A method for the quantitative analysis of lysergic diethylamide and analogs thereof

Abstract

The present application belongs to the technical field of drug detection, and particularly relates to a quantitative analysis method of LSD and analogues thereof. The quantitative analysis method comprises the following steps: (S1) respectively weighing LSD, 1cP-LSD, 1P-LSD and 1V-LSD standard samples, and dissolving and preparing into standard solutions with a series of concentrations by using acetonitrile; (S2) performing GC-FID detection on the standard solutions in step S1 by using a gas chromatograph equipped with a hydrogen flame ionization detector, taking the concentration as the abscissa and the peak area as the ordinate, establishing a standard curve of each substance, and taking a phenyl-polydimethylsiloxane as the chromatographic column, wherein the phenyl content is 20-40%; (S3) performing homogenization treatment on a sample to be detected, adding acetonitrile as an extraction solvent, oscillating extraction after sealing, centrifugation, filtering the supernatant through a filter membrane to obtain a sample solution, performing GC-FID detection on the sample solution by using the same parameters in step S2, and bringing the peak area into the standard curve in step S2 to quantitatively analyze the concentration of LSD and analogues thereof.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, specifically relating to a quantitative analysis method for ergot diethylamine and its analogues. Background Technology

[0002] Lysergic acid diethylamine (LSD) is a potent semi-synthetic hallucinogen, first synthesized in 1938 by Swiss chemist Albert Hoffmann. Its hallucinogenic effects were discovered in 1943, and it is classified as a Class I psychotropic drug in my country. LSD is often seized in various carriers, commonly including blotting paper and candy. When blotting paper is used as a carrier, it closely resembles the style of postage stamps, hence the common term "stamp." A single "stamp" typically contains 50–200 µg of LSD.

[0003] To evade legal sanctions, criminals modify the lysergic acid skeleton to obtain lysergic acid diethylamine analogs. These LSD analogs are widely circulated on platforms such as the dark web, posing a significant challenge to forensic identification and drug enforcement. Examples include cyclopropionyl lysergic acid diethylamine (1cP-LSD), propionyl lysergic acid diethylamine (1P-LSD), and valerate lysergic acid diethylamine (1V-LSD). Their structural formulas are shown in Table 1, and basic information is shown in Table 2.

[0004] Table 1. Chemical structures of lysergic acid diethylamine and its analogues

[0005]

[0006] Table 2 Structural information of lysergic acid diethylamine and its analogues

[0007]

[0008] 1P-LSD was first detected in 2015, followed by 1cP-LSD and 1V-LSD. On June 16, 2024, the Ministry of Public Security, the National Health Commission, and the National Medical Products Administration jointly issued the "Announcement on Adding 46 Substances, Including Bromophene, to the Supplementary List of Controlled Varieties of Non-Pharmaceutical Narcotic Drugs and Psychotropic Substances," listing 1cP-LSD, 1P-LSD, and 1V-LSD under control. The announcement took effect on July 1, 2024. Currently, there is no standard method for simultaneously detecting LSD, 1cP-LSD, 1P-LSD, and 1V-LSD. Therefore, researching rapid, sensitive, and accurate qualitative and quantitative analytical methods for LSD and its analogues is of great significance to law enforcement practice.

[0009] CN116391128A discloses a quantitative method for lysergic acid diethylamide (LSD) and 2,3-dihydro-3-hydroxy-2-oxoergot diethylamine (OH-LSD) in human plasma. This patent uses LC-MS / MS analysis to measure, identify, and quantify LSD and its main metabolite OH-LSD in samples. The patent employs liquid chromatography-tandem mass spectrometry (LC-MS / MS), and the detection targets are limited to LSD and its metabolites, excluding 1cP-LSD, 1P-LSD, 1V-LSD, and similar substances.

[0010] CN121805579A discloses a time-resolved fluorescence immunochromatographic assay kit for the simultaneous detection of mescaline and ergot diethylamine. The kit detects mescaline and ergot diethylamine based on the principle of competitive immunochromatography and does not involve LSD analogues.

