Determination of 2-[N-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-L-alanyl]-(1S,3S,5S)-2- azabicyclo[3.3.0]octane-3-carboxylic acid (Hoe 498) and its hydrolysis product in serum and urine.

A highly sensitive and specific enzymatic assay for the quantitative determination of 2-[N-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-L-alanyl]-(1S,3S, 5S)-2-azabicyclo[3.3.0]octane-3-carboxylic acid (Hoe 498) and its hydrolysis product Hoe 498-diacid in serum and a GLC method for the simultaneous determination of both compounds in urine are described. Both methods involve extraction from the respective biological fluid using disposable C 18 columns. In serum, the enzyme-inhibiting properties of Hoe 498-diacid (Hoe 498 can readily be converted to its hydrolysis product) are utilized for the determination of both compounds concurrently. The serum extract is dissolved in buffer solution and incubated with human serum as angiotensin converting enzyme (peptidyl-dipeptide-hydrolase, EC 3.4.15.1) source and hip-his-leu substrate. The hippuric acid liberated is quantitated by HPLC. The urine extract is treated with diazomethane followed by trifluoroacetic anhydride to convert Hoe 498 and Hoe 498-diacid to their methylester, trifluoroacetyl derivatives, which are then determined simultaneously by GLC using a nitrogen specific detector.