Method for measuring k value

Abstract

The present invention provides a method for measuring the K value of fresh food, the method comprising: (a) a step for loading, onto a carrier having zirconium dioxide, an acidic liquid specimen that is prepared from fresh food and that contains ATP-related compounds, and allowing phosphate group-containing compounds in the liquid specimen to adsorb onto the carrier; (b) a step for measuring, under acidic conditions, the absorbance of a liquid phase that is collected after the step (a) at any wavelength within the range of 240 nm to 260 nm; and (c) a step for calculating the ratio of the absorbance of the liquid phase measured in the step (b) to the absorbance of the original liquid specimen at any wavelength within the range. The present invention also provides a kit and a liquid chromatograph for measuring the K value, each suitable for use in the method.

Description

How to measure K value

[0001] The present invention relates to a method for measuring a K value, a method for evaluating the freshness of fresh food, and a kit and device that can be used for these methods.

[0002] The K value is used as a scientific index indicating the freshness of fresh seafood. High-performance liquid chromatography (Japan Agricultural Standards (JAS) 0023:2022) is a known method for measuring the K value. In this method, a diluted solution of perchloric acid is added to a sample requiring freshness testing to inactivate endogenous enzymes that decompose ATP-related substances and extract the ATP-related substances, which are then neutralized and measured by high-performance liquid chromatography (HPLC). While this method can measure the K value with high accuracy, it requires an HPLC analyzer and takes approximately one hour to complete the measurement.

[0003] On the other hand, Patent Document 1 discloses a method for determining the freshness of meat, which is characterized by comparative observation of the size and density of spots of nucleic acid-related compounds after filter paper electrophoresis, and determining that the meat is fresh when the size and density of the spot remaining at the origin are relatively smaller than those of the spots that have moved. Although this method is relatively simple in that it determines freshness without separating individual ATP-related substances, it requires an electrophoresis device and an image analyzer.

[0004] Furthermore, Non-Patent Document 1 discloses a method in which ATP-related substances are roughly divided into two fractions using an ion exchange column, and the K value is calculated using the absorbance at 250 nm of both fractions. Although this method does not require the separation of individual ATP-related substances, it requires the use of solvents with different compositions for adsorption of ATP-related substances and elution of each fraction, making the operation complicated.

[0005] Patent No. 4291381

[0006] Hitoshi Uchiyama et al., Bulletin of the Japanese Society of Scientific Fisheries 50(2), 263-267 (1984)

[0007] An object of the present invention is to provide a means for enabling simple and rapid measurement of K value.

[0008] The present inventors have discovered that a carrier containing zirconium dioxide exhibits high adsorption capacity for ATP, ADP, AMP, and inosinic acid (IMP) under acidic conditions, but exhibits little or no adsorption capacity for inosine (HxR) and hypoxanthine (Hx). Based on the properties of this carrier, the inventors have found that the K value can be derived from the ratio of the absorbance at a specific wavelength of an acidic sample solution containing an ATP-related substance to the absorbance of the liquid phase after solid-phase extraction of the acidic sample solution using a carrier containing zirconium dioxide.

[0009] The present disclosure provides the following: Item 1. A method for measuring the K value of fresh food, comprising the steps of: (a) loading an acidic sample solution containing ATP-related substances prepared from fresh food onto a carrier having zirconium dioxide to adsorb phosphate group-containing compounds in the sample solution onto the carrier; (b) measuring the absorbance of the liquid phase recovered after step (a) at any wavelength within a range of 240 nm to 260 nm under acidic conditions; and (c) calculating the ratio of the absorbance of the liquid phase measured in step (b) to the absorbance of the original sample solution at any wavelength within said range. Item 2. The method of Item 1, wherein the pH of the sample solution is 3.5 or less. Item 3. The method of Item 1 or 2, wherein the absorbance is measured at a pH of 3.5 or less. Item 4. The method of any one of Items 1 to 3, wherein the sample solution is prepared from fresh food by extraction with an aqueous solution containing 0.1 to 10% by volume of acid. Item 5. Item 6. The method according to any one of Items 1 to 4, wherein the sample liquid is prepared from fresh food by extraction with an aqueous solution containing 0.1 to 10% by volume of perchloric acid. Item 7. The method according to any one of Items 1 to 5, wherein the sample liquid is pretreated with a hydrophobic carrier. Item 8. The kit according to Item 7, wherein the carrier is packed in a spin column or a syringe column. Item 9. A liquid chromatograph for measuring K values, comprising a carrier having zirconium dioxide and an absorbance measuring device. Item 10. The liquid chromatograph according to Item 9, comprising a column packed with a carrier having zirconium dioxide, a liquid delivery pump, and an absorbance measuring device. Item 11. The liquid chromatograph according to Item 9 or 10, which is a microfluidic device.

[0010] According to the present invention, the K value can be measured simply and quickly without separating and quantifying individual ATP-related substances, thereby making it possible to evaluate the freshness of fresh foods such as marine products (fish, mollusks, shellfish, crustaceans, etc.) and meat.

[0011] FIG. 1 is a diagram showing the decomposition process of ATP-related substances in fresh foods such as seafood. FIG. 2 is a diagram showing an example of a measurement method in the present disclosure. FIG. 3 is a diagram showing an example of a measurement method in the present disclosure. FIG. 4 is a diagram showing an example of a liquid chromatograph in the present disclosure. FIG. 5 is a diagram showing an example of a liquid chromatograph in the present disclosure. FIG. 6 is an HPLC chromatogram (upper row) of an ATP-related substance mixture in which ATP-related substances are dissolved in a 1% aqueous perchloric acid solution, and ZrO ２ The lower part shows an HPLC chromatogram of the mixture of the flow-through liquid and the washing liquid after separation by the spin column. ２ 1 shows an HPLC chromatogram of a mixture of the eluate and the wash solution after separation using a spin column. １８ The lower part shows an HPLC chromatogram of a mixture of the flow-through liquid and the washing liquid after separation using a spin column. The upper part shows an HPLC chromatogram of a mixture of ATP-related substances in which ATP-related substances are dissolved in a 1% aqueous solution of perchloric acid, and the ZrO ２ 1 is an HPLC chromatogram (bottom) of a mixture of the flow-through liquid and washing liquid after separation using a syringe-type column. It shows the absorption spectra of each ATP-related substance at pH 1, pH 7, and pH 12. It is a diagram showing an HPLC chromatogram (left) when the K value of a fish meat extract is measured by HPLC, and the K value by the HPLC method and the K value by the measurement method in the present disclosure (right). It is a diagram showing the technique used to measure the K value of fish meat in Example 4, which is an example of the measurement method in the present disclosure.

