Method for determining the content of etomidate in e-liquid

Abstract

The application discloses a kind of belongs to analytical detection technical field, specifically related to a kind of determination method of etomidate content in electronic cigarette oil, comprising the following steps: using step-by-step dilution method to pretreat the electronic cigarette oil to be measured to obtain test sample;Using high performance liquid chromatography tandem mass spectrometry technique to determine the content of etomidate in the test sample;The chromatographic conditions in the high performance liquid chromatography tandem mass spectrometry technique include: chromatographic column: n-octadecyl column;Mobile phase A phase: 4mmol / L-6mmol / L ammonium acetate+0.05% (v / v)-0.15% (v / v) formic acid aqueous solution;Mobile phase B phase: methanol.The determination method of etomidate content in the electronic cigarette oil can not only greatly save the solvent usage amount, but also can ensure the accuracy of detection, reduce the influence of matrix effect.

Description

Technical Field

[0001] This invention belongs to the field of analytical testing technology, specifically relating to a method for determining the content of etomidate in e-cigarette liquid. Background Technology

[0002] Etomidate, with the molecular formula C14H16N2O2, CAS: 33125-97-2, and molecular weight: 244.29, belongs to the imidazole derivative class and is mainly used clinically for general anesthesia and sedation. [1-2] The drug. There are generally two ways in which drug users ingest etomidate: one is to remove some tobacco from cigarettes and spray etomidate onto the tobacco; the other is to add etomidate to ordinary e-cigarette liquid.

[0003] Aside from a few review articles and commentaries, no case reports on the use of etomidate, a substitute drug, have been found in China. In recent years, South Korean researchers have reported on cases related to etomidate use. In 2019, Jung YK et al. detected etomidate and its metabolite etomidate acid in the urine of individuals who had used etomidate. In 2021, Yum H et al. detected etomidate and metometase in the blood of individuals who died from veterinary use of etomidate and metometase. In 2022, Park YJ et al. detected etomidate and its metabolite etomidate acid in the hair of laboratory animals. Current technologies for detecting etomidate in e-cigarette liquids mostly use high-performance liquid chromatography (HPLC), but the complex composition of e-cigarette liquids may interfere with the detection of etomidate. Therefore, there is an urgent need to develop a more accurate method for the quantitative detection of etomidate in e-cigarette liquids. Summary of the Invention

[0004] The purpose of this invention is to provide a method for determining the etomidate content in e-cigarette liquid, which can accurately detect the etomidate content in e-cigarette liquid.

[0005] The following technical solutions are used to achieve the above objectives.

[0006] The first aspect of this invention provides a method for determining the content of etomidate in electronic e-cigarette liquid, comprising the following steps:

[0007] The test sample was obtained by pretreatment of the e-liquid to be tested using a stepwise dilution method.

[0008] The content of etomidate in the test sample was determined by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS); the chromatographic conditions of the HPLC-MS / MS technique included:

[0009] Chromatographic column: n-octadecyl column;

[0010] Mobile phase A phase: 4mmol / L-6mmol / L ammonium acetate + 0.05% (v / v)-0.15% (v / v) formic acid aqueous solution;

[0011] Mobile phase B phase: methanol.

[0012] In some embodiments, the step of pretreating the electronic cigarette liquid to be tested by using the stepwise dilution method to obtain the test sample includes the following steps:

[0013] S1, adding methanol solution containing methoxyphenamine internal standard to the electronic cigarette liquid to be tested for ultrasonic treatment;

[0014] S2, sequentially diluting the electronic cigarette liquid after ultrasonic treatment with methanol solution containing methoxyphenamine internal standard to a final concentration of 8 μg / mL-12 μg / mL, and filtering to obtain the test sample.

[0015] In some embodiments, the dilution multiple is 10-30 times.

