CN114478389A - Isotopic psychoactive substance labeled compound, preparation method, application and application thereof - Google Patents

Isotopic psychoactive substance labeled compound, preparation method, application and application thereof Download PDF

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CN114478389A
CN114478389A CN202210133884.2A CN202210133884A CN114478389A CN 114478389 A CN114478389 A CN 114478389A CN 202210133884 A CN202210133884 A CN 202210133884A CN 114478389 A CN114478389 A CN 114478389A
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mdmb
indazole
dimethyl
pinaca
butanamide
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孙喆明
林何
石寒天
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Third Research Institute of the Ministry of Public Security
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/54—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings condensed with carbocyclic rings or ring systems
    • C07D231/56—Benzopyrazoles; Hydrogenated benzopyrazoles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/05—Isotopically modified compounds, e.g. labelled
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N2030/042—Standards
    • G01N2030/045—Standards internal

Abstract

The isotopically active substance labeled compound provided by the invention can be used as an internal standard for measuring the content of a psychoactive substance N- (3, 3-dimethyl-butyramide-2-yl) -1- (5-1-alkenyl amyl) -indazole-3-methyl butyrate and a metabolite N- (3, 3-dimethyl-butyramide-2-yl) -1- (5-1-alkenyl amyl) -indazole-3-butyric acid in a hair inspection material, and the isotopically active substance labeled compound is used for measuring the content of the N- (3, 3-dimethyl-butyramide-2-yl) -1- (5-1-alkenyl amyl) -indazole-3-butyl acid according to the retention time and ion pair matching The acid methyl ester and the metabolite N- (3, 3-dimethyl-butyramide-2-yl) -1- (5-1-allyl amyl) -indazole-3-butyric acid thereof are qualitatively detected, and specifically, the corresponding compound is quantitatively detected according to the chromatographic peak area thereof. The isotope labeled compound is used as an internal standard substance, can reduce the matrix effect of a detection material, and has good application prospect in aspects such as judicial identification and the like.

Description

Isotopic psychoactive substance labeled compound, preparation method, application and application thereof
Technical Field
The invention belongs to the technical field of preparation and application of standard substances of mental active substances, and particularly relates to an isotopic mental active substance labeled compound, a preparation method, application and application thereof.
Background
Aiming at the detection of trace and trace substances in a detected material, an isotope dilution mass spectrometry method of a liquid chromatogram-mass spectrometer combined with an isotope labeling standard substance internal standard is the most common and efficient detection means. The isotope labeled standard substance is used as an internal standard, and the isotope labeled internal standard and a compound to be detected have completely consistent chemical properties except molecular weight, so that the isotope dilution mass spectrometry can achieve high detection precision and sensitivity, and is an indispensable tool for accurately detecting trace psychoactive substances.
The method for detecting trace amount of methyl N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-alkenyl amyl) -indazole-3-butyrate (MDMB-4en-PINACA) and metabolite N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-alkenyl amyl) -indazole-3-butyric acid (MDMB-4en-PINACA metabolite) in the biological test material at present is generally liquid phase mass spectrometry, and the content of MDMB-4en-PINACA and metabolite thereof in the test material is determined by a standard curve method by selecting a compound with chemical properties similar to those of MDMB-4en-PINACA and metabolite thereof as an internal standard. The closer the internal standard is in nature to the target, the lower its matrix effect. The internal standards currently selected are generally the more common D9-APINACA or D5-diazepam.
Therefore, it is an urgent problem to be solved by those skilled in the art to provide an isotopically active substance labeled compound for improving the accuracy and sensitivity of detection of the psychoactive substance MDMB-4en-PINACA and its metabolites in the test material.
Disclosure of Invention
The main purpose of the present invention is to solve the problems of low detection accuracy and sensitivity of the psychoactive substances in the prior art, and in one aspect, the present invention provides an isotopically active substance labeled compound, which has the following structural formula:
Figure BDA0003503749070000021
wherein R is1、R2、R3、R4At least one is D, R is methyl or hydrogen, theContains methyl N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-en-tyl) -indazole-3-butanoate (MDMB-4en-PINACA) and its metabolite N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-en-tyl) -indazole-3-butanoate (MDMB-4en-PINACA metabolite).
