High-Performance Thin-Layer Chromatography-Densitometry-Tandem ESI-MS to Evaluate Phospholipid Content in Exosomes of Cancer Cells.

The question of whether exosome lipids can be considered as potential cancer biomarkers faces our current limited knowledge of their composition. This is due to the difficulty in isolating pure exosomes, the variability of the biological sources from which they are extracted, and the uncertainty of the methods for lipid characterization. Here, we present a procedure to isolate exosomes and obtain a deep, repeatable, and rapid phospholipid (PL) composition of their lipid extracts, from embryonic murine fibroblasts (NIH-3T3 cell line) and none (B16-F1) and high (B16-F10) metastatic murine skin melanoma cells. The analytical method is based on High Performance Thin-Layer Chromatography with Ultraviolet and fluorescence densitometry and coupled to Electrospray (ESI)-tandem Mass Spectrometry (MS). Under the conditions described in this work, separation and determination of PL classes, (sphingomyelins, SM; phosphatidylcholines, PC; phosphatidylserines, PS; and phosphatidylethanolamines, PE) were achieved, expressed as µg PL/100 µg exosome protein, obtained by bicinchoninic acid assay (BCA). A detailed structural characterization of molecular species of each PL class was performed by simultaneous positive and negative ESI-MS and MS/MS directly from the chromatographic plate, thanks to an elution-based interface.

Changes in fluorescent emission due to non-covalent interactions as a general detection procedure for thin-layer chromatography.

Changes in fluorescence emission due to non-covalent analyte-fluorophore interactions in silica gel plates are studied and used as a general detection procedure for thin-layer chromatography (TLC). The presence of the analyte modifies the microenvironment of the fluorophore and thus changes the balance between radiative (k(r)) and non-radiative (k(nr)) emission constants. A model is proposed for analyte-fluorophore induced electrostatic interactions, which depend on analyte polarizability and are responsible for fluorescence enhancements. As consequence of these induced interactions, the analyte creates an apolar environment that prevents non-fluorescent decay mechanisms, decreasing k(nr). On the other hand, the effect of an increase in refractive index on k(r) is investigated, as it contributes to some extent to fluorescence enhancements in silica gel medium. Changes in fluorescence emission should be regarded as a general property of fluorophores in the presence of analytes, and criteria that fluorophores should meet to be used as sensitive TLC probes are discussed here.

Globotriaosylceramide-related biomarkers of fabry disease identified in plasma by high-performance thin-layer chromatography - densitometry- mass spectrometry.

Identification of 19 molecular species of globotriaosylceramides (Gb3) in extracts from a Fabry's plasma patient and a healthy control was performed by High-Performance Thin-Layer Chromatography (HPTLC)-densitometry and online coupling to Mass Spectrometry (MS). Separation was carried out on LiChrospher plates using Automated Multiple Development (AMD). Densitometry was performed on twin plates by combining detection in the visible at 550 nm, through previous on-plate orcinol derivatization, and by Ultraviolet 190 nm, using a non-impregnated plate. The latter was directly coupled to an ion-trap mass spectrometer through an automated elution-based interface. Gb3 molecular species, which were identified by HPTLC- Electrospray Mass Spectrometry (+)-MS and confirmed by MS/MS or HPTLC-Atmospheric Pressure Chemical Ionization Mass Spectrometry (+)-MS, are: five isoforms of saturated Gb3; seven isoforms of methylated Gb3; and seven species with two additional double bonds. Twelve of these species were previously reported as biomarkers of Fabry's lysosomal disorder using a Liquid Chromatography-MS-based method, and the other seven are structurally similar, closely related to them. Saturated Gb3 isoforms migrated on LiChrospher plate in one of the separated peaks corresponding to the migration zone of ceramide trihexosides standard. Instead, methylated and unsaturated Gb3 species co-migrated with sphingomyelin species. Ion intensity ESI-MS profiles show that saturated Gb3 species in Fabry's plasma were in higher concentration than in control sample. Before applying the Thin-Layer Chromatography (TLC)-MS interface on HPTLC separated peaks, its positioning precision was first studied using ceramide tri-hexosides as model compound. This provided information on Gb3 peak broadening and splitting during its migration.

High-Performance Thin-Layer Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry for Identifying Neutral Lipids and Sphingolipids in Complex Samples.

High-performance thin-layer chromatography was directly combined with electrospray mass spectrometry (ESI-MS) for structural identification issues below the level of lipid classes in complex samples through a portable, automated, elution-based interface. For samples as diverse as biodiesel and human plasma, separation conditions using Automated Multiple Development were selected in each case to provide lipid classes as zones narrow enough to ensure a direct transfer of them to ESI-MS. The respective zone of interest can be selected at will. ESI+ spectra of neutral lipids and sphingolipids showed sodium adducts when recorded from the plate. By using the described technique and ion-trap technology, the respective sodium adducts were fragmented. Sodium remained as the charge of the fragment ions and, thus, was useful for their structural identification through MSn. In this way, composition profiles of each class by ESI+-MS, and further identification of individual lipids and the molecular species belonging to each of them, were obtained by MS/MS and/or high-resolution MS. Thus, mono and diacylglycerides in ESI+ and fatty acids (in ESI-) were identified as low-concentration impurities in a fatty acid methyl ester-based biodiesel sample. Likewise, molecular species of sphingomyelins and globotriaosylceramides were unequivocally identified in human plasma samples.

