Validation of an analytical method for the titration of injectable lyophilized Amphotericin B

Abstract

Introduction: The injectable lyophilized Amphotericin B is used as an antifungal against a wide spectrum of fungi.

Objective: Validate an analytical method by high-performance liquid chromatography for the titration of the injectable lyophilized Amphotericin B.

Methods: The method was based on the separation of the active substance through an Apollo-C18 chromatographic column (5 μm) (250 x 4.6 mm), with ultraviolet detection at 380 nm, for which a mobile phase composed of a mixture of 650 mL of Acetonitrile: Methanol: Tetrahydrofuran (39:17:9) and 350 mL of EDTA Buffer (2.5 mmol/L) was used, with a flow rate of 1.5mL/min. The calibration curve was performed in the range of 0.01 mg/mL to 0.03 mg/mL, (from 50% to 150% of the amount proposed in the technique).

Results: The method was linear, with a correlation coefficient equal to 0.9998; the statistical test for interception and slope was considered non-significant. A recovery of 100.48% was obtained in the range of concentrations studied and the Cochran and Student's t tests were not significant. The coefficient of variation in the repeatability study was equal to 0.07% for the 6 replicates tested, while in the intermediate precision analyses the Fischer and Student's t tests were not significant. In the specificity study, no interference from additional peaks was observed near the retention time of the main product. The analytical method was linear, precise, specific and accurate in the range of concentrations studied.

Conclusions: The analytical method validated by high-performance liquid chromatography for the quantification of the active ingredient of the injectable lyophilized Amphotericin B, turned out to be linear, precise, accurate and specific, in the range of concentrations established between 0.01 mg/mL and 0.03 mg/mL.