Method and kit for detecting fentanyl/morphine compound

文档序号：1361377 发布日期：2020-08-11 浏览：8次中文

阅读说明：本技术 一种检测芬太尼/吗啡类化合物的方法及试剂盒 (Method and kit for detecting fentanyl/morphine compound ) 是由郭磊王凯徐斌吴剑峰朱颖洁谢剑炜于 2019-02-01 设计创作，主要内容包括：本发明涉及一种检测样品中芬太尼类化合物/吗啡类化合物的方法,还涉及一种试剂盒,以及该试剂盒用于检测样品中芬太尼类化合物/吗啡类化合物的用途。该方法可以快速定性检测样品中是否含有芬太尼类化合物和/或吗啡类化合物,也可以定量检测样品中的微量芬太尼类化合物。该方法具有灵敏、快速、易操作等优点,可用于现场快检、毒物检测、法医鉴定等多领域。(The invention relates to a method for detecting fentanyl compounds/morphine compounds in a sample, and also relates to a kit and application of the kit for detecting fentanyl compounds/morphine compounds in the sample. The method can be used for rapidly and qualitatively detecting whether the sample contains the fentanyl compound and/or the morphine compound or quantitatively detecting a trace amount of the fentanyl compound in the sample. The method has the advantages of sensitivity, rapidness, easy operation and the like, and can be used in the fields of on-site rapid detection, poison detection, forensic identification and the like.)

1. Gold nanoparticles and providing I^-The use of a combination of ionic substances for the detection of fentanyl in a sample, or for the manufacture of a kit for the detection of fentanyl in a sample,

preferably, the gold nanoparticles are bare gold nanoparticles or core-shell gold nanoparticles, such as porous core-shell gold nanoparticles;

preferably, the diameter of the gold nanoparticles is about 1-200nm, for example: about 10-150nm, about 30-70 nm, about 50-55nm, about 40-60 nm;

preferably, said providing I^-The ionic substance is a substance containing I^-Salts of ions or of ions capable of preparing I^-Chemical starting materials for ions, e.g. containing I^-Inorganic salts of ions including MgI₂KI and the like;

preferably, the fentanyl compound is a compound represented by formula I or a pharmaceutically acceptable salt thereof:

wherein: r₁Is hydrogen or R₆-ethyl radical, wherein R₆Is phenyl, C₃-C₆Cycloalkyl, 5-6 membered heterocyclyl, 5-6 membered heteroaryl, or ester group, said R₆Optionally substituted with one or more substituents selected from: alkyl, alkenyl, alkoxy, hydroxy, halogen, haloalkyl, amino and nitro;

R₂is alkyl, alkenyl, alkoxy, ester group, hydroxyl, halogen, haloalkyl, amino or nitro;

R₃is an alkyl, ester or acyl group, said R₃Optionally substituted with one or more substituents selected from: alkyl, alkenyl, alkoxy, hydroxy, halogen, haloalkyl, amino and nitro;

R₄is alkyl or acyl, said R₄Optionally substituted with one or more substituents selected from: alkyl, alkenyl, alkoxy, hydroxy, halogen, haloalkyl, amino and nitro;

R₅is phenyl, C₃-C₆Cycloalkyl, 5-6 membered heterocyclyl or 5-6 membered heteroaryl, said R₅Optionally substituted with one or more substituents selected from: alkyl, alkenyl, alkoxy, hydroxy, halogen, haloalkyl, amino and nitro;

preferably, the pharmaceutically acceptable salt is citrate or hydrochloride;

preferably, the fentanyl compound is selected from: fentanyl, 3-methylfentanyl, carfentanil, sufentanil, remifentanil, acetylfentanil, valerylfentanil, acrylfentanil, isobutyrylfentanil, 4-fluorobutyrylfentanil, alfentanil, furofentanil, norfentanyl, and combinations thereof;

preferably, the sample is a biological sample (e.g., urine) or a chemical sample (e.g., a bulk drug (including heroin, procaine, acetaminophen, caffeine, etc.), a pharmaceutical excipient (including D-mannitol), a food (including solid food, liquid food), a food additive).

2. A method for detecting a fentanyl compound in a sample, comprising the acts of:

adding nano gold particles into a sample to be detected, mixing, and adding a providing agent I^-Mixing the ionic substances to obtain a mixture;

collecting a Surface Enhanced Raman Spectroscopy (SERS) of the mixture;

alternatively, the presence or absence of fentanyl in the biological sample is determined if the surface enhanced Raman spectrum of the mixture is at wavenumber of about 990-^-1Determining that the fentanyl compound exists in the biological sample when the characteristic peak appears;

alternatively, the fentanyl compound in the sample is quantified by using the intensity of the characteristic peak,

optionally, before adding the gold nanoparticles into the sample to be detected, pretreating the sample;

preferably, the gold nanoparticles are bare gold nanoparticles or core-shell gold nanoparticles, such as porous core-shell gold nanoparticles;

preferably, the diameter of the gold nanoparticles is about 1-200nm, for example: about 10-150nm, about 30-70 nm, about 50-55nm, about 40-60 nm;

preferably, the fentanyl compound is a compound represented by formula I or a pharmaceutically acceptable salt thereof:

R₂is alkyl, alkenyl, alkoxy, ester group, hydroxyl, halogen, haloalkyl, amino or nitro;

R₃is an alkyl, ester or acyl group, said R₃Is optionally selectedSubstituted with one or more substituents selected from: alkyl, alkenyl, alkoxy, hydroxy, halogen, haloalkyl, amino and nitro;

R₄is alkyl or acyl, said R₄Optionally substituted with one or more substituents selected from: alkyl, alkenyl, alkoxy, hydroxy, halogen, haloalkyl, amino and nitro;

preferably, the pharmaceutically acceptable salt is citrate or hydrochloride;

preferably, the sample is a biological sample (such as urine) or a chemical sample (such as a raw material drug (including heroin, procaine, acetaminophen, caffeine and the like), a pharmaceutical excipient (including D-mannitol), a food (including a solid food, a liquid food), and a food additive);

preferably, the concentration of gold nanoparticles in the mixture is about 0.05-5nM, about 0.1-4nM, about 0.25-2nM, about 0.25-1.5nM or about 0.25-1nM, for example: about 0.1nM, about 0.2nM, about 0.25nM, about 0.3nM, about 0.5nM, about 0.8nM, about 1nM, about 1.2nM, about 1.5nM, about 1.8nM, about 2nM, about 2.2nM, about 2.5nM, about 2.8nM, about 3.0nM, about 3.2nM, about 3.4nM, about 3.6nM, about 3.8nM, about 4nM, about 4.2nM, about 4.5nM, about 4.8 nM;

preferably, I is provided in the mixture^-The concentration of ionic species is about 50-400mM, such as about 100-200 mM.

3. The assay of claim 2, wherein: the sample is a biological sample (e.g., urine), and the concentration of gold nanoparticles in the mixture is about 0.25-2nM, about 0.25-1.5nM, about 0.5-1.5nM, or about 0.8-1.2nM, e.g.: about 0.9nM, about 1nM, about 1.1 nM.

4. The assay of claim 3, wherein the method of pretreating the sample comprises the acts of:

diluting the sample with water (e.g., ultrapure water), preferably at a dilution factor of 5-10;

adjusting the pH of the diluted sample to 9-11, e.g., 10;

purifying the sample by using C18 (octadecylsillyl silica gel, octadecyl bonded silica gel) to obtain a sample to be detected;

preferably, the pH of the biological sample is adjusted with NaOH solution.

5. The assay of claim 4, wherein: the method for purifying the sample by using the C18 comprises the following operations:

rinsing C18 with methanol and/or water (e.g., ultra pure water), respectively;

contacting the sample with C18 to remove liquid from the sample;

rinsing C18 with water, and discarding eluate;

eluting C18 with mixed solution of methanol and formic acid aqueous solution to obtain eluent, i.e. sample to be detected,

preferably, the volume ratio of methanol to aqueous formic acid in the mixture of methanol and aqueous formic acid is 1 (0.8-1.2), for example 1: 1;

preferably, the concentration of the aqueous formic acid solution is 0.08-0.12 v/v%, for example 0.1 v/v%.

6. The assay of claim 2, wherein: the sample is a chemical sample (such as bulk drug (including heroin, procaine, acetaminophen, caffeine, etc.), pharmaceutic adjuvant (including D-mannitol), food (including solid food, liquid food), food additive),

the concentration of gold nanoparticles in the mixture is about 0.1-2nM, e.g., about 0.25-1.5nM, about 0.1-0.5nM, about 0.2-0.3nM, about 0.2-0.4nM, about 0.6-0.8nM, about 1.2-1.8 nM.

7. The assay of claim 6, wherein the method of pretreating the sample comprises the acts of:

the sample is dissolved with a solvent such as water (preferably ultrapure water) to obtain a sample to be measured.

