CN113267589B - Analysis method of 16 synthetic cannabinoids and metabolites thereof in hair - Google Patents
Analysis method of 16 synthetic cannabinoids and metabolites thereof in hair Download PDFInfo
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- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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Abstract
The invention discloses an analysis method of 16 synthetic cannabinoids and metabolites thereof in hair, which comprises the following steps: s1, preparing a mixed reference substance solution and a mixed internal standard solution; s2, taking the pre-treated hair sample to be detected and the blank hair sample to prepare a sample solution to be detected and a standard curve solution to be detected with standard concentration; and S3, respectively carrying out liquid chromatography-tandem mass spectrometry on the sample solution to be detected and a standard curve solution to be detected with the standard concentration, drawing a standard curve by taking the mass concentration of each analyte in the hair as a horizontal coordinate and the peak area ratio of the quantitative ions of the analytes to the internal standard as a vertical coordinate, and calculating the content of 16 synthetic cannabinoids and metabolites thereof in the hair sample to be detected. The invention fills the gap that the hair is used as a biological detection material for detecting the novel synthetic cannabinoids and metabolites thereof, and provides related technical support for relevant fields such as judicial identification and the like.
Description
Technical Field
The invention relates to the technical field of detection of cannabinoids and metabolites thereof, in particular to an analysis method of 16 synthetic cannabinoids and metabolites thereof in hair.
Background
According to the results of the european drug and drug addiction monitoring center (EMCDDA) and the european early warning system (EMS), synthetic cannabinoids are still a new class of psychoactive substances with the fastest growth rate. The compounds are derivatives simulating traditional narcotic Tetrahydrocannabinol (THC), have strong addiction, low price and strong concealment, are not easy to detect, and are often used as smoking substitutes of traditional narcotics. The synthetic cannabinoids have a greater potency than THC and bind to the receptor with an affinity 80-100 times greater than THC. Common acute adverse reactions such as restlessness, anxiety, hypertension, psychosis, tachycardia, epilepsy, myocardial infarction, renal dysfunction and the like. The synthetic cannabinoids began to flood since 2004. In recent years, the abuse of the tobacco is more and more common in China, and the tobacco is added into electronic cigarette oil and herbal spices by lawbreakers. As far as the present is concerned, there are about 50 of the synthetic cannabinoids that have been regulated in our country, and there are still a considerable number of synthetic cannabinoids that are not listed in the regulatory list. At present, due to the large number of illegal abuse of synthetic cannabinoids, the number of cases which cause acute poisoning and death is increasing, and the public health safety problems of various countries are seriously threatened.
In recent years, various methods for analyzing synthetic cannabinoids and metabolites thereof have been reported. Among them, immunoassay methods for analyzing synthetic cannabinoids have been limited due to insufficient specificity and the continuous appearance of derivatives of synthetic cannabinoids, despite the development of immunoassay methods. The literature reports analysis of synthetic cannabinoids in urine, blood and saliva, and the main analytical methods used include liquid chromatography tandem mass spectrometry, gas chromatography tandem mass spectrometry and the like. Compared with blood and urine samples, the saliva sample has simpler components, convenient material taking and no destructiveness, and is widely concerned by people. However, most of these methods can analyze only a few synthetic cannabinoids, and the sample pretreatment method is time-consuming. In general, after ingestion of synthetic cannabinoids, the parent compound of the synthetic cannabinoid is generally not detectable in the urine. Therefore, the uptake of synthetic cannabinoids is largely dependent on the detection of metabolites. Hair is an important test material for detecting drug abuse. So far, there are relatively few documents related to the analysis of synthetic cannabinoids in hair, mainly for mother medicines, and few metabolites.
Accordingly, it is an object of the present invention to provide a method for detecting 16 synthetic cannabinoids and their metabolites in hair.
Disclosure of Invention
In order to achieve the purpose, the invention provides a method for inspecting the contents of 16 synthetic cannabinoids and metabolites thereof in hair by an LC-MS/MS method. The hair is used as biological material, and has the advantages of easy collection, stability, easy storage, long detection window, and capability of reflecting long-term medical history. The hair analysis is more systematic and standardized after decades of development, and the result can be used as a reference basis of a court.