[0016] In some embodiments, in step S2, the following steps are included:

[0017] Diluting the electronic cigarette liquid after ultrasonic treatment with methanol solution containing methoxyphenamine internal standard to a concentration of 4 mg / mL-6 mg / mL;

[0018] Diluting the diluted electronic cigarette liquid solution with methanol solution containing methoxyphenamine internal standard to a final concentration of 0.1 mg / mL-0.5 mg / mL;

[0019] Further diluting the diluted electronic cigarette liquid solution with methanol solution containing methoxyphenamine internal standard to a final concentration of 8 μg / mL-12 μg / mL.

[0020] In some embodiments, in step S2, the following steps are included:

[0021] Diluting the electronic cigarette liquid after ultrasonic treatment with methanol solution containing methoxyphenamine internal standard to a concentration of 4.8 mg / mL-5.2 mg / mL;

[0022] Further diluting the diluted electronic cigarette liquid solution with methanol solution containing methoxyphenamine internal standard to a final concentration of 0.2 mg / mL-0.3 mg / mL.

[0023] Further diluting the diluted electronic cigarette liquid solution with methanol solution containing methoxyphenamine internal standard to a final concentration of 9.8 μg / mL-10.2 μg / mL.

[0024] In some embodiments, the electronic cigarette oil after dilution in step S2 is filtered with a 0.2-0.3 pm polytetrafluoroethylene membrane to obtain the test sample.

[0025] In some embodiments, the methoxyphenamine internal standard is dissolved in methanol at a concentration of 0.8-1.2 ng / mL.

[0026] In some embodiments, the n-octadecyl column is an Agilent Poroshell 120EC-C18 2.1 x 50 mm, 1.9 pm.

[0027] In some embodiments, the chromatographic conditions include:

[0028] Mobile phase A: 4.5-5.5 mmol / L ammonium acetate + 0.07-0.13% (v / v) formic acid in water.

[0029] In some embodiments, the chromatographic conditions further include:

[0030] The elution is performed in gradient mode with the following elution program:

[0031] 0-1.0 min, 95% A; 1.0-4.5 min, 95%-15% A; 4.5-7.0 min, 15% A; 7.01-10.00 min, 95% A.

[0032] In some embodiments, the chromatographic conditions include:

[0033] Chromatographic column: Agilent Poroshell 120EC-C18 2.1 x 50 mm, 1.9 pm;

[0034] Mobile phase A: 4.8-5.2 mmol / L ammonium acetate + 0.09-0.11% (v / v) formic acid in water.

[0035] Mobile phase B: methanol.

[0036] Flow rate: 0.2-0.3 mL / min, injection volume: 4-6 pL.

[0037] Column temperature: 34-36°C.

[0038] In the present application, the inventors found that in the method for determining the content of etomidate in electronic cigarette oil, by using stepwise dilution method for pretreatment of the electronic cigarette oil to be tested to obtain the test sample, combined with the use of high performance liquid chromatography tandem mass spectrometry technology, improvement of chromatographic conditions and selection of specific chromatographic column, etc. Operations, not only can greatly save the amount of solvent used, but also can ensure the accuracy of detection, and make the recovery of etomidate detection higher, and the relative average deviation RSD between different batches of electronic cigarette oil is smaller, reduce the influence of matrix effect, improve the accuracy and precision of the determination of etomidate content in electronic cigarette oil. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is the signal response graph obtained by detecting the electronic cigarette oil test sample under the chromatographic condition of methanol: 5mmol / L ammonium acetate + 0.1% formic acid buffer as the mobile phase.

[0040] Figure 2 is the signal response graph obtained by detecting the electronic cigarette oil test sample under the chromatographic condition of acetonitrile: 20mmol / L ammonium acetate + 0.1% formic acid buffer as the mobile phase.

[0041] Figure 3 (1) and Figure 3 (2) are the result graphs obtained by continuously performing the same test twice using chromatographic column Phenomenex Kinetex Biphenyl 100x4.6mm, 2.6μm.

