Method for detecting dephosphorylated nummulina velutipes and toad tryptamine in urine sample

Abstract

The invention relates to the technical field of emergency detection, in particular to a method for detecting dephosphorylated nummulina velutipes and toad tryptamine in a urine sample. According to the method, a to-be-detected urine sample is detected by adopting an Online SPE-LC-MS / MS method, a liquid chromatographic column is a BEHC18 chromatographic column, and a solid-phase extraction column is an online HLB small column. According to the method, online purification and enrichment of the sample can be automatically completed, the analysis time is short, the requirement for the dosage of the urine sample is small, complex sample pretreatment is not needed, the detection limit can reach 0.03 ng / mL, and the method has good sensitivity and accuracy and can meet the analysis requirement of the actual sample. The method is of great significance to identification of food poisoning sources and symptomatic treatment of patients.

Description

This application claims priority to application number 2025109348723, application date: July 8, 2025, invention title "A method for detecting dephosphorylated psilocybin and bufotenine in urine samples". Technical Field

[0001] This invention relates to the field of emergency detection technology, and more specifically, to a method for detecting dephosphorylated psilocybin and bufotenine in urine samples. Background Technology

[0002] Psilocybin (selosine), dephosphorylated psilocybin (selosine), and bufotenine are a class of tryptamine compounds with hallucinogenic effects, commonly found in hallucinogenic fungi such as those in the genera *Psilocybe*, *Chlorophyllum*, *Gymnocarpus*, and *Hylocereus*. Their structural formulas are as follows: Psilocytoin (Ⅰ) Dephosphorylated psiocytoin (Ⅱ) Bufotenamine (Ⅲ).

[0003] These compounds generally act on the central nervous system. Ingestion can cause sweating, drooling, tearing, noticeable color hallucinations, respiratory failure, and cardiovascular reactions. Severe cases may include pupillary constriction, blurred vision, muscle spasms, diarrhea, slowed heart rate, decreased blood pressure, and even coma and death. These compounds are rapidly metabolized in bodily fluids. Studies have shown that psilocybin is undetectable in urine and plasma collected 24 hours after oral administration. This is because psilocybin is immediately hydrolyzed in the body to its active metabolite, dephosphorylated psilocybin, with a half-life of 163 minutes. Approximately 67% of dephosphorylated psilocybin exists in urine as psilocybin glucuronide conjugate (PCG), with only 1.5–3.4% excreted unchanged in urine.

[0004] Because the levels of dephosphorylated psilocybin in the urine of poisoned patients are low (generally ng / mL), and the urine matrix is ​​complex, containing inorganic salts, proteins, and high concentrations of metabolites that interfere with detection, detection is difficult and conventional detection methods cannot detect it, thus delaying the optimal period for detection and treatment.

[0005] Currently, there is no rapid detection method for neurotoxins from mushrooms in the urine of poisoned patients. Since psilocybin and dephosphorylated psilocybin mainly exist in urine as PCG, it is generally necessary to enzymatically digest the urine overnight beforehand, followed by protein precipitation or solid-phase extraction combined with liquid chromatography-tandem mass spectrometry for determination. While protein precipitation is simple to operate, it has poor purification efficiency and significant matrix effects; conventional solid-phase extraction using small columns is cumbersome, time-consuming, and labor-intensive. In sudden emergencies, results are often required within a short timeframe, and these methods are insufficient to meet the requirements for rapid detection in emergency poisoning situations. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for detecting dephosphorylated psilocybin and bufotenine in urine samples.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: This invention provides a method for detecting dephosphorylated psilocybin and bufotenine in urine samples. The method employs Online SPE-LC-MS / MS, wherein the liquid chromatography column is a BEHC column. 18 The chromatographic column and solid-phase extraction column are online HLB columns.

[0008] Based on the above technical solution, the present invention can be further improved as follows.

[0009] Furthermore, the liquid chromatography column has dimensions of 2.1 mm × 100 mm and a diameter of 1.7 µm; the mobile phase A of the liquid chromatography is ammonium formate-formic acid aqueous solution, and the mobile phase B of the liquid chromatography is acetonitrile.

[0010] Furthermore, in the mobile phase A of the liquid chromatography, the concentration of ammonium formate is 5 mmol / L, and the volume percentage of formic acid in the formic acid water is 0.1%. The flow rate of the liquid chromatography is 0.15-0.25 mL / min, the column temperature is 35-45℃, and the injection volume is 1.5-2.5 mL. The liquid chromatography uses gradient elution. Based on the total volume of mobile phase A and mobile phase B being 100%, the elution program is as follows:

[0011] Furthermore, there are two solid-phase extraction columns, namely SPE column 1 and SPE column 2, both of which have a specification of 25µm and 2.1×20mm; the mobile phase A of the online solid-phase extraction is a pure aqueous solution, and the mobile phase B of the online solid-phase extraction is methanol; Online solid-phase extraction employs gradient elution. Assuming the total volume of the mobile phase in online solid-phase extraction is 100%, the elution procedure and the corresponding processes for each elution stage are as follows:

[0012] Furthermore, in the Online SPE-LC-MS / MS method, the mass spectrometry conditions are as follows: Ionization source: Electrospray ionization source, ESI+; Detection method: Multiple reaction monitoring (MRM); Capillary voltage: 3500 (+) 2500 (-); Nozzle voltage: (+) 1500, (-) 1500; Drying gas temperature: 250℃; Drying gas flow rate: 15 L / min; Atomizer pressure: 35 psi; Sheath gas temperature: 350 ℃; Sheath gas flow rate: 11 L / min.

