used

Biotage:

» Biotage® VacMaster™-20 Sample Processing Station
(with 16 mm rack), p/n 121-2016, fitted with polypropylene stopcocks (p/n 121-0009-PP)
» Biotage® VacMaster™ LVE Kit (PFAS) 1, 3, 6 mL SPE Cartridge (p/n 121-2190)
» ISOLUTE® 101 500 mg/6 mL SPE Cartridges, p/n 101-0050-C
» TurboVap® LV Automated Solvent Evaporation System, p/n 415000
» TurboVap® LV Multi Rack (48 Positions, 10–20 mm Tubes), p/n 414964

Wellington laboratories:

» USEPA Method 537.1 PDSL (linear) stocks, 1.2 mL, p/n EPA-537PDSL-R1
» USEPA Method 537 Internal Standard Mix, 1.2 mL, p/n EPA-537IS
» USEPA Method 537.1 Surrogate Mix, 1.2 mL, p/n EPA-537SS-R1

Restek:

» PFAS Delay Cartridge, 5 μm, 50 x 2.1 mm, p/n 27854
» Raptor C18 LC Cartridge, 5 μm, 50 x 2.1 mm, p/n 9304552

Sigma-Aldrich:

» Ammonium Acetate, ACS Reagent Grade ≥ 97%, p/n 238074-25G

Honeywell:

» Water, ACS Certified, HPLC Grade, p/n AH365-4
» Methanol, Burdick & Jackson™, LC-MS Grade, p/n LC230-4

VWR:

» 15 mL Polypropylene Centrifuge Tubes with Caps, p/n 21008-670
» 250 mL Polypropylene Wide Mouth Bottles, p/n 414004-125

ACROS organics:

» Tris(hydroxymethyl)aminomethane, p/n 140505000

Analytes

Listing of Target Analytes, Surrogate Standards, and Internal Standards.

Summary of SPE method

SPE cartridge format

ISOLUTE® 101 500 mg/6 mL p/n 101-0050-C

Sample pre-treatment

Add 0.94 g of tris(hydroxymethyl)aminomethane and 660 μL of concentrated HCl to each 250 mL sample, check pH is 7 ± 0.5. Add surrogate standards.

Conditioning

Condition each cartridge with methanol (15 mL)

Equilibration

Equilibrate each cartridge with reagent water (18 mL + 3 mL)

Sample loading

Load sample (250 mL) at a flow rate of 15 mL/min

Wash

Rinse the cartridge with reagent water (sample bottle rinsate, 2 x 7.5 mL)

Dry

Dry the cartridge for 5 minutes using a vacuum of -10 to -15 in. Hg.

Elution

Elute the analytes with methanol (sample bottle rinsate 2 x 4 mL)

Post extraction

Evaporate to dryness and reconstitute in methanol/water (96/4, v/v, 990 μL). Add IS and mix prior to analysis.

Sample preparation procedure

1. Clean all parts of the VacMaster™ system per the procedure given in Appendix A.
2. Set up and fill new sample containers with 250 mL of water.
3. Add 0.94 g of tris(hydroxymethyl)aminomethane and 660 μL of concentrated HCl to each of the sample containers.
4. Verify the pH of the sample is 7 ± 0.5.
5. Prepare for the determination of the initial sample volume by either marking the level of the sample on the container or by weighing the sample container.
6. Add 10 μL of the undiluted Surrogate Standard Mix to each of the sample containers. If the mixes used were different than the ones outlined in this note, adjust the concentration or spiking amounts as needed.
7. Using the VacMaster™ LVE Kit, place one end of the cleaned tubing into the bottom of each of the sample containers, and secure in position using the clips provided.
8. Load ISOLUTE® 101 cartridges onto the VacMaster™. Seal any unused positions using VacMaster™ Port Sealing Plugs (p/n 121-0005)
9. Rinse each cartridge with 15 mL of methanol and apply vacuum to pull it to waste. Do not allow the sorbent to go dry.
10.  Rinse each cartridge with 18 mL of reagent water and apply vacuum to send it to waste. Do not allow the water level to drop below the top of the packing.
11. Add 3 mL of reagent water to each tube and attach the cartridge adapters to the cartridges.
12. Load the samples onto the cartridges using a flow rate of 15 mL/min.
13. Once the sample has been fully loaded, rinse the sample containers using 7.5 mL of reagent water, swirl to ensure the full rinsing of the container, and load the aliquot onto the cartridge. Repeat this step one additional time.
14.  Dry the cartridge for 5 minutes using a vacuum of -10 to -15 in. Hg.
15. Load 15 mL centrifuge tubes into the rack corresponding to each of the cartridge positions and load into the VacMaster™.
16. Rinse each sample container using 4 mL of methanol and swirl to ensure the full rinsing of the container. Load the aliquot through the appropriate cartridge and collect at a dropwise rate. Repeat this step one additional time.
17. Determine the initial sample volume by either using a graduated cylinder and filling the sample container to the original mark or by taking an additional weight of the container.
18. Transfer the centrifuge tubes to the TurboVap® LV system and concentrate the samples under nitrogen according to the parameters in Table below.
19. Reconstitute each extract using 990 μL of 96% methanol/4% water within the centrifuge tubes.
20. Add 10 μL of the internal standard mix and mix thoroughly.
21. Transfer each extract to a 1 mL storage vial.
22. Transfer a 300 μL aliquot of each extract to an autosampler vial.
23.  Load the extract onto a calibrated LC-MS/MS system and process using the conditions given in the below sections.
    
