Method for detecting nicotine content in tobacco leaves

Abstract

The invention belongs to the technical field of tobacco detection, and particularly discloses a method for detecting nicotine content in tobacco leaves. The method comprises the following steps: S1, drying and crushing the tobacco leaves, adding a methanol solution of sodium hydroxide for extraction, and centrifuging and filtering through a membrane to obtain a nicotine extracting solution; s2, the nicotine extracting solution is detected through a liquid chromatograph, during detection, mobile phases comprise a mobile phase A containing 90% by volume of water and 10% by volume of methyl alcohol and a mobile phase B containing 90% by volume of methyl alcohol and 10% by volume of water, constant-speed elution is carried out, alkaloid components are determined through comparison with the peak appearance time of a standard substance, and then the concentration of each alkaloid component is calculated according to a standard curve. Sodium hydroxide is combined with acidic groups in nicotine to form soluble salts so as to achieve the purpose of separating and extracting nicotine, and the accuracy of nicotine content determination by liquid chromatography is improved by using an ultraviolet detection method.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco detection and relates to a method for detecting nicotine content in tobacco leaves. Background Art

[0002] Nicotine is one of the important alkaloids in tobacco leaves and has also been detected in plants of the genus Nicotiana in the Solanaceae family. It is also a highly volatile botanical insecticide that can prevent and control pest larvae on various vegetables and fruit trees. The existing methods for extracting and separating nicotine from tobacco leaves include ethanol-water ultrasonic extraction, acid extraction and release, methanol extraction, and acetic acid-water extraction. The mainstream detection methods include gas chromatography, gas chromatography-mass spectrometry, gas chromatography-hydrogen flame detector, high performance liquid chromatography-photodiode array detector, and ultra high performance liquid chromatography-mass spectrometry.

[0003] CN111337604 A discloses a method for determining the nicotine content in tobacco products, which uses gas chromatography to analyze the nicotine content of the extract. Although gas chromatography is widely used in determining the nicotine content in tobacco leaves, it also has problems such as easy conversion of nicotine and low detection limit and detection limit. Summary of the invention

[0004] The invention provides a method for detecting the nicotine content in tobacco leaves, wherein sodium hydroxide is combined with the acidic groups in nicotine to form soluble salts so as to achieve the purpose of separating and extracting nicotine, and an ultraviolet detection method is used to improve the accuracy of liquid chromatography determination of nicotine content.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme: a method for detecting nicotine content in tobacco leaves, comprising the following steps: S1. Dry and crush tobacco leaves, add a methanol solution of sodium hydroxide to extract, and centrifuge through a membrane to obtain a nicotine extract; S2. The nicotine extract is detected by liquid chromatography. During the detection, the mobile phase includes a mobile phase A of 90% by volume water + 10% methanol and a mobile phase B of 90% by volume methanol + 10% water. Gradient elution is performed. The alkaloid components are qualitatively identified by comparing the peak time with that of the standard. The concentration of each alkaloid component is calculated according to the standard curve. The unit is mg / mL. The calculation formula of the alkaloid component content is as follows: Alkaloid content (mg / g DW) = ; Wherein: y is the nicotine concentration calculated by the standard curve, in mg / mL; L1 is the volume of the extract, in mL; M is the sample weight, in g; m is the sample dry matter rate, specifically 0.95.

[0006] Optionally, the concentration of sodium hydroxide in the methanol solution of sodium hydroxide is 0.1 mol / L; methanol adopts 75% methanol aqueous solution.

[0007] Optionally, the amount of the methanol solution of sodium hydroxide added is 50-100 times that of the tobacco leaf powder.

[0008] Optionally, during extraction in S1, ultrasonic shaking is performed for 20-60 minutes.

[0009] Optionally, liquid chromatography detection is performed with an ultraviolet detector, a C18 chromatographic column, a column temperature of 40° C., and a detection wavelength of 259 nm.

