Rapid identification method of cannabinoid compound based on paper spray mass spectrometry

Abstract

The invention belongs to the field of analytical chemistry and mass spectrometry detection, and discloses a rapid identification method of cannabinoid compounds based on paper spray mass spectrometry, which comprises the following steps: dropwise adding a to-be-detected sample solution on a cuprous ion modified cellulose paper base, and carrying out paper spray mass spectrometry detection to obtain the cannabinoid compounds. By monitoring a protonated molecule ion peak [M + H] < + > and a Cu < + > adduct ion peak [M + 63Cu] < + > / [M + 65Cu] < + > in a mass spectrogram, strictly controlled key isomers CBD and delta9-THC can be distinguished, and the method can be used for accurately identifying whether a complex matrix contains a cannabinoid compound with a 1, 5-diene structure or not and distinguishing the cannabinoid compound from a cannabinoid isomer without the cannabinoid compound with the 1, 5-diene structure. Meanwhile, whether CBC, CBL, delta9-THC or delta8-THC and other cannabinoid compounds are contained or not is prompted when CBD exists. The method does not need to depend on a high-resolution mass spectrum or a secondary mass spectrum, does not need to use noble metal ions and metal organic complexes, can realize rapid, accurate and low-cost identification within tens of seconds only under the condition of a low-resolution mass spectrum and a primary mass spectrum, and has a good prospect in non-clinical diagnosis scenes such as scientific research, supervision and detection and the like.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry and mass spectrometry detection technology, and particularly relates to a method for rapid identification of cannabinoid compounds. Background Technology

[0002] marijuana( Cannabis sativa L. Cannabinoids are an important fiber and oilseed crop, cultivated in multiple climate zones worldwide. Their bioactive secondary metabolites—cannabinoids—have broad application potential in medicine, food, and industry. Cannabinoids are one of the important secondary metabolites of this plant, with over one hundred members identified, among which cannabidiol (CBD) is the most prominent. Cannabidiol CBD) and Δ9-tetrahydrocannabinol (Δ 9-Tetrahydrocannabinol CBD and Δ9-THC have attracted the most attention. Although both have been reported to have pharmacological activities, CBD has no significant psychoactive activity, while Δ9-THC is the main psychoactive component. Rapid and accurate detection and differentiation between CBD and cannabinoids such as Δ9-THC is of practical significance.

[0003] At the analytical detection level, traditional chromatographic-based methods, such as gas chromatography (GC), high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), or high-performance liquid chromatography-mass spectrometry (HPLC-MS), can achieve the separation and identification of cannabinoids, but chromatographic separation is time-consuming and cannot meet the needs of rapid screening. Direct perfusion mass spectrometry can rapidly detect samples without chromatographic separation, but it has problems such as sample contamination of the ion source, strict requirements for sample pretreatment, and easy cross-contamination between samples; to avoid residual interference between samples, a long rinsing time is usually required. Open-type mass spectrometry analysis methods (such as paper spray mass spectrometry and real-time direct analysis mass spectrometry) also do not require chromatographic separation. They can directly ionize samples under atmospheric pressure or open conditions, and introduce the generated ions into the mass spectrometer for analysis and detection. These methods usually require only simple or no sample pretreatment. Compared with direct perfusion mass spectrometry, they not only reduce instrument contamination and cross-contamination between samples, but also enable direct and rapid detection of samples, with the advantages of simplicity and efficiency. However, such methods typically rely on protonated molecular ion signals ([M+H)). + In complex matrices, detection processes are often accompanied by high background noise and low signal-to-noise ratios, while lacking sufficient structural specificity, thus significantly limiting the reliability and sensitivity of qualitative discrimination. For example, in identifying cannabinoid isomers (CBD, CBC, CBL, Δ8-THC, and Δ9-THC), mass spectrometry alone cannot distinguish them without chromatographic separation. This is because these compounds have the same molecular formula (C6H2O). 21 H 30O2) and similar physicochemical properties produce [M+H] molecules with the same mass-to-charge ratio in primary mass spectrometry. + The peaks are highly similar to those of protonated molecular ions, and their secondary mass spectrometry fragmentation patterns are also very similar. Therefore, even with high-resolution mass spectrometry and secondary mass spectrometry, it is difficult to accurately distinguish them based solely on the protonated molecular ion signal. To improve detection specificity, some studies have attempted to utilize selective complexation of metal ions (such as Ag) + This can be used to assist in the detection and differentiation of cannabinoids. For example, using silver-based paper spray mass spectrometry, both CBD and Δ9-THC are associated with Ag. + They form addition ion peaks with the same mass-to-charge ratio, thus producing the same [M+Ag] peak. + Although the signals cannot be distinguished between CBD and Δ9-THC at the primary mass spectrometry level, the [M+Ag] of CBD and Δ9-THC are [missing information]. + The peaks exhibit different secondary mass spectrometric fragments, allowing for the differentiation of CBD and Δ9-THC at the secondary mass spectrometry level. However, this type of method requires confirmation by secondary mass spectrometry, complicating the detection process and increasing equipment costs. Other compounds containing olefin structures or polar functional groups (such as hydroxyl and carboxyl groups) can also react with Ag. + Addition occurs, further complicating cannabinoid identification. Meanwhile, silver, being a precious metal, is expensive, further limiting the feasibility of this strategy in large-scale applications. Therefore, there is an urgent need to develop a rapid detection method that can reliably identify cannabinoids using only conventional low-resolution mass spectrometry (MS / MS), without relying on high-resolution or secondary mass spectrometry capabilities. Summary of the Invention

[0004] The technical problem to be solved by this invention is that existing detection methods suffer from problems such as complex operation, long time consumption, high cost and insufficient specificity in complex matrices. This invention proposes a method that can quickly, accurately and economically detect and distinguish cannabinoids containing 1,5-diene structures and cannabinoids without 1,5-diene structures and their isomers.

