Simultaneous spectrofluorimetric and spectrophotometric determination of melatonin and pyridoxine in pharmaceutical preparations by multivariate calibration methods

Partial least-squares (PLS) calibration and principal component regression (PCR) methods were utilized for the simultaneous spectrofluorimetric and spectrophotometric determination of pyridoxine (PY) and melatonin (MT). Since emission and adsorption spectra of these drugs overlap, PY and MT cannot be directly determined by fluorimetric nor by spectrophotometric methods. Full-spectrum multivariate calibration PLS and PCR methods were developed for both fluorimetry and spectrophotometry. The conditions were optimized for fluorimetric as well as for spectrophotometric determination of both drugs. The simultaneous determination of PY and MT was carried out in mixtures by recording the emission fluorescence spectrum between 324 and 500 nm (λ_ex 285 nm) for fluorimetry, and by recording the absorption spectrum between 250 and 350 nm for spectrophotometry (λ_max(PY) 310 nm, λ_max(MT) 278 nm). The experimental calibration matrixes were designed orthogonally. At the optimum conditions, dynamic ranges were 0.04–1.3 and 0.1–4 μg ml^–1 for fluorimetry and 1–22 and 1–24 μg ml^–1 for spectrophotometry for MT and PY, respectively. The calibration concentrations were prepared in the dynamic ranges. The parameters of the chemometrics procedure for the simultaneous determination of MT and PY were optimized, and the proposed methods were validated with prediction set. Finally the procedures were successfully applied to simultaneous spectrofluorimetric and spectrophotometric determination of PY and MT in synthetic mixtures and in a pharmaceutical formulation.