A method for detecting 5-hydroxymethylfurfural in coffee beans

Abstract

The application provides a method for detecting 5-hydroxymethylfurfural in coffee beans, and the method effectively eliminates the interference of a complex matrix and ensures good separation of components by optimizing a sample pretreatment process and a chromatographic gradient elution condition; wherein, aiming at the high lipid matrix of roasted coffee beans, a hexane vortex degreasing step is innovatively introduced at the initial stage of extraction, which efficiently intercepts and removes lipid interferents, successfully solves the chemical background charge competition at the electrospray ion source, and ensures the authenticity and accuracy of quantitative results; and the high performance liquid chromatography gradient elution program used can make the target HMF quickly complete elution focusing and symmetric peak, and in addition, by optimizing the multiple reaction monitoring channel parameters of tandem mass spectrometry, the background chemical noise caused by the complex matrix is greatly reduced. The detection method can accurately qualitatively and quantitatively detect HMF in coffee beans, has good chromatographic separation and linear relationship, high precision and accuracy, and is suitable for quality detection of furfural hazards in coffee beans.

Description

Technical Field

[0001] This invention belongs to the field of food analysis technology, specifically relating to a method for detecting 5-hydroxymethylfurfural in coffee beans. Background Technology

[0002] 5-Hydroxymethylfurfural (HMF) is an important byproduct formed during food heat processing or long-term storage, resulting from the dehydration of reducing sugars via the Maillard reaction or the caramelization of sugars under acidic conditions. During coffee bean roasting, the formation, accumulation, and degradation of HMF exhibit extremely complex dynamic changes with increasing temperature and time. HMF content is not only a key process marker for monitoring the maturity, degree of roasting, and color and flavor quality of coffee, but also, as a potential cytotoxic and genotoxic heat-processed hazard, its residue level is an important indicator for assessing food safety and health quality.

[0003] When analyzing and detecting HMF in complex coffee bean matrices (especially roasted coffee bean samples), most existing technologies generally suffer from the following technical bottlenecks: Conventional HPLC-UV / DAD methods lack the high selectivity of mass spectrometry, making them prone to peak overlap and baseline drift when dealing with complex high-fat and high-polyphenol coffee matrices, and their sensitivity is insufficient, making it difficult to monitor microscopic dynamic evolution; GC-MS methods are limited by the strong polarity and thermal instability of HMF, requiring time-consuming and error-prone derivatization pretreatment, which cannot meet the needs of high-throughput analysis. Summary of the Invention

[0004] In order to overcome at least one of the problems existing in the prior art, the present invention aims to provide a method for detecting 5-hydroxymethylfurfural in coffee beans.

[0005] This invention innovatively provides a method for detecting HMF in roasted coffee beans based on high performance liquid chromatography-triple quadrupole tandem mass spectrometry. Through a specific pretreatment purification system and precise chromatography-mass spectrometry coupling conditions, rapid and specific qualitative and quantitative analysis of HMF in roasted coffee beans with complex high-fat matrix is ​​achieved.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for detecting 5-hydroxymethylfurfural in coffee beans, the method comprising a defatting step, a solvent extraction step, and a high-performance liquid chromatography-mass spectrometry step; The defatting step includes: adding coffee bean powder to n-hexane, vortexing to mix and centrifuging, discarding the n-hexane, and obtaining defatted coffee bean powder.

[0007] Preferably, the ratio of coffee bean powder to hexane is (0.01-0.3) g: 1 mL.

[0008] More preferably, the ratio of coffee bean powder to n-hexane is 0.01g:1mL, 0.02g:1mL, 0.03g:1mL, 0.04g:1mL, 0.05g:1mL, 0.06g:1mL, 0.07g:1mL, 0.08g:1mL, 0.09g:1mL, 0.10g:1mL, 0.11g:1mL, 0.12g:1mL, 0.13g:1mL, 0.14g:1mL, or 0.15g:1mL. The values ​​are any one of the following: 0.16g:1mL, 0.17g:1mL, 0.18g:1mL, 0.19g:1mL, 0.20g:1mL, 0.21g:1mL, 0.22g:1mL, 0.23g:1mL, 0.24g:1mL, 0.25g:1mL, 0.26g:1mL, 0.27g:1mL, 0.28g:1mL, 0.29g:1mL, 0.30g:1mL, or any range of any two values.

