A method for simultaneous determination of ten carbonyl compounds in tobacco flavors based on UPLC

Abstract

The invention discloses a method for simultaneously determining the contents of ten carbonyl compounds in tobacco flavors based on UPLC; the method uses ultra-high performance liquid chromatography, specially selected HSS T3 chromatographic columns, DNPH-HCL (2,4-dinitrophenylhydrazine hydrochloride) pre-column derivatization and ternary mobile phase for gradient elution, and is supplemented by other suitable experimental means to achieve simultaneous analysis and determination of ten carbonyl compounds in tobacco flavors, including formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone, butyraldehyde, acetoin and diacetyl; compared with the prior art in which only eight carbonyl compounds can be determined, the technical effect is significant, and the ten carbonyl compounds in tobacco flavors can be quickly and effectively separated completely.

Description

Technical Field

[0001] The invention relates to the technical field of analytical determination methods for carbonyl compounds in tobacco flavors, and in particular to a method for simultaneously determining the contents of ten carbonyl compounds in tobacco flavors based on UPLC. Background Art

[0002] In the prior art, the application of tobacco flavors in traditional cigarettes and e-cigarette oils has been quite mature. Whether it is the improvement of the taste and quality of tobacco in the flavoring process, or the blending of different flavors in the e-liquid, it plays a vital role. However, during its combustion and atomization process, carbonyl compounds are a class of harmful substances that have a strong irritating effect on the human sensory system and respiratory system; especially carbonyl compounds such as formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone, butyraldehyde, acetoin and diacetyl. Countries have formulated corresponding regulations to control their content. Therefore, it is particularly important to establish an efficient and rapid analysis method for these ten carbonyl compounds in tobacco flavors.

[0003] Due to the diversity and complexity of tobacco flavor matrices, qualitative and quantitative analysis is very difficult; further, it is necessary to separate the peaks of ten substances separated from each other from this complex matrix for qualitative and quantitative analysis, which is undoubtedly more difficult to handle and more complex to analyze; and in the prior art, the document with application number 201510523425.5 discloses a method for determining 8 carbonyl compounds in electronic cigarette liquid, which uses a DNPH solution prepared with dilute hydrochloric acid to react with the carbonyl compounds in the electronic cigarette liquid for derivatization reaction, uses Tris base to adjust the pH of the reaction solution, maintains the stability of the reaction product, and uses HPLC analysis to determine the 8 carbonyl compounds in the electronic cigarette liquid; however, compared with the above-mentioned ten substances that need to be analyzed, acetoin and diacetyl cannot be analyzed and determined in this method, and the analysis time of the entire method is long. It is also relatively long; in addition, there is a document with application number 201510425214.8 that discloses a method for determining the main carbonyl compounds in cigarette tobacco using the UPLC-IE method. The method analyzes the main carbonyl compounds in cigarette tobacco by ultra-high performance liquid chromatography isocratic elution (UPLC-IE), but this method can only determine "formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone, butyraldehyde", but cannot determine the two carbonyl compounds of acetoin and diacetyl; it can be seen that in the prior art, due to the high complexity of the tobacco flavor matrix, it is impossible to simultaneously analyze and determine the ten carbonyl compounds of formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone, butyraldehyde, acetoin and diacetyl, so it is necessary to establish an analytical method for accurately and quickly determining the ten carbonyl compounds in tobacco flavors. Summary of the invention

[0004] In view of the problem existing in the above-mentioned technology that it is impossible to effectively separate and accurately and quickly determine the ten carbonyl compounds in tobacco flavors, the present application provides a technical solution to solve the problem.

[0005] To achieve the above object, the present invention provides a method for simultaneously determining the contents of ten carbonyl compounds in tobacco flavors based on UPLC, comprising the following steps:

[0006] (1) Preparation of derivatization reagents

[0007] Weigh 2.00 g of DNPH-HCL in a 2 L beaker, add 500 mL of acetonitrile and 40 mL of phosphoric acid aqueous solution respectively, dissolve, add 500 mL of water and mix well to obtain a derivatization reagent; transfer to a brown reagent bottle and store away from light;

[0008] (2) Preparation of standard solution

[0009] Weigh 0.1000 g of acetoin and diacetyl respectively into a 10 mL brown volumetric flask, dilute with acetonitrile and dilute to the mark to obtain an acetoin solution and a diacetyl solution, and store at -18°C in the dark; then weigh 0.1 mL of the acetoin solution and 0.1 mL of the diacetyl solution into a 25 mL brown volumetric flask, add 20 mL of a derivatization reagent, shake well, react at room temperature for 20 min, add 1 mL of pyridine, and dilute to the mark with acetonitrile to obtain a first mixed standard stock solution;

