A method for detecting norfentanyl in urine

Abstract

The present invention provides a detection method for norfentanyl in urine. The present invention uses ligand TP-CF3-COOH and metal Cu to prepare an organic metal framework to prepare a portable fluorescent test paper, which can be applied to the detection of norfentanyl in urine. For sudden on-site rapid detection needs, the instrument equipment only needs fluorescent test paper, an ultraviolet flashlight and a mobile phone, the reagent pack only needs acetonitrile and sodium chloride, and the overall detection time is short. The minimum detection limit of norfentanyl in the detection method of the present invention is 0.532 ng / L, and the detection range is 1 ng / L-100 ng / L. The detection method of the present invention only requires a simple material package and an ultraviolet lamp. The fluorescent color RGB value is tested by a mobile phone APP or other smart device. After comparison with the standard curve, the concentration of norfentanyl in the urine sample can be obtained quickly without the need for large-scale equipment.

Description

Technical Field

[0001] The invention belongs to the technical field of norfentanyl detection, and particularly relates to a method for detecting norfentanyl in urine. Background Art

[0002] Fentanyl, a potent opioid sedative, plays an important role in medical applications, including sedatives, analgesics, and veterinary sedation. However, its potent potency also carries a high risk of abuse. After entering the body, fentanyl is rapidly and extensively metabolized primarily by the cytochrome P450 family of metabolizing enzymes (CYP3A4) in the liver, with a biological half-life of 2-4 hours. The primary metabolite undergoes N-dealkylation to produce the inactive norfentanyl. Approximately 75% of fentanyl is excreted in the urine within 24 hours, primarily as norfentanyl. This suggests that testing for norfentanyl can effectively complement fentanyl detection.

[0003] Currently, most experimental methods focus on the qualitative and quantitative detection of fentanyl. However, technical approaches for detecting norfentanyl, such as liquid chromatography-mass spectrometry (LC-MS / MS), often require complex pretreatment methods and long detection times, resulting in inadequate results for some field samples. Furthermore, kits based on the principle of specific antigen-antibody binding can also be used for norfentanyl detection, but these methods are primarily designed for simple matrices and are prone to false positives in complex matrices, making them incapable of accurate qualitative and quantitative detection.

[0004] The inventor's previous patent CN116514651A reported an amphiphilic aggregation-induced luminescence material fluorescent probe, which uses the compound TP-CF3-COOH and can be used for the detection of norfentanyl in drinking water and serum. However, this detection can only be performed in an aqueous solution with a simple solvent or a serum solution with fewer interfering substances, which places extremely high demands on the sample itself for on-site rapid detection. At the same time, this fluorescent probe is a fluorescence detection method that is carried out by intermolecular charge transfer and aggregation state changes between fentanyl and norfentanyl, resulting in fluorescence quenching and fluorescence blue shift. This quenching mode fluorescent probe detection method is susceptible to complex sample background interference. Therefore, this method cannot be used for urine samples that are more easily obtained. This is because the background interference of urine samples is strong, and there are extremely strong requirements for the anti-interference ability and specific enrichment ability of the detection method.