[0012] The following description is based on representative embodiments or specific examples, and the present invention is not limited to such embodiments or specific examples. In this specification, numerical ranges expressed using "to" or "-" mean ranges that include the numerical values ​​at both ends as upper and lower limits, unless otherwise specified. The upper and lower limits of each numerical range exemplified in this specification can be arbitrarily combined. All numerical values ​​include numbers that are rounded to that number by one digit less. For example, "1.0" includes numbers that are rounded to that number by rounding, i.e., numbers greater than or equal to 0.95 and less than 1.05.

[0013] [Definition] In the present disclosure, ATP-related substances refer to adenosine triphosphate (ATP) and its decomposition products, adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosinic acid (IMP), inosine (HxR), and hypoxanthine (Hx). In the muscles of seafood and other animals, ATP is sequentially decomposed into ADP, AMP, IMP, HxR, and Hx over a period of several hours to several days after death. It is known that the decomposition of IMP to HxR is rate-limiting in this series of ATP decomposition processes (see FIG. 1).

[0014] The K value is an index of ATP decomposition that takes this into account, and is calculated by the following formula:

[0015] In the above formula, the numbers in parentheses indicate the molar concentration of each component. The numerator is the sum of the molar concentrations of HxR and Hx, which are degradation products after the rate-limiting step has been exceeded. A high K value indicates that the decomposition of ATP has progressed beyond the rate-limiting step, i.e., the freshness has decreased.

[0016] [Method for measuring K value] In one aspect, the present disclosure provides a method for measuring the K value of fresh food (hereinafter also simply referred to as the measurement method), comprising the steps of: (a) loading an acidic sample solution containing an ATP-related substance prepared from fresh food onto a carrier having zirconium dioxide, and allowing phosphate group-containing compounds in the sample solution to be adsorbed onto the carrier; (b) measuring the absorbance of the recovered liquid phase at any wavelength within a range of 240 nm to 260 nm under acidic conditions; and (c) calculating the ratio of the absorbance of the liquid phase measured in step (b) to the absorbance of the original sample solution at any wavelength within the range.

[0017] The acidic sample solution containing ATP-related substances prepared from fresh food is a preparation from fresh food containing animal muscle tissue, such as fresh seafood (fish, mollusks, shellfish, crustaceans, etc.) or fresh meat (meat from livestock, etc.), which is the test sample whose freshness is to be evaluated, for example, an extract of fresh food. The extract can be prepared by immersing the test sample in an appropriate aqueous medium. For example, the extract can be prepared by immersing a finely chopped test sample in an aqueous medium, stirring or homogenizing it, and then centrifuging to recover the supernatant, or by filtering it through a filter such as filter paper and recovering the filtrate. The extract can also be prepared by swabbing or scraping the surface of the test sample with an appropriate tool (such as a plastic or glass rod) and then immersing the tool in an appropriate aqueous medium.

[0018] As shown in Figure 1, fresh foods are preferably those in which ATP is decomposed via IMP, such as fish, mollusks (squids and octopuses), arthropods (shrimps and crabs), echinoderms (sea urchin gonads), and livestock meat (chicken, pork, beef, etc.).

[0019] The pH of the aqueous medium used to prepare the extract may be acidic, neutral, or alkaline, as long as it is capable of dissolving the ATP-related substances in the test sample, and is preferably acidic. An acidic aqueous medium denatures and insolubilizes proteins in the test sample during extraction, thereby preventing the ATP-related substances in the test sample from being unintentionally decomposed by endogenous enzymes that use the ATP-related substances as substrates. Furthermore, an alkaline aqueous medium, for example, having a pH of 7 to 10, preferably 7.5 to 10, more preferably 8 to 9, has an optimum pH of 5 to 8 for endogenous enzymes, and therefore can inhibit the activity of endogenous enzymes and suppress unintended decomposition of ATP-related substances.

[0020] The acidic aqueous medium used to prepare the extract is an aqueous solution containing an acid, and may contain, for example, at least one acid selected from the group consisting of perchloric acid, trichloroacetic acid, dichloroacetic acid, trifluoroacetic acid, hydrochloric acid, acetic acid, and formic acid, at a concentration of, for example, 0.1 to 10% by volume, preferably 0.1 to 5% by volume, more preferably 0.3 to 1% by volume, and even more preferably 1% by volume. The acid is preferably at least one selected from the group consisting of perchloric acid, trichloroacetic acid, hydrochloric acid, and acetic acid. When multiple acids are contained, the above concentration refers to the total concentration of all acids. Extraction using an aqueous perchloric acid solution can be performed as described in Japanese Agricultural Standards (JAS) 0023:2022.

[0021] The extract prepared from the test sample may be subjected to a pretreatment in which the extract is loaded onto a hydrophobic carrier (e.g., octadecylsilylated silica gel, octylsilylated silica gel, etc.) to adsorb impurity components onto the hydrophobic carrier, and the liquid that passes through the hydrophobic carrier may then be used as the sample liquid in step (a), or the liquid that passes through the hydrophobic carrier and the washing liquid for the hydrophobic carrier after adsorption may be combined and used as the sample liquid in step (a).

[0022] In step (a), a sample liquid is used whose pH is acidic, preferably pH 3.5 or less, for example, within the range of -0.5 to 3.5, -0.5 to 3, -0.5 to 2.5, -0.5 to 2, 0 to 3.5, 0 to 3, 0 to 2.5, 0 to 2, 0.5 to 3.5, 0.5 to 3, 0.5 to 2.5, or 0.5 to 2. A liquid to be used as the sample liquid, such as the above-mentioned extract, hydrophobic carrier flow-through liquid, or a mixture of the flow-through liquid and a washing liquid, may be used in step (a) after adjusting its pH to within the above range by adding an appropriate acid or alkali. When the pH of the liquid to be used as the sample liquid is acidic, it can be used as the sample liquid in step (a) without adjusting the pH.

