Analysis method for coffee components

Abstract

To easily and rapidly analyze trigonelline and pyrocatechol as coffee components.SOLUTION: A method for analyzing coffee components includes: a preparation step of preparing a sample liquid containing coffee components extracted from coffee beans; a dilution step of diluting the sample liquid; and a detection step of detecting the coffee components by performing liquid chromatography on the diluted sample liquid. In the detection step, the sample liquid is caused to pass through a column filled with a filler having a pentafluorophenyl propyl group, and then, the trigonelline and the pyrocatechol in the sample liquid having passed through are detected by a photo diode array ultraviolet visible absorbance detector.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for analyzing coffee components. [Background technology]

[0002] Functional ingredients in food products have long been studied. Among these, coffee components extracted from coffee beans have also been studied. In recent years, the coffee components trigonelline and pyrocatechol have been reported to be involved in the prevention or improvement of lifestyle-related diseases such as dementia, diabetes, and cancer. Similarly, chlorogenic acid has also been reported to have antioxidant properties and anti-aging effects.

[0003] Liquid chromatography is commonly used as a method for analyzing functional components such as coffee components. For example, in Patent Document 1, chlorogenic acid, which is highly hydrophobic, is separated by reversed-phase chromatography, while trigonelline, a hydrophilic compound, is retained by hydrophilic interaction chromatography, a type of normal-phase chromatography, because it is not retained by reversed-phase chromatography. In addition, in Patent Document 2, both trigonelline and chlorogenic acid are simultaneously separated by reversed-phase chromatography using an ion-pair reagent. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6259146 [Non-patent literature]

[0005] [Non-Patent Document 1] Kana ARAI et al. ANTLYTICAL SCIENCES, August 2015, Volume 3, p. 831-835 Summary of the Invention [Problem to be solved by the invention]

[0006] However, the method of Patent Document 1 requires two separate liquid chromatographies, which is time-consuming and labor-intensive. Hydrophilic interaction chromatography, in particular, suffers from poor analytical stability, i.e., poor reproducibility of retention times. To improve this, sufficient column equilibration time must be ensured. The method of Patent Document 2 uses an ion-pair reagent, which requires a conditioning period during which the stationary phase (packing material) is modified with the ion-pair reagent. This conditioning period lasts approximately two to three hours, which requires a long time before analysis can begin. Furthermore, since this method is not compatible with gradient elution, which changes the solvent ratio of the mobile phase during analysis to complete the analysis in a short time, the analysis itself takes a long time because an isocratic elution method is used, in which the solvent ratio of the mobile phase is kept constant.

[0007] Furthermore, the above two documents do not disclose the analysis of the important pyrocatechol, nor do they disclose the simultaneous analysis of both trigonelline and pyrocatechol, which are effective against lifestyle-related diseases.

[0008] Therefore, an object of the present invention is to provide a method for easily analyzing the coffee components trigonelline and pyrocatechol in a short period of time. [Means for solving the problem]

[0009] The analytical method of a first aspect of the present invention is a method for analyzing coffee components, and includes, in order: a preparation step of preparing a sample liquid containing coffee components extracted from coffee beans; a dilution step of diluting the sample liquid; and an analysis step of performing liquid chromatography on the diluted sample liquid to analyze trigonelline and pyrocatechol. In the analysis step, the sample liquid is passed through a column packed with a packing material having pentafluorophenylpropyl groups, and the passed sample liquid is then detected using a photodiode array ultraviolet-visible absorbance detector. [Effects of the Invention]

[0010] According to the analytical method of the first aspect, the coffee components trigonelline and pyrocatechol can be analyzed simply and quickly. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows chromatograms of Example 1 and Comparative Example 1 obtained by analyzing a mixed standard solution at a wavelength of 270 nm. The vertical axis indicates peak intensity, and the horizontal axis indicates retention time. The upper part of the chromatogram shows the results of Comparative Example 1 (using a C18 stationary phase), and the lower part shows the results of Example 1 (using a PFPP stationary phase). [Figure 2] Figure 2 shows a chromatogram of the mixed standard solution analyzed at a wavelength of 325 nm. [Figure 3] FIG. 3 shows the calibration curves for trigonelline and pyrocatechol. [Figure 4] FIG. 4 shows chromatograms from Example 2 in which coffee and a mixed standard solution were analyzed at a wavelength of 270 nm. The vertical axis represents peak intensity, and the horizontal axis represents retention time. The upper row of the chromatogram shows the results for coffee, and the lower row shows the results for the standard solution. [Figure 5] FIG. 5 is a chromatogram of Example 2 in which coffee and the mixed standard solution were analyzed at a wavelength of 325 nm. [Figure 6] These are UV spectra of the separated solution and individual standard solutions at each coffee peak, with the solid line showing the results for coffee and the dashed line showing the results for the individual standard solutions. DETAILED DESCRIPTION OF THE INVENTION

[0012] 1. First embodiment The analysis method of the first embodiment of the present invention comprises, in order, a preparation step, a dilution step, and a detection step. Each step will be described in detail below.

