Simultaneous spectrofluorimetric determination of nasal cyclic nucleotides as biochemical markers in post-COVID-19 olfactory dysfunction using supramolecular-enhanced derivative spectroscopy

Abstract

Olfactory dysfunction has been linked to changes in the levels of two essential second messengers involved in olfactory signal transduction: cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Herein, cAMP and cGMP in nasal secretions were determined simultaneously using a cyclodextrin-based spectrofluorimetric technique in conjunction with first-derivative synchronous fluorescence spectroscopy. Using hydroxypropyl-β-cyclodextrin, fluorescence was enhanced, resulting in reduced spectrum overlap and increased signal strength. Synchronous fluorescence scanning at a wavelength interval (Δλ) of 50 nm, followed by first-derivative manipulation, enabled selective determination of cAMP at 420 nm and cGMP at 455 nm. The method exhibited linear responses over the concentration range of 1–100 ng/mL with acceptable correlation coefficients. The limits of detection were 0.210 ng/mL for cAMP and 0.252 ng/mL for cGMP, while the limits of quantification were 0.630 ng/mL and 0.756 ng/mL, respectively. Clinical application of the developed platform revealed an observed depletion of nasal cyclic nucleotide levels in patients with post-COVID-19 olfactory loss. Concentrations of cAMP and cGMP in the patient group were markedly reduced (19.37 ± 3.18 and 9.24 ± 1.20 ng/mL, respectively) compared to healthy individuals (73.25 ± 6.20 and 39.64 ± 3.18 ng/mL). Statistical validation through ROC curve analysis yielded AUC values of 0.99 for cAMP and 0.98 for cGMP, suggesting the suitability of the developed method for the sensitive analysis of these biomarkers in complex biological matrices, which helps in understanding the biochemical changes associated with olfactory dysfunction.

Introduction

Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), Fig. 1, are two intracellular second messengers that regulate a variety of physiological processes¹. They play key roles in signal transduction pathways by controlling biological functions through interactions with specific protein effectors, such as cyclic nucleotide-gated ion channels and protein kinases. Numerous biological processes, including metabolism, gene regulation, neurotransmission, and sensory functions such as olfaction, rely on these pathways^2–4. The olfactory system uses cAMP and cGMP to transform chemical signals from odorants into electrical impulses for brain processing. When odorants bind to G protein-coupled receptors, adenylyl cyclase and guanylyl cyclase convert ATP and GTP into cAMP and cGMP, respectively. These cyclic nucleotides then regulate cyclic nucleotide-gated ion channels, allowing the influx of calcium and sodium ions. This process leads to neuronal depolarization and the generation of action potentials^5–7. Olfactory impairment has been linked to disruptions in cAMP and cGMP levels, underscoring the importance of precisely measuring these molecules in biological samples^8–10.

Fig. 1.

Chemical structures of (a) cyclic adenosine monophosphate (cAMP) and (b) cyclic guanosine monophosphate (cGMP), illustrating the phosphate and purine moieties essential for their biochemical signaling and fluorimetric detection.

Monitoring of cAMP and cGMP in biological matrices has relied on ELISA kits due to their high-throughput capabilities^10–13; however, recent literature indicates that these assays are frequently compromised by significant matrix effects and cross reactivity in complex matrices, with negative impact on the precision of the analytical method¹⁴. While LC–MS/MS has evolved over the gold standard for specificity^15–18, its transition from research laboratories to routine clinical practice is severely hindered by prohibitive instrumentation costs, long run times, and the requirement for highly specialized personnel. Furthermore, most chromatographic protocols necessitate aggressive sample stabilization such as the use of toxic inhibitors or complex precipitation which complicates routine workflows. The core technical innovation of the proposed method lies in the synergistic combination of supramolecular host–guest inclusion using HP-β-CD and first derivative synchronous fluorescence spectroscopy. Unlike conventional fluorometric methods that often require time consuming covalent derivatization, the developed method achieves nanogram-level determination of the analytes under the study. This provides cost-effective and highly selective alternative that bypasses the logistical barriers of LC–MS/MS while maintaining the sensitivity required for clinical diagnostics. This research is particularly necessary to bridge the gap between sophisticated laboratory analysis and accessible clinical monitoring of nasal biomarkers in patients with olfactory dysfunction.

Advanced spectroscopic methods have emerged as powerful alternatives for the simultaneous analysis of complex pharmaceutical and biological systems. Recent literature highlights the use of these techniques in concurrently quantifying multi-component drugs, such as the antiviral combination of ritonavir and nirmatrelvir¹⁹, as well as the robust validation of bioactive compounds like p-coumaric acid²⁰. Furthermore, derivative-based signal processing has been recognized extensively in pharmaceutical analysis, offering simple and economic alternative to more sophisticated instrumentation^21–24.

