An overview on lipids in nuts and oily fruits: oil content, lipid composition, health effects, lipidomic fingerprinting and new biotechnological applications of their by-products.

Tree nuts and oily fruits are used as a diet complement and are highly consumed worldwide. The production and consumption of these foods have been increasing, and an enormous global market value is forecasted for 2023. Besides their high nutritional value and lipid content, they provide health benefits to fat metabolism, heart, skin, and brain. The industrial by-products of these oily foods represent promising raw materials for many industries. However, the lipidomic analysis of nuts and oily fruits is still in its early stages. State-of-the-art analytical approaches for the lipid profiling and fingerprinting of nuts and oily fruits have been developed using high-performance liquid chromatography and high-resolution mass spectrometry for the accurate identification and structural characterization at the molecular species level. It is expected to bring a new understanding of these everyday foods' nutritional and functional value. This review comprises the oil content and lipid composition of various nuts and oily fruits, particularly those mostly consumed worldwide and having recognized beneficial health effects, biological activities associated with the lipids from different oily foodstuffs, analytical methodologies to analyze lipids in nuts and oily fruits, and the potential biotechnological applications of their industrial by-products for a lipid-based commercial valorization.

Unravelling the fatty acid profiles of different polychaete species cultured under integrated multi-trophic aquaculture (IMTA).

Polychaetes can be successfully employed to recover otherwise wasted nutrients present in particulate organic matter (POM) of aquaculture effluents. The present study describes the fatty acid (FA) profile of four different polychaete species cultured in sand filters supplied with effluent water from a marine fish farm. The FA profile of cultured and wild Hediste diversicolor was compared and revealed a ≈ 24.2% dissimilarity, with cultured biomass displaying a higher content in two essential n-3 highly unsaturated FA (HUFA) (EPA [20:5 n-3] and DHA [22:6 n-3]-eicosapentaenoic and docosahexaenoic acid, respectively). The comparison of the FA profile of cultured H. diversicolor with that of other polychaete species whose larvae successfully settled on the sand filters (Diopatra neapolitana, Sabella cf. pavonina and Terebella lapidaria) revealed that their FA profile, which is here described for the first time, displayed high levels of EPA and DHA (≈ 1.5-4.8 and 1.0-1.1 µg mg-1 DW, respectively). The highest concentration of total FA per biomass of polychaete was recorded in H. diversicolor and T. lapidaria, with both species being the ones whose FA profiles revealed a lowest level of dissimilarity and more closely resembled that of the aquafeed used in the fish farm. In the present work it was demonstrated that it is possible to produce polychaetes biomass with high nutritional value through an eco-design concept such as integrated multi-trophic aquaculture (IMTA). Indeed, this framework promotes a cleaner production and, in this specific case, allowed to recover essential fatty acids that are commonly wasted in aquaculture effluents.

Coping with Starvation: Contrasting Lipidomic Dynamics in the Cells of Two Sacoglossan Sea Slugs Incorporating Stolen Plastids from the Same Macroalga.

Several species of sacoglossan sea slugs are able to sequester chloroplasts from algae and incorporate them into their cells. However, the ability to maintain functional "stolen" plastids (kleptoplasts) can vary significantly within the Sacoglossa, giving species different capacities to withstand periods of food shortage. The present study provides an insight on the comparative shifts experienced by the lipidome of two sacoglossan sea slug species, Elysia viridis (long-term retention of functional chloroplasts) and Placida dendritica (retention of non-functional chloroplasts). A hydrophilic interaction liquid chromatography-mass spectrometry approach was employed to screen the lipidome of specimens from both species feeding on the macroalga Codium tomentosum and after 1-week of starvation. The lipidome of E. viridis was generally unaffected by the absence of food, while that of P. dendritica varied significantly. The retention of functional chloroplasts by E. viridis cells allows this species to endure periods of food shortage, while in P. dendritica a significant reduction in the amount of main lipids was the consequence of the consumption of its own mass to endure starvation. The large proportion of ether phospholipids (plasmalogens) in both sea slug species suggests that these compounds may play a key role in chloroplast incorporation in sea slug cells and/or be involved in the reduction of the oxidative stress resulting from the presence of kleptoplasts.

