Extraction and identification method of geranyl primumoside and linaloyl primumoside

Abstract

The invention provides an extraction and identification method of geranyl primumoside and linaloyl primumoside. The method comprises the following steps: 1) extracting geranyl primumoside and linaloyl primumoside in agricultural products; (2) carrying out chromatographic separation on geranyl primumoside and linaloyl primumoside by virtue of a liquid chromatography-tandem mass spectrometry method; (3) monitoring four pairs of mass ion pairs 447.2 gt in an ESI-mode through a liquid chromatography-tandem mass spectrometry method; 315.2 gt, 447.2 gt, 315.2 gt 161.1, 447.2 gt, 447.2 gt; 89.0, 447.2 gt, 447.2 gt; 149.2, and the strength is greater than 149.2; 4) comparing the intensity of each mass ion pair of the chromatographic peak at the same retention time, wherein the intensity is 447.2 gt; the strength of 149.2 is greater than 447.2 gt; when the specific value is greater than 1, the compound corresponding to the chromatographic peak is judged to be geranyl primuloside; when the gt is 447.2 gt; the strength of 149.2 is less than 447.2 gt; and when the intensity is less than 1, the compound corresponding to the chromatographic peak is judged to be linalyl primuroside. The method has the advantages that the extraction operation is simple, the analysis is rapid and efficient, the period is short, and the identification sensitivity of the geranyl primuroside and the linalyl primuroside in the agricultural products is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of compound extraction and identification, and specifically relates to a method for extracting and identifying geranyl primveroside and linalyl primveroside.

Background Art

[0002] Aroma is one of the important indicators affecting the quality of agricultural products. In addition to the free state, aroma substances in agricultural products also exist in large amounts as non-fragrant and hardly volatile glycoside aroma precursors. During the growth or processing of agricultural products, the glycoside aroma precursors are released into free aroma under the action of endogenous glycosidases. Therefore, the types and contents of glycoside aroma precursors directly affect the quality of agricultural products.

[0003] Glycoside aroma precursors are a class of sugar-containing derivatives formed by the condensation of the hydroxyl group of sugar and the hydroxyl group of aroma. Common ligands include benzyl alcohol, phenethyl alcohol, geraniol, and linalool, and sugar structures include glucose, primverose, etc. At present, a variety of glycoside aroma precursors have been found in agricultural products, such as benzyl glucoside, benzyl primveroside, phenethyl glucoside, phenethyl primveroside, geranyl primveroside, and linalyl primveroside, etc. Among them, geranyl primveroside and linalyl primveroside are a pair of structural isomers with the same molecular weight and similar structures, which are difficult to identify.

[0004] The determination methods of glycoside aroma precursors include hydrolysis method, derivatization method, direct determination method, etc., but they have disadvantages such as inaccurate qualitative analysis, long period, and complex operation. The liquid chromatography-tandem mass spectrometry method can directly determine one of geranyl primveroside and linalyl primveroside, but due to the same qualitative mass spectrometry ion pair information, these two glycosides cannot be distinguished.

[0005] For example, the patent with the application number CN202210984085 discloses a method for detecting enantiomers of linalool primveroside in tea, the patent with the application number CN202010983355 discloses a method for simultaneously determining 6 glycoside aroma precursors in pomelo, and the patent with the application number CN201810179791 discloses a method for directly detecting glycoside-bound aroma precursor substances in tea; in the above prior arts, geranyl primveroside and linalyl primveroside in the sample are both identified by comparing with the standard product, that is, liquid chromatography-tandem mass spectrometry technology is used to separate geranyl primveroside and linalyl primveroside contained in the sample and obtain the retention time, and qualitative analysis is carried out by the consistency of the retention time with the standard product.

[0006] That is, although liquid chromatography-tandem mass spectrometry technology is used to detect glycoside aroma precursors in the prior art, they are all identified by the retention time of the standard product, and this method also has problems such as complex operation and long period.

Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for extracting and identifying geranyl primveroside and linalyl primveroside, which has the advantages of simple extraction operation, rapid and efficient analysis, short cycle, and significantly improved identification sensitivity of geranyl primveroside and linalyl primveroside in agricultural products.

