Simultaneous determination of glycyl-L-histidyl-L-lysine and its metabolite, L-histidyl-L-lysine, in rat plasma by high-performance liquid chromatography with post-column derivatization.

A selective and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of glycyl-L-histidyl-L-lysine (GHK), a liver-cell growth factor isolated from human plasma, and its metabolite, L-histidyl-L-lysine (HK), in rat plasma. Both high selectivity and sensitivity were achieved by the use of solid-phase extraction with a Bond-Elut Certify cartridge, ion-pair chromatography with 1-pentanesulfonate on a 5-microm Capcell Pak C18 UG120 column (250x4.6 mm I.D.) with a guard column, and by post-column derivatization with o-phthalaldehyde (OPA). GHK and HK were extracted from 0.1 ml of rat plasma after addition of o-phenanthroline to protect against degradation. The limit of detection for GHK and HK were 50 and 15 ng/ml, respectively, and the calibration curves were linear in the range 0.1-5.0 microg/ml. The developed method was applied to the pharmacokinetic study of GHK after a single dose was administered intravenously to rats. GHK was rapidly degraded to HK, which was eliminated rapidly.