Insights on Ultrafiltration-Based Separation for the Purification and Quantification of Methotrexate in Nanocarriers.

The evaluation of encapsulation efficiency is a regulatory requirement for the characterization of drug delivery systems. However, the difficulties in efficiently separating nanomedicines from the free drug may compromise the achievement of accurate determinations. Herein, ultrafiltration was exploited as a separative strategy towards the evaluation of methotrexate (MTX) encapsulation efficiency in nanostructured lipid carriers and polymeric nanoparticles. The effect of experimental conditions such as pH and the amount of surfactant present in the ultrafiltration media was addressed aiming at the selection of suitable conditions for the effective purification of nanocarriers. MTX-loaded nanoparticles were then submitted to ultrafiltration and the portions remaining in the upper compartment of the filtering device and in the ultrafiltrate were collected and analyzed by HPLC-UV using a reversed-phase (C18) monolithic column. A short centrifugation time (5 min) was suitable for establishing the amount of encapsulated MTX in nanostructured lipid carriers, based on the assumption that the free MTX concentration was the same in the upper compartment and in the ultrafiltrate. The defined conditions allowed the efficient separation of nanocarriers from the free drug, with recoveries of >85% even when nanoparticles were present in cell culture media and in pig skin surrogate from permeation assays.

Halophyte plants from sustainable marine aquaponics are a valuable source of omega-3 polar lipids.

Marine aquaponics is a promising sustainable approach for the production of profitable crops such as halophytes. However, the effect of this culture approach on the lipid composition of halophytes remains unknown. In this work, we contrasted the polar lipidome of Salicornia ramosissima and Halimione portulacoides when produced in marine aquaponics (effluent from a super-intensive flatfish aquaculture production), with that of conspecifics from donor wild populations. Phospholipids and glycolipids were identified and quantified by LC-MS and MS/MS and their profile statistically analysed. Halophytes produced in aquaponics have higher levels of glycolipids with n-3 fatty acids (DGDG 36:3; SQDG 36:3; MGDG 36:6) compared with the donor wild populations. In the case of H. portulacoides, a significant increase of phospholipids bearing n-3 fatty acids (most in PC and PE) was also recorded. These lipids have potential applications in food, feed and pharmaceutical industries, contributing to the valorization of halophytes produced under sustainable aquaculture practices.

Exercise training counteracts urothelial carcinoma-induced alterations in skeletal muscle mitochondria phospholipidome in an animal model.

Cancer associated body wasting is the cause of physical disability, reduced tolerance to anticancer therapy and reduced survival of cancer patients and, similarly to cancer, its incidence is increasing. There is no cure for this clinical condition, and the pathophysiological process involved is largely unknown. Exercise training appears as the gold standard non-pharmacological therapy for the management of this wasting syndrome. Herein we used a lipidomics approach based on liquid chromatography coupled with high-resolution mass spectrometry (LC-HR-MS) to study the effect of exercise in the modulation of phospholipids profile of mitochondria isolated from gastrocnemius muscle of a pre-clinical model of urothelial carcinoma-related body wasting (BBN induced), submitted to 13 weeks of treadmill exercise after diagnosis. Multivariate analysis showed a close relationship between the BBN exercise group and both control groups (control sedentary and control exercise), while the BBN sedentary group was significantly separated from the control groups and the BBN exercise group. Univariate statistical analysis revealed differences mainly in phosphatidylserine (PS) and cardiolipin (CL), although some differences were also observed in phosphatidylinositol (PI, LPI) and phosphatidylcholine (PC) phospholipids. PS with shorter fatty acyl chains were up-regulated in the BBN sedentary group, while the other species of PS with longer FA and a higher degree of unsaturation were down-regulated, but the BBN exercise group was mostly similar to control groups. Remarkably, exercise training prevented these alterations and had a positive impact on the ability of mitochondria to produce ATP, restoring the healthy phospholipid profile. The remodelling of mitochondria phospholipid profile in rats with urothelial carcinoma allowed confirming the importance of the lipid metabolism in mitochondria dysfunction in cancer-induced skeletal muscle remodelling. The regulation of phospholipid biosynthetic pathways observed in the BBN exercise group supported the current perspective that exercise is an adequate therapeutic approach for the management of cancer-related muscle remodeling.

