A preliminary screening method for identifying heroin association

Abstract

The present application belongs to the technical field of dangerous goods detection and analysis, and particularly relates to a preliminary screening method for discriminating heroin correlation. The present application adopts ultra-high performance liquid chromatography to gradient elute 8 alkaloids in heroin, combines ultraviolet absorption spectrum and retention time to confirm 8 alkaloid peaks, and calculates the concentration of each alkaloid through the peak area external standard method of each alkaloid. The concentration values of the 8 alkaloids are combined into specific ratio combinations, 14 specific alkaloid concentration ratio groups are combined, and z-score standardization is performed on these ratio combinations to eliminate the dimensional differences between the concentrations of each alkaloid and the influence of heroin hydrolysis on the concentration content of each alkaloid in heroin. Finally, the Pearson coefficient method is used to calculate the correlation between the real seized samples of heroin. The discrimination result of the present application is accurate, the cost is low, the operation is simple, and the present application can be used as a preliminary screening method for discriminating heroin correlation.

Description

Technical Field

[0001] This invention belongs to the field of hazardous materials detection and analysis technology, specifically relating to a preliminary screening method for determining the association with heroin. Background Technology

[0002] Heroin, as one of the most common and widely consumed drugs, poses a significant threat to society. Identifying the correlation between heroin samples is beneficial for analyzing heroin trafficking routes and improving the ability of law enforcement agencies to link cases. Analyzing specific impurities, byproducts, adulterants, and diluents in illicit heroin samples can provide strategic and tactical intelligence. Strategic intelligence provides information about the processing and geographical origin of drugs, while tactical intelligence identifies the correlation between two or more cases.

[0003] Morphine is extracted from the poppy plant and is the most abundant alkaloid in poppies. Mixing morphine with acetic anhydride and heating it can convert it into heroin through acetylation. However, because drug manufacturing plants cannot completely extract and purify morphine under ideal conditions, some opium alkaloids remain impurities in illegally synthesized heroin, including acetylcodeine, codeine, morphine, and O. 3 - Monoacetylmorphine, O 6 - Monoacetylmorphine, papaverine, noscapine, etc. Furthermore, heroin degrades during transportation and storage due to factors such as light and humidity, naturally transforming into monoacetylmorphine. If the alkaloid content is directly standardized using the z-score method for correlation determination, the effects of sample hydrolysis and dimensions cannot be eliminated in practical applications, leading to errors in correlation evaluation.

[0004] Currently, the primary method for determining heroin correlation is liquid chromatography-mass spectrometry (LC-MS), which involves semi-quantitative analysis of various intermediate-acidic impurities in heroin samples followed by Pearson correlation analysis. However, because there are no standards for these intermediate-acidic impurities, and high heroin concentrations can severely contaminate the LC-MS system, it can negatively impact the analysis of heroin, morphine, and oxytocin. 6 Qualitative and quantitative analysis of components such as monoacetylmorphine can cause interference. When using this method, special equipment is often required, which has high requirements for equipment and high cost, making it unfavorable for the implementation of heroin correlation identification.

[0005] Therefore, how to accurately determine the association of heroin using a simple and easy-to-implement method is a technical problem that needs to be solved. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a preliminary screening method for determining heroin correlation. Based on ultra-high performance liquid chromatography (UHPLC), this invention employs gradient elution of eight alkaloids in heroin to obtain chromatograms. After confirming the peaks of the eight alkaloids, the concentration of each alkaloid is calculated using the external standard method based on its peak area. Specific ratio combinations of the eight alkaloid concentrations are then performed to form 14 alkaloid combinations. The ratio results of these 14 alkaloid combinations are then standardized using z-fraction to eliminate dimensional differences between alkaloid concentrations and the influence of heroin hydrolysis on the concentration of each alkaloid in heroin. Finally, the Pearson coefficient method is used to calculate the correlation between heroin samples. This invention provides accurate results, is low-cost, and simple to operate, making it a viable preliminary screening method for determining heroin correlation.

[0007] The present invention is specifically implemented through the following technical solutions.

[0008] This invention provides a preliminary screening method for determining heroin-related associations, comprising the following steps:

[0009] Multiple sample solutions were prepared using acetonitrile as solvent; each sample solution was analyzed by ultra-high performance liquid chromatography (UHPLC) to obtain chromatograms.

[0010] Eight alkaloids were used as standards, and standard solutions of different concentrations were prepared. These solutions were then analyzed by ultra-high performance liquid chromatography (UHPLC). Under the same UHPLC conditions, chromatograms of the standards at different concentrations were obtained. A standard curve regression equation was obtained by plotting the peak area of ​​the standard chromatograms on the ordinate and the concentration on the abscissa. The eight alkaloids were morphine, codeine, and O... 3 - Monoacetylmorphine, O 6 - Monoacetylmorphine, acetylcodeine, heroin, papaverine, and noscapine; among them, acetylcodeine is abbreviated as ACC, codeine as COD, morphine as MOR, and O 3 Monoacetylmorphine (abbreviated as O) 3 O 6 Monoacetylmorphine (abbreviated as O) 6 Papaverine is abbreviated as PAP, noscapine as NOS, and heroin as HER.