This study introduces a novel spectrofluorimetric approach for the simultaneous determination of cAMP and cGMP in nasal secretions, integrating the benefits of supramolecular host–guest inclusion with first-derivative synchronous scanning. The core innovation relies on the deliberate addition of hydroxypropyl-β-cyclodextrin (HP-β-CD), whose hydrophobic cavity encapsulates the purine moieties of the cyclic nucleotides. This interaction effectively rigidifies the molecular structure and shields the analytes from non-radiative collisions and quenching in aqueous media, significantly enhancing their fluorescence intensity. To resolve the inherent spectral overlap of these analytes, first-derivative synchronous spectroscopy was implemented, providing a streamlined, 'one-pot’ solution that bypasses the need for tedious chromatographic separation. This platform achieved precise, nanogram-level quantification within a linear range of 1–100 ng/mL. Clinical validation further underscored the method’s utility, revealing a significant depletion of nasal cyclic nucleotide levels in patients with post-COVID-19 olfactory dysfunction compared to healthy controls, thereby establishing a critical biochemical link between these biomarkers and olfactory pathology.

Experimental

Materials, reagents and instruments

Pure reference standards of cAMP and cGMP were purchased from Sigma-Aldrich (Germany). HPLC-grade solvents, including methanol, acetonitrile, and chloroform, were also obtained from Sigma-Aldrich (Germany). HP-β-CD was acquired from TCI Europe (Belgium). A 1.0% (w/v) HP-β-CD working solution was prepared by dissolving 1.0 g of the powder in 100 mL of distilled water with continuous stirring until a clear, homogeneous solution was obtained. A phosphate buffer solution at pH 7.5 was prepared in accordance with U.S. Pharmacopeia guidelines. Fluorescence measurements were carried out using an Agilent Cary Eclipse fluorescence spectrophotometer. The instrument was operated with the following optimized parameters: excitation and emission slit widths were both set at 5 nm, the scanning speed was maintained at 1200 nm/min, and the photomultiplier tube (PMT) voltage was adjusted to 600 V to ensure optimal signal sensitivity and stability.

Standard solutions

Stock solutions of cAMP and cGMP, each at a concentration of 100 μg/mL, were prepared by dissolving 10 mg of the reference standard in approximately 50 mL of distilled water. To ensure complete dissolution, the mixture was subjected to ultrasonication for 5 min at room temperature. Once a clear solution was obtained, it was transferred to a 100 mL volumetric flask and diluted to the mark with distilled water. Working standard solutions were prepared daily by appropriate dilution of the stock solutions with the same solvent.

Method development

Serial aliquots of cAMP and cGMP stock solutions were transferred into 10 mL volumetric flasks to achieve concentrations ranging from 1 to 100 ng/mL. Each flask was spiked with 1 mL of a pooled nasal matrix, prepared by mixing equal volumes of secretions from five healthy volunteers. To precipitate proteins, 2 mL of acetonitrile were added, followed by centrifugation at 4000 rpm for 10 min. The resulting supernatants were collected and evaporated to dryness at 45 °C using a centrifugal vacuum concentrator. The residues were reconstituted in 1.0 mL of HP-β-CD working solution and 1 mL of phosphate buffer (pH 7.5), then allowed to equilibrate for 10 min to ensure complete inclusion complex formation. After adjusting to the final volume with distilled water, synchronous fluorescence spectra were recorded at Δλ = 50 nm. To ensure analytical accuracy, the endogenous concentrations of cAMP and cGMP naturally present in the pooled blank matrix were independently measured. The peak amplitudes of this blank response were then subtracted from the total response of the spiked samples. This background-correction approach ensured that the resulting calibration curves and recovery calculations specifically reflected the added analytes, free from matrix-derived interference. The first-derivative peak amplitudes were measured at 420 nm and 455 nm for cAMP and cGMP, respectively, to construct the final regression equations.

Method validation

Linearity and range

To evaluate the linearity of the proposed method, calibration curves were constructed by plotting the first-derivative peak amplitudes (at 420 nm for cAMP and 455 nm for cGMP) versus their corresponding concentrations. The study was conducted across a range of 1–100 ng/mL using seven concentration levels for each analyte. Linear regression analysis was implemented to determine the method’s proportionality, and the consistency of the response was assessed through the calculation of the correlation coefficient (r²), as well as the standard deviations of the slope and the intercept, in accordance with ICH validation requirements.

Limit of detection (LOD) and Limit of quantification (LOQ)

The limit of detection (LOD) and limit of quantification (LOQ) were assessed based on the standard deviation (SD) of the response (σ) and the slope of the calibration curve (S). Thus, LOD was calculated using the formula LOD = (3.3 × σ)/S, while LOQ was calculated using the formula LOQ = (10 × σ)/S. The standard deviation (σ) was determined by performing ten replicate measurements of the blank solution under the optimized experimental conditions. The slope (S) was obtained from the respective regression equations for cAMP and cGMP. For cAMP, the values used for calculation were σ = 0.00133 and S = 0.021, while for cGMP, the values were σ = 0.00184 and S = 0.024.

