Method for detecting total sugar in electronic cigarette liquid

Abstract

The invention discloses a method for detecting total sugar in electronic cigarette liquid. The method comprises the following steps: (1) pre-treating a sample; (2) LC-MS / MS detection is carried out; and (3) determining a standard curve and quantifying. By accurately measuring the total sugar content in the tobacco tar and combining the verified correlation model, whether the tobacco tar has the risk of burning smell or not can be rapidly judged, and large-scale suction actual measurement is not needed. The method has the advantages of high detection efficiency, low sample consumption and low pre-screening cost, is suitable for batch preliminary screening or conventional risk pre-judgment before marketing approval, can intercept high-risk tobacco tar in advance, and reduces subsequent actual measurement pressure.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemical analysis, more particularly to a method for detecting total sugar in electronic cigarette liquid. BACKGROUND

[0002] Research shows that the total sugar content in electronic cigarette liquid is significantly positively correlated with the generation of paste taste (a key quality defect). When the total sugar content exceeds 0.25% (mass fraction), there is a significant risk of causing paste taste problems. However, the current colorimetric detection method faces serious interference in actual application - electronic cigarette liquid itself contains a large amount of propylene glycol, glycerol (commonly used solvents) and various flavor components. These matrix components may produce background signals (interference intensity can reach the target detection level) equivalent to the total sugar color reaction in the blank experiment, resulting in the inability to accurately quantify the total sugar content. This limitation may cause two types of misjudgment risks: Missed true risk: if the total sugar content in the tobacco tar is actually more than 0.25% (but is masked by interference), it may be incorrectly determined as "no paste taste risk", allowing tobacco tar with potential paste taste problems to enter the market; False positive misjudgment: if the blank interference signal is mistaken for a total sugar positive signal (even if the actual total sugar content does not exceed the standard), it may lead to qualified tobacco tar without paste taste risk being incorrectly labeled as "high risk", and thus cannot be normally marketed.

[0003] Due to the low background content of total sugar in electronic cigarette liquid, the above interference problem further amplifies the judgment deviation, seriously affecting the effective identification of paste taste risk and product quality control.

[0004] Tobacco tar, as the core component of electronic cigarettes, needs to be approved by China Tobacco before it can be marketed. In order to ensure product compliance and market acceptance, products on the market must ensure no paste taste risk (paste taste is a key quality defect affecting user experience). If the paste taste risk test is not completed before the product is approved for marketing, the problem may not be detected until after the product is approved, which may result in the product being rejected or returned to work due to paste taste problems, causing significant waste of human, material and time resources. Therefore, the paste taste risk test must be a key pre-approval step before marketing.

[0005] Currently, the industry mainly uses the following two methods to conduct paste taste risk testing before marketing approval: Method one: combination of machine suction and manual suction for actual measurement verification Directly observe whether the burnt taste appears by simulating a real smoking scene and performing systematic smoking test on the cartridge made of the tobacco tar. Specifically, at least 100 cartridge samples are extracted, each cartridge is continuously smoked for 1-2 hours (mainly by machine smoking and supplemented by manual smoking to cover different smoking conditions), and the burnt taste risk is comprehensively evaluated. This method is direct and reliable, but has limitations such as long test period, large labor investment and large sample consumption, and is suitable for verification steps after high-risk screening.

[0006] Method two: rapid detection based on the correlation between total sugar content and burnt taste Studies have shown that the total sugar content in the tobacco liquid is strongly correlated with the burnt taste risk - when the total sugar content exceeds 0.2%, the burnt taste problem may be caused. By accurately determining the total sugar content in the tobacco tar and combining the verified correlation model, it can be quickly judged whether the tobacco tar has a burnt taste risk without large-scale actual measurement. This method has the advantages of high detection efficiency, low sample consumption and low pre-screening cost, and is suitable for batch screening before listing or routine risk prediction, which can intercept high-risk tobacco tar in advance and reduce subsequent actual measurement pressure.

