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. 2026 Aug 5;16(39):44852–44862. doi: 10.1039/d6ra04719j

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

Ali Alqahtani a, Sumaiah D Alrubiaan b, Roaya S Alqurashi b, Mohamad A Omar c,✉
PMCID: PMC13439739  PMID: 42559233

Abstract

The kynurenine pathway, the main route of tryptophan catabolism, contributes to the pathogenesis of essential hypertension by promoting immune activation and endothelial dysfunction. Within cardiovascular disease, kynurenine (KYN) acts both as a diagnostic biomarker and as a mechanistic mediator. This study aimed to develop a sensitive fluorometric method for plasma KYN determination evaluating its clinical relevance in hypertensive cohorts. Because native KYN exhibits weak intrinsic fluorescence, derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was employed to generate a highly fluorescent derivative with enhanced spectroscopic properties, thereby improving the sensitivity of fluorometric detection. The resulting KYN–NBD derivative was measured at excitation and emission wavelengths of 485 and 555 nm, respectively. The reaction produced a stable KYN–NBD complex with a quantum yield of 0.63 compared with 0.22 for underivatized KYN, markedly improving method sensitivity. Validation according to ICH guidelines demonstrated linearity over 100–800 ng mL−1 (r2 = 0.9996), high sensitivity (LOD: 10 ng mL; LOQ: 30 ng mL−1), and suitable accuracy and precision (recovery: 98.62–99.71%; RSD < 0.79%). The method additionally showed excellent selectivity without interference from confounding substances. To demonstrate its clinical applicability in a complex biological matrix, the validated assay was successfully applied to plasma samples obtained from 48 participants enrolled in a clinical case-control study, addressing the need for a simple and sensitive method suitable for routine plasma KYN determination. Plasma KYN levels were significantly higher in hypertensive individuals than in normotensive controls (510 ± 34 vs. 375 ± 25 ng mL−1, p < 0.001), correlating with systolic and diastolic blood pressure.

Development and validation of an NBD-F-based fluorometric method for the determination of plasma kynurenine and its clinical relevance in essential hypert.graphic file with name d6ra04719j-ga.webp

1. Introduction

Kynurenine (KYN), a major tryptophan metabolite produced, is an essential mediator linking metabolism, immunity, and inflammation. KYN is the first stable intermediate of the kynurenine pathway, the major route of tryptophan metabolism initiated by indoleamine-2,3-dioxygenase (IDO). It serves as a precursor for biologically active metabolites, including kynurenic acid, 3-hydroxykynurenine, anthranilic acid, and quinolinic acid, which collectively regulate immune responses, oxidative stress, and vascular function. Elevated KYN levels are documented across infectious, neurodegenerative, malignant, and cardiovascular disorders.1–7 In cardiovascular conditions like essential hypertension, KYN is associated with endothelial dysfunction, vascular remodeling, and oxidative stress, highlighting its involvement in disease pathophysiology.8–11 Mechanistically, KYN pathway activation yields metabolites driving vascular dysfunction. Compounds like 3-hydroxykynurenine and quinolinic acid promote oxidative stress, compromising endothelial nitric oxide synthase (eNOS) functionality. This depletes bioavailable nitric oxide (NO) and disrupts downstream signaling. This imbalance is amplified by eNOS uncoupling, generating superoxide instead of NO, which drives vascular stiffening, increased peripheral resistance, and reduced vasodilation.12–14 Conversely, kynurenic acid exhibits limited, context-dependent antioxidant properties.15

Concurrently, KYN binds the aryl hydrocarbon receptor (AhR), which regulates immune and vascular systems.16–18 AhR activation upregulates endothelial adhesion molecules, promoting inflammation and leukocyte attachment. In vascular smooth muscle cells, it enhances remodeling, migration, and proliferation, causing structural changes. This sustained signaling shifts gene expression toward a pro-constrictive state, disrupting vascular tone homeostasis and increasing hypertension susceptibility. In parallel, IDO activity promotes regulatory T-cell development. However, a Treg/Th17 imbalance sustains chronic inflammation, prolonging endothelial dysfunction and increasing vascular tone.19–23