8. A kit, comprising:

the gold nano-particles are prepared by the following steps,

providing I^-The species of the ions are selected such that,

c18(octadecylsilyl silica gel, octadecyl-bonded silica gel),

optionally, the kit further comprises one or more selected from the group consisting of: methanol, a pH regulator, formic acid or an aqueous formic acid solution;

optionally, the kit further comprises instructions;

optionally, the kit further comprises one or more titration plates;

preferably, the gold nanoparticles are bare gold nanoparticles or core-shell gold nanoparticles, such as porous core-shell gold nanoparticles;

preferably, the diameter of the gold nanoparticles is about 1-200nm, for example: about 10-150nm, about 30-70 nm, about 50-55nm, about 40-60 nm;

preferably, the C18 is a C18 membrane, a C18 filler or a C18 solid phase extraction column;

preferably, the pH regulator is NaOH or a solution containing NaOH;

preferably, the concentration of the aqueous formic acid solution is 0.08-0.12 v/v%, such as 0.1 v/v%;

preferably, the specification at least describes the detection method according to any one of claims 3 to 5.

9. Use of a kit according to claim 8 for the detection (including qualitative or quantitative) of fentanyl in a sample, or

Use in the detection (including qualitative detection or quantification) of a fentanyl compound in a sample using Surface Enhanced Raman Spectroscopy (SERS);

preferably, the sample is a biological sample (e.g., urine);

preferably, the sample is a chemical sample (e.g., bulk drug (including heroin, procaine, acetaminophen, caffeine, etc.), pharmaceutical excipient (including D-mannitol), food (including solid food, liquid food), food additive);

preferably, the fentanyl compound is a compound represented by formula I or a pharmaceutically acceptable salt thereof:

wherein: r₁Is hydrogen or R₆-ethyl radical, wherein R₆Is phenyl, C₃-C₆Cycloalkyl, 5-6 membered heterocyclyl, 5-6 membered heteroaryl, or ester group, said R₆Optionally substituted with one or more substituents selected from: alkyl, alkeneAlkyl, alkoxy, hydroxy, halogen, haloalkyl, amino, and nitro;

R₂is alkyl, alkenyl, alkoxy, ester group, hydroxyl, halogen, haloalkyl, amino or nitro;

R₃is an alkyl, ester or acyl group, said R₃Optionally substituted with one or more substituents selected from: alkyl, alkenyl, alkoxy, hydroxy, halogen, haloalkyl, amino and nitro;

R₄is alkyl or acyl, said R₄Optionally substituted with one or more substituents selected from: alkyl, alkenyl, alkoxy, hydroxy, halogen, haloalkyl, amino and nitro;

preferably, the pharmaceutically acceptable salt is citrate or hydrochloride;

preferably, the fentanyl compound is selected from: fentanyl, 3-methylfentanyl, 3-methylthiofentanyl, α -methylfentanyl, carfentanil, sufentanil, remifentanil, acetylfentanyl, acetyl- α -methylfentanyl, valerylfentanyl, acrylfentanyl, butyrylfentanyl, isobutyrylfentanyl, 4-fluorobutyrylfentanyl, 4-fluoroisobutyrylfentanyl, Alfentanil, furanfentanyl, norfentanyl, cyclopropylfentanyl, cyclopentylfentanyl, β -hydroxyfentanyl, β -hydroxy-3-methylfentanyl, 1-phenylcyclohexylamine, Alfentanil (Alfentanil), Thiafentanil, 1-piperidinocyclohexanone carbonitrile (PCC), 4-pilinuno-N-phenylenethyl-4-piperidine (ANPP), and combinations thereof; preferably, the fentanyl-based compound is selected from: fentanyl, 3-methylfentanyl, carfentanil, sufentanil, remifentanil, acetylfentanil, valerylfentanil, acrylfentanil, isobutyrylfentanil, 4-fluorobutyrylfentanil, alfentanil, furofentanil, norfentanyl, and combinations thereof.

10. Gold nanoparticles and providing Cl^-Use of a combination of ionic substances for detecting fentanyl and/or morphine compounds in a sample, or

Use in the preparation of a kit for the detection of fentanyl and/or morphine compounds in a sample, or

Use for the qualitative detection of fentanyl and/or morphine compounds in a sample,

preferably, the gold nanoparticles are bare gold nanoparticles or core-shell gold nanoparticles, such as porous core-shell gold nanoparticles;

preferably, the diameter of the gold nanoparticles is about 1-200nm, for example: about 10-150nm, about 30-70 nm, about 50-55nm, about 40-60 nm;

preferably, said providing Cl^-The ionic substance being Cl^-Salts of ions or capable of preparing Cl^-Chemical starting materials for ions, e.g. containing Cl^-Inorganic salts of ions, including NaCl, MgCl₂Etc.;

preferably, the fentanyl compound is a compound represented by formula I or a pharmaceutically acceptable salt thereof:

R₂is alkyl, alkenyl, alkoxy, ester group, hydroxyl, halogen, haloalkyl, amino or nitro;

R₃is an alkyl, ester or acyl group, said R₃Optionally one or more selected fromSubstituent group substitution: alkyl, alkenyl, alkoxy, hydroxy, halogen, haloalkyl, amino and nitro;

R₄is alkyl or acyl, said R₄Optionally substituted with one or more substituents selected from: alkyl, alkenyl, alkoxy, hydroxy, halogen, haloalkyl, amino and nitro;

preferably, the pharmaceutically acceptable salt is citrate or hydrochloride;

preferably, the morphine compound is a compound shown as a formula II or a pharmaceutically acceptable salt thereof,

wherein: r₁Is hydrogen, alkyl, phenyl, C₃-C₆Cycloalkyl, 5-6 membered heterocyclyl or 5-6 membered heteroaryl, wherein alkyl, phenyl, C₃-C₆The cycloalkyl, 5-6 membered heterocyclyl or 5-6 membered heteroaryl is optionally substituted with one or more substituents selected from the group consisting of: halogen, alkoxy, hydroxy, nitro, haloalkyl, amino;

represents a single or double bond;

R₂is hydroxy, alkoxy, methylene, oxo or acyloxy;

R₃is hydroxy, alkoxy or acyloxy;

R₄is alkyl or alkenyl, wherein R₄Optionally substituted with one or more substituents selected from: halogen, alkoxy, hydroxy, nitro, haloalkyl, amino;

R₅is hydroxy or alkyl, wherein alkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, alkoxy, hydroxy, nitro, haloalkyl, amino;

optionally, an ethyl bridge is formed between positions 6 and 14;

preferably, the pharmaceutically acceptable salt is a sulfate, hydrochloride, tartrate, citrate, hydrobromide, hydroiodide or lactate salt; preferably, the morphinoid compound is selected from: morphine, 3-monoacetylmorphine, 6-monoacetylmorphine, heroin, codeine, thienorphine, 030418, ciprofloxacin, buprenorphine, normorphine, hydromorphine, methylmorphine, oxymorphone, dihydromorphine, nicotinic morphine, ethylmorphine, benzylmorphine, morphorphine, morphine methanesulfonate, dihydroetorphine, etorphine hydrochloride, acetylcodeine, nicotinylcodeine, dihydrocodeine, acetyldihydrocodeine, hydrocodone, dihydrocodeinone, oxymorphone, hydromorphone, methyl dihydromorphone, naloxone, naltrexone, nalmefene, and combinations thereof;

preferably, the morphinoid compound is selected from: morphine, 6-monoacetylmorphine, heroin, codeine, thienorphine, 030418, naloxone, naltrexone, nalmefene and combinations thereof;

11. A method for detecting fentanyl and/or morphine in a sample, comprising the acts of:

adding nano gold particles into a sample to be detected, mixing, and adding Cl^-Mixing the ionic substances; obtaining a mixture;

collecting a Surface Enhanced Raman Spectroscopy (SERS) of the mixture;

optionally, determining whether fentanyl and/or morphine compounds are present in the biological sample if the mixture has a surface enhanced Raman spectrum at wavenumbers of about 990-1005cm^-1Determining that the fentanyl compound exists in the biological sample when the characteristic peak appears; if the surface enhanced Raman spectrum of the mixture is about 620-635cm at a wavenumber^-1Determining that the morphine compound exists in the biological sample when a characteristic peak appears;

optionally, the intensities of the characteristic peaks are used for quantifying the fentanyl type compound and/or the morphine type compound in the sample,

optionally, before adding the gold nanoparticles into the sample to be detected, pretreating the sample;

preferably, the gold nanoparticles are bare gold nanoparticles or core-shell gold nanoparticles, such as porous core-shell gold nanoparticles;

preferably, the diameter of the gold nanoparticles is about 1-200nm, for example: about 10-150nm, about 30-70 nm, about 50-55nm, about 40-60 nm;

preferably, said providing Cl^-The ionic substance being Cl^-Salts of ions orIs capable of preparing Cl^-Chemical starting materials for ions, e.g. containing Cl^-Inorganic salts of ions, including NaCl, MgCl₂Etc.;

preferably, the fentanyl compound is a compound represented by formula I or a pharmaceutically acceptable salt thereof:

