The following products were used: tryptamine (98%, w/w), sodium hydroxide (98%, w/w), hydrochloric acid (37%, w/w), glacial trichloroacetic acid (99%, w/w), and glacial acetic acid (99%, w/w). We also used other biogenic amines such as dopamine (DOP), tyramine (TYR), cadaverine (CAD), spermine (SPM), spermidine (SPD), agmatine (AGM), putrescine (PUT), and histamine (HIS) to study the interferences with tryptamine (TRYP). As solvents, we used distilled water, methanol (MeOH), acetonitrile (ACN) and ethanol (EtOH). All these products and solvents were of analytical grade and were provided by Sigma-Aldrich.

Fluorescence spectra were recorded using a Perkin Elmer LS-45 spectrofluorometer interfaced with a computer equipped with FL-Winlab software. For weighing, we used a Sartorius precision balance with an accuracy of 0.1 mg. A 100 to 1000 μL micropipette, flasks and beakers with varying volumes from 5 to 100 mL, a Consort C6010 pH meter, and a Thermo Scientific SL16R centrifuge were used. The software programs OriginPro 8.5 and ChemDraw Ultra 8.0 were helpful for data processing and molecular schematic drawings, respectively. During all experiments, a quartz cuvette with five polished faces was chosen, and for the spectrofluorometer, we set the slit to 10 nm and the voltage to 600 volts.

A stock solution of tryptamine with a concentration of 10⁻² M was prepared in a 50 mL flask using methanol. From this stock solution, dilutions were made to obtain desired concentrations for the working solutions. The solutions were protected from light with aluminum foil and stored in a refrigerator.

Canned fish (sardines in vegetable oil) and white tuna in water (albacore), purchased from a local supermarket, were stored in a freezer after opening. However, to prevent the risk of biogenic amine levels evolving, all samples purchased were kept in a freezer.

We applied the solid-phase extraction (SPE) procedure to recover tryptamine from the canned fish. After grinding, 10 g of the ground material was mixed with 80% pure methanol and 20% distilled water in a 100 mL volumetric flask. The mixture was homogenized for 10 minutes using a magnetic stirrer. The homogenate was then centrifuged at 4000 rpm for 5 minutes at room temperature. The supernatant was decanted, filtered and stored in the refrigerator until use.

Tryptamine is a biogenic amine that exhibits natural fluorescence. We examined the excitation and emission spectra of a 10⁻⁷ M tryptamine solution in both aqueous and organic media ( Figure 1 ). The resulting curves were well-resolved. The excitation spectra were characterized by two peaks, one at 216 nm and the other at 278 nm, while the emission spectrum was characterized by a single peak, with a maximum between 352 nm and 371 nm. Figure 1 shows that when transitioning from an aqueous medium to ethanol, there was a hypsochromic shift in the emission wavelength, accompanied by an increase in fluorescence intensity. This can be explained by the fact that tryptamine was highly soluble in ethanol.

Using a 1 M HCl solution and a 1 M NaOH solution, we adjusted the desired pH value between 1 and 13 for a 10⁻⁷ M aqueous tryptamine solution. We recorded the excitation and emission spectra for each pH value ( Figure 2(A) ). This figure shows that pH variation does not affect the shape of the emission spectrum. However, there was a bathochromic shift of the band when transitioning from acidic to basic conditions. This is not surprising, as it has already been proven that pH is a factor that strongly affects the fluorescence signal of organic compounds in aqueous media [40] [41] . Additionally, protonation or deprotonation of functional groups profoundly modifies the fluorophores, sometimes altering the shape of excitation or emission spectra and the fluorescence intensity.

Regarding fluorescence intensity ( Figure 2(B) ), in acidic conditions, there is an increase in fluorescence intensity up to pH 4, followed by a slight decrease in intensity until pH 7. In basic conditions, an increase in intensity was observed up to pH 10. Beyond this pH value, a sharp decrease in fluorescence intensity was noted, reaching pH 13. At pH 10, the intensity is higher compared to other pH values on the curve ( Figure 2(B) ). Therefore, pH 10 was chosen as the optimal pH for the subsequent analytical work in aqueous media.

For each temperature value set between 25˚C and 80˚C, we recorded the excitation and emission fluorescence curves of a 10⁻⁷ M aqueous tryptamine solution at regular intervals of 5˚C or 10˚C. The results showed that increasing the temperature does not affect either the maxima of the excitation and emission wavelengths or the shape of the spectra ( Figure 3(A) ). However, we observed that fluorescence intensity decreases as the temperature increases ( Figure 3(B) ). We can concluded that tryptamine fluorescence in water was inhibited at higher temperatures. This can be explained by an increase in thermal agitation and a higher number of collisions between solvent molecules, which may lead to a decrease in fluorescence quantum yield and fluorescence lifetime, resulting from a greater efficiency of non-radiative processes [42] .

To gain more insight into the stability of tryptamine in aqueous media, we studied the kinetics by monitoring the evolution of the fluorescence signal over time at the analytical excitation (277 nm) and emission (366 nm) wavelengths ( Figure 4 ). The results of this study showed that tryptamine is relatively stable at the optimal pH.

