Method for detecting flavor substances in tobacco extract

Abstract

The invention discloses a method for detecting flavor substances in a tobacco extract, which comprises the following steps: adding a sample into a headspace bottle by adopting a headspace solid-phase microextraction-GC / MS (Gas Chromatography-Mass Spectrometer) combined method, then adding a saturated chloride aqueous solution, sealing and uniformly shaking, and then detecting by adopting a solid-phase microextraction-GC / MS combined instrument. According to the method, the saturated chloride is added to dilute the sample to increase the ion concentration in the solution, so that volatile aroma substances are promoted to be separated out from the sample, and the one-time detection sensitivity of the three aroma substances including terpinene, 1, 1, 6-trimethyl-1, 2, 3, 4-tetrahydronaphthalene and (E)-1-(2, 3, 6-trimethylphenyl) butyl-1, 3-diene is higher.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tobacco detection, and more particularly to a method for detecting flavor substances in tobacco extract. BACKGROUND

[0002] Tobacco source flavor raw materials (such as tincture, pure oil, essential oil and alcohol extract, i.e. extract, etc.) are important raw materials for cigarette flavoring, among which extract is the most common. Accurate detection of volatile and semi-volatile aroma substances in tobacco source flavor raw materials is of great significance for flavor blending and quality control. 1,2,3,4-tetrahydro-1,1,6-trimethylnaphthalene, (E)-1-(2,3,6-trimethylphenyl) but-1,3-diene and terpinolene are all important flavor substances in tobacco flavor raw materials, and the contents of 1,2,3,4-tetrahydro-1,1,6-trimethylnaphthalene, (E)-1-(2,3,6-trimethylphenyl) but-1,3-diene and terpinolene in extract are relatively low, and there is a lack of a simple method for detecting the above three substances with high sensitivity at one time in the prior art.

[0003] Headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC / MS) has become a widely used detection method in the tobacco industry due to its simple operation and high sensitivity.

[0004] However, for tobacco source flavor raw materials, especially alcohol extract, especially high propylene glycol content ethanol extract, due to its high viscosity and large density, if the undiluted sample is directly used for HS-SPME-GC / MS detection, the chromatographic peak response of most volatile substances will be generally low, and some low content substances cannot be detected at all, and the detection sensitivity is obviously insufficient.

[0005] In the prior art, there are reports of using saturated saline water (sodium chloride) as a diluent to improve detection sensitivity, but the effect is not good when detecting 1,2,3,4-tetrahydro-1,1,6-trimethylnaphthalene, (E)-1-(2,3,6-trimethylphenyl) but-1,3-diene and terpinolene. Therefore, there is an urgent need for a HS-SPME-GC / MS pretreatment method for these indicators in tobacco flavoring with optimized dilution ratio to improve detection sensitivity. SUMMARY

[0006] To solve the above technical problems, the present application provides a method for detecting flavor substances in tobacco extract, which can improve the detection sensitivity of flavor substances in tobacco flavoring.

[0007] The technical scheme provided by the present application is as follows: A method for detecting flavoring substances in a tobacco extract, which adopts a headspace solid phase microextraction-GC / MS combined method, adds a sample into a headspace bottle, then adds a saturated chloride aqueous solution, seals and shakes uniformly, and then performs solid phase microextraction-gas chromatography-tandem mass spectrometry detection.

[0008] The chloride is selected from calcium chloride.

[0009] The dilution multiple of the sample is 5-7, that is, the mass ratio of the sample to the saturated chloride is 1: (5-7).

[0010] The three flavoring substances 1,2,3,4-tetrahydro-1,1,6-trimethylnaphthalene, (E)-1-(2,3,6-trimethylphenyl)buta-1,3-diene, and terpinolene are detected at one time.

[0011] The extraction temperature of the solid phase microextraction is 60℃, and the extraction time is 50 min.

[0012] The mass of the flavoring material used for detection is 0.9 g.

[0013] The extraction head of the headspace bottle is a DVB / CAR / PDMS fiber.

[0014] The oscillation speed of the solid phase microextraction is 250 rpm, the purge gas is high-purity nitrogen (N2), and the flow rate is 20 mL / min.

[0015] The gas chromatography conditions are as follows: The chromatographic column is an Agilent DB-5MS capillary column (30 m x 0.25 mm x 0.25 μm); The injection port temperature is 250℃; The carrier gas is high-purity helium (He) with a flow rate of 1.0 mL / min; The injection mode is split injection with a split ratio of 10:1; The column temperature program is as follows: the initial temperature is 50℃, maintained for 2 min, then increased to 280℃ at a rate of 8℃ / min, and maintained for 30 min; The total running time is 60.75 min.

