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 ninhydrinderivatized 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 10 4 Lmol -1 cm -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.

Effectiveness of Biological Assays as an Alternative Method to Determine the Potency of Antibiotics: A Review

“…In this study, agar plates were inoculated with Staphylococcus aureus, and a well was designated for the standard amikacin sulfate (100 μL), while other wells contained amikacin sulfate injection with concentrations of 2, 4, 8 and 16 ug/mL. The bioassay conducted in this study demonstrated linearity within the concentration range from 1 to 16 μg/mL (bioassay, r = 0.9994) and the results were compared with a spectrophotometric assay, which showed linearity within the concentration range of 10 to 50 μg/ mL (spectrophotometric, r = 0.9998) [18]. This study highlights the application of the agar well method in evaluating the antimicrobial activity of amikacin sulfate injection and demonstrates its comparability to the spectrophotometric assay within their respective concentration ranges.…”

“…A comparative analysis was conducted between biological and ninhydrin-derivatized spectrophotometric assays for amikacin sulfate injection. The precision, linearity, coefficient and accuracy were highly appreciated, indicating its reliability and ability to provide accurate results for the quantification of amikacin sulfate in the injection [18].…”

“…In a study conducted by Vaikosen et al, the agar well method was introduced to evaluate and validate the antimicrobial activity of amikacin sulfate injection [18]. In this study, agar plates were inoculated with Staphylococcus aureus, and a well was designated for the standard amikacin sulfate (100 μL), while other wells contained amikacin sulfate injection with concentrations of 2, 4, 8 and 16 ug/mL.…”

Spectrophotometric Fingerprinting and Chemical Determination of Streptomycin, Amikacin, Neomycin, and Gentamycin Sulphate by Condensing With Ninhydrin Reagent

“…All of the chemicals used were of analytical quality. The method reported by Vaikosen et al, [19], with few modifications, was adopted in the study.…”

“…With a few modifications, the procedures described by Vaikosen et al, [19], were followed. After properly transferring an aliquot amount of each aminoglycoside's working standard solution into a series of 10 ml calibrated volumetric flasks, 1 ml of the ninhydrin reagent was added and cooked in a boiling hot water bath for 15 min.…”