[0005] The cannabinoids of this invention include cannabidiol (CBD). Cannabidiol CBD), cannabinol ( Cannabigerol CBG), cannabinol ( Cannabino l, CBN), cannabinoids ( Cannabichromene CBC), cannabicycloenol (CBC) Cannabicyclol , CBL), Δ9-tetrahydrocannabinol (Δ 9-Tetrahydrocannabinol Δ9-THC), Δ8-tetrahydrocannabinol (Δ 8-Tetrahydrocannabinol Rapid identification of CBD, CBG, CBN, CBC, CBL, Δ9-THC, and Δ8-THC, and other cannabinoids with similar structural features. The chemical structural formulas, elemental compositions, and molecular weights of CBD, CBG, CBN, CBC, CBL, Δ9-THC, and Δ8-THC are shown below. Figure 1as shown.

[0006] To solve the above technical problems, the technical scheme provided by the present application is: A rapid identification method of cannabinoid compounds based on paper spray mass spectrometry, comprising the following steps: dropping a to-be-tested sample solution on a cellulose paper base modified by cuprous ions, performing paper spray mass spectrometry detection, and simultaneously monitoring protonated molecular ion peaks [M+H] + and Cu + adduct ion peaks [M+ 63 Cu] + / [M+ 65 Cu] + ; if [M+H] + peaks and [M+ 63 Cu] + / [M+ 65 Cu] + peaks are simultaneously detected, it is determined that the to-be-tested sample contains cannabinoid compounds with a 1,5-diene structure; if only [M+H] + peaks are detected, it is determined that the to-be-tested sample contains cannabinoid compounds without a 1,5-diene structure and does not contain cannabinoid compounds with a 1,5-diene structure.

[0007] The above method further comprises the following steps: the solvent of the to-be-tested sample solution is methanol; the to-be-tested sample solution contains at least one solute selected from the group consisting of CBD, CBG, CBN, CBC, CBL, Δ9-THC and Δ8-THC; and the concentration of the solute in the to-be-tested sample solution is 10-100 µg / mL.

[0008] Further, the cellulose paper base modified by cuprous ions is prepared by the following method: cellulose chromatography paper is soaked in a cuprous chloride acetonitrile solution with a concentration of 5-15 mmol / L, the paper base is taken out after ultrasonic treatment, and is completely dried to obtain a cellulose paper base loaded with cuprous ions on the surface.

[0009] Further, the cellulose chromatography paper has an isosceles triangle shape with a base length of 7±0.5 mm and a height of 13±0.5 mm. The cellulose paper base prepared by the present application, in combination with the specific shape and size, can effectively reduce background noise, improve signal-to-noise ratio, and be more conducive to maintaining stable detection signals.

[0010] Further, the cannabinoid compounds with a 1,5-diene structure include CBD or CBG, and the cannabinoid compounds without a 1,5-diene structure include CBC, CBL, CBN, Δ9-THC or Δ8-THC.

[0011] Furthermore, the rapid identification method does not add any other metal ions, reagents, or complexes.

[0012] Furthermore, the paper spray mass spectrometry employs first-order mass spectrometry, and the detection conditions include: using positive ion mode, and a scanning range of [missing information]. m / z 100~500, monitored ions include: m / z 315 (CBD, CBC, CBL, Δ9-THC or Δ8-THC of [M+H]) + ), m / z 311 (CBN's [M+H]) + ), m / z 377 / 379 (CBD's [M+]) 63 Cu] + / [M+ 65 Cu] + ), m / z 317 (CBG's [M+H]) + )and m / z 379 / 381 (CBG's [M+]) 63 Cu] + / [M+ 65 Cu] + ),each m / z The deviation is within ±0.4; the criterion for detecting ion peaks is a signal-to-noise ratio (S / N) ≥ 3.

[0013] Furthermore, the detection conditions for the paper spray mass spectrometry also include: atomizing gas flow rate of 0.4~0.6 L / min, heating block temperature of 200~400 ℃, DL tube temperature of 200~300 ℃, and interface voltage of 3.5~4.5 kV.

[0014] Furthermore, if simultaneously detected m / z 315 [M+H] + Fenghe m / z 377 / 379 of [M+] 63 Cu] + / [M+ 65 Cu] + If a peak is detected, the sample is determined to contain cannabidiol; if a peak is detected simultaneously... m / z 317's [M+H] + Fenghe m / z 379 / 381 of [M+ 63 Cu] + / [M+ 65 Cu] + If a peak is detected, the sample is determined to contain cannabinol; if only one peak is detected... m / z 311 of [M+H] +If a peak is detected, the sample is determined to contain cannabinol; if only one peak is detected... m / z 315 [M+H] + If a peak is detected, the sample is determined to contain at least one of cannabinoids, cannabidiol, Δ9-tetrahydrocannabinol, and Δ8-tetrahydrocannabinol, and not cannabidiol.

