Non-targeted detection method for drug components in electronic cigarette

Abstract

The invention belongs to the field of analysis and detection. The invention provides a non-targeted detection method for drug components in electronic cigarettes, which comprises the following steps: pretreating a to-be-detected sample, weighing the sample, adding 50% methanol, performing ultrasonic treatment, filtering with a filter membrane, taking the filtrate for ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) detection, and comparing the data detected by the UPLC-Q-TOF-MS with the data detected by the UPLC-Q-TOF-MS to obtain the drug components in the electronic cigarettes. And importing into analysis software, comparing with real standard substance secondary mass spectrum information in an MS-DIAL public database, screening, and judging that the first ion and the second ion can be successfully matched to be positive. According to the method, non-targeted screening is achieved, the method is not limited to a drug component database of targeted analysis in a laboratory or an existing standard substance, the possible false negative probability of targeted screening can be effectively reduced, a sample can be directly injected and analyzed after being treated, the method does not depend on synchronous injection of the standard substance, and the method can be used for large-scale screening of a common inspection and detection mechanism; the efficiency of identifying the drug components in the electronic cigarette is greatly improved, the detection time is short, and the effects in the aspects of detection limit, precision and recovery rate are excellent.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection, specifically relating to a non-targeted detection method for drug components in e-cigarettes. Background Technology

[0002] Currently, drug testing techniques mainly include morphological testing, chemical analysis, spectroscopy, chromatography, mass spectrometry, and immunoassay. The main targets for detection are synthetic cannabinoids, synthetic opioids, etomidate, etc. The samples are mainly seized drugs, biological samples, and environmental samples such as water and soil.

[0003] Morphological examination examines macroscopic features quickly and economically, but relies on experience and requires intact samples. Chemical analysis methods are characterized by inexpensive reagents, simple operation, fast reaction speed, intuitive conclusions, no need for special equipment, and few location restrictions, but lack specificity and sensitivity. Spectroscopy, especially nuclear magnetic resonance spectroscopy, has advantages such as non-destructive detection, small sample requirements, and applicability to various sample forms including solids and liquids. It can be used for structural analysis and has unique advantages when standard substances are scarce, but the instrument and maintenance costs are extremely high. Chromatography, especially gas chromatography and liquid chromatography, has advantages such as strong separation ability, accurate quantification, and wide applicability, but requires standard substances for comparison. Mass spectrometry has high sensitivity and high flexibility and is the core technology for drug testing, but the instrument cost is high and the detection environment requirements are high. Immunoassay has advantages such as high sensitivity, strong selectivity, simple operation, no need for special sample treatment, time saving, and low consumables. However, because the identification of each type of poison requires special reagents, and the reagents often have a certain time-limited effect, the promotion and application of this method are somewhat limited.

[0004] Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) combines the advantages of strong chromatographic separation and stable retention time with the high sensitivity and accurate confirmation of mass spectrometry, and is currently widely accepted as the "gold standard" in the field of drug testing technology. Screening for synthetic cannabis, synthetic opioids, and etomidate in e-cigarettes can be performed using UHPLC-MS / MS, with sample extraction using organic solvents and C0.05... 18 Gradient elution is performed using a reversed-phase column. Screening results are determined by comparing the retention times of samples and standards, as well as primary and secondary mass spectrometry data. Common mass spectrometry acquisition methods include triple quadrupole multiple reaction monitoring (MRM) mode and data-dependent scanning mode of high-resolution mass spectrometry. The triple quadrupole MRM method requires standard reference comparison, while current high-resolution mass spectrometry techniques often use standard references to establish a database for targeted screening before comparison.

[0005] The existing technology has the following drawbacks: (1) It is basically a targeted screening, which is powerless against new drug components or those not tested in the laboratory, and is prone to false negative results; (2) Screening methods require standard substances. Screening for multiple drug components requires corresponding standard substances, but drug standard substances are strictly controlled and are very difficult to obtain and use. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a non-targeted detection method for drug components in e-cigarettes. This detection method is a non-targeted screening method that is not limited to a database of drug components for targeted analysis in the laboratory or existing standard substances. It does not rely on the simultaneous injection of standard substances, which greatly improves the efficiency of identifying drug components in e-cigarettes. The quantitative analysis method developed accordingly has a short detection time and excellent performance in terms of detection limit, precision, and recovery rate.