[0011] The peaks of eight alkaloids in the chromatograms of multiple sample solutions to be analyzed were confirmed. The confirmed peak areas were substituted into the standard curve regression equation of the corresponding alkaloids to calculate the concentrations of the eight alkaloids in the multiple sample solutions to be analyzed.

[0012] Eight alkaloids from multiple samples were combined to obtain 14 alkaloid combinations. The ratios of these 14 combinations were calculated based on the concentrations of the eight alkaloids. The 14 alkaloid combinations were: ACC / HER, ACC / (O... 3 +O 6 ), O 3 / O 6 ACC / (O 3 +O 6 +HER), ACC / (MOR+O) 3 +O 6 +HER), (O 3 +O 6 ) / HER、(COD+MOR) / (O 3 +O 6 +HER), HER / (O 3 +O 6 ), (COD+ACC) / (MOR+O) 3 +O 6 +HER), (COD+MOR+O) 3 +O 6 +HER) / ACC、HER / (COD+MOR+ACC+O 3 +O 6 (MOR+O) 3 +O 6 +HER) / (COD+ACC)、(PAP+NOS) / (HER+O 3 +O 6 +MOR) and NOS / PAP.

[0013] The ratio results of 14 alkaloid combinations in multiple samples to be analyzed were standardized by z-score, and the Pearson correlation coefficient was calculated based on the z-score standardized results. The heroin association was determined based on the Pearson correlation coefficient.

[0014] Preferably, when performing ultra-high performance liquid chromatography (UHPLC), the chromatographic conditions for the sample solution and the standard solution are the same, specifically: C18 column, packing particle size 1.7 μm, column length 100 mm, inner diameter 2.1 mm; column temperature: 40℃; flow rate: 1.0 mL / min; injection volume: 1 μL; detection wavelength: 210 nm; mobile phase: phase A is acetonitrile, phase B is sodium dihydrogen phosphate buffer. The specific preparation method of sodium dihydrogen phosphate buffer is as follows: sodium dihydrogen phosphate and 85% concentrated phosphoric acid are mixed, dissolved and diluted with ultrapure water; the volume ratio of sodium dihydrogen phosphate to 85% concentrated phosphoric acid is 1.1 g: 1 mL; the volume ratio of 85% concentrated phosphoric acid to the diluted solution is 1:500; the gradient elution parameters are as follows (by volume percentage):

[0015] 0.0~9.0min, 98%~70%B; 9.0min~9.5min, 70%~20%B; 9.5min~12.0min, 20%B; 12.0min~12.5min, 20%~98%B; 12.5min~16.0min, 98%B.

[0016] Preferably, the specific preparation method of the standard solution is as follows: using acetonitrile as the solvent, heroin, acetylcodeine and O... 6 Monoacetylmorphine was dissolved in acetonitrile to prepare first mixed standard solutions of different concentrations. The heroin concentrations in the first mixed standard solutions were 0.25 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, 0.01 mg / mL, and 0.005 mg / mL, respectively. In each first mixed standard solution, the concentrations of heroin, acetylcodeine, and O... 6 - The mass ratio of monoacetylmorphine is 5:3:5.

[0017] Using methanol as a solvent, morphine, codeine, and O 3 Monoacetylmorphine, noscapine, and papaverine were dissolved in methanol to prepare second mixed standard solutions of different concentrations. The concentrations of morphine in the second mixed standard solutions were 0.05 mg / mL, 0.02 mg / mL, 0.01 mg / mL, 0.005 mg / mL, 0.002 mg / mL, and 0.001 mg / mL, respectively. In each second mixed standard solution, the concentrations of morphine, codeine, and O... 3 The mass ratio of monoacetylmorphine, noscapine, and papaverine is 1:1:1:1:1.

[0018] Preferably, the standard curve regression equation is: morphine standard curve regression equation is y=7392.7x-326.6, correlation coefficient R0 2 =0.9987; the regression equation for the codeine standard curve is y=9608.6x-2981.5, and the correlation coefficient R0 is 0.9987. 2 =0.9993; O 3 The standard curve regression equation for monoacetylmorphine is y = 6239x - 2033.7, with a correlation coefficient R0. 2 =0.9994; O 6 The standard curve regression equation for monoacetylmorphine is y = 7996.7x + 6857, R0 2 =0.9998; the standard curve regression equation for acetylcodeine is y = 8167.7x + 3821.3, R0 2 =0.9998; the standard curve regression equation for heroin is y=6111.7x+4581.5, R0 2=0.9998; the regression equation for the papaverine standard curve is y=7225.3x-1062.4, R0 2 =0.9994; the regression equation of the noscapine standard curve is y=15836.3x-5110.9, R0.9 2 =0.9995.

[0019] Preferably, when confirming the peaks of eight alkaloids in the chromatograms of multiple sample solutions to be analyzed, the specific method is as follows: The retention times and UV spectra of the target alkaloids in the chromatograms of multiple sample solutions to be analyzed are compared with the retention times and UV spectra of the chromatograms of the eight alkaloid standards. The peak of the target alkaloid in the chromatogram of the sample solution to be analyzed is considered confirmed when both of the following conditions (a) and (b) are met: (a) the relative error between the retention time of the target alkaloid and the retention time of the standard chromatogram is less than 2%; (b) the UV spectra of the target alkaloid and the standard are extracted separately in a Waters Empower ultra-high performance liquid chromatography workstation, and the characteristic UV absorption wavelengths and absorption peak shapes of the target alkaloid and the standard are consistent. This avoids interference from other substances such as acetaminophen, piracetam, and other common adulterants.