[0011] LSD analogues contain multiple structural isomers, making complete separation and accurate quantification difficult. For example, LSD, MiPLA, and LAMPA are isomers of each other; 1P-LSD and 1P-MiPLA are isomers of each other; and 1cP-LSD and 1cP-MiPLA are isomers of each other. These isomers have the same molecular weight and highly similar mass spectrometry fragmentation patterns, making them difficult to distinguish using only mass spectrum library searches. In existing methods, when using conventional GC-MS programs (such as a heating rate of 10 °C / min), some isomer peaks overlap or the resolution is insufficient, making accurate identification impossible (Tusiewicz K et al., Molecules, 2024, 29(23): 5717-5734). This problem is particularly prominent when the sample contains multiple isomers simultaneously.

[0012] Gas chromatography (GC) is one of the most commonly used methods for the quantitative analysis of narcotics. It offers advantages such as stable response, wide linear range, good reproducibility, and relatively low cost, making it particularly suitable for the accurate quantitative analysis of target components in seized substances. Currently, GC-FID is widely used for the determination of the content of various drugs and their metabolites, and serves as an important means of routine quality control in laboratories. GC also has potential applications in the analysis of LSD and its analogues, providing reliable quantitative data support in forensic identification and law enforcement practice. LSD, 1cP-LSD, 1P-LSD, and 1V-LSD have highly similar molecular structures and physicochemical properties, exhibiting extremely similar retention behaviors on conventional gas chromatography columns. In particular, 1cP-LSD and 1V-LSD, with their similar molecular weights and chemical structures, are difficult to separate on conventional columns. Insufficient separation directly affects the accuracy and reliability of the quantitative results.

[0013] Currently, there is a lack of a method that can simultaneously solve the above problems: avoiding false positive results caused by alcoholysis, achieving complete separation of LSD from its various structural isomers (especially 1cP-LSD and 1V-LSD), and possessing good quantitative accuracy, sensitivity, and reproducibility. Therefore, developing an accurate and reliable quantitative detection method for LSD and its analogues is of significant practical importance for drug enforcement and forensic identification. Summary of the Invention

[0014] To address the lack of an effective quantitative detection method for LSD and its analogues 1cP-LSD, 1P-LSD, and 1V-LSD in existing technologies, this invention proposes a quantitative analysis method for ergot diethylamine and its analogues. By optimizing parameters such as solvent, temperature, and chromatographic column, this invention achieves rapid separation and quantitative analysis of these four substances (1cP-LSD, 1P-LSD, and 1V-LSD). The method exhibits good linearity (r > 0.998) in the range of 0.01–0.5 mg / mL, with a limit of quantitation as low as 0.01 mg / mL; the spiked recoveries are 101.7%–109.9% (RSD ≤ 1.27%); and the RSDs for repeatability (n=6) and reproducibility (n=6) are both less than 2%. This method demonstrates excellent accuracy, precision, and stability. This invention provides the following technical solutions to address the aforementioned technical problems:

[0015] A quantitative analytical method for ergot diethylamine and its analogues includes the following steps:

[0016] (S1) Weigh out LSD, 1cP-LSD, 1P-LSD and 1V-LSD standards respectively, and dissolve them in acetonitrile to prepare standard solutions of a series of concentrations;

[0017] (S2) The standard solution from step S1 was analyzed by GC-FID using a gas chromatograph equipped with a flame ionization detector. A standard curve was established for each substance with concentration on the x-axis and peak area on the y-axis. The chromatographic column was phenyl-polydimethylsiloxane, in which phenyl content was 10-40%.

[0018] (S3) The sample to be tested is homogenized, acetonitrile is added as the extraction solvent, sealed and shaken for extraction, centrifuged, the supernatant is filtered through a membrane to obtain the sample solution, and GC-FID detection is performed using the same parameters as in step S2. The peak area is then entered into the standard curve of step S2 to quantitatively analyze the concentration of ergot diethylamine and its analogues.

[0019] Further, in step S1, the series concentrations are 0.0005 mg / mL, 0.001 mg / mL, 0.0025 mg / mL, 0.005 mg / mL, 0.01 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.25 mg / mL, and 0.5 mg / mL, respectively.