[0007] The purpose of this invention is to provide a non-targeted detection method for drug components in electronic cigarettes, comprising the following steps: (1) Pretreatment of the sample to be tested: Weigh the sample, add 50% methanol, sonicate, filter through a filter membrane, and use the filtrate for detection by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS). (2) Import the data detected by UPLC-Q-TOF-MS into the analysis software and compare and screen it with the secondary mass spectrometry information of real standards in the MS-DIAL database. If both primary and secondary ions can be successfully matched, it is judged as positive.

[0008] This invention employs an ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) detection method. By comparing and screening the secondary mass spectrometry information with real standards in a database, drug components in e-cigarettes can be qualitatively detected.

[0009] Furthermore, the allowable tolerances for the precise mass numbers of primary and secondary ions are 0.01 and 0.025 Da, respectively.

[0010] Furthermore, the chromatographic conditions for UPLC-Q-TOF-MS were set as follows: C18 chromatographic column; The column temperature is 35-45℃; Mobile phase A is a 0.1% formic acid solution, and mobile phase B is a methanol-acetonitrile solution containing 0.1% formic acid. The volume ratio of methanol to acetonitrile in mobile phase B is 1:1. The flow rate is 0.1-0.5 mL / min. -1 ; The injection volume is 1-10 μL; Gradient elution procedure: From 0 to 1 minute, A is 85-95%. From 1 to 22 minutes, A decreased from 85-95% to 5%-20%, and remained at this level for 3 minutes. After 25-26 minutes, A increases from 5%-20% to 85-95%, and is maintained for 5 minutes.

[0011] Furthermore, the mass spectrometry conditions for UPLC-Q-TOF-MS were set as follows: The ionization method is electrospray ionization (ESI) source, positive ion mode; Electrospray voltage: 5500 V, de-clustering voltage: 80 V; Air curtain pressure: 35 psi, impact air pressure: 8 psi; The temperature of the spray gas and auxiliary heating gas is 550℃; IDA mode, with 10 candidate precursor ions; Preferably, the TOF MS scan range is 100~1000 Da, the collision energy is 10±0 V, and the cumulative time is 0.25 s; the TOFMS / MS scan range is 50~1000 Da, the collision energy is 35±15 V, and the cumulative time is 0.05 s.

[0012] Further, in step (1), the ratio of sample to 50% methanol is 0.05-0.35 g : 20-100 mL. The ultrasonic treatment frequency is 15-50 kHz, the power is 50-350 W, and the ultrasonic treatment time is 5 min-1 h. Microporous membrane filtration is specifically performed using a 0.01-0.35 μm microporous membrane. The C18 chromatographic column is a 100 mm × 2.1 mm, 1.8 μm C18 column.

[0013] Furthermore, the chromatographic conditions for quantitative determination are set as follows: The chromatographic column, column temperature, injection volume, and flow rate are the same as the chromatographic conditions in the above steps; Mobile phase A is a 0.1% formic acid solution, and mobile phase B is acetonitrile; Gradient elution procedure: From 0 to 5 minutes, A decreases from 65-95% to 15-35%, and remains at this level for 3 minutes. At 8.0-8.1 min, A increases from 15-35% to 65-95% and is maintained for 2 min.

[0014] Compared with the prior art, the present invention has the following advantages: (1) This invention establishes for the first time a method for non-targeted detection of drug components in e-cigarettes using UPLC-Q-TOF-MS. The non-targeted screening method is not limited to the drug component database or existing standard substances for laboratory targeted analysis, which can effectively reduce the probability of false negatives in targeted screening. (2) After processing, the sample can be directly injected for analysis without relying on the simultaneous injection of standard substances. Therefore, it can be used for large-scale screening by ordinary testing and inspection institutions, which greatly improves the efficiency of identifying drug components in e-cigarettes. The quantitative analysis method developed thereafter has a short detection time and excellent performance in terms of detection limit, precision and recovery rate. (3) The results obtained by the detection method of the present invention have a high degree of matching with the identification of drug components in e-cigarettes, and the detection limit can reach 7.5 μg·g. -1 The relative standard deviation (RSD) is approximately 2.1%, and the method is simple, low-cost, and easy to operate in batches.

[0015] In addition, it can also help professional drug testing laboratories reduce the use of controlled reference materials. Attached Figure Description

[0016] Figure 1 The total ion chromatogram (TIC) of the sample used for non-targeted screening in Example 1 of this invention; Figure 2 The mass spectrum of the component measured at retention time RT is 12.991 min; Figure 3 In the figure, A, B, and C are the extracted ion chromatograms (EIC) of the standard substance, positive sample, and negative sample, respectively, during the quantitative determination (m / z = 245.128 5). Detailed Implementation

[0017] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention. Example 1

[0018] Our hospital cooperated with the Wenzhou Municipal Market Supervision and Administration Bureau in carrying out a special rectification campaign against illegal e-cigarettes, and used this invention to non-targeted screen positive samples.