The invention provides an analysis method of 16 synthetic cannabinoids and metabolites thereof in hair, which comprises the following steps:
s1, preparing a mixed reference substance solution and a mixed internal standard solution, wherein the internal standard in the mixed internal standard solution is (+/-) -11-nor-9-carboxyl-delta 9 -tetrahydrocannabinol-D 3 And JWH-018N- (4-hydroxypentyl) methylaolite-D 5 ；
S2, taking the pre-treated hair sample to be detected and the blank hair sample to prepare a sample solution to be detected and a standard curve solution to be detected with standard concentration;
and step S3: respectively carrying out liquid chromatography-tandem mass spectrometry on the sample solution to be detected and a standard curve solution to be detected with standard concentration, drawing a standard curve by taking the mass concentration of each analyte in the hair as a horizontal ordinate and taking the peak area ratio of the quantitative ions of the analytes to the internal standard as a vertical coordinate, and calculating the content of 16 synthetic cannabinoids and metabolites thereof in the hair sample to be detected.
Preferably, the 16 synthetic cannabinoids and metabolites thereof are 5F-MDMB-PICA, 4F-MDMB-BUTINACA, 5F-CUMYL-PINCA, B-4 en-PINCA, 5F-ADB, 4F-MDMB-BICA, CUMYL-4CN-BINACA, ADB-BUTINACA, 5Cl-APINACA, 5 Cl-AB-PINCA, 5F-AMB-PICA, AMB-4en-PICA, 5F-MPP-PICA, 8978 zxUPft 8978-AB-CHMFMDMC, EG-018, AMB-FUBICA, 5F- PICA metabiolate 2, 5F-MDMB-PICA metabiolate 7, 5F-ADB metabiolate 2 and PYMB-4-PINACEAc. Wherein (±) -11-nor-9-carboxy- Δ 9 -tetrahydrocannabinol-D 3 Is an internal standard of 5F-MDMB-PICA, 4F-MDMB-BUTINACA, 5F-CUMYL-PINCA, MDMB-4 en-PINCA, 5F-ADB, 4F-MDMB-BICA, CUMYL-4CN-BINACA, ADB-BUTINACA, 5Cl-APINACA, 5 Cl-AB-PINCA, 5F-AMB-PICA, AMB-4en-PICA, 5F-MPP-PICA, 3,5-AB-CHMFUPPYCA, EG-018, AMB-BIFUCA, JWH-018N- (4-hydroxypentyl) methylabate-D 5 Internal standards for 5F-MDMB- PICA methacrylate 2, 5F-MDMB-PICA methacrylate 7, 5F-ADB methacrylate 2, and MDMB-4en-PINACA butanoic acid.
Preferably, in step S3, the liquid chromatography conditions are as follows:
and (3) chromatographic column: waters Acquity
HSS T 3 column,100mm × 2.1mm,1.8 μm; mobile phase A:20mmol/L ammonium acetate buffer solution containing 0.1% formic acid, mobile phase B: acetonitrile; mixing the mobile phase A and the mobile phase B in different volumes, and performing gradient elution;
flow rate: 0.3mL/min.
Further, the specific process of the gradient elution is as follows:
the volume ratio of mobile phase A to mobile phase B is kept constant at 50; in 1-6 minutes, uniformly and gradually changing the volume ratio of the mobile phase A to the mobile phase B from 50 to 10; the volume ratio of the mobile phase A to the mobile phase B is kept constant at 10; in 9-9.1 minutes, uniformly and gradually changing the volume ratio of the mobile phase A to the mobile phase B from 10; the volume ratio of mobile phase a to mobile phase B was kept constant at 50.
Preferably, in step S3, the mass spectrometry conditions are as follows:
the detection system is used for detecting the positive ion mode of the mass spectrum of the electrospray ionization device, selecting an ion monitoring mode, collecting and analyzing data by adopting an analysis software1.5 multi-workstation, monitoring the multi-ion mode, directly feeding the sample by a mass spectrometer at the ion source temperature of 500 ℃, then carrying out parent ions and daughter ions, screening the cluster removing voltage and the collision energy to obtain the maximum ion intensity, and keeping the stability of the collision dissociation energy.
Preferably, in step S1, the preparation method of the mixed control solution is: precisely transferring a proper amount of each reference substance solution, and adding methanol to dilute the reference substance solution into mixture reference substance solutions with the mass concentrations of 5, 10, 25, 50, 100 and 200ng/mL respectively;
preferably, in step S1, the method for preparing the mixed internal standard solution is as follows: precision fetching (+/-) -11-nor-9-Carboxy-delta 9 Proper amount of-THC-D3 and JWH-018N- (4-hydroxyphenylyl) methylabolite-D5, and methanol is added to prepare a mixed internal standard solution with the mass concentration of 0.4 ng/mL.