[0023] The sample solution is preferably an aqueous solution containing at least one acid selected from the group consisting of perchloric acid, trichloroacetic acid, dichloroacetic acid, trifluoroacetic acid, hydrochloric acid, acetic acid, and formic acid, and may contain, for example, at least one acid selected from the above group at a concentration of 0.1 to 10% by volume, preferably 0.1 to 5% by volume, more preferably 0.3 to 1% by volume, and even more preferably 1% by volume. The acid is preferably at least one selected from the group consisting of perchloric acid, trichloroacetic acid, hydrochloric acid, and acetic acid. When multiple types of acids are contained, the above concentration is the total concentration of all the acids.

[0024] The zirconium dioxide-containing carrier contains zirconium dioxide at least on its surface. The zirconium dioxide-containing carrier holds zirconium dioxide on at least the surface portion of the substrate with enough strength to prevent dissociation during solid-phase extraction. The carrier may be a substrate coated with zirconium dioxide. The substrate may be any substrate commonly used as a substrate for solid-phase extraction carriers (also called solid phases, separation materials, or adsorbents), such as silica gel, polymers (styrene-divinylbenzene-based polymers, divinylbenzene-based polymers, methacrylates, etc.), magnesium silicate, alumina, graphite carbon, activated carbon, cellulose, polyamide, etc. The carrier may be, for example, silica gel coated with zirconium dioxide. The carrier may be coated with titanium dioxide in addition to zirconium dioxide. The carrier may also be composed of zirconium dioxide, for example, a porous body composed of zirconium dioxide, or may be composed of zirconium dioxide and titanium dioxide, for example, a mixture of a porous body composed of zirconium dioxide and a porous body composed of titanium dioxide.

[0025] The carrier may have any shape commonly used for solid-phase extraction, such as a particulate or monolithic shape, and may be packed into a column in the form of a spin column, a tip, a syringe, a cartridge, a luer device, a multi-well plate, or the like.

[0026] The carrier is used in step (a) after conditioning with an appropriate solvent, for example, an aqueous solution of the same composition as the acid-containing aqueous solution used in preparing the extract. Loading the sample solution onto the carrier in step (a) can be performed using a method commonly used in solid-phase extraction. For example, the sample solution is injected into one end of a column packed with the carrier, and the sample solution is passed through the carrier by centrifugation, pressurization, or natural flow, or by suction at the other end under reduced pressure, thereby allowing the phosphate group-containing compounds in the sample solution to be adsorbed onto the carrier. Alternatively, for example, by mixing a particulate carrier with the sample solution, the phosphate group-containing compounds in the sample solution can be adsorbed onto the carrier. Among the ATP-related substances in the sample solution, ATP, ADP, AMP, and IMP containing phosphate groups are adsorbed onto the carrier and removed from the sample solution, leaving behind HxR and Hx, which do not contain phosphate groups.

[0027] The liquid phase is the liquid recovered after step (a). The liquid phase includes the sample liquid after loading onto the carrier in step (a), and may further include a washing solution for the carrier after step (a). The liquid that can be used to wash the carrier (washing liquid) is, for example, an aqueous solution having the same composition as the acid-containing aqueous solution used to prepare the extract. The effluent recovered from the other end of the column loaded with the sample liquid can be used as is as the liquid phase in step (b). Alternatively, after passing the sample liquid, the column can be washed by passing a washing liquid through it, and the recovered effluent and washing liquid can be combined and used as the liquid phase in step (b). Alternatively, a mixture of the particulate carrier and the sample liquid can be allowed to stand or centrifuged, and the supernatant can be recovered and used as the liquid phase in step (b). Alternatively, a mixture of the particulate carrier and the sample liquid can be allowed to stand or centrifuged, the supernatant can be recovered, and the carrier can be washed with a washing liquid, and the recovered supernatant and washing liquid can be combined and used as the liquid phase in step (b).

[0028] In step (b), the absorbance of the liquid phase is measured under acidic conditions at any wavelength within the range of 240 nm to 260 nm, for example, at 245 nm, 250 nm, 254 nm, or 260 nm. The absorbance measurement can be performed preferably at any wavelength within the range of 240 nm to 255 nm, more preferably at any wavelength within the range of 240 nm to 250 nm. As shown in the examples below, the absorbance of each component of ATP-related substances is approximately the same at wavelengths within the above range under acidic conditions, and therefore, absorbance can be used as an indicator of concentration regardless of the type of ATP-related substance.

[0029] The absorbance of the liquid phase can be measured under acidic conditions, preferably at a pH of 3.5 or less, for example, within the range of pH −0.5 to 3.5, −0.5 to 3, −0.5 to 2.5, −0.5 to 2, 0 to 3.5, 0 to 3, 0 to 2.5, 0 to 2, 0.5 to 3.5, 0.5 to 3, 0.5 to 2.5, or 0.5 to 2. The liquid phase is basically acidic since it is derived from an acidic sample solution, and pH adjustment by addition of an acid or alkali is not essential for measuring absorbance, but the pH may be adjusted within the above range.

[0030] It is preferable that no pH adjustment is performed between steps (a) and (b). In this case, the pH of both the sample liquid in step (a) and the liquid phase in step (b) is 3.5 or less, for example, within the range of −0.5 to 3.5, −0.5 to 3, −0.5 to 2.5, −0.5 to 2, 0 to 3.5, 0 to 3, 0 to 2.5, 0 to 2, 0.5 to 3.5, 0.5 to 3, 0.5 to 2.5, or 0.5 to 2.

[0031] Both the sample liquid in step (a) and the liquid phase in step (b) are preferably aqueous solutions containing at least one acid selected from the group consisting of perchloric acid, trichloroacetic acid, dichloroacetic acid, trifluoroacetic acid, hydrochloric acid, acetic acid, and formic acid. In this case, these liquids may contain, for example, at least one acid selected from the above group at a concentration of 0.1 to 10% by volume, preferably 0.1 to 5% by volume, more preferably 0.3 to 1% by volume, and even more preferably 1% by volume.

