CN111487336B - Method for analyzing 37 fentanyl novel psychoactive substances in hair - Google Patents
Method for analyzing 37 fentanyl novel psychoactive substances in hair Download PDFInfo
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- G—PHYSICS
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- 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
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Abstract
The invention discloses an analysis method of 37 fentanyl novel psychoactive substances in hair, which comprises the following steps: (1) preparing a solution; (2) preparing a sample; (3) and (3) respectively carrying out liquid chromatography-tandem mass spectrometry on the sample solution to be detected, the standard curve solution to be detected and the blank solution to be detected, which are prepared in the step (2), and establishing an analysis method for measuring 37 fentanyl new psychoactive substances in the sample solution. The invention establishes a high-sensitivity and high-selectivity HPLC-MS/MS qualitative and quantitative analysis method by taking fentanyl new psychoactive substances as research objects and hair as a biological detection material, applies the method to the detection and analysis of specific cases, and provides related technical support for related fields such as judicial identification and the like.
Description
Technical Field
The invention relates to the technical field of fentanyl new psychoactive substance detection, in particular to an analysis method of 37 fentanyl new psychoactive substances in hair.
Background
New Psychoactive Substances (NPS) refer to emerging drugs that are similar in chemical structure to, but not identical to, other traditional psychotropic drugs and are "legal" substitutes for internationally regulated drugs. The new mental active substances mainly comprise phenethylamines, cathinone, piperazines, fentanyl and the like by taking a chemical structure as a basic classification basis. The synthesis of opioids (particularly fentanyl-type psychoactive substances) as one of the large groups of new psychoactive substances, which have chemical structures and properties similar to those of the traditional controlled drugs, but which are covered by the "legal" coat, has become a new target for lawless persons. However, few studies are currently conducted on the analysis method for synthesizing the novel opioid psychoactive substances, so that the establishment of the analysis method with high sensitivity and strong selectivity is particularly important for improving the judicial identification level and fighting against illegal crimes.
At present, the manufacturing, selling, smuggling and abuse problems of new mental active substances are more and more prominent on the global scale, and become a recognized problem in the international drug inhibition field. Fentanyl as a mu-opioid receptor agonist is frequently used with other drugs such as heroin, ***e, methamphetamine, etc. due to its ability to produce a sense of joy, addiction and dependence. Moreover, the risk of abuse is extremely high due to the more potent and low lethal dose. At present, the number of people who are fatalities due to the abuse of fentanyl substances is increased sharply, so that the public health of each country is seriously threatened, and people need to pay attention to the fentanyl substances widely.
In recent years, various methods for analyzing fentanyl substances have been reported, and the methods mainly include immunoassay, gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. The biological test material for detecting fentanyl substances reported at present mainly comprises body fluids such as blood samples, urine samples and saliva. The scientists of Melissa et al used for the separation and analysis of 18 fentanyl substances in whole blood samples by LC-MS/MS method and applied the practical cases. Saliva was analyzed for fentanyl and demethylfentanyl by scientists such as Bista. Scientists such as Shanher, etc. used on-line solid phase extraction to analyze 7 fentanyl species in dry spots. However, the fentanyl substance is high in metabolic speed in blood and urine, and the detection time limit is short. Therefore, when the information about whether the fentanyl substance is abused for a long time is provided for the object to be detected, other detection materials are also needed. In recent years, in the fields of forensic toxicology, clinical chemistry and the like, hair has become a basic biological matrix and can replace conventional samples such as blood, urine and the like for drug detection. The hair sample has more unique use value in the field of court toxicity analysis, and is mainly shown in the following steps: (1) the hair detection device has a longer detection window, can provide long-term medication information, and can reflect the medication history compared with the condition that urine and blood can only reflect 2 to 4 days of medication; (2) the original drug can be generally detected in the hair, which is helpful for determining the exogenous target substance; (3) the collection of the sample is noninvasive and easy to obtain. The characteristic that the hair analysis can provide unique information makes the hair analysis important supplement of other biological detection materials and plays an increasingly important role in the aspects of abuse of new psychoactive substances and the like.
Therefore, an analysis method using hair as a biological sample and fentanyl-based novel psychoactive substances as a research object is urgently needed.
Disclosure of Invention
The invention aims to provide an analysis method of 37 fentanyl new psychoactive substances in hair, which takes the fentanyl new psychoactive substances as research objects and the hair as a biological test material, establishes a high-sensitivity and high-selectivity HPLC-MS/MS qualitative and quantitative analysis method, is applied to detection and analysis of specific cases, and provides related technical support for related fields such as judicial identification.