Coralyne cation, a fluorescent probe for general detection in planar chromatography.

A large number of analytes, including non-fluorescent ones, can be sensitively detected by fluorescence scanning densitometry using silica gel HPTLC plates impregnated with a solution of coralyne cation. This is carried out by the variation, increase or decrease, that the corresponding analyte induces on native coralyne emission at a given excitation wavelength. A similar phenomenon was previously described for berberine cation, and Reichardt's dye probes. However, the sensitivity of coralyne in HPTLC detection of non-fluorescent, structurally different analytes (e.g., long-chain alkanes, alcohols, alkylbromides, neutral lipids) is superior to that of the above-mentioned probes. In this work, the analytical viability of this phenomenon for HPTLC detection using coralyne as a probe is explored, and fluorescent responses of a number of analytes on the coralyne system are rationalized in the light of a previously proposed model. This establishes that the resulting intensity for a probe in the presence of a given compound can be explained as a balance between radiative (contribution of non-specific interactions) and non-radiative processes (specific interactions), the latter producing fluorescence quenching. Experimental results and proposed model suggest that this phenomenon may be general for practically all kinds of analytes.

Molecular weight distributions of industrially-produced poly-(epsilon-caprolactams) by gel permeation chromatography.

Gel permeation chromatography with differential refractometry is used to obtain molecular weight distributions (MWD) of poly-(epsilon-caprolactams). Elution is carried out using an m-cresolchlorobenzene mixture (50:50, v/v) at 50 degrees C. MW values are obtained by a Hamielec-based calibration method, using broad-MWD poly-(epsilon-caprolactam) standards with the same chemical nature and similar MWD to the samples. Relative errors for the number-average MW (Mn) using this calibration method range from 0.4% (in the low polyamide MW range) to 20% (in the high polyamide MW range). These values are much lower than those obtained from narrow-MWD polystyrene calibration, which range from 39% to 78%. Similar values have been obtained for the other usual average MW parameters. The ability to obtain repeatability parameters for a given confidence interval and the utilization of statistical criteria for chromatogram rejection allow this method to be used in quality control for MWD of poly-(epsilon-caprolactams). Thus, production variables are related to polyamide-6 behavior in its ulterior treatment. Typical relative standard deviation percentages (for n=6) of a polyamide sample range from 1.9% (for Mn) to 3.3% (for M(z+1)).

Fluorescence detection by intensity changes for high-performance thin-layer chromatography separation of lipids using automated multiple development.

Changes in emission of berberine cation, induced by non-covalent interactions with lipids on silica gel plates, can be used for detecting and quantifying lipids using fluorescence scanning densitometry in HPTLC analysis. This procedure, referred to as fluorescence detection by intensity changes (FDIC) has been used here in combination with automated multiple development (HPTLC/AMD), a gradient-based separation HPTLC technique, for separating, detecting and quantifying lipids from different families. Three different HPTLC/AMD gradient schemes have been developed for separating: neutral lipid families and steryl glycosides; different sphingolipids; and sphingosine-sphinganine mixtures. Fluorescent molar responses of studied lipids, and differences in response among different lipid families have been rationalized in the light of a previously proposed model of FDIC response, which is based on ion-induced dipole interactions between the fluorophore and the analyte. Likewise, computational calculations using molecular mechanics have also been a complementary useful tool to explain high FDIC responses of cholesteryl and steryl-derivatives, and moderate responses of sphingolipids. An explanation for the high FDIC response of cholesterol, whose limit of detection (LOD) is 5 ng, has been proposed. Advantages and limitations of FDIC application have also been discussed.

General contribution of nonspecific interactions to fluorescence intensity.

Many chemical compounds, including nonfluorescent ones, induce changes in the fluorescence spectra of certain probes, such as berberine cation and Reichardt's betaine, both in the absence and the presence of solvent, that affect almost exclusively emission intensity. In this work, the application of fluorescence detection by intensity changes (FDIC) to HPLC and TLC chromatographic systems with fluorescence detectors has been studied. FDIC detection is of special interest in detecting nonfluorescent analytes, either in HPLC or in TLC mode. It does not involve covalent interactions, and the dielectric permittivity (epsilon) of the medium plays an important role. The balance between nonspecific and specific interactions produces either an increase or a decrease in fluorescence intensity. Therefore, the influence of chromatographic conditions and chemical structure of analytes on the sign and magnitude of fluorescence peaks for sample detection in HPLC and TLC systems has been discussed. In general, probe nature and concentration determine response and detection sensitivity for a given sample in TLC and HPLC. As solubility and fluorescence properties in solvents determine the operating conditions for a FDIC probe in HPLC mode, nature and flows of mobile phase and solvent are important for chromatographic response and detection sensitivity.