Review of literature reveals KYN determination is predominantly performed using chromatographic techniques, including HPLC,24,25 LC-MS/MS,26 and GC-MS for rat brain tissues.27 Although these methods provide reliable quantification, they are associated with prolonged analysis times, labor-intensive sample pretreatment, and substantial solvent consumption. A direct fluorometric assay has been reported for rapid screening of KYN in urine samples;28 however, it was validated only in synthetic urine without evaluating plasma matrix effects, limiting its applicability to clinical plasma analysis. Direct spectroscopic determination of KYN is challenging because of its weak native fluorescence and interference from endogenous plasma constituents. Direct spectroscopic determination of KYN is challenging because of its weak native fluorescence and interference from endogenous plasma constituents, which limit analytical sensitivity and selectivity. To overcome these limitations, the proposed NBD-F derivatization-based fluorometric method converts KYN into a highly fluorescent derivative while eliminating the chromatographic step, thereby substantially reducing analysis time and avoiding mobile phase preparation and column maintenance. The achieved LOD of 10 ng mL−1 is markedly lower than that reported for HPLC-UV methods and is sufficient to quantify KYN across the lower end of its physiological concentration range. Although LC-MS/MS provides superior sensitivity and specificity, its high instrumentation cost and operational complexity restrict its routine use in clinical biochemistry laboratories. Notably, to the best of our knowledge, the present method is the first to undergo full bioanalytical validation in authentic human plasma. The method demonstrated excellent linearity, accuracy, precision, and selectivity, supporting its suitability for both research and clinical applications. Furthermore, it was applied to investigate the clinical significance of circulating KYN in patients with essential hypertension, providing a sensitive and practical approach for routine plasma KYN determination.

2. Experimental

2.1. Materials, reagents, and instruments

Pure-grade KYN reference standard (98.79%) and the fluorogenic modifier NBD-F were both procured from Sigma-Aldrich (USA). To prepare the working derivatization reagent, a 1% (w/v) stock concentration was generated by dissolving 100 mg of NBD-F in 10 mL of HPLC-grade acetonitrile. Additionally, HPLC-grade dimethyl sulfoxide (DMSO) and acetonitrile were sourced from Sigma-Aldrich (USA) for chemical processing.

Phosphate buffer solutions covering the pH range from 6 to 8, and borate buffer solutions within the pH range of 8.5–10 were prepared following the United States Pharmacopeia (USP) procedure.29 Pooled human plasma used for method development and validation was obtained from healthy adult volunteers recruited through hospital of Al-Azhar University, Damietta, Egypt. Individual plasma samples used for the clinical application were collected from participants enrolled in the proposed clinical study. Fluorometric data acquisition was executed utilizing an Agilent Cary Eclipse fluorescence spectrophotometer equipped with dedicated monochromators for both excitation and emission pathways.

2.2. Standard solutions

Primary stock concentrations of KYN were generated at 100 µg mL−1 by dissolving the analyte in a 5% aqueous DMSO matrix.

2.3. Establishment of the fluorometric method

To establish the analytical calibration framework, progressive volumes of the KYN stock solution were accurately delivered into a series of 10 mL volumetric flasks to obtain final added KYN concentrations of 100–800 ng mL−1. Each calibration standard contained 1.0 mL of pooled human plasma. Because kynurenine is an endogenous plasma metabolite and analyte-free plasma is not readily available, pooled plasma containing endogenous KYN was used for calibration. The same pooled plasma batch was used throughout the calibration procedure to maintain a constant endogenous KYN background in all calibration standards, and calibration was performed using a matrix-matched standard-addition approach. Matrix proteins were then precipitated by adding 1.5 mL of 10% (w/v) trichloroacetic acid (TCA),30 followed by centrifugation at 6000 rpm for 10 min. The resulting clear supernatant was transferred to a reaction vessel and mixed with 1.0 mL of borate buffer (pH 8.5) and 1.0 mL of 1% (w/v) NBD-F solution. The reaction mixture was incubated at 50 °C for 15 min, allowed to cool to room temperature, and subsequently treated with 0.5 mL of 1 N HCl. The final volume was adjusted to 10 mL with distilled water. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 555 nm. A reagent blank processed under identical conditions was used as the reference. The proposed derivatization reaction between KYN and NBD-F is illustrated in Fig. 1.

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

Fig. 1

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2.4. Method development and optimization studies

Prior to validation, single-variable experiments were conducted to optimize derivatization parameters. Each factor was investigated sequentially by varying one parameter while maintaining all others constant.

2.4.1. Investigation of pH and buffer capacity

To identify the optimal environment for the nucleophilic substitution reaction, the effect of pH was evaluated across a range of 6–8 utilizing a phosphate buffer system, and a range of 8.5–10 utilizing a borate buffer system. Following the identification of the appropriate pH environment, the volume of the buffering medium was varied systematically from 0.25 to 2.0 mL to evaluate the influence of ionic strength and buffering capacity on the fluorescence response.

2.4.2. Optimization of NBD-F parameters

The concentration of NBD-F was varied across a percentage range of 0.2–2.2% (w/v) to determine the concentration required for optimal reaction yields while avoiding any concentration-dependent quenching phenomena. Concurrently, the volume of the NBD-F working solution was modulated between 0.25 and 2.0 mL to investigate its impact on the formation efficiency of the fluorescent derivative.

2.4.3. Evaluation of temperature and incubation time

To determine the thermodynamic parameters of the reaction, the mixture was incubated across a wide temperature range spanning from 25 to 70 °C. At each evaluated temperature, the incubation time was progressively monitored over a specified time interval from 5 to 30 min to establish the exact time required for complete derivatization and to ensure the stability of the formed complex.

