Method for detecting norfentanyl in urine

Abstract

The invention provides a method for detecting norfentanyl in urine, which is characterized in that a ligand TP-CF3-COOH and metal Cu are used for preparing an organic metal framework to prepare portable fluorescent test paper, and the portable fluorescent test paper can be applied to detection of norfentanyl in urine. For the sudden field rapid detection requirement, instruments and equipment only need fluorescent test paper, an ultraviolet flashlight and a mobile phone, a reagent bag only needs acetonitrile and sodium chloride, and the overall detection time is short. According to the detection method disclosed by the invention, the lowest detection limit of mefentanyl is 0.532 ng / L, and the detection range is 1 ng / L-100 ng / L. According to the detection method disclosed by the invention, the fluorescence color RGB value is tested through a mobile phone APP or other intelligent equipment by virtue of a simple material bag and an ultraviolet lamp, and after comparison with a standard curve, the concentration of the norfentanyl in the urine sample can be quickly obtained without large-scale equipment and instruments.

Description

Technical Field

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

[0002] As a potent opioid sedative, fentanyl plays an important role in the medical field including sedatives, analgesics, and veterinary sedatives. However, its powerful potency also brings a very high risk of abuse. After fentanyl enters the human body, it is mainly metabolized rapidly and extensively by the cytochrome P450 family of metabolic enzymes (CYP3A4) in the liver, with a biological half-life of 2 - 4 h. The main metabolite is generated by N-demethylation to form inactive norfentanyl. Approximately 75% of fentanyl is mainly excreted in urine within 24 h in the form of norfentanyl. This indicates that detecting norfentanyl can effectively complement the deficiencies in fentanyl detection.

[0003] Currently, the focus of most experimental methods is on the qualitative and quantitative detection of fentanyl. For the technical means of detecting norfentanyl, such as liquid chromatography-tandem mass spectrometry (LC-MS / MS), most require complex pretreatment methods and a long overall detection time, and it is impossible to obtain timely result feedback for some on-site samples. Secondly, test kits based on the principle of specific binding of antigen and antibody can also be used for the detection of norfentanyl, but this method is mostly used for simple matrices and is prone to false positives in complex matrices, and accurate qualitative and quantitative detection cannot be carried out.

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

[0005] In order to overcome the limitations of the monomer TP-CF3-COOH material in the detection of norfentanyl in complex samples, the following methods can be used. 1. The impurities of the analyte can be removed by performing complex sample pretreatment, but this method will increase the complexity and time of the pretreatment process and the anti-interference ability of the material itself has not changed, resulting in poor results; 2. More complex modifications to the existing material groups. This method has a long cycle, and the relevant anti-interference groups have not been reported, requiring long-term exploration. 3. According to literature reports, the detection of the fluorescence on-mode has higher anti-interference ability than the detection of fluorescence quenching. Therefore, it is considered to construct TP-CF3-COOH and other materials into an on-mode fluorescence detection mode, but the difficulty of this method lies in the strong fluorescence characteristics of the aggregation-induced luminescence material under the condition of intramolecular motion restriction (RIM). Therefore, it is very difficult to construct it as a material in the initial fluorescent dark state and successfully light it up after adding the analyte. Although there have been reports of fluorescent detection methods that combine AIE molecules with metals to construct a light-on mode, there are no reports on the use of MOF materials prepared by combining TP-CF3-COOH as a monomer and metal for the detection of norfentanyl. The synthesis of dark-state MOF also faces the difficulties of the pore structure causing the AIE monomers to aggregate and trigger the strong fluorescence mode and whether the AIE monomers can form a regular MOF crystal structure after combining with metals. In addition, according to the existing mechanism, the detection of fentanyl and norfentanyl by TP-CF3-COOH monomers is based on spatial specific binding, molecular conformational changes and intermolecular charge transfer. For MOF materials, the main mechanism that may be effective is spatial specific binding and intermolecular charge transfer, and intermolecular charge transfer and metal charge transfer are competitive. Whether the path can be opened and whether ON-OFF can occur is also unpredictable. CN115707954A discloses a SERS substrate based on MOF materials for quantitative detection of fentanyl. The method uses deuterated fentanyl as an internal standard and can quantitatively detect the content of fentanyl in complex biological samples. The substrate includes NH2-MIL-101 / AuNPs obtained by co-incubation of NH2-MIL-101 and HAuCl4. The mechanism of this patent is not that MOF and the analyte directly detect and convert signals, but that the instrument has a specific detection fingerprint for fentanyl. The role of the MOF material is to make the analyte better adhere to the detection substrate after mixing with the analyte, thereby achieving signal enhancement for the existing SERS detection method. During the detection process, it is necessary to carry a SERS instrument for detection, which is not convenient for sudden on-site detection needs. Summary of the invention

