Oxylipins - involved in inflammatory processes - are reported in several diseases, in biological, pharmacological, and physiological fields. To face the structural complexity of oxylipins, the study of isomers and isobars species relied on Selected Reaction Monitoring (SRM) and Multiple Reaction Monitoring (MRM) in tandem mass spectrometry such as triple quadrupole, quadrupole-Time of Flight (TOF). Unfortunately, false positive signals in cellular matrix could occur using MRM or SRM mode since the MS/MS spectrum of each molecule is not acquired with the previous mode to help molecule confirmation. Using the versatile ability of LTQ-Orbitrap (R) Velos Pro mass spectrometer, we developed a novel method based on data dependent acquisition (DDA) workflow for oxylipins analysis. To reach sufficient data points per peak and a better sensitivity to quantify oxylipins traces, an optimization of the acquisition frequency was carried out both on linear trap and Orbitrap analyzers. A segmentation of the chromatographic profile and an optimization of the collision energies by HCD (higher energy collision dissociation) for each eicosanoid increased the acquisition frequency significantly and the detection threshold: around 2 pg for some prostanoids and 0.02-2 pg for some leukotrienes and oxidized species. We validated our method in terms of specificity (RSD <10%), sensitivity, accuracy and precision. The intra and interday accuracy were between 86.56% and 114.93%. Besides, a relative standard deviation less than 15% as intraand inter-day precision were obtained for almost all molecules. A linear range between 2.5 and 12,500 pg was reached. DDA approach on LTQ-Orbitrap<(R)> constitutes an alternative to MRM mode on triple quadrupole for eicosanoids quantification in complex matrices. Finally, this method helped us to compare for the first time the amount of prostanoids released by J774 and THP-1 macrophages under lipopolysaccharide (LPS) stimulation.

The presence of a new ceramide subclass, the 1-O-acyl omega-linoleoyloxy ceramides [1-O-E (EO) Cer], has been previously highlighted in reconstructed human epidermis (RHE). These ceramides are double esterified on two positions. The first is the 1-O position of the sphingoid base moiety with a long to very long chain of acyl residues (1-O-E), and the second is the position of the ω-hydroxyl group of the fatty acid moiety with linoleic acid (EO). Considering its chemical structure and hydrophobicity, this subclass can contribute to the skin barrier. Thus, it is important to determine whether this subclass is also present in native human stratum corneum (SC). This work compares ceramide structures of this novel subclass between RHE (in vitro) and two sources of human SC (in vivo and ex vivo) using normal-phase high-performance liquid chromatography coupled to high-resolution mass spectrometry (NP-HPLC/HR-MSn). The results confirm the presence of this double esterified ceramide subclass [1-O-E (EO) Cer] in human SC. The molecular profile obtained from the RHE was very close to that found in the human SC (in vivo and ex vivo). In addition, thanks to the targeted MS2/MS3 analysis, a new ceramide subclass was discovered and characterized in the three studied samples. We propose to name it [A-1-O-E (EO) Cer] because in these ceramides species, the fatty acid—esterified with the sphingoid base on the 1-O position—is hydroxylated on the α position. These results highlight the potential of both the analytical method and the characterization approach employed in this study.

Linoleoyl macrogolglyceride (LM) and oleoyl macrogolglyceride (OM) are pharmaceutical ingredients, obtained from corn and apricot kernel oils respectively. This study aims to know the detailed chemical composition of LM and OM, in order to understand their roles in pharmaceutical formulations. These two products were analyzed by non-aqueous reversed phase high-performance liquid chromatography (NARP-HPLC), using Vintage Series KR C18 column (250 × 4.6 mm, 5 µm) and non-aqueous acetonitrile/acetone mixture as mobile phase. The ionization source used was atmospheric pressure photoionization (APPI) and the analyzer was LTQ-Orbitrap ® (hybrid analyzer: double linear ion trap coupled to a Fourier transform orbital trap). LM and OM consist of complex mixtures, constituted of mono-(MG), di-(DG) and triglycerides (TG) and mono-(MPEGE) and di PEG-6 esters (DPEGE) of linoleic acid (18:2) for LM and of oleic acid (18:1) for OM. NARP-HPLC-APPI method allowed the separation and the identification of the glyceride classes (MGs, DGs and TGs) and the PEG esters of different chain lengths (PEG-chain lengths of fatty acid moieties and number of units of ethylene oxide), at the same time and in one single run, for both products LM and OM. The comparative study between LM and OM showed that, a higher presence of linoleic esters for LM, and a higher presence of oleic esters for OM.