[0007] Direct determination of total sugar content refers to directly determining the content of total sugar by using monosaccharide or total sugar as a control. Currently commonly used methods mainly include ultraviolet-visible spectrophotometry, high-performance gel permeation chromatography, near-infrared spectroscopy and iodometric method, etc. Ultraviolet-visible spectrophotometry is to hydrolyze total sugar into monosaccharide under the action of concentrated acid, form sugar aldehyde derivatives through dehydration condensation, combine with corresponding color developing agents to develop color, and then determine the absorbance after color development to quantify. Common color developing methods include phenol-sulfuric acid method, anthrone-sulfuric acid method, 3-5-dinitrosalicylic acid-sulfuric acid method, etc. This kind of color developing method is a general quantitative method, which can produce color reaction as long as the compound contains carbonyl group, and has the problems of false positive, large interference and low accuracy. High-performance gel permeation chromatography first needs to establish a method for analyzing the molecular weight and distribution of total sugar, determine the characteristic chromatographic peak, and then separate the quality marker by ultrafiltration to obtain the standard. The selection and separation of the marker are often limited, and the sensitivity of the method is generally low. Near-infrared spectroscopy is not only related to the chemical components of the sample to be measured, but also affected by the size, shape and density of the sample. SUMMARY

[0008] Therefore, the present application provides a method for detecting total sugar in electronic cigarette liquid. This method uses phenol-sulfuric acid method for hydrolysis and derivation, and uses high-resolution mass spectrometry for qualitative analysis of the color developing substance. Then, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) is used for testing, which reduces interference and improves the sensitivity of analysis. The minimum detection concentration is 0.003 μg / mL.

[0009] To achieve the above object, the present application adopts the following technical solutions: A method for detecting total sugar in electronic cigarette liquid, comprising the following steps: (1) Sample pretreatment The phenol-sulfuric acid method is used for hydrolysis and derivation treatment of the total sugar standard curve solution and the electronic cigarette liquid, respectively. (2) LC-MS / MS detection After hydrolysis and derivation treatment, the electronic cigarette liquid is directly detected by an ultra-high performance liquid chromatograph after separation by a chromatographic column. The chromatographic conditions include setting the injection volume, mobile phase composition, flow rate, column temperature, and gradient elution program. The mass spectrometry conditions include using an electrospray ionization ion source, setting the ionization voltage, collision energy, and scanning range, and using the MS / MS mode for qualitative and quantitative analysis of target compounds. The mobile phase composition includes: mobile phase A: 0.1% formic acid solution, and mobile phase B: acetonitrile. (3) Standard curve and quantification Under the set LC-MS / MS analysis conditions, the response value of the total sugar standard curve solution is determined, and the standard curve is drawn.

[0010] Preferably, in the phenol-sulfuric acid method of step (1), 4% phenol solution and sulfuric acid are used. Preparation of 4% phenol solution: weigh 4g of phenol, add first-grade water to 100mL, mix well, and obtain.

[0011] Preferably, the parent ion of the color-developing substance generated by hydrolysis and derivation of the total sugar standard curve solution is m / z: 281.0, and the fragment ions are m / z: 201.0 and m / z: 80.0.

[0012] Preferably, the chromatographic column in step (2) is HSS T3 2.5um 3.0*150mm.

[0013] Preferably, the injection volume in step (2) is 3ul.

[0014] Preferably, the flow rate in step (2) is 0.4ml / min.

[0015] Preferably, the column temperature in step (2) is 40℃.

[0016] Preferably, the gradient elution procedure in step (2) is: 0 min, A 85%, B 15%; 1.0 min, A 85%, B 15%; 7.0 min, A 10%, B 90%; 7.1 min, A 85%, B 15%; 10.0 min, A 85%, B 15%.

[0017] Preferably, the ionization voltage in step (2) comprises a capillary voltage and a cone voltage; the capillary voltage is negative mode 2.0 kv, and the cone voltage is 20 ev.

[0018] Preferably, the temperature of the electrospray ionization ion source in step (2) is 120 DEG C.

