Method for qualitatively and quantitatively detecting alkyl nitrite substances by NMR (nuclear magnetic resonance) method

Abstract

The invention relates to the technical field of drug detection and chemical analysis, in particular to a qualitative and quantitative detection method for alkyl nitrite substances by an NMR (nuclear magnetic resonance) method, and aims to solve the problems that when the alkyl nitrite substances are detected in the prior art, the alkyl nitrite substances are complex in matrix and cannot be accurately qualitatively detected, and the detection accuracy is poor. In order to solve the problems that in the prior art, a sample to be detected is directly detected after being dissolved in a deuterated reagent at room temperature by utilizing the low-temperature detection characteristic of NMR (nuclear magnetic resonance), and by combining a hydrogen nuclear magnetic resonance spectrum, a one-dimensional selective excitation total correlation spectrum and a diffusion sequencing spectrum, the content of the alkyl nitrite substances is determined. Reliable qualification in a complex matrix is realized; meanwhile, absolute quantification is realized by adopting a stable and easily available internal standard substance 1, 4-dimethoxybenzene, and a deconvolution fitting algorithm is introduced to process signal overlapping in combination with a quantitative nuclear magnetic resonance hydrogen spectrum and a quantitative nuclear magnetic resonance carbon spectrum, so that the anti-interference capability and the result credibility of quantitative detection are improved.

Description

Technical Field

[0001] This invention relates to the field of drug detection and chemical analysis technology, and in particular to a qualitative and quantitative method for the NMR detection of alkyl nitrite esters. Background Technology

[0002] Alkyl nitrites, chemically classified as esters, contain unstable nitroso (–O–N=O) functional groups, resulting in extremely high volatility, thermal instability, and photosensitivity. In limited studies on the detection of "Rush," alkyl nitrites are metabolized very rapidly in the body, entering the bloodstream and taking effect within tens of seconds. They are quickly metabolized and broken down into corresponding alcohols by the liver, kidneys, lungs, intestinal mucosa, and blood vessels within just over 10 minutes. This leads to a very short detection window for alkyl nitrites, indirectly reflecting the difficulty in obtaining evidence of their presence.

[0003] Due to the unique physicochemical properties of alkyl nitrites, current detection methods for these substances are quite limited. Their high volatility renders conventional infrared spectroscopy unsuitable; their small molecular weight makes soft ionization difficult; and their ultraviolet absorption is close to the cutoff wavelength, so commonly used liquid chromatography-mass spectrometry (LC-MS) and high-performance liquid chromatography (HPLC) cannot determine alkyl nitrites. Furthermore, the high-temperature vaporization process at the injection port of gas chromatography (GC) or gas chromatography-mass spectrometry (GC-MS) causes significant decomposition of unstable alkyl nitrites. Therefore, headspace sampling combined with gas chromatography (such as HS-GC) or HS-GC-MS is currently the preferred method for their determination.

[0004] Because HS-GC is less sensitive than HS-GC-MS, HS-GC-MS is currently a more commonly used alternative, but it still has limitations. First, headspace equilibrium temperature and time need to be strictly controlled, otherwise it will affect the gas-liquid partition equilibrium and lead to poor reproducibility; second, the injection port for sample vaporization is exposed to high temperatures, which may cause the decomposition of alkyl nitrites, resulting in inaccurate quantification, which is unacceptable in forensic content identification; in addition, "Rush" often contains multiple solvents and fragrances, with a complex matrix, and co-escaped components may cause overlapping chromatographic peaks or mass spectrometry interference, interfering with accurate qualitative analysis; finally, quantification still relies on high-purity, easily decomposable alkyl nitrite ester standards, which are costly and whose long-term reliability is difficult to guarantee.

[0005] With the increasing number of "Rush" cases in recent years, the number of samples involved has also risen rapidly. The large number of cases indicates that "Rush" abuse is spreading rapidly, necessitating further improvements in the detection efficiency of alkyl nitrites in "Rush." ​​Therefore, developing a detection method that avoids high temperatures, is highly resistant to matrix interference, does not rely on high-purity standards, and can simultaneously achieve confirmatory qualitative and accurate quantitative analysis of alkyl nitrites is of urgent practical significance in the field of drug identification. Summary of the Invention

[0006] To address the aforementioned problems, this invention aims to provide a qualitative and quantitative detection method for alkyl nitrite esters using NMR. Existing technologies for detecting "Rush" suffer from inaccurate quantification due to thermal decomposition of alkyl nitrite esters caused by high-temperature sample introduction, reliance on high-purity standard materials, and the complexity of the "Rush" matrix, which hinders accurate characterization. This invention utilizes NMR technology for qualitative and quantitative detection of alkyl nitrite esters, fundamentally avoiding the thermal decomposition problem. Furthermore, the combination of multidimensional pulse sequencing and data processing techniques effectively overcomes the limitations of complex matrix interference and standard material dependence.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: On the one hand, the present invention provides a qualitative detection method for alkyl nitrite esters by NMR, comprising the following steps: The sample to be tested is dissolved with a deuterated reagent and mixed well to obtain a deuterated solution; The deuterated solution was transferred to a nuclear magnetic resonance spectrometer, and the hydrogen nuclear magnetic resonance spectrum and diffusion ordering spectrum of the sample to be tested were collected respectively. Based on the proton nuclear magnetic resonance spectrum of the sample to be tested, the characteristic peaks of suspected alkyl nitrite esters are identified and recorded as target characteristic peaks; One-dimensional selective excitation full correlation spectrum of target characteristic peaks is collected; By combining the spectral data of the proton nuclear magnetic resonance spectrum, one-dimensional selective excitation total correlation spectrum, and diffusion ordering spectrum of the sample to be tested, and comparing them with the proton nuclear magnetic resonance spectrum of standard alkyl nitrite esters, the presence of alkyl nitrite esters in the sample to be tested can be determined based on the comparison results.