[0013] Furthermore, the following steps are included: S1. Pre-treat the urine sample to be tested and add a mixed internal standard solution of dephosphorylated psilocybin and bufotenine; S2. Perform the Online SPE-LC-MS / MS detection on the pretreated urine sample; S3. Based on the peak area values ​​of the dephosphorylated psilocybin internal standard and the bufotenine internal standard obtained from the detection of the urine sample to be tested, calculate the content of dephosphorylated psilocybin and bufotenine in the urine sample to be tested.

[0014] Furthermore, in step S1, the pretreatment method is to mix the urine sample to be tested with the mixed internal standard solution, add water to make up the volume and filter.

[0015] Furthermore, the urine sample to be tested is 0.5 mL, and the total volume of the solution after dilution is 5-15 mL.

[0016] Furthermore, the mixed internal standard solution uses dephosphorylated psilocybin-d 10 The solution was prepared by mixing the solution with toad tryptamine-d4 hydrochloride solution.

[0017] Furthermore, in the mixed internal standard solution, dephosphorylated psilocybin-d 10 The total mass concentration of bufotenine-d4 was 10-50 ng / mL.

[0018] The beneficial effects of this invention are as follows: (1) The method for detecting dephosphorylated psilocybin and bufotenine in urine samples of the present invention uses the Online SPE-LC-MS / MS method to detect the urine samples to be tested. The operation is simple and can automatically complete the online purification and enrichment of the samples. The analysis time is short, and it only takes 10 minutes to realize the ultra-trace rapid detection and analysis of neurotoxins in cases of poisoning by accidental ingestion of hallucinogenic fungi. (2) The method for detecting dephosphorylated psilocybin and bufotenine in urine samples of the present invention requires a small amount of urine sample, only water is needed for dilution, and no complicated sample pretreatment is required, which further improves the detection efficiency. (3) The detection method of dephosphorylated psilocybin and bufotenine in urine samples of the present invention has a detection limit of 0.03 ng / mL, which can effectively improve the sensitivity and efficiency of emergency poisoning detection and meet the actual needs of emergency poisoning detection. (4) The method for detecting dephosphorylated psilocybin and bufotenine in urine samples of the present invention has a recovery rate of 75%-108% and a relative standard deviation (RSD) of less than 20% for the compounds, indicating that the method has good reproducibility and accuracy and can meet the analytical requirements of actual samples. (5) The detection method of dephosphorylated psilocybin and bufotenine in urine samples of the present invention has strong applicability and can be widely promoted. It is of great significance for the identification of food poisoning sources and the symptomatic treatment of patients. Attached Figure Description

[0019] Figure 1 The standard curve MRM chromatogram in Example 1 is a method for detecting dephosphorylated psilocybin and bufotenine in urine samples according to the present invention. Figure 2 The method for detecting dephosphorylated psilocybin and bufotenine in urine samples according to the present invention is illustrated in Example 1, with the spiked urine sample MRM chromatogram. Figure 3 The method for detecting dephosphorylated psilocybin and bufotenine in urine samples according to the present invention is illustrated in Example 1, which shows the flow path of the dual-column alternating online solid-phase extraction module. Figure 4 The standard MRM chromatogram of the HLB online solid-phase extraction column used in Example 3 is shown in the method for detecting dephosphorylated psilocybin and bufotenine in urine samples according to the present invention. Figure 5 In the method for detecting dephosphorylated psilocybin and bufotenine in urine samples according to the present invention, C is used in Example 3. 18 Standard MRM chromatogram during online solid-phase extraction using a small column; Figure 6 This is a comparison chart of the recovery rates of spiked urine samples under different pretreatment methods in Example 4, which is part of the method for detecting dephosphorylated psilocybin and bufotenine in urine samples according to the present invention. Detailed Implementation

[0020] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0021] The method for detecting dephosphorylated psilocybin and bufotenine in urine samples of the present invention employs an Online SPE-LC-MS / MS method, wherein the liquid chromatography column is a BEHC. 18 The chromatographic column and solid-phase extraction column are online HLB columns.

