A screening method for fentanyl-like substances

Abstract

The invention belongs to the technical field of detection and identification of fentanyl-like substances, and specifically discloses a screening method for fentanyl-like substances. The phase chromatography-quadrupole / time-of-flight high-resolution mass spectrometry established by the invention can detect 143 fentanyl-like substances in 10 minutes, has the characteristics of strong practicability, high accuracy, high sensitivity, simple and rapid operation, and can meet the detection technical requirements of public security drug control, criminal investigation, post office customs and airport customs for fentanyl monitoring. Each laboratory does not need to spend a lot of money to purchase standard samples of fentanyl-like substances, and the screening and detection of fentanyl substances can be completed by copying the 143 fentanyl primary and secondary mass spectrometry libraries of the invention into its software, and has good application value and application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of fentanyl-like substance detection and identification, in particular to a method for screening fentanyl-like substances. Background Art

[0002] Fentanyl is an opioid substance, a synthetic, powerful narcotic analgesic with a potency 50-100 times that of morphine. Even a small amount of it can cause harm to the human body and even endanger life. Due to the wide variety of fentanyl substances and their strong medicinal properties, they are targeted by international drug traffickers and become an important ingredient in their manufacture of new drugs. They have been listed as the third generation of drugs popular around the world after traditional drugs and synthetic drugs. In the United States, the abuse and addiction of opioids such as fentanyl has become a social problem with great influence. In 2017, 64,000 people died due to the abuse of opioids such as fentanyl, and the death rate has exceeded the abnormal death rate from firearms, traffic accidents, etc., ranking first.

[0003] At present, there are no literature reports on the detection of fentanyl as a whole category at home and abroad. Among the foreign literature reports on fentanyl, most are reports on the simultaneous detection of dozens to nearly 100 drugs, including fentanyl-like substances, but the number of detected varieties is less than 30, and most of them use triple quadrupole tandem mass spectrometers. Domestic detection reports are almost all about the 27 types of fentanyl that my country implemented control before the whole category was listed in May 2019.

[0004] There are many problems at present:

[0005] (1) Standard samples are difficult to obtain. Due to the high sensitivity of this type of substance, there are few reports at home and abroad, and standard samples are difficult to obtain.

[0006] (2) There is a lack of necessary standard support. Listing fentanyl-like substances as a whole category is an innovative measure for China to deal with drug problems. However, there is currently no inspection and identification standard for fentanyl-like substances. It is urgent to promote the construction of a fentanyl-like substance monitoring system based on suspicious substances and spectral analysis, comprehensively improve the level of monitoring and early warning, on-site inspection, inspection and identification, and hazard assessment of fentanyl-like substances and other new drugs, and provide scientific and efficient technical support for daily supervision and law enforcement.

[0007] (3) There is a shortage of professional talents. The non-target screening of unknown substances is technically difficult and difficult to promote. In the absence of a target, conducting screening for unknown fentanyl-like substances is like looking for a needle in a haystack. It places high demands on technical personnel and is a difficult challenge.

[0008] (4) It is necessary to solve the problem of target detection and full coverage of types. In the early stage, 27 fentanyl-like substances were listed, but new drugs continue to emerge. The three ministries of the state jointly announced that fentanyl-like substances will be listed as a whole category from May 1. This is not only an innovative measure for China's drug problem governance, but also a higher challenge for fentanyl-like substance detection and identification technology. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides a method for screening fentanyl-like substances.