[0008] The present invention is implemented as follows:

[0009] A method for extracting and identifying geranyl primveroside and linalyl primveroside, the steps of the method are as follows:

[0010] 1) Extraction of geranyl primveroside and linalyl primveroside in agricultural products;

[0011] 2) Chromatographic separation of geranyl primveroside and linalyl primveroside by liquid chromatography-tandem mass spectrometry;

[0012] 3) By liquid chromatography-tandem mass spectrometry, in the ESI - mode, monitor the intensities of 4 pairs of mass ion pairs 447.2>315.2, 447.2>161.1, 447.2>89.0, 447.2>149.2;

[0013] 4) Compare the intensities of each mass ion pair of the chromatographic peak at the same retention time. When the intensity of 447.2>149.2 is greater than the intensity of 447.2>161.1 and the ratio is greater than 1, it is determined that the compound corresponding to the chromatographic peak is geranyl primveroside; when the intensity of 447.2>149.2 is less than the intensity of 447.2>161.1 and less than 1, it is determined that the compound corresponding to the chromatographic peak is linalyl primveroside.

[0014] Furthermore, the chromatographic and mass spectrometric conditions are as follows:

[0015] Chromatographic column: ACQUITY UPLC BEH C18 (2.1×100mm, 1.7μm);

[0016] Chromatographic conditions: The aqueous phase (phase A) of the mobile phase is 5 mmol / L ammonia aqueous solution, and the organic phase (phase B) is acetonitrile; the flow rate of the mobile phase is 0.4 mL / min, the column oven temperature is 40°C, the sample tray temperature is 15°C, and the injection volume is 2 μL; the gradient elution program is 1 min, 10% B; 6 min, 40% B; 7 min, 50% B; 10 min, 50% B; 10.1 min, 90% B; 11 min, 10% B; 13 min, 10%.

[0017] Mass spectrometry conditions: ESI electrospray ionization source; negative ion mode; multiple reaction monitoring (MRM); atmospheric pressure ionization (API) gas is nitrogen with purity > 95%; collision gas is argon with purity > 99.999%; interface voltage 4.5 kv; interface temperature 400 °C; desolvation temperature 650 °C; heating block temperature 400 °C; DL temperature 150 °C; drying gas flow rate 10 L / min, nebulizing gas 3 L / min, heating gas 10 L / min;

[0018] Furthermore, the mass spectrometry parameters for collecting geranylprimveroside and linalylprimveroside are as follows:

[0019] Table 1 Mass spectrometry parameters for collecting geranylprimveroside and linalylprimveroside

[0020]

[0021] The present invention has the following advantages:

[0022] 1. The present invention can be applied to the identification of geranylprimveroside and linalylprimveroside in agricultural products. The mobile phase composition adopted in the present invention is: under the mobile phase with 5 mmol / L ammonia aqueous solution as the aqueous phase and acetonitrile as the organic phase; the monitored ions are: monitoring the parent ion mass-to-charge ratio 447, fragment ions 315.2, 161.1, 149.2, 89.0 in the ESI negative mode; the collected signals are: simultaneously collecting the ion pair signals of 447.2>315.2, 447.2>161.1, 447.2>89.0, 447.2>149.2, and recording their intensities; the setting of the above conditions makes the limits of detection (LOD) of geranylprimveroside and linalylprimveroside in agricultural products be 0.89 ng / mL and 1.06 ng / mL respectively, significantly improving the identification sensitivity of geranylprimveroside and linalylprimveroside in agricultural products.

[0023] 2. The present invention first proposes a method for identifying isomeric compounds (geranylprimveroside and linalylprimveroside) by the difference in the intensities of fragment ions generated by liquid chromatography-tandem mass spectrometry, that is, geranylprimveroside and linalylprimveroside are identified by the difference in the intensity ratio of 447.2>149.2 and 447.2>161.1. The identification process does not require the preparation of a standard curve by configuring a standard product, and the process is simple and has a short cycle.

Description of the Drawings

[0024] The present invention will be further described below with reference to the drawings in conjunction with the embodiments.

[0025] Figure 1 It is a schematic diagram of the mass spectrometry fragmentation modes of geranylprimveroside and linalylprimveroside in the embodiments of the present invention. The left side is the mass spectrometry fragmentation mode of geranylprimveroside, and the right side is the mass spectrometry fragmentation mode of linalylprimveroside.

[0026] Figure 2 This is the mass chromatogram of geranyl primveroside and linalyl primveroside in the sensitivity experiment of the embodiments of the present invention. The mass chromatogram of 0.89 ng / mL geranyl primveroside is on the left, and the mass chromatogram of 1.06 ng / mL linalyl primveroside is on the right.

