CN111487336A - Method for analyzing 37 fentanyl novel psychoactive substances in hair - Google Patents
Method for analyzing 37 fentanyl novel psychoactive substances in hair Download PDFInfo
- Publication number
- CN111487336A CN111487336A CN202010286659.3A CN202010286659A CN111487336A CN 111487336 A CN111487336 A CN 111487336A CN 202010286659 A CN202010286659 A CN 202010286659A CN 111487336 A CN111487336 A CN 111487336A
- Authority
- CN
- China
- Prior art keywords
- fentanyl
- solution
- hair
- mobile phase
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- 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
-
- 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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
Abstract
The invention discloses an analysis method of 37 fentanyl new psychoactive substances in hair, which comprises the following steps of (1) solution preparation, (2) sample preparation, and (3) liquid chromatography-tandem mass spectrometry analysis is respectively carried out 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), so as to establish an analysis method for determining the 37 fentanyl new psychoactive substances in the sample solution.
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, cocaine, 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 greater action, the 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 are reported, wherein the methods mainly comprise immunoassay, gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry, biological detection materials for detecting the fentanyl substances reported at present mainly comprise body fluids such as blood samples, urine samples and saliva, scientists such as Melissa carry out separation analysis on 18 fentanyl substances in whole blood samples by L C-MS/MS method and apply to practical cases, scientists such as Bista carry out analysis on fentanyl and demethylated fentanyl in saliva, scientists such as Shanher use carry out analysis on 7 fentanyl substances in dry blood spots by online solid phase extraction, but the fentanyl substances have high metabolic speed in blood and urine and short detection time limit, so that when information about whether a detected object abuses the fentanyl substances for a long time is provided, other detection materials are required to be adopted, and in recent years, hairs are easy to become a basic biological matrix, can replace conventional fields such as medical science, clinical toxicology and clinical chemistry, can only provide important information about 2 hair poison, and provide unique hair poison information which can be used for hair, and can be used for a long-time, thus the hair sample has the characteristics of providing a long-time, and the hair poison and the hair-based on the hair-based biological analysis, the characteristics of the hair-and the hair-abused biological detection, the hair-free and the hair-free-and-point hair-applied biological detection result of the hair-applied biological analysis and the hair-applied biological detection method, and the hair-applied.
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 HP L C-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:
preparing mixed reference substance solution by 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/m L respectively;
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-d5Demethylated fentanyl-d5And U-47700-d3Adding 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 10ng/m L;
(2) sample preparation:
precisely weighing 20mg of a pre-treated hair sample to be detected, placing the hair sample to be detected in a 2m L test tube, adding a proper amount of grinding beads, and the mixed internal standard solution prepared in the step (1) with the amount of 1m L, carrying out freeze grinding in a homogenizer, then centrifuging, and filtering the supernatant through a 0.22 mu m microporous filter 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 2m L test tube, respectively adding a mixed reference substance solution prepared in the step (1) of 10 mu L, adding an appropriate amount of grinding beads and a mixed internal standard solution prepared in the step (1) of 1m L, carrying out freeze grinding in a homogenizer, then centrifuging, and filtering supernate through a 0.22 mu m microfiltration membrane to obtain a standard curve solution to be detected;
precisely weighing 20mg of the blank hair sample subjected to pretreatment, placing the blank hair sample into a test tube with the diameter of 2m L, adding a proper amount of grinding beads, and the mixed internal standard solution prepared in the step (1) with the diameter of 1m L, carrying out freeze grinding in a homogenizer, then centrifuging, and filtering the supernate through a microfiltration membrane with the diameter of 0.22 mu m 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 (acetylmethylfentanyl), deacrylfentanyl (4-ANPP), acryloylfentanyl (acetylfentanyl), butyrylfentanyl (butylfentanyl), isobutyrylfentanyl (isobutrylfentanyl), p-fluorofentanyl (para-fluorofentanyl), o-fluorofentanyl (ortho-fluorofentanyl), p-fluorobutyrylfentanyl (PFBF), 4-fluoroisobutyrylfentanyl (4-fluoroisopropylfentanyl), β -hydroxytetrafentanyl (β -hydroxyfentanyl), cis-3-methylfenfefenfefenyl (fes-3-methylfentanyl), thiofentanyl (hyacinyl-3-methylfentanyl), thiofentanyl (tetrahydrofuranthryl-3-methoxyfentanyl), thiofentanyl (tetrahydrofuryl-3-methoxyfentanyl), N-methoxyfentanyl (mercaptofentanyl), N-3-methoxyfentanyl (mercaptofentanyl), N-4800, N-methylanthryl (4-3-methoxyfentanyl), N-methoxyfentanyl (3-4800), N-methoxyfentanyl (4-methoxyfentanyl), N-methoxyfentanyl, N-3-methoxyfentanyl, N-8600, N-methoxyfentanyl, N-3-methoxyfentanyl, N-8600, N-methoxyfentanyl, N-3-methoxyfentanyl, N.
