Rapid evaporative ionization mass spectrometry: A survey through 15 years of applications.

Rapid evaporative ionization mass spectrometry (REIMS) is a relatively recent MS technique explored in many application fields, demonstrating high versatility in the detection of a wide range of chemicals, from small molecules (phenols, amino acids, di- and tripeptides, organic acids, and sugars) to larger biomolecules, that is, phospholipids and triacylglycerols. Different sampling devices were used depending on the analyzed matrix (liquid or solid), resulting in distinct performances in terms of automation, reproducibility, and sensitivity. The absence of laborious and time-consuming sample preparation procedures and chromatographic separations was highlighted as a major advantage compared to chromatographic methods. REIMS was successfully used to achieve a comprehensive sample profiling according to a metabolomics untargeted analysis. Moreover, when a multitude of samples were available, the combination with chemometrics allowed rapid sample differentiation and the identification of discriminant features. The present review aims to provide a survey of literature reports based on the use of such analytical technology, highlighting its mode of operation in different application areas, ranging from clinical research, mostly focused on cancer diagnosis for the accurate identification of tumor margins, to the agri-food sector aiming at the safeguard of food quality and security.

A miniaturized comprehensive approach for total lipidome analysis and vitamin D metabolite quantification in human serum.

The balance between the different lipid molecules present in biological fluids accurately reflects the health state of the organism and can be used by medical personnel to finely tune therapy to a single patient, a process known as precision medicine. In this work, we developed a miniaturized workflow for the analysis of different lipid classes at the intact level, as well as their fatty acid constituents, starting from human serum. Fatty acids were identified by using flow-modulated comprehensive gas chromatography coupled to mass spectrometry (FM-GC × GC-MS), and their relative amount as well as the ratio of specific FA classes was determined by using FM-GC × GC with a flame ionization detector. Ultra-high-performance liquid chromatography coupled to tandem mass spectrometry was used for the simultaneous quantification of vitamin D metabolites and assessment of different intact lipid classes. An MRM method was developed for the quantification of five vitamin D metabolites (vitamin D2, vitamin D3, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 24R,25-dihydroxyvitamin D3), and validated in terms of LoD, LoQ, accuracy, and precision, also using a certified reference material. At the same time, a combination of SCAN, precursor ion scan, and neutral loss scan, in both positive and negative modes, was used for the identification of 81 intact lipid species, such as phospholipids, cholesteryl esters, and triacylglycerols, in less than 25 min. In order to easily monitor the lipid composition and speed up the identification process, a two-dimensional map of the lipidome was generated, by plotting the molecular weight of the identified molecules versus their retention time. Moreover, a relative quantification was performed within each lipid class identified. The combination of untargeted and targeted data could provide useful information about the pathophysiological condition of the organism and evaluate, in a tailored manner, an efficient action.

Direct analysis in real time of high-quality extra virgin olive oils for the rapid and automatic identification of origin trademark.

BACKGROUND: Following our previous research on the differentiation of Italian extra virgin olive oils (EVOOs) by rapid evaporative ionization mass spectrometry coupled to a tandem high resolution mass analyser, the present study deals with the evaluation of another direct mass spectrometry (direct-MS) approach for the rapid and automatic discrimination of EVOOs. In particular, direct analysis in real time (DART-MS) was explored as an ambient MS (AMS) source for the building of a top-quality Italian EVOOs database and fast identification of unknown samples. A single quadrupole detector (QDa) was coupled with DART, taking advantage of a cost-saving, user-friendly and less sophisticated instrumental setup. Particularly, quickstrip cards, located on a moving rail holder, were employed, allowing for the direct analysis of 12 EVOO spots in a total analysis time of 6 min. The aim was to develop a reliable statistical model by applying principal component and linear discriminant analyses to clusterize and classify EVOOs according to geographical origin and cultivar, as main factors determining their nutritional and sensory profiles. RESULTS: Satisfactory results were achieved in terms of identification reliability of unknown EVOOs, as well as false positive risk, thus confirming that the use of AMS combined with chemometrics is a powerful tool against fraudulent activities, without the need for mass accuracy data, which would increase the analysis cost. CONCLUSION: A DART ionization source with a compact and reliable QDa MS analyser allowed for rapid fingerprinting analysis. Furthermore, MS spectra provided quali-quantitative information successfully related to EVOO differentiation. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Non-psychoactive cannabinoids identification by linear retention index approach applied to a hand-portable capillary liquid chromatography platform.