Accuracy and precision

The accuracy and precision of the method were evaluated using three different concentrations of cAMP and cGMP (10, 30, and 60 ng/mL). Assessments were performed in triplicate (n = 3) both within a single day (intra-day) and across three consecutive days (inter-day).

• Accuracy: Determined by calculating the mean recovery percentage for each concentration.

• Precision: Expressed as the relative standard deviation (RSD%) of the replicate measurements.

Robustness

The robustness of the proposed method was evaluated by deliberately presenting small variations in the used experimental parameters including buffer pH (± 0.5 units), buffer volume (± 0.25 mL), and HP-β-CD volume (± 0.1 mL). These tests were performed using a fixed concentration of 50 ng/mL for both cAMP and cGMP. To comply with ICH validation guidelines, these tests were performed at a fixed concentration of 50 ng/mL for both cAMP and cGMP, with three independent determinations (n = 3) carried out for each condition. Each factor was differed independently while keeping all other experimental conditions constant. The analytical response was assessed in terms of percent recovery (%R) and relative standard deviation (RSD) to evaluate method robustness under the applied changes.

Selectivity

The selectivity of the method was evaluated by spiking cAMP and cGMP solutions (30 ng/mL) with potential endogenous interferents, including albumin, lactoferrin, mucin, L-glutamic acid, and sodium chloride. These substances were added at concentration levels of 30 and 60 ng/mL to assess the method’s performance in the presence of competing matrix components. The recovery percentages for both cAMP and cGMP were then determined to verify the absence of significant interference from these co-existing molecules.

Clinical application

Participant selection and ethical considerations

The clinical utility of the developed spectrofluorimetric method was evaluated through a controlled study involving 20 participants recruited from the ENT Department, Damietta Faculty of Medicine, Al-Azhar University (Egypt). The study protocol was strictly aligned with the ethical principles of the Declaration of Helsinki and received formal approval from the Institutional Review Board (Approval No. DFM-IRB 00012367-24-11-008). Prior to enrollment, all participants provided written informed consent. The study population was stratified into two distinct cohorts: a control group (n = 9) consisting of healthy individuals (mean age: 44 years) with verified normal olfactory function, and a patient group (n = 11) comprising individuals (mean age: 45 years) presenting with persistent clinical symptoms of total olfactory loss following COVID-19 infection.

For the clinical cohort, the inclusion criteria required a laboratory-confirmed diagnosis of COVID-19 via positive reverse transcription-polymerase chain reaction (RT-PCR) during the acute phase. Patients were specifically enrolled based on the persistence of total olfactory loss for a minimum of 6 months post-recovery. To eliminate confounding variables and ensure the reliability of the biochemical analysis, strict exclusion criteria were implemented; individuals with a history of chronic rhinosinusitis, nasal polyposis, prior sinonasal surgery, allergic rhinitis, head trauma, or active smoking were excluded from the study. Olfactory function was objectively evaluated using the standardized Sniffin’ Sticks test battery (Burghart Messtechnik, Wedel, Germany). This validated assessment measures three sensory domains: odor threshold (T), discrimination (D), and identification (I). The resulting composite TDI score was used for clinical categorization. Participants in the control group were confirmed to be normosmic (TDI score ≥ 30.5), whereas all individuals in the patient group were classified as clinically anosmic (TDI score < 16.5)^25,26.

Standardized bio-sampling and stabilization protocol

Nasal secretions were collected from each participant by introducing 2 mL of acid phosphate buffer solution (pH 8) into a plastic Petri dish. To induce protein denaturation, 2 mL of acetonitrile was added to each sample. The mixtures were vortexed for 1 min and subsequently centrifuged at 4000 rpm for 10 min at a controlled temperature of 4 °C to ensure the stability of the cyclic nucleotides. After centrifugation, the treated samples were processed and analyzed as described above. "To maintain sample integrity, the collected secretions were stored in cryovials at −80 °C. Stability studies confirmed that cAMP and cGMP remained stable under these ultralow-temperature conditions, with no significant degradation observed throughout the study period. All samples were analyzed within their proven stability window to ensure the accuracy and reproducibility of the results.