[0042] Figure 4 (1) and Figure 4 (2) are the result graphs obtained by continuously performing the same test twice using chromatographic column Agilent Poroshell 120EC-C182.1x50mm, 1.9μm. DETAILED DESCRIPTION

[0043] In order to facilitate the understanding of the present application, the present application will be described more fully below. The present application can be realized in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0044] The experimental methods in the following examples not specified in the specific conditions are usually carried out according to the conventional conditions or according to the conditions recommended by the manufacturer. The various common chemical reagents used in the examples are commercially available products.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0046] The technical solutions of the present application will be described in detail below in combination with specific embodiments.

[0047] Instruments and reagents

[0048] Agilent 1290 II-6470 QQQ LC-MS / MS liquid chromatography-tandem mass spectrometry instrument (Agilent, USA); electronic balance, adjustable pipette, range 20 μL-1000 μL (Eppendorf).

[0049] Etomidate (10 mg, purity≧98.5%, purchased from Shanghai Yuansisil Biological Technology Co., Ltd.); methoxyphenamine (100 mg, content 99.9%, purchased from China Institute for Drug Control). Acetonitrile, methanol and formic acid are all chromatographically pure (Merck, USA). Ammonium acetate HPLC, purity≥97% (J.T. Baker, USA), and experimental water is ultrapure water.

[0050] Example 1

[0051] The present embodiment provides a preparation method of a test sample for determining the content of etomidate in electronic cigarette oil, comprising the following steps:

[0052] 1. Preparation of methanol solution containing methoxyphenamine internal standard

[0053] Accurately weigh 10 mg of methoxyphenamine in a 10 mL volumetric flask, add an appropriate amount of methanol to dissolve and dilute to the mark, to obtain a methoxyphenamine internal standard stock solution with a concentration of 1.0 mg / mL.

[0054] Accurately transfer 10 μL of the methoxyphenamine internal standard stock solution into a 10 mL volumetric flask, and dilute to the mark with methanol to obtain a methoxyphenamine internal standard intermediate stock solution of 1.0 μg / mL.

[0055] Accurately transfer 1 mL of the methoxyphenamine internal standard intermediate stock solution into a 1000 mL volumetric flask, and dilute to the mark with methanol to obtain a methanol solution containing methoxyphenamine internal standard of 1.0 ng / mL.

[0056] 2. Preparation of test sample of electronic cigarette oil to be tested

[0057] Precisely weigh 50 mg of e-liquid sample, place it in a 10 mL volumetric flask, add 7 mL of methanol solution containing 1.0 ng / mL methoxyphenamine internal standard, ultrasonic for 10 min, then dilute to the mark with methanol solution containing methoxyphenamine internal standard, obtain a solution with a concentration of 5 mg / mL of e-liquid, take 0.5 mL of e-liquid solution with a concentration of 5 mg / mL, place it in a 10 mL volumetric flask, then dilute to the mark with methanol solution containing methoxyphenamine internal standard, obtain a solution with a concentration of 0.25 mg / mL of e-liquid, take 0.4 mL of e-liquid solution with a concentration of 0.25 mg / mL, place it in a 10 mL volumetric flask, then dilute to the mark with methanol solution containing methoxyphenamine internal standard, obtain a solution with a concentration of 10 μg / mL of e-liquid, filter with a 0.22 μm polytetrafluoroethylene (PTFE) membrane to obtain the test sample.

[0058] Example 2

[0059] The present embodiment provides a method for determining the content of etomidate in e-liquid, comprising the following steps:

[0060] 1. Reagent preparation

[0061] 1.1 Preparation of etomidate standard solution

[0062] Precisely weigh 10 mg of etomidate in a 10 mL volumetric flask, dilute to the mark with methanol, shake well, and obtain a standard stock solution of etomidate with a concentration of 1.0 mg / mL.

[0063] Precisely pipette 100 μL of etomidate standard stock solution into a 10 mL volumetric flask, dilute to the mark with methanol, shake well, and obtain an etomidate standard stock solution with a concentration of 10 μg / mL.

[0064] Precisely pipette 20 μL of etomidate standard stock solution into a 10 mL volumetric flask, dilute to the mark with methanol, shake well, and obtain an etomidate standard working solution with a concentration of 20 ng / mL.