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 preparation method of the extraction solution comprises the following steps of taking 230m L of ammonium formate solution with pH of 5.3 and concentration of 2 mmol/L, adding 125m L of methanol and 145m L of acetonitrile, and mixing 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 × g, and the centrifugation time is 3 min.
Preferably, in step (3), the column of the liquid chromatography: WatersAcquity
HSST 3column (100mm × 2.1.1 mm, 1.8 μm), mobile phase A20 mmol/L ammonium acetate buffer solution containing 0.1% formic acid, mobile phase B acetonitrile, flow rate 0.2m L/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 4.0min, the mobile phase A is 72.0%, and the mobile phase B is 28.0%;
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%, and the mobile phase B is 95.0%;
at 15.0min, 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 L C-MS/MS method, takes the hair as biological detection material, has the advantages of easy collection, stability, easy storage, long detection window, capability of reflecting long-time medication history and the like, ensures that hair analysis is more systematic and normalized, and the result can be used as reference basis of court.
Drawings
FIG. 1 is a chromatogram of 37 targets at a mass concentration of LL OQ 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 may be combined with each other without conflict.
Medicine preparation:
37 control substances are fentanyl (fentanyl), norfentanyl (norfentanyl), alfentanyl (alfentanyl), acetylfentanyl (acetylfentanyl), acetylnorfentanyl (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-methylfenfectamyl), furan (furanyl), furan (3-methylfentanyl), urea-3-methylfentanyl (tetrahydrofuran-4800), N-3-methoxyfentanyl (3-norfentanyl, N-methylanthryl (4-methylanthryl), N-methylanthryl-3-methoxyfentanyl (tetrahydrofuran-4800), N-3-methoxyfentanyl (N-methoxyfentanyl), N-methoxyfentanyl, N-methoxy.
Methanol and acetonitrile (UP L C) were purchased from Sigma-Aldrich (St. L ouis, MO, USA), ammonium formate (UP L C) was purchased from Fluka (Buchs, Switzerland), formic acid (4.014784.0250, AR) and ammonium acetate (UP L C) were purchased from CNW (UK), deionized water was prepared from 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).
ACQUITYUP L C I-C L ASS liquid chromatograph (Waters corporation, USA) tandem QTRAP 6500 Plus triple quadrupole linear ion trap composite Mass spectrometer (SCIEX corporation, USA)
HSS T3(100mm × 2.1.1 mm, 1.8 μm), Pre-column Waters Acquity
HSS T3(100mm × 2.1.1 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:
preparing mixed reference substance solution by 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/m L respectively;
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-d5Demethylated fentanyl-d5And U-47700-d3Adding 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 10ng/m L;
(2) sample preparation:
precisely weighing 20mg of a pre-treated hair sample to be detected, placing the hair sample to be detected in a 2m L test tube, adding a proper amount of grinding beads, and the mixed internal standard solution prepared in the step (1) with the amount of 1m L, carrying out freeze grinding in a homogenizer, then centrifuging, and filtering the supernatant through a 0.22 mu m microporous filter 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 2m L test tube, respectively adding a mixed reference substance solution prepared in the step (1) of 10 mu L, adding an appropriate amount of grinding beads and a mixed internal standard solution prepared in the step (1) of 1m L, carrying out freeze grinding in a homogenizer, then centrifuging, and filtering supernate through a 0.22 mu m microfiltration membrane to obtain a standard curve solution to be detected;
precisely weighing 20mg of the blank hair sample subjected to pretreatment, placing the blank hair sample into a test tube with the diameter of 2m L, adding a proper amount of grinding beads, and the mixed internal standard solution prepared in the step (1) with the diameter of 1m L, carrying out freeze grinding in a homogenizer, then centrifuging, and filtering the supernate through a microfiltration membrane with the diameter of 0.22 mu m 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 to take 230m L of ammonium formate solution with pH of 5.3 and concentration of 2 mmol/L, add 125m L of methanol and 145m L of acetonitrile, and mix to obtain the extraction solution.
