Development and validation of an NBD-F-based fluorometric method for the determination of plasma kynurenine and its clinical relevance in essential hypertension

Figures

• 

  Fig. 1. Schematic representation of the nucleophilic substitution reaction between KYN and NBD-F.

• 

  Fig. 2. Stoichiometric analysis of the KYN–NBD reaction using Job's method of continuous variation.

• 

  Fig. 3. (a) Effect of borate buffer pH on the derivatization of KYN with NBD-F, showing maximum fluorescence intensity (FI) at pH 8.5. (b) Effect of borate buffer volume (pH 8.5) on the derivatization of KYN with NBD-F, showing maximum fluorescence intensity (FI) at 1.0 mL.

• 

  Fig. 4. (a) Effect of NBD-F concentration (0.2–2.2%, w/v) on the derivatization of KYN, showing maximum fluorescence intensity (FI) at 1.0% (w/v). (b) Effect of NBD-F volume (0.25–2.0 mL) on the derivatization of KYN, showing maximum fluorescence intensity (FI) at 1.0 mL.

• 

  Fig. 5. (a) Effect of reaction temperature on the derivatization of KYN with NBD-F, showing maximum fluorescence intensity (FI) at 50 °C. (b) Effect of reaction time on the derivatization of KYN with NBD-F, showing maximum fluorescence intensity (FI) after 15 min.

• 

  Fig. 6. Fluorescence emission spectra of the KYN–NBD adduct and reagent blank, showing the enhancement of fluorescence intensity (FI) following derivatization.

• 

  Fig. 7. Fluorescence emission spectra of the KYN–NBD adduct over the concentration range of 100–800 ng mL−1, showing concentration-dependent enhancement of fluorescence intensity (FI) with a maximum emission at 555 nm.

• 

  Fig. 8. Fluorescence intensity (FI) of potential interfering compounds relative to KYN, demonstrating the selectivity of the proposed method.