[0008] Furthermore, the steps for acquiring the proton nuclear magnetic resonance spectrum of the sample to be tested specifically include: applying a 30° hard pulse with a pulse sequence of zg30 to the deuterated solution to obtain the proton nuclear magnetic resonance spectrum of the sample to be tested.

[0009] Furthermore, the specific steps for acquiring the diffusion ordination spectrum of the sample to be tested include: applying a DOSY pulse with a pulse sequence of ledbpgp2s to the deuterated solution to obtain the diffusion ordination spectrum of the sample to be tested.

[0010] Furthermore, the steps for acquiring the one-dimensional selective excitation full correlation spectrum of the target characteristic peak specifically include: applying a 1D select TOCSY pulse with the pulse sequence seldigpzs to the deuterated solution to selectively excite the target characteristic peak and obtain the one-dimensional selective excitation full correlation spectrum of the target characteristic peak.

[0011] Furthermore, the specific steps for comparing the proton NMR spectrum, one-dimensional selective excitation total correlation spectrum, and diffusion ordering spectrum of the sample to be tested with the proton NMR spectrum of standard alkyl nitrite esters include: Obtain the characteristic peaks of the proton nuclear magnetic resonance spectrum of standard alkyl nitrite esters, and denote them as standard characteristic peaks; Based on the proton nuclear magnetic resonance spectrum of the sample to be tested, the chemical shift, peak shape and integral area of ​​the target characteristic peak are obtained and compared with the chemical shift, peak shape and integral area of ​​the standard characteristic peak. The comparison result is recorded as the first comparison result. The one-dimensional selective excitation fully correlated spectrum is compared with the proton nuclear magnetic resonance spectrum of the sample to be tested to obtain the chemical shift, peak shape and integral area of ​​other proton peaks belonging to the same spin system as the target characteristic peak. The chemical shift, peak shape and integral area of ​​other proton peaks belonging to the same spin system as the standard characteristic peak are compared with the chemical shift, peak shape and integral area of ​​other proton peaks belonging to the same spin system as the standard characteristic peak. The comparison result is recorded as the second comparison result. The diffusion ordering spectrum is compared with the proton nuclear magnetic resonance spectrum of the sample to be tested to obtain the diffusion coefficient of the target characteristic peak, and then compared with the diffusion coefficient of the standard characteristic peak. The comparison result is recorded as the third comparison result.

[0012] Furthermore, the specific steps for determining whether the sample to be tested contains alkyl nitrite esters include: If the first comparison result, the second comparison result, and the third comparison result are all consistent, it is determined that the sample to be tested contains alkyl nitrite esters. If any of the first, second, and third comparison results are inconsistent, it is determined that no alkyl nitrite esters are detected in the sample to be tested.

[0013] On the other hand, the present invention also provides a quantitative detection method for alkyl nitrite esters by NMR. When the qualitative detection method described above determines that the sample contains alkyl nitrite esters, quantitative nuclear magnetic resonance (NMR) technology is used to quantitatively detect the alkyl nitrite esters in the sample. The quantitative detection method specifically includes the following steps: An internal standard solution containing a known concentration of internal standard is added to the deuterated solution, and the mixture is stirred to prepare a quantitative test solution. The quantitative test solution was transferred to the nuclear magnetic resonance spectrometer, and the quantitative nuclear magnetic resonance hydrogen spectrum and quantitative nuclear magnetic resonance carbon spectrum of the quantitative test solution were collected. The deconvolution fitting algorithm was used to separate the overlapping signals in the quantitative nuclear magnetic resonance hydrogen spectrum and the quantitative nuclear magnetic resonance carbon spectrum, and the quantitative peaks of nitrite alkyl esters in the quantitative nuclear magnetic resonance hydrogen spectrum and the quantitative nuclear magnetic resonance carbon spectrum were selected respectively. The selected quantitative peak and the internal standard peak of the internal standard were integrated separately. Based on the integration area ratio of the quantitative peak and the internal standard peak, and combined with the internal standard method calculation formula, the concentration and content of alkyl nitrite esters based on quantitative nuclear magnetic resonance hydrogen spectrum and quantitative nuclear magnetic resonance carbon spectrum were obtained respectively. The average concentration and content of alkyl nitrite esters were calculated separately, and these were taken as the final concentration and final content of alkyl nitrite esters.

[0014] Furthermore, the method for preparing an internal standard solution containing an internal standard of known concentration specifically includes: weighing a known mass of 1,4-dimethoxybenzene as an internal standard, and diluting it with deuterated chloroform to obtain an internal standard solution.

[0015] Further, the steps of transferring the quantitative test solution to the nuclear magnetic resonance spectrometer and acquiring the quantitative proton NMR spectrum and quantitative carbon NMR spectrum of the quantitative test solution specifically include: The quantitative test solution was subjected to a 90° hard pulse scan with a pulse sequence of zg90 to obtain the quantitative proton NMR spectrum; The quantitative test solution was subjected to inverted gated decoupled pulse scanning with a pulse sequence of zgig30 to obtain the quantitative carbon NMR spectrum.

[0016] Furthermore, the quantitative peak of alkyl nitrite esters in the quantitative proton NMR spectrum was selected as the CH peak; The CH3 peak is selected as the quantitative peak for alkyl nitrite esters in quantitative carbon NMR spectra.

[0017] The improvement of the present invention compared with the prior art is that, 1. This invention is based on nuclear magnetic resonance spectroscopy (NMR). The entire detection process is carried out at room temperature or even low temperature, completely avoiding the problem of inaccurate quantification caused by thermal decomposition of alkyl nitrite esters due to high temperature at the injection port in traditional methods such as GC-MS. Furthermore, only deuterated reagents are needed to dissolve the sample before direct detection, eliminating cumbersome pretreatment steps such as extraction, concentration, and derivatization. This simplifies the operation process, shortens the detection time, and improves detection efficiency, which is beneficial for the rapid screening of target substances in forensic identification. This invention has developed a rapid qualitative and quantitative detection method for alkyl nitrite esters in "Rush" using nuclear magnetic resonance spectroscopy. This detection method performed well in 10 real samples, detecting isobutyl nitrite in 9 samples, with a concentration range of 519~769 mg / mL and a content range of 64%~97%.

