Tobacco volatile matter detection method based on combination of headspace solid-phase microextraction and salting-out

Abstract

The invention belongs to the technical field of tobacco detection, and particularly discloses a tobacco volatile matter detection method based on headspace solid-phase microextraction and salting-out combination. Comprising the following steps: S1, removing main veins from sample tobacco leaves, drying, preparing into powder, sealing, and storing in a refrigerator for later use; s2, the tobacco leaf powder is placed in a headspace bottle, an internal standard compound is added, then a boiling salt solution is added, microextraction fibers aged in advance are inserted, water bath extraction and adsorption are carried out, and after extraction is completed, the microextraction fibers are inserted into a GC-MS injection port for desorption; and S3, performing qualitative and quantitative treatment on the volatile substances by adopting GC-MS. The method adopts an HS-SPME method for extraction, does not introduce organic solvent interference, and is simple and rapid to operate, low in cost, high in detection sensitivity and high in detection efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco detection, and relates to a tobacco volatile matter detection method based on the combination of headspace solid phase microextraction and salting out. Background Art

[0002] The aroma of tobacco products comes from tobacco leaves, especially cigar leaves that are rolled after two or more fermentations. Aroma has become an important indicator for evaluating its sensory properties. Good quality cigars basically have no smell of smoke, but instead have sweet, nutty, coffee aromas, etc. Therefore, the detection of volatiles (or aroma substances) in tobacco leaves is particularly important.

[0003] Most reports on cigar tobacco aroma extraction still use organic solvent extraction methods. For example, CN102565233A discloses a method for determining volatile and semi-volatile secondary metabolites in fresh tobacco leaves, which uses a methanol-chloroform mixed solvent to ultrasonically extract volatile substances in tobacco powder. This method uses organic solvents with high toxicity, complex pretreatment methods, inconvenient operation and high cost. Summary of the invention

[0004] The present invention provides a tobacco volatiles detection method based on headspace solid phase microextraction (HS-SPME) combined with salting out, which is simple, fast and low-cost to operate. The HS-SPME method is used for extraction without introducing organic solvent interference, and the detection efficiency and sensitivity are high.

[0005] In order to achieve the above objectives, the present invention adopts the following technical solution: a method for detecting tobacco volatiles based on headspace solid phase microextraction and salting out, comprising the following steps: S1. Remove the main veins of the sample tobacco leaves and make them into powder after drying; S2. Put tobacco leaf powder into a headspace bottle, add internal standard compound, then add boiling saturated salt solution, insert pre-aged microextraction fiber, extract and adsorb in water bath, and insert microextraction fiber into GC-MS injection port for analysis after extraction; S3. Use GC-MS to carry out qualitative and quantitative processing of volatile substances.

[0006] Optionally, the tobacco is fresh tobacco leaves or tobacco products. The tobacco products are preferably cigars or cigarettes.

[0007] Optionally, if the powder in S1 is not used immediately, it needs to be stored in a refrigerator at a temperature of -20 to -80°C. When the raw material is fresh leaves, the temperature is controlled at -80°C, and the storage temperature of tobacco leaf products is controlled at -20°C.

[0008] Optionally, the internal standard compound is cyclohexanone.

[0009] Optionally, the salt solution is a saturated salt solution, and the salt is specifically NaCl, CuSO4 or NaHSO3.

[0010] Optionally, the salt solution is a saturated NaHSO3 solution.

[0011] Optionally, before using the microextraction fiber, it is aged at 250°C for 30 min; during water bath extraction and adsorption, the temperature is 60-70°C and the time is 40-55 min; the desorption temperature is 230°C and the time is 3-5 min.

[0012] Optionally, during GC-MS detection, the GC conditions are: injection port temperature 230°C, carrier gas is high-purity helium, column flow rate 1.0 mL / min, column box 40°C, non-split injection; the temperature program is 60°C for 2 min, then increased to 260°C at 4°C / min, and maintained at 260°C for 5 min.

[0013] Optionally, during GC-MS detection, the MS conditions are: electron bombardment ion source, electron energy 70 eV, ion source temperature 230° C., and mass scanning range 35-400 m / z.

[0014] The invention also relates to the application of the detection method in tobacco aroma extraction.

[0015] The present invention has the following beneficial effects: 1. When extracting volatiles from tobacco, the present invention utilizes headspace solid phase microextraction, and combines salting-out technology at the same time. By adding a boiling saturated salt solution, the density and polarity of the aqueous phase are increased, which plays a role in increasing the precipitation of essential oils and improving the extraction efficiency. In addition, when selecting NaHSO3 solution, since NaHSO3 has a good effect of inhibiting enzyme activity, it can significantly inhibit the enzyme activity during the extraction of fresh leaf aroma, ensuring that the aroma is not transformed during the extraction process, and can shorten the extraction time and increase the types and concentrations of volatiles that can be detected. The extraction does not use organic solvents, and the determination of volatile substances will not bring interference from organic solvents, and the micro-extraction fiber can be reused 100-120 times, which means that the micro-extraction fiber can extract 100-120 samples, greatly saving the cost of extraction, and is easy to operate and highly safe.