R₂is alkyl, alkenyl, alkoxy, ester group, hydroxyl, halogen, haloalkyl, amino or nitro;

R₃is an alkyl, ester or acyl group, said R₃Optionally substituted with one or more substituents selected from: alkyl, alkenyl, alkoxy, hydroxy, halogen, haloalkyl, amino and nitro;

R₄is alkyl or acyl, said R₄Optionally substituted with one or more substituents selected from: alkyl, alkenyl, alkoxy, hydroxy, halogen, haloalkyl, amino and nitro;

preferably, the pharmaceutically acceptable salt is citrate or hydrochloride;

preferably, the fentanyl compound is selected from: fentanyl, 3-methylfentanyl, 3-methylthiofentanyl, α -methylfentanyl, carfentanil, sufentanil, remifentanil, acetylfentanyl, acetyl- α -methylfentanyl, valerylfentanyl, acrylfentanyl, butyrylfentanyl, isobutyrylfentanyl, 4-fluorobutyrylfentanyl, 4-fluoroisobutyrylfentanyl, Alfentanil, furanfentanyl, norfentanyl, cyclopropylfentanyl, cyclopentylfentanyl, β -hydroxyfentanyl, β -hydroxy-3-methylfentanyl, 1-phenylcyclohexylamine, Alfentanil (Alfentanil), Thiafentanil, 1-piperidinocyclohexanone carbonitrile (PCC), 4-pilinuno-N-phenylenethyl-4-piperidine (ANPP), and combinations thereof; preferably, the fentanyl compound is selected from: fentanyl, 3-methylfentanyl, carfentanil, sufentanil, remifentanil, acetylfentanil, valerylfentanil, acrylfentanil, isobutyrylfentanil, 4-fluorobutyrylfentanil, alfentanil, furofentanil, norfentanyl, and combinations thereof;

preferably, the morphine compound is a compound shown as a formula II or a pharmaceutically acceptable salt thereof,

represents a single or double bond;

R₂is hydroxy, alkoxy, methylene, oxo or acyloxy;

R₃is hydroxy, alkoxy or acyloxy;

R₄is alkyl or alkenyl, wherein R₄Optionally substituted with one or more substituents selected from: halogen, alkoxy, hydroxy, nitro, haloalkyl, amino;

R₅is hydroxy or alkyl, wherein alkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, alkoxy, hydroxy, nitro, haloalkyl, amino;

optionally, an ethyl bridge is formed between positions 6 and 14;

preferably, the morphinoid compound is selected from: morphine, 6-monoacetylmorphine, heroin, codeine, thienorphine, 030418, naloxone, naltrexone, nalmefene and combinations thereof;

preferably, the sample is a chemical sample (such as bulk drug (including heroin, procaine, acetaminophen, caffeine, etc.), pharmaceutical excipients (including D-mannitol), food (including solid food, liquid food), food additive);

preferably, the concentration of gold nanoparticles in the mixture is about 0.05-5nM, about 0.1-4nM, about 0.25-2nM, about 0.25-1.5nM, about 0.1-0.5nM, about 0.2-0.3nM, about 0.2-0.4nM, about 0.6-0.8nM, about 1.2-1.8nM or about 0.25-1nM, for example: about 0.1nM, about 0.2nM, about 0.25nM, about 0.3nM, about 0.5nM, about 0.8nM, about 1nM, about 1.2nM, about 1.5nM, about 1.8nM, about 2nM, about 2.2nM, about 2.5nM, about 2.8nM, about 3.0nM, about 3.2nM, about 3.4nM, about 3.6nM, about 3.8nM, about 4nM, about 4.2nM, about 4.5nM, about 4.8 nM;

preferably, the concentration of gold nanoparticles in the mixture is about 0.1-2nM, e.g., about 0.25-1.5nM, about 0.1-0.5nM, about 0.2-0.3nM, about 0.2-0.4nM, about 0.6-0.8nM, about 1.2-1.8 nM;

preferably, Cl is provided in the mixture^-The concentration of ionic species is about 50-400mM, such as about 100-200 mM;

preferably, the method of pretreating the sample comprises the following operations:

the sample is dissolved with a solvent such as water (preferably ultrapure water) to obtain a sample to be measured.

12. A method for the qualitative detection of fentanyl and/or morphine in a sample, comprising the following operations:

preparing a standard solution of fentanyl compounds and/or morphine compounds;

adding nano gold particles into the standard solution, mixing, and adding Cl^-Mixing the ionic species of (a) to obtain a first mixture;

collecting a Surface Enhanced Raman Spectroscopy (SERS) of the first mixture;

performing Principal Component Analysis (PCA) analysis or partial least squares-discriminant analysis (PLS-DA) analysis on the Surface Enhanced Raman Spectroscopy (SERS) of the first mixture, and establishing a PCA analysis model or a PLS-DA analysis model;

adding nano gold particles into a sample to be detected, mixing, and adding Cl^-Mixing the ionic species of (a) to obtain a second mixture;

collecting a Surface Enhanced Raman Spectroscopy (SERS) of the second mixture;

determining whether the sample contains fentanyl and/or morphine based on PCA analysis model or PLS-DA analysis model,

optionally, before adding the gold nanoparticles into the sample to be detected, pretreating the sample;

preferably, the method of pretreating the sample comprises the following operations:

dissolving the sample with a solvent, such as water (preferably ultrapure water), to obtain a sample solution to be tested,

preferably, the gold nanoparticles are bare gold nanoparticles or core-shell gold nanoparticles, such as porous core-shell gold nanoparticles;

preferably, the diameter of the gold nanoparticles is about 1-200nm, for example: about 10-150nm, about 30-70 nm, about 50-55nm, about 40-60 nm;

preferably, the fentanyl compound is a compound represented by formula I or a pharmaceutically acceptable salt thereof:

R₂is alkyl, alkenyl, alkoxy, ester group, hydroxyl, halogen, haloalkyl, amino or nitro;

R₃is an alkyl, ester or acyl group, said R₃Optionally substituted with one or more substituents selected from: alkyl, alkenyl, alkoxy, hydroxy, halogen, haloalkyl, amino and nitro;

R₄is alkyl or acyl, said R₄Optionally substituted with one or more substituents selected from: alkyl, alkenyl, alkoxy, hydroxy, halogen, haloalkyl, amino and nitro;

R₅is phenyl, C₃-C₆Cycloalkyl, 5-6 membered heterocyclyl or 5-6 membered heteroAryl radical, said R₅Optionally substituted with one or more substituents selected from: alkyl, alkenyl, alkoxy, hydroxy, halogen, haloalkyl, amino and nitro;

preferably, the pharmaceutically acceptable salt is citrate or hydrochloride;

preferably, the morphine compound is a compound shown as a formula II or a pharmaceutically acceptable salt thereof,

represents a single or double bond;

R₂is hydroxy, alkoxy, methylene, oxo or acyloxy;

R₃is hydroxy, alkoxy or acyloxy;

R₄is alkyl or alkenyl, wherein R₄Optionally substituted with one or more substituents selected from: halogen, alkoxy, hydroxy, nitro, haloalkyl, amino;

R₅is hydroxy or alkyl, wherein alkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, alkoxy, hydroxy, nitro, haloalkyl, amino;

optionally, an ethyl bridge is formed between positions 6 and 14;

preferably, the pharmaceutically acceptable salt is a sulfate, hydrochloride, tartrate, citrate, hydrobromide, hydroiodide or lactate salt;

preferably, the morphinoid compound is selected from: morphine, 3-monoacetylmorphine, 6-monoacetylmorphine, heroin, codeine, thienorphine, 030418, ciprofloxacin, buprenorphine, normorphine, hydromorphine, methylmorphine, oxymorphone, dihydromorphine, nicotinic morphine, ethylmorphine, benzylmorphine, morphorphine, morphine methanesulfonate, dihydroetorphine, etorphine hydrochloride, acetylcodeine, nicotinylcodeine, dihydrocodeine, acetyldihydrocodeine, hydrocodone, dihydrocodeinone, oxymorphone, hydromorphone, methyl dihydromorphone, naloxone, naltrexone, nalmefene, and combinations thereof;

preferably, the morphine compound is selected from: morphine, 6-monoacetylmorphine, heroin, codeine, thienorphine, 030418, naloxone, naltrexone, nalmefene and combinations thereof;

preferably, the concentration of gold nanoparticles in the first mixture or second mixture is about 0.1-2nM, e.g., about 0.25-1.5nM, about 0.1-0.5nM, about 0.2-0.3nM, about 0.2-0.4nM, about 0.6-0.8nM, about 1.2-1.8 nM;

preferably, Cl is provided in the mixture^-The concentration of ionic species is about 50-400mM, such as about 100-200 mM.