[0032] Furthermore, the acidic aqueous medium used in preparing the extract, the liquid for washing the carrier in the pretreatment, and the liquid for washing the carrier after step (a) are preferably aqueous solutions of the same composition, i.e., aqueous solutions containing the same type of acid at the same concentration, which allows the preparation of the sample solution, the adsorption onto the carrier, the washing of the carrier, and the measurement of absorbance to be all performed using the same acidic aqueous solution.

[0033] In step (c), the ratio of the absorbance of the liquid phase measured in step (b) to the absorbance of the original sample solution is calculated. Since the absorbance of the original sample solution corresponds to the total concentration of all ATP-related substances and the absorbance of the liquid phase corresponds to the total concentration of HxR and Hx, the ratio of the absorbance of the liquid phase to the absorbance of the original sample solution corresponds to the K value. The absorbance of the original sample solution is preferably measured at the same pH as the liquid phase and at the same wavelength as the liquid phase. When the carrier flow-through liquid and the washing solution in step (a) are combined to form the liquid phase, the absorbance of the original sample solution is preferably measured after adding the same volume of an appropriate dilution liquid as the washing solution, for example, an aqueous solution having the same composition as the aqueous acid-containing solution used in preparing the extract solution, to the sample solution before being subjected to step (a) so that the dilution ratio is the same.

[0034] When the sample liquid is an extract of fresh food, the K value obtained by the above-mentioned measurement method can be used as an index of the freshness of the fresh food. Therefore, in one aspect, the present disclosure provides a method for evaluating or inspecting the freshness of fresh food (hereinafter also simply referred to as a freshness evaluation method), which includes the above-mentioned steps (a) to (c).

[0035] In certain embodiments, the measurement method and freshness evaluation method include the steps of: (a1) loading a sample liquid having a pH of 3.5 or less, which contains an extract obtained by extracting fresh food using an acidic aqueous medium, onto a carrier containing zirconium dioxide, and allowing phosphate group-containing compounds in the sample liquid to be adsorbed onto the carrier; (b1) measuring the absorbance of the liquid phase recovered after step (a1) at any wavelength within a range of 240 nm to 260 nm; and (c1) calculating the ratio of the absorbance of the liquid phase measured in step (b1) to the absorbance of the sample liquid at any wavelength within the range before being subjected to step (a1).

[0036] In another embodiment, the measurement method and freshness evaluation method include the steps of: (a2) loading a sample liquid having a pH of 3.5 or less, which contains an extract extracted from fresh food using an acidic aqueous medium, onto a carrier containing zirconium dioxide to adsorb phosphate group-containing compounds in the sample liquid onto the carrier; (b2) measuring the absorbance at a wavelength in the range of 240 nm to 260 nm for each of the liquid phase recovered after step (a2) and the sample liquid before being subjected to step (a2); and (c2) calculating the ratio of the absorbance of the liquid phase to the absorbance of the sample liquid. An example of this embodiment is shown in FIG. 2.

[0037] In another embodiment, the measurement method and freshness evaluation method include the steps of: (a3-1) loading a sample solution having a pH of 3.5 or less, containing an extract extracted from fresh food using an acidic aqueous medium, onto a carrier containing zirconium dioxide, thereby adsorbing phosphate group-containing compounds in the sample solution onto the carrier; (a3-2) loading a sample solution having a pH of 3.5 or less, containing an extract extracted from fresh food using an acidic aqueous medium, onto a hydrophobic carrier, thereby adsorbing impurity components in the sample solution onto the carrier; (b3) measuring the absorbance at a wavelength within a range of 240 nm to 260 nm for each of the liquid phases recovered after step (a3-1) and step (a3-2); and (c3) calculating the ratio of the absorbance of the liquid phase obtained from step (a3-1) to the absorbance of the liquid phase obtained from step (a3-2). An example of this embodiment is shown in FIG. 3. According to this embodiment, a fresh food extract containing precipitate can be used without requiring prior separation procedures such as centrifugation or filtration.

[0038] [Kit] In one aspect, the present disclosure provides a kit for measuring K values ​​or evaluating the freshness of fresh food, comprising a carrier having zirconium dioxide. The kit preferably comprises the carrier packed in a spin column or a syringe column. In addition to the carrier, the kit may also comprise an aqueous medium for preparing an extract, preferably an acidic aqueous medium, a carrier washing liquid, a dilution liquid, a container for liquid phase recovery, a pretreatment filter, a pretreatment column packed with a hydrophobic carrier, and the like. The kit can be used in the above-mentioned measurement method, and each of its components and its method of use are as described in the above-mentioned measurement method section.

[0039] [Liquid Chromatograph] In one aspect, the present disclosure provides a liquid chromatograph for measuring K values ​​or evaluating the freshness of fresh foods, which is equipped with a carrier containing zirconium dioxide and an absorbance measurement device (e.g., a UV detector, a photodiode array detector, etc.). The liquid chromatograph can be used in the above-mentioned measurement method, and each of its components and its method of use are as described in the above-mentioned measurement method section.

[0040] The liquid chromatograph preferably comprises a column packed with a carrier containing zirconium dioxide, a liquid delivery pump or suction device, and an absorbance measurement device. One end of the column is connected to a mobile phase reservoir via a flow path, and the other end of the column is connected to a flow cell of the absorbance measurement device via a flow path. The liquid delivery pump is connected to a position upstream of the column near the reservoir, and the suction device is connected to a position downstream of the column near the end of the flow path, and both are responsible for delivering the mobile phase. An appropriate mobile phase, for example, an aqueous solution having the same composition as the aqueous solution containing the acid used in preparing the extract, is delivered into the flow path, and the sample solution is injected from the upstream side of the column. The absorbance is measured downstream of the column, and the ratio to the absorbance of the original sample solution is calculated to measure the K value.

[0041] In one embodiment, the liquid chromatograph comprises a column packed with a carrier containing zirconium dioxide, a liquid feed pump or suction device, and two absorbance measurement devices connected upstream and downstream of the column. As shown in Figure 4, an extract injected through an inlet upstream of the column first passes through the flow cell of the first absorbance measurement device, and the absorbance at any wavelength within the range of 240 nm to 260 nm is measured. The extract then passes through the column to adsorb the phosphate group-containing compound onto the carrier, and then passes through the flow cell of the second absorbance measurement device, and the absorbance at any wavelength within the range of 240 nm to 260 nm is measured, and the K value is calculated from the ratio of the two absorbances.