[0006] To address the disadvantages in the detection of norfentanyl in urine in the prior art, where the detection effect is easily interfered by complex matrices and the detection results are not satisfactory, the present invention proposes a method for detecting norfentanyl in urine based on metal-organic framework materials of aggregation-induced emission materials. By preparing a portable fluorescence test strip, it can be applied to the detection of norfentanyl in urine, providing a new technical support and reliance for the on-site rapid detection of norfentanyl. For the on-site rapid detection requirements of emergencies, only a fluorescence test strip, an ultraviolet flashlight with a built-in battery, and a mobile phone are needed for the instrument and equipment, and only acetonitrile and sodium chloride are needed for the reagent kit, and the overall detection time is short. Specifically, the present invention provides the following technical solutions to achieve the above object: A method for detecting norfentanyl in urine, comprising the following steps: (S1) Prepare an organometallic framework Cu@TP-CF3-COOH from a ligand TP-CF3-COOH and a water-soluble copper source under acidic conditions. The structure of TP-CF3-COOH is shown in the following formula (I): (I); (S2) Add the organometallic framework to a solvent to obtain a stock solution of a fluorescent probe; soak a carrier in the stock solution of the fluorescent probe and dry it to obtain a fluorescent probe test strip; (S3) Drop different concentrations of norfentanyl standard urine solutions treated onto the fluorescent probe test strip, air-dry it, and then irradiate it with an ultraviolet lamp. Establish a standard curve based on the G / R value of the photo and the change in norfentanyl concentration; (S4) Drop the treated urine sample to be tested onto the fluorescent probe test strip, air-dry it, and then irradiate it with an ultraviolet lamp. Calculate the concentration of norfentanyl in the urine sample to be tested based on the G / R value and the standard curve.

[0007] Further, 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, with the water-soluble copper source calculated as Cu. Preferably, the molar ratio of TP-CF3-COOH to the water-soluble copper source is 3-3.5:1.

[0008] Further, in step (S1), the reaction medium is selected from at least one of water, DMF, DMSO, THF, and ethyl acetate; the acidic condition is to add nitric acid to make the pH of the system 2-4.

[0009] Further, in step (S1), the preparation conditions are heating at 50 - 90 °C for 10 - 30 h, preferably heating at 60 - 80 °C for 15 - 20 h. After the reaction, blue-green crystals are formed, which are the metal-organic framework. After the crystals are washed and dried, the metal-organic framework is obtained. There are no special limitations on washing and drying. For example, washing is performed with at least one of DMF, DMSO, and ethanol, and drying is vacuum drying.

[0010] Further, in step (S2), the solvent is selected from at least one of acetonitrile and THF, the concentration of the metal-organic framework in the fluorescent probe stock solution is 0.01 - 1 mg / mL, preferably 0.1 - 0.3 mg / mL; the carrier is at least one of filter paper and cellulose paper. There are no special limitations on the soaking time and drying. For example, soaking is for 10 - 30 min, and drying is in an oven at a constant temperature of 30 - 40 °C.

[0011] Further, in steps (S3) and (S4), for the different-concentration norfentanyl standard urine solutions and the urine samples to be tested, the treatment is to mix the urine samples, organic solvents, and salts evenly, and then let them 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 the urine sample, organic solvent, and salt is 0.5 - 1 mL : 0.5 - 1 mL : 0.5 - 1 g.

