Carotene analysis method

Abstract

To analyze α-carotene and β-carotene in a distinctive manner.SOLUTION: There is provided an analysis method for analyzing carotene by liquid chromatography, and includes passing a measurement sample containing α-carotene and β-carotene through a column filled with filler having an isobutyl group and an octadecyl group to separate the α-carotene and β-carotene from each other.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] Among various vitamins that work to keep the body functioning normally, vitamin A is mainly retinol, and is produced by the breakdown of carotene in the body. Most carotene is composed of α-carotene or β-carotene. The bioavailability of α-carotene and β-carotene is estimated to be 1 / 24 and 1 / 12, respectively (see Non-Patent Document 1), and the bioavailability of the two is significantly different. Therefore, in order to investigate the activity of vitamin A in food and drink, it is important to individually quantify the amounts of α-carotene and β-carotene contained in the food and drink. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Ministry of Health, Labor and Welfare, "Dietary Reference Intakes for Japanese (2020 Edition)", pp. 171-172, https: / / www.mhlw.go.jp / content / 10904750 / 000586553.pdf, [Retrieved May 11, 2023], Internet Summary of the Invention [Problem to be solved by the invention]

[0004] However, even if one attempts to measure foods and drinks containing α-carotene and β-carotene by mass spectrometry, it is difficult to distinguish and analyze the two because α-carotene and β-carotene are isomers with very similar structures. Also, even in the case of measurement by liquid chromatography, when a general reversed-phase column (specifically, a C18 column filled with silica gel surface-modified with octadecyl groups) is used, the dynamics of the two in the mobile phase are similar, and the retention times of the two are almost the same, making it difficult to separate the two.

[0005] Therefore, an object of the present invention is to provide a method capable of distinguishing and analyzing α-carotene and β-carotene. [Means for solving the problem]

[0006] The analytical method of the first aspect of the present invention is a method for analyzing carotene by liquid chromatography, in which a measurement sample containing α-carotene and β-carotene is passed through a column packed with a packing material having isobutyl groups and octadecyl groups to separate α-carotene and β-carotene. Effect of the Invention

[0007] According to the analytical method of the first aspect, α-carotene and β-carotene in a measurement sample can be analyzed separately. [Brief description of the drawings]

[0008] [Figure 1] 1 is a mass chromatogram obtained in Example 1. The vertical axis indicates peak area, and the horizontal axis indicates retention time. [Diagram 2] 2 shows the calibration curves prepared in Example 1, with the left graph showing the calibration curve for α-carotene and the right graph showing the calibration curve for β-carotene. The vertical axis shows the peak area, and the horizontal axis shows the concentration (ng / mL). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] 1. First embodiment In the analysis method according to the first embodiment of the present invention, liquid chromatography mass spectrometry is performed on a measurement sample. That is, liquid chromatography is performed on the measurement sample, and then mass spectrometry is performed on the measurement sample separated by liquid chromatography.

[0010] The measurement sample contains both α-carotene and β-carotene (see the structural formula below), and may be, for example, a vegetable component or a liquid containing the same. When the measurement liquid is a liquid (sample liquid), it may be diluted with an organic solvent such as methanol or water, as necessary. In addition, as a pretreatment, sodium sulfate or the like may be added for salting out, or centrifugation may be performed.

[0011] [ka]

[0012] In liquid chromatography, the measurement sample is mixed with the mobile phase (eluent) and passed through a column to separate the components of the measurement sample, α-carotene and β-carotene, over time. As an apparatus for liquid chromatography, a known liquid chromatograph analyzer may be used, and for example, the Nexera series manufactured by Shimadzu Corporation is commercially available.

[0013] A column used in liquid chromatography is filled with a packing material having an isobutyl group and an octadecyl group. That is, a measurement sample is passed through a column filled with a packing material having an isobutyl group and an octadecyl group. This allows α-carotene and β-carotene to be retained in the column for different retention times, and then separated. Such a packing material is a packing material having an octadecyl group (a so-called C18 stationary phase) to which an isobutyl group has been further introduced. More specifically, it is obtained by chemically modifying silica particles surface-modified with an octadecyl group with an isobutyl group. Furthermore, it is preferable that the packing material is end-capped with a trimethylsilyl group or the like. Specific examples of packing materials having an isobutyl group and an octadecyl group include Kinetex XB-C18 manufactured by Phenomenex.

[0014] The mobile phase may be, for example, an organic solvent such as methanol, ethanol, acetonitrile, 2-propanol, acetone, chloroform, or tetrahydrofuran, preferably methanol and / or ethanol, more preferably a mixed solvent of methanol and ethanol. This allows the retention times of α-carotene and β-carotene to be shifted more reliably, and these peaks to be separated more reliably. In the case of a mixed solvent, the ratio of ethanol in the mixed solvent may be, for example, 5 (v / v)% or more, preferably 10 (v / v)% or more, and, for example, 50 (v / v)% or less, preferably 25 (v / v)% or less.