To assess the effectiveness of the proposed method, the analytical performance was determined under optimal conditions in different solvents, namely water (pH 10), acetonitrile, methanol, and ethyl acetate. Linear correlations were observed in all solvents, with correlation coefficients (r²) ranging from 0.9977 to 0.9996. These correlation coefficients, which are close to unity, indicate the high precision of our measurements ( Figure 5 ).

From the calibration curves, the analytical parameters, including the detection limits (LOD), quantification limits (LOQ) and relative standard deviations (RSD), were determined ( Table 1 ).

^aExcitation (𝜆_ex) and emission (𝜆_em) wavelengths, ^bLinear correlation coefficients, ^cLinearity range, ^dLOD = Limit of detection, defined as the analyte concentration giving a signal-to-noise ratio (S/N) equal to 3 (IUPAC criterion), ^eLOQ = Limit of Quantification, defined as the analyte concentration giving a signal-to-noise (S/N) ratio of 10 (IUPAC criterion), ^fRelative Standard Deviation (n = 6).

From the obtained linear calibration curves, we determined the analytical parameters ( Table 1 ). The relative standard deviations (RSD) ranged from 0.56% to 5.56%, depending on the solvent. These values demonstrate the good reproducibility of our measurements. The limits of detection (LOD) and limits of quantification (LOQ) vary from 0.09 ng/mL to 1.16 ng/mL and from 0.30 ng/mL to 3.87 ng/mL, respectively. These low limits highlight the high sensitivity of the method. These values indicate that this method can be applied to the analysis of trace amounts of tryptamine in food products.

The direct fluorimetric method we developed has detection limits lower than those reported by authors using various methods, such as ultrasonic-assisted dispersive liquid-liquid microextraction (UA-DLLME) combined with high-performance liquid chromatography (HPLC) with fluorescence detection (FLD) (LOD = 3.2 ng/mL) [43] , capillary electrophoresis with UV detection (500 ng/mL) [44] , LPME-HPLC with UV detection (LOD = 20 ng/mL) [45] , DLLME-SFO-LC with UV detection (LOD = 5 ng/mL) [46] and DLLME-GC with mass spectrometry (LOD = 1.7 ng/mL) [36] . Therefore, the LOD and LOQ values obtained in this study are very low, suggesting that direct fluorescence can be considered an appropriate and sensitive method for determining tryptamine in canned fish.

We recorded the excitation and emission spectra of tryptamine in the standard solution and in the canned sardines in vegetable oil and canned white tuna samples ( Figure 6 ). These spectra showed no spectral difference in terms of shape or wavelength. Therefore, we can conclude that not only was tryptamine present in the fish, but methanol proved to be more effective in extracting tryptamine from fish than trichloroacetic acid.

To determine the amount of tryptamine in canned fish, the standard addition curve was established. This curve was nearly parallel to the tryptamine calibration curve ( Figure 7 ). This good parallelism indicated that the matrix effect was very negligible in all our measurements. From this curve, the recovery rate of tryptamine (%R) was determined using the following equation:

$\%R=\frac{C_t}{C_0+C_a}\times 100$

In this formulation, C_t represents the concentration of tyramine found using the calibration line, C_a is the added concentration, and C₀ is the blank concentration.

^a: Added concentration, ^b: Found concentration, ^c: Percent recovery, ^d: Recovery interval, ^e: Relative standard deviation.

In all the samples, satisfactory recovery percentages, ranging from 94.35% to 107.14%, were found ( Table 2 ). These values, close to 100%, demonstrated the effectiveness of the extraction method. Similarly, the very low relative standard deviations (RSD), ranging from 0.8% to 1.42%, show the good reproducibility of the measurements. The values obtained were therefore in accordance with international standards for analytical method validation [47] .

From the standard addition curves, the mass concentration (C₀) of tryptamine in each fish sample was determined. The determination of C₀ allowed us to calculate the mass percentage of tryptamine in the different canned fish samples. Taking dilution effects into account, knowing the concentration C₀ enables us to determine the mass rates of tryptamine in milligrams per kilogram of fish (mg/kg) for each sample studied. For both types of samples, the mass rates of tryptamine were obtained as follows: 86.0 mg/kg for canned sardines in vegetable oil and 39.88 mg/kg for canned white tuna. These values show that for every kilogram of sardines in vegetable oil consumed, 86.0 mg of pure tryptamine was ingested. Other researchers have conducted work on the determination of tryptamine in fish using HPLC. For example, Bilgin et al. [48] detected tryptamine in about 42.85% of all their study samples, with values ranging from ND to 190.61 mg/Kg. Furthermore, Zhai et al. [49] reported that tryptamine was detected in the range of 10.12 to 20.15 mg/kg in fish samples. High levels of tryptamine may be linked to the use of low-quality raw fish. Köse et al. [50] reported that tryptamine was detected in marinated anchovies at 55.8 mg/kg. In their study, tryptamine levels were generally higher than 20 mg/kg, especially in marinated anchovies. Some technological processes, such as marination, fermentation and salting, can increase the possibility of biogenic amine formation. A low pH of 4.0 to 5.5, which can be reached in salted anchovies, is favorable to increased activity of amino acid decarboxylase [51] .

