Method for measuring glycoside in tobacco through enzyme hydrolysis-gas chromatography-mass spectrum combination

Abstract

The invention discloses a method for measuring glycoside in tobacco through ultrasonic extraction-macroporous resin solid-phase extraction purification-enzyme hydrolysis-gas chromatography-mass spectrum combination. According to the method, beta-D-glucosidase is used for directly performing enzyme hydrolysis on the glycoside which is subjected to macroporous resin solid-phase extraction purification after being extracted ultrasonically, and then qualitative and relatively quantitative analyses are conducted through gas chromatography-mass spectrum combination; the method is accurate, reliable, capable of accurately identifying 24 kinds of glycoside and relatively quantifying 28 kinds of glycoside and applicable to simultaneous measurement of glycoside ingredients in different types of tobacco, a nonselective and destructive hydrolysis pattern of acid hydrolysis is overcome, and in addition, substance confirmation through commercial standard spectrum libraries and retention indexes in literatures is facilitated.

Description

technical field

[0001] The invention belongs to the field of chemical analysis of tobacco, and in particular relates to a method for measuring glycosides in tobacco by combining enzyme hydrolysis-gas chromatography-mass spectrometry. Background technique

[0002] Glycosides are an important class of latent aroma substances. They are secondary metabolites formed during the growth and development of tobacco. They are mainly composed of reduced hydroxyl groups of monosaccharides or disaccharides and aroma substances (terpene alcohols, fatty alcohols) Classes, C13 norisoprene derivatives or phenylpropane derivatives, etc.) bonded stable bonded state compounds. It is not only an important precursor of volatile aroma components in tobacco (it can release free aroma components through enzymatic hydrolysis or combustion cleavage), but also has a wide range of biological functions (such as the main form of storage and transportation of aroma components, protecting cells Vulnerable s...

Examples

Embodiment 1

[0029] The method for the determination of glycosides in tobacco by enzymatic hydrolysis-gas chromatography-mass spectrometry of the present invention comprises the following steps:

[0030] (1) Extraction: Accurately weigh 4.0000 g of tobacco powder into a stoppered Erlenmeyer flask, add 250 μg of phenyl-β-D-glucopyranoside internal standard 1, and add 40 mL of methanol for ultrasonic extraction for 30 min (100 Hz at room temperature ), let stand for 15 minutes after the ultrasonication, and filter the supernatant, add 40 mL of methanol to the residue for ultrasonic extraction for 30 minutes (100 Hz at room temperature), combine the filtrates after the ultrasonication, and concentrate the methanol to 2 mL in a rotary concentrator;

[0031] (2) Purification: After dissolving and mixing with 25 mL of deionized water, transfer it to the XAD-2 macroporous resin glass column. The adsorbed glycosides are first rinsed with 50 mL of water to remove water-soluble sugars, amino acids an...

Embodiment 2

[0040] Tobacco glycosides were determined by enzymatic hydrolysis-gas chromatography-mass spectrometry for flue-cured tobacco, sun-cured tobacco and oriental tobacco by using the method of Example 1 of the present invention. The varieties of the three types of tobacco are K326, Kenner and Basma, and the parts are all in the middle leaves. All the tobacco leaves have not been rebaked and alcoholized. The chromatogram and glycoside content of relative quantitative determination are as Figure 2-5 and shown in Table 2.

[0041]

[0042]

technical field

[0001] The invention belongs to the field of chemical analysis of tobacco, and in particular relates to a method for measuring glycosides in tobacco by combining enzyme hydrolysis-gas chromatography-mass spectrometry. Background technique

[0002] Glycosides are an important class of latent aroma substances. They are secondary metabolites formed during the growth and development of tobacco. They are mainly composed of reduced hydroxyl groups of monosaccharides or disaccharides and aroma substances (terpene alcohols, fatty alcohols) Classes, C13 norisoprene derivatives or phenylpropane derivatives, etc.) bonded stable bonded state compounds. It is not only an important precursor of volatile aroma components in tobacco (it can release free aroma components through enzymatic hydrolysis or combustion cleavage), but also has a wide range of biological functions (such as the main form of storage and transportation of aroma components, protecting cells Vulnerable s...

Examples

Embodiment 1

[0029] The method for the determination of glycosides in tobacco by enzymatic hydrolysis-gas chromatography-mass spectrometry of the present invention comprises the following steps:

[0030] (1) Extraction: Accurately weigh 4.0000 g of tobacco powder into a stoppered Erlenmeyer flask, add 250 μg of phenyl-β-D-glucopyranoside internal standard 1, and add 40 mL of methanol for ultrasonic extraction for 30 min (100 Hz at room temperature ), let stand for 15 minutes after the ultrasonication, and filter the supernatant, add 40 mL of methanol to the residue for ultrasonic extraction for 30 minutes (100 Hz at room temperature), combine the filtrates after the ultrasonication, and concentrate the methanol to 2 mL in a rotary concentrator;

[0031] (2) Purification: After dissolving and mixing with 25 mL of deionized water, transfer it to the XAD-2 macroporous resin glass column. The adsorbed glycosides are first rinsed with 50 mL of water to remove water-soluble sugars, amino acids an...

Embodiment 2

[0040] Tobacco glycosides were determined by enzymatic hydrolysis-gas chromatography-mass spectrometry for flue-cured tobacco, sun-cured tobacco and oriental tobacco by using the method of Example 1 of the present invention. The varieties of the three types of tobacco are K326, Kenner and Basma, and the parts are all in the middle leaves. All the tobacco leaves have not been rebaked and alcoholized. The chromatogram and glycoside content of relative quantitative determination are as Figure 2-5 and shown in Table 2.

[0041]

[0042]

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