Plackett-Burman and Box-Behnken designs as chemometric tools for micro-determination of l-Ornithine

Plackett-Burman (PB) and Box-Behnken (BB) screening and response surface factorial designs were used to evaluate spectrophotometric and spectrofluorimetric approaches for the determination of l-Ornithine (ORN) as per se and in dietary supplements. Both approaches were based on the derivatization of the primary amino group of ORN via Hantzsch condensation reaction producing yellow coloured adducts (dihydrolutidine derivative). The reaction product was determined spectrophotometrically (method A) at λ_max=327nm and spectrofluorimetrically (method B) at 480nm (λ_em) after excitation at 325nm (λ_ex). A multivariate scheme was tailored to investigate the process numerical variables; reaction temperature, heating time, reagent volume, and pH implementing PB as a screening design followed by BB as an optimization strategy. Categorical factors including diluting solvent and sequence of addition were kept invariable. Responses of the reaction systems were the maximum absorbance (Y1) and maximum fluorescence intensity (Y2), correspondingly. Quality tools as well as ANOVA testing, before and after response transformation were used to decide upon the substantial variables. Following the optimization of reaction variables using desirability plots, calibration graphs were found to be rectilinear in the range of 6-14μg/mL and 0.4-1.2μg/mL for methods A and B, respectively. Both methods proved to be sensitive with detection limits (DL) of 337 and 85ng/mL, and quantitation limits (QL) of 1086 and 283ng/mL, for methods A and B, respectively. An interference study was performed using potential foreign species. No significant interference effect was observed on any of the proposed procedures. System performance was addressed following ICH guidelines and considering parameters such as linearity, detection and quantification limits, accuracy and precision, robustness and specificity.