[0015] Furthermore, if the sample is determined to contain cannabidiol, it can be further identified whether the sample containing cannabidiol contains other cannabinoid compounds (such as at least one of CBC, CBL, Δ9-THC, and Δ8-THC) that do not have a 1,5-diene structure but have the same molecular weight of 314 Da. The specific operation includes the following steps: The sample to be tested m / z The theoretical concentration of cannabidiol was calculated using linear equation (a) based on the measured peak height response intensity of 315, and then the theoretical concentration was calculated using linear equation (b). m / z The theoretical peak height response intensity value is 377. Then, this theoretical peak height response intensity value is compared with the sample to be tested. m / z Compare the measured peak height response intensity values ​​of 377; like m / z If the difference between the theoretical peak height response intensity value and the measured peak height response intensity value is within 20%, then the sample is determined to contain only cannabidiol; if... m / z If the theoretical peak height response intensity value of 377 is more than 20% higher than the measured peak height response intensity value, it is determined that the sample contains other cannabinoid compounds (such as at least one of CBC, CBL, Δ9-THC and Δ8-THC) that do not have a 1,5-diene structure but have the same molecular weight of 314 Da. The linear equation (a): y =1.93×10 5 x +4.30×10 4 , R 2 =0.9992, the linear equation (b): y =1.10×10 4 x +1.98×10 6 , R 2 =0.9937, where y This represents the peak height response intensity value. x For concentration.

[0016] The method of the present application can distinguish between cannabinoid compounds with 1,5-diene structure and recognized as non-psychoactive (such as CBD, CBG) and other cannabinoid compounds without 1,5-diene structure. Some of the latter compounds have significant psychoactivity (such as Δ9-THC, Δ8-THC), and some have lower or reported limited psychoactivity (such as CBC, CBL, CBN). The mechanism is that cuprous ions can selectively π-complex with 1,5-diene structure and preferentially form stable [M+ 63 Cu] + / [M+ 65 Cu] + diagnostic ions, thereby significantly improving structural selectivity and discrimination reliability.

[0017] Taking isomers CBD and Δ9-THC as examples, the specific mechanism is as follows: Firstly, the CBD molecule contains 1,5-diene structure, which can form a stable complex with Cu + , in addition to the [CBD+H] + ( m / z 315) commonly seen in traditional mass spectrometry methods, a characteristic [CBD+Cu] + peak ( m / z 377 / 379) is also produced, wherein, m / z 377 and m / z 379 correspond to copper stable isotopes 63 Cu and 65 Cu addition peaks, respectively. Thus, even under low-resolution mass spectrometry conditions, CBD can be quickly and accurately determined in the first mass spectrum, significantly improving the qualitative reliability. Similarly, CBG can also be identified by [CBG+H] + ( m / z 317) and [CBG+Cu] + ( m / z 379 / 381), wherein, m / z 379 and Figure 1 381 correspond to copper stable isotopes 63 Cu and 65 Cu addition peaks, respectively. Secondly, cannabinoids without 1,5-diene structure such as CBC, CBL, CBN, Δ9-THC or Δ8-THC cannot form Cu + addition peaks, and only exhibit corresponding protonated ion peaks, thereby realizing the identification of isomers CBD and Δ9-THC in the first mass spectrum of low-resolution mass spectrometry. Thirdly, compared with unmodified paper-based materials, cuprous ion-modified cellulose paper-based materials can effectively reduce background noise and improve signal-to-noise ratio, thereby being superior to conventional paper spray mass spectrometry methods in the detection of these cannabinoids.

[0018] Compared with the prior art, the present application has the following advantages: 1. The method of the present application only needs to use low-resolution mass spectrometry (without expensive high-resolution mass spectrometry), and can realize low-cost detection (without using noble metals or metal organic complexes) under the condition of primary mass spectrometry (without using secondary mass spectrometry), so as to quickly and accurately identify whether the cannabinoid compound contains a 1,5-diene structure, and then distinguish the cannabinoid containing the structure from the cannabinoid not containing the structure; the whole process only takes a few seconds, and even in a complex matrix, by comprehensively utilizing the two discriminators of molecular weight information and specific chemical structure characteristics, the presence of the protonated molecular ion peak [M+H] can be monitored, so as to quickly and intuitively confirm whether the cannabinoid containing the 1,5-diene structure exists. + and Cu + adduct ion peak [M+ 63 Cu] + / [M+ 65 Cu] + , quickly and intuitively confirm whether the cannabinoid containing the 1,5-diene structure exists.

[0019] 2. The method of the present application can realize the differentiation of the strictly controlled key isomers CBD and Δ9-THC, and effectively overcome the limitations of the existing detection means (such as only relying on molecular weight, using adduct peaks with unknown composition, needing the help of secondary mass spectrometry or high-resolution mass spectrometry, relying on silver ions and other noble metals); the method can be used for rapid screening of cannabinoid compounds, and has good application prospect in scientific research, regulatory detection and other non-clinical diagnosis scenes.

[0020] 3. The present application uses cellulose paper base modified by cuprous ion for paper spray mass spectrometry analysis, which can effectively reduce background interference and improve signal-to-noise ratio, can realize the differentiation of the strictly controlled key isomers CBD and Δ9-THC, can be used for accurately identifying whether the cannabinoid compound containing the 1,5-diene structure exists in a complex matrix and distinguishing from the cannabinoid isomer not containing the structure, and at the same time, can prompt whether the cannabinoid compounds such as CBC, CBL, Δ9-THC or Δ8-THC exist when CBD exists, can be used for analyzing cannabinoid compounds such as CBD, CBG, CBN, CBC, CBL, Δ9-THC and Δ8-THC, and has the advantages of simple method, low production cost and being conducive to industrialization promotion. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creating any creative labor.