13. A kit, comprising:

the gold nano-particles are prepared by the following steps,

providing Cl^-The species of the ions are selected such that,

optionally, the kit further comprises instructions;

optionally, the kit further comprises one or more titration plates;

preferably, the gold nanoparticles are bare gold nanoparticles or core-shell gold nanoparticles, such as porous core-shell gold nanoparticles;

preferably, the diameter of the gold nanoparticles is about 1-200nm, for example: about 10-150nm, about 30-70 nm, about 50-55nm, about 40-60 nm;

preferably, the specification describes at least the detection method according to claim 11 or 12.

14. Use of a kit according to claim 13 for the detection (including qualitative or quantitative) of fentanyl and/or morphine in a sample, or

Use in the detection (including qualitative detection or quantification) of a fentanyl compound in a sample using Surface Enhanced Raman Spectroscopy (SERS);

preferably, the fentanyl compound is a compound represented by formula I or a pharmaceutically acceptable salt thereof:

R₂is alkyl, alkenyl, alkoxy, ester group, hydroxyl, halogen, haloalkyl, amino or nitro;

R₃is an alkyl, ester or acyl group, said R₃Optionally substituted with one or more substituents selected from: alkyl, alkenyl, alkoxy, hydroxy, halogen, haloalkyl, amino and nitro;

R₄is alkyl or acyl, said R₄Optionally substituted with one or more substituents selected from: alkyl, alkenyl, alkoxy, hydroxy, halogen, haloalkyl, amino and nitro;

preferably, the pharmaceutically acceptable salt is citrate or hydrochloride;

preferably, the morphine compound is a compound shown as a formula II or a pharmaceutically acceptable salt thereof,

represents a single or double bond;

R₂is hydroxy, alkoxy, methylene, oxo or acyloxy;

R₃is hydroxy, alkylOxy or acyloxy;

R₄is alkyl or alkenyl, wherein R₄Optionally substituted with one or more substituents selected from: halogen, alkoxy, hydroxy, nitro, haloalkyl, amino;

R₅is hydroxy or alkyl, wherein alkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, alkoxy, hydroxy, nitro, haloalkyl, amino;

optionally, an ethyl bridge is formed between positions 6 and 14;

preferably, the pharmaceutically acceptable salt is a sulfate, hydrochloride, tartrate, citrate, hydrobromide, hydroiodide or lactate salt;

preferably, the morphinoid compound is selected from: morphine, 6-monoacetylmorphine, heroin, codeine, thienorphine, 030418, naloxone, naltrexone, nalmefene and combinations thereof.

Technical Field

The invention belongs to the field of analytical chemistry, and relates to a method for detecting a fentanyl compound/morphine compound in a sample, a kit and application of the kit for detecting the fentanyl compound/morphine compound in the sample.

Background

Fentanyl and morphine are opioid receptor agonists, and are commonly used clinically to relieve pain in cancer patients and other chronic pain. Currently, 1400 fentanyl homologs and 250 Morphine homologs have been reported (ForeniciToxicol., 2018,36, 12-32; https:// en. wikipedia. org/wiki/Morphine). Some have pharmaceutical uses, of which the fentanyl homologue, carfentanil, has the strongest analgesic activity and is 100 times more potent than fentanyl (Arzneimitelforschung, 1976,26,1521-1531), while fentanyl has 50-100 times more potent than morphine. In addition, fentanyl and morphine compounds can produce euphoria while relieving pain, and are extremely easy to addict. However, very minute intake of fentanyl can cause toxicity in the body, half the lethal dose of fentanyl is 0.03mg/kg (monkey, intravenous) or 3.1mg/kg (rabbit, intravenous), (Arzneimitelforkung, 1976,26, 1548) -1551.), and 2mg of carfentanyl is sufficient to render an adult lethal (Carfentanil: A Dangerous New Factor in the U.S. Opioid Crisis, 2016).

Currently, social and public safety issues with abuse of fentanyl/morphine compounds have raised concerns in countries around the world. For example, as a semi-synthetic compound derived from morphine, the number of deaths from heroin abuse in the U.S. increased 5-fold from 2010 to 2016, reaching nearly 1.9 million in 2017 (addict. behav.,2017,74, 63-66; NCHS Data Brief,2017,294, 1-8); on the other hand, fentanyl compounds have replaced heroin and become the leading killer of drug abuse deaths in 2017 in the U.S., the number of deaths reaches 2.9 million (NCHS Data Brief,2017,294,1-8), and in most abuse deaths, a large amount of heroin is mixed with a small amount of fentanyl, carfentanil or other homologs of fentanyl. Meanwhile, fentanyl has the characteristics of easy synthesis and designable structure (ACS chem. Neurosci.,2018,9,2428-2437), and the new fentanyl compound appears endlessly, such as non-medicinal fentanyl-oxfentanyl appearing in 2016, which brings great challenges to the supervision department. Fentanyl and all its homologs have been cataloged by a number of countries.

In response to abuse, poisoning and even death events, from drug materials to biomedical samples, a field test method for rapidly distinguishing fentanyl/morphine compounds in different matrixes is needed to help correctly identify the species of the poison and distinguish the fact that the poison is "genuine". For example, the fentanyl and the morphine compounds in the raw materials of the medicine or various auxiliary materials can be correctly and quickly distinguished; as another example, for biomedical samples, it is desirable to discriminate the particular form of an analyte in conjunction with metabolic conditions. For example, heroin has a very short plasma half-life in humans, only 1.3-7.8min (curr. clin. pharmacol.2006,1, 109-. Therefore, fentanyl/morphine should be used as a form of detection for the fentanyl/heroin mixture in a urine sample.

At present, methods for rapidly screening fentanyl/morphine compounds mainly comprise a colorimetric method, an immunological method, an infrared spectrum, an ion mobility spectrum, a normal-pressure mass spectrum, a Surface Enhanced Raman Spectrum (SERS) method and the like. The SERS technology has the advantages of high sensitivity, high resolution, simplicity, rapidness, suitability for direct detection of a water phase system and the like, can provide general spectral information of compound types and fingerprint spectral information of a certain compound at the same time, and is particularly suitable for trace analysis. Currently, few reports are made on SERS detection of fentanyl/morphine compounds, and SERS detection in simple matrixes such as aqueous solutions, methanol solutions and the like is mainly used (anal. chem.2018,90, 12678-.

Disclosure of Invention

The first aspect of the present invention relates to gold nanoparticles (AuNPs) and provides I^-Use of a combination of ionic substances for the detection of fentanyl in a sample, or for the manufacture of a kit for the detection of fentanyl in a sample.

In certain embodiments, the use according to the first aspect of the invention, wherein the sample is detected using surface enhanced raman spectroscopy.

The second aspect of the invention relates to a method for detecting a fentanyl compound in a sample, which comprises the following operations:

adding nano gold particles into a sample to be detected, mixing, and adding a providing agent I^-Mixing the ionic substances to obtain a mixture;

collecting a Surface Enhanced Raman Spectroscopy (SERS) of the mixture;

alternatively, the fentanyl compound in the sample is quantified by using the intensity of the characteristic peak,

optionally, the sample is pretreated before adding the gold nanoparticles to the sample to be tested.

In certain embodiments, a method of detecting a fentanyl compound in a sample according to the second aspect of the present invention, wherein the concentration of nanogold particles in the mixture is from about 0.05 to about 5nM, from about 0.1 to about 4nM, from about 0.25 to about 2nM, from about 0.25 to about 1.5nM or from about 0.25 to about 1nM, for example: about 0.1nM, about 0.2nM, about 0.25nM, about 0.3nM, about 0.5nM, about 0.8nM, about 1nM, about 1.2nM, about 1.5nM, about 1.8nM, about 2nM, about 2.2nM, about 2.5nM, about 2.8nM, about 3.0nM, about 3.2nM, about 3.4nM, about 3.6nM, about 3.8nM, about 4nM, about 4.2nM, about 4.5nM, about 4.8 nM.

In certain embodiments, a method of detecting a fentanyl compound in a sample as described in the second aspect of the invention, wherein I is provided in the mixture^-The concentration of ionic species is about 50-400mM, such as about 100-200 mM.

In certain embodiments, a method of detecting a fentanyl compound in a sample according to the second aspect of the present invention, wherein the sample is a biological sample (e.g., urine), and the concentration of gold nanoparticles in the mixture is from about 0.25 to about 2nM, from about 0.25 to about 1.5nM, from about 0.5 to about 1.5nM, or from about 0.8 to about 1.2nM, for example: about 0.9nM, about 1nM, about 1.1 nM.

In certain embodiments, the method for detecting fentanyl in a sample according to the second aspect of the present invention, wherein the sample is pretreated before the gold nanoparticles are added to the sample to be detected.