In order to achieve the purpose, the invention adopts the technical scheme that:
provides an analysis method of 37 fentanyl novel psychoactive substances in hair, which comprises the following steps:
(1) solution preparation:
preparation of mixed control solution: precisely transferring 37 reference substances, and diluting with methanol to obtain mixed reference substance solutions with mass concentrations of 4, 10, 20, 100, 200, 400, 1000, 2000, 2500 and 4000ng/mL respectively for later use;
preparing an extraction solution: mixing the ammonium formate solution with methanol and acetonitrile to obtain an extraction solution;
preparing a mixed internal standard solution: accurate removal of fentanyl-d 5 Demethylfentanyl-d 5 And U-47700-d 3 Adding the extraction solution into a proper amount of the mixed internal standard solution to prepare a mixed internal standard solution with the mass concentration of 10 ng/mL;
(2) sample preparation:
precisely weighing 20mg of a pre-treated hair sample to be detected, placing the hair sample to be detected in a 2mL test tube, adding an appropriate amount of grinding beads and 1mL of the mixed internal standard solution prepared in the step (1), carrying out freeze grinding in a homogenizer, then centrifuging, and filtering the supernate through a 0.22 mu m microporous membrane to obtain a sample solution to be detected;
precisely weighing 20mg of a blank hair sample subjected to pretreatment, placing the blank hair sample into a 2mL test tube, respectively adding 10 μ L of the mixed reference substance solution prepared in the step (1), then adding an appropriate amount of grinding beads and 1mL of the mixed internal standard solution prepared in the step (1), carrying out freeze grinding in a homogenizer, then centrifuging, and filtering the supernatant through a 0.22 μm microporous filter membrane to obtain a standard curve solution to be measured;
precisely weighing 20mg of a blank hair sample subjected to pretreatment, placing the blank hair sample into a 2mL test tube, adding an appropriate amount of grinding beads and 1mL of the mixed internal standard solution prepared in the step (1), carrying out freeze grinding in a homogenizer, then centrifuging, and filtering the supernate through a 0.22-micrometer microporous membrane to obtain a blank solution to be detected;
(3) and (3) respectively carrying out liquid chromatography-tandem mass spectrometry on the sample solution to be detected, the standard curve solution to be detected and the blank solution to be detected, which are prepared in the step (2), and establishing an analysis method for measuring 37 fentanyl new psychoactive substances in the sample solution.
Preferably, the 37 control substances are fentanyl (fentanyl), norfentanyl (norfentanyl), alfentanyl (alfentanyl), acetylfentanyl (acetylfentanyl), acetylmethylfentanyl (acetylnorfentanyl), deacrylfentanyl (4-ANPP), acryloylfentanyl (acetylfentanyl), butyrylfentanyl (butyrylfentanyl), isobutyrylfentanyl (isobutoxyfentanyl), p-fluorofentanyl (para-fluorofentanyl), o-fluorofentanyl (ortho-fluorofentanyl), p-fluorobutyrylfentanyl (PFBF), 4-fluoroisobutyrylfentanyl (4-fluoroisobutyrylfentanyl), β -hydroxyfentanyl (β -hydroxyfentanyl), cis-3-methylfentanyl (cis-3-methylfentanyl), furylfentanyl (sulfenyl), remifentanyl (hydracrylic), remifentanyl (hydracrylic (4-fluorofentanyl), remifentanyl (hydracrylic acid (4-hydroxyfentanyl), remifentanyl (hydracrylic acid (propionic acid) (e), propionic acid (propionic acid) (PFBF), propionic acid (propionic acid) (Fe-3-propionic acid) (Fe-3-propionic acid) (e) and remifentanyl) as well as a) as a compound, Valerylfentanyl, carfentanil, norcarfentanil, methoxyacetylfentanyl, cyclopropylfentanyl, U-47700, N-demethyl U-47700(N-desmethyl U-47700), U-50488, U-48800, W-18, fentanyl-d 5, beta-hydroxy-3-methylfentanyl (beta-hydroxy-3-methylfentanyl), beta-hydroxy-fentanyl (beta-hydroxyfentanyl), trans-3-methylfentanyl (trans-3-methylfentanyl), alpha-methylfentanyl (alpha-methylfentanyl), 3-methylthiofentanyl (3-methylfentanyl), thiofentanyl (thioantenanyl), and tetrahydrofentanyl (THF-F).
Preferably, the fentanyl-d 5 is an internal standard for fentanyl and fentanyl analogs, the demethylfentanyl-d 5 is an internal standard for demethylfentanyl, acetyldemethylfentanyl, and demethylcarfentanyl, and U-47700-d3 is an internal standard for opioids.
Preferably, the specific preparation method of the extraction solution is as follows: 230mL of ammonium formate solution with pH of 5.3 and concentration of 2mmol/L is taken, and 125mL of methanol and 145mL of acetonitrile are added and mixed to obtain the extraction solution.
