CN113834886B - Detection method for rapidly and accurately quantifying nitrogen-oxygen heterocyclic compounds in green tea and evaluation method for dryness degree of green tea - Google Patents
Detection method for rapidly and accurately quantifying nitrogen-oxygen heterocyclic compounds in green tea and evaluation method for dryness degree of green tea Download PDFInfo
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Abstract
The invention discloses a method for rapidly and accurately detecting nitrogen-oxygen heterocyclic compounds in green tea and a method for evaluating the dryness degree of the green tea, and particularly relates to a method for rapidly analyzing the nitrogen-oxygen heterocyclic compounds in the green tea by combining a metal bath-assisted arrow-shaped solid phase microextraction technology with a gas chromatography-triple quadrupole tandem mass spectrometry technology. The detection method of the nitrogen-oxygen heterocyclic compounds provided by the invention can realize simultaneous qualitative and quantitative detection of 7 nitrogen-oxygen heterocyclic compounds in tea, has good selectivity and high sensitivity, solves the problems of low level of the nitrogen-oxygen heterocyclic compounds in green tea and serious influence of complex matrixes of the tea on analysis interference of target objects, and can realize rapid and accurate determination of the nitrogen-oxygen heterocyclic compounds in the green tea. The green tea drying degree evaluation method screens and proposes furan aldehyde as a key index compound for evaluating the box type hot air drying degree (water content).
Description
Technical Field
The invention relates to the field of chemical analysis, in particular to a method for quickly and accurately detecting nitrogen-oxygen heterocyclic compounds in green tea and a method for evaluating the dryness degree of the green tea.
Background
Green tea is one of the main tea types of tea in China, and is popular with consumers due to the excellent quality of high and fresh aroma and strong and mellow taste, such as West lake Longjing tea, xinyang Maojian tea, biluochun tea and the like. Currently, in the research of green tea aroma components, over 200 aroma components including alcohol, aldehyde, ketone, ester, acid, nitrogen-oxygen heterocyclic compounds and the like are separated and identified, wherein the nitrogen-oxygen heterocyclic compounds such as pyrazine, pyridine, pyrrole, furan, furfural and the like have outstanding aroma characteristics and low aroma threshold value, and have important contribution to the formation of green tea characteristic quality. Kumazawa et al found that 2-acetyl-1-pyrroline, 2-ethyl-3,5-dimethylpyrazine, tetramethylpyrazine, 2-acetyl-2-thiazoline, 2,3-diethyl-5-methylpyrazine had very high dilution of aroma (FD) factor values and was a characteristic aroma component of pan-fired green tea. In addition, baba et al found that 4-hydroxy-2,5-dimethyl-3 (2H) -furanone, 3-hydroxy-4,5-dimethyl-2 (5H) -furanone, 2-isopropyl-3-methylpyrazine, and 2-vinyl-3,5-dimethylpyrazine all showed very high FD values in three green tea varieties of Longjing, maofeng, and Biluochun, and are key aroma components of green tea. In the food flavor list published by the American food Flavor and Extract Manufacturers Association (FEMA), nitrogen-oxygen heterocyclic compounds occupy a prominent position, and derivatives thereof are widely present in baked foods, have important contributions to the formation of different food characteristic flavors and have wide development prospects.
The content of nitrogen-oxygen heterocyclic compounds in the tea is very low, and the direct analysis cannot be carried out frequently. Therefore, the pretreatment technology is particularly important, and is directly related to the quality of the analysis method, and influences the reliability, accuracy and analysis throughput of the result. At present, the pretreatment technology of nitrogen-oxygen heterocyclic compounds in food mainly comprises simultaneous distillation extraction, solvent extraction, liquid-liquid extraction (LLE), solid-phase extraction (SPE), stirring rod adsorption extraction (SBSE) and the like. Keim et al extracted by LLE and purified the azacyclo compound in the white spirit by silica gel, the method introduces an extraction step of removing most esters and alcohols, effectively improves the detection of azacyclo compound, but too many pretreatment steps lead to the loss of target. The method is simple and quick, has good repeatability, and has high selection of target compounds, which is the development trend and the requirement of the modern sample pretreatment technology. In the detection analysis of nitrogen heterocyclic compounds, gas chromatography-Flame Ionization Detector (FID) and nitrogen-phosphorus detector (NPD) were used for the first time, and the sensitivity to low levels of substances was insufficient, and although gas chromatography can effectively separate different components, reliable detection of some specific components was difficult, and thus a combined mass spectrometry technique was required. The GC-QqQ-MS/MS has the advantages of retention time and tandem mass spectrum confirmation, greatly improves the detection accuracy, can eliminate the interference of complex matrixes in food in a multi-reaction monitoring (MRM) mode, has good selectivity and high sensitivity, and becomes a rapid, accurate and reliable analysis tool in modern analysis laboratories.