[0010] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0011] A method for screening fentanyl-like substances comprises the following steps:

[0012] S1. Collect M types of fentanyl that can be found with written records, and establish a primary target library of theoretical accurate mass numbers of fentanyl parent ions based on the molecular formulas of the M types of fentanyl;

[0013] S2. Classify M types of fentanyl into N types according to their structural formulas, find a representative fentanyl in each type and obtain its standard, and obtain N representative secondary mass spectra of fentanyl by HPLC-QTof detection, and analyze the fragmentation mechanism of each fragment ion using electronic theory and energy theory based on the N representative secondary mass spectra of fentanyl, and summarize the fragmentation rules and characteristics of each type of fentanyl;

[0014] S3. According to the fragmentation law and characteristics of each type of fentanyl, the secondary fragment ions of MN types of fentanyl for which standard substances cannot be obtained are theoretically derived, and the secondary fragment ion spectral library of fentanyl is established by combining N representative fentanyls and MN types of fentanyl secondary fragment ions for screening and detection of the entire type of fentanyl substances;

[0015] S4. Establishing the quantitative and qualitative detection of samples containing fentanyl substances by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry using N fentanyl-like substances as existing standards.

[0016] Furthermore, the step S4 specifically includes:

[0017] S41. Prepare N representative standard stock solutions of fentanyl substances: Take N representative fentanyl substances and dilute them to 10 mL with methanol to prepare standard stock solutions with a concentration of 10 mg / L. Store at 4°C in the dark.

[0018] S42. Prepare N representative mixed standard stock solutions of fentanyl substances: pipette 200 μL or 20 μL of the above N representative standard stock solutions of fentanyl substances into 10 mL volumetric flasks respectively, dilute to the mark with acetonitrile to prepare a mixed standard stock solution with a concentration of 200 μg / L, and store at 4°C in the dark;

[0019] S43, prepare N representative mixed standard working solutions of fentanyl substances: dilute N representative mixed standard stock solutions of fentanyl substances with 9 times the volume of acetonitrile aqueous solution to a mixed standard working solution of appropriate concentration, and store at 4° C. in the dark;

[0020] S44. After pretreatment of the sample containing fentanyl, it was detected by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, and the chromatographic conditions were as follows: chromatographic column: Kinetex C18 2.6μm 100A100×2.1mm, mobile phase A was 0.08% formic acid aqueous solution, and mobile phase B was acetonitrile; flow rate: 0.4mL / min; chromatographic column temperature: 40°C; injection volume: 10μL; gradient elution program: 0-5min, 5%-80%B; 5-8min, 80%B; 8-8.1min, 80%-5%B; 8.1-10.5min, 5%B; mass spectrometry conditions were as follows: ionization mode: electrospray ionization ESI, positive ion scanning mode; mass spectrometry calibration method: sodium formate solution external standard calibration; calibration cycle: sodium formate calibration solution and other test solutions were injected; acquisition mode: TOFMS-IDA-TOF MS / MS; scanning range: m / z 50-650 for both primary and secondary; electrospray voltage IS: 5500V; ion source temperature TEM: 550℃; nebulizer gas pressure GS1: 345kPa; auxiliary gas pressure GS2: 414kPa; curtain gas pressure CUR: 276kPa; declustering voltage DP: 80V; collision energy CE: 30±5;

[0021] S45. Establishment of high-resolution mass spectrometry library: Input the chemical formula and Chinese and English names of M fentanyl-like substances, and use the high-resolution mass spectrometry software to calculate the theoretical accurate mass number [M+H] of each compound parent ion. + , and construct a primary compound library; separately use N representative fentanyl substance mixed standard working solutions, collect their secondary fragment ion spectra in Product Ion IDA mode, import them into the high-resolution mass spectral library, and construct a secondary compound library; the remaining MN kinds of fentanyl without standards use the secondary fragment ions of MN kinds of fentanyl to establish a secondary compound library, and complete the construction of M kinds of fentanyl primary and secondary compound libraries;

[0022] S46. Identification and confirmation of N types of fentanyl: In the TOF MS mode, if the retention time of the fentanyl to be tested in the sample is within ±2.5% of the retention time of one of the N representative fentanyl substance standard stock solutions of the corresponding concentration; the deviation between the measured value and the theoretical value of the accurate mass number of the parent ion of the test substance is less than or equal to 5ppm; if the abundance ratio of the secondary fragment ion is consistent with that of the library ion, then the sample is judged to contain the representative fentanyl substance;

[0023] S47. Identification and confirmation of MN types of fentanyl: In TOF MS mode, the deviation between the measured and theoretical values ​​of the accurate mass number of the parent ion of the analyte in the sample is less than or equal to 5 ppm; if the secondary fragments of the compound contain one of the secondary fragment ions of the MN types of fentanyl and all the ions of its corresponding compound, it is determined that the sample contains this fentanyl-like substance.