[0019] Preferably, the total sugar comprises monosaccharides (such as glucose, fructose), disaccharides (such as sucrose, maltose), oligosaccharides and polysaccharides (such as starch, cellulose).

[0020] It can be known from the above technical solution that, compared with the prior art, the present application has the following beneficial effects: By accurately determining the total sugar content in the tobacco tar, and combining the verified correlation model, it can be quickly judged whether the tobacco tar has a paste taste risk without large-scale smoking measurement. The method has the advantages of high detection efficiency, less sample consumption, and low pre-screening cost, is suitable for batch preliminary screening or regular risk prediction before market approval, can intercept high-risk tobacco tar in advance, and reduces subsequent measurement pressure. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0022] Figure 1 The first mass spectrum for color developing substance identification; Figure 2 The second mass spectrum for color developing substance identification; Figure 3 Typical spectrum; Figure 4 In the figure, from top to bottom, the corresponding spectrum of the injection amount 10 μL, 1 μL, 3 μL, respectively; Figure 5 In the figure, from top to bottom, the corresponding spectrum of the flow rate 0.7 mL / min, 0.2 mL / min, 0.5 mL / min, respectively; Figure 6 In the figure, from top to bottom, the corresponding spectrum of the column temperature 45 DEG C, 25 DEG C, 40 DEG C, respectively. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0024] This invention first utilizes the phenol-sulfuric acid method to hydrolyze and derivatize total sugars, generating chromogenic substances, which are then qualitatively identified by high-resolution mass spectrometry (LC-QTOF). (See attached image.) Figure 1 and Figure 2 The precursor ion was determined to be m / z: 281.0, and the fragment ions were m / z: 201.0 and m / z: 80.0. Following qualitative analysis, the following procedures were performed: Detection method: 1. Solution preparation (the amount of solution prepared can be adjusted according to actual needs).

[0025] 1.1 Sample diluent: ultrapure water.

[0026] 1.2 Mobile phase A: 0.1% formic acid solution.

[0027] 1.3 Mobile phase B: Acetonitrile solution.

[0028] 1.4 Preparation of 4% phenol solution: Weigh 4g of phenol, add primary water to make up to 100mL, and mix well; concentrated sulfuric acid is used. 1.5 Preparation of reference stock solution: Accurately weigh 0.03 g of anhydrous glucose reference standard, add primary water to make up to 50 mL, mix well to obtain an anhydrous glucose standard stock solution of 0.6 mg / mL.

[0029] 1.6 Preparation of anhydrous glucose standard curve solution: Accurately measure 0.02 ml, 0.04 ml, 0.06 ml, 0.08 ml, and 0.10 ml of the reference stock solution, respectively, dilute with primary water to 1 mL, place in 10 mL centrifuge tubes, and mix well to obtain anhydrous glucose standard curve solutions with concentrations of 0.012 mg / mL, 0.024 mg / mL, 0.036 mg / mL, 0.048 mg / mL, and 0.060 mg / mL.

[0030] 1.7 Precision take anhydrous glucose standard curve 0.10 mL, dilute to 1 mL with first grade water, mix well to obtain anhydrous glucose standard curve test solution with concentrations of 0.24 ppm, 0.48 ppm, 0.72 ppm, 0.96 ppm and 1.20 ppm. Precision take 42 uL of the above 0.24 ppm anhydrous glucose standard curve test solution, dilute to 1 mL with first grade water to obtain 0.01 ppm anhydrous glucose standard curve test solution.

[0031] 1.8 Preparation of sample solution: weigh 0.5 g of tobacco tar, dilute to 10 mL with first grade water, mix well, then precision take 0.20 mL of the solution, dilute to 10 mL with first grade water, mix well, and transfer 1.0 mL of the above solution to a 10 mL centrifuge tube to obtain the sample solution (the dilution factor can be adjusted according to the type of sample).

[0032] 1.9 Hydrolysis and derivatization step: add 0.5 ml of 4% phenol solution to each of the anhydrous glucose standard curve test solution and sample solution, mix well, quickly add 4.5 ml of sulfuric acid, mix well, incubate in a 40°C water bath for 30 minutes, remove, place in an ice water bath for 5 minutes, remove, and dilute the derived sample with first grade water by 10 times.