[0012] Further, in step (S4), the urine sample to be tested is selected from human urine and livestock urine (cattle urine, horse urine, sheep urine, pig urine); the incubation conditions are incubation at 23 - 30 °C.

[0013] 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 a metal-organic framework made from a water-soluble Cu source and TP-CF3-COOH, and the chemical structure of TP-CF3-COOH is shown in the following formula (I): (I).

[0014] The present invention also provides a kit for on-site and rapid detection of norfentanyl in urine, a fluorescent probe test strip, a centrifuge tube (Eppendorf tube), a solvent, and a sodium salt in aliquots; the fluorescent probe test strip is prepared by a preparation method including the following steps: (S1) Prepare the metal-organic framework Cu@TP-CF3-COOH from the ligand TP-CF3-COOH and a water-soluble copper source under acidic conditions. The structure of TP-CF3-COOH is shown in the following formula (I): (I); (S2) Add the metal-organic framework into a solvent to obtain a stock solution of the fluorescent probe; soak the carrier in the stock solution of the fluorescent probe and then dry it to obtain a fluorescent probe test strip.

[0015] Further, 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.

[0016] The present invention also provides a method for on-site and rapid detection of norfentanyl in urine, which includes the following steps: Take a urine sample and add it into a centrifuge tube, add a solvent and a sodium salt, let it stand for salting out, then drop the supernatant onto a filter paper, after air drying, take a photo of the test paper under an ultraviolet lamp, obtain the RGB values of the test paper photo, calculate the G / R value, compare it with the standard curve, and obtain the concentration of norfentanyl in the urine sample.

[0017] Further, 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.

[0018] By using TP-CF3-COOH and metal Cu to support the metal-organic framework, the present invention significantly improves the sensitivity of the fluorescent probe for detecting norfentanyl in urine samples. The lowest detection limit of the detection method of the present invention for fentanyl is 0.532 ng / L. The detection method of the present invention only requires simple material kits and an ultraviolet lamp. By testing the RGB values of the fluorescence color through a mobile phone APP or other intelligent devices and comparing with the standard curve, the concentration of norfentanyl in urine samples can be obtained quickly without large-scale equipment and instruments. Description of the Drawings

[0019] Figure 1 It is the scanning electron microscope photo of Cu@TP-CF3-COOH obtained in Example 1; Figure 2 It is the SEM energy spectrum surface scan map of Cu@TP-CF3-COOH obtained in Example 1; Figure 3 It is the FT-IR diagram of Cu@TP-CF3-COOH obtained in Example 1; Figure 4 It is the X-ray diffraction spectrum diagram of Cu@TP-CF3-COOH obtained in Example 1; Figure 5 It is the nitrogen adsorption-desorption isotherm and pore size distribution diagram of Cu@TP-CF3-COOH obtained in Example 1; Figure 6 It is the fluorescence spectrum of Cu@TP-CF3-COOH obtained in Example 1; Figure 7It is a diagram of dropping different standard concentration norfentanyl urine solutions in Example 1 onto a Cu@TP-CF3-COOH fluorescent test strip; Figure 8 Standard curve of norfentanyl urine solution in Example 1; Figure 9 It is a schematic diagram of rapid urine sample detection with the aid of fluorescence using a portable material kit containing a fluorescent probe test strip; Figure 10 It is a digital picture of Cu@TP-CF3-COOH in Example 1, Co@TP-CF3-COOH in Comparative Example 1, and Mg@TP-CF3-COOH in Comparative Example 2. Detailed implementation mode

[0020] The present invention will be described in detail below in conjunction with the specific implementation modes.

[0021] The urine is from the urine of cows in Inner Mongolia Ningcheng Ranch (obtained in compliance with animal welfare requirements).

[0022] Example 1 (I) Preparation of Cu@TP-CF3-COOH (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.

