Net analyte signal (NAS)-based multivariate calibration methods were employed for simultaneous determination of anthazoline and naphazoline. The NAS vectors calculated from the absorbance data of the drugs mixture were used as input for classical least squares (CLS), principal component and partial least squares regression PCR and PLS methods. A wavelength selection strategy was used to find the best wavelength region for each drug separately. As a new procedure, we proposed an experimental design-neural network strategy for wavelength region optimization. By use of a full factorial design method, some different wavelength regions were selected by taking into account different spectral parameters including the starting wavelength, the ending wavelength and the wavelength interval. The performance of all the multivariate calibration methods, in all selected wavelength regions for both drugs, was evaluated by calculating a fitness function based on the root mean square error of calibration and validation. A three-layered feed-forward artificial neural network (ANN) model with back-propagation learning algorithm was employed to model the nonlinear relationship between the spectral parameters and fitness of each regression method. From the resulted ANN models, the spectral regions in which lowest fitness could be obtained were chosen. Comparison of the results revealed that the net NAS-PLS resulted in lower prediction error than the other models. The proposed NAS-based calibration method was successfully applied to the simultaneous analyses of anthazoline and naphazoline in a commercial eye drop sample.