[0016] The mass spectrometry (MS) conditions are as follows: The electron impact (EI) mode, the ionization energy is 70 eV; The ion source temperature is 230℃; The transfer line temperature is 280℃; Scanning modes: Two types, Full Scan, with a mass range of m / z 33-400; Selected Ion Monitoring (SIC), with a mass range of m / z 50-550; Solvent delay: 0 min.

[0017] Compared to existing technologies, the present invention provides a method for detecting aroma compounds in tobacco extracts. This method employs headspace solid-phase microextraction-GC / MS. After adding the sample to a headspace vial, a saturated chloride aqueous solution is added. The vial is sealed, shaken, and then analyzed using solid-phase microextraction-gas chromatography-tandem mass spectrometry. The present invention increases the ion concentration in the solution by adding saturated calcium chloride to dilute the sample, thereby promoting the precipitation of volatile aroma compounds from the sample. The sensitivity for detecting three aroma compounds—terpinene, 1,1,6-trimethyl-1,2,3,4-tetrahydronaphthalene, and (E)-1-(2,3,6-trimethylphenyl)but-1,3-diene—is significantly higher than that of existing technologies. Detailed Implementation

[0018] To enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0019] The sources of the substances used in the embodiments of this invention are as follows: Sample information: The self-made alcohol extract of Yunnan tobacco, high in propylene glycol (propylene glycol content of 25%), has a refractive index (20 ℃) ​​of 1.4855 and a relative density (20 / 20 ℃) ​​of 1.3080.

[0020] The remaining substances were commercially available and of analytical grade.

[0021] The solid-phase microextraction-gas chromatography-tandem mass spectrometry system used in this invention is a Gerstel multi-functional sample introduction platform - Agilent 7890A / 5975C. Example 1 Accurately weigh 0.9 g of sample into a 20 mL headspace vial, add 4.5 g of saturated calcium chloride aqueous solution, seal and shake well, then perform solid-phase microextraction-gas chromatography-tandem mass spectrometry (HS-SPME-GC / MS) under the following conditions: Extraction fiber: DVB / CAR / PDMS 50 / 30 μm, 1 cm; Extraction temperature: 60℃; Extraction time: 50 min; Oscillation speed: 250 rpm; Desorption time: 5 min; Desorption temperature: 250℃; Chromatographic column: DB-5MS 30m × 0.25mm × 0.25μm; Carrier gas: He, 1.0 mL / min; Column temperature: 50℃ for 2 min, then increase to 280℃ at 8℃ / min and hold for 30 min; MS conditions: EI 70eV, ion source 230℃, transfer line 280℃, full scan m / z 33-400, SiC m / z 50-550.

[0022] Example 2 Step 1) Accurately weigh 0.9 g of sample into a 20 mL headspace vial, add 0.9 g of saturated calcium chloride aqueous solution, seal and shake well, then perform solid-phase microextraction-gas chromatography-tandem mass spectrometry (HS-SPME-GC / MS) under the following conditions: Extraction fiber: DVB / CAR / PDMS 50 / 30 μm, 1 cm; Extraction temperature: 60℃; Extraction time: 50 min; Oscillation speed: 250 rpm; Desorption time: 5 min; Desorption temperature: 250℃; Chromatographic column: DB-5MS 30m × 0.25mm × 0.25μm; Carrier gas: He, 1.0 mL / min; Column temperature: 50℃ for 2 min, then increase to 280℃ at 8℃ / min and hold for 30 min; MS conditions: EI 70eV, ion source 230℃, transfer line 280℃, full scan m / z 33-400, SiC m / z 50-550.

[0023] Example 3 Accurately weigh 0.9 g of sample into a 20 mL headspace vial, add 6.3 g of saturated calcium chloride aqueous solution, seal and shake well, then perform solid-phase microextraction-gas chromatography-tandem mass spectrometry (HS-SPME-GC / MS) under the following conditions: Extraction fiber: DVB / CAR / PDMS 50 / 30 μm, 1 cm; Extraction temperature: 60℃; Extraction time: 50 min; Oscillation speed: 250 rpm; Desorption time: 5 min; Desorption temperature: 250℃; Chromatographic column: DB-5MS 30m × 0.25mm × 0.25μm; Carrier gas: He, 1.0 mL / min; Column temperature: 50℃ for 2 min, then increase to 280℃ at 8℃ / min and hold for 30 min; MS conditions: EI 70eV, ion source 230℃, transfer line 280℃, full scan m / z 33-400, SiC m / z 50-550.