[0022] This invention, for the first time, combines online solid-phase extraction (SPE) with liquid chromatography-mass spectrometry (LC-MS). By optimizing chromatographic conditions, SPE parameters, and pretreatment conditions, an online SPE-LC-MS / MS method was established for the detection of bufotenine and psilocybin in poisoned urine samples. Compared to conventional methods, this method is simple to operate, can automatically complete online sample purification and enrichment, and has a short analysis time—only 10 minutes—to achieve ultra-trace rapid detection and analysis of neurotoxins from hallucinogenic mushroom poisoning cases. Furthermore, this method uses less organic reagent, making it environmentally friendly.

[0023] The detection method of this invention can also effectively improve the accuracy and sensitivity of the analysis. The sample volume is only 0.5 mL, and the detection limit can reach 0.03 ng / mL. It can effectively improve the sensitivity and efficiency of emergency poisoning detection and meet the actual needs of emergency poisoning detection.

[0024] In the detection method of the present invention, preferably, the liquid chromatography column has a specification of 2.1 mm × 100 mm and a diameter of 1.7 µm; more preferably, the liquid chromatography column is a Waters ACQUITYUPLC BEH C. 18 Experimental analysis revealed that C 18 The column is better at separating isomers than the T3 column.

[0025] Preferably, the mobile phase A of the liquid chromatography is an ammonium formate-formic acid aqueous solution, and the mobile phase B of the liquid chromatography is acetonitrile; more preferably, in the mobile phase A of the liquid chromatography, the concentration of ammonium formate is 5 mmol / L, and the volume percentage of formic acid in the formic acid aqueous solution is 0.1%.

[0026] More preferably, the flow rate of the liquid chromatography is 0.15-0.25 mL / min, the column temperature is 35-45℃, and the injection volume is 1.5-2.5 mL; the liquid chromatography uses gradient elution, and the elution program is shown in Table 1, with the total volume of the mobile phase of the liquid chromatography being 100%.

[0027] Table 1 The above elution procedure has a good elution effect.

[0028] Further preferred settings include an injection volume of 2 mL, a flow rate of 0.2 mL / min, and a column temperature of 40 °C.

[0029] In the detection method of the present invention, preferably, the solid phase extraction column is Waters HLB Direct Connect HP, a water-wettable reversed-phase adsorbent with a hydrophilic-lipophilic balance, and has a specification of 25µm and 2.1×20mm; this solid phase extraction column has a good effect on tryptophan-type mushroom toxins.

[0030] In one embodiment of the present invention, the specific connection method of the switching valve and the online SPE column 1, online SPE column 2 and liquid chromatography system in the dual-column switching fully automatic online solid phase extraction process is as follows: the online solid phase extraction (quaternary pump) system and the liquid chromatography separation (binary pump) system are connected by a ten-way valve, and the sample is enriched and analyzed online by switching the valve. The two columns, online SPE column 1 and online SPE column 2, are in parallel.

[0031] Based on the above connection method, the specific switching steps of the present invention are as follows: The online solid-phase extraction (SPE) process is controlled by a quaternary pump, while the ultra-high performance liquid chromatography (UHPLC) analysis process is controlled by a binary pump. Alternating sample loading and elution are achieved via a switching valve and two online SPE columns, designated as Online SPE Column 1 and Online SPE Column 2.

[0032] The specific analytical procedure is as follows: A 2 mL sample is taken using a multi-functional autosampler and first loaded into the quantitative loop. Then, the sample is pushed to the online SPE column 1 for enrichment. At this time, the online SPE column 2 is connected to the liquid chromatography flow path. The analyte from the previous injection on SPE column 2 is eluted by the chromatographic mobile phase to the analytical column. After the current sample is enriched on SPE column 1, the quaternary pump mobile phase flow rate is reduced to 0.01 mL / min. At this point, the valve switches to connect the online SPE column 1 to the liquid chromatography flow path. Since the mobile phase A (pure water) from the online solid-phase extraction is not retained on the solid-phase extraction column and has been discharged as waste after enrichment, SPE column 1 is only connected to the chromatographic system after the valve switch. At this point, only the enriched target analyte and the mobile phase of the binary chromatography system enter the column. The analyte in SPE column 1 is eluted by the chromatographic mobile phase to the analytical column and then detected by tandem mass spectrometry. Meanwhile, the online SPE column 2 is connected to the quaternary pump online solid-phase extraction flow path, the SPE column 2 is cleaned and activated, and the next round of sample analysis begins.

[0033] During the above analysis, dephosphorylated psilocybin and bufotenine in a single sample were consistently enriched in the same online SPE column.

[0034] As can be seen from the above process, this invention uses two online HLB columns of identical specifications and packing materials for alternating online solid-phase extraction (SPE). While online SPE column 1 is being analyzed by the tandem mass spectrometer, online SPE column 2 is being cleaned and reactivated, ready for the next analysis. There is no idle waiting time in the liquid chromatography-tandem mass spectrometry (LC-MS / MS). The essence of alternating columns lies in continuous injection and zero-dead-time cleaning / activation, significantly improving throughput.