[0013] (Preparation process) In this step, a sample liquid containing coffee components is prepared.

[0014] Coffee components are extracted from coffee beans by roasting the coffee beans at a high temperature (e.g., 150°C to 250°C), grinding them, and contacting the grounds with water or hot water. Specific examples of such coffee components include trigonelline, pyrocatechol, chlorogenic acid, caffeine, and caffeic acid. In addition to these, impurities adhering to the coffee beans are also contained in the sample solution.

[0015] In the first embodiment, both trigonelline and pyrocatechol, which are expected to have preventive or ameliorative effects against lifestyle-related diseases, are analyzed simultaneously as coffee components. Preferably, chlorogenic acid, which is expected to have anti-aging effects, is also analyzed simultaneously. That is, the three components of trigonelline, pyrocatechol, and chlorogenic acid are analyzed simultaneously. More preferably, the five main components of coffee, trigonelline, pyrocatechol, chlorogenic acid, caffeine, and caffeic acid, are analyzed simultaneously.

[0016] (Dilution process) In this step, the sample liquid obtained in the preparation step is diluted, thereby preparing a diluted sample liquid (diluted sample liquid).

[0017] The solvent for dilution may be water or an organic solvent, with water being preferred.

[0018] The dilution concentration may be determined appropriately depending on the concentration of the extracted coffee components. When analyzing coffee obtained by general extraction, the coffee may be diluted, for example, 2 times or more, preferably 5 times or more, and for example, 100 times or less, preferably 50 times or less. This allows coffee components to be detected with high sensitivity while preventing contamination of equipment such as columns.

[0019] (Detection process) In this step, the diluted sample solution is subjected to liquid chromatography, which separates and detects the coffee components.

[0020] In liquid chromatography, a sample solution is mixed with an eluent (mobile phase) and passed through a column to separate the components in the sample solution over time. Liquid chromatography can be performed using a known liquid chromatograph analyzer, such as the Nexera series manufactured by Shimadzu Corporation.

[0021] Examples of the eluent include organic solvents and water. Examples of the organic solvent include acetonitrile, methanol, ethanol, isopropanol, acetone, chloroform, and tetrahydrofuran. These organic solvents can be used alone or in combination. Acetonitrile is preferred. This allows for reliable detection of coffee components.

[0022] In the first embodiment, a gradient elution method is preferably employed. That is, the concentration of the organic solvent in the mobile phase is gradually increased from the beginning of the analysis. A buffer solution such as a phosphate buffer solution can be used to adjust the concentration of the organic solvent. This allows for improved peak separation of each component while shortening the time required to complete the analysis.

[0023] The column equipped in the liquid chromatograph is packed with a packing material having pentafluorophenylpropyl groups. That is, the sample liquid is passed through a column packed with a packing material having pentafluorophenylpropyl groups (pentafluorophenyl groups). Such packing materials can be obtained, for example, by chemically bonding pentafluorophenylpropyl groups to silica gel. Specific examples include Shim Pack Scepter PFPP (registered trademark) from Shimadzu Corporation and InertSustain PFP from GL Sciences. In the first embodiment, the column packing material (stationary phase) has pentafluorophenylpropyl groups. Compared to the mixed solution of acidic buffer and organic solvent used as the mobile phase, the pentafluorophenylpropyl groups are more hydrophobic, exhibiting the separation behavior of reversed-phase chromatography. Caffeine is believed to exhibit π-π interactions and CH / π interactions in addition to hydrophobic interactions, resulting in greater retention than a C18 stationary phase. Pyrocatechol, chlorogenic acid, and caffeic acid are more hydrophobic than the mobile phase, but the pentafluorophenylpropyl group is also highly hydrophobic, so an interaction occurs between the two due to the affinity between their hydrophobic groups. Pyrocatechol is less hydrophobic than chlorogenic acid and caffeic acid, which have a catechol skeleton, and its retention is lower compared to these three components. On the other hand, the pentafluorophenylpropyl group is highly polarized with respect to the highly hydrophilic trigonelline. Since trigonelline is also polarized as an electron-donating compound with a positively charged pyridine ring structure, it is presumed that this is due to an interaction between polar molecules due to Coulomb force. The first embodiment is not limited to the above mechanism.