Based on the complexity of tea aroma components and the special performances of low content, volatility, instability and the like of nitrogen-oxygen heterocyclic compounds, a rapid analysis method integrating high selectivity and high sensitivity is urgently needed to be developed, and theoretical guidance is provided for the improvement of high-quality green tea processing technology and technology.
Disclosure of Invention
Aiming at the defects in the field, the invention provides a method for rapidly and accurately detecting the heterocyclic nitrogen oxide compounds in the green tea, which can realize simultaneous qualitative and quantitative detection of 7 heterocyclic nitrogen oxide compounds in the green tea, has good selectivity and high sensitivity, solves the problems that the heterocyclic nitrogen oxide compounds in the green tea are low in level and difficult to detect, and the complicated matrix of the tea has serious influence on the analysis interference of a target object, and can rapidly and accurately detect the heterocyclic nitrogen oxide compounds in the green tea.
A method for rapidly and accurately detecting nitrogen-oxygen heterocyclic compounds in green tea in a quantitative mode is a detection method for rapidly analyzing the nitrogen-oxygen heterocyclic compounds in the green tea by combining a metal bath assisted arrow-shaped solid phase micro-extraction technology with a gas chromatography-triple quadrupole tandem mass spectrometry technology, and comprises the following steps:
(a) Preparation of standard working solutions:
taking standard substances of 2-methylfuran, 2-n-butylfuran, 2-n-pentylfuran, furan aldehyde, pyrrole, acetylpyrazine and acetylpyrrole as targets, preparing a 20mg/L standard stock solution by using ethanol to fix the volume, and then diluting the stock solution to 5000ng/mL, 2000ng/mL, 1000ng/mL, 500ng/mL, 300ng/mL, 100ng/mL, 50ng/mL, 20ng/mL, 10ng/mL and 5ng/mL step by step, wherein the concentration of the 2-methylpyrazine-d 6 internal standard is a working solution with the concentration of 50ng/mL for later use;
(b) Metal bath assisted arrow-shaped solid phase microextraction technology for extracting nitrogen-oxygen heterocyclic compounds
1) Aging of the extraction fiber head: loading the extracted fiber head into an Arrow-shaped solid phase microextraction (SPME Arrow) handle to form an SPME Arrow manual sample injector, inserting into a sample inlet with the temperature of 270 ℃, aging for 30min Zhong Housu, returning the fiber head, and pulling out the sample inlet;
2) Preparation of a standard sample: transferring a series of working solutions with different concentrations in the step (a) into a 20mL headspace bottle, and screwing down the working solutions by using a polytetrafluoroethylene sealing cap;
preparing a sample to be tested: accurately weighing 1.0g of tea sample into a 20mL headspace bottle, adding 3950 muL of distilled water and 50 muL of 4mg/L isotope 2-methylpyrazine-d 6 to ensure that the concentration of an isotope internal standard substance in a system is 50ng/mL, and screwing down by using a polytetrafluoroethylene sealing cap;
the preparation process of the tea sample comprises the following steps:
(I) Spreading fresh tea leaves: the indoor temperature is 25-28 ℃, and the humidity is 70-80%;
(II) roller type enzyme deactivation: the temperatures of the roller enzyme deactivating machines 1, 2 and 3 are respectively 280 ℃, 275 ℃ and 275 ℃;
(III) rolling: rotating at 50-52r/min, air-kneading for 5min, lightly kneading for 5min, loosening and pressing for 2min, medium pressing for 5min, heavily kneading for 3min, and air-kneading for 5min;
(IV) drying at 110 ℃ for 15min by gross fire, respectively weighing 80g of the tea leaves, placing the tea leaves in a stainless steel sieve tray, then placing the stainless steel sieve tray in a box type hot air dryer, drying at 115 ℃ for 5-50 min, and taking out the dried tea leaves to obtain the tea sample;
3) Nitrogen-oxygen heterocyclic compound extraction: putting the headspace bottle in the step 2) into a metal bath for heating, puncturing a diaphragm by adopting a Carbon WR SPME Arrow fiber head at the temperature of 60 ℃, and adsorbing for 30min above the headspace bottle;
4) Analyzing a sample: after extraction, inserting the SPME Arrow extraction needle into a GC sample inlet, performing resolution for 4min at 200 ℃, and performing gas chromatography-triple quadrupole tandem mass spectrometry (GC-QqQ-MS/MS) analysis;
(c) Gas chromatography-triple quadrupole tandem mass spectrometry technology analysis condition optimization