[0018] 2. This invention utilizes conventional methods... 1 By combining H-NMR, 1D select TOCSY, and DOSY, a multi-dimensional qualitative verification system was established. This invention can not only rely on... 1 H-NMR spectroscopy compares the chemical shifts, peak shapes, and integral areas of characteristic peaks. It can also verify the coupling relationship of associated proton signals and the diffusion coefficient of characteristic peaks, enabling accurate characterization of the target substance from multiple levels. This allows for high-confidence accuracy in complex mixed matrices like "Rush," effectively overcoming interference from complex matrices, effectively eliminating false positives and false negatives, and providing reliable qualitative analysis. This provides conclusive evidence for the nature of cases, case filing, prosecution, and other procedures in forensic identification.

[0019] 3. This invention selects 1,4-dimethoxybenzene, a chemically stable substance with a single-peak signal that does not overlap with the target characteristic peak, as an internal standard. This eliminates the need for expensive standard materials, achieving absolute quantification and solving the problems of high cost, difficulty in obtaining, and easy decomposition and instability of standard materials. A deconvolution fitting algorithm is introduced to handle signal overlap caused by complex matrices, achieving overlap... 1 Quantitative analysis of H-QNMR, combined with 13 C-QNMR performs dual quantitative verification to ensure the accuracy of the results and meet the requirements of forensic science for the accuracy, reproducibility, and reliability of expert evidence. Attached Figure Description

[0020] Figure 1 The sample to be tested in this invention 1 H-NMR spectrum and standard isobutyl nitrite 1 Comparison of H-NMR spectra.

[0021] Figure 2 This is a 1D select TOCSY excitation pattern of the sample to be tested according to the present invention.

[0022] Figure 3 This is the DOSY spectrum of the sample to be tested according to the present invention.

[0023] Figure 4 These are photographs of 10 samples suspected to be "Rush" from this invention.

[0024] Figure 5 The sample to be tested in this invention 13 C-NMR spectrum and standard isobutyl nitrite 13 Comparison of C-NMR spectra. Detailed Implementation

[0025] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0026] Due to the increasing number of "Rush"-related cases, seven alkyl nitrites have been included in the "List of Hazardous Chemicals," clearly classifying them as chemical substances harmful to human health. The seven alkyl nitrites are listed in Table 1 below. Because alkyl nitrites contained in "Rush" are metabolized very rapidly in the body, quickly decomposing into corresponding alcohols within just over 10 minutes, existing detection methods for alkyl nitrites suffer from slow detection speed and inaccurate quantification. Therefore, this invention proposes a qualitative and quantitative detection method for alkyl nitrites using NMR (Nuclear Magnetic Resonance Spectroscopy). This method is simple, rapid, and accurate, requiring no complex pretreatment, and possesses high resolution and sensitivity. Based on NMR spectroscopy, it achieves qualitative and quantitative detection of alkyl nitrites in "Rush," avoiding thermal decomposition of alkyl nitrites during detection. Furthermore, by combining multidimensional pulse sequences, it overcomes the interference problem caused by complex matrices in "Rush," thereby achieving accurate qualitative and quantitative detection of alkyl nitrites in "Rush."

[0027] Table 1. Seven alkyl nitrites listed in the Hazardous Chemicals Catalogue: ; Example 1: This example uses isobutyl nitrite in "Rush" as an example, taking "Rush" as the sample to be tested, and introduces the qualitative detection method of isobutyl nitrite in the sample using NMR. Specifically: This qualitative detection method utilizes nuclear magnetic resonance hydrogen spectroscopy (NMR spectroscopy). 1 This method combines three spectra—H-NMR, one-dimensional selective excitation total correlation spectroscopy (1Dselect TOCSY), and diffusion ordering spectroscopy (DOSY)—to achieve rapid qualitative analysis of isobutyl nitrite in complex matrices. 1 By analyzing the characteristic peak shape, chemical shift, and integral area of ​​H-NMR, combined with the coupling relationship of the associated proton signal from 1D select TOCSY and the diffusion coefficient of the characteristic peak of DOSY, matrix interference can be effectively eliminated, thereby enabling rapid and accurate identification of whether isobutyl nitrite is present in the sample. The specific steps for the qualitative detection of isobutyl nitrite in the sample using NMR in this embodiment are as follows: S1: Dissolve the sample to be tested with a deuterated reagent, mix well, and obtain a deuterated solution.

[0028] Accurately measure 100 μL of “Rush” sample and 0.6 mL of deuterated reagent, mix the “Rush” sample and deuterated reagent to obtain deuterated solution; The preferred deuteration reagent is deuterated chloroform (CDCl3, 99.9%), purchased from the Cambridge Isotope Laboratory. As a weakly polar solvent, deuterated chloroform is suitable for dissolving isobutyl nitrite due to its low polarity, adhering to the "like dissolves like" principle and ensuring the stability of isobutyl nitrite molecules in the deuterated solution. Furthermore, deuterated chloroform has excellent dissolving power, effectively dissolving most ester compounds and forming a homogeneous solution, avoiding localized concentration unevenness in the deuterated solution. In addition, deuterated chloroform... 1 Only trace proton signals remain in the H-NMR spectrum, which will not cause overlap or interference with the characteristic peaks of isobutyl nitrite; and deuterated chloroform is low in cost, making it suitable for large-scale forensic identification or routine screening.

[0029] S2: Transfer the deuterated solution to a nuclear magnetic resonance spectrometer and collect the hydrogen nuclear magnetic resonance spectrum and diffusion ordering spectrum of the sample to be tested.