The aim of the present research was the application of the linear retention index (LRI) system for the identification of non-psychoactive cannabinoids using a portable LC instrument. The miniaturization, viz. the use of very low quantities of mobile phase, enabled the development of a compact mobile system to be used for in situ analysis, also according to a green and cost-saving approach. In particular, new capillary LC (cap-LC) methods coupled with UV detection were developed for the analysis of extracts of Cannabis sativa L. Two setups were explored to achieve the efficient separation of twenty-four cannabinoids: a single column setup which exploited a sub-2 µm packing to increase the chromatographic resolution, and a dual-column setup based on the serial connection of two different stationary phases, each coupled to an UV detector. The latter allowed the determination of two LRI values for each analyte, thus increasing the identification power. Moreover, since two different wavelengths were used on the LED-based UV detectors, the ratio of the absorbances measured on each chromatographic trace represented a third identification criterion, thus fulfilling the recommendations of the Scientific Working Group for The Analysis of Seized Drugs (SWDRUG) about the categories of analytical techniques to be used and the minimum number of parameters required for the unambiguous identification of drugs. The obtained results could be used for the development of a novel analytical method for fast and automatic in situ forensic investigations and hemp breeding programs, also minimizing the consumption of both sample and solvent.

Rapid Evaporative Ionization Mass Spectrometry-Based Lipidomics for Identification of Canine Mammary Pathology.

The present work proposes the use of a fast analytical platform for the mass spectrometric (MS) profiling of canine mammary tissues in their native form for the building of a predictive statistical model. The latter could be used as a novel diagnostic tool for the real-time identification of different cellular alterations in order to improve tissue resection during veterinary surgery, as previously validated in human oncology. Specifically, Rapid Evaporative Ionization Mass Spectrometry (REIMS) coupled with surgical electrocautery (intelligent knife-iKnife) was used to collect MS data from histologically processed mammary samples, classified into healthy, hyperplastic/dysplastic, mastitis and tumors. Differences in the lipid composition enabled tissue discrimination with an accuracy greater than 90%. The recognition capability of REIMS was tested on unknown mammary samples, and all of them were correctly identified with a correctness score of 98-100%. Triglyceride identification was increased in healthy mammary tissues, while the abundance of phospholipids was observed in altered tissues, reflecting morpho-functional changes in cell membranes, and oxidized species were also tentatively identified as discriminant features. The obtained lipidomic profiles represented unique fingerprints of the samples, suggesting that the iKnife technique is capable of differentiating mammary tissues following chemical changes in cellular metabolism.

Elucidation of the Lipid Composition of Hemp (Cannabis sativa L.) Products by Means of Gas Chromatography and Ultra-High Performance Liquid Chromatography Coupled to Mass Spectrometry Detection.

The growing demand in natural matrices that represent a source of dietary and nutraceutical molecules has led to an increasing interest in Cannabis sativa, considered to be a multipurpose, sustainable crop. Particularly, the considerable content in essential fatty acids (FAs) makes its derived-products useful food ingredients in the formulation of dietary supplements. In this research, the FA and triacylglycerol (TAG) composition of hempseed oils and flours were investigated using gas chromatography coupled to mass spectrometry and flame ionization detection as well as liquid chromatography coupled to mass spectrometry (LC-MS), respectively. Furthermore, a recently introduced linear retention index (LRI) approach in LC was successfully employed as a useful tool for the reliable identification of TAG species. A total of 30 FAs and 62 glycerolipids were positively identified in the investigated samples. Relative quantitative analyses confirmed linoleic acid as the most abundant component (50-55%). A favorable omega6/omega3 ratio was also measured in hemp-derived products, with the α-linolenic acid around 12-14%. Whereas, γ-linolenic acid was found to be higher than 1.70%. These results confirm the great value of Cannabis sativa as a source of valuable lipids, and the further improvement of the LRI system paves the way for the automatization of the identification process in LC.

Identification of high-value generating molecules from the wastes of tuna fishery industry by liquid chromatography and gas chromatography hyphenated techniques with automated sample preparation.