Lipidomic Profiling of the Olive (Olea europaea L.) Fruit towards Its Valorisation as a Functional Food: In-Depth Identification of Triacylglycerols and Polar Lipids in Portuguese Olives.

Olives (Olea europaea L.) are classic ingredients in the Mediterranean diet with well-known health benefits, but their lipid composition has not been fully addressed. In this work, we characterised triacylglycerol (TAG) and polar lipid profiles of the olive pulp while using a complementary methodological approach that was based on solid-phase extraction to recover the neutral lipid (NL) and the polar lipid-rich fractions. The TAG profile was analysed in the NL-fraction by C30 reversed-phase liquid chromatography (LC) and the polar lipid profile by normal-phase hydrophilic interaction liquid chromatography (HILIC), with both being coupled to electrospray ionization-mass spectrometry (ESI-MS) and ESI-MS/MS. This approach identified 71 TAG ions that were attributed to more than 350 molecular species, with fatty acyl chain lengths from C11:0 to C26:0, including different polyunsaturated acyl chains. The polar lipids included 107 molecular species that belonged to 11 lipid classes that comprised phospholipids, glyceroglycolipids, glycosphingolipids, and betaine lipids. In addition to polyunsaturated fatty acids, some of the phospholipids, glycolipids, and glycosphingolipids that were identified in the olive pulp have been described as biologically active molecules. Lipidomic phenotyping of the olive pulp has led to the discovery of compounds that will allow for a better assessment of its nutritional value and new applications of bioactive lipid components in this functional food.

Lipidomics Reveals Similar Changes in Serum Phospholipid Signatures of Overweight and Obese Pediatric Subjects.

Obesity is a public health problem and a risk factor for pathologies such type 2 diabetes mellitus, cardiovascular diseases, and nonalcoholic fatty liver disease. Given these clinical implications, there is a growing interest to understand the pathophysiological mechanism of obesity. Changes in lipid metabolism have been associated with obesity and obesity-related complications. However, changes in the lipid profile of obese children have been overlooked. In the present work, we analyzed the serum phospholipidome of overweight and obese children by HILIC-MS/MS and GC-MS. Using this approach, we have identified 165 lipid species belonging to the classes PC, PE, PS, PG, PI, LPC, and SM. The phospholipidome of overweight (OW) and obese (OB) children was significantly different from normal-weight children (control). Main differences were observed in the PI class that was less abundant in OW and OB children and some PS, PE, SM, and PC lipid species are upregulated in obese and overweight children. Although further studies are needed to clarify some association between phospholipid alterations and metabolic changes, our results highlight the alteration that occurs in the serum phospholipid profile in obesity in children.

Oxidized phosphatidylserine mitigates LPS-triggered macrophage inflammatory status through modulation of JNK and NF-kB signaling cascades.