[0035] Preferably, the mobile phase A of the online solid-phase extraction is a pure aqueous solution, and the mobile phase B is methanol. The online solid-phase extraction uses gradient elution. With the total volume of the mobile phase in the online solid-phase extraction being 100%, the elution procedure and the corresponding process for each elution stage are shown in Table 2. Table 2 In the table above, after SPE column 2 is activated, it waits for the next round of sample injection. Before loading the new sample, the online solid phase extraction still needs to rinse the injection port. After rinsing and injection, the sample is loaded at 0.51 min, and the sample is enriched in SPE column 2. Then, the elution is completed according to the above procedure.

[0036] The above-mentioned online solid-phase extraction elution method is effective in separating the peaks of bufotenine and dephosphorylated psilocybin, and has the best retention effect.

[0037] Preferably, in the Online SPE-LC-MS / MS method of the present invention, the mass spectrometry conditions are as follows: Ionization source: Electrospray ionization source, ESI+; Detection method: Multiple reaction monitoring (MRM); Capillary voltage: 3500(+) 2500(-); Nozzle voltage: (+)1500, (-)1500; Drying gas temperature: 230-270℃; Drying gas flow rate: 10-20L / min; Atomizer pressure: 30-40psi; Sheath gas temperature: 300-400℃; Sheath gas flow rate: 10-12L / min.

[0038] Preferably, the detection method of the present invention specifically includes the following steps: S1. Pre-process the urine sample to be tested and add a mixed internal standard solution of dephosphorylated psilocybin and bufotenine.

[0039] Preferably, the pretreatment method involves mixing the urine sample to be tested with the mixed internal standard solution, adding water to make up the volume, and filtering. Experiments have shown that direct dilution and injection can meet the detection requirements, making the sample pretreatment method simpler and more efficient.

[0040] Preferably, the urine sample to be tested is 0.5 mL, and the total volume of the solution after dilution is 5-15 mL.

[0041] Preferably, the mixed internal standard solution uses dephosphorylated psilocybin-d 10 The solution was prepared by mixing the solution with toad tryptamine-d4 hydrochloride solution.

[0042] Preferably, in the mixed internal standard solution, dephosphorylated psilocybin-d 10 The total mass concentration of bufotenine-d4 was 10-50 ng / mL.

[0043] S2. Perform Online SPE-LC-MS / MS analysis on the pretreated urine samples.

[0044] Preferably, the detection method of the present invention uses a dual-column alternating online solid-phase extraction module (Guangzhou Zhida Laboratory Technology Co., Ltd.) for online solid-phase extraction.

[0045] S3. Based on the peak area values ​​of the internal standards of dephosphorylated psilocybin and bufotenine in the urine sample, calculate the content of dephosphorylated psilocybin and bufotenine in the urine sample.

[0046] Preferably, the specific calculation can be performed by constructing a standard curve, which can be obtained by detecting dephosphorylated psilocybin standards and bufotenine standards at different concentration gradients.

[0047] The detection method of this invention achieves a recovery rate of 75%-108% for both toxins, and the relative standard deviation (RSD) for the compounds is less than 20%, indicating good reproducibility and accuracy, meeting the analytical requirements of practical samples. Furthermore, compared with literature methods, this method is faster, simpler to operate, and more sensitive.

[0048] The present invention will be illustrated by specific embodiments below.

[0049] The instruments used in this embodiment were: a 1290Ⅱ-6495C liquid chromatography-tandem triple quadrupole mass spectrometer (Agilent Technologies, Inc.), a dual-column alternating online solid-phase extraction module (Guangzhou Zhida Laboratory Technology Co., Ltd.), and a Waters ACQUITYUPLC BEH C. 18 Chromatographic column (1.7µm, 2.1 mm × 100 mm, Waters Corporation, USA), Waters HLBDirect Connect HP (25µm, 2.1 × 20 mm, Waters Corporation, USA).

[0050] The reagents used in the examples were: chromatographically pure methanol, chromatographically pure acetonitrile, chromatographically pure formic acid, chromatographically pure ammonium formate, and ultrapure water. The needle filter was made of polytetrafluoroethylene (PTFE) with a pore size of 0.22 µm.

[0051] The standard solutions used in the examples were: acetonitrile dephosphorylated psilocybin solution (100 µg / mL, Tianjin Alta Technology Co., Ltd.), bufotenine standard solution (5 mg, purity 98.8%, Shanghai Anpu Cuishi Standard Technology Service Co., Ltd.), bufotenine-d4 hydrochloride solution (100 µg / mL, Tianjin Alta Technology Co., Ltd.), and dephosphorylated psilocybin-d... 10 (10µg / mL, Tianjin Alta Technology Co., Ltd.)

[0052] Simultaneously, 10 µg / mL mixed standard stock solutions of dephosphorylated psilocybin and bufotenine were prepared using methanol, as well as bufotenine-d4 and dephosphorylated psilocybin-d4. 10 The mixed internal standard stock solution, at 1 µg / mL, can be stored at -18°C protected from light for up to one week. Before use, dilute with pure water to the required concentration as needed.