[0024] The main innovations of the present invention are: first, it realizes the transition from non-target screening to target screening. For the non-target screening of unknown substances, there are problems of high technical difficulty and difficulty in promotion. In the context of no target at all, conducting screening for unknown fentanyl-like new psychoactive substances is like looking for a needle in a haystack. It has high requirements for technical personnel and is a difficult challenge. This project sorts out the current global fentanyl searches through a full database search, finds all 143 types of fentanyl, and realizes the conversion of non-target detection to target detection, which is an important technological breakthrough. Second, by classifying the 143 types of fentanyl for target detection, one standard product is purchased for each type, and a total of 29 representative standard products are purchased. Each fragment ion of the 29 fentanyls is subjected to mass spectrometry analysis according to energy theory and electronic theory, and no related reports have been found at home and abroad. Third, based on the mass spectrometry analysis of each fragment ion of 29 types of fentanyl, the mass spectrometry fragmentation rules and characteristics of the entire class of fentanyl were summarized. Based on the summarized rules and characteristics, the secondary fragment ions of 114 types of fentanyl without standards were theoretically derived, and a secondary fragment ion library of 114 types of fentanyl without standards was established, completing the screening and detection of 114 types of fentanyl without standards, solving the problem of not being able to purchase standards for an entire class of fentanyls, and establishing a scientific and complete high-resolution mass spectrometry screening and detection method for 143 entire classes of fentanyls, filling domestic and international gaps and reaching an international leading level.

[0025] Compared with the prior art, the phase chromatography-quadrupole / time-of-flight high-resolution mass spectrometry established by the present invention can complete the detection of 143 fentanyl-like substances in 10 minutes, has the characteristics of strong practicability, high accuracy, high sensitivity, simple and rapid operation, and can meet the detection technology requirements of public security drug control, criminal investigation, post office customs and airport customs for fentanyl monitoring. Each laboratory does not need to spend a lot of money to purchase standard samples of fentanyl-like substances. The screening and detection of fentanyl substances can be completed by copying the 143 fentanyl primary and secondary mass spectrometry libraries of the present invention into its software, and has good application value and application prospects. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0027] This embodiment is specifically a screening method for fentanyl-like substances, which is described in detail below.

[0028] A total of 143 fentanyl compounds were collected by searching the U.S. Scifinder database, the PubChem Compound database of the National Center for Biotechnology Information (NCBI), the China National Knowledge Infrastructure (CNKI) scientific literature database, the British ChemSpider website, the U.S. ChemBlink website, the ChemNet website, the CaymanChem website, the United Nations Office on Drugs and Crime (UNODC) Early Warning Advisory (EWA) documents and notices, Google, Baidu and Wikipedia. The Chinese and English names, molecular formulas, and theoretical accurate mass information of the parent ions of the 143 fentanyls are listed in Table 1. The molecular formulas and English names were copied to the PeakView software of the 5600+ high-resolution mass spectrometer, and the summed ion +H mode was entered to automatically generate a list of theoretical accurate mass numbers of the 143 fentanyls, completing the establishment of the primary target library of 143 fentanyls.

[0029] Table 1

[0030]

[0031]

[0032]

[0033]

[0034]

[0035] Note: The varieties marked in red are 29 representative fentanyl substances for which standard products have been purchased.

