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 BEH-filled C18 chromatographic column, and a solid phase extraction column is an online HLB (Hydrophile Lipophile Balance) 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

Technical Field

[0001] The present invention relates to the field of emergency detection technology, and in particular to a method for detecting dephosphopsilin and toad tryptamine in urine samples. Background Art

[0002] Psilocybin, dephosphopsilin, and toad tryptamine are a class of tryptamine compounds with hallucinogenic effects. They are often found in hallucinogenic fungi such as Psilocybin, Pleurotus, Gymnocybe, and Mycorrhizum. Their structural formula is as follows: Psilocybin (Ⅰ) Psilocybin dephosphate (Ⅱ) Toad serotonin (Ⅲ).

[0003] These compounds generally act on the central nervous system. Accidental ingestion can cause sweating, drooling, tearing, pronounced color hallucinations, respiratory failure, cardiovascular reactions, and, in severe cases, pupil constriction, blurred vision, muscle spasms, diarrhea, slowed heart rate, low blood pressure, and even coma and death. These compounds are rapidly metabolized in human body fluids. Studies have shown that the parent compound psilocybin is undetectable in urine and plasma collected 24 hours after oral administration. This is because psilocybin is immediately hydrolyzed into its active metabolite, dephosphopsilin, which has a half-life of 163 minutes. Approximately 67% of dephosphopsilin in urine exists as dephosphopsilin glucuronide conjugate (PCG), with only 1.5–3.4% of the dose excreted unchanged in the urine.

[0004] Because the level of dephosphopsilin in the urine of poisoned patients is low (generally in the ng / mL range), and the urine matrix is complex, containing inorganic salts, proteins, and high concentrations of metabolites that interfere with detection, detection is difficult and cannot be detected using conventional detection methods, thus delaying the optimal detection and treatment period.

[0005] Currently, there is no rapid method for detecting neurotoxins in the urine of poisoned patients. Because psilocybin and dephosphopsilin exist primarily in urine as PCG, urine must generally be enzymatically hydrolyzed overnight before detection using protein precipitation or solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry. While protein precipitation is simple to perform, it suffers from poor purification and significant matrix effects. Conventional solid-phase extraction cartridge methods are cumbersome, time-consuming, and labor-intensive. In emergencies, test results often need to be obtained within a short period of time, and these methods struggle to meet the requirements for rapid poisoning emergency testing. Summary of the Invention

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

[0007] The technical solution of the present invention to solve the above technical problems is as follows: The present invention provides a method for detecting dephosphorylated psilocybin and toad tryptamine in urine samples, wherein the urine sample to be tested is detected by an Online SPE-LC-MS / MS method, wherein the liquid chromatography column is a C filled with BEH. 18 Chromatographic column, solid phase extraction column is an online HLB column.

[0008] On the basis of the above technical solution, the present invention can also be improved as follows.

[0009] Furthermore, the specifications of the liquid chromatography column are 2.1 mm×100 mm, 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 liquid chromatography flow rate is 0.15-0.25 mL / min, the column temperature is 35-45°C, and the injection volume is 1.5-2.5 mL. The liquid chromatography adopts gradient elution. Based on the total volume of the mobile phase A and the mobile phase B of the liquid chromatography being 100%, the elution procedure is as follows: 0.0-1.0 min, 5% mobile phase B; 1.0-4.0 min, 95% mobile phase B; 4.0-6.0 min, 95% mobile phase B; 6.0-8.0 min, 100% mobile phase B; 8.0-8.1 min, 5% mobile phase B; 8.1-10.0 min, 5% mobile phase B.

[0011] Furthermore, the specifications of the solid phase extraction column are 25µm, 2.1×20mm; the mobile phase A of the online solid phase extraction is pure aqueous solution, and the mobile phase B of the online solid phase extraction is methanol; Online solid phase extraction uses gradient elution. Taking the total volume of the mobile phase of online solid phase extraction as 100%, the elution procedure is as follows: 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 ion 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.