Preferably, in step (2), the pretreatment is: placing the hair sample in a test tube with a plug, sequentially cleaning with ultrapure water for 3 times and acetone for 3 times, collecting the last acetone cleaning solution, drying, redissolving with the extraction solution prepared in the step (1) to remove external pollution factors, cleaning the hair, drying at room temperature, and cutting into 2-3mm sections for later use.
Preferably, in the step (2), the homogenizer is a Bead raptor 24Elite multifunctional sample homogenizer, the grinding speed of the freeze grinding is 6m/s, the grinding time is 20s, the time interval between two times of grinding is 40s, and the cycle number is 10.
Preferably, in the step (2), the centrifugal force of the centrifugation is 14000 Xg, and the centrifugation time is 3 min.
Preferably, in step (3), the column of the liquid chromatography: waters Acquity
HSS T3 column (100 mm. times.2.1 mm, 1.8 μm); mobile phase A: 20mmol/L ammonium acetate buffer solution containing 0.1% formic acid, mobile phase B: acetonitrile; flow rate: 0.2 mL/min.
Preferably, the elution gradient for the chromatographic analysis in step (3) is as follows:
at 0min, the mobile phase A is 85.0%, and the mobile phase B is 15.0%;
at 1.0min, the mobile phase A is 85.0%, and the mobile phase B is 15.0%;
at 4.0min, the mobile phase A is 72.0 percent, and the mobile phase B is 28.0 percent;
at 5.0min, the mobile phase A is 72.0 percent, and the mobile phase B is 28.0 percent;
at 10.0min, the mobile phase A is 70.0 percent, and the mobile phase B is 30.0 percent;
at 13.0min, mobile phase A is 55.0%, and mobile phase B is 45.0%;
at 13.5min, the mobile phase A is 5.0 percent, and the mobile phase B is 95.0 percent;
at 14.5min, the content of mobile phase A is 5.0%, and the content of mobile phase B is 95.0%;
at 15.0min, the mobile phase A is 85.0%, and the mobile phase B is 15.0%;
at 16.0min, mobile phase A was 85.0% and mobile phase B was 15.0%.
Preferably, in the step (3), the mass spectrum is detected by adopting a positive ion mode of an electrospray ionization mass spectrometer, an ion monitoring mode is selected, and analysis software 1.5 multiple workstations are adopted for data collection and analysis and multi-ion mode monitoring; the ion source temperature of the mass spectrometer is 500 ℃, and the parent ions and the child ions, the cluster removing voltage and the collision energy are screened after direct sample injection to obtain the maximum ion strength and keep the collision dissociation energy stable.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the invention inspects 37 fentanyl substances and novel synthetic opioid substances in hair by an LC-MS/MS method, takes the hair as a biological detection material, has the advantages of easy collection, stability, easy storage, long detection window, capability of reflecting the long-time medication history and the like, enables the hair analysis to be more systematic and standardized, and can be used as a reference basis of court.
Drawings
FIG. 1 is a chromatogram of the mass concentration LLOQ of 37 kinds of targets in hair.
Detailed Description
The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting.
It should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other without conflict.
Medicine preparation:
37 control substances are fentanyl (fentanyl), demethylfentanyl (norfentanyl), alfentanyl (alfentanyl), acetylfentanyl (acetylfentanyl), acetyldemethylfentanyl (acetylmethylfentanyl), deacetylfentanyl (4-ANPP), acryloylfentanyl (acylfentanyl), butyrylfentanyl (butyrylfentanyl), isobutyrylfentanyl (isobutrylfentanyl), p-fluorofentanyl (para-fluorofentanyl), o-fluorofentanyl (ortho-fluorofentanyl), p-fluorobutyrylfentanyl (PFBF), 4-fluoroisobutyrylfentanyl (4-fluoro-isobutryltanyl), β -hydroxyfentanyl (β -hydroxyfentanyl), cis-3-methylfentanyl (cis-3-methylfenfect), furanfentanyl (cyanofentanyl), remifentanyl (carnyl), remifentanil (4-hydroxyfentanyl), remifentanil (carnyl), and their salts (salts, valerylfentanyl (valyl fentanyl), carfentanyl (carfentanil), norcarfentanyl (norcarpentanil), methoxyacetylfentanyl (methoxyacetylfentanyl), cyclopropylfentanyl (cyclopropyfentanyl), U-47700, N-demethyl U-47700(N-desmethyl U-47700), U-50488, U-48800, W-18, fentanyl-d 5, demethylfentanyl-d 5 and U-47700-d3 are all available from Cerilliant, USA. The controls β -hydroxy-3-methylfentanyl (β -hydroxy-3-methylfentanyl), β -hydroxy-fentanyl (β -hydroxyfentanyl), trans-3-methylfentanyl (trans-3-methylfentanyl), α -methylfentanyl (α -methylfentanyl), 3-methylthiofentanyl (3-methylthiofentanyl), thiofentanyl (thiofentanyl) and tetrahydrofurfentanyl (THF-F) were from the public police department of Shanghai, the criminal investigation department.
