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Dried blood spot (DBS) is a minimally invasive sampling technique that has several advantages over conventional venipuncture/arterial blood sampling. More recently, DBS has also been applied for lipidomics analysis, but this is an area that requires further research. The few works found in the literature on lipidomics of DBS samples performed the analysis in adult samples, leaving pediatric ages unmapped. The objective of this study was to assess the variability of the lipid profile (identified by high-resolution C18 RP-LC-MS/MS) of DBS at pediatric age (0-10 days, 2-18 months, and 3-13 years) and to identify age-related variations. The results revealed that the lipidomic signature of the three age groups is significantly different, especially for a few species of neutral lipids and phosphatidylcholines. The main contributors to the differentiation of the groups correspond to 3 carnitine (Car), 2 cholesteryl ester (CE), 2 diacylglycerol (DG), 2 triacylglycerol (TG), 3 phosphatidylcholine (PC), 1 ether-linked PC, 1 phosphatidylethanolamine (PE), 1 ether-linked PE and 1 phosphatidylinositol (PI) species, all with statistically significant differences. Additionally, lipid species containing linoleic acid (C18:2) were shown to have significantly lower levels in the 0-10 days group with a gradual increase in the 2-18 month, reaching the highest concentrations in the 3-13 year group. The results of this study highlighted the adaptations of the lipid profile at different pediatric ages. These results may help improve understanding of the evolution of lipid metabolism throughout childhood and should be investigated further.

Dried blood spots (DBS) are being considered as an alternative sampling method of blood collection that can be used in combination with lipidomic and other omic analysis. DBS are successfully used in the clinical context to collect samples for newborn screening for the measurement of specific fatty acid derivatives, such as acylcarnitines, and lipids from whole blood for diagnostic purposes. However, DBS are scarcely used for lipidomic analysis and investigations. Lipidomic studies using DBS are starting to emerge as a powerful method for sampling and storage in clinical lipidomic analysis, but the major research work is being done in the pre- and analytical steps and procedures, and few in clinical applications. This review presents a description of the impact factors and variables that can affect DBS lipidomic analysis, such as the type of DBS card, haematocrit, homogeneity of the blood drop, matrix/chromatographic effects, and the chemical and physical properties of the analyte. Additionally, a brief overview of lipidomic studies using DBS to unveil their application in clinical scenarios is also presented, considering the studies of method development and validation and, to a less extent, for clinical diagnosis using clinical lipidomics. DBS combined with lipidomic approaches proved to be as effective as whole blood samples, achieving high levels of sensitivity and specificity during MS and MS/MS analysis, which could be a useful tool for biomarker identification. Lipidomic profiling using MS/MS platforms enables significant insights into physiological changes, which could be useful in precision medicine., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