As a preferred embodiment, in the step (2), the pretreatment comprises the steps of putting a hair sample into a test tube with a plug, washing the hair sample for 3 times by using ultrapure water and 3 times by using acetone, collecting acetone washing liquid for the last time, drying the hair by using the acetone washing liquid, redissolving the hair by using the extraction solution prepared in the step (1) to remove external pollution factors, drying the hair at room temperature after washing the hair, cutting the hair into 2-3mm sections for later use, 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 the two grinding is 40s, the cycle time is 10 times, in the step (2), the centrifugal force of the centrifugation is 14000 × g, and the centrifugation time is 3 min.
As a preferred embodiment, the liquid chromatography column: waters Acquity
HSS T3column (100mm × 2.1.1 mm, 1.8 μm), mobile phase A20 mmol/L ammonium acetate buffer solution containing 0.1% formic acid, mobile phase B acetonitrile, flow rate 0.2m L/min, gradient elution as 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-d5For internal standards of fentanyl and fentanyl analogs, demethylfentanyl-d5For the metabolites demethylfentanyl, acetyldemethylfentanyl and demethylcarfentanil internal standards, U-47700-d3Is 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 direct mass spectrum sample injection of 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 table 2, so that the fragment ions have the maximum response value, 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 with different concentrations and containing all target substances; 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 the intra-day precision and the inter-day precision, and the precision and the accuracy are specifically measured by examining the precision and the accuracy of L OQ, low, medium and high concentrations, taking blank hair, adding a proper amount of a mixed reference solution to obtain quality control samples with the mass concentration of 2, 5, 10, 500 and 2000pg/mg and 6 parts of each concentration, pre-treating the samples according to the preparation method in the step (2), carrying out sample injection analysis according to the conditions of an instrument in the step (3), calculating the intra-day precision and the accuracy, expressing the accuracy in a bias manner, comparing the percentage between the obtained value and the actual value by linear calculation through linear calculation of the concentration, continuously measuring for 4 days, simultaneously measuring with a standard curve, calculating the concentration of the quality control samples by using the standard curve of the current day, calculating the inter-day precision, and examining the change of the repeated experiments on the same day and different days.
As a preferred embodiment, the specific process of the linear range 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 the 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) The linear equation for each compound was calculated from 6 points with a minimum concentration of L OQ (S/N.gtoreq.10) and ensures that the concentration of the actual sample falls within the linear range.
The methodological verification index also comprises extraction recovery rate and matrix effect, and the specific process of the extraction recovery rate and the matrix effect comprises the steps of dividing samples into three groups according to a method provided by Matuszewski and the like to calculate the extraction recovery rate and the matrix effect, adding a mixed reference substance solution with a certain concentration before extraction in the preparation method of the hair reference step (2) with different sources in the group I, adding a mixed reference substance solution with a corresponding concentration after extraction in the preparation method of the hair reference step (2) with different sources in the group II, preparing a mixed reference substance solution with a corresponding concentration in the group III, adding a proper amount of blank hair with different sources into the mixed reference substance solution to obtain quality control pgg samples with mass concentrations of 2/mg, 5/mg (low concentration), 5 pgmg/mg (medium concentration) and 30ng/m L (high concentration), adding 6 samples of each concentration in each group, analyzing according to the instrument condition in the step (3), recording and extracting the recovery rate A and the matrix effectⅠ/AⅡThe matrix effect is AⅡ/AⅢ。
1. Selectivity is
Comparing the chromatogram of each compound in the hair sample confirms that the endogenous substance and the possible same drug are not interfered by the target substance and the internal standard, the peak emergence time of each compound is distributed between 2.4 and 15.23min, and 4 pairs of isomers are basically separated, the peak area can be respectively confirmed, and the chromatogram when the mass concentration of each target substance is LL OQ is shown in figure 1.