[0023] (2) Preparation of Cu@TP-CF3-COOH: Dissolve the TP-CF3-COOH (105 mg, 0.18 mmol) and Cu(NO3)2·3H2O (153 mg, 0.63 mmol) obtained in step (1) in a solution of DMF / H2O (50 mL, 4:1) and acidify the system with concentrated HNO3 to make the pH = 2. Heat this solution at 80 °C for 18 h to form blue-green crystals. Centrifuge and wash 5 times with 20 mL of DMF, and then wash 3 times with 15 mL of ethanol. After washing, dry it under vacuum at 60 °C for standby (209 mg, 81%) to obtain an organic metal framework, expressed as Cu@TP-CF3-COOH.

[0024] Figure 1 It is a scanning electron microscope photograph of Cu@TP-CF3-COOH obtained in Example 1. It can be seen that it presents a uniform columnar three-dimensional structure. The diameter of a single columnar structure is about 250 - 300 nm, and the length is between 1.6 - 2.3 μm, with a smooth surface.

[0025] Figure 2This is a SEM spectrum scan of Cu@TP-CF3-COOH obtained in Example 1. The content of each element is shown in Table 1 below. It shows that the material is composed of carbon, hydrogen, oxygen, fluorine and copper, and is evenly distributed.

[0026] Table 1 Element content of Cu@TP-CF3-COOH .

[0027] Figure 3 This is the FT-IR image 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 has a peak at 526 cm -1 Compared with TP-CF3-COOH, the vibration peak of Cu@TP-CF3-COOH at 1397 cm -1 -COO appeared - The symmetric stretching vibration peak of -COO at 1550 - The asymmetric stretching vibration peaks indicate that in Cu@TP-CF3-COOH, the copper ions form a planar tetragonal coordination environment with two carboxyl oxygen atoms from the TP-CF3-COOH ligand and two water molecules. In addition, the -OH peak of the TP-CF3-COOH ligand at 2600-2800 disappears, accompanied by the disappearance of the Cu(NO3)2·H2O water peak at 3400, both of which indicate the successful synthesis of Cu@TP-CF3-COOH.

[0028] 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 show that the material has good crystallinity.

[0029] Figure 5 The nitrogen adsorption-desorption isotherm and pore size distribution diagram of Cu@TP-CF3-COOH obtained in Example 1. 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, and the average diameter of Cu@TP-CF3-COOH obtained in Example 1 is 17.95 Å.

[0030] Figure 6 It is the fluorescence spectrum of Cu@TP-CF3-COOH obtained in Example 1. It is proved that when norfentanyl with a concentration of 20 μg / L is added, an obvious fluorescence spectrum can be detected, while in the pure solution of Cu@TP-CF3-COOH, there is no fluorescence intensity. Therefore, it shows that the Cu@TP-CF3-COOH material can be used for the detection experiment of norfentanyl.

[0031] (II) Establish a standard working curve (1) Prepare the stock solution of Cu@TP-CF3-COOH fluorescent probe: Take 1 mg of Cu@TP-CF3-COOH prepared in Example 1 and add it to 10 mL of acetonitrile. After the solid is completely dissolved, ultrasonicate at room temperature for 30 minutes to obtain a 100 mg / L fluorescent probe stock solution.

[0032] (2) Prepare the portable fluorescent test strip of Cu@TP-CF3-COOH Immerse a round filter paper with a diameter of 1.5 cm into 20 mL of the above-mentioned stock solution for 10 minutes, then take it out and dry it in a constant temperature oven at 37 °C for 6 h.

[0033] (3) Prepare the norfentanyl standard solution: Prepare the norfentanyl standard solution with urine, and 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 respectively; (4) Establish a standard working curve: Pretreat the urine sample. Take 0.5 mL of the standard solution urine sample in (3), add 0.5 mL of acetonitrile, vortex for 1 minute, then add 1 g of sodium chloride, vortex for 10 seconds, and then take the supernatant. Take 6 pieces of the round filter paper prepared in (2) for fluorescence detection, and no obvious fluorescence is shown. Then take 5 μL of the pretreated urine sample in (3) and titrate it onto the round filter paper respectively, and wait for about 30 seconds to air dry naturally. Take a photo of the test strip under a 365 nm ultraviolet lamp, as Figure 7 shown. Then use the mobile phone photo to capture the RGB value of the obtained fluorescence color, calculate G / R, establish a standard curve with the norfentanyl concentration as the abscissa and the G / R value as the ordinate, as Figure 8 shown. There is a good linear relationship between the G / R of the fluorescent test strip and the norfentanyl concentration, which is Y = 0.1143x + 2.8867, R 2= 0.998. According to the standard deviation δ = 2.03% obtained from 10 measurements, the lowest detection limit of this probe for methylfentanyl was calculated to be 0.532 ng / L.