Complex lipids, phospholipids (PLs) and triacylglycerides (TAGs), are prone to modifications induced by reactive nitrated species and reactive oxygen species, generating a range of nitrated, nitrosated or nitroxidized derivatives, as nitro PLs and nitro TAGs. These modified lipids (epilipids) have been reported in vitro and in vivo using lipidomics approaches. However, their detection in living systems remains a challenge hampered by its complexity, high structural diversity, and low abundance. The advances in high-resolution mass spectrometry combined with the higher sensitivity of the instruments like Orbitrap-based mass spectrometers opened new opportunities for the detection of these modified complex lipids. This review summarizes the challenges and findings behind the identification of nitrated, nitrosated and nitroxidized PLs and TAGs fragmentation fingerprints based on collision-induced dissociation (CID) and higher energy CID (HCD) MS/MS approaches. Following what has already been reported for nitrated fatty acids, these complex lipids are found to act as endogenous mediators with potential electrophilic properties and can express bioactivities such as anti-inflammatory and antioxidant actions. This information can be used to design untargeted and targeted lipidomics strategies for these modified complex lipids in biological samples as well as in pathological, food and industrial settings, further unveiling their biological and signalling roles., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Cholesteryl esters (CE) are prone to oxidation under increased oxidative stress conditions, but little is known about oxidized CE species (oxCE). To date, only a few oxCE have been identified, however, mainly based on the detection of molecular ions by mass spectrometry (MS) or target approaches for specific oxCE. The study of oxCE occurring from radical oxidation is still scarcely addressed. In this work, we made a comprehensive assessment of oxCE derivatives and their specific fragmentation patterns to identify detailed structural features and isomer differentiation using high-resolution C18 HPLC-MS- and MS/MS-based lipidomic approaches. The LC-MS/MS analysis allowed us to pinpoint oxCE structural isomers of long-chain and short-chain species, eluting at different retention times ( t R ). Data analysis revealed that oxCE can be modified either in the fatty acyl moiety or in the cholesterol ring. The location of the hydroxy/hydroperoxy group originates characteristic fragment ions, namely the unmodified cholestenyl cation ( m / z 369) for the isomer with oxidation in the fatty acyl chain or ions at m / z 367 and m / z 385 (369 + 16) when oxygenation occurs in the cholesterol ring. Additionally, we identified CE 18:2 and 20:4 aldehydic and carboxylic short-chain products that showed a clear fragmentation pattern that confirmed the modification in the fatty acyl chain. Specific fragmentation fingerprinting allowed discrimination of the isobaric short-chain species, namely carboxylic short-chain products, from hydroxy aldehyde short-chain products, with a hydroxycholesterol moiety. This new information is important to identify different oxCE in biological samples and will contribute to unraveling their role in biological conditions and diseases such as cardiovascular disease.

Frying allows cooking food while promoting their organoleptic properties, imparting crunchiness and flavor. The drawback is the oxidation of triacylglycerides (TAGs), leading to the formation of primary oxidized TAGs. Although they have been associated with chronic and degenerative diseases, they are precursors of pleasant flavors in fried foods. Nevertheless, there is a lack of knowledge about the oxidation species present in foods and their involvement in positive/negative health effects. In this work, high-resolution (HR) C 30 reversed-phase (RP)-liquid chromatography (LC)-tandem HR mass spectrometry (MS/MS) was used to identify primary oxidation TAGs resulting from heating triolein (160 °C, 5 min). This allows simulating the initial heating process of frying oils usually used to prepare fried foods, such as chips, crisps, and snacks. Beyond hydroxy, dihydroxy, hydroperoxy, and hydroxy-hydroperoxy derivatives, already reported in phospholipids oxidized by Fenton reaction, new compounds were identified, such as dihydroxy-hydroperoxy-triolein derivatives and positional isomers (9/10- and 9/12-dihydroxy-triolein derivatives). These compounds should be considered when proposing flavor formation pathways and/or mitigating lipid-derived reactive oxygen species occurring during food frying.

