JP6773951B2 - How to prepare saliva sample - Google Patents

Description

The present invention relates to a method of collecting a certain amount of saliva from a subject to prepare a saliva sample having excellent storage stability and handleability, and a method of detecting an organic acid from the saliva sample obtained by the method.

Saliva is a non-invasive and easily collectable biological sample, and some biomolecules such as organic acids in saliva can function as biomarkers. Therefore, like blood, saliva is also useful as a sample for clinical examinations and the like. Saliva is usually collected by collecting it in a container as saliva or by absorbing it in a filter paper or swab. Examples of the swab for collecting saliva include a swab containing an applicator for collecting and then releasing saliva and Kaijima bicomponent fiber (see, for example, Patent Document 1), and rubbing the collected saliva on a medium or the like. Disclosed are swabs that can be released by (see, eg, Patent Document 2). Further, a swab used for collecting a biological sample for analyzing DNA and stored together with a desiccant (see, for example, Patent Document 3) is also disclosed.

On the other hand, some organic acids in biological samples are useful as biomarkers, and methods for measuring these are disclosed. For example, for the quantification of 3-hydroxybutyric acid (3-HB), which is useful as a biomarker for evaluating fatty acid catabolism in the liver, enzyme spectrophotometry is used to detect NADH after adding NAD and D-3-hydroxybutyric acid dehydrogenase. Is used (see, for example, Non-Patent Document 1 or 2). Another method for quantifying NADH is high-sensitivity HPLC (high performance liquid chromatography) in which 3HB is enzymatically converted to acetacetic acid (AA) and then derivatized with p-nitrobenzenediazonium fluoroboric acid, and this derivative is detected by spectrophotometry or ultraviolet rays. Chromatography) Methods of quantification by assay have been reported (see, for example, Non-Patent Documents 3 or 4). Further, as a method for directly detecting 3-HB, a method for detecting 3-HB by a gas chromatography-mass spectrometry (GC-MS) method after derivatization (see, for example, Non-Patent Document 5 or 6) or without derivatization. In addition, a method of detecting by LC-MS / MS (liquid chromatography-tandem mass spectrometry) (LC-P-ESI-MS / MS) by electrospray ionization (ESI) in the positive ion mode (for example, Non-Patent Document 7). (See.) Has also been reported, but the detection sensitivity of these methods is as low as that of the enzyme spectrophotometric method.

On the other hand, in patients with digestive disorders, abnormal nutritional metabolism occurs due to decreased metabolism, digestion, absorption capacity, or overnutrition. Amino acids are of high importance as an energy source because of abnormal glucose and lipid metabolism in many digestive disorders. In particular, branched-chain amino acids (BCAA: valine, leucine, isoleucine) are catabolized in the mitochondria of skeletal muscle instead of sugars and lipids during starvation, hyponutrition, long-term exercise and metabolic diseases, and serve as an energy source. BCAAs catabolized in skeletal muscle are metabolized to acetyl-CoA and succinyl-CoA. BCAAs bind CoA that cannot cross the mitochondrial membrane by a common enzymatic reaction within the mitochondria. Specifically, BCAA is converted from branched-chain α-keto acid (from valine to α-ketoisovaleric acid) in mitochondria by BCAT (BCAA amino acid transtransferase) and BCKDH complex (branched chain α-keto acid dehydrogenase complex). , From leucine to α-ketoisocaproic acid (KIC), from isoleucine to α-keto-β-methylvaleric acid), then to CoA form (α-ketoisovaleric acid to isobutyryl CoA, KIC to isovaleryl CoA, α-keto It is metabolized from -β-methylketo acid to α-methylbutyryl CoA). CoA binding is essential in the BCAA catabolism process, but only valine has CoA removed by a specific enzymatic reaction during the catabolism pathway, and the intermediate metabolic product 3-hydroxyisobutyric acid (3-HIB). Can be done. Since 3-HIB to which CoA is not bound has a small molecular weight, it passes through the mitochondrial membrane and a part of it leaks out of the cell. In fact, in starved and cirrhotic patients, serum 3-HIB levels increase with skeletal muscle atrophy.