[0009] Preferably, the vortex mixing time is 4-10 min. More preferably, the vortex mixing time is any value or a range of any two values ​​from 4, 5, 6, 7, 8, 9, to 10 min.

[0010] Preferably, the step of discarding n-hexane includes: discarding the upper n-hexane defatting liquid after centrifugation, and then ventilating and drying to remove the residual n-hexane.

[0011] Preferably, the solvent extraction step includes: adding defatted coffee bean powder to an acetonitrile aqueous solution, vortexing and centrifuging sequentially, and then taking the supernatant solution to obtain a 5-hydroxymethylfurfural extract.

[0012] More preferably, the concentration of acetonitrile in the acetonitrile aqueous solution is 10% (v / v).

[0013] Preferably, the chromatographic conditions for the high-performance liquid chromatography-mass spectrometry (HPLC-MS) step include: The liquid chromatography column used is a reversed-phase octadecylsilane-bonded silica gel column.

[0014] Preferably, the chromatographic conditions for the high-performance liquid chromatography-mass spectrometry (HPLC-MS) step include: Mobile phase A is a 0.1% (v / v) aqueous solution of formic acid; mobile phase B is pure acetonitrile.

[0015] Preferably, the chromatographic conditions for the high-performance liquid chromatography-mass spectrometry (HPLC-MS) step include: The elution method is gradient elution, and the gradient elution procedure is as follows: From 0 to 2 minutes, phase A was 95% and phase B was 5%. Between 2.0 and 2.1 minutes, phase A decreased from 95% to 10%, while phase B increased from 5% to 90%. 2.1–3.0 min, Phase A is 10%, Phase B is 90%; From 3.0 to 3.1 min, phase A increased from 10% to 95%, while phase B decreased from 95% to 5%. 3.1–5.0 min, Phase A is 95%, Phase B is 5%.

[0016] Preferably, the chromatographic conditions of the high-performance liquid chromatography-mass spectrometry (HPLC-MS) step further include at least one of the following: 1) Column temperature: 30-50℃; 2) Injection volume: 1-5 μL; 3) Flow rate: 0.2 - 0.3 mL / min.

[0017] The high-performance liquid chromatography gradient elution program employed in this invention enables the target analyte HMF to be eluted, focused, and symmetrically eluted in approximately 0.92 minutes. Simultaneously, within 2.0 to 3.0 minutes, by rapidly increasing the organic phase ratio to 90%, strongly retained impurities remaining in the column are quickly flushed out. The total cycle time for a single injection analysis and column equilibration is strictly controlled within 5.0 minutes. The pretreatment process eliminates the need for cumbersome derivatization reactions, and the short system reequilibration time significantly improves the detection efficiency for large batches of samples.

[0018] Preferably, the mass spectrometry in the high performance liquid chromatography-mass spectrometry (HPLC-MS) step is performed using a triple quadrupole mass spectrometer. The conditions for the mass spectrometry include at least one of the following: a) Electrospray ionization source, positive ion mode scanning; b) The temperature for removing the solvent gas is 500 ℃; c) Ion source temperature 150℃; d) The capillary voltage is set to 3.0 kV; e) The conical orifice air flow rate is set to 50 liters / hour.

[0019] This invention significantly reduces background chemical noise caused by complex matrices by optimizing the multi-reaction monitoring channel parameters of tandem mass spectrometry.

[0020] Preferably, the method further includes the following steps: preparing a reference solution and a test solution, performing high performance liquid chromatography-mass spectrometry (HPLC-MS), and recording the chromatograms; and calculating the 5-hydroxymethylfurfural content in the test solution using the external standard method.

[0021] Preferably, the coffee bean varieties include Arabica, Robusta, or Liberica.

