DOE assisted RP-HPLC method development and validation for estimation of L-Cysteine and cystine with dansyl chloride derivatization in presence of amino acid mixture

The aim of the present study is to apply Design of Experiments (DoE), to develop an assay and optimize the derivatization reaction. DoE study type is Response Surface, Sub type is Split-plot, Design is D-optimal method was used in the study for the separation of L-Cysteine and L-Cystine in presence of amino acids by RP-HPLC. DoE Response Surface Split-plot method was used in this study. DoE allows to interpret the results with better outcome and enhanced understanding. Pre-column derivatizing agent Dansyl Chloride was used for dansylation of amino acids; 10mm Ammonium Acetate buffer P^H 6.3, Acetonitrile was used as mobile phase in Eclipce XDB C18 column (150mm x 4.6mm, 5µm). Derivatizing reagent volume, derivatization reaction heating time, and temperature was evaluated as variables in the DoE. The wavelength for all amino acids is 222nm. Conclusively DoE as an efficient tool for optimization of dansylation reaction for the separation of L-Cysteine and L-Cystine in presence of amino acids. Method development was established and the design is validated. The proposed method has adequate reproducibility, specificity and accurate for the estimation of L-Cystine and L-Cystine in the presence of amino acids in routine analysis.