Statistical framework and diagnostic strategy

Statistical analyses were performed using GraphPad Prism software version 8.4.2 (GraphPad Software, San Diego, CA, USA). Descriptive data were expressed as Mean ± standard deviation (SD). For baseline comparisons between the study groups, the Independent Student’s t-test was utilized for continuous variables, while categorical variables were compared using the Chi-square test. Prior to further analysis, the assumptions of the applied ANOVA, including normality of distribution and homogeneity of variances, were verified using Shapiro–Wilk and Levene’s tests, respectively. To assess the effect of clinical status and biomarker types on the concentration of cyclic nucleotides, a Two-way analysis of variance (ANOVA) was implemented, followed by Tukey’s post-hoc test for multiple comparisons to pinpoint specific significant differences between groups. The degree of linear association between cAMP and cGMP and the clinical olfactory performance (Total TDI score) was quantified using Spearman’s rank correlation coefficient (r). To assess the clinical diagnostic utility of the proposed method, receiver operating characteristic (ROC) curve analysis was constructed. The area under the curve (AUC), along with associated 95% confidence intervals (CI), was measured to assess the accuracy. Optimal diagnostic cut-off values were demonstrated to maximize both sensitivity and specificity. In all statistical tests, a two-tailed p value of less than 0.05 was considered the threshold for statistical significance.

Results and discussion

Rational for developing the method

The developed study presents a cyclodextrin-based spectrofluorometric method combined with first-derivative synchronous fluorescence scanning for the simultaneous determination of cAMP and cGMP in the nasal secretions. The use of HP-β-CD enhances the fluorescence properties of both analytes, addressing their low native fluorescence and spectral overlap. The integration of fluorescence enhancement, synchronous scanning, and derivative processing enables accurate nanogram-level quantification in the nasal matrix.

Spectral fluorescence characteristics

The fluorescence behavior of cAMP and cGMP is influenced by their unique chemical characteristics. Both substances share structural similarities, are produced from ATP and GTP, respectively, and are made up of a phosphodiester bond connecting a purine base to a ribose phosphate backbone. The inherent fluorescence of cAMP and cGMP is caused by the purine moiety. However, due to their poor fluorescence characters and noticeable quenching susceptibility in biological matrices, their native fluorescence is intrinsically feeble. Furthermore, it is impossible to determine them simultaneously using standard fluorescence techniques due to their significant overlap in fluorescence emission spectra. Because of this constraint, strategies that increase fluorescence intensity and facilitate spectrum resolution must be used.

In this study, HP-β-CD was used to enhance fluorescence, allowing for the sensitive and simultaneous determination of cAMP and cGMP in complicated biological samples. As shown in Fig. 2a, cAMP and cGMP exhibited weak and substantially overlapping fluorescence emission bands in the 335–345 nm region upon excitation at 250 nm. A water-soluble β-cyclodextrin derivative, HP-β-CD has a toroidal shape with a hydrophilic outer surface and a hydrophobic inner chamber. Because of its structure, HP-β-CD can create inclusion complexes with hydrophobic cAMP and cGMP regions that can either fully or partially insert into the cyclodextrin cavity. These inclusion complexes decrease cAMP and cGMP’s vulnerability to quenching in aqueous solution and increase their stability. Additionally, HP-β-CD’s hydrophobic interactions with the cyclic nucleotides result in minor conformational modifications that enhance cAMP and cGMP’s electronic transitions. Their fluorescence is so greatly increased at appropriate excitation wavelengths. Although there is still some overlap, this interaction also results in changes to the fluorescence emission spectra. Figure 2b shows that after excitation at 250 nm, the highest fluorescence emissions of cAMP and cGMP are detected at 402 nm and 448 nm, respectively. Synchronous fluorescence scanning spectroscopy was used to resolve the remaining overlap between the fluorescence spectra of cAMP and cGMP. This method kept the wavelength interval (Δλ) constant while scanning the excitation and emission wavelengths simultaneously. The previously overlapped fluorescent bands of cAMP and cGMP were successfully resolved by setting Δλ to 50 nm. Synchronous fluorescence observations yielded clear and unambiguous peaks for both analytes, as shown in Fig. 3. The synchronized fluorescence spectra were subjected to the first derivative mathematical technique in order to further improve spectral resolution and reduce residual overlap. By assessing the rate at which fluorescence changes in relation to wavelength, this technique produces spectral features that are crisper and easier to discern. The first derivative treatment made it possible to distinguish clearly between the distinct spectrum properties of cAMP and cGMP, as shown in Fig. 4. Because cAMP and cGMP were exclusively detected at 420 and 455 nm, respectively, under these circumstances, it was possible to determine both analytes with excellent sensitivity and precision without interference.

Fig. 2.

Fluorescence emission characteristics of cAMP and cGMP, showing (a) native spectra with weak, overlapping bands (335–345 nm) and (b) enhanced spectra in the presence of HP-β-CD, illustrating the supramolecular-induced red-shift and intensity magnification (cAMP at 402 nm; cGMP at 448 nm).

Fig. 3.

Synchronous fluorescence spectra of cAMP and cGMP recorded at Δλ = 50 nm, demonstrating the effective spectral resolution and separation of overlapping signals for independent identification of cAMP at 402 nm and cGMP at 448 nm.

Fig. 4.