[0012] Further, the following steps are included: S1. Pre-treating the urine sample to be tested, and adding a mixed internal standard solution of dephosphorylated psilocybin and toad tryptamine; S2. performing the online SPE-LC-MS / MS test on the urine sample after the pretreatment; S3. Calculate the contents of dephosphopsilin and toad tryptamine in the urine sample according to the peak area values of the dephosphopsilin internal standard and toad tryptamine internal standard obtained in the urine sample.

[0013] 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.

[0014] Furthermore, the urine sample to be tested is 0.5 mL, and the total volume of the solution after constant volume is 5-15 mL.

[0015] Furthermore, the mixed internal standard solution uses dephosphorylated psilocybin-d 10 The solution is prepared with toad tryptamine-d4 hydrochloride solution.

[0016] Furthermore, in the mixed internal standard solution, dephosphorylated psilocybin-d 10 The total mass concentration of toad tryptamine-d4 is 10-50 ng / mL.

[0017] The beneficial effects of the present invention are: (1) The method for detecting dephospho-psilocybin and toad tryptamine in urine samples of the present invention adopts an online SPE-LC-MS / MS method to detect the urine sample to be tested. The method is simple to operate, can automatically complete the online purification and enrichment of the sample, and has a short analysis time. It only takes 10 minutes to achieve ultra-trace rapid detection and analysis of neurotoxins in cases of accidental hallucinogenic mushroom poisoning; (2) The method for detecting dephosphopsilocybin and toad tryptamine in urine samples of the present invention requires a small amount of urine sample and only requires dilution with water, without the need for complex sample pretreatment, thereby further improving the detection efficiency; (3) The detection method of dephosphopsilin and toad tryptamine 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 poisoning emergency detection and meet the actual needs of poisoning emergency detection; (4) The method for detecting dephosphopsilocybin and toad tryptamine in urine samples of the present invention has an average recovery rate of 75%-108%, and the relative standard deviation (RSD) of the compounds is less than 20%, indicating that the method has good reproducibility and accuracy and can meet the analytical requirements of actual samples; (5) The method for detecting dephospho-psilocybin and toad tryptamine in urine samples of the present invention has strong applicability and can be widely promoted, which is of great significance for the identification of food poisoning sources and the symptomatic treatment of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The method for detecting dephosphopsilin and toad tryptamine in urine samples of the present invention, and the standard curve MRM chromatogram in Example 1; Figure 2 The method for detecting dephosphopsilin and toad tryptamine in urine samples of the present invention, and the MRM chromatogram of the spiked urine sample in Example 1; Figure 3 Schematic diagram of the flow path of the dual-column alternating online solid phase extraction module in Example 1 for the detection method of dephosphorylated psilocybin and toad tryptamine in urine samples of the present invention; Figure 4 This is the standard MRM chromatogram of the method for detecting dephosphopsilin and toad tryptamine in urine samples of the present invention when using the HLB online solid phase extraction cartridge in Example 3; Figure 5 The method for detecting dephosphorylated psilocybin and toad tryptamine in urine samples of the present invention is as follows: 18 Standard MRM chromatogram for online solid phase extraction cartridge; Figure 6 2 is a comparative chart of the recoveries of spiked urine samples under different pretreatment methods in Example 4, showing the method for detecting dephosphorylated psilocybin and toad tryptamine in urine samples of the present invention. DETAILED DESCRIPTION

[0019] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0020] The method for detecting dephosphorylated psilocybin and toad tryptamine in urine samples of the present invention adopts an online SPE-LC-MS / MS method to detect the urine sample to be tested, wherein the liquid chromatography column is a C filled with BEH 18 Chromatographic column, solid phase extraction column is an online HLB column.

[0021] The detection method presented here is the first to utilize online solid-phase extraction (SPE) combined with liquid chromatography-mass spectrometry (LC-MS / MS). By optimizing chromatographic conditions, online SPE parameters, and pretreatment conditions, an online SPE-LC-MS / MS method for the detection of toadstryptamine and dephosphorylated psilocybin in poisoned urine samples has been established. Compared to conventional methods, this method is simple to operate, capable of automated online sample purification and enrichment, and offers a short analysis time, achieving ultra-trace rapid detection of neurotoxins in cases of hallucinogenic mushroom poisoning in just 10 minutes. Furthermore, this method utilizes minimal organic reagents, making it environmentally friendly.