[0005] To overcome the limitations of the monomeric TP-CF3-COOH material in detecting norfentanyl in complex samples, several approaches are possible. 1. Complex sample pretreatment can be performed to remove analyte impurities, but this method increases the complexity and time of the pretreatment process and, because the material's inherent anti-interference ability remains unchanged, results in poor results. 2. More complex modifications can be made to existing material groups. This approach is time-consuming, and no relevant anti-interference groups have been reported, requiring long-term exploration. 3. According to literature reports, fluorescence on-mode detection has higher anti-interference capabilities than fluorescence quenching detection. Therefore, considering constructing TP-CF3-COOH and other materials into an on-mode fluorescence detection mode, this approach is difficult. However, the difficulty lies in the strong fluorescence characteristics of aggregation-induced emission materials under restricted intramolecular motion (RIM). Therefore, whether it can be constructed into a material with an initial dark fluorescence state and successfully illuminated upon addition of the analyte is very difficult. Although there have been reports of fluorescent detection methods that combine AIE molecules with metals to create a light-on mode, there have been no reports of using MOF materials prepared by combining TP-CF3-COOH as a monomer with a metal for the detection of norfentanyl. The synthesis of dark-state MOFs also faces challenges: the pore structure causes AIE monomers to aggregate, leading to a strong fluorescence mode; and whether a regular MOF crystal structure can be formed after the AIE monomers combine with the metal. Furthermore, according to existing mechanisms, the detection of fentanyl and norfentanyl by TP-CF3-COOH monomers is based on spatially specific binding, molecular conformational changes, and intermolecular charge transfer. Therefore, for MOF materials, the main mechanisms that may be effective are spatially specific binding and intermolecular charge transfer. Since intermolecular charge transfer and metal charge transfer compete, whether this pathway can be opened up and whether an ON-OFF state can occur is also unpredictable. CN115707954A discloses a SERS substrate based on MOF materials for the quantitative detection of fentanyl. This method uses deuterated fentanyl as an internal standard and can quantitatively detect the fentanyl content in complex biological samples. The substrate comprises NH2-MIL-101 / AuNPs, obtained by co-incubating NH2-MIL-101 and HAuCl4. The patented mechanism is not a direct detection and signal conversion between the MOF and the analyte. Instead, the instrument has a specific detection fingerprint for fentanyl. The MOF material, after mixing with the analyte, allows the analyte to adhere better to the detection substrate, thereby enhancing the signal of existing SERS detection methods. However, the SERS instrument must be carried during the detection process, which is inconvenient for sudden on-site detection needs. Summary of the Invention

[0006] In order to solve the disadvantages of the existing technology for the detection of norfentanyl in urine, the detection effect is easily interfered by complex matrices, and the detection results are not ideal, the present invention proposes a detection method for norfentanyl in urine based on a metal organic framework material of aggregation-induced luminescence material. By preparing a portable fluorescent test paper, it can be applied to the detection of norfentanyl in urine, providing new technical support and support for the on-site rapid detection of norfentanyl. For the demand for sudden on-site rapid detection, the instrument and equipment only need fluorescent test paper, a UV flashlight with its own battery, and its own mobile phone. The reagent package only needs acetonitrile and sodium chloride, and the overall detection time is short. Specifically, the present invention provides the following technical solutions to achieve the above purpose:

[0007] A method for detecting norfentanyl in urine comprises the following steps:

[0008] (S1) The ligand TP-CF3-COOH and a water-soluble copper source are prepared under acidic conditions to form an organic metal framework Cu@TP-CF3-COOH, wherein the structure of TP-CF3-COOH is shown in the following formula (I):

[0009] (I);

[0010] (S2) adding the organic metal framework to a solvent to obtain a fluorescent probe stock solution; immersing the carrier in the fluorescent probe stock solution and drying the carrier to obtain a fluorescent probe test paper;

[0011] (S3) dropping the treated norfentanyl standard urine solutions of different concentrations onto fluorescent probe test paper, air-drying, and irradiating with ultraviolet light, and establishing a standard curve based on the G / R value of the photograph and the change in norfentanyl concentration;

[0012] (S4) dropping the treated urine sample onto a fluorescent probe test paper, air-drying the sample, and then irradiating the sample with ultraviolet light. The concentration of norfentanyl in the urine sample is calculated based on the G / R value and the standard curve.

[0013] Furthermore, in step (S1), the water-soluble copper source is selected from at least one of copper nitrate, copper chloride, and copper sulfate; the molar ratio of TP-CF3-COOH to the water-soluble copper source is 3-5:1, and the water-soluble copper source is calculated as Cu. Preferably, the molar ratio of TP-CF3-COOH to the water-soluble copper source is 3-3.5:1.

[0014] Furthermore, in step (S1), the reaction medium is selected from at least one of water, DMF, DMSO, THF, and ethyl acetate; and the acidic condition is to add nitric acid to make the system pH 2-4.

[0015] Furthermore, in step (S1), the preparation conditions are to heat to 50-90°C for 10-30h, preferably to 60-80°C for 15-20h. After the reaction, blue-green crystals are formed, which are the organic metal framework. After the crystals are washed, they are dried to obtain the organic metal framework. There is no particular limitation on washing and drying, for example, washing with at least one of DMF, DMSO, and ethanol, and drying is vacuum drying.

