Method for determining methoxybenzene compounds in tea leaves based on multiple headspace solid-phase microextraction

Abstract

The invention relates to the technical field of chemical analysis, in particular to a method for determining methoxybenzene compounds in tea based on multiple headspace solid-phase microextraction. The method comprises the following steps: 1) performing headspace solid-phase microextraction on methoxybenzene compounds in tea leaves for many times; extraction conditions are as follows: 0.05-0.40 g of tea leaves are added into a 20mL headspace bottle, water with the mass being 3-7 times that of the tea leaves is used as a solvent, and extraction is performed for 4-6 times; the extraction temperature is 30-70 DEG C; the extraction time of each time is 30 to 70 minutes; and 2) determining the methoxybenzene compound by gas chromatography-mass spectrometry, obtaining a fitted equation according to the peak area and the extraction times, and further obtaining the content of the methoxybenzene compound. According to the method, the methoxybenzene compounds in the tea leaves can be fully extracted, and the problem of inaccurate quantification caused by the difference of the release rate of the methoxybenzene compounds is avoided.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis, and in particular to a method for determining methoxybenzene compounds in tea based on multiple headspace solid phase microextraction. Background Art

[0002] Tea aroma is a comprehensive expression of the interaction of volatile odor active ingredients. It is one of the most important factors in measuring tea quality and directly determines the market value of tea and consumer choice. Methoxybenzene compounds are considered to be a class of substances formed by microbial degradation and methylation of catechins, as well as thermal degradation during post-fermentation. Most of them have a stale taste and are the key aroma compounds that form the unique stale flavor of Pu'er tea. Therefore, accurate quantification of the content of methoxybenzene compounds in Pu'er tea is of great significance for the quantitative evaluation of its quality.

[0003] Multiple headspace solid phase microextraction (MHS-SPME) is a step-by-step headspace extraction technique for quantitative analysis of volatile components in complex solid and liquid samples. MHS-SPME technology calculates the total amount of target analytes by performing multiple consecutive extractions on the same sample to achieve complete extraction. Therefore, there is no need to consider the impact of differences in matrix composition on a single extraction, and avoid overcoming the influence of matrix effects. When a limited number of extractions can be performed to completely extract, the total amount of target analytes extracted each time can be obtained by simply adding together. However, in general, it is difficult to achieve a process of complete sample extraction through multiple consecutive solid phase microextractions. Summary of the invention

[0004] To solve at least one of the problems of the prior art, the present invention provides a method for determining methoxybenzene compounds in tea leaves, which accurately determines the content of methoxybenzene compounds in tea leaves (e.g., Pu'er tea) based on multiple headspace solid phase microextraction-gas chromatography-mass spectrometry. Headspace solid phase microextraction takes a short time, is easy to operate, does not require solvents, is green, and is easy to automate. The method of the present invention can fully extract methoxybenzene compounds in tea leaves, avoiding the problem of inaccurate quantification due to differences in the release rates of methoxybenzene compounds.

[0005] A method for determining methoxybenzene compounds in tea, comprising:

[0006] 1) Multiple headspace solid phase microextraction of methoxybenzene compounds in tea;

[0007] Extraction conditions: add 0.05-0.40g tea leaves to a 20mL headspace bottle, use 3-7 times the weight of tea leaves in water as solvent, extract 4-6 times; extraction temperature is 30-70℃; each extraction time is 30-70min;

[0008] 2) Gas chromatography-mass spectrometry (GC-MS) was used to determine methoxybenzene compounds, and a fitting equation was obtained based on the peak area and the number of extractions, and then the content of methoxybenzene compounds was obtained using standard substances.

[0009] Preferably, the tea leaves are crushed to 40-80 mesh, more preferably 60 mesh. Studies have found that tea leaves within this particle size range are more conducive to dissolving methoxybenzene compounds.

[0010] Preferably, the tea leaves are ground at low temperature.

[0011] Preferably, the tea leaves are dark teas, including Pu'er tea, Liubao tea and the like.

[0012] Preferably, 0.10-0.20 g of tea leaves are added to a 20 mL headspace bottle. Studies have found that when the amount of tea leaves is within this range, the methoxybenzene compounds released during the extraction time are more conducive to saturating the extraction head, thereby improving the accuracy of the measurement results.

[0013] Preferably, the amount of solvent used is 4-6 times the mass of tea leaves. Studies have found that when the amount of solvent water is within this range, the methoxybenzene compounds released during the extraction time are more conducive to saturating the extraction head, thereby improving the accuracy of the measurement results.

[0014] Ultrapure water is preferably used in the present invention.