[0022] The detection method of the present invention can also effectively improve the accuracy and sensitivity of the analysis. The sample dosage is only 0.5mL, and the detection limit can reach 0.03ng / mL. It can effectively improve the sensitivity and efficiency of poisoning emergency detection and meet the actual poisoning emergency detection needs.

[0023] In the detection method of the present invention, preferably, the specification of the liquid chromatography column is 2.1 mm×100 mm, 1.7 μm; further preferably, the liquid chromatography column is Waters ACQUITY UPLC BEH C 18 ; Through experimental analysis, it was found that C 18 The column has better separation effect on isomers than the T3 column.

[0024] Preferably, 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; further 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 water is 0.1%.

[0025] Further preferably, the liquid chromatography flow rate is 0.15-0.25 mL / min, the column temperature is 35-45°C, and the injection volume is 1.5-2.5 mL; the liquid chromatography uses gradient elution, and based on the total volume of the liquid chromatography mobile phase as 100%, the elution program is as follows: 0.0-1.0 min, 5% mobile phase B; 1.0-4.0 min, 95% mobile phase B; 4.0-6.0 min, 95% mobile phase B; 6.0-8.0 min, 100% mobile phase B; 8.0-8.1 min, 5% mobile phase B; 8.1-10.0 min, 5% mobile phase B. The above-mentioned program has a good elution effect.

[0026] More preferably, the injection volume is 2 mL, the flow rate is 0.2 mL / min, and the column temperature is 40°C.

[0027] In the detection method of the present invention, preferably, the solid phase extraction column is Waters HLB Direct Connect HP, with a specification of 25µm and 2.1×20mm; this solid phase extraction column has a good effect on tryptamine mushroom toxins with stronger fat solubility.

[0028] Preferably, 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; the online solid phase extraction adopts gradient elution, and the total volume of the mobile phase of the online solid phase extraction is 100%, and the elution procedure is as follows: The above-mentioned elution method of online solid phase extraction has a good effect on the peak separation of toad tryptamine and dephosphorylated psilocybin, and the retention effect is the best.

[0029] 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 ion monitoring MRM; Capillary voltage: 3500(+) 2500(-); Nozzle voltage: (+)1500, (-)1500; Drying gas temperature: 230-270℃; Drying gas flow rate: 10-20L / min; Nebulizer pressure: 30-40psi; Sheath gas temperature: 300-400℃; Sheath gas flow rate: 10-12L / min.

[0030] Preferably, the detection method of the present invention specifically comprises the following steps: S1. Pre-treat the urine sample to be tested and add a mixed internal standard solution of dephosphorylated psilocybin and toad tryptamine.

[0031] Preferably, 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; through experiments, it is found that direct dilution and injection can meet the detection requirements, making the sample pretreatment method simpler and more efficient.

[0032] Preferably, the urine sample to be tested is 0.5 mL, and the total volume of the solution after constant volume is 5-15 mL.

[0033] Preferably, the mixed internal standard solution is dephosphorylated psilocybin-d 10 The solution is prepared with toad tryptamine-d4 hydrochloride solution.

[0034] Preferably, in the mixed internal standard solution, dephosphorylated psilocybin-d 10 The total mass concentration of toad tryptamine-d4 is 10-50 ng / mL.

[0035] S2. Perform online SPE-LC-MS / MS detection on the pre-treated urine sample.

[0036] 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, and the process is as follows: A 2 mL water sample is first taken with a multifunctional autosampler and transferred to an online SPE column via flow switching for online enrichment. During enrichment, the two SPE columns are rotated according to system settings. Interfering substances are first cleaned with solvent, and the cleaning solution is discarded. The target components on the SPE column are then gradient eluted using a mobile phase. Simultaneously, the flow path is switched to the liquid chromatography system for sample analysis. After elution, the SPE column is cleaned with a mixed solvent and conditioned before the next sample is analyzed, completing the entire injection cycle.

