Synchronous spectrofluorimetric determination of tryptophan and kynurenine in human plasma using hydroxypropyl-β-cyclodextrin complexation for investigating their role in major depressive disorder

Major depressive disorder (MDD) is recently associated with immunological activation and deregulation of the kynurenine pathway, which redirects tryptophan metabolism away from serotonin production and towards neuroactive metabolites. Precise determination of tryptophan and kynurenine in biological samples is important to investigate their association with MDD pathophysiology. This study presents a highly sensitive spectrofluorimetric method for determining tryptophan and kynurenine in human plasma utilizing hydroxypropyl-β-cyclodextrin (HP-β-CD) inclusion complexation and synchronous fluorescence scanning. Furthermore, the work represents the first report describing the simultaneous determination of these analytes using HP-β-CD-assisted synchronous spectrofluorimetry in human plasma, offering a cost-effective alternative to conventional chromatographic techniques. HP-β-CD enhanced the fluorescence intensity of tryptophan and kynurenine and provided distinct emission peaks, minimizing spectral overlap. Synchronous fluorescence spectra were integrated by scanning both excitation and emission monochromators simultaneously at a constant wavelength offset (Δλ) of 50 nm and the fluorescence peak values were measured at 400 nm for selective tryptophan determination and 455 nm for selective kynurenine determination. The method exhibited linear relationship over a concentration range of 1-100 µM, with limits of detection (LOD) of 0.20 µM for tryptophan and 0.22 µM for kynurenine, and limits of quantification (LOQ) of 0.60 µM and 0.66 µM, respectively. Quantum yield evaluations, performed in triplicate (n = 3), confirmed significant fluorescence enhancement by HP-β-CD, increasing tryptophan quantum yield from 0.170 ± 0.006 to 0.520 ± 0.011 and kynurenine quantum yield from 0.090 ± 0.004 to 0.320 ± 0.009 (p < 0.001). The method demonstrated high accuracy, with mean recovery rates of 99.25% for tryptophan and 98.87% for kynurenine, and low RSD values across intra- and inter-day measurements. The method was applied to human plasma samples and the findings exhibited significantly decreased tryptophan levels in MDD patients (41.20 ± 3.22 µM) compared to healthy controls (62.50 ± 6.20 µM, p = 0.003), and elevated kynurenine levels (3.50 ± 1.20 µM vs. 1.20 ± 0.20 µM, p = 0.004).