Bell R R, Bombardt P A, DuCharme D W, Kolaja G J, Packwood W H, Bothwell B E, Satoh P S
Upjohn Company, Kalamazoo, MI 49001.
Biomed Chromatogr. 1994 Sep-Oct;8(5):224-9. doi: 10.1002/bmc.1130080506.
A high-performance liquid chromatographic (HPLC) assay method has been developed for the quantitative determination of iothalamate and p-aminohippuric acid (PAH) concentrations in serum and urine samples in the male rat. Glomerular filtration rate (GFR) was measured as clearance of iothalamate, while effective renal blood flow (ERBF) was measured as clearance of PAH. The method is simple, rapid and sensitive and detects iothalamate and PAH in rat serum and urine following administration of bolus doses and continuous infusions of iothalamate and PAH. Samples of serum and urine were deproteinized with two volumes of acetonitrile containing the internal standard, and an aliquot chromatographed on a C18 reversed-phase column. The mobile phase was comprised of 0.1 M sodium phosphate with 1.2 mM tetrabutylammonium phosphate: methanol, 85:15 (v/v), at a flow rate of 1.0 mL/min. The analytical column eluate was monitored with a UV detector at 254 nm with quantitation achieved using peak-height ratios. The precision of the method was 6.6 and 3.6% for iothalamate in serum and urine, and 5.6 and 4.9% for PAH in serum and urine, respectively. The lower limit of quantitation was 0.63 microgram/mL for iothalamate and 1.25 microgram/mL for PAH in serum, and 3.1 microgram/mL for iothalamate and 1.5 microgram/mL for PAH in urine. Recovery of iothalamate from serum and urine was 99.9 and 93.5%, respectively. Recovery of PAH from serum and urine was 99.8 and 92.6%, respectively. The present study demonstrated that non-radioactive iothalamate and PAH can be measured simultaneously using a HPLC assay to measure GFR and ERBF in the male rat.
已开发出一种高效液相色谱（HPLC）分析方法，用于定量测定雄性大鼠血清和尿液样本中碘他拉酸盐和对氨基马尿酸（PAH）的浓度。肾小球滤过率（GFR）通过碘他拉酸盐的清除率来测量，而有效肾血流量（ERBF）通过PAH的清除率来测量。该方法简单、快速且灵敏，可在静脉推注剂量以及持续输注碘他拉酸盐和PAH后检测大鼠血清和尿液中的碘他拉酸盐和PAH。血清和尿液样本用含内标的两倍体积乙腈进行脱蛋白处理，取一份等分试样在C18反相柱上进行色谱分析。流动相由0.1M磷酸钠与1.2mM磷酸四丁铵：甲醇，85:15（v/v）组成，流速为1.0mL/min。用紫外检测器在254nm处监测分析柱洗脱液，使用峰高比进行定量。该方法对血清和尿液中碘他拉酸盐的精密度分别为6.6%和3.6%，对血清和尿液中PAH的精密度分别为5.6%和4.9%。血清中碘他拉酸盐的定量下限为0.63微克/毫升，PAH为1.25微克/毫升；尿液中碘他拉酸盐的定量下限为3.1微克/毫升，PAH为1.5微克/毫升。碘他拉酸盐从血清和尿液中的回收率分别为99.9%和93.5%。PAH从血清和尿液中的回收率分别为99.8%和92.6%。本研究表明，使用HPLC分析法可同时测量非放射性碘他拉酸盐和PAH，以测定雄性大鼠的GFR和ERBF。