[0065] 1.2 Preparation of mobile phase A: 5 mmol / L ammonium acetate and 0.1% formic acid buffer

[0066] Weigh 0.39 g of ammonium acetate, dissolve in 1000 mL of ultrapure water, add 1 mL of formic acid, mix well, filter, ultrasonic, and use.

[0067] 1.3 Preparation of test sample

[0068] The same as Example 1.

[0069] 2. Detect the content of etomidate in e-liquid by high performance liquid chromatography tandem mass spectrometry:

[0070] 2.1 Liquid chromatography conditions

[0071] Mobile phase A phase: 5 mmol / L ammonium acetate + 0.1% (v / v) formic acid aqueous solution; mobile phase B phase: methanol; flow rate was 0.25 mL / min, injection volume was 5.0 μL, column (Agilent Poroshell 120 EC-C18 2.1 x 50 mm, 1.9 μm) was connected with C18 guard column; column temperature was 35 °C, gradient elution: 0-1.0 min, 95% A; 1.0-4.5 min, 95%-15% A; 4.5-7.0 min, 15% A; 7.01-10.00 min, 95% A.

[0072] 2.2 Mass spectrometry conditions

[0073] a) Ion source: electrospray ionization - positive ion mode (ESI+);

[0074] b) Detection mode: multiple reaction monitoring (MRM);

[0075] c) Capillary voltage: 3000 V, nozzle voltage: 0 V;

[0076] d) Sheath gas temperature: 380 °C, sheath gas flow rate: 12 L / min, drying gas temperature: 350 °C, drying gas flow rate: 5 L / min, atomizer pressure: 30 Psi;

[0077] e) Ion residence time: 50 msec; mass spectrometry parameters are shown in Table 2.1.

[0078] Table 2.1 Mass spectrometry parameters

[0079]

[0080] Note: 1) is a quantitative ion pair.

[0081] Example 3 Methodology investigation of the method for determining the content of etomidate in electronic cigarette liquid of the present application

[0082] 3.1 Mobile phase selection

[0083] The strengths of the signal responses of etomidate were compared between the mobile phase acetonitrile: 20 mmol / L ammonium acetate + 0.1% (v / v) formic acid buffer and the mobile phase methanol: 5 mmol / L ammonium acetate + 0.1% (v / v) formic acid buffer, and it was found through a pre-test of 0.1 ng / mg etomidate that the signal response of the mobile phase methanol: 5 mmol / L ammonium acetate + 0.1% (v / v) formic acid buffer was stronger than that of the mobile phase acetonitrile: 20 mmol / L ammonium acetate + 0.1% (v / v) formic acid buffer. Figure 1 is a signal response graph obtained by detecting the electronic cigarette liquid test sample under the chromatographic condition of the mobile phase methanol: 5 mmol / L ammonium acetate + 0.1% (v / v) formic acid buffer, Figure 2is the signal response graph obtained by detecting the e-liquid sample under the chromatographic conditions of the mobile phase acetonitrile: 20 mmol / L ammonium acetate + 0.1% (v / v) formic acid buffer Figure 1 and Figure 2 It can be seen that the signal response of the mobile phase methanol: 5 mmol / L ammonium acetate + 0.1% (v / v) formic acid buffer is 36% higher than that of the mobile phase acetonitrile: 20 mmol / L ammonium acetate + 0.1% (v / v) formic acid buffer.