In this study, we focused on biogenic amines with primary amine groups such as histamine (HIS), spermidine (SPD), cadaverine (CAD), putrescine (PUT), dopamine (DOPA), tyramine (TYR), and agmatine (AGM). These amines are present in many foods (plant products and fish) [52] [53] and could interfere with tryptamine within our studied wavelength range. We studied the interference effects of these biogenic amines, which are likely to be present in food along with the biogenic amine of interest. A fixed concentration of 10⁻⁷ M (0.0160 µg/mL) of tryptamine was used in the study ( Figure 8 ). To evaluate the interference effects, we progressively added concentrations of biogenic amines that may interfere with tryptamine. The influence of dopamine (DOPA), cadaverine (CAD), spermine (SPM), spermidine (SPD), agmatine (AGM), putrescine (PUT), histamine (HIS) and tyramine (TYR) on the spectra and fluorescence intensities of tryptamine was examined.

The addition of these amines did not alter the shape of the spectra or shift the maximum emission wavelength. However, a decrease in fluorescence signal was observed for dopamine (DOPA), cadaverine (CAD), spermine (SPM), spermidine (SPD), putrescine (PUT), histamine (HIS), and tyramine (TYR), whereas agmatine (AGM) led to an increase in fluorescence intensity at the added concentrations. We summarized the tolerance limit (LT) values and the mass percentage of each interfering species in Table 3 .

In our case, the tolerance limit (TL) is defined as the threshold concentration of the interferent at which the percentage variation in the fluorescence signal of tryptamine does not exceed ± 5%. This can be written as:

$\left|\Delta \text{F}\left(\text{\%}\right)\right|=\frac{{\text{F}}_0-\text{F}}{{\text{F}}_0}\times 100=5\text{\%}$

In this expression, ∆F (%) represents the percentage variation in the tyramine signal; F₀ and F represent the fluorescence signal of tyramine in the absence and presence of interfering species, respectively. In our case, the value of F₀ is equal to 279.21 relative fluorescence intensity units.

$\left|\Delta \text{F}\left(\text{\%}\right)\right|=\frac{{\text{F}}_0-\text{F}}{{\text{F}}_0}\times 100=5\%$

From this expression, we can deduce the limit values corresponding to this precision. Therefore, we can have two limit values, F₁ and F₂.

If $\left|\Delta \text{F}\left(\text{\%}\right)\right|\ge 0$, we write $\frac{{\text{F}}_0-{\text{F}}_1}{{\text{F}}_0}\times 100=+5\%$we will have: ${\text{F}}_1=\frac{95\times {\text{F}}_0}{100}=265.25$.

If $\left|\Delta \text{F}\left(\text{\%}\right)\right|\le 0$, we write $\frac{{\text{F}}_0-{\text{F}}_2}{{\text{F}}_0}\times 100=-5\%$ we will have: ${\text{F}}_2=\frac{105\times {\text{F}}_0}{100}=293.17$.

Thus, concentrations that do not interfere with the emission of tryptamine will correspond to intensities between F₁ and F₂. Outside of this range, the precision of the method exceeds ± 5%; the measurement thus becomes less precise.

From the intersection of the curve F = f [AB] with the lines y = F₁ or y = F₂, we can determine the tolerance limits (TL) at X₁ or X₂, respectively. If there is no intersection, the amine does not interfere with the tryptamine assay. Table 3 summarizes the concentration ranges tested, the different tolerance limit values, and the corresponding mass rates at the tolerance limits for each interfering species relative to tryptamine.

Fixed concentration = ${\left[\text{TRYP}\right]}_0$= 0.0160 µg/mL; $\infty$: non-interfering; Mass rates = $\frac{\left[\text{AB}\right]}{{\left[\text{TRYP}\right]}_0}\times 100$.

On the one hand, Table 3 shows that dopamine, histamine, and tyramine are the amines that interfere the least with the analysis of tryptamine. No interference was noted in the quantification of tryptamine when the sample also contained agmatine.

On the other hand, the table reveals that cadaverine and putrescine interfere much more with the determination of tryptamine, followed by spermine and spermidine. Nevertheless, it is possible to measure tryptamine with an accuracy of ±5% in a sample containing cadaverine, as long as the mass of cadaverine does not exceed 40% of that of tryptamine. Similarly, for tryptamine quantification with an accuracy of ±5% in a sample containing putrescine, the mass of putrescine must not exceed 60% of that of tryptamine. In a sample containing spermine or spermidine, tryptamine can be quantified with the same precision even at much higher concentrations (121% for spermine and 153% for spermidine). Tyramine interferes at a mass percentage of 429%, histamine at 461%, and dopamine at 593%.