Chromatographic separation conditions: a chromatographic column VF-WAXms at 260 ℃ and 30m multiplied by 250 μm multiplied by 0.25 μm; temperature programming: the initial column temperature is 40 ℃, the temperature is kept for 3min, the temperature is increased to 120 ℃ at the speed of 3 ℃/min, the temperature is kept for 2min, and then the temperature is increased to 240 ℃ at the speed of 10 ℃/min, and the temperature is kept for 3min; carrier gas: helium with purity of 99.999%; carrier gas flow rate: 1.0mL/min; n is a radical of hydrogen 2 The collision airflow rate is 1.5mL/min; he quenching gas is 2.25mL/min; the split ratio is as follows: 5:1;
the retention time and mass spectrum conditions of the internal standard substance, 2-methylfuran, 2-n-butylfuran, 2-n-pentylfuran, furan aldehyde, pyrrole, acetylpyrazine and acetylpyrrole are optimized, and the parameter conditions of an optimized Multiple Reaction Monitoring (MRM) mode are as follows:
other mass spectral parameters: transmission line temperature: 250 ℃; the ion source temperature is 230 ℃; solvent retardation: 2min; an ionization mode: an EI source; ionization energy: 70eV; q2 collision gas: nitrogen with the purity more than or equal to 99.999 percent;
(d) Qualitative and quantitative analysis of nitrogen-oxygen heterocyclic compounds
And (3) qualitative analysis: according to the retention time and the characteristic ion peak of each standard substance;
quantitative analysis is carried out by adopting an internal standard method: performing linear regression analysis by using the working solution in the step (a) and according to the information of the selected characteristic qualitative ions and the selected characteristic quantitative ions by using the concentration ratio of the target compound to the internal standard as a horizontal coordinate and the peak area ratio as a vertical coordinate to obtain a standard working curve; under the same condition, the content of the target volatile matter in the tea sample to be detected is calculated by substituting the ratio of the peak area of the target volatile matter in the sample to be detected to the peak area of the internal standard into the corresponding standard working curve.
In step (d):
the standard working curve for 2-methylfuran was y =0.002239 x-1.448717 x 10 -4 ，R 2 =0.9990, linear range is 10-1000 ng/mL;
the standard working curve for 2-n-butylfuran is y =0.658498 x +0.067972 2 =0.9967, linear range is 5-500 ng/mL;
the standard working curve for 2-n-pentylfuran was y =0.686733 x-0.084885, r 2 =0.9909, linear range is 5-1000 ng/mL;
the standard working curve for furan aldehyde is y =0.438733 x +0.025218 2 =0.9995, linear range is 10-1000 ng/mL;
the standard working curve for pyrrole is y =0.054700 x +0.099804 2 =0.9984, linear range is 10-500 ng/mL;
the standard working curve of acetylpyrazine is y =0.109487 x +0.028905 2 =0.9986, linear range is 5-1000 ng/mL;
the standard working curve for acetylpyrrole is y =0.144857 x +0.118640 2 =0.9964, linear range 5-1000 ng/mL.
The invention also provides a green tea drying degree evaluation method, which adopts the detection method for quickly and accurately quantifying the nitrogen-oxygen heterocyclic compounds in the green tea, takes furan aldehyde as a key index compound for evaluating the drying degree of the box-type hot air, takes the furan aldehyde content detected by tea samples under different drying time lengths as an x variable, and takes the drying degree with water content as an investigation index as a y variable for regression analysis to obtain a drying degree fitting curve; and under the same condition, calculating to obtain the corresponding green tea drying degree by substituting the furan aldehyde content detected by the tea sample to be detected into a drying degree fitting curve.
The dryness fit curve is y =10 -5 x 2 –0.0018x+0.1133，R 2 ＝0.9672。
Compared with the prior art, the invention has the main advantages that:
1. compared with the traditional water bath heating mode, the metal bath heating mode has the advantages of high heating speed, accurate temperature control, temperature uniformity of less than or equal to +/-0.3 ℃ and temperature stability of +/-0.3 ℃.
2. According to the invention, the arrow-shaped solid-phase microextraction technology is adopted to extract the nitrogen-oxygen heterocyclic compounds in the green tea, the pretreatment of the sample is simple and quick, the complex pretreatment process is avoided, no organic solvent is needed, and the method is green and environment-friendly; SPME Arrow has higher sensitivity, high extraction efficiency and long service life than the traditional SPME under the same material.
3. The invention establishes a GC-QqQ-MS/MS method for simultaneously qualitatively and quantitatively analyzing nitrogen-oxygen heterocyclic compounds in green tea, and introduces stable isotope (2-methylpyrazine-d 6) as an internal standard substance under the condition of optimizing MRM mode parameters, thereby effectively eliminating the interference of complex matrixes, enhancing the response signal of target substances, reducing the noise of a base line, realizing the base line separation of the target substances and greatly improving the analysis sensitivity.
