Selective high-performance liquid chromatographic assays for hydralazine and its metabolites in plasma of man

Selective high-performance liquid chromatographic assays for hydralazine (I), hydralazine pyruvic acid hydrazone (II) and the acetylation metabolites, namely s-triazolo[3,4-alpha]-phthalazine (V) and 3-hydroxymethyl (VI) and 3-methyl-s-triazolo[3,4-alpha]phthalazine (VII) in human plasma were developed. Utilizing the fluorescence of these compounds or their derivatives the limits of detection could be extended down to 5 nmole/l (1 ng/ml) for I, 1 nmole/l (0.2 ng/ml) for II and 0.5 nmole/l (0.1 ng/ml) for V--VII. The intra-assay coefficients of variation for the assays ranged from 2 to 7% over the concentration range 5.0 to 0.05 micromole/l and the inter-assay variability in the slope of the standard curves ranged rom 4 to 8%. An improved method for measuring the sum of I plus all its hydrazones (apparent I) was also developed. On addition of I to fresh plasma at 37 degrees, half the added I was converted to II within 15 min and there was no detectable level of I, 2 h after the addition. The plasma level--time course of I, and its metabolites in a healthy volunteer (slow acetylator) following separate oral and intravenous administrations of I indicated that I contributed only a small fraction (4.3 and 4.7% respectively) to the area under the plasma level--time curve of apparent hydralazine.