Modifying QuEChERS for complicated matrices- High Fat Samples30 Jun 2020
This post is part of a series on QuEChERS. Here are links to the previous two posts, in case you may wish to catch up before reading this one:
(See Jana’s post here for further details on dry samples: QuECHERS approach optimization for low-moisture matrices - case of honey and brown rice flour
As in my last post for this series, I suggest using the following for reference
For greater detail on modifications: “A review of recent developments and trends in the QuEChERS sample preparation approach”, Rejczak & Tuzimski, De Gruyter Open Chemistry, 2015, 1, 13, 980 -1010. https://doi.org/10.1515/chem-2015-0109
And again, here is a link to the USDA database at https://fdc.nal.usda.gov/ to obtain a listing of protein, total lipids, fatty acids, carbohydrates, sugars, and cholesterol (a sterol).
Before we start discussing more modifications to QuEChERS, I wanted to highlight some characteristics of QuEChERS sorbents that may help us in solving specific challenges with matrices. There are primary characteristics of each sorbent that we usually use to select a sorbent for a particular sample matrix. There are also other more secondary interactions that can complement the primary effects of another sorbent, as shown in the table below.
|Sorbent||Primary Action||Secondary Action|
|PSA||sugars, fatty acids, organic acids||polar pigments such as anthocyanines, and some sterols, ionic lipid components|
|C18-EC||nonpolar interferences, long chain hydrocarbons, lipids, waxes||proteins, starches, long chain fatty acids and long chain organic acids, some pigments, some sugars|
|GCB||pigments||some lipid components such as sterols, and planar polyphenols, flavonoids|
For example, “lipids” and “sterols” are listed as potential primary, and secondary interactions in the chart above. Lipids are composed of fats and oils, including those commonly known as triglycerides, as well as some waxes, fatty acids and sterol compounds (such as cholesterol) , and ionic lipids (such as phospholipids). To remove lipids from a sample, you would primarily use C18-EC sorbent, but looking at the secondary interactions in the chart, PSA and GCB also have affinity for some lipid components, such as sterols and ionic/charged lipid molecules. Consequently, the sorbents are often used in pairs to take advantage of this and to also remove other matrix components along the way. For lipid removal, it is very common to see PSA and C18-EC used together. The C18 may remove the bulk of the lipids, but the PSA complements it by removing some additional lipid molecules, possibly the ones that are smaller or more polar that C18 may not do as well. GCB can also retain and remove nonpolar components of lipid such as sterols, but is less common because of possible interaction with planar analytes.
High Fat Samples
Samples containing high amount of lipids or solid fats are perhaps the most challenging matrices. Here are the techniques I would try for lipid removal, starting with most preferred. (Please note the “preference” is my opinion.)
dSPE with PSA/MgSO4/C18-EC – This is a demonstration of the classic QuEChERS technique and represents the example we just discussed above for a sample containing lipids and sterols. PSA and C18-EC sorbent are often the perfect combination to remove this matrix interference. GCB can also be added if necessary, preferably if none of the analytes are planar molecules. Sometimes GCB is used in small amounts even with planar analytes, although caution is recommended. If this is attempted, a small amount of chlorophyll remaining in the extract indicates that planar analytes still remain intact in the extract. Here's an example of this technique using PSA/MGSO4/C18-EC.
- Avocado: Analyzing avocado: How to deal with lack of water and keep the fats out
- Animal Fat: “An Evaluation method for determination of non-polar pesticide residues in animal fat samples by using dispersive solid-phase extraction clean-up and GC-MS”, Castillo et al, Analytical and Bioanalytical Chemistry, 2011, 400, 5, 1315-28. https://www.researchgate.net/publication/49792170_An_evaluation_method_for_determination_of_non-polar_pesticide_residues_in_animal_fat_samples_by_using_dispersive_solid-phase_extraction_clean-up_and_GC-MS
Cooling/Freezing the QuEChERS extract- As described on the QuEChERS website mentioned earlier, (QuEChERS: About the method), extract aliquots from Stage 1 can be stored in a freezer, anywhere from 2 hours to overnight, to precipitate out the fats and waxes. Remaining supernatants may still need to undergo dSPE to remove other matrix interferences before analysis by GC or LC. As with all techniques, validation tests should be performed to ensure adequate recovery of the target analytes and efficiency of the method. In particular with this technique, it is important to make sure that the target analytes aren’t precipitated along with the lipid layer. Sometimes an acceptable approach is to add surrogate or internal standard prior to this step to take this into account. Here are some examples of this technique.