The present work aims to a promising re-utilization of the massive waste derived from the tuna fishing industry, for which by-products can represent more than 50% of the original material. Due to the considerable content in polyunsaturated fatty acids and noble proteins, such wastes can be used as primary source of functional ingredients in the production of nutraceuticals. The composition of the lipid and protein tuna fractions was investigated by means of gas chromatography-mass spectrometry and high-performance liquid chromatography-tandem mass spectrometry methods (in wastes and edible parts), and a preliminary characterization of potential bioactive peptides was achieved. Automated sample preparation allowed speeding up the analytical workflow, while allowing for highly sensitive and selective lipid characterization. The ω3 fatty acid content was found higher in waste products compared to the muscle, in terms of fatty acids as well as complex lipids. As for peptides, extraction by isoelectric solubilization/precipitation was performed, followed by enzymatic digestion and high-performance liquid chromatography-tandem mass spectrometry analysis. Furthermore, the use of bioinformatics tools highlighted the presence of potential antimicrobial peptides in the samples investigated.

Miniaturized LC in Molecular Omics.

Miniaturized LC has evolved at an exponential rate over the last 50 years. In the past decade, it has received considerable attention in the field of bioanalytical separation science and technology due to the need to measure different classes of biomolecules present in a variety of matrixes on a global scale to gain a deeper understanding of complex biological processes. This field has become a dominant area underpinning the molecular omics research (e.g., proteomics, metabolomics, lipidomics, and foodomics), allowing key insights into the function and mechanism of small to very large biomolecules on a molecular level. This Feature highlights the recent advances in molecular omics focusing on miniaturized LC technology combined with mass spectrometry-based platforms, with a particular emphasis on the strategies adopted and applications using new and sensitive nanoscale analytical methodologies.

Rapid and miniaturized qualitative and quantitative gas chromatography profiling of human blood total fatty acids.

The qualitative and quantitative profiling of fatty acids in human blood is a useful tool in disease prevention and health care, two concepts that are intimately related. In fact, fatty acid (FA) analysis can provide in-depth information on a specific metabolic state of individuals. The goal of the present research consisted of the development of a rapid and miniaturized analytical strategy for the complete characterization of the fatty acid profile in human blood. Sample collection was carried out by using the dried blood spot approach, while fatty acid derivatization to methyl esters was performed directly by using sodium methoxide and boron trifluoride. The following figures of merit were defined: intra- and inter-day repeatability, linearity range, limits of detection, and quantification. Additionally, the accuracy of the developed method was evaluated in the analysis of a certified reference human plasma sample. Apart from blood, the analytical procedure was also applied to samples of human serum and plasma. During the final stage of the research, the developed analytical method was performed in a fully automated manner. Graphical abstract.

Evaluation of matrix effect in one-dimensional and comprehensive two-dimensional liquid chromatography for the determination of the phenolic fraction in extra virgin olive oils.

Olea europaea, meaning "European olive," is a small tree belonging to the family Oleaceae, occurring in the Mediterranean Basin. Olive oil is an essential component of a balanced diet because of its nutritional value. Among micronutrients, phenolic compounds did show important beneficial effects for human health. The majority of the research studies on the phenol content are carried out by liquid chromatography combined to photodiode array and/or mass spectrometry detection; however, because of matrix complexity, one-dimensional liquid chromatography cannot be sometimes sufficient to obtain rewarding separations, requiring more advanced analytical techniques. In this work, comprehensive two-dimensional liquid chromatography, incorporating RP-Amide and C18 stationary phases, in the first and second dimension, respectively, both under reversed phase conditions, was investigated for the determination of the phenolic fraction in extra virgin olive oil samples. As far as detection is concerned, triple quadrupole mass spectrometry was employed under multi reaction monitoring mode offering superior selectivity and sensitivity. The reduction of matrix effects, when using comprehensive two-dimensional liquid chromatography with respect to conventional one-dimensional liquid chromatography, was assessed by comparing the slopes of calibration curves built from standard solutions and spiked olive oil samples.

Lipid profile of fish species by liquid chromatography coupled to mass spectrometry and a novel linear retention index database.

In a previous article, Rigano et al. established a new linear retention index system for the identification of triacylglycerols by liquid chromatography methods only on the basis of the retention behavior and independently from many experimental parameters. In that work, a database of 209 compounds was built, but only 54 of them, typical of vegetable oils, were confirmed by mass spectrometry. The aim of the present research is to extend the applicability of the novel approach to more complex samples, such as fish lipid extracts, and assess the complementarity between mass spectromtery and retention information to achieve univocal identification. With this purpose, a new software was implemented to make the identification process easy and automatic as in gas chromatography-mass spectrometry where the retention index filter is added in the spectral search to discriminate between compounds with similar mass spectrometry spectra. A total of 69 species were identified and, thanks to their baseline separation obtained by an ultra high performance liquid chromatography method, a semiquantification was also performed. The species under investigation were Dicentrarchus labrax, coming from aquaculture and the wild. Some differences in their native lipid composition were observed, probably related to a different diet. A major number of samples would be necessary to confirm such a preliminary finding.