4. The method for detecting the heterocyclic nitrogen oxide compounds in the green tea overcomes the defects of the prior art, and provides a rapid analysis method for the heterocyclic nitrogen oxide compounds in the green tea, which integrates high selectivity and high sensitivity.
Drawings
FIG. 1 is a graph showing the trend of nitrogen-oxygen heterocycles in different drying periods;
FIG. 2 is a graph of the furan aldehyde content and the dryness (water content) of the sample.
Detailed Description
The invention is further described with reference to the following drawings and specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are conducted under conditions not specified, usually according to conventional conditions, or according to conditions recommended by the manufacturer.
1. Instruments and reagents
1.1 instruments
7890A-7000C gas chromatography-mass spectrometer (Agilent Technologies, USA); arrow-shaped solid phase microextraction handles and extraction fiber heads (Agilent Technologies, usa); the Arrow-shaped solid phase microextraction fiber head mainly adopts Carbon WR SPME Arrow; AL 204 electronic analytical balance (mettler-toledo instruments shanghai ltd); headspace bottles (20mL, agilent Technologies); sealing caps (polytetrafluoroethylene, agilent Technologies); a metal bath heating device (Zhejiang Naddy scientific instruments Co., ltd.).
1.2 materials and reagents
Preparation of dried green tea sample: fresh tea leaves are harvested in middle ten days of 2020 in 4 months, shengzhou city in Zhejiang province, the variety is Chinese tea 108, and the tenderness is from one bud and one leaf to two buds and two leaves. The specific processing technology is as follows: spreading (indoor temperature 25-28 deg.C, humidity 70-80%) → roller type enzyme deactivating (temperature of roller type enzyme deactivating machine 1, 2,3 groups respectively is 280 deg.C, 275 deg.C.) → rolling (rotation speed 50-52r/min, air kneading 5 min-light kneading 5 min-loose pressing 2 min-middle pressing 5 min-heavy kneading 3 min-air kneading 5 min) → drying with hair fire (110 deg.C, 15 min), weighing 80g respectively, placing in stainless steel sieve tray, drying in box type hot air drying machine at 115 deg.C for 5min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min respectively, and taking out.
Nitrogen oxygen heterocyclic compound: 2-Methylfuran (98%) was purchased from Shanghai Aladdin Biotechnology Ltd; 2-n-butyl furan (98%), furan aldehyde (more than or equal to 99.5%), pyrrole (99%) and acetylpyrazine (98%) are purchased from Shanghai Mielin Biotech, inc.; 2-n-pentylfuran, acetylpyrrole (> 98%) purchased from Chiloeve (Shanghai) chemical industry development Limited; 2-methylpyrazine-d 6 (99.5%) was purchased from CDN Isotips.
Reagent: ethanol (AR, purity ≥ 99.7%) was purchased from Meclin reagent Inc.; deionized water was supplied by Waohaha.
2. Experimental methods
(a) Preparation of standard working solutions:
taking standard substances of 2-methylfuran, 2-n-butylfuran, 2-n-pentylfuran, furan aldehyde, pyrrole, acetylpyrazine and acetylpyrrole as targets, using ethanol to perform constant volume to prepare a standard stock solution with the concentration of 20mg/L, and then gradually diluting the standard stock solution to 5000ng/mL, 2000ng/mL, 1000ng/mL, 500ng/mL, 300ng/mL, 100ng/mL, 50ng/mL, 20ng/mL, 10ng/mL and 5ng/mL, wherein the internal standard concentration (2-methylpyrazine-d 6) is constant to be a working solution with the concentration of 50ng/mL for later use.
(b) Metal bath assisted arrow-shaped solid phase microextraction technology for extracting nitrogen-oxygen heterocyclic compounds
I: aging of the extraction fiber head: and (3) placing the extracted fiber head into an Arrow-shaped solid phase microextraction (SPME Arrow) handle to form an SPME Arrow manual sample injector, inserting into a sample inlet with the temperature of 270 ℃, aging for 30min Zhong Housu, returning the fiber head, and pulling out the sample inlet.
II: preparing a standard sample and a sample to be detected: transferring a series of nitrogen-oxygen heterocyclic working solutions (5000 ng/mL, 2000ng/mL, 1000ng/mL, 500ng/mL, 300ng/mL, 100ng/mL, 50ng/mL, 20ng/mL, 10ng/mL and 5 ng/mL) with different concentrations, wherein the internal standard concentration (2-methylpyrazine-d 6) is constantly 50ng/mL and screwing the internal standard concentration into a 20mL headspace bottle by using a polytetrafluoroethylene sealing cap, accurately weighing 1.0g of tea sample into the 20mL headspace bottle, adding 3950 mu L of purified water and 50 mu L of isotope 2-methylpyrazine-d 6 (4 mg/L) to ensure that the concentration of the isotope internal standard substance in the system is 50ng/mL, and screwing the isotope internal standard substance by using the polytetrafluoroethylene sealing cap.