[0027] Figure 3 This is the mass chromatogram of 100 ng / mL geranyl primveroside and linalyl primveroside in the embodiments of the present invention. The mass chromatogram of geranyl primveroside is on the left, and the mass chromatogram of linalyl primveroside is on the right.

[0028] Figure 4 This is the chromatogram of each ion pair of the mixed standard of 500 ng / mL geranyl primveroside and linalyl primveroside in the embodiments of the present invention. The chromatogram of each ion pair of component 1 is on the left, and the chromatogram of each ion pair of component 2 is on the right.

[0029] Figure 5 This is the mass chromatogram of each component in the Shoumei white tea sample in the embodiments of the present invention. The mass chromatogram of component 1 is on the left, and the mass chromatogram of component 2 is on the right.

Specific Embodiments

[0030] The present invention relates to a method for extracting and identifying geranyl primveroside and linalyl primveroside, which specifically includes the following steps:

[0031] 1. Sample extraction

[0032] Weigh about 2 g (accurate to 0.01 g) of the sample into a 50 mL polypropylene centrifuge tube, add 10 mL of methanol, homogenize at 10000 r / min for 2 min, centrifuge at 4500 r / min for 5 min, take 2 mL of the supernatant into a 10 mL polypropylene centrifuge tube, add 50 mg of QuEchERS purification reagent C18, 100 mg of PSA, and 100 mg of GCB, mix well, let stand, and take the supernatant through a 0.22 μm filter membrane;

[0033] 2. Chromatographic and mass spectrometric conditions

[0034] Chromatographic column: ACQUITY UPLC BEH C18 (2.1×100 mm, 1.7 μm).

[0035] Chromatographic conditions: The aqueous phase (phase A) of the mobile phase is 5 mmol / L ammonia water solution, and the organic phase (phase B) is acetonitrile. The flow rate of the mobile phase is 0.4 mL / min, the temperature of the column oven is 40 °C, the temperature of the sample tray is 15 °C, and the injection volume is 2 μL. The gradient elution program is 1 min, 10% B; 6 min, 40% B; 7 min, 50% B; 10 min, 50% B; 10.1 min, 90% B; 11 min, 10% B; 13 min, 10%.

[0036] Mass spectrometry conditions: ESI electrospray ionization source; negative ion mode; multiple reaction monitoring (MRM); atmospheric pressure ionization (API) gas (nitrogen, purity > 95%); collision gas (argon, purity > 99.999%); interface voltage 4.5 kv; interface temperature 400 °C; desolvation temperature 650 °C; heating block temperature 400 °C; DL temperature 150 °C; drying gas flow rate 10 L / min, nebulizing gas 3 L / min, heating gas 10 L / min.

[0037] Table 1 Mass spectrometry parameters for collecting geranyl primveroside and linaloolyl primveroside

[0038]

[0039] 3. Instrumental analysis

[0040] Take 1 mL of the purified sample and perform injection analysis using liquid chromatography-tandem mass spectrometry. Using the chromatographic and mass spectrometry conditions provided in step 2, monitor the ion pair signals of the mass chromatogram. Record the ion pair intensities of 447.2>315.2, 447.2>161.1, 447.2>89.0, and 447.2>149.2 for each chromatographic peak respectively.

[0041] 4. Result identification

[0042] By identifying the chromatographic peaks that generate 4 ion pairs, the chromatographic peaks are determined. Compare the intensities of 447.2>161.1 and 447.2>149.2 in the same chromatographic peak. When the intensity of 447.2>149.2 is greater than the intensity of 447.2>161.1 and the ratio is greater than 1, it is determined that the compound corresponding to the chromatographic peak is geranyl primveroside; when the intensity of 447.2>149.2 is less than the intensity of 447.2>161.1 and less than 1, it is determined that the compound corresponding to the chromatographic peak is linaloolyl primveroside.