[0024] The column temperature is, for example, 50°C or lower, preferably 30°C or lower, more preferably 28°C or lower, and for example, 5°C or higher, preferably 10°C or higher. By setting the column temperature below the upper limit, peak separation is improved. In particular, by setting the column temperature to 30°C or lower, the separation between the impurity peak and the trigonelline peak becomes clear, allowing for more accurate quantitative analysis of trigonelline.

[0025] The detector used is a photodiode array (semiconductor element) ultraviolet-visible absorbance detector (hereinafter abbreviated as "photodiode array detector"). Specifically, the sample liquid passing through the column (in other words, the separated liquid, which is the liquid containing the coffee components separated over time) is detected by the photodiode array detector. Specifically, a predetermined light (e.g., a mixture of tungsten lamp light and D2 lamp light) is irradiated onto the sample liquid in the flow cell, the transmitted light is dispersed by a diffraction grating, and the light at each wavelength is detected by the photodiode array detector. The detected light is then analyzed or processed, producing a graph that shows the light intensity (particularly the absorbance of the separated liquid) according to retention time, as a chromatogram. This chromatogram is obtained for multiple wavelengths arbitrarily set by the photodiode array detector. Furthermore, since the UV spectrum (e.g., absorption spectrum) of the separated liquid at a specific retention time can be obtained, a UV spectrum can be obtained for each separated coffee component. That is, by using a photodiode array detector, chromatograms for multiple wavelengths can be obtained, and UV spectra for each of the separated coffee components can also be obtained.

[0026] Preferably, there are multiple wavelengths to be detected, including, for example, a wavelength of 260 nm to 280 nm (hereinafter referred to as the first wavelength) and a wavelength of 315 nm to 335 nm (hereinafter referred to as the second wavelength). In particular, trigonelline, pyrocatechol, chlorogenic acid, caffeine, and caffeic acid all absorb light at the first wavelength, making spectroscopic analysis possible. Furthermore, in particular, chlorogenic acid and caffeic acid strongly absorb light at the second wavelength, improving the detection accuracy of these two components.

[0027] In the chromatogram obtained by this step, particularly in the chromatogram detected at the first wavelength, peaks corresponding to trigonelline, pyrocatechol, chlorogenic acid, caffeine, and caffeic acid are observed in different retention time ranges, and in particular, the peaks of the above five components are observed between retention times of 3 minutes and 15 minutes.

[0028] This allows simultaneous analysis of all coffee components in the sample solution, specifically, trigonelline and pyrocatechol, preferably, three components (trigonelline, pyrocatechol, and chlorogenic acid), and most preferably, five components (trigonelline, pyrocatechol, chlorogenic acid, caffeine, and caffeic acid).

[0029] In the first embodiment, the quantitative analysis of the coffee components is made possible by creating a calibration curve. Specifically, for each coffee component, multiple standard solutions of known concentrations are prepared, and the detection process is performed to obtain chromatograms of the multiple concentrations, thereby creating a calibration curve for each component (a graph plotting the relationship between peak intensity and known concentration). The peak intensity of a coffee component in a sample solution of unknown concentration is matched to this calibration curve, allowing the concentration of the coffee component to be determined. In the first embodiment, the calibration curve has excellent linearity, allowing the concentration of the coffee component to be measured with excellent accuracy.

[0030] In the first embodiment, the coffee components can be identified by outputting an absorption spectrum, such as a UV spectrum. Specifically, a UV spectrum is acquired for a specific peak in a chromatogram, and the acquired UV spectrum is compared with the UV spectra of known coffee components. This makes it possible to reliably determine whether a specific peak in a chromatogram is a peak due to the coffee component of interest. This is effective when a sample solution contains a variety of impurities and peaks of various impurities are detected in the chromatogram, because it allows accurate identification of the coffee component of interest.

[0031] According to the analytical method of the first embodiment, coffee components can be analyzed from a sample solution, particularly trigonelline, pyrocatechol, chlorogenic acid, caffeine, and caffeic acid, simultaneously separated, detected, and quantified. Furthermore, the method is simple because it allows simultaneous analysis of coffee components in a single liquid chromatography run using a single column. Furthermore, it does not require the use of ion-pair reagents or extensive conditioning time, allowing for analysis in a short time. Furthermore, the column can be reused because the stationary phase is not denatured by ion-pair reagents. Furthermore, the retention times and peak areas in the resulting chromatograms are highly reproducible, resulting in high analytical accuracy. Furthermore, it is possible to create a calibration curve with high linearity and minimal variation, allowing for accurate measurement of the concentrations of coffee components. Furthermore, because the peaks of impurities and coffee components can be detected separately and identified using UV spectra, coffee components can be more accurately identified and quantified.