2. Detection limit and quantification limit
The hair samples with mass concentrations of 0.5, 1, 2, 2.5 and 5pg/mg are respectively considered, the precision (RSD) of the L OQ quality control hair sample is less than 20% by taking the mass concentration of L OD when the signal-to-noise ratio S/N is more than or equal to 3 and the mass concentration of L OQ.6 and 80-120% of RSD when the signal-to-noise ratio S/N is more than or equal to 10, experimental results show that the L OD of each compound in the hair is 0.5-2.5pg/mg and the LL OQ is 2-5pg/mg, which can sufficiently meet the requirement of daily test cases, and the experimental results are shown in Table 3.
3. Linear range
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 R2. 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 good2) > 0.99, results are shown in table 3;
TABLE 3 regression equation, Linear Range, correlation coefficient, L OD and LL OQ 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%, respectively, 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 substances in the hair sample, and compared with other ultrasonic methods and the like, the method is simple to operate and consumes less time. 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 effects
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 2000 pg/mg. As a result, the extraction recovery rate of each target object is 89.42-119.68%, the matrix effect range is 44.81-119.77%, wherein the matrix effect of other compounds meets the requirement except that the ME of W-18 is 44.81-54.11%. Due to the difference of chemical structures, the W-18 retention time is 15.23min, and the final unstable stage of gradient elution can 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 methodological validation, the invention was 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, the police inquires 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 respectively as follows: 183.91pg/mg, 131.68pg/mg and 31.48 pg/mg. And according to the experimental examination result, the police carries out further investigation on the reported person. The final evidence shows that the case is related to 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 present case relates to the use of fentanyl, 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 (10)
1. A method for analyzing 37 fentanyl novel psychoactive substances in hair, which is characterized by comprising the following steps:
(1) solution preparation:
preparing mixed reference substance solution by 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/m L respectively;
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-d5Demethylated fentanyl-d5And U-47700-d3Adding 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 10ng/m L;
(2) sample preparation:
precisely weighing 20mg of a pre-treated hair sample to be detected, placing the hair sample to be detected in a 2m L test tube, adding a proper amount of grinding beads, and the mixed internal standard solution prepared in the step (1) with the amount of 1m L, carrying out freeze grinding in a homogenizer, then centrifuging, and filtering the supernatant through a 0.22 mu m microporous filter 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 2m L test tube, respectively adding a mixed reference substance solution prepared in the step (1) of 10 mu L, adding an appropriate amount of grinding beads and a mixed internal standard solution prepared in the step (1) of 1m L, carrying out freeze grinding in a homogenizer, then centrifuging, and filtering supernate through a 0.22 mu m microfiltration membrane to obtain a standard curve solution to be detected;
precisely weighing 20mg of the blank hair sample subjected to pretreatment, placing the blank hair sample into a test tube with the diameter of 2m L, adding a proper amount of grinding beads, and the mixed internal standard solution prepared in the step (1) with the diameter of 1m L, carrying out freeze grinding in a homogenizer, then centrifuging, and filtering the supernate through a microfiltration membrane with the diameter of 0.22 mu m 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.