[0015] Preferably, the extraction temperature is 50-60° C. Studies have found that when the extraction temperature is within this range, the methoxybenzene compounds released during the extraction time are more conducive to saturating the extraction head and improving the accuracy of the measurement results.

[0016] Preferably, each extraction time is 40-50 minutes. Studies have found that within this extraction time range, the methoxybenzene compounds released during the extraction time are more conducive to saturating the extraction head and improving the accuracy of the measurement results.

[0017] Preferably, the extraction conditions further include: a stirring speed of 180-220 rpm and a balancing time of 4-6 min.

[0018] Further preferably, the extraction conditions also include: stirring speed of 200 rpm and equilibration time of 5 min.

[0019] Preferably, the process further comprises decomposing the annealing agent at 240-260° C. for 4-6 min, more preferably decomposing the annealing agent at 250° C. for 5 min.

[0020] The methoxybenzene compounds of the present invention include 1,2-dimethoxybenzene, 3,4-dimethoxytoluene, 1,2,3-trimethoxybenzene, 4-ethyl-1,2-dimethoxybenzene, 1,2,4-trimethoxybenzene, 1,2,3-trimethoxy-5-methylbenzene and 1,2,3,4-tetramethoxybenzene.

[0021] Specifically, the method for determining methoxybenzene compounds in tea leaves comprises:

[0022] S1: Accurately weigh 0.10-0.20g of crushed tea leaves (crushed to 60 mesh at low temperature) into a 20mL headspace bottle, and add ultrapure water 4-6 times the weight of the tea leaves; place the headspace bottle in a 50-60℃ water bath and stir at a stirring speed of 180-220rpm for 4-6min; insert the extraction head and extract for 40-50min;

[0023] S2: Insert the extraction head into the GC-MS injection port, decompose at 250℃ for 4-6min, and analyze;

[0024] Then the extraction head is inserted into the headspace bottle again, and extraction is performed according to step S1; the extraction is repeated 5 times;

[0025] The fitting equation was obtained according to the peak area and the number of extractions, and then the content of methoxybenzene compounds was obtained using standard substances.

[0026] The determination method provided by the present invention has the following beneficial effects:

[0027] 1) Fully extract methoxybenzene compounds in tea

[0028] The release of methoxybenzene compounds from Pu'er tea is a slow process. A single solid phase microextraction can only enrich a part of the substances. Multiple extractions bring in the linear relationship equation between the logarithm of the extraction peak area and the number of extractions, and calculate the total peak area of ​​methoxybenzene compounds to achieve the purpose of sufficient extraction and analysis of methoxybenzene compounds.

[0029] 2) Avoid the problem of inaccurate quantification due to differences in the release rates of methoxybenzene compounds.

[0030] Since the release pattern of methoxybenzene compounds in tea is different from that of standard samples, there is a certain error in the quantification of the content of methoxybenzene compounds in tea using standard samples. The use of multiple headspace solid phase microextraction can effectively avoid the problem of poor quantitative accuracy caused by differences in release rate.

[0031] The method of the invention can simply, quickly and accurately detect the content of methoxybenzene compounds in tea leaves, and is particularly suitable for accurate quantification of methoxybenzene compounds in dark teas such as Pu'er tea and Liubao tea. DETAILED DESCRIPTION

[0032] In order to make the above-mentioned purpose, features and advantages of the present invention more obvious and easy to understand, the specific implementation of the present invention is described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0033] Gas chromatography-mass spectrometry: Thermo Trace 1300GC coupled with ISQ MS.

[0034] Headspace solid phase microextraction or fiber head: 65 μm PDMS / DVB solid phase microextraction extraction head, Supelco, USA.

[0035] Example 1

[0036] A method for determining methoxybenzene compounds in tea, comprising:

[0037] S1: Accurately weigh 0.10g of crushed Pu'er tea (crushed to 60 mesh at low temperature) into a 20mL headspace bottle, and add ultrapure water 5 times the weight of the tea leaves; place the headspace bottle in a 50℃ water bath and stir at a stirring speed of 200rpm for 5min; insert the extraction head and extract for 40min;

[0038] S2: Insert the extraction head into the GC-MS injection port, decompose at 250℃ for 5min, and analyze;

[0039] Then the extraction head is inserted into the headspace bottle again, and extraction is performed according to step S1; the extraction is repeated 5 times;

[0040] The fitting equation was obtained according to the peak area and the number of extractions, and the content of methoxybenzene compounds was obtained.

[0041] The GC-MS analysis conditions are as follows:

[0042] GC conditions: Capillary column: HP-5MS (30 m×0.25 μm×0.25 mm).