[0020] The characteristic UV absorption spectra of each alkaloid in liquid phase were as follows: morphine 209.2 nm, 285 nm; codeine 210.4 nm, 285 nm; O 3 - Monoacetylmorphine 205.6nm, 280.2nm; O 6 - Monoacetylmorphine 208nm, 283.8nm; Acetylcodeine 209.2nm, 283.8nm; Heroin 204.5nm, 279nm; Papaverine 199.8nm, 250.5nm, 308.8nm; Noscapine 212.7nm, 312.4nm.

[0021] Preferably, the specific method for preparing multiple sample solutions to be analyzed is as follows: using acetonitrile as a solvent, multiple samples to be analyzed are dissolved by sonication, centrifuged, and the supernatant is diluted with sodium dihydrogen phosphate buffer to obtain multiple sample solutions to be analyzed. The volume ratio of sample to acetonitrile is 2.9 mg to 3.1 mg: 1 mL; the supernatant is diluted with sodium dihydrogen phosphate buffer, and the volume ratio of supernatant to sodium dihydrogen phosphate buffer is 1:9.

[0022] The preferred z-score standardization formula is:

[0023] ;

[0024] in, For the sample Standardized data, This is the ratio result for each combination of alkaloids. =1, 2, 3…14, It is the mean of the ratios of the 14 alkaloid combinations. It is the standard deviation of the ratio results of the 14 alkaloid combinations.

[0025] Preferred method for calculating Pearson correlation coefficient:

[0026] ;

[0027] in, The Pearson correlation coefficient is used. For the sample Standardized data, For the sample Standardized data, The calculation method and Same, for The mean, for The mean, The number of alkaloid combinations. =14.

[0028] Preferably, the correlation determination uses 0.98 as the correlation threshold, and 0.99 > 0.98. A value greater than 0.98 indicates a strong correlation. A value greater than 0.99 indicates a very strong correlation.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This invention employs ultra-high performance liquid chromatography (UHPLC) to perform gradient elution of eight alkaloids in a heroin sample to be analyzed, obtaining a chromatogram. The eight alkaloids are morphine, codeine, and O. 3 - Monoacetylmorphine, O 6The study identified eight alkaloids in the chromatograms: monoacetylmorphine, acetylcodeine, heroin, papaverine, and noscapine. The concentrations of each alkaloid were calculated using the external standard method based on their peak areas. These eight alkaloid concentrations were then combined in specific ratios to form 14 alkaloid combinations. The ratios of these 14 combinations were then standardized using z-fractions to eliminate dimensional differences between alkaloid concentrations and the influence of heroin hydrolysis on the concentrations of each alkaloid in heroin. Finally, the Pearson coefficient method was used to calculate the correlation between heroin samples. This invention can be compared with liquid chromatography-mass spectrometry (LC-MS). Using LC-MS analysis results as a reference, the correlation analysis results of this invention showed a matching degree of 96.32%, which is superior to correlation analysis methods that directly standardize alkaloid ratios. This invention provides accurate results, is low-cost, and simple to operate, making it a suitable initial screening method for heroin correlation identification. This invention reduces instrument analysis and maintenance costs, making it particularly easy to implement for laboratories with limited budgets. Attached Figure Description

[0031] Figure 1 This is a standard curve of concentration-peak area for MOR.

[0032] Figure 2 This is a standard curve of COD concentration versus peak area.

[0033] Figure 3 For O 3 The concentration-peak area standard curve.

[0034] Figure 4 For O 6 The concentration-peak area standard curve.

[0035] Figure 5 This is the concentration-peak area standard curve for ACC.

[0036] Figure 6 This is a standard curve of HER concentration-peak area.

[0037] Figure 7 This is a standard curve of PAP concentration versus peak area.

[0038] Figure 8 This is a standard curve of NOS concentration versus peak area.

[0039] Figure 9 For gradient elution to obtain 0.05 mg / mL H MIX3 Chromatograms of a series of standard solutions.

[0040] Figure 10 M 0.05 mg / mL obtained by gradient elution MIX5 Chromatograms of a series of standard solutions.

[0041] Figure 11 A chromatogram of a heroin seizure sample. Detailed Implementation

[0042] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention. Unless otherwise specified, the experimental methods and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials described are commercially available.

[0043] This invention employs ultra-high performance liquid chromatography (UHPLC) to perform gradient elution of eight alkaloids in a heroin sample to be analyzed, obtaining a chromatogram. The eight alkaloids are morphine, codeine, and O. 3 - Monoacetylmorphine, O 6 The study identified eight alkaloids in the chromatograms: monoacetylmorphine, acetylcodeine, heroin, papaverine, and noscapine. The concentrations of each alkaloid were calculated using the external standard method based on their peak areas. These eight alkaloid concentrations were then combined in specific ratios to form 14 alkaloid combinations. The ratios of these 14 combinations were then standardized using z-fraction to eliminate dimensional differences between alkaloid concentrations and the influence of heroin hydrolysis on the concentrations of each alkaloid in heroin. Finally, the Pearson coefficient method was used to calculate the correlation between heroin samples. Compared to directly standardizing the eight alkaloids using z-fraction and then calculating the correlation between heroin samples, this invention first combines the eight alkaloid concentrations to obtain 14 alkaloid combinations, calculates the ratios of these 14 combinations, and then standardizes the ratios using z-fraction, resulting in a higher accuracy in the correlation results.

