GC-MS (gas chromatography-mass spectrometry) detection method for malic acid derivatives in tobacco

Abstract

The invention belongs to the technical field of chemical analysis of tobaccos, and particularly relates to a GC-MS detection method for malic acid derivatives in tobaccos. The GC-MS detection method for the malic acid derivatives in the tobacco comprises the steps that a sample to be detected is extracted and then subjected to a derivatization reaction to obtain a liquid to be detected, gas chromatography-mass spectrometry testing is conducted, detected malic acid is 2-hydroxy-3-isopropyl malic acid and 2-isopropyl malic acid, the temperature of a sample inlet is 290-300 DEG C, programmed heating is adopted, the temperature is kept at 60-70 DEG C for 10-15 min, the temperature is kept at 60-70 DEG C for 20-30 min, and the temperature is kept at 60-70 DEG C for 20-30 min. 2-hydroxy-3-isopropyl malic acid and 2-isopropyl malic acid serve as raw materials, the raw materials are heated to 290-310 DEG C at the heating rate of 5-8 DEG C / min, the temperature is kept for 10-15 min at the temperature of 290-310 DEG C, scanning is conducted in an SRM scanning mode, ion pairs of the 2-hydroxy-3-isopropyl malic acid and the 2-isopropyl malic acid are 191.1 / 73.1 and 73.1 / 45 respectively, the retention time of the 2-hydroxy-3-isopropyl malic acid and the retention time of the 2-isopropyl malic acid are 0.3-0.5 min, and the collision energy is 8-10 eV. Through derivatization reaction after extraction and selection of specific sample introduction temperature, temperature rising program and mass spectrum parameters during gas chromatography-mass spectrometry analysis, the detection method disclosed by the invention has relatively high accuracy and sensitivity.

Description

Technical Field

[0001] The invention relates to the technical field of tobacco chemical analysis, and in particular to a GC-MS detection method for malic acid derivatives in tobacco. Background Art

[0002] 2-Hydroxy-3-isopropylmalic acid (3-Isopropylmalic acid) and 2-isopropylmalic acid (2-Isopropylmalic acid) are two important organic acids, which are widely present in plants, participate in multiple biosynthetic pathways, and have important physiological functions and pharmacological effects.

[0003] 2-Hydroxy-3-isopropylmalic acid is the precursor of isopentanol, which is a higher alcohol and an important aroma component of plants. It promotes the growth and pollination of pollen tubes of plants. 2-Isopropylmalic acid is the precursor of leucine, which is an essential amino acid and plays an important role in the growth, development and stress resistance of plants. In addition, 2-hydroxy-3-isopropylmalic acid and 2-isopropylmalic acid also have multiple pharmacological activities such as antioxidant, anti-inflammatory, antibacterial and anti-cancer, and are an important source of plant medicines. Therefore, 2-hydroxy-3-isopropylmalic acid and 2-isopropylmalic acid have high research value.

[0004] The currently disclosed detection methods for malic acid are mainly liquid chromatography or gas chromatography, which uses a chromatographic column to separate the compounds to be tested in the extract. The Chinese patent application number CN115993408A, which was published on April 21, 2023, discloses a method for determining four organic acids, citric acid, malic acid, lactic acid, and acetic acid, in tobacco flavors and fragrances. The extract obtained by extraction is pre-treated by a chromatographic column and then ion exclusion liquid chromatography is used to qualitatively analyze the organic acid components. However, due to the complex matrix in plant samples, a large amount of solvent is required for pretreatment when using liquid chromatography or gas chromatography for detection, and a large amount of eluent is required for chromatographic analysis, resulting in the detection of 2-hydroxy-3-isopropyl malic acid and 2-isopropyl malic acid facing the problems of excessive solvent consumption and long analysis time, and the quantitative analysis results of ion exclusion liquid chromatography are not accurate, and even cannot be quantitatively analyzed.