Preferably, in step S2, the pretreatment specifically comprises: cleaning the hair sample with acetone for multiple times, drying and re-dissolving with methanol to remove external pollution factors, and drying at room temperature to obtain a cleaned hair sample; the cleaned hair sample was cut to 1-2mm with scissors.
Preferably, in step S2, the preparation method of the sample solution to be tested comprises: precisely weighing 20mg of the pre-treated hair sample to be detected, placing the pre-treated hair sample to be detected in a 2mL grinding tube containing grinding beads, adding 1mL of the mixed internal standard solution, grinding at the temperature of below 4 ℃, carrying out ultrasonic treatment for 10min, centrifuging to obtain supernatant, and filtering by a microporous filter membrane to obtain the sample solution to be detected.
Preferably, in step S2, the preparation method of the standard curve solution to be tested comprises: precisely weighing 20mg of the blank hair sample subjected to pretreatment, placing the blank hair sample into 2mL of grinding tubes containing grinding beads, respectively adding 10 mu L of the mixture reference substance solutions with different concentrations, adding 1mL of the mixed internal standard solution, grinding at the temperature of below 4 ℃ and carrying out ultrasound for 10min, centrifuging to obtain a supernatant, and filtering by a microporous filter membrane to obtain the standard curve solution to be detected.
Preferably, the grinding parameters are: the speed is 6m/s; the time is 20s; the residence time was 40s; the cycle was 10 times.
Preferably, the parameters of the centrifugation are: the centrifugal force was 13500 Xg, and the centrifugation time was 5min.
The invention provides a method for inspecting 16 synthetic cannabinoids and metabolites thereof in hair, fills the gap that the hair is used as a biological detection material for detecting novel synthetic cannabinoids and metabolites thereof, and provides related technical support for relevant fields such as judicial identification and the like.
The method adopts the freezing grinding technology, so that the synthetic cannabinoids and metabolites thereof in the hair sample are released, and compared with other methods such as alkaline hydrolysis, ultrasound and the like, the method has the advantages of simple operation and shorter time consumption.
Compared with other detection methods for synthetic cannabinoids, the method is a method capable of detecting the maximum number of novel synthetic cannabinoids at the same time.
In addition, the method effectively separates 1 pair of isomers, is simple, high in sensitivity and good in selectivity, and is particularly suitable for daily case treatment.
Drawings
FIG. 1 is a chromatogram of LLOQ in hair at a mass concentration of each object.
Detailed Description
The present invention will be described in detail and specifically with reference to the following examples to facilitate better understanding of the present invention, but the following examples do not limit the scope of the present invention.
The instrument comprises the following steps:
XW-80A vortex mixer (Shanghai medical university instrumentation plant)
MinSpin high speed centrifuge (Eppendorf, germany)
AFS-10 ultrapure water preparation system (Germany Merck company)
Shanghai Jingxin JXFSPRP-CLN refrigerated grinding instrument (Shanghai Jingxin Co., ltd.)
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
HSS T3 (100 mm. Times.2.1mm, 1.8 μm), pre-column: waters Acquity
HSS T3 (100 mm. Times.2.1 mm,1.8 μm), data analysis was performed using a MultiQuant 3.0.2 workstation.
Drugs and devices:
comparison products: 5F-MDMB-PICA, 4F-MDMB-BUTINACA, 5F-CUMYL-PINCA, MDMB-4 en-PINCA, 5F-ADB, 4F-MDMB-BICA, CUMYL-4CN-BINACA, ADB-BUTINACA, 5Cl-APINACA, 5 Cl-AB-PIA, 5F-AMB-PICA, AMB-4en-PICA, 5F-MPP-PICA, 3,5-AB-CHMFUPPYCA, EG-018, AMB-BIFUCA, 5F-MDMB- PICA methyl 2, 5F-MDMB-PICA methyl 7, 5F-ADB methyl 2, MDMB-4 en-PIA butyl acid standard products are available from Camyman Chemicals, USA.
Deuterated Internal Standard (IS): (±) -11-nor-9-carboxy- Δ 9 -tetrahydrocannabinol-D 3 [ i.e., (+ -) -11-nor-9-Carboxy-. DELTA. 9 -THC-D 3 ]And JWH-018N- (4-hydroxypentyl) methylabate-D 5 Standards were purchased from Cerliiant reagent, usa.