[0010] Accurately weigh 0.0250 g of DNPH derivatives of formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone and butyraldehyde respectively into a 25 mL brown volumetric flask, and dissolve to volume with 50% acetonitrile to obtain the second mixed standard stock solution;

[0011] Accurately measure 0.5 mL of the first mixed standard stock solution and 0.3 mL of the second mixed standard stock solution respectively, dissolve them in a 50 mL brown volumetric flask and dilute to the mark with 50% acetonitrile to obtain the mixed standard mother solution;

[0012] Accurately measure 0.3mL, 0.5mL, 1mL, 3mL, and 5mL of the mixed standard mother solution into a 10mL brown volumetric flask, and dissolve it to the mark with 50% acetonitrile to obtain a gradient standard solution;

[0013] (3) Preparation of test samples

[0014] Weigh 0.8000g of electronic cigarette liquid into a 10mL brown volumetric flask, add 8mL of derivatization reagent, shake well, react at room temperature for 20min, add 0.4mL of pyridine, and dilute to the mark with acetonitrile; then filter with a PTFE filter membrane to obtain the sample filtrate to be tested;

[0015] UPLC conditions:

[0016] The chromatographic column used was HSS T3, with specifications of 2.1 × 100 mm, 2.7 μm;

[0017] Mobile phase: Phase A is 0.1% formic acid solution: acetonitrile = 80:20; Phase B is 0.1% formic acid solution: acetonitrile = 50:50; Phase C is 0.1% formic acid acetonitrile solution; Gradient elution conditions are as follows:

[0018]

[0019] Flow rate 0.3mL / min; column temperature 32℃; detection wavelength 365nm; injection volume 10μL; the gradient standard solution and the sample filtrate to be tested are measured according to the above steps. Among them, the acetonitrile, formic acid, phosphoric acid, and pyridine used in the above experimental method are all liquid chromatography pure reagents; the purity of DNPH-HCL is ≥98%; the purity of acetoin, formaldehyde, acetaldehyde, diacetyl, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone and butyraldehyde used in the standard solution is ≥98.0%. More preferably, a blank sample test is first performed to eliminate the interference of the matrix.

[0020] The beneficial effects of the present invention are as follows: the present invention discloses a method for simultaneously determining the contents of ten carbonyl compounds in tobacco flavors based on UPLC; the method uses ultra-high performance liquid chromatography, specially selected HSS T3 chromatographic columns, and adopts DNPH-HCL (2,4-dinitrophenylhydrazine hydrochloride) pre-column derivatization and ternary mobile phase for gradient elution, assisted by other suitable experimental means, to achieve simultaneous analysis and determination of ten carbonyl compounds in tobacco flavors, including formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone, butyraldehyde, acetoin and diacetyl; compared with the prior art that can only determine eight of the carbonyl compounds, the technical effect is significant, and the ten carbonyl compounds in tobacco flavors can be quickly and effectively separated completely. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The liquid chromatogram obtained by testing ten carbonyl compound standard reference substances of the present invention;

[0022] Figure 2 This is a liquid chromatogram of the No. 1 electronic cigarette liquid sample of the present invention;

[0023] Figure 3 This is a liquid chromatogram of the No. 2 electronic cigarette liquid sample of the present invention;

[0024] Figure 4 This is a liquid chromatogram of the No. 3 electronic cigarette liquid sample of the present invention;

[0025] Figure 5 This is a liquid chromatogram of the No. 4 electronic cigarette liquid sample of the present invention;

[0026] Figure 6 This is a liquid chromatogram of the No. 5 electronic cigarette liquid sample of the present invention;

[0027] Figure 7 This is a liquid chromatogram of the No. 6 electronic cigarette liquid sample of the present invention. DETAILED DESCRIPTION

[0028] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with the accompanying drawings.

[0029] In the following description, general election example details are given to provide a deeper understanding of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all embodiments. It should be understood that the specific embodiments are only used to explain the present invention and are not used to limit the present invention.

[0030] It should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the existence of features, integers, steps, operations, elements or components, but do not exclude the existence or addition of one or more other features, integers, steps, operations, elements, components or combinations thereof.

[0031] As described in the background technology, due to the complexity of the matrix of tobacco flavors in the prior art, the UPLC-IE method and the HPLC method can only effectively separate eight substances therein, and there is also a problem of insufficient disclosure in the methods, which will not be elaborated here; it can be seen that the separation of target substances in complex matrices is very difficult, and it is also a problem that technical personnel in this field have long wanted to solve but have not been able to solve; the present invention, however, adopts a solution that is different from the technical means in the prior art, and can effectively separate ten substances at the same time; in order to solve this problem, the present application discloses a technical solution.