Stratum corneum lipids are responsible for the skin’s barrier function. They are the final product of epidermis lipid biosynthesis. During this process, lipids evolve from simple to complex structures in three main levels respectively (stratum basal level, stratum granulosum level, and stratum corneum level). Our aim was to simultaneously analyze and characterize the structure of total epidermis lipids. A powerful analytical method (normal-phase liquid chromatography coupled with high-resolution mass spectrometry (NPLC/HR-MSn)) was developed in order to separate, in a single run, lipid classes with a wide polarity range. Chromatographic conditions were particularly designed to analyze lipids of intermediate polarity such as ceramides. Rich information was obtained about the molecular structure of keratinocyte differentiation biomarkers such as ceramides, glucosylceramides, and sphingomyelins and the microstructures of reconstructed human epidermis lipids using HR-MSn. A new subclass of ceramides, 1-O-Acyl Omega-linoleoyloxy ceramides [1-O-E (EO) Cer] has been highlighted. This class is double esterified on the 1-O-position of sphingoid base with long to very long chain acyl residues (1-O-E) and on the position of ω-hydroxyl group of fatty acid with the linolenic acid (EO). Considering its chemical structure and hydrophobicity, this subclass can contribute to the skin barrier. In addition, we detected a new epidermis sphingomyelins. Our lipidomic approach offers a direct access to epidermis biomarkers.

Objective Although xerosis is a common skin disorder among the population, there is no in vivo global study focusing on xerotic skin. Hence, the objective of this study was to characterize xerotic skin from the surface to the molecular scale with in vivo and non-invasive approaches. Methods For this purpose, 15 healthy volunteers with normal skin and 19 healthy volunteers with xerotic skin were selected by a dermatologist, thanks to a visual scorage. Firstly, the skin surface was characterized with biometric measurements. Then, the state of skin dryness was assessed by in vivo confocal microscopy. The molecular signature of xerotic skin was then determined by in vivo confocal Raman microspectroscopy. Finally, an identification of stratum corneum (SC) lipids was performed using Normal phase liquid chromatography (NP-LC) coupled to two detectors: Corona and High Resolution/Mass Spectroscopy (HR/MS). Results Results obtained at the skin surface displayed an increase in the transepidermal water loss (TEWL) and a decrease in the hydration rate in xerotic skin. Confocal microscopy revealed an alteration of the cell shape in xerotic skin. Moreover, confocal Raman microspectroscopy demonstrated directly in vivo and non-invasively the lack of organization and conformation of lipids in this skin. Finally, HPLC analyses revealed that the three ceramide sub-classes (NdS, NS and EOP) significantly decrease in xerosis. Altogether, these results identify parameters for the characterization of xerotic skin compared to normal. Conclusion This study highlighted discriminative parameters from the surface to the molecular level in vivo and non-invasively between xerotic and normal skins. These results will be useful for the development of new cosmetic active ingredients dedicated to xerotic skin.