Preferably, the structural formula of the isotopically active substance labeled compound is:
Figure BDA0003503749070000022
in another aspect of the present invention, there is provided a method for preparing the isotopically active substance-labeled compound, comprising the steps of:
(1) carrying out Friedel-crafts acylation reaction on N- (3-methyl-butyryl benzylamine-2-yl) formamide and N-5-fluoropentyl indole under the catalysis of aluminum trichloride to generate MDMB-4 en-PINACA;
Figure BDA0003503749070000023
(2) deprotection of the intermediate in step (1) with basic conditions produces N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-enepentyl) - indazole 4,5,6, 7-D4-3-butanoic acid (D4-MDMB-4en-PINACA metabolite).
In a further aspect, the present invention provides the use of the isotopically active substance labeled compound described above in detecting the content of the psychoactive substance methyl N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-enpentyl) -indazole-3-butyrate (MDMB-4en-PINACA) and its metabolite methyl N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-enpentyl) -indazole-3-butyrate (MDMB-4 en-pinacametalate) in biological samples or wastewater.
In the above application, the method for detecting the content of the psychoactive substance MDMB-4en-PINACA and the metabolite thereof in the biological detection material or the sewage by using the isotopic psychoactive substance labeled compound comprises the following steps:
(1) setting the detection conditions of liquid chromatography-mass spectrometry;
(2) drawing a standard curve: adding MDMB-4en-PINACA, D4-MDMB-4en-PINACA, MDMB-4en-PINACA methylate and D4-MDMB-4en-PINACA methylate standard solutions with different proportions into a negative detection material corresponding to a sample to be detected, performing LC-MS/MS detection after the same pretreatment steps as the sample to be detected, and making a standard curve on the peak area ratio and the concentration of the MDMB-4en-PINACA methylate to the D4-MDMB-4en-PINACA, and the MDMB-4en-PINACA methylate to the D4-MDMB-4en-PINACA methylate; wherein the internal standard is the isotopically labeled compound of claim 1 or 2.
(3) Pretreatment and determination of a sample to be detected: adding the isotopic psychoactive substance labeled compound as described in claim 2 into a sample to be detected as an internal standard, performing LC-MS/MS detection after the same pretreatment steps as those used for drawing a standard curve to obtain quantitative parameters of the sample to be detected and the internal standard, and calculating by using a formula of the standard curve of an internal standard method to obtain the content of the sample to be detected.
Preferably, in step (1), the mobile phase for the liquid chromatography-mass spectrometry detection is: acetonitrile (0.01% formic acid), B: water (0.01% formic acid, 5% ammonium formate), gradient: 1min 10% A,2min 50% A,4min 90% A,6min 95% A; a chromatographic column: poroshell120 PFP 3.0x100mm 1.9.9 um; column temperature: 30 ℃; flow rate: 0.5 mL/min; sample introduction amount: 5 mu L of the solution; an ion source: electrospray ion source, positive mode (ESI +); the spraying voltage is 3500V; ion source temperature: 340 ℃; collision gas: nitrogen gas.
Preferably, in the step (2), the mass concentration of the internal standard substance in the mixed standard solution is 0.1-99.9%.
Preferably, in the step (3), the mass concentration of the internal standard substance in the sample to be tested is 0.1% -99.9%.
Preferably, in the step (3), the internal standard method formula is calculated as,
Figure BDA0003503749070000031
wherein X is the concentration of a sample to be detected, Y is the peak area obtained by liquid chromatography-mass spectrometry detection, b0Is the intercept of the standard curve and is, b1is the slope of the standard curve.
Preferably, the mass content of the psychoactive substance MDMB-4en-PINACA and metabolites thereof in the above biological samples or the above sewage is 10-9％-10％。
In a further aspect, the present invention provides a method for detecting the content of psychoactive substances MDMB-4en-PINACA and metabolites thereof in biological test materials or wastewater, said method comprising detecting psychoactive substances using isotopically active substance-labeled compounds D4-MDMB-4en-PINACA and D4-MDMB-4en-PINACA metabolite as internal standards.