[0084] 3.2 Selection of chromatographic column

[0085] In this paper, the pre-test of 0.1 ng / mg etomidate was carried out on Agilent Poroshell 120EC-C18 2.1×50mm, 1.9μm and Phenomenex Kinetex Biphenyl 100×4.6mm, 2.6μm to evaluate the resolution, sensitivity, tailing factor and stability of the target etomidate. Among them, Figure 3 (1) and Figure 3 (2) are two continuous experiments using the same chromatographic column Phenomenex Kinetex Biphenyl 100×4.6mm, 2.6μm, from Figure 3 (1) and Figure 3 (2) it is found that the chromatographic column is not sensitive enough, there is peak broadening, and the chromatographic efficiency is poor. Figure 4 (1) and Figure 4 (2) are two continuous experiments using the same chromatographic column Agilent Poroshell 120EC-C18 2.1×50mm, 1.9μm, it is found that the chromatographic column is sensitive enough, the chromatographic peak is sharp, there is no peak broadening, and the chromatographic efficiency meets the requirements. Therefore, Agilent Poroshell 120EC-C18 2.1×50mm, 1.9μm has higher chromatographic efficiency and better stability than Phenomenex Kinetex Biphenyl 100×4.6mm, 2.6μm.

[0086] 3.3 Calibration curve, detection limit and quantitative limit test

[0087] Accurately pipette 50 mg of series of blank e-liquid into 10 mL volumetric flask, add 0, 10, 20, 50, 100, 250, 1000 ng etomidate standard solution respectively, and then follow the method of Example 2. Take etomidate mass as the abscissa, and the corresponding etomidate quantitative ion characteristic peak area as the ordinate to draw the calibration curve, and obtain the linear equation Y = 18949071.349967X - 277. The correlation coefficient, detection limit, recovery rate, and precision are shown in Table 3.1.

[0088] The test shows that the calibration curve of etomidate has good linear relationship, the linear range is 0.2 ng / mg to 20 ng / mg, the linear coefficient R 2 is greater than 0.99, which meets the determination requirements, the detection limit is 0.1 ng / mg, and the quantification limit is 0.2 ng / mg.

[0089] 3.4 Precision test and standard addition recovery test

[0090] Respectively take 6 portions of 50 mg of blank e-liquid sample into series of centrifuge tubes with cover, and add 100 ng of etomidate standard solution, and then follow the method of Example 2 to operate, and determine the content by the calibration curve method, compare with the theoretical value to calculate the recovery rate and relative standard deviation, and the results are shown in Table 3.1.

[0091] As shown in Table 3.1, the standard addition recovery rate of etomidate is 97.2%, and the RSD is 4.8%, and the accuracy and precision of the method can meet the quantitative analysis requirements.

[0092] Table 3.1 Method detection limit, calibration curve, standard addition recovery, and precision

[0093]

[0094] 3.5 Matrix effect test

[0095] Take 10 kinds of blank e-liquid matrixes of different sources, add etomidate standard solution of different mass concentrations, and prepare low, medium, and high quality control samples of three different mass concentrations. The added mass concentration of the target etomidate is 1 ng / mg, 5 ng / mg, and 20 ng / mg, and the sample is analyzed according to the method of Example 2. Each mass concentration sample is determined in parallel for 6 times, the peak area of the target to be detected is set as a; and the etomidate standard solution of the same mass concentration is analyzed at the same time, the peak area of the target to be detected is set as b. The matrix effect = a / b x 100%. The matrix effect is calculated by the peak area ratio of the added target of the blank e-liquid of different matrixes, and the results are shown in Table 3.2, the range is 86.4% to 105.5%, the average is 96.0%, and the matrix effect is not obvious.

[0096] Table 3.2 Etomidate matrix effect test

[0097]

[0098] Test Example 1 Influence of test samples prepared by different pretreatment methods on determination results of etomidate in e-liquid

[0099] In this test example, the preparation method of the test sample of the e-liquid to be measured in Reference Example 1 was used, and the e-liquid to be measured was pretreated by one-step dilution method and step-by-step dilution method, repeated 6 times. The dilution process and the influence on the determination results of etomidate in e-liquid by one-step dilution method are shown in Table 4.1, and the dilution process and the influence on the determination results of etomidate in e-liquid by step-by-step dilution method are shown in Table 4.2.

[0100] The accuracy can be calculated by the method of standard addition recovery: standard addition recovery = |measured value of standard addition sample - measured value of sample| / standard addition amount x 100%, since the blank e-liquid matrix is added, the test measured value is 0.