Nitrated lipids have been detected in vitro and in vivo, usually associated with a protective effect. While nitrated fatty acids have been widely studied, few studies reported the nitration and nitroxidation of the phospholipid classes phosphatidylcholine, and phosphatidylethanolamine. However, no information regarding nitrated and nitroxidized phosphatidylserine can be found in the literature. This work aims to identify and characterize the nitrated and nitroxidized derivatives of 1-palmitoyl-2-oleoyl- sn -3-glycero-phosphoserine (POPS), obtained after incubation with nitronium tetrafluoroborate, by liquid chromatography (LC) coupled to mass spectrometry (MS) and tandem MS (MS/MS). Several nitrated and nitroxidized products were identified, namely, nitro, nitroso, nitronitroso, and dinitro derivatives, as well as some nitroxidized species such as nitrosohydroxy, nitrohydroxy, and nitrohydroperoxy. The fragmentation pathways identified were structure-dependent and included the loss of HNO and HNO₂ for nitroso and nitro derivatives, respectively. Combined losses of PS polar head group plus HNO or HNO₂ and carboxylate anions of modified fatty acyl chain were also observed. The nitrated POPS also showed antiradical potential, demonstrated by the ability to scavenge the ABTS ●+ and DPPH ● radicals. Overall, this in vitro model of nitration based on LC-MS/MS provided additional insights into the nitrated and nitroxidized derivatives of PS and their fragmentation fingerprinting. This information is a valuable tool for targeted analysis of these modified PS in complex biological samples, to further explore the new clues on the antioxidant potential of nitrated POPS.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Photodynamic therapy combines visible light and a photosensitizer (PS) in the presence of molecular oxygen to generate reactive oxygen species able to modify biological structures such as phospholipids. Phosphatidylethanolamines (PEs), being major phospholipid constituents of mammalian cells and membranes of Gram-negative bacteria, are potential targets of photosensitization. In this work, the oxidative modifications induced by white light in combination with cationic porphyrins (Tri-Py(+)-Me-PF and Tetra-Py(+)-Me) were evaluated on PE standards. Electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS) were used to identify and characterize the oxidative modifications induced in PEs (POPE: PE 16:0/18:1, PLPE: PE 16:0/18:2, PAPE: PE 16:0/20:4). Photo-oxidation products of POPE, PLPE and PAPE as hydroxy, hydroperoxy and keteno derivatives and products due to oxidation in ethanolamine polar head were identified. Hydroperoxy-PEs were found to be the major photo-oxidation products. Quantification of hydroperoxides (PE-OOH) allowed differentiating the potential effect in photodamage of the two porphyrins. The highest amounts of PE-OOH were notorious in the presence of Tri-Py(+)-Me-PF, a highly efficient PS against bacteria. The identification of these modifications in PEs is an important key point in the understanding cell damage processes underlying photodynamic therapy approaches., (Copyright © 2013 John Wiley & Sons, Ltd.)

Phosphatidylserine (PS) is an aminophospholipid that is prone to glycation. In oxidative conditions, glycated PS may lead to the formation of Amadori compounds and advanced glycated end products (AGEs), which are known to accumulate in diabetic patients. Nevertheless, there have been no studies that identified products from the oxidative reaction of glycated PS. In this study, glycated 1-palmitoyl-2-oleoyl-PS was synthesized and further oxidized by Fenton reagent. The AGES formed were structurally characterized by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in negative mode. The oxidation products from glycated PS that we have found include products arising from the oxidation of the fatty acyl chains (hydroperoxides, hydroxides and keto derivatives), and arising from oxidative cleavage of serine polar head and lyso-glycated PS. Oxidation in C6 of glucose lead to the formation of glucuronyl-PS. In addition, new products arising from oxidative cleavage of glucose moiety (between C1C2, C2C3 and C3C4 bonds) were identified as PS-AGES. The current findings add substantially to the best of our knowledge of PS glycoxidation products, opening new perspectives for the detection of these products in complex biological matrices., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Phosphatidylethanolamines (PE) are one of the major components of cells membranes, namely in skin and in retina, that are continuously exposed to solar UV radiation being major targets of photooxidation damage. In addition, due to the presence of the free amine group, PE can also undergo glycation, in hyperglycemic conditions which may increase the susceptibility to oxidation. The aim of this study is to develop a model, based on mass spectrometry (MS) analysis, to identify photooxidative degradation of selected PE (POPE: PE 16:0/18:1, PLPE: PE 16:0/18:2, PAPE: PE 16:0/20:4) and glycated PEs due to UV irradiation. Photooxidation products were analysed by electrospray ionization MS (ESI-MS) and tandem MS (ESI-MS/MS) in positive and negative mode. Emphasis is placed in the influence of glycation in the generation of distinct photooxidation products. ESI-MS spectra of PE after UV photo-irradiation showed mainly hydroperoxy derivatives, due to oxidation of unsaturated fatty acyl chains. Glycated PE gave rise to several new photooxidation products formed due to oxidative cleavages of the glucose moiety, namely between C1 and C2, C2 and C3, and C5 and C6 of this sugar unit. These new products were identified by ESI-MS/MS in positive mode showing distinct neutral loss depending on the different structure of the polar head group. These new identified advanced glycated photooxidation products may have a deleterious role in the etiology of diabetic retinopathy and in diabetic retinal microvascular complications., (Copyright © 2013 John Wiley & Sons, Ltd.)