The isotopically active substance labeled compound D4-N- (3, 3-dimethyl-butyramide-2-yl) -1- (5-1-alkenyl amyl) -indazole-3-methyl butyrate and metabolite N- (3, 3-dimethyl-butyramide-2-yl) -1- (5-1-alkenyl amyl) -indazole-3-methyl butyrate provided by the invention can be used for the psychoactive substance N- (3, 3-dimethyl-butyramide-2-yl) -1- (5-1-alkenyl amyl) -indazole-3-methyl butyrate and metabolite N- (3, 3-dimethyl-butyramide-2-yl) -1- (5-1-alkenyl amyl) methyl butyrate in sewage detection materials The internal standard for measuring the content of the base) -indazole-3-butyric acid can reduce the matrix effect of the detection material, and has good application prospect in aspects such as judicial identification and the like.
Drawings
FIG. 1 shows the high resolution mass spectrum of D4-MDMB-4en-PINACA in example 1.
FIG. 2 is a schematic representation of the method of example 1 for D4-MDMB-4 en-PINCA1H-NMR spectrum.
FIG. 3 shows the results of D4-MDMB-4en-PINACA in example 113C-NMR spectrum.
FIG. 4 is a high resolution mass spectrum of D4-MDMB-4en-PINACAmetabolite in example 2.
FIG. 5 shows D4-MDMB-4en-PINACA metabolite in example 21H-NMR spectrum.
FIG. 6 shows the preparation of D4-MDMB-4en-PINACA metabolite in example 213C-NMR spectrum.
FIG. 7 shows the chromatogram for the detection of the hair MDMB-4 en-PINCA and MDMB-4 en-PINCA metablate samples in example 3.
FIG. 8 shows the chromatogram for the detection of the hair MDMB-4 en-PINCA and MDMB-4 en-PINCA metablate samples in example 3.
FIG. 9 shows the MDMB-4en-PINACA standard curve for hair from example 3.
FIG. 10 is a graph of the MDMB-4en-PINACA metabolite standard curve of hair in example 3.
FIG. 11 shows the chromatogram for the detection of the MDMB-4 en-PINCA and MDMB-4 en-PINCA metablate samples in example 4.
FIG. 12 sample detection chromatogram 2 for MDMB-4en-PINACA and MDMB-4en-PINACA metablate in example 4.
FIG. 13 is a graph of the MDMB-4en-PINACA standard curve for hair from example 4.
FIG. 14 is a graph of the MDMB-4en-PINACA metabolite standard curve of hair in example 4.
FIG. 15 shows the chromatogram for detection of the MDMB-4 en-PINCA and MDMB-4 en-PINCA metablate samples in example 5.
FIG. 16 is a chromatogram of the detection of the hair MDMB-4 en-PINCA and MDMB-4 en-PINCA metablate samples from example 5.
FIG. 17 shows the MDMB-4en-PINACA standard curve for hair from example 5.
FIG. 18 is a hair MDMB-4en-PINACA metabolate standard curve of example 5.
Fig. 19 is a structural formula of an isotopically active substance labeled compound of the present invention.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further explained by combining the specific drawings.
In the context of the present invention, "matrix effect" is understood to mean the influence and interference of substances in the test material, other than the test substance MDMB-4en-PINACA and its metabolites, on the analytical process and the test results.
In the context of the present invention, the mass concentration of MDMB-4en-PINACA and its metabolites in the hair is 10-9％-10％。
The isotopically active substance labeled compound D4-3, 3-dimethyl-2- (1- (pent-4-en-1-yl) -1H-indazole-3-carboxamide) methyl butyrate provided by the invention has the following structural formula:
Figure BDA0003503749070000051
the metabolite N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-enpentyl) -indazole-3-butanoic acid, having the following structural formula:
Figure BDA0003503749070000052
the invention is further illustrated by the following specific examples.
Example 1
Preparation method of D4-3, 3-dimethyl-2- (1- (pent-4-en-1-yl) -1H-indazole-3-carboxamide) methyl butyrate (D4-MDMB-4 en-PINACA).