[0032] 2. Aspects It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0033] (Item 1) A method for analyzing coffee components according to one embodiment includes, in order, a preparation step of preparing a sample liquid containing coffee components extracted from coffee beans, a dilution step of diluting the sample liquid, and a detection step of performing liquid chromatography on the diluted sample liquid to detect the coffee components. In the detection step, the sample liquid may be passed through a column packed with a filler having pentafluorophenylpropyl groups, and trigonelline and pyrocatechol in the passed sample liquid may then be detected using a photodiode array ultraviolet-visible absorbance detector.

[0034] (Item 2) In the analysis method described in item 1, the wavelength of light used to detect the coffee components by the photodiode array ultraviolet-visible absorbance detector may be at least 260 nm or more and 280 nm or less, and 315 nm or more and 335 nm or less.

[0035] (Item 3) In the analytical method according to item 1 or 2, the temperature of the column may be 30° C. or lower.

[0036] (Item 4) In the analysis method according to any one of items 1 to 3, trigonelline, pyrocatechol, chlorogenic acid, caffeine, and caffeic acid may be detected simultaneously in the detection step. [Example]

[0037] The present invention will now be described in detail with reference to examples, but the scope of the present invention is not limited to these examples.

[0038] Example 1 (Analysis of mixed standard solution) A mixed standard solution containing 10 mg / L each of trigonelline, pyrocatechol, chlorogenic acid, caffeine, and caffeic acid was prepared, and liquid chromatography was performed under the following conditions. The column was packed with a packing material containing pentafluorophenylpropyl groups (PFPP), and a photodiode array detector was used. The chromatogram at a detection wavelength of 270 nm is shown in Figure 1, and the chromatogram at a detection wavelength of 325 nm is shown in Figure 2.

[0039] Device name: Nexera lite (ultra-high performance liquid chromatograph, manufactured by Shimadzu Corporation) Column: Shim-pack Scepter PFPP-120 (150 mm x 4.6 mm ID, 3 μm) Flow rate: 1.0mL / min Mobile phase: A) 20mmol / L (Sodium) phosphate buffer (pH 2.6) B) Acetonitrile Gradient elution: 0%B (0.00-1.00 min), 10%B (4.00 min), 20%B (10.00-12.00 min), 70%B (12.01-13.00 min), 0%B (13.01-18.00 min) Mixer: 180 μL Column temperature: 25℃ Injection volume: 5μL Detector: Photodiode array (PDA) ultraviolet-visible absorbance detector ("SPD-M40", Shimadzu Corporation) Detection wavelength: 270nm, 325nm

[0040] Five peaks were confirmed in Figures 1 and 2, enabling the separation and detection of five coffee components (trigonelline, pyrocatechol, chlorogenic acid, caffeine, and caffeic acid). Furthermore, because each peak met the optimum retention time recommended by the liquid chromatograph, which was 3 minutes or more, it was clear that the five components were properly retained and separated on the column. Furthermore, because the retention times of the five components were within 13.5 minutes, it was clear that the analysis could be completed in a short time.

[0041] In addition, individual standard solutions containing only one of the coffee components were also prepared, and the above-mentioned liquid chromatography was performed to match each component with its peak. By doing so, it was confirmed that peak "1" was derived from trigonelline, peak "2" from pyrocatechol, peak "3" from chlorogenic acid, peak "4" from caffeine, and peak "5" from caffeic acid.

[0042] <Comparative Example 1> The procedure was the same as in Example 1, except that the column was changed to a column packed with a C18 stationary phase (Shim-pack Scepter C18-120 (150 mm × 4.6 mm ID, 3 μm)). The chromatogram at a detection wavelength of 270 nm is shown in FIG. 1, and the chromatogram at a detection wavelength of 325 nm is shown in FIG. 2.

[0043] In Comparative Example 1, the peak "1", i.e., the peak representing trigonelline, was detected at a retention time of 2.1 minutes, which was inappropriate because it did not satisfy the optimum retention time of 3 minutes or more.

[0044] (Reproducibility of retention time) A mixed standard solution containing 1 mg / L of each of the five components was prepared, and the same analysis as above was repeated six times. The relative standard deviation (RSD) of the retention time and peak area of ​​each peak was calculated. The results are shown in Table 1 below.

