阅读说明：本技术 马尿酸类分析用的分离管柱、马尿酸类分析用的液相色谱仪、以及马尿酸类的分析方法 (Separation column for analysis of equine uric acid, liquid chromatograph for analysis of equine uric acid, and method for analysis of equine uric acid ) 是由 山口忠行 藤村大树 大槻秀幸 于 2018-12-28 设计创作，主要内容包括：马尿酸类分析用的分离管柱(12)是填充有填充剂的分离管柱,所述填充剂中123μmol/g以上的β-环糊精化学键结于二氧化硅基材。通过使用使123μmol/g以上的β-环糊精化学键结于二氧化硅基材的物质作为分离管柱(12)的填充剂,即使不使用含有环糊精的流动相,也能够实现马尿酸、邻甲基马尿酸、间甲基马尿酸、对甲基马尿酸、苦杏仁酸分离的分离。(The separation column (12) for maleic acid analysis is a separation column packed with a filler in which beta-cyclodextrin of 123. mu. mol/g or more is chemically bonded to a silica substrate. By using a substance in which beta-cyclodextrin of 123. mu. mol/g or more is chemically bonded to a silica base as a filler for a separation column (12), separation of hippuric acid, o-methylhippuric acid, m-methylhippuric acid, p-methylhippuric acid, and mandelic acid can be achieved without using a mobile phase containing cyclodextrin.)

1. A separation column for analyzing hippuric acid is filled with a filler, wherein the filler is formed by chemically bonding beta-cyclodextrin with a silicon dioxide substrate, and the beta-cyclodextrin is more than 123 mu mol/g.

2. The separation column of claim 1, wherein the beta-cyclodextrin chemically bonded to the silica substrate is 272 μmol/g or less.

3. A liquid chromatograph for analyzing hippuric acid, comprising:

a separation column for maleic acid analysis, filled with a filler in which β -cyclodextrin of 123 μmol/g or more is chemically bonded to a silica substrate;

a mobile phase liquid feeding part for feeding the mobile phase to the separation column;

a sample injection unit configured to inject a sample into the mobile phase flowing from the mobile phase delivery unit toward the separation column; and

a detector connected downstream of the separation column for detecting components in the effluent from the separation column.

4. The liquid chromatograph of claim 3, wherein the beta-cyclodextrin chemically bonded to the silica substrate of the separation column is 272 μmol/g or less.

5. The liquid chromatograph of claim 3, wherein the mobile phase comprises aqueous phosphoric acid or formic acid, and acetonitrile.

6. The liquid chromatograph of claim 5, wherein the detector comprises a mass spectrometer.

7. A method for analyzing an equoic acid, comprising subjecting a sample containing at least two of equuric acid, o-methylhippuric acid, m-methylhippuric acid, p-methylhippuric acid and mandelic acid to chromatography using, as a separation column, a separation column for analyzing an equoic acid packed with a filler in which at least 123. mu. mol/g of beta-cyclodextrin is chemically bonded to a silica base.

8. The assay of claim 7, wherein the beta-cyclodextrin chemically bonded to the silica substrate of the separation column is 272 μmol/g or less.

9. The assay of claim 7, wherein the mobile phase used in the chromatographic assay comprises aqueous phosphoric acid or formic acid, and acetonitrile.

Technical Field

The present invention relates to a separation column for hippuric acid analysis, a liquid chromatograph for hippuric acid analysis, and a method for hippuric acid analysis.

Background

Toluene and xylene are used in large amounts as solvents for paints and adhesives, and examples of poisoning caused by work and poisoning caused by juvenile diluent play have been reported. In japan, it is regulated that special health diagnosis is required for workers who are engaged in organic solvent business by the labor health standard method, that is, metabolites in urine (toluene is hippuric acid and xylene is methylhippuric acid) which are indicators of exposure to organic solvents are measured, and these measurements of metabolites in urine are examined by the high performance liquid chromatography ultraviolet-ultraviolet (HPLC-UV) method in clinical laboratories.