2.4.4. Optimization of the acidification step

To suppress background fluorescence resulting from the potential hydrolysis of NBD-F into NBD-OH under alkaline conditions, a reaction quenching step was introduced post-incubation. The addition of a 0.5 mL volume of 1 N hydrochloric acid (HCl) was incorporated and evaluated to minimize reagent blank interference.

2.5. Method validation

To ensure regulatory compliance, the analytical method underwent validation controlled by ICH approaches, assessing linearity, accuracy, precision, and selectivity.31 The linear response was verified by analyzing reference KYN solutions across a spectrum of 100–800 ng mL−1 under the adopted operational parameters.

Furthermore, the method was validated according to the EMA bioanalytical method validation guideline and the FDA bioanalytical method validation guidance for plasma-based assays.32,33 The validation included the assessment of matrix effect using the post-extraction addition approach at low (200 ng mL−1) and high (600 ng mL−1) levels, extraction recovery following TCA protein precipitation, carryover, dilution integrity, analyte stability under bench-top, freeze–thaw, and long-term storage conditions, and robustness by introducing minor deliberate variations in pH, NBD-F concentration, temperature, and incubation time.

The limit of detection (LOD) and limit of quantification (LOQ) were measured using the following equations:2.5.where SDr represents the residual standard deviation of the regression line, and the slope is derived from the linear regression analysis.

The fidelity and reproducibility of the developed method were evaluated at three KYN concentrations (200, 400, and 600 ng mL−1). Accuracy was determined by calculating percentage recovery during intra-day and inter-day analyses. Precision was assessed using intra-day repeatability and inter-day intermediate precision and expressed as the relative standard deviation (RSD). Method selectivity was investigated using structurally related biomolecules and endogenous primary amine-containing compounds that could interfere with KYN quantification. The tested compounds included anthranilic acid, 3-hydroxykynurenine, tyrosine, phenylalanine, tryptophan, serotonin, lysine, ornithine, dopamine, adrenaline, and glucosamine. Individual stock solutions (200 ng mL−1) were analyzed under the optimized assay conditions. Fluorescence responses were measured under identical optical settings (excitation 485 nm, emission 555 nm) to evaluate potential spectral overlap and interference with KYN determination.

2.6. Clinical study

The clinical study was conducted as a cross-sectional case-control investigation to quantify circulating KYN concentrations and evaluate their clinical significance in patients with essential hypertension compared with healthy normotensive controls.

2.6.1. Sample size assessment

The required cohort size was determined utilizing the conventional statistical equation designated for the comparison of two distinct independent means,34 detailed below:2.6.1.where Zα/2 is the value corresponding to a 95% confidence interval (1.96), Zβ denotes the statistical power set at 80% (0.84), σ signifies the estimated standard deviation of circulating KYN levels within the plasma matrix, and Δ represents the minimal threshold for clinically meaningful variance between the hypertensive cohort and the control groups.

Assuming plasma KYN levels in patients with hypertension, the reported σ values range from 180 to 220 ng mL−1, whereas the mean Δ between hypertensive patients and healthy controls is approximately 100 ng mL−1. To maintain clinical validity, these projected variables were integrated into the current statistical estimation. Consequently, the minimum required cohort was determined to be 20 subjects per arm. To account for potential attrition, dropouts, or pre-analytical sample degradation (such as hemolyzed specimens), this baseline estimate was expanded by 10%, culminating in a final enrollment of 24 participants per group, with a collective study population of 48 individuals.

2.6.2. Participant demographics and selection protocol

A total of 48 participants were enrolled from the affiliated hospital of Al-Azhar University in Damietta, Egypt. The study included 24 patients with essential hypertension and 24 healthy normotensive controls matched for age and sex. Eligible participants were 30–65 years old. Hypertension was diagnosed according to the European Society of Cardiology/European Society of Hypertension criteria as systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg on at least two clinical visits or current use of antihypertensive medication. Healthy controls had blood pressure <130/80 mmHg, no history of cardiovascular disease, and no antihypertensive treatment. Exclusion criteria for both groups included cardiovascular diseases other than essential hypertension, diabetes mellitus, chronic kidney disease, active inflammatory, infectious, or autoimmune disorders, malignancy, immunosuppressive therapy, pregnancy, and lactation.

2.6.3. Plasma sample collection and preparation

Following an overnight fasting period of no less than 10 h to mitigate dietary influences on circulating metabolites, a 5 mL volume of peripheral venous blood was drawn from each subject into EDTA-containing tubes. To assure the preservation of metabolic profiles, the collected specimens were instantly transferred to an ice bath and subjected to sedimentation via centrifugation at 6000 rpm for 18 min to isolate the plasma matrix. To prevent analyte degradation associated with recurrent freeze thaw sequences, the isolated plasma layer was immediately partitioned into sterile cryovials. Standardized processing of all specimens was accomplished within one hour of venipuncture to maintain metabolic stability and guarantee procedural uniformity. The determination of plasma KYN levels was subsequently executed using the newly developed fluorometric protocol.