[0036] The 143 fentanyl substances were sorted by molecular weight and divided into 21 categories according to molecular weight and structural differences. As many standard substances as possible were purchased, and at least one standard substance was purchased in each category. A total of 29 standard substances were purchased, which can fully cover the 21 categories. The secondary mass spectra of the 29 fentanyl substances were obtained through HPLC-QTof detection. The fragmentation mechanism of each fragment ion was analyzed using electronic theory and energy theory based on the secondary mass spectra of the 29 fentanyl substances.

[0037] Based on the fragmentation mechanisms of 29 fentanyl compounds, the fragmentation rules and characteristics of fentanyl compounds are summarized:

[0038] (1) The parent ions of fentanyl compounds are very stable. For most compounds, the parent ions can only be effectively fragmented after the CE value reaches 30V.

[0039] (2) Induced fragmentation (i) is the main fragmentation mode for most fentanyl compounds, and the ions formed have the highest abundance. For example, m / z 188, 84, 105, 194, 111, 238, etc., all induced fragmentation originates from amide and piperidine groups, and the induction ability of amide groups is significantly stronger than that of piperidine groups. Except for compounds with other substituents at the para position of piperidine groups, the fragments with the highest abundance ratio all come from induced fragmentation of amide groups.

[0040] (3) Compounds such as fentanyl, heptanoyl fentanyl, and cyclopropyl fentanyl will undergo single H rearrangement (rH) to lose propionyl, heptanoyl, and cyclopropyl acyl groups to form m / z 281 fragment ions. Although the abundance ratio of this ion is not high, it is specific and regular. Both straight-chain alkane substituents and cycloalkane substituents have similar specific ions. The more carbon numbers of the straight-chain alkane substituents, the higher the ion abundance. For example, the abundance ratio of the m / z 281 ion in heptanoyl fentanyl is significantly higher than that in fentanyl.

[0041] (4) Fentanyl, heptanoylfentanyl, cyclopropylfentanyl and other compounds can all undergo double H rearrangement (r2H) and then break the double bond to form fragment ions m / z 146, 132 or 216. Although the abundance ratio of double H rearrangement ions is not high, they are also specific and regular. Most fentanyl contains such ions, especially the m / z 216 ion obtained by double H rearrangement. For compounds with different substituents on the amide, the fragments obtained by double H rearrangement will be different, which has obvious specificity and helps to determine the group composition of the substituents, such as m / z 272 for heptanoylfentanyl, m / z 228 for cyclopropylfentanyl, m / z 264 in phenylfentanyl, and m / z 232 in methoxyacetylfentanyl.

[0042] (5) Product ions formed by elimination reaction (re) are also a very common fragmentation mode. For example, m / z 134 and 140 are both product ions of elimination reaction. Phenethyl substituents are more likely to undergo elimination reaction than thiophenethyl substituents (higher abundance ratio).

[0043] (6) A comparative study of 29 fentanyl secondary fragment ions showed that the double H rearrangement (r2H) ion m / z 146 and the elimination reaction (re) 134 were the two fragments produced by phenethyl-substituted fentanyl, showing significant regularity.

[0044] (7) Compounds such as sufentanil and carfentanil that contain a methoxy substituent at the para-piperidinyl group have a large number of fragmentation ions. The induced fragmentation ion is not necessarily the ion with the highest abundance ratio. It is more likely to undergo a mono-H rearrangement (rH) to lose a neutral molecule of methanol, such as m / z 355 for sufentanil and m / z 385 for carfentanil. Similarly, compounds such as remifentanil that contain a methyl formate substituent at the para-piperidinyl group also have a large number of fragmentation ions and are more likely to undergo a mono-H rearrangement (rH) to lose a neutral molecule of methyl formate or methanol, such as m / z 317 and 345.

[0045] (8) Compounds containing hydroxyl substituents, such as β-hydroxyfentanyl and β-hydroxythiofentanyl, produce characteristic dehydrated ions, such as m / z 335 for β-hydroxyfentanyl and m / z 341 for β-hydroxythiofentanyl.