[0016] Furthermore, in step (S2), the solvent is selected from at least one of acetonitrile and THF, the concentration of the organic metal framework in the fluorescent probe stock solution is 0.01-1 mg / mL, preferably 0.1-0.3 mg / mL, and the carrier is at least one of filter paper and cellulose paper. There are no specific restrictions on the soaking time and drying time, for example, soaking time is 10-30 minutes, and drying is performed in a constant temperature oven at 30-40°C.

[0017] Furthermore, in steps (S3) and (S4), the standard urine solutions of different concentrations of norfentanyl are treated, and the urine sample to be tested is treated by uniformly mixing the urine sample, an organic solvent, and a salt, allowing the mixture to stand for salting out and stratification, and collecting the supernatant. The organic solvent is selected from at least one of acetonitrile and tetrahydrofuran, and the salt is selected from at least one of sodium chloride, potassium chloride, sodium nitrate, sodium sulfate, potassium nitrate, and potassium sulfate; and the ratio of urine sample, organic solvent, and salt is 0.5-1 mL: 0.5-1 mL: 0.5-1 g.

[0018] Furthermore, in step (S4), the urine sample to be tested is selected from human urine, livestock urine (cow urine, horse urine, sheep urine, pig urine); and the incubation condition is 23-30°C.

[0019] The present invention also provides the use of Cu@TP-CF3-COOH in the detection of norfentanyl in urine samples. Cu@TP-CF3-COOH is an organic metal framework made of a water-soluble Cu source and TP-CF3-COOH. The chemical structure of TP-CF3-COOH is shown in the following formula (I):

[0020] (I).

[0021] The present invention also provides a kit for on-site, rapid detection of norfentanyl in urine, comprising a fluorescent probe test paper, a centrifuge tube (Eppendorf tube), a solvent, and packaged sodium salt; the fluorescent probe test paper is prepared by a preparation method comprising the following steps:

[0022] (S1) The ligand TP-CF3-COOH and a water-soluble copper source are prepared under acidic conditions to form an organic metal framework Cu@TP-CF3-COOH, wherein the structure of TP-CF3-COOH is shown in the following formula (I):

[0023] (I);

[0024] (S2) adding the organic metal framework to a solvent to obtain a fluorescent probe stock solution; immersing the carrier in the fluorescent probe stock solution and drying the carrier to obtain a fluorescent probe test paper.

[0025] Furthermore, the sodium salt is selected from at least one of sodium chloride, sodium sulfate, and sodium nitrate, and the solvent is selected from at least one of acetonitrile and tetrahydrofuran.

[0026] The present invention also provides a method for on-site, rapid detection of norfentanyl in urine, comprising the following steps: taking a urine sample and adding it to a centrifuge tube, adding a solvent and a sodium salt, standing for salting out, dripping the supernatant onto filter paper, air-drying, taking a photograph of the test paper under ultraviolet light, etc., obtaining the RGB value of the test paper photograph, calculating the G / R value, and comparing it with a standard curve to obtain the norfentanyl concentration in the urine sample.

[0027] Furthermore, the wavelength of the ultraviolet light is 300-400 nm, such as 365 nm. The ratio of urine, solvent and sodium salt is 0.5-1 mL: 0.5-1 mL: 0.05-0.1 g.

[0028] By incorporating TP-CF3-COOH and Cu as support for an organic metal framework, the present invention significantly improves the sensitivity of a fluorescent probe for detecting norfentanyl in urine samples. The detection method achieves a minimum detection limit of 0.532 ng / L for methylfentanyl. The method requires only a simple material kit and a UV lamp. By measuring the RGB values ​​of the fluorescent color using a mobile phone app or other smart device and comparing them with a standard curve, the concentration of norfentanyl in a urine sample can be rapidly determined without the need for extensive equipment or instrumentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a scanning electron microscope photograph of Cu@TP-CF3-COOH obtained in Example 1;

[0030] Figure 2 This is the SEM energy spectrum scan of Cu@TP-CF3-COOH obtained in Example 1;