    

TurboVap® LV Concentration Protocol.

*The nozzle position was adjusted such that it was as far to the right as possible to give the user a clear view of the vortex within the tube.

LC-MS/MS conditions

Shimadzu Nexera X2 UHPLC system

» CBM-20A Communications Controller
» DGU-20A5R Degassing Unit
» LC-30AD Solvent Delivery Unit
» SIL-30AC Autosampler
» CTO-30A Cartridge Over
» SPD-20A UV-Vis Detector

Cartridges

» Restek PFAS Delay Cartridge, 5 μm, 50 x 2.1 mm, p/n 27854
» Restek Raptor C18 LC Cartridge, 5 μm, 50 x 2.1 mm, p/n 9304552

Mobile phases

A: 20 mM Ammonium Acetate in Water
B: Methanol

Flow rate

0.6 mL/min

Injection volume

10 μL

Cartridge temperature

35 ˚C

AB SCIEX Triple Quad™ 5500

Note: The PFAS conversion kit from SCIEX was installed and used for this testing with the exception of the included UHPLC cartridges.

Curtain gas

35

Collision gas

8

Ion spray voltage

-4500

Temperature

350 ˚C

Ion source gas 1

50

Ion source gas 2

50

Polarity

Negative

For a complete listing of MRM Transitions, see Appendix B

Results

System calibration

For the work being done here, a total of nine points were used in the calibration covering a range of 0.2-100 ppt in the sample. The lowest three points were below the calculated MRL. The curve was forced through zero as required by method 537.1 and achieved excellent linearity across the calibration range.

Figure 1. Calibration curves for PFOS and PFOA. Calibration curves for the remaining target analytes in Table 1 are shown in Appendix B.

Demonstration of peak asymmetry factor

Method 537.1 only requires that the first two eluting peaks on a single mid-level calibration standard have their peak asymmetry factors calculated and that the results of this calculation must fall within a range of 0.8-1.5. For the work done here, the scope of the investigation was broadened and six of the early eluting compounds were examined across all calibration levels. The results are given in Table below and shown graphically in Figure 2.

Figure 2. Peak asymmetry factor with the range of acceptance shown in white.


Each of the first six eluting compounds across all calibration levels were able to meet and exceed the asymmetry factor requirements proving that the chromatographic conditions are acceptable.

Determination of the Minimum Reporting Level (MRL) and Detection Limits (DL)

A target MRL of 2 ng/L was selected and seven replicate laboratory fortified blanks (LFBs) were created and ran at that concentration. Table below lists the recoveries of the replicates, showing the averages and deviations for each compound along-side the calculated half-range for the prediction interval of results (HRPIR), the upper and lower bounds for the PIR, and the resulting DL calculations. It should be noted that the constant used to calculate the HRPIR and the DL was adjusted to accommodate the increased degrees of freedom for this experiment.

*Analytes were used in salt form and calculated concentrations were corrected to compensate where needed.

Figure 3. Upper and Lower calculated PIR limits with the range of acceptance shown in white. Those compounds with an asterisk were used in salt form.
 
Based on the data obtained, the calculated Upper and Lower PIR were all well within the specified boundaries and the MRL concentrations given in Table 5 are all deemed acceptable. The DL was calculated from this data set and is found to be lower than the DL given in Table 5 of EPA Method 537.1.

Demonstration of low system background

An investigation into the background of the complete process was done in three steps. The first step was to run blank injections of a mixture of 96% methanol/4% water on the analytical system (system blank). The second step was to load centrifuge tubes containing a similar volume of methanol as would result from the extraction process onto the evaporation system, allowing them to concentrate to dryness, be reconstituted, and then run on the analytical system (evaporation blank). The third and final step was to create a full Laboratory Reagent Blank (LRB), extract and concentrate it, reconstitute it, and run it on the analytical system. By separating the process into three separate steps it becomes easier to determine what, if any, contribution to the overall background each of the steps has. The result of these tests are shown in Table below and selected data are shown graphically in Figures 4 and 5.