[0016] 2. The method provided by the present invention can comprehensively detect volatile substances in tobacco leaves. Using GC-MS technology, most volatile substances in tobacco leaves can be detected and analyzed in a non-targeted manner. GC-MS detection has high sensitivity and can also have a good response to certain important aroma substances with small content but low threshold. It is particularly suitable for the determination of volatile substances in cigar tobacco leaves, and can also be used for the detection of volatile substances in cigarette tobacco or other tobacco. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the total ion current diagram in Example 1 of the present invention. DETAILED DESCRIPTION

[0018] The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0019] The method provided by the present invention can be used for the extraction and detection of volatiles in tobacco samples. The tobacco samples can be fresh tobacco leaves or tobacco products. The following description will be made using cigar tobacco leaves as an example.

[0020] Example 1: Determination of Volatile Substances in Cigar Tobacco Leaves 1. Sample preparation and aroma extraction After sampling the fermented cigar tobacco leaves, the samples were vacuum freeze-dried and ground into powder for later use. Weigh 0.5 g of tobacco leaf powder into a 20 mL headspace bottle, add 0.3 mL of the internal standard compound cyclohexanone (the concentration of cyclohexanone in the final solution is 189.4 μg / mL) to the headspace bottle with a syringe, then add 5 mL of boiling ultrapure water, insert the microextraction fiber (aged at 250°C for 30 min in advance), and then immediately extract and adsorb in a 70°C water bath for 55 min. After the extraction is completed, the microextraction fiber is immediately inserted into the GC-MS injection port for 5 min of analysis.

[0021] 2. GC-MS: GC conditions: injection port temperature 230℃, carrier gas high-purity helium, column flow rate 1.0 mL / min, column box 40℃, non-split injection. Temperature program 60℃ for 2 min, then increase to 260℃ at 4℃ / min, and keep at 260℃ for 5 min. MS conditions: electron bombardment ion source, electron energy 70 eV, ion source temperature 230℃, mass scan range 35-400 m / z. Total ion flow diagram is shown in Figure 1 .

[0022] 3. Qualitative and quantitative analysis of volatile substances: The volatile components were qualitatively analyzed with reference to the NIST14 database and the retention index (RI) value of the substance.

[0023] The internal standard method was used for quantification: volatile component content (μg / g DW) = (volatile component peak area × internal standard content) / internal standard peak area.

[0024] Example 2 Same as Example 1, except that 5 mL of boiling ultrapure water was replaced by 5 mL of boiling saturated NaCl solution.

[0025] Example 3 Same as Example 1, except that 5 mL of boiling ultrapure water is replaced by 5 mL of boiling saturated CuSO4 solution.

[0026] Example 4 Same as Example 1, except that 5 mL of boiling ultrapure water was replaced by 5 mL of boiling saturated NaHSO 3 solution.

[0027] The volatile substance contents of tobacco leaves measured in Examples 1 to 4 are shown in Tables 1 and 2.

[0028] Table 1

[0029] Table 2

[0030] As can be seen from Table 1 and Table 2, the treatment comparison between Example 1 and Example 2-Example 4 shows that the salting-out method can significantly increase the extraction of tobacco aroma essential oil during SPME and significantly increase the types of identified substances. The comparison between Examples 2-4 shows that saturated NaHSO3 as a salting-out solution can significantly enhance the precipitation of tobacco essential oil and increase the total amount of essential oil extracted by SPME.

[0031] The above embodiments describe the preferred implementation of the present invention, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for detecting tobacco volatiles based on headspace solid phase microextraction and salting out, characterized in that: The following steps are involved: S1. Drying the sample tobacco leaves into powder; S2. Put tobacco leaf powder into a headspace bottle, add internal standard compound, then add boiling saturated salt solution, insert pre-aged microextraction fiber, extract and adsorb in water bath, and insert microextraction fiber into GC-MS injection port for analysis after extraction; S3. Use GC-MS to carry out qualitative and quantitative processing of volatile substances.

2. The detection method according to claim 1, characterized in that: The tobacco is fresh tobacco leaves or tobacco products.

3. The detection method according to claim 1, characterized in that: The powder in S1 is stored in a refrigerator when ready for use, and the temperature of the refrigerator is -20-80°C.

4. The detection method according to claim 1, characterized in that: The internal standard compound is cyclohexanone.

5. The detection method according to claim 1, characterized in that: The salt in the salt solution is NaCl, CuSO4 or NaHSO3.

6. The detection method according to claim 5, characterized in that: The salt solution is NaHSO3.

7. The detection method according to claim 1, characterized in that: Before using the microextraction fiber, it was aged at 250°C for 30 minutes; the water bath extraction adsorption temperature was 60-70°C and the time was 40-55 minutes; the desorption temperature was 230°C and the time was 3-5 minutes.

8. The detection method according to any one of claims 1 to 7, characterized in that During GC-MS detection, the GC conditions were as follows: injection port temperature 230°C, high-purity helium as carrier gas, column flow rate 1.0 mL / min, column box 40°C, and splitless injection; the temperature program was maintained at 60°C for 2 min, then increased to 260°C at 4°C / min, and maintained at 260°C for 5 min.

9. The detection method according to any one of claims 1 to 7, characterized in that During GC-MS detection, the MS conditions were as follows: electron bombardment ion source, electron energy 70 eV, ion source temperature 230 °C, and mass scanning range 35-400 m / z.

10. Application of the detection method according to any one of claims 1 to 9 in tobacco aroma extraction.

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