[0044] The selection of specific alkaloids and their combinations is very important, especially in illicit heroin samples, O 3 - Monoacetylmorphine and O 6 The content of monoacetylmorphine is closely related to its processing level. For example, the acetic anhydride method involves reacting morphine base with acetic anhydride in a molar ratio of approximately 1:3 at 85℃~95℃ for 6~8 hours, mainly producing intermediate O. 3 - Monoacetylmorphine is then converted to diacetylmorphine, i.e., heroin base. The acetyl chloride process first produces O... 3 - Monoacetylmorphine and O 6 A mixture of monoacetylmorphine, further acetylated to heroin, suffers from poor reaction selectivity, making yield control difficult. Furthermore, a "homemade method" using a trifluoroacetic anhydride / acetic acid system to acetylate morphine can shorten the reaction time to half an hour, and compared to the acetic anhydride method, O 3 - Monoacetylmorphine and O6 - Monoacetylmorphine production increased, while heroin production slightly decreased. Therefore, the characteristic content of these synthetic intermediates and byproducts is an important basis for inferring heroin manufacturing processes and plays a crucial role in linking cases. Similarly, the differences in the content of natural alkaloids derived from poppies, such as papaverine and noscapine, are important indicators for identifying geographical origin. Heroin in Asia mainly originates from two major drug-producing regions: Southeast Asia and Southwest Asia. Southwest Asian heroin samples are typically characterized by higher levels of noscapine and papaverine, while Southeast Asian heroin drugs show lower levels of papaverine and noscapine. Morphine, as the end product of heroin hydrolysis, has relatively stable chemical properties, but its content is easily affected by the storage environment. 3 - Monoacetylmorphine and O 6 Monoacetylmorphine, as an intermediate in heroin synthesis, reflects the degree of heroin hydrolysis and details of the manufacturing process; analyzing its changes allows for tracing the degradation process of the sample. Acetylcodeine, a byproduct of heroin synthesis, indicates the characteristics of the synthesis process through its ratio to heroin. Codeine, a natural opioid alkaloid, can compensate for compositional variations caused by sample hydrolysis through its combined ratio with morphine.

[0045] The contents of this invention will be described in detail below.

[0046] A preliminary screening method for determining heroin-related associations includes the following steps:

[0047] Step 1, Sample pretreatment method:

[0048] Weigh 15 mg of each sample to be analyzed into centrifuge tubes (the allowable range is 15 ± 0.5 mg). Add 5 mL of acetonitrile, sonicate to dissolve (100% power, 25℃, 15 min), and then centrifuge at 4000 rpm for 10 min to obtain multiple solutions. Take 0.1 mL of each solution, add 0.9 mL of sodium dihydrogen phosphate buffer, filter through a filter membrane, and obtain multiple sample solutions to be analyzed. The sodium dihydrogen phosphate buffer is prepared as follows: weigh 2.2 g of sodium dihydrogen phosphate, add 2 mL of 85% concentrated phosphoric acid, dissolve in ultrapure water and dilute to 1000 mL, sonicate, and let stand until needed.

[0049] Step 2: Inject the multiple sample solutions to be analyzed prepared in Step 1 into an ultra-high performance liquid chromatograph to obtain chromatograms of the multiple sample solutions to be analyzed.

[0050] The ultra-high performance liquid chromatography (UHPLC) conditions were as follows: ACQUITY UPLC CSH C18 column with specifications of 1.7 μm particle size, 100 mm column length, and 2.1 mm inner diameter; column temperature: 40℃; flow rate: 1.0 mL / min; injection volume: 1 μL; detection wavelength: 210 nm; gradient elution; mobile phase: phase A was acetonitrile, and phase B was sodium dihydrogen phosphate buffer. The sodium dihydrogen phosphate buffer was prepared by weighing 2.2 g of sodium dihydrogen phosphate, adding 2 mL of 85% concentrated phosphoric acid, dissolving in ultrapure water and diluting to 1000 mL, sonicating, and allowing to stand until needed. The gradient elution program, expressed as a volume percentage, is shown in Table 1.

[0051] Table 1 Gradient elution program

[0052]

[0053] Step 3: Take 5mg of heroin, 3mg of acetylcodeine, and O... 6 - Mix 5 mg of monoacetylmorphine standard and dissolve it in 2 mL of acetonitrile to obtain a mixed standard stock solution, denoted as H. MIX3 Take morphine, codeine, and O2 respectively. 3 - Mix 2.5 mg each of monoacetylmorphine, noscapine, and papaverine as standard substances, dissolve in 5 mL of methanol to obtain a mixed standard stock solution, denoted as M. MIX5 H was prepared using sodium dihydrogen phosphate buffer. MIX3 After dilution, heroin concentrations of 0.25 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, 0.01 mg / mL, and 0.005 mg / mL were obtained. MIX3 A series of mixed standard solutions. M was prepared using sodium dihydrogen phosphate buffer. MIX5 After dilution, morphine concentrations of 0.05 mg / mL, 0.02 mg / mL, 0.01 mg / mL, 0.005 mg / mL, 0.002 mg / mL, and 0.001 mg / mL were obtained, respectively. MIX5 A series of mixed standard solutions. The sodium dihydrogen phosphate buffer used here is the same as that used in step 2.

