Simultaneous Determination of Kynurenine and Kynurenic Acid by High-Performance Liquid Chromatography Photoirradiation System Using a Mobile Phase Containing 18-crown-6

Abstract

A high-performance liquid chromatography (HPLC) system has been developed for the fluorometric determination of kynurenine (KYN) and kynurenic acid (KYNA) in human serum using a mobile phase containing 18-crown-6. A retention time of KYNA was adjusted with pH of phosphate buffer in 18-crown-6. KYN and KYNA were separated on a CAPCELLPAK C18 (250 × 4.6 mm i.d.). The mobile phase consisted of 35 mmol/L phosphate buffer (pH 8.0)/methanol (85/15, v/v) containing 35 mmol/L hydrogen peroxide and 10 mmol/L 18-crown-6. The retention times of KYN and KYNA were 18and 24 minutes, respectively. The calibration graphs of KYN and KYNA were linear over the range 180 to 2900 and 1 to 84 nmol/L by injecting a 50-μL volume of KYN and KYNA, respectively. Pretreatment of serum was achieved by deproteinization only. The mean recoveries of KYN and KYNA from serum were more than 97%.

Developmental and Nutritional Dynamics of Malpighian Tubule Autofluorescence in the Asian Tiger Mosquito Aedes albopictus

“…In contrast, AF spectra from the distal portions encompass the overall 410-610 nm interval, with a variable contribution from the 500-610 nm component depending on the type of sample under consideration. These results are consistent with the few examples of AF spectra reported in the literature for tryptophan and its metabolites as pure compounds, which anyway show some variability in the peak position and shape that can be ascribed to the effects of solvents as compared to the molecular micro-environmental conditions in granules [35,36]. Also, the literature reports on the AF microspectrofluorometric in situ characterization of the MTs of Drosophila, showing wide spectra in the 430-550 nm interval or a narrower band peaking just below 450 nm in the wild-type Oregon-R strain and in the vermillion brown eye-color mutants, respectively.…”

“…The well-established role of tryptophan, KYN and other derivatives in regulating immune, inflammatory, and neurological processes in humans has promoted the exploration of their autofluorescence (AF) spectral properties for the development of fast and cost-effective analytical techniques aimed at quantifying these compounds in the field of biomedicine [ 35 , 36 ]. Furthermore, a family of brominated derivatives of KYN, likely responsible for the skin biofluorescence of certain shark species, has been optically characterized as a foundation for addressing new questions regarding antimicrobial and photoprotective roles [ 37 ].…”

A review of chromatographic methods for bioactive tryptophan metabolites, kynurenine, kynurenic acid, quinolinic acid, and others, in biological fluids

“…Other than the aforementioned HPLC methods using aqueous Zn 2+ , in 1988, a method involving post-column photo-irradiation system followed by fluorescence detection was reported by Watanabe and Mawatari group (Figure 2b) (Atsumi et al, 2019;Mawatari et al, 1988Mawatari et al, , 1989Mawatari et al, , 1991Tabara et al, 1987). Note: KYN: kynurenine; KYNA: kynurenic acid; LOD: limit of detection; LOQ: limit of quantification.…”

“…Watanabe's group demonstrated that some Trp metabolites are In a recent report, a crown ether, namely, 18-crown-5, was added to the mobile phase to enhance the separation of KYN and KYNA for simultaneous separation in human plasma (Figure 4b) (Atsumi et al, 2019). In addition, the addition of 18-crown-5 to the mobile phase reduced the interfering peaks.…”