A cross-validation study was performed by analyzing 20 clinical plasma samples (10 hypertensive and 10 normotensive) in parallel using the proposed fluorometric method and a previously validated HPLC-UV method.25 Method agreement was evaluated using Passing–Bablok regression and Bland–Altman analysis.

2.6.4. Ethical considerations

The proposed study was approved by the Institutional Review Board of the Faculty of Medicine, Al-Azhar University, Damietta (Approval No. DFM-IRB 00012367-26-06-014). Adhering to the Declaration of Helsinki, all participants provided written informed consent prior to enrollment.

3. Results and discussion

3.1. Basis for method development

Growing evidence supporting KYN as a cardiovascular biomarker highlights the need for a sensitive analytical approach. In this study, a straightforward and accurate method was developed to quantify KYN in complex biological matrices, particularly at low physiological levels. A fluorescence-based assay was employed due to its high sensitivity for detecting low-abundance analytes. Because native KYN exhibits weak fluorescence, derivatization with NBD-F was applied to generate a stable fluorescent product, enabling improved quantification in plasma samples.

3.2. Reaction mechanism of KYN derivatization with NBD-F

As illustrated in Fig. 1, the chemical modification of KYN utilizing NBD-F proceeds via a nucleophilic substitution pathway. This process is initiated by a nucleophilic attack from the primary amine functional group of KYN targeting the electron-poor carbon atom on the NBD-F ring, which is highly activated by the strongly electron-withdrawing nitro group. This interaction subsequently drives the elimination of a hydrogen fluoride molecule, yielding a stable covalent bond that generates the highly fluorescent KYN–NBD complex.

3.3. Structural characterization using 1H NMR spectroscopy

The chemical structures of KYN and the KYN–NBD derivative were verified by 1H NMR spectroscopy. As shown in Fig. S1, the 1H NMR spectrum of KYN displayed characteristic aromatic proton signals at δ 6.2–7.6 ppm and aliphatic methylene signals at δ 2.2–4.0 ppm. Broad exchangeable peaks at δ 12–13 ppm were assigned to the amino and amide protons. After reaction with NBD-F, the KYN–NBD spectrum (Fig. S2) showed additional aromatic signals at δ 7.0–9.0 ppm, corresponding to the benzofurazan ring of the NBD group. The amino proton signals exhibited reduced intensity and downfield shifts, indicating covalent modification of the primary amino group. The aliphatic proton signals remained within δ 2.5–4.0 ppm, demonstrating that the KYN backbone was preserved after derivatization. These spectral changes confirm the successful formation of the fluorescent KYN–NBD derivative through nucleophilic substitution with NBD-F.

3.4. Quantum yield evaluation and stoichiometric characterization of the KYN–NBD adduct by Stern–Volmer and Job's method

The quantum yield (Φ) of KYN, NBD-F, and the KYN–NBD derivative was executed to characterize the photophysical behavior of the fluorophore-labeled KYN–NBD complex. Quinine sulfate in 0.1 M H2SO4 (Φref = 0.546) was used as the reference standard. The Φ values were calculated using the following equation:353.4.where φx and φref represent the corresponding values of the sample and reference, Fx and Fref are the fluorescence signal, Ax and Aref denote the respective optical densities recorded at the excitation wavelength, which were strictly maintained below 0.1 to mitigate inner filter phenomena), ηx and ηref represent the refractive indices of the respective solvent media (distilled water and 0.1 M H2SO4).

The Φ of KYN was found to be 0.22, whereas the Φ of NBD-F was 0.09. After derivatization, the KYN–NBD derivative exhibited a pronounced rise in the Φ and found to be 0.63, exceeding that of native KYN. This pronounced increase demonstrates the efficiency of KYN derivatization with NBD-F. This significant fluorescence enhancement is attributed to the restriction of non-radiative decay pathways upon the covalent coupling of the benzofurazan moiety to the primary amine of KYN.

The thermodynamic stability of the KYN–NBD derivative was evaluated by calculating the chemical formation constant (Ka). Because free NBD-F exhibits minimal fluorescence, increasing KYN concentrations produced concentration-dependent fluorescence enhancement at 555 nm. The binding affinity was determined using an adapted reciprocal association equation:363.4.where F0 is the initial fluorescence intensity of NBD-F, F is the measured fluorescence intensity in the presence of KYN, Fmax is the maximum plateaued fluorescence intensity of the fully formed complex, and [KYN] represents the molar concentration of kynurenine acting as the binding partner. The calculated association constant (Ka) was found to be 3.27 × 104 M−1, indicating a robust chemical affinity and highly efficient product formation between KYN and NBD-F.

The reaction stoichiometry governing the derivatization of KYN with NBD-F was characterized employing Job's method of continuous variation,37 with the structural profiles illustrated in Fig. 2. Throughout this optimization protocol, the aggregate molarity of the two core reactants was maintained at a fixed threshold, while their individual mole fractions were modulated systematically to monitor the resulting changes in fluorescence intensity. The generated profiles revealed a prominent inflection maximum corresponding to a mole fraction of 0.5, establishing a clear 1 : 1 reaction ratio for the synthesized KYN–NBD adduct. The lack of additional maxima further demonstrated that higher-order complexes were not generated, indicating the selectivity of the derivatization process.