[0054] H MIX3 Series of mixed standard solutions and M MIX5 A series of mixed standard solutions were injected into an ultra-high performance liquid chromatograph (UHPLC) under the same conditions as in step 2, and the elution program was the same as in Table 1. Chromatograms of each mixed standard solution at its corresponding concentration were obtained. Figure 9 H is 0.05 mg / mL MIX3 Chromatogram of the mixed standard solution. Figure 10 M is 0.05 mg / mL MIX5Chromatogram of the mixed standard solution. Figure 11 A chromatogram of a seized heroin sample.

[0055] Based on the obtained chromatogram, a standard curve regression equation is obtained by plotting peak area on the ordinate and concentration on the abscissa. The linear correlation coefficient should be greater than 0.998. The corresponding standard curve regression equation is:

[0056] The standard curve regression equation for morphine is y = 7392.7x - 326.6, and the correlation coefficient R0 is [value missing]. 2 =0.9987; the regression equation for the codeine standard curve is y=9608.6x-2981.5, and the correlation coefficient R0 is 0.9987. 2 =0.9993; O 3 The standard curve regression equation for monoacetylmorphine is y = 6239x - 2033.7, with a correlation coefficient R0. 2 =0.9994; O 6 The standard curve regression equation for monoacetylmorphine is y = 7996.7x + 6857, R0 2 =0.9998; the standard curve regression equation for acetylcodeine is y = 8167.7x + 3821.3, R0 2 =0.9998; the standard curve regression equation for heroin is y=6111.7x+4581.5, R0 2 =0.9998; the regression equation for the papaverine standard curve is y=7225.3x-1062.4, R0 2 =0.9994; the regression equation of the noscapine standard curve is y=15836.3x-5110.9, R0.9 2 =0.9995. The standard curves are shown below. Figures 1-8 As shown.

[0057] Step 4: Confirm the peaks of the eight alkaloids in the chromatograms of the multiple sample solutions to be analyzed in Step 2. The specific method for confirmation is as follows: compare the retention times and UV spectra of the target alkaloids in the chromatograms of the multiple sample solutions to be analyzed with the retention times and UV spectra of the chromatograms of the eight alkaloid standards. The peak of the target alkaloid in the chromatogram of the sample solution to be analyzed is considered confirmed when both of the following conditions (a) and (b) are met: (a) the relative error between the retention time of the target alkaloid and the retention time of the standard chromatogram is less than 2%; (b) the UV spectra of the target alkaloid and the standard are extracted separately in the Waters Empower ultra-high performance liquid chromatography workstation, and the characteristic UV absorption wavelengths and absorption peak shapes of the target alkaloid and the standard are consistent. The reference retention times and UV absorption spectral parameters of the eight alkaloids in the ultra-high performance liquid chromatograms are shown in Table 2.

[0058] Table 2. Reference retention times and UV absorption spectra of eight alkaloids on ultra-high performance liquid chromatography (UHPLC) chromatograms.

[0059]

[0060] Refer to the retention times in Table 2 to identify the peaks in the chromatogram of the sample solution to be analyzed. On the one hand, the relative error between the retention times of each target alkaloid in the sample and the standard should be less than 2%. On the other hand, the UV spectra of each target alkaloid in the sample and the standard should be compared; the characteristic UV absorption wavelengths and peak shapes of both must be consistent. The UV absorption spectral parameters in Table 2 are used as a reference to help identify the positions of the eight alkaloids in the chromatogram of the sample solution when the retention time of the sample solution drifts or the sample composition is complex.

[0061] Substitute the peak area of ​​the confirmed target alkaloid into the regression equation of the standard curve corresponding to step 3, and calculate the mass concentration of the target alkaloid in the sample solution using the external standard method. Using this method, the concentrations of the eight alkaloids in each sample to be analyzed are calculated. Note: The peak area of ​​the target alkaloid in the sample solution should be within the linear range of the standard curve; otherwise, the sample size should be appropriately increased or decreased before re-measurement.

[0062] Eight alkaloids were combined to obtain 14 alkaloid combinations. The ratios of these 14 combinations were calculated, and the 14 alkaloid combinations are ACC / HER, ACC / (O... 3 +O 6 ), O 3 / O 6 ACC / (O 3 +O 6 +HER), ACC / (MOR+O) 3 +O 6 +HER), (O 3 +O 6 ) / HER、(COD+MOR) / (O 3 +O 6 +HER), HER / (O 3 +O 6 ), (COD+ACC) / (MOR+O) 3 +O 6 +HER), (COD+MOR+O) 3 +O 6 +HER) / ACC、HER / (COD+MOR+ACC+O 3 +O 6 (MOR+O) 3 +O 6+HER) / (COD+ACC)、(PAP+NOS) / (HER+O 3 +O 6 +MOR) and NOS / PAP. Acetyl codeine is abbreviated as ACC, codeine as COD, morphine as MOR, and O 3 Monoacetylmorphine (abbreviated as O) 3 O 6 Monoacetylmorphine (abbreviated as O) 6 Papaverine is abbreviated as PAP, noscapine as NOS, and heroin as HER.