[0037] S3. Calculate the contents of dephosphopsilin and toad tryptamine in the urine sample according to the peak area values of the dephosphopsilin internal standard and toad tryptamine internal standard obtained in the urine sample.

[0038] Preferably, the specific calculation can be performed by constructing a standard curve, and the standard curve can be obtained by detecting dephosphopsilin standards and toad tryptamine standards with different concentration gradients.

[0039] The detection method of the present invention achieved an average recovery rate of 75%-108% for both toxins, with a relative standard deviation (RSD) of less than 20% for each compound. This demonstrates the method's excellent reproducibility and accuracy, meeting the analytical requirements of real-world samples. Furthermore, compared with methods reported in the literature, this method demonstrated a shorter time consumption, simpler operation, and higher sensitivity.

[0040] The present invention is illustrated below by means of specific examples.

[0041] The instruments used in the examples were: 1290Ⅱ-6495C liquid chromatography-tandem triple quadrupole mass spectrometer (Agilent, USA), dual column alternating online solid phase extraction module (Guangzhou Zhida Laboratory Technology Co., Ltd.), Waters ACQUITYUPLC BEH C 18 Chromatographic columns (1.7 μm, 2.1 mm × 100 mm, Waters Corporation, USA), Waters HLB DirectConnect HP (25 μm, 2.1 × 20 mm, Waters Corporation, USA).

[0042] The reagents used in the examples were: chromatographically pure methanol, chromatographically pure acetonitrile, chromatographically pure formic acid, chromatographically pure ammonium formate, and ultrapure water for the experiments. The syringe filter was made of polyvinylidene fluoride (PVDF) with a pore size of 0.22 µm.

[0043] The standard solutions used in the examples were: dephospho-psilocybin solution in acetonitrile (100 μg / mL, Tianjin Alta Technology Co., Ltd.), toad tryptamine standard solution (5 mg, purity 98.8%, Shanghai Anpu Cuishi Standard Technology Service Co., Ltd.), toad tryptamine-d4 hydrochloride solution (100 μg / mL, Tianjin Alta Technology Co., Ltd.), dephospho-psilocybin-d 10 (10 µg / mL, Tianjin Alta Technology Co., Ltd.).

[0044] At the same time, methanol was used to prepare 10 μg / mL mixed standard stock solutions of dephosphorylated psilocybin and toad tryptamine, toad tryptamine-d4 and dephosphorylated psilocybin-d 10 Mix the internal standard stock solution (1 µg / mL) and store at -18°C in the dark. It can be stored for 1 week. Dilute with purified water to the desired concentration before use.

[0045] Example 1 Detection of dephospho-psilocybin and toad tryptamine in urine samples This example uses the method of the present invention to detect dephosphopsilin and toad tryptamine in spiked urine samples at different concentrations. The specific detection process is as follows: (1) Sample pretreatment: Take 0.5 mL of urine sample and place it in a 15 mL centrifuge tube. Add 20 µL of internal standard solution and dilute to 10 mL with water. After filtering through a 0.22 µm filter membrane, place the sample in a sample injection bottle for testing.

[0046] (2) Liquid chromatography conditions: The chromatographic column used in this example was a Waters ACQUITY UPLC BEH C 18 The column was equipped with an 8-well plate (2.1 mm × 100 mm, 1.7 µm). The mobile phase A was 5 mM ammonium formate-0.1% formic acid aqueous solution, and the mobile phase B was acetonitrile. The flow rate was 0.2 mL / min. The column temperature was 40 °C. The injection volume was 2 mL. The mobile phase gradient was as follows: 0.0-1.0 min, 5% B; 1.0-4.0 min, 95% B; 4.0-6.0 min, 95% B; 6.0-8.0 min, 100% B; 8.0-8.1 min, 5% B; 8.1-10.0 min, 5% B.

[0047] (3) Online solid phase extraction conditions: The pre-treated sample was enriched using an online solid phase extraction HLB cartridge. The online solid phase extraction mobile phase A was pure aqueous solution, and the mobile phase B was methanol. The online solid phase extraction mobile phase gradient settings were shown in Table 1.