[0031] Figure 3 is the FT-IR spectrum of Cu@TP-CF3-COOH obtained in Example 1;

[0032] Figure 4 is the X-ray diffraction spectrum of Cu@TP-CF3-COOH obtained in Example 1;

[0033] Figure 5 is the nitrogen adsorption-desorption isotherm and pore size distribution of Cu@TP-CF3-COOH obtained in Example 1;

[0034] Figure 6 is the fluorescence spectrum of Cu@TP-CF3-COOH obtained in Example 1;

[0035] Figure 7 This is a graph of the Cu@TP-CF3-COOH fluorescent test paper coated with different standard concentrations of norfentanyl urine solution in Example 1;

[0036] Figure 8 Norfentanyl urine solution standard curve in Example 1;

[0037] Figure 9 The middle is a schematic diagram of a portable material package containing fluorescent probe test strips for rapid urine sample testing using fluorescence;

[0038] Figure 10 These are digital images of Cu@TP-CF3-COOH of Example 1, Co@TP-CF3-COOH of Comparative Example 1, and Mg@TP-CF3-COOH of Comparative Example 2. DETAILED DESCRIPTION

[0039] The present invention is described in detail below with reference to specific embodiments.

[0040] The urine was sourced from cattle urine at Ningcheng Ranch in Inner Mongolia (obtained in accordance with animal welfare requirements).

[0041] Example 1

[0042] (1) Preparation of Cu@TP-CF3-COOH

[0043] (1) Preparation of TP-CF3-COOH: The synthesis of the ligand molecule TP-CF3-COOH can be prepared by referring to the method in patent CN202310744554.1.

[0044] (2) Preparation of Cu@TP-CF3-COOH: TP-CF3-COOH (105 mg, 0.18 mmol) obtained in step (1) and Cu(NO3)2·3H2O (153 mg, 0.63 mmol) were dissolved in a DMF / H2O solution (50 mL, 4:1) and acidified with concentrated HNO3 to pH 2. The solution was heated at 80°C for 18 h to form blue-green crystals. The crystals were washed five times with 20 mL of DMF by centrifugation and then three times with 15 mL of ethanol. After washing, the crystals were dried at 60°C in a vacuum vacuum (209 mg, 81%) for use. The resulting organic metal framework was expressed as Cu@TP-CF3-COOH.

[0045] Figure 1This is a scanning electron microscope image of the Cu@TP-CF3-COOH obtained in Example 1. It can be seen that it exhibits a uniform columnar three-dimensional structure. The diameter of a single columnar structure is about 250-300 nm, the length is between 1.6-2.3 μm, and the surface is smooth.

[0046] Figure 2 This is a SEM scan of the Cu@TP-CF3-COOH obtained in Example 1. The element contents are shown in Table 1 below. This shows that the material is composed of carbon, hydrogen, oxygen, fluorine, and copper, and is evenly distributed.

[0047] Table 1 Element content of Cu@TP-CF3-COOH

[0048] .

[0049] Figure 3 This is the FT-IR graph of Cu@TP-CF3-COOH obtained in Example 1. The reactants Cu(NO3)2·H2O and TP-CF3-COOH and the product Cu@TP-CF3-COOH were analyzed by Fourier transform infrared spectroscopy: Figure 4 It can be seen that Cu@TP-CF3-COOH is at 526cm -1 Compared with TP-CF3-COOH, the vibration peak of Cu@TP-CF3-COOH at 1397 cm -1 -COO appears - The symmetrical stretching vibration peak of -COO at 1550 - The asymmetric stretching vibration peaks indicate that the copper ion in Cu@TP-CF3-COOH forms a planar tetragonal coordination environment with two carboxyl oxygen atoms from the TP-CF3-COOH ligand and two water molecules. Furthermore, the disappearance of the -OH peak of the TP-CF3-COOH ligand at 2600-2800 nm, along with the disappearance of the Cu(NO3)2·H2O water peak at 3400 nm, confirm the successful synthesis of Cu@TP-CF3-COOH.

