The sample was weighed on electronic balance (XSR204, Mettler Toledo, Greifensee, Switzerland). FL spectra were acquired using a Varioskan Flash multimode microplate reader (3001, Thermo Fisher Scientific, Vantaa, Finland). The reaction system was maintained at in a biochemical incubator (KB53, BINDER GmbH, Tuttlingen, Germany). Supernatant was obtained in constant temperature large capacity centrifuge (DL-5-B, Shanghai anting scientific instrument factory, Shanghai, China). Ultrapure water (18.2 MΩ cm) was prepared using a Millipore Simplicity water purification system (Merck Millipore, MA, United States). Mass spectrometric analysis was conducted using an electrospray ionization (ESI) equipped X500R quadrupole time-of-flight (QTOF) mass spectrometer (AB Sciex, Framingham, MA, United States). The data were processed using Origin 2024 (OriginLab Corporation, Northampton, MA, United States).

The wolfberries were purchased from distinct manufacturers. SO₂ fluorescent probe was obtained from Suzhou Zhuoxin Yayi Technology Co., Ltd. (Jiangsu, China). Sodium sulfite anhydrous (Na₂SO₃, ≥98.0% purity) was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China). HPLC-grade acetonitrile (CH₃CN, ≥99.9%) was acquired from Macklin Biochemical Co., Ltd. (Shanghai, China). HEPES buffer (20 mM, pH 7.4) was acquired from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Anionic reagent was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

The fluorescent probe was dissolved in acetonitrile as stock solution (2.0 mM). Stock solutions of common anions (234 mM) including sulfite and other typical anions (Cl⁻, HCO₃ ⁻, H₂PO₄ ⁻, S₂O₃ ^2-, F⁻, Br⁻, I⁻, SO₄ ^2-, PO₄ ^3-, NO₃ ⁻, C₂O₄ ^2-, CH₃COO⁻, NO₂ ⁻, HSO₃ ⁻) were prepared by dissolving corresponding salts in HEPES buffer (20 mM, pH 7.4).

Varying concentrations of sulfite were mixed with 3 μL of the probe (60 μM) in 1.5 mL EP tubes. The volume was brought to 100 μL with HEPES buffer (20 mM, pH 7.4), and the solution was incubated at 35 °C for 15 min. Fluorescence intensity (λex/λem = 495/516 nm) was recorded on an enzyme label analyzer. A blank solution devoid of SO₃ ^2- was prepared and measured concurrently. Each assay was replicated three times (n = 3).

Specifically, 3 g of the sample was precisely weighed and mixed with 50 mL of HEPES buffer (20 mM, pH 7.4). The solution was allowed to stand for 15 min prior to centrifugation (5000 r/min, 5 min) to obtain the supernatant (test solution). For the detection assay, an aliquot of 5 μL of this test solution was combined with 3 μL of the probe (60 μM) in a 1.5 mL EP tube. The mixture was brought to a total volume of 100 μL with HEPES buffer, reacted at 35 °C for 15 min, and subsequently analyzed for fluorescence intensity (λex/λem = 495/516 nm) using an enzyme label analyzer.

ESI-MS spectral analyses (positive ion mode) of the fluorescent probe solution (1 μg/mL) and the reaction solution of the probe with SO₃ ^2- (100 mM) confirmed the proposed plausible mechanism. The plausible mechanism of fluorescent probe were shown in Figure 1. The peak of fluorescent probe at m/z 551.1319 (calcd m/z 551.1313 [M + Na]⁺), the peak of reaction product at m/z 333.0768 (calcd m/z 333.0763 [M + H]⁺).

The MS/MS spectra of the probe and reaction product were shown in Supplementary Figures S1, S2; Supplementary Table S1, the [M + Na]⁺ ion of probe at m/z 551.1305 was generated as the base peak, and ions at m/z 453.0937 [M-C₅H₆O₂+Na]⁺, m/z 355.0569 [M-(C₅H₆O₂)*2+Na]⁺ were observed. The [M + H]⁺ ion of reaction product at m/z 333.0766 served as the base peak, and ions at m/z 289.0860 [M-CHO₂+H]⁺, m/z 271.0761 [M-CHO₂-H₂O + H]⁺ were observed.

The stable reaction system was established in the HEPES buffer (20 mM, pH 7.4). In the experiment, the probe (0.2 mM) was co-incubated with SO₃ ^2- (2.34 mM) in HEPES buffer (20 mM, pH 7.4). First, one of the excitation or emission wavelengths was fixed, and the mixed solution was detected to obtain fluorescence intensity. The excitation and emission wavelengths were then screened based on the maximum fluorescence intensity. As shown in Figure 2b, the excitation and emission wavelengths were 495 nm and 516 nm, respectively. As shown in Figure 2c, the probe itself is non-fluorescent and colorless, upon addition of SO₃ ^2- to the solution, it exhibits an enhanced fluorescence signal and a bright yellow color. Meanwhile, the results of ultraviolet absorption spectra (Figure 2a) and the visual color changes were mutually corroborative of each other.

