Modifying QuEChERS for complicated matrices- High Fat Samples

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:

QuEChERS dSPE selection-which one is best?

Modifying QuECHERS for complicated matrices- Dry Samples

(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 to use the following for reference

From the official QuEChERS website (, maintained by CVUA Stuttgart, these specific documents:

For Extraction (Stage 1):

For dSPE Cleanup (Stage 2):

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.

And again, here is a link to the USDA database at 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
MgSO4 excess water
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 are some examples of this technique using PSA/MGSO4/C18-EC.


Cooling/Freezing the QuEChERS extract– As described on the QuEChERS website mentioned earlier, (, 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.


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 are some examples of this technique.


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.


Additional Resources:


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.


Tags: , , , , ,

2 Responses to “Modifying QuEChERS for complicated matrices- High Fat Samples”

  1. Tim Strutt says:

    Hi Nancy,
    A look at recent work be Steven Lehotay (USDA, the original lead author of QuEChERS) et al, might useful for analysts who are required to carry out automated analysis of pesticides etc. in a a wide range of matrices. A recent paper is here:
    (High-Throughput Mega-Method for the Analysis of Pesticides, Veterinary Drugs, and Environmental Contaminants by Ultra-High-Performance Liquid Chromatography−Tandem Mass Spectrometry and Robotic Mini-Solid-Phase Extraction Cleanup + Low-Pressure Gas Chromatography−Tandem Mass Spectrometry, Part 1: Beef – Sergio H. Monteiro, Steven J. Lehotay*, Yelena Sapozhnikova, Ederina Ninga, and Alan R. Lightfield Publication Date:May 22, 2020

    The UPLC-MS/MS method just requires ultracentrifugation and automated solvent exchange of a small aliquot of the MeCN/H2O extract with eluent, the GC–MS/MS method takes the remaining solution and then uses salting-out and Automated μ-SPE ITSP Cleanup (Instrument Top Sample Prep) with a CTC-PAL autosampler.


  2. Thanks for your input, Tim!

Leave a Reply

Restek Domestic Customer Service



Your Full Name

Your Email

Company Name


Spam Block (Please leave this blank)

all fields required

Thank you

Your message has been sent. We will be in touch shortly.

Message not sent

Sorry, your message could not be sent at this time. Please try again later, or contact Restek or your local Restek representative via phone.