According to the reports of fuji economic shares, inc, the world's clinical examination market reaches $ 704 billion (USD) (7 million 2000 billion yen) in 2020, and the average annual growth rate since 2015 has gone by 2.4%. The devices in the tri-polar market in U.S. and U.S. are in need of more or less renewal, but the examination environment in emerging countries (east Europe, Russia, China, south America, Africa) is in preparation, so that the enhancement of the examination system including the novel introduction of the devices is accelerated, and the clinical examination market is expected to be driven in the future.

In japan, large clinical laboratory companies (SRL, BML, LSI, medince, FALCO biosystems (FALCO biosystems), the institute of microorganisms in the east of the river, the institute of health science), and the like have been responsible for analysis of metabolites in urine. These clinical laboratory companies have been actively introduced with ultra high-speed Liquid Chromatography (LC) systems, and adopted novel analytical methods for the purpose of improving efficiency of analytical services and productivity. These urine metabolite measurements were examined by HPLC-UV method in clinical examination companies.

Disclosure of Invention

[ problems to be solved by the invention ]

In the examination by the HPLC-UV method, a C18 column was used as a separation column, and a reverse phase analysis was performed using a liquid containing a phosphate buffer and acetonitrile as a mobile phase. However, in such a reverse phase analysis, it is not possible to separate all of the components to be analyzed, such as hippuric acid, o-methylhippuric acid, m-methylhippuric acid, p-methylhippuric acid, and mandelic acid.

Therefore, an analysis using a liquid containing cyclodextrin (cyclodextrin) as a mobile phase was also carried out. However, cyclodextrin and a buffer are likely to precipitate as salts, which causes troubles in a separation column and a device, and also causes a problem that analysis using a mass spectrometer cannot be performed.

The present invention has been made in view of the above problems, and an object thereof is to enable separation and analysis of a hippuric acid without using a mobile phase containing cyclodextrin.

[ means for solving problems ]

The separation column for analyzing the maleic acid compound is a separation column filled with a filler, wherein the filler is formed by chemically bonding beta-cyclodextrin with a silicon dioxide substrate, and the beta-cyclodextrin is more than 123 mu mol/g. The present inventors have confirmed that by using a substance in which β -cyclodextrin of 123 μmol/g or more is chemically bonded to a silica base as a filler of a separation column, even without using a mobile phase containing cyclodextrin, separation of hippuric acid, o-methylhippuric acid, m-methylhippuric acid, p-methylhippuric acid, and mandelic acid can be performed. Therefore, when the separation column of the present invention is used, separation and analysis of the hippuric acid can be performed without using a mobile phase containing cyclodextrin.

In addition, in experiments conducted by the present inventors, it was confirmed that the separation of the equoic acids was improved as the chemical bonding amount of β -cyclodextrin to the silica substrate was increased. On the other hand, in the above experiment, it was also confirmed that the more the amount of chemical bonding of β -cyclodextrin to the silica substrate, the longer the precipitation time of each component from the separation column. In order to efficiently perform the examination of a plurality of samples, it is necessary to shorten the time required for the analysis of one sample, and the time required for the analysis of one sample is preferably within 5 minutes, more preferably within 4 minutes.

The present inventors have confirmed that by adjusting the amount of β -cyclodextrin chemically bonded to the silica substrate to 272 μmol/g or less, all of hippuric acids, i.e., hippuric acid, o-methylhippuric acid, m-methylhippuric acid, p-methylhippuric acid, and mandelic acid, which are components to be analyzed, can be separated from the separation column within 4 minutes and detected. That is, from the viewpoint of realizing high-speed analysis of the maleic acids, the separating agent for a separation column of the present invention is preferably one in which β -cyclodextrin is chemically bonded to a silica substrate at a ratio of 123 μmol/g to 272 μmol/g.

The liquid chromatograph for analyzing the maleic acid compound of the present invention comprises: the separation column described above; a mobile phase liquid feeding part for feeding the mobile phase to the separation column; a sample injection unit configured to inject a sample into the mobile phase flowing from the mobile phase delivery unit toward the separation column; and a detector connected downstream of the separation column for detecting components in the effluent from the separation column. Since a separation column packed with a filler in which β -cyclodextrin of 123 μmol/g or more is chemically bonded to a silica substrate is used, separation and analysis of the hippuric acid can be performed without using a mobile phase containing cyclodextrin.