Recent evidence suggests that phosphatidylserine (PS) and its oxidized species drive the clearance of apoptotic cells by macrophages with putative immune response modulation. However, it is not clear whether PS and oxidized PS differentially modulate at molecular level the functional responses of macrophages. Therefore, we proposed in this work to explore this question by evaluating the influence of PS oxidation products on the macrophages inflammatory status. Thus, we determined the effects of oxidized 1-palmitoyl-2-linoleoyl-phosphatidylserine (oxPLPS) and PLPS on RAW 264.7 macrophages production of the pro-inflammatory mediator nitric oxide (NO) and on the levels of the inducible NO synthase (Nos2) and Il1ß mRNA. The ability of PLPS and oxPLPS to modulate the lipopolysaccharide (LPS)-triggered macrophage activation was also analyzed. Finally, the effects of PLPS species over canonical inflammation-associated signaling pathways, such as nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) were also disclosed. The results obtained showed that both PLPS and oxPLPS species are deprived of intrinsic pro-inflammatory activity. Exquisitely, only oxPS were found to significantly inhibit NO production and iNos and IL1ß genes transcription induced by LPS. At a molecular level, these effects were partially due to attenuation of LPS-induced c-Jun-N-terminal kinase (JNK) phosphorylation and p65 NF-κB nuclear translocation. Overall our data suggest that oxPLPS, but not native PLPS, mitigates pro-inflammatory signaling in macrophages, contributing to containment of inflammation during apoptotic cell engulfment.

Decoding the Fatty Acid Profile of Bacillus licheniformis I89 and Its Adaptation to Different Growth Conditions to Investigate Possible Biotechnological Applications.

Bacillus licheniformis I89 is a Gram-positive bacterium, a producer of the lantibiotic lichenicidin. No information is available on its fatty acid (FA) composition. Bacillus species are rich in branched FA (BrFA), claimed to be beneficial to human health and to treat diseases. Herein, the FA profile of B. licheniformis I89 was evaluated under different growth conditions: at two growth temperatures (37 and 50 °C) and at different growth phases (lag, exponential, and stationary), using gas chromatography-mass spectrometry. The FA profile revealed predominant BrFA of the iso-series and anteiso-series (i-15:0, ai-15:0, i-16:0, i-17:0, and ai-17:0) and low amounts of saturated FA (14:0, 16:0, and 18:0). Comparing the FA profiles at different temperatures, in the lag phase, at 50 °C, there was a decrease of ai-17:0 and a decrease of i-15:0 in the exponential phase, in comparison with 37 °C. In all growth phases, there was a decrease of ai-15:0 and an increase of i-17:0. From the lag to the stationary phase, at 50 °C, there was a decrease of ai-17:0 and i-16:0, whereas i-15:0 increased, while at 37 °C, there was an increase of i-15:0 and i-16:0, and a decrease in ai-15:0 and ai-17:0. B. licheniformis I89 can adapt its FA profile, at moderate temperatures, by changing the iso-FA and anteiso-FA composition and the iso/anteiso ratio. This nonpathogenic bacterium species can be used as a source of BrFA with putative beneficial health effects for gut protection and with reported antitumor properties, foreseeing its use for producing compounds with biotechnological applications.

Lipidomic signature of Bacillus licheniformis I89 during the different growth phases unravelled by high-resolution liquid chromatography-mass spectrometry.

Bacillus licheniformis I89 is a non-pathogenic, Gram-positive bacterium, frequently found in soil. It has several biotechnological applications as producer of valuable compounds such as proteases, amylases, surfactants, and lantibiotics. Herein, it is reported the identification of the polar lipidome of B. licheniformis I89 during the different growth phases (lag, exponential and stationary) at 37 °C. The analytical approach relied on hydrophilic interaction liquid chromatography coupled to electrospray ionization mass spectrometry (HILIC-ESI-MS), accurate mass measurements and tandem mass spectrometry (MS/MS). In the lipidome of B. licheniformis I89 were identified four phospholipid classes: phosphatidylethanolamine, phosphatidylglycerol, lysyl-phosphatidylglycerol, and cardiolipin; two glycolipid classes: monoglycosyldiacylglycerol and diglycosyldiacylglycerol; and two phosphoglyceroglycolipid classes: mono-alanylated lipoteichoic acid primer and lipoteichoic acid primer. The same lipid species were identified at the different growth phases, but there were significant differences on the relative abundance of some molecular species. There was a significant increase in the 30:0 lipid species and a significant decrease in the 32:0 lipid species, between exponential and stationary phases, when compared to lag phase. No differences were observed between exponential and stationary phases. The lipidomic-based approach used herein is a very promising tool to be employed in the study of bacterial lipid composition, which is a requirement to understand its metabolism and response to growth conditions.