Exploration of Rapid Evaporative-Ionization Mass Spectrometry as a Shotgun Approach for the Comprehensive Characterization of Kigelia Africana (Lam) Benth. Fruit.

Rapid evaporative-ionization mass spectrometry (REIMS) coupled with an electroknife as a sampling device was recently employed in many application fields to obtain a rapid characterization of different samples without any need for extraction or cleanup procedures. In the present research, REIMS was used to obtain a metabolic profiling of the Kigelia africana fruit, thus extending the applicability of such a technique to the investigation of phytochemical constituents. In particular, the advantages of REIMS linked to a typical electrosurgical handpiece were applied for a comprehensive screening of this botanical species, by exploiting the mass accuracy and tandem MS capabilities of a quadrupole-time of flight analyzer. Then, 78 biomolecules were positively identified, including phenols, fatty acids and phospholipids. In the last decade, Kigelia africana (Lam.) Benth. fruit has attracted special interest for its drug-like properties, e.g., its use for infertility treatments and as anti-tumor agent, as well as against fungal and bacterial infections, diabetes, and inflammatory processes. Many of these properties are currently correlated to the presence of phenolic compounds, also detected in the present study, while the native lipid composition is here reported for the first time and could open new directions in the evaluation of therapeutic activity.

Rapid evaporative ionization mass spectrometry coupled with an electrosurgical knife for the rapid identification of Mediterranean Sea species.

The topic of food analysis and safety has attracted increasing interest in recent decades owing to recent scandals concerning fraudulent activities (mislabeling, sophistication, adulteration, etc.) that can undermine human health. Among them, seafood fraud has probably the strongest relationship with food safety, an activity that goes beyond economic interests. This article explores the capabilities of an innovative instrumental setup, called the "iKnife," as a powerful tool in this specific research area, where until now genomics and proteomics have been the workhorses in analytical approaches. iKnife, which means "intelligent knife," is the name of a recent technology based on rapid evaporative ionization mass spectrometry (REIMS). REIMS is an emerging technique able to characterize different samples rapidly, affording a comprehensive profile usable as a fingerprint, without the need for preliminary extraction or cleanup procedures. In detail, a REIMS source is coupled to a high-resolution tandem mass spectrometer; such coupling allows one to maximize the amount of information (discriminant features) collected for a single analysis, as well as to focus on target analytes to achieve enhanced sensitivity and selectivity. A database was created from 18 marine species typical of the Mediterranean Sea, all caught in the very small area of the Strait of Messina, and reliable identification was achieved for each species with confidence higher than 99%. One big model and three submodels were built by principal component analysis and linear discriminant analysis for unambiguous key variable identification within each class (e.g., Cephalopoda), order (e.g., Perciformes), or family (e.g., Carangidae). Graphical abstract.

Determination of the polyphenolic fraction of Pistacia vera L. kernel extracts by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry detection.

Pistachio (Pistacia vera L.) belongs to the Anacardiaceae family and it is a small tree species. It is native of the Middle East and Central Asia, but currently, it is cultivated also in California and in some Mediterranean countries, such as Greece and Italy. The most important pistachio producers are Iran, the USA, and Turkey. Besides being a delicious nut, pistachio, due to its wholesome nutritional properties, could be considered as a functional food. According to the results of several studies, pistachios have been proven to have various groups of valuable phytochemicals such as anthocyanins, flavan-3-ols, proanthocyanidins, flavonols, isoflavones, flavanones, stilbenes, and phenolic acids, possessing excellent biological activities. The most common analytical technique employed for their analysis is represented by liquid chromatography coupled to photodiode array and mass spectrometry detection. However, conventional LC can present some limits especially in terms of resolving power. In this contribution, as a powerful alternative, comprehensive two-dimensional liquid chromatography (LC×LC) was applied to the determination of the polyphenolic fraction of pistachio kernels from different geographical origins. A 150-mm micro-bore cyano column (2.7 µm dp) and 50-mm superficially porous C18 silica column (2.7 µm dp) in the first (1D) and second (2D) dimensions were employed, respectively. For boosting orthogonality, a shift 2D gradient was investigated leading to an increase in the overall peak capacity. The newly developed LC×LC method showed satisfactory linearity, sensitivity, precision and accuracy, which was then applied to sample quantitative analysis. A total of 51 different polyphenolic compounds were determined in the four samples investigated and 18 out of them are hereby reported for the first time.

