A novel quality-by-design assisted HPLC-DAD method for the simultaneous quantification of tryptophan, tryptophol, and voriconazole for early diagnosis and prognosis of fungal infections decoding quorum sensing phenomenon

Abstract

For the first time, a comprehensive analytical approach is introduced that simultaneously quantifies a metabolic precursor (tryptophan), a quorum-sensing biomarker for fungal infections (tryptophol), and an antifungal drug (voriconazole) within a single platform using reversed-phase high-performance liquid chromatography coupled with diode array detection (HPLC-DAD). The method utilizes a Pursuit PFP column featuring a unique pentafluorinated structure, with a mobile phase of methanol: water (60:40, v/v), a flow rate of 1.0 mL/min, and a detection wavelength set at 254.0 nm. An Analytical Quality-by-Design (AQbD) methodology was employed, incorporating a full factorial design for optimal method development. Validation was performed in accordance with ICH guidelines, demonstrating exceptional linearity (2.0-60.0 μg/mL) for all target analytes, along with high precision, accuracy, and system suitability. Furthermore, the method proved robust and versatile when applied to complex matrices, including spiked human serum and pharmaceutical tablet formulations. Noteworthy is the integration of green and white chemistry principles for evaluating the method's sustainability, representing a significant advancement in analytical technique development. Assessment of greenness, blueness, and whiteness with AGREE, ComplexGAPI index, BAGI and RGB 12 Tools, respectively. This innovative analytical platform provides a powerful tool for the early detection and real-time therapeutic monitoring of fungal infections. By enabling the simultaneous analysis of a metabolic marker, a quorum-sensing specific biomarker, and an antifungal agent, the method advances personalized medicine. It offers a novel, efficient, and sustainable solution for the personalized management of fungal infections, enhancing both diagnostic and therapeutic strategies.

Keywords: Analytical quality-by-design; Fungal infections; HPLC-DAD; Spiked human serum; Sustainable analysis; Tryptophan; Tryptophol; Voriconazole.