[0022] The beneficial effects of this invention are: This invention establishes a method for detecting 5-hydroxymethylfurfural in roasted coffee beans based on high-performance liquid chromatography-triple quadrupole tandem mass spectrometry. Targeting the high-fat matrix of roasted coffee beans, this invention innovatively introduces a hexane vortex defatting step at the initial stage of extraction. Rigorous pre-extraction spiked comparison experiments confirmed that, using the pure solvent working solution (peak area 123,510) as a baseline, the total peak area of ​​the sample measured using this method was 3,166,638. After deducting the inherent background value (3,019,327), the net peak area response value for spiked recovery was 147,311, resulting in an actual spiked recovery rate of 119.3%, fully meeting the accuracy requirements (80% ~ 120%) for trace residue analysis. In contrast, the conventional extraction comparison, omitting the defatting step, had a total peak area of ​​2,730,234. After deducting the undefatted background value (2,726,482), the net peak area for spiked recovery was only 3,752, with an actual spiked recovery rate of only 3.0%. The data discrepancy scientifically confirms that the present invention can efficiently intercept and remove lipid interferences, successfully eliminate the chemical background charge competition at the electrospray ion source, and achieve a qualitative leap in method accuracy from 3.0% to 119.3%. Attached Figure Description

[0023] Figure 1 Multiple reaction monitoring chromatogram of spiked sample that has not been defatted with n-hexane; Figure 2 Multiple reaction monitoring chromatogram of spiked sample for hexane defatting. Detailed Implementation

[0024] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from conventional commercial channels or prepared and isolated through simple synthesis; unless otherwise specified, the processes employed are conventional processes in the art.

[0025] Example 1 This embodiment provides a method for detecting 5-hydroxymethylfurfural in roasted coffee beans, the specific steps of which are as follows: (1) Sample pretreatment steps Accurately weigh 0.2 g (accurate to 0.0001 g) of uniformly ground, medium-roasted coffee beans (Catim washed coffee beans purchased from Yonghui Manor in Pu'er, Yunnan, production batch number: 20240820, roasted under normal medium-roasting conditions such as roasting at 200℃ for about 10 minutes, pulverized and passed through a 60-mesh sieve, average particle size ≤250 μm) into a centrifuge tube. First, a specific purification procedure for the high-lipid matrix was introduced: 2.0 mL of n-hexane was added to the centrifuge tube as a defatting solvent, and the tube was vortexed at a maximum speed of 3000 r / min for 5 minutes to fully dissolve the high concentration of lipid components in the coffee powder into the organic phase; then, the tube was centrifuged at 4000 rpm for 5 minutes, and the upper layer of n-hexane defatting liquid was completely removed and discarded using a pipette; the centrifuge tube containing the sample was placed in a fume hood and allowed to stand for 10 minutes to allow the residual n-hexane solvent to completely evaporate to dryness.

[0026] After the lipid matrix was removed and the solvent had completely evaporated, 5.0 mL of extraction solvent (10% acetonitrile aqueous solution by volume) was added to the solid residue. The mixture was then vortexed at a maximum speed of 3000 r / min for 6 minutes to achieve initial mixing. Subsequently, the centrifuge tubes were placed in a 40 ℃ water bath ultrasonic cleaner (ultrasonic frequency of 40 kHz) for ultrasonic-assisted extraction for 30 minutes, utilizing the cavitation effect of ultrasound to promote the complete dissolution of HMF in the matrix pores. After extraction, the mixture was centrifuged at 10000 rpm for 10 minutes to ensure complete sedimentation of insoluble particles and sample residues. The supernatant was accurately transferred and linearly diluted with 10% acetonitrile aqueous solution (recommended dilution factor of 10-fold) according to the actual expected HMF content in the sample. The diluted solution was filtered through a 0.22 μm pore size polyethersulfone organic microporous membrane and collected in a liquid chromatography-mass spectrometry injection vial to obtain the test solution.