N- (3-methyl-butyrylbenzyl-2-yl) formamide (750mg,3mmol) was dissolved in 50mL of anhydrous oxygen-free carbon tetrachloride at-20 deg.C, aluminum trichloride (400mg,3mmol) was added, then N-pent-4-enyl indole (200mg, 1mmol) in anhydrous oxygen-free carbon tetrachloride (25mL) was added dropwise, stirred and slowly warmed to room temperature, held at room temperature for 12 hours, heated to 40 deg.C and heated for 2 hours. After cooling, the reaction was quenched by addition of ammonium chloride solution, the solvent was removed by rotary evaporation, dissolved in 150 mL of ethyl acetate, and the insoluble material was removed by filtration through celite, eluting with 30mL of diethyl ether-ethyl acetate 1:1 solution washing of diatomaceous earth, combining the organic phases, washing with 5% aqueous hydrochloric acid, extraction of the aqueous phase with ethyl acetate, and drying of the combined organic phases with anhydrous magnesium sulphate. The drying agent was removed by filtration and the solvent removed by rotary evaporation. Purification by silica gel column chromatography (petroleum ether: ethyl acetate 4:1) gave D4-3, 3-dimethyl-2- (1- (pent-4-en-1-yl) -1H-indazole-3-carboxamide) butyric acid methyl ester (D4-MDMB-4en-PINACA) as a white solid (207mg, 54%).
1H NMR(600MHz,Methanol-d4)δ5.51(ddt,J＝16.8,10.1,6.3Hz,1H),4.78–4.63(m,2H), 4.57(s,1H),4.30(s,1H),4.17(t,J＝6.6Hz,2H),3.46(s,3H),1.84–1.66(m,4H),0.77(s,9H). 13C NMR(151MHz,cd3od)δ173.05,164.18,142.40,138.48,137.37,127.57,123.80,123.47, 122.60,116.14,110.75,61.23,52.46,49.69,35.82,31.80,29.99,27.09.HR-MS(ESI/TOF)m/z: Calcd.for C20H24D4N3O3[M+H]+ 362.2382; 362.2381 shows the spectra in figures 1, 2 and 3.
Example 2
D4-3, 3-dimethyl-2- (1- (pent-4-en-1-yl) -1H-indazole-3-carboxamide) butanoic acid (D4-MDMB-4en-PINACA metabolite).
Methyl D4-3, 3-dimethyl-2- (1- (pent-4-en-1-yl) -1H-indazole-3-carboxamide) butanoate (D4-MDMB-4en-PINACA) (362mg, 1mmol), dissolved in 5% lithium hydroxide in methanol-water 1:1, heated to 60 ℃ and stirred overnight. The reaction mixture was washed with ether, the aqueous phase was adjusted to pH 1 with 30% hydrochloric acid, a large amount of solid precipitated, filtered, air-dried, and recrystallized from acetone-petroleum ether to give D4-3, 3-dimethyl-2- (1- (pent-4-en-1-yl) -1H-indazole-3-carboxamide) butanoic acid (D4-MDMB-4en-PINACA metabolite) as a white solid (223mg, 62%).
1H NMR(600MHz,Methanol-d4)δ8.23–8.10(m,1H),7.60(d,J＝8.6Hz,1H),7.42(ddd,J ＝8.4,6.9,1.0Hz,1H),7.34–7.11(m,1H),4.47(t,J＝7.0Hz,2H),2.32(t,J＝7.4Hz,2H),2.19(d, J＝2.0Hz,6H),2.11(s,3H),1.97(td,J＝14.9,14.0,6.6Hz,2H),1.77(s,7H),1.59(p,J＝7.5Hz, 2H).13C NMR(151MHz,cd3od)δ174.13,164.20,142.46,138.51,137.50,127.56,123.82,123.45, 122.65,116.14,110.80,61.17,49.69,35.65,31.83,30.01,27.20.HR-MS(ESI/TOF)m/z:Calcd.for C19H22D4N3O3[M+H]+ 348.2225; 348.2225 is Found. The spectra are shown in figures 4,5 and 6.
Example 3
Methyl N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-enepentyl) -indazole-3-butanoate and its metabolite N- (3, content of 3-dimethyl-butanamide-2-yl) -1- (5-1-enpentyl) -indazole-3-butyric acid (MDMB-4en-PINACA metallate).
