In vivo distribution of free long-chain sphingoid bases in the human stratum corneum by high-performance liquid chromatographic analysis of strippings.

Conditions were established for the in vivo determination of free long-chain bases (phytosphingosine, sphingosine and sphinganine) in human stratum corneum layers by reversed-phase HPLC analysis of adhesive-tape strippings. Long-chain bases were first extracted from individual strippings with pure methanol in an ultrasound bath, then o-phthalaldehyde derivatives were separated on a C18 column with a gradient from methanol-water (80:20, v/v) to pure methanol. By performing a second series of strippings for protein determination it was possible to express the amounts of free long-chain bases per milligram of protein for each individual stratum corneum layer. Co-elution of endogenous phytosphingosine, sphingosine and spinganine was demonstrated in separate experiments by addition of standards to typical strips. From a study of three human volunteers, average concentrations of free long-chain bases in the stratum corneum were found to be 2.8, 1.2 and 0.5 nmol/mg of protein for phytosphingosine, sphingosine and sphinganine, respectively.