[0027] (2) High performance liquid chromatography separation conditions The test solution prepared in step (1) above was injected into a high-performance liquid chromatography (HPLC) system using an autosampler to separate the target analyte from residual impurities. The chromatographic conditions were set as follows: Chromatographic column: A reversed-phase octadecylsilane bonded silica column (Waters ACQUITY UPLC BEH C18 column, 2.1 mm × 50 mm, packing particle size 1.7 μm) was used to achieve rapid focusing and elution of the target analyte by utilizing the high-pressure column efficiency of the micro-diameter column. Column temperature: strictly controlled between 30 ℃ and 50 ℃ (preferably 40 ℃) to stabilize the distribution coefficient of solute between the stationary phase and the mobile phase; Injection volume: 1 μL to 5 μL (preferably 2 μL) to prevent mass spectrometer saturation caused by overload; Flow rate: set to 0.2 mL / min to 0.3 mL / min (preferably 0.2 mL / min), injection chamber temperature set to 4℃ to 10℃ (preferably 6℃); Mobile phase system: Mobile phase A is a 0.1% (v / v) aqueous solution of formic acid (used to improve the protonation ionization efficiency of the target analyte in positive ion mode), and mobile phase B is chromatographically pure acetonitrile; Gradient elution procedure: Existing conventional chromatographic methods for detecting HMF mostly employ isocratic elution or long-cycle slow linear gradients. The typical single analysis cycle in this field is often as long as 15 to 30 minutes, and it is difficult to completely elute strongly retained hydrophobic impurities in complex coffee matrices. To overcome the shortcomings of low analytical throughput and matrix residue, and to ensure the complete elution of co-extracted strongly retained polyphenols and residual lipids, while achieving rapid peak elution of HMF, this invention, based on the significant difference in the partition coefficients of the target analyte and matrix components on the stationary phase, employs the following precise time-series step gradient elution procedure: From 0 to 2.0 minutes, the volume percentage of mobile phase B (acetonitrile) remains constant at 5%. During this time, the highly polar HMF focuses on the stationary phase and elutes sharply around 0.92 minutes, achieving initial separation from the weakly polar matrix. From 2.0 to 2.1 minutes, the proportion of mobile phase B linearly increases from 5% to 90% within 0.1 minutes. From 2.1 to 3.0 minutes, the proportion of mobile phase B is maintained at a high organic phase level of 90%. Strong elution intensity is used to rapidly flush out strongly retained impurities such as caffeine and polyphenols remaining in the column, preventing column contamination and retention time drift caused by continuous injection. From 3.0 to 3.1 minutes, the proportion of mobile phase B linearly decreases from 90% to 5% within 0.1 minutes. From 3.1 to 5.0 minutes, the proportion of mobile phase B remains at 5%, and the column is rebalanced to restore the stationary phase to its initial state, preparing for the next sample injection. The total cycle time for a single injection analysis is strictly controlled within 5.0 minutes.

[0028] (3) Triple quadrupole mass spectrometry detection conditions The eluent from the chromatographic column is directly introduced into a triple quadrupole mass spectrometer for highly selective detection. The core parameters and operating modes of the mass spectrometer are set as follows: Ion source and scanning mode: An electrospray ion source is used, operating in positive ion scanning mode; Detection mode: The multi-reaction monitoring mode is adopted, which uses a two-stage quadrupole mass screening to achieve highly specific fracture fragment monitoring and completely eliminates the interference of background mass noise. Ion source parameters: Ion source temperature is set to 150 ℃, desolvation gas (high-purity nitrogen) temperature is strictly controlled at 500 ℃, capillary voltage is set to 3.0 kV, and cone orifice gas flow rate is set to 50 L / h to ensure efficient drying of atomized droplets and high ionization yield of target molecules; Characteristic ion pairs and reaction parameters (key invention features): For HMF (molecular weight 126), the protonated hydrogenated molecular ion formed in positive ion mode has a mass-to-charge ratio (m / z) of 127.0. Two characteristic reaction channels were set up for mass spectrometry: Quantitative channel: The precursor ion (mother ion) has a mass-to-charge ratio of 127.0. When it passes through the second-stage collision chamber, a collision energy of 14 to 16 electron volts (preferably 15 electron volts) is applied, causing it to undergo specific dehydration and breakage. The characteristic product ion (daughter ion) has a mass-to-charge ratio of 109.0. This channel has a high response value and a clean background, making it suitable for integration quantification using the external standard method. The cone voltage is set to 30 volts. Qualitative channel: The precursor ion mass-to-charge ratio is also 127.0. A collision energy of 19 to 21 electron volts (preferably 20 electron volts) is applied in the collision chamber to further break down the precursor ion, causing it to lose functional groups such as carbonyl groups. The mass-to-charge ratio of the characteristic product ion is monitored to be 81.0. This channel is used for synergistic qualitative confirmation. The cone voltage is also set to 30 volts.