[0015] The flow rate of the mobile phase is, for example, 5 mL / min or less, preferably 1 mL / min or less, more preferably 0.4 mL / min or less, for example, 0.1 mL / min or more. By setting the flow rate of the mobile phase to the above upper limit or less, the separation of the peak derived from α-carotene and the peak derived from β-carotene in the chromatogram (total ion chromatogram, mass chromatogram, etc.) is improved. On the other hand, by setting the flow rate of the mobile phase to the above lower limit or more, the analysis time can be shortened.

[0016] In addition, either an isocratic method or a gradient method may be used in the liquid chromatography of the first embodiment. When an isocratic method is used, it is preferable to increase the ratio of the organic solvent and wash the column after the analysis.

[0017] The column temperature is, for example, 20° C. or more, preferably 30° C. or more, and for example, 60° C. or less, preferably 50° C. or less. By setting the temperature within the above range, peak separation becomes even better.

[0018] As a result, the measurement sample that has passed through the column is separated into α-carotene and β-carotene according to their respective retention times. Specifically, α-carotene passes through the column first, and then β-carotene passes through the column after a predetermined time.

[0019] Next, in mass spectrometry, the α-carotene and β-carotene separated in the measurement sample are each ionized, and ions having a specific m / z (mass-to-charge ratio) are selected or separated and detected.

[0020] In mass spectrometry, examples of ionization methods include electron ionization (EI), electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), etc. In the first embodiment, from the viewpoint of more reliably ionizing and detecting α-carotene and β-carotene, APCI is preferably used.

[0021] Examples of the method of mass separation for the ionized α-carotene and β-carotene, i.e., the type of mass spectrometer, include a quadrupole mass spectrometer, a magnetic sector mass spectrometer, a time-of-flight mass spectrometer, an ion trap (including an orbitrap) mass spectrometer, and an ion cyclotron resonance mass spectrometer. A tandem mass spectrometer consisting of a plurality of spectrometers may also be used. That is, in the first embodiment, liquid chromatography tandem mass spectrometry (LC / MS / MS) may be adopted. Examples of the types of tandem mass spectrometry (MS / MS) include a triple quadrupole (QQ) mass spectrometer, a tandem time-of-flight (TOF-TOF) mass spectrometer, a quadrupole-time-of-flight (Q-TOF) mass spectrometer, a quadrupole-ion trap (Q-IT) mass spectrometer, a quadrupole-ion cyclotron resonance (Q-ICR) mass spectrometer, and an ion trap-time-of-flight (IT-TOF) mass spectrometer.

[0022] In the first embodiment, it is preferable to employ a tandem mass spectrometry method, and in particular, it is preferable to use a triple quadrupole mass spectrometer, which can obtain a mass spectrum in which specific fragment ions derived from α-carotene and β-carotene are separated and detected, and from the spectrum, it is possible to more accurately identify and quantify α-carotene and β-carotene.

[0023] In tandem mass spectrometry using a triple quadrupole, only precursor ions are selected or passed through the first quadrupole (first mass separation unit: Q1), the precursor ions are fragmented in the next collision chamber to generate multiple types of product ions (a type of fragment ion), only specific product ions are selected or passed through the second quadrupole (second mass separation unit: Q2), and finally the product ions are detected in the detection unit. At this time, it is preferable to detect at least ions with m / z of about 119.0 and ions with m / z of about 123.0 as product ions. Particularly preferably, in the case of identifying α-carotene, an ion with m / z of about 537.5 is selected as the precursor ion in Q1, and an ion with m / z of about 119.0 is selected as the product ion in Q2, while in the case of identifying β-carotene, an ion with m / z of about 537.5 is selected as the precursor ion, and an ion with m / z of about 119.0 is selected as the product ion. As a result, since α-carotene and β-carotene generate these m / z ions in the collision chamber, the presence of α-carotene and β-carotene can be detected. In particular, in the cleavage of α-carotene in the ion collision chamber, many ions with m / z of 123.0 are generated, whereas in the cleavage of β-carotene, many ions with m / z of 119.0 are generated. Therefore, a peak derived from α-carotene appears prominently in the mass spectrum at m / z around 119.0, and a peak derived from β-carotene appears prominently in the mass spectrum at m / z around 123.0, so that α-carotene and β-carotene can be detected with high sensitivity. In selecting m / z, the polarity of the ions is set to + (plus). In the first embodiment, "near" refers to a range of ±0.5, and for example, near 119.0 is a range of 118.5 to 119.5.

[0024] As an apparatus used for mass spectrometry, a known mass spectrometer may be used, for example, the LCMS series manufactured by Shimadzu Corporation. Various conditions in the mass spectrometer, such as flow rate, temperature, collision energy, etc., may be appropriately set according to the recommendations of each apparatus.

