Separation and spectrofluorometric assay of the β‐adrenergic blocker sotalol from blood and urine

A sensitive spectrofluorometric assay of sotalol·HCl (d,l‐4‐(2‐isopropylamino‐1‐hydroxyethyl)methanesulfonanilide hydrochloride; MJ 1999), a potent and specific β‐adrenergic blocking agent, was developed. The compound fluoresces in alkali at 250/350 nm. and in acid at 235/309 nm. The maximum extractability was found at a pH of 9, where the substance is primarily the zwitterion with potentiometric pKa' values of 8.30 and 9.80, where the former has been spectrophotometrically assigned to the dissociation of the conjugated sulfanilino group. A mixture of n‐amyl alcohol and chloroform (1 : 3, v/v) at a volume ratio of organic solvent and pH 9 buffer solution of 8 : 1 extracted 85% of the compound, which was completely reextracted into 5.2 N HCl. The fluorescence of the monocharged molecule was measured at 235/309 nm. in the acid solution. The sensitivity of the assay was 0.1 mcg./ml. in plasma and 2.5 mcg./ml. in urine. Statistical analyses of assays of sotalol‐spiked plasmas, urines, and acidic extraction blanks showed no dose × day interactions and no significant variations of the slopes of calibration curves within 3‐4 days of assay; no significant assay differences when the drug was stored under refrigeration in water, acidic extraction blanks, whole blood, plasma, serum, and urine for 2‐6 days; and no significant effects from blood and urine obtained from different dogs. The recovery of the drug was 69.4% from plasma and 81% from urine. The sensitivity of 0.1 mcg./ml. in plasma and 2.5 mcg./ml. in urine and the reproducibility of 2% at a midrange concentration of 0.5 mcg./ml. in plasma and of 2.2% at a midrange concentration of 10 mcg./ml. in urine allow the assay to be applicable for monitoring blood levels and urinary excretion of the drug when administered in therapeutic amounts.