[0053] Example 1: Detection of dephosphorylated psilocybin and bufotenine in urine samples This embodiment uses the method of the present invention to detect dephosphorylated psilocybin and bufotenine in urine samples spiked with different concentrations: The sample pretreatment method in this embodiment is as follows: take 0.5 mL of urine sample into a 15 mL centrifuge tube, add 10 µL of internal standard solution, dilute to 10 mL with water, filter through a 0.22 µm filter membrane, and then place the sample in a sample vial for testing.

[0054] The pretreated sample was enriched using an online solid-phase extraction (HLB) column. The overall flow diagram for this dual-column switching fully automated online solid-phase extraction method is shown below. Figure 3 .like Figure 3 As shown, the process is as follows: First, 2 mL of sample 1 is taken by the multi-functional autosampler and transported to the online SPE column 1 for online enrichment by switching the flow path; after enrichment, the flow path system is switched from the online solid phase extraction flow path controlled by the quaternary pump to the liquid chromatography flow path controlled by the binary pump by the ten-way valve. The binary solvent manager uses the mobile phase to perform gradient elution of the target components on the SPE column 1 to analyze sample 1.

[0055] When the flow path of SPE column 1 is switched to the flow path of the liquid chromatography system, the online SPE column 2 is cleaned and activated, waiting to start the loading and online enrichment of sample 2. The specific process is the same as described above.

[0056] After elution, SPE column 1 is cleaned with a mixed solvent by a quaternary solvent manager and activated before the analysis of sample 3, while online SPE column 2 begins liquid chromatography analysis.

[0057] Through the above cycle, multiple samples can be continuously injected and analyzed. The terms "Sample 1", "Sample 2", and "Sample 3" are only used to distinguish different samples in the cycle of injection analysis and do not refer to any specific sample. That is, there is no mutual influence between the multiple samples, and they do not need to be analyzed in a certain order.

[0058] In the above process, the mobile phase A used for online solid-phase extraction is pure aqueous solution, and the mobile phase B is methanol. The mobile phase gradient settings for online solid-phase extraction are shown in Table 2 above.

[0059] The chromatographic column used in the above process was a Waters ACQUITYUPLC BEH C. 18 (2.1 mm × 100 mm, 1.7 µm), the chromatographic mobile phase A was 5 mM ammonium formate-0.1% formic acid aqueous solution, the mobile phase B was acetonitrile, the flow rate was 0.2 mL / min, the column temperature was 40 ℃, the injection volume was 2 mL, and the chromatographic mobile phase gradient was set as shown in Table 1 above.

[0060] The mass spectrometry conditions in this embodiment are as follows: Ionization source: Electrospray ionization source, ESI+; Detection method: Multiple reaction monitoring (MRM); Capillary voltage: 3500(+) 2500(-); Nozzle voltage: (+) 1500, (-) 1500; Drying gas temperature: 250 ℃; Drying gas flow rate: 15 L / min; Nebulizer pressure: 35 psi; Sheath gas temperature: 350 ℃; Sheath gas flow rate: 11 L / min.

[0061] The mass spectrometry parameters of the compounds are shown in Table 3: Table 3 Mass Spectrometry Parameters of Compounds Note: Items marked with * are quantitative ion pairs. The preparation of the standard solution in this embodiment is as follows: Weigh 5 mg of bufotenine and dilute to 10 mL with methanol to prepare a stock solution with a bufotenine concentration of 500 µg / mL. Take 100 µL of dephosphorylated psilocybin stock solution (100 µg / mL) and 20 µL of bufotenine stock solution (500 µg / mL), dilute to 10 mL with methanol to prepare a mixed standard stock solution with a concentration of 1 µg / mL. Take 10 µL of bufotenine-d4 stock solution (100 µg / mL) and dephosphorylated psilocybin-d4 stock solution (100 µg / mL) and... 10 Prepare a 1µg / mL mixed internal standard stock solution by diluting 100µL of methanol to 1mL. Store at -18℃ protected from light for up to one week. Dilute with pure water to the required concentration before use.

[0062] Take 10 µL of the mixed standard stock solution (1 µg / mL) and dilute to 10 mL with pure water to prepare a 1 ng / mL mixed standard working solution. Take 10 µL of the mixed internal standard stock solution (1 µg / mL) and dilute to 1 mL with pure water to prepare a 10 ng / mL mixed internal standard working solution.

[0063] The standard curve plotting process in this embodiment is as follows: 20µL, 50µL, 100µL, 200µL, 500µL, 1000µL, and 2000µL of mixed standard working solution (1ng / mL) were respectively transferred to 10µL of mixed internal standard working solution (10ng / mL), and then diluted to 10mL with pure water to prepare a series of mixed standards with concentrations of 0.002ng / mL, 0.005ng / mL, 0.01ng / mL, 0.02ng / mL, 0.05ng / mL, 0.1ng / mL, and 0.2ng / mL, respectively.