[0043] Inlet temperature: 250℃; splitless mode, splitless time 5min; constant flow mode, flow rate 1.0mL / min; programmed temperature rise: initial temperature 50℃, hold for 3min, then increase to 180℃ at 4℃ / min, hold for 5min, then increase to 250℃ at 15℃. The total time is 45.17min.

[0044] MS conditions: EI source, ionization energy 70 eV; transfer line temperature 250 °C; ion source temperature 250 °C; solvent delay 3 min, mass spectrometry scanning range 35-350 Hz.

[0045] When the target component is consistent under other conditions, for a certain amount of sample, the extraction peak area and the number of extractions show an exponential decay relationship, and the total amount of the target analyte can be obtained by summing up the formula.

[0046] The linear relationship equation between the logarithm of the extraction peak area and the number of extractions is:

[0047] LqCy i =(i-1)lnβ+lnA 1 (1)

[0048] A i is the peak area of ​​the i-th extraction sample, β can be calculated by lnA i The slope of the peak area with the number of extractions (il) is obtained. Therefore, the peak area decreases exponentially with the number of extractions. Only 3-4 HS-SPME processes are needed to determine the constant β. The total peak area can be calculated by the following formula:

[0049] A T =A 1 / (1-β) (2)

[0050] Thus, the total amount of each analyte in the whole system can be obtained.

[0051] The determination coefficient R of the fitting equation obtained by multiple extractions 2 Both are greater than 0.960.

[0052] The following screens the Pu'er tea dosage, water addition amount, water bath temperature and extraction time.

[0053] 1) Pu'er tea dosage

[0054] Accurately weigh 0.05g, 0.10g, 0.20g, 0.30g, and 0.40g of Pu'er tea, respectively, and place them in 20mL headspace bottles. Add 5 times of ultrapure water, heat in a water bath at 50°C, extract for 50min, stir and extract 5 times, and perform GC-MS analysis. The fitting equation obtained based on the peak area and extraction times is the determination coefficient R 2 The closer it is to 1, the better the equation fitting effect is, and the more accurate the total peak area of ​​methoxybenzene compounds calculated in the end is. The results of the investigation on the dosage of Pu'er tea are shown in Table 1.

[0055] Table 1 Results of investigation on tea dosage

[0056]

[0057] The results in Table 1 show that when the mass of Pu'er tea is too small, the methoxybenzene compounds released during the extraction time cannot saturate the extraction head. The coefficient of determination R of the fitting equation obtained by multiple extractions 2 The determination coefficient R is between 0.817 and 0.932, and the determination coefficient R 2 The closer to 1, the more 0.10-0.20g tea addition amount R 2 The optimal value is that as the quality of tea leaves increases again, the release regularity of methoxybenzene compounds released during the extraction time becomes worse, resulting in a determination coefficient R 2 Therefore, the quality of tea leaves is controlled at 0.10-0.20g.

[0058] 2) Amount of water added

[0059] Weigh 0.10g of Pu'er tea accurately and place it in a 20mL headspace bottle. Add ultrapure water 3, 4, 5, 6, and 7 times the weight of the tea, heat in a 50℃ water bath, stir and extract 5 times, analyze by GC-MS, and obtain the fitting equation based on the peak area and extraction times. The results of the water addition investigation are shown in Table 2.

[0060] Table 2 Results of water addition

[0061]

[0062] The results in Table 2 show that the increase in the amount of water added is conducive to the release of methoxybenzene compounds. When the amount of water added is 5 times the mass of tea leaves, the determination coefficient R 2 The closer it is to 1, the more water is added, the lower the release rate of methoxybenzene compounds tends to be, and the extraction head cannot be saturated within the extraction time. Therefore, the amount of water added is controlled at 4-6 times the mass of the tea leaves.

[0063] 3) Water bath temperature

[0064] Weigh 0.10g of Pu'er tea accurately and place it in a 20mL headspace bottle. Add ultrapure water 5 times the weight of the tea. Set the water bath heating temperature to 30℃, 40℃, 50℃, 60℃, and 70℃, respectively. Stir and extract 5 times. The extraction time is 50min. GC-MS analysis is performed. The fitting equation is obtained based on the peak area and the number of extractions. The results of the water bath temperature investigation are shown in Table 3.

[0065] Table 3 Water bath temperature investigation results

[0066]

[0067] The results in Table 3 show that when the water bath temperature is too low, the methoxybenzene compounds released during the extraction time cannot saturate the extraction head, and the coefficient of determination R of the fitting equation obtained by multiple extractions 2 Poor, with the increase of water bath temperature, the coefficient of determination R2 The closer to 1, the R 2 The optimal value is that as the water bath temperature increases again, the release regularity of the methoxybenzene compounds released during the extraction time becomes worse, resulting in a determination coefficient R 2 Therefore, the water bath temperature was controlled at 50-60°C.

