Automated PFAS sample preparation for ES 04056.1 with Biotage® PrepXpert-8

Introduction

Per- and polyfluoroalkyl substances (PFAS) have been extensively produced and applied across industrial and consumer sectors for decades, resulting in their designation as a priority class of contaminants in environmental monitoring programs. As regulatory expectations continue to advance, laboratories are increasingly required to implement standardized and defensible methods for PFAS determination. ES 04056.1 provides a structured analytical framework for the extraction and quantification of PFAS in water samples, supporting consistency and reliability. This application note presents a
validated workflow aligned with ES 04056.1, using the Biotage® PrepXpert-8 automated solid-phase extraction system with EVOLUTE® PFAS cartridges to deliver reproducible, high-quality results. The workflow, supported by the TurboVap® LV system, demonstrates a robust and automated approach suitable for routine PFAS analysis under ES 04056.1.
 

Analytes

Table 1. Listing of target analytes, extracted labeled standards, and internal standards

Solution preparation

Ammonia methanol solution

1. Add 400 μL of NH4OH for every 99.6 mL of methanol to a clean beaker.

    

2. Agitate to homogenize the solution.

    

3. Prepare fresh solution daily before extraction.

Sample preparation procedure

1. Clean the Biotage® PrepXpert-8 automated SPE extractor system using the technique given in Appendix A. If the cleanliness of a particular channel is suspect, it is recommended that the cleaning method be run multiple times and that the cleanliness be verified by extracting blanks.

2. Set up and fill new sample containers with water. To aid with the sample volume determination later, either mark the meniscus of the fluid on each sample container or obtain a weight of each sample.

3. Adjust sample pH to 3 using glacial acetic acid.

4. Spike the extracted labeled standards into each sample at a concentration corresponding to the mid-point of the calibration curve.

5. If necessary to perform statistical studies, spike a known amount of PFAS target standard into the water samples to achieve the desired concentration.

6. Attach each prepared water sample to the sample bottle caps on the Biotage® PrepXpert-8 system and ensure that the sip tube is angled correctly with the ability to drain all liquid from the sample bottle.

7. Attach the EVOLUTE® PFAS SPE cartridges onto the adapters of the Biotage® PrepXpert-8 system.

8. Load 15 mL centrifuge tubes onto the collection vessel rack and ensure that it is installed on the Biotage® PrepXpert-8 automated tray.

9. Program in the parameters for the extraction of samples by ES 04056.1 given in Table 2, adjusting for the preferred sample volumes, and run the method. The protocol will result in an approximately 10 mL extract.

10. 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.

11. Transfer the centrifuge tubes to the TurboVap® LV system and concentrate the samples to a final volume less than 0.5 mL using nitrogen according to the parameters in Table 3.

12. Add methanol to each extract to achieve a final volume of 0.5 – 1.0 mL.

13. Add an appropriate amount of internal standard to each extract and mix to homogenize.

14. Load the extract onto a calibrated LC-MS/MS system and process using the conditions given in the below sections.

*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

Agilent 1290 Infinity II LC System:

• 1290 Infinity II Multicolumn Thermostat, G7116B

• 1290 Infinity II Multisampler, G7167B

• 1290 Infinity II High Speed Pump, G7120A

Columns

• InfinityLab PFC Delay Column, 4.6 x 30 mm, p/n 5062-8100 
• ZORBAX Eclipse Plus C18 Guard Column, 2.1 x 5 mm 1.8 µm, p/n 821725-901 
• ZORBAX RRHD Eclipse Plus C18, 95 Å, 2.1 x 50 mm, 1.8 µm, p/n 959757-902 

   

Mobile phases

A: 20 mM ammonium acetate in water

B: Methanol

LC gradient:

• Flow rate: 0.4 mL/min
• Injection volume: 5 μL
• Column temperature: 50 ˚C

Agilent 6470 MS/MS, G6470B

• Gas temperature: 230 ˚C
• Gas flow: 4 L/min
• Nebulizer: 20 psi
• Sheath gas temperature: 375 ˚C
• Sheath gas flow: 12 L/min
• Capillary voltage (Positive): 3500 V
• Capillary voltage (Negative): 3500 V
• Nozzle voltage (Positive): 500 V
• Nozzle voltage (Negative): 0 V
  

Results

System calibration

For the work being done here, a total of five points were used in the calibration covering a range of 0.2-20 ppt. The calibration curve was forced through zero and achieved excellent R2 values.

