Utilization of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) for spectrochemical determination of L-ornithine: A multivariate optimization-assisted approach

Abstract

A simple and highly sensitive univariate calibration strategy based on ultraviolet-visible (UV-Vis) absorption spectroscopy and assisted by multivariate screening and optimization was utilized for the determination of l-ornithine (l-ORN) as such and in the alimentary supplements. l-ORN, an OTC marketed amino acid, is widely used for bodybuilding and might be abused by athletes. A nucleophilic substitution reaction using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) was the basis of the current investigation. Plackett-Burman design (PBD) and a response surface optimizer as screening and fine-tuning strategies, respectively, were instigated. Four numerical variables, reaction time (RT), temperature (Temp), pH and reagent volume (RV), and one categorical variable, the diluting solvent (DS), were considered. Absorbance of the yellow-colored adduct at 469 nm was the response studied. Pareto analysis, along with analysis of variance (ANOVA) were used to ascertain the significant variables (screening phase) and their domains (optimization phase). Response transformation and stepwise analysis were employed when necessary. Probability, cube and individual value plots were used to get an insight into the statistical impact of the variables tested. Multiple responses' optimization was performed using Derringer's function. Calibration curves were linear in the range of 5-50 g mL-1. Job's technique of continuous variation showed that the stoichiometric ratio is 2:1 (NBD-Cl:l-ORN). The proposed technique was successfully applied to the dietary supplements of l-ORN, inferring no interference from adjuvants and excipients. Analytical performance of this technique was validated conforming to the ICH standards.