[0020] Preferably, in step S2, the phenyl group accounts for 30-35% of the phenyl-polydimethylsiloxane chromatographic column.

[0021] This invention utilizes acetonitrile as the extraction solvent, which is its first inventive point. Many existing technologies use methanol as the extraction solvent. However, when using methanol as the extraction solvent, the inventors found that the responses of 1cP-LSD, 1P-LSD, and 1V-LSD were relatively small during GC analysis, while the response of LSD was significantly larger. This is because under high-temperature conditions, 1cP-LSD, 1P-LSD, and 1V-LSD undergo deacylation reactions, losing cyclopropionyl, propionyl, and valerate groups, thus converting to LSD, affecting the accuracy of qualitative and quantitative analysis. Methanol, as a protic solvent, can provide H₂O. + This promotes the occurrence of the above reactions. The present invention uses acetonitrile as the extraction solvent. Firstly, it has good solubility for LSD and its analogues; secondly, acetonitrile is an aprotic polar solvent and cannot provide acidic protons. When using acetonitrile as the extraction solvent, the above reactions are essentially not observed within a short analysis time; thirdly, acetonitrile has low viscosity, making it easier to vaporize rapidly at the injection port, reducing the interference of solvent residue on separation. Low viscosity also helps to form a narrower initial sample bandwidth, thereby obtaining sharper, more symmetrical chromatographic peaks; finally, acetonitrile provides a clean background response to the FID detector, without generating impurity peaks that interfere with the quantification of the target analyte; simultaneously, its low UV absorption characteristics allow the same sample solution to be used simultaneously for LC-UV or LC-MS analysis, exhibiting good method compatibility.

[0022] For GC quantification, the resolution of the target analytes needs to be greater than 1.5 to achieve accurate analysis and quantification. This invention selects a moderately polar phenyl-polydimethylsiloxane column, i.e., phenyl content 20-35%. 1cP-LSD and 1V-LSD can separate the four target analytes, and the peak shapes are good, meeting the quantification requirements.

[0023] Further, in step S2, the parameters for GC-FID detection are as follows: the temperature program is an initial temperature of 200-220℃, held for 2-5 min, then the temperature is increased to 300-320℃ at a rate of 10-20℃ / min, and held for 20-30 min; the injection port temperature is 250-300℃, the detector temperature is 330-360℃, the split ratio is 15-25:1, the injection volume is 5-10 μL, and the column flow rate is 1-5 mL / min.

[0024] LSD and its analogues, including four substances, exhibit long retention times on the chromatographic column, which increases both analysis time and the risk of instability. This invention sets the initial temperature of the temperature program to 200-220℃. Compared to 180℃, an initial temperature of 200℃ has no significant impact on peak shape and separation, and therefore does not affect quantitative analysis. Under these conditions, the four target analytes can be completely separated within 30 minutes.

[0025] In a preferred embodiment of the present invention, the parameters for GC-FID detection are as follows: initial temperature 200 ℃, held for 2 min, temperature increased to 320 ℃ at 20 ℃ / min, held for 24 min, injection port temperature 280 ℃, detector temperature 350 ℃, split ratio 10:1, injection volume 1 µL, and column flow rate 1.0 mL / min.

[0026] Further, in step S3, the ratio of the sample to be tested to acetonitrile is 10-100 mg: 10 mL, the sample is sealed and shaken for 10-30 min, and then centrifuged at 4000-8000 rpm for 5-10 min.

[0027] Further, in step S3, the extraction solvent is a mixture of acetonitrile and toluene, and 3-5 mg / mL of triethylamine is added; preferably, the volume ratio of acetonitrile to toluene is 3-5:1. The inventors unexpectedly discovered that using the above-mentioned solvent for extraction can improve the tailing problem caused by the inability to separate 1cP-LSD and 1V-LSD. When the concentrations of 1cP-LSD and 1V-LSD are close, this tailing problem does not lead to serious detection errors. However, when the concentrations of 1V-LSD and 1cP-LSD differ significantly, such as a 1:10 ratio, the tailing phenomenon becomes severe, affecting the detection results. Using the above-mentioned mixture of acetonitrile and toluene, with the addition of a small amount of triethylamine, can avoid this phenomenon.