Phospholipids (PL) are components of increased value in nutritional applications, especially those rich in polyunsaturated fatty acids (PUFA) such as α‐linolenic acid (ALA, 18:3 n‐3) and linoleic acid (LA, 18:2 n‐6). Currently, the oil processing industry generates by‐products in the form of oilseed and plant cakes that can be valorized for food/feed applications. With the aim of diversifying PL sources and improving their identification in terms of fatty acid (FA) composition, plant cakes obtained from five different sources (camelina, flaxseed, hemp, sesame, walnut) were extracted and analyzed by lipidomics approach using three analytical methods of mass spectrometry (MS): (i) shotgun; (ii) reverse phase liquid chromatography (RPLC) in multiple reaction monitoring (MRM) mode; and (iii) normal phase liquid chromatography (NPLC) with data dependant scan MS fragmentation. We obtained complementary information: while shotgun allowed identifying PL species, RPLC associated to MRM enabled to access high sensitivity and to focus on minority species. Then, NPLC in data dependant scan led to the identification of lysophospholipids and cardiolipin species besides phosphatidylcholine (PC). Among, the five plant cakes analyzed, camelina cake showed the highest percentage of ALA. If ALA was also found in flaxseed and hemp cakes, it was in a lesser extent. Moreover, ALA was located on the sn‐2 position of the glycerol backbone, a valuable position for nutritional applications.Practical applications: The food industry generates large amounts of by‐products, and most of them remain unused. Among them, degumming pastes, i.e., by‐products from the oil refining process, could be valuable sources of phospholipids (PL). These phospholipids present interesting properties, as emulsifiers, thickening agents, or antioxidants, for food or cosmetic industries. Moreover, depending on the plant source, the fatty acid composition in phospholipids could be different from that of the oil (mainly composed of triglycerides instead of phospholipids), especially for α‐linolenic acid (ALA, 18:3 n‐3). Among, the five plant cakes analyzed (camelina, flaxseed, hemp, sesame, walnut), camelina cake showed the highest percentage of ALA. If ALA was also found in flaxseed and hemp cakes, it was in a lesser extent. Moreover, ALA was located in the sn‐2 position of the glycerol, a valuable position for nutritional applications. On the whole, using powerful and complementary analytical procedures for PL characterization allowed widening the potential sources of this type of lipids.Workflow of the plant cake lipid analysis based on three analytical methods of mass spectrometry.

Chromatographic profiling of plant metabolites is therefore a good tool for quality control of such herbal medicinal products. Our objective was to propose a protocol for sample preparation and liquid chromatographic profiling of non-polar metabolites for quality assessment of African herbal medicinal products. The methodology is based on the chemometric assessment of liquid chromatographic profiles of non-polar metabolites issued from several batches of leaves of Combretum micranthum and Mitracarpus scaber. Metabolic profiling is carried out by non-aqueous liquid chromatography on porous carbon graphite, coupled with mass spectrometry, after extraction with dichloromethane and removal of chlorophyll. Our method using liquid chromatography, coupled to mass spectrometry can detect non-polar metabolites already identified in the two herbal drugs. Chemometric data analysis of chromatographic profiles using the PLS-discriminant analysis with or without orthogonal signal correction, allowed a distinction between the two herbal drugs. (C) 2016 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.

The use of animals for scientific research is increasingly restricted by legislation, increasing the demand for human skin models. These constructs present comparable bulk lipid content to human skin. However, their permeability is significantly higher, limiting their applicability as models of barrier function, although the molecular origins of this reduced barrier function remain unclear. This study analyses the stratum corneum (SC) of one such commercially available reconstructed skin model (RSM) compared with human SC by spectroscopic imaging and chromatographic profiling. Total lipid composition was compared by chromatographic analysis (HPLC). Raman spectroscopy was used to evaluate the conformational order, lateral packing and distribution of lipids in the surface and skin/RSM sections. Although HPLC indicates that all SC lipid classes are present, significant differences are observed in ceramide profiles. Raman imaging demonstrated that the RSM lipids are distributed in a non-continuous matrix, providing a better understanding of the limited barrier function.