[0025] As a result, a total ion chromatogram, mass chromatogram, and mass spectrum are obtained for the measurement sample separated by liquid chromatography. Among these, for example, in the mass chromatogram, peaks of ions (precursor ions, product ions) derived from α-carotene and β-carotene are observed at different retention times. Specifically, in liquid chromatography for a measurement sample containing α-carotene and β-carotene, α-carotene elutes from the column before β-carotene and reaches the mass spectrometer, so that in the mass chromatogram, a peak derived from α-carotene appears at an earlier retention time than a peak derived from β-carotene.

[0026] In particular, in a triple quadrupole liquid chromatography mass spectrometry, two major peaks appear in a mass spectrum in which the m / z of the precursor ion is selected at around 537.5 and the m / z of the product ion is selected at around 119.0. Similarly, two major peaks appear in a mass spectrum in which the m / z of the precursor ion is selected at around 537.5 and the m / z of the product ion is selected at around 123.0. In these mass spectra, the two peaks are ions derived from α-carotene and β-carotene, respectively, and the peak appearing at the earlier retention time is the α-carotene peak, and the peak appearing at the later retention time is the β-carotene peak.

[0027] From the above, when the analytical method of the first embodiment is performed on an unknown measurement sample and two peaks are observed in the obtained mass spectrum, it can be determined that the measurement sample contains both α-carotene and β-carotene.

[0028] The calculation (quantitative analysis) of the concentrations of α-carotene and β-carotene is possible by creating a detection line. Specifically, measurement samples containing α-carotene and β-carotene of known concentrations are prepared at a plurality of concentrations, and the above-mentioned mass spectrometry is performed on these to create a calibration curve plotting the relationship between the concentration and peak intensity (peak area, etc.) for each of α-carotene and β-carotene. The concentrations of α-carotene and β-carotene can be determined by corresponding each peak intensity in the measurement samples containing α-carotene and β-carotene of unknown concentrations to this calibration curve.

[0029] According to the analysis method of the first embodiment, α-carotene and β-carotene can be individually quantified, and the analysis can be performed in a short time.

[0030] 2. Second embodiment The analytical method of the second embodiment of the present invention is a method of analyzing a measurement sample by liquid chromatography. In other words, the analytical method of the second embodiment is a method in which only liquid chromatography is performed as an analytical method without performing mass spectrometry.

[0031] The measurement conditions are the same as those of the liquid chromatography of the first embodiment. The analysis method of the second embodiment provides a chromatogram in which the peaks derived from α-carotene and β-carotene are separated from each other. From this chromatogram, α-carotene and β-carotene can be identified. Furthermore, by measuring the intensities of these peaks and referring to a calibration curve prepared in advance, the concentrations of α-carotene and β-carotene can be calculated individually.

[0032] 3. 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) One embodiment of a method for analyzing carotene is a method for analyzing carotene by liquid chromatography, in which a measurement sample containing α-carotene and β-carotene may be passed through a column filled with a packing material having an isobutyl group and an octadecyl group to separate α-carotene and β-carotene.

[0034] (Item 2) In the analytical method according to item 1, mass spectrometry may be performed on the measurement sample separated by liquid chromatography.

[0035] (Item 3) In the analysis method according to item 2, the measurement sample may be ionized by atmospheric pressure chemical ionization in the mass spectrometry.

[0036] (Item 4) In the analytical method according to item 2 or 3, ions having m / z of about 119.0 and ions having m / z of about 123.0 may be detected in the mass spectrometry.

[0037] (Item 5) In the analysis method according to any one of items 2 to 4, α-carotene may be identified based on a peak in a mass chromatogram obtained by detecting ions having an m / z of about 123.0 in the mass spectrometry, and β-carotene may be identified based on a peak in a mass chromatogram obtained by detecting ions having an m / z of about 119.0.

[0038] (Item 6) In the analysis method according to any one of items 1 to 5, the mobile phase may be a mixed solvent of methanol and ethanol. EXAMPLES

[0039] The present invention will now be described in detail with reference to examples and comparative examples, but the scope of the present invention is not limited thereto.

[0040] <Example 1> (Analysis of sample liquid) 5 g of anhydrous sodium sulfate and 10 mL of ethanol were added to 1 g of commercially available vegetable juice, and the mixture was shaken for 5 minutes, then centrifuged for 5 minutes to collect the supernatant. The precipitate from the centrifugation was subjected to extraction processing two more times, and the supernatant was collected. These supernatants were mixed, and ethanol was added to make a constant volume of 40 mL. 40 mL of this extract was diluted 100 times with ethanol, centrifuged for 10 minutes, and the supernatant was collected to prepare a measurement sample. Chromatography mass spectrometry was performed on this measurement sample under the following conditions. The column used was a Kinetex XB-C18 column with an isobutyl group introduced (end-capped with a trimethylsilyl group).

