Trace analysis method for pyrroloquinoline quinone

Abstract

To provide an analysis method capable of reducing loss of PQQ in a process of preparing a sample for measurement from an object sample such as blood and more accurately determining the quantity of PQQ in the sample for measurement.SOLUTION: An analysis method has: a reaction stage for causing pyrroloquinoline quinone or its salt and acetone to react on each other in an object sample to produce acetone-added pyrroloquinoline quinone or its salt; a purification stage for purifying the acetone-added pyrroloquinoline quinone or its salt through a solid-phase adsorbent treatment to obtain a sample for measurement; and a quantitative analysis stage for determining the quantity of the pyrroloquinoline quinone and its salt contained in an object to be measured by a fluorescence detection device or mass spectroscope based upon a chromatogram of the acetone-added pyrroloquinoline quinone or its salt included in the sample for measurement by a chromatography method.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for microanalyzing pyrroloquinoline quinone. [Background technology]

[0002] Pyrroloquinoline quinone (hereinafter simply referred to as "PQQ") is a compound formed by the condensation of a pyrrole ring and a quinoline ring, resulting in an o-quinone structure. PQQ is known to function as an electron carrier, and when incorporated into aminoadipate semialdehyde dehydrogenase (AASDH), which is involved in the metabolism of the essential amino acid lysine, it enables AASDH to undergo redox reactions. Therefore, PQQ is considered to be a coenzyme for AASDH. This makes it the third redox coenzyme after nicotinamide (pyridine nucleotide) and flavin, and it has the potential to become a novel vitamin.

[0003] PQQ is also believed to have many important physiological activities, such as promoting cell proliferation, anti-cataract, preventing and treating liver disease, promoting wound healing, anti-allergic, inhibiting reverse transcriptase, inhibiting glyoxylase I, and anti-cancer, and the industrial importance of its use is increasing.

[0004] PQQ has been known to be widely present in bacteria, molds, yeasts, and other fungi, but in recent years it has been reported that it is widely present not only in bacteria but also in plants such as rice and mammals. Although it has been reported to be detected in various tissues and organs in mammals, mammals do not have a PQQ synthesis pathway, so it is believed that mammals ingest PQQ from food.

[0005] In order to further study the effects of PQQ on living organisms, discover further effects, and develop medicines, supplements, food and beverage products, and related products that can more effectively utilize the effects of PQQ, it is desirable to develop a method for detecting PQQ contained in biological samples, etc. For example, Non-Patent Document 1 discloses a method for measuring the PQQ concentration in plasma using high-performance liquid chromatography with chemiluminescence detection (HPLC-CL). Also, Non-Patent Document 2 discloses a method for separating acetone adducts of PQQ by HPLC and detecting them with a UV detector. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Mizuho Fukuda et al., J Pharm Biomed Anal. Vol 145, pp814-820, 2017 [Non-patent document 2] Kenji. Kano et al., Analytical Science, Vol 7 pp 737, 1991 Summary of the Invention [Problem to be solved by the invention]

[0007] However, the method described in Non-Patent Document 1 requires special equipment and special internal standards because it utilizes chemiluminescence, making it less versatile and the operation is likely to be complicated. Furthermore, the method described in Non-Patent Document 2 analyzes acetone adducts of PQQ by HPLC-UV, but is not intended for biological samples containing impurities. In fact, when biological samples containing impurities are targeted, this method has problems with sensitivity in trace analysis.

[0008] The present invention has been made in consideration of the above problems, and aims to provide an analytical method that reduces the loss of PQQ in the process of preparing a measurement sample from a target sample such as blood, and that can more accurately quantify PQQ in the measurement sample. [Means for solving the problem]

[0009] As a result of intensive investigations to solve the above-mentioned problems, the present inventors have found that the above-mentioned problems can be solved by converting pyrroloquinoline quinone using acetone to a more stable acetone-added pyrroloquinoline quinone (hereinafter also referred to as "ACTPQQ"), followed by a predetermined purification step and a quantitative analysis step, thereby completing the present invention.