[0048] Table 1 Online solid phase extraction gradient elution conditions like Figure 3 As shown, the dual-column switching fully automatic online solid-phase extraction process is as follows: first, a 2 mL water sample is taken by the multifunctional automatic sampler, and the water sample is transported to the online SPE column 1 through the flow switching for online enrichment. During enrichment, according to the system settings, the 1 and 2 columns are rotated for enrichment; the quaternary solvent manager uses the solvent to clean the SPE column for interferences, and the cleaning liquid is discharged to waste; after cleaning, the binary solvent manager uses the mobile phase to perform gradient elution on the target components on the SPE column. The sample loading and elution program is shown in Table 2. At the same time as elution, the flow path is switched to the liquid chromatography system flow path for sample analysis. After the elution is completed, the SPE column is cleaned with a mixed solvent by the quaternary solvent manager and activated before the next sample analysis to complete the entire injection cycle. The overall flow path diagram of the online SPE is shown in Figure 2 .

[0049] (4) Mass spectrometry conditions: Ionization source: electrospray ionization source, ESI+; Detection mode: multiple reaction ion monitoring (MRM); Capillary voltage: 3500(+) 2500(-); Nozzle voltage: (+)1500, (-)1500; Drying gas temperature: 250°C; Drying gas flow rate: 15 L / min; Nebulizer pressure: 35 psi; Sheath gas temperature: 350°C; Sheath gas flow rate: 11 L / min.

[0050] Table 2 Mass spectrometry parameters of compounds Note: The ions marked with * are quantitative ion pairs. (5) Preparation of standard solution: Weigh 5 mg of toad tryptamine and dilute to 10 mL with methanol to prepare a toad tryptamine stock solution with a concentration of 500 μg / mL. Take 100 μL of dephosphorylation psilocybin stock solution (100 μg / mL) and 20 μL of toad tryptamine stock solution (500 μg / mL), dilute to 10 mL with methanol, and prepare a 1 μg / mL mixed standard stock solution. Take 10 μL of toad tryptamine-d4 stock solution (100 μg / mL) and 20 μL of dephosphorylation psilocybin-d 10 Prepare a 1µg / mL mixed internal standard stock solution by adding 100µL of 10µg / mL HCl to 1mL of methanol. Store at -18°C in the dark for 1 week. Dilute with purified water to the desired concentration before use.

[0051] Prepare a 1 ng / mL mixed standard working solution by taking 10 μL of the mixed standard stock solution (1 μg / mL) and diluting to 10 mL with pure water. Prepare a 10 ng / mL mixed internal standard working solution by taking 10 μL of the mixed internal standard stock solution (1 μg / mL) and diluting to 1 mL with pure water.

[0052] (6) Drawing of standard curve: pipette 20uL, 50uL, 100uL, 200uL, 500uL, 1000uL, 2000uL of mixed standard solution (1ng / mL) respectively, add 10uL of mixed internal standard solution (10ng / mL), dilute to 10mL with pure water, and prepare the concentrations to be 0.002ng / mL, 0.005ng / mL, 0.01ng / mL, 0.02ng / mL, 0.05ng / mL, respectively. 0.1ng / mL, 0.2ng / mL mixed standard series.

[0053] (7) Calculation: The content of the compound in the sample is calculated according to formula (1): X=C×V / V0……………………(1) Where: X represents the content of dephosphopsilin and toad tryptamine in urine, in nanograms per milliliter (ng / mL); C represents the concentration of dephosphopsilin and toad tryptamine in the sample in the standard curve according to the internal standard method, in nanograms per milliliter (ng / mL); V represents the fixed volume of the sample solution to be tested, in milliliters (mL), where V=10; V0 represents the urine sampling volume, milliliters (mL), where V0=0.5; The calculation result is rounded to 2 significant figures.

[0054] The standard curve MRM chromatogram of this example is as follows Figure 1 As shown in the figure, the MRM chromatogram of spiked urine sample (spiked concentration 0.2 ng / mL) is as follows Figure 2 As shown. Figure 1 and Figure 2 It can be seen that this method can obtain chromatographic peaks corresponding to the two substances with good separation, indicating that the method can detect dephosphopsilin and toad tryptamine contained in the urine sample.