[0034] Example 2 The Cu@TP-CF3-COOH provided by the present invention can be used as a material for rapid on-site detection of norfentanyl in urine samples. Figure 9 The figure shows a schematic diagram of rapid urine sample detection by fluorescence with a portable material kit containing a fluorescence probe test strip. The material includes 0.5 mL of acetonitrile solution dispensed in a 1.5 mL EP tube, 1 g of pre-dispensed sodium chloride, a common ultraviolet lamp, and a fluorescence test strip loaded with Cu@TP-CF3-COOH. This material kit can be pre-packaged in an 8*15 cm self-sealing bag and can be stored for a long time at room temperature.

[0035] Take 20 μL of 1000 μg / mL norfentanyl standard solution into a vial for injection, blow with nitrogen for 5 minutes, then add urine until the solution volume is 2 mL, mix well and vortex for 1 minute; take 20 μL of the above solution, add urine until the volume is 8 mL, and vortex for 30 seconds; take 100 μL of the above solution, add urine until the volume is 1 mL. This solution is intended to be used as an on-site urine sample.

[0036] In the case of sudden on-site detection, take 0.5 mL of urine sample, add it to an EP tube containing 0.5 mL of acetonitrile, mix well and add 1 g of sodium chloride. After standing for salting out, take 5 μL of the supernatant and titrate it onto a round filter paper, and wait for about 30 seconds to air dry naturally. Take a photo of the test strip under a 365 nm ultraviolet lamp. Subsequently, use a mobile phone photo to capture the RGB value of the obtained fluorescence color, calculate the G / R value, and compare it with the standard curve obtained in Example 1 (2) to obtain the content of norfentanyl in the urine sample as 24.96 ng / L. It can be seen that the method for detecting norfentanyl in urine of the present invention does not require large-scale equipment and instruments, only the above material kit, an ultraviolet lamp, and a mobile phone that can recognize RGB values, and can quickly and on-site quantitatively detect norfentanyl in urine samples. In case of an on-site emergency, on-site detection is urgently needed to make a judgment on the possible cause in the shortest time. This material kit is easy to operate. Not only can on-site detection personnel determine the problem in time for rescue, but ordinary people can also quickly judge whether their own sudden situation is caused by accidental contact with fentanyl drugs through simple training, so as to save themselves. Therefore, highly selective and sensitive detection of norfentanyl in urine on-site is of great significance for its screening and first aid care.

[0037] Comparative Example 1 Other conditions and operations are the same as those in Example 1, except that in step (1)(2), Cu(NO3)2 is replaced with an equimolar amount of Co(NO3)2.

[0038] Comparative Example 2 Other conditions and operations were the same as those in Example 1, except that in step (i)(2), Cu(NO3)2 was replaced with an equimolar amount of Mg(NO3)2.

[0039] Figure 10 Digital pictures 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.