In the method for analyzing hippuric acids of the present invention, a sample containing at least two components selected from hippuric acid, o-methylhippuric acid, m-methylhippuric acid, p-methylhippuric acid and mandelic acid is subjected to a chromatographic analysis using the liquid chromatograph.

In the present invention, as the mobile phase, a liquid containing no cyclodextrin and no buffer, for example, a liquid containing phosphoric acid water or formic acid water and acetonitrile can be used. Thus, the occurrence of a failure of the column or the apparatus due to the precipitation of the salt can be prevented.

As described above, by using a liquid containing no cyclodextrin and no buffer as the mobile phase, a mass spectrometer can be used as the detector.

[ Effect of the invention ]

The separation column of the present invention is a separation column filled with a filler in which β -cyclodextrin of 123 μmol/g or more is chemically bonded to a silica substrate, and therefore, separation and analysis of a maleic acid compound can be performed without using a mobile phase containing cyclodextrin.

The liquid chromatograph of the present invention uses a separation column packed with a filler in which β -cyclodextrin of 123 μmol/g or more is chemically bonded to a silica base material, and therefore, separation and analysis of a maleic acid compound can be performed without using a mobile phase containing cyclodextrin.

The analysis method of the present invention uses a separation column packed with a packing material in which β -cyclodextrin of 123 μmol/g or more is chemically bonded to a silica substrate to perform chromatography, and therefore, separation and analysis of a hippuric acid can be performed without using a mobile phase containing cyclodextrin.

Drawings

FIG. 1 is a schematic view showing the configuration of an embodiment of a liquid chromatograph for analyzing a maleic acid compound.

FIG. 2 is a chromatogram showing the relationship between the chemical bond amount of beta-cyclodextrin to a silica substrate and the degree of separation of maleic acids in a column packing.

Detailed Description

Hereinafter, a separation column for analyzing a hippuric acid, a liquid chromatograph, and a method for analyzing a hippuric acid according to the present invention will be described with reference to the drawings.

FIG. 1 shows an example of a flow path structure of a liquid chromatograph for analyzing a maleic acid compound.

The liquid chromatograph of the present embodiment includes: a liquid feeding pump 4, a liquid feeding pump 6, a mixer 8, a sample injection section 10, a separation column 12, an ultraviolet absorbance detector (UV)14, and a Mass Spectrometer (MS) 16. The liquid feeding pump 4 feeds phosphoric acid water or formic acid water, and the liquid feeding pump 6 feeds acetonitrile. The liquids sent by the liquid sending pump 4 and the liquid sending pump 6 are mixed in the mixer 8 and flow through the analysis channel 2 connected downstream of the mixer 8.

The analysis channel 2 is provided with a sample injection section 10, a separation column 12, a UV 14, and a MS16 in this order from the upstream side. The liquid-feeding pumps 4 and 6 constitute a mobile phase liquid-feeding unit for feeding the mobile phase to the separation column 12 via the analysis channel 2. The sample injection unit 10 is for injecting a sample into a mobile phase that is sent to the separation column 12 through a mobile phase sending unit including a liquid sending pump 4 and a liquid sending pump 6. The sample injected from the sample injection part 10 contains at least two components selected from hippuric acid, o-methylhippuric acid, m-methylhippuric acid, p-methylhippuric acid, and mandelic acid.

The separation column 12 is a separation column for a maleic acid analysis packed with a filler in which β -cyclodextrin of 123 μmol/g or more is chemically bonded to a silica substrate. All the components of hippuric acid, o-methylhippuric acid, m-methylhippuric acid, p-methylhippuric acid and mandelic acid can be separated through the separation column 12.

