Improved Selectivity of a High-Performance Liquid Chromatography Assay for Debrisoquine and Its 4-Hydroxy Metabolite from Urine

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Abstract
Debrisoquine is an antihypertensive drug that displays polymorphic metabolism and has been used to determine hydroxylator status in human subjects. A high-performance liquid chromatographic method for the simultaneous urine analysis of debrisoquine (D) and its primary metabolite, 4-hydroxydebrisoquine (4-OHD), is described. A cyanopropyl (CN) extraction column and CN analytical column were used with a mobile phase consisting of 20% acetonitrile/20 mM sodium dihydrogen orthophosphate (pH = 7.1) at a flow rate of 1.5 ml/min. The peaks of interest, as well as the internal standard, were eluted from the column in less than 14 min. The method described was validated for within-day and between-day accuracy and precision for both D and 4-OHD. Coefficients of variation at all concentrations tested were < 4.1% for D and < 9.3% for 4-OHD. The assay was used to evaluate D/4-OHD ratios, thereby determining hydroxylation status in 65 healthy male volunteers.