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

Fig. 2

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3.5. Method development and optimization studies

3.5.1. pH study

The influence of pH on the derivatization of KYN with NBD-F was investigated using phosphate buffer (pH 6–8) and borate buffer (pH 8.5–10). As shown in Fig. 3a, maximum fluorescence intensity was obtained at pH 8.5, indicating optimal conditions for the nucleophilic substitution reaction. At pH 10, partial hydrolysis of NBD-F forms NBD-OH, increasing background fluorescence and potentially interfering with the derivative signal.38 Therefore, after cooling, 0.5 mL of 1 N HCl was added to minimize this interference. Borate buffer volumes ranging from 0.25 to 2.0 mL were then evaluated under identical conditions. As shown in Fig. 3b, fluorescence intensity increased with buffer volume, reaching a maximum at 1.0 mL. Further increases produced no additional enhancement, indicating that the optimal protonation state and ionic strength had been achieved for stable fluorescence emission. Accordingly, pH 8.5 and 1.0 mL borate buffer were selected for subsequent experiments.

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. 3

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3.5.2. Effect of NBD-F concentration and volume

The effect of NBD-F concentration on KYN derivatization was evaluated over the range of 0.2–2.2% (w/v). As shown in Fig. 4a, fluorescence intensity increased with reagent concentration, reaching a plateau at 1% (w/v), indicating optimal derivatization without concentration-dependent quenching. The effect of reagent volume was then investigated using 1% (w/v) NBD-F. As shown in Fig. 4b, fluorescence intensity increased with NBD-F volume up to 1.0 mL, reflecting efficient derivative formation under the selected conditions.

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. 4

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3.5.3. Effect of temperature

The influence of reaction temperature on the derivatization of KYN with NBD-F was investigated over the range of 25–70 °C. As illustrated in Fig. 5a, fluorescence intensity increased progressively with temperature, reaching a maximum at 50 °C. The initial increase in fluorescence can be attributed to accelerated nucleophilic substitution kinetics between KYN and NBD-F, promoting more efficient formation of the fluorescent KYN–NBD derivative. At temperatures above 50 °C, the fluorescence response gradually decreased, which may be attributed to partial thermal degradation of the fluorescent derivative and/or increased side reactions that reduce derivatization efficiency. Therefore, 50 °C was selected as the optimum reaction temperature, providing the highest fluorescence response while maintaining derivative stability.

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. 5

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3.5.4. Effect of reaction time

The effect of reaction time on the derivatization process was examined over the range of 5–30 min. As shown in Fig. 5b, fluorescence intensity increased steadily with reaction time, reaching a maximum after 15 min. This behavior indicates the progressive formation of the fluorescent KYN–NBD derivative until completion of the derivatization reaction. Extending the incubation period beyond 15 min produced no further enhancement in fluorescence intensity, suggesting that the reaction had reached completion and that prolonged heating offered no analytical advantage while potentially increasing the likelihood of derivative degradation or secondary reactions. Accordingly, an incubation time of 15 min was selected as the optimum reaction period for subsequent experiments.

3.6. Fluorometric quantification of plasma KYN via NBD-F derivatization

The quantification of KYN was achieved via an indirect approach involving chemical modification with the fluorogenic probe NBD-F, which yielded a highly fluorescent KYN–NBD complex. The photophysical properties and emission profile of this generated KYN–NBD derivative was assessed through comparison against a blank matrix control, with the corresponding spectral data depicted in Fig. 6. The blank exhibited minimal background fluorescence with no significant excitation or emission peaks. In contrast, the KYN–NBD derivative showed a distinct excitation maximum at 485 nm and a strong emission peak at 555 nm. In addition, the fluorescence emission spectra of the derivative at KYN concentrations ranging from 100 to 800 ng mL−1 are displayed in Fig. 7. A progressive enhancement in the emission signal was observed alongside elevating levels of KYN, establishing a clear concentration-dependent fluorometric profile. To evaluate the matrix effect on method sensitivity, the calibration curve slope obtained from the matrix-matched standard-addition method in plasma was compared directly with that in pure solvent. A negligible difference was observed between the two slopes, confirming that the plasma matrix did not compromise analytical sensitivity.