[0046] Furthermore, based on the fragmentation mechanisms and rules of the 29 fentanyls, the fragmentation mechanisms and pathways of the remaining 114 fentanyls without standards were speculated, and possible secondary fragment ions of the 114 compounds were obtained. The speculated mechanisms and speculated secondary fragment ions are shown in Table 2.

[0047] Table 2

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062] The secondary fragment ions of 29 fentanyls and the theoretically predicted secondary fragment ions of 114 fentanyls without standards were combined to construct a secondary fragment ion library of 143 fentanyls, which are listed in Table 1.

[0063] An ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry quantitative and qualitative detection method was established by purchasing 29 representative fentanyl-like substance standards, and a screening method for 143 fentanyls was also established.

[0064] Instruments and reagents

[0065] TripleTOF 5600+ liquid chromatography-quadrupole / time-of-flight mass spectrometer and Analyst TF1.7 workstation (AB SCIEX, USA); analytical balances MSA225S-1CE-DA (Sartorius, Germany, sensitivity 0.01 mg) and JY3002 (Shanghai Precision Scientific Instruments, Inc., sensitivity 0.01 g); vortex mixer MS 3Digital (IKA, Germany); ultrapure water system PRO VF-T (Sartorius, Germany); centrifuge Hermle Z323K (Hermle, Germany); pipette (Eppendorf, Germany).

[0066] Acetonitrile and methanol (chromatographic grade) were purchased from Thermo Scientific, USA; formic acid (chromatographic grade) was purchased from Beijing Dicoma Technology Co., Ltd.

[0067] Norfentanyl, Butyryl norfentanyl, Norsufentanil, 4-Anilino-N-phenethylpiperidine / 4-ANPP, Norcarfentanil, N-methylnorcarfentanil, 2,3-seco-Fentanyl, β-hydroxyfentanyl, β-Hydroxythiofentanyl, N-benzyl furanyl norfentanyl, Thienyl fentanyl, Ocfentanil / 2-fluoro methoxyacetyl fentanyl The concentration of standard solutions of fentanyl, furanylfentanyl, remifentanil, benzyl carfentanil, sufentanil, alfentanil, fentanyl, despropionyl para-Fluorofentanyl, phenyl fentanyl, cyclopropyl fentanyl and methoxyacetyl fentanyl were all 100 mg / L, and the solvent was methanol, which was purchased from Tianjin Alta Technology Co., Ltd.

[0068] Thiofentanyl, α-methyl thiofentanyl, 4-methoxy acetyl fentanyl, 4-methoxyacrylfentanyl, 4-methoxymethoxyacetyl fentanyl, 4-methoxytetrahydrofuran fentanyl and heptanoylfentanyl solid standards, all with a purity greater than 95%, were purchased from Shanghai Yuansi Biaowu Technology Co., Ltd.

[0069] Preparation of standard solution

[0070] 29 fentanyl-like substances standard stock solutions: Accurately pipette 1 mL of each of the 22 100 mg / L liquid standard solutions in 3.4.1, dilute to 10 mL with methanol, and prepare standard stock solutions with a concentration of 10 mg / L; Accurately weigh 1 mg (accurate to 0.01 mg) of each of the 7 solid standard substances in 3.4.1, dilute to 10 mL with methanol, and prepare standard stock solutions with a concentration of 100 mg / L, and store at 4°C in the dark.

[0071] Mixed standard stock solutions of 29 fentanyl-like substances: Pipette 200 μL or 20 μL of the above 29 standard stock solutions into 10 mL volumetric flasks respectively, dilute to the mark with acetonitrile to prepare a mixed standard stock solution with a concentration of 200 μg / L, and store at 4°C in the dark.

[0072] Mixed standard working solution of 29 fentanyl-like substances: Dilute the mixed standard stock solution with acetonitrile aqueous solution (1:9, v / v) to a mixed standard working solution of appropriate concentration and store at 4°C in the dark.