[0005] The Chinese patent with the authorization announcement number CN109212066B published on June 18, 2021 discloses a method for determining non-fatty organic acids in tobacco and tobacco products. The tobacco leaves are ground into powder and ultrasonically extracted with a dichloromethane extractant to obtain an extract for silane derivatization reaction, followed by GC-MS / MS testing and analysis. Non-fatty organic acids include 3-hydroxypropionic acid, 2-hydroxybenzoic acid, cinnamic acid, lactic acid, etc. GC-MS / MS testing and analysis can greatly reduce solvent consumption and reduce analysis time, but the detection method does not disclose detection methods for 2-hydroxy-3-isopropylmalic acid and 2-isopropylmalic acid, and the detection and analysis of the two malic acids still have the problem of poor accuracy of quantitative detection and analysis. Summary of the invention

[0006] The invention provides a GC-MS detection method for malic acid derivatives in tobacco, which solves the problem of poor accuracy in quantitative detection and analysis of two malic acids, 2-hydroxy-3-isopropylmalic acid and 2-isopropylmalic acid, in the prior art.

[0007] In order to solve the above technical problems, the technical solution of the GC-MS detection method of malic acid derivatives in tobacco of the present invention is:

[0008] A GC-MS detection method for malic acid derivatives in tobacco comprises the following steps: extracting a sample to be tested in an extractant, performing a derivatization reaction with a derivatization reagent after extraction to obtain a test liquid, and taking the test liquid for testing and analysis by gas chromatography-mass spectrometry; the malic acid is 2-hydroxy-3-isopropyl malic acid and 2-isopropyl malic acid;

[0009] The gas chromatography conditions are as follows: the injection port temperature is 290-300°C, the temperature is programmed, 60-70°C is maintained for 10-15 minutes, then the temperature is increased to 290-310°C at a heating rate of 5-8°C / min, and maintained at 290-310°C for 10-15 minutes;

[0010] The mass spectrometry conditions are: scanning in SRM scanning mode, the ion pair of the 2-hydroxy-3-isopropylmalic acid is 191.1 / 73.1, the retention time is 0.3 to 0.5 min, and the collision energy is 8 to 10 eV; the ion pair of the 2-isopropylmalic acid is 73.1 / 45, the retention time is 0.3 to 0.5 min, and the collision energy is 8 to 10 eV.

[0011] The present invention improves the prior art and provides a GC-MS detection method for malic acid derivatives in tobacco. Through a derivatization reaction after extraction, a specific injection port temperature, a heating program and mass spectrometry parameters are selected during gas chromatography-mass spectrometry analysis, so that the detection and analysis of 2-hydroxy-3-isopropyl malic acid and 2-isopropyl malic acid have a higher spiked recovery rate and a lower detection limit, and the accuracy and sensitivity are high. The method can be used for the accurate quantitative determination of 2-hydroxy-3-isopropyl malic acid and 2-isopropyl malic acid.

[0012] In order to further improve the extraction effect of the compounds to be detected in the sample, preferably, the extractant is water.

[0013] In order to further improve the extraction efficiency of the compounds to be detected, preferably, the extraction is ultrasonic extraction, the ultrasonic extraction temperature is 25-30° C., and the ultrasonic extraction time is 30-60 min.

[0014] In order to further completely extract the compounds to be detected in the sample, preferably, the mass volume ratio of the sample to be detected and the extractant is 20 mg: (1.5-2) mL.

[0015] In order to further facilitate the quantification of the compounds to be detected to improve the detection sensitivity, preferably, the derivatization reagent is a methoxyamine pyridine standard solution and N,O-bis(trimethylsilyl)trifluoroacetamide.

[0016] In order to further improve the degree of derivatization of the compound to be detected, preferably, the derivatization reaction is to extract the extract, dry it, vortex it with the methoxyamine pyridine standard solution, incubate it on a shaking table once, and then add N, O-bis(trimethylsilyl) trifluoroacetamide for a second incubation on a shaking table.

[0017] In order to further improve the derivatization efficiency of the detected compound, preferably, the temperature of the first shaking incubation is 37-40°C, the time is 90-100 min, and the temperature of the second shaking incubation is 60-70°C, the time is 50-60 min.

[0018] In order to further improve the separation effect, preferably, the chromatographic column used in the gas chromatography is Agilent DB-5MS.

[0019] In order to further improve the mass spectrum peak response, preferably, the gas chromatograph uses helium as the carrier gas with a flow rate of 1.0 to 1.5 mL / min, adopts split mode injection, and the split ratio is 10:1 to 50:1.