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

Fig. 6

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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. 7

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3.7. Method validation

Method validation was performed according to ICH guidelines, and the results are summarized in Table 1. Matrix-matched calibration using pooled plasma established a linear range of 100–800 ng mL−1 with excellent linearity (r2 = 0.9996). The method showed high sensitivity, with LOD and LOQ values of 10 ng mL−1 and 30 ng mL−1, respectively. The bioanalytical validation parameters presented in Table 2 confirmed the reliability of the proposed method for plasma analysis in accordance with the EMA and FDA guidelines. The matrix factor was within the acceptable range (85–115%) at 200 ng mL−1 and high 600 ng mL−1 levels. Extraction recovery was 91.4 ± 2.1% and 92.8 ± 1.8% at 200 and 600 ng mL−1, respectively. No carryover signal exceeding 20% of the lower concentration level was observed after injection of the highest calibration standard (800 ng mL−1). Dilution integrity was confirmed for 1600 ng mL−1 samples diluted 1 : 2 and 1 : 4 with blank plasma, yielding recoveries of 99.1 ± 2.0% and 98.7 ± 1.7%, respectively. The analyte remained stable after 6 h at room temperature, three freeze thaw cycles, and 30 days of storage at −20 °C, with all measured concentrations remaining within ±15% of the nominal values. Robustness testing demonstrated that deliberate variations in pH (±0.2 units), NBD-F concentration (±0.1%), temperature (±2 °C), and incubation time (±2 min) did not significantly affect method performance, with all %RSD values remaining below 3%.

Table 1. Analytical performance and regression characteristics of the proposed fluorometric method for KYN determinationa.

Parameters Analytical findings
Excitation wavelength (λex, nm) 485
Emission wavelength (λem, nm) 555
Linearity range (ng mL−1) 100–800
Slope (b) 0.0032 ± 0.0001
Intercept (a) 0.1440 ± 0.0025
Coefficient of determination (r2) 0.9996
Limit of detection (LOD, ng mL−1) 10
Limit of quantification (LOQ (ng mL−1) 30
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a

Values are the mean of three independent calibration blocks (n = 3).

Table 2. Bioanalytical validation parameters of the proposed method, including carryover, dilution integrity, analyte stability (bench-top, freeze–thaw, and long-term), and robustnessa.

Validation parameter Condition/Level Result Acceptance criterion
Matrix factor 200 ng mL−1 96.3 ± 3.1 85–115%
600 ng mL−1 97.8 ± 2.4
Extraction recovery 200 ng mL−1 91.4 ± 2.1 85–115%
600 ng mL−1 92.8 ± 1.8
Carryover Blank plasma after 800 ng mL−1 standard <5% of LOQ signal ≤20% of LOQ
Dilution integrity 1 : 2 dilution (from 1600 ng mL−1) Recovery: 99.1 ± 2.0% 85–115%
1 : 4 dilution (from 1600 ng mL−1) Recovery: 98.7 ± 1.7% 85–115%
Bench-top stability 6 h, room temperature, (200 ng mL−1) 98.1 ± 1.9% 85–115%
6 h, room temperature, (600 ng mL−1) 97.4 ± 2.1% 85–115%
Freeze–thaw stability (3 cycles, −20 °C) 200 ng mL−1 96.8 ± 2.3% 85–115%
600 ng mL−1 97.2 ± 1.8% 85–115%
Long-term stability (30 days, −20 °C) 200 ng mL−1 95.3 ± 2.7% 85–115%
600 ng mL−1 96.1 ± 2.2% 85–115%
Robustness pH ± 0.2 units RSD < 2.1% RSD ≤ 3%
NBD-F concentration ± 0.1% RSD < 1.8% RSD ≤ 3%
Temperature ± 2 °C RSD < 2.4% RSD ≤ 3%
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a

Extraction recovery determined by TCA protein precipitation. Matrix factor calculated by post-extraction addition approach. Acceptance criterion for matrix factor: 85–115% (EMA/FDA bioanalytical guidance). All stability results expressed as mean recovery (%) ± SD (n = 3) relative to freshly prepared QC samples. Robustness expressed as %RSD across triplicate measurements under each perturbed condition. Acceptance criterion for stability recovery and dilution integrity: 85–115% (EMA/FDA bioanalytical guidance). Acceptance criterion for robustness: RSD ≤ 3%.

The accuracy and precision of the proposed method were evaluated using KYN quality control samples at 200, 400, and 600 ng mL−1 (Table 3). Intra-day recovery ranged from 99.14% to 99.71%, while inter-day recovery ranged from 98.62% to 99.33%. Intra-day precision (%RSD) was 0.522–0.699%, and inter-day precision ranged from 0.620% to 0.788%. These results demonstrate excellent accuracy, reproducibility, and precision of the proposed analytical method.

Table 3. Intra-day and inter-day accuracy and precision of the proposed fluorometric method for KYN determination.

KYN (ng ml−1) Intra-day (n = 3) Inter-day (n = 3)
Pure founda (ng mL−1) ± SD Accuracy recovery (%) Precision RSD (%) Pure founda (ng mL−1) ± SD Accuracy recovery (%) Precision RSD (%)
200 198.27 ± 0.25 99.14 0.52 197.24 ± 0.05 98.62 0.62
400 398.24 ± 0.28 99.56 0.69 396.56 ± 0.08 99.14 0.72
600 598.25 ± 0.32 99.71 0.62 596.24 ± 0.07 99.33 0.79
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a

Average of three independent determinations.