[0022] Figure 2 Schematic diagram of cannabinoid chemical structure analyzed in the present application; Figure 3 Schematic diagram of the preparation of subcopper ion modified paper base and the process of paper spray mass spectrometry for the analysis of cannabis samples in the present application; m / z Standard curve between CBD concentration and mass spectrometry response intensity established by the present application using subcopper ion modified paper base combined with paper spray mass spectrometry, wherein figure (a) is [CBD+H] + m / z 315Linear relationship diagram of response intensity and concentration, figure (b) is [CBD+ 63 Cu] + Figure 4 377Linear relationship diagram of response intensity and concentration; Figure 5 Mass spectrum diagram of CBD and Δ9-THC obtained by paper spray mass spectrometry analysis using subcopper ion modified paper base in the present application, wherein figure (a) is the mass spectrum diagram of CBD, and figure (b) is the mass spectrum diagram of Δ9-THC; Figure 6 Mass spectrum diagram of CBG obtained by paper spray mass spectrometry analysis using subcopper ion modified paper base in the present application; Figure 7 Mass spectrum diagram of Δ9-THC obtained by paper spray mass spectrometry analysis using blank paper base and subcopper ion modified paper base in the present application, wherein figure (a) is the mass spectrum diagram of Δ9-THC matrix spiked sample loaded on blank paper base, and figure (b) is the mass spectrum diagram of Δ9-THC matrix spiked sample loaded on subcopper ion paper base; Figure 8 Mass spectrum diagram of CBC obtained by paper spray mass spectrometry analysis using subcopper ion modified paper base in the present application; Figure 9 Mass spectrum diagram of CBL obtained by paper spray mass spectrometry analysis using subcopper ion modified paper base in the present application; Figure 10 Mass spectrum diagram of Δ8-THC obtained by paper spray mass spectrometry analysis using subcopper ion modified paper base in the present application; Figure 11 Mass spectrum diagram of CBN obtained by paper spray mass spectrometry analysis using subcopper ion modified paper base in the present application; Figure 12 Mass spectrum diagram of CBD and Δ9-THC standard mixed solution obtained by paper spray mass spectrometry analysis using subcopper ion modified paper base in the present application; Figure 13 Mass spectrum diagram of commercial full-spectrum CBD oil 1 obtained by paper spray mass spectrometry analysis using subcopper ion modified paper base in the present application; Figure 14The mass spectrum of commercially available full-spectrum CBD oil 2 obtained by paper spray mass spectrometry using the paper base modified by cuprous ions according to the present application; Figure 4 The chromatograms of commercially available full-spectrum CBD oil 1 and commercially available full-spectrum CBD oil 2 analyzed by high performance liquid chromatography according to the present application; The above Figure 6 、 5 The samples used in 7-10 are as follows: 50 μL of cannabinoid standard stock solution (1 mg / mL, solvent: methanol) is accurately pipetted, diluted to 1 mL with methanol, and vortexed to obtain a standard solution with a concentration of 50 μg / mL. The standard solutions of CBD, Δ9-THC, Δ8-THC, CBG, CBC, CBL and CBN are prepared by this method. Figures 11-14 100 mg of hemp sample without cannabinoids is weighed, 1 mL of methanol is added, vortexed for 3 min, and ultrasonically extracted for 10 min. The obtained extract is filtered through a 0.22 μm organic nylon filter membrane to obtain a matrix solution. 10 μL of the matrix solution is accurately pipetted, 50 μL of Δ9-THC standard solution (1 mg / mL) is added, diluted to 1 mL with methanol, and mixed thoroughly to obtain a matrix spiked sample solution.

[0023] Figure 1 The sample preparation and detection conditions are shown in the specific examples. DETAILED DESCRIPTION

[0024] In order to facilitate the understanding of the present application, the present application will be described in more detail below in conjunction with the drawings and preferred embodiments, but the scope of protection of the present application is not limited to the following specific embodiments.

[0025] Unless otherwise defined, all the professional terms used herein have the same meaning as generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing the specific embodiments and are not intended to limit the scope of protection of the present application.

[0026] Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the present application can be purchased on the market or can be prepared by existing methods.

[0027] The specific embodiments of the present application include the following steps: I. Chemical structural formula of the detected cannabinoids and methodological definition (1) The detected cannabinoids include CBD, CBG, CBN, CBC, CBL, Δ9-THC and Δ8-THC, the chemical structural formula, elemental composition and molecular weight of which are shown in Figure 2 .

[0028] (2) The "paper spray mass spectrometry" method refers to the ionization / detection process in which the test solution is dropped onto a triangular paper substrate and data is directly collected by high-pressure spraying, such as... m / z As shown.

[0029] (3) Detection conditions: "Scanning range is..." m / z "100~500" refers to the mass-to-charge ratio scanned by the mass spectrometer. m / z The range is 100~500.

[0030] (4) The "[M+H]" claimed in this invention + The "peak" refers to the protonated molecular ion peak formed when the analyte molecule M combines with a proton during mass spectrometry ionization. "[M+" 63 Cu] + / [M+ 65 Cu] + The "peak" refers to the peak of the analyte molecule M during mass spectrometry ionization, where it interacts with a single-valent copper ion (Cu). + The addition peak is formed by the combination of ) and .

[0031] II. Materials and Reagents Paper base: Cellulose paper (quantitative filter paper or equivalent material).

[0032] Cuprous salt: Cuprous chloride (CuCl) is preferred; cuprous thiophene-2-carboxylic acid or cuprous acetate are also acceptable; cuprous iodide (CuI) is not preferred.

[0033] Solvent: Acetonitrile (ACN) was used as the paper-based modification solvent; the sample was extracted with methanol.

[0034] III. Paper Base Finishing and Pretreatment (1) A cuprous ion modified solution (10 mmol / L) was prepared by dissolving CuCl in acetonitrile.

[0035] (2) The paper substrate was impregnated with the modification solution, ultrasonically treated for 10 min, and then removed and dried in an oven at 45-55 ℃ to obtain Cu. + To decorate the paper base.

