Establishment and Validation of High-Performance Liquid Chromatography-Tandem Mass Spectrometry for Simultaneous...

Hypertension is a major cause of premature death worldwide despite the availability of a number of antihypertensive medication monotherapies. Therefore, several polytherapies have been prescribed, among which the combination of amlodipine with indapamide is often the preferred choice to control blood pressure, especially in the elderly, because of its efficacy and safety. To ensure the quality of this combination drug, a versatile procedure for the simultaneous quantification of components is required. Thus, this study aims to develop a liquid chromatography - tandem mass spectrometric procedure and validate according to FDA and EMA guidelines to determine amlodipine and indapamide in human plasma.

A liquid-liquid extraction with tert-butyl methyl ether and ethyl acetate (1:1) was applied to extract the compounds. Amlodipine was ionized with positive electrospray ionization and detected by multiple reaction monitoring mode, while indapamide was ionized with negative electrospray ionization and detected by selected ion monitoring mode. Samples were chromatographically analyzed on a C18 column (150 × 4.6 mm; 3.5 μm), eluted by the mobile phase of methanol and 0.025% formic acid (90:10, v/v).

Linearity ranged from 0.29- to 17.14-ng/mL amlodipine, from 1.14- to 68.57-ng/mL indapamide. The lower limit of quantitation of amlodipine and indapamide is 0.29- and 1.14-ng/mL, respectively. The validation using furosemide as an internal standard showed that the specificity, intra- and interday precision and accuracy, matrix effect, sample carryover, dilution, and stability were in the acceptable range.

The method met validation criteria of US-FDA and EMA guidelines, thereby recommended for application in in vivo bioavailability and bioequivalence assessment of fixed-dose combinations of amlodipine with indapamide.