This study focuses on the mechanism of sitamaquine-resistance in Leishmania donovani. Sitamaquine accumulated 10 and 1.4 fold more in cytosol than in membranes of wild-type (WT) and of sitamaquine-resistant (Sita-R160) L. donovani promastigotes, respectively. The sitamaquine accumulation was a concentration-dependent process in WT whereas a saturation occurred in Sita-R160 suggesting a reduced uptake or an increase of the sitamaquine efflux. Membrane negative phospholipids being the main target for sitamaquine uptake, a lipidomic analysis showed that sitamaquine-resistance did not rely on a decrease of membrane negative phospholipid rate in Sita-R160, discarding the hypothesis of reduced uptake. However, sterol and phospholipid metabolisms were strongly affected in Sita-R160 suggesting that sitamaquine-resistance could be related to an alteration of phosphatidylethanolamine-N-methyl-transferase and choline kinase activities and to a decrease in cholesterol uptake and of ergosterol biosynthesis. Preliminary data of proteomics analysis exhibited different protein profiles between WT and Sita-160R remaining to be characterized.

The quality assessment of African traditional herbal medicinal products is a difficult challenge since they are complex mixtures of several herbal drug or herbal drug preparations. The plant source is also often unknown and/or highly variable. Plant metabolites chromatographic profiling is therefore an important tool for quality control of such herbal products. The objective of this work is to propose a protocol for sample preparation and gas chromatographic profiling of non-polar metabolites for quality control of African traditional herbal medicinal products. The methodology is based on the chemometric assessment of chromatographic profiles of non-polar metabolites issued from several batches of leaves of Combretum micranthum and Mitracarpus scaber by high temperature gas chromatography coupled to mass spectrometry, performed on extracts obtained in refluxed dichloromethane, after removal of chlorophyll pigments. The method using high temperature gas chromatography after dichloromethane extraction allows detection of most non-polar bioactive and non-bioactive metabolites already identified in leaves of both species. Chemometric data analysis using Principal Component Analysis and Partial Least Squares after Orthogonal Signal Correction applied to chromatographic profiles of leaves of Combretum micranthum and Mitracarpus scaber showed slight batch to batch differences, and allowed clear differentiation of the two herbal extracts.

A comparison of electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) for the analysis of a wide range of lipids has been performed on standard mixtures and extracts of Leishmania donovani promastigotes resistant to Amphotericin B (AmB). Calibration model, precision, limits of detection and quantification (LOD and LOQ) were assessed for each source. APPI provided the highest signal, signal-to-noise (S/N), and sensitivity for non-polar and low-polarity lipids, while ESI and APCI gave better results for the most polar ones. The linear model was valid for all lipids, except for one class with APPI, six classes with ESI, and eleven classes with APCI. LODs ranged from 0.2 to 20 mu g mL(-1) for ESI, from 0.1 to 10 mu g mL(-1) for APCI, and from 0.02 to 9.5 mu g mL(-1) for APPI. LOQs ranged from 0.2 to 61 mu g mL(-1) for ESI, from 0.4 to 31 mu g mL(-1) for APCI, and from 0.1 to 29 mu g mL(-1) for APPI. Each source provided similar lipid composition and variations in a comparison of three different L. donovani samples: miltefosine-treated, miltefosine-resistant and treated miltefosine-resistant parasites. A treated miltefosine-resistant sample was finally analyzed with each ion source in order to verify that the same lipid molecular species are detected. (C) 2012 Elsevier B.V. All rights reserved.

Lipidomic studies often use liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) for separation, identification, and quantification. However, due to the wide structural diversity of lipids, the most apolar part of the lipidome is often detected with low sensitivity in ESI. Atmospheric pressure (APPI) can be an alternative ionization source since normal-phase solvents are known to enhance photoionization of these classes. In this paper, we intend to show the efficiency of APPI to identify different lipid classes, with a special interest on sphingolipids. In-source APPI fragmentation appears to be an added value for the structural analysis of lipids. It provides a detailed characterization of both the polar head and the non polar moiety of most lipid classes, and it makes possible the detection of all lipids in both polarities, which is not always possible with ESI.