[0036] (3) The adequacy of drying can be determined by the background ion [Cu+2ACN]. + = m / z 145 / 147 Decision: If detected m / z If the temperature reaches 145 / 147, continue drying to avoid residual acetonitrile and Cu. + Coordination competition inhibition [cannabinoid + Cu] + form.

[0037] IV. Sample Loading and Testing Conditions (1) Drop the solution to be tested (standard solution or cannabinoid sample extraction solution, such as commercially available full-spectrum CBD oil extraction solution, typically 10-20 μL, the concentration of cannabinoids in the standard solution is 1-200 μg / mL, and the concentration of cannabinoids in the cannabinoid sample extraction solution is 10-100 μg / mL) onto the modified paper substrate.

[0038] (2) Paper spray mass spectrometry high-pressure spray collects full scan (monitoring m / z 315, m / z 377 / 379, m / z 317, m / z 379 / 381, Figure 3 311, etc.).

[0039] (3) CBD standard curve: as shown in m / z , [M+H] + ( m / z 315) peak height response intensity is approximately linear with CBD concentration in the range of 1-100 μg / mL; [M+Cu] + ( m / z 377) peak height response intensity is approximately linear with CBD concentration in the range of 10-200 μg / mL, and the standard curve obtained is used to determine whether other cannabinoids with a molecular weight of 314 Da exist in the CBD sample.

[0040] V. Identification rules and data interpretation (1) CBD direct identification: the spectrum simultaneously appears protonated molecular ion peak [M+H] + = m / z 315 and Cu + addition ion peak [M+ 63 Cu] + / [M+ 65 Cu] + = Figure 4 377 / 379, i.e. CBD is determined to be present. The mass spectrum obtained by detecting the CBD standard solution with copper ion paper spray mass spectrometry is shown in m / z (a), in which m / z 315.3 is the CBD protonated molecular ion peak [M+H] + , m / z 377.2 / 379.2 is the CBD Cu + addition ion peak [M+ 63 Cu] + / [M+ 65 Cu] + .

[0041] (2) CBG direct identification: the spectrum simultaneously appears protonated molecular ion peak [M+H] + = m / z317 and Cu + Adduct ion peak [M+ 63 Cu] + / [M+ 65 Cu] + = Figure 5 379 / 381, which indicates that CBG is contained. The mass spectrum obtained by detecting the CBG standard solution by paper-based copper ion spray mass spectrometry is shown in FIG. 3. As shown in FIG. 3, the peak at 317.4 is the protonated molecular ion peak [M+H] of CBG, and the peaks at 379.3 / 381.3 are the adduct ion peaks [M+Cu] of CBG and Cu, m / z m / z 317.4 is the protonated molecular ion peak [M+H] + of CBG, m / z 379.3 / 381.3 are the adduct ion peaks [M+Cu] + of CBG and Cu, 63 + / [M+ 65 Cu] + .

[0042] (3) Identification of other cannabinoids with 1,5-diene structure: the protonated molecular ion peak [M+H] + of the to-be-detected molecule M and the adduct ion peak [M+Cu] + of M and Cu, 63 + / [M+ 65 Cu] + appear in the spectrum at the same time, which indicates that the cannabinoid with 1,5-diene structure is contained.

[0043] (4) Detection of Δ9-THC and its isomers: only m / z 315 appears, the signal-to-noise ratio is greater than 3 (S / N>3), and Figure 4 377 / 379 do not appear, which preliminarily indicates that Δ9-THC, CBC, CBL or Δ8-THC is contained, as shown in (b) and Figures 6-9 m / z

[0044] (5) Detection of other cannabinoids without 1,5-diene structure: only the protonated molecular ion peak [M+H] + appears, the signal-to-noise ratio is greater than 3 (S / N>3), and the corresponding adduct ion peak [M+Cu] + of Cu, 63 + / [M+ 65 Cu] + does not appear, which preliminarily indicates that the cannabinoid without 1,5-diene structure is contained. For example, only Figure 10 311 appears, the signal-to-noise ratio is greater than 3 (S / N>3), and the adduct ion peak [M+Cu] + of Cu, 63 + / [M+​​​​​​​65 Cu] + Preliminary identification suggests it is CBN, such as m / z As shown.

[0045] (6) To identify whether other isomers of cannabinoids (such as Δ9-THC, CBC, CBL, and Δ8-THC) were present in CBD samples, a series of concentration gradients of CBD standard solutions (1, 5, 10, 20, 50, 100, and 200 µg / mL) were tested. The results showed that in the range of 1–100 µg / mL, [CBD+H] + ( m / z 315) Peak height response intensity is approximately linear with concentration; within the range of 10–200 µg / mL, [CBD+ 63 Cu] + ( Figure 3 377) Peak height response intensity is approximately linear with concentration, such as m / z As shown. Based on this, it can be determined through measurement. Figure 3 315 peak high response intensity Figure 3 The linear equation shown in Figure (a) is: y =1.93×10 5 x +4.30×10 4 , R 2 =0.9992, where y This represents the peak height response intensity value. x The theoretical concentration of CBD is calculated based on the concentration of CBD, and then the theoretical concentration of CBD is used... m / z The linear equation shown in Figure (b) (linear equation (b):) y =1.10×10 4 x +1.98×10 6 , R 2 =0.9937, where y This represents the peak height response intensity value. x Calculated for concentration m / z The theoretical peak height response intensity of 377, and thus the measured... m / z The composition of the sample is determined by comparing the high response intensity of peak 377: if m / z If the relative error between the theoretical peak height response intensity value and the measured peak height response intensity value is within 20%, then the sample is determined to contain only cannabidiol; if... m / z377 Theoretical peak height response intensity value is more than 20% higher than the measured peak height response value, it is determined that the sample to be tested still contains cannabichromene, cannabicyclol, Δ9-tetrahydrocannabinol or Δ8-tetrahydrocannabinol. The application of this method needs to limit the CBD concentration in the sample to be within the range of 10~100 µg / mL.