[0010] Optionally, the injection volume is 10 μL, the elution rate is 1.0 mL / min, and the gradient elution program is: 0 min-1.0 min (0% B)-4.0 min (15% B)-8.0 min (35% B)-15.0 min (40% B)-30 min (0% B).

[0011] Optionally, 0.15% sodium acetate was added to mobile phase A to adjust the pH to 6.8. Optionally, the pH of mobile phase B is controlled at 3.8.

[0012] The invention also relates to the application of the detection method in the detection of nicotine content in cigar tobacco leaves.

[0013] Optionally, the nicotine includes but is not limited to nornicotine (NOR), anabasine (ANAB), anabasine (ANAT), and nicotine (NIC).

[0014] The present invention has the following beneficial effects: 1. The present invention has found that nicotine is miscible with water at temperatures below 60°C and above 210°C to form hydrates. It is also miscible with alcohols and ethers such as ether and ethanol, and is easily soluble in most organic solvents. It forms salts after combining with acids or bases. In addition, nicotine has different optical isomers, and the natural product is a levorotatory isomer with different maximum ultraviolet absorption intensities, usually between 240-260nm. The present invention uses methanol and ultrasonic vibration extraction to extract various compounds in tobacco leaves to the greatest extent. In this process, the sodium hydroxide in the solution also plays a role, reacting with the acidic groups of the nicotine molecules in a certain proportion to form a soluble sodium salt. After sufficient dissolution, a nicotine extract is obtained, which reduces the leaching of other interfering substances and greatly improves the extraction efficiency.

[0015] 2. The extracted solution of the present invention adopts liquid chromatography to analyze the nicotine content of the extract, and the ultraviolet detection method can be used to improve the accuracy of liquid chromatography to determine the nicotine content and avoid the interference of its optical isomers and related substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the liquid chromatogram of the sample in Example 1 of the present invention.

[0017] Figure 2 This is the gas chromatogram of the sample in Example 2 of the present invention.

[0018] Figure 3 This is the liquid chromatogram of the sample in Example 3 of the present invention. DETAILED DESCRIPTION

[0019] 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.

[0020] Example 1 (1) For fermented cigar tobacco leaves, weigh 0.5 g tobacco leaf powder into a 50 mL centrifuge tube, add 50 mL of methanol solution containing 0.1 mol / L 75% sodium hydroxide, extract by ultrasonic vibration for 60 min, and centrifuge through a membrane to obtain a nicotine extract.

[0021] (2) The nicotine extract in step (1) was subjected to liquid chromatography, equipped with a UV detector, a C18 chromatographic column, a column temperature of 40°C, and a detection wavelength of 259 nm. The injection volume was 10 μL, the mobile phase A (90% water + 10% methanol, pH 6.8, 0.15% sodium acetate), the mobile phase B (90% methanol + 10% water, pH 3.8), and the gradient elution program was: 0min-1.0min (0% B)-4.0min (15% B)-8.0min (35%B)-15.0min (40%B)-30min (0% B). The elution rate was uniform at 1.0 mL / min. The alkaloid components were qualitatively identified by comparing the elution time with that of the standard, and then the concentration (mg / mL) of each alkaloid component was calculated based on the standard curve. The chromatogram is shown in Figure 1 .

[0022] Example 2 (1) For fermented cigar tobacco leaves, weigh 0.5 g tobacco leaf powder into a 50 mL centrifuge tube, add 50 mL of methanol solution containing 0.1 mol / L 75% sodium hydroxide, extract by ultrasonic vibration for 60 min, and centrifuge through a membrane to obtain a nicotine extract.

[0023] (2) The nicotine extract in step (1) was placed on a gas chromatograph equipped with a flame ionization detector and an FFAP column. The temperature was raised from an initial column temperature of 50°C at a rate of 9°C / min to 230°C and maintained for 2 minutes. The alkaloid components were identified by comparing the peak time with that of the standard, and the concentration (mg / mL) of each alkaloid component was calculated based on the standard curve. The chromatogram is shown in Figure 2 .