Given the complexity of the TCM matrices, the anti-interference capability of the probe was evaluated by detecting various common anions, including Cl⁻, HCO₃ ⁻, H₂PO₄ ⁻, S₂O₃ ^2-, F⁻, Br⁻, I⁻, SO₄ ^2-, PO₄ ^3-, NO₃ ⁻, C₂O₄ ^2-, CH₃COO⁻, NO₂ ⁻ and HSO₃ ⁻. The results indicated that the probe specifically recognizes SO₃ ^2- with negligible response to other anions (Figures 2d,e). Overall, the probe showed high selectivity for SO₃ ^2-/HSO₃ ⁻, supporting its applicability to sulfite detection in complex TCM matrices.

Next, the storage stability was evaluated by examining the probe stock solution after storage at −20 °C for 1, 3, 5 and 7 days. The relative standard deviation (RSD) of the fluorescence intensity was 2.95%, demonstrating that the probe exhibits reliable storage stability for up to 7 days (Figure 2f).

Reaction temperature, incubation time and probe concentration were optimized to achieve faster, more complete and more sensitive detection of SO₃ ^2-. The study primarily verified the temperature effect. Data indicate that the reaction between the probe and SO₃ ^2- tends toward completion at 35 °C (Figures 3a,b). Furthermore, the optimal reaction time was examined by assessing the reaction at time points of 5, 15, 25 and 35 min. Results showed that 15 min was sufficient for the detection system to react completely. The standard curve showed a better goodness of fit within 15 min (Figure 3c). Finally, the concentration of fluorescent probe was screened by fixing the amount of SO₃ ^2-. As indicated in Figure 3d, through the fluorescence intensity detection of the probe-SO₃ ^2- reaction, 60 μM was determined to be the optimal probe concentration in the tested range (40, 60, 80, 100, 200 μM).

To improve the accuracy and reliability of detection results, the key parameters of the sample extraction process were optimized with the recovery rate and RSD as the evaluation indices. The SO₃ ^2- concentrations for the low, medium and high spiking levels were 7 μM, 25 μM and 50 μM. The 7 μM SO₃ ^2- was derived by integrating the sample extraction process, with reference to the Chinese Pharmacopoeia, which stipulates that the sulfur dioxide residues should not exceed 150 mg/kg.

Moreover, matrix interference was observed in the sample extraction solution during fluorescence probe detection experiments, and the addition of 50 mL of this extraction solution mitigated such interference (Supplementary Figure S3). Meanwhile, there was no significant difference between the standard curve constructed in HEPES buffer (20 mM, pH 7.4) and that constructed in mixed blank wolfberry matrix (Supplementary Figure S4).

The pretreatment parameters considered included sample forms (whole wolfberry, powder) and extraction conditions such as extraction modes (static, shaking, ultrasonic), extraction time, and extraction solvent. As shown in Figure 4a, the recovery rates of intact wolfberries spiked with low, medium, and high concentrations of SO₃ ^2- were all above 60%. Additionally, Table 1 indicated that the RSD of the average recovery rates for the three concentrations in intact wolfberries was much lower than in powdered samples. Thus, subsequent experiments were conducted using intact wolfberries for further optimization.

Figure 4b showed that the recovery rates of low, medium and high concentrations of SO₃ ^2- stabilized at around 65%. Table 1 showed that compared with the shaking and ultrasonic methods, the RSD of the average recovery rates for the three concentrations the static soaking method is lower, indicating that the static method is relatively stable across low, medium and high concentrations.

Figure 4c showed that 15 min was the optimal extraction time, as determined by comparing the recovery rates of 15, 30, 45 and 60 min. As shown in Figure 4d; Table 1, the HEPES buffer (20 mM, pH 7.4) was selected as the optimal extraction solvent because it yielded higher recovery rates and a relatively lower RSD compared with other solvents.

In addition, the fluorescence intensity exhibited a linear correlation with SO₃ ^2- concentration (R ² = 0.9912, y = 2.7361x-12.4778, 5–75 μM). The limit of detection (LOD = 3 σ/k, σ = 1.3532, k = 2.7361) was calculated to be 1.5 μM.

All the results confirmed that the optimal conditions were as follows: accurately weigh 3 g of sample, precisely add 50 mL of HEPES buffer (20 mM, pH 7.4), let stand for 15 min, centrifuged, and use the supernatant as the test solution. At the end of the experiment, the low, middle, high recovery rates of 10 batches of samples were detected using this method. As shown in Table 2, the average recovery rate ranged between 89.5% and 100.8%.

Finally, the practicality of the new method was verified by the synchronous detection of wolfberry using both the fluorescent probe method and the titration method, and the results showed that According to the limit value of sulfur dioxide residues in wolfberry specified in the Pharmacopoeia of the People’s Republic of China, the fluorescent probe method can be used to obtain the test results, on the basis of which the qualification of samples is determined. The detailed results are presented in Table 3.