Liquid Chromatographic-Electrospray Ionization-Mass Spectrometry Method for the Simultaneous Determination of Gramicidin, Neomycin & Triamcinolone Acetonide and Characterization of Novel Forced Degradation Products

Abstract

The dosage form iotrim (gramicidin, neomycin and triamcinolone) is a combination of two antibiotics (gramicidin and neomycin) and a steroid (triamcinolone). The antibiotics work by killing the bacteria that cause infections. The steroid blocks the action of chemical messengers (prostaglandins) that make the affected area red, swollen and itchy. Consequently, there was still a need to develop a simple, less time consuming and economical method for the simultaneous determination of gramicidin, neomycin and triamcinolone acetonide. The current work is an effort to develop a fast and reproducible LC-MS technique for the simultaneous estimation of gramicidin, neomycin and triamcinolone acetonide. The objective of the present procedure was to validate and develop a precise and accurate liquid chromatography-mass spectrometry (LC-MS) technique for the simultaneous quantification of gramicidin, neomycin and triamcinolone acetonide. Gramicidin, neomycin and triamcinolone acetonide were monitored on Shimadzu-8045 mass spectrometer equipped with electro spray ionization interface. The retention times of gramicidin, neomycin and triamcinolone acetonide were found at 9.145 min, 7.273 min and 2.435 min, respectively. The limit of detection (LOD) results for gramicidin, neomycin and triamcinolone acetonide were observed to be 0.15, 1.5 and 0.6 μg/mL, respectively while the limit of quantification (LOQ) results were observed to be 0.5, 5, 2 μg/mL concentration, respectively. The linear range for gramicidin, neomycin and triamcinolone acetonide were found in the concentration ranges from 1.25-7.5 μg/mL, 12.5-75 μg/mL and 5-30 μg/mL with regression coefficient of 0.9991, 0.9996, 0.9999, respectively. Accuracy values for gramicidin, neomycin and triamcinolone acetonide were found to be in the range of 98.64%, 99.4%, 99.5% respectively. The % RSD for six replicates in precision was less than 2%. According to ICH Q2(R1) recommendations, this method was successfully tested with LC-MS to confirm the chemical structures of newly produced degradation products of triamcinolone acetonide and neomycin. The developed process was validated efficaciously as per ICH guidelines.