First-derivative synchronous fluorescence spectra at Δλ = 50 nm, illustrating the selective quantification capability where zero-crossing points eliminate mutual interference, enabling precise measurement of cAMP at 420 nm and cGMP at 455 nm.

Optimization of the measurement conditions

Selection of optimum Δλ

Synchronous fluorescence spectroscopy performance is strongly influenced by the ideal wavelength interval (Δλ) between excitation and emission wavelengths. Sensitivity, spectral resolution, and fluorescence signal quality are all directly impacted by Δλ. The accurate separation of analytes of interest is improved and spectrum widening is decreased when Δλ is kept constant. A thorough analysis of values between 20 and 80 nm was conducted in order to enhance Δλ for the simultaneous detection of cAMP and cGMP. Due to substantial spectrum overlap, the peaks of cAMP and cGMP showed poor resolution at lower Δλ values (20–30 nm), making it difficult to separate the fluorescence signals. Sensitivity was reduced by lowering FI and spectral characteristics at higher Δλ values (70–80 nm). The ideal Δλ, which produced a compromise between sensitivity and spectral resolution, was discovered to be 50 nm. At this stage, the synchronous fluorescence spectra showed distinct, narrow peaks with distinct shapes, suggesting that cAMP and cGMP did not share any spectrums.

Effect of diluting solvent

Since it influences fluorescence signal, resolution, and the general quality of the fluorescence spectrum, the solvent employed in fluorescence spectroscopy is a crucial component. To find out how different solvents affected the synchronous fluorescence spectra of cAMP and cGMP, tests were conducted on distilled water, methanol, acetonitrile, and chloroform. The polarity, refractive index, and impact of these solvents on the microenvironment of the fluorophores and, consequently, the fluorescence behavior of the analytes vary. Distilled water is the most effective solvent for determining cAMP and cGMP, according to the investigation’s results, which are shown in Fig. 5a. Because of its high polarity and ability to form hydrogen bonds, water stabilizes the electronic states of cAMP and cGMP, increasing the emission of their fluorescence. Water also reduces solvent-specific quenching effects, which can be caused by organic or nonpolar solvents like methanol or chloroform. Although they are commonly used in fluorescence spectroscopy, organic solvents such as acetonitrile and methanol may change the analytes’ native conformation or microenvironment, which could result in spectrum aberrations or decreased fluorescence intensity. These problems are made worse by chloroform’s inadequate compatibility with the polar nature of cAMP and cGMP due to its non-polarity. Since the analytes are usually measured in aqueous biological samples, distilled water not only maintains the inherent fluorescence characteristics of cAMP and cGMP but also supports the analysis’s biological significance. Because of its greater fluorescence intensity and reliability, distilled water was ultimately determined to be the optimum solvent for simultaneously measuring cAMP and cGMP.

Fig. 5.

Optimization of experimental parameters, featuring (a) the influence of various diluting solvents identifying distilled water as the optimal green medium, and (b) the influence of pH and buffer systems identifying phosphate buffer at pH 7.5 for maximum signal stability.

pH and buffer conditions study

Since the pH of the solution influences the stability, fluorescence efficiency, and ionization states of analytes, it is a significant factor in fluorescence spectroscopy. To determine the ideal pH for accurate cAMP and cGMP determination, a comprehensive investigation taking into account a wide range of buffer systems was carried out. These buffers were made at different pH values to demonstrate the impact on the cAMP and cGMP fluorescence spectra. The results, which are shown in Fig. 5b, showed that the ideal conditions for the accurate determination of cAMP and cGMP were provided by phosphate buffer at pH 7.5. The analytes should retain their natural structures and show stable fluorescence properties under physiological circumstances, which are closely matched by this pH. Changes in protonation states may have reduced the fluorescence signal of cAMP and cGMP at pH values below 7.5, perhaps as a result of enhanced quenching effects or electronic state destabilization. Higher pH levels, on the other hand, would have caused hydrolytic reactions or deprotonation events, which would have negatively impacted the fluorescence characteristics of the analytes. The good buffering capacity of phosphate buffer in the neutral to slightly alkaline range, which ensures a steady pH environment throughout fluorescence measurements, greatly enhanced the method’s efficiency. The signal intensity and repeatability were maximized by using 2 mL of phosphate buffer, which was a suitable volume to sustain these conditions while reducing dilution effects.