[0073] Sample preparation

[0074] Chocolate, medicines and white powder: weigh 0.2 g (accurate to 0.01 g) of the sample into a 50 mL plastic centrifuge tube, add 10 mL of acetonitrile, vortex on a vortexer for 2 min, centrifuge at 4 000 r / min for 5 min, take 2 mL of the supernatant and filter through a membrane (0.20 μm, organic phase) for testing.

[0075] Protein and milk beverages: weigh 0.2 g (accurate to 0.01 g) of the sample into a 50 mL plastic centrifuge tube, add 10 mL of acetonitrile, vortex on a vortexer for 2 min, centrifuge at 4 000 r / min for 5 min, take 2 mL of the supernatant and filter through a membrane (0.20 μm, organic phase) for testing.

[0076] Glucose powder: weigh 0.2 g (accurate to 0.01 g) of the sample and add 10 mL of acetonitrile aqueous solution (1:9, v / v) to dissolve. Vortex on a vortexer for 2 min. Take 2 mL of the supernatant and filter it through a membrane (0.20 μm, organic phase) for testing.

[0077] Drinking water, fruit and vegetable beverages, health drinks, tea beverages, and alcoholic beverages: Weigh 0.2 g (accurate to 0.01 g) of the test solution, add 10 mL of acetonitrile aqueous solution (1:9, v / v) to dissolve, vortex on a vortexer for 2 min, take 2 mL of the supernatant and filter through a membrane (0.20 μm, organic phase) for testing.

[0078] Chromatographic and mass spectrometric conditions

[0079] Chromatographic conditions

[0080] Chromatographic column: Kinetex C18 2.6μm 100A 100×2.1mm (inner diameter), mobile phase A is formic acid aqueous solution (containing 0.08% formic acid), mobile phase B is acetonitrile; flow rate: 0.4mL / min; column temperature: 40℃; injection volume: 10μL. Gradient elution program: 0-5min, 5%-80%B; 5-8min, 80%B; 8-8.1min, 80%-5%B; 8.1-10.5min, 5%B.

[0081] Mass spectrometry conditions

[0082] Ionization mode: electrospray ionization (ESI), positive ion scanning mode; mass spectrometry calibration method: sodium formate solution external standard calibration; calibration cycle: sodium formate calibration solution and other test solutions are injected; acquisition mode: TOF MS-IDA-TOFMS / MS (simultaneous acquisition of primary and secondary); scanning range: m / z 50-650 for both primary and secondary; electrospray voltage (IS): 5500V; ion source temperature (TEM): 550℃; nebulizer gas pressure (GS1): 345kPa; auxiliary gas pressure (GS2): 414kPa; curtain gas pressure (CUR): 276kPa; declustering voltage (DP): 80V; collision energy (CE): 30±5.

[0083] The chemical formulas and Chinese and English names of 143 fentanyl-like substances were input, and the theoretical accurate mass number ([M+H] + ), and constructed into a primary compound library, see Table 1.

[0084] The standard working solutions of 29 fentanyl-like substances were used separately, and their secondary fragment ion spectra were collected in the Product Ion IDA mode, which were imported into the high-resolution mass spectral library to construct a secondary compound library. The secondary fragment ions derived from Table 2 were used to establish the secondary compound library for the remaining 114 fentanyls without standards, and the construction of the primary and secondary compound libraries of 143 fentanyls was completed.

[0085] Identification and confirmation of 29 types of fentanyl (qualitative)

[0086] In TOF MS mode (primary), if the retention time of the analyte in the sample solution deviates from the retention time of the standard solution of the corresponding concentration within ±2.5%; the deviation between the measured value and the theoretical value of the accurate mass number of the parent ion of the analyte is less than or equal to 5ppm; and the abundance ratio of the secondary fragment ion is consistent with that of the library ion, it can be determined that the sample contains the fentanyl-like substance.