[0019] 1. Instruments and reagents The X500B ultra-high performance liquid chromatography-time-of-flight mass spectrometer, including a binary pump, column oven, autosampler, and time-of-flight mass spectrometer (QTOF), is manufactured by Ibrance Corporation, USA. Milli-Q Integral 5 ultrapure water system, Merck Millipore GmbH, Germany.

[0020] Etomidate standard with a purity of 99.8%, China National Institutes for Food and Drug Control; Methanol and acetonitrile were of chromatographic grade, and formic acid was of mass spectrometric grade, from Merck, Germany.

[0021] The samples were obtained from random inspections conducted by the Wenzhou Municipal Market Supervision and Administration Bureau as part of its special campaign to crack down on illegal electronic cigarettes.

[0022] 2. Weigh 0.2 g of sample and place it in a 100 mL volumetric flask. Add 60 mL of 50% (volume fraction) methanol, sonicate for 15 min, cool to room temperature, dilute to the mark with 50% methanol, shake well, filter with a 0.22 μm nylon microporous membrane, and take the filtrate for ultra-high performance liquid chromatography-time-of-flight mass spectrometry non-target screening.

[0023] 3. Instrument conditions 3.1 Chromatographic conditions for non-targeted screening Waters ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm); The column temperature is 40 ℃; Mobile phase A is a 0.1% formic acid solution, and mobile phase B is a methanol-acetonitrile (volume ratio 1:1) solution containing 0.1% formic acid; The flow rate is 0.3 mL·min -1 ; The injection volume was 5 μL; Gradient elution procedure: 0~1.0 min, A is 95%; 1.0~22.0 min, A decreased from 95% to 5%, and was maintained for 3 min; At 25.0~26.0 min, A increased from 5% to 95% and was maintained for 5 min.

[0024] 3.2 Chromatographic conditions for quantitative determination The chromatographic column, column temperature, flow rate, and injection volume are the same as the chromatographic conditions in 3.1 above; Mobile phase A is a 0.1% formic acid solution, and mobile phase B is acetonitrile; Gradient elution procedure: From 0 to 5.0 min, A decreased from 70% to 24% and remained at that level for 3 min; At 8.0~8.1 min, A increased from 24% to 70% and remained at that level for 2 min.

[0025] 3.3 Mass Spectrometry Conditions Electrospray ionization (ESI) source, positive ion mode; The electrospray voltage is 5500 V, and the de-clustering voltage is 80 V; The curtain air pressure is 35 psi, and the impact air pressure is 8 psi. The temperature of the spray gas and auxiliary heating gas is 550 ℃; IDA mode, with 10 candidate precursor ions; The TOF MS scan range is 100~1000 Da, the collision energy is 10±0 V, and the accumulation time is 0.25 s; The TOF MS / MS scan range is 50~1000 Da, the collision energy is 35±15 V, and the accumulation time is 0.05 s.

[0026] Figure 1 The TIC (Total Ion Chromatography) chromatogram is shown for samples from non-targeted screening. The mass spectrum of the component measured at RT (Retention Time) of 12.991 min is shown below. Figure 2 As shown.

[0027] 4. Data Analysis The above non-targeted screening data were converted into a new format using software and imported into MS-DIAL analysis software. Using the MS-DIAL database containing secondary mass spectrometry information for tens of thousands of real standards as a library, non-targeted screening was performed. The allowable tolerances for the precise mass numbers of primary and secondary ions were 0.01 and 0.025 Da, respectively. A positive result was defined as a match between both primary and secondary ions. The screened e-cigarette components are shown in Table 1.

[0028] Table 1 Non-targeted screening results

[0029] Among the components detected in the table, betaine is a common alkaloid in tobacco, nicotine is a component in e-liquid that relieves nicotine cravings, dioctyl phthalate is a common plasticizer, and etomidate is an anesthetic that was included in the list of Class II psychotropic drugs on October 1, 2023.

[0030] Precision testing Accurately weigh 20 mg of etomidate reference standard, dissolve and dilute it to 20 mL with 50% methanol to prepare a solution of 1 mg / mL. -1 The standard stock solution was then serially diluted with 50% methanol to prepare mass concentrations of 50, 100, 200, 300, 400 and 500 ng·mL. -1 A series of standard solutions.

