Determination of captopril and its disulphides in whole human blood and urine by high-performance liquid chromatography with ultraviolet detection and precolumn derivatization

An assay that measures the total, reduced, and protein-bound captopril, the orally active antihypertensive drug, in whole human blood and urine has been developed. The procedure involves a precolumn derivatization of the drug via its sulfhydryl group with 1-benzyl-2-chloropyridinium bromide followed by solid-phase extraction and reversed-phase high-performance liquid chromatography separation with ultraviolet detection at 314 nm. Oxidized and protein-bound captopril is converted to reduced form by the use of triphenylphosphine and derivatized and quantified in the same manner. The proposed method offers the possibility of determining the in vivo redox status of captopril in blood of patients orally given a standard dose of at least 12.5 mg of captopril as part of the treatment of hypertensive disease and/or congestive heart failure. In the recommended procedure the sulfhydryl form of captopril is trapped with minimal oxidation by derivatizing blood samples at the time of collection. This is attained by drawing blood directly into tubes containing solutions of 1-benzyl-2-chloropyridinium bromide. The method enables also the determination of urinary excretion of captopril and its disulphides after oral administration of the drug. Accurate determinations are possible over a concentration range of 10 to 500 ng/ml captopril in blood, 50 to 1200 ng/ml captopril in urine and 10 to 1000 ng/ml captopril disulphide and 50 to 3000 ng/ml captopril disulphide in blood and urine, respectively. The detection and quantification limits for both blood and urine are 0.3 and 10 ng/ml, respectively.