[0087] Identification and confirmation of 114 types of fentanyl (qualitative)

[0088] In the TOF MS mode (primary), if the deviation between the measured value of the accurate mass number of the parent ion of the analyte in the sample solution and the theoretical value is less than or equal to 5 ppm; if the secondary fragments of the compound contain all the ions of the corresponding compounds in Table 2 (114 fentanyls), it can be determined that the sample contains the fentanyl-like substance.

[0089] The 29 fentanyl mixed standard stock solutions were diluted with acetonitrile and water (1:9, v / v) to standard working solutions with concentrations of 1, 2, 5, 10 and 20 μg / L. The solutions were determined under the conditions specified in Section 3.4.4. The 29 fentanyl solutions showed a linear relationship in the range of 1 to 20 μg / L. The linear range and correlation coefficients are shown in Table 3. The correlation coefficients (r) of all 29 fentanyl solutions were greater than 0.995.

[0090] Table 3

[0091]

[0092]

[0093] Furthermore, in order to verify the feasibility of the screening method of this embodiment, the quantitative limit and detection limit of the method of the present invention were determined.

[0094] Limit of quantitation and limit of detection: According to the EU document SANTE / 11813 / 2017, the limit of quantitation (LOQ) is the minimum spike concentration level at which the recovery rate and RSD in the spike recovery test meet the performance requirements (recovery rate 70% to 120% and RSD ≤ 20%). The minimum spike level of this method is 0.05 mg / kg. The average recovery rate and RSD of 29 fentanyls in 4 matrices meet the above requirements, and the signal-to-noise ratio (S / N) is greater than 10. Therefore, the limit of quantitation (LOQ) of this method is determined to be 0.05 mg / kg; the limit of detection (LOD) is determined according to the signal-to-noise ratio (S / N) ≥ 3, which is 0.01 mg / kg.

[0095] Furthermore, to verify the accuracy and precision of the method, blank hypoglycemic drugs, Lulu, glucose powder and Zhenlu health drink samples were selected for 0.05, 0.1 and 0.5 mg / kg 3 Six parallel samples (n=6) were spiked at each level for recovery experiments. The method was used for detection. The accuracy and precision data are shown in Tables 4 to 15.

[0096] Table 4

[0097]

[0098]

[0099] Table 5

[0100]

[0101] Table 6

[0102]

[0103]

[0104] Table 7

[0105]

[0106]

[0107] Table 8

[0108]

[0109]

[0110] Table 9

[0111]

[0112]

[0113] Table 10

[0114]

[0115] Table 11

[0116]

[0117] Table 12

[0118]

[0119]

[0120] Table 13

[0121]

[0122]

[0123] Table 14

[0124]

[0125]

[0126] Table 15

[0127]

[0128]

[0129] From the results in Tables 4 to 15, it can be seen that the recoveries of 29 fentanyls in the four matrices were 86.1% to 109.1%, 88.5% to 109.4%, 86.8% to 112.1% and 89.1% to 112.9%, respectively, and the relative standard deviation (RSD) was 1.9% to 19.8%, indicating that the method has high accuracy and good reproducibility, and is suitable for the detection of samples such as medicines, glucose powder, beverages, drinking water and alcohol.

[0130] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for screening fentanyl-like substances, characterized in that: The following steps are involved: S1. Collect M types of fentanyl that can be found with written records, and establish a primary target library of theoretical accurate mass numbers of fentanyl parent ions based on the molecular formulas of the M types of fentanyl; S2. Classify M types of fentanyl into N types according to their structural formulas, find a representative fentanyl in each type and obtain its standard, and obtain N representative secondary mass spectra of fentanyl by HPLC-QTof detection, and analyze the fragmentation mechanism of each fragment ion using electronic theory and energy theory based on the N representative secondary mass spectra of fentanyl, and summarize the fragmentation rules and characteristics of each type of fentanyl; S3. According to the fragmentation law and characteristics of each type of fentanyl, the secondary fragment ions of MN types of fentanyl for which standard substances cannot be obtained are theoretically derived, and the secondary fragment ion spectral library of fentanyl is established by combining N representative fentanyls and MN types of fentanyl secondary fragment ions for screening and detection of the entire type of fentanyl substances; S4. Establishing the quantitative and qualitative detection of samples containing fentanyl substances by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry using N fentanyl-like substances as existing standards.

