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-Silm) column. The
gradient elution was from heptane to acetone/butanol 90:10 v/v in 4%/min at 78 lL/
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 lL/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.