Stratum corneum ceramide profiles in vitro, ex vivo, and in vivo: characterization of the α-hydroxy double esterified ceramides.

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.

Comprehensive characterization and simultaneous analysis of overall lipids in reconstructed human epidermis using NPLC/HR-MSn: 1-O-E (EO) Cer, a new ceramide subclass.

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.

Comparison of structure and organization of cutaneous lipids in a reconstructed skin model and human skin: spectroscopic imaging and chromatographic profiling.

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.

High-temperature micro liquid chromatography for lipid molecular species analysis with evaporative light scattering detection.

The need for a rapid and sensitive chromatographic technique for analyzing lipid molecular species, has led to the development of an high-temperature micro liquid chromatographic system (HTLC) coupled to an evaporative light scattering detector. The increased diffusion coefficients and reduced viscosity at higher temperatures allowed lipids to be analyzed rapidly with solvents differing from those classically used in lipids chemistry. Hypercarb, a reverse phase material, was used for its different properties including heat resistance in high temperature micro HPLC. We have investigated the temperature effect on kinetic performances in HTLC, established pure solvents eluent strength at high temperature and studied different classes of lipids with seven pure solvents. We found that it was possible to use alcohols solvents in the mobile phase to elute lipids without the use of chlorinated solvents. A quick and simple method was developed to analyze a complex lipid simple, ceramide type III and type IV.

Sensitive method for the detection of 22 benzodiazepines by gas chromatography-ion trap tandem mass spectrometry.

A gas chromatography-ion trap tandem mass spectrometry method for simultaneous detection of 22 benzodiazepines is presented. Four operating modes were first optimized: the electron impact ionization and chemical ionization modes were compared on both underivatized and trimethylsilylated drugs. Results were compared in terms of sensitivity in MS-MS experiments. The trimethylsilylation of benzodiazepines including a protic functional group allows decreasing their detection threshold by a factor of 10-100. In terms of sensitivity, the comparison between both ionization modes shows that the most efficient one depends on the benzodiazepine considered. The use of an ion trap analyzer allows switching from an ionization mode to another one during the chromatographic process. It also provides a great selectivity owing to the MS-MS and multiple reaction monitoring acquisition modes. The detection thresholds are in the range 10-500 pg/microl for all the studied benzodiazepines but the three "triazolo" ones: estazolam, alprazolam and triazolam, have a detection threshold of 1 ng/microl. The applicability of the method on whole blood and urine extracts was demonstrated on an example implying five benzodiazepines among the most frequently encountered in forensic toxicology: nordazepam, oxazepam, bromazepam, flunitrazepam and prazepam.

Adsorption-desorption effects in ion trap mass spectrometry using in situ ionization.

Quadrupole mass spectrometers were compared for the GC-MS analysis of six molecules frequently encountered in analytical toxicology: diazepam, alprazolam, triazolam, LSD (lysergic acid diethylamide), trimethylsilylated LSD and trimethylsilylated buprenorphine. Experiments performed with ion trap detectors using in situ ionization led to important chromatographic peak tailing for the most polar compounds; it was assumed to result from adsorption-desorption of neutral molecules in the mass spectrometer. This study showed that the degree of peak tailing is correlated with analyte polarity, with materials coating ion trap surfaces and with analysis temperature and that this anomaly can be greatly reduced using passivated surfaces and a high temperature of analysis.

Development and validation of a gas chromatography-mass spectrometry method for the simultaneous determination of buprenorphine, flunitrazepam and their metabolites in rat plasma: application to the pharmacokinetic study.