The selectivity results demonstrated that none of the investigated compounds produced significant interference under the optimized assay conditions. All potential interferents, including anthranilic acid, 3-hydroxykynurenine, tyrosine, phenylalanine, tryptophan, serotonin, lysine, ornithine, dopamine, adrenaline, and glucosamine, were evaluated at 200 ng mL−1. Fluorescence responses were measured at an excitation wavelength of 485 nm and an emission wavelength of 555 nm. As shown in Fig. 8, none of the investigated compounds generated appreciable fluorescence at the selected wavelength pair, and all recorded emission signals remained below 4% of the response obtained for KYN, confirming the excellent selectivity of the proposed method for plasma KYN determination.

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

Fig. 8

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3.8. Clinical application

Clinical application of the method revealed elevated plasma KYN in essential hypertension patients versus normotensive controls as presented in Table 4. Normotensive individuals exhibited a mean concentration of 375 ± 25 ng mL−1, whereas the hypertensive cohort demonstrated a marked increase to 510 ± 34 ng mL−1 (p < 0.001). Statistical assessment revealed that circulating KYN levels positively correlated with both systolic (r = 0.48, p = 0.006) and diastolic (r = 0.42, p = 0.018) blood pressure parameters, demonstrating a moderate yet highly meaningful linear association. These findings indicate that rising KYN levels are associated with increasing blood pressure, consistent with upregulated IDO pathway activity under chronic inflammatory and oxidative stress conditions. Persistent activation of the kynurenine pathway has been implicated in endothelial dysfunction through reduced NO bioavailability, enhanced oxidative stress, and vascular inflammation, all of which contribute to vascular remodeling and hypertensive vascular injury. Accordingly, the elevated plasma KYN concentrations observed in the present study may reflect these underlying pathophysiological processes, supporting the potential utility of KYN as a biomarker of hypertension-associated vascular dysfunction.

Table 4. Plasma KYN concentrations and their correlation with blood pressure parameters in normotensive controls and patients with essential hypertension.

Parameter Controls (n = 24) Hypertensive patients (n = 24) p-Value
Plasma KYN (ng mL−1, mean ± SD) 375 ± 25 510 ± 34 0.0002
Systolic blood pressure (mmHg, mean ± SD) 118 ± 9 152 ± 11 —
Diastolic blood pressure (mmHg, mean ± SD) 74 ± 6 92 ± 8 —
Correlation of KYN with SBP (r) — — 0.48 (p = 0.006)
Correlation of KYN with DBP (r) — — 0.42 (p = 0.018)
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Cross-validation demonstrated excellent agreement between the proposed fluorometric method and the reference HPLC-UV method,21 as summarized in Table 5. Passing–Bablok regression yielded a slope of 1.02 (95% CI: 0.97–1.07) and an intercept of −4.8 ng mL−1 (95% CI: −18.6 to 9.0 ng mL−1), indicating the absence of systematic bias. Bland–Altman analysis showed a mean difference of −2.3 ng mL−1, with 95% limits of agreement ranging from −21.4 to 16.8 ng mL−1, which were within clinically acceptable limits over the concentration range of 320–580 ng mL−1.

Table 5. Cross-validation of the proposed fluorometric method with the reference HPLC-UV method21 using 20 clinical plasma samplesa.

Statistical measure Result Interpretation
Number of sample pairs n = 20 (10 hypertensive + 10 normotensive) —
Concentration range analyzed (ng mL−1) 320–580 Covers physiological range for both groups
Passing–Bablok slope (95% CI) 1.02 (0.97–1.07) No proportional bias (CI includes 1.0)
Passing–Bablok intercept—ng mL−1 (95% CI) −4.8 (−18.6 to 9.0) No constant bias (CI includes 0)
Bland–Altman mean difference (ng mL−1) −2.3 Negligible systematic difference
Bland–Altman 95% limits of agreement (ng mL−1) −21.4 to +16.8 Within ±5% of mean KYN; clinically acceptable
Pearson r 0.991 (p < 0.001) Excellent correlation between methods
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a

Passing–Bablok regression: non-parametric method for method comparison; slope = 1 and intercept = 0 indicate no proportional or constant bias, respectively. Bland–Altman analysis: mean difference = systematic bias; 95% limits of agreement define the range within which 95% of individual differences fall. A mean difference of −2.3 ng mL−1 represents <0.6% of the mean KYN concentration in the study cohort (∼440 ng mL−1), confirming excellent agreement.