[0029] (4) Qualitative and quantitative data analysis steps Qualitative confirmation method: Under the same instrument conditions, compare the chromatographic retention time of the target peak in the sample test solution with the retention time of the standard working solution (consistency deviation within ±2.5%). At the same time, compare the relative abundance ratio of the two sets of characteristic mass spectrometry reaction ion pairs in the quantitative channel (127.0>109.0) and the qualitative channel (127.0>81.0). If the relative deviation of the ion abundance ratio of the sample peak compared with the ion abundance of the standard at the same concentration meets the internationally recognized residual analysis standard, then the presence of HMF in the sample is confirmed.

[0030] External standard method for quantification: Accurately weigh HMF reference standard with a purity of ≥98%, dissolve it in a 90:10 water / acetonitrile mixture to prepare a high-concentration reference standard stock solution. Immediately before use, serially dilute the reference standard stock solution with blank coffee matrix extract (i.e., a blank solution without exogenous spikes but undergoing the same pretreatment defatting process) to prepare a series of matrix-matched reference standard solutions with concentration gradients of 0.05 μg / mL, 0.1 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1.0 μg / mL, and 5.0 μg / mL. In actual testing, the test solution obtained from the pretreatment step is used as the test solution for determination.

[0031] The samples were sequentially injected into a chromatography-mass spectrometry (GC-MS) instrument for analysis, and the peak areas corresponding to the quantitative channels (127.0 > 109.0) were recorded. A matrix-matched standard curve equation with excellent linear correlation coefficient (R² ≥ 0.999) was constructed by using the mass concentration of HMF in the reference solution (μg / mL) as the abscissa (x) and the corresponding peak area response value as the ordinate (y). The actual peak area of ​​the test sample solution after subtracting the background was substituted into this standard curve equation to calculate the mass concentration of the target analyte in the extract. Then, based on the sample weight (0.2 g) and the final dilution volume, the actual residual HMF content (mg / kg) in the roasted coffee bean sample was calculated.

[0032] This method significantly reduces background chemical noise from complex matrices by optimizing the multi-reaction monitoring channel parameters of tandem mass spectrometry. The method exhibits excellent linearity within a concentration range of 0.05 μg / mL to 5.0 μg / mL, with a univariate linear regression equation for the constructed standard curve: y = 116275.8x + 2337.7, and a linear correlation coefficient of R² = 0.9999. The method has a limit of detection (LOD) of 0.015 μg / mL and a limit of quantitation (LOQ) of 0.05 μg / mL (equivalent to a LOD of 0.038 mg / kg and a LOQ of 0.125 mg / kg in solid samples), enabling precise capture and microscopic monitoring of subtle differences in HMF content in coffee beans of different roast levels.

[0033] Comparative Example 1 The detection method for Comparative Example 1 is the same as that in the Example, except that: defatting is not performed, and roasted coffee bean powder is directly added to 5.0 mL of extraction solvent (10% acetonitrile aqueous solution by volume) for extraction and high performance liquid chromatography-mass spectrometry.