[0033] 2. Chromatographic conditions Table 1

[0034] Table 2: Mass spectrometry parameters

[0035] Table 3: MRM parameters

[0036] 2.1 Injection analysis: injection analysis: ultrapure water solution, standard curve solution, sample solution.

[0037] 2.2 System suitability acceptable standard: Linear correlation coefficient R of total sugar component 2 ≥ 0.99.

[0038] Verification conclusion: 1.1 Specificity 1.1.1 Investigate whether the products obtained after hydrolysis and derivatization of fructose, glucose and pullulan are consistent; investigate whether the detection of derivatives is interfered in the tobacco tar matrix.

[0039] 1.1.2 Solution preparation: Fructose 0.74 μg / ml standard solution: weigh 100 mg of fructose into a 10 mL volumetric flask, add ultrapure water to dissolve and dilute to the mark, shake well; take the above solution, dilute to 44.4 ug / mL with ultrapure water; the following hydrolysis and derivation steps are operated to obtain.

[0040] Glucose 0.74 μg / ml standard solution: weigh 100 mg of glucose into a 10 mL volumetric flask, add ultrapure water to dissolve and dilute to the mark, shake well; take the above solution, dilute to 44.4 ug / mL with ultrapure water; the following hydrolysis and derivation steps are operated to obtain.

[0041] Pullulan 0.5 μg / ml standard solution: weigh 100 mg of pullulan into a 10 mL volumetric flask, add ultrapure water to dissolve and dilute to the mark, shake well; take the above solution, dilute to 30 ug / mL with ultrapure water; the following hydrolysis and derivation steps are operated to obtain.

[0042] E-liquid sample solution: weigh about 0.5 g of e-liquid into a 10 ml capacity, add ultrapure water to dilute to the mark, shake well to obtain; take 0.2 mL of the above solution, add 9.8 mL of ultrapure water and mix well for use; the following hydrolysis and derivation steps are operated to obtain.

[0043] E-liquid sample solution with standard addition: weigh about 0.5 g of e-liquid into a 10 ml capacity, add ultrapure water to dilute to the mark, shake well to obtain; take 0.2 mL of the above solution, take 0.5 mL of 600 μg / mL pullulan solution, add 9.3 mL of ultrapure water and mix well for use; the following hydrolysis and derivation steps are operated to obtain.

[0044] Hydrolysis and derivation steps: take 1.00 mL of the test solution into a 10 mL centrifuge tube, add 0.5 ml of 4% phenol solution, mix well, quickly add 4.5 ml of sulfuric acid, mix well, incubate in a 40℃ water bath for 30 minutes, take out, place in an ice water bath for 5 minutes, take out, dilute the derived sample with first grade water by 10 times.

[0045] 1.1.3 Injection analysis: injection analysis of blank solution and the above test solution.

[0046] 1.1.4 Acceptable standard: the blank solution has no interference with the detection components; the e-liquid matrix has no interference with the detection components.

[0047] 1.1.5 Experimental results: Table 4:

[0048] Table 5: Interference of matrix on compound detection

[0049] Conclusion: The retention times of fructose, glucose, and pullulan were 4.70 min, 4.70 min, and 4.70 min, respectively; it was indicated that the color-developing substances formed after the hydrolysis and derivatization of monosaccharides (fructose and glucose) and pullulan were consistent; the recovery rate of the pullulan content measured with glucose as the standard was 108.2%, and the subsequent verification was performed with pullulan as the standard.

[0050] The blank solution had no interference with the detection components, and the recovery rate of the standard addition solution in the electronic cigarette liquid was 80.5%, which was within the acceptable standard range of 80%-120%, indicating that the electronic cigarette liquid matrix had no interference with the detection components.

[0051] 1.2 Linearity 1.2.1 Operation method: sequentially inject the linear solution, take the peak area ratio of the components in the linear solution as the ordinate, and take the component concentration in the standard working solution as the abscissa to draw the standard curve.