Downstream of the separation column 12, UV 14 and MS16 are connected as detectors for detecting components separated by the separation column 12. In the liquid chromatograph, since a liquid containing no cyclodextrin and no buffer is used as a mobile phase, no salt is precipitated in the analysis system. Therefore, analysis using the MS16 can be performed. Further, the MS16 is not an essential constituent component, and thus only the UV 14 may be provided as a detector.

Fig. 2 is a chromatogram showing the results of the verification with respect to the relationship between the chemical bond amount of β -cyclodextrin to the silica substrate in the packing of the separation column 12 of the liquid chromatograph and the degree of separation of the maleic acid compounds. In the figure, (A) shows a case where the amount of beta-cyclodextrin chemically bonded to the silica substrate is 58. mu. mol/g, (B) shows a case where the amount of beta-cyclodextrin chemically bonded to the silica substrate is 123. mu. mol/g, (C) shows a case where the amount of beta-cyclodextrin chemically bonded to the silica substrate is 207. mu. mol/g, and (D) shows a case where the amount of beta-cyclodextrin chemically bonded to the silica substrate is 272. mu. mol/g.

In the validation, a separation column with an inner diameter of 3mm and a length of 100mm was used. The silica gel of the packing packed in the separation column had a particle size of about 2.2 μm. Using a mixture of 0.1% phosphoric acid water and acetonitrile at 9: the liquid mixed at the ratio of 1 was used as a mobile phase, the flow rate was 0.8mL/min, and the temperature of the separation column 12 (the set temperature of the column oven) was 40 ℃. The individual chromatograms (A) to (D) were obtained by measuring the absorbance of light having a wavelength of 230nm by UV 14. In the peaks of each of the spectra (a) to (D), "", is creatinine (creatinine), "1" is o-methylhippuric acid, "2" is hippuric acid, "3" is m-methylhippuric acid, "4" is mandelic acid, and "5" is p-methylhippuric acid.

As shown in fig. 2, the greater the amount of chemical bonds between β -cyclodextrin and the silica matrix, the more the separation of "1" o-methylhippuric acid, "2" hippuric acid, "3" m-methylhippuric acid, "4" mandelic acid, and "5" p-methylhippuric acid increases. That is, the greater the amount of chemical bonding of β -cyclodextrin to the silica matrix, the greater the separation of the hippuric acids.

In addition, when the chemical bonding amount of beta-cyclodextrin to the silica matrix is 58 μmol/g, peak 1 is combined with peak 2, peak 3 is combined with peak 4, and o-methylhippuric acid and hippuric acid, and m-methylhippuric acid and mandelic acid are not separated. On the other hand, when the chemical bonding amount of beta-cyclodextrin to the silica matrix is 123. mu. mol/g, five peaks of 1 to 5 appear, and the maleic acids are separated. Therefore, it was found that separation and analysis of the equoic acids can be performed by adjusting the chemical bonding amount of β -cyclodextrin to the silica matrix to 123 μmol/g or more, even if cyclodextrin is not contained in the mobile phase.

As described above, the retention of the maleic acids increases as the amount of chemical bonding of β -cyclodextrin to the silica matrix increases, but the time until the measurement target component is precipitated from the separation column 12 increases due to the increase in the retention. In order to efficiently analyze the analyte, the time required until all of the components to be measured are detected is preferably within 5 minutes, more preferably within 4 minutes. As is clear from the verification data in fig. 2, when the chemical bond amount of β -cyclodextrin to the silica matrix is 272 μmol/g, p-methylhippuric acid, which is the slowest to precipitate from the separation column 12, among hippuric acids is detected within 4 minutes, and when the chemical bond amount of β -cyclodextrin to the silica matrix is 272 μmol/g or less, high-speed analysis can be realized.

In the verification in fig. 2, a mixed solution of phosphoric acid water and acetonitrile was used as the mobile phase, but the same result was obtained by using formic acid water instead of phosphoric acid water.

[ description of symbols ]

2: analytical flow path

4. 6: liquid feeding pump

8: mixing device

10: sample injection part

12: separation tubular column

14: ultraviolet absorbance detector (UV)

16: mass Spectrometer (MS).