(1) Liquid chromatography-mass spectrometry detection conditions:
a) the instrument model is as follows: agilent 1290-6470 QQQ;
b) a chromatographic column: poroshell120 PFP 3.0x100mm 1.9.9 um;
c) column temperature: 30 ℃;
d) mobile phase: acetonitrile (0.01% formic acid), B: water (0.01% formic acid, 5% ammonium formate), gradient: 1min 50% A,2min 50% A,4min 90% A,6min 95% A;
e) flow rate: 0.5 mL/min;
f) sample introduction amount: 5 mu L of the solution;
g) an ion source: electrospray ion source, positive mode (ESI +);
h) the spraying voltage is 3500V;
i) ion source temperature: 340 ℃;
j) collision gas: nitrogen gas.
The ion pairs and corresponding conditions are shown in table 1:
TABLE 1
Figure BDA0003503749070000071
(2) Pretreatment and detection of samples
The hair sample is cleaned by ultrapure water, detergent water and acetone, dried and then cut into pieces, each of which is weighed at 20.0mg, 1mL of methanol (containing 10ng/mL of D4-MDMB-4en-PINACA and 10ng/mL of D4-MDMB-4en-PINACA methyl acetate) is added, after grinding, ultrasonic treatment is carried out for 30min in a frozen ultrasonic instrument, centrifugation is carried out for 5min at 4000r, 800 muL of supernatant is taken, evaporation is carried out under water bath air flow at 60 ℃, 80 muL of methanol is used for redissolution, 5 muL of supernatant is filtered by a 0.22 muL filter membrane, and LC-MS/MS analysis is carried out to obtain the graph as shown in figures 7 and 8. Wherein the quantitative ion pair is 348.2/232.0, the peak area ratio of MDMB-4en-PINACA and the internal standard D4-MDMB-4en-PINACA in two detections is 29599/58699, 39086/78513, and the peak area ratio of MDMB-4en-PINACA metablate and the internal standard D4-MDMB-4en-PINACA metablate is 7481/156380, 35925/743680.
(3) Drawing of standard curve
Processing blank negative hair sample with ultrapure water, liquid detergent and waterKetone cleaning, air drying, cutting, weighing 20.0mg each, adding 1mL methanol (containing 10ng/mL D4-MDMB-4en-PINACA and 10ng/mL D4-MDMB-4en-PINACA metablate), adding 20 μ L MDMB-4en-PINACA standard control with concentration of 0, 100, 200, 400, 1000, 2000, 20000ng/mL, preparing hair addition samples with concentration of 0, 0.1, 0.2, 0.4, 1.0, 2.0, 10, 20ng/mg, preparing 3 parts each in parallel, vortexing for 3min, soaking for 30min at room temperature, performing LC-MS/MS detection after sample pretreatment, and making standard curves for the ratio and concentration of MDMB-4en-PINACA, D4-pinacb-4 en-PINACA to obtain the area of nacn peak shown in fig. 9. The formula of the standard curve is Y2.947854X-0.436116, wherein X is the concentration of the sample to be detected, Y is the peak area obtained by liquid chromatogram-mass spectrum detection, b0-0.436116 is the intercept of the standard curve, b12.947854 is the slope of the standard curve.
Standard curves were made for the peak area ratios and concentrations of MDMB-4en-PINACA metablate and D4-MDMB-4en-PINACA metablate to give the profile shown in FIG. 10. The formula of the standard curve is Y0.936315X 0.153274, wherein X is the concentration of the sample to be detected, Y is the peak area obtained by liquid chromatogram-mass spectrum detection, b0-0.153274 is the intercept of the standard curve, b10.936315 is the slope of the standard curve.
According to the quantitative relation between the peak area ratio and the concentration in the standard curve and the calculation formula:
Figure BDA0003503749070000081
and calculating that the content of the MDMB-4en-PINACA in the sample to be detected is 0.32ng/mg, and the content of the MDMB-4en-PINACA metablate is 0.22 ng/mg.
Example 4
Methyl N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-enepentyl) -indazole-3-butanoate and its metabolite N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-enepentyl) -indazole-3-butanoate from wastewater were examined with D4-3, 3-dimethyl-2- (1- (pent-4-en-1-yl) -1H-indazole-3-carboxamide) butyric acid methyl ester (D4-MDMB-4 en-PINCA) and D4-3, 3-dimethyl-2- (1- (pent-4-en-1-yl) -1H-indazole-3-butanoate (D4-MDMB-4 en-PINCA metallate) as internal standards, content of 3-dimethyl-butanamide-2-yl) -1- (5-1-enpentyl) -indazole-3-butyric acid (MDMB-4en-PINACA metallate).