[0028] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0029] I. The quantitative analysis method of this invention can rapidly and accurately perform quantitative analysis on four substances: LSD and its analogues 1cP-LSD, 1P-LSD, and 1V-LSD, filling the gap in the simultaneous quantitative detection of these four substances. The method of this invention can achieve complete separation of the four target analytes within 30 minutes, with a short analysis time, making it suitable for rapid detection of large batches of samples. The method of this invention has good repeatability and reproducibility, and can reliably and stably complete the testing of samples.

[0030] Second, this invention uses acetonitrile as the extraction solvent, which, under high-temperature gas chromatography analysis conditions, essentially prevents 1cP-LSD, 1P-LSD, and 1V-LSD from undergoing deacylation reactions to convert to LSD, thus ensuring the accuracy of quantitative analysis. This invention uses a moderately polar capillary column, enabling complete separation of 1cP-LSD and 1V-LSD, with good peak shapes for the four target analytes, meeting the requirements for accurate quantification. Attached Figure Description

[0031] Figure 1 These are gas chromatograms of four substances: LSD, 1cP-LSD, 1P-LSD, and 1V-LSD. Detailed Implementation

[0032] LSD (purity ≥99.8%), 1cP-LSD (purity ≥99.7%), 1P-LSD (purity ≥99.5%), and 1V-LSD (purity ≥99.3%) are chemical reference standards jointly developed by the Shanghai Institute of Criminal Science and Technology and Shanghai Yuansi Standard Technology Co., Ltd.

[0033] The Agilent GC8890 gas chromatograph is equipped with a high-purity hydrogen generator and a 7693A autosampler; the Shimadzu LC-40D X3 liquid chromatograph is equipped with an SPD-M40 diode array detector, a SIL-40C x3 autosampler, and a CTO-40S column oven.

[0034] Example 1

[0035] Weigh each standard powder and dilute to 10 mL volumetric flasks with acetonitrile to obtain four single standard stock solutions with a concentration of 1 mg / mL for each target analyte. Take appropriate amounts of each target analyte stock solution and dilute with acetonitrile to prepare a series of standard solutions with concentrations of 0.0005, 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, and 0.5 mg / mL for later use.

[0036] GC test conditions: SH-35 MS (30 m × 0.25 mm × 0.25 µm, 35% phenyl-65% polydimethylsiloxane) capillary column; temperature program: initial temperature 200 ℃, hold for 2 min, increase to 320 ℃ at 20 ℃ / min, hold for 24 min; injector temperature 280 ℃; detector temperature 350 ℃; split ratio 10:1; injection volume 1 µL; column flow rate 1.0 mL / min; external standard method for quantification.

[0037] Under the above GC test conditions, the gas chromatograms of the four target analytes are as follows: Figure 1 As shown. Among them Figure 1In the diagram, a, b, c, and d correspond to LSD, 1cP-LSD, 1P-LSD, and 1V-LSD, respectively. It can be seen that the heating rate is 20 ℃ / min. Under these conditions, the four target substances can be completely separated within 30 min.

[0038] According to GB / T 35655-2017, the standard solutions of the four target analytes were diluted to obtain a series of samples with low mass concentrations for determination. Each concentration point was measured repeatedly six times, and the lowest detection result with a relative standard deviation (RSD) of less than 2% was taken as the limit of quantitation. A series of mixed standard solutions with different concentrations were prepared for determination. A curve was plotted with the concentration (mg / mL) of each component as the abscissa and the peak area as the ordinate, and the curve was fitted linearly. The limit of quantitation and linear range results of the GC method in Example 1 are shown in Table 3.

[0039] Table 3 Retention time, linear range, linear equation, correlation coefficient, and limit of quantitation for GC method

[0040]

[0041] After homogenization, 100 mg of the sample was weighed into a capped centrifuge tube, 10 mL of acetonitrile was added, the tube was sealed and shaken for 10 min, centrifuged at 5000 r / min for 5 min, and the supernatant was filtered through a membrane to obtain the sample solution for instrument analysis.

[0042] Using Quadrex 007-20 (20 m × 0.18 mm × 0.2 µm, 20% phenyl-80% polydimethylsiloxane), the results were similar to those using SH-35 MS, with LOQs also being 0.01 mg / mL.