[0042] In another embodiment, the liquid chromatograph may further include a column packed with a hydrophobic carrier in addition to a column packed with a carrier containing zirconium dioxide, a liquid feed pump or suction device, and two absorbance measurement devices. This liquid chromatograph is connected in the following order from upstream: a column packed with a hydrophobic carrier, a flow cell of a first absorbance measurement device, a column packed with a carrier containing zirconium dioxide, and a flow cell of a second absorbance measurement device. As shown in Figure 5, an extract injected from an inlet upstream of the column first passes through a column packed with a hydrophobic carrier to undergo pretreatment in which impurity components are adsorbed onto the hydrophobic carrier, followed by absorbance measurement with the first absorbance measurement device, adsorption of a phosphate group-containing compound, and absorbance measurement with the second absorbance measurement device, and the K value is calculated from the ratio of the two absorbances.

[0043] The liquid chromatograph in the present disclosure may be a microfluidic device. A microfluidic device is a device in which a microscale flow path is formed on a substrate such as a resin or glass substrate. By forming a flow path including a column in the microfluidic device, filling the column portion with a carrier, and connecting it to a liquid delivery pump and an absorbance measurement device, the microfluidic device can be used as an ultra-compact liquid chromatograph.

[0044] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0045] [Materials and Methods] The reagents, instruments, analytical conditions, etc. used in this example are as follows: Unless otherwise specified, % in a solution means % by volume. Reagents: Perchloric acid (60%) (special reagent grade, Fujifilm Wako Pure Chemical Industries, Ltd.), potassium dihydrogen phosphate (special reagent grade, Fujifilm Wako Pure Chemical Industries, Ltd.), dipotassium hydrogen phosphate (special reagent grade, Fujifilm Wako Pure Chemical Industries, Ltd.), ATP: adenosine 5'-triphosphate disodium hydrate (>98.0%, Tokyo Chemical Industry Co., Ltd.), ADP: adenosine 5'-diphosphate disodium (>98.0%, Tokyo Chemical Industry Co., Ltd.), AMP: adenosine 5'-monophosphate disodium (>95.0%, Tokyo Chemical Industry Co., Ltd.), IMP: inosine 5'-monophosphate disodium hydrate (>98.0%, Tokyo Chemical Industry Co., Ltd.), HxR: inosine (>98.0%, Tokyo Chemical Industry Co., Ltd.), Hx: hypoxanthine (>98.0%, Tokyo Chemical Industry Co., Ltd.) Spin column: GL Sciences Inc. MonoSpin Phospholipid S type (ZrO ２ Spin column, carrier used: silica monolith coated with zirconium dioxide and titanium dioxide) GL Sciences Inc. MonoSpin TiO S type (TiO ２ Spin column, carrier used: titanium dioxide coated silica monolith) GL Sciences Inc. MonoSpin C18 S type (C １８ Spin column: Support used: Silica monolith bonded with octadecyl groups) Syringe column: GL Sciences InertSep Phospholipid Remover (50 mg / 1 mL) (ZrO ２Called a syringe-type column, carrier used: a mixed carrier of 80-90% zirconium dioxide (zirconia) porous particles and 10-20% titanium dioxide (titania) porous particles) Biopsy trephine: KAI Medical BP series (φ2.0 x 7.0 mm) Homogenizer: Disposable homogenizer Biomasher II (Nippi Corporation) 10% perchloric acid aqueous solution, 5% perchloric acid aqueous solution, and 1% perchloric acid aqueous solution: Prepared by diluting perchloric acid (60%) with water. Centrifugal device: KUBOTA tabletop micro refrigerated centrifuge Model 3520 (used at a rotation speed of 2,000 to 3,000 rpm) TOMY Seiko small micro centrifuge Multi Spin (used at a rotation speed of 6,000 rpm) Ultraviolet-visible spectrophotometer: Shimadzu UV-1800 ultraviolet-visible spectrophotometer HPLC device: Shimadzu Prominence series liquid chromatograph (HPLC) HPLC analysis conditions: Column: YMC-Triart C18 (particle size 5 μm, pore size 12 nm, inner diameter 4.6 mm × length 150 mm or 250 mm) (YMC Co., Ltd.), mobile phase: 50 mM potassium phosphate buffer (pH 6.8), flow rate: 1.0 mL / min, column temperature: 40°C, measurement wavelength: 254 nm, analysis time: 30 minutes. Absorbance measurement: cuvette: UVette 220 nm-1,600 nm (plastic cuvette, optical path length 10 mm) (Eppendorf). ATP-related substance mixture: Standard compounds (0.10 mmol) of ATP, ADP, AMP, IMP, HxR, and Hx were weighed and dissolved in water (10 mL) to prepare 10 mM solutions. 1.0 mL of each of the six ATP-related substance solutions (10 mM) was taken and mixed, and water (4.0 mL) was added to make a total volume of 10 mL to prepare a 1.0 mM ATP-related substance mixture. The 1.0 mM ATP-related substance mixture was further diluted 10-fold with water to prepare a 0.10 mM ATP-related substance mixture.

[0046] Example 1 Adsorption Test of ATP-Related Substances The following test was carried out using a 10 μM standard sample solution (1% perchloric acid solution, pH 1) prepared by mixing a 0.10 mM ATP-related substance mixture solution, a 10% perchloric acid aqueous solution, and water in a ratio of 1:1:8.

[0047] (1) Adsorption conditioning under acidic conditions: ZrO ２ The spin column was placed in a 1.5 mL Eppendorf tube, and washed by applying 1% perchloric acid solution (0.30 mL) and centrifugation (2,000-6,000 rpm, 2 min). This procedure was repeated twice. Separation: The conditioned spin column was placed in a new 1.5 mL Eppendorf tube. 10 μM standard sample solution (0.10 mL) was applied, and centrifugation (2,000-6,000 rpm, 2 min) was performed. Furthermore, 1% perchloric acid solution (0.10 mL) was applied to the spin column, and centrifugation (2,000-6,000 rpm, 2 min) was performed. HPLC analysis: The collected solution (total 0.20 mL) in the Eppendorf tube and the 10 μM standard sample solution (0.10 mL) were mixed with an equal volume of 250 mM dipotassium hydrogen phosphate solution, and the mixture was filtered through a 0.45 μm filter. Half of the filtrate was analyzed by HPLC.