[0052] 1.2.2 Acceptable standard: the linear correlation coefficient R of the component standard curve 2 ≥ 0.99.

[0053] 1.2.3 Experimental results: Table 6

[0054] Conclusion: the linear correlation coefficient of the component was 0.9988, which met the acceptable standard of ≥ 0.99, indicating good linearity.

[0055] 1.3 Quantitative limit and detection limit 1.3.1 Solution preparation: Quantitative limit test solution: take the linear minimum point as the quantitative limit test solution.

[0056] Detection limit test solution: dilute with an appropriate amount of 50% methanol solution to a total sugar concentration of 0.0033 μg / ml. The dilution concentration can be adjusted according to the actual test results until the appropriate concentration is found.

[0057] 1.3.2 Acceptable standard: The S / N of the total sugar component in the quantitative limit solution was ≥ 10:1.

[0058] The S / N of the total sugar component in the detection limit solution was ≥ 3:1.

[0059] 1.3.3 Experimental results: Table 7

[0060] Conclusion: The total sugar limit of quantification solution of 0.01 ug / ml has a S / N of 260, meeting the requirement of ≥10:1.

[0061] The total sugar limit of detection solution of 0.003 ug / ml has a S / N of 62, meeting the requirement of ≥3:1.

[0062] 1.4 Accuracy 1.4.1 Solution Preparation: 1.4.1.1 Total Sugar Standard Solution: Take 100 mg of pullulan into a 10 mL volumetric flask, add ultrapure water to dissolve and dilute to the mark, shake well; take the above solution, dilute to 600 ug / mL with ultrapure water.

[0063] 1.4.1.2 Sample Solution: Weigh about 0.5 g of e-liquid into a 10 ml capacity, add ultrapure water to dilute to the mark, shake well.

[0064] 1.4.1.3 Hydrolysis and Derivation Step: Take 1.00 mL of the solution to be tested into a 10 mL centrifuge tube, add 0.5 ml of 4% phenol solution, mix well, quickly add 4.5 ml of sulfuric acid, mix well, incubate in a 40°C water bath for 30 minutes, take out, place in ice water bath for 5 minutes, take out, dilute the derived sample with primary water by 10 times.

[0065] 1.4.1.4 Prepare accuracy solutions according to the following table: Table 8

[0066] 1.4.2 Operation Method: The accuracy solutions are sequentially injected, the peak area is recorded, and the concentration of each component in the solution is calculated according to the standard curve.

[0067] 1.4.3 Calculation Formula: 。

[0068] 1.4.4 Acceptable Standard: The recovery rate of each accuracy solution should be within 80%-120%.

[0069] 1.4.5 Experimental Results: Table 9

[0070] Conclusion: The recovery rate of each concentration of the spiked solution is 80.9%-88.9%, which meets the acceptable standard of 80%-120%.

[0071] 1.5. Precision 1.5.1 Reproducibility: Solution Preparation: Prepare 6 precision solutions according to accuracy solution-4.

[0072] Operation method: The solutions were injected in turn, and the peak area was recorded. According to the standard curve, the concentration of each component in the solution was calculated.

[0073] 1.5.2 Acceptable standard: the RSD of the concentration of each component in 6 precision solutions was ≤10.0%.

[0074] 1.5.3 Intermediate precision: the operation under the repeatability item was repeated by different personnel.

[0075] 1.5.4 Acceptable standard: the RSD of the concentration of each component in 12 precision solutions was ≤10.0%.

[0076] 1.5.5 Experimental results: Table 10

[0077] Table 11

[0078] Conclusion: The RSD of the total sugar component in the 6 repeatability test solutions was 3.7%, which met the acceptable standard of ≤10.0%.

[0079] The maximum RSD of the total sugar component in the 12 test solutions was 5.6%, which met the acceptable standard of ≤10.0%, indicating that the method precision was good.