From this, 3-HIB is expected to be used as an index reflecting the amino acid catabolism state in skeletal muscle, that is, as a BCAA catabolism marker in skeletal muscle. However, a method capable of detecting a trace amount of 3-HIB in a biological sample with sufficient sensitivity has not been reported so far. For example, as a method for directly detecting 3-HIB, it is detected by the LC-MS / MS method (LC-N-ESI-MS / MS) by electrospray ionization (ESI) in the negative ion mode without derivatization. Although a method (see, for example, Non-Patent Document 7) has been reported, the method has low sensitivity and therefore requires a large amount of serum, for example, to detect 3-HIB in serum. Further, it has been reported that when 3-HB in blood was detected by the GC-MS method, 3-HIB could not be separated from 3-HB by chromatography (for example, non-patent documents). 8).

As a method for detecting organic acids other than 3-HB and 3-HIB, after derivatizing malonic acid to di- (1-methyl-3-piperidinyl) malonic acid, LC-P-ESI-MS / MS) is used. A method for detecting (see, for example, Non-Patent Document 9) has been reported. In this method, by introducing an amine residue into malonic acid to form a malonic acid ester, ionization was promoted and the detection sensitivity was significantly increased.

Moreover, as a metabolite of leucine which is one of BCAA, 3-hydroxyisovaleric acid (3-HMB) can be mentioned. Like other BCAAs, leucine is metabolized in the mitochondria via KIC to the CoA body (isovaleryl CoA), but about 5 to 10% of KIC passes through the mitochondrial membrane and is 3-bye in the cytoplasm by KIC-dioxygenase. It is metabolized to HMB. The effects of 3-HMB include promotion of protein synthesis by the mTOR pathway, suppression of protein degradation by suppression of the ubiquitin / proteasome pathway, and muscle synthesis by promotion of cholesterol synthesis by hydroxymethylglutaryl-CoA (HMG-CoA) converted from HMB-CoA. Sheath stabilization is known. Due to these actions, 3-HMB is expected to be used as a marker for BCAA-derived liver protein (albumin, etc.) synthesis and a marker for skeletal muscle hypertrophy. In fact, since 3-HMB is used as a supplement for the purpose of muscle-building effect, in order to examine the blood dynamics when 3-HMB is ingested, 3 in the blood of rats after ingestion of 3-HMB. It has been reported that the −HMB concentration was measured by LC-N-ESI-MS / MS without derivatization (see, for example, Non-Patent Document 10). In addition, a method of simultaneously measuring 3-HB and 3-HMB without derivatization by HILIC (hydrophilic interaction chromatography) -MS / MS has also been reported, but in the measurement method, 3-HMB Since the detection limit is 0.4 μM and the amount of 3-HMB in a biological sample (blood, saliva, etc.) is smaller than that of 3-HB or 3-HMB, a method for more sensitive detection is required (). For example, see Non-Patent Document 11).

Further, as an organic acid having a structure similar to that of 3-HB or the like, there is 2-hydroxybutyric acid (2-HB). 2-HB is produced by α-ketobutyric acid, which is a metabolite of threonine and methionine, as a by-product of catabolism by NADH-dependent LDH and 2-HB dehydrogenase (α-HBDH), which is an isozyme of LDH. In addition, since α-ketobutyric acid is metabolized to propionyl-CoA, which is a precursor of succinyl-CoA, an increase in 2-HB may be due to increased catabolism of α-ketobutyric acid by LDH or α-HBDH, or from α-ketobutyric acid to propionyl-CoA. Caused by metabolic deficiency. In addition, α-ketobutyric acid is also associated with glutathione production because it is also produced as a by-product of the metabolism of cysteine, a precursor amino acid of glutathione, a potent antioxidant, from cystathione. It has been reported that 2-HB can be used as an early marker for predicting insulin resistance and glucose intolerance in non-diabetic patients (see, for example, Non-Patent Document 12). This is because lipid peroxidation and oxidative stress in the liver are involved in the onset of insulin resistance and glucose intolerance, and it is due to the increase in NADH and NAD ⁺ associated with the increase in glutathione production in the liver and lipid peroxidation. Conceivable. In this study, 2-HB in serum and whole blood was measured by UHPLC-MS or HILIC-MS / MS device, and it was determined from the concentration in serum (1 μg / mL = about 9.6 μM or more). Measurement with a small amount of biological sample (serum, etc.) has not been studied so far (see, for example, Non-Patent Document 11 and Non-Patent Document 2).

