Vibrational spectroscopies for the analysis of cutaneous permeation: experimental limiting factors identified in the case of caffeine penetration.

Caffeine is utilised as a reference for permeation studies in dermatology and cosmetology. The present work aimed to monitor the permeation of a caffeine solution through the skin. For this purpose, Raman and infrared studies were performed. Raman microspectroscopy permitted a dynamic follow-up of the caffeine diffusion. In complementary, infrared microimaging provided information of the caffeine localization in the skin by applying multivariate statistical processing on skin tissue sections. Herein, we prove the possibility of tracking low concentrations of caffeine through the skin and we highlight some experimental limitations of vibrational spectroscopies.

Retention behaviour of polyunsaturated fatty acid methyl esters on porous graphitic carbon.

Retention with porous graphitic carbon was investigated with 25 structures of fatty acid methyl esters (FAMEs) with two different mobile phases: CH(3)CN:CHCl(3) 60:40 (v/v) and CH(3)OH:CHCl(3) 60:40 (v/v) with both 0.1% triethylamine (TEA) and an equimolar amount of HCOOH. Preliminary results showed that the use of TEA/HCOOH led to the response increase of saturated FAMEs with evaporative light scattering detection. No increase was observed for unsaturated one. These modifiers may slightly reduce the retention of FAMEs but did not significantly modify the separation factor with porous graphitic carbon. Thermodynamic parameters were calculated for each structure using Van't Hoff plot measured over the temperature range from 10 to 50 degrees C, with the both mobile phase conditions. All the studied compounds were found linked by the same retention mechanism on porous graphitic carbon. Quantitative in silico analysis of the retention using a molecular mechanics calculation demonstrated a good correlation between the retention factors and the molecular interaction energy values (r>0.93). Especially the Van der Waals energy was predominant, and the contribution of electrostatic energy was negligible for the quantitative analysis of the retention. The results indicate that Van der Waals force, hydrophobic interaction, is predominant for the retention of FAMEs on this packing material. The relative retention for highly unsaturated homologues can be changed by the selection of the weak solvent CH(3)CN or CH(3)OH. Then isomers differing only in the position of the carbon double bond on the alkyl chain can be separated and their behaviour is summarised as the closer the carbon double bonds to the FAME polar head, the more the retention decreases. Finally, the more important the number of carbon double bonds in the alkyl chain is, the smaller the retention is.

Microanalytical systems for separations of stratum corneum ceramides.

The small amount of lipids from human skin obtained with noninvasive sampling method led us to investigate microanalytical separation techniques. The lipid class analysis was performed with a micro polyvinyl alcohol-silica (PVA-Sil) column. The gradient elution was from heptane to acetone/butanol 90:10 v/v in 4%/min at 78 microL/min. In addition an evaporative light scattering detector (ELSD) was modified for micro-LC. All solvents contained 0.1% of triethylamine and formic acid in stoichiometric amount, which increased the ELSD response. In these conditions, the cholesterol eluted before free fatty acid, and squalene and triglycerides close to the dead volume. The various ceramide classes eluted following the order of the increased number of hydroxyl groups. The LOD for ceramides was 2.2 ng. The advantages of this method are the use of a normal stationary phase more reliable due to its chemical stability, its surface homogeneity and its development in microchromatography without chlorinated solvents which offers small LOD and the whole profile of lipids present in stratum corneum (SC). A method using a narrow-bore PVA-Sil column was used to collect ceramide fraction. Then the molecular species were analysed with a porous graphitic carbon column in capillary LC using a gradient from CH3OH/CHCl3 70:30 v/v to CHCl3 at 2%/min with a flow rate at 5 microL/min. The LOD obtained for ceramide was 1 ng. Both methods were assessed with SC samples obtained by rinsing a 5.7 cm2 area of the forearm with 25 mL of ethanol.

Application of a xenon arc lamp as a light source for evaporative light scattering detection.