Leishmaniasis is a widespread parasitic disease principally treated by intravenous drugs. Hexadecylphosphocholine (miltefosine) has recently proved its efficacy by oral route. Although its mechanism of action has been investigated, and principally relies on perturbations of the metabolism of lipids and especially phospholipids, further studies need to be conducted to detect precisely which metabolic pathways are impacted. For this purpose, the present work proposes a complete lipidomic study focused on membrane phospholipids of clones of Leishmania donovani non-treated (NT), treated (T) and resistant (R) to miltefosine. Firstly, a separation of phospholipids in normal phase high-performance liquid chromatography (NP-HPLC) was coupled to a mass spectrometer (MS) equipped with an electrospray (ESI) ion source, and response was compared to evaporative light scattering detection (ELSD). Secondly, a quantification of phospholipid classes was performed using NP-HPLC/ESI/MS on NT, T and R clones of L. donovani . Thirdly, full-scan acquisitions of analyzed samples were compared using orthogonal signal correction-partial least square-discriminant analysis (OSC-PLS-DA) to highlight phospholipid molecular species of interest between the three types of clones. Structural determination of the most relevant species has finally been performed using tandem mass spectrometry. A first hypothesis on the effect of miltefosine on lipid metabolic pathways is then proposed.

OBJECTIVES This study focuses on the mechanism of interaction of sitamaquine with Leishmania donovani membranes, and its accumulation within the parasites. METHODS A biomimetic model of the outer layer of a Leishmania plasma membrane was used to examine the interactions of sitamaquine with lipids. The plasma membranes of L. donovani promastigotes were depleted of sterol using cholesterol oxidase, in order to assess the importance of sterols in drug-membrane interactions. Sterols were quantified and sitamaquine susceptibility was assessed using the MTT test. Kinetics of sitamaquine accumulation and efflux were measured under different conditions. RESULTS Sitamaquine interacts first with phospholipid anionic polar head groups and then with phospholipid acyl chains to insert within biological membranes and accumulates rapidly in the Leishmania cytosol according to a sterol-independent process. The rapid sitamaquine efflux observed was related to an energy-dependent mechanism since the intracellular amount of sitamaquine was enhanced three times in the absence of glucose and the efflux was inhibited in energy-depleted conditions. (1)H NMR analysis of motile lipid showed that sitamaquine did not affect lipid trafficking in Leishmania. CONCLUSIONS We propose that sitamaquine rapidly accumulates in Leishmania by diffusion along an electrical gradient and is concentrated in the cytosol by an energy- and sterol-independent process. The affinity of sitamaquine for membranes was transitory and an energy-dependent efflux was demonstrated, suggesting the presence of an as yet uncharacterized transporter.

The surface of polymeric nanocapsules used as ultrasound contrast agents (UCAs) was modified with PEGylated phospholipids in order to escape recognition and clearance by the mononuclear phagocyte system and achieve passive tumor targeting. Nanocapsules consisted of a shell of poly(lactide-co-glycolide) (PLGA) encapsulating a liquid core of perfluorooctyl bromide (PFOB). They were decorated with poly(ethylene glycol-2000)-grafted distearoylphosphatidylethanolamine (DSPE-PEG) incorporated in the organic phase before the solvent emulsification-evaporation process. The influence of DSPE-PEG concentration on nanocapsule size, surface charge, morphology, hydrophobicity and complement activation was evaluated. Zeta potential measurements, Hydrophobic interaction chromatography and complement activation provide evidence of DSPE-PEG presence at nanocapsule surface. Electronic microscopy reveals that the core/shell structure is preserved up to 2.64 mg of DSPE-PEG for 100 mg PLGA. In vivo ultrasound imaging was performed in mice bearing xenograft tumor with MIA PaCa-2 cells, either after an intra-tumoral or intravenous injection of nanocapsules. Tumor was observed only after the intra-tumoral injection. Despite the absence of echogenic signal in the tumor after intravenous injection of nanocapsules, histological analysis reveals their accumulation within the tumor tissue demonstrating that tissue distribution is not the unique property required for ultrasound contrast agents to be efficient.