[0032] The present invention provides a method for simultaneously determining the contents of ten carbonyl compounds in tobacco flavors based on UPLC, comprising the following steps:

[0033] (1) Preparation of derivatization reagents

[0034] Weigh 2.00 g DNPH-HCL (2,4-dinitrophenylhydrazine hydrochloride) in a 2L beaker, add 500 mL acetonitrile and 40 mL phosphoric acid aqueous solution respectively, dissolve, add 500 mL water and mix evenly to obtain a derivatization reagent; transfer to a brown reagent bottle and store away from light; the shelf life is 1 week;

[0035] (2) Preparation of standard solution

[0036] Weigh 0.1000g of acetoin and diacetyl respectively into a 10mL brown volumetric flask, dilute with acetonitrile and dilute to the scale (i.e., 10mL) to obtain an acetoin solution and a diacetyl solution, and store at -18°C in the dark; then measure 0.1mL of the acetoin solution and 0.1mL of the diacetyl solution into a 25mL brown volumetric flask, add 20mL of a derivatization reagent, shake well, react at room temperature for 20min, add 1mL of pyridine, and dilute to the scale (i.e., 25mL) with acetonitrile to obtain a first mixed standard stock solution; in the prior art, there are no corresponding DNPH derivative reference substances for acetoin and diacetyl. This method can obtain the corresponding derivatives for analysis, which is also a key means to complete this determination;

[0037] Accurately weigh 0.0250 g of DNPH derivatives of formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone and butyraldehyde respectively into a 25 mL brown volumetric flask, and dissolve them in 50% acetonitrile to the mark (i.e., 25 mL) to obtain the second mixed standard stock solution;

[0038] Accurately measure 0.5 mL of the first mixed standard stock solution and 0.3 mL of the second mixed standard stock solution in a 50 mL brown volumetric flask and dissolve them to the mark (i.e., 50 mL) with 50% acetonitrile to obtain a mixed standard mother solution;

[0039] Accurately measure 0.3mL, 0.5mL, 1mL, 3mL, and 5mL of the mixed standard mother solution into a 10mL brown volumetric flask, and dissolve it to the mark with 50% acetonitrile to obtain a gradient standard solution;

[0040] (3) Preparation of test samples

[0041] Weigh 0.8000g of electronic cigarette liquid into a 10mL brown volumetric flask, add 8mL of derivatization reagent, shake well, react at room temperature for 20min, add 0.4mL of pyridine, and dilute to the mark with acetonitrile; then filter with a PTFE filter membrane to obtain the sample filtrate to be tested;

[0042] UPLC conditions:

[0043] The chromatographic column used was HSS T3, with specifications of 2.1 × 100 mm, 2.7 μm;

[0044] Mobile phase: Phase A is 0.1% formic acid solution: acetonitrile = 80:20; Phase B is 0.1% formic acid solution: acetonitrile = 50:50; Phase C is 0.1% formic acid acetonitrile solution; Gradient elution conditions are as follows: The 0.1% and fraction ratios shown are volume percentages, which are common knowledge in the art and are not described in detail;

[0045]

[0046] Flow rate: 0.3 mL / min; column temperature: 32°C; detection wavelength: 365 nm; injection volume: 10 μL; the gradient standard solution and the sample filtrate to be tested were measured according to the above steps.

[0047] Preferably, acetonitrile, formic acid, phosphoric acid, and pyridine are all HPLC-grade reagents; the purity of DNPH-HCL is ≥98%; the purity of acetoin, formaldehyde, acetaldehyde, diacetyl, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone, and butyraldehyde used in the standard solution is ≥98.0%. More preferably, a blank sample test is first performed to eliminate the interference of the matrix.

[0048] Example

[0049] The above method was used to analyze and test 6 batches of tobacco flavors on the market; the results are as follows:

[0050]

[0051]

[0052] Note: - indicates not detected; the formaldehyde content in the result is the converted formaldehyde content;

[0053] For details, see Figure 1-Figure 7 , wherein the abbreviations used in the figure are: Acn: represents acetoin-DNPH; For: represents formaldehyde-DNPH; Ace: represents acetaldehyde-DNPH; Dia: represents diacetyl-DNPH; Acet: represents acetone-DNPH; Acr: represents acrolein-DNPH; Pio: represents propionaldehyde-DNPH; Cro: represents crotonaldehyde-DNPH; Buta: represents 2-butyraldehyde-DNPH; Tyra: represents butyraldehyde-DNPH. Figure 1 This is the liquid chromatogram obtained by testing ten carbonyl compound standard reference substances using this method; Figure 2 This is the liquid chromatogram of the No. 1 electronic cigarette liquid sample. Figure 3 This is the liquid chromatogram of the No. 2 e-cigarette liquid sample. Figure 4 This is the liquid chromatogram of the No. 3 e-cigarette liquid sample. Figure 5 This is the liquid chromatogram of the No. 4 electronic cigarette liquid sample. Figure 6 This is the liquid chromatogram of the No. 5 electronic cigarette liquid sample. Figure 7 This is the liquid chromatogram of the No. 6 electronic cigarette liquid sample; it can be seen from the peaks in the figure that the ten substances can be accurately separated, and it can be seen that the matrix components in the tobacco flavor are very complex, which has a great interference on the target components and is not conducive to accurate qualitative and quantitative analysis. In this scheme, ternary gradient elution is used in combination with other experimental methods, so the peaks of the ten carbonyl compounds can be clearly and accurately separated, which is very difficult; at the same time, from Figure 1It can be seen that it only takes 33 minutes to successfully separate the peaks of ten carbonyl compounds. The technical effect is far superior to the existing technology and has great significance and value for practical applications.

