Thin-layer chromatographic detection of cyclosporine and its metabolites in whole blood using rhodamine B and alpha-cyclodextrin.

High-performance thin-layer chromatography (HPTLC) has advantages for the analysis of cyclosporine (CsA) and its metabolites in peripheral blood not shared by the radioimmunoassay (RIA) or high-performance liquid chromatography (HPLC) methods. While separation by HPTLC and HPLC is based on relative hydrophobicity, HPTLC (unlike HPLC) is capable of concurrent multisample analysis without expensive instrumentation. As distinguished from RIA, HPTLC detects CsA and metabolites as separate components, and does not use radioactive reagents. A novel rhodamine B/alpha-cyclodextrin stain was developed and the characteristic retention factors (Rf values), as determined by the ratio of the migration distance of a component in relation to the solvent front, were determined for the mobile phase heptane: pyridine: ethyl acetate (100: 75: 1, v/v) on aminopropyl-bonded silica gel HPTLC plates: 0.65, cyclosporin D (CsD); 0.60, CsA; 0.50, dihydrocyclosporin C (dhCsC); 0.42, metabolites M-21 and M-17; 0.40, M-1; 0.35, M-E; 0.25, M-D; and 0.22, M-A. Metabolite M-18 showed migration similar to that of M-17 using a mobile phase of heptane: pyridine: acetonitrile (5:2:1, v/v) in the 0.60-0.50 range. The metabolite profiles were obtained in 8 patients receiving the drug for the first time. The HPTLC analytical technique identifies CsA and its metabolites in peripheral blood and offers advantages for pharmacologic monitoring of transplant patients.