The standard tungsten-halogen light source used in a commercial evaporative light scattering detector (ELSD) was replaced with a 180 W xenon arc lamp. The xenon arc lamp possesses a broader spectrum in the UV region than the halogen source. The influence of the UV transmittance of five selected solvents was studied with a size-exclusion chromatography column. This solvent parameter was not observed to influence the ELSD response between the two light source settings. With the solvents studied, better sensitivity was obtained with the xenon arc lamp than the halogen lamp. This high-energy source was applied to ceramide III analysis with an octadecyl-grafted silica column and methanol:tetrahydrofuran 97:3 as the mobile phase, and the sensitivity of the quantification of ceramide III increased 16-fold for injected amounts of 14 approximately 140 ng. The molecular species in a sample of naturally occurring ceramides was analyzed using two C18 columns at 40 degrees C and gradient elution from 100% acetonitrile to 100% isopropanol in 30 min. The increased ELSD sensitivity achieved when using the xenon arc lamp allowed both the minor and major ceramide species to be observed, in contrast to the results achieved when the halogen lamp was used, where the increased photomultiplier voltage needed to observed the signals from the minor species caused the signals from the major ceramide species to occur above the detector response window.

Atmospheric pressure photoionization coupled to porous graphitic carbon liquid chromatography for the analysis of globotriaosylceramides. Application to Fabry disease.

Globotriaosylceramides (Gb(3)) are biological compounds implicated in Fabry disease, a lysosomal storage disease due to the deficient activity of alpha-D-galactosidase A, which results in an accumulation of Gb(3) in many organs. The naturally occurring samples are composed of mixtures of several molecular species differing by the structure of the alkyl chains and the nature of the sphingoid base. Atmospheric pressure photoionization mass spectrometry (APPI-MS) proved to be an efficient method for the analysis of globotriaosylceramide molecular species, both in direct injection and by coupling with liquid chromatography (LC). In the positive ion mode, in-source fragmentations yield very precious information that can be used to determine the structure of the alkyl chains. In the negative ion mode, the chloroform solvent participates to the analyte ionization by forming an adduct with chloride ions generated in situ. Combination of LC on a Porous Graphitic Carbon stationary phase and APPI-MS allowed the detection of a great number of species from biological samples isolated from Fabry patients. This method could be an interesting analytical tool for the biochemical investigation of (sphingo) lipid metabolism.

Fast fingerprinting by MALDI-TOF mass spectrometry of urinary sediment glycosphingolipids in Fabry disease.

Fabry disease (FD) is an X-linked inborn error of glycosphingolipid (GSL) metabolism, caused by a deficiency of the lysosomal alpha-galactosidase A, which results in high levels in lysosomes and biological fluids of globotriaosylceramide (Gb3) and digalactosylceramide (Ga2), also known as galabiosylceramide. We report here a detailed study of the molecular species of GSLs in urinary samples obtained from hemizygous and heterozygous patients by use of matrix-assisted laser desorption ionisation and tandem mass spectrometry (MALDI-MS-MS). Twenty-two and fifteen molecular species were identified in the globotriaosylceramide and digalabiosylceramide series, respectively. The major sphingoid base was sphingosine (d18:1), and dihydrosphingosine (C18:0) and sphingadienine (d18:2) were also present. The molecular profiles obtained by MALDI-TOF-MS enabled us to show significant differences between GSLs composition for young, adult or atypic hemizygote and heterozygote patients. Thus, MALDI-TOF-MS and MS-MS proved a powerful tool for screening a population of patients with clinical signs suggestive of FD by direct and rapid GSL fingerprinting and identification, and for study of the biological processes occurring in glycosphingolipid accumulation.

Wheat digalactosyldiacylglycerol molecular species profiling using porous graphitic carbon stationary phase.

The potential of porous graphitic carbon stationary phase (PGC) was assessed for the separation of molecular species of digalactosyldiacylglycerol (DGDG). Detection was by an evaporative light scattering detector (ELSD). A conventional optimization strategy allowed definition of a quaternary non-aqueous mobile phase and separation of 9 wheat DGDG molecular species with isocratic elution: methanol/toluene/tetrahydrofuran/chloroform 64.3/21.5/13.7/0.5 v/v with 0.1% of triethylamine and a stoichiometric amount of formic acid. The molecular species were identified by LC/MS. The chromatographic behavior of DGDG on PGC was then compared to previous studies. The addition of a carbon double bond on the alkyl chain decreased the retention. This contribution was less important when the number of unsaturations increased in the alkyl chain. The consequence of this retention behavior with PGC was an elution order of molecular species which did not agree with the partition number as observed with C18 grafted stationary phases.

Adaptation of an evaporative light-scattering detector to micro and capillary liquid chromatography and response assessment.

A commercially available evaporative light-scattering detection (ELSD) system was adapted for micro and capillary LC. Therefore the various parameters involved in the droplet formation during the nebulization step in the ELSD system were studied. It was shown that the velocity term in the Nukiyama Tanasawa equation remains constant, leading to droplets of the same order of magnitude for narrow bore and capillary columns. Consequently, the ELSD modification was performed by decreasing the internal diameter of the effluent capillary tube in the nebulizer nozzle and by keeping its external diameter constant. Next, response curves for a conventional and the developed micro and capillary LC were compared as to investigate why a linear ELSD response is often obtained when used in micro or capillary LC. By splitting the flow rate post column, we showed that the nebulization process was not at the origin of the phenomenon. For ceramide III and tripalmitin, the response curves were found to be non-linear. However the curvature was less significant when the columns internal diameter decreased. Calculated particle size profiles for micro or capillary LC suggest that the particle entering the detection chamber are bigger than under conventional LC conditions. Last, triethylamine and formic acid were used to increase the response of the detector. The response enhancement, expected from previous studies, was established for the two lipids involved in this study.

Retention behaviour of unsaturated fatty acid methyl esters on porous graphitic carbon.

The eluotropic strength of binary mobile phases was calculated for three homologous series of cis, trans, and cis-cis unsaturated fatty acid methyl esters (FAMEs). Binary mobile phases with chloroform, dichloromethane, or tetrahydrofuran as strong solvent and methanol or acetonitrile as weak solvent were tested. The volume fraction of strong solvent in the binary phases was between 0.3 and 0.8. Curves of eluotropic strength versus volume fraction of strong solvents showed similar trends to previously published results for saturated homologues. Correlation coefficients of the plots of eluotropic strength values for saturated versus unsaturated FAMEs were close to 1.0. Therefore these similarities validate the model of eluotropic strength previously established with saturated FAMEs as relevant for unsaturated FAMEs. The separation factors between cis and trans homologues always showed elution of the cis before the trans homologue. The difference in retention is due primarily to the geometry of the molecule. The retention is lowered more by the addition of a first carbon double bond than by the addition of a second one, independently of the mobile phase composition.

Glycolipid class profiling by packed-column subcritical fluid chromatography.

The potential of packed-column subcritical fluid chromatography (SubFC) for the separation of lipid classes has been assessed in this study. Three polar stationary phases were checked: silica, diol, and poly(vinyl alcohol). Carbon dioxide (CO2) with methanol as modifier was used as mobile phase and detection performed by evaporative light scattering detection. The influence of methanol content, temperature, and pressure on the chromatographic behavior of sphingolipids and glycolipids were investigated. A complete separation of lipid classes from a crude wheat lipid extract was achieved using a modifier gradient from 10 to 40% methanol in carbon dioxide. Solute selectivity was improved using coupled silica and diol columns in series. Because the variation of eluotropic strength depending on the fluid density changes, a normalized separation factor product (NSP) was used to select the nature, the number and the order of the columns to reach the optimum glycolipid separation.

Isolation of ceramide fractions from skin sample by subcritical chromatography with packed silica and evaporative light scattering detection.

Separative method of lipid classes from the stratum corneum was developed with packed silica and supercritical CO2 containing 10% of methanol at 15 degrees C, 15 MPa and 3 ml min(-1). The elution order of lipid classes was first esterified cholesterol, triglycerides, squalene co-eluted in a single peak, then free fatty acids, free cholesterol, ceramides and finally glycosylceramides. The ceramides were eluted in several fractions which depended on the number of hydroxyl groups in the molecule, i.e. more hydroxyl groups were contained in ceramides, more important was the retention. Moreover, the retention was not altered by the presence of carbon double bond and variation of the alkyl chain length. The ceramide response with the evaporative light scattering detector was improved by turning the influence of the solvent nature on the response to advantage. Therefore, addition of various solvents with or without triethylamine and formic acid were tested in post-column due to the incompatibility of such modifiers with silica stationary phase. Thereby the solvent conditions for the separation and the detection can be adjusted almost independently. The response was greatly increased by post-column addition of 1% (v/v) triethylamine and its equivalent amount of formic acid in dichloromethane introduced at 0.1 ml min(-1) into the mobile phase. This device had allowed the detection of 400 ng of ceramide with a S/N = 21, whereas no peak was observed in absence of the post-column addition. Finally, the method was applied to the treatment of skin sample which led to highly enriched ceramide fraction.

Eluotropic strength in non-aqueous liquid chromatography with porous graphitic carbon.

Porous graphitic carbon is an attractive packing for the chromatographic analysis of highly hydrocarbonaceous compounds with non-aqueous mobile phase. An eluotropic-strength scale of 10 pure organic solvents was established using the methylene selectivity from the fatty acid methyl ester homologous series (chain length between 18 and 31 carbon atoms). Eight binary mobile phases combining a weak solvent: methanol or acetonitrile with a strong solvent: toluene, chloroform, dichloromethane or tetrahydrofuran at different volume fractions phi of strong solvents (ranging from 0.3 to 1.0) were tested and their eluotropic strengths were then compared with those of pure solvents. The curves of the eluotropic strength versus the volume fraction of the strong solvent followed two different trends: linear or curved. The knowledge of the pure solvent strength is not sufficient to predict the eluotropic strength of solvent in the mixture. Then modelling of the eluotropic strength for binary mobile phases was envisaged in order to provide a prediction tool. This model was assessed for the establishment of the composition of eight iso-eluotropic mobile phases. Good assessment was found except in the case of toluene with acetonitrile where the difference between the predicted and the real value was the highest.

Structure-retention diagrams of ceramides established for their identification.

Molecular species analysis of ceramides was carried out using porous graphitic carbon with gradient elution: chloroform-methanol from 45:55 to 85:15 with a slope at 2.7%/min. These conditions gave a linear relationship between retention data and structure of ceramides. It was demonstrated that linearity occurred when a high slope value of linear gradient elution was used. Thereby the linear diagram was evolved by plotting the adjusted retention time against the total number of carbon atoms of ceramide molecules. Each line represents one ceramide class. Such a Structure-Retention Diagram describes ceramide retention and thus constitutes an identification method using only retention data. This Structure-Retention Diagram was assessed and compared to another obtained from octadesyl-grafted silica in terms of their reproducibility, precision and ability to provide ceramide identification. Better identification was obtained using the results from both Structure-Retention Diagrams. This approach with a two-dimensional separation system allowed to take advantage of the specificity of both identification models.

Mechanism of response enhancement in evaporative light scattering detection with the addition of triethylamine and formic acid.

Triethylamine with an equimolar amount of formic acid added to the mobile phase provides an enhancement of the evaporative light scattering detector (ELSD) response. After characterization of the influence of various parameters on the ELSD response, a sequential strategy was defined to elucidate this response enhancement. The response enhancement was more marked at low mobile phase flow rate, and was highly dependent on solutes and solvents. The influence of drift tube temperature on response enhancement with various solutes demonstrated that triethylamine and formic acid mainly act as mass amplifiers by the inclusion of triethylamine-formic acid clusters inside the droplets.