Comparative application of biological and ninhydrin- derivatized spectrophotometric assays in the evaluation and validation of amikacin sulfate injection

Abstract

Instrumental techniques are preferred over bioassay methods for antibiotic quantification mainly due to speed and ability to quantify metabolites in biological samples; however, the potency and biological activity of these drugs cannot be assessed. Two methods - agar well diffusion (bio-assay) and spectrophotometric methods were used to evaluate amikacin sulfate injection. Agar plates were inoculated with S. aureus inoculum; zones of inhibition from its susceptibility to amikacin were obtained, while spectrophotometric absorption at 650 nm of ninhydrin- derivatized amikacin in phosphate buffer (pH 8) was measured. Methods performance showed linearity from 1 - 16 μgmL^-1 (bioassay, r = 0.9994) and 10-50 μgmL^-1 (spectrophotometric, r = 0.9998). Molar absorptivity was 2.595 x 10⁴ Lmol^-1cm^-1. Limits of detection and quantification were 1.07 and 3.24 μgmL^-1 respectively for bioassay method, while corresponding values for spectrophotometric method were 0.98 and 2.97 μg mL^-1. Relative standard deviations were ≤ 2.0% for both methods, with recoveries from 95.93 - 100.25%. Amikacin in brands ranged from 97.53 ± 2.68 to 100.84 ± 1.82%, student’s t-test was ≤ 2.78 (n = 4) with respect to label claim for both methods. Experimental paired t-test (t = 2.07; n = 4) and F-test (F = 3.94; n = 4) values indicated no significant difference between both methods, hence comparable and can jointly be used in quality control assessment of antibiotics.

Keywords:
Biological activity; Potency; Derivatizaton; Aminoglycosides; Molar absorptivity; Ninhydrin