[0064] calculate: The content of compounds in the sample is calculated according to formula (1): X=C×V / V0……………………(1) In the formula: X represents the content of dephosphorylated psilocybin and bufotenine in urine, in nanograms per milliliter (ng / mL). C represents the concentration of dephosphorylated psilocybin and bufotenine in the sample according to the internal standard method in the standard curve, in nanograms per milliliter (ng / mL). V represents the final volume of the sample solution, in milliliters (mL), where V = 10. V0 represents the urine sample volume, in milliliters (mL), where V0 = 0.5; The calculation result should be rounded to two significant figures.

[0065] The standard curve MRM chromatogram of this embodiment is as follows: Figure 1 As shown, the MRM chromatogram of the spiked urine sample (spiked concentration 0.2 ng / mL) is as follows. Figure 2 As shown. According to Figure 1 and Figure 2 It can be seen that this method can obtain chromatographic peaks corresponding to the two substances and has good separation, indicating that the dephosphorylated psilocybin and bufotenine contained in the urine sample were detected.

[0066] The addition concentrations and determination concentrations of bufotenine and dephosphorylated psilocybin in this embodiment are shown in Table 4. As can be seen from Table 4, the method of the present invention can accurately quantify the two substances.

[0067] Table 4 Concentrations of bufotenine and dephosphorylated psilocybin in urine Example 2: Validation of detection results under different chromatographic conditions Since chromatographic conditions not only affect the separation efficiency and peak shape of the target analyte, but also its ionization efficiency, thus influencing the detector's response value and sensitivity, this embodiment investigates different chromatographic conditions to verify that the chromatographic conditions used in the detection method of this invention are optimal.

[0068] Specifically, this embodiment uses a 10 ng / mL mixed standard solution to compare Waters ACQUITYUPLCBEH C 18 Peak elution of two chromatographic columns, namely Waters ACQUITYUPLC HSS T3 (2.1 mm × 100 mm, 1.7 µm) and Waters ACQUITYUPLC HSS T3 (2.1 mm × 100 mm, 1.7 µm), under four different mobile phase systems (A: 0.1% formic acid water-methanol, B: 0.1% formic acid water-acetonitrile, C: 0.1% formic acid water containing 5 mmol / L ammonium formate-methanol, D: 0.1% formic acid water containing 5 mmol / L ammonium formate-acetonitrile).

[0069] The results showed that bufotenine and dephosphorylated psilocybin exhibited peak tailing when using the HSS T3 column; BEH C 18 The chromatographic column achieved peak separation for the isomers bufotenine and dephosphorylated psilocybin. Furthermore, when mobile phase A was selected, the peak shapes of each target analyte were narrower and the response was highest; see details below. Figure 1 This is because adding an appropriate amount of formic acid to the mobile phase can inhibit the dissociation of sample components, increase the retention of components on the stationary phase, and improve the peak shape of the sample. Adding a buffer salt system shifts the ionic equilibrium of the analyte to a "neutral" state, which is beneficial for its distribution in the reversed-phase system.

[0070] Therefore, based on the experimental results of this embodiment, it can be concluded that using BEH C 18 The optimal detection chromatographic conditions are: chromatographic column, 0.1% formic acid aqueous solution containing 5 mmol / L ammonium formate-acetonitrile as mobile phase (chromatographic conditions of Example 1).

[0071] Example 3: Validation of Detection Results of Online Solid-Phase Extraction Column and Sample Loading Mobile Phase To prevent the loss of the target analyte, water / buffer solution is generally used as the loading solvent. The loading solution contains organic solvent, which helps to remove unwanted impurities from the sample. Generally, the proportion of organic phase in the loading solution does not exceed 10% (V / V).

[0072] This embodiment uses two different online solid-phase extraction columns and three different mobile phases for detection to verify that the online solid-phase extraction column and mobile phase of the present invention are optimal. In this embodiment, a 0.05 ng / mL mixed standard solution is used as the test sample for detection.

[0073] Specifically, the online solid-phase extraction columns used in this embodiment are: Waters XBridge C 18 (10µm, 2.1×30mm) and Waters HLB Direct Connect HP (25µm, 2.1×20mm).

[0074] The three different mobile phases for sample loading were: A: 0.1% formic acid water, B: pure water, and C: 0.1% formic acid water-acetonitrile (95+5).

[0075] according to Figure 4 It can be seen that when using an HLB online solid-phase extraction column for online enrichment of samples and loading with pure water, bufotenine and dephosphorylated psilocybin showed good peak separation and optimal retention. This is because the molecular structures of bufotenine and dephosphorylated psilocybin contain hydrophobic aromatic rings with indole rings, which makes them more lipophilic. HLB is a hydrophilic-lipophilic solid-phase extraction column, while C... 18 Column packing has less polarity than other adsorbents, making it more suitable for non-polar compounds. Therefore, as... Figure 5 As shown, bufotenine and dephosphorylated psilocybin at C 18 Solid-phase extraction columns have poor retention rates.