[0024] Comparative Example 1 Accurately weigh 0.9 g of sample into a 20 mL headspace vial, add 4.5 g of saturated sodium chloride aqueous solution, seal and shake well, then perform solid-phase microextraction-gas chromatography-tandem mass spectrometry (HS-SPME-GC / MS) under the following conditions: Extraction fiber: DVB / CAR / PDMS 50 / 30 μm, 1 cm; Extraction temperature: 60℃; Extraction time: 50 min; Oscillation speed: 250 rpm; Desorption time: 5 min; Desorption temperature: 250℃; Chromatographic column: DB-5MS 30m × 0.25mm × 0.25μm; Carrier gas: He, 1.0 mL / min; Column temperature: 50℃ for 2 min, then increase to 280℃ at 8℃ / min and hold for 30 min; MS conditions: EI 70eV, ion source 230℃, transfer line 280℃, full scan m / z 33-400, SiC m / z 50-550.

[0025] Comparative Example 2 Accurately weigh 0.9 g of sample into a 20 mL headspace vial, add 4.5 g of saturated potassium chloride aqueous solution, seal and shake well, then perform solid-phase microextraction-gas chromatography-tandem mass spectrometry (HS-SPME-GC / MS) under the following conditions: Extraction fiber: DVB / CAR / PDMS 50 / 30 μm, 1 cm; Extraction temperature: 60℃; Extraction time: 50 min; Oscillation speed: 250 rpm; Desorption time: 5 min; Desorption temperature: 250℃; Chromatographic column: DB-5MS 30m × 0.25mm × 0.25μm; Carrier gas: He, 1.0 mL / min; Column temperature: 50℃ for 2 min, then increase to 280℃ at 8℃ / min and hold for 30 min; MS conditions: EI 70eV, ion source 230℃, transfer line 280℃, full scan m / z 33-400, SiC m / z 50-550.

[0026] Comparative Example 3 Accurately weigh 0.9 g of sample into a 20 mL headspace vial, add 4.5 g of saturated magnesium chloride aqueous solution, seal and shake well, then perform solid-phase microextraction-gas chromatography-tandem mass spectrometry (HS-SPME-GC / MS) under the following conditions: Extraction fiber: DVB / CAR / PDMS 50 / 30 μm, 1 cm; Extraction temperature: 60℃; Extraction time: 50 min; Oscillation speed: 250 rpm; Desorption time: 5 min; Desorption temperature: 250℃; Chromatographic column: DB-5MS 30m × 0.25mm × 0.25μm; Carrier gas: He, 1.0 mL / min; Column temperature: 50℃ for 2 min, then increase to 280℃ at 8℃ / min and hold for 30 min; MS conditions: EI 70eV, ion source 230℃, transfer line 280℃, full scan m / z 33-400, SiC m / z 50-550.

[0027] Comparative Example 4 Accurately weigh 0.9 g of sample into a 20 mL headspace vial, add 4.5 g of deionized water, seal and shake well, then perform solid-phase microextraction-gas chromatography-tandem mass spectrometry (HS-SPME-GC / MS) under the following conditions: Extraction fiber: DVB / CAR / PDMS 50 / 30 μm, 1 cm; Extraction temperature: 60℃; Extraction time: 50 min; Oscillation speed: 250 rpm; Desorption time: 5 min; Desorption temperature: 250℃; Chromatographic column: DB-5MS 30m × 0.25mm × 0.25μm; Carrier gas: He, 1.0 mL / min; Column temperature: 50℃ for 2 min, then increase to 280℃ at 8℃ / min and hold for 30 min; MS conditions: EI 70eV, ion source 230℃, transfer line 280℃, full scan m / z 33-400, SiC m / z 50-550.

[0028] Comparative Example 5 Accurately weigh 0.9 g of sample into a 20 mL headspace vial, add 4.5 g of n-hexane, seal and shake well, then perform solid-phase microextraction-gas chromatography-tandem mass spectrometry (HS-SPME-GC / MS) under the following conditions: Extraction fiber: DVB / CAR / PDMS 50 / 30 μm, 1 cm; Extraction temperature: 60℃; Extraction time: 50 min; Oscillation speed: 250 rpm; Desorption time: 5 min; Desorption temperature: 250℃; Chromatographic column: DB-5MS 30m × 0.25mm × 0.25μm; Carrier gas: He, 1.0 mL / min; Column temperature: 50℃ for 2 min, then increase to 280℃ at 8℃ / min and hold for 30 min; MS conditions: EI 70eV, ion source 230℃, transfer line 280℃, full scan m / z 33-400, SiC m / z 50-550.