[0024] Example 3 (1) For fermented cigar tobacco leaves, weigh 0.5 g tobacco leaf powder into a 50 mL centrifuge tube, add 50 mL of methanol solution containing 0.1 mol / L 75% sodium hydroxide, extract by ultrasonic vibration for 60 min, and centrifuge through a membrane to obtain a nicotine extract.

[0025] (2) The nicotine extract in step (1) was subjected to liquid chromatography, equipped with a UV detector, a C18 chromatographic column, a column temperature of 40°C, and a detection wavelength of 259 nm. The injection volume was 10 μL, mobile phase A (ultrapure water, pH 6.8, 0.15% sodium acetate), mobile phase B (methanol, pH 3.8), 30% B constant elution, and elution rate of 1.0 mL / min. The alkaloid components were qualitatively identified by comparing the elution time with that of the standard, and then the concentration (mg / mL) of each alkaloid component was calculated based on the standard curve. The chromatogram is shown in Figure 3 .

[0026] The nicotine contents measured in Examples 1 to 3 above are shown in Table 1.

[0027] Table 1

[0028] Note: * indicates not detected.

[0029] By comparing Example 1 with Example 2, it can be found that the gas phase method has more interferences. Since nicotine is not the only volatile substance in tobacco leaves, the qualitative work of nicotine using the gas phase method is more complicated and not as direct and clear as the liquid chromatography method of Example 1. By comparing Example 1 with Example 3, it can be found that Example 1 can separate the four nicotines well through the optimized design of the mobile phase elution gradient and the design of the mobile phase ratio, while Example 3 with the commonly used mobile phase ratio can not achieve good separation of the four nicotines.

[0030] 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 nicotine content in tobacco leaves, characterized in that: The following steps are involved: S1. Dry and crush tobacco leaves, add a methanol solution of sodium hydroxide to extract, and centrifuge through a membrane to obtain a nicotine extract; S2. The nicotine extract is detected by liquid chromatography. During the detection, the mobile phase includes a mobile phase A of 90% by volume water + 10% methanol and a mobile phase B of 90% by volume methanol + 10% water. Gradient elution is performed. The alkaloid components are qualitatively identified by comparing the peak time with that of the standard. The concentration of each alkaloid component is calculated according to the standard curve. The unit is mg / mL. The calculation formula of the alkaloid component content is as follows: Alkaloid content (mg / g DW) = ; Where: y is the nicotine concentration calculated by the standard curve, in mg / mL; L1 is the volume of the extract, in mL; M is the sample weight, in g; m is the dry matter ratio of the sample, specifically 0.

95.

2. The detection method according to claim 1, characterized in that: The concentration of sodium hydroxide in the methanol solution of sodium hydroxide is 0.1 mol / L; methanol adopts 75% methanol aqueous solution.

3. The detection method according to claim 2, characterized in that: The amount of sodium hydroxide methanol solution added is 50-100 times that of tobacco leaf powder.

4. The detection method according to claim 1, characterized in that: When extracting from S1, ultrasonic shaking is used for 20-60 minutes.

5. The detection method according to claim 1, characterized in that: Liquid chromatography was used for detection, equipped with a UV detector, a C18 chromatographic column, a column temperature of 40 °C, and a detection wavelength of 259 nm.

6. The detection method according to claim 5, characterized in that: The injection volume was 10 μL, the elution rate was 1.0 mL / min, and the gradient elution program was: 0 min-1.0 min (0% B)-4.0 min (15% B)-8.0 min (35% B)-15.0 min (40% B)-30 min (0% B).

7. The detection method according to any one of claims 1 to 6, characterized in that: To mobile phase A, 0.15% sodium acetate was added to adjust the pH to 6.

8.

8. The detection method according to any one of claims 1 to 6, characterized in that: The pH of mobile phase B was controlled at 3.

8.

9. Use of the detection method according to any one of claims 1 to 8 in the detection of nicotine content in cigar tobacco leaves.

10. The use according to claim 9, characterized in that: The nicotine includes but is not limited to nornicotine (NOR), anabasine (ANAB), anabasine (ANAT), and nicotine (NIC).

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