[0043] The following will be combined with the appendix Figures 1-5The technical solutions of the present invention will be clearly and completely described in conjunction with the specific embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention. For those not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer shall be followed. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0044] Example 1

[0045] 1. Method principle

[0046] Geranyl primveroside and linalyl primveroside are a pair of constitutional isomers with the same molecular weight and similar structures. Determined by liquid chromatography tandem mass spectrometry, their fragmentation patterns are similar and can produce the same fragment ions. According to the subtle differences in the structures of the two components, the present invention invents a method for monitoring the same fragment ions under specific conditions and identifying them based on the differences in the contents of the fragment ions. Under the conditions of the present invention, geranyl primveroside produces more fragment ions with a mass-to-charge ratio of 149.1 and fewer fragment ions with a mass-to-charge ratio of 161.1; however, linalyl primveroside is the opposite, producing fewer fragment ions with a mass-to-charge ratio of 149.1 and more fragment ions with a mass-to-charge ratio of 161.1. Their fragmentation patterns are as Figure 1 shown.

[0047] 2. Method sensitivity

[0048] The sensitivity of geranyl primveroside and linalyl primveroside was verified by measuring the signal intensity of the standard solution of the analyte diluted step by step. The detection limit was confirmed when the signal-to-noise ratio (S / N) was 3, and the quantification limit was confirmed when the signal-to-noise ratio (S / N) was 10.

[0049] When the concentration of geranyl primveroside was 0.89 ng / mL (monitoring ion pair 447.2>149.2), the signal-to-noise ratio (S / N) was 3.4. When the concentration of linalyl primveroside was 1.06 ng / mL (monitoring ion pair 447.2>161.1), the signal-to-noise ratio (S / N) was 3.1. Therefore, the sensitivity of this method was confirmed to be 0.89 ng / mL for geranyl primveroside and 1.06 ng / mL for linalyl primveroside. The mass chromatograms of geranyl primveroside and linalyl primveroside Figure 2 shown.

[0050] 3. Method verification

[0051] The present invention is verified by injecting standards of geranyl-primveroside and linalyl-primveroside. Under the experimental conditions of this method, the verification is carried out on: (1) standards of 100 ng / mL geranyl-primveroside or 100 ng / mL linalyl-primveroside. By identifying the chromatographic peaks generating 4 ion pairs, it is found that there is 1 chromatographic peak generating 4 ion pairs for each single standard, and the intensities of 447.2>161.1 and 447.2>149.2 of the chromatographic peak are obtained, as Figure 3 shown.

[0052] The relevant data obtained are shown in Table 2:

[0053] Table 2 Intensities and ratios of different ion pairs of the same chromatographic peak in the standard

[0054]

[0055] In the verification results, the ratio of 447.2>149.2 to 447.2>161.1 of component 1 is 8.1×10 -3 , which is much less than 1, and it can be determined as linalyl-primveroside. The ratio of 447.2>149.2 to 447.2>161.1 of component 2 is 33.7, and it can be determined as geranyl-primveroside. The determination results are consistent with the standards used in the experiment. It is proved that the method is feasible and effective.

[0056] (2) Verification of the mixed standard of 500 ng / mL geranyl-primveroside and linalyl-primveroside. By identifying the chromatographic peaks generating 4 ion pairs, it is found that there are two chromatographic peaks generating 4 ion pairs, and the intensities of 447.2>161.1 and 447.2>149.2 of each chromatographic peak are obtained, as Figure 4 shown.

[0057] The relevant data obtained are shown in Table 3:

[0058] Table 3 Intensities and ratios of different ion pairs of the same chromatographic peak in the mixed standard solution of 500 ng / mL geranyl-primveroside and linalyl-primveroside

[0059]

[0060] In the verification results, the ratio of 447.2>149.2 to 447.2>161.1 of component 1 is 5.0×10 -3 , which is much less than 1, and it can be determined as linalyl-primveroside. The ratio of 447.2>149.2 to 447.2>161.1 of component 2 is 58.7, and it can be determined as geranyl-primveroside. The reliability of the determination results is also verified by the spiking method in the experiment.

[0061] 4. Analysis of actual samples

[0062] Taking Shoumei white tea as an example, the components of geranyl primveroside and linaloolyl primveroside contained in it were identified. The sample was extracted, analyzed and identified by instrumental determination. By identifying the chromatographic peaks that produced 4 ion pairs, it was found that there were two chromatographic peaks that produced 4 ion pairs, and the intensities of 447.2>161.1 and 447.2>149.2 of the chromatographic peaks were obtained, as Figure 5 shown.