The performance of charged aerosol detection (CAD) was compared to evaporative light scattering detection (ELSD) for the analysis of Leishmania membrane phospholipid (PL) classes by NP-HPLC. In both methods, a PVA-Sil column was used for the determination of the major Leishmania membrane PLs, phosphatidic acid, phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylethathanolamine, phosphatidylserine, lysophosphatidylethathanolamine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine in the same analysis. Although the response of both detection methods can be fitted to a power function, CAD response can also be described by a linear model with determination coefficients (R(2)) ranging from 0.993 to 0.998 for an injected mass of 30 ng to 20.00 microg. CAD appeared to be directly proportional when a restricted range was used and it was found to be more sensitive at lowest mass range than ELSD. With HPLC-ELSD the limits of detection (LODs) were between 71 and 1195 ng and the limits of quantification (LOQs) were between 215 and 3622 ng. With HPLC-CAD, the LODs were between 15 and 249 ng whereas the limits of quantification (LOQs) were between 45 and 707 ng. The accuracy of the methods ranged from 62.8 to 115.8% and from 58.4 to 110.5% for ELSD and CAD, respectively. The HPLC-CAD method is suitable to assess the influence of miltefosine on the composition of Leishmania membrane phospholipids.

We present here an easy method to modify the Surface chemistry of polymeric microcapsules of perfluorooctyl bromide used as ultrasound contrast agents (UCAs). Capsules were obtained by a solvent emulsification-evaporation process with phospholipids incorporated in the organic phase before emulsification. Several phospholipids were reviewed: fluorescent, pegylated and biotinylated phospholipids. The influence of phospholipid concentration oil microcapsule size and morphology was evaluated. Only a fraction of the phospholipids is associated to microcapsules, the rest being dissolved With the surfactant in the aqueous phase. Microscopy shows that phospholipids are present within the shell and that the core/shell structure is preserved Lip to 0.5 mg fluorescent phospholipids, Lip to about 0.25 mg pegylated phospholipids or biotinylated phospholipids (for 100 mg of polymer, poly(lactide-co-glycolide) (PLGA)). HPLC allows quantifying phospholipids associated to capsules: they correspond to 10% of pegylated phospholipids introduced in the organic phase. The presence of pegylated lipids at the Surface of capsules was confirmed by X-ray photon electron spectroscopy (XPS). The pegylation did not modify the echographic signal arising from Capsules. Finally biotinylated microcapsules incubated with neutravidin tend to aggregate, which confirms the presence of biotin at the Surface. These results are encouraging and future work will consist of nanocapsule surface modification for Molecular imaging. (C) 2008 Elsevier Ltd. All rights reserved.

Polyethylene glycol (PEG) chains covalently linked to phospholipids are often used in the preparation of lipid or even polymer colloidal particles to avoid recognition and clearance by the reticuloendothelial system and to increase their plasmatic half-life. To the best of our knowledge, no direct method allows yet to quantify these pegylated phospholipids. The aim of this work was to develop a method for the quantification of a typical pegylated phospholipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], DSPE-PEG2000, associated to polymeric microcapsules of perfluorooctyl bromide (PFOB). Reverse phase high-performance liquid chromatography (HPLC) was used, coupled with a corona charged aerosol detection (HPLC–CAD). Calibrations standards consisted of plain microcapsules and pegylated phospholipids (DSPE-PEG2000) in the concentration range of 2.23–21.36μg/mL (0.22–2.14μg injected). Calibration curve was evaluated with two different model, linear and power model. The power model describes experimental values better than the linear model, for pegylated phospholipids with the CAD detector. The correlation coefficient for the power model was 0.996, and limits of detection and quantification obtained were 33 and 100ng, respectively. This method proved to be selective and sensitive; the accuracy of the method ranged from 90 to 115% and the relative standard deviation was ≤5.3%. Pegylated phospholipids associated to microcapsules, as well as the phospholipids and total phospholipids in the suspensions were successfully quantified in three different preparations of microcapsules.