A sensitive bioanalytical method for the simultaneous determination of amlodipine and glibenclamide

Abstract

Type 2 diabetes mellitus triggers hypertension as a complication. The use of amlodipine and glibenclamide drugs simultaneously results in a synergistic and effective lowering of blood sugar and blood pressure. In the testing of bioavailability and bioequivalence, as well as the monitoring of drug concentrations in the blood, a sensitive bioanalytical approach that meets existing reference requirements, such as the European Medicines Agency (EMA) recommendation, is required. Presently, there is no simultaneous bioanalytical method of amlodipine and glibenclamide that meets EMA requirements. This study aimed to develop a sensitive bioanalytical method that fulfills EMA requirements for determining the levels of amlodipine and glibenclamide simultaneously. Amlodipine and glibenclamide in plasma were extracted with acetonitrile at 10°C. The derivatization was conducted using 0.08% 4-chloro-7-nitrobenzofurazan at pH 8.6 with Teorell and Stenhagen buffer for 20 min at 70°C, followed by the addition of 0.1 N sulfuric acid. High-performance liquid chromatography analysis used a LiChrospher RP 18 column with a size of 125×40 mm ID; mobile phase, acetonitrile: 0.01% phosphoric acid (52:48); flow rate of 1 mL/min; and emission and excitation wavelength for glibenclamide and amlodipine at 346 and 300 nm and 535 and 480 nm, respectively. The concentration ranges were 0.1-20 ng/mL for amlodipine and 1-200 ng/mL for glibenclamide. The average ranges of percentage coefficient of variation and percentage difference were 1.76%-14.62% and 4.48%-11.18% for amlodipine and 0.56%-11.92% and 2.92%-12.75% for glibenclamide. This sensitive and simultaneous bioanalytical method for amlodipine and glibenclamide fulfills the EMA requirements.