(1) Liquid chromatography-mass spectrometry detection conditions: same as in example 3.
(2) Pretreatment and detection of samples
Filtering the sewage sample by filter paper, taking 100mL, adding 2mL of methanol (containing 10ng/mL D4-MDMB-4en-PINACA and 10ng/mL D4-MDMB-4en-PINACA methylate), passing each 50mL of methanol through SPE column at the speed of 5mL/min, rinsing with 5mL of methanol after finishing the sample loading, finally eluting with 5mL of 5% ammonia water-acetonitrile solution, volatilizing the eluent under 40 ℃ water bath air flow, redissolving with 80 muL of methanol, passing through 0.22 muL of filter membrane, and carrying out LC-MS/MS analysis by 15 muL of filter membrane to obtain the graph shown in figures 11 and 12. Wherein the quantitative ion pair is 348.2/232.0, the peak area ratio of MDMB-4en-PINACA and the internal standard D4-MDMB-4en-PINACA in two detections is 9241/23730, 8987/24300, and the peak area ratio of MDMB-4en-PINACA metablate and the internal standard D4-MDMB-4en-PINACA metablate is 42510/57918, 43840/58351.
(3) Drawing of standard curve
Adding 1mL of methanol (containing 10ng/mL of D4-MDMB-4 en-PINCA and 10ng/mL of D4-MDMB-4 en-PINCA methyl acetate) into 50mL of a negative sewage sample, adding 20 μ L of MDMB-4 en-PINCA standard reference substances with the concentrations of 0, 1.25, 2.5, 5, 12.5, 25, 125 and 250ng/mL, preparing sewage adding samples with the concentrations of 0, 0.5, 1.0, 2.0, 5.0, 10, 50 and 100ng/L, preparing 3 parts of each concentration in parallel, vortexing for 3min, standing and soaking for 30min at room temperature, treating according to a sample pretreatment NAC process, and performing LC-MS/MS detection, and making standard curves on the ratio and the concentration of MDMB-4 en-PIA and D MDMB 4-4 en-PINCA to obtain a peak area shown in figure 13. The formula of the standard curve is Y0.579400X-0.403656, wherein X is the concentration of the sample to be detected, Y is the peak area obtained by liquid chromatogram-mass spectrum detection, b0-0.403656 is the intercept of the standard curve, b10.579400 is the slope of the standard curve.
Making standard curve for the peak area ratio and concentration of MDMB-4en-PINACA metablate and D4-MDMB-4en-PINACA metablate to obtainTo the map shown in fig. 14. The formula of the standard curve is 0.188053X 0.166259, wherein X is the concentration of the sample to be detected, Y is the peak area obtained by liquid chromatogram-mass spectrum detection, b0-0.166259 is the intercept of the standard curve, b10.188053 is the slope of the standard curve.
According to the quantitative relation between the peak area ratio and the concentration in the standard curve and the calculation formula:
Figure BDA0003503749070000091
and calculating that the content of the MDMB-4 en-PINCA in the sample to be detected is 1.35ng/mg, and the content of the MDMB-4 en-PINCA metabolite is 4.82 ng/mg.
Example 5
Methyl D4-3, 3-dimethyl-2- (1- (pent-4-en-1-yl) -1H-indazole-3-carboxamide) butanoate (D4-MDMB-4en-PINACA) and D4-3, 3-dimethyl-2- (1- (pent-4-en-1-yl) -1H-indazole-3-carboxamide) butanoate (D4-MDMB-4en-PINACA metallate) were used as internal standards to detect methyl N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-en-enpentyl) -indazole-3-butanoate and its metabolite N- (3, content of 3-dimethyl-butanamide-2-yl) -1- (5-1-enpentyl) -indazole-3-butyric acid (MDMB-4en-PINACA metallate).
(2) Liquid chromatography-mass spectrometry detection conditions: same as in example 3.