[0076] Example 4: Validation of detection results using different pretreatment methods This example compares the extraction effects of direct dilution of spiked urine samples and dilution after protein precipitation using different organic reagents. Specifically, this example uses acetonitrile, methanol, and an acetonitrile-methanol mixture to dilute the urine samples, wherein the volume ratio of the acetonitrile-methanol mixture is V... 乙腈 ∶V 甲醇 The ratio is 5:1.

[0077] Since the pH value of the loading solution and the acidity coefficient (pKa) of the compounds determine their form in solution, thus affecting the solid-phase extraction efficiency, this example also investigated the overall recovery of each target compound in 1.0 ng / mL spiked urine samples under two different pH conditions: one group was adjusted to pH 2-3 with formic acid; the other group was not added with formic acid, and the measured pH value was 6-7.

[0078] Experimental results are as follows Figure 6 As shown, Figure 6 The specific experimental groups are as follows: A1: Dilute with pure water; A2: Dilute with pure water and adjust the pH to approximately 2 with formic acid; B1: Protein was precipitated with methanol and then diluted with pure water; B2: After precipitating the protein with methanol, dilute with pure water and adjust the pH to ≈2 with formic acid; C1: Protein was precipitated with acetonitrile and then diluted with pure water; C2: After precipitating the protein with acetonitrile, dilute with pure water and adjust the pH to ≈2 with formic acid; D1: Protein was precipitated using an acetonitrile-methanol mixed solution and then diluted with pure water; D2: After precipitating the protein with an acetonitrile-methanol mixture, dilute with pure water and adjust the pH to approximately 2 with formic acid.

[0079] The experimental results show that the peak shape is best and the recovery rate is highest when urine samples are directly diluted with water without adjusting the pH value.

[0080] This is because a certain proportion of organic phase remains in the loading solution after the protein is precipitated by the organic phase. Bufotenine and psilocybin are highly lipid-soluble and do not easily retain on the HLB column. Furthermore, since both are weakly basic alkaloids, when the sample dilution is alkaline or neutral, they exist mainly in molecular form in solution, which can improve their adsorption capacity on the HLB column; however, when the sample dilution is acidic, they exist in ionic form in solution, exhibiting the behavior of polar compounds. Charged ions easily interact with the solid-phase extraction column packing material, resulting in poor peak shape and decreased recovery rate. Based on the investigation results, the final urine pretreatment method was to directly dilute and load the sample with pure water.

[0081] Example 5 Methodological Validation This embodiment verifies the detection method of the present invention, including determining the limit of detection and limit of quantitation, and verifying the precision and accuracy. The specific experimental process is as follows: (1) Standard curve, correlation coefficient, limit of detection and limit of quantitation: A mixed standard solution of bufotenine and dephosphorylated psilocybin, along with an internal standard, was added to a blank water sample to prepare a 0.002–0.1 ng / mL mixed standard solution. Analysis was performed using the method of this invention (Example 1). Each experimental group was run in triplicate (n=6). A standard curve was plotted with the mass concentration of the compound on the x-axis and the ratio of the peak area of ​​the quantitative ion to the internal standard on the y-axis. The specific standard curve equation and linear correlation coefficient R are described below. 2 As shown in Table 3.

[0082] In accordance with the requirements for the determination of the limit of detection in GB / T 27417-2017 "Guideline for Conformity Assessment and Validation of Chemical Analysis Methods", a blank urine sample was added with a standard solution of a concentration close to the minimum mass concentration. The limit of detection was determined to be 3 times the signal noise and the limit of quantitation was determined to be 10 times the signal noise. The relevant results are shown in Table 5.

[0083] Experimental results showed that bufotenine and dephosphorylated psilocybin exhibited good linearity within the concentration range of 0.002–0.1 ng / mL, with R0 2 All values ​​were greater than 0.99. The limits of detection for bufotenine and dephosphorylated psilocybin in urine were 0.01 ng / mL and 0.03 ng / mL, respectively, and the limits of quantification were 0.03 ng / mL and 0.1 ng / mL, respectively.

[0084] Table 5. Linear equations, correlation coefficients, method detection limits, and quantitation limits for bufotenol and dephosphorylated psilocybin in urine samples. (2) Precision and accuracy: Three different concentrations of mixed standard additives (low, medium, and high) were added to blank urine samples. The concentrations of bufotenine spiked were 0.05 ng / mL, 0.1 ng / mL, and 0.2 ng / mL, respectively; the concentrations of dephosphorylated psilocybin spiked were 0.2 ng / mL, 0.5 ng / mL, and 1.0 ng / mL, respectively.

[0085] For the solutions of the above concentrations, sample pretreatment was performed as follows: 0.5 mL of sample was placed in a 15 mL centrifuge tube, 10 µL of internal standard solution was added, and the volume was adjusted to 10 mL with water. The sample was then filtered through a 0.22 µm filter membrane and placed in a sample vial for instrumental analysis.