Japanese Unexamined Patent Publication No. 2014-16367 Japanese Patent Application Laid-Open No. 2007-523663 Japanese Unexamined Patent Publication No. 2014-10132

Williamson, 2 others, Biochemical Journal, 1962, Vol. 82, p. 90. Laun, 6 others, Clinical and Experimental Medicine, 2001, Volume 1, page 201. Yamato, 4 others, BIOLOGICAL and PHARMACEUTICAL BULLETIN, 2003, Vol. 26, p. 397. Yamato, 8 others, Analytical Biochemistry, 2009, Vol. 384, p. 195. Heil, 9 others, Biomedical Sciences and Applications, 2000, Vol. 379, p. 313. Hassan and one others, Journal of Analytical Toxicology, 2009, Vol. 33, p. 502. Sunny, 9 others, American Journal of Physiology-Endocrinology and Metabolism, 2010, Vol. 298, p. E1226. Des Rosiers, 6 others, Analytical Biochemistry, 1988, Vol. 173, p. 96. Honda, 7 others, Journal of Lipid Research, 2009, Vol. 50, p. 2124. Deshpande, 6 others, Biomedical Chromatography, 2013, Vol. 27, pp. 142-147. Sorensen, 6 others, Clinical Biochemistry, 2013, Vol. 46, pp. 1877-1883. Gall, 11 others, PLoS One, 2010, Volume 5, e10883. Shiina, 2 others, Chemistry Letters, 2002, Vol. 31, p. 286. Taguchi, "Introduction to Quality Engineering: Designing Quality into Products and Process", published by Asian Productivity Organization in 1986.

An object of the present invention is to provide a method for collecting a certain amount of saliva from a subject to prepare a saliva sample having excellent storage stability and handleability, and a method for detecting an organic acid from the saliva sample obtained by the method. And.

