Automated determination of amoxycillin in biological fluids by column switching in ion-pair reversed-phase liquid chromatographic systems with post-column derivatization.

Amoxycillin, a polar aminopenicillin, is rather unstable in biological fluids. Degradation can be prevented by fast sample pretreatment and storage at -70 degrees C or below. After pH adjustment, it is stable in biological fluids for over 16 h. The samples were handled by automated chromatography overnight. The chromatographic system consisted of a small guard column, two analytical columns separated by a switching valve, a post-column reactor and a fluorescence detector. The chromatographic events and the calculation of results were handled by a computing integrator. The chromatography was based on ion-pairing principles. An efficient clean-up of the biological fluids was obtained by a heart cut from the first column, where the neutral mobile phase contained hexyl sulphate. In the second column the organic anion was exchanged for a large quaternary ammonium compound; amoxycillin was then retained as an ion pair. The composition of the mobile phases had to be designed carefully in order to avoid a disturbance of the chromatographic performance on the last column. An adequate selectivity and sensitivity was obtained by a post-column derivatization with fluorescamine. Detection limits were 10 and 25 ng/ml for plasma and urine, respectively, and the inter-assay precisions at low levels (350 and 2000 ng/ml for plasma and urine, respectively) were ca. 5% (R.S.D.).