[0030] Specifically, during NMR data acquisition, the deuterated solution is placed in the NMR tube. The NMR tube is first inserted into the rotor and then pneumatically or mechanically fed into the center of the magnet, ensuring that the NMR tube is in the region with the most uniform magnetic field and the highest sensitivity of the radio frequency coil. This sample introduction method avoids positional deviations and vibration interference caused by manual placement. Subsequently, the NMR spectrometer automatically performs field locking, shimming, and tuning. The deuterated solution is then used to... 2 The H signal serves as the field-locking signal, eliminating magnetic field drift, ensuring the reproducibility of chemical shifts, and facilitating comparison with the database. Subsequently, by adjusting the current of a series of shimming coils, the magnetic field is uniformly distributed around the NMR tube. Good shimming results in a symmetrical, narrow Lorentz peak shape and allows the separation of characteristic peaks with similar chemical shifts, making it particularly suitable for identifying the target analyte isobutyl nitrite in complex matrices. Finally, the resonant frequency of the probe circuit is adjusted, and impedance matching is achieved to ensure efficient transfer of radio frequency energy to the NMR tube and reception of the return signal, improving the signal-to-noise ratio and detection sensitivity, which is beneficial for the detection of weak signals.

[0031] Subsequently, a 30° hard pulse was applied to the deuterated solution in the NMR tube using a NMR spectrometer, with the pulse sequence being zg30, to obtain the proton NMR spectrum of the sample. 1 H-NMR spectrum).

[0032] Finally, DOSY pulses were applied to the deuterated solution in the NMR tube, with the pulse sequence ledbpgp2s, to obtain the DOSY spectrum of the sample. DOSY spectroscopy (diffusion ordering spectroscopy) is a special NMR technique. Unlike chromatographic separation, DOSY separates molecules based on the difference in self-diffusion coefficients in solution, without requiring a physical separation process.

[0033] S3: Based on the proton nuclear magnetic resonance spectrum of the sample to be tested, identify the characteristic peak of suspected isobutyl nitrite and record it as the target characteristic peak.

[0034] Specifically: based on the hydrogen nuclear magnetic resonance spectrum of the sample to be tested ( 1 (H-NMR spectrum), observe the chemical shifts and peak shapes of each characteristic peak on the hydrogen nuclear magnetic resonance spectrum, identify the characteristic peak suspected to be isobutyl nitrite in the hydrogen nuclear magnetic resonance spectrum, and record this characteristic peak as the target characteristic peak.

[0035] S4: Acquire the one-dimensional selective excitation full correlation spectrum of the target characteristic peaks; Specifically, a 1D select TOCSY pulse sequence of seldigpzs is applied to the deuterated solution to selectively excite the target characteristic peak, obtaining a one-dimensional selective excitation fully correlated spectrum (1D select TOCSY spectrum) of the target characteristic peak. The 1D select TOCSY pulse combines selective excitation and fully correlated spectrum to first selectively excite a specific proton, and then transmit the magnetization vector of that proton to all other protons belonging to the same coupling network through the TOCSY mixing sequence.

[0036] S5: Combine the proton nuclear magnetic resonance spectrum, one-dimensional selective excitation total correlation spectrum and diffusion ordering spectrum of the sample to be tested with the proton nuclear magnetic resonance spectrum of standard isobutyl nitrite, and determine whether the sample to be tested contains isobutyl nitrite based on the comparison results. First, 100 μL of isobutyl nitrite was accurately measured and dissolved in 0.6 mL of deuterated chloroform to obtain a standard solution. The isobutyl nitrite (IBN, 95%) used in the standard solution was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The proton nuclear magnetic resonance (HNMR) spectrum of the standard solution was collected to obtain the characteristic peak of the HNMR spectrum of the standard isobutyl nitrite, which was recorded as the standard characteristic peak. The standard characteristic peak was determined to be a methylene group directly linked to a nitrosyl group, with a broad peak shape and a chemical shift between 4.3 ppm and 4.8 ppm. Based on the HNMR spectrum of the sample to be tested, the chemical shift, peak shape, and integrated area of ​​the target characteristic peak were obtained and compared with those of the standard characteristic peak. The comparison result was recorded as the first comparison result.

[0037] Specifically, for the obtained test sample 1 Fourier transform was performed on the raw FID signal of the H-NMR spectrum, and then the results were analyzed based on the sample. 1 1H-NMR spectroscopy was used to obtain the chemical shift, peak shape, and integrated area of ​​the target characteristic peak. The chemical shift, peak shape, and integrated area of ​​the target characteristic peak were compared with those of the standard isobutyl nitrite using the 1H NMR spectrum. 1 H-NMR spectrum and standard isobutyl nitrite1 Refer to the attached H-NMR spectrum comparison diagram. Figure 1 As shown. Due to the sample to be tested... 1 H-NMR spectrum and standard isobutyl nitrite 1 The H-NMR spectra show the same spin system, and this shared spin system is a characteristic specific to the structure, consistent with both standard isobutyl nitrite and suspected isobutyl nitrite. Therefore, both... 1 The differences in the H-NMR spectra are not particularly large. Figure 1 It can also be seen that the sample to be tested... 1 H-NMR spectrum and standard isobutyl nitrite 1 The differences in the H-NMR spectra are not significant. Based on this premise, Table 2 below compares the standard isobutyl nitrite. 1 The standard characteristic peaks in the H-NMR spectrum were analyzed for NMR assignment, and Table 2 below also shows the NMR assignments for the samples to be tested. 1 NMR assignment analysis of the target characteristic peak of isobutyl nitrite in H-NMR spectrum.