The Contribution of Carotenoids, Phenolic Compounds, and Flavonoids to the Antioxidative Properties of Marine Microalgae Isolated from Mediterranean Morocco.

This study aimed to investigate the potential of four sea water microalgae, isolated and cultivated at M'diq Bay in Morocco, as a new source of natural antioxidants. These microalgae belong to different classes, including Phaedactylium tricornitum (Bacillariophyceae), Nannochloropsis gaditana (Eustigmatophyceae), Nannochloris sp (Trebouxiophyceae), and Tetraselmis suecica (Chlorodendrophycea). The antioxidant properties were screened by the use of in vitro assays, namely 2,2-difenil-1-picrylhydrazyl, Ferric reducing antioxidant power, and Ferrous ions chelating activity, and compoundidentification was carried out in methanol and acetone extracts of both dried and fresh microalgae biomass by HPLC-PDA-MS analysis. Among the investigated microalgae, Phaedactylium tricornutum was the richest one regarding its carotenoid (especially all-E-fucoxanthin) and phenolic (especially protocatechuic acid) contents, as well as antioxidant activity (65.5%), followed by Nannochloris sp, Tetraselmis suicica, and Nannochloropsis gaditana, with antioxidant activity of 56.8%, 54.9%, and 51.1%, respectively.

Proposal of a Linear Retention Index System for Improving Identification Reliability of Triacylglycerol Profiles in Lipid Samples by Liquid Chromatography Methods.

The retention index system was proposed in 1958 by Kováts. It is based on the correlation between the retention time of the analytes and the ones of a series of reference standards, making retention data dependent on the chromatographic phenomenon only, viz., on the three-term interaction analyte-stationary phase-mobile phase. Particularly in GC, because the mobile phase has a negligible influence, the retention of the analytes depends almost entirely on the stationary phase and it is as independent as possible from operating conditions. This makes retention index databases usable in samples identification at both the intra- and interlaboratory levels. The scope of this research was to create a similar identification system in LC, where the identification of unknowns is still a challenge because of the unavailability of spectral databases related to the low reproducibility of mass spectra generated by means of the atmospheric pressure ionization technique normally interfaced to LC. Lipid compounds, specifically triacylglycerols, were selected as target analytes mainly due to their regular chromatographic LC profile under reversed phase conditions. The odd carbon chain number triacylglycerol series from trinonain to trinonadecanoin was chosen as the basis of the retention index scale and a database of 209 triacylglycerols was built. For this purpose an ultra high-performance LC method, able to maximize the baseline separation of triacylglycerols in different real-world samples (vegetable oils, fish, and milk samples) was developed.

Novel comprehensive multidimensional liquid chromatography approach for elucidation of the microbosphere of shikimate-producing Escherichia coli SP1.1/pKD15.071 strain.

Shikimic acid is a intermediate of aromatic amino acid biosynthesis and the preferred starting material for production of the most commonly prescribed anti-influenza drug, Tamiflu. Its six-membered carbocyclic ring is adorned with several chiral centers and various functionalities, making shikimic acid a valuable chiral synthon. When microbially-produced, in addition to shikimic acid, numerous other metabolites are exported out of the cytoplasm and accumulate in the culture medium. This extracellular matrix of metabolites is referred to as the microbosphere. Due to the high sample complexity, in this study, the microbosphere of shikimate-producing Escherichia coli SP1.1/pKD15.071 was analyzed by liquid chromatography and comprehensive two-dimensional liquid chromatography coupled to photodiode array and mass spectrometry detection. GC analysis of the trimethylsilyl derivatives was also carried out in order to support the elucidation of the selected metabolites in the microbosphere. The elucidation of the metabolic fraction of this bacterial strain might be of valid aid for improving, through genetic changes, the concentration and yield of shikimic acid synthesized from glucose. Graphical abstract.

Comprehensive lipid profiling in the Mediterranean mussel (Mytilus galloprovincialis) using hyphenated and multidimensional chromatography techniques coupled to mass spectrometry detection.