[0010] That is, the present invention is as follows. [1] a reaction step of reacting pyrroloquinoline quinone or a salt thereof with acetone in a target sample to produce acetone-added pyrroloquinoline quinone or a salt thereof; a purification step of purifying the acetone-added pyrroloquinoline quinone or a salt thereof with a solid-phase adsorbent to obtain a measurement sample; Based on a chromatogram of the acetone-added pyrroloquinoline quinone and its salt contained in the measurement sample by a chromatography method, Target sample and a quantitative analysis step of quantifying the amount of pyrroloquinoline quinone and its salt contained in the solution using a fluorescence detection device or a mass spectrometer. Analysis method. [2] The base used in the solid-phase adsorbent treatment step is a substituted pyridine. The analytical method described in [1]. [3] The substituted pyridine includes methylpyridine. 〔2〕 The analytical method described in [4] The detection device is a fluorescence detection device. The analytical method according to any one of [1] to [3]. [5] The measurement sample is derived from blood. The analytical method according to any one of [1] to [4]. [Effects of the Invention]

[0011] According to the present invention, it is possible to provide an analytical method that can reduce the loss of PQQ in the process of preparing a measurement sample from a target sample such as blood and more accurately quantify PQQ in the measurement sample. Such an analytical method can contribute to the development of health foods, pharmaceuticals, etc. related to PQQs. [Brief explanation of the drawings]

[0012] [Figure 1] ACTPQQ aqueous solution: This is a chromatograph obtained by HPLC analysis of the measurement sample in Example 1. [Figure 2] 1 is a chromatograph obtained by HPLC analysis of a measurement sample in Comparative Example 1: PQQ aqueous solution. [Figure 3] 1 shows a chromatogram obtained by HPLC analysis of a measurement sample in Example 3 for PQQ in fetal bovine serum. [Figure 4] FIG. 1 shows a calibration curve of ACTPQQ concentration obtained in Example 3. [Figure 5] 1 shows a chromatogram of PQQ in rabbit plasma obtained by HPLC analysis of a measurement sample in Example 4. [Figure 6] FIG. 1 shows the time course of PQQ concentration in rabbit plasma in Example 4. [Figure 7] 1 shows a chromatogram of PQQ in human plasma obtained by HPLC analysis of a measurement sample in Example 5. [Figure 8] FIG. 1 shows a calibration curve of ACTPQQ concentration obtained in Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0013] The following describes in detail an embodiment of the present invention (hereinafter referred to as "the present embodiment"); however, the present invention is not limited to this embodiment, and various modifications are possible without departing from the spirit of the present invention.

[0014] 1.Analysis method The analytical method of this embodiment includes a reaction step of reacting pyrroloquinoline quinone or a salt thereof with acetone in a target sample to produce acetone-added pyrroloquinoline quinone or a salt thereof, a purification step of treating the acetone-added pyrroloquinoline quinone or a salt thereof with a solid-phase adsorbent to purify the acetone-added pyrroloquinoline quinone or a salt thereof to obtain a measurement sample, and a chromatography step of determining the acetone-added pyrroloquinoline quinone or a salt thereof based on a chromatogram of the acetone-added pyrroloquinoline quinone and a salt thereof contained in the measurement sample. Target sample and a quantitative analysis step of quantifying the amounts of pyrroloquinoline quinone and salts thereof contained in the solution using a fluorescence detection device or a mass spectrometer.

[0015] Here, the term "target sample" refers to the object to be analyzed for PQQ, such as a blood sample, food, or drink, as described below. The term "measurement sample" refers to a sample from which impurities have been removed so that the sample can be subjected to chromatography.

[0016] Generally, ultra-high sensitivity analysis of trace components in a target sample is performed by mass spectrometry such as MS. However, when attempting to analyze PQQ contained in a target sample by mass spectrometry, it has been found that the ionization of PQQ is difficult to proceed, resulting in low sensitivity for trace analysis.

[0017] In contrast, in this embodiment, the PQQ contained in the target sample is converted into an acetone adduct, and then quantified using a fluorescence detection device or a mass spectrometer, thereby improving the sensitivity of the trace analysis of PQQ.

[0018] 1.1.Reaction process The reaction step is a step of reacting pyrroloquinoline quinone or a salt thereof with acetone in a target sample to produce acetone-added pyrroloquinoline quinone or a salt thereof. The reaction method is not particularly limited, but an example includes a method in which pyrroloquinoline quinone or a salt thereof is mixed with acetone to cause an aldol reaction. The aldol reaction may be carried out in a solution, a suspension, or a gel, but is preferably carried out in water.