Methanol and acetonitrile (UPLC) were purchased from Sigma-Aldrich (st. louis, MO, USA). Ammonium formate (UPLC) was purchased from Fluka (Buchs, Switzerland). Formic acid (4.014784.0250, AR) and ammonium acetate (UPLC) were purchased from CNW (UK). Deionized water was prepared from a Milli-Q (Millipore, MA, USA) water purification system. Blank hair was obtained from laboratory volunteers.
The instrument conditions were as follows:
XW-80A vortex mixer (Shanghai medical university apparatus), Bead Ruptor 24Elite multifunctional sample homogenizer (OMNI corporation, USA), MinnSpin high speed centrifuge (Eppendorf corporation, Germany), AFS-10 ultrapure water preparation system (Merck, Germany).
ACQUITY UPLC I-CLASS liquid chromatograph (Waters, USA) tandem QTRAP 6500Plus triple quadrupole linear ion trap composite mass spectrometer (SCIEX, USA). A chromatographic column: waters Acquity
HST 3(100 mm. times.2.1 mm, 1.8 μm), pre-column: waters Acquity
HSST 3(100 mm. times.2.1 mm, 1.8 μm), data analysis was performed using a MultiQuant 3.0.2 workstation.
This example provides a method for analyzing 37 fentanyl-based psychoactive substances in hair, comprising the following steps:
(1) solution preparation:
preparation of mixed control solution: precisely transferring 37 reference substances, adding methanol to dilute into mixed reference substance solutions with mass concentrations of 4, 10, 20, 100, 200, 400, 1000, 2000, 2500 and 4000ng/mL respectively for later use;
preparing an extraction solution: mixing the ammonium formate solution with methanol and acetonitrile to obtain an extraction solution;
preparing a mixed internal standard solution: accurate removal of fentanyl-d 5 Demethylated fentanyl-d 5 And U-47700-d 3 Adding the extraction solution into a proper amount of the mixed internal standard solution to prepare a mixed internal standard solution with the mass concentration of 10 ng/mL;
(2) sample preparation:
precisely weighing 20mg of a pre-treated hair sample to be detected, placing the hair sample to be detected into a 2mL test tube, adding a proper amount of grinding beads, mixing with 1mL of the mixed internal standard solution prepared in the step (1), carrying out freeze grinding in a homogenizer, then centrifuging, and filtering the supernatant through a 0.22 mu m microporous membrane to obtain a sample solution to be detected;
precisely weighing 20mg of a blank hair sample subjected to pretreatment, placing the blank hair sample into a 2mL test tube, respectively adding 10 μ L of the mixed reference substance solution prepared in the step (1), then adding an appropriate amount of grinding beads and 1mL of the mixed internal standard solution prepared in the step (1), carrying out freeze grinding in a homogenizer, then centrifuging, and filtering the supernatant through a 0.22 μm microporous filter membrane to obtain a standard curve solution to be measured;
precisely weighing 20mg of a blank hair sample subjected to pretreatment, placing the blank hair sample into a 2mL test tube, adding a proper amount of grinding beads, mixing with 1mL of the mixed internal standard solution prepared in the step (1), carrying out freeze grinding in a homogenizer, then centrifuging, and filtering the supernatant through a 0.22-micrometer microporous membrane to obtain a blank solution to be detected;
(3) and (3) respectively carrying out liquid chromatography-tandem mass spectrometry on the sample solution to be detected, the standard curve solution to be detected and the blank solution to be detected, which are prepared in the step (2), and establishing an analysis method for measuring 37 fentanyl new psychoactive substances in the sample solution.
As a preferred embodiment, the specific preparation method of the extraction solution is as follows: 230mL of ammonium formate solution with pH of 5.3 and concentration of 2mmol/L is taken, and 125mL of methanol and 145mL of acetonitrile are added and mixed to obtain the extraction solution.
As a preferred embodiment, in the step (2), the pretreatment is: putting a hair sample into a test tube with a plug, sequentially washing the hair sample with ultrapure water for 3 times and acetone for 3 times, collecting acetone washing liquid at the last time, re-dissolving the acetone washing liquid with the extraction solution prepared in the step (1) after drying, removing external pollution factors, drying the hair at room temperature after washing, and cutting the hair into sections of 2-3mm for later use; in the step (2), the homogenizer is a Bead raptor 24Elite multifunctional sample homogenizer, the grinding speed of the freezing grinding is 6m/s, the grinding time is 20s, the interval time of the two grinding is 40s, and the cycle number is 10; in the step (2), the centrifugal force of the centrifugation is 14000 Xg, and the centrifugation time is 3 min.