[0046] Example 1: A rapid identification method of cannabinoid compounds based on paper spray mass spectrometry, comprising the following steps: 1. Materials and reagents CBD, Δ9-THC, Δ8-THC, CBG and CBN cannabinoid standard samples are cannabinoid methanol solutions with a concentration of 1 mg / mL (purity ≥ 99%), purchased from Sigma Aldrich Trading Co., Ltd.; CBC (purity ≥ 99%) is purchased from Shanghai Taos Biological Technology Co., Ltd.; CBL (purity ≥ 98%) is purchased from Beijing Inokai Technology Co., Ltd.; Methanol and acetonitrile are chromatography / mass spectrometry grade, purchased from Beijing Inokai Technology Co., Ltd.; CuCl is analytical pure, purchased from Shanghai Adama Reagent Co., Ltd.; Quantitative filter paper is used as a paper-based substrate, purchased from Hangzhou Special Paper Co., Ltd.

[0047] 2. Paper-based modification and instrument conditions Dissolve CuCl in acetonitrile to prepare a 10 mmol / L solution, soak the filter paper, and take out the paper substrate after ultrasonic treatment for 10 min. Dry at 45~55 ℃ until the background ion [Cu+2ACN] + m / z 145 / 147) signal completely disappears, confirming complete solvent evaporation. The detection instrument is Shimadzu MS / MS-8040, in which the paper spray mass spectrometry adopts positive ion mode, with the following parameters: full scan (Scan) and selected ion monitoring (SIM); atomization gas flow 0.5 L / min; heating block temperature 400 ℃; DL tube temperature 250 ℃; interface voltage 4.5 kV; scan range m / z 100~500. The monitored characteristic ions include: m / z 315 ([CBD / Δ9-THC / Δ8-THC / CBC / CBL+H] + ), m / z 311 ([CBN+H] + ), m / z 317 ([CBG+H] + ), m / z 377 / 379 ([CBD+ 63 Cu] + / [CBD+ 65 Cu] + ), Figure 11 379 / 381 ([CBG+​63 Cu] + / [CBG+ 65 Cu] + For each test, 15 µL of sample solution was added to the modified paper substrate.

[0048] 3. Sample preparation Accurately transfer 60 µL of a 1 mg / mL CBD standard solution and 20 µL of a 1 mg / mL Δ9-THC standard solution, and dilute to 1 mL with methanol to prepare a mixed solution with a CBD to Δ9-THC molar ratio of approximately 3:1.

[0049] 4. Test Results Sample test results are as follows m / z As shown.

[0050] CBD confirmed: Appearance in mass spectrometry m / z 315([CBD+H] + )and Figure 3 377 / 379 ([CBD+ 63 Cu] + / [CBD+ 65 Cu] + ), confirming the relationship between CBD and Cu + Adducts are present.

[0051] Inferences based on Δ9-THC, Δ8-THC, CBC, or CBL: According to the CBD standard curve. m / z The linear equation (a) shown in Figure (a) is derived from... Figure 3 The theoretical concentration of CBD was calculated to be 69 µg / mL based on the measured peak height response intensity of 315; according to the CBD standard curve... m / z The linear equation shown in Figure (b) is calculated from the theoretical concentration of CBD of 69 µg / mL. m / z The theoretical peak height response intensity of 377 is 2.74 × 10⁻⁶. 6 The actual measured value in the spectrum m / z The peak height response intensity of 377 is 0.38 × 10⁻⁶. 6 The theoretical value is significantly higher than the measured value (relative error of 86%, greater than 20%), indicating that... m / z 315 is not entirely contributed by CBD; it is inferred that Δ9-THC, Δ8-THC, CBC, or CBL coexist in the sample.

[0052] CBG investigation: No detection detected m / z Peaks 317 and 379 / 381 (detection criterion: S / N≥3) rule out the possibility of CBG presence.

[0053] CBN investigation: No detection detectedm / z 311 peak (detection criterion S / N≥3), excluding the possibility of the presence of CBN.

[0054] 5. Conclusion This example shows that the method of the present application can be used to confirm the presence of CBD under the conditions of the first mass spectrum using [M+H] + and [M+ 63 Cu] + / [M+ 65 Cu] + The adduct peak confirms the presence of CBD, and can be used to indicate the coexistence of Δ9-THC, Δ8-THC, CBC or CBL through the relative error between the theoretical value and the measured value, so that rapid and accurate qualitative analysis can be achieved without chromatographic separation.

[0055] Example 2: A rapid identification method for cannabinoid compounds based on paper spray mass spectrometry, comprising the following steps: 1. Sample and detection conditions Two commercially available full-spectrum CBD oil samples (CBD oil sample 1 and CBD oil sample 2) were selected. According to the CBD concentration indicated on the label, assuming that the CBD extraction recovery rate was 100%, the theoretical concentration of CBD in the sample after extraction and dilution was ensured to be in the range of 10-100 µg / mL for paper spray mass spectrometry analysis. The specific operation was as follows: 100 mg of full-spectrum CBD oil sample was weighed, 1 mL of methanol was added, and vortex mixing was performed for 3 min, followed by ultrasonic extraction for 10 min. The obtained extract was filtered through a 0.22 µm organic nylon filter membrane to obtain a 100 mg / mL full-spectrum CBD oil stock solution. 10 µL of the full-spectrum CBD oil stock solution was diluted to 1 mL with methanol to prepare a 1 mg / mL full-spectrum CBD oil analysis sample solution. The paper spray mass spectrometry detection conditions were the same as in Example 1, and the following ions were monitored: m / z 315、 m / z 317、 m / z 311、 m / z 377 / 379、 Figure 12 379 / 381, and the theoretical value and the measured value were compared in combination with the CBD standard curve, and the mass spectrometry detection results are shown in m / z 、 13 .