2. The method for analyzing 37 fentanyl-based neopsychoactive substances in hair according to claim 1, wherein the 37 control substances are fentanyl (fentanyl), demethylfentanyl (norfentanyl), alfentanyl (alfentanyl), acetylfentanyl (acetylfentanyl), acetyldemethylfentanyl (acetylfentanyl), deacetylfentanyl (4-ANPP), acrylfentanyl (acylfentanyl), butyrylfentanyl (butyrylfentanyl), isobutyrylfentanyl (isobutrylfentanyl), p-fluorofentanyl (para-fluorofentanyl), o-fluorofentanyl (ortho-fluorofentanyl), p-fluorobutyrylfentanyl (PFBF), 4-fluoroisobutyrylfentanyl (4-fluorobutyrylfentanyl-isopropyl-fentanyl), 25-hydroxyfentanyl (β -hydroxyfentanyl), tetrahydrofencetanyl-3-methyl-3-ethyl-3-norfentanyl, N-methoxyfentanyl (3-norfentanyl), N-carboxyfentanyl (carboxyfentanyl, N-carboxyfentanyl (carboxyfentanyl), N-carboxyfentanyl (carboxyfentanyl, N-carboxyfentanyl.
3. The method for analyzing 37 fentanyl-based psychoactive substances in hair according to claim 2, 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.
4. The method for analyzing 37 fentanyl-type novel psychoactive substances in hair according to claim 1, wherein the extraction solution is prepared by mixing 230m L of ammonium formate solution with pH of 5.3 and concentration of 2 mmol/L with 125m L of methanol and 145m L of acetonitrile to obtain the extraction solution.
5. 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.
6. 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 BeadRuptor 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.
7. 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 × g, and the centrifugation time is 3 min.
8. The method for analyzing 37 fentanyl-type psychoactive substances in hair according to claim 1, wherein in step (3), the liquid chromatography column: waters Acquity
HSST 3column (100mm × 2.1.1 mm, 1.8 μm), mobile phase A20 mmol/L ammonium acetate buffer solution containing 0.1% formic acid, mobile phase B acetonitrile, flow rate 0.2m L/min.
9. The method for analyzing 37 fentanyl-type psychoactive substances in hair according to claim 7, wherein the gradient of elution in 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 4.0min, the mobile phase A is 72.0%, and the mobile phase B is 28.0%;
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%, and the mobile phase B is 95.0%;
at 15.0min, mobile phase A was 85.0% and mobile phase B was 15.0%.
10. 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010286659.3A CN111487336B (en) | 2020-04-13 | 2020-04-13 | Method for analyzing 37 fentanyl novel psychoactive substances in hair |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010286659.3A CN111487336B (en) | 2020-04-13 | 2020-04-13 | Method for analyzing 37 fentanyl novel psychoactive substances in hair |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111487336A true CN111487336A (en) | 2020-08-04 |
| CN111487336B CN111487336B (en) | 2022-08-19 |
Family
ID=71795036
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010286659.3A Active CN111487336B (en) | 2020-04-13 | 2020-04-13 | Method for analyzing 37 fentanyl novel psychoactive substances in hair |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111487336B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112255343A (en) * | 2020-10-16 | 2021-01-22 | 宁波市疾病预防控制中心 | Method and kit for measuring fentanyl drugs in biological sample |
| 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 |
| CN113267589A (en) * | 2021-03-18 | 2021-08-17 | 司法鉴定科学研究院 | Analysis method of 16 synthetic cannabinoids and metabolites thereof in hair |
| CN113376277A (en) * | 2021-06-08 | 2021-09-10 | 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 |
| CN115047109A (en) * | 2022-06-17 | 2022-09-13 | 三明海关综合技术服务中心 | Method for detecting fentanyl new psychoactive substances in food |