As a preferred embodiment, the liquid chromatography column: waters Acquity
HSS T 3 column (100 mm. times.2.1 mm, 1.8 μm); mobile phase A: 20mmol/L ammonium acetate buffer solution containing 0.1% formic acid, mobile phase B: acetonitrile; flow rate: 0.2mL/min, gradient elution is shown in Table 1;
TABLE 1 gradient elution procedure
3 internal standards were used to calculate the peak area ratios for each compound, of which fentanyl-d 5 For internal standards of fentanyl and fentanyl analogs, demethylfentanyl-d 5 For the metabolites desmethylfentanyl, acetyldesmethylfentanyl and desmethylcarfentanyl internal standards, U-47700-d 3 Is an opioid internal standard;
the mass spectrum is detected by adopting a positive ion mode of an electrospray ionization mass spectrometer (Applied Biosystems/MDS SCIEX, Toronto, Canada), an ion monitoring mode is selected, and analysis software 1.5(Waters) multi-workstation is adopted for data collection and analysis and multi-ion mode monitoring; the ion source temperature of the mass spectrometer is 500 ℃, and the parent ions and the daughter ions, as well as the Declustering Potential (DP) and the Collision Energy (CE) are screened to obtain the maximum ion intensity and keep the Collision dissociation energy stable; through respectively and directly carrying out mass spectrum sample injection on each target analyte, fragment ions, declustering voltage and collision energy of 37 fentanyl substances, novel synthetic opioids and 3 internal standard compounds are sequentially determined as shown in a table 2, so that the fragment ions have maximum response values, and each compound is calculated through two fragment ions;
TABLE 2 Mass Spectrometry parameters, Retention time and ion ratio of the Compounds
And (3) verification of methodology:
methodological validation indices commonly used in the quantitative analysis of biopharmaceuticals are validated according to the guidelines of the hair analysis society and the literature of Peters et al, and include: selectivity, detection limit, quantitation limit, precision, accuracy, linear range, and stability.
The selectivity is generally detected by a blank matrix, and the specific processes are as follows: blank hair from 10 volunteers, free of fentanyl and novel synthetic opioids, was analyzed to ensure that there was no interference from other substances during the peak time of the target component, and in addition, possible co-drug induced interference was investigated.
The specific processes of the detection limit and the quantification limit are as follows: adding blank hair into the mixed reference solution to prepare hair samples containing all target substances with different concentrations; and taking the mass concentration when the signal-to-noise ratio S/N is larger than or equal to 3 as a detection limit, taking the mass concentration when the signal-to-noise ratio S/N is larger than or equal to 10 as a quantification limit, and taking the quantification limit as the minimum concentration of a linear range to carry out experimental investigation on the precision and accuracy of 6 parallel hair samples.
The precision comprises intra-day precision and inter-day precision; the precision and accuracy are as follows: carrying out precision and accuracy investigation on four concentrations of LOQ, low, medium and high, taking blank hair, adding a proper amount of mixed reference substance solution to obtain quality control samples with mass concentrations of 2, 5, 10, 500 and 2000pg/mg, wherein each concentration is 6 parts of sample; performing sample pretreatment according to the preparation method in the step (2), performing sample injection analysis according to the instrument condition in the step (3), and calculating the precision and accuracy in the day; the accuracy is expressed in terms of bias, and the percentage between the value obtained by linear calculation and the true value is compared through linear calculation of the concentration; continuously measuring for 4 days, measuring simultaneously with the standard curve, calculating the concentration of the quality control sample according to the standard curve of the day, and calculating the day precision; precision the changes after repeated experiments on the same and different days were examined.
As a preferred embodiment, said linear rangeThe specific process is as follows: adding a proper amount of mixed reference substance solution into blank hair to obtain hair samples with different mass concentrations, performing sample pretreatment according to the preparation method in the step (2), performing sample injection analysis according to the instrument condition in the step (3), performing regression by adopting a weighted least square method by adopting the mass concentration of a target in whole blood as a horizontal coordinate and the peak area ratio of the target to an internal standard as a vertical coordinate, obtaining a linear equation, and calculating a correlation coefficient (R) 2 ) (ii) a The linear equation for each compound was calculated from 6 points with the lowest concentration being LOQ (S/N.gtoreq.10) and ensuring that the concentration of the actual sample falls within the linear range.