In certain embodiments, a method for detecting a fentanyl compound in a sample according to the second aspect of the present invention, wherein the method for pretreating the sample comprises the following operations:

diluting the sample with water (e.g., ultrapure water), preferably at a dilution factor of 5-10;

adjusting the pH of the diluted sample to 9-11, e.g., 10;

the sample was purified with C18(octadecylsilyl silica gel, octadecyl silica gel) to obtain a sample to be tested.

In certain embodiments, a method for detecting fentanyl in a sample as described in the second aspect of the present invention, wherein the pH of the biological sample is adjusted with a NaOH solution.

the sample is dissolved with a solvent such as water (preferably ultrapure water) to obtain a sample to be measured.

In certain embodiments, a method for detecting a fentanyl compound in a sample according to the second aspect of the present invention, wherein the method for purifying the sample with C18 comprises the following operations:

rinsing C18 with methanol and/or water (e.g., ultra pure water), respectively;

contacting the sample with C18 to remove liquid from the sample;

rinsing C18 with water, and discarding eluate;

and eluting the C18 by using a mixed solution of methanol and a formic acid aqueous solution to obtain an eluent, namely the sample to be detected.

In certain embodiments, the method for detecting a fentanyl compound in a sample according to the second aspect of the present invention, wherein the volume ratio of methanol to the aqueous formic acid solution in the mixture of methanol and aqueous formic acid solution is 1 (0.8-1.2), for example 1: 1.

In certain embodiments, a method for detecting a fentanyl compound in a sample as described in the second aspect of the present invention, wherein the concentration of the aqueous formic acid solution is from 0.08 to 0.12 v/v%, for example 0.1 v/v%.

In certain embodiments, the method for detecting a fentanyl compound in a sample according to the second aspect of the present invention, wherein the sample is a biological sample (e.g., urine), and the sample is pretreated before adding the gold nanoparticles to the sample to be detected, comprises the following operations:

diluting the sample with water (e.g., ultrapure water), preferably at a dilution factor of 5-10;

adjusting the pH of the diluted sample to 9-11, e.g., 10;

the sample was purified with C18(octadecylsilyl silica gel, octadecyl silica gel) to obtain a sample to be tested.

In certain embodiments, the method for detecting fentanyl in a sample according to the second aspect of the present invention, wherein the sample is a chemical sample (e.g., drug substance (including heroin, procaine, acetaminophen, caffeine, etc.), pharmaceutical excipient (including D-mannitol), food (including solid food, liquid food), food additive), and the concentration of the gold nanoparticles in the mixture is about 0.1 to 2nM, such as about 0.25 to 1.5nM, about 0.1 to 0.5nM, about 0.2 to 0.3nM, about 0.2 to 0.4nM, about 0.6 to 0.8nM, about 1.2 to 1.8 nM.

In certain embodiments, the method for detecting fentanyl compounds in a sample according to the second aspect of the present invention, wherein the sample is a chemical sample (e.g., crude drug (including heroin, procaine, acetaminophen, caffeine, etc.), pharmaceutical excipients (including D-mannitol), food (including solid food, liquid food), food additives), and the sample is pretreated before adding the gold nanoparticles to the sample to be detected, wherein the method for pretreating the sample comprises the following operations: the sample is dissolved with a solvent such as water (preferably ultrapure water) to obtain a sample to be measured.

A third aspect of the invention relates to a kit comprising:

the gold nano-particles are prepared by the following steps,

providing I^-The species of the ions are selected such that,

c18(octadecylsilyl silica gel, octadecyl bonded silica gel).

In certain embodiments, the kit of the third aspect of the invention further comprises one or more selected from the group consisting of: methanol, pH regulator, formic acid or aqueous formic acid solution.

In certain embodiments, the kit of the third aspect of the invention further comprises instructions.

In certain embodiments, the kit of the third aspect of the invention further comprises one or more titration plates (e.g. 96-well plates).

In certain embodiments, the kit according to the third aspect of the present invention, wherein the pH adjusting agent is preferably NaOH or a solution containing NaOH, and the concentration of the aqueous formic acid solution is preferably 0.08-0.12 v/v%, such as 0.1 v/v%, and the specification preferably describes at least the detection method according to the second aspect of the present invention.

A fourth aspect of the invention relates to the use of a kit according to the third aspect of the invention for detecting (including qualitatively detecting or quantifying) a fentanyl compound in a sample, or

Use in the detection (including qualitative detection or quantification) of fentanyl compounds in a sample using Surface Enhanced Raman Spectroscopy (SERS) methods.

In certain embodiments, the use of the fourth aspect of the invention, wherein the sample is a biological sample (e.g., urine).

In certain embodiments, the use according to the fourth aspect of the invention, wherein the sample is a chemical sample (e.g. bulk drug (including heroin, procaine, acetaminophen, caffeine, etc.), pharmaceutical excipient (including D-mannitol), food (including solid food, liquid food), food additive).

A fifth aspect of the invention relates to gold nanoparticles and provision of Cl^-Use of a combination of ionic substances for the detection (including qualitative or quantitative) of fentanyl and/or morphine compounds in a sample, or

The use in the manufacture of a kit for the detection (including qualitative or quantitative) of fentanyl and/or morphine in a sample.

In certain embodiments, the use according to the fifth aspect of the invention, wherein the sample is detected using surface enhanced raman spectroscopy.

In certain embodiments, the use of the fifth aspect of the invention, wherein the gold nanoparticles and the providing Cl are^-Use of a combination of ionic substances for the qualitative detection of fentanyl and/or morphine compounds in a sample.

In certain embodiments, the use of the fifth aspect of the invention, wherein the sample is a chemical sample (e.g. a drug substance (including heroin, procaine, acetaminophen, caffeine, etc.), a pharmaceutical excipient (including D-mannitol), a food product (including solid food, liquid food), a food additive).

A sixth aspect of the invention relates to a method for detecting fentanyl and/or morphine in a sample, comprising the acts of:

adding nano gold particles into a sample to be detected, mixing, and adding Cl^-Mixing the ionic substances; obtaining a mixture;

collecting a Surface Enhanced Raman Spectroscopy (SERS) of the mixture;

optionally, the intensities of the characteristic peaks are used for quantifying the fentanyl type compound and/or the morphine type compound in the sample,

optionally, the sample is pretreated before adding the gold nanoparticles to the sample to be tested.

In certain embodiments, the method for detecting fentanyl and/or morphine in a sample according to the sixth aspect of the present invention, wherein the sample is a chemical sample (e.g., crude drug (including heroin, procaine, acetaminophen, caffeine, etc.), pharmaceutical excipient (including D-mannitol), food (including solid food, liquid food), food additive).

In certain embodiments, the method for detecting fentanyl and/or morphine in a sample as described in the sixth aspect of the invention, wherein the concentration of nanogold particles in said mixture is from about 0.05 to about 5nM, from about 0.1 to about 4nM, from about 0.25 to about 2nM, from about 0.25 to about 1.5nM, from about 0.1 to about 0.5nM, from about 0.2 to about 0.3nM, from about 0.2 to about 0.4nM, from about 0.6 to about 0.8nM, from about 1.2 to about 1.8nM or from about 0.25 to about 1nM, such as: about 0.1nM, about 0.2nM, about 0.25nM, about 0.3nM, about 0.5nM, about 0.8nM, about 1nM, about 1.2nM, about 1.5nM, about 1.8nM, about 2nM, about 2.2nM, about 2.5nM, about 2.8nM, about 3.0nM, about 3.2nM, about 3.4nM, about 3.6nM, about 3.8nM, about 4nM, about 4.2nM, about 4.5nM, about 4.8 nM;

in certain embodiments, the method for detecting fentanyl and/or morphine in a sample according to the sixth aspect of the invention, wherein the concentration of nanogold particles in the mixture is from about 0.1 to about 2nM, e.g., from about 0.25 to about 1.5nM, from about 0.1 to about 0.5nM, from about 0.2 to about 0.3nM, from about 0.2 to about 0.4nM, from about 0.6 to about 0.8nM, from about 1.2 to about 1.8 nM.

In certain embodiments, the method for detecting fentanyl and/or morphine in a sample according to the sixth aspect of the invention, wherein the mixture is provided with Cl^-The concentration of ionic species is about 50-400mM, such as about 100-200 mM.

In certain embodiments, the method for detecting fentanyl and/or morphine in a sample according to the sixth aspect of the present invention, wherein the method for pretreating the sample comprises the following operations:

the sample is dissolved with a solvent such as water (preferably ultrapure water) to obtain a sample to be measured.