[0029] Comparative Example 6 Accurately weigh 0.9 g of sample into a 20 mL headspace vial, seal it, and perform solid-phase microextraction-gas chromatography-tandem mass spectrometry (HS-SPME-GC / MS) under the following conditions: Extraction fiber: DVB / CAR / PDMS 50 / 30 μm, 1 cm; Extraction temperature: 60℃; Extraction time: 50 min; Oscillation speed: 250 rpm; Desorption time: 5 min; Desorption temperature: 250℃; Chromatographic column: DB-5MS 30m × 0.25mm × 0.25μm; Carrier gas: He, 1.0 mL / min; Column temperature: 50℃ for 2 min, then increase to 280℃ at 8℃ / min and hold for 30 min; MS conditions: EI 70eV, ion source 230℃, transfer line 280℃, full scan m / z 33-400, SiC m / z 50-550.

[0030] The peak areas of the three main substances detected in the examples and comparative examples are listed in the table below:

[0031] As can be seen from the table above, the pretreatment detection methods using saturated calcium chloride to dilute the samples in Examples 1 and 3 showed significantly larger peak areas for the three aroma compounds—terpinene, 1,1,6-trimethyl-1,2,3,4-tetrahydronaphthalene, and (E)-1-(2,3,6-trimethylphenyl)but-1,3-diene—than those using other diluents. This indicates that the detection methods in Examples 1 and 3 of this invention have higher sensitivity for detecting these three tobacco aroma compounds. This is mainly because, compared to other solvents, the saturated calcium chloride used in Examples 1 and 3 increases the ion concentration in the solution, thereby promoting the precipitation of volatile aroma compounds from the sample for enrichment by the solid-phase microextraction head, thus improving detection sensitivity.

[0032] The table above also shows that the peak areas of three aroma compounds—terpenoids, 1,1,6-trimethyl-1,2,3,4-tetrahydronaphthalene, and (E)-1-(2,3,6-trimethylphenyl)but-1,3-diene—detected by different dilution factors of saturated calcium chloride aqueous solution to the aroma raw material sample varied, with the highest peak area observed at a 7-fold dilution, followed by a 5-fold dilution. If a further increase in dilution factor is desired to improve the peak area, given that the aroma sample weighed in this application is 0.9 g, a higher dilution factor would result in an excessively high liquid level in the headspace vial. During shake extraction, the risk of liquid splashing from the sample vial into the solid-phase microextraction head in the headspace vial would be greater. Therefore, a dilution factor of 7-fold for the sample using saturated calcium chloride aqueous solution is recommended.

[0033] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not 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 aroma substances in tobacco extracts, employing headspace solid-phase microextraction-GC / MS, characterized in that: After adding the sample to the headspace vial, add a saturated chloride aqueous solution, seal and shake well, and then perform solid-phase microextraction-gas chromatography-tandem mass spectrometry for detection.

2. The method for detecting aroma substances in tobacco extract as described in claim 1, characterized in that: The chloride is selected from calcium chloride.

3. The method for detecting aroma substances in tobacco extract as described in claim 2, characterized in that: The sample was diluted 5-7 times, meaning the mass ratio of the sample to saturated chloride was 1:(5-7).

4. The method for detecting aroma substances in tobacco extract as described in claim 1, characterized in that: It is used to detect three aroma compounds in a single step: 1,2,3,4-tetrahydro-1,1,6-trimethylnaphthalene, (E)-1-(2,3,6-trimethylphenyl)but-1,3-diene, and terpinene.

5. The method for detecting aroma substances in tobacco extract as described in claim 1, characterized in that: The solid-phase microextraction was performed at an extraction temperature of 60°C for 50 minutes, and the mass of the fragrance raw material used for testing was 0.9 g.

6. The method for detecting aroma substances in tobacco extract as described in claim 1, characterized in that: The extraction head of the headspace vial is made of DVB / CAR / PDMS fiber.

7. The method for detecting aroma substances in tobacco extract as described in claim 1, characterized in that: The oscillation speed of the solid-phase microextraction was 250 rpm; the purging gas was high-purity nitrogen (N2) at a flow rate of 20 mL / min.

8. The method for detecting aroma substances in tobacco extract as described in claim 1, characterized in that... The gas chromatography conditions are as follows: Chromatographic column: Agilent DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm); Inlet temperature: 250℃; Carrier gas: High-purity helium (He), flow rate 1.0 mL / min; Injection mode: split injection, split ratio 10:1; Column temperature program: Initial temperature 50 ℃, hold for 2 min, then increase to 280 ℃ at a rate of 8 ℃ / min and hold for 30 min; Total running time: 60.75 min.

9. The method for detecting aroma substances in tobacco extract as described in claim 1, characterized in that, The mass spectrometry (MS) conditions are as follows: Electron impact (EI) mode, ionization energy 70 eV; Ion source temperature: 230 ℃; Transmission line temperature: 280 ℃; Scanning modes: Two types, Full Scan, with a mass range of m / z 33-400; Selected Ion Monitoring (SIC), with a mass range of m / z 50-550; Solvent delay: 0 min.

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