The methodological verification indexes further comprise extraction recovery rate and matrix effect, and the specific processes of the extraction recovery rate and the matrix effect are as follows: the samples were divided into three groups according to the method proposed by Matuszewski et al to calculate the extraction recovery and matrix effect; group I: adding a mixed reference substance solution with a certain concentration before extracting hair from different sources according to the preparation method in the step (2); group II: adding mixed reference substance solutions with corresponding concentrations after extraction according to the preparation method of the step (2) for hair with different sources; group III: preparing a mixed reference substance solution with corresponding concentration; adding blank hairs from different sources into a proper amount of the mixed reference substance solution to obtain quality control samples with mass concentrations of 2pg/mg, 5pg/mg (low concentration), 5pg/mg (medium concentration) and 30ng/mL (high concentration), wherein each concentration comprises 6 samples; analyzing sample introduction according to the instrument condition in the step (3), and recording the peak area (A); the extraction recovery rate is A Ⅰ /A II The matrix effect is A II /A III 。
1. Selectivity is selected
And comparing the chromatograms of the compounds in the hair sample to confirm that the endogenous substance and the possible same medicine have no interference on the target substance and the internal standard. The peak emergence time of each compound is distributed between 2.4 min and 15.23min, 4 pairs of isomers are basically separated, and the peak areas can be respectively confirmed. The chromatogram when the target substance concentration was LLOQ is shown in FIG. 1.
2. Detection limit and quantification limit
The hair samples with mass concentrations of 0.5, 1, 2, 2.5 and 5pg/mg were examined, respectively, and the mass concentration with a signal-to-noise ratio S/N of 3 or more was LOD, and the mass concentration with a signal-to-noise ratio S/N of 10 or more was LOQ. The precision (RSD) of 6 LOQ quality control hair samples is less than 20%, and the accuracy is between 80% and 120%. The results of the experiments show that the LOD of each compound in hair is 0.5-2.5pg/mg, the LLOQ is 2-5pg/mg, which is enough to meet the daily test protocol, and the results of the experiments are shown in Table 3.
3. Linear range of
By inspecting hair samples with mass concentration of 2, 5, 10, 50, 100, 200, 500, 1000, 2000, 2500pg/mg, the peak area ratio and concentration of the target compound and the internal standard are calculated according to the weighted (1/x) least square method to obtain a linear regression equation and R 2 . The results show that the respective compounds in the hair samples are well linear in the corresponding concentration ranges and that the correlation coefficient (R) is good 2 ) > 0.99, results are shown in table 3;
TABLE 3 regression equation, Linear Range, correlation coefficient, LOD and LLOQ for each target in hair
4. Precision and accuracy
The results of precision and accuracy within day were obtained by examining quality control samples at mass concentrations of 2, 5, 10, 500, 2000pg/mg, 6 samples per concentration (n is 6). The results were obtained with day precision (n-24) by continuous measurement for 4 days, simultaneously with the daily calibration curve. The results show that the daily precision of each compound is 0.8-13.32%, the daytime precision is 3.02-12.41%, and the accuracy is 85.63-116.1%, which indicates that the method has good precision and accuracy, and the experimental results are shown in Table 4;
TABLE 4 precision and accuracy test results
5. Stability of
The experiment examines the stability of the method after the quality control sample is extracted and placed for 24 hours at 4 ℃. The stability of the method is investigated by preparing 2, 5, 10, 500, 2000pg/mg hair samples and calculating the accuracy of the quality control samples according to the standard curve on the day of the following. The results show that the accuracy of the stability of the quality control samples is 77.44% -113.71%, and the results are shown in table 5;
TABLE 5 stability test results
The method adopts the freeze grinding technology as a pretreatment method to release the fentanyl substance in the hair sample, and compared with other ultrasonic methods and the like, the method has the advantages of simple operation and shorter time consumption. Compared with other fentanyl substance detection methods, the method disclosed by the invention can be used for simultaneously detecting the fentanyl substance in the largest number at present. In addition, the invention effectively separates 4 pairs of isomers, has simple method, high sensitivity and good selectivity, and is particularly suitable for daily case treatment.
6. Extraction recovery and matrix effect
And calculating to obtain the extraction recovery rate and the matrix effect by recording the peak areas of the quality control samples with the corresponding mass concentrations of 2, 5, 10, 500 and 2000pg/mg in each group. The result shows that the extraction recovery rate of each target object is 89.42-119.68%, the matrix effect range is 44.81-119.77%, wherein the ME except W-18 is 44.81-54.11%, and the matrix effect of other compounds meets the requirement. Due to the difference of chemical structures, the retention time of W-18 is 15.23min, and the final unstable stage of gradient elution is likely to cause lower matrix effect. The method is to be further examined for W-18, and the extraction recovery rate and the matrix effect result are shown in tables 6 and 7;
TABLE 6 test results of extraction recovery
TABLE 7 results of matrix Effect test
Application example 1
After methodology verification, the invention is applied to practical case analysis: in a case, a male reported by his colleagues may have a history of drug abuse; after receiving the report, a police inquires about the reported person and collects a hair sample of the reported person; the hair samples were then sent to a forensic laboratory for analysis.
In this case, the test person first divided the hair to be examined into 3 sections (S1: 0-3 cm; S2: 3-6 cm; S3: 6-9cm), and confirmed whether 37 fentanyl and novel synthetic opioids were contained therein by the method of the present invention.