Tuning culturing conditions towards the production of neutral lipids from lubricant-based wastewater in open mixed bacterial communities.

Production of bacterial lipid-based biofuels using inexpensive substrates, as wastes, is an emerging approach. In this work, a selective process using carbon feast-famine cycles was applied to obtain an indigenous microbial community of hydrocarbon-degrading and lipid-accumulating bacteria, using a real lubricant-based wastewater as carbon source. In the conditions applied, the enriched bacterial community, dominated by members of the genus Rhodococcus, Pseudomonas and Acinetobacter, was able to degrade almost all hydrocarbons present in the wastewater within 24 h' incubation and to accumulate, although in low levels, triacylglycerol (TAG) (<5% of cell dry weight (CDW)) and polyhydroxyalkanoates (PHA) (3.8% ±â€¯1.1% of the CDW) as well as an unknown lipid (29% ±â€¯6% of CDW), presumably a wax ester-like compound. The influence of culture conditions, namely carbon and nitrogen concentrations (and C/N ratio) and cultivation time, on the amount and profile of produced storage compounds was further assessed using a statistical approach based on a central composite circumscribed design and surface response methodology. The regression analysis of the experimental design revealed that only nitrogen concentration and C/N ratio are significant for neutral lipid biosynthesis (p < 0.05). Maximum neutral lipid content, i.e. 33% (CDW basis), was achieved for the lowest carbon and nitrogen concentrations evaluated (10 g COD L-1 and 0.02 g N L-1). PHA accounted for less than 5% of CDW. In these conditions, neutral lipid content was mainly composed by TAG, about 70% (w/w). TAG precursors, namely monoacylglycerols (MAG), diacylglycerols (DAG) and fatty acids (FA), accounted for 22% of total neutral lipids and WE for about 7%. Nevertheless, according to the applied response surface model, further improvement of neutral lipids content is still possible if even lower nitrogen concentrations are used. The fatty acids detected in TAG extracts ranged from myristic acid (C14:0) to linoleic acid (C18:2), being the most abundant palmitic acid (C16:0), stearic acid (C18:0) and oleic acid (C18:1). This study shows the feasibility of combining treatment of hydrocarbon contaminated wastewater, herein demonstrated for lubricant-based wastewater, with the production of bacterial neutral lipids using open mixed bacterial communities. This approach can decrease the costs associated to both processes and contribute to a more sustainable waste management and production of lipid-based biofuels.

Polar Lipids from Olives and Olive Oil: A Review on Their Identification, Significance and Potential Biotechnological Applications.

Polar lipids are minor components of olives and olive oil and include a myriad of molecules such as phospholipids and glycolipids. Even though sensitive and high-resolution analytical approaches have been used to unveil the polar lipidome of these matrices, new insights on their composition are needed. In this review, we will describe the findings on the identification and characterization of polar lipids from olives and olive oil and the underlying analytical challenges. The significance of polar lipids will also be discussed as potential markers of identity and traceability of olives and olive oil and in detecting adulteration of olive oil. Their potential impact on nutrition and health will be presented as a valuable source of bioactive compounds and as promising ingredients for different uses from olive-derived industrial by-products.

Polar lipidome profiling of Salicornia ramosissima and Halimione portulacoides and the relevance of lipidomics for the valorization of halophytes.

