Eco-friendly spectrofluorimetric and HPLC-fluorescence methods for simultaneous determination of melatonin and zolpidem in pharmaceuticals

Two eco-friendly, facile, rapid, and highly sensitive analytical methodologies were developed for the concurrent estimation of melatonin and zolpidem tartrate in raw materials and dosage forms. The first approach is a green first derivative synchronous spectrofluorimetric method (Method I), utilizing a wavelength interval (Δλ) of 60 nm and capturing derivative signals at 265.0 nm and 339.0 nm for melatonin and zolpidem, respectively. Method I exhibited excellent linearity over the ranges of 8.0-70.0 ng/mL for melatonin and 10.0-80.0 ng/mL for zolpidem, with detection limits of 1.62 ng/mL and 1.19 ng/mL. The second method (Method II) involves high-performance liquid chromatography coupled with fluorescence detection (HPLC-FD), employing a C18 column and a mobile phase of methanol:0.05% triethylamine (70:30, v/v, pH 5.5) at a flow rate of 1.0 mL/min. Fluorescence detection was performed at 383 nm following excitation at 243 nm. This method achieved linearity within 150.0-1500.0 ng/mL for melatonin and 50.0-700.0 ng/mL for zolpidem, with detection limits of 18.87 ng/mL and 8.86 ng/mL, respectively. Both methods were rigorously validated in accordance with ICH Q2(R1) guidelines, demonstrating high accuracy, precision, and robustness in analyzing commercial dosage forms. Greenness and sustainability were quantitatively assessed using the AGREE and eco-scale metrices, while method applicability and performance were evaluated using the Blue Applicability Grade Index (BAGI), affirming both techniques as efficient and environmentally sustainable tools for routine quality control.