The seventh aspect of the invention relates to a method for qualitatively detecting fentanyl and/or morphine compounds in a sample, comprising the following operations:

preparing a standard solution of fentanyl compounds and/or morphine compounds;

adding nano gold particles into the standard solution, mixing, and adding Cl^-Mixing the ionic species of (a) to obtain a first mixture;

collecting a Surface Enhanced Raman Spectroscopy (SERS) of the first mixture;

adding nano gold particles into a sample to be detected, mixing, and adding Cl^-Mixing the ionic species of (a) to obtain a second mixture;

collecting a Surface Enhanced Raman Spectroscopy (SERS) of the second mixture;

determining whether the sample contains fentanyl and/or morphine based on PCA analysis model or PLS-DA analysis model,

optionally, the sample is pretreated before adding the gold nanoparticles to the sample to be tested.

In certain embodiments, the method for qualitatively detecting a fentanyl and/or morphine compound in a sample according to the seventh aspect of the present invention, wherein the method for pretreating the sample comprises the following operations:

the sample is dissolved with a solvent such as water (preferably ultrapure water) to obtain a sample to be measured.

In certain embodiments, the method for qualitatively detecting fentanyl and/or morphine in a sample according to the seventh aspect of the present invention, wherein the sample is a chemical sample (e.g., crude drug (including heroin, procaine, acetaminophen, caffeine, etc.), pharmaceutical excipient (including D-mannitol), food (including solid food, liquid food), food additive).

In certain embodiments, the method for the qualitative detection of fentanyl and/or morphine in a sample according to the seventh aspect of the invention, wherein the concentration of nanogold particles in said first or second mixture is from about 0.1 to about 2nM, such as from about 0.25 to about 1.5nM, from about 0.1 to about 0.5nM, from about 0.2 to about 0.3nM, from about 0.2 to about 0.4nM, from about 0.6 to about 0.8nM, from about 1.2 to about 1.8 nM.

In certain embodiments, the method for the qualitative detection of fentanyl and/or morphine in a sample according to the seventh aspect of the present invention, wherein Cl is provided in said mixture^-The concentration of ionic species is about 50-400mM, such as about 100-200 mM.

An eighth aspect of the present invention relates to a kit comprising:

the gold nano-particles are prepared by the following steps,

providing Cl^-The species of the ions are selected such that,

optionally, the kit further comprises instructions;

optionally, the kit further comprises one or more titration plates (e.g. 96-well plates).

In certain embodiments, the kit according to the eighth aspect of the present invention, wherein the instructions at least describe the detection method according to the seventh aspect of the present invention.

A ninth aspect of the invention relates to the use of a kit according to the eighth aspect of the invention for detecting (including qualitatively detecting or quantifying) fentanyl and/or morphine in a sample, or

Use in the detection (including qualitative detection or quantification) of fentanyl compounds in a sample using Surface Enhanced Raman Spectroscopy (SERS) methods.

In certain embodiments, the kit of the eighth aspect of the present invention, wherein the sample is a chemical sample (e.g., crude drug (including heroin, procaine, acetaminophen, caffeine, etc.), pharmaceutical excipient (including D-mannitol), food (including solid food, liquid food), food additive).

In certain embodiments, the nanogold particles of the invention are bare gold nanoparticles or core-shell gold nanoparticles, such as, for example, porous core-shell gold nanoparticles.

In certain embodiments, the gold nanoparticles of the present invention have a diameter of about 1 to 200nm, for example: about 10-150nm, about 30-70 nm, about 50-55nm, about 40-60 nm.

In some implementationsIn the scheme, the invention provides^-The ionic species may be a species comprising I^-Salts of ions, e.g. containing I^-Inorganic salts of ions including MgI₂KI, etc. In the kit of the present invention, the providing I^-The salts of the ions may be present in solution or in solid form. When the solid exists in the form of solid, the solid can be directly added into a liquid sample to be detected during use, or can be dissolved by water and then used. In certain embodiments, the fentanyl-based compound of the present invention is a compound of formula I:

R₂is alkyl, alkenyl, alkoxy, ester group, hydroxyl, halogen, haloalkyl, amino or nitro;

R₃is an alkyl, ester or acyl group, said R₃Optionally substituted with one or more substituents selected from: alkyl, alkenyl, alkoxy, hydroxy, halogen, haloalkyl, amino and nitro;

R₄is alkyl or acyl, said R₄Optionally substituted with one or more substituents selected from: alkyl, alkenyl, alkoxy, hydroxy, halogen, haloalkyl, amino and nitro;

In certain embodiments, the pharmaceutically acceptable salt of the fentanyl compounds of the present invention is citrate or hydrochloride.

In certain embodiments, the fentanyl-based compounds of the present invention are selected from: fentanyl, 3-methylfentanyl, 3-methylthiofentanyl, α -methylfentanyl, carfentanil, sufentanil, remifentanil, acetylfentanyl, acetyl- α -methylfentanyl, valerylfentanyl, acrylfentanyl, butyrylfentanyl, isobutyrylfentanyl, 4-fluorobutyrylfentanyl, 4-fluoroisobutyrylfentanyl, Alfentanil, furanfentanyl, norfentanyl, cyclopropylfentanyl, cyclopentylfentanyl, β -hydroxyfentanyl, β -hydroxy-3-methylfentanyl, 1-phenylcyclohexylamine, Alfentanil (Alfentanil), thiafentanel, 1-Piperidinocyclohexanecarbonitrile (PCC), 4-pilinano-N-phenylethyyl-4-piperidine (ANPP), and combinations thereof.

In certain embodiments, the fentanyl-based compounds of the present invention are selected from: fentanyl, 3-methylfentanyl, carfentanil, sufentanil, remifentanil, acetylfentanil, valerylfentanil, acrylfentanil, isobutyrylfentanil, 4-fluorobutyrylfentanil, alfentanil, furofentanil, norfentanyl, and combinations thereof.

In certain embodiments, the sample of the invention is a biological sample (e.g., urine) or a chemical sample (e.g., a drug substance (including heroin, procaine, acetaminophen, caffeine, etc.), a pharmaceutical excipient (including D-mannitol), a food product (including solid food, liquid food), a food additive).

In certain embodiments, the C18 of the present invention is a C18 membrane, a C18 packing, or a C18 solid phase extraction column.

In certain embodiments, the providing of Cl as described herein^-The ionic species may be Cl-containing^-Salts of ions, e.g. containing Cl^-Inorganic salts of ions, including NaCl, MgCl₂And the like. In the kit of the present invention, the providing I^-The salts of the ions may be present in solution or in solid form. When in solid form, the composition can be usedIt can be directly added into liquid sample to be measured, or dissolved in water for use. In certain embodiments, the morphinoid compounds of the present invention are compounds of formula II or a pharmaceutically acceptable salt thereof,

represents a single or double bond;

R₂is hydroxy, alkoxy, methylene, oxo or acyloxy;

R₃is hydroxy, alkoxy or acyloxy;

R₄is alkyl or alkenyl, wherein R₄Optionally substituted with one or more substituents selected from: halogen, alkoxy, hydroxy, nitro, haloalkyl, amino;

R₅is hydroxy or alkyl, wherein alkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, alkoxy, hydroxy, nitro, haloalkyl, amino;

optionally, an ethyl bridge is formed between positions 6 and 14.

In certain embodiments, the pharmaceutically acceptable salt of the morphinoid compound of the present invention is a sulfate, hydrochloride, tartrate, citrate, hydrobromide, hydroiodide or lactate salt;

in certain embodiments, the morphine compounds of the present invention are selected from the group consisting of: morphine, 3-monoacetylmorphine, 6-monoacetylmorphine, heroin, codeine, thienorphine, 030418, ciprofloxacin, buprenorphine, normorphine, hydromorphine, methylmorphine, oxymorphone, dihydromorphine, nicotinic morphine, ethylmorphine, benzylmorphine, morphorphine, morphine methanesulfonate, dihydroetorphine, etorphine hydrochloride, acetylcodeine, nicotinylcodeine, dihydrocodeine, acetyldihydrocodeine, hydrocodone, dihydrocodeinone, oxymorphone, hydromorphone, methyl dihydromorphone, naloxone, naltrexone, nalmefene, and combinations thereof.

In certain embodiments, the morphinoid compounds of the present invention are selected from: morphine, 6-monoacetylmorphine, heroin, codeine, thienorphine, 030418, naloxone, naltrexone, nalmefene and combinations thereof.

In the present invention, the gold nanoparticles are gold microparticles with a diameter of about 1-200nm, such as: about 10-150nm, about 30-70 nm, about 50-55nm, about 40-60 nm. The gold nanoparticles can be bare gold nanoparticles, core-shell gold nanoparticles and preferably porous core-shell gold nanoparticles.

