This paper shows the potential of excitation-emission fluorescence spectroscopy and several second-order methods, such as parallel factor analysis (PARAFAC), multiway partial least-squares (N-PLS) or bilinear least-squares (BLLS), as a multicalibration technique for the analysis of leucovorin (LV) and irinotecan (CPT-11). Although CPT-11 presents native fluorescence, leucovorin has little native fluorescence; however, under irradiation with short-wavelength UV light in the presence of traces of hydrogen peroxide, leucovorin was converted into a highly fluorescent compound. This reaction has been used for the sensitive and selective determination of both compounds. The convenience of analysing the total luminescence spectrum information when using multivariate calibration methods on fluorescence data is demonstrated. Direct determination of mixtures of both drugs in urine was accomplished on the basis of excitation-emission matrices (EEMs) and the three-way multivariate methods.
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