[0031] Samples and standard solutions were taken for quantitative determination. The content was determined by external standard method using etomidate adduct ion [M+H]+ (m / z= 245.1285) as the quantitative ion.

[0032] Figure 3 In the figure, A, B, and C are the EIC (extracted ion chromatograms) of the standard substance, positive sample, and negative sample, respectively, during the quantitative determination (m / z=245.128 5).

[0033] Adding etomidate standard solution to negative samples to achieve a signal-to-noise ratio (S / N) of 3 resulted in a method detection limit of 7.5 μg·g. -1 .

[0034] 2, 10, and 20 mL of standard stock solution (equivalent to 20%, 100%, and 200% of the sample content) were added to the positive samples, respectively. Six parallel samples were prepared for each addition level, and the measurements were repeated six times on the same day. The relative standard deviation (RSD) of the measured values ​​was calculated to assess the intra-day precision of the method. Measurements were taken once a day for six consecutive days, and the RSD of the measured values ​​over the six days was calculated to assess the inter-day precision of the method. The results are shown in Table 2.

[0035] Table 2 Precision Test Results Data Table

[0036] The test data above show that the detection limit of the non-targeted screening method for drug components in e-cigarettes of this invention can reach 7.5 μg·g⁻¹. -1 The recoveries at low, medium, and high spiked levels were 92.2%, 109%, and 94.1%, respectively, indicating excellent accuracy in quantitative analysis. The intra-day relative standard deviation (RSD) reached 2.1%, and the inter-day RSD reached 4.7%, indicating excellent intra-day and inter-day precision in quantitative analysis. This invention can effectively screen for unknown drug components in e-cigarettes and can effectively reduce the probability of false negatives in targeted screening. The quantitative analysis method established based on non-targeted screening is accurate and reliable.

[0037] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes or modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention and should be defined by the claims.

Claims

1. A method of non-targeted detection of a drug component in an electronic cigarette, characterized in that, Comprising the following steps, (1) Pretreatment of the sample to be tested: weigh the sample, add 50% methanol, ultrasonic treatment, filter membrane filtration, take the filtrate for UPLC-Q-TOF-MS detection; (2) The data of UPLC-Q-TOF-MS detection is imported into the analysis software, and the secondary mass spectrum information of the real standard in the MS-DIAL database is compared and screened, and the one and two level ions that can be matched successfully are judged as positive.

2. The method of claim 1, wherein, The chromatographic conditions of UPLC-Q-TOF-MS are set, and the parameter settings are as follows: C18 chromatographic column; The column temperature is 35-45℃; The mobile phase A is 0.1% formic acid solution, and the B is methanol-acetonitrile solution containing 0.1% formic acid, wherein the volume ratio of methanol to acetonitrile is 1:1; Flow rate 0.1-0.5 mL min -1 ; The injection amount is 1-10μL; Gradient elution program: 0~1min, A is 85-95%, 1~22min, A is reduced from 85-95% to 5%-20%, and maintained for 3min, 25~26min, A is increased from 5%-20% to 85-95%, and maintained for 5min.

3. The method of claim 1, wherein, The mass spectrometry conditions of UPLC-Q-TOF-MS are set, and the parameter settings are as follows: The ionization mode is electrospray ionization ESI source, positive ion mode; Electrospray voltage: 5500 V; De-clustering voltage: 80 V; Gas curtain gas pressure: 35 psi; Collision gas pressure: 8 psi; Spray gas temperature: 550℃; IDA mode.

4. The method of claim 3, wherein, The TOF MS scan range is 100~1 000 Da, the collision energy is 10±0 V, and the accumulation time is 0.25 s; The TOF MS / MS scan range is 50~1 000 Da, the collision energy is 35±15 V, and the accumulation time is 0.05 s.

5. The method of claim 1, wherein, In step (1), the dosage ratio of sample to 50% methanol is 0.05-0.35g:20-100mL, the ultrasonic frequency of ultrasonic treatment is 15-50kHZ, the power is 50-350W, the ultrasonic treatment time is 5min-1h, and the pore size of filter membrane filtration is 0.01-0.35μm.

6. The method of claim 2, wherein, The chromatographic condition parameter settings for quantitative determination of drug components in electronic cigarettes are as follows: The chromatographic column, column temperature, and injection amount are the same as described in claim 2; The mobile phase A is 0.1% formic acid solution, and the B is acetonitrile; Gradient elution program: 0-5min, A is reduced by 15-35% from 65-95%, and maintained for 3min, 8.0~8.1 min, A is increased from 15-35% to 65-95%, and maintained for 2 min.

Images