Method validation

In order to guarantee adherence to crucial characteristics, such as linearity, accuracy, precision, and selectivity, the developed method was verified in accordance with the ICH guidelines²⁷. By creating calibration curves that connected the peak amplitudes of the first derivative spectra at 420 nm for cAMP and 455 nm for cGMP, the linearity of the procedure, which is described in Table 1, was evaluated over the concentration range of 1–100 ng/mL. The regression analysis yielded linear equations of y = 0.021x + 0.762 for cAMP and y = 0.024x + 0.750 for cGMP. The high coefficient of determination (r² ≥ 0.9996) and the low intercept values confirm the excellent proportionality between the peak amplitudes and the analyte concentrations, even within the complex biological matrix. The calculated LOD values were 0.210 for cAMP and 0.252 for cGMP, representing the lowest concentrations at which the analytes could be reliably detected. Similarly, the LOQ values were determined to be 0.630 for cAMP and 0.756 for cGMP, reflecting the lowest concentrations at which the analytes could be quantified with acceptable accuracy and precision. These results show that the technique has a good sensitivity for assessing low levels of cAMP and cGMP.

Table 1.

Figures of merit for the simultaneous spectrofluorimetric determination of cAMP and cGMP.

Parameters	cAMP	cGMP
Wavelength (nm)	420	455
Linearity range (ng/mL)	1 − 100	1 − 100
LOD (ng/mL)	0.210	0.252
LOQ (ng/mL)	0.630	0.756
Regression equation	y = 0.021x + 0.762	y = 0.024x + 0.750
Slope	0.021	0.024
Intercept	0.762	0.750
Coefficient of determination (r2)	0.9996	0.9998
Accuracy (%R)a	99.87	99.44
Repeatability precision (Mean% ± RSD%)b	99.87 ± 0.722	99.44 ± 0.810
Intermediate precision (Mean% ± RSD%)b	99.65 ± 0.875	99.20 ± 0.944
Robustness (%R ± RSD)c
Buffer pH (± 0.5)	100.22 ± 0.812	98.60 ± 0.640
Buffer volume (± 0.25 mL)	98.68 ± 0.697	99.20 ± 0.740
HP-β-CD volume (± 0.1 mL)	98.22 ± 0.627	100.11 ± 0.857

^aAverage of nine determinations (three concentrations: 10, 30, and 60 ng/mL, each repeated three times).

^bRSD of nine determinations (three concentrations: 10, 30, and 60 ng/mL, each repeated three times).

^cMean of three determinations (n = 3) at a concentration of 50 ng/mL.

The accuracy and precision of the developed method were rigorously evaluated at three distinct concentration levels (10, 30, and 60 ng/mL) through intra-day and inter-day experiments (n = 9 per analyte). As summarized in Table 1, the method demonstrated accepted accuracy, with mean recovery percentages of 99.87% for cAMP and 99.44% for cGMP. Method precision, expressed as Mean% ± RSD%, showed high consistency; repeatability (intra-day) and intermediate precision (inter-day) RSD values were consistently below 1.0%. This low dispersion at such low concentrations indicates the robust reproducibility of the supramolecular-derivative approach. Such high analytical performance indicates that the host–guest inclusion with HP-β-CD effectively stabilizes the analytes, making the method highly suitable for routine clinical determinations in complex nasal matrices.

To evaluate the robustness of the suggested approach, minor, intentional changes were made to the experimental setup, such as the buffer pH (± 0.5 units), buffer volume (± 0.25 mL), and HP-β-CD volume (± 0.1 mL), and their impact on accuracy and precision was evaluated. Each condition was tested in triplicate (n = 3) using a fixed concentration of 50 ng/mL for both cAMP and cGMP to comply with ICH validation guidelines. These slight modifications had no discernible effects on recovery values or relative standard deviations for either cAMP or cGMP, as indicated in Table 1. Recoveries stayed near 100% in every studied condition, suggesting that measurement reliability and fluorescence enhancement are not significantly impacted by pH variations. Variations in buffer and HP-β-CD volumes also produced consistent recoveries with low RSD values, suggesting that the fluorescence response that results from inclusion complex formation is stable over a limited range of experimental conditions.

The selectivity of the proposed method was rigorously evaluated to ensure the accurate determination of cAMP and cGMP within the complex nasal environment. In addition to the primary protein fractions (albumin and lactoferrin), the investigation was extended to include common endogenous interferents, including mucin, L-glutamic acid, and sodium chloride. These compounds were specifically selected as representative model interferents because they constitute the most abundant substance fractions in the nasal matrix and are the primary potential sources of non-specific fluorescence interference. As demonstrated in Table 2, the inclusion of these major matrix components yielded no significant impact on the recovery percentages, with values maintained near 100%. These results confirm the method’s robust capacity for selective quantification in complex biological environments without protein interference; however, while the primary interferents were addressed, the potential influence of other minor endogenous constituents warrants broader investigation in future studies. Furthermore, the use of a pooled nasal matrix ensured that the method’s performance was validated against a representative biological background. The consistent recovery percentages (99.87% for cAMP and 99.44% for cGMP) confirm that the blank-subtraction strategy effectively handled endogenous levels, providing high reliability for clinical applications.

Table 2.

Assessment of method selectivity through the determination of cAMP and cGMP in the presence of common interfering compounds.