[0056] To evaluate and compare the accuracy of the results obtained in this invention, the cannabinoid composition of these two full-spectrum CBD oils was analyzed using classic high-performance liquid chromatography-diode array detector (HPLC-DAD). Chromatographic separation was performed on an Agilent Zorbax SB-C18 column (4.6 × 250 mm, 5 µm) at a monitoring wavelength of 220 nm. The mobile phase consisted of 0.1% formic acid aqueous solution (phase A) and 0.1% formic acid acetonitrile solution (phase B), with a flow rate set at 1.0 mL / min. The gradient elution program was as follows: 0–5 min, 60% B; 5–30 min, 60%–80% B; 30–35 min, 80% B; 35–36 min, 80%–90% B; 36–42 min, 90% B; immediately switched to 60% B at 42.1 min, and maintained at 60% B from 42.1–52 min to restore the initial conditions. The total run time was 52 min, and the injection volume was 10 µL. During sample preparation, 150 µL of full-spectrum CB oil stock solution was taken and diluted to 1.0 mL with methanol to obtain a sample solution with a concentration of 15 mg / mL. The resulting solution was mixed and then directly injected for chromatographic analysis.

[0057] 2. Test Results CBD and Δ9-THC determination: CBD was detected in both full-spectrum oil samples. Figure 12 Peaks 315 and 377 / 379 indicate the presence of a CBD. According to... m / z Figure 3 315 Measured Peak Height Response Intensity and Standard Curve Figure 3 The linear equation (a) yielded a CBD concentration of 29.6 µg / mL. Based on the calculated CBD concentration and the standard curve... m / z The linear equation (b) is calculated m / z The theoretical peak height response intensity value of 377 is 2.31 × 10⁻⁶. 6 The peak response intensity is 0.67 × 10⁻⁶ higher than the actual peak height response intensity. 6 (Relative error 70%) indicates Figure 13 315 is not entirely contributed by CBD; it is speculated that oil sample 1 contains cannabinoids with a molecular weight of 314 Da, such as Δ9-THC, CBC, CBL, or Δ8-THC. m / z Zhongyou Figure 3 315 Measured Peak Height Response Intensity and Standard Curve Figure 3 The linear equation (b) yielded a CBD concentration of 40.8 µg / mL. Based on the calculated CBD concentration and the standard curve... m / z The linear equation (b) is calculated m / z The theoretical peak height response intensity value of 377 is 2.43 × 10⁻⁶. 6The peak response intensity is 1.93 × 10⁻⁶ higher than the actual peak height response intensity. 6 (Relative error 21%) indicates m / z 315 is not entirely contributed by CBD; it is speculated that oil sample 2 contains cannabinoids with a molecular weight of 314 Da, such as Δ9-THC, CBC, CBL, or Δ8-THC.

[0058] CBG Confirmation: Both full-spectrum CBD oil samples tested positive for CBG. m / z Peaks 317 and 379 / 381 confirm the existence of CBG.

[0059] CBN investigation: No detection detected Figure 14 Peak 311 (detection criterion: S / N≥3) excludes the possibility of CBN.

[0060] Verification: The results of liquid chromatography detection show that ( m / z In addition to CBD, the two oil samples also contained Δ9-THC, CBC and CBG (the chromatographic peaks of CBG and CBD partially overlapped), which is consistent with the paper spray mass spectrometry results and verifies the accuracy of the results obtained by this invention.

[0061] 3. Conclusion This embodiment demonstrates that, in complex matrices, the method of the present invention can utilize [M+H]. + With [M+ 63 Cu] + / [M+ 65 Cu] + The signal is used to directly identify cannabinoids containing a 1,5-diene structure, such as CBD and CBG; while for cannabinoids without a 1,5-diene structure, such as CBN, the identification mainly relies on [M+H]. + Ions were confirmed. Furthermore, by comparing the characteristic peaks of CBD ( ​ 377) The high relative error between theoretical and measured values ​​can further determine whether other isomers of cannabinoids such as Δ9-THC, Δ8-THC, CBC, and CBL may also be present in the sample when CBD is detected. Compared with unmodified paper-based spray mass spectrometry, cuprous modified paper significantly reduces background interference and improves the signal-to-noise ratio, thereby enhancing the clarity and reliability of complex sample detection.

[0062] In summary, this invention provides a rapid method for identifying cannabinoids. Cuprous chloride and other cuprous salts are dissolved in acetonitrile to modify a cellulose paper base, which is then dried to obtain a cuprous ion-modified paper base. During detection, the sample is added to the modified paper base and analyzed using paper spray mass spectrometry. Cannabinoids containing 1,5-diene structures, such as CBD and CBG, not only produce conventional protonated molecular ion peaks ([M+H)... + It also forms M and Cu + Addition ion peak ([M+)63 Cu] + / [M+ 65 Cu] + ), constituting a double determination index, thereby significantly enhancing the qualitative reliability. For cannabinoids such as Δ9-THC, Δ8-THC, CBN, CBC, CBL, etc. that do not contain a 1,5-diene structure, only the protonated ion peak is observed, without the generation of Cu + adduct ion signal, thereby realizing the differentiation of cannabinoids containing a 1,5-diene structure and cannabinoids isomers not containing a 1,5-diene structure. At the same time, the subcopper modified paper base can effectively inhibit the interference of background ions and improve the signal-to-noise ratio. Compared with the prior art, the present method does not need to rely on high-resolution mass spectrometry or secondary mass spectrometry, nor does it need to use noble metal ions and metal organic complexes, and can realize rapid, accurate and low-cost identification within tens of seconds under the condition of low-resolution mass spectrometry primary mass spectrometry, and has good prospects in scientific research and supervision and detection, etc. non-clinical diagnostic scenarios.