Buprenorphine (BUP), a synthetic opioid analgesic, is frequently abused alone, and in association with benzodiazepines. Fatalities involving buprenorphine alone seem very unusual while its association with benzodiazepines, such as flunitrazepam (FNZ), has been reported to result in severe respiratory depression and death. The quantitative relationship between these drugs remain, however, uncertain. Our objective was to develop an analytical method that could be used as a means to study and explore, in animals, the toxicity and pharmacological interaction mechanisms between buprenorphine, flunitrazepam and their active metabolites. A procedure based on gas chromatography-mass spectrometry (GC-MS) is described for the simultaneous analysis of buprenorphine, norbuprenorphine (NBUP), flunitrazepam, N-desmethylflunitrazepam (N-DMFNZ) and 7-aminoflunitrazepam (7-AFNZ) in rat plasma. The method was set up and adapted for the analysis of small plasma samples taken from rats. Plasma samples were extracted by liquid-liquid extraction using Toxi-tubes A. Extracted compounds were derivatized with N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA), using trimethylchlorosilane (TMCS) as a catalyst. They were then separated by GC on a crosslinked 5% phenyl-methylpolysiloxane analytical column and determined by a quadrupole mass spectrometer detector operated under selected ion monitoring mode. Excellent linearity was found between 0.125 and 25 ng/microl plasma for BUP, 0.125 and 12.5 ng/microl for NBUP and N-DMFNZ, 0.125 and 5 ng/microl for FNZ, and between 0.025 and 50 ng/microl for 7-AFNZ. The limit of quantification was 0.025 ng/microl plasma for 7-AFNZ and 0.125 ng/microl for the four other compounds. A good reproducibility (intra-assay CV=0.32-11.69%; inter-assay CV=0.63-9.55%) and accuracy (intra-assay error=2.58-12.73%; inter-assay error=0.83-11.07%) were attained. Recoveries were 71, 67 and 81%, for BUP, FNZ and N-DMFNZ, respectively, and 51% for NBUP and 7-AFNZ, with CV ranging from 5.4 to 13.9%, and were concentration-independent. The GC-MS method was successfully applied to the pharmacokinetic study of BUP, NBUP, FNZ, DMFNZ and 7-AFNZ in rats, after administration of BUP and FNZ.

Low pressure chemical ionization in ion trap mass spectrometry.

This article presents the specificities of low pressure chemical ionization in ion trap mass spectrometry. One main feature is the ability to perform chemical ionization with liquid reagents as readily as with "conventional" gases (methane, isobutane and ammonia). The reactivities and analytical applications of gas and liquid reagents are summarized from literature data and are compared when possible.

A selective and sensitive method for quantitation of lysergic acid diethylamide (LSD) in whole blood by gas chromatography-ion trap tandem mass spectrometry.

A gas chromatography-ion trap tandem mass spectrometry (GC-ion trap MS-MS) method for detection and quantitation of LSD in whole blood is presented. The sample preparation process, including a solid-phase extraction step with Bond Elut cartridges, was performed with 2 mL of whole blood. Eight microliters of the purified extract was injected with a cold on-column injection method. Positive chemical ionization was performed using acetonitrile as reagent gas; LSD was detected in the MS-MS mode. The chromatograms obtained from blood extracts showed the great selectivity of the method. GC-MS quantitation was performed using lysergic acid methylpropylamide as the internal standard. The response of the MS was linear for concentrations ranging from 0.02 ng/mL (detection threshold) to 10.0 ng/mL. Several parameters such as the choice of the capillary column, the choice of the internal standard and that of the ionization mode (positive CI vs. EI) were rationalized. Decomposition pathways under both ionization modes were studied. Within-day and between-day stability were evaluated.

Establishing high temperature gas chromatographic profiles of non-polar metabolites for quality assessment of African traditional herbal medicinal products.

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.

Atmospheric pressure photoionization as a powerful tool for large-scale lipidomic studies.

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.

Comparison of electrospray ionization, atmospheric pressure chemical ionization and atmospheric pressure photoionization for a lipidomic analysis of Leishmania donovani.

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 µg mL(-1) for ESI, from 0.1 to 10 µg mL(-1) for APCI, and from 0.02 to 9.5 µg mL(-1) for APPI. LOQs ranged from 0.2 to 61 µg mL(-1) for ESI, from 0.4 to 31 µg mL(-1) for APCI, and from 0.1 to 29 µg 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.

The performance of PEGylated nanocapsules of perfluorooctyl bromide as an ultrasound contrast agent.

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.

Phospholipid decoration of microcapsules containing perfluorooctyl bromide used as ultrasound contrast agents.

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 on 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 up to 0.5 mg fluorescent phospholipids, up 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.