[0038] Table 2. NMR assignment analysis of isobutyl nitrite: ; Table 2 The peaks indicated by 'br.s' represent quantitative peaks; 'd' represents doublets; and 'hept' represents septets. Table 2 shows that isobutyl nitrite contains hydrogen atoms in three different chemical environments, thus exhibiting three sets of characteristic peaks. The 3-CH2 group, directly bonded to the nitrite ester bond, experiences a strong electron-withdrawing effect from the oxygen atom, resulting in a low electron cloud around the hydrogen nucleus and a chemical shift of δ = 4.52 ppm. Simultaneously, due to the rotational nature of the ester bond, the conventional doublet transforms into a broad singlet, a highly typical characteristic signal shared by isobutyl nitrite. The 2-CH group, close to the nitrite ester bond, is coupled by six hydrogen atoms from both 1-CH3 and 4-CH3, exhibiting a characteristic septet with a chemical shift of δ = 2.01 ppm and a coupling constant J = 6.87. The 1-CH3 and 4-CH3 groups have the highest electron clouds around their hydrogen nuclei and are coupled by one hydrogen atom from 2-CH, exhibiting a characteristic doublet with a chemical shift of δ = 0.97 ppm and a coupling constant J = 6.87. The integrals of the three characteristic peaks should be close to 2:1:6, the peak shapes should be completely consistent when they can be identified, and the difference in coupling constants should be less than 5%.

[0039] Secondly, based on the selective excitation of the target characteristic peaks, the one-dimensional selective excitation fully correlated spectrum of the target characteristic peaks is obtained (see attached figure). Figure 2As shown. When selectively exciting the target characteristic peaks, three target characteristic peaks (suspected characteristic peaks of isobutyl nitrite) with chemical shifts of 0.971, 2.021, and 4.524 were selected for excitation, yielding three corresponding 1D select TOCSY spectra. These three 1D select TOCSY spectra show that the three target characteristic peaks with chemical shifts of 0.971, 2.021, and 4.524 belong to the same spin system and are mutually coupled. This achieves the identification and separation of three sets of suspected isobutyl nitrite target characteristic peaks from the proton nuclear magnetic resonance spectrum of the sample with a complex matrix. (Appendix) Figure 2 The results show clear separation of the target characteristic peak after selective excitation, proving the reliability of this qualitative detection method. The one-dimensional selective excitation fully correlated spectrum of the target characteristic peak is compared with the proton nuclear magnetic resonance spectrum of the sample to obtain the chemical shift, peak shape, and integrated area of ​​other proton peaks belonging to the same spin system as the target characteristic peak. These are then compared with the chemical shift, peak shape, and integrated area of ​​other proton peaks belonging to the same spin system as the standard characteristic peak; the comparison result is recorded as the second comparison result.

[0040] Then, based on the obtained DOSY spectrum, the DOSY spectrum is referenced in Appendix Figure 3 As shown, the diffusion coefficient of the target characteristic peak can be obtained through DOSY spectroscopy. The diffusion coefficient is a physical constant. In the complex matrix of DOSY spectroscopy, different molecules will have different diffusion coefficients. Taking isobutyl nitrite as an example, isobutyl nitrite has three different hydrogen atoms. For the same substance, all three hydrogen atoms will have the same diffusion coefficient. This allows isobutyl nitrite in the test sample to be separated from other matrices, achieving virtual separation. After comparing the diffusion ordering spectrum with the proton NMR spectrum of the test sample to obtain the diffusion coefficient of the target characteristic peak, the diffusion coefficient of the target characteristic peak is compared with the diffusion coefficient of the standard characteristic peak. The comparison result is recorded as the third comparison result. By verifying the consistency of the diffusion coefficients of the target characteristic peak and the standard characteristic peak, it is confirmed from the perspective of physical properties whether the target characteristic peak and the standard characteristic peak belong to the same substance, avoiding misjudgment.

[0041] Finally, the first, second, and third comparison results are compared: if the first, second, and third comparison results are consistent, it is determined that the sample contains isobutyl nitrite; if any one of the first, second, and third comparison results is inconsistent, it is determined that isobutyl nitrite is not detected in the sample.

[0042] Example 2: Based on the detection of isobutyl nitrite in the sample of Example 1 above, this example uses NMR to further quantify isobutyl nitrite in the sample. Specifically: This quantitative detection method utilizes quantitative nuclear magnetic resonance (NMR) spectroscopy (H1N).1 H-QNMR) and quantitative carbon NMR (H-QNMR) 13 C-QNMR, combined with a deconvolution fitting algorithm. For quantitative detection of substances with uncomplicated matrices, existing techniques typically use... 1 Quantification can be performed using H-QNMR, but for complex matrices like "Rush," relying solely on H-QNMR is insufficient. 1 Quantitative analysis using H-QNMR is inaccurate because... 1 The chemical shift width of H-QNMR is only 15ppm~20ppm, which is too low in resolution and easily causes overlap. 13 The chemical shift width of C-QNMR can reach 200 ppm, which can effectively reduce the risk of chemical shift overlap. In the quantitative stage, this translates to better integration and calculation. This is the quantitative detection method of this invention. 1 Based on H-QNMR, combined with 13 The main reason for C-QNMR is that it is solved through deconvolution fitting algorithms. 1 To address the overlap issue in H-QNMR and further avoid interference and improve result accuracy, additional methods were used... 13 C-QNMR further validated the results by quantifying them from different dimensions, thus solving the problems mentioned in the background technology, such as dependence on standard substances, interference from complex matrices, and inaccurate quantification.

[0043] The specific steps for quantitatively detecting isobutyl nitrite in "Rush" using NMR in this embodiment are as follows: Step 1: Add an internal standard solution containing a known concentration of internal standard to the deuterated solution, mix well, and prepare a quantitative test solution.

[0044] Specifically, since the singlet signal (δ=6.84 ppm) of 1,4-dimethoxybenzene (1,4-DB) does not interfere with the characteristic peak of isobutyl nitrite, 1,4-dimethoxybenzene was chosen as the internal standard to prepare the internal standard solution. 1,4-Dimethoxybenzene (1,4-DB, 99.0%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. 100 mg of 1,4-dimethoxybenzene was accurately weighed and dissolved in 10 mL of deuterated chloroform to prepare a 10 mg / mL internal standard solution.

