Determination of amoxicillin in body fluids by reversed-phase liquid chromatography coupled with a post-column derivatization procedure

Abstract

Quantitative methods for determination of amoxicillin in body fluids are described. They comprise separation by reversed-phase chromatography (LiChrosorb RP-8, 5 micron) of the aqueous supernatants obtained from plasma or urine after purification steps involving protein precipitation followed by extraction in the case of plasma, or a double extraction procedure in the case of urine, post-column derivatization with air segmentation, and finally measurement of the UV absorbance at 310 nm. The derivatization involves formation of the mercuric mercaptide of penicillenic acid and is specific for compounds with an intact penicillanic acid ring system. Detection limits achieved on injecting 200 microliter of plasma and 20 microliter of urine are about 25 ng/ml and 200 ng/ml, respectively, but it is possible to improve the sensitivity further by injecting larger volumes. Precisions (srel) obtained for determination of 0.10 and 0.45 migrogram/ml in plasma were 3.72 and 1.40%, respectively. Some problems regarding column stability originating from the injection of biological samples are discussed.