# MALDI-TOF Mass Spectrometry Applications for Food Fraud Detection
## 2 MALDI Applications
### Oils  
Olive oil is one of the main ingredients in the Mediterranean diet [56]. The International Olive Council defines two categories of olive oil [57], the first comprising oils suitable for consumption, such as extra-virgin, virgin, and ordinary olive oil, the second comprising oils that must be further processed prior to ingestion. Extra-virgin olive oil (EVOO) is the most valuable, since it is obtained from olive fruits using mechanical processes or other physical means [58] that lead to a product of unmatched value, characterized by unique features, such as nutritional quality, health benefits, and pleasant flavor. These features make EVOOs expensive products that are continuously counterfeited in many countries [59] with oils produced from cheaper fruits/seeds or with other lower quality olive oils or even mislabeling virgin/refined olive oils. Of course, analytical methods are crucial for detecting EVOO frauds [60] and many scientists are committed to the development of MALDI-TOF MS applications. A matrix-less laser desorption/ionization (LDI) approach using a stainless-steel target plate was proposed [61] for the fast analysis of diluted soy, sunflower, and extra-virgin olive oil samples. MS spectra characterized by oil-specific profiles and free of MALDI-matrix peaks were obtained, allowing the easy discrimination of the oil under study. Most of the \(m/z\) ions present in the spectrum were easily attributed to tri/diacylglycerols compounds, some of which were found to be diagnostic ions for olive (\(m/z\) 907.77) and sunflower oil (903.79 and 901.78), thus permitting the detection of adulteration of olive with sunflower oil. All the diagnostic ions were potentially attributable to different compounds, namely OOO, SOL, SLO, LSO (907.77), OLL, LOL, SLLn, LSLn, SLLn, OOLn, OLnO (903.79), LLL, OLLn, OLn, and LOLn (901.78), with O being oleic acid, S, stearic acid, L, linoleic acid, and Ln, linolenic acid. Over the following years, the same research group published three more papers focused on the development of MALDI methods for the challenging detection of hidden hazelnut oil in extra virgin olive oil, each time using different classes of compounds as markers for the adulteration. In the first work [62], the polar fraction of oils was enriched and characterized using hydrophilic liquid chromatography micro-columns coupled with MALDI. Lysophosphatidylcholine (LPC) (16:0/0:0), LPC (18:1/0:0), and LPC (18:2/0:0) were identified and used as diagnostic compounds for the presence of hazelnut oil in EVOO to a level of 5%. The second application [63] exploited a modified Bligh-Dyer method for the selective extraction of phospholipids, present in seed oils at much higher concentration levels and then used as markers for hazelnut in olive oil. The solution resulting from the combination of \(\alpha\)-Cyano-4-hydroxycinnamic acid (176 mM) and tributylamine (equimolar) was used as both extraction solvent and MALDI matrix. The method was capable to detect adulterations at a 1% contamination level. The last work [64] used cold acetone precipitation followed by in-solution tryptic digestion and final MALDI analysis for the revealing of hazelnut peptide markers arising from the main hazelnut proteins Cor a 9, Cor a 11, and Cor a 1. These markers can potentially be used for the determination of hazelnut traces in oils and processed foods. SDS-PAGE analysis confirmed the presence of hazelnut proteins in hazelnut extracts with molecular masses in the range of 10-60 kDa. [PERSON] and colleagues [65] reported that the adulteration of extra virgin olive oil with solvent-extracted hazelnut oil can be directly traced down to a 1% level by SDS-PAGE analysis of hazelnut proteins, even if a MALDI analysis was indeed necessary to confirm the identity of the alleged allergens, i.e., two oleosin isoforms and Cor a 9. MALDI-TOF, combined with unsupervised hierarchical clustering, principal component analysis, and [PERSON]'s correlation analysis, was also successfully adopted [66] to detect very low amounts (0.5%) of corn oil in EVOO.