[0045] [Table 1]

[0046] The standard deviation for retention time was less than 0.1% for all five components, and the standard deviation for peak area was less than 0.7% for all five components. These results indicate that the variation in both retention time and peak area was very low, demonstrating excellent reproducibility.

[0047] (Creating a calibration curve) For each of the five components, individual standard solutions with different concentrations were prepared, and the same analysis was performed as above to create a calibration curve. The calibration curve concentration range and contribution rate are shown in Table 2 below. The calibration curves for trigonelline and pyrocatechol are shown in Figure 3.

[0048] [Table 2]

[0049] In Table 2 and Figure 3, the contribution rate for all five components was 0.9999% or more, and excellent linearity was obtained, indicating that they can be effectively used as calibration curves.

[0050] <Example 2> (Coffee Analysis) 10 g of commercially available ground coffee beans were extracted with 150 mL of hot water, and the resulting extract was filtered through a 0.2 μm membrane filter. This extract was then diluted 10-fold with ultrapure water to prepare a sample solution. Liquid chromatography was performed using this sample solution under the same analytical conditions as in Example 1. The chromatogram (solid line) at a detection wavelength of 270 nm is shown in Figure 4 . The chromatogram (solid line) at a detection wavelength of 325 nm is shown in Figure 5 . Furthermore, the UV spectrum from 200 nm to 400 nm was measured for the peaks "1" to "4." The results are shown in Figure 6 . The concentrations of each component were determined based on the calibration curve. The analysis and concentration determination of the sample solution were repeated six times, and the relative standard deviations were calculated. The results are shown in Table 3 below.

[0051] [Table 3]

[0052] (Analysis of standard solution) A mixed standard solution containing 20 mg / L trigonelline, 2 mg / L pyrocatechol, 20 mg / L chlorogenic acid, 20 mg / L caffeine, and 2 mg / L caffeic acid was prepared, and liquid chromatography was performed under the same analytical conditions as in Example 1. Chromatograms (dashed lines) for this mixed standard solution are shown in Figures 4 and 5. Furthermore, UV spectra were measured from 200 nm to 400 nm for the individual standard solutions of trigonelline, pyrocatechol, chlorogenic acid, and caffeine. The results are shown in Figure 6.

[0053] (Consideration) As shown in Figures 4 and 5, peaks "1" to "4" were also confirmed in the chromatogram of the coffee components extracted from coffee beans. Furthermore, as shown in Figure 6, the spectra of each individual standard solution and the UV spectra of peaks "1" to "4" were completely consistent. From these, it was found that peaks "1" to "4" correspond to trigonelline, pyrocatechol, chlorogenic acid, and caffeine, respectively, and that these components are contained in coffee. Furthermore, since no peak corresponding to caffeic acid was confirmed, it can be concluded that caffeic acid is not contained in this coffee. Furthermore, the quantitation performance was excellent, with a small standard deviation. Furthermore, a small peak (see arrow) was detected before peak "1" in Figure 4. This is due to impurities, and it was found that the analytical method of the present invention was able to separate the impurities.

[0054] Example 3 Coffee samples roasted for different times were prepared and subjected to the liquid chromatography described above in the same manner as in Example 1. The column temperature was changed to 25°C, 30°C, and 35°C, and the analysis was performed. As a result, it was confirmed that at 25°C, the trigonelline peak and the peak of the impurities immediately preceding it were clearly separated. At 30°C, the trigonelline peak and the peak of the impurities were close to each other, with the bases of both peaks slightly overlapping. At 35°C, both peaks overlapped.

Claims

1. a preparation step of preparing a sample liquid containing coffee components extracted from coffee beans; a dilution step of diluting the sample liquid; and a detection step of detecting the coffee components by performing liquid chromatography on the diluted sample solution, In the detection step, the sample liquid is passed through a column packed with a filler having a pentafluorophenylpropyl group, and then trigonelline and pyrocatechol in the passed sample liquid are detected using a photodiode array ultraviolet-visible absorbance detector.

2. 2. The analytical method according to claim 1, wherein the wavelengths of light used to detect the coffee components by the photodiode array ultraviolet-visible absorbance detector are at least 260 nm to 280 nm and 315 nm to 335 nm.

3. 3. The analytical method according to claim 1, wherein the temperature of the column is 30° C. or less.

4. The analytical method according to any one of claims 1 to 3, wherein trigonelline, pyrocatechol, chlorogenic acid, and caffeine are detected simultaneously in the detection step.

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