3.9. Performance comparison: current fluorometric assay vs. reported coumarin-based chemosensor for KYN

The proposed NBD-F fluorometric assay was compared with a previously reported coumarin-based chemosensor28 for KYN determination as summarized in Table 6. The NBD-F method showed superior sensitivity, applicability to biological samples, and clinical relevance. It exhibited a linear range of 100–800 ng mL−1 (≈0.49–3.9 µM) with an LOD of 10 ng mL−1 (≈0.049 µM). In comparison, the coumarin-based method provided a linear range of 1–20 µM (≈208–4160 ng mL−1) and an LOD of 0.7 µM (≈146 ng mL−1). Thus, the proposed method achieved approximately 14-fold higher sensitivity, attributed to the high fluorogenic efficiency of NBD-F and optimized derivatization conditions. Unlike the coumarin sensor, which was validated only in synthetic urine, the proposed method was successfully validated in human plasma using a simple protein precipitation and derivatization procedure with minimal matrix interference. Clinically, plasma KYN levels were significantly higher in patients with essential hypertension (510 ± 34 ng mL−1) than in healthy controls (375 ± 25 ng mL−1, p < 0.001), supporting the association between elevated KYN, increased IDO activity, and immune-metabolic dysregulation. The previous method did not include clinical validation or pathophysiological investigation.

Table 6. Comparison of the analytical performance and clinical applicability of the proposed NBD-F-based fluorometric method and the previously reported coumarin-based chemosensor assay for KYN determination.

Parameter Proposed NBD-F-based fluorescence method Previously reported coumarin-based chemo sensor28 Remarks
Analyte matrix Human plasma Synthetic urine Present method applicable to complex biological matrices
Derivatization/detection system NBD-F derivatization (fluorogenic reaction) 3-Formyl-4-(ethylthio)-7-(diethylamino)-coumarin chemosensor NBD-F shows higher fluorogenic efficiency
Linearity range 100–800 ng mL−1 (≈0.49–3.9 µM) 1–20 µM (≈208–4160 ng mL−1) NBD-F method demonstrates improved working range for physiological KYN levels
LOD (ng mL−1) 10 ng mL−1 (≈0.049 µM) 0.7 µM (≈146 ng mL−1) NBD-F method exhibits superior analytical sensitivity
Sample preparation Simple plasma protein precipitation and derivatization Synthetic urine spiking and direct detection NBD-F approach suitable for clinical plasma use
Matrix applicability Real human plasma (endogenous KYN) Synthetic urine (model samples) Demonstrates greater translational value
Clinical relevance Elevated plasma KYN in hypertension: 510 ± 34 vs. 375 ± 25 ng mL−1 (p < 0.001) Not evaluated Only current method establishes clinical association
Overall evaluation High sensitivity, simple operation, clinically validated Analytical feasibility only NBD-F method provides analytical and clinical advantages
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3.10. Limitations and future prospectives

The proposed NBD-F method provides high sensitivity, selectivity, and analytical robustness for accurate plasma KYN quantification at physiological concentrations. However, the derivatization step increases sample preparation time, limiting its suitability for fully automated, high-throughput analysis. Although the method showed low interference and excellent performance, validation was conducted in a single laboratory, and inter-laboratory reproducibility remains to be established. Clinically, the study was limited by a small, single-center cohort, reducing generalizability. In addition, no multivariable analysis was performed to account for potential confounders, including age, sex, BMI, and renal function. The study also included only patients with essential hypertension, excluding secondary hypertension and other cardiovascular diseases. Future multicenter studies with larger cohorts, longitudinal follow-up, and multivariable analyses are needed to confirm the clinical utility and generalizability of the method. Integration with multiplex fluorescence assays may further improve characterization of tryptophan pathway alterations in cardiovascular and inflammatory diseases.

4. Conclusion

A simple, sensitive, and validated fluorometric method for the determination of plasma kynurenine based on NBD-F derivatization was successfully developed. The method demonstrated excellent linearity, accuracy, precision, selectivity, robustness, and a low detection limit, while eliminating the need for chromatographic separation, thereby reducing analysis time and instrument requirements. Clinical application revealed significantly higher plasma kynurenine concentrations in patients with essential hypertension than in normotensive controls, together with positive correlations between kynurenine levels and blood pressure. These results support the association between kynurenine pathway activation and hypertension-related vascular dysfunction and highlight the potential of plasma kynurenine as a clinically relevant biomarker. Although further validation in larger multicenter cohorts is required, the proposed method provides a practical analytical platform for routine plasma kynurenine determination and future investigations of kynurenine pathway alterations in cardiovascular and inflammatory diseases.

Conflicts of interest

The authors declare no competing interests.

Supplementary Material

RA-016-D6RA04719J-s001

Acknowledgments

The authors extend their appreciation to the Deanship of Research and Graduate Studies at King Khalid University for funding this work through a Small Research Project under grant number RGP.1/87/46.

Data availability

The datasets used during the current study are included in the manuscript.

Supplementary information (SI) is available. See DOI: https://doi.org/10.1039/d6ra04719j.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

RA-016-D6RA04719J-s001
RA-016-D6RA04719J-s001.pdf (129.2KB, pdf)

Data Availability Statement

The datasets used during the current study are included in the manuscript.

Supplementary information (SI) is available. See DOI: https://doi.org/10.1039/d6ra04719j.

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