(2) Pretreatment and detection of samples
Filtering urine sample with filter paper, taking 10mL, adding 0.2mL methanol (containing 10ng/mL D4-MDMB-4en-PINACA and 10ng/mL D4-MDMB-4en-PINACA methylate), loading each 5mL SPE column at the speed of 5mL/min, rinsing with 5mL methanol after loading, eluting with 5mL 5% ammonia-acetonitrile solution, evaporating the eluent under 40 ℃ water bath air flow, redissolving with 80 μ L methanol, filtering with 0.22 μ L filter membrane, and analyzing with LC-MS/MS to obtain the graph shown in figures 15 and 16. Wherein the quantitative ion pair is 348.2/232.0, the peak area ratio of MDMB-4en-PINACA and the internal standard D4-MDMB-4en-PINACA in two detections is 46180/27779, 46063/27825, and the peak area ratio of MDMB-4en-PINACA metablate and the internal standard D4-MDMB-4en-PINACAmetabolite is 6092/249909, 10500/257039.
(3) Drawing of standard curve
Adding 0.1mL of methanol (containing 10ng/mL of D4-MDMB-4 en-PINCA and 10ng/mL of D4-MDMB-4 en-PINCA methyl acetate) into 5mL of a negative urine sample, adding 20 μ L of MDMB-4 en-PINCA standard control with the concentration of 0, 1.25, 2.5, 5, 12.5, 25, 125, 250 μ g/mL, preparing 3 urine samples with the concentration of 0, 5, 10, 20, 50, 100, 500, 1000ng/mL in parallel, vortexing for 3min, performing LC-MS/MS detection after sample pretreatment process, and making a standard curve for the peak area ratio and concentration of the MDMB-4 en-PINCA and the D4-MDMB-4 en-PINCA to obtain a graph shown in figure 17. The formula of the standard curve is Y0.059264X-0.500630, wherein X is the concentration of the sample to be detected, Y is the peak area obtained by liquid chromatogram-mass spectrum detection, b0-0.059264 is the intercept of the standard curve, b10.500630 is the slope of the standard curve.
Standard curves were made for the peak area ratios and concentrations of MDMB-4en-PINACA metablate and D4-MDMB-4en-PINACA metablate to give the profile shown in FIG. 18. The formula of the standard curve is 0.018206X 0.170602, wherein X is the concentration of the sample to be detected, Y is the peak area obtained by liquid chromatogram-mass spectrum detection, b0-0.170602 is the intercept of the standard curve, b10.018206 is the slope of the standard curve.
According to the quantitative relation between the peak area ratio and the concentration in the standard curve and the calculation formula:
Figure BDA0003503749070000101
and calculating the content of MDMB-4 en-PINCA in the sample to be detected to be 36.4ng/mL and the content of MDMB-4 en-PINCA metabolite in the sample to be detected to be 11.1 ng/mL.
When the isotopic psychoactive substance labeled compound provided by the invention is used for detecting a psychoactive substance MDMB-4 en-PINCA in a detection material, a proper amount of the isotopic psychoactive substance labeled compound D4-MDMB-4 en-PINCA is added into the detection material, appropriate pretreatment is carried out according to the detection requirement, then liquid chromatography-mass spectrometry (LC-MS/MS) detection is carried out, the isotopic psychoactive substance labeled compound is used as an internal standard, and qualitative and quantitative detection of the substance to be detected is realized by comparing the peak area ratios of a target substance and the substance to be detected in a Multiple Reaction Monitoring (MRM) mode, and the detectable psychoactive substance is MDMB-4 en-PINCA, so that the isotopic psychoactive substance labeled compound has strong specificity and high sensitivity.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
In this specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.

Claims (11)

1. An isotopically active substance labeled compound, wherein said compound has the formula:
Figure FDA0003503749060000011
wherein R is1、R2、R3、R4At least one of D and R is methyl or hydrogen, and the psychoactive substance contains N- (3, 3-dimethyl-butyramide-2-yl) -1- (5-1-alkenyl amyl) -indazole-3-butyric acid methyl ester and metabolite N- (3, 3-dimethyl-butyramide-2-yl) -1- (5-1-alkenyl amyl) -indazole-3-butyric acid.