[0019] The reaction temperature is preferably 0 to 80°C, more preferably 15 to 50°C. A reaction temperature of 0°C or higher is industrially preferred because it increases the reaction rate. Furthermore, a reaction temperature of 80°C or lower reduces the pressure resistance required of the reaction vessel, thereby eliminating the need for an expensive, highly pressure-resistant pressurized vessel, which is industrially preferred. Furthermore, the reaction time is preferably 1 minute to 48 hours, more preferably 5 to 60 minutes.

[0020] The amount of acetone used in the reaction system is preferably 0.1 to 40 parts by mass, more preferably 0.5 to 35 parts by mass, and even more preferably 1.0 to 30 parts by mass, relative to 1 part by mass of pyrroloquinoline quinone.

[0021] The pH during the aldol reaction is preferably more than 7, more preferably 8 to 14, and even more preferably 9 to 10. In this case, a base may be used to make the system alkaline. Such a base is not particularly limited, but examples thereof include sodium hydroxide and potassium hydroxide.

[0022] Furthermore, when the reaction is stopped, the pH of the system may be neutral or acidic, preferably acidic. The acid used to make the system acidic is not particularly limited, but examples thereof include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, acetic acid, citric acid, oxalic acid, succinic acid, and tartaric acid.

[0023] 1.1.1. Measurement target The acetone-added pyrroloquinoline quinone used in the analytical method of this embodiment is represented by the following formula (1). One to three of the three carboxyl groups possessed by the acetone-added pyrroloquinoline quinone may form a salt with an alkali metal salt or the like. The alkali metal is not particularly limited, but examples thereof include sodium and potassium. [ka]

[0024] Next, pyrroloquinoline quinones to be analyzed in the analytical method of this embodiment are shown below. PQQ includes oxidized PQQ represented by the following formula (2) and reduced PQQ represented by the following formula (3). Depending on the environment, the PQQ contained in the target sample can vary from a state in which oxidized PQQ is relatively abundant to a state in which reduced PQQ is relatively abundant. For example, when the target sample is a solution, reduction tends to proceed easily in the solution, so reduced PQQ tends to be present in a relatively large amount. On the other hand, when the target sample is exposed to an oxidizing environment, oxidized PQQ tends to be present in a relatively large amount. [ka]

[0025] In the reaction step of the analytical method of this embodiment, acetone-added pyrroloquinoline quinone or a salt thereof is produced from both oxidized PQQ and reduced PQQ, and therefore, the analytical method of this embodiment allows the total amount of oxidized PQQ and reduced PQQ to be quantified.

[0026] The salt of pyrroloquinoline quinone to be analyzed is not particularly limited, and examples thereof include salts with metals such as alkali metals and alkaline earth metals; and salts with non-metals such as ammonium cations. In particular, disodium salt, which is one of the alkali metal salts, is widely used in foods and is therefore important as an analytical target.

[0027] 1.1.2.Target Sample The target sample is not particularly limited as long as it contains pyrroloquinoline quinone, and examples thereof include samples that particularly require trace analysis, such as blood samples, tissue samples, and natural material samples; oral medications and supplements such as capsules, tablets, powders, and granules; foods and beverages such as drinks, jellies, gummies, retort foods, and other foods; and topical agents such as cosmetics, cleansers, and other topical agents.

[0028] Among these, the target sample is preferably derived from blood, and examples of such target samples include, but are not limited to, whole blood, plasma, or serum, or target samples derived from these. Among these, plasma or serum, or target samples derived from these, are preferred. For example, when taking or using medicines, supplements, food and beverages, topical preparations, etc. containing PQQ as described above, analyzing the amount of PQQ in the blood can further advance research into the uptake efficiency of PQQ and the blood concentration at which PQQ effectively exerts its functions. Therefore, targeting such samples can contribute to the development of health foods, pharmaceuticals, etc. related to PQQs. Furthermore, since blood-derived target samples generally contain many impurities and may contain a low amount of PQQ, the microanalysis of this embodiment is more effective.

[0029] The content of pyrroloquinoline quinone or a salt thereof in a target sample is preferably 0.0005 to 100 nM, more preferably 0.005 to 50 nM, and even more preferably 0.05 to 10 nM. According to the analysis method of this embodiment, even a target sample containing such a relatively small amount of pyrroloquinoline quinone or a salt thereof can be easily quantitatively analyzed.