Experimental results show that sufentanil is detected in 3 sections of samples after the hair sample to be detected is segmented, and the mass concentrations are as follows: 183.91pg/mg, 131.68pg/mg and 31.48 pg/mg. And according to the experimental examination result, the police further investigates the person to be reported. The last evidence shows that the case is related to the abuse of fentanyl, consistent with the experimental results.
Application example 2
After methodological validation, the invention was applied to practical case analysis: in the case of a woman who is physically ill-suited to hospital interrogation, the doctor advises him to go to a forensic laboratory for examination; the experimenter collected his hair sample for analysis after interrogation.
In this case, the experimenter took a hair sample of the identified person 3cm from the root of the hair and confirmed by the method of the present invention whether 37 fentanyl-based substances and the novel synthetic opioid were contained therein.
As a result of the experiment, fentanyl was detected in the hair sample at a mass concentration of 8.02 pg/mg. From the results of the experiment, the experimenter further confirmed that the woman had performed a surgery a little before. The results show that the case relates to the use of fentanyl substances and is consistent with the experimental results.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Claims (6)
1. A method for analyzing 37 fentanyl novel psychoactive substances in hair, which is characterized by comprising the following steps:
(1) solution preparation:
preparation of mixed control solution: precisely transferring 37 reference substances, adding methanol to dilute into mixed reference substance solutions with mass concentrations of 4, 10, 20, 100, 200, 400, 1000, 2000, 2500 and 4000ng/mL respectively for later use;
preparing an extraction solution: 230mL of ammonium formate solution with pH of 5.3 and concentration of 2mmol/L is taken, and 125mL of methanol and 145mL of acetonitrile are added and mixed to obtain an extraction solution;
preparing a mixed internal standard solution: accurate removal of fentanyl-d 5 Demethylated fentanyl-d 5 And U-47700-d 3 Adding the extraction solution into a proper amount of the mixed internal standard solution to prepare a mixed internal standard solution with the mass concentration of 10 ng/mL;
(2) sample preparation:
precisely weighing 20mg of a pre-treated hair sample to be detected, placing the hair sample to be detected in a 2mL test tube, adding an appropriate amount of grinding beads and 1mL of the mixed internal standard solution prepared in the step (1), carrying out freeze grinding in a homogenizer, then centrifuging, and filtering the supernate through a 0.22 mu m microporous membrane to obtain a sample solution to be detected;
precisely weighing 20mg of a blank hair sample subjected to pretreatment, placing the blank hair sample into a 2mL test tube, respectively adding 10 μ L of the mixed reference substance solution prepared in the step (1), then adding an appropriate amount of grinding beads and 1mL of the mixed internal standard solution prepared in the step (1), carrying out freeze grinding in a homogenizer, then centrifuging, and filtering the supernatant through a 0.22 μm microporous filter membrane to obtain a standard curve solution to be measured;
precisely weighing 20mg of a blank hair sample subjected to pretreatment, placing the blank hair sample into a 2mL test tube, adding a proper amount of grinding beads, mixing with 1mL of the mixed internal standard solution prepared in the step (1), carrying out freeze grinding in a homogenizer, then centrifuging, and filtering the supernatant through a 0.22-micrometer microporous membrane to obtain a blank solution to be detected;
(3) respectively carrying out liquid chromatography-tandem mass spectrometry on the sample solution to be detected, the standard curve solution to be detected and the blank solution to be detected, which are prepared in the step (2), and establishing an analysis method for determining 37 fentanyl new psychoactive substances in the sample solution;
a column of the liquid chromatography: waters Acquity
HSS T3 column, 100mm × 2.1mm, 1.8 μm; a mobile phase A: 20mmol/L ammonium acetate buffer solution containing 0.1% formic acid, streamAnd (3) moving phase B: acetonitrile; flow rate: 0.2 mL/min;
the elution gradient for the chromatographic analysis was as follows:
at 0min, the mobile phase A is 85.0%, and the mobile phase B is 15.0%;
at 1.0min, the mobile phase A is 85.0 percent, and the mobile phase B is 15.0 percent;
at 4.0min, the mobile phase A is 72.0%, and the mobile phase B is 28.0%;
at 5.0min, the mobile phase A is 72.0 percent, and the mobile phase B is 28.0 percent;