[0048] Results: The HPLC chromatogram is shown in Figure 6. Under acidic conditions, compounds with phosphate groups (ATP, ADP, AMP, IMP) among ATP-related substances are absorbed by ZrO ２ The compounds were adsorbed to the spin column and removed 100%. On the other hand, compounds without phosphate groups (HxR, Hx) were eluted 100% without being adsorbed.

[0049] (2) Adsorption conditioning under neutral conditions: ZrO ２The spin column was placed in a 1.5 mL Eppendorf tube, and water (0.30 mL) was applied and washed by centrifugation (2,000-6,000 rpm, 2 min). This procedure was repeated twice. Separation procedure: The conditioned spin column was placed in a new 1.5 mL Eppendorf tube. A neutralized sample solution (0.10 mL) prepared by adjusting a 10 μM standard sample solution to pH 7 with 1 N sodium hydroxide was applied, and the column was centrifuged (2,000-6,000 rpm, 2 min). Water (0.10 mL) was then applied to the spin column, and the column was centrifuged (2,000-6,000 rpm, 2 min). HPLC analysis: Half of the solution collected in the Eppendorf tube (total 0.20 mL) or 0.05 mL of the neutralized sample solution was analyzed by HPLC.

[0050] Results: The HPLC chromatogram is shown in Figure 7. Under neutral conditions, compounds with phosphate groups (ATP, ADP, AMP, IMP) were cleaved by ZrO ２ The compounds were adsorbed to the spin column and completely removed. On the other hand, the elution rates of HxR and Hx, which do not have a phosphate group, were 64% and 94%, respectively, and a portion of them was adsorbed to the column. ２ It was found that an acidic solution was necessary for the separation procedure using a spin column.

[0051] (3) Pre-conditioning with a hydrophobic carrier: C １８The spin column was placed in a 1.5 mL Eppendorf tube, and washed by applying 1% perchloric acid solution (0.30 mL) and centrifugation (2,000-6,000 rpm, 2 min). This procedure was repeated twice. Separation: The conditioned spin column was placed in a new 1.5 mL Eppendorf tube. 10 μM standard sample solution (0.10 mL) was applied, and centrifugation (2,000-6,000 rpm, 2 min) was performed. Furthermore, 1% perchloric acid solution (0.10 mL) was applied to the spin column, and centrifugation (2,000-6,000 rpm, 2 min) was performed. HPLC analysis: The collected solution (total 0.20 mL) in the Eppendorf tube and the 10 μM standard sample solution (0.10 mL) were mixed with an equal volume of 250 mM dipotassium hydrogen phosphate solution, and the mixture was filtered through a 0.45 μm filter. Half of the filtrate was analyzed by HPLC.

[0052] Results: The HPLC chromatogram is shown in Figure 8. １８ In the spin column, all ATP-related substances were eluted with a recovery rate of 94% to 98% without adsorbing to the column. １８ The spin column can be used as a filter to remove unwanted substances such as proteins contained in the fish meat extract.

[0053] (4) Syringe-type column use conditioning: ZrO ２The syringe column was placed in the suction device, and 1% perchloric acid solution (0.30 mL) was applied and washed by suction (-0.06 MPa, 3 min). This procedure was repeated twice. Separation: A new 10 mL tube and the conditioned syringe column were placed in the suction device. 10 μM standard sample solution (0.15 mL) was applied, and suction (-0.06 MPa, 3 min) was performed. Furthermore, 1% perchloric acid solution (0.15 mL) was applied to the syringe column, and suction (-0.06 MPa, 3 min) was performed (twice in total). HPLC analysis: The solution collected in the 10 mL tube (total 0.45 mL) and the 10 μM standard sample solution (0.10 mL) were each mixed with an equal volume of 250 mM dipotassium hydrogen phosphate solution and filtered through a 0.45 μm filter. Half of the former filtrate and one-third of the latter filtrate were analyzed by HPLC.

[0054] Results: The HPLC chromatogram is shown in Figure 9. Among the ATP-related substances, compounds with phosphate groups (ATP, ADP, AMP, IMP) were detected by ZrO ２ More than 99% of the compounds were adsorbed to the syringe column, and less than 1% of each compound was eluted. On the other hand, compounds without phosphate groups (HxR, Hx) were not adsorbed and more than 99% were eluted. (5) TiO ２ Comparison with spin column ZrO ２ Instead of a spin column, TiO ２ The same method as in (1) was used except that a spin column was used. ２ An adsorption test of ATP-related substances was carried out using a spin column. The elution rate of each ATP-related substance (the percentage of the substance not adsorbed to the column but recovered in the flow-through liquid and washing liquid) is shown in Table 1. ２ It was revealed that in the spin column, some of the ATP, ADP, AMP, and IMP, which have phosphate groups, were eluted without being adsorbed, and that HxR and Hx, which do not have phosphate groups, were also adsorbed. ２ Spin column and TiO ２Although spin columns are commonly modified with metal oxides known to have the ability to bind to phosphate groups, the adsorption of ATP-related substances differs depending on the type of metal oxide. ２ While the spin column has favorable adsorption performance for K value measurement, TiO ２ It was shown that the spin column is not suitable as an adsorption carrier for measuring the K value.

[0055] Example 2. Measurement of K Values ​​Using ATP-Related Substances (1) Absorption Spectra of Each ATP-Related Substance Figure 10 shows the absorption spectra of each ATP-related substance when dissolved at the same concentration (0.1 mM) in a 1% perchloric acid solution (pH 1), a 100 mM phosphate buffer solution (pH 7), and a 100 mM tripotassium phosphate solution (pH 12). At all pH values, ATP, ADP, and AMP exhibited spectra that were consistent with each other, and IMP and HxR also exhibited spectra that were consistent with each other. In contrast, Hx exhibited a spectrum similar to IMP and HxR in acidic conditions but different spectra in neutral and alkaline conditions.