- Adipose tissue from rats: “QuEChERS-Based Method for Pesticides Analysis in Adipose Tissue Associated with Rat Ovaries”, Journal of Analytical Toxicology. June 2017, 41, 5, 399-406. https://academic.oup.com/jat/article/41/5/399/3069334
- Peanuts: “Extension of the QuEChERS Method for Pesticide Residues in Cereals to Flaxseeds, Peanuts, and Doughs”, Agric. Food Chem. 2010, 58, 10, 5950-5958. https://pubs.acs.org/doi/10.1021/jf902988b
SPE cartridge (cSPE) cleanup- In some cases, the amount of fat is too high to efficiently remove with the above mentioned techniques. Using cartridge SPE gives the analyst an option to use more sorbent that removes more of the fatty matrix. This can be accomplished with PSA or C18 sorbent or a combination of the two. Often a pass-through technique can be used that is quite simple, discussed in the following blog post. In this case, the sample extract is passed straight through the cartridge and the fatty matrix remains on the cartridge. It’s great because you don’t have the extra load, rinse and subsequent elution steps typically involved with SPE. Fatty Acid Removal from QuEChERS-Type Extracts with Quick and Easy PSA Cleanup Cartridge Pass
Here's an example of this technique.
- Tuna fish: Are fatty acids overwhelming your QuEChERS dSPE PSA cleanup and causing issues in your GC analysis? Get more cleanup capacity with cartridge SPE Cleanup!
- Soybean, Bovine liver: “Waters Food Testing Application Notes Booklet”, Waters Corporation document ID 720005049EN, 2017, downloadable from this webpage: https://www.waters.com/waters/library.htm?locale=en_US&lid=134800757
- CBD oil (mycotoxins): High-Throughput Analysis of Mycotoxins in Cannabis CBD Oil Pairs Simplified Cleanup with LC-MS_MS Sensitivity
Hexane/nonpolar solvent partitioning- If the above procedures are not sufficient to remove lipids from a sample, another option is to incorporate some amount of hexane or other nonpolar solvents into the QuEChERS extraction (Stage 1) or perhaps even following QuEChERS dSPE (Stage 2). Here are some examples of using this concept at Stage 1.
- Olive oil: “Chlorinated Pesticide Residues in Olive Oil Rtx-CLPesticides2” chromatogram GC-FF01043 (See Notes at the bottom re: QuEChERS procedure).
- Milk: Fatty Acid Removal from QuEChERS-Type Extracts with Quick and Easy PSA Cleanup Cartridge Pass Through
- Peanut butter (Aflatoxins): “A High Sensitivity LC/MS/MS Method with QuEChERS Sample Pre-treatment for Analysis of Aflatoxins in Peanut Butter Samples”, Shimadzu Corporation Document ID PO-CON1541E, 2015, downloadable from this webpage: https://www.shimadzu.com/an/literature/lcms/apo115045.html
- Smoked Salmon (for PAH analysis, uses partition with acetone/ethyl acetate/isooctane): “Determination of Parent and Substituted Polycyclic Aromatic Hydrocarbons in High-Fat Salmon Using a Modified QuEChERS Extraction, Dispersive SPE and GC-MS”, Agric. Food Chem. 2011, 59,15, 8108-8116. https://pubs.acs.org/doi/10.1021/jf201745a
- "How to choose the right dSPE for QuEChERS” video
- “Did you know QuEChERS was made to be modified” video
- "QuEChERS Sample Preparation Procedures"
- "QuEChERS Made Even Easier"
- “QuEChERS-Where to start”
- “The QuEChERS Method- Background Information and Recent Developments”, Anastassiades, CVUA Stuttgart, 2006, CRL-SRM, 1st Joint CRL-Workshop. https://www.eurl-pesticides.eu/library/docs/srm/1stws2006_lecture_anastassiades_QuEChERS.pdf
- “Dispersive Solid Phase Extraction for the Analysis of Veterinary Drugs Applied to Food Samples: A Review”, Islas et al, J. Analytical Chemistry. V.2017. 2017 Oct 18. doi: 10.1155/2017/8215271
Thanks for reading our discussion of high fat sample matrices. Please look for the next post on samples containing high amounts of sugars and starches.