Sensitive Highhyphen;Performance Liquid Chromatographic Assay for Determination of Amiloride in Biologic Fluids Using an Ionhyphen;Pair Extraction Method

摘要

The paucity of data on disposition of the potassium-retaining diuretic amiloride is mainly owing to the lack of sensitive and specific analytic methods for measuring the drug in biologic fluids. Poor lipophilicity and extremely low plasma concentrations impose severe constraints. A method is described for analysing plasma and urine concentrations of amiloride using an ion-pair extraction method and high-performance liquid chromatography (HPLC) with fluorescence detection. Amiloride and internal standard triamterene form an ion-pair with bromothymol blue at pH 7.6. The ion-pairs are extracted into diethyl ether: dichloromethane (2:1), and back-extraction into 0.1percnt; tetrabutylammonium hydroxide liberates the drugs from their ion pairs. Analyses are performed using HPLC with a reverse-phase C18column and a mobile phase of 11percnt; acetonitrile and 0.5percnt; triethylamine adjusted to pH 3. Amiloride is quantified using fluorescence detection (366-nm excitation, 418-nm emission cutoff). Retention times are 2.2 and 6.0 min for amiloride and triamterene, respectively. The sensitivity limit is 0.2 ng/ml, and recovery is 82percnt; for amiloride and 71percnt; for triamterene. Calibration curves are linear (range 0.25ndash;25 ng/ml for plasma and 0.05ndash;2.0 mUg/ml for urine). Inter- and intraday assay variability is 8percnt; and precision is within 5percnt;. The assay is of sufficient sensitivity and specificity for the study of the pharmacokinetics of amiloride in humans.