[0050] Figure 4 This is the X-ray diffraction spectrum of Cu@TP-CF3-COOH obtained in Example 1. It can be seen that the prepared Cu@TP-CF3-COOH has high crystallinity. The carboxyl ligand of TP-CF3-COOH has a large spatial occupancy, so a strong (111) peak is generated at 4.8°. In addition, the 9.6° (222) peak and the 12.08° (220) peak indicate that TP-CF3-COOH has spatial coordination of copper ions. These results indicate that the material has good crystallinity.

[0051] Figure 5The nitrogen adsorption-desorption isotherm and pore size distribution of Cu@TP-CF3-COOH obtained in Example 1 are shown. Figure 5 A is the nitrogen adsorption-desorption isotherm. The results show that the BET surface area of ​​Cu@TP-CF3-COOH is 492.6050 m² / g. This indicates that the Cu@TP-CF3-COOH material has a loose and porous structure. Figure 5 B is the pore size distribution diagram. The average diameter of Cu@TP-CF3-COOH obtained in Example 1 is 17.95 Å.

[0052] Figure 6 This is the fluorescence spectrum of Cu@TP-CF3-COOH obtained in Example 1. This demonstrates that a significant fluorescence spectrum can be detected when 20 μg / L of norfentanyl is added, whereas no fluorescence intensity is observed in a pure Cu@TP-CF3-COOH solution. This demonstrates that the Cu@TP-CF3-COOH material can be used in norfentanyl detection experiments.

[0053] (2) Establishing a standard working curve

[0054] (1) Preparation of Cu@TP-CF3-COOH fluorescent probe stock solution:

[0055] 1 mg of Cu@TP-CF3-COOH prepared in Example 1 was added to 10 mL of acetonitrile. After the solid was completely dissolved, ultrasonic treatment was performed at room temperature for 30 minutes to obtain a 100 mg / L fluorescent probe stock solution.

[0056] (2) Preparation of Cu@TP-CF3-COOH portable fluorescent test paper

[0057] A circular filter paper with a diameter of 1.5 cm was immersed in 20 mL of the above-mentioned stock solution for 10 minutes, then taken out and dried in a constant temperature oven at 37°C for 6 hours.

[0058] (3) Preparation of norfentanyl standard solution:

[0059] Prepare norfentanyl standard solutions using urine to prepare standard solutions with norfentanyl concentrations of 0 ng / L, 1 ng / L, 20 ng / L, 40 ng / L, 60 ng / L, 80 ng / L, and 100 ng / L;

[0060] (4) Establish a standard working curve:

[0061] Urine sample pretreatment: Take 0.5mL (3) standard solution urine sample, add 0.5mL acetonitrile, vortex for 1 minute, then add 1g sodium chloride, vortex for 10 seconds, and then take the supernatant. Take 6 pieces of (2) prepared circular filter paper for fluorescence detection, showing no obvious fluorescence. Then take 5μL of the treated (3) urine sample and titrate it onto the circular filter paper respectively, wait for about 30 seconds to dry naturally. Take a photo of the test paper under 365nm ultraviolet light, as shown Figure 7 Then, the fluorescence color RGB value obtained by mobile phone photos was used to calculate G / R, and a standard curve was established with norfentanyl concentration as the horizontal axis and G / R value as the vertical axis, as shown in the figure below. Figure 8 As shown in the figure, there is a good linear relationship between the G / R of the fluorescent test paper and the concentration of norfentanyl, which is Y=0.1143x+2.8867, R 2 =0.998. Based on the standard deviation δ = 2.03% obtained from 10 measurements, the minimum detection limit of this probe for p-methylfentanyl was calculated to be 0.532 ng / L.

[0062] Example 2

[0063] The present invention provides Cu@TP-CF3-COOH, which can be used as a material for rapid on-site detection of norfentanyl in urine samples. Figure 9 The diagram below shows a portable kit containing fluorescent probe test strips for rapid urine testing using fluorescence. The kit includes 0.5 mL of acetonitrile solution in a 1.5 mL EP tube, 1 g of pre-packaged sodium chloride, a standard UV lamp, and fluorescent test strips loaded with Cu@TP-CF3-COOH. The kit can be pre-packaged in 8 x 15 cm ziplock bags and stored long-term at room temperature.