[0043] Example 2

[0044] Four substances were added to blotting paper without the target analytes at concentrations of 0.01 mg / mL, 0.05 mg / mL, and 0.25 mg / mL, respectively. Each assay was performed in triplicate. GC conditions and sample pretreatment were the same as in Example 1. The recoveries were calculated. The results are shown in Table 2. The average recoveries of each target analyte in the GC method ranged from 101.7% to 109.9%, with relative standard deviations (RSD, n=3) ranging from 0.06% to 1.27%, meeting the recovery requirements.

[0045] Table 4. Recovery rates and relative standard deviations of various substances by GC method (n=3)

[0046]

[0047] Example 3

[0048] Four target substances were added to blotting paper that did not contain the target substances, so that the concentration of the four target substances was 0.05 mg / mL. Six samples were prepared independently and repeatedly. The GC conditions were the same as in Example 1. The samples were continuously tested on the GC machine, and each sample was tested once. The relative standard deviation (RSD, n=6) was calculated. The results are shown in Table 3. The RSD of the GC method was less than 2%, which met the repeatability requirements of the method.

[0049] Table 5 GC Test

[0050]

[0051] As can be seen, the GC method established in this invention is an accurate, rapid, sensitive, and well-separated method for the quantitative analysis of four substances: LSD, 1cP-LSD, 1V-LSD, and 1P-LSD. It is suitable for the qualitative and quantitative analysis of LSD, 1cP-LSD, 1V-LSD, and 1P-LSD in seized items, meeting the requirements of relevant identification work and providing a reference for the identification of LSD and other ergotamine substances in related cases. However, the separation degree between 1cP-LSD and 1V-LSD is still relatively low, resulting in a higher recovery rate for 1cP-LSD. It is possible that because 1V-LSD has a higher retention time (RT) in the GC chromatogram, and the separation between 1V-LSD and 1cP-LSD is not high, the leading edge of the later peak (1cP-LSD) will inevitably extend below the tail of 1V-LSD (tailing phenomenon). When the integration algorithm of the chromatography workstation performs vertical cutting, it will incorporate part of the later peak into the earlier peak, resulting in a higher peak area of ​​the leading edge (1V-LSD).

[0052] Example 4

[0053] To verify the above hypothesis, the test was repeated with LSD, 1P-LSD, 1V-LSD, and 1cP-LSD spiked at concentrations of 0.05 mg / mL, 0.05 mg / mL, 0.05 mg / mL, and 0.5 mg / mL, respectively, indicating a decrease in the concentration of 1V-LSD. The results are shown in Table 6 below.

[0054] Table 6. Results of simultaneous GC detection of four substances

[0055]

[0056] It is evident that when the concentration ratio of 1V-LSD to 1cP-LSD is 1:10, the tailing phenomenon becomes severe.

[0057] Example 5

[0058] The other conditions were the same as in Example 4, except that acetonitrile was replaced with a 3:1 volume mixture of acetonitrile and toluene, and the mixture contained 3 mg / mL of triethylamine. The spiking concentrations of LSD, 1P-LSD, 1V-LSD, and 1cP-LSD were 0.05 mg / mL, 0.05 mg / mL, 0.05 mg / mL, and 0.5 mg / mL, respectively. The results are shown in Table 7 below.

[0059] Table 7. Results of simultaneous GC detection of four substances

[0060]

[0061] As can be seen, in Example 5, the recovery rates of each substance returned to normal levels, indicating that the mixed solvent of acetonitrile and toluene, as well as the addition of a small amount of triethylamine, can significantly improve the tailing phenomenon of 1cP-LSD and 1V-LSD.

[0062] Example 6

[0063] Other conditions are the same as in Example 1. The difference is that the initial temperature in the GC test is changed to 180°C. Peak shape and separation effect are not significantly affected and will not affect quantitative analysis, but the time for complete separation of the four target substances is longer.

