Application and validation of a LC/fluorescence method for the determination of amoxicillin in sheep serum and tissue cage fluid

Abstract

A LC method with fluorescence detection after pre-column mercury dichloride derivation was developed and validated for the quantitative determination of amoxicillin in sheep blood serum and tissue cage fluid at levels down to 100 and 200 ng/mL, respectively. Spiked blood serum and tissue cage fluid samples were deproteinized, derivatized with mercury dichloride and extracted prior to reversed phase LC analysis with fluorescence spectrophotometric detection at an excitation wavelength of 355 nm and an emission wavelength of 435 nm. Separation was carried out on a C 18 column with a mobile phase consisting of phosphate buffer, octanesulphonate sodium (OCT), and acetronitrile. A regression model using 1/concentration weighting was found the most appropriate for quantification. The intra-day precision for serum was 1.65–8.74% and for tissue cage fluid was 2.48–6.27%. The inter-day precision for serum was 0.39–3.57% and for tissue cage fluid was 0.44–2.54%. The overall precision over 3 days for blood serum using of 108 replicates was 1.70–9.44% and for tissue cage fluid using of 54 replicates was 2.51–6.76%. Studies of amoxicillin stability in blood serum and tissue cage fluid indicated that amoxicillin was stable after 4 weeks storage at −85 °C. The method was successfully applied for the determination of amoxicillin in blood serum and tissue cage fluid samples collected from rams after intravenous administration.