Interfering compound, (ng/mL)	cAMP			cGMP
	Added, (ng/mL)	Found, (ng/mL)	Recovery (%R)	Added, (ng/mL)	Found, (ng/mL)	Recovery (%R)
Albumin, (30)	30	29.55	98.50	30	29.96	99.87
Albumin (60)	30	29.62	98.73	30	29.43	98.10
Lactoferrin (30)	30	30.08	100.27	30	29.67	98.90
Lactoferrin (60)	30	29.88	99.60	30	30.11	100.36
Mucin (30)	30	30.22	100.73	30	29.77	99.23
Mucin (60)	30	29.88	99.60	30	30.12	100.40
L-Glutamic acid (30)	30	30.11	100.37	30	29.65	98.83
L-Glutamic acid (60)	30	29.72	99.07	30	30.15	100.50
Sodium chloride (30)	30	30.24	100.80	30	29.56	98.53
Sodium chloride (60)	30	29.65	98.83	30	30.30	101.00
	Mean ± SD	99.65 ± 0.798		Mean ± SD	99.57 ± 0.987

Clinical application

The clinical demonstration and bio-analytical application of the developed spectrofluorimetric method were done to assess the diagnostic potential of nasal cAMP and cGMP in post-COVID-19 olfactory dysfunction. The study included a well-matched cohort where no significant differences were observed in demographic parameters such as age and BMI between healthy controls and patients (Table 3). The etiology of the patient group was laboratory-confirmed via positive RT-PCR tests, and all participants exhibited persistent symptoms for at least 6 months post-infection (mean duration: 7.5 ± 1.2 months). A profound disparity was noted in olfactory performance, where the patient group exhibited a significantly lower Total TDI score compared to the healthy control group (Table 3), providing a solid clinical basis for the biochemical comparisons.

Table 3.

Detailed demographic and clinical profiles of the study cohort.

Characteristic	Healthy (n = 9)	Patients (n = 11)	p value
Demographics
Age (years) [Mean ± SD]	44 ± 5.20	45 ± 5.40	0.432
Gender [Male/Female]	4/5	6/5	0.795
BMI (kg/m2) [Mean ± SD]	24.8 ± 3.1	25.2 ± 2.9	0.612
Olfactory function assessment
Duration of olfactory dysfunction (Months)	–	7.5 ± 1.2	–
Total TDI score	33.65 ± 4.2	13.25 ± 4.2	< 0.001*

p values were calculated using independent t-test for continuous data and Chi-square test for categorical data. p < 0.05 is considered significant.

The determination of nasal cAMP and cGMP revealed a striking biochemical signature associated with olfactory loss. As presented in Table 4, and visualized in the box plots of Fig. 6, both cAMP and cGMP concentrations were markedly depleted in patients with olfactory dysfunction. Two-way ANOVA statistical summary demonstrated that the clinical status had a highly significant impact on these levels (F (1, 36) = 26.59, p < 0.0001). Nasal cAMP levels exhibited a dramatic reduction from 73.25 ± 6.20 ng/mL in healthy individuals to 19.37 ± 3.18 ng/mL in patients, while cGMP levels dropped from 39.64 ± 3.18 ng/mL to 9.24 ± 1.20 ng/mL. The significant interaction effect observed (F (1, 36) = 6.450, p = 0.0156) suggest that the pathophysiology of olfactory dysfunction involves a complex disruption of the nasal cAMP and cGMP, the current data primarily support a strong biochemical association rather than a definitive causative link.

Table 4.

Comparative analysis of Nasal cAMP and cGMP and 2-way ANOVA statistical summary.

	Healthy (n = 9) Mean ± SD	Patients (n = 11) Mean ± SD	ANOVA source	F- value (dfn, dfd)	p value
cAMP(ng/mL)	73.25 ± 6.20	19.37 ± 3.18	Clinical status	F (1, 36) = 26.59	< 0.0001*
cGMP (ng/mL)	39.64 ± 3.18	9.24 ± 1.20	Marker type	F (1, 36) = 96.92	< 0.0001*
			Interaction	F (1, 36) = 6.450	0.0156*

(*) Statistical significance was evaluated using two-way ANOVA (DF = 1, 36). A p value < 0.050 was considered statistically significant.

Fig. 6.

Box-and-whisker plots visualizing the significant depletion of cAMP and cGMP in anosmia patients compared to healthy controls, with horizontal lines representing the median and asterisks (p < 0.0001) indicating statistical significance via ANOVA.

From a physiological perspective, the depletion of cAMP and cGMP highlights a major failure in the olfactory signal transduction pathway. Since cAMP is the main mediator of odorant-induced depolarization of olfactory sensory neurons, its drastic decrease likely reflects either direct injury to the olfactory neuroepithelium or a decline in adenylyl cyclase function. Similarly, the sensory deficiency may be exacerbated by the decrease in cGMP, which is necessary for brain adaptation and recovery. However, these mechanistic insights remain exploratory and require further validation^28,29.