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

Determination of the minimum reporting level (MRL) and detection limits (DL)

A target MRL of 2 ng/L was selected and eight replicate laboratory fortified blanks (LFBs) were created and run at that concentration. The resulting data was then used to calculate the DL for all target compounds. Figure 2 illustrates the calculated detection limit (DL) for all ES 04056.1 target compounds ranging between 0.12 ng/L to 0.28 ng/L. All calculated DL were well below 1/3 concentration of the MRL concentration.

Figure 2. MRL and DL recoveries. Compounds marked with an asterisk were analysed in salt form. The data for individual compounds is shown in Appendix D.

Initial demonstration of precision and accuracy (IDP, IDA)

Four LFB samples each were prepared at a concentration of 10 ng/L. The resulting data was used to determine the precision and accuracy of the sample preparation process, Figure 3 below illustrates the accuracy while Figure 4 illustrates the precision; all target compounds exhibited tight recoveries, recovering on average between 97%-105% of the spiked amount and had calculated CV of 5.1% or less. These results fall well within the limits defined by ES 04056.1 of ± 25% of the nominal value with relative standard deviations under 20%.

Figure 3. Initial demonstration of accuracy (10 ng/L, n=4). Compounds marked with an asterisk were analysed in salt form.

Figure 4. Initial demonstration of precision (10 ng/L, n=4). Compounds marked with an asterisk were analysed in salt form. The data for individual compounds is shown in Appendix E.

Demonstration of low system background

A study was conducted to investigate each part of the extraction process and any their potential contribution to PFAS background. This sequential process is visualized below and highlights each component of the full extraction process as they are screened for PFAS background contribution before finally being combined into a single process when extracting the Reagent Water Blank samples.

The result of these tests are given in Appendix F and selected data are shown below in Figures 5-8.

Figure 5. Contribution of the TurboVap® LV to the PFAS Background. Compounds marked with an asterisk were analysed in salt form.

Figure 6. Contribution of the EVOLUTE® PFAS SPE cartridges to the PFAS Background. Compounds marked with an asterisk were analysed in salt form.

Figure 7. Contribution of the Biotage® PrepXpert-8 to the PFAS Background. Compounds marked with an asterisk were analysed in salt form.

Figure 8. PFAS background for full LRB extraction using EVOLUTE® PFAS SPE cartridges. Compounds marked with an asterisk were analysed 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 resulting data for the Biotage® PrepXpert-8, the EVOLUTE® PFAS SPE cartridges, and the TurboVap® LV blanks, only trace levels of PFAS are observed, however these trace levels are all well below the lowest curve point of the calibration and will not interfere with water sample extractions. When evaluating the LRB sample extractions, it can be seen that there are indications of the presence of a PFAS background at very low levels. However, even at the highest concentrations detected, all levels are much lower than the 1/3 MRL limit indicating that the background is acceptable.

Examination of system carryover

To simulate an influent sample, four LFB samples were created with concentrations which were more than two times greater than the highest point on the calibration curve. These samples were extracted, and the clean up procedure given in Appendix A was run three times. To ensure that the system background was adequately reduced, a set of four LRB samples were extracted immediately after the cleaning procedure and analysed. The LRB data obtained from this study is presented in Appendix F.
The trace levels of PFAS targets that were observed were non-detect for all compounds. These results show that the cleaning method given in Appendix A is sufficient to clean the Biotage® PrepXpert-8 system; however, if higher than desired concentrations of any PFAS compounds remain, it is suggested to run additional cleaning methods or examine the use of alternative cleaning solvents to help re-establish the system background.

Conclusion

The results of this study confirm that the Biotage® PrepXpert-8 system, in combination with the TurboVap® LV, provides a reliable and efficient platform for the extraction of the PFAS analytes outlined in ES 04056.1. The testing demonstrated that the system is PFAS free, eliminating concerns about background contamination and ensuring accurate extraction and monitoring of all target compounds.

With exceptional precision and accuracy, as well as the calculated DL; the Biotage® PrepXpert-8 proves to be a robust solution that can easily meet and exceed the demands of the method. This validated workflow provides laboratories with a reliable, automated solution for PFAS analysis under ES 04056.1.

Appendix A: Cleaning procedure

For the best results, it is recommended that this procedure be completed before the use of the Biotage® PrepXpert-8 each day and at the end of each extraction prior to proceeding with the next set of samples.

1. Ensure that an empty cleaning cartridge and sample bottle are installed onto each position on the automated extraction system.

2. Load and run the cleaning method outlined in the table below.

3. Remove the cleaning cartridges and sample bottles, disposing of the remaining solvent from the sample bottles.

4. Using methanol in a squeeze bottle, clean the adapters, sample bottle rinse heads, and the sip tubes.

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 to F

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Litrature number: AN1022

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