[0020] In order to further improve the ionization efficiency, preferably, the ion source of the mass spectrometer is EI, the ion source temperature is 290-300°C, and the transmission line temperature is 280-290°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the extracted ion chromatogram of 2-hydroxy-3-isopropylmalic acid in Example 1 under SRM scanning mode;

[0022] Figure 2 This is the extracted ion chromatogram of 2-isopropylmalic acid in Example 1 in SRM scanning mode. DETAILED DESCRIPTION

[0023] The technical concept of the GC-MS detection method of malic acid derivatives in tobacco provided by the present invention is as follows:

[0024] A GC-MS detection method for malic acid derivatives in tobacco comprises the following steps: extracting a sample to be tested in an extractant, performing a derivatization reaction with a derivatization reagent after extraction to obtain a test liquid, and taking the test liquid for testing and analysis by gas chromatography-mass spectrometry; the malic acid is 2-hydroxy-3-isopropyl malic acid and 2-isopropyl malic acid;

[0025] The gas chromatography conditions are as follows: the injection port temperature is 290-300°C, the temperature is programmed, 60-70°C is maintained for 10-15 minutes, then the temperature is increased to 290-310°C at a heating rate of 5-8°C / min, and maintained at 290-310°C for 10-15 minutes;

[0026] The mass spectrometry conditions are: scanning in SRM scanning mode, the ion pair of the 2-hydroxy-3-isopropylmalic acid is 191.1 / 73.1, the retention time is 0.3 to 0.5 min, and the collision energy is 8 to 10 eV; the ion pair of the 2-isopropylmalic acid is 73.1 / 45, the retention time is 0.3 to 0.5 min, and the collision energy is 8 to 10 eV.

[0027] The GC-MS detection method of malic acid derivatives in tobacco provided by the present invention uses a derivatization reaction after extraction to make the compounds to be detected more stable and more suitable for chromatographic analysis, and is convenient for quantification and separation, thereby improving the detection sensitivity of the compounds to be detected; and by selecting a specific injection port temperature, a heating program and mass spectrometry parameters during gas chromatography-mass spectrometry analysis, the detection and analysis of 2-hydroxy-3-isopropyl malic acid and 2-isopropyl malic acid have a higher spiked recovery rate and a lower detection limit, thereby improving the accuracy and sensitivity of the detection and analysis, and can be used for the accurate quantitative determination of 2-hydroxy-3-isopropyl malic acid and 2-isopropyl malic acid.

[0028] The molecular formulas of the 2-hydroxy-3-isopropyl malic acid (left) and 2-isopropyl malic acid (right) are as follows:

[0029]

[0030] The following is a further description of the implementation of the present invention in conjunction with specific examples. The 2-hydroxy-3-isopropylmalic acid (CAS No.: 16048-89-8) and 2-isopropylmalic acid (CAS No.: 3237-44-3) standards were purchased from Sigma-Aldrich; the tobacco leaves were from tobacco K326, purchased from the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences; the methoxyamine pyridine standard solution (20 mg / mL) was purchased from Shanghai Keether Chemical Technology Co., Ltd.; the N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) was purchased from Sigma-Aldrich; the centrifuge was a Thermo Heraeus Fresco 21 centrifuge; the ultrasound used a Kunshan KQ-700DE ultrasound instrument; the sample was weighed using a Mettler Toledo XSR105DU balance.

[0031] 1. Specific embodiment of the GC-MS detection method of malic acid derivatives in tobacco of the present invention

[0032] The GC-MS detection method of malic acid derivatives in tobacco of this embodiment is as follows:

[0033] The dried tobacco leaves (Nicotiana tabacum K326) were ground into powder, 20.0 mg of tobacco leaf powder was weighed into a 2 mL centrifuge tube, 1.5 mL of water was added, and the extraction system was ultrasonically extracted at room temperature (25°C) under ice bath conditions for 30 min to obtain an extract. The extract was centrifuged at 4°C and 14000 rpm for 10 min, 500 μL of the supernatant was taken into a sample bottle and concentrated to dryness in a centrifugal freeze dryer; 100 μL of methoxyamine-pyridine standard solution was added to the freeze-dried extract, vortexed for 2 min to re-dissolve, and incubated at a constant temperature shaker at 37°C for 90 min, and finally 100 μL of BSTFA reagent was added and incubated at a constant temperature shaker at 60°C for 60 min, and the test solution was obtained after returning to room temperature, and the test solution was taken for GC-MS detection.