| CN115236230A (en) * | 2022-07-21 | 2022-10-25 | 司法鉴定科学研究院 | Method for detecting wild Wuyuyu caused hallucinogenic component mescaline in hair |
| CN115452995A (en) * | 2022-09-23 | 2022-12-09 | 司法鉴定科学研究院 | Method for testing drugs in single hair with sub-millimeter length |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001089359A (en) * | 1999-09-24 | 2001-04-03 | Toko Yakuhin Kogyo Kk | Gel or gel preparation for nasal drip of analgesic component |
| CN103728403A (en) * | 2013-08-30 | 2014-04-16 | 宜昌人福药业有限责任公司 | Method for detecting relevant substance in hydrochloric acid remifentanil active ingredient |
| JP2015129190A (en) * | 2005-05-25 | 2015-07-16 | プロジェニックス ファーマシューティカルズ，インコーポレーテッド | (r)-n-methylnaltrexone, processes for its synthesis and its pharmaceutical use |
| CN105209459A (en) * | 2013-05-17 | 2015-12-30 | 奥古斯特沃尔夫博士有限两合公司-医药 | Perhydroquinoxaline derivatives useful as analgesics |
| US20160370388A1 (en) * | 2015-06-19 | 2016-12-22 | Amy J. Reisinger | Rapid and Sensitive Method of Forensic Toxicology in Post-Mortem Subjects Using Oral Fluid Testing |
-
2020
- 2020-04-13 CN CN202010286659.3A patent/CN111487336B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001089359A (en) * | 1999-09-24 | 2001-04-03 | Toko Yakuhin Kogyo Kk | Gel or gel preparation for nasal drip of analgesic component |
| JP2015129190A (en) * | 2005-05-25 | 2015-07-16 | プロジェニックス ファーマシューティカルズ，インコーポレーテッド | (r)-n-methylnaltrexone, processes for its synthesis and its pharmaceutical use |
| CN105209459A (en) * | 2013-05-17 | 2015-12-30 | 奥古斯特沃尔夫博士有限两合公司-医药 | Perhydroquinoxaline derivatives useful as analgesics |
| CN103728403A (en) * | 2013-08-30 | 2014-04-16 | 宜昌人福药业有限责任公司 | Method for detecting relevant substance in hydrochloric acid remifentanil active ingredient |
| US20160370388A1 (en) * | 2015-06-19 | 2016-12-22 | Amy J. Reisinger | Rapid and Sensitive Method of Forensic Toxicology in Post-Mortem Subjects Using Oral Fluid Testing |
Non-Patent Citations (5)
| Title |
|---|
| ALBERTO SALOMONE 等: "Detection of Fentanyl Analogs and Synthetic Opioids in Real Hair Samples", 《JOURNAL OF ANALYTICAL TOXICOLOGY》 * |
| EMMA PARTRIDGE 等: "A Validated Method for the Screening of 320 Forensically Significant Compounds in Blood by LC/QTOF, with Simultaneous Selected Compounds", 《JOURNAL OF ANALYTICAL TOXICOLOGY》 * |
| GIULIO MANNOCCHI 等: "Development and validation of fast UHPLC-MS/MS screening method for 87 NPS and 32 other drugs of abuse in hair and nails: application to real cases", 《ANALYTICAL AND BIOANALYTICAL CHEMISTRY》 * |
| MARIE KATRINE KLOSE NIELSEN 等: "Simultaneous screening and quantification of 52 common pharmaceuticals and drugs of abuse in hair using UPLC–TOF-MS", 《FORENSIC SCIENCE INTERNATIONAL》 * |
| 施妍 等: "血液中20种芬太尼类物质UPLC-MS/MS法的建立及应用", 《法医学杂志》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112255343A (en) * | 2020-10-16 | 2021-01-22 | 宁波市疾病预防控制中心 | Method and kit for measuring fentanyl drugs in biological sample |
| WO2022077808A1 (en) * | 2020-10-16 | 2022-04-21 | 宁波市疾病预防控制中心 | Method and kit for measuring fentanyl drug in biological sample |
| 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 |
| CN113267589A (en) * | 2021-03-18 | 2021-08-17 | 司法鉴定科学研究院 | Analysis method of 16 synthetic cannabinoids and metabolites thereof in hair |
| CN113376277A (en) * | 2021-06-08 | 2021-09-10 | 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 |
| CN115047109A (en) * | 2022-06-17 | 2022-09-13 | 三明海关综合技术服务中心 | Method for detecting fentanyl new psychoactive substances in food |
| CN115236230A (en) * | 2022-07-21 | 2022-10-25 | 司法鉴定科学研究院 | Method for detecting wild Wuyuyu caused hallucinogenic component mescaline in hair |
| CN115236230B (en) * | 2022-07-21 | 2024-01-19 | 司法鉴定科学研究院 | Method for detecting wild Wu Yuyu-induced magic ingredient of maskanin in hair |
| CN115452995A (en) * | 2022-09-23 | 2022-12-09 | 司法鉴定科学研究院 | Method for testing drugs in single hair with sub-millimeter length |