Marine algae are one of the most important sources of high‐value compounds such as polar lipids, omega‐3 fatty acids, photosynthetic pigments, or secondary metabolites with interesting features for different niche markets. Acetabularia acetabulum is a macroscopic green single‐celled alga, with a single nucleus hosted in the rhizoid. This alga is one of the most studied dasycladalean species and represents an important model system in cell biology studies. However, its lipidome and pigment profile have been overlooked. Total lipid extracts were analyzed using hydrophilic interaction liquid chromatography‐high resolution mass spectrometry (HILIC‐HRMS), tandem mass spectrometry (MS/MS), and high‐performance liquid chromatography (HPLC). The antioxidant capacity of lipid extracts was tested using DPPH and ABTS assays. Lipidomics identified 16 polar lipid classes, corresponding to glycolipids, betaine lipids, phospholipids, and sphingolipids, with a total of 191 lipid species, some of them recognized by their bioactivities. The most abundant polar lipids were glycolipids. Lipid classes less studied in algae were identified, such as diacylglyceryl‐carboxyhydroxymethylcholine (DGCC) or hexosylceramide (HexCer). The pigment profile of A. acetabulum comprised carotenoids (17.19%), namely cis‐neoxanthin, violaxanthin, lutein and β,β‐carotene, and chlorophylls a and b (82.81%). A. acetabulum lipid extracts showed high antioxidant activity promoting a 50% inhibition (IC50) with concentrations of 57.91 ± 1.20 μg · mL−1 (438.18 ± 8.95 μmol Trolox · g−1 lipid) in DPPH and 20.55 ± 0.60 μg · mL−1 in ABTS assays (918.56 ± 27.55 μmol Trolox · g−1 lipid). This study demonstrates the potential of A. acetabulum as a source of natural bioactive molecules and antioxidant compounds. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) (exossomes, microvesicles and apoptotic bodies) have been well acknowledged as mediators of intercellular communications in prokaryotes and eukaryotes. Lipids are essential molecular components of EVs but at the moment the knowledge about the lipid composition and the function of lipids in EVs is limited and as for now none lipidomic studies in Giardia EVs was described. Therefore, the focus of the current study was to conduct, for the first time, the characterization of the polar lipidome, namely phospholipid and sphingolipid profiles of G. lamblia trophozoites, microvesicles (MVs) and exosomes, using C18—Liquid Chromatography—Mass Spectrometry (C18-LC-MS) and Tandem Mass Spectrometry (MS/MS). A total of 162 lipid species were identified and semi-quantified, in the trophozoites, or in the MVs and exosomes belonging to 8 lipid classes, including the phospholipid classes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), cardiolipins (CL), the sphingolipid classes sphingomyelin (SM) and ceramides (Cer), and cholesterol (ST), and 3 lipid subclasses that include lyso PC (LPC), lyso PE (LPE) and lyso PG (LPG), but showing different abundances. This work also identified, for the first time, in G. lamblia trophozoites, the lipid classes CL, Cer and ST and subclasses of LPC, LPE and LPG. Univariate and multivariate analysis showed clear discrimination of lipid profiles between trophozoite, exosomes and MVs. The principal component analysis (PCA) plot of the lipidomics dataset showed clear discrimination between the three groups. Future studies focused on the composition and functional properties of Giardia EVs may prove crucial to understand the role of lipids in host-parasite communication, and to identify new targets that could be exploited to develop novel classes of drugs to treat giardiasis. [ABSTRACT FROM AUTHOR]