[0045] It should be noted that QNMR generally uses internal standard quantification. The basic principles for selecting an internal standard are high purity, easy solubility in deuterated reagents, and non-interference of the quantitative signal with the analyte. (Refer to...) Figure 1 and Figure 5 According to isobutyl nitrite (IBN) 1 H-NMR and 13 For C-NMR, 1,4-DB with a single-peak signal was ultimately selected as the internal standard.

[0046] When preparing the quantitative test solution, take 100 μL of deuterated dissolution solution, add 500 μL of internal standard solution to the deuterated dissolution solution, mix the two well, and then take the supernatant into the NMR tube to obtain the quantitative test solution.

[0047] Step 2: Transfer the quantitative test solution to the nuclear magnetic resonance spectrometer and collect the quantitative nuclear magnetic resonance hydrogen spectrum and quantitative nuclear magnetic resonance carbon spectrum of the quantitative test solution.

[0048] Specifically, the NMR tube is transferred into the NMR spectrometer, and quantitative nuclear magnetic resonance (NMR) hydrogen spectroscopy is used. 1 When performing pulse scanning of the quantitative test solution using H-QNMR, a 90° hard pulse was used, with the pulse sequence being zg90. The parameters were set as follows: spectral width 15 ppm, relaxation delay (D1) 30 s (to ensure complete spin recovery), 32 scans, and sampling time 8.19 s. 1 Spectral data of H-QNMR.

[0049] Quantitative nuclear magnetic resonance carbon spectroscopy ( 13 When performing pulse scanning of the quantitative test solution using C-QNMR, an inverted gated decoupling pulse was used with a pulse sequence of zgig30. The parameters were set as follows: spectral width 238 ppm, relaxation delay 30 s, and 1024 scans. 13 C-QNMR spectral data.

[0050] Step 3: Using the deconvolution fitting algorithm, the overlapping signals in the quantitative nuclear magnetic resonance hydrogen spectrum and the quantitative nuclear magnetic resonance carbon spectrum are separated, and the quantitative peaks of nitrite alkyl esters in the quantitative nuclear magnetic resonance hydrogen spectrum and the quantitative nuclear magnetic resonance carbon spectrum are determined respectively.

[0051] Specifically, the chromatographic data was processed using Mestnova software, with manual Fourier transform, phase correction, and baseline smoothing. To address signal overlap in complex matrices, a deconvolution fitting algorithm was employed to separate overlapping peaks. A Lorentz-Gaussian mixture fitting was selected, and its deconvolution process can be recorded and reproduced, further reducing errors. This data processing method does not rely on perfect chromatographic separation and can still accurately integrate some overlapping peaks, making it suitable for the high-precision quantification required in forensic science.

[0052] Based on isobutyl nitrite 1 ¹H NMR revealed that the integral of 3-CH₂, which exhibits rotation, was inaccurate, and that 1-CH₃ and 4-CH₃ were easily interfered with by residual isopropanol. Therefore, 2-CH₂ was ultimately chosen as the integral. 1 The quantitative peak of H-QNMR; similarly, in 13The 2-CH and 3-CH2 fractions of isobutyl nitrite in C-NMR are easily interfered with, leading to inaccurate integration. Therefore, 1-CH3 and 4-CH3 (δ=18.97 ppm) were selected as the fractions. 13 Quantitative peaks of C-QNMR.

[0053] Step 4: Integrate the selected quantitative peak and the internal standard peak of the internal standard respectively. Based on the integration area ratio of the quantitative peak and the internal standard peak, and combined with the internal standard method calculation formula, obtain the concentration and content of alkyl nitrite esters based on quantitative nuclear magnetic resonance hydrogen spectrum and quantitative nuclear magnetic resonance carbon spectrum respectively.

[0054] Specifically, the peak of 1,4-dimethoxybenzene with a single-peak signal is selected as the internal standard peak, and in step three, 2-CH is selected as... 1 The quantitative peaks of H-QNMR were selected as 1-CH3 and 4-CH3. 13 The quantitative peak of C-QNMR was determined. The internal standard peak and the quantitative peak of isobutyl nitrite were integrated, and the concentration C was calculated based on the ratio of the integrated areas of the internal standard peak and the quantitative peak. x and content P x Direct integration can reduce errors, thereby improving quantitative accuracy. Concentration C x and content P x Calculate separately according to formula (1) and formula (2): (mg / mL) (1); (mg / mg) (2); Will 1 H-QNMR and 13 Substituting the integral values ​​of the internal standard peak and the quantitative peak of C-QNMR into formulas (1) and (2), where C x I x N x M x W x P x These represent the concentration, integrated peak area, number of hydrogen nuclei, molar mass, weighed mass, and content of the analyte (x), respectively; I std N std M std W std P std V represents the integrated peak area, number of hydrogen nuclei, molar mass, weighed mass, and content of the internal standard (std), respectively. x The volume of the analyte (x) is represented; the concentration of isobutyl nitrite can be obtained according to formula (1), and the content of isobutyl nitrite can be obtained according to formula (2).

[0055] In the above calculation process, due to1 H-QNMR and 13 Each of the C-QNMR peaks has a quantitative peak, therefore 1 H-QNMR calculations yielded a concentration and content. 13 C-QNMR also yielded a concentration and content, 1 H-QNMR and 13 The mean value of the calculation results obtained from C-QNMR was used as the final concentration and final content of isobutyl nitrite.

[0056] In summary, all spectra in Examples 1 and 2 were acquired using a Bruker Avence Neo Ascend 600MHz nuclear magnetic resonance spectrometer (HR-BBO600S3-BBF / H / D-5.0-Z probe), with the temperature controlled at 300K. The sampling parameters are shown in Table 3 below. Table 3 NMR experimental parameters: ; Example 3: This example verifies the quantitative calculation results in Example 2 by using methods such as linear range, spiked recovery rate, and intra-day / inter-day precision.

