An article by JB Renaud, JP Walsh, and MW Sumarah at two institutions in Canada was published in the September 2022 edition of Toxins. The research group developed a reference material for monitoring chronic exposure to Aflatoxin B1, a carcinogen produced by some mold species, which can cause liver cancer. Their paper was entitled “Optimization of Aflatoxin B1-Lysine Analysis for Public Health Exposure Studies.”
In March 2022, we reported on a study published by researchers at the University of Ghent on the measurement of 24 mycotoxins from dried blood Mitra microsamples. This study demonstrated sample stability on the Mitra microsampling device, even when compared to liquid blood samples. No cases of mycotoxin exposure were missed when testing 20 dried blood microsamples. Indeed, the authors of the study stated, “VAMS sampling can serve as an excellent alternative to conventional venous sampling to perform a quantitative screening of mycotoxin exposure.”
The cause of the mutagenicity of AFB1 is well understood. Ironically, it is due to the action of natural detoxification by the cytokine P450 system in the liver, leading to an unstable epoxide moiety of the toxin. This epoxide moiety then reacts with guanidine moieties on DNA molecules and, as a result, inter-chelates with DNA, which then leads to mutation events.
As well as binding to DNA, the AFB1 epoxide, which readily hydrolyses to a dialdehyde, covalently binds to lysine moieties on proteins. For example, it is thought that AFB1 reacts specifically with two specific lysine residues on human serum albumin (HSA) and, as a result, can be exploited as a biomarker for chronic exposure studies of the toxin.
The specific nature of the biomarker is the lysine adduct (AFB1-Lys), which is released by ex vivo enzymatic proteolytic activity using pronase (a mixture of proteases) in the sample preparation event.
One of the most popular analytical techniques for analysis of AFB1-Lys is LC-MS/MS. It should be noted that there are several challenges to ensuring reliable assays are developed. The first challenge is making sure that sample cleanup us sufficiently effective that ion suppression / enhancement (SSE%) is minimized.
Another challenge is the need for effective analytical standards. Finally, the cold-chain shipping of protein samples (blood samples) can be cost prohibitive, so alternative methods for cheaper, simpler transport and storage are in demand.
It was these challenges that motivated Renaud et al to develop an AFB1-serum albumin (SA) reference material and to compare the results of using this SA on dried matrix microsamples vs. wet serum.
The study authors were able to create and characterize an AFB1-Lys adduct serum albumin reference material to be used in method development and validation. Even though the Mitra-VAMS devices showed promise, the researchers determined that they needed a sample volume of around 100 µL, which was more volume than the 20 µL VAMS tips on the Mitra devices provided. The authors report that they plan to use the method for interlaboratory comparisons.
This paper demonstrates the importance of understanding every step in a development process. The use of two separate IS to control both rates of degradation and SSE% is a perfect example of such a control. Moreover, the volumetric nature of the Mitra devices based on VAMS are a practical improvement over traditionally collected DBS samples, as has been shown in many other studies.
Finally, one possible solution to overcoming the limited volume observation would be to collect 3-4 30 µL Mitra microsamples and pool the digestates. Because there was a concentration step built into the sample preparation, it is conceivable that pooled digestates from 3-4 microsamples may solve this limitation.
This article was summarized for our readers by James Rudge, PhD, Microsampling Technical Director, Trajan. This is curated content. To learn more about the important research outlined in this blog, visit the original article in Toxins.
Image Credits: iStock, Trajan