Cardiolipins (CL) are anionic dimeric phospholipids bearing four fatty acids, found in inner mitochondrial membrane as structural components and are involved in several processes as oxidative phosphorylation or apoptotic signalling. As other phospholipids, CL can be modified by reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can modulate various cellular functions. Modifications of CL by RNS remain largely unstudied although other nitrated lipids are emerging as bioactive molecules. In this work, we developed a C30-LC-HRMS/MS methodology to identify the nitrated and nitroxidized tetralinoleoyl-cardiolipin (TLCL), using a biomimetic model of nitration, and to disclose specific fragmentation pathways under HCD MS/MS. Using this lipidomics approach, we were able to separate and identify nitro, nitroso, nitronitroso, and nitroxidized TLCL derivatives, comprising 11 different nitrated compounds. These products were identified using accurate mass measurements and the fragmentation pattern acquired in higher-energy collision dissociation (HCD)-tandem MS/MS experiments. These spectra showed classifying fragmentation pathways, yielding phosphatidic acid (PA−), lysophosphatidic acid (LPA−), and carboxylate fragment ions with the modifying moiety. Remarkably, the typical neutral losses associated with the added moieties were not observed. In conclusion, this work has developed a new method for the identification of nitroso, nitrated and nitroxidized cardiolipin products by using a C30LC-MS platform method, potentially allowing their detection in biological samples. Image 1 • C30 LC-MS allows to separate nitro-oxidation products of nitroxidized TLCL. • C30 LC-MS separated and identified 11 different nitrated compounds. • HCD-MS/MS showed classifying fragmentation pathways. • HCD-MS/MS fragmentation confirmed the identity of each isomer after LC separation. [ABSTRACT FROM AUTHOR]

Nitrated phospholipids have recently been detected in vitro and in vivo and associated with beneficial health effects. They were identified and quantified in biological samples by lipidomics methodologies using liquid chromatography-collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) acquired with the linear ion trap mass spectrometer. Only a few studies have used higher-energy collision dissociation (HCD)-MS/MS in high-resolution Orbitraps to characterize nitrated phosphatidylserines and nitrated cardiolipins, highlighting the marked differences in the fragmentation patterns when using CID or HCD fragmentation methods. In this study, we aimed to evaluate the fragmentation of nitrated phosphatidylcholine and nitrated phosphatidylethanolamine species under HCD-MS/MS. We studied the effect of normalized collision energy (NCE) in the fragmentation pattern to identify the best acquisition conditions and reporter ions to detect nitrated phospholipids. The results showed that the intensity of the typical neutral loss of nitrous acid (HNO2) diminishes with increasing NCE, becoming non-detectable for a higher NCE. Thus, the loss of HNO2 could not be the most suitable ion/fragment for the characterization of nitrated phospholipids under HCD. In HCD-MS/MS new fragment ions were identified, corresponding to the nitrated fatty acyl chains, NO2-RCOO−, (NO2-RCOOH-H2O + H)+, and (NO2-RCOOH + H)+, suggested as potential reporter ions to detect nitrated phospholipids when using the HCD-MS/MS lipidomics analysis. [ABSTRACT FROM AUTHOR]

Nitrated lipids have been detected in vitro and in vivo, usually associated with a protective effect. While nitrated fatty acids have been widely studied, few studies reported the nitration and nitroxidation of the phospholipid classes phosphatidylcholine, and phosphatidylethanolamine. However, no information regarding nitrated and nitroxidized phosphatidylserine can be found in the literature. This work aims to identify and characterize the nitrated and nitroxidized derivatives of 1-palmitoyl-2-oleoyl-sn-3-glycero-phosphoserine (POPS), obtained after incubation with nitronium tetrafluoroborate, by liquid chromatography (LC) coupled to mass spectrometry (MS) and tandem MS (MS/MS). Several nitrated and nitroxidized products were identified, namely, nitro, nitroso, nitronitroso, and dinitro derivatives, as well as some nitroxidized species such as nitrosohydroxy, nitrohydroxy, and nitrohydroperoxy. The fragmentation pathways identified were structure-dependent and included the loss of HNO and HNO2 for nitroso and nitro derivatives, respectively. Combined losses of PS polar head group plus HNO or HNO2 and carboxylate anions of modified fatty acyl chain were also observed. The nitrated POPS also showed antiradical potential, demonstrated by the ability to scavenge the ABTS●+ and DPPH● radicals. Overall, this in vitro model of nitration based on LC-MS/MS provided additional insights into the nitrated and nitroxidized derivatives of PS and their fragmentation fingerprinting. This information is a valuable tool for targeted analysis of these modified PS in complex biological samples, to further explore the new clues on the antioxidant potential of nitrated POPS. [ABSTRACT FROM AUTHOR]