Methanol and acetonitrile (UPLC) were purchased from Sigma-Aldrich (st. Louis, MO, USA).
Ammonium formate (UPLC) was obtained from Fluka (Buchs, switzerland).
Formic acid (98%, 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.
Microfiltration membranes (national chemical reagents, ltd.).
[ examples ] A method for producing a compound
The embodiment provides a method for rapidly analyzing 16 synthetic cannabinoids and metabolites thereof in hair, which comprises the following steps:
step S1, solution preparation:
step S11, preparation of a mixed reference solution: precisely transferring a proper amount of each reference substance solution, and adding methanol to dilute the reference substance solution into mixture reference substance solutions with the mass concentrations of 5, 10, 25, 50, 100 and 200ng/mL respectively;
step S12, preparing a mixed internal standard solution:
precision shifting (+/-) -11-nor-9-Carboxy-delta 9 -THC-D 3 And JWH-018N- (4-hydroxypentyl) methylabate-D 5 Adding methanol into the mixture to prepare a mixed internal standard solution with the mass concentration of 0.4 ng/mL.
S2, preparing a sample solution to be detected and a standard curve solution to be detected:
step S21, preparation of a sample solution to be detected:
precisely weighing 20mg of a pre-treated hair sample to be detected, placing the pre-treated hair sample to be detected in a 2mL grinding tube (containing grinding beads), adding 1mL of mixed internal standard solution prepared in the step S12, grinding the hair sample at the temperature below 4 ℃ by using a Shanghai Jingxin JXFSPR-CLN freeze grinder (Shanghai Jingxin Co., ltd.), performing ultrasonic treatment for 10min and centrifuging for 5min, taking about 200 mu L of supernatant, filtering the supernatant by using a 0.22 mu m microporous filter membrane (national chemical reagent Co., ltd.), and taking filtrate to obtain a sample solution to be detected.
The pretreatment comprises the following specific steps: cleaning the hair sample with acetone for 3 times, drying, re-dissolving with methanol to remove external pollution factors, and drying at room temperature to obtain a cleaned hair sample; cut the cleaned hair sample to 1-2mm with scissors.
Grinding parameters: the speed is 6m/s; the time is 20s; the residence time was 40s; the cycle was 10 times.
Centrifugal parameters: the centrifugal force was 13500 Xg.
Step S22, preparing a standard curve solution to be detected:
precisely weighing 20mg of a blank hair sample subjected to pretreatment, placing the blank hair sample into 2mL of grinding tubes (containing grinding beads), respectively adding 10 μ L of mixture reference substance solutions with the mass concentrations of 5, 10, 25, 50, 100 and 200ng/mL prepared in the step S11, respectively, adding 1mL of the mixed internal standard solution prepared in the step S12, then grinding the hair sample at the temperature below 4 ℃ by using a Shanghai Jing SpRP-CLN cryo-grinder (Shanghai Jing Xin Co., ltd.), carrying out ultrasonic treatment on the hair sample for 10min, centrifuging for 5min, and filtering supernate by using a 0.22 μm microporous membrane to obtain a standard curve solution to be detected.
The pretreatment, grinding parameters and centrifugation parameters are the same as those in step S21.
And S3, respectively carrying out liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis on the standard curve solution to be detected prepared in the step S2, drawing a standard curve by taking the mass concentration of each analyte in the hair as a horizontal coordinate and the peak area ratio of the quantitative ions of the analytes to the internal standard as a vertical coordinate, and calculating the content of 16 synthetic cannabinoids and metabolites thereof in the hair sample to be detected.
The instrument conditions were as follows:
the chromatographic conditions were as follows:
and (3) chromatographic column: waters Acquity
HSS T3column (100 mm. Times.2.1mm, 1.8 μm); a mobile phase A:20mmol/L ammonium acetate buffer solution containing 0.1% formic acid, mobile phase B: acetonitrile; flow rate: 0.3mL/min, gradient elution is shown in Table 1 below.