[0086] For each concentration level, three parallel samples were analyzed, and the average spiked recoveries and relative standard deviations were calculated. The results are shown in Table 6. The experimental results show that the spiked recoveries ranged from 75% to 108%, the relative standard deviations of the compounds were less than 20%, and the precision and accuracy met the relevant requirements of GB / T 27417-2017 "Guideline for Conformity Assessment and Validation of Chemical Analysis Methods".

[0087] Table 6. Spiked recoveries and relative standard deviations of bufotenine and dephosphorylated psilocybin in urine samples (n=6) Example 6: Practical Application of the Detection Method In this embodiment, the method of the present invention was used to analyze urine samples from patients who suffered wild mushroom poisoning in Hubei Province in 2023-2024, and no pathogens were detected. Based on the epidemiological investigation results, this is because the mushrooms consumed by the poisoned individuals were not hallucinogenic.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting dephosphorylated psilocybin and bufotenine in urine samples, characterized in that, The urine samples were analyzed using an Online SPE-LC-MS / MS method, with a BEHC column used in the liquid chromatography. 18 The chromatographic column and solid-phase extraction column are online HLB columns.

2. The method for detecting dephosphorylated psilocybin and bufotenine in urine samples according to claim 1, characterized in that, The liquid chromatography column has dimensions of 2.1 mm × 100 mm and a diameter of 1.7 µm; the mobile phase A of the liquid chromatography is ammonium formate-formic acid aqueous solution, and the mobile phase B of the liquid chromatography is acetonitrile.

3. The method for detecting dephosphorylated psilocybin and bufotenine in urine samples according to claim 2, characterized in that, In the mobile phase A of the liquid chromatography, the concentration of ammonium formate is 5 mmol / L, and the volume percentage of formic acid in the formic acid solution is 0.1%. The flow rate of the liquid chromatography is 0.15-0.25 mL / min, the column temperature is 35-45℃, and the injection volume is 1.5-2.5 mL. The liquid chromatography uses gradient elution. Based on the total volume of mobile phase A and mobile phase B being 100%, the elution program is as follows: 。 4. The method for detecting dephosphorylated psilocybin and bufotenine in urine samples according to claim 1, characterized in that, The solid-phase extraction column consists of two columns, namely SPE column 1 and SPE column 2, both of which have a diameter of 25µm and a length of 2.1×20mm. The mobile phase A for online solid-phase extraction is a pure aqueous solution, and the mobile phase B for online solid-phase extraction is methanol. Online solid-phase extraction employs gradient elution. Assuming the total volume of the mobile phase in online solid-phase extraction is 100%, the elution procedure and the corresponding processes for each elution stage are as follows: 。 5. The method for detecting dephosphorylated psilocybin and bufotenine in urine samples according to claim 4, characterized in that, In the Online SPE-LC-MS / MS method, the mass spectrometry conditions are as follows: Ionization source: Electrospray ionization source, ESI+; Detection method: Multiple reaction monitoring (MRM); Capillary voltage: 3500 (+) 2500 (-); Nozzle voltage: (+) 1500, (-) 1500; Drying gas temperature: 250 ℃; Drying gas flow rate: 15 L / min; Atomizer pressure: 35 psi; Sheath gas temperature: 350 ℃; Sheath gas flow rate: 11 L / min.

6. A method for detecting dephosphorylated psilocybin and bufotenine in urine samples according to any one of claims 1-5, characterized in that, Includes the following steps: S1. The urine sample to be tested is pretreated and a mixed internal standard solution containing dephosphorylated psilocybin and bufotenine is added. S2. Perform the Online SPE-LC-MS / MS detection on the pretreated urine sample; S3. Based on the peak area values ​​of the dephosphorylated psilocybin internal standard and the bufotenine internal standard obtained from the detection of the urine sample to be tested, calculate the content of dephosphorylated psilocybin and bufotenine in the urine sample to be tested.

7. The method for detecting dephosphorylated psilocybin and bufotenine in urine samples according to claim 6, characterized in that, In step S1, the pretreatment method is to mix the urine sample to be tested with the mixed internal standard solution, add water to make up the volume and filter.

8. The method for detecting dephosphorylated psilocybin and bufotenine in urine samples according to claim 7, characterized in that, The urine sample to be tested is 0.5 mL, and the total volume of the solution after dilution is 5-15 mL.

9. The method for detecting dephosphorylated psilocybin and bufotenine in urine samples according to claim 7, characterized in that, The mixed internal standard solution used was dephosphorylated psilocybin-d 10 The solution was prepared by mixing the solution with toad tryptamine-d4 hydrochloride solution.

10. The method for detecting dephosphorylated psilocybin and bufotenine in urine samples according to claim 9, characterized in that, In the mixed internal standard solution, dephosphorylated psilocybin-d 10 The total mass concentration of bufotenine-d4 was 10-50 ng / mL.

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