[0034] The 2-hydroxy-3-isopropylmalic acid standard was configured into a 1000 ng / mL reference stock solution, and the 2-isopropylmalic acid standard was configured into a 500 ng / mL reference stock solution, which were diluted with methanol to form a series of reference solutions of different concentrations. The same derivatization method was used to obtain reference test solutions, and the reference test solutions were taken for GC-MS detection.

[0035] The chromatographic conditions for GC-MS detection were as follows: the gas chromatograph was Thermo TRACE 1310, the autosampler was Thermo TriPlus RSH Smart, and the chromatographic column was Agilent DB-5MS (30m×0.25mm, 0.25μm); high-purity helium (99.999%) was used as the carrier gas with a flow rate of 1.0mL / min; the injection was carried out in split mode with a split ratio of 50:1; the injection port temperature was 300°C, and the temperature was programmed to maintain at 70°C for 10min, then increased to 310°C at a heating rate of 5°C / min, and maintained at 310°C for 10min.

[0036] The mass spectrometry conditions during GC-MS detection are as follows: the mass spectrometer adopts a triple quadrupole mass spectrometer, specifically Thermo TSQ9000, the ion source is EI, the ion source temperature is 300°C, the transfer line temperature is 280°C, and the SRM scanning mode is used for scanning. The characteristic ions of the compounds to be tested are automatically optimized in the Chromeleon 7 software. The characteristic ions of the compounds to be tested and their corresponding collision energy parameters and retention time parameters are shown in Table 1, wherein the ion pair of 2-hydroxy-3-isopropylmalic acid is 191.1 / 73.1, the retention time is 0.3 min, and the collision energy is 10 eV; the ion pair of the 2-isopropylmalic acid is 73.1 / 45, the retention time is 0.3 min, and the collision energy is 10 eV.

[0037] Table 1 Characteristic ions of mass spectrometry analysis and their corresponding collision energy parameters and retention time parameters

[0038]

[0039]

[0040] 2. Experimental Examples

[0041] Linear range and detection limit test: The test results of a series of reference solutions with different concentrations diluted with methanol were analyzed, and the linear equations, linear ranges, correlation coefficients and detection limits of 2-hydroxy-3-isopropylmalic acid and 2-isopropylmalic acid were obtained as shown in Table 2.

[0042] Table 2 Linearity test data of 2-hydroxy-3-isopropylmalic acid and 2-isopropylmalic acid

[0043]

[0044] It can be seen from Table 2 that the fitting curve obtained by the detection method provided by the present invention is R 2The value of 5-nitropropylidene guanidine dihydrochloride was 0.9976-0.9993, indicating that the response peak area of ​​2-hydroxy-3-isopropylmalic acid and 2-isopropylmalic acid in the tested concentration range has a good linear relationship with their concentration, and the detection limits of 2-hydroxy-3-isopropylmalic acid and 2-isopropylmalic acid are 0.001 ng / mL and 0.01 ng / mL, respectively, which have low detection limits.

[0045] Intra-day and inter-day precision test: The tobacco leaf sample (tobacco K326) was sampled continuously for 3 days according to the test method provided by the present invention, and measured 6 times a day to examine its precision. The test results of intra-day precision and inter-day precision are shown in Table 3.

[0046] Table 3 Intra-day and inter-day precision test results

[0047] name Intraday RSD (n=6) Daily RSD (n=4) 2-Hydroxy-3-isopropylmalic acid 1.53 3.91 2-Isopropylmalic acid 1.85 1.49

[0048] It can be seen from the test results in Table 3 that the intra-day and inter-day RSDs are both less than 5.0%, indicating that the instrument is in good and stable condition, further indicating that the test method provided by the present invention has the advantages of fast analysis speed, high precision and good stability.

[0049] Spiked recovery test: The tobacco leaf sample (tobacco K326) was tested for the spiked recoveries of 2-hydroxy-3-isopropylmalic acid and 2-isopropylmalic acid standards according to the test method provided by the present invention. The results are shown in Table 4.