[0063] The relevant data obtained are shown in Table 4:

[0064] Table 4 Intensities and ratios of different ion pairs in Shoumei white tea

[0065]

[0066] According to the component identification method of the present invention, when the ratio of 1447.2>149.2 to 447.2>161.1 of component 1 is 7.0×10 -4 , which is much less than 1, it is determined that the compound corresponding to this chromatographic peak is linaloolyl primveroside; when the ratio of 1447.2>149.2 to 447.2>161.1 of component 2 is 50.0, which is much greater than 1, it is determined that the compound corresponding to this chromatographic peak is geranyl primveroside.

[0067] In summary, the present invention has the following advantages:

[0068] 1. The present invention can be applied to the identification of geranyl primveroside and linaloolyl primveroside contained in agricultural products. The mobile phase composition adopted by the present invention is: under the mobile phase with 5 mmol / L ammonia aqueous solution as the aqueous phase and acetonitrile as the organic phase; the monitored ions are: monitoring the parent ion mass-to-charge ratio 447, fragment ions 315.2, 161.1, 149.2, 89.0 in the ESI negative mode; the collected signals are: simultaneously collecting the ion pair signals of 447.2>315.2, 447.2>161.1, 447.2>89.0, 447.2>149.2 and recording their intensities; the setting of the above conditions makes the detection limits (LOD) of geranyl primveroside and linaloolyl primveroside 0.89 ng / mL and 1.06 ng / mL respectively, significantly improving the identification sensitivity of geranyl primveroside and linaloolyl primveroside in agricultural products.

[0069] 2. The present invention first proposes a method for identifying isomeric compounds (geranyl primveroside and linaloolyl primveroside) by the difference in the intensities of fragment ions generated by liquid chromatography-tandem mass spectrometry, that is, geranyl primveroside and linaloolyl primveroside are identified by the difference in the intensity ratio of 447.2>149.2 to 447.2>161.1. The identification process does not require the preparation of a standard curve by configuring a standard product, and the process is simple and the cycle is short.

[0070] Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments we described are illustrative only and not used to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope protected by the claims of the present invention.

Claims

1. A method for extracting and identifying geranyl primulosides and linalyl primulosides, characterized in that: The method steps are as follows: 1) Extraction of geranyl primulosides and linalyl primulosides from agricultural products; 2) separating geranyl primulosides and linalyl primulosides by liquid chromatography-tandem mass spectrometry; 3) Liquid chromatography tandem mass spectrometry, ESI - In mode, the intensities of four mass ion pairs 447.2>315.2, 447.2>161.1, 447.2>89.0, and 447.2>149.2 were monitored; 4) Compare the intensities of the mass ion pairs of the chromatographic peaks at the same retention time. When the intensity of 447.2>149.2 is greater than the intensity of 447.2>161.1, and the ratio is greater than 1, the compound corresponding to the chromatographic peak is determined to be geranyl primulosidoside; when the intensity of 447.2>149.2 is less than the intensity of 447.2>161.1, and less than 1, the compound corresponding to the chromatographic peak is determined to be linalyl primulosidoside.

2. The method for extracting and identifying geranyl primulosides and linalyl primulosides according to claim 1, characterized in that: The chromatographic mass spectrometry conditions are as follows: Chromatographic column: ACQUITY UPLC BEH C18 (2.1×100 mm, 1.7 μm); Chromatographic conditions: the aqueous phase (phase A) of the mobile phase was 5 mmol / L ammonia solution, and the organic phase (phase B) was acetonitrile; the mobile phase flow rate was 0.4 mL / min, the column oven temperature was 40°C, the sample tray temperature was 15°C, and the injection volume was 2 μL; the gradient elution program was 1 min, 10% B; 6 min, 40% B; 7 min, 50% B; 10 min, 50% B; 10.1 min, 90% B; 11 min, 10% B; 13 min, 10%. Mass spectrometry conditions: ESI electrospray ion source; negative ion mode; multiple reaction monitoring (MRM); atmospheric pressure ionization (API) gas is nitrogen, purity>95%; collision gas is argon, purity>99.999%; interface voltage 4.5kV; Interface temperature 400°C; desolvation temperature 650°C; heating block temperature 400°C; DL temperature 150°C; drying gas flow 10L / min, nebulizing gas 3L / min, heating gas 10L / min.

3. The method for extracting and identifying geranyl primulosides and linalyl primulosides according to claim 1 or 2, characterized in that: The mass spectrometry parameters for collecting geranyl primulosides and linalyl primulosides are as follows: Table 1 Mass spectrometry parameters for collecting geranyl primulosides and linalyl primulosides

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