[0056] In acidic conditions, the spectra of the six ATP-related substances intersect at roughly the same point (around 245-250 nm), and it has been shown that measuring the absorbance near this wavelength makes it possible to calculate the total concentration of ATP-related substances, even in mixtures. Even in neutral conditions, the absorbance near 250 nm is similar, so it is thought that it is possible to calculate the total concentration, although it is expected that the error will be larger than in acidic conditions. On the other hand, in alkaline conditions, the spectra of each component are different, making it thought to be difficult to calculate the total concentration from the absorbance at a single wavelength.

[0057] (2) Comparison with K value by HPLC method Six types of solutions were prepared by mixing 10 mM solutions of each ATP-related substance in the following volumes, and then adding 9.0 mL of water to adjust the total concentration of the ATP-related substances to 1 mM. K=10%: ATP:ADP:AMP:IMP:HxR:Hx=0.78mL:0.04mL:0.04mL:0.04mL:0.05mL:0.05mL K=20%: ATP:ADP:AMP:IMP:HxR:Hx=0.50mL:0.04mL:0.04mL:0.22mL:0.10mL:0.10mL K=30%: ATP:ADP:AMP:IMP:HxR:Hx=0.20mL:0.10mL:0.10mL:0.30mL:0.20mL:0.10mL K=40%: ATP:ADP:AMP:IMP:HxR:Hx = 0.00mL:0.04mL:0.04mL:0.52mL:0.20mL:0.20mL K = 50%: ATP:ADP:AMP:IMP:HxR:Hx = 0.00mL:0.00mL:0.10mL:0.40mL:0.25mL:0.25mL K = 60%: ATP:ADP:AMP:IMP:HxR:Hx = 0.00mL:0.00mL:0.10mL:0.30mL:0.20mL:0.40mL Each of the six types of solutions above was mixed with 10% perchloric acid aqueous solution and water in a ratio of 1:1:8 to prepare model samples (samples 1 to 6, all pH 6.0) with theoretical K values ​​of 10, 20, 30, 40, 50, or 60. 1. A solution containing ATP-related substances at a total concentration of 100 μM was prepared and the following tests were carried out.

[0058] Conditioning: ZrO ２The spin column was placed in a 1.5 mL Eppendorf tube, and washed by applying 1% perchloric acid aqueous solution (0.30 mL) and centrifuging (2,000-6,000 rpm, 2 min). This procedure was repeated twice. Separation procedure: The conditioned spin column was placed in a new 1.5 mL Eppendorf tube. A model sample (0.10 mL) was applied, and centrifuged (2,000-6,000 rpm, 2 min). A 1% perchloric acid aqueous solution (0.10 mL) was then applied to the spin column, and centrifuged (2,000-6,000 rpm, 2 min). The solution collected in the Eppendorf tube (0.20 mL in total) was designated Solution II. Absorbance measurement: The absorbance was measured at wavelengths ranging from 240 nm to 260 nm for Solution I (0.20 mL), prepared by diluting a model sample (0.10 mL) with 1% aqueous perchloric acid (0.10 mL), and for Solution II (0.20 mL) separated by a spin column. If the absorbance exceeded 2.0, the sample was diluted appropriately with 1% aqueous perchloric acid and the absorbance was measured. The K value (%) was calculated by multiplying the absorbance of Solution II by the absorbance of the solution (%) by 100. HPLC analysis: Equal volumes of 250 mM dipotassium hydrogen phosphate solution were mixed with each of Solutions I and II, and the mixture was filtered through a 0.45 μm filter. Half of the filtrate was analyzed by HPLC. The peak areas of each ATP-related substance in the resulting chromatogram were used to calculate the molar extinction coefficient (Lmol / L) of each ATP-related substance at pH 6.8 and 254 nm. －１ cm －１ The molar concentration ratio of each ATP-related substance was determined from the value divided by the following formula: ATP = 14,100, ADP = 14,100, AMP = 14,100, IMP = 10,800, HxR = 10,400, Hx = 10,000), and the K value was calculated from this molar concentration ratio.

[0059] Results: The K values ​​calculated from the absorbance at each wavelength and the K values ​​determined by the HPLC method are shown in Table 2. The K values ​​calculated from the absorbance at 240-250 nm had an error of within ±2.5% compared to the K values ​​determined by the HPLC method, and the K value calculated from the absorbance at 245 nm in particular agreed well with the K value determined by the HPLC method.

[0060] Example 3. Measurement of K Value Using Fish Meat Extract. Three samples (approximately 10 mg to 20 mg) were collected from fish meat (yellowtail fillets, tuna sashimi, red sea bream sashimi, and salmon fillets) cooled to 4°C using a biopsy trephine. The collected samples were placed in 1.5 mL Eppendorf tubes, and 0.50 mL of 5% perchloric acid solution cooled to 4°C was added. The samples were then homogenized for approximately 30 seconds while cooled to 4°C. The mixture was centrifuged (3,000 g, 4°C, 7 min) to precipitate insoluble matter such as proteins. The clear supernatant (0.10 mL) was removed and diluted with 0.40 mL of water to prepare analytical sample solutions (all pH 1). Next, ZrO was measured using the same method as in Example 2(2), except that the model sample was replaced with the analytical sample solution and the wavelengths for absorbance measurement were 245 nm, 254 nm, and 260 nm. ２ Spin column conditioning, separation, absorbance measurement, and HPLC analysis were performed.

[0061] Results: Figure 11 shows the HPLC chromatogram, the K value calculated from the absorbance at each wavelength, and the K value determined by the HPLC method. ２ The K values ​​calculated from the absorbance of the sample solution prepared using the spin column were not significantly different from the K values ​​obtained by HPLC at any wavelength. Among the model samples in Example 2 (2), Samples 1 to 3, which had particularly high ATP, ADP, and AMP contents, the K values ​​calculated from the absorbance measured at 250 to 260 nm tended to be smaller than the K values ​​obtained by HPLC. In contrast, this tendency was not observed in the fish meat extract of this example. From this, it is considered preferable to measure the K value of a sample with a high ATP, ADP, and AMP content, for example, very fresh fish meat, in which case the absorbance measurement should be performed in the range of 240 to 250 nm. However, when measuring the K value of an actual sample such as the fish meat used in this example, any wavelength in the range of 240 to 260 nm can be used.