[0030] 1.2. Purification process The purification step is a step of purifying the acetone-added pyrroloquinoline quinone or its salt with a solid-phase adsorbent to obtain a measurement sample. Examples of purification treatment using a solid-phase adsorbent include a method of passing a target sample through the solid-phase adsorbent to adsorb impurities onto the solid-phase adsorbent and obtain the acetone-added pyrroloquinoline quinone or its salt to be measured, and a method of passing a target sample through the solid-phase adsorbent to adsorb the acetone-added pyrroloquinoline quinone or its salt to be measured onto the solid-phase adsorbent, separating it from unadsorbed impurities, and then desorbing and eluting the acetone-added pyrroloquinoline quinone or its salt from the solid-phase adsorbent.

[0031] Among these, a preferred method is to pass a target sample through a solid-phase adsorbent, adsorb the acetone-added pyrroloquinoline quinone or its salt to be measured on the solid-phase adsorbent, separate it from non-adsorbed impurities, and then desorb and elute the acetone-added pyrroloquinoline quinone or its salt from the solid-phase adsorbent. This tends to reduce the loss of PQQ in the process of preparing a measurement sample from a target sample such as blood.

[0032] The type of solid-phase adsorbent and the composition of the washing and elution solvents can be selected appropriately depending on various conditions, such as the type of target sample. The solid-phase adsorbent is not particularly limited, but examples include a polymer bound to a hydrophobic functional group, a column packed with octadecylsilyl (ODS), or a cation exchange column packed with a cation exchanger. Among these, an ODS column is preferred.

[0033] In the following, an example will be given in which the acetone-added pyrroloquinoline quinone or a salt thereof to be measured is adsorbed onto a solid-phase adsorbent, but the analysis method of this embodiment is not limited to this.

[0034] 1.2.1. Conditioning of solid-phase adsorbents Before passing acetone-added pyrroloquinoline quinone or a salt thereof through the solid-phase adsorbent, the solid-phase adsorbent may be conditioned by passing a predetermined solution through it.

[0035] Such a conditioning solution is not particularly limited, but examples thereof include lower alcohols such as methanol, water, aqueous acid solutions, etc. The conditioning solutions may be used in combination or separately.

[0036] When using the conditioning solution separately, can pass through lower alcohol first, then through water, and finally through aqueous acid solution, or can pass through any other order.Among these, it is preferred to pass through lower alcohol first, then through water, and finally through aqueous acid solution.This tends to reduce the loss of PQQ in the process of preparing measurement sample from target sample such as blood.

[0037] The acid contained in the acid aqueous solution is not particularly limited, but examples thereof include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, acetic acid, citric acid, oxalic acid, succinic acid, tartaric acid, etc. The pH of the acid aqueous solution is preferably 1 to 6, more preferably 1 to 5, and even more preferably 1 to 4.

[0038] 1.2.2. Adsorption onto solid-phase adsorbents By passing a target sample containing acetone-added pyrroloquinoline quinone or a salt thereof through the solid-phase adsorbent, the acetone-added pyrroloquinoline quinone or a salt thereof contained in the target sample can be adsorbed onto the solid-phase adsorbent.

[0039] In this case, the target sample is preferably acidic, and the pH is preferably 1 to 6, more preferably 1 to 5, and even more preferably 1 to 4. When the reaction of such an acidic target sample is stopped using an acid in the above reaction step, the reaction solution may be used as is.

[0040] Methods for passing the target sample through the solid-phase adsorbent include the pressurization method, in which pressure is applied from the inlet side of the solid-phase adsorbent, the vacuum method, in which vacuum and suction are applied from the outlet side of the solid-phase adsorbent, and the centrifugation method, in which centrifugal force can be applied.

[0041] A washing solution may be passed through the solid-phase adsorbent after the sample has been passed through to wash away impurities. Examples of the washing solution include water and aqueous acid solutions. Examples of aqueous acid solutions include those exemplified in the conditioning section.

[0042] 1.2.3. Elution from solid-phase adsorbents By passing the eluate through the solid-phase adsorbent, the acetone-added pyrroloquinoline quinone or a salt thereof can be desorbed and eluted from the solid-phase adsorbent, thereby obtaining a measurement sample. Note that the measurement sample thus obtained contains acetone-added pyrroloquinoline quinone or a salt thereof, with impurities separated from it.