[0064] Place 20 μL of a 1000 μg / mL norfentanyl standard solution in a vial and purge with nitrogen for 5 minutes. Then, add urine to a volume of 2 mL and mix thoroughly by vortexing for 1 minute. Add 20 μL of this solution to a volume of 8 mL and vortex for 30 seconds. Finally, add 100 μL of this solution to a volume of 1 mL. This solution is intended to be used as a field urine sample.

[0065] For sudden on-site testing, take a 0.5 mL urine sample and add it to an EP tube containing 0.5 mL of acetonitrile. After mixing, add 1 g of sodium chloride. After standing for salting out, take 5 μL of the supernatant and titrate it onto a circular filter paper. Allow to air-dry for approximately 30 seconds. Take a photo of the test paper under a 365 nm UV lamp. Then, use a mobile phone to capture the RGB values ​​of the fluorescent color, calculate the G / R value, and compare it to the standard curve obtained in Example 1 (ii). The content of norfentanyl in the urine sample is 24.96 ng / L. It can be seen that the method of detecting norfentanyl in urine of the present invention does not require the use of large-scale equipment or instruments. Only the above-mentioned material package, a UV lamp, and a mobile phone capable of identifying RGB values ​​are required for rapid, on-site quantitative detection of norfentanyl in urine samples. On-site emergencies urgently require on-site testing to determine the possible cause in the shortest possible time. The kit is easy to use, allowing on-site testing personnel to promptly identify problems and initiate emergency response. It also allows ordinary people, after simple training, to quickly determine whether their own emergencies are due to accidental exposure to fentanyl and thus help themselves. Therefore, highly selective and sensitive urine detection for norfentanyl is crucial for on-site screening and emergency care.

[0066] Comparative Example 1

[0067] Other conditions and operations are the same as those in Example 1, except that in step (1) (2), Cu(NO3)2 is replaced by an equimolar amount of Co(NO3)2.

[0068] Comparative Example 2

[0069] Other conditions and operations are the same as those in Example 1, except that in step (1) (2), Cu(NO3)2 is replaced by an equimolar amount of Mg(NO3)2.

[0070] Figure 10 These are digital images of Cu@TP-CF3-COOH of Example 1, Co@TP-CF3-COOH of Comparative Example 1, and Mg@TP-CF3-COOH of Comparative Example 2.

[0071] The inventors discovered that among MOFs constructed from TP-CF3-COOH and various metals, only Cu@TP-CF3-COOH can achieve a non-fluorescent state in the solid state and then illuminate upon addition of a sample containing norfentanyl. Therefore, it can be used for the detection of norfentanyl in urine samples based on aggregation-induced emission (AIEL). To overcome the limitations of TP-CF3-COOH in detecting norfentanyl in complex samples, we considered using TP-CF3-COOH as a luminescent monomer and combining it with other systems to construct an on-mode fluorescence detection method. The complex construction process requires consideration of the aggregation-induced emission properties of TP-CF3-COOH itself and the mechanism of norfentanyl detection: a triple cooperative mechanism involving spatially specific binding, conformational changes, and intermolecular charge transfer. The ultimate goal is to have the complex initially dark and then trigger illumination based on the detection mechanism, which is extremely difficult to achieve. Although AIE materials found in MOF materials in the literature can be used for light-up detection, there are no reports on the use of MOF materials prepared by combining TP-CF3-COOH as a monomer with a metal for the detection of norfentanyl. We speculate that after TP-CF3-COOH is made into a MOF material, the metal charge transfer may cause the composite material to be in a dark state. After the addition of norfentanyl, the intermolecular charge transfer is formed, competing with the metal charge transfer, opening up the original aggregation-induced fluorescence pathway, thereby causing the ON-OFF reaction. The metal charge transfer composed of the Cu group can realize the dark state of the aggregation-induced luminescence material in the MOF structure, and based on this, it is prepared as a portable fluorescent test paper. Subsequent experiments also verified that it can be used for the light-up detection of norfentanyl in complex matrices such as urine.