TABLE 1 gradient elution procedure
| Time (min) | Flow rate (mL/min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 0.3 | 50 | 50 |
| 1 | 0.3 | 50 | 50 |
| 6 | 0.3 | 10 | 90 |
| 9 | 0.3 | 10 | 90 |
| 9.1 | 0.3 | 50 | 50 |
| 10 | 0.3 | 50 | 50 |
The mass spectrometry conditions were as follows:
1 internal standard is used for calculating the peak area ratio of each compound, and JWH-018N- (4-hydroxypentyl) metablate-D5 is the internal standard for synthesizing the cannabinoids and metabolites thereof.
The detection system is positive ion mode detection of electrospray ionization mass spectrometry (Applied Biosystems/MDS SCIEX, toronto, canada), an ion monitoring mode is selected, analysis software1.5 (Waters) multi-workstation is adopted for data collection and analysis, multi-ion mode monitoring is carried out, the temperature of a mass spectrometer ion source is 500 ℃, mother ions and daughter ions are carried out after direct sample injection, and the maximum ion intensity is obtained by screening of Declustering Potential (DP) and Collision Energy (CE), so that stable Collision and dissociation energy is kept.
Fragment ions, declustering voltage, collision energy and retention time of 16 synthetic cannabinoids and metabolites thereof and 1 internal standard compound are sequentially established by directly injecting each target analyte by mass spectrometry (see table 2), so that the fragment ions have maximum response values, and each compound is calculated by two fragment ions.
TABLE 2 Mass Spectrometry parameters, retention time for the Compounds
[ methodological validation ]
Methodological validation indices commonly used in The quantitative analysis of validated biopharmaceuticals based on The Society of Hair Testing (SoHT) guidelines and The literature of Peters et al are: selectivity, linear range, limit of detection (LOD), limit of quantification (LOQ), precision (intra-day precision and inter-day precision), accuracy (intra-day accuracy and inter-day accuracy), and stability. In addition, extraction recovery rate, matrix effect, and the like are often desired.
1. Selectivity is
Selectivity is typically measured by a blank solution to be tested (without any standards and internal standards). In this experiment, blank hair from 10 volunteers, free of synthetic cannabinoids and their metabolites, was analysed to ensure that there was no interference from other substances during the peak time of the target component. In addition, possible interferences caused by the same drugs were investigated.
Preparing a blank solution to be detected:
precisely weighing 20mg of the blank hair sample subjected to pretreatment, placing the blank hair sample into a 2mL grinding tube (containing grinding beads), grinding the blank hair sample by using a Shanghai Jingxin JXFSPRP-CLN refrigerated grinding instrument (Shanghai Jingxin Co., ltd.) at the temperature of below 4 ℃, carrying out ultrasonic treatment on the hair sample for 10min, centrifuging the hair sample for 5min, and filtering the supernatant through a 0.22 mu m microporous membrane to obtain the blank solution to be detected. Wherein the pretreatment, the grinding parameters and the centrifugation parameters are the same as the step S21 in the embodiment.
As a result: by comparing the chromatograms of the compounds in the hair sample, it was confirmed that the endogenous and possibly the same drug did not interfere with the target and internal standards. The peak emergence time of each compound is distributed between 2.57 and 8.68min, and 1 pair of isomers are basically separated, and the peak areas can be respectively confirmed. The chromatogram at LLOQ is shown in FIG. 1.
2. LOD and LOQ
A proper amount of the mixed reference solution is added to the blank hair to obtain quality control samples with the mass concentrations of 0.5 pg/mg, 1 pg/mg, 2 pg/mg, 6 parallel samples are obtained at each concentration point, and the quality control sample solution is prepared according to the method for preparing the sample solution to be detected in the step S21 of the embodiment. And taking the mass concentration with the signal-to-noise ratio S/N being more than or equal to 3 as a detection limit, taking the mass concentration with the signal-to-noise ratio S/N being more than or equal to 10 as a quantification limit, taking the quantification limit as the minimum concentration of a linear range, and carrying out experimental investigation on the precision and accuracy of 6 parallel hair samples.
As a result: the precision (RSD) of 6 LOQ quality control hair samples is less than 20 percent, and the accuracy is between 80 and 120 percent. The experimental results show that the LOD of each compound in hair is 0.5-5pg/mg, and the LLOQ is 1-10pg/mg, which is enough to meet the daily test case requirement. The results are shown in Table 3.
3. Linearity
Taking the mass concentration of each analyte in hair as a horizontal coordinate, taking the peak area ratio of quantitative ions of the analytes to an internal standard as a vertical coordinate, performing regression operation by adopting a weighted least square method to obtain a linear equation, and calculating a correlation coefficient (R) 2 ). The linear equation for each compound was calculated from 5-8 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.
