Determination of amoxycillin in human plasma by direct injection and coupled-column high-performance liquid chromatography

This work reports the use of multidimensional HPLC by coupling a restricted access medium (RAM) bovine serum albumin (BSA) octadecyl column (100×4.6 mm I.D., 10 μm particle size and 120 Å pore size) to an octadecyl Hypersil column (150×4.6 mm I.D., 5 μm particle size and 120 Å pore size) to the analysis of amoxycillin in human plasma by direct injection. Ion pairing was necessary to extract amoxycillin with good recovery from the plasma proteins. To prepare the spiked samples, aliquots (60 μl) of the appropriated standard solutions were added to each culture tube containing an 180 μl of plasma and a solution of 0.30 m M tetrabuthylammonium phosphate (60 μl). They were vortexed for 15 s and then 290 μl were transferred to autosampler vials. Aliquots (250 μl) of the spiked plasma samples were injected to a column-switching HPLC system. An analysis time of 25 min with no time spent on sample preparation was achieved. The developed method showed good selectivity, sensitivity, accuracy and precision for direct analysis of this polar low wavelength ultraviolet absorption antibiotic using only 180 μl of human plasma. The validated method proved to be reliable and sensitive for the determination of amoxycillin in plasma samples of five healthy volunteers to whom test and reference formulations were administered as an oral dose (500 mg).