[0068] 4) Extraction time

[0069] 0.10g of Pu'er tea was accurately weighed and placed in a 20mL headspace bottle, and ultrapure water 5 times the weight of the tea was added. The water bath heating temperature was set to 50°C, and the extraction time was set to 30min, 40min, 50min, 60min, and 70min. Stir and extract 5 times, GC-MS analysis, and the fitting equation was obtained based on the peak area and the number of extractions. The results of the extraction time investigation are shown in Table 4.

[0070] Table 4 Extraction time investigation results

[0071]

[0072] The results in Table 4 show that when the extraction time is too short, the methoxybenzene compounds released during the extraction time cannot saturate the extraction head, and the coefficient of determination R of the fitting equation obtained by multiple extractions 2 Poor, with the extension of extraction time, the coefficient of determination R 2 The closer to 1, the extraction time R is 40-50min 2 The optimal value is that as the extraction time is extended again, the release regularity of methoxybenzene compounds becomes worse, resulting in a determination coefficient R 2 Therefore, the extraction time is controlled at 40-50 minutes.

[0073] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all 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 determining methoxybenzene compounds in tea, characterized in that: include: 1) Multiple headspace solid phase microextraction of methoxybenzene compounds in tea; Extraction conditions: add 0.05-0.40g tea leaves to a 20mL headspace bottle, use 3-7 times the weight of tea leaves in water as solvent, extract 4-6 times; extraction temperature is 30-70℃; each extraction time is 30-70min; 2) Gas chromatography-mass spectrometry was used to determine methoxybenzene compounds, and a fitting equation was obtained based on the peak area and the number of extractions, and then the content of methoxybenzene compounds was obtained using standard substances.

2. The method for determining methoxybenzene compounds in tea leaves according to claim 1, characterized in that: The tea leaves are crushed to 40-80 mesh, preferably 60 mesh; Preferably, the tea leaves are dark teas, including Pu'er tea and Liubao tea.

3. The method for determining methoxybenzene compounds in tea leaves according to claim 1 or 2, characterized in that: Add 0.10-0.20 g of tea leaves to a 20 mL headspace vial.

4. The method for determining methoxybenzene compounds in tea leaves according to any one of claims 1 to 3, characterized in that: The amount of solvent used is 4-6 times the mass of tea leaves.

5. The method for determining methoxybenzene compounds in tea leaves according to any one of claims 1 to 4, characterized in that: The extraction temperature is 50-60℃.

6. The method for determining methoxybenzene compounds in tea leaves according to any one of claims 1 to 5, characterized in that: Each extraction time is 40-50 minutes.

7. The method for determining methoxybenzene compounds in tea leaves according to any one of claims 1 to 6, characterized in that: The extraction conditions also include: stirring speed 180-220 rpm, equilibrium time 4-6 min; Preferably, the extraction conditions further include: a stirring speed of 200 rpm and a balancing time of 5 min.

8. The method for determining methoxybenzene compounds in tea leaves according to any one of claims 1 to 7, characterized in that: It also includes decomposing the annealing agent at 240-260°C for 4-6 minutes, more preferably decomposing the annealing agent at 250°C for 5 minutes.

9. The method for determining methoxybenzene compounds in tea leaves according to claim 1, characterized in that: include: S1: Accurately weigh 0.10-0.20g of crushed tea leaves into a 20mL headspace bottle, and add ultrapure water 4-6 times the weight of the tea leaves; place the headspace bottle in a 50-60℃ water bath and stir at a stirring speed of 180-220rpm for 4-6min; insert the extraction head and extract for 40-50min; S2: Insert the extraction head into the GC-MS injection port, decompose at 250℃ for 4-6min, and analyze; Then the extraction head is inserted into the headspace bottle again, and extraction is performed according to step S1; the extraction is repeated 5 times; The fitting equation was obtained according to the peak area and the number of extractions, and then the content of methoxybenzene compounds was obtained using standard substances.

10. The method for determining methoxybenzene compounds in tea leaves according to any one of claims 1 to 9, characterized in that: The methoxybenzene compounds include 1,2-dimethoxybenzene, 3,4-dimethoxytoluene, 1,2,3-trimethoxybenzene, 4-ethyl-1,2-dimethoxybenzene, 1,2,4-trimethoxybenzene, 1,2,3-trimethoxy-5-methylbenzene and 1,2,3,4-tetramethoxybenzene.

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