Some halophytes are currently used as gourmet plant ingredients for human consumption. The polar lipidome of the succulent organs of Salicornia ramosissima (fresh branch tips) and Halimione portulacoides (leaves) were characterized in-depth, with more than two hundred lipid species being identified in both halophytes. The lipid species identified were distributed over five classes of phospholipids, three classes of glycolipids and one class of glycosphingolipids. Despite the existence of some species-specific differences between the polar lipidome, phospholipids and glycolipids show a high content of n-3 fatty acids in both S. ramosissima and H. portulacoides. These results highlights the advantage of employing mass spectrometry based lipidomic platform towards the valorization of halophytes as a source of valuable nutrients and bioactives, fostering potential applications in the fields of healthy and functional food products, and for nutraceutical and pharmaceutical uses.

Gas-phase structural characterization of neuropeptides Y Y1 receptor antagonists using mass spectrometry: Orbitrap vs triple quadrupole.

Collision induced dissociation of triple quadrupole mass spectrometer (CID-QqQ) and high-energy collision dissociation (HCD) of Orbitrap were compared for four neuropeptides Y Y1 (NPY Y1) receptor antagonists and showed similar qualitative fragmentation and structural information. Orbitrap high resolution and high mass accuracy HCD fragmentation spectra allowed unambiguous identification of product ions in the range 0.04-4.25 ppm. Orbitrap mass spectrometry showed abundant analyte-specific product ions also observed on CID-QqQ. These results show the suitability of these product ions for use in quantitative analysis by MRM mode. In addition, it was found that all compounds could be determined at levels >1 µg L-1 using the QqQ instrument and that the detection limits for this analyzer ranged from 0.02 to 0.6 µg L-1. Overall, the results obtained from experiments acquired in QqQ show a good agreement with those acquired from the Orbitrap instrument allowing the use of this relatively inexpensive technique (QqQ) for accurate quantification of these compounds in clinical and academic applications.

Kleptoplasty does not promote major shifts in the lipidome of macroalgal chloroplasts sequestered by the sacoglossan sea slug Elysia viridis.

Sacoglossan sea slugs, also known as crawling leaves due to their photosynthetic activity, are highly selective feeders that incorporate chloroplasts from specific macroalgae. These "stolen" plastids - kleptoplasts - are kept functional inside animal cells and likely provide an alternative source of energy to their host. The mechanisms supporting the retention and functionality of kleptoplasts remain unknown. A lipidomic mass spectrometry-based analysis was performed to study kleptoplasty of the sacoglossan sea slug Elysia viridis fed with Codium tomentosum. Total lipid extract of both organisms was fractionated. The fraction rich in glycolipids, exclusive lipids from chloroplasts, and the fraction rich in betaine lipids, characteristic of algae, were analysed using hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-LC-MS). This approach allowed the identification of 81 molecular species, namely galactolipids (8 in both organisms), sulfolipids (17 in C. tomentosum and 13 in E. viridis) and betaine lipids (51 in C. tomentosum and 41 in E. viridis). These lipid classes presented similar lipidomic profiles in C. tomentosum and E. viridis, indicating that the necessary mechanisms to perform photosynthesis are preserved during the process of endosymbiosis. The present study shows that there are no major shifts in the lipidome of C. tomentosum chloroplasts sequestered by E. viridis.

Data on coffee composition and mass spectrometry analysis of mixtures of coffee related carbohydrates, phenolic compounds and peptides.

The data presented here are related to the research paper entitled "Transglycosylation reactions, a main mechanism of phenolics incorporation in coffee melanoidins: inhibition by Maillard reaction" (Moreira et al., 2017) [1]. Methanolysis was applied in coffee fractions to quantify glycosidically-linked phenolics in melanoidins. Moreover, model mixtures mimicking coffee beans composition were roasted and analyzed using mass spectrometry-based approaches to disclose the regulatory role of proteins in transglycosylation reactions extension. This article reports the detailed chemical composition of coffee beans and derived fractions. In addition, it provides gas chromatography-mass spectrometry (GC-MS) chromatograms and respective GC-MS spectra of silylated methanolysis products obtained from phenolic compounds standards, as well as the detailed identification of all compounds observed by electrospray mass spectrometry (ESI-MS) analysis of roasted model mixtures, paving the way for the identification of the same type of compounds in other samples.