[0062] Example 4: Extraction and analysis using a 1% perchloric acid solution By using a 1% perchloric acid solution as the extraction solution for ATP-related substances, all operations, including the extraction from fish meat, the removal of insoluble matter in the extract using a filter column, the conditioning and separation using the spin column, and the measurement of absorbance of the recovered solution, can be performed using only a 1% perchloric acid solution. The following describes the method used to measure the K value of fish meat using a 1% perchloric acid solution. All operations were performed at room temperature, and a small tabletop microcentrifuge was used for centrifugation.

[0063] Preparation of analytical sample solution: Extraction of ATP-related substances. Samples (approximately 20 mg to 40 mg) were collected from fish meat (yellowtail and salmon, two specimens each) using a biopsy trephine. The collected samples were placed in 5 mL Eppendorf tubes, and 1% aqueous perchloric acid solution (2.5 mL) was added. The samples were then homogenized for approximately 30 seconds using a homogenizer. The samples were centrifuged (6,000 rpm, 2 min) to precipitate insoluble matter such as proteins, and the supernatant was used as the analytical sample solution (both pH 1).

[0064] Filter column treatment: Removal of insoluble materials from the extraction solution A filter column (0.45 μm) was placed in a 1.5 mL Eppendorf tube. 0.10 mL of the analytical sample solution was applied, followed by centrifugation (6,000 rpm, 2 min). 0.10 mL of 1% perchloric acid solution was then applied to the filter column, followed by centrifugation (6,000 rpm, 2 min). The solution collected in the Eppendorf tube (0.20 mL in total) was designated Solution I.

[0065] Spin column separation: Separation of ATP-related substances Conditioning: ZrO ２The spin column was placed in a 1.5 mL Eppendorf tube, and 1% perchloric acid solution (0.30 mL) was applied and centrifuged (6,000 rpm, 2 min) to wash. This procedure was repeated twice. Separation: The conditioned spin column was placed in a new 1.5 mL Eppendorf tube. 0.10 mL of the analytical sample solution was applied, and centrifuged (6,000 rpm, 2 min). 0.10 mL of 1% perchloric acid solution was then applied to the spin column, and centrifuged (6,000 rpm, 2 min). The solution collected in the Eppendorf tube (0.20 mL in total) was designated Solution II.

[0066] Absorbance measurement: Calculation of K value The absorbance at 245 nm of Solution I (0.20 mL) and Solution II (0.20 mL) prepared as described above was measured. The K value was calculated by the formula: K value (%) = (absorbance of Solution II / absorbance of Solution I) x 100. Three samples were collected from the same fish meat specimen, and the K value of each sample was calculated using the procedure described above. The average of the three K values ​​obtained was used as the K value for that fish meat specimen.

[0067] Comparison of K values ​​by the spin column method and K values ​​by the HPLC method The K value measurement results by the spin column method (three values ​​and their average) and the K values ​​calculated by the HPLC method are shown in Table 3. It was shown that the K values ​​calculated by this spin column method were in good agreement with the K values ​​calculated by the conventional HPLC method, for samples ranging from those with low K values ​​and high freshness to those with high K values ​​and reduced freshness.

[0068] Example 5. Adsorption test of ATP-related substances using various acidic solutions. Following the procedure described in Example 1, ATP-related substance mixtures (10 μM) were prepared using 1% aqueous perchloric acid, 1% aqueous trichloroacetic acid, 1% aqueous hydrochloric acid, and 1% aqueous acetic acid. The ATP-related substance mixtures from each acidic solution were subjected to spin column separation according to the method described in Example 1, and the eluted recovered solution was analyzed by HPLC. The elution rates of ATP-related substances in the recovered solution eluted using each acidic solution are shown in Table 4.

[0069] As already shown in Figure 6, when a 1% aqueous solution of perchloric acid was used, compounds having a phosphate group among ATP-related substances (ATP, ADP, AMP, IMP) were dissolved in ZrO ２ The compounds were adsorbed to the carrier spin column and were removed almost 100%. On the other hand, compounds without phosphate groups (HxR, Hx) were not adsorbed and were eluted almost 100%. Similarly, when 1% trichloroacetic acid aqueous solution, 1% hydrochloric acid aqueous solution, and 1% acetic acid aqueous solution were used, compounds with phosphate groups (ATP, ADP, AMP, IMP) were also removed by ZrO ２ Almost 100% of the compounds without phosphate groups (HxR, Hx) were removed by the carrier spin column, and almost 100% of the compounds without phosphate groups (HxR, Hx) were eluted. From these results, it can be seen that ZrO ２ It was demonstrated that ATP-related substances can be separated using a carrier spin column.

Claims

1. A method for measuring the K value of fresh food, comprising the steps of: (a) loading an acidic sample solution containing ATP-related substances prepared from fresh food onto a carrier having zirconium dioxide, and allowing phosphate group-containing compounds in the sample solution to be adsorbed onto the carrier; (b) measuring the absorbance of the liquid phase recovered after step (a) under acidic conditions at any wavelength within the range of 240 nm to 260 nm; and (c) calculating the ratio of the absorbance of the liquid phase measured in step (b) to the absorbance of the original sample solution at any wavelength within said range.

2. The method according to claim 1, wherein the pH of the sample solution is 3.5 or less.

3. The method of claim 1, wherein the absorbance measurement is carried out at a pH of 3.5 or less.

4. The method of claim 1, wherein the sample solution is prepared from fresh food by extraction with an aqueous solution containing 0.1 to 10% by volume of acid.

5. The method of claim 1, wherein the sample solution is prepared from fresh food by extraction with an aqueous solution containing 0.1 to 10% by volume of perchloric acid.

6. The method according to claim 1, wherein the sample liquid is pretreated with a hydrophobic carrier.

7. A kit for measuring K value, comprising a carrier having zirconium dioxide.

8. The kit according to claim 7, wherein the carrier is packed in a spin column or a syringe column.

9. A liquid chromatograph for measuring K values, equipped with a carrier containing zirconium dioxide and an absorbance measuring device.

10. The liquid chromatograph according to claim 9, comprising a column packed with a carrier containing zirconium dioxide, a liquid delivery pump, and an absorbance measuring device.

11. The liquid chromatograph of claim 9, which is a microfluidic device.

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