As a result: the linear regression equation and R are obtained by examining hair samples with mass concentrations of 1, 2, 5, 10, 20, 50, 100, 200pg/mg and calculating the peak area ratio and concentration of each analyte and internal standard according to a weighted (1/x) least squares method 2 . The results show that the respective compounds in the hair samples have good linear relationship in the corresponding concentration range and correlation coefficient (R) 2 ) > 0.99, and the results are shown in Table 3.
TABLE 3 regression equation, linear Range, correlation coefficient, LOD and LLOQ for each target in Whole blood
4. Precision and accuracy
And (3) carrying out precision and accuracy examination on the LOQ, low, medium and high quality control samples, taking blank hair, adding a proper amount of mixed reference substance solution to obtain quality control samples with the mass concentrations of 1, 2, 5, 10, 20, 50 and 150pg/mg, and obtaining 6 parallel samples at each concentration point. A quality control sample solution was prepared by the method of preparing a sample solution to be tested in step S21 of example. Sample analysis was performed according to the method of example step S3, and the in-day precision and accuracy (n = 6) were calculated. Measurements were performed for 4 consecutive days, and day-to-day precision and accuracy were calculated (n = 24). The precision is expressed by Relative Standard Deviation (RSD), the quality control sample and the standard curve are measured simultaneously, the closeness degree between the concentrations of the quality control sample parallel samples is calculated by the standard curve of the day, and the change of the precision after the experiment is repeated on the same day and different days is examined. Accuracy is expressed in bias, by linearly calculating concentration, comparing the percentage between the linearly calculated value and the true value.
As a result: the precision of each compound in the day is 0.11-10.9%, the precision in the day is 2.5-12.6%, the accuracy in the day is 87.0-110.7%, and the accuracy in the day is 95.0-105.9%, which indicates that the method has good precision and accuracy.
5. Extraction recovery and matrix effects
The extraction recovery and matrix effect were calculated according to the method proposed by Matuszewski et al, dividing the samples into three groups:
group I: accurately weighing 20mg of hair from different sources after pretreatment respectively, placing the hair into 2mL of grinding tubes (containing grinding beads), adding a proper amount of mixed reference substance solution respectively to obtain quality control samples with the mass concentrations of 1, 2, 5, 10, 20, 50 and 150pg/mg, adding 1mL of mixed internal standard solution, grinding the hair by using a Shanghai Jing JXFSPRP-CLN refrigerated grinding instrument (Shanghai Jing Xin Co., ltd.) at the temperature below 4 ℃, carrying out ultrasonic treatment on the ground hair for 10min, centrifuging for 5min, and filtering the supernatant by using a 0.22 mu m microporous filter membrane to obtain the hair conditioner.
And (II) group: precisely weighing 20mg of hair from different pre-treated sources, respectively, placing the hair into a 2mL grinding tube (containing grinding beads), adding 1mL of mixed internal standard solution, grinding the hair by using a Shanghai Jingxin JXFSPRP-CLN cryo-grinder (Shanghai Jingxin Co., ltd.) at the temperature below 4 ℃, carrying out ultrasonic treatment for 10min after grinding the hair sample, centrifuging for 5min, filtering the supernatant by using a 0.22 mu m microporous filter membrane, and respectively adding a proper amount of mixed reference solution to obtain samples with the mass concentration of 1, 2, 5, 10, 20, 50 and 150 pg/mg.
Group III: preparing mixed reference substance solution with corresponding mass concentration of 1, 2, 5, 10, 20, 50 and 150 pg/mg.
Each group of 6 samples with each concentration is injected according to the method in the step S3Analysis, peak area (a) was recorded. Extraction recovery = a Ⅰ /A Ⅱ Matrix effect = A Ⅱ /A Ⅲ 。
As a result: 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 1, 2, 5, 10, 20, 50 and 150 pg/mg. The result shows that the extraction recovery rate range of each analyte is 36.1% -93.3%, the matrix effect range is 19.1% -110.0%, wherein the analytes 5F-MDMB-PICA, 4F-MDMB-BICA and 5F-MPP-PICA generate ion inhibition in hair matrix, the ME range is 19.1% -49.9%, and the matrix effects of other compounds are all in accordance with requirements. According to the reports of related documents, the ion inhibition phenomenon is found in the synthesis of cannabinoids in hair matrix. The matrix effect of the analyte is greatly affected by the concentration, with lower concentrations being more susceptible to ion inhibition. We also speculate that the competition during ionization is influenced by the physicochemical properties of the analyte, with the 5F-MDMB-PICA, 4F-MDMB-BICA, 5F-MPP-PICA compounds being relatively less polar and the other compounds being relatively more polar. The 5F-MDMB-PICA, 4F-MDMB-BICA, 5F-MPP-PICA compounds have reduced ionization efficiency, resulting in ion inhibition.