[0063] Step 5: Perform z-fractional standardization on the ratio results of the 14 alkaloid combinations of multiple samples to be analyzed obtained in Step 4 to eliminate the dimensional differences between the concentrations of each alkaloid and the influence of hydrolysis on the content of each alkaloid in heroin. Then, use the Pearson coefficient method to determine the correlation of multiple heroin samples.

[0064] z-score standardization formula:

[0065] ;

[0066] in, For the sample Standardized data, This is the ratio result for each combination of alkaloids. =1, 2, 3…14, It is the mean of the ratios of the 14 alkaloid combinations. It is the standard deviation of the ratio results of the 14 alkaloid combinations.

[0067] Pearson correlation coefficient calculation:

[0068] ;

[0069] in, The Pearson correlation coefficient is used. For the sample Standardized data, For the sample Standardized data, The calculation method and Same, for The mean, for The mean, The number of alkaloid combinations. =14.

[0070] The correlation determination uses 0.98 as the correlation threshold; 0.99 > 0.98. A value greater than 0.98 indicates a strong correlation. A value greater than 0.99 indicates a very strong correlation.

[0071] Example:

[0072] Twenty samples were selected and subjected to the above treatment. The ratios of the 14 alkaloid combinations obtained are shown in Table 3.

[0073] Table 3. Ratios of alkaloid content in 20 samples

[0074]

[0075] The ratio results of the 14 alkaloid combinations in Table 3 were then standardized using z-fractions to eliminate the dimensional differences between the concentrations of each alkaloid and the effect of hydrolysis on the content of each alkaloid in heroin. The results are shown in Table 4.

[0076] Table 4. Standardized results of z-fraction ratios for 14 alkaloid combinations

[0077]

[0078] Pearson correlation coefficient was used to analyze the standardized z-score results in Table 4. The calculations and results are shown in Table 5.

[0079] Table 5 Pearson Correlation Coefficients result

[0080]

[0081] Using a correlation coefficient greater than 0.98 as the dividing point, 0.99 > A value greater than 0.98 indicates a strong correlation. A correlation greater than 0.99 is considered extremely strong. Table 5 shows that samples 2 and 3 are extremely strongly correlated; samples 4, 12, and 19 are extremely strongly correlated; samples 5 and 11 are extremely strongly correlated; samples 6 and 11 are extremely strongly correlated; samples 7, 8, and 9 are extremely strongly correlated; samples 5 and 19 are strongly correlated; samples 8 and 20 are strongly correlated; samples 10 and 19 are strongly correlated; and samples 12 and 19 are extremely strongly correlated. Analysis of heroin samples from different regions of Northwest China, combined with information from seized cases, revealed numerous instances of extremely strong correlations between heroin samples seized by multiple branches of the Urumqi Public Security Bureau; and multiple heroin drug cases in Weinan, Yulin, Xianyang, and other cities and counties in Shaanxi Province also showed extremely strong correlations. Of particular concern is the strong correlation between heroin samples from Xianyang and multiple heroin samples from Urumqi. This suggests that heroin illegally sold and circulated in Shaanxi Province and Xinjiang originates from the same upstream source, belonging to a large-scale drug distribution channel. Furthermore, when this technology was applied to analyze heroin characteristics nationwide, several heroin samples from Shaanxi, Gansu, and Urumqi, Xinjiang, showed strong correlations, as did several heroin samples from Chongqing, Guizhou, and Yunnan. Combined with geographical location, these are inferred to belong to heroin distribution routes in Northwest and Southwest China, respectively. Therefore, this invention's technology is of significant importance for correlation analysis and case investigation in cross-provincial drug trafficking cases.

[0082] Comparative example:

[0083] Compared with the industry standard liquid chromatography-mass spectrometry (LC-MS), 15 characteristic impurities in the above 20 samples were extracted using LC-MS. Retention time, characteristic ion pairs, and ion pair abundance ratio were used as qualitative criteria, and peak area of ​​quantitative ion pairs was used as quantitative criteria. After preprocessing the peak areas of the 15 characteristic impurities, the correlation between samples was determined using the Pearson correlation coefficient method. The peak areas of the 15 characteristic impurities are shown in Tables 6 and 7.

[0084] Preprocessing is performed on the peak areas of characteristic impurities. This preprocessing involves normalization followed by square root extraction.

[0085] ;

[0086] In the formula: The number of characteristic impurities, =15; Characteristic impurities chromatographic peak area. =1… ; To The results of the preprocessed data are shown in Table 8.

[0087] Pearson correlation coefficient calculate:

[0088] ;

[0089] In the formula, The number of characteristic impurities, =15, For the sample middle Preprocessed data, To test sample Q Preprocessed data, for The mean, for The mean. The calculation method and same.

[0090] LC-MS Pearson correlation coefficient The results are shown in Table 9.

[0091] Table 6 Peak areas of 15 characteristic impurities by LC-MS

[0092]

[0093] Table 7 (Continued from Table 6)

[0094]

[0095] Table 8. Results of normalization and square root treatment of LC-MS peak areas

[0096]

[0097] Table 9. Results of LC-MS Pearson correlation coefficient r

[0098]

[0099] Using a correlation coefficient greater than 0.98 as the dividing point, A result greater than 0.98 is considered positive. It can be observed that there is a strong correlation between No. 4 and No. 5, No. 6 and No. 11, No. 7 and No. 8, No. 8 and No. 9, and No. 12 and No. 19.

