Analytical HPLC Method Validation of Amiloride and Its Pharmacokinetic Study in Humans

Abstract

This study was aimed to validate a reliable analytical method for the pharmacokinetic study of amiloride in human plasma by a high performance liquid chromatography (HPLC) system with UV detection. Triamterene was used as an internal standard. After extraction with ethylacetate, the supernatant was evaporated. Then, the residue was reconstituted and an aliquot was injected onto the HPLC system. Separation was performed on a Capcell Pak C₁₈ UG120 column (4.6 mm × 150 mm, 5 µm particles) with a mobile phase of 12% acetonitrile containing 0.4% glacial acetic acid and UV detection at a wavelength of 360 nm. The intra- and inter-day precision expressed as the relative standard deviation was less than 15%. The flow rate of mobile phase was 1 mL/min and the retention time of amiloride and internal standard, triamterene, was found to be 3.06 and 8.80 min, respectively. The lower limit of quantification (LOQ) was 0.2 ng/mL of amiloride using 1 mL of plasma. The calibration curve was linear in the concentration range of 0.2–50 ng/mL (r ² = 1.0000). The mean accuracy was 85.1–101.7%. The coefficient of variation (precision) in the intra- and inter-day validation was 2.1–12.5 and 2.4–13.7%, respectively. The pharmacokinetics of amiloride was evaluated after a single oral administration of 10 mg to healthy volunteers. The AUC_0–72hr, C_max, T_max, and T_1/2 were 267.7 ± 60.0 ng·hr/mL, 22.9 ± 5.4 ng/mL, 3.0 ± 0.8 hr, and 13.6 ± 2.4 hr, respectively. These results demonstrated that this method was highly feasible and reproducible for pharmacokinetic studies of amiloride in eight volunteers after oral adminis-tration (10 mg as amiloride HCl).

Keywords:

• Amiloride
• HPLC
• Human plasma
• UV detection

ACKNOWLEDGMENT

This research was supported by a grant (04142KFDA451) from Korea Food & Drug Administration.

Manuscript 6290