[0101] Table 4.1 Influence of one-step dilution method on determination results of etomidate in e-liquid

[0102] Table 4.2 Influence of step-by-step dilution method on determination results of etomidate in e-liquid

[0103]

[0104] From the above Tables 4.1 and 4.2, it can be seen that compared with the method of directly using one-step dilution, the pretreatment of the e-liquid to be measured by step-by-step dilution method can greatly save methanol solvent, reduce the influence of toxic and harmful substances on the environment, and the accuracy is also improved, with an average accuracy of 97.2%.

[0105] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, but as long as the combinations of the technical features do not contradict, they should be considered within the scope of the present disclosure.

[0106] The above-described embodiments only express several embodiments of the present application, which are described in detail and specifically, but should not be construed as limiting the scope of the patent. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the protection scope of the present patent should be subject to the appended claims.

Claims

1. A method for determining the etomidate content in electronic e-cigarette liquid, characterized in that, Includes the following steps: The test sample was obtained by pretreatment of the e-liquid to be tested using a stepwise dilution method. The content of etomidate in the test sample was determined by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS); the chromatographic conditions of the HPLC-MS / MS technique included: Chromatographic column: n-octadecyl column; Mobile phase A: 4 mmol / L~6 mmol / L ammonium acetate + 0.05% v / v~0.15% v / v formic acid aqueous solution; Mobile phase B: methanol; The n-octadecyl column is an Agilent Poroshell 120 EC-C18; The chromatographic conditions also include: Gradient elution mode is used, and the elution procedure is as follows: 0~1.0 min, 95% A; 1.0~4.5 min, 95%~15% A; 4.5~7.0 min, 15% A; 7.01~10.00 min, 95% A.

2. The determination method as described in claim 1, characterized in that, The step of pretreatment of the e-liquid to obtain the test sample using the stepwise dilution method includes the following steps: S1. Add a methanol solution containing methoxyphenamine internal standard to the e-liquid to be tested and perform ultrasonication. S2. The ultrasonically diluted e-liquid was gradually diluted with a methanol solution containing methoxyphenamine internal standard until the final concentration of the e-liquid was 8 μg / mL to 12 μg / mL. The sample was then filtered to obtain the test sample.

3. The determination method as described in claim 2, characterized in that, The dilution factor is 10 to 30 times.

4. The determination method as described in claim 3, characterized in that, Step S2 includes the following steps: The ultrasonically treated e-liquid was diluted with a methanol solution containing methoxyphenamine internal standard to a concentration of 4 mg / mL to 6 mg / mL. The diluted e-cigarette liquid solution was diluted with a methanol solution containing methoxyphenamine internal standard to a final concentration of 0.1 mg / mL to 0.5 mg / mL. The diluted e-cigarette oil solution was then diluted with a methanol solution containing methoxyphenamine internal standard to a final concentration of 8 μg / mL to 12 μg / mL.

5. The determination method as described in claim 2, characterized in that, In step S2, after the e-cigarette oil is diluted, it is filtered through a 0.2μm~0.3μm polytetrafluoroethylene membrane to obtain the test sample.

6. The determination method as described in claim 2, characterized in that, In a methanol solution containing methoxyphenamine as an internal standard, the concentration of methoxyphenamine is 0.8 ng / mL to 1.2 ng / mL.

7. The determination method according to any one of claims 1 to 6, characterized in that, The chromatographic conditions include: Mobile phase A: 4.5 mmol / L~5.5 mmol / L ammonium acetate + 0.07% v / v~0.13% v / v formic acid aqueous solution.

8. The determination method as described in claim 7, characterized in that, The chromatographic conditions include: Column: Agilent Poroshell 120 EC-C18 2.1×50mm, 1.9μm; Mobile phase A: 4.8 mmol / L~5.2 mmol / L ammonium acetate + 0.09% v / v~0.11% v / v formic acid aqueous solution; Mobile phase B: methanol; Flow rate: 0.2 mL / min ~ 0.3 mL / min, injection volume: 4 μL ~ 6 μL; Column temperature: 34℃~36℃.

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