[0050] Table 4 Spiked recovery test results

[0051]

[0052]

[0053] As can be seen from Table 5, the spiked recoveries of 2-hydroxy-3-isopropylmalic acid and 2-isopropylmalic acid are between 88.00% and 115.40%, indicating that the malic acid detection method of the present invention has high reliability.

[0054] Quantitative test: The extracted ion chromatograms of 2-hydroxy-3-isopropylmalic acid and 2-isopropylmalic acid measured by the malic acid derivative detection method in Example 1 under the SRM scanning mode are as follows: Figure 1 and Figure 2 The peak time results of 2-hydroxy-3-isopropyl malic acid and 2-isopropyl malic acid are shown in Table 5. The contents of 2-hydroxy-3-isopropyl malic acid and 2-isopropyl malic acid are 9.091 and 89.57 μg / mL, respectively.

[0055] Table 5 Peak times of 2-hydroxy-3-isopropylmalic acid and 2-isopropylmalic acid

[0056] Peak time (min) 2-Hydroxy-3-isopropylmalic acid 29.29 2-Isopropylmalic acid 29.39

[0057] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A GC-MS detection method for malic acid derivatives in tobacco, characterized in that: The following steps are involved: The sample to be tested is extracted in an extractant, and after the extraction, a derivatization reagent is used for a derivatization reaction to obtain a test liquid, and the test liquid is tested and analyzed by gas chromatography-mass spectrometry; the malic acid is 2-hydroxy-3-isopropyl malic acid and 2-isopropyl malic acid; The gas chromatography conditions are as follows: the injection port temperature is 290-300°C, the temperature is programmed, 60-70°C is maintained for 10-15 minutes, then the temperature is increased to 290-310°C at a heating rate of 5-8°C / min, and maintained at 290-310°C for 10-15 minutes; The mass spectrometry conditions are: scanning in SRM scanning mode, the ion pair of the 2-hydroxy-3-isopropylmalic acid is 191.1 / 73.1, the retention time is 0.3 to 0.5 min, and the collision energy is 8 to 10 eV; the ion pair of the 2-isopropylmalic acid is 73.1 / 45, the retention time is 0.3 to 0.5 min, and the collision energy is 8 to 10 eV.

2. The GC-MS detection method for malic acid derivatives in tobacco according to claim 1, characterized in that: The extractant is water.

3. The GC-MS detection method of malic acid derivatives in tobacco according to claim 2, characterized in that: The extraction is ultrasonic extraction, the ultrasonic extraction temperature is 25-30° C., and the ultrasonic extraction time is 30-60 min.

4. The GC-MS detection method for malic acid derivatives in tobacco according to claim 1, characterized in that: The mass volume ratio of the sample to be tested and the extractant is 20 mg: (1.5-2) mL.

5. The GC-MS detection method of malic acid derivatives in tobacco according to claim 2, characterized in that: The derivatization reagents are methoxyamine pyridine standard solution and N,O-bis(trimethylsilyl)trifluoroacetamide.

6. The GC-MS detection method of malic acid derivatives in tobacco according to claim 5, characterized in that: The derivatization reaction is to dry the extract obtained by extraction, vortex with a methoxyamine-pyridine standard solution, incubate on a shaking table once, and then add N,O-bis(trimethylsilyl)trifluoroacetamide for a second incubation on a shaking table.

7. The GC-MS detection method for malic acid derivatives in tobacco according to claim 6, characterized in that: The temperature of the first shaking incubation is 37-40° C., and the time is 90-100 min. The temperature of the second shaking incubation is 60-70° C., and the time is 50-60 min.

8. The GC-MS detection method for malic acid derivatives in tobacco according to claim 1, characterized in that: The chromatographic column used in the gas chromatography is Agilent DB-5MS.

9. The GC-MS detection method for malic acid derivatives in tobacco according to claim 1, characterized in that: The gas chromatograph uses helium as carrier gas with a flow rate of 1.0 to 1.5 mL / min, adopts split mode for sample injection, and has a split ratio of 10:1 to 50:

1.

10. The GC-MS detection method for malic acid derivatives in tobacco according to claim 1, characterized in that: The ion source of the mass spectrometer is EI, the ion source temperature is 290-300°C, and the transmission line temperature is 280-290°C.

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