2. The isotopically active substance labeled compound of claim 1, wherein the isotopically active substance labeled compound has the formula:
Figure FDA0003503749060000012
3. a method for preparing an isotopically active substance-labeled compound according to claim 1 or 2, comprising the steps of:
(1) carrying out Friedel-crafts acylation reaction on N- (3-methyl-butyryl benzylamine-2-yl) ammonium formate and N-5-fluoropentyl indole under the catalysis of aluminum trichloride to generate a benzyl protected intermediate;
(2) deprotection of the intermediate in step (1) with basic conditions produces N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-enepentyl) -indazole 4,5,6, 7-D4-3-butanoic acid (D4-MDMB-4en-PINACA metabolite).
4. Use of an isotopically labeled compound according to claim 1 or 2 for detecting the content of the drug N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-enpentyl) -indazole-3-butyric acid methyl ester and its metabolite N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-enpentyl) -indazole-3-butyric acid in hair.
5. The use according to claim 4, characterized in that the method for detecting the content of the drug N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-enpentyl) -indazole-3-butyric acid methyl ester and its metabolite N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-enpentyl) -indazole-3-butyric acid in hair using the isotopically labeled compound of claim 1 or 2 comprises the following steps:
(1) setting the detection conditions of liquid chromatography-mass spectrometry;
(2) drawing a standard curve: adding MDMB-4en-PINACA, D4-MDMB-4en-PINACA, MDMB-4en-PINACA methylate and D4-MDMB-4en-PINACA methylate standard solutions in different proportions into a negative detection material corresponding to a sample to be detected, performing LC-MS/MS detection after the same pretreatment steps as the sample to be detected, and making standard curves of peak area ratios and concentrations of the MDMB-4en-PINACA methylate and the D4-MDMB-4en-PINACA, the MDMB-4en-PINACA methylate and the D4-MDMB-4en-PINACA methylate, wherein the internal standard substance is the isotopic drug labeling compound according to claim 1 or 2;
(3) pretreatment and determination of a sample to be detected: adding the isotope drug labeled compound in claim 2 into a sample to be detected as an internal standard, performing LC-MS/MS detection after the same pretreatment steps as those used for drawing a standard curve to obtain quantitative parameters of the sample to be detected and the internal standard, and calculating by using a formula of the standard curve of an internal standard method to obtain the content of the sample to be detected.
6. The method according to claim 5, wherein in the step (1), the mobile phase for the liquid chromatography-mass spectrometry detection is as follows: acetonitrile (0.01% formic acid), B: water (0.01% formic acid, 5% ammonium formate), gradient: 1min 10% A,2min 50% A,4min 90% A,6min 95% A; a chromatographic column: poroshell120 PFP 3.0x100mm 1.9.9 um; column temperature: 30 ℃; flow rate: 0.5 mL/min; sample injection amount: 5 mu L of the solution; an ion source: electrospray ion source, positive mode (ESI +); the spraying voltage is 3500V; ion source temperature: 340 ℃; collision gas: nitrogen gas.
7. The method according to claim 5, wherein in the step (2), the mass concentration of the internal standard substance in the mixed standard solution is 0.1-99.9%.
8. The method according to claim 5, wherein in the step (3), the mass concentration of the internal standard substance in the sample to be tested is 0.1-99.9%.
9. The method of claim 5, wherein in step (3), the internal standard method formula is calculated as:
Figure FDA0003503749060000021
wherein X is the concentration of a sample to be detected, Y is the peak area obtained by liquid chromatography-mass spectrometry detection, b0Is the intercept of the standard curve, b1Is the slope of the standard curve.
10. The use according to claim 4, wherein the bioassay material contains N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-eny ρ entyl) -indazole-3-butyric acid methyl ester and its metabolite N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-eny ρ entyl) -indazole-3-butyric acid in an amount of 10-9％-10％。
11. A method for detecting the content of the drug N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-enpentyl) -indazole-3-butyric acid methyl ester and the metabolite N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-enpentyl) -indazole-3-butyric acid in biological detection materials, which is characterized in that the method comprises the steps of detecting the drug N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-enpentyl) -indazole-3-butyric acid methyl ester and the metabolite N- (3, 3-dimethyl-butanamide-2-yl) -1- (5-1-enpentyl) -indazole-3-butanoic acid.
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CN113321611A (en) * 2021-02-01 2021-08-31 无锡诺平医药科技有限公司 Isotope mental active substance labeled compound and preparation method and application thereof

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