[0055] The added concentrations and measured concentrations of toad tryptamine and dephosphopsilin of this example are shown in Table 3. It can be seen from Table 3 that the method of the present invention can accurately quantitatively detect the two substances.

[0056] Table 3 Concentrations of toad tryptamine and dephosphorylated psilocybin in urine Example 2 Verification of the test results under different chromatographic conditions Since chromatographic conditions not only affect the separation effect and chromatographic peak shape of the target, but also the ionization efficiency of the target, thereby affecting the response value and sensitivity of the detector, this example investigates different chromatographic conditions to verify that the detection method of the present invention uses the optimal chromatographic conditions.

[0057] Specifically, this example uses a 10 ng / mL mixed standard solution to compare the Waters ACQUITY UPLCBEH C 18 Peak elution patterns of two chromatographic columns, ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.7 µm) and Waters ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.7 µm), using four different mobile phase systems (A: 0.1% formic acid in water-methanol, B: 0.1% formic acid in water-acetonitrile, C: 0.1% formic acid in water containing 5 mmol / L ammonium formate in methanol, and D: 0.1% formic acid in water containing 5 mmol / L ammonium formate in acetonitrile).

[0058] The results showed that when using HSS T3 column, toad tryptamine and dephosphorylated psilocybin showed peak tailing; when using BEH C 18 The chromatographic column achieves peak separation of the isomers of toad tryptamine and dephosphorylated psilocybin. At the same time, when the mobile phase is selected as system A, the peak shape of each target compound is narrower and the response is the highest. 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 can shift the ion balance of the analyzed compound toward a "neutral" form, which is beneficial for distribution in the reversed-phase system.

[0059] Therefore, according to the test results of this embodiment, it can be concluded that using BEH C 18 The optimal chromatographic conditions for detection are as follows: a chromatographic column and 0.1% formic acid water containing 5 mmol / L ammonium formate-acetonitrile as the mobile phase (chromatographic conditions of Example 1).

[0060] Example 3 Verification of the test results of online solid phase extraction column and loading mobile phase To prevent the loss of the target substance, water / buffer solution is generally used as the loading solvent during the loading process. The organic solvent contained in the loading solution helps to remove unwanted impurities from the sample. Generally, the proportion of organic phase in the loading does not exceed 10% (V / V).

[0061] This example uses two different online solid phase extraction columns and three different loading mobile phases to test and verify that the online solid phase extraction column and loading mobile phase of the present invention are optimal. This example uses a 0.05 ng / mL mixed standard solution as the test sample for testing.

[0062] Specifically, the online solid phase extraction columns used in this example are: Waters XBridge C 18 (10µm, 2.1×30mm) and Waters HLB Direct Connect HP (25µm, 2.1×20mm).

[0063] The three different loading mobile phases were: A: 0.1% formic acid water, B: pure water, and C: 0.1% formic acid water-acetonitrile (95+5).

[0064] according to Figure 4 It can be seen that when the HLB online solid phase extraction column was used to enrich the sample online and pure water was used for loading, the peak separation effect of toad tryptamine and dephosphoryl psilocybin was good and the retention effect was the best. The reason is that the molecular structure of toad tryptamine and dephosphoryl psilocybin contains indole ring hydrophobic aromatic ring structure, which makes the two more fat soluble. HLB is a hydrophilic and lipophilic solid phase extraction column, while C 18 The polarity of column packing is smaller than that of other adsorbents, and it has a stronger applicability to non-polar compounds. Figure 5 As shown, toad tryptamine and dephosphorylated psilocybin in C 18 The retention effect of solid phase extraction cartridges is poor.

[0065] Example 4 Verification of test results of different pre-treatment methods This example compares the extraction effects of directly diluting spiked urine samples and selecting different organic reagents to precipitate proteins before diluting and loading the sample. Specifically, this example uses acetonitrile, methanol, and acetonitrile-methanol mixed solutions to dilute urine samples, where the volume ratio of the acetonitrile-methanol mixed solution is V 乙腈 ∶V 甲醇 It is 5:1.