[0043] Such an eluent is not particularly limited, but examples thereof include an aqueous base solution, a lower alcohol such as methanol, acetonitrile, water, etc. The eluents may be used in combination or separately.

[0044] When using separate eluents, the aqueous base solution may be passed through first, and then water or lower alcohol may be passed through, or any other washing order may be used.Among these, it is preferred to pass the aqueous base solution first, and then water or lower alcohol may be passed through.This tends to reduce the loss of PQQ in the process of preparing a measurement sample from a target sample such as blood.

[0045] The base contained in the aqueous base solution is not particularly limited, but for example, organic bases such as triethylamine, pyridine, substituted pyridine, etc.; inorganic bases such as sodium hydroxide, potassium hydroxide, etc. are included.Among these, organic bases are preferred, heterocyclic aromatic compounds such as pyridine, substituted pyridine, etc. are preferred, and substituted pyridine is more preferred.By using such bases, the loss of PQQ tends to be reduced during the process of preparing a measurement sample from a target sample such as blood.

[0046] Furthermore, since the base used in the eluate is contained in the measurement sample, it is preferable that the base have little effect on the quantitative analysis step described below. From this perspective, substituted pyridines are preferable to pyridine as bases that have little effect on the quantitative analysis step of acetone-added pyrroloquinoline quinone or a salt thereof. The reason for this is not particularly limited, but it is thought that the use of substituted pyridines allows the quantitative analysis step of acetone-added pyrroloquinoline quinone or a salt thereof to be carried out without being affected by such impurities.

[0047] The substituted pyridine is not particularly limited, but examples thereof include methylpyridine, ethylpyridine, butylpyridine, dimethylpyridine, etc. Among these, methylpyridine is preferred.

[0048] The content of the base in the eluate is preferably 0.1 to 15% by mass, more preferably 2.0 to 10% by mass, based on the total amount of the eluate. When the content of the base is within the above range, loss of PQQ tends to be reduced during the process of preparing a measurement sample from a target sample such as blood.

[0049] 1.2.4. Dilution or concentration The measurement sample obtained as described above may be diluted or concentrated to a concentration suitable for use in the quantitative analysis step described below. Dilution may be performed using the eluent described above, such as an aqueous base solution, a lower alcohol such as methanol, acetonitrile, or water. Concentration may be performed by evaporating the solution under reduced pressure or by spraying a gas onto the sample to evaporate the solution and concentrate the sample.

[0050] In addition to the above method, all solvents may be removed from the measurement sample obtained as described above, and then the sample may be redissolved in an arbitrary solvent to readjust the measurement sample with the arbitrary solvent and concentration, and then the sample may be subjected to the quantitative analysis step described below.

[0051] The measurement sample obtained as described above contains acetone-added pyrroloquinoline quinone or a salt thereof obtained by reacting pyrroloquinoline quinone or a salt thereof with acetone, and other impurities.

[0052] 1.3.Quantitative analysis process The quantitative analysis step is carried out based on a chromatogram of acetone-added pyrroloquinoline quinone and its salts contained in the measurement sample by a chromatography method, Target sample The amount of pyrroloquinoline quinone and its salts contained in the solution is determined using a fluorescence detection device or a mass spectrometer.

[0053] Here, the term "chromatogram" refers to a chart obtained by plotting the signal of each component obtained at each elution time as signal intensity (vertical axis) per time (horizontal axis).

[0054] The quantitative analysis method for acetone-added pyrroloquinoline quinone and its salts contained in a measurement sample by chromatography can be performed by conventional methods, such as the standard addition method, the internal standard method, and the absolute calibration curve method. The quantitative method can be adopted taking into consideration the required accuracy and correction. Among these, the standard addition method is preferred from the viewpoint of minimizing the influence of interference and increasing analytical accuracy.

[0055] The detector used in this embodiment is a fluorescence detector or a mass spectrometer. Among these, a fluorescence detector is preferred. Use of such a detector tends to further improve the detection accuracy of acetone-added pyrroloquinoline quinone and its salts in the measurement sample. Furthermore, since a fluorescence detector does not need to ionize acetone-added pyrroloquinoline quinone in the measurement sample, the detection accuracy tends to further improve. In particular, a method using a fluorescence detector is suitable when a substituted pyridine is used as the eluent.