Measurements of the effects of wine maceration with oak chips using an electronic tongue.

The use of oak products as a cheaper alternative to expensive wood barrels was recently permitted in Europe, which led to a continuous increase in the use of oak chips and staves in winemaking. The feasibility of the potentiometric electronic tongue as a tool for monitoring the effects of wine maceration with oak chips was evaluated. Four types of commercially available oak chips subjected to different thermal treatments and washing procedures and their mixture were studied. Ethanolic extracts of the chips were analysed using electrospray mass spectrometry and 28 phenolic and furanic compounds were identified. The electronic tongue comprising 22 potentiometric chemical sensors could distinguish artificial wine solutions and Cabernet Sauvignon wine macerated with different types of oak chips, quantify total and non-flavonoid phenolic content, as well as the concentrations of added oak chips. Using measurements at two pH levels, 3.2 and 6.5, improved the accuracy of quantification.

Transglycosylation reactions, a main mechanism of phenolics incorporation in coffee melanoidins: Inhibition by Maillard reaction.

Under roasting conditions, polysaccharides depolymerize and also are able to polymerize, forming new polymers through non-enzymatic transglycosylation reactions (TGRs). TGRs can also occur between carbohydrates and aglycones, such as the phenolic compounds present in daily consumed foods like coffee. In this study, glycosidically-linked phenolic compounds were quantified in coffee melanoidins, the polymeric nitrogenous brown-colored compounds formed during roasting, defined as end-products of Maillard reaction. One third of the phenolics present were in glycosidically-linked form. In addition, the roasting of solid-state mixtures mimicking coffee beans composition allowed the conclusion that proteins play a regulatory role in TGRs extension and, consequently, modulate melanoidins composition. Overall, the results obtained showed that TGRs are a main mechanism of phenolics incorporation in melanoidins and are inhibited by amino groups through Maillard reaction.

Characterization of phospholipid nitroxidation by LC-MS in biomimetic models and in H9c2 Myoblast using a lipidomic approach.

Under nitroxidative stress conditions, lipids are prone to be modified by reaction with reactive nitrogen species (RNS) and different modifications were reported to occur in fatty acids. However, in the case of phospholipids (PL) studied under nitroxidative stress conditions, only nitroalkene derivatives of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), were reported when using both in vitro biomimetic conditions and in vivo model system of type 1 diabetes mellitus. Therefore, in order to further explore other nitroxidative modifications of PL, a biomimetic model of nitroxidation combined with liquid chromatography mass spectrometry (MS) and MS/MS approaches were used to characterize the nitrated and nitroxidized derivatives of PCs and PEs. Single and multiple nitrated derivatives of phospholipids (PLs) such as nitroso and dinitroso, nitro, dinitro, and nitronitroso derivatives, together with nitroxidized derivatives were identified. Further, the specific MS/MS fragmentation pathways of these products were studied. Product ions arising from loss of HNO and HNO2, from the combined loss of HNO (or HNO2) and polar head groups, [NOn-FA+On+H]+ and [NOn-FA+On-H]- (n=1-2) product ions corresponding to the modified fatty acyl chains were observed, depending on each modification. The knowledge obtained from the study of the MS/MS fragmentation pattern has allowed us to identify nitrated PCs, including NO2-PC, (NO2)2-PCs, (NO2)(NO)-PC, NO-PC; nitrated PEs, NO2-PEs; and nitroxidized PCs, (NO2)(2O)-PC in H9c2 cells under starvation, but not under ischemia or control conditions. The physiological relevance of this nitrated and nitroxidized PCs and PEs species observed exclusively in cardiomyoblast cells (H9c2) under starvation is still unknown but deserves to be explored.