III: nitrogen-oxygen heterocyclic compound extraction: the headspace bottle was heated in a metal bath and adsorbed onto the headspace bottle for 30min at 60 ℃ using a Carbon WR SPME Arrow fiber tip to pierce the septum.
IV: analyzing a sample: after extraction, the SPME Arrow extraction needle was inserted into the GC inlet and analyzed at 200 ℃ for 4min for GC-QqQ-MS/MS analysis.
(c) Gas chromatography-triple quadrupole tandem mass spectrometry technology analysis condition optimization
Chromatographic separation conditions: chromatographic column VF-WAXms,260 ℃,30m × 250 μm × 0.25 μm; temperature programming: the initial column temperature is 40 ℃, the temperature is kept for 3min, the temperature is increased to 120 ℃ at the speed of 3 ℃/min, the temperature is kept for 2min, and then the temperature is increased to 240 ℃ at the speed of 10 ℃/min, and the temperature is kept for 3min; carrier gas: helium (99.999% pure); carrier gas flow rate: 1.0mL/min; n is a radical of 2 The collision airflow rate is 1.5mL/min; he quenching gas 2.25mL/min; the split ratio was 5:1.
Optimizing mass spectrum conditions: the optimized mass spectrum parameters mainly comprise parent ions, daughter ions and collision energy. Firstly, performing full scanning on standard stock solution between m/z 40-400, determining retention time and primary mass spectrogram of 7 nitrogen-oxygen heterocyclic compounds, and selecting proper ions from the primary mass spectrogram as parent ions; secondly, respectively carrying out secondary mass spectrometry on 7 nitrogen-oxygen heterocyclic compounds for the selected parent ions in a product ion scanning mode to obtain fragment ion information, and determining qualitative and quantitative ion pairs: the compound name 2-methylfuran qualitative and quantitative ion pair is 82 → 53 and 82 → 39, 2-n-butylfuran qualitative and quantitative ion pair is 81 → 51 and 81 → 53, 2-n-pentylfuran qualitative and quantitative ion pair is 81 → 51 and 81 → 53, furan aldehyde qualitative and quantitative ion pair is 96 → 67 and 96 → 39, pyrrole qualitative and quantitative ion pair is 67 → 39 and 67 → 41, acetylpyrazine qualitative and quantitative ion pair is 122 → 80 and 122 → 94, acetylpyrrole qualitative and quantitative ion pair is 109 → 66 and 109 → 94; then the collision energy (5, 10, 15, 20, 25, 30, 35, 40, 45, 50V) of the secondary mass spectrum is optimized, and finally the parameter condition of the optimal Multiple Reaction Monitoring (MRM) mode is shown in the table 1, wherein the parameter condition enables the target object to be more accurately characterized and can effectively reduce the matrix interference.
TABLE 1 optimization results of nitrogen-oxygen heterocyclic compound retention time, qualitative ion, quantitative ion and Collision Energy (CE)
Other mass spectral parameters: transmission line temperature: 250 ℃; the ion source temperature is 230 ℃; solvent retardation: 2min; an ionization mode: an EI source; ionization energy: 70eV; q2 collision gas: nitrogen (purity is more than or equal to 99.999%).
(d) Qualitative and quantitative analysis of pyrazine compounds
And (3) qualitative analysis: according to the retention time and the characteristic ion peak of each standard.
Quantitative analysis was performed by internal standard method: 5000ng/mL, 2000ng/mL, 1000ng/mL, 500ng/mL, 300ng/mL, 100ng/mL, 50ng/mL, 20ng/mL, 10ng/mL and 5ng/mL are respectively prepared, wherein the nitrogen-oxygen heterocyclic compound mixed standard solution with the internal standard concentration (2-methylpyrazine-d 6) being constant at 50ng/mL is subjected to linear regression analysis by taking the concentration ratio of the target compound to the internal standard as the abscissa and the peak area ratio as the ordinate according to the information of the selected characteristic qualitative ions and the quantitative ions to obtain a standard working curve. Under the same condition, the content of the target volatile matter in the tea sample to be detected is calculated by substituting the ratio of the peak area of the target volatile matter in the sample to be detected to the peak area of the internal standard into the corresponding standard curve.
3. Sensory evaluation
Tea samples under different drying time periods are evaluated strictly by experts with tea evaluation experience for many years according to GB/T23776-2018, the aroma of the tea is mainly evaluated, and the aroma type and intensity are repeatedly determined by combining hot smell, warm smell and cold smell.