[0056] When using a fluorescence detection device, excitation wavelengths and fluorescence wavelengths that can detect acetone-added pyrroloquinoline quinone and its salts should be used. Specifically, the excitation wavelength should be set to 300 to 400 nm, and the fluorescence wavelength should be set to 420 to 550 nm.

[0057] The chromatography method that can be used in this embodiment may be selected taking into consideration the required accuracy and correction, and examples thereof include liquid chromatography (LC) such as high-performance liquid chromatography (HPLC). The combination of column and eluent used in this method is not particularly limited. The HPLC device includes a separation column, a pump that delivers the separation solution to the separation column, and a detector. The HPLC device may also include other elements, such as an autosampler and a heater.

[0058] A reversed-phase column can be used as the separation column. Examples of reversed-phase columns include columns packed with octadecylsilylated silica gel packing material (ODS column, C8 column, C2 column), and columns in which these are combined with an ion exchange resin, with ODS columns being particularly preferred. In particular, when performing analysis by HPLC, it is preferable to use a column packed with octadecylsilylated silica gel packing material with a particle size of 5.0 μm or less (ODS column), and more preferably an ODS column with a particle size of 1.7 to 5.0 μm.

[0059] The eluent is not particularly limited, but examples thereof include phosphate buffer, acetate buffer, formate buffer, carbonate buffer, and a mixed buffer thereof. Furthermore, an organic solvent may be added as needed. Examples of such organic solvents include, but are not particularly limited to, acetonitrile and methanol. Furthermore, an ion pair method may be used in which an ion pair reagent is added to the eluent in order to increase the degree of separation. Examples of the ion pair reagent include, but are not particularly limited to, ammonium salts, sulfonic acid compounds, and the like.

[0060] The elution method is not particularly limited, and examples thereof include isocratic elution, in which the composition of the mobile phase (eluent) is not changed during elution, and gradient elution, in which the composition of the mobile phase (eluent) is changed during elution. The elution method can be appropriately selected depending on the separation ability.

[0061] The analytical method of this embodiment has the following advantages and features in PQQ quantitative analysis. First, derivatization into an acetone adduct stabilizes PQQ, increasing the recovery rate in the purification process. Second, derivatization into an acetone adduct improves the sensitivity of quantitative analysis. Furthermore, using a specified eluent can prevent substances that may be confused with PQQ from being mixed into the measurement sample. [Example]

[0062] The present invention will be described in more detail below using examples and comparative examples, but the present invention is not limited to the following examples.

[0063] The preparation method for each sample used in the analysis is described below. Unless otherwise specified, the reagents used in the examples were Wako special grade reagents. BioPQQ manufactured by Mitsubishi Gas Chemical Company was used for the pyrroloquinoline quinone dinatrium used in the experiments. Sep-Pac C18 manufactured by Waters was used as the solid-phase adsorbent.

[0064] [HPLC analysis conditions] Liquid delivery unit: (Shimadzu Corporation) Column: Inert Sustein C18 (GL Sciences, length 150mm, inner diameter 4.6mm) Detector: Fluorescence detector, excitation 355nm, emission 460nm HPLC eluent: 0.4% phosphoric acid / 27% methanol in water Column temperature: 30℃ Eluent flow rate: 1mL / min, Introduced amount: 20μL Analysis time: 30min

[0065] 250mM carbonate buffer: 15.95 g of Na2CO3 and 8.4 g of NaHCO3 were dissolved in distilled water and the total volume was adjusted to 1 L.

[0066] Example 1: Analysis of acetone adduct PQQ standard Acetone adduct PQQ standard (ACTPQQ) was added to pure water to a concentration of 500 μg / L to prepare a target sample.

[0067] A solid-phase adsorbent (C18 column, 500 mg, 3 mL) was placed in a vacuum manifold and used under reduced pressure to achieve a pressure difference of 3-5 inHg with the atmosphere. Specifically, the solid-phase adsorbent was conditioned by passing 4 mL of methanol, 4 mL of water, and 2 mL of 0.1 M hydrochloric acid through it in that order. Next, 300 μL of the target sample prepared as described above was passed through it to adsorb ACTPQQ onto the solid-phase adsorbent, and 2 mL of water was passed through it to wash it. After that, 600 μL of a 5% aqueous solution of methylpyridine was passed through the solid-phase adsorbent to elute ACTPQQ from the solid-phase adsorbent, and 600 μL of methanol was passed through it twice to obtain a measurement sample.