That is, the present invention relates to the following methods for preparing saliva samples [1] to [13], the following methods [14] to [18] for detecting organic acids, and the following [19] for measuring changes in organic acid concentration over time. It provides a method.
[1] The first method for preparing a saliva sample according to the present invention comprises an absorption step of absorbing saliva into a saliva absorbing member and a drying step of drying the saliva absorbing member having absorbed saliva in the absorption step. In the absorption step, the saliva absorbing member is made to absorb a predetermined amount of saliva, and after the drying step, a region in which a predetermined amount of saliva-derived solid content is adsorbed is cut from the saliva absorbing member. To prepare a saliva sample in which a predetermined amount of solid content derived from saliva is held in a saliva absorbing member in a dry state, the saliva absorbing member is in the shape of a strip or a rod, and in the absorption step, One end of the saliva absorbing member is brought into contact with saliva, and saliva is sucked up by a capillary phenomenon to cause the saliva absorbing member to absorb saliva, and after the drying step, the saliva-derived solid content of the saliva absorbing member is adsorbed. Of the areas that are being sucked up, saliva is obtained by cutting a predetermined area or volume of area from a part that is 8 mm or more away from the tip of the sucked saliva toward the end side in contact with saliva. It is characterized in that it is collected as a sample.
[2] In the method for preparing a saliva sample according to the above [1], it is preferable that the saliva is contained in a saliva container in a liquid state.
[3] In the method for preparing a saliva sample according to [1] or [2], it is preferable to carry out the absorption step by bringing the saliva absorbing member into contact with saliva in the oral cavity of the subject.
[4] In the method for preparing a saliva sample according to [3], the saliva absorbing member has a strip shape or a rod shape, and in the absorption step, one end of the saliva absorbing member is put into the mouth of the subject. By sucking saliva by the capillary phenomenon, the saliva absorbing member absorbs saliva, and after the drying step, the saliva-derived solid content of the saliva absorbing member is adsorbed in the oral cavity of the subject. in an area not in direct contact with the saliva, and sucked obtained towards the site which is located more than 8mm from the tip of the saliva to the contacted end portion side direction saliva, a predetermined region of the surface integral or volume fraction It is preferable to collect the cut saliva sample as a saliva sample.
[5] In the method for preparing a saliva sample according to any one of [1] to [4], an antibacterial agent may be contained in a saliva absorbing member that has absorbed saliva in the absorbing step before the drying step. preferable.
[6] In the method for preparing a saliva sample according to the above [1] or [2], it is preferable that the saliva absorbing member absorbs a mixture of saliva and an antibacterial agent in the absorption step.
[7] In the method for preparing a saliva sample according to any one of [1] to [4], it is preferable that the saliva absorbing member holds an antibacterial agent in advance.
[8] The second method for preparing a saliva sample according to the present invention is an absorption step of absorbing saliva into a saliva absorbing member and a solution containing the saliva absorbing member having absorbed saliva in the absorption step containing an antibacterial agent. It has an antibacterial agent treatment step of immersing it in, and in the absorption step, the saliva absorbing member absorbs a predetermined amount of saliva, and after the absorption step, a predetermined amount of saliva is derived from the saliva absorbing member. A solution containing a predetermined amount of saliva-derived solids and a saliva absorbing member by immersing the recovered solid content by cutting the adsorbed region in the solution containing the antibacterial agent. A saliva sample is prepared, and the saliva absorbing member is in the shape of a strip or a rod. In the absorption step, one end of the saliva absorbing member is brought into contact with saliva, and saliva is sucked up by a capillary phenomenon. After the drying step, the end of the region where saliva-derived solids of the saliva absorbing member are adsorbed and brought into contact with saliva from a portion 8 mm or more away from the tip of the sucked saliva. It is characterized in that a saliva sample obtained by cutting a region of a predetermined area or volume toward the portion side is collected.
[9] In the method for preparing a saliva sample according to any one of [5] to [8], the antibacterial agent is preferably a quaternary ammonium salt-based antibacterial agent.
[10] In the method for preparing a saliva sample according to any one of [1] to [9], the amount of saliva absorbed by the saliva absorbing member in the absorption step may be the maximum amount of saliva absorbed by the saliva absorbing member. preferable.
[11] In the method for preparing a saliva sample according to any one of [1] to [10], the predetermined amount is preferably 1 to 50 μL.
[12] In the method for preparing a saliva sample according to any one of [1] to [11], it is preferable that the saliva absorbing member is made of a synthetic resin fiber.
[13] In the method for preparing a saliva sample according to any one of [1] to [7], it is preferable that the drying in the drying step is natural drying.
[14] In the method for detecting an organic acid in saliva according to the present invention, a saliva sample is prepared by the method for preparing a saliva sample according to any one of [1] to [13] above, and the obtained saliva sample is used as a polar organic substance. An extraction step of extracting a saliva-derived organic acid from the saliva sample by immersing it in a solvent, and a polar organic solvent from which the organic acid has been extracted after the extraction step are used as a saliva-derived organic acid detection sample. The recovery step of separating and recovering from the saliva sample and the organic acid in the saliva-derived organic acid detection sample recovered by the recovery step are described in the following general formulas (1) -1 to (1) -3 (formula (1)). Of -1 to 3, R ¹ and R ² are represented by either one of a hydroxyalkyl group having 1 to 6 carbon atoms and the other representing a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. It has a detection step of synthesizing a derivative by ester-bonding with a derivatizing reagent and detecting the derivative by an electrospray ionized LC-MS / MS (liquid chromatography-tandem mass analysis) method in a positive ion mode. It is characterized by that.

[ 15 ] In the method for detecting an organic acid in saliva according to [ 14 ], it is preferable that the polar organic solvent is acetonitrile.
[ 16 ] In the method for detecting an organic acid in saliva according to [ 14 ] or [ 15 ], it is preferable to immerse the saliva sample in a polar organic solvent containing an internal standard substance in advance in the extraction step. ..
[ 17 ] In the method for detecting an organic acid in saliva according to any one of [ 14 ] to [ 16 ], the derivatizing reagents are 2-pyridinemethanol, 1-piperidineethanol, and 2- (2-hydroxyethyl). It is preferably one or more selected from the group consisting of -1-methylpyrrolidine.
[ 18 ] In the method for detecting an organic acid in saliva according to any one of [ 14 ] to [ 17 ], the organic acid is 3-hydroxybutyric acid, 3-hydroxyisobutyric acid, 3-hydroxyisovaleric acid, 2-. It is preferably one or more selected from the group consisting of hydroxybutyric acid and lactic acid.
[ 19 ] The method for measuring the change over time in the organic acid concentration according to the present invention is the saliva according to any one of [ 14 ] to [ 18 ] above for a plurality of saliva samples prepared over time from the same subject. A method for detecting the organic acid in the sample is performed, and the change over time in the saliva concentration of the subject is examined.