6. Stability of
Adding a proper amount of the mixed reference substance solution into blank hair to obtain quality control samples with mass concentrations of 1, 2, 5, 10, 20, 50 and 150pg/mg, preparing the quality control sample solution according to the method for preparing the sample solution to be detected in the step S21 of the embodiment by using 6 samples with each concentration, observing the stability of the samples after respectively placing the samples under an automatic sample injection rod at 4 ℃ for 24 hours, 48 hours and 72 hours, and calculating the accuracy of the quality control samples according to the standard curve of the day of follow-up to observe the stability of the method.
As a result: the stability accuracy of the quality control sample under 24h, 48h and 72h is respectively 88.5-108.5%, 88.9-112.6% and 91.1-113.3%.
The method adopts the freezing grinding technology as a pretreatment method to release the synthetic cannabinoids and metabolites thereof in the hair sample, and has the advantages of simple operation and shorter time consumption compared with other methods such as alkaline hydrolysis, ultrasound and the like. Compared with other detection methods for synthetic cannabinoids, the method can detect the most novel synthetic cannabinoids at the same time. In addition, the method effectively separates 1 pair of isomers, is simple, high in sensitivity and good in selectivity, and is particularly suitable for daily case treatment.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Claims (9)
1. A method for analyzing 16 synthetic cannabinoids and metabolites thereof in hair, comprising the steps of:
s1, preparing a mixed reference substance solution and a mixed internal standard solution, wherein the internal standard in the mixed internal standard solution is (+/-) -11-nor-9-carboxyl-delta 9 -tetrahydrocannabinol-D 3 And JWH-018N- (4-hydroxypentyl) methylabate-D 5 ；
S2, taking the pre-treated hair sample to be detected and the blank hair sample to prepare a sample solution to be detected and a standard curve solution to be detected with standard concentration;
and step S3: respectively carrying out liquid chromatography-tandem mass spectrometry on a sample solution to be detected and a standard curve solution to be detected with standard concentration, drawing a standard curve by taking the mass concentration of each analyte in hair as a horizontal coordinate and the peak area ratio of quantitative ions of the analytes to an internal standard as a vertical coordinate, and calculating the content of 16 synthetic cannabinoids and metabolites thereof in the hair in a hair sample to be detected;
the liquid chromatography conditions were as follows:
a chromatographic column: watersAcquity
HSST 3 column,100mm × 2.1mm,1.8 μm; mobile phase A:20mmol/L acetic acidAmmonium buffer solution containing 0.1% formic acid, mobile phase B: acetonitrile; mixing the mobile phase A and the mobile phase B in different volumes, and performing gradient elution;
flow rate: 0.3mL/min of the water-soluble polymer,
the specific process of the gradient elution is as follows:
the volume ratio of mobile phase A to mobile phase B is kept constant at 50; in 1-6 minutes, uniformly and gradually changing the volume ratio of the mobile phase A to the mobile phase B from 50 to 10; the volume ratio of the mobile phase A to the mobile phase B is kept constant at 10; in 9-9.1 minutes, uniformly and gradually changing the volume ratio of the mobile phase A to the mobile phase B from 10 to 50; the volume ratio of the mobile phase A to the mobile phase B is kept constant at 50;
the 16 synthetic cannabinoids and their metabolites are 5F-MDMB-PICA, 4F-MDMB-BUTINACA, 5F-CUMYL-PINCA, MDMB-4 en-PINCA, 5F-ADB, 4F-MDMB-BICA, CUMYL-4CN-BINACA, ADB-BUTINACA, 5Cl-APINACA, 5Cl-AB-PINACA, 5F-AMB-PICA, AMB-4en-PICA, 5F-MPP-PICA, 3,5-AB-CHMFUPPYCA, EG-018, AMB-FUBICA, 5F-MDMB-PICAmetabolite2, 5F-MDMB-PIC-Ametabolite 7, 5F-ADBmeta-2, and MDMB-4-PINACA tanoic acid.
