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Automation of PFAS Samples in Milk Matrices using SPE
This basic method provides the core methodology for translating a workflow into OneLab-executable script(s) as an attempt to fully or semi-automate a specific procedure. It demonstrates the benefits of automation and highlights OneLab capabilities and best practices to promote solution adoption, helping transition from manual to a more automated approach. It can be used alone or serves as a building block for a more complex workflow and is easily adaptable to users' requirements.
Overview
Per- and Polyfluoroalkyl Substances (PFAS) are a group of man-made chemicals that have multiple fluorine atoms attached to an alkyl chain. PFAS were found to be applicable due to their high stability and hydrophobic nature, which led to their use in many areas for various applications such as packaging, surfactants, and firefighting fluids. However, PFAS were found to be toxic and potent carcinogens (1). As such, there is a pressing need to reduce the usage and prevalence of PFAS in all materials, and one important area is to reduce PFAS in food. As PFAS compounds are resistant to degradation and bioaccumulate in the tissues of mammals (2), they can be transferred from food products to humans through consumption. The European Food Safety Authority (EFSA) predicts food to be a predominant channel for PFAS exposure. Milk, being the staple food product for toddlers and children, would affect them disproportionately compared to adults, and as such, there is a need to monitor PFAS levels in milk matrices (4).
Milk is a complex food matrix, and to analyze trace levels of PFAS, a solid-phase extraction (SPE) protocol using Oasis WAX cartridges can be used to improve extraction recovery and obtain samples with minimized matrix interference. Oasis WAX is a mixed-mode reverse phase-weak anionic exchange polymer that can bind to PFAS that are hydrophobic with anionic side groups. Milk matrices are washed away during the wash step, and finally, PFAS is eluted with basic organic solvents.
Figure 1: Sample preparation workflow for PFAS in milk using OASIS WAX
Preparing milk samples for analysis using an LC-MS/MS involves a 4-part workflow requiring many pipetting tasks and is a time-consuming process. It is, therefore, desirable to automate as many parts of the workflow as possible to improve throughput and reduce human experimental errors. The protocol shown here automates the priming of OASIS WAX cartridges, loading, rinsing, and eluting the sample, thus improving experimental efficiency while reducing errors.
Figure 2: Graphical illustration of the automated SPE sample extraction and LC-MS analysis workflow using the Oasis WAX for PFAS 6 cc cartridges on the Andrew+ Pipetting Robot configured with the Extraction+ Connected Device.
PFAS contamination from the setup is minimized due to several reasons. The method contains a methanol washing step for each new pipette tip, ensuring the pipette tips used are PFAS-free.
Secondly, there is no contact between the OASIS WAX cartridges and the collection vials, ensuring no environmental contamination from the exterior of the cartridges. Finally, there are little to no human actions taken during the protocol, decreasing the competency of the user required to perform PFAS analysis at the lowest levels required to meet regulatory requirements.
Assay notes
This procedure is designed as a general protocol and may need to be adjusted to suit individual sample needs or quantities. Sample preparation steps need to be performed before placing the samples into the sample vials. Likewise, drying down and reconstitution need to occur after the protocol finishes, prior to LC-MS analysis.
Figure 3: Samples and required solutions at the start.
Figure 4: Andrew+ OneLab deck set up for the automated “PFAS Testing in Milk using Oasis WAX for PFAS - 12_samples” assay. Domino/Device locations on Andrew+ deck: [1] 5ml Tip Rack Holder Domino, [2] Collection Labware Rack Domino with Eppendorf® 5 mL snap-cap tube, racked, [3] 15ml Conical Centrifuge Tube Domino, [4] 50ml Conical Centrifuge Tube Domino, [5] Extraction+ Device with OASIS WAX for PFAS 6cc cartridges, [6] 50ml Conical Centrifuge Tube Domino, [7] 250ml Duran Bottle Domino
Protocol specifications
Automated PFAS Testing in Milk using Oasis WAX for PFAS - 12 samples:
- Estimated time of execution: 1h 06m 49s
- Hands-on time: 0s
- Tip consumption:
- 30× 100-5000 μL tips
Ordering information
Andrew+ System Components: Dominos, Devices, Electronic Pipettes & Tips
- Andrew+ Pipetting Robot
- OneLab software
- 5000 µL Tip Rack Holder Domino | p/n 186009599
- 15ml Conical Centrifuge Tube Domino | p/n 186010087
- 2× 50ml Conical centrifuge Tube Domino | p/n 186009614
- 250mL Duran Bottle Domino | p/n 186010193
- Extraction+ Base Kit with Plate Gripper | p/n 176005201
- Extraction+ 6cc Cartridge Kit including 1x Collection Labware Rack Domino | p/n 176005206
- Andrew Alliance Bluetooth Electronic Pipette, 1-ch 5000 μL | p/n 186009608
Recommended consumables
- Sartorius, Optifit Tips, 100-5000 μL (x30) | p/n 700013291
- 2× DURAN® 250 mL clear glass laboratory bottle | p/n 218013651
- 12× Eppendorf Tubes® 5 mL snap-cap centrifuge tube | p/n 0030119401
- 10× VWR® 50 mL conical centrifuge tube | p/n 525-0610
- 12× Eppendorf Tubes® 15mL conical centrifuge tubes | p/n 0030122194
- Oasis WAX for PFAS Analysis 6 cc Vac Cartridge, 150mg Sorbent per Cartridge, 30µm Particle Size, 30/pk | p/n 186009345
- or Oasis WAX for PFAS Analysis 6 cc Vac Cartridge, 150mg Sorbent per Cartridge, 30µm Particle Size, 300/pk | p/n 186009344
References
- Temkin, A. M., Hocevar, B. A., Andrews, D. Q., Naidenko, O. V., & Kamendulis, L. M. (2020). Application of the key characteristics of carcinogens to per and polyfluoroalkyl substances. International Journal of Environmental Research and Public Health, 17(5), 1668. https://doi.org/10.3390/ijerph17051668
- Cousins, I. T., DeWitt, J. C., Glüge, J., Goldenman, G., Herzke, D., Lohmann, R., Ng, C. A., Scheringer, M., & Wang, Z. (2020). The high persistence of pfas is sufficient for their management as a chemical class. Environmental Science: Processes & Impacts, 22(12), 2307–2312. https://doi.org/10.1039/d0em00355g
- Macheka-Tendenguwo, L. R., Olowoyo, J. O., Mugivhisa, L. L., & Abafe, O. A. (2018). Per- and polyfluoroalkyl substances in human breast milk and current analytical methods. Environmental Science and Pollution Research, 25(36), 36064–36086. https://doi.org/10.1007/s11356-018-3483-z
- PFAS in food: EFSA assesses risks and sets tolerable intake. European Food Safety Authority. (n.d.). https://www.efsa.europa.eu/en/news/pfas-food-efsa-assesses-risks-and-sets-tolerable-intake#:~:text=The%20threshold%20%E2%80%93%20a %20group%20tolerable,of%20these%20substances%20in%20food