A novel high throughput 96-well-based fluorimetric method to measure amikacin in pharmaceutical formulations:...

Area of Science:

• Analytical Chemistry
• Pharmaceutical Analysis
• Spectrofluorimetry

Background:

• Amikacin, an aminoglycoside antibiotic, requires derivatization for quantification due to lack of chromophore.
• Existing analytical methods for amikacin can be time-consuming and require significant sample volumes.

Purpose of the Study:

• To develop a simple, sensitive, and rapid spectrofluorimetric method for amikacin sulfate quantification.
• To optimize the method using design of experiments (DOE) for robustness and reproducibility.
• To validate the method according to International Conference of Harmonization (ICH) guidelines.

Main Methods:

• Spectrofluorimetry utilizing a 96-well plate reader.
• Design of Experiments (DOE) including univariate and multivariate analysis (central composite design) to optimize pH, temperature, and reagent concentration.
• Validation of the method for linearity, limit of detection (LOD), limit of quantification (LOQ), and precision (intraday and interday).

Main Results:

• The developed method is linear from 1.9 to 10 μg/ml with R² = 0.9991.
• LOD and LOQ were determined as 0.649 μg/ml and 1.9 μg/ml, respectively.
• Intraday and interday precision (%RSD) were below 5%, demonstrating high reproducibility.
• Quantification of amikacin in liposomes and commercial formulations (Amicin®).
• High-throughput analysis of 96 samples in 125 seconds (1.3 sec/sample).

Conclusions:

• A robust, sensitive, and rapid microplate-based spectrofluorimetric method for amikacin sulfate measurement was successfully developed and validated.
• The method offers a cost-effective and convenient alternative for analyzing amikacin in pharmaceutical formulations.
• This high-throughput approach significantly reduces analysis time compared to existing methods.