[0057] First, standard substances were added to the matrix in varying amounts to simulate and verify the accuracy of results within a concentration range of approximately 2% to 29% at concentrations ranging from 0.02 mg / mL to 0.29 mg / mL. Specifically: 10 μL, 20 μL, 50 μL, 100 μL, and 200 μL of isobutyl nitrite were added to 500 μL of internal standard solution to prepare linearity verification solutions with concentrations of 0.02 mg / mL, 0.04 mg / mL, 0.09 mg / mL, 0.17 mg / mL, and 0.29 mg / mL, respectively. The linearity was good (R2>0.999), and the linearity determination results are shown in Table 4 below.

[0058] Table 4. Linear range of QNMR detection for isobutyl nitrite: ; As can be seen from Table 4, whether 1 H-QNMR or 13 The C-QNMR results were consistent with the actual added concentrations and contents. For example, for the linear sample with a concentration of 0.02 mg / ml, the quantitative 1H NMR spectrum was 0.019 and the quantitative 1C NMR spectrum was 0.0202.

[0059] Secondly, each linear sample was processed in parallel for six replicates within the same day and then measured to calculate repeatability (intra-day precision). For each day within the six days, one copy of each sample was processed and measured to calculate reproducibility (inter-day precision). Repeatability and reproducibility assess the stability of a sample's detection, avoiding significant differences in results between tests of the same sample at different times within the same day and between tests at different times across different days. The specific results for the recovery rate, repeatability, and reproducibility of isobutyl nitrite spiked are shown in Table 5 below.

[0060] Table 5. Recovery, repeatability, and reproducibility of isobutyl nitrite spiked ester: ; As shown in Table 5, the intraday precision was less than 2%, and the interday precision was less than 4.1%. 1 H-QNMR and 13 The quantitative peak of C-QNMR was within the range of 0.02–0.29 mg / mL, and the relative error of the spiked recovery was less than 10%, indicating good accuracy.

[0061] Example 4: In this example, the qualitative detection method from Example 1 and the quantitative detection method from Example 2 were used to perform qualitative and quantitative detection on 10 suspected "Rush" samples. Photos of these 10 suspected "Rush" samples are attached. Figure 4 All samples were seized and submitted for testing from cases handled by the police. In this embodiment, these 10 suspected "Rush" samples were labeled as Y1 to Y10. The existing HS-GCMS method was used to perform qualitative testing on these 10 suspected "Rush" samples as a control group. The qualitative testing method in Example 1 and the quantitative testing method in Example 2 were used to perform qualitative and quantitative testing on the samples as a verification group.

[0062] Specifically, the basic physical properties of all samples were observed and perceived. It was found that all samples had a distinct gasoline-like smell, and most samples produced a large number of bubbles after being opened. The "Rush" drug itself has strong volatility and a special smell similar to gasoline or solvents. In other words, the physical properties of all samples are basically consistent with those of the "Rush" drug.

[0063] Subsequently, qualitative tests were performed on the samples. The qualitative test results of the verification group and the control group are shown in Table 6 below. The results in Table 6 show that isobutyl nitrite was not detected in sample Y5 in both the verification group and the control group, while isobutyl nitrite was detected in all other samples.

[0064] Table 6 Comparison of qualitative results of isobutyl nitrite determination in samples by NMR and HS-GCMS methods: ; Finally, the quantitative detection method of Example 2 was used to quantitatively detect the remaining 9 samples (excluding Y5). The results are shown in Table 7 below. Table 7 shows that the concentration of isobutyl nitrite in the 9 samples ranged from 519 mg / mL to 769 mg / mL, and the content ranged from 64% to 97% (mass ratio). RD in Table 7 represents... 1 H-QNMR and 13 The discrepancy between the concentration and content results calculated by C-QNMR can prove that 1 H-QNMR and 13 The concentration and content results calculated by C-QNMR have little deviation, so the average value can be taken as the final concentration and final content of isobutyl nitrite.

[0065] Table 7 1 H-QNMR and 13 C-QNMR determination of the concentration and content of isobutyl nitrite in the sample: ; Therefore, this invention utilizes the low-temperature detection characteristics of NMR, allowing the sample to be dissolved in a deuterated reagent at room temperature before direct detection, completely avoiding chemical decomposition caused by high temperatures, thus ensuring the authenticity and accuracy of the detection results. 1 The combination of ¹H-NMR, 1D select TOCSY, and DOSY enables reliable qualitative analysis in complex matrices. Simultaneously, the use of a stable and readily available internal standard, 1,4-dimethoxybenzene, allows for absolute quantification without the need for target analyte standards. 1 H-QNMR and 13 C-QNMR introduces a deconvolution fitting algorithm to handle signal overlap, thereby improving the anti-interference ability and reliability of quantitative detection results.

[0066] In summary, this invention is the first to utilize NMR to simultaneously perform qualitative and quantitative analysis of isobutyl nitrite in 10 genuine "Rush" samples. IBN was detected in 9 samples, with concentrations ranging from 519 to 69 mg / mL and contents ranging from 64% to 97%. This verifies the practicality of the detection and analysis method developed in this invention, enabling rapid analysis of isobutyl nitrite in "Rush" even without high-purity standard materials. It meets the requirements of grassroots law enforcement units for rapid result identification and provides a new method and approach for identifying alkyl nitrite esters.

[0067] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A qualitative detection method for alkyl nitrite esters by NMR, characterized in that: Includes the following steps: The sample to be tested is dissolved with a deuterated reagent and mixed well to obtain a deuterated solution; The deuterated solution was transferred to a nuclear magnetic resonance spectrometer, and the hydrogen nuclear magnetic resonance spectrum and diffusion ordering spectrum of the sample to be tested were collected respectively. Based on the proton nuclear magnetic resonance spectrum of the sample to be tested, the characteristic peaks of suspected alkyl nitrite esters are identified and recorded as target characteristic peaks; One-dimensional selective excitation full correlation spectrum of target characteristic peaks is collected; By combining the spectral data of the proton nuclear magnetic resonance spectrum, one-dimensional selective excitation total correlation spectrum, and diffusion ordering spectrum of the sample to be tested, and comparing them with the proton nuclear magnetic resonance spectrum of standard alkyl nitrite esters, the presence of alkyl nitrite esters in the sample to be tested can be determined based on the comparison results.