[0072] Comparative Example 3

[0073] Other conditions and operations are the same as in Example 1, with the difference being that TP-CF3-COOH is used instead of Cu@TP-CF3-COOH as a fluorescent probe. It was found that the detection of norfentanyl in urine samples could not be performed because the fluorescence spectrum did not change significantly in proportion to the salt environment in the urine solution. The inventors previously reported in patent CN116514651A a fluorescent probe based on amphiphilic aggregation-induced emission material, which uses the compound TP-CF3-COOH and can be used to detect norfentanyl in drinking water and serum. However, this detection can only be performed in aqueous solutions with simple solvents or serum solutions with fewer interfering substances, which places extremely high demands on the sample itself for on-site rapid detection. At the same time, this fluorescent probe is a fluorescence detection method that is performed by intermolecular charge transfer and aggregation state changes between fentanyl and norfentanyl, resulting in fluorescence quenching and fluorescence blue shift. This quenching mode fluorescent probe detection method is susceptible to interference from complex sample backgrounds. Therefore, this method cannot be used for urine samples that are easier to obtain. This is because the background interference of urine samples is strong, and it places extremely high demands on the anti-interference ability and specific enrichment ability of the detection method.

Claims

1. A method for detecting norfentanyl in urine, characterized in that: The following steps are involved: (S1) The ligand TP-CF3-COOH and a water-soluble copper source are prepared under acidic conditions to form an organic metal framework Cu@TP-CF3-COOH, wherein the structure of TP-CF3-COOH is shown in the following formula (I): (I) (S2) adding the organic metal framework to a solvent to obtain a fluorescent probe stock solution; immersing the carrier in the fluorescent probe stock solution and drying the carrier to obtain a fluorescent probe test paper; (S3) dropping the treated norfentanyl standard urine solutions of different concentrations onto fluorescent probe test paper, air-drying, and irradiating with ultraviolet light, and establishing a standard curve based on the G / R value of the photograph and the change in norfentanyl concentration; (S4) dropping the treated urine sample onto a fluorescent probe test paper, air-drying the sample, and then irradiating the sample with ultraviolet light. The concentration of norfentanyl in the urine sample is calculated based on the G / R value and the standard curve.

2. The detection method according to claim 1, wherein In step (S1), the water-soluble copper source is selected from at least one of copper nitrate, copper chloride, and copper sulfate; the molar ratio of TP-CF3-COOH to the water-soluble copper source is 3-5:1, and the water-soluble copper source is calculated as Cu.

3. The detection method according to claim 2, characterized in that In steps (S3) and (S4), the treatment is to mix the urine sample, organic solvent, and salt evenly, let it stand for salting out and stratification, and take the supernatant; the organic solvent is selected from at least one of acetonitrile and tetrahydrofuran, and the salt is selected from at least one of sodium chloride, potassium chloride, sodium nitrate, sodium sulfate, potassium nitrate, and potassium sulfate; the ratio of urine sample, organic solvent, and salt is 0.5-1 mL: 0.5-1 mL: 0.5-1 g.

4. The detection method according to claim 1, wherein In step (S1), the reaction medium is selected from at least one of water, DMF, DMSO, THF, and ethyl acetate; and the acidic condition is to add nitric acid to make the system pH 2-4.

5. The detection method according to claim 1, wherein In step (S1), the preparation condition is to heat to 50-90°C for 10-30h.

6. The detection method according to claim 1, characterized in that In step (S2), the solvent is selected from at least one of acetonitrile and THF, the concentration of the organic metal framework in the fluorescent probe stock solution is 0.01-1 mg / mL; and the carrier is at least one of filter paper or cellulose paper.

7. The detection method according to claim 6, characterized in that The concentration of the organic metal framework in the fluorescent probe stock solution is 0.1-0.3 mg / mL.

8. The detection method according to claim 1, wherein In step (S4), the urine sample to be tested is selected from human urine and livestock urine; and the incubation condition is 23-30°C.

9. The detection method according to claim 8, characterized in that Livestock urine is cow urine, horse urine, sheep urine or pig urine.

10. Use of Cu@TP-CF3-COOH in the detection of norfentanyl in urine samples, characterized in that: Cu@TP-CF3-COOH is an organic metal framework made of a water-soluble Cu source and TP-CF3-COOH. The chemical structure of TP-CF3-COOH is shown in the following formula (I): (I)。

Images