2. The method of analyzing 16 synthetic cannabinoids and their metabolites in hair as claimed in claim 1, wherein (+ -) -11-nor-9-carboxy- Δ ™ 9 -tetrahydrocannabinol-D 3 Is an internal standard of 5F-MDMB-PICA, 4F-MDMB-BUTINACA, 5F-CUMYL-PINCA, MDMB-4 en-PINCA, 5F-ADB, 4F-MDMB-BICA, CUMYL-4CN-BINACA, ADB-BUTINACA, 5Cl-APINACA, 5 Cl-AB-PINCA, 5F-AMB-PICA, AMB-4en-PICA, 5F-MPP-PICA, 3,5-AB-CHMFUPPYCA, EG-018, AMB-BIFUCA, JWH-018N- (4-hydroxypentyl) methyl acetate-D 5 Internal standards of 5F-MDMB-PICAmetabolite2, 5F-MDMB-PICAmetabolite7, 5F-ADBmetabiolate 2 and MDMB-4en-PINACA butanic acid.
3. The method for analyzing 16 synthetic cannabinoids and their metabolites in hair as claimed in claim 1, wherein in step S3, the mass spectrometric conditions are as follows:
the detection system is used for detecting the positive ion mode of the mass spectrum of the electrospray ionizer, selecting an ion monitoring mode, collecting and analyzing data by adopting multiple workstations of Analystsofare1.5, monitoring the multiple ion mode, directly feeding the sample by a mass spectrometer with the ion source temperature of 500 ℃, then carrying out the screening of parent ions and daughter ions, the cluster removal voltage and the collision energy to obtain the maximum ion intensity, and keeping the stability of the collision dissociation energy.
4. The method for analyzing 16 synthetic cannabinoids and their metabolites in hair as claimed in claim 1, wherein in step S1, the mixed control solution is formulated by: precisely transferring a proper amount of each reference substance solution, and adding methanol to dilute the reference substance solution into mixture reference substance solutions with the mass concentrations of 5, 10, 25, 50, 100 and 200ng/mL respectively.
5. The method for analyzing 16 synthetic cannabinoids and their metabolites in hair as claimed in claim 1, wherein in step S1, the mixed internal standard solution is formulated by: precision fetching (+/-) -11-nor-9-Carboxy-delta 9 -THC-D 3 And JWH-018N- (4-hydroxypentyl) methylaolite-D 5 Adding methanol into the mixture to prepare a mixed internal standard solution with the mass concentration of 0.4 ng/mL.
6. The method for analyzing 16 synthetic cannabinoids and their metabolites in hair as claimed in claim 1, wherein in step S2, the pre-treatment comprises the specific steps of: cleaning the hair sample with acetone for multiple times, drying, redissolving with methanol, and drying at room temperature to obtain a cleaned hair sample; the washed hair samples were cut to 1-2mm.
7. The method for analyzing 16 synthetic cannabinoids and metabolites thereof as claimed in claim 1, wherein in step S2, the sample solution to be tested is prepared by: precisely weighing 20mg of the preprocessed hair sample to be detected, placing the hair sample to be detected into 2mL of grinding tubes containing grinding beads, adding 1mL of the mixed internal standard solution, grinding and ultrasonically treating the hair sample at the temperature of below 4 ℃, centrifuging the hair sample to be detected to obtain supernatant, and filtering the supernatant through a microporous filter membrane to obtain the sample solution to be detected.
8. The method for analyzing 16 synthetic cannabinoids and metabolites thereof in hair as claimed in claim 1, wherein in step S2, the standard curve solution to be tested is prepared by the following steps: precisely weighing 20mg of the blank hair sample subjected to pretreatment, placing the blank hair sample into 2mL of grinding tubes containing grinding beads, respectively adding 10 mu L of the mixture reference substance solutions with different concentrations, adding 1mL of the mixed internal standard solution, grinding and ultrasonically treating at the temperature of below 4 ℃, centrifuging to obtain a supernatant, and filtering by a microporous filter membrane to obtain the standard curve solution to be detected.
9. The method for the analysis of 16 synthetic cannabinoids and their metabolites in hair as claimed in claim 7 or 8, wherein the parameters of the milling are: the speed is 6m/s; the time is 20s; the residence time was 40s; the cycle was 10 times.
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