Detection of Regulated Ergot Alkaloids in Food Matrices by Liquid Chromatography-Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometry

The aim of our group is the proposal of new analytical methodology that enables an efficient control of contaminant and residues in different matrices through the unique cubicle identification of compounds. We tried to solve some challenges related to food safety using advanced analytical platforms and sustainable sample treatment. Recent research of mycotoxins highlight advancements in detection methods as massive spectrometry and biosensors.

It also explores the health impact and strategies for contamination reduction, aiming to improve food safety and the develop efficient detoxification processes in agricultural practices. Mass spectrometry is key for detecting chemical residuals and contaminants, including mycotoxins in food. Recent advances in mass spectrometry have shift research from target to non-target analytical method, with the commercialization of ion mobility, high-resolution mass spectrometry contributing to this growing trend.

It allows the analytical quantification of each regulated ergot alkaloid. The chromatographic analysis of these compounds may lead to misquantification, especially if the peaks are not resolved enough. So the implementation of ion mobility spectrometry provide another dimension to quantify them based on their mobility values.

Our interest in future research is not only to continue addressing the control of emerging contaminant in foods, but also to focus on exposomic and human biomonitoring. It is crucial to understand the connection between environmental exposure and health, measuring chemicals, toxin, and other metabolites in biological fluids. To begin, prepare two 1.5-milliliter amber vials with 500 microliters of intermediate solution, and another vial with a mixture of solvents as washing solution.

While conditioning the liquid chromatography column, write the work list, including the two solutions. In the method file column of the work list, load the acquisition method file, saved with the required parameters. Create a folder to save all acquired data files.

Route all samples to the selected folder in the sample path column. Go to the upper toolbar of the software and run the work list. Once the work list is executed, open the qualitative software.

To load all the analyzed samples, select file, followed by open work list. Right click on a sample corresponding to the EA solution. Go to edit chromatogram, then select type, followed by extracted ion chromatogram.

Enter the theoretical molecular mass of the protonated ion for each EA, then click add and OK.Six extracted ion chromatograms will appear, corresponding to six EAs and their epimers. In each extracted ion chromatogram, two peaks corresponding to the main EA and its epimer will appear. Right click while selecting the whole area of one peak, and the ion mobility spectrum will pop up.

Go to the x axis of the ion mobility spectrum, right click, and click on collision cross section. If the CCS value does not appear, right click on the mobility spectrum and select copy to mobilogram, then right click again on the mobility spectrum and select assigned charge state. Introduce the exact mass and the charge of the ion.

Then based on fragmentation data from literature and databases, compare and select the most intense product ion as a complimentary identification point. Note the retention time, CCS value, and exact mass of the main ion adduct for each EA to create a quantitation method. Next, open the data processing software and click on the method management tab, then click on analyte settings.

Enter the name of each EA alongside its retention time, CCS value, and the mass of the protonated adducts. Adjust the tolerance for each identification point as necessary while using the default settings. To begin, condition the liquid chromatography column and configure the acquisition parameters.

Weigh one gram of sample in a 50-milliliter centrifuge tube. Add four milliliters of the extraction solution, and vortex the sample for one minute. Centrifuge the sample for five minutes at 9, 750 G and four degrees Celsius.

Transfer the entire supernatant to a 15-milliliter centrifuge tube containing 150 milligrams of sorbent mixture, and vortex the sample for one minute. After centrifuging the sample, collect the supernatant and transfer it to a four-milliliter amber glass vial. Evaporate the extract under a gentle stream of nitrogen, then reconstitute the sample in 750 microliters of methanol water mixture.

Using a two-milliliter syringe, filter the sample through a 0.22-micrometer nylon filter into a 1.5-milliliter amber chromatographic vial. To begin, prepare the ergot alkaloid, or EA samples, and obtain the matrix-matched calibration curve using liquid chromatography. Open the quantitative software and navigate to the task quick tab.

Click on import batch. Once the batch is imported, click on process batch below import batch. After data processing is complete, go to review screening and check for issues related to automatic integration, such as incorrect chromatographic peaks being too close to each other.

If so, narrow or widen the values referring to retention time tolerance, CCS value tolerance, and/or mass error initially established in the quantitative method file, and reprocess the data. If automatic peak integration still selects incorrect peaks, manually select the area in the chromatogram that appears in the review screening window. In the batch management tab, navigate to the batch concentration window, and specify a concentration value for each level established earlier.

Once done, click save concentrations. Click on quantify batch, and adjust the parameters such as the model to fit calibration data, waiting, and forcing the model to cross zero. Once the quantification of ergot alkaloids is complete, click on the quantitation tab, then right click on the batch folder at the far left and select generate report.

Finally, choose analyte quantitation and export the report in the desired format. Chromatographic separation revealed clear peaks for each EA epimer pair except for ergotamine and ergotamine, which showed slight overlap due to their close retention times. Ion mobility spectrometry allowed baseline separation of ergotamine and ergotamine, providing distinguishable CCS values.

Signal suppression or enhancement effect analysis showed minimal matrix interference with a minus 32%to 18%range, allowing accurate quantification using the standard calibration curve, except for ergametranine that required from the matrix-matched calibration curve. Method accuracy was confirmed by recovery rates for all analytes within 72 to 117%except for ergotamine, which showed recovery values of 43 to 45%Limit of quantification values ranged from 0.65 to 2.6 nanograms per milliliter, confirming the method sensitivity.