[0064] Comparative Example 1

[0065] Other conditions were the same as in Example 1, except that acetonitrile was replaced with methanol. When methanol was used as the extraction solvent, the responses of 1cP-LSD, 1P-LSD, and 1V-LSD were relatively small during GC analysis, while the response of LSD was significantly larger. This is because under high-temperature conditions, 1cP-LSD, 1P-LSD, and 1V-LSD undergo deacylation reactions, losing cyclopropionyl, propionyl, and valerate groups, thus converting to LSD, affecting the accuracy of qualitative and quantitative analysis. Methanol, as a protic solvent, can provide H₂O. + This promotes the occurrence of the above reactions.

[0066] Comparative Example 2

[0067] Other conditions were the same as in Example 1, except that the SH-35 MS (35% phenyl-65% polydimethylsiloxane) was replaced with an HP-5 column (5% phenyl-95% polydimethylsiloxane). 1cP-LSD and 1V-LSD were completely inseparable.

[0068] Comparative Example 3

[0069] Other conditions were the same as in Example 1, except that the SH-35 MS (35% phenyl-65% polydimethylsiloxane) was replaced with an HP-50+ column (50% phenyl-50% polydimethylsiloxane). Diffusion of the analyte (LSD analogue) within the stationary phase membrane became extremely difficult. This is likely due to the increased phenyl content, which significantly enhanced the π-π packing forces, resulting in the target analyte being eluted only at higher temperatures.

Claims

1. A quantitative analysis method for ergot diethylamine and its analogues, characterized in that, Includes the following steps: (S1) Weigh out LSD, 1cP-LSD, 1P-LSD and 1V-LSD standards respectively, and dissolve them in acetonitrile to prepare standard solutions of a series of concentrations; (S2) The standard solution from step S1 was analyzed by GC-FID using a gas chromatograph equipped with a flame ionization detector. A standard curve was established for each substance with concentration on the x-axis and peak area on the y-axis. The chromatographic column was phenyl-polydimethylsiloxane, in which phenyl content was 10-40%. (S3) The sample to be tested is homogenized, the extraction solvent is added, the sample is sealed and extracted by shaking, centrifugation is performed, the supernatant is filtered through a membrane to obtain the sample solution, and the sample solution is detected by GC-FID using the same parameters as in step S2. The peak area is then entered into the standard curve of step S2 to quantitatively analyze the concentration of ergot diethylamine and its analogues. The extraction solvent is acetonitrile.

2. The quantitative analysis method according to claim 1, characterized in that, In step S1, the series concentrations are 0.0005 mg / mL, 0.001 mg / mL, 0.0025 mg / mL, 0.005 mg / mL, 0.01 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.25 mg / mL, and 0.5 mg / mL, respectively.

3. The quantitative analysis method according to claim 1, characterized in that, In step S2, the phenyl group in the phenyl-polydimethylsiloxane chromatographic column accounts for 30-35%.

4. The quantitative analysis method according to claim 1, characterized in that, In step S2, the parameters for GC-FID detection are as follows: the initial temperature is 200-220℃, held for 2-5 min, and then the temperature is increased to 300-320℃ at a rate of 10-20℃ / min, held for 20-30 min; the injection port temperature is 250-300℃, the detector temperature is 330-360℃, the split ratio is 15-25:1, the injection volume is 5-10 μL, and the column flow rate is 1-5 mL / min.

5. The quantitative analysis method according to claim 1, characterized in that, The parameters for GC-FID detection are as follows: initial temperature 200℃, hold for 2 min, increase to 320℃ at 20℃ / min, hold for 24 min, injection port temperature 280℃, detector temperature 350℃, split ratio 10:1, injection volume 1µL, and column flow rate 1.0mL / min.

6. The quantitative analysis method according to claim 1, characterized in that, In step S3, the ratio of the sample to be tested to acetonitrile is 10-100 mg: 10 mL. The sample is sealed and shaken for 10-30 min, then centrifuged at 4000-8000 rpm for 5-10 min.

7. The quantitative analysis method according to claim 1, characterized in that, In step S3, the extraction solvent is a mixture of acetonitrile and toluene, and 3-5 mg / mL of triethylamine is added.

8. The quantitative analysis method according to claim 7, characterized in that, The volume ratio of acetonitrile to toluene is 3-5:1.

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