DEVELOPMENT OF A HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ASSAY FOR G418 SULFATE (GENETICIN)

We have developed a chromatographic assay with high sensitivity and sp ecificity to quantify G418 sulfate (Geneticin), an antibiotic used rou tinely in molecular genetics experiments for selecting eukaryotic tran sformants. With this method, G418 in tissues and plasma samples can be quantitated without the confounding factors often associated with bio logical assays, After removal of proteins in homogenized tissue or pla sma samples with methanol (2:1, vol/vol), the amino group of G418 was derivatized with 1-fluoro-2,4-dinitrobenzene (DNFB) to form the UV-vis ible G418-DNFB product, The DNFB-derivatized G418 was separated on a r eversed-phase C-18 column with an acetonitrile and water gradient as t he mobile phase, Under these assay conditions, the detection limit for G418 sulfate in buffer, plasma, and tissues was recorded at 78 ng/ml and the linearity was recorded for concentrations up to 100 mu g/ml. T he data obtained from this analysis indicate that this assay can be us ed for the quantitative determination of G418 sulfate in plasma and ti ssue samples.