Separating column for separating hippuric acid and mandelic acid, liquid chromatograph for separating hippuric acid and mandelic acid, and method for analyzing hippuric acid and mandelic acid

Abstract

A separating column (12) for hippuric acid analysis is filled with a filler in which 123 ?mol/g or more of ?-cyclodextrin is chemically bonded to a silica matrix. By using such a filler for the separating column (12) in which 123 ?mol/g or more of ?-cyclodextrin is chemically bonded to the silica matrix, hippuric acid, o-methyl hippuric acid, m-methyl hippuric acid, p-methyl hippuric acid, and mandelic acid can be separated without using a mobile phase containing cyclodextrin.

Claims

1. A method for analyzing hippuric acid, o-methyl hippuric acid, m-methyl hippuric acid, p-methyl hippuric acid, and mandelic acid, comprising the step of: subjecting a sample containing hippuric acid, o-methyl hippuric acid, m-methyl hippuric acid, p-methyl hippuric acid, and mandelic acid to chromatographic analysis using a separating column filled with a filler in which 272 ?mol/g of ?-cyclodextrin is chemically bonded to a silica matrix, and separating all components of hippuric acid, o-methyl hippuric acid, m-methyl hippuric acid, p-methyl hippuric acid, and mandelic acid individually by the separating column.

2. The method according to claim 1, wherein a mobile phase used in the chromatographic analysis contains phosphoric acid water or formic acid water and acetonitrile.

3. The method according to claim 1, further comprising the step of detecting separation of all the components of hippuric acid, o-methyl hippuric acid, m-methyl hippuric acid, p-methyl hippuric acid, and mandelic acid within 4 minutes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a configuration diagram schematically showing an example of a liquid chromatograph for hippuric acid analysis.

(2) FIG. 2 is a chromatogram showing the relationship between the amount of ?-cyclodextrin chemically bonded to a silica matrix of a filler for separating column and the degree of separation of hippuric acids.

EMBODIMENTS OF THE INVENTION

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

(4) FIG. 1 is an example of a channel configuration of a liquid chromatograph for hippuric acid analysis.

(5) A liquid chromatograph of this example includes liquid delivery pumps 4 and 6, a mixer 8, a sample injector 10, a separating column 12, an ultraviolet absorbance detector (UV) 14, and a mass spectrometer (MS) 16. The liquid delivery pump 4 delivers phosphoric acid water or formic acid water, and the liquid delivery pump 6 delivers acetonitrile. Liquids delivered by the liquid delivery pumps 4 and 6 are mixed in the mixer 8, and the mixture flows through an analysis channel 2 connected to a downstream side of the mixer 8.

(6) The sample injector 10, the separating column 12, the UV 14 and the MS 16 are provided in this order from the upstream side in the analysis channel 2. The liquid delivery pumps 4 and 6 constitute a mobile phase liquid delivery unit for delivering a mobile phase to the separating column 12 through the analysis channel 2. The sample injector 10 injects a sample into the mobile phase which is delivered to the separating column 12 by the mobile phase liquid delivery unit including the liquid delivery pumps 4 and 6. The sample injected by the sample injector 10 contains at least two components of hippuric acid, o-methyl hippuric acid, m-methyl hippuric acid, p-methyl hippuric acid, and mandelic acid.

(7) The separating column 12 is a separating column for hippuric acid analysis which is filled with a filler in which 123 ?mol/g or more of ?-cyclodextrin is chemically bonded to a silica matrix. The separating column 12 can separate all components of hippuric acid, o-methyl hippuric acid, m-methyl hippuric acid, p-methyl hippuric acid, and mandelic acid.

(8) The UV 14 and the MS16 as a detector for detecting the components separated in the separating column 12 are connected to a downstream side of the separating column 12. In the liquid chromatograph, a liquid containing no cyclodextrin and buffer solution is used as a mobile phase, whereby the precipitation of salts does not occur in an analytical system. This allows analysis using the MS 16. Since the MS 16 is not an essential component, only the UV 14 may be provided as a detector.