The task of lipid analysis and profiling is taking centre stage in many research fields and as a consequence, there has been an intense effort to develop suitable methodologies to discover, identify, and quantify lipids in the systems investigated. Given the high complexity and diversity of the lipidome, researchers have been challenged to afford thorough knowledge of all the lipid species in a given sample, by gathering the data obtained by complementary analytical techniques. In this research, an "omic" approach was developed to quickly fingerprint lipids in the Mediterranean mussel (Mytilus galloprovincialis), by exploiting multidimensional and hyphenated techniques. In detail, two-dimensional comprehensive hydrophilic interaction liquid chromatography coupled to reversed-phase liquid chromatography afforded both class-type separation and lipid assignment within the total lipid species in the sample, by the coupling of a 2.1-mm I.D. partially porous stationary phase in the first dimension, to a short (50 mm) monodisperse octadecylsilica secondary column; individual molecular species were afterwards identified by means of their ion trap-time of flight mass spectra obtained by electrospray ionization. More than 200 neutral and polar lipids were identified, and among the latter, phosphatydylcholine and phosphatydylethanolamine were the most represented classes, together with their mono-acylated forms, plasmanyl and plasmenyl derivatives. Subsequently, separation of the saturated and unsaturated isomers of the fatty acids (including the saturated C16:0 and the polyunsaturated C22:6) in the offline collected phospholipid fractions was accomplished by gas chromatography analysis of the corresponding methyl esters, on a 200 m × 0.25 mm, 0.2 µm d f ionic liquid column.

Monoacylglycerol and diacylglycerol production by hydrolysis of refined vegetable oil by-products using an immobilized lipase from Serratia sp. W3.

Unlike the synthetic surfactants, mono- and diacylglycerols have the advantage to be biodegradable and non-toxic. In the present work, the hydrolysis of lipid fraction by-products of refined vegetable oils was performed by Serratia sp. W3 lipase immobilized on CaCO3 by combined adsorption and precipitation. This support was selected out of four carriers as it exhibited the finest activity support (950 U/g) and the most satisfactory behavior at use. The immobilized preparation with CaCO3 was stable and active in the whole range of pH (4 to 9) and temperature (37 to 55°C), yielding a 75% degree of hydrolysis at optimal environmental conditions of pH 8.5 and temperature 55°C. Thin-layer chromatography, gas chromatography, and liquid chromatography methods were evaluated to determine the analytical characterization of hydrolysis products. For monoacylglycerols and diacylglycerol fractions identified in the samples, a novel approach by liquid chromatography method was employed, through a homemade linear retention index database and a dedicated software. The adopted approach allowed the use of basic instrumentation set-ups, without the need of sophisticated detectors, such as mass spectrometers. Thus, it could be an effective alternative to produce emulsifiers from cheap vegetable oils.

Nano Liquid Chromatography Directly Coupled to Electron Ionization Mass Spectrometry for Free Fatty Acid Elucidation in Mussel.

Recently the miniaturization of liquid chromatography (LC) systems and progresses in mass spectrometry instrumentation have enabled direct introduction of the effluent coming from a nanoLC column into the high-vacuum region of an electron ionization source. In the present research, a nanoLC system was directly coupled to an electron ionization mass spectrometer (EI-MS) without any interface or modification of the ion source. The advantage with respect to atmospheric pressure ionization techniques, normally coupled with LC, is major identification power because of a more extensive and reproducible fragmentation pattern, without any matrix effect or mobile-phase interference. In particular, a nanoLC/EI-MS method was developed for elucidation of the free fatty acid profile in mussel samples, avoiding a previous derivatization step, required when gas chromatographic analysis is involved. A total of 20 fatty acids were reliably identified through the comparison with commercial libraries. A quantitative determination was also carried out by using the response factors approach along with the internal standard method, allowing for quantification of 14 fatty acids. Among them, palmitic acid resulted the most abundant, followed by ω6 arachidonic acid. The quantitative data were compared with those obtained by a well-established technique, such as gas chromatography with flame ionization detection (GC-FID). Both nanoLC/EI-MS and GC-FID methods were validated and similar results were obtained in terms of limit of detection and quantification, resulting in the picomole range, and sensitivity as well was not significantly different, as demonstrated by comparing the slope values of the calibration curves (p < 0.05, from a t-test).