High‐pressure liquid chromatographic assay for hydralazine in human plasma

A specific high‐performance liquid chromatographic assay for hydralazine in human plasma was developed. Plasma hydralazine is reacted with 10 μl of p‐anisaldehyde for 7 min at room temperature to form hydralazine p‐anisaldehyde hydrazone. This derivative is extracted into ethyl acetate, and the solvent is removed by evaporation. The residue is reconstituted in 100 μl of methanol, and 90 μl is injected onto a reversed‐phase column. The mobile phase is 32% acetonitrile in 0.75 M acetate buffer, pH 3.4, at a flow rate of 2 ml/min. The retention time of hydralazine p‐anisaldehyde hydrazone is 6.5 min. The average coefficient of variation over 10‐200 ng/ml is 5.5%, and the sensitivity limit is 5 ng/ml. Under the assay conditions, hydralazine pyruvic acid hydrazone, a known plasma metabolite of hydralazine, yields <0.1% hydralazine. Detectable plasma hydralazine levels of 5‐20 ng/ml were found 10‐30 min after a 0.5‐mg/kg oral dose of hydralazine hydrochloride was given to a male volunteer.