HPLC Determination of Utibapril and its Diacid FPL 63674XX in Rodent Laboratory Diet Using Selective Extraction and Gradient Elution Chromatography

Utibapril is a novel thiadiazoline that is currently under investigation as an antihypertensive agent. The major degradation product, a diacid FPL 63674XX, is biologically active. An HPLC method has been developed to simultaneously determine both utibapril and its diacid in rodent laboratory diet used to dose animals in long term toxicology studies. The method is based on liquid-solid extraction of the compounds followed by direct injection of the extract. Gradient elution chromatography is utilized to better separate the diacid from interferences. Recovery of utibapril and the diacid from the lab diet was found to be 97.6 +/- 0.44% and 99.7 +/- 2.8%, respectively (n = 3) at 0.43% w/w levels. The separation is achieved on a octadecylsilane column using a flow rate of 0.75 ml/min and a column temperature of 70-degrees-C. The UV detector was set at 260 nm. A linear and step gradient program was established using mobile phases consisting of 0.05 M phosphate buffer, pH 2.5 and 40:60 0.05 M phosphate buffer, pH 2.5 - acetonitrile. An injection volume of 20-mu-l was utilized and the chromatographic run time was set at 38 min. The assay method shows linearity for utibapril over a 0.25 - 2% w/w range (r = 0.9998, n = 5) and for the diacid over a 0.0043 - 0.43% w/w range (r = 0.9960, n = 5). Accuracy and precision for both compounds were in the 1-5% range over the linear ranges specified above. The method was applied to an actual rodent diet sample formulated to contain 0.43% w/w utibapril.