[0100] Therefore, using the LC-MS correlation discrimination results as a reference, in this embodiment, among 20 heroin samples and a total of 190 sample groups, 12 correlation groups were identified using the method of this invention, and 5 correlation groups were identified using the LC-MS method. All 5 correlation groups can be verified using the method of this invention. Therefore, the method of this invention can be used as a preliminary screening method for correlation discrimination.

[0101] In summary, the analysis results of 20 heroin samples using the method of this invention, compared with those obtained by liquid chromatography-mass spectrometry (LC-MS), showed a matching degree of 96.32%. This demonstrates that the method provided by this invention is low-cost, has a high matching degree, and can provide more accurate alkaloid comparison information in the samples. For suspected heroin samples with excessively high initial screening concentrations, they can be diluted before LC-MS analysis, reducing the maintenance costs of the LC-MS instrument. Therefore, the ultra-high performance liquid chromatography (UHPLC) method of this invention can serve as a preliminary screening method for heroin correlation identification, quickly identifying suspicious targets. By combining information from various cases to link and analyze them, the direction of investigation can be clarified, and the upstream and downstream links and key figures in the drug crime chain can be identified, which is crucial for in-depth case analysis.

[0102] Literature Chan KW, Tan GH, Wong RCS. Statistical Validation for the Profiling of Heroin by Associating Simulated Postcut Samples with the CorrespondingPrecut Sample. JF orensic S Ci 2013;58, among the 11 specific alkaloid ratio combinations recorded, there was no O 3 - Monoacetylmorphine, O 6 The correlation results for alkaloids such as monoacetylmorphine, papaverine, and noscapine showed a 50% false negative rate and an even higher false negative rate. The correlation screening method of this invention showed a 96.32% match with the semi-quantitative LC-MS results for 15 acidic impurities, thus demonstrating higher reliability. Furthermore, compared to the ratio method for 11 specific alkaloids, the cumulative contribution rate of the principal components PC1 and PC2 increased from 77.4% to 95.6%, with the X3 variable O... 3 / O 6 The eigenvalue of 0.94 in the PC2 direction indicates that this variable contributes the most to data variation.

[0103] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, it is intended to include any modifications and variations that fall within the scope of the claims and their equivalents.

Claims

1. A preliminary screening method for determining heroin-relatedness, characterized in that, Includes the following steps: Multiple sample solutions to be analyzed were prepared using acetonitrile as the solvent; Multiple sample solutions were analyzed by ultra-high performance liquid chromatography (UHPLC) to obtain chromatograms of the sample solutions. Eight alkaloids were used as standards to prepare standard solutions of different concentrations. These solutions were then analyzed by ultra-high performance liquid chromatography (UHPLC) to obtain chromatograms at different concentrations. A standard curve regression equation was obtained by plotting the peak area (ordinate) and concentration (abscissa) of the standard chromatograms for the eight alkaloids. The eight alkaloids were morphine, codeine, and O... 3 - Monoacetylmorphine, O 6 - Monoacetylmorphine, acetylcodeine, heroin, papaverine, and noscapine; The peaks of eight alkaloids in the chromatograms of multiple sample solutions to be analyzed were confirmed. The confirmed peak areas were substituted into the standard curve regression equation of the corresponding alkaloids to calculate the concentrations of the eight alkaloids in the multiple sample solutions to be analyzed. Eight alkaloids from multiple samples were combined to obtain 14 alkaloid combinations. The ratios of these 14 combinations were calculated based on the concentrations of the eight alkaloids. The 14 alkaloid combinations were: ACC / HER, ACC / (O... 3 +O 6 ), O 3 / O 6 ACC / (O 3 +O 6 +HER), ACC / (MOR+O) 3 +O 6 +HER), (O 3 +O 6 ) / HER、(COD+MOR) / (O 3 +O 6 +HER), HER / (O 3 +O 6 ), (COD+ACC) / (MOR+O) 3 +O 6 +HER), (COD+MOR+O) 3 +O 6 +HER) / ACC、HER / (COD+MOR+ACC+O 3 +O 6 (MOR+O) 3 +O 6 +HER) / (COD+ACC)、(PAP+NOS) / (HER+O 3 +O 6 +MOR) and NOS / PAP; where ACC is acetylcodeine, COD is codeine, MOR is morphine, and O 3 For O 3 - Monoacetylmorphine, O 6 For O 6 - Monoacetylmorphine, PAP stands for papaverine, NOS stands for noscapine, and HER stands for heroin; The ratio results of 14 alkaloid combinations in multiple samples to be analyzed were standardized by z-fraction, and the Pearson correlation coefficient was calculated based on the z-fraction standardized results. The correlation between the multiple samples to be analyzed was determined based on the Pearson correlation coefficient. The specific preparation method for the standard solution is as follows: using acetonitrile as the solvent, heroin, acetylcodeine, and O... 6 Monoacetylmorphine was dissolved in acetonitrile to prepare first mixed standard solutions of different concentrations. The heroin concentrations in the first mixed standard solutions were 0.25 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, 0.01 mg / mL, and 0.005 mg / mL, respectively. In each first mixed standard solution, the concentrations of heroin, acetylcodeine, and O... 6 - The mass ratio of monoacetylmorphine is 5:3:5; Using methanol as a solvent, morphine, codeine, and O 3 Monoacetylmorphine, noscapine, and papaverine were dissolved in methanol to prepare second mixed standard solutions of different concentrations. The concentrations of morphine in the second mixed standard solutions were 0.05 mg / mL, 0.02 mg / mL, 0.01 mg / mL, 0.005 mg / mL, 0.002 mg / mL, and 0.001 mg / mL, respectively. In each second mixed standard solution, the concentrations of morphine, codeine, and O... 3 - The mass ratio of monoacetylmorphine, noscapine, and papaverine is 1:1:1:1:1; When confirming the peaks of eight alkaloids in the chromatograms of multiple sample solutions to be analyzed, the specific method is as follows: the retention time and ultraviolet spectrum of the target alkaloid in the chromatograms of multiple sample solutions to be analyzed are compared with the retention time and ultraviolet spectrum of the chromatograms of the eight alkaloid standards. The peak of the target alkaloid in the chromatogram of the sample solution to be analyzed is considered confirmed when both of the following conditions (a) and (b) are met: (a) the relative error between the retention time of the target alkaloid and the retention time of the standard chromatogram is less than 2%; (b) the ultraviolet spectra of the target alkaloid and the standard are extracted in the Waters Empower ultra-high performance liquid chromatography workstation, and the characteristic ultraviolet absorption wavelength and absorption peak shape of the target alkaloid and the standard are consistent.