| CN115452995B (en) * | 2022-09-23 | 2024-01-19 | 司法鉴定科学研究院 | Method for inspecting medicine in single sub-millimeter length hair |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111487336B (en) | 2022-08-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111487336B (en) | Method for analyzing 37 fentanyl novel psychoactive substances in hair | |
| US9816980B2 (en) | Apparatus and corresponding method for sampling and analyzing drugs and respective metabolites in breath air, particularly suitable for performing road drug tests | |
| Gentili et al. | Simultaneous detection of amphetamine-like drugs with headspace solid-phase microextraction and gas chromatography–mass spectrometry | |
| WO2012122128A2 (en) | Detection of cancer by volatile organic compounds from breath | |
| Min et al. | Determination of dl-amino acids, derivatized with R (−)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2, 1, 3-benzoxadiazole, in nail of diabetic patients by UPLC–ESI-TOF-MS | |
| Wang et al. | Determination of 17 illicit drugs in oral fluid using isotope dilution ultra-high performance liquid chromatography/tandem mass spectrometry with three atmospheric pressure ionizations | |
| CN106841427B (en) | A kind of tandem mass spectrum kit detecting PKU and CAH | |
| CN112630311A (en) | Metabolic markers and kits for detecting affective disorders and methods of use | |
| Larabi et al. | Development and validation of liquid chromatography‐tandem mass spectrometry targeted screening of 16 fentanyl analogs and U‐47700 in hair: Application to 137 authentic samples | |
| Jang et al. | Simultaneous determination of LSD and 2-oxo-3-hydroxy LSD in hair and urine by LC–MS/MS and its application to forensic cases | |
| Świądro et al. | Development of a new method for drug detection based on a combination of the dried blood spot method and capillary electrophoresis | |
| Murtada et al. | A simple poly (styrene-co-divinylbenzene)-coated glass blood spot method for monitoring of seven antidepressants using capillary liquid chromatography-mass spectrometry | |
| Shi et al. | Detection of a new tert-leucinate synthetic cannabinoid 5F-MDMB-PICA and its metabolites in human hair: application to authentic cases | |
| Fan et al. | Analysis of synthetic cannabinoids in wastewater of major cities in China | |
| CN113325103A (en) | Analysis method for simultaneously measuring gelsemium, gelsemine and gelsemine in hair | |
| CN114813989B (en) | Method for simultaneously extracting and detecting melatonin, N-acetylserotonin, 6-hydroxy melatonin, cortisol and cortisone in hair | |
| KR20090027896A (en) | Method for analysis of polyamines in urine or plasma using liquid chromatography/electronspray ionization-tandem mass spectrometry along with amine carbamylated derivatization | |
| CN113267589B (en) | Analysis method of 16 synthetic cannabinoids and metabolites thereof in hair | |
| CN113820424A (en) | HPLC-MS/MS method for simultaneously determining concentration of 14 antidepressants in human plasma | |
| CN110146615A (en) | A method of surveying nicotine in serum, 3- (pyrrolidin-2-yl) pyridine, pyridazole ketone and testosterone concentration simultaneously | |
| Chromiec et al. | New green DI-SPME/CE-MS method for quantitative analysis of selected illicit drugs in human whole blood | |
| CN114544826B (en) | Application of reagent for detecting histidine in blood plasma in preparation of depression detection kit | |
| CN109342598A (en) | A kind of method and its detection kit for identifying cerebral hemorrhage biomarker | |
| CN113640415B (en) | Method for detecting content of alpha-androstanol in truffle sporocarp | |
| CN114544822B (en) | Application of reagent for detecting lysophosphatidylcholine (22:0) in blood plasma in preparation of depression detection kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |