Separation and characterisation of sphingoceramides by high-performance liquid chromatography-electrospray ionisation mass spectrometry.

We developed a simple and reliable analytical method for the quantification and the characterization of ceramides extracted from biological samples by high-performance liquid chromatography (HPLC) coupled to electrospray ionisation tandem mass spectrometry (ESI/MS/MS). The chromatographic separation of analytes was carried out in a RP8 column, eluting with a methanol-water mixture in gradient elution mode. The separated lipids were detected by total ion monitoring and characterised by MS/MS spectra; quantitative analysis was performed by integrating the extracted ion peaks obtained in the negative ion mode. Good repeatability was obtained for retention time (0.3-2%), peak area ratio (A(S)/A(IS), 2-8%), as well as limit of detection (LOD, 5-26 pg) and quantification (LOQ, 13-53 pg). The method was validated for the analysis of N-palmitoyl-D-erythro-sphingosine (Cer16), N-stearoyl-D-erythro-sphingosine (Cer18), N-tetracosanoyl-D-erythro-sphingosine (N24:0, lignoceric ceramide, Cer24:0), and N-tetracos-15'-enoyl-D-erythro-sphingosine (N24:1, nervonic ceramide, Cer24:1), giving good results. Lipid mixtures, extracted from skin and epidermal cells, were analysed for their content of the studied ceramides.

Evaluation of teicoplanin chiral stationary phases of 3.5 and 5 microm inside diameter silica microparticles by polar-organic mode capillary electrochromatography.

Different types of fused-silica capillaries of 75 microm inside diameter (ID) were packed, namely type A and B, and evaluated for the direct resolution of racemates of several basic compounds by enantioselective capillary electrochromatography (e-CEC). Type A was packed with a chiral stationary phase (CSP) containing teicoplanin (TE) mixed with silica microparticles (3:1 w/w) while type B contained only the TE-CSP. In both cases, particles of different sizes (3.5 and 5 microm ID) were employed. A polar-organic mobile phase containing methanol-acetonitrile (60-40% v/v and 0.05% w/v ammonium acetate was used. Several beta-blockers (alprenolol, oxprenolol, metoprolol, pindolol, salbutamol, propranolol, atenolol, acebutolol) were baseline-enantioresolved with both capillary types, in very short times.

Enantiomeric separation of acidic compounds of pharmaceutical interest by capillary electrochromatography employing glycopeptide antibiotic stationary phases.

Enantiomeric separation of some selected acidic compounds of pharmaceutical interest belonging to the group of non-steroidal anti-inflammatory drugs were separated by capillary electrochromatography employing silica based glycopeptide antibiotic stationary phases, namely vancomycin or a teicoplanin derivatives (Hepta-Tyr). The vancomycin stationary phase allowed to achieve the chiral resolution of some racemic studied compounds only using mobile phases containing ammonium formate at a relatively low pH 2.5-3.5 and acetonitrile. Employing the teicoplanin derivative stationary phase, good enantiomeric resolution was achieved eluting with mobile phases containing sodium phosphate pH 6-acetonitrile. Enantiomers were moved to the detector because a relatively high reversed electroosmotic flow (due to the positive charge of the stationary phase) and to the electrophoretic mobility of analytes.

Use of short-end injection capillary packed with a glycopeptide antibiotic stationary phase in electrochromatography and capillary liquid chromatography for the enantiomeric separation of hydroxy acids.

A new chiral stationary phase (CSP) was prepared by reacting MDL 63,246 (Hepta-Tyr), a glycopeptide antibiotic belonging to the teicoplanin family, with 5-microm diol-silica particles. The CSP mixed with 5-microm amino silica particles (3:1) was packed into 75-microm fused-silica capillaries for only 6.6 cm and used for electrochromatographic experiments analyzing several hydroxy acid enantiomers. A reversed electroosmotic flow carried both analytes and mobile phase towards the anode in a short time (1-3 min), being baseline resolved all the studied analytes. In order to achieve the fastest enantiomeric resolution of the studied hydroxy acids, the effect of several experimental parameters such as mobile phase composition (organic modifier type and concentration, pH of the buffer and ionic strength), capillary temperature and applied voltage on enantioresolution factor, retention time, enantioselectivity were evaluated. The packed capillary column allowed the separation of mandelic acid enantiomers in less than 72 s with resolution factor Rs=2.18 applying a voltage of 30 kV and eluting with a mobile phase composed by 50 mM ammonium acetate (pH 6)-water-acetonitrile (1:4:5, v/v). The CSP was also tested in the capillary liquid chromatography mode resolving all the studied enantiomers applying 12 bar pressure to the mobile phase [50 mM ammonium acetate (pH 6)-water-methanol-acetonitrile, 1:4:2:3, v/v)], however, relatively long analysis times were observed (12-20 min).

A glycopeptide antibiotic chiral stationary phase for the enantiomer resolution of hydroxy acid derivatives by capillary electrochromatography.

Separation of hydroxy acid enantiomers was achieved by using capillary electrochromatography (CEC) employing a chiral stationary phase (CSP) based on MDL 63,246 (Hepta-Tyr), a macrocyclic antibiotic of the teicoplanin family. The chiral selector was chemically bonded to 5 num diol-modified silica particles and the CSP mixed with amino silica (3:1 w/w) was packed into a 75 num ID fused-silica capillary. The CEC experiments were carried out by using an aqueous reversed-phase mode for the enantiomeric resolution of hydroxy acid compounds. Good enantioresolution was achieved for mandelic acid (MA), m-hydroxymandelic acid (m-OH-MA), p-OH-MA, and 3-hydroxy-4-methoxymandelic acid (3-OH-4-MeO-MA). The CEC system was less enantioselective towards 2-phenyllactic acid (2-PhL) and 3-PhL while mandelic acid methyl ester (MA-Et-Est) enantiomers were not resolved. Several experimental parameters, such as organic solvent type and concentration, buffer pH, capillary temperature, on enantioresolution factor, retention time, and retention factor were studied.

Separation of delta-, gamma- and alpha-tocopherols by CEC.

In this study capillary electrochromatography (CEC) was used for the separation of three tocopherols (TOHs), namely delta-, gamma- and alpha-TOH and the antioxidant compound, butylated hydroxytoluene (BHT). The CEC experiments were carried out using an octadecylsilica (ODS) stationary phase packed, in our laboratory, in a fused-silica capillary (100 microm I.D., 365 microm O.D. x 33 cm of total length and 24.6 or 8.4 cm effective length). The mobile phase was composed by a mixture of methanol (MeOH) and acetonitrile (ACN), at different concentrations and 0.01% (w/v) of ammonium acetate. Retention time (t(R)), retention factor (k), resolution (R(s)) of the three TOHs were strongly influenced by the organic solvent composition of the run buffer and by the effective length of the capillary. Optimum experimental conditions were found even employing the short effective length of the capillary achieving the baseline separation of the studied analytes in a relatively short time (less than 5 min). The optimized method was applied to the qualitative analysis of vitamin E (alpha-TOH) present in a human serum extract.

Use of vancomycin silica stationary phase in packed capillary electrochromatography: III. enantiomeric separation of basic compounds with the polar organic mobile phase.

The separation of basic compounds into their enantiomers was achieved using capillary electrochromatography in 50 or 75 microm inner diameter (ID) fused-silica capillaries packed with silica a stationary phase derivatized with vancomycin and mobile phases composed of mixtures of polar organic solvents containing 13 mM ammonium acetate. Enantiomer resolution, electroosmotic flow, and the number of theoretical plates were strongly influenced by the type and concentration of the organic solvent. Mobile phases composed of 13 mM ammonium acetate dissolved in mixtures of acetonitrile/methanol, ethanol, n-propanol, or isopropanol were tested and the highest enantioresolutions were achieved using the first mobile phase, allowing the separation of almost all investigated enantiomers (9 from 11 basic compounds). The use of capillaries with different ID (50 and 75 microm ID) packed with the same chiral stationary phase revealed that a higher number of theoretical plates and higher enantioresolution was achieved with the tube with lowest ID.