[0034] Methodological Validation 1. Accuracy and Precision Figure 1 The image shows the HMF multiple reaction monitoring chromatogram of the spiked sample of Comparative Example 1 (without hexane defatting), demonstrating the mass spectrometry results of the spiked sample of the comparative example extracted directly with 10% acetonitrile aqueous solution without the hexane defatting step. Figure 1 The horizontal axis represents the retention time, ranging from 0 to 5.0 minutes; the vertical axis represents the detector signal strength, ranging from 0 to 1.0 × 10⁻⁶. 6Counting per second. At the front of the chromatogram, the target analyte HMF elutes within the retention time range of 0.92 min to 1.10 min, forming the target mass spectrometry response peak. The core technical feature revealed by this chromatogram lies in the dynamic changes in its baseline microstructure: within the high-proportion organic phase elution range of reversed-phase chromatography (3.5 min to 5.0 min), the chromatogram exhibits extremely violent, noisy, and dense spike-like high-frequency noise fluctuations, with a significant increase in the overall baseline response abundance. This chaotic baseline waveform directly confirms that high-abundance, strongly retained lipid impurities and hydrophobic macromolecules in roasted coffee beans are co-extracted into the mass spectrometry system and concentrated in the electrospray ionization source during high-proportion organic phase elution. The concentrated spray ionization of lipid components in this region caused continuous mass background noise and strong charge competition with HMF eluting around 1.0 min at the microscopic level. This confirmed the source of the catastrophic matrix inhibition effect in both space and time (resulting in a total peak area response of only 2,730,234 for the spiked sample group, and a net peak area of ​​only 3,752 after deducting the inherent background value of 2,726,482, resulting in an extremely low actual spiked recovery of only 3.0%).

[0035] Figure 2 The image shows the HMF multiple reaction monitoring chromatogram of the spiked sample from Example 1 (hexane defatting), demonstrating the mass spectrometry results of the spiked sample from the example, which was analyzed after strictly following the pretreatment scheme described in this invention and introducing hexane vortex defatting purification. Figure 2 The physical quantity on the horizontal axis, the physical quantity on the vertical axis, and the range span. Figure 1 To ensure complete consistency and facilitate intuitive scientific comparison. Figure 2 In this study, the target analyte HMF also eluted within the range of 0.92 to 1.10 minutes, exhibiting more symmetrical, sharper peak shapes without tailing. Figure 1 The key feature that provides a stark technical contrast lies in the baseline performance during the later stages of elution: within the high-proportion organic phase elution range of the same retention time of 3.5 to 5.0 minutes, the chromatographic baseline is extremely smooth, clean, and stable, without any noticeable distortion. Figure 1Despite the chaotic, jagged background interference and significant high-frequency noise, the baseline signal intensity remained at an extremely low instrument background level. This significant difference in spectral traces clearly indicates that the hexane vortex defatting operation introduced in the pretreatment step efficiently and thoroughly intercepted and removed the highly interfering lipid matrix, such as coffee oil, from the roasted coffee beans before exogenous extraction. The chemical environment around the electrospray ionization source was fundamentally purified, thus successfully eliminating the matrix inhibition effect, restoring the ionization efficiency of trace target analytes to normal, and ensuring accurate overlap and quantitative release of the spiked signal (resulting in a total peak area response of 3,166,638 for the spiked sample and an actual spiked recovery rate of 119.3% after deducting the inherent background).

[0036] 2. Stability Six independent parallel pretreatment and determinations were performed on the same coffee sample, and the relative standard deviation (RSD) of HMF content was only 2.3%, with ideal spiked recovery, ensuring the reproducibility and robustness of the test results when dealing with complex food processing matrices.

[0037] In summary, this invention effectively eliminates interference from complex matrices and ensures good separation of components by optimizing sample pretreatment processes and chromatographic gradient elution conditions. Specifically, for the high-lipid matrix of roasted coffee beans, an innovative hexane vortex defatting step is introduced at the initial extraction stage, efficiently intercepting and removing lipid interferences and successfully eliminating chemical background charge competition at the electrospray ionization source, ensuring accurate and reliable quantitative results. Furthermore, the high-performance liquid chromatography gradient elution program allows the target analyte HMF to rapidly elute and focus, achieving symmetrical peak formation. The pretreatment process eliminates the need for cumbersome derivatization reactions, resulting in a short system rebalancing time and significantly improving the detection efficiency of large batches of samples. Additionally, by optimizing the multi-reaction monitoring channel parameters of tandem mass spectrometry, background chemical noise from complex matrices is significantly reduced. The detection method of this invention can accurately qualitatively and quantitatively identify HMF in coffee beans, exhibiting good chromatographic separation and linearity, high precision and accuracy, and is suitable for the quality detection of furfural, a hazardous substance in coffee beans.