[0068] [Comparative example 1: PQQ standard analysis] A PQQ standard was added to pure water to give a concentration of 500 μg / L to prepare a target sample, and the same procedure as in Example 1 was then carried out to obtain a measurement sample.

[0069] The measurement samples obtained in Example 1 and Comparative Example 1 were further diluted 10-fold with pure water and subjected to HPLC analysis. 0.4% phosphoric acid / 35% aqueous methanol was used as the HPLC eluent. The results of the HPLC analysis are shown in Table 1. The chromatogram of Example 1 is shown in FIG. 1, and the chromatogram of Comparative Example 1 is shown in FIG. 2.

[0070] [Table 1]

[0071] As shown in Table 1, when fluorescent detection was performed in HPLC analysis, it was confirmed that ACTPQQ had a higher peak area than PQQ. This indicates that analytical sensitivity can be improved by using ACTPQQ. Furthermore, it was found that the recovery rate after solid-phase extraction was high for ACTPQQ, with little loss.

[0072] Comparative Example 2: Pyridine used without derivatization A measurement sample was obtained by the same procedure as in Comparative Example 1, except that methylpyridine was replaced with pyridine and PQQ was eluted from the solid-phase adsorbent. The obtained measurement sample was further diluted 10 times with pure water and subjected to HPLC analysis in the same manner as in Comparative Example 1. As a result, it was found that another peak was present at the elution position of PQQ, which interfered with the detection of PQQ. Furthermore, the presence of this other peak increased the peak area, making it impossible to obtain the correct peak area, and the apparent recovery rate changed significantly to 120%.

[0073] Comparative Example 3: No derivatization, elution with pyridine solution A target sample was prepared by adding PQQ to 0.1 M hydrochloric acid to a concentration of 0.5 mg / L. A solid-phase adsorbent (C18 column, 2 g, 12 mL) was then placed on a vacuum manifold and depressurized to a pressure difference of 3-5 inHg with respect to the atmosphere. Specifically, the solid-phase adsorbent was conditioned by passing 4 mL of methanol, 4 mL of water, and 2 mL of 0.1 M hydrochloric acid through the adsorbent in that order. Next, 10 mL of the target sample prepared as described above was passed through the adsorbent to adsorb PQQ onto the solid-phase adsorbent, followed by washing with 20 mL of 1 mM hydrochloric acid. Subsequently, 3 mL of 5% pyridine was passed through the adsorbent to elute PQQ from the solid-phase adsorbent, yielding a measurement sample. HPLC analysis revealed no detectable PQQ, with a recovery rate of 0%.

[0074] Example 2: Analysis of PQQ acetone reaction 250 μL of PQQ aqueous solution, 130 μL of 250 mM carbonate buffer, and 20 μL of acetone were placed in a 2 mL test tube and heated at 50° C. for 30 minutes, after which 100 μL of 1 M hydrochloric acid was added to stop the reaction.

[0075] A solid-phase adsorbent (C18 column, 500 mg, 3 mL) was placed in a vacuum manifold and used under reduced pressure to achieve a pressure difference of 3-5 inHg with the atmosphere. Specifically, the solid-phase adsorbent was conditioned by passing 4 mL of methanol, 4 mL of water, and 2 mL of 1 M hydrochloric acid through it in that order. Next, 400 μL of the target sample prepared as described above was passed through it to adsorb ACTPQQ onto the solid-phase adsorbent, and 2 mL of water was passed through it to wash it. After that, 400 μL of a 5% aqueous solution of methylpyridine was passed through the solid-phase adsorbent to elute PQQ from the solid-phase adsorbent, and 400 μL of methanol was passed through it twice to obtain a measurement sample.

[0076] The measurement samples obtained in Example 2 were diluted 10-fold with pure water and subjected to HPLC analysis. The results are summarized for each PQQ concentration in the initial PQQ aqueous solution in Table 2. As shown in Table 2, the average recovery rate by column purification was 70%. The concentration dependence was linear with R2 = 0.9992.