2. The preliminary screening method for determining heroin association according to claim 1, characterized in that, When performing ultra-high performance liquid chromatography (UHPLC), the chromatographic conditions for the sample solution and the standard solution were the same, specifically: C18 column, packing particle size 1.7 μm, column length 100 mm, inner diameter 2.1 mm; column temperature: 40℃; flow rate: 1.0 mL / min; injection volume: 1 μL; detection wavelength: 210 nm; mobile phase: phase A is acetonitrile, phase B is sodium dihydrogen phosphate buffer. The specific preparation method of sodium dihydrogen phosphate buffer is as follows: sodium dihydrogen phosphate and 85% concentrated phosphoric acid are mixed, dissolved and diluted with ultrapure water; the volume ratio of sodium dihydrogen phosphate to 85% concentrated phosphoric acid is 1.1 g: 1 mL; the volume ratio of 85% concentrated phosphoric acid to the diluted solution is 1:500; the gradient elution parameters are as follows (volume percentage): 0.0~9.0min, 98%~70%B; 9.0min~9.5min, 70%~20%B; 9.5min~12.0min, 20%B; 12.0 min ~ 12.5 min, 20% ~ 98% B; 12.5min~16.0min, 98%B.

3. The preliminary screening method for determining heroin association according to claim 1, characterized in that, The standard curve regression equation for morphine is y = 7392.7x - 326.6, and the correlation coefficient R0 is [value missing]. 2 =0.9987; the regression equation for the codeine standard curve is y=9608.6x-2981.5, and the correlation coefficient R0 is 0.9987. 2 =0.9993; O 3 The standard curve regression equation for monoacetylmorphine is y = 6239x - 2033.7, with a correlation coefficient R0. 2 =0.9994; O 6 The standard curve regression equation for monoacetylmorphine is y = 7996.7x + 6857, R0 2 =0.9998; the standard curve regression equation for acetylcodeine is y = 8167.7x + 3821.3, R0 2 =0.9998; the standard curve regression equation for heroin is y=6111.7x+4581.5, R0 2 =0.9998; the regression equation for the papaverine standard curve is y=7225.3x-1062.4, R0 2 =0.9994; the regression equation of the noscapine standard curve is y=15836.3x-5110.9, R0.9 2 =0.9995.

4. The preliminary screening method for determining heroin association according to claim 1, characterized in that, The specific preparation method for multiple sample solutions to be analyzed is as follows: using acetonitrile as solvent, multiple samples to be analyzed are dissolved by sonication, centrifuged, and the supernatant is diluted with sodium dihydrogen phosphate buffer to obtain multiple sample solutions to be analyzed; the volume ratio of sample to acetonitrile is 2.9mg~3.1mg:1mL; the supernatant is diluted with sodium dihydrogen phosphate buffer, and the volume ratio of supernatant to sodium dihydrogen phosphate buffer is 1:

9.

5. The preliminary screening method for determining heroin association according to claim 1, characterized in that, The standardized formula for z-scores is: ； in, For the sample Standardized data, This is the ratio result for each combination of alkaloids. =1, 2, 3…14, It is the mean of the ratios of the 14 alkaloid combinations. It is the standard deviation of the ratio results of the 14 alkaloid combinations.

6. The preliminary screening method for determining heroin association according to claim 1, characterized in that, Pearson correlation coefficient calculation: ； in, The Pearson correlation coefficient is used. For the sample Standardized data, For the sample Standardized data, The calculation method and same, for The mean, for The mean, The number of alkaloid combinations. =14.

7. The preliminary screening method for determining heroin association according to claim 6, characterized in that, The correlation determination uses 0.98 as the correlation threshold; 0.99 > 0.

98. A value greater than 0.98 indicates a strong correlation. A value greater than 0.99 indicates a very strong correlation.

Images