[0038] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for detecting 5-hydroxymethylfurfural in coffee beans, characterized in that, The method includes a defatting step, a solvent extraction step, and a high-performance liquid chromatography-mass spectrometry step. The defatting step includes: adding coffee bean powder to n-hexane, vortexing to mix and centrifuging, discarding the n-hexane, and obtaining defatted coffee bean powder.

2. The method for detecting 5-hydroxymethylfurfural in coffee beans according to claim 1, characterized in that, The ratio of coffee bean powder to hexane is (0.01-0.3) g: 1 mL.

3. The method for detecting 5-hydroxymethylfurfural in coffee beans according to claim 1, characterized in that, The vortex mixing time is 4-10 minutes.

4. The method for detecting 5-hydroxymethylfurfural in coffee beans according to claim 1, characterized in that, The solvent extraction step includes: adding defatted coffee bean powder to an acetonitrile aqueous solution, vortexing and centrifuging sequentially, and then taking the supernatant solution to obtain a 5-hydroxymethylfurfural extract.

5. The method for detecting 5-hydroxymethylfurfural in coffee beans according to claim 1, characterized in that, The chromatographic conditions for the high-performance liquid chromatography-mass spectrometry (HPLC-MS) step include: The liquid chromatography column used is a reversed-phase octadecylsilane-bonded silica gel column.

6. The method for detecting 5-hydroxymethylfurfural in coffee beans according to claim 1, characterized in that, The chromatographic conditions for the high-performance liquid chromatography-mass spectrometry (HPLC-MS) step include: Mobile phase A is a 0.1% (v / v) aqueous solution of formic acid; mobile phase B is pure acetonitrile.

7. The method for detecting 5-hydroxymethylfurfural in coffee beans according to claim 1, characterized in that, The chromatographic conditions for the high-performance liquid chromatography-mass spectrometry (HPLC-MS) step include: The elution method is gradient elution, and the gradient elution procedure is as follows: From 0 to 2 minutes, phase A was 95% and phase B was 5%. Between 2.0 and 2.1 minutes, phase A decreased from 95% to 10%, while phase B increased from 5% to 90%. 2.1–3.0 min, Phase A is 10%, Phase B is 90%; From 3.0 to 3.1 min, phase A increased from 10% to 95%, while phase B decreased from 95% to 5%. 3.1–5.0 min, Phase A is 95%, Phase B is 5%.

8. The method for detecting 5-hydroxymethylfurfural in coffee beans according to claim 1, characterized in that, The chromatographic conditions for the high-performance liquid chromatography-mass spectrometry (HPLC-MS) step also include at least one of the following: 1) Column temperature: 30-50℃; 2) Injection volume: 1-5 μL; 3) Flow rate: 0.2 - 0.3 mL / min.

9. The method for detecting 5-hydroxymethylfurfural in coffee beans according to claim 1, characterized in that, The mass spectrometry in the high performance liquid chromatography-mass spectrometry (HPLC-MS) step was performed using a triple quadrupole mass spectrometer. The conditions for the mass spectrometry include at least one of the following: a) Electrospray ionization source, positive ion mode scanning; b) The temperature for removing the solvent gas is 500 ℃; c) Ion source temperature 150℃; d) The capillary voltage is set to 3.0 kV; e) The conical orifice air flow rate is set to 50 liters / hour.

10. The method for detecting 5-hydroxymethylfurfural in coffee beans according to any one of claims 1-9, characterized in that, The procedure also includes the following steps: preparing reference solution and test solution, performing high performance liquid chromatography-mass spectrometry (HPLC-MS), and recording the chromatograms; and calculating the 5-hydroxymethylfurfural content in the test solution using the external standard method.

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