[0077] [Table 2]

[0078] Example 3: Microanalysis of PQQ in fetal bovine serum A target sample was obtained by adding a predetermined amount of PQQ to fetal bovine serum (FBS). 250 μL of the target sample, 130 μL of 250 mM carbonate buffer, and 20 μL of acetone were placed in a 2 mL test tube and heated at 50°C for 30 minutes. Then, 100 μL of 1 M hydrochloric acid was added to stop the reaction.

[0079] Measurement samples were then obtained by the same procedure as in Example 2. The measurement samples obtained in Example 3 were then diluted 10-fold with pure water and subjected to HPLC analysis. The results are summarized for each PQQ concentration in the initial PQQ aqueous solution and are shown in Table 3. A chromatogram at a concentration of 121 nM is also shown in FIG. 3. The lower limit of detection was 0.6 nM. It was found that the method of this embodiment enables highly sensitive analysis. The following experiment was performed using the calibration curve obtained in Example 3 (FIG. 4).

[0080] [Table 3]

[0081] Example 4: Administration to rabbits Capsules of BioPQQ (manufactured by Mitsubishi Gas Chemical Co., Inc.) were administered to rabbits. Blood was collected from the ears over time from the start of administration to prepare plasma. The plasma sample thus obtained was used as the control sample for an acetone addition reaction, followed by solid-phase adsorption treatment and detection by HPLC in the same manner as in Example 2. The ACTPQQ concentration was calculated according to the regression equation of the calibration curve in Figure 4 of Example 3: y = 9897.4 + 195531. This demonstrates that the method of this embodiment in rabbits can be used to analyze blood kinetics.

[0082] The results of the HPLC analysis of Example 4 are shown in Table 4 and Figure 6. Figure 5 shows the chromatogram of the sample after 3 hours.

[0083] [Table 4]

[0084] Example 5: Analysis of PQQ in human plasma A target sample was obtained by adding a predetermined amount of PQQ to normal human plasma (Cosmo Bio Co., Ltd.). 250 μL of the target sample, 130 μL of 250 mM carbonate buffer, and 20 μL of acetone were placed in a 2 mL test tube and heated at 50°C for 30 minutes. To stop the reaction, 100 μL of 1 M hydrochloric acid was added.

[0085] Measurement samples were then obtained by the same procedure as in Example 2. The measurement samples obtained in Example 5 were then diluted 10-fold with pure water and subjected to HPLC analysis. The results are summarized for each PQQ concentration in the initial PQQ aqueous solution and are shown in Table 5 and FIG. 8. A chromatogram at a concentration of 100 nM is also shown in FIG. 7. As shown in Example 5, this embodiment demonstrates that human plasma can also be used as a measurement target.

[0086] [Table 5]

[0087] [Reference example] A sample of the acetone adduct of PQQ was diluted to a concentration of 0.2 nM. 20 μL of this sample was analyzed under the same conditions as the HPLC analysis conditions described above, except that a UV detector with a UV detection wavelength of 259 nm was used instead of a fluorescence detector. However, no chromatographic peaks were detected with the UV detector. Analysis was performed under the same conditions, except that a fluorescence analyzer was used instead, and a chromatographic peak was detected. The peak area calculated from the chromatogram was 8.13 × 10 4 In this way, even low-concentration samples that are difficult to detect with a UV detector can be detected using a fluorescence detection device. [Industrial Applicability]

[0088] The analytical method of the present invention has industrial applicability as a method for quantitatively measuring PQQ contained in foods and the like.

Claims

1. a reaction step of reacting pyrroloquinoline quinone or a salt thereof with acetone in a target sample to produce acetone-added pyrroloquinoline quinone or a salt thereof; a purification step of purifying the acetone-added pyrroloquinoline quinone or a salt thereof with a solid-phase adsorbent to obtain a measurement sample; and a quantitative analysis step of quantifying the amounts of the pyrroloquinoline quinone and its salt contained in the target sample using a fluorescence detection device or a mass spectrometer based on a chromatogram of the acetone-added pyrroloquinoline quinone and its salt contained in the measurement sample obtained by a chromatography method. Analysis method.

2. The base used in the solid-phase adsorbent treatment step is a substituted pyridine. The analytical method according to claim 1 .

3. The substituted pyridine includes methylpyridine. The analytical method according to claim 2.

4. The detection device is a fluorescence detection device. The analytical method according to any one of claims 1 to 3.

5. The measurement sample is derived from blood. The analytical method according to any one of claims 1 to 4.

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