[0066] Because the pH of the sample solution and the compound's pKa determine its form in solution and thus affect solid-phase extraction efficiency, this example also examined the overall recovery of each target compound in a 1.0 ng / mL spiked urine sample under two different pH conditions: one in which formic acid was added to adjust the pH to 2-3; and one in which formic acid was not added and the measured pH was 6-7.

[0067] The experimental results are as follows Figure 6 As shown, Figure 6 The details of the experimental groups are as follows: A1: Dilute with pure water; A2: Dilute with pure water and adjust pH to 2 with formic acid; B1: methanol precipitation of protein followed by dilution with pure water; B2: Methanol precipitation of protein followed by dilution with pure water, and adjustment of pH to 2 with formic acid; C1: acetonitrile precipitation of protein followed by dilution with pure water; C2: After protein precipitation with acetonitrile, dilute with pure water and adjust pH to 2 with formic acid; D1: protein precipitation with acetonitrile-methanol mixed solution followed by dilution with pure water; D2: After protein precipitation with acetonitrile-methanol mixed solution, it was diluted with pure water and the pH was adjusted to 2 with formic acid.

[0068] According to the experimental results, it can be seen that when the urine sample is directly diluted with water without adjusting the pH value, the peak shape is the best and the recovery rate is the highest.

[0069] This is because a certain proportion of organic phase still exists in the sample after protein precipitation from the organic phase. Toad tryptamine and dephosphorylated psilocybin are highly lipid-soluble and difficult to retain on the HLB column. Furthermore, since both are weakly alkaline alkaloids, when the sample diluent is alkaline or neutral, they exist primarily in molecular form in the solution, which improves adsorption capacity on the HLB column. However, when the sample diluent is acidic, they exist in the solution as ions, behaving like polar compounds. Charged ions easily interact with the solid-phase extraction column packing, resulting in poor peak shape and reduced recovery. Based on the investigation results, the final urine pretreatment choice was to directly dilute the sample with pure water.

[0070] Example 5 Methodology Verification This example verifies the detection method of the present invention, including determining the detection limit and quantification limit, precision and accuracy. The specific experimental process is as follows: (1) Standard curve, correlation coefficient, detection limit and quantification limit: A mixed standard solution of toad tryptamine and dephosphorylated psilocybin and an internal standard were added to a blank water sample to prepare a 0.002-0.1 ng / mL mixed standard solution. The method of the present invention (Example 1) was used for analysis. Three parallel experiments were performed for each experimental group (n=6). A standard curve was drawn with the mass concentration of the compound as the horizontal axis and the peak area ratio of the quantification ion to the internal standard as the vertical axis. The specific standard curve equation and linear correlation coefficient R 2 As shown in Table 3.

[0071] In accordance with the requirements for method detection limit determination in GB / T 27417-2017 “Guidelines for Validation and Verification of Chemical Analysis Methods for Conformity Assessment”, blank urine samples were spiked with standard solutions close to the minimum mass concentration. Three times the signal-to-noise ratio was determined as the detection limit, and 10 times the signal-to-noise ratio was determined as the quantification limit. The relevant results are shown in Table 4.

[0072] The experimental results showed that the linearity of toad tryptamine and dephosphorylated psilocybin was good in the concentration range of 0.002~0.1ng / mL, and R 2Both were greater than 0.99. The detection limits of toad tryptamine and dephosphopsilin in urine were 0.01 ng / mL and 0.03 ng / mL, respectively, and the quantitative limits were 0.03 ng / mL and 0.1 ng / mL, respectively.

[0073] Table 4 Linear equations, correlation coefficients, method detection limits, and quantification limits of toad tryptamine and dephosphopsilocybin in urine samples (2) Precision and accuracy: Mixed standard spiked samples of three different concentrations, low, medium and high, were added to blank urine samples. The spiked concentrations of toad tryptamine were 0.05 ng / mL, 0.1 ng / mL and 0.2 ng / mL; the spiked concentrations of dephosphopsis mushroom were 0.2 ng / mL, 0.5 ng / mL and 1.0 ng / mL.