2. The method for screening fentanyl-like substances according to claim 1, characterized in that: The step S4 specifically includes: S41. Prepare N representative fentanyl standard stock solutions: Take N representative fentanyl and dilute them to 10 mL with methanol to prepare a standard stock solution with a concentration of 10 mg / L, and store it at 4°C in the dark; S42. Prepare N representative mixed standard stock solutions of fentanyl substances: pipette 200 μL or 20 μL of the above N representative standard stock solutions of fentanyl substances into 10 mL volumetric flasks respectively, dilute to the mark with acetonitrile to prepare a mixed standard stock solution with a concentration of 200 μg / L, and store at 4°C in the dark; S43, prepare N representative mixed standard working solutions of fentanyl substances: dilute N representative mixed standard stock solutions of fentanyl substances with 9 times the volume of acetonitrile aqueous solution to a mixed standard working solution of appropriate concentration, and store at 4° C. in the dark; S44. After pretreatment of the sample containing fentanyl, it was detected by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, and the chromatographic conditions were as follows: chromatographic column: Kinetex C18 2.6μm 100A100×2.1mm, mobile phase A was 0.08% formic acid aqueous solution, and mobile phase B was acetonitrile; flow rate: 0.4mL / min; chromatographic column temperature: 40°C; injection volume: 10μL; gradient elution program: 0-5min, 5%-80%B; 5-8min, 80%B; 8-8.1min, 80%-5%B; 8.1-10.5min, 5%B; mass spectrometry conditions were as follows: ionization mode: electrospray ionization ESI, positive ion scanning mode; mass spectrometry calibration method: sodium formate solution external standard calibration; calibration cycle: sodium formate calibration solution and other test solutions were injected; acquisition mode: TOFMS-IDA-TOF MS / MS; scanning range: m / z 50-650 for both primary and secondary; electrospray voltage IS: 5500V; ion source temperature TEM: 550℃; nebulizer gas pressure GS1: 345kPa; auxiliary gas pressure GS2: 414kPa; curtain gas pressure CUR: 276kPa; declustering voltage DP: 80V; collision energy CE: 30±5; S45. Establishment of high-resolution mass spectrometry library: Input the chemical formula and Chinese and English names of M fentanyl-like substances, and use the high-resolution mass spectrometry software to calculate the theoretical accurate mass number [M+H] of each compound parent ion. + , and construct a primary compound library; separately use N representative fentanyl substance mixed standard working solutions, collect their secondary fragment ion spectra in Product Ion IDA mode, import them into the high-resolution mass spectral library, and construct a secondary compound library; the remaining MN kinds of fentanyl without standards use the secondary fragment ions of MN kinds of fentanyl to establish a secondary compound library, and complete the construction of M kinds of fentanyl primary and secondary compound libraries; S46. Identification and confirmation of N types of fentanyl: In the TOF MS mode, if the retention time of the fentanyl to be tested in the sample is within ±2.5% of the retention time of one of the N representative fentanyl substance standard stock solutions of the corresponding concentration; the deviation between the measured value and the theoretical value of the accurate mass number of the parent ion of the test substance is less than or equal to 5ppm; if the abundance ratio of the secondary fragment ion is consistent with that of the library ion, then the sample is judged to contain the representative fentanyl substance; S47. Identification and confirmation of MN types of fentanyl: In TOF MS mode, the deviation between the measured and theoretical values ​​of the accurate mass number of the parent ion of the analyte in the sample is less than or equal to 5 ppm; if the secondary fragments of the compound contain one of the secondary fragment ions of the MN types of fentanyl and all the ions of its corresponding compound, it is determined that the sample contains this fentanyl-like substance.

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