4. Results and analysis
4.1 quantitative standard curve drawing, lowest detection limit, quantitative limit and recovery rate determination of nitrogen-oxygen heterocyclic compounds
For accurate quantification, GC-QqQ-MS/MS analysis is carried out on a series of nitrogen-oxygen heterocyclic compound standard solutions with different concentration gradients, linear regression analysis is carried out by taking the concentration ratio of a target compound to an internal standard as a horizontal coordinate and the peak area ratio as a vertical coordinate, and a standard working curve is obtained. Under the same condition, by mixing the sample to be testedAnd (4) substituting the ratio of the peak area of the target volatile matter in the product to the peak area of the internal standard into the corresponding standard curve to calculate the content of the target volatile matter in the tea sample to be detected. As shown in Table 2, all the standard curves show a good linear relationship in the concentration range of 5-1000ng/mL, and the correlation coefficient R 2 Are both higher than 0.99, and the accuracy of the analysis method and the accuracy of the operation are well verified. In addition, in order to ensure that the quantitative result is within the reliable detection concentration, the LODs and LOQs of nitrogen-oxygen heterocyclic compounds are examined by taking 3 times of signal-to-noise ratio as the lowest detection Limit (LODs) of the method and 10 times of signal-to-noise ratio as the lowest quantification Limit (LOQs). The results show that LOD and LOQ of various nitrogen-oxygen heterocycles have no obvious difference in magnitude and can be reduced to the level of ng/mL, wherein LODs are between 0.11 and 1.28ng/mL, LOQs is between 0.37 and 4.26ng/mL, and the ideal sensitivity of the instrument is fully demonstrated.
TABLE 2 Linear concentration range, detection limit and quantitation limit of nitrogen-oxygen heterocyclic compounds
To further verify the reliability of the analysis method, the recovery rate of the nitrogen-oxygen heterocyclic compound was measured. As shown in Table 3, the calculated normalized recovery rate was 89.21% -117.32% and the RSD value was 0.42% -5.38% when 100ng/mL of the azacyclo compound standard was added to the blank matrix. The results fully show that the analysis method has stronger reliability.
TABLE 3 recovery of nitrogen-oxygen heterocycles and determination of RSD (%) value
| Name of Compound | Addition level of (ng/mL) | Recovery rate of added standard | RSD |
| 2- |
100 | 106.04％ | 1.27％ |
| 2-n- |
100 | 117.32％ | 0.42％ |
| 2-n- |
100 | 116.86％ | 2.76 |
| Furanal aldehyde | |||
| 100 | 94.57％ | 5.38％ | |
| Azole compounds | 100 | 89.21％ | 4.55 |
| Acetylpyrazine | |||
| 100 | 97.19％ | 4.21 | |
| Acetyl pyrrole | |||
| 100 | 1.06.14％ | 1.55％ |
4.2 qualitative and quantitative analysis of nitrogen-oxygen heterocyclic compounds in green tea
Through detection and analysis of the nitrogen-oxygen heterocyclic compounds of green tea in different drying periods, 7 nitrogen-oxygen heterocyclic compounds including 2-methylfuran, 2-n-butylfuran, 2-n-pentylfuran, furan aldehyde, pyrrole, acetylpyrazine and acetylpyrrole are identified, and the results are shown in figure 1. The content of 2-methylfuran in samples of green tea under different drying time lengths is 46.43-75.41 ng/mL, which is slowly increased along with the drying time length, and reaches the maximum in 50 min; the content of the 2-n-butyl furan is between 6.51 and 18.21ng/mL, the regularity is not obvious along with the change of drying time, and the content reaches the highest value under 35 min; the content of the 2-n-pentylfuran is 219.59-390.08 ng/mL, the content is increased and then reduced along with the drying time, and the content reaches the highest after 35 min; the furan aldehyde content is between 13.79 and 98.47ng/mL, the furan aldehyde content is increased and then reduced along with the drying time, the furan aldehyde content reaches the highest content at 45min and then is slightly reduced; the content of pyrrole is 13.69-144.42 ng/mL, which is increased and then reduced along with the drying time, and the content reaches the highest value under 45min and then rapidly decreases; the content of the acetylpyrazine is 11.71-531.57 ng/mL, the acetylpyrazine rapidly increases in 35min along with the extension of the drying time, the increasing speed is up to 15 times, and the content reaches the highest in 45 min; the content of the acetyl pyrrole is 157.53-1454.36 ng/mL, which is slowly increased along with the drying time and reaches the maximum in 50 min.