[0074] The solutions of each concentration were pretreated by taking 0.5 mL of sample into a 15 mL centrifuge tube, adding 10 µL of internal standard solution, and diluting the volume to 10 mL with water. The sample was filtered through a 0.22 µm filter membrane and placed in a sample injection bottle for testing.

[0075] Three replicates were analyzed for each concentration level, and the average spiked recoveries and relative standard deviations were calculated, as shown in Table 5. The experimental results showed that the sample 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 requirements of GB / T 27417-2017, "Guidelines for Validation and Validation of Chemical Analysis Methods for Conformity Assessment."

[0076] Table 5 Recovery and relative standard deviation of spiked bufotryptamine and dephosphopsilocybin in urine samples (n=6) Example 6 Practical Application of the Detection Method This example used the method of the present invention to test urine samples from patients poisoned by ingesting wild mushrooms in Hubei Province from 2023 to 2024, and found no positive results. Based on the epidemiological survey results, this is because none of the poisoned individuals consumed hallucinogenic mushrooms.

[0077] 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 in the scope of protection of the present invention.

Claims

1. A method for detecting dephosphorylated psilocybin and toad tryptamine in urine samples, characterized in that: The urine samples were tested using the online SPE-LC-MS / MS method, where the liquid chromatography column was C filled with BEH. 18 Chromatographic column, solid phase extraction column is an online HLB column.

2. The method for detecting dephosphopsilin and toad tryptamine in urine according to claim 1, wherein: The specifications of the liquid chromatography column are 2.1 mm×100 mm, 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 dephosphopsilin and toad tryptamine in urine according to claim 2, wherein: 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 liquid chromatography flow rate is 0.15-0.25 mL / min, the column temperature is 35-45°C, and the injection volume is 1.5-2.5 mL. The liquid chromatography adopts gradient elution. Based on the total volume of the mobile phase A and the mobile phase B of the liquid chromatography being 100%, the elution procedure is as follows: 0.0-1.0 min, 5% mobile phase B; 1.0-4.0 min, 95% mobile phase B; 4.0-6.0 min, 95% mobile phase B; 6.0-8.0 min, 100% mobile phase B; 8.0-8.1 min, 5% mobile phase B; 8.1-10.0 min, 5% mobile phase B.

4. The method for detecting dephosphopsilin and toad tryptamine in urine according to claim 1, wherein: The specifications of the solid phase extraction column are 25µm, 2.1×20mm; the mobile phase A of the online solid phase extraction is pure aqueous solution, and the mobile phase B of the online solid phase extraction is methanol; Online solid phase extraction uses gradient elution. Taking the total volume of the mobile phase of online solid phase extraction as 100%, the elution procedure is as follows: 。 5. The method for detecting dephosphopsilin and toad tryptamine in urine 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 ion monitoring MRM; Capillary voltage: 3500(+) 2500(-); nozzle voltage: (+)1500, (-)1500; drying gas temperature: 250°C; drying gas flow rate: 15 L / min; nebulizer pressure: 35 psi; sheath gas temperature: 350°C; sheath gas flow rate: 11 L / min.

6. The method for detecting dephosphopsilin and toad tryptamine in urine according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1. Pre-treating the urine sample to be tested, and adding a mixed internal standard solution containing dephosphopsilin and toad tryptamine as internal standards; S2. performing the online SPE-LC-MS / MS test on the urine sample after the pretreatment; S3. Calculate the contents of dephosphopsilin and toad tryptamine in the urine sample according to the peak area values of the dephosphopsilin internal standard and the toad tryptamine internal standard obtained by detection.

7. The method for detecting dephosphopsilin and toad tryptamine in urine 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 dephosphopsilin and toad tryptamine in urine according to claim 7, wherein: The urine sample to be tested is 0.5 mL, and the total volume of the solution after constant volume is 5-15 mL.

9. The method for detecting dephosphopsilin and toad tryptamine in urine according to claim 7, wherein: The mixed internal standard solution is dephosphorylated psilocybin-d 10 The solution is prepared with toad tryptamine-d4 hydrochloride solution.

10. The method for detecting dephosphopsilocybin and toad tryptamine 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 toad tryptamine-d4 is 10-50 ng / mL.

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