Bethune C, Bui T, Liu M L, Kay M A, Ho R J
Department of Pharmaceutics, University of Washington, Seattle 98195, USA.
Antimicrob Agents Chemother. 1997 Mar;41(3):661-4. doi: 10.1128/AAC.41.3.661.
We have developed a chromatographic assay with high sensitivity and specificity to quantify G418 sulfate (Geneticin), an antibiotic used routinely in molecular genetics experiments for selecting eukaryotic transformants. With this method, G418 in tissues and plasma samples can be quantitated without the confounding factors often associated with biological assays. After removal of proteins in homogenized tissue or plasma 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-visible G418-DNFB product. The DNFB-derivatized G418 was separated on a reversed-phase C18 column with an acetonitrile and water gradient as the 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 micrograms/ml. The data obtained from this analysis indicate that this assay can be used for the quantitative determination of G418 sulfate in plasma and tissue samples.
我们开发了一种具有高灵敏度和特异性的色谱分析方法，用于定量硫酸庆大霉素（遗传霉素），这是一种在分子遗传学实验中常用于筛选真核转化体的抗生素。通过这种方法，可以在不存在通常与生物学分析相关的混杂因素的情况下，对组织和血浆样本中的硫酸庆大霉素进行定量。在用甲醇（2:1，体积/体积）去除匀浆组织或血浆样本中的蛋白质后，硫酸庆大霉素的氨基用1-氟-2,4-二硝基苯（DNFB）进行衍生化，形成紫外可见的硫酸庆大霉素-DNFB产物。将经DNFB衍生化的硫酸庆大霉素在以乙腈和水梯度为流动相的反相C18柱上进行分离。在这些分析条件下，缓冲液、血浆和组织中硫酸庆大霉素的检测限为78 ng/ml，浓度高达100μg/ml时呈线性。从该分析获得的数据表明，该分析方法可用于血浆和组织样本中硫酸庆大霉素的定量测定。