2. The qualitative detection method for alkyl nitrite esters by NMR according to claim 1, characterized in that: The specific steps for acquiring the proton nuclear magnetic resonance spectrum of the sample to be tested include: applying a 30° hard pulse with a pulse sequence of zg30 to the deuterated solution to obtain the proton nuclear magnetic resonance spectrum of the sample to be tested.

3. The qualitative detection method for alkyl nitrite esters by NMR according to claim 2, characterized in that: The specific steps for acquiring the diffusion ordination spectrum of the sample to be tested include: applying DOSY pulses with a pulse sequence of ledbpgp2s to the deuterated solution to obtain the diffusion ordination spectrum of the sample to be tested.

4. The qualitative detection method for alkyl nitrite esters by NMR according to claim 3, characterized in that: The specific steps for acquiring the one-dimensional selective excitation full correlation spectrum of the target characteristic peak include: applying a 1D select TOCSY pulse with the pulse sequence seldigpzs to the deuterated solution to selectively excite the target characteristic peak and obtain the one-dimensional selective excitation full correlation spectrum of the target characteristic peak.

5. The qualitative detection method for alkyl nitrite esters by NMR according to claim 4, characterized in that: The specific steps for comparing the proton NMR spectrum, one-dimensional selective excitation total correlation spectrum, and diffusion ordering spectrum of the sample to be tested with the proton NMR spectrum of a standard alkyl nitrite ester include: Obtain the characteristic peaks of the proton nuclear magnetic resonance spectrum of standard alkyl nitrite esters, and denote them as standard characteristic peaks; Based on the proton nuclear magnetic resonance spectrum of the sample to be tested, the chemical shift, peak shape and integral area of ​​the target characteristic peak are obtained and compared with the chemical shift, peak shape and integral area of ​​the standard characteristic peak. The comparison result is recorded as the first comparison result. The one-dimensional selective excitation fully correlated spectrum is compared with the proton nuclear magnetic resonance spectrum of the sample to be tested to obtain the chemical shift, peak shape and integral area of ​​other proton peaks belonging to the same spin system as the target characteristic peak. The chemical shift, peak shape and integral area of ​​other proton peaks belonging to the same spin system as the standard characteristic peak are compared with the chemical shift, peak shape and integral area of ​​other proton peaks belonging to the same spin system as the standard characteristic peak. The comparison result is recorded as the second comparison result. The diffusion ordering spectrum is compared with the proton nuclear magnetic resonance spectrum of the sample to be tested to obtain the diffusion coefficient of the target characteristic peak, and then compared with the diffusion coefficient of the standard characteristic peak. The comparison result is recorded as the third comparison result.

6. The qualitative detection method for alkyl nitrite esters by NMR according to claim 5, characterized in that: The specific steps for determining whether a sample contains alkyl nitrites include: If the first comparison result, the second comparison result, and the third comparison result are all consistent, it is determined that the sample to be tested contains alkyl nitrite esters. If any of the first, second, and third comparison results are inconsistent, it is determined that no alkyl nitrite esters are detected in the sample to be tested.

7. A quantitative detection method for alkyl nitrite esters by NMR, characterized in that: When the qualitative detection method according to any one of claims 1-6 determines that the sample contains alkyl nitrites, quantitative nuclear magnetic resonance (NMR) technology is used to quantitatively detect the alkyl nitrites in the sample. The quantitative detection method specifically includes the following steps: An internal standard solution containing a known concentration of internal standard is added to the deuterated solution, and the mixture is stirred to prepare a quantitative test solution. The quantitative test solution was transferred to the nuclear magnetic resonance spectrometer, and the quantitative nuclear magnetic resonance hydrogen spectrum and quantitative nuclear magnetic resonance carbon spectrum of the quantitative test solution were collected. The deconvolution fitting algorithm was used to separate the overlapping peaks in the quantitative nuclear magnetic resonance hydrogen spectrum and the quantitative nuclear magnetic resonance carbon spectrum, and the quantitative peaks of alkyl nitrite esters in the quantitative nuclear magnetic resonance hydrogen spectrum and the quantitative nuclear magnetic resonance carbon spectrum were selected respectively. The selected quantitative peak and the internal standard peak of the internal standard were integrated separately. Based on the integration area ratio of the quantitative peak and the internal standard peak, and combined with the internal standard method calculation formula, the concentration and content of alkyl nitrite esters based on quantitative nuclear magnetic resonance hydrogen spectrum and quantitative nuclear magnetic resonance carbon spectrum were obtained respectively. The average concentration and content of alkyl nitrite esters were calculated separately, and these were taken as the final concentration and final content of alkyl nitrite esters.

8. The method for quantitative detection of alkyl nitrite esters by NMR according to claim 7, characterized in that: The specific method for preparing an internal standard solution containing an internal standard of known concentration includes: weighing a known mass of 1,4-dimethoxybenzene as the internal standard, and diluting it with deuterated chloroform to obtain the internal standard solution.

9. The method for quantitative detection of alkyl nitrite esters by NMR according to claim 8, characterized in that: The specific steps for transferring the quantitative test solution to the nuclear magnetic resonance spectrometer and acquiring the quantitative proton and carbon NMR spectra of the quantitative test solution include: The quantitative test solution was subjected to a 90° hard pulse scan with a pulse sequence of zg90 to obtain the quantitative proton NMR spectrum; The quantitative test solution was subjected to inverted gated decoupled pulse scanning with a pulse sequence of zgig30 to obtain the quantitative carbon NMR spectrum.

10. The method for quantitative detection of alkyl nitrite esters by NMR according to claim 9, characterized in that: The CH peak is selected for quantitative analysis of alkyl nitrite esters in proton NMR spectroscopy. The CH3 peak is selected as the quantitative peak for alkyl nitrite esters in quantitative carbon NMR spectra.

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