[0080] 1.6 Solution stability 1.6.1 The repeatability Sample 1 was used as the solution stability test sample, and was stored at 10°C for 1, 3, and 5 days before being repeatedly tested to confirm the stability of the solution. The average of the repeatability test results was used as the 0 time result. The storage time could be adjusted according to the actual situation.

[0081] 1.6.2 Acceptable standard: the ratio of the test results after storage to the 0 time results should be within the range of 90%-110%.

[0082] 1.6.3 Experimental results: Table 12

[0083] Conclusion: After the test solution was stored at 10°C for 3 days, the ratio of the total sugar concentration in the solution to the 0 time was within the range of 102.6%, which met the acceptable standard of within the range of 90%-110%. It was indicated that the test solution was stable within 3 days at 10°C.

[0084] The various embodiments described in this specification are implemented in a progressive manner, each embodiment focusing on the differences from other embodiments, and the same or similar parts between embodiments can be mutually referred to. For the apparatus disclosed by the embodiments, since it corresponds to the method disclosed by the embodiments, the description is relatively simple, and the relevant parts can be referred to the description of the method part.

[0085] The above description of disclosed embodiments enables one of ordinary skill in the art to make or use the application. Various modifications to these embodiments will be readily apparent to those of ordinary skill in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Therefore, the application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting total sugar in e-liquid, characterized in that, Comprising the following steps: (1) Sample pretreatment The total sugar standard curve solution and the electronic cigarette liquid were hydrolyzed and derivatized by phenol-sulfuric acid method respectively; (2) LC-MS / MS detection The electronic cigarette liquid treated by hydrolysis and derivatization was directly separated by a chromatographic column and then detected by a mass spectrometer after using an ultra-high performance liquid chromatograph; Among them, the chromatographic conditions are set as follows: injection volume, mobile phase composition, flow rate, column temperature and gradient elution program; The mass spectrometry conditions are set as follows: using electrospray ionization ion source, setting ionization voltage, collision energy and scanning range; using MS / MS mode for qualitative and quantitative analysis of target compounds; The mobile phase composition is as follows: mobile phase A: 0.1% formic acid solution, mobile phase B: acetonitrile; (3) Standard curve and quantification Under the set LC-MS / MS analysis conditions, the response value of the total sugar standard curve solution is determined, and the standard curve is drawn; then the response value of the electronic cigarette liquid is compared with the standard curve, and the content of each compound in the sample is calculated. 2.The method of claim 1, wherein, In step (1), concentrated sulfuric acid and 4% phenol solution are used in the phenol-sulfuric acid method; 4% phenol solution preparation: weigh 4g of phenol, add first-grade water to 100mL, mix well to obtain. 3.The method of claim 1, wherein, The parent ion of the color-developing substance generated by hydrolysis and derivatization of the total sugar standard curve solution is m / z: 281.0, and the fragment ions are m / z: 201.0 and m / z: 80.

0. 4.The method of claim 1, wherein, In step (2), the chromatographic column is HSS T3 2.5um 3.0*150mm, and the column temperature is 40℃.

5. The method for detecting total sugar in e-liquid according to claim 1, characterized in that, In step (2), the injection volume is 3ul. 6.The method of claim 1, wherein, In step (2), the flow rate is 0.4ml / min.

7. The method according to claim 1, characterized in that, In step (2), the gradient elution program is as follows: 0min, A 85%, B 15%; 1.0min, A 85%, B 15%; 7.0min, A 10%, B 90%; 7.1min, A 85%, B 15%; 10.0min, A 85%, B 15%. In step (2), the ionization voltage includes capillary voltage and cone voltage; the capillary voltage is negative mode 2.0kv, and the cone voltage is 20ev. In step (2), the temperature of the electrospray ionization ion source is 120℃. 8.The method of claim 1, wherein the total sugar in the e-liquid is detected by the method. The total sugar includes monosaccharides, disaccharides, oligosaccharides and polysaccharides. 9.The method of claim 1, wherein, ​ 10. The method for detecting total sugar in e-liquid according to any one of claims 1-9, characterized in that, ​

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