at 10.0min, the mobile phase A is 70.0 percent, and the mobile phase B is 30.0 percent;
at 13.0min, mobile phase A is 55.0%, and mobile phase B is 45.0%;
at 13.5min, the mobile phase A is 5.0 percent, and the mobile phase B is 95.0 percent;
at 14.5min, the mobile phase A is 5.0%, and the mobile phase B is 95.0%;
at 15.0min, the mobile phase A is 85.0%, and the mobile phase B is 15.0%;
at 16.0min, the mobile phase A is 85.0%, and the mobile phase B is 15.0%;
the 37 control products were fentanyl (fentanyl), norfentanyl (norfentanyl), alfentanyl (alfentanyl), acetylfentanyl (acetylfentanyl), acetylnorfentanyl (acetylfentanyl), deacetylfentanyl (acetylnorfentanyl), deacetylfentanyl (4-ANPP), acryloylfentanyl (acylfentanyl), butyrylfentanyl (butyrylfentanyl), isobutyrylfentanyl (isobutrylfentanyl), p-fluorofentanyl (para-fluorofentanyl), o-fluorofentanyl (ortho-fluorofentanyl), p-fluorobutyrylfentanyl (PFBF), 4-fluoroisobutyrylfentanyl (4-fluoro-isobutryltanyl), β -hydroxyfentanyl (β -hydroxyfentanyl), cis-3-methylfentanyl (cis-3-methylfentanyl), furanofentanyl (nicotinamide), ricin (nicotinamide), remifentanyl (remifentanyl), remifentanyl (remillic acid), remifentanyl (remifentanyl), remifentanyl (propionic acid), propionic acid (propionic acid) (2) (propionic acid) (2) (propionic acid) (2) (propionic acid) (2) (propionic acid) (2) (propionic acid) was), propionic acid) (propionic acid) of (propionic acid) (propionic acid) (was obtained, but not (propionic acid) (was obtained, but not (r) of (propionic acid) (was obtained, but not (r) of (r) (was obtained, but not (propionic acid) (was obtained, but not (, Valerylfentanyl, carfentanyl, norcarfentanyl, methoxyacetylfentanyl, cyclopropylfentanyl, U-47700, N-demethyl-U-47700, U-50488, U-48800, W-18, beta-hydroxy-3-methylfentanyl, beta-hydroxy-3-methylthiofentanyl, trans-3-methylfentanyl, alpha-methylfentanyl, 3-methylthiofentanyl, thiofentanyl, and tetrahydrofuran (THF-F).
2. The method for analyzing 37 fentanyl-based psychoactive substances in hair according to claim 1, wherein said fentanyl-d 5 is an internal standard for fentanyl and fentanyl analogs, said demethylfentanyl-d 5 is an internal standard for demethylfentanyl, acetyldemethylfentanyl and demethylcarfentanil, and U-47700-d3 is an internal standard for opioids.
3. The method for analyzing 37 fentanyl-based novel psychoactive substances in hair according to claim 1, wherein in the step (2), the pretreatment is: placing the hair sample in a test tube with a plug, sequentially cleaning with ultrapure water for 3 times and acetone for 3 times, collecting the last acetone cleaning solution, drying, redissolving with the extraction solution prepared in the step (1) to remove external pollution factors, cleaning the hair, drying at room temperature, and cutting into 2-3mm sections for later use.
4. The method for analyzing 37 fentanyl-type novel psychoactive substances in hair, as claimed in claim 1, wherein in the step (2), the homogenizer is a Bead raptor 24Elite multifunctional sample homogenizer, the grinding speed of the freeze grinding is 6m/s, the grinding time is 20s, the interval time between two grinding is 40s, and the cycle number is 10.
5. The method for analyzing 37 fentanyl-type psychoactive substances in hair according to claim 1, wherein in the step (2), the centrifugal force of the centrifugation is 14000 Xg, and the centrifugation time is 3 min.
6. The method for analyzing 37 fentanyl novel psychoactive substances in hair, according to the claim 1, is characterized in that in the step (3), the mass spectrum is detected by adopting a positive ion mode of an electrospray ionization mass spectrum, an ion monitoring mode is selected, and data collection and analysis are carried out by adopting an analysis software 1.5 multi-workstation and multi-ion mode monitoring is carried out; the ion source temperature of the mass spectrometer is 500 ℃, and the parent ions and the child ions, the cluster removing voltage and the collision energy are screened after direct sample injection to obtain the maximum ion strength and keep the collision dissociation energy stable.
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| CN112782305A (en) * | 2020-12-29 | 2021-05-11 | 苏州海科医药技术有限公司 | Method for analyzing sufentanil concentration in blood plasma sensitively and rapidly and suitable for pharmacokinetic research |
| CN113267589B (en) * | 2021-03-18 | 2022-11-04 | 司法鉴定科学研究院 | Analysis method of 16 synthetic cannabinoids and metabolites thereof in hair |
| CN113376277B (en) * | 2021-06-08 | 2023-03-17 | 西安海关技术中心 | High-resolution mass spectrometry detection method for fentanyl drugs in health care products |
| CN114705780A (en) * | 2022-04-07 | 2022-07-05 | 中国计量科学研究院 | Preparation method and application of fentanyl drug hair standard substance |
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