(9) FIG. 2 is a chromatogram showing the verification results of the relationship between the amount of ?-cyclodextrin chemically bonded to the silica matrix of the filler for the separating column 12 of the liquid chromatograph and the degree of separation of hippuric acids. In FIG. 2(A), the amount of ?-cyclodextrin chemically bonded to the silica matrix is 58 ?mol/g; in FIG. 2(B), the amount of ?-cyclodextrin chemically bonded to the silica matrix is 123 ?mol/g; in FIG. 2(C), the amount of ?-cyclodextrin chemically bonded to the silica matrix is 207 ?mol/g; and in FIG. 2(D), the amount of ?-cyclodextrin chemically bonded to the silica matrix is 272 ?mol/g.

(10) In the verification, a separating column having an inner diameter of 3 mm and a length of 100 mm was used. The particle size of silica gel of the filler with which the separating column is filled is about 2.2 ?m. A liquid in which 0.1% phosphoric acid water and acetonitrile were mixed at a ratio of 9:1 was used as a mobile phase. The flow rate of the liquid was set to 0.8 mL/min, and the temperature of the separating column 12 (the set temperature of a column oven) was set to 40? C. Each of the chromatograms (A) to (D) was obtained by measuring the absorbance of light having a wavelength of 230 nm in the UV 14. Of the peaks in each of the chromatograms (A) to (D), * represents creatinine; 1 represents o-methyl hippuric acid; 2 represents hippuric acid; 3 represents m-methyl hippuric acid; 4 represents mandelic acid; and 5 represents p-methyl hippuric acid.

(11) As shown in FIG. 2, it is found that, as the amount of ?-cyclodextrin chemically bonded to the silica matrix is increased, the separation of 1 o-methyl hippuric acid, 2 hippuric acid, 3 m-methyl hippuric acid, 4 mandelic acid, and 5 p-methyl hippuric acid is improved. That is, as the amount of ?-cyclodextrin chemically bonded to the silica matrix is increased, the degree of separation of the hippuric acids is improved.

(12) When the amount of ?-cyclodextrin chemically bonded to the silica matrix is 58 ?mol/g, the peaks 1 and 2 are bonded to each other, the peaks 3 and 4 are bonded to each other, and o-methyl hippuric acid and hippuric acid are not separated from each other, and m-methyl hippuric acid and mandelic acid are not separated from each other. Meanwhile, when the amount of ?-cyclodextrin chemically bonded to the silica matrix is 123 ?mol/g, five peaks 1 to 5 appear, and the hippuric acids are separated. Therefore, it is found that, by adjusting the amount of ?-cyclodextrin chemically bonded to the silica matrix to 123 ?mol/g or more, the hippuric acids can be separated and analyzed without cyclodextrin being contained in the mobile phase.

(13) As described above, as the amount of ?-cyclodextrin chemically bonded to the silica matrix is increased, the retention of the hippuric acids is increased, but the increased retention causes an increased time until a component to be measured is eluted from the separating column 12. In order to perform analysis with high efficiency, it is ideal that the time until all components to be measured are detected is 5 minutes or less, and more preferably 4 minutes or less. According to the verification data in FIG. 2, it is found that, when the amount of ?-cyclodextrin chemically bonded to the silica matrix is 272 ?mol/g, p-methyl hippuric acid with the slowest elution from the separating column 12 among the hippuric acids is detected within 4 minutes, whereby, when the amount of ?-cyclodextrin chemically bonded to the silica matrix is 272 ?mol/g or less, high-speed analysis can be realized.

(14) In the verification of FIG. 2, the mixed solution of phosphoric acid water and acetonitrile is used as the mobile phase, but the same results can be obtained by using formic acid water instead of the phosphoric acid water.

DESCRIPTION OF REFERENCE SIGNS

(15) 2: Analysis channel 4, 6: Liquid delivery pump 8: Mixer 10: Sample injector 12: Separating column 14: Ultraviolet absorbance detector (UV) 16: Mass spectrometer (MS)