[0041] [LC conditions] Device name: Ultra-high performance liquid chromatograph Nexera X3 (Shimadzu Corporation) Column: Kinetex XB-C18 (100mm x 3.0mm I.D., 1.7µm) Mobile phase: A) Methanol B) Ethanol Time program conditions: Isocratic method B concentration 15% (0-7 minutes), 100% (7.01-12 minutes) Flow rate: 0.5mL / min Column temperature: 40℃ Injection volume: 5μL

[0042] [MS conditions] Instrument name: Triple quadrupole mass spectrometer LCMS-8050 (Shimadzu Corporation) Ionization mode: APCI Nebulizer gas flow rate: 3L / min Drying gas flow rate: 10L / min DL temperature: 200℃ Interface temperature: 300℃ Heat block temperature: 200℃ Measurement mode: multiple reaction monitoring (MRM), conditions see Table 1 below

[0043] [Table 1]

[0044] The mass chromatogram obtained at this time is shown in Figure 1. As shown in Figure 1, the two peaks are observed separately, which shows that α-carotene and β-carotene can be measured separately. Furthermore, the measurement was completed in less than 10 minutes, which shows that the analysis was performed quickly. When the above-mentioned liquid chromatography mass spectrometry method was performed on the standard sample solution of α-carotene and the standard sample solution of β-carotene, it was confirmed that the peak at a retention time of approximately 6.1 minutes was due to α-carotene, and the peak at a retention time of approximately 6.5 minutes was due to β-carotene.

[0045] 1, the peak originating from m / z537.45>123.15 in the α-carotene peak (retention time 6.1 min) is 1.7 times larger in area than the peak originating from m / z537.45>119.10, and the sensitivity is good, so it is preferable to detect α-carotene based on the ion originating from m / z537.45>123.15. On the other hand, the peak originating from m / z537.45>119.10 in the β-carotene peak (retention time 6.5 min) is 4.6 times larger in area than the peak originating from m / z537.45>123.15, and the sensitivity is good, so it is preferable to detect β-carotene based on the ion originating from m / z537.45>119.10.

[0046] (Creating a calibration curve) Five types of standard samples containing α-carotene and β-carotene with known concentrations were prepared. These were analyzed in the same manner as in Example 1 above. The quantification and reproducibility are shown in Table 2. A calibration curve was also created based on these results, which is shown in Figure 2. Table 2 and Figure 2 show that α-carotene and β-carotene can be quantified accurately over a wide range.

[0047] [Table 2]

[0048] <Example 2> The same procedure as in Example 1 was carried out under LC conditions, except that the flow rate of the mobile phase was changed to 0.4 mL / min. In the obtained mass chromatogram, the peaks of α-carotene and β-carotene were observed at retention times of around 7.7 minutes and 8.1 minutes, respectively. The resolution of the peaks of α-carotene and β-carotene (USP method) was 1.505. On the other hand, the resolution in Example 1 was 1.085. This shows that the peak separation in Example 2 is better.

[0049] <Comparative Examples 1 to 4> The LC conditions were the same as in Example 1, except that the columns were changed to (1) AQUITY UPLC BEH C18, (2) Shim-pack Scepter PFPP-120, (3) Kinetex 2.6uBiphenyl 100A, or (4) Kinetex PFP 100A. When the mass chromatograms were checked, the α-carotene peak and the β-carotene peak were not separated for any of the columns, making it difficult to distinguish between the two.

Claims

1. 1. A method for analyzing carotene by liquid chromatography, comprising the steps of: A method for analyzing carotene, comprising passing a measurement sample containing α-carotene and β-carotene through a column packed with a packing material having an isobutyl group and an octadecyl group, thereby separating α-carotene and β-carotene.

2. The method for analyzing carotene according to claim 1, wherein mass spectrometry is performed on the measurement sample separated by liquid chromatography.

3. 3. The method for analyzing carotene according to claim 2, wherein the measurement sample is ionized by atmospheric pressure chemical ionization in the mass spectrometry.

4. 3. The method for analyzing carotene according to claim 2, wherein in the mass spectrometry, ions with m / z in the vicinity of 119.0 and ions with m / z in the vicinity of 123.0 are detected.

5. 5. The method for analyzing carotene according to claim 4, wherein, in the mass spectrometry, α-carotene is identified based on a peak in a mass chromatogram obtained by detecting ions having an m / z of about 123.0, and β-carotene is identified based on a peak in a mass chromatogram obtained by detecting ions having an m / z of about 119.

0.

6. 2. The method for analyzing carotene according to claim 1, wherein the mobile phase is a mixed solvent of methanol and ethanol.

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