In certain embodiments, the bare gold nanoparticles of the present invention are commercially available or can be prepared by methods commonly used in the art. For example, gold nanoparticles of various sizes are prepared from chloroauric acid by a reduction method. The reduction methods include, but are not limited to, the following: PVP (new warrior of shin-shi, gold Shihao, PVP) protective reduction method for preparing gold nanoparticles, rare metal materials and engineering, 2003, 32(1):50-53), phosphomolybdic acid photocatalytic reducing agent method (jowar Hei, Wuying, Niuhuahong, etc., phosphomolybdic acid as photocatalytic reducing agent for preparing gold nanoparticles, spectroscopic laboratory, 2007, 3 rd stage, 334 page 337), trisodium citrate reduction method (G.fresn, Nature Phys. Sci.1973,241,20.), ultraviolet light-induced reduction method (Raney Dong, Lvcheng, Jinan, etc., research on preparation of gold sol and activity thereof by ultraviolet light-induced reduction, light scattering science, 2005,17(14): 329) 331), sol-template method (Shao Guini, Zhang Xingtang, Liu ice, etc., sol-template method for preparing one-dimensional nano-gold material, modern SERS, chemical engineering, 26 (1): 44-46, Sodium borohydride reduction, hydroxylamine hydrochloride reduction, starch reduction, glucose reduction, cyclodextrin reduction, and the like. Preferably, the preparation method adopts a trisodium citrate reduction method.

In some embodiments, the core-shell gold nanoparticles of the present invention are prepared by wrapping SiO2 shells with different thicknesses on the surface of bare gold nanoparticles, according to the methods disclosed in j.f.li, y.f.huang, y.ding, z.l.yang, s.b.li, x.s.zhou, f.r.fan, w.zhang, z.y.zhou, d.y.wu, b.ren, z.l.wang, z.q.tie, Nature,2010,464,392, or according to the methods disclosed in nat.protoc.2012,8, 52-65.

In certain embodiments, the kits of the invention, wherein the gold nanoparticles provide I^-Ionic species or providing Cl^-The ionic species are suitably packaged, individually in vials, sachets and/or any container suitable for use in the detection method.

In certain embodiments, the kit of the invention, wherein the providing I^-The ionic species are suitably packaged, individually in vials, sachets and/or any container suitable for use in the detection method.

In certain embodiments, the kit of the invention, wherein said providing Cl^-The ionic species are suitably packaged, individually in vials, sachets and/or any container suitable for use in the detection method.

In certain embodiments, the kit of the present invention, wherein the C18(octadecyl bonded silica gel) can be a C18 membrane, a C18 packing, or a C18 solid phase extraction column. In use, the sample may be purified by placing a C18 film or C18 packing material in a suitable container, such as a chromatograph.

In certain embodiments, the kits of the present invention, wherein the methanol, pH adjuster, formic acid or aqueous formic acid solution is suitably packaged, each in a vial, a pouch, and/or any container suitable for use in a detection method.

In certain embodiments, the method for detecting fentanyl in a sample according to the second aspect of the present invention comprises a detection scheme as shown in figure 1-a.

In certain embodiments, the method for qualitatively detecting fentanyl and/or morphine compounds in a sample according to the seventh aspect of the present invention comprises performing Principal Component Analysis (PCA) or partial least squares-discriminant analysis (PLS-DA) analysis on the collected Surface Enhanced Raman Spectroscopy (SERS) using SIMCA-P software (e.g., ver.11.5) to establish a PCA analysis model or a PLS-DA analysis model.

In certain embodiments, the method for qualitatively detecting fentanyl and/or morphine in a sample according to the seventh aspect of the present invention comprises a detection scheme as shown in fig. 1-B.

In some embodiments, in the kit of the present invention, the gold nanoparticles may be a prepared solution (referred to as a raw solution) or a concentrated solution of the raw solution.

In certain embodiments, the invention provides^-The ionic substances may also be those capable of preparing I^-When the ionic chemical raw materials are used, the raw materials are mixed and then react to prepare the chemical raw materials containing I^-A substance of ions.

In certain embodiments, the providing of Cl as described herein^-The ionic species may also be capable of producing Cl^-When the ionic chemical raw materials are used, the raw materials are mixed and then react to prepare the Cl-containing chemical raw material^-A substance of ions.

The term "alkyl" as used herein refers to a saturated straight or branched chain monovalent hydrocarbon radical, preferably having 1 to 12 carbon atoms, more preferably having 1 to 10, 1 to 8, 1 to 6,1 to 4 or 1 to 3 carbon atoms. Typical examples of "alkyl" include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, tert-pentyl, neopentyl, hexyl, heptyl, octyl and the like.

The term "alkenyl" as used in the present invention refers to an ethylenically unsaturated straight or branched chain monovalent hydrocarbon radical containing at least one carbon-carbon double bond (-C ═ C-), preferably having from 2 to 12 carbon atoms, more preferably having from 2 to 10,2 to 8, 2 to 6,2 to 4 or 2 to 3 carbon atoms. Representative examples of "alkenyl" include, but are not limited to, ethenyl, propenyl, allyl, prop-2-en-1-yl, but-1-enyl, but-2-enyl, pent-1-yl, pent-2-yl, 1, 3-pentadienyl, hex-1-yl, hex-2-enyl, 1, 3-hexadienyl, heptenyl, octenyl, and the like.

The term "C" as used in the present invention₃-C₆Cycloalkyl "means a saturated cyclic hydrocarbon group having 3 to 6 carbon atoms and having a single ring, preferably having 3 to 4 carbon atoms or 5 to 6 carbon atoms. Typical examples of "cycloalkyl" include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

The term "ester group" as used herein means-C (O) OR¹⁰Wherein R is¹⁰Selected from alkyl and C as defined herein₃-C₆A cycloalkyl group. Typical examples of "ester groups" include, but are not limited to, -C (O) OCH₃，-C(O)OC₂H₅And the like.

The term "alkoxy" as used herein means the group-OR¹¹Wherein R is¹¹Is alkyl or C as defined in the invention₃-C₆A cycloalkyl group. Typical examples of "alkoxy" include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1, 2-dimethylbutoxy, cyclohexyloxy, cyclobutyloxy, cyclopentyloxy, cyclopropyloxy, and the like.

The term "amino" as used in the present invention means-NH₂。

The term "hydroxy" as used in the present invention means-OH.

The term "nitro" as used herein means-NO₂。

The term "halogen" as used herein means fluorine, chlorine, bromine or iodine. Preferred halogen groups are fluorine, chlorine or bromine.

The term "haloalkyl" as used herein means an alkyl group which is mono-or polysubstituted with halogens, such as fluorine, chlorine, bromine or iodine. Preferred haloalkyl groups are chloromethyl, chloroethyl, dichloroethyl, trifluoromethyl, difluoromethyl, monofluoromethyl, and the like.

The term "5-6 membered heteroaryl" as used herein denotes a heteroaromatic ring group having 5-6 ring members, including monocyclic heteroaromatic rings and polycyclic aromatic rings wherein the monocyclic aromatic ring is fused to one or more other aromatic rings. Heteroaryl groups have one or two or more heteroatoms selected from O, S or N. Typical examples of "heteroaryl" include, but are not limited to, furyl, imidazolyl, thienyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, triazolyl, and the like.

The term "5-6 membered heterocyclyl" as used herein denotes a cycloalkyl group as defined herein comprising one, two or more heteroatoms independently selected from N, O and S. Typical examples of "heterocyclyl" include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperazinyl, piperidinyl, morpholinyl, and the like.

The term "acyl" as used herein means the group-C (O) R¹²Wherein R is¹²Selected from hydrogen and alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylamino, dialkylamino, aryl or heteroaryl groups as defined herein. Typical examples of "acyl" include, but are not limited to, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, cyclohexylcarbonyl, benzoyl and the like.

The term "acyloxy", as used herein, means-OC (O) R¹²wherein-C (O) R¹²Is an acyl group as defined herein. Typical examples of "acyloxy" include, but are not limited to, formyloxy, acetyloxy, propionyloxy, and the like.

In the present invention, the unit "M" represents mol/L, "μ M" represents μmol/L, "nM" represents nmol/L, and "mM" represents mmol/L.

As used herein, the term "about" when used to modify a value or range of values means that the value or range of values and the range of values or ranges of values within acceptable tolerances of those skilled in the art, e.g., the range of values is 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, etc.

In the present invention, the term "Active Pharmaceutical Ingredient (API)" refers to any substance or mixture of substances used in the manufacture of drugs, and when used in the manufacture of drugs, it becomes an Active Ingredient of the drugs.

In the present invention, the term "pharmaceutical excipient" refers to excipients and additives used in the manufacture of pharmaceutical products and in the formulation of pharmaceutical formulations. Common pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrating agents, permeation enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculating and deflocculating agents, filter aids, release retardants, and the like.

In the present invention, the term "food" refers to various products and materials for human consumption or drinking and to articles that are conventionally both food and traditional Chinese medicinal materials, but do not include articles intended for therapeutic purposes. Generally, solid food, liquid food such as nonalcoholic beverage, and beverage containing alcohol (including white spirit, beer, wine, yellow wine, rice wine, medicated wine, etc.) are included.

In the present invention, the term "food additive" refers to an artificial or natural substance, including a nutrient supplement, added to food for the purpose of improving the quality and color, flavor, taste of food, and for the purpose of preservation, freshness and processing.