According to the method for preparing a saliva sample according to the present invention, a certain amount of saliva can be easily collected from a subject to prepare a saliva sample having excellent storage stability and handleability.
Further, the method for detecting an organic acid and the method for measuring a change in organic acid concentration with time according to the present invention are obtained by using a specific derivatizing reagent for an organic acid in a saliva sample prepared by the method for preparing a saliva sample according to the present invention. It is a method of detecting by LC-P-ESI-MS / MS after derivatization using, and by these methods, 3-HIB, 3-HB, 3-HMB, 2-HB, from a very small amount of saliva, Organic acids such as lactic acid can be detected with high sensitivity.

It is a figure which showed the typical P-ESIMS spectrum of 3-HB (2PM-3-HB) derivatized with 2-pyridinemethanol. In Reference Example 1, it is the figure which showed the total ion chromatogram (upper) and MS chromatogram (lower) of the sample containing only 3-HB derivatized with 2-pyridinemethanol. It is a figure which showed the MS spectrum of FIG. 2A. In Reference Example 1, it is the figure which showed the total ion chromatogram (upper) and MS chromatogram (lower) of the sample containing only 3-HIB derivatized with 2-pyridinemethanol. It is a figure which showed the MS spectrum of FIG. 2C. In Reference Example 1, it is a figure which showed the product ion chromatogram when m / z 196 of the sample containing 3-HB and 3-HIB derivatized with 2-pyridinemethanol was used as a precursor ion. It is a figure which showed the MS / MS spectrum of the 3-HIB peak of FIG. 2E. In Reference Example 2, it is the figure which showed the total ion chromatogram (upper) and MS chromatogram (lower) of the sample containing only 3-HB derivatized with 1-piperidine ethanol. It is a figure which showed the MS spectrum of FIG. 3A. In Reference Example 2, it is the figure which showed the total ion chromatogram (upper) and MS chromatogram (lower) of the sample containing only 3-HIB derivatized with 1-piperidine ethanol. It is a figure which showed the MS spectrum of FIG. 3C. In Reference Example 2, it is a figure which showed the product ion chromatogram when m / z 216 of the sample containing 3-HB and 3-HIB derivatized with 1-piperidine ethanol was used as a precursor ion. It is a figure which showed the MS / MS spectrum of the 3-HIB peak of FIG. 3E. In Reference Example 3, the total ion chromatogram (upper) and MS chromatogram (lower) of a sample containing only 3-HB derivatized with 2- (2-hydroxyethyl) -1-methylpyrrolidine are shown in the figure. is there. It is a figure which showed the MS spectrum of FIG. 4A. In Reference Example 3, the total ion chromatogram (upper) and MS chromatogram (lower) of a sample containing only 3-HIB derivatized with 2- (2-hydroxyethyl) -1-methylpyrrolidine are shown. is there. It is a figure which showed the MS spectrum of FIG. 4C. In Reference Example 3, the product ion chromatogram when m / z 216 of a sample containing 3-HB and 3-HIB derivatized with 2- (2-hydroxyethyl) -1-methylpyrrolidine was used as a precursor ion is shown. It is a figure shown. It is a figure which showed the MS / MS spectrum of the 3-HIB peak of FIG. 4E. In Reference Example 4, it is a figure which showed the typical SRM chromatogram of 3-HB (2PM-3-HB) derivatized with 2-pyridinemethanol and the [ ¹³ C ₄ ] isotope. Reference Example 5, 2-pyridine derivatized with methanol 3-HB and 3-HIB and 3-HMB and total ion chromatogram of a sample containing 2-HB and ^3-HB- 13 _{C 4} (1 stage) , Product ion chromatogram with m / z 196 as precursor ion (second stage), Product ion chromatogram with m / z 200 as precursor ion (third stage), m / z 210 as precursor ion It is a figure which showed the product ion chromatogram (4th stage) at the time of. In Reference Example 6, it is a figure which showed the measurement result of 3-HB concentration and 3-HIB concentration in serum left at room temperature after blood collection. In Reference Example 6, it is a figure which showed the measurement result of 3-HB concentration and 3-HIB concentration in saliva left at room temperature after collection. In Reference Example 7, it is a figure which showed the result of having investigated the correlation between the serum concentration of 3-HB and the saliva (saliva) concentration. In Reference Example 7, it is the figure which showed the result of having investigated the correlation between the serum concentration of 3-HIB and the saliva (saliva) concentration. In Reference Example 7, it is a figure which showed the result of having investigated the correlation between the serum concentration of 2-HB and the saliva (saliva) concentration. In Reference Example 8, it is a figure which showed the exