Use of pH gradients in continuous-flow systems and multivariate regression techniques applied to the determination of methionine and cysteine in pharmaceuticals

The simultaneous spectrophotometric determination of methionine and cysteine in presence of cystine and other compounds in pharmaceuticals, using a multivariate calibration method, was studied. The method is based on the reaction between the analytes and the o-phthalaldehyde-N-acetyl-l-cysteine (OPA-NAC) reagent performed in a continuous-flow system (FI). The FI system allows the generation of a local pH gradient in order to produce spectral and/or kinetic changes in the UV-Vis spectra of the amino acid-OPA-NAC derivatives. This information is used to improve the prediction ability of the Partial Least-Squares (PLS) models. The performance of two FI assemblies, the selection of variables and the pretreatment of the data were studied. A series of eighteen synthetic solutions containing different concentrations of methionine, cysteine and cystine was prepared and used as a calibration set to study the PLS models. In addition, solutions of a commercial formulation containing the three amino acids were prepared and the content of methionine and cysteine was predicted. The results obtained using the PLS models were compared with those obtained using an HPLC method.