The sensory evaluation results at different drying times are shown in table 4, from which it can be seen that the aroma of tea leaves at different drying times shows a gradual process of foot fire → slightly high fire → old fire, the aroma score reaches the highest (88 points) at 20min of drying time and then shows a decreasing trend. The furan aldehyde content under different drying time lengths is taken as an x variable, the drying degree (water content is taken as a survey index) is taken as a y variable to carry out regression analysis, as shown in figure 2, the furan aldehyde content and the drying degree (water content) show a binomial relation,regression equation y =10 -5 x 2 –0.0018x+0.1133(R 2 = 0.9672). The water content is used as the degree index of drying temperature and time, and the requirement of the national standard (GB/T14456.1-2017) on the water content of green tea (pan-fired green tea, baked green tea and steamed green tea) is less than or equal to 7 percent. Therefore, under the specified temperature condition, furan aldehyde can be used as a key indicator compound for evaluating the drying degree (water content) of the box-type hot air, and the drying degree of furan aldehyde content between 28.5997 and 37.3692ng/mL is more suitable (7% ≧ corresponding water content is ≧ 6%).
TABLE 4 sensory evaluation results at different drying durations
| Number of | Aroma comment | Scoring fragrance |
| E-5min | High fever at foot | 86.7 |
| E-10min | Slightly higher fire | 86.3 |
| E-15min | Slightly high fire attack and chestnut flavor after cooling | 87.3 |
| E-20min | Slightly higher fire attack with chestnut flavor | 88.0 |
| E-25min | A strong fire with a slight chestnut flavor | 86.0 |
| E-30min | High fire | 83.7 |
| E-35min | High fire | 80.0 |
| E-40min | High fire, old fire | 79.7 |
| E-45min | Old fire | 79.0 |
| E-50min | Old fire | 78.0 |
Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the above description of the present invention, and equivalents also fall within the scope of the invention as defined by the appended claims.
Claims (10)
1. A method for rapidly and accurately quantifying nitrogen and oxygen heterocyclic compounds in green tea is characterized in that a method for rapidly analyzing the nitrogen and oxygen heterocyclic compounds in the green tea by combining a metal bath assisted arrow-shaped solid phase microextraction technology with a gas chromatography-triple quadrupole tandem mass spectrometry technology comprises the following steps:
(a) Preparation of standard working solutions:
taking standard products of 2-methylfuran, 2-n-butylfuran, 2-n-pentylfuran, furan aldehyde, pyrrole, acetylpyrazine and acetylpyrrole as targets, using ethanol to perform constant volume to prepare a standard stock solution of 20mg/L, and then diluting the standard stock solution to 5000ng/mL, 2000ng/mL, 1000ng/mL, 500ng/mL, 300ng/mL, 100ng/mL, 50ng/mL, 20ng/mL, 10ng/mL and 5ng/mL step by step, wherein the concentration of the 2-methylpyrazine-d 6 internal standard solution is constantly determined to be a working solution of 50 zxft 3763/mL for later use;
(b) Metal bath assisted arrow-shaped solid phase microextraction technology for extracting nitrogen-oxygen heterocyclic compounds
1) Aging of the extraction fiber head: loading the extracted fiber head into an Arrow-shaped solid phase microextraction handle to form an SPME Arrow manual sample injector, inserting into a sample inlet with the temperature of 270 ℃, aging for 30min Zhong Housu, returning the fiber head, and pulling out the sample inlet;
2) Preparation of a standard sample: moving a series of working solutions with different concentrations in the step (a) into a 20mL headspace bottle, and screwing down the working solutions by using a polytetrafluoroethylene sealing cap;
preparing a sample to be tested: accurately weighing 1.0g tea sample into a 20mL headspace bottle, adding 3950 mu L of distilled water and 50 mu L of 4mg/L isotope 2-methylpyrazine-d 6, ensuring the concentration of an isotope internal standard substance in a system to be 50ng/mL, and screwing up by using a polytetrafluoroethylene sealing cap;
the preparation process of the tea sample comprises the following steps:
(I) Spreading fresh tea leaves: the indoor temperature is 25-28 ℃, and the humidity is 70-80%;
(II) roller type enzyme deactivation: the temperatures of 1, 2 and 3 groups of roller fixation machines are respectively 280 ℃, 275 ℃ and 275 ℃;
(III) rolling: rotating at 50-52r/min, air kneading for 5min, lightly kneading for 5min, loosely pressing for 2min, medium pressing for 5min, heavily kneading for 3min, and air kneading for 5min;
(IV) drying at 110 ℃ for 15min with gross fire, respectively weighing 80g of the tea, placing the tea in a stainless steel sieve tray, then placing the stainless steel sieve tray in a box type hot air dryer, drying at 115 ℃ for 5 to 50min, and taking out the tea to obtain the tea sample;