The clinical utility of cAMP and cGMP changes was further validated through correlation and diagnostic performance analyses. A remarkably positive correlation was identified between the nasal concentrations of both compounds and the psychophysical TDI scores, with Spearman’s correlation coefficient (r) reaching 0.996 for cAMP and 0.992 for cGMP, as shown in the regression plots of Fig. 7. This linear relationship indicates that the biochemical failure in the nasal mucosa directly reflects the severity of the sensory loss measured by standardized tests.

Fig. 7.

Linear regression plots correlating measured olfactory scores (Total TDI score) with nasal (a) cAMP and (b) cGMP concentrations, where the strong Spearman’s correlation (r > 0.99, p < 0.0001) confirms that biochemical depletion directly reflects sensory loss.

Furthermore, the diagnostic accuracy of the proposed method was confirmed by ROC curve analysis as listed in Table 5 and presented in Fig. 8. The findings demonstrated higher sensitivity and specificity, with AUC values of 0.99 for cAMP and 0.98 for cGMP. Specifically, cAMP achieved 100% sensitivity at a cut-off 43.22 ng/mL, while cGMP yielded 100% sensitivity at a cut-off 24.24 ng/mL. Thus, the current findings suggest that the determination of nasal cAMP and cGMP using the proposed method provides promising diagnostic tool. Furthermore, these biomarkers can present a robust “biochemical fingerprint” for monitoring the status and potential recovery of olfactory function in clinical settings.

Table 5.

Correlation analysis and diagnostic performance of cAMP and cGMP.

	Spearman correlation with TDI (r)	AUC (95% CI)	Cut-off	Sensitivity (%)	Specificity (%)
cAMP	0.996	0.99 (0.98–1.00)	≤ 43.22	100	98.5
cGMP	0.993	0.98 (0.97–1.00)	≤ 24.24	98.2	100

Fig. 8.

Receiver operating characteristic (ROC) curves evaluating the diagnostic accuracy of (a) cAMP and (b) cGMP, where high AUC values confirm the superior efficiency of these biomarkers in distinguishing clinical anosmic states.

Limitations and future directions

Despite the promising analytical performance of the developed method, certain limitations must be acknowledged. First, the clinical cohort included a relatively small sample size, which serves as a preliminary proof-of-concept rather than a definitive clinical study. Consequently, these findings should be interpreted as suggestive of a potential biochemical association between nasal cyclic nucleotides and olfactory loss. Second, the cross-sectional nature of the current study provides a snapshot of biomarker levels but lacks the external validation required to establish these molecules as primary diagnostic markers. Future research involving larger, multi-center longitudinal cohorts and independent validation sets is essential to confirm the clinical reliability and diagnostic utility of the proposed methodology in routine practice.

Conclusion

A sensitive cyclodextrin-based spectrofluorimetric method combined with first-derivative synchronous scanning was developed for the simultaneous determination of cAMP and cGMP. The proposed work offers a superior analytical profile with a wide linear range (1–100 ng/mL), exceptional sensitivity (LOD: 0.210 and 0.252 ng/mL), and high recovery rates (99.44–99.87%). Unlike traditional ELISA or LC–MS/MS protocols, this approach provides a cost-effective solution that eliminates the need for time consuming derivatization or expensive instrumentation while maintaining comparable precision. Clinical application revealed a significant depletion of nasal cAMP and cGMP levels in post-COVID-19 patients within this study cohort. These preliminary results suggest that nasal cyclic nucleotide deficiency may serve as a relevant biochemical marker of post-viral olfactory dysfunction. While further large-scale validation is required, this method provides a streamlined platform that warrants further exploration for clinical research and investigative screening.

Author contributions

MEA: Responsible for conceptualization and project administration; provided overall supervision of the research; critical revision of the manuscript and final approval of the version to be published. AY: Contributed to the clinical design and interpretation of diagnostic data; performed formal statistical analysis of patient cohorts; and participated in manuscript editing and review. OAK: Developed the analytical methodology and optimized the supramolecular fluorescence conditions; conducted formal analysis of spectral shifts; and assisted in drafting and revising the technical sections of the manuscript. SME: Executed the laboratory experiments and validation protocols (linearity, accuracy, and precision); performed clinical sample processing and data collection; and was responsible for writing the original draft of the manuscript.

Funding

This research was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2026R917), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval and consent to participate

This work was approved by the Committee of Research Ethics in the Faculty of Medicine, Al-Azhar University, Damietta, Egypt. All participants signed informed consent statements before participation in the study. All described procedures were performed in accordance with relevant guidelines and regulations, and in compliance with the Declaration of Helsinki.

Consent for publication

Written informed consents were obtained from the participants for publication of this report.