The invention has the beneficial technical effects

The use of the first aspect of the invention, the method for detecting fentanyl compounds in samples of the second aspect of the invention, or the kit of the third aspect of the invention can realize rapid and selective detection of trace fentanyl compounds in complex samples. SERS selective detection of as low as 0.05% fentanyl in heroin subjects (fentanyl concentrations 10ng/mL, 50ng/mL) was achieved in chemical samples of fentanyl/heroin mixtures; in chemical samples of fentanyl and 4 common raw material medicines or medicinal auxiliary materials, SERS (surface enhanced Raman scattering) selective detection of fentanyl as low as 0.002% in the medicinal auxiliary materials can be realized (the concentration of fentanyl is 10 ng/mL). In urine samples, SERS selective detection of as low as 0.01% fentanyl in morphine samples (fentanyl concentration of 50ng/mL) was achieved. The method has the advantages of sensitivity, rapidness, easy operation and the like, and can be used in the fields of on-site rapid detection, poison detection, forensic identification and the like.

The use of the fifth aspect of the present invention, the detection method of the sixth aspect or the seventh aspect, or the kit of the eighth aspect, can rapidly and qualitatively detect whether a sample contains a fentanyl compound and/or a morphine compound, and the method has the advantages of sensitivity, rapidness, easy operation, and the like, and can be used in the fields of rapid field detection, poison detection, forensic identification, and the like.

Drawings

FIG. 1-A is a flow chart of a method for detecting fentanyl in a sample;

FIG. 1-B is a flow chart of a method for qualitatively detecting fentanyl and/or morphine in a sample;

FIG. 2-UV-Vis spectrum of AuNPs;

FIG. 2-B is a transmission electron micrograph of AuNPs;

FIG. 3-A Structure of fentanyl-based compounds;

FIG. 3-B Structure of morphine-like compounds;

FIG. 4-Raman spectrum of fentanyl;

FIG. 4-B SERS spectra of fentanyl compounds;

FIG. 4-C Raman spectra of morphine compounds;

FIG. 4-SERS spectra of morphine compounds;

FIG. 5-A different classes of inorganic salts vs. fentanyl 1000cm^-1(ii) SERS peak and codeine 622cm^-1The influence of SERS spectrum peak intensity is detected, wherein the concentration of fentanyl and codeine is 100 ng/mL;

FIG. 5-B NaCl vs. fentanyl 1000cm at different concentrations^-1(ii) SERS peak and codeine 622cm^-1Influence of SERS spectral peak intensity of fentanyl and codeineThe concentration is 100 ng/mL;

FIG. 6-A Main component analysis (PCA) differentiation results after wet testing of fentanyl/morphine, wherein the concentrations of fentanyl and morphine are both 500 ng/mL;

FIG. 6-B the degree of contribution of different Raman shifts of fentanyl/morphine compounds to PCA discrimination;

FIG. 6-results of PCA differentiation after dry detection of fentanyl/morphine compounds; the concentrations of fentanyl and morphine compounds are both 500ng/mL (dry detection refers to a method of dropping a mixture of a sample and AuNPs on a silicon wafer, volatilizing to dryness and then carrying out SERS detection);

FIG. 6-partial least squares (PLS-DA) differentiation results after wet assay of D fentanyl/morphine compounds; the concentration of fentanyl and morphine compounds is 500 ng/mL;

FIG. 6-PCA discrimination results after wet detection of E fentanyl/morphine; the concentration of the fentanyl and the morphine compound is 100 ng/mL;

FIG. 6-PCA discrimination results after dry detection of fentanyl/morphine; the concentration of the fentanyl and the morphine compound is 100 ng/mL;

FIG. 6-G PLS-DA differentiation results after wet assay of fentanyl/morphine; the concentration of the fentanyl and the morphine compound is 100 ng/mL;

in FIGS. 6-A through 6-G, the legends have the meanings given in ■ for fentanyl, ● for 3-methylfentanyl, ▲ for carfentanil, ◆ for sufentanil, ● for remifentanil, □ for morphine, ○ for 6-monoacetylmorphine, △ for heroin, ◇ for codeine;represents thienorphine;the representation of 030418 is represented by,

FIG. 7-A PCA differentiation results for fentanyl/morphine mixtures at different concentrations, wherein the legends have the meanings given below for ■ for fentanyl and ● for 3-methyl-phenTainie, ▲ for carfentanil, ◆ for sufentanil, ● for remifentanil, □ for morphine, ○ for 6-monoacetylmorphine, △ for heroin, ◇ for codeine;represents thienorphine;represents 030418;represents a fentanyl/morphine ratio of 10: 1;represents a fentanyl/morphine ratio of 5: 1;represents a fentanyl/morphine ratio of 1: 1;represents a fentanyl/morphine ratio of 1: 5;representing a fentanyl/morphine ratio of 1: 10);

FIGS. 7-B PCA differentiation results for 8 fentanyl and 3 morphine compounds, where the legends have the meanings given below for ■ for fentanyl, ● for 3-methylfentanyl, ▲ for carfentanil, ◆ for sufentanil;represents remifentanil, □ represents morphine, ○ represents 6-monoacetylmorphine, △ represents heroin, ◇ represents codeine;represents thienorphine;represents 030418;represents 8 fentanyl compounds, including acetylfentanyl, valerylfentanyl, acryloylfentanyl, isobutyrylfentanyl, 4-fluorobutyrylfentanyl, alfentanil, furofentanyl, norfentanyl;representative of 3 morphine compounds including naloxone, naltrexone, nalmefene);

FIGS. 7-C PLS-DA differentiation of 8 fentanyl and 3 morphine compounds, wherein the legends have the same meaning as in FIG. 7-B;

FIG. 7-D PCA differentiation results for mixtures of fentanyl/heroin at different concentrations, where the legends have the meanings given below for ■ for fentanyl, ● for 3-methylfentanyl, ▲ for carfentanil, ◆ for sufentanil, ● for remifentanil, □ for morphine, ○ for 6-monoacetylmorphine, △ for heroin, ◇ for codeine;represents thienorphine;represents 030418;represents a heroin/fentanyl ratio of 500: 1;represents a heroin/fentanyl ratio of 100: 1; ● represents a heroin/fentanyl ratio of 50: 1;represents a heroin/fentanyl ratio of 10: 1;represents a heroin/fentanyl ratio of 5: 1;represents a heroin/fentanyl ratio of 1: 1;represents a heroin/fentanyl ratio of 1: 5;representing a heroin/fentanyl ratio of 1: 10);

FIG. 8-A fentanyl (50ng/mL)1000cm after addition of different concentrations of heroin^-1Intensity change of SERS spectrum peak;

FIG. 8-B fentanyl (10ng/mL)1000cm after addition of different concentrations of heroin^-1(iii) intensity variation of SERS peak;

FIG. 9 is a SERS spectrum of fentanyl and drug or drug adjuvant at a concentration ratio of 1: 2000; (● 1000cm^-1；○622cm^-1)

FIG. 10 is a graph showing the effect of different pretreatment methods on the fentanyl SERS signal in urine; (● 1000cm^-1)

FIG. 11 fentanyl (50ng/mL)1000cm in urine after addition of different concentrations of morphine^-1(iii) intensity variation of SERS peak;

FIG. 12 is a standard curve for quantitative determination of 5 fentanyl compounds in urine.

Detailed Description

The following examples are presented to further illustrate the essence of the present invention, and it should be understood that the following examples are only illustrative of the present invention, but not intended to limit the scope of the present invention. The following examples, which do not indicate specific conditions, were conducted according to conventional conditions or as recommended by the manufacturer. The medicines or reagents used are not indicated by manufacturers, and are all conventional products which can be obtained commercially.

Although many of the materials and methods of operation used in the examples below are well known in the art, the invention is described in detail herein. It will be apparent to those skilled in the art that the materials and methods of operation used in the following examples are well known in the art, unless otherwise specified.

In the following examples:

d-mannitol was purchased from Alfa Aesar (USA);

norfentanil was purchased from shanghai shao profound co ltd;

fentanyl, morphine, codeine were purchased from the chinese drug biologies institute (beijing);

heroin, caffeine, 6-monoacetylmorphine from national narcotics laboratory (Beijing);

procaine and acetaminophen are purchased from the institute of food and drug testing, china (beijing);

acetylfentanyl, valerylfentanyl, acrylfentanyl, isobutyrylfentanyl, 4-fluorobutyrylfentanyl, furfentanyl, and olfentanyl are gifted by the forensic test and identification center of the public bureau of Beijing;

carfentanil, sufentanil, 3-methylfentanil, remifentanil, thienorphine, 030418, naloxone, naltrexone, nalmefene were from the military medical research institute of the national military academy of liberty military.

The structural formulae of fentanyl and morphine used in the following examples are shown below:

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Method and kit for detecting fentanyl/morphine compound

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