3) Nitrogen-oxygen heterocyclic compound extraction: putting the headspace bottle in the step 2) into a metal bath for heating, puncturing a diaphragm by adopting a Carbon WR SPME Arrow fiber head at the temperature of 60 ℃, and adsorbing for 30min above the headspace bottle;
4) Analyzing a sample: after extraction, inserting the SPME Arrow extraction needle into a GC sample inlet, performing resolution for 4min at 200 ℃, and performing gas chromatography-triple quadrupole tandem mass spectrometry technology analysis;
(c) Gas chromatography-triple quadrupole tandem mass spectrometry technology analysis condition optimization
Chromatographic separation conditions: a chromatographic column VF-WAXms,260 ℃,30m x 250μm x 0.25μm; temperature programming: the initial column temperature is 40 ℃, the temperature is kept for 3min, the temperature is increased to 120 ℃ at the speed of 3 ℃/min, the temperature is kept for 2min, and then the temperature is increased to 240 ℃ at the speed of 10 ℃/min, and the temperature is kept for 3min; carrier gas: helium with purity of 99.999%; flow rate of carrier gas: 1.0mL/min; n is a radical of 2 The collision airflow rate is 1.5mL/min; he quench gas 2.25mL/min; the split ratio is as follows: 5:1;
and optimizing retention time and mass spectrum conditions of the internal standard substance, 2-methylfuran, 2-n-butylfuran, 2-n-pentylfuran, furan aldehyde, pyrrole, acetylpyrazine and acetylpyrrole, wherein the optimized parameter conditions of the multiple reaction monitoring mode are as follows:
other mass spectral parameters: transmission line temperature: 250 ℃; the ion source temperature is 230 ℃; solvent retardation: 2min; an ionization mode: an EI source; ionization energy: 70eV; q2 collision gas: nitrogen with purity more than or equal to 99.999 percent;
(d) Qualitative and quantitative analysis of nitrogen-oxygen heterocyclic compounds
And (3) qualitative analysis: according to the retention time and the characteristic ion peak of each standard substance;
quantitative analysis was performed by internal standard method: performing linear regression analysis by using the working solution in the step (a) and according to the information of the selected characteristic qualitative ions and the selected characteristic quantitative ions by using the concentration ratio of the target compound to the internal standard as a horizontal coordinate and the peak area ratio as a vertical coordinate to obtain a standard working curve; under the same condition, the content of the target volatile matter in the tea sample to be detected is calculated by substituting the ratio of the peak area of the target volatile matter in the sample to be detected to the peak area of the internal standard into the corresponding standard working curve.
2. The method of claim 1, wherein in step (d) the standard working curve of 2-methylfuran is y =0.002239 x-1.448717 x 10 -4 ，R 2 =0.9990, linear range 10 to 1000ng/mL.
3. The detection method according to claim 1, wherein in step (d), the standard working curve of 2-n-butylfuran is y =0.658498 x +0.067972, R 2 And the linear range is 5 to 500ng/mL, and the linear range is 0.9967.
4. The assay of claim 1 wherein in step (d) the standard working curve for 2-n-pentylfuran is y =0.686733 x-0.084885R 2 =0.9909, linear range 5 to 1000ng/mL.
5. The detection method according to claim 1, wherein in step (d), the standard working curve of furan aldehyde is y =0.438733 x +0.025218 2 =0.9995, linear range 10 to 1000ng/mL.
6. The method of claim 1, wherein in step (d), the standard working curve of pyrrole is y =0.054700 x +0.099804, R 2 =0.9984, and the linear range is 10 to 500 ng/mL.
7. The detection method as claimed in claim 1, wherein in step (d), the standard working curve of acetylpyrazine is y =0.109487 x +0.028905 2 =0.9986, linear range 5 to 1000ng/mL.
8. The detection method according to claim 1, wherein in step (d), the standard working curve of acetylpyrrole is y =0.144857 x +0.118640, R 2 =0.9964, linear range 5 to 1000ng/mL.
9. A green tea dryness degree evaluation method is characterized in that a detection method of any one of claims 1~8 is adopted, furan aldehyde is used as a key index compound for evaluating the drying degree of box-type hot air, the furan aldehyde content detected by tea samples under different drying time lengths is used as an x variable, and the dryness degree taking the water content as an investigation index is used as a y variable for carrying out regression analysis to obtain a dryness degree fitting curve; and under the same condition, calculating to obtain the corresponding green tea drying degree by substituting the furan aldehyde content detected by the tea sample to be detected into a drying degree fitting curve.
10. The method for evaluating dryness of green tea according to claim 9, wherein the dryness fitting curve is y =10 -5 x 2 – 0.0018x + 0.1133，R 2 = 0.9672。
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