*Analytes were used in salt form and calculated concentrations were corrected to compensate where needed.
NOTE: Where “N.D.” is indicative of the inability of the target peak to be separated from the system background.


Figure 4. Contribution of the TurboVap® LV to the PFAS Background. Those compounds with an asterisk were used in salt form.


Figure 5. PFAS Background for full LRB. Those compounds with an asterisk were used in salt form.

For those results which were generated using only the analytical system, all target analytes were N.D. (unable to be separated from the noise in the baseline) and so were not listed out in the previous tables.

When examining the data resulting for both the TurboVap® LV and the full LRB (which includes the Biotage® VacMaster™ manifold, Large Volume Loading Kit, and the ISOLUTE® 101 cartridges as well as the TurboVap® LV) there are clear indications of the presence of a PFAS background. However, even at the highest concentrations detected, all levels are much lower than the 1/3 MRL limit specified within EPA Method 537.1 indicating that the background is acceptable and will not interfere with future sample runs.

Initial Demonstration of Precision and Accuracy (IDP, IDA)

To determine the precision and accuracy of the sample preparation process, eight LFB samples were prepared at concentrations of 20 ppt. The data is shown in Table below and illustrated in Figures 6 and 7.
The results show that the average recovery for each target analyte was within 15% of the nominal value; falling well within the criteria of ± 30% stated within Method 537.1. Additionally, the relative standard deviation (RSD) for each analyte fell under 11% on average; again being much less than the 20% requirement set forth within the method.

*Analytes were used in salt form and calculated concentrations were corrected to compensate where needed.
 
Figure 6. Initial Demonstration of Accuracy with range of acceptance shown in white (20 ng/L, n=8). Those compounds with an asterisk were used in salt form.

Figure 7. Initial Demonstration of Precision with range of acceptance shown in white (20 ng/L, n=8). Those compounds with an asterisk were used in salt form.

Examination of system carryover

To simulate an influent sample, eight LFB samples were created with concentrations which were much higher that the calibration curve. These samples were split into two groups of four. The first group of four samples were extracted and, immediately following them, a LRB was extracted. For the second group, again four samples were extracted followed by a set of four LRB samples however, between the two extractions the cleaning procedure given in Appendix A was run three times. The sets of LFB and LRB samples were analyzed and the data for the LRB runs is given in Table 8 and illustrated in Figure 8.
The graph shown in Figure 8 indicates that without a cleaning procedure between influent and effluent sample extractions, carryover should be expected for some of the PFAS compounds contained within the test. Additionally, while the cleaning procedure did reduce the background to a point below the 1/3 MRL limit on average for all compounds, there is one instance where the compound NEtFOSAA was found to be above the limit. This indicates that further cleaning should be done in the future for influent samples of this concentration.

Figure 8. Results of carryover study following eight, 400 ng/L LFB samples; Four LRB samples extracted without cleaning procedure and four with the cleaning procedure in Appendix A. Those compounds with an asterisk were used in salt form.

Results of carryover study following eight, 400 ng/L LFB samples; Four LRB samples extracted without cleaning procedure and four with the cleaning procedure in Appendix A.

*Analytes were used in salt form and calculated concentrations were corrected to compensate where needed.

Conclusion

With the scrutiny being given to the presence of PFAS compounds in the environment, it is essential to find reliable products which can meet the requirements of EPA Method
537.1. This application note has shown that the Biotage® VacMaster™ vacuum manifold with accessories, ISOLUTE® 101 SPE cartridges and the TurboVap® LV can be used to easily meet and exceed the demands of the method.

Ordering information

Appendix A

Biotage® VacMaster™ cleaning procedure

For the best results, it is recommended that this procedure be completed before the use of the Biotage® VacMaster™ each day and at the end of each extraction prior to proceeding with the next set of samples.

1. Ensure that a cartridge and cartridge adapter is installed onto each VacMaster™ position slated to be cleaned.
2. Fill a clean beaker with 50 mL of methanol and place no more than four of the LVE Kit lines into the beaker.
3. Apply vacuum to the manifold and pull the methanol through the positions into the waste container.
4. Remove the cartridge and discard.
5. Using methanol in a squeeze bottle, clean the exterior of the LVE Kit’s lines, the cartridge adapters, the stopcock, and the metal cannula. Discard all rinsate.
6. Repeat this up to three times for all positions which require cleaning.

Note: In situations where the previous sample was highly concentrated, the above cleaning procedure may need to be repeated multiple times. If there is concern regarding potential carryover contamination regardless of the cleaning procedure, a laboratory reagent blank should be run in that position to ensure its cleanliness.

Appendix B

MRM transitions

Appendix C

Calibration curves

Figure 9. Calibration curves for the target analytes in Table 1, covering a concentration range of 0.2-100 ppt.

Literature number: AN958