[0040] The inventors found that among the MOFs made of TP-CF3-COOH and many metals, only Cu@TP-CF3-COOH could achieve a non-fluorescent state in the solid state and could light up fluorescence after adding a sample of norfentanyl. Therefore, it can be used for the detection of norfentanyl in urine samples based on aggregation-induced emission materials. To overcome the limited detection of norfentanyl by the TP-CF3-COOH material in complex samples, we considered using TP-CF3-COOH as a luminescent monomer and constructing a turn-on mode fluorescence detection method by compounding with other systems. During the construction of the composite, the aggregation-induced emission characteristics of TP-CF3-COOH itself and the mechanism of detecting norfentanyl need to be considered: the triple cooperative mechanism of spatial specific binding, conformational change, and intermolecular charge transfer. The ultimate goal is to make the composite in a dark state in the initial state, and then, according to the detection mechanism, trigger it to light up, which is extremely difficult to achieve. Although the AIE materials in the MOF materials found in the literature can be used for turn-on detection, there is no report on the use of the MOF materials prepared by combining TP-CF3-COOH as a monomer with metals for the detection of norfentanyl. We speculated that after the TP-CF3-COOH was made into a MOF material, the metal charge transfer might make the composite material in a dark state, and after adding norfentanyl, due to the formation of intermolecular charge transfer and the competitive effect with the metal charge transfer, the original aggregation-induced fluorescence pathway was opened, thus causing the ON-OFF phenomenon. The metal charge transfer formed by the Cu group could make the aggregation-induced emission material in a dark state in the MOF structure, and based on this, a portable fluorescence test strip was prepared, and subsequent experiments also verified that it could be used for the turn-on detection of norfentanyl in complex matrices such as urine.

[0041] Comparative Example 3 Other conditions and operations are the same as those in Example 1, except that TP-CF3-COOH is used instead of Cu@TP-CF3-COOH as the fluorescent probe. It is found that the detection of norfentanyl in urine samples cannot be carried out because it is greatly affected by the salt environment in the urine solution and there is no significant proportional change in the fluorescence spectrum. The inventor's previous patent CN116514651A reported an amphiphilic aggregation-induced emission material fluorescent probe, in which the compound TP-CF3-COOH can be used for the detection of norfentanyl in drinking water and serum. However, this detection can only be carried out in an aqueous solution with a simple solvent or a serum solution with fewer interfering substances, which has extremely high requirements for the sample itself for on-site rapid detection. At the same time, this fluorescent probe is a fluorescence detection method based on the intermolecular charge transfer and the change of aggregation state between fentanyl and norfentanyl, resulting in fluorescence quenching and fluorescence blue shift. This fluorescence detection method with this quenching mode is easily interfered by the complex sample background. Therefore, this method cannot be used for urine samples that are easier to obtain because the background interference of urine samples is strong and there are extremely high requirements for the anti-interference ability and specific enrichment ability of the detection method.

Claims

1. A detection method for norfentanyl in urine, characterized in that, It includes the following steps: (S1) Prepare an organometallic framework Cu@TP-CF3-COOH from a ligand TP-CF3-COOH and a water-soluble copper source under acidic conditions. The structure of TP-CF3-COOH is shown in the following formula (I): (I) (S2) Add the organometallic framework to a solvent to obtain a stock solution of the fluorescent probe; soak a carrier in the stock solution of the fluorescent probe and dry it to obtain a fluorescent probe test strip. (S3) Drop different concentrations of processed norfentanyl standard urine solutions onto the fluorescent probe test strips, air-dry them, and then irradiate them with an ultraviolet lamp. Establish a standard curve based on the G / R value of the photos and the change in norfentanyl concentration. (S4) Drop the processed urine sample to be tested onto the fluorescent probe test strip, air-dry it, and then irradiate it with an ultraviolet lamp. Calculate the concentration of norfentanyl in the urine sample to be tested 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, with the water-soluble copper source calculated as Cu.

3. The detection method according to claim 2, wherein In steps (S3) and (S4), the "processing" is to mix the urine sample, an organic solvent, and a salt evenly, let it stand for salting-out and layering, 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 the urine sample, the organic solvent, and the salt is 0.5-1 mL: 0.5-1 mL: 0.5-1 g.

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

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

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, and the concentration of the organometallic framework in the stock solution of the fluorescent probe is 0.01-1 mg / mL; the carrier is at least one of filter paper and cellulose paper.

7. The detection method according to claim 6, wherein The concentration of the organometallic framework in the stock solution of the fluorescent probe 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; the incubation condition is to incubate at 23-30 °C.

9. The detection method according to claim 8, wherein Livestock urine is bovine urine, equine urine, ovine urine, or porcine 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 organometallic framework made from 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