[0054] The advantages of the present invention are:

[0055] Through ultra-high performance liquid chromatography, specially selected HSS T3 chromatographic column, DNPH-HCL (2,4-dinitrophenylhydrazine hydrochloride) pre-column derivatization and ternary mobile phase for gradient elution, supplemented by other suitable experimental means, the simultaneous analysis and determination of ten carbonyl compounds in tobacco flavors, including formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone, butyraldehyde, acetoin and diacetyl, is achieved; compared with the existing technology that can only determine eight of the carbonyl compounds, the technical effect is significant, and the ten carbonyl compounds in tobacco flavors can be quickly and effectively separated.

[0056] The above disclosures are only several specific embodiments of the present invention, but the present invention is not limited thereto, and any changes that can be conceived by those skilled in the art should fall within the protection scope of the present invention.

Claims

1. A method for simultaneously determining the contents of ten carbonyl compounds in tobacco flavors based on UPLC, wherein the ten carbonyl compounds are acetoin, diacetyl, formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone and butyraldehyde, characterized in that: The following steps are involved: (1) Configuration of derivatization reagents Weigh 2.00 g of DNPH-HCL in a 2 L beaker, add 500 mL of acetonitrile and 40 mL of phosphoric acid aqueous solution respectively, dissolve, add 500 mL of water and mix well to obtain a derivatization reagent; transfer to a brown reagent bottle and store away from light; (2) Preparation of standard solution Weigh 0.1000 g of acetoin and diacetyl respectively into a 10 mL brown volumetric flask, dilute with acetonitrile and dilute to the mark to obtain acetoin solution and diacetyl solution, and store at -18°C in the dark; then weigh 0.1 mL of acetoin solution and 0.1 mL of diacetyl solution into a 25 mL brown volumetric flask, add 20 mL of derivatization reagent, shake well, react at room temperature for 20 min, add 1 mL of pyridine, and dilute to the mark with acetonitrile to obtain the first mixed standard stock solution; Accurately weigh 0.0250 g of DNPH derivatives of formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone and butyraldehyde respectively into a 25 mL brown volumetric flask, and dissolve to volume with 50% acetonitrile to obtain the second mixed standard stock solution; Accurately measure 0.5 mL of the first mixed standard stock solution and 0.3 mL of the second mixed standard stock solution in a 50 mL brown volumetric flask and dissolve to volume with 50% acetonitrile to obtain the mixed standard mother solution; Accurately measure 0.3mL, 0.5mL, 1mL, 3mL, and 5mL of the mixed standard mother solution into a 10mL brown volumetric flask, and dissolve it to the mark with 50% acetonitrile to obtain a gradient standard solution; (3) Preparation of test samples Weigh 0.8000g of electronic cigarette liquid into a 10mL brown volumetric flask, add 8mL of derivatization reagent, shake well, react at room temperature for 20min, add 0.4mL of pyridine, and dilute to the mark with acetonitrile; then filter with a PTFE filter membrane to obtain the sample filtrate to be tested; UPLC conditions: The chromatographic column used was HSS T3, with specifications of 2.1 × 100 mm, 2.7 µm; Mobile phase: Phase A is 0.1% formic acid solution: acetonitrile = 80:20; Phase B is 0.1% formic acid solution: acetonitrile = 50:50; Phase C is 0.1% formic acid acetonitrile solution; Gradient elution conditions are as follows: Flow rate: 0.3mL / min; column temperature: 32℃; detection wavelength: 365nm; injection volume: 10µL; the gradient standard solution and the sample filtrate to be tested were measured according to the above steps.

2. The method for simultaneously determining the contents of ten carbonyl compounds in tobacco flavors based on UPLC according to claim 1, characterized in that: Acetonitrile, formic acid, phosphoric acid, and pyridine are all HPLC-grade reagents; DNPH-HCL purity ≥ 98%; acetoin, formaldehyde, acetaldehyde, diacetyl, acetone, acrolein, propionaldehyde, crotonaldehyde, 2-butanone, and butyraldehyde used in the standard solution; The purity is ≥98.0%.

Images