Claims

1. A method for rapid identification of cannabinoid compounds based on paper spray mass spectrometry, characterized by, The method comprises the following steps: The sample solution was dropped onto a cellulose paper substrate modified with cuprous ions, and paper spray mass spectrometry was performed. The protonated molecular ion peak [M+H] was simultaneously monitored in the mass spectrum. + With Cu + Addition ion peak [M+] 63 Cu] + / [M+ 65 Cu] + If [M+H] are detected simultaneously + peak and [M+ 63 Cu] + / [M+ 65 Cu] + If a peak is detected, the sample is determined to contain cannabinoid compounds with a 1,5-diene structure. If only the [M+H] + peak is detected, it is determined that the sample under test contains a cannabinoid compound that does not have a 1,5-diene structure.

2. The rapid identification method according to claim 1, characterized in that, The solvent of the sample solution to be detected is methanol, and the sample solution to be detected contains at least one solute of cannabidiol, cannabigerol, cannabinol, cannabichromene, cannabicitran, delta-9-tetrahydrocannabinol and delta-8-tetrahydrocannabinol, and the concentration of the solute in the sample solution to be detected is 10-100 µg / mL.

3. The rapid identification method according to claim 1, characterized in that, The cuprous ion modified cellulose paper base is prepared by the following method: cellulose chromatographic paper is soaked in a cuprous chloride acetonitrile solution with a concentration of 5-15 mmol / L, the paper base is taken out after ultrasonic treatment, and is completely dried to obtain a cellulose paper base with a surface loaded with cuprous ions.

4. The rapid identification method according to claim 3, characterized in that, The cellulose chromatographic paper has an isosceles triangle shape with a bottom side length of 7±0.5 mm and a height of 13±0.5 mm.

5. The rapid identification method of claim 1, wherein, The cannabinoid compound with a 1,5-diene structure includes cannabidiol or cannabigerol, and the cannabinoid compound without a 1,5-diene structure includes cannabinol, cannabichromene, cannabicitran, delta-9-tetrahydrocannabinol or delta-8-tetrahydrocannabinol.

6. The rapid identification method of claim 1, wherein, The rapid identification method does not add other metal ions, reagents and complexes.

7. The rapid identification method according to any one of claims 1 to 6, characterized in that, The paper spray mass spectrum adopts a primary mass spectrum, and the detection conditions include: using a positive ion mode, a scanning range of 100~500, and monitoring ions including: m / z 100~500, monitoring ions including: m / z 315、 m / z 311、 m / z 377 / 379、 m / z 317 and m / z 379 / 381, each m / z deviation is within ±0.4; the determination condition of the detected ion peak is that the signal-to-noise ratio S / N≥3.

8. The rapid identification method according to claim 7, characterized in that, The detection conditions of the paper spray mass spectrometry further include: an atomization gas flow of 0.4-0.6 L / min, a heating block temperature of 200-400 ℃, a DL tube temperature of 200-300 ℃, and an interface voltage of 3.5-4.5 kV.

9. The rapid identification method according to claim 7, characterized in that, If both are detected m / z 315 [M+H] + Fenghe m / z 377 / 379 of [M+] 63 Cu] + / [M+ 65 Cu] + If a peak is detected, the sample is determined to contain cannabidiol; if a peak is detected simultaneously... m / z 317's [M+H] + Fenghe m / z 379 / 381 of [M+ 63 Cu] + / [M+ 65 Cu] + If a peak is detected, the sample is determined to contain cannabinol; if only one peak is detected... m / z 311 of [M+H] + If a peak is detected, the sample is determined to contain cannabinol; if only one peak is detected... m / z 315 [M+H] + If a peak is detected, the sample is determined to contain at least one of cannabinoids, cannabicycloenols, Δ9-tetrahydrocannabinol, and Δ8-tetrahydrocannabinol.

10. The rapid identification method according to claim 9, characterized in that, If it is determined that the sample to be detected contains cannabidiol, it is further identified whether the sample containing cannabidiol also contains at least one of cannabichromene, cannabicitran, delta-9-tetrahydrocannabinol and delta-8-tetrahydrocannabinol, and the specific operation comprises the following steps: The measured peak height response intensity value of the sample to be measured m / z 315The measured peak height response intensity value of the sample to be measured m / z 377The measured peak height response intensity value of the sample to be measured m / z 377The measured peak height response intensity value of the sample to be measured If m / z 377 the relative error between the theoretical peak height response intensity value and the measured peak height response intensity value is within 20%, it is determined that the sample to be tested contains only cannabidiol; if m / z 377 the theoretical peak height response intensity value is more than 20% higher than the measured peak height response value, it is determined that the sample to be tested also contains cannabichromene, cannabicyclol, Δ9-tetrahydrocannabinol or Δ8-tetrahydrocannabinol; m / z m / z 377 The case that the theoretical peak height response intensity value is more than 20% lower than the measured peak height response value is less common in the detection of common cannabinoids, and therefore is not considered. the linear equation (a): y = 1.93 x 10 5 x + 4.30 x 10 4 , R 2 = 0.9992, the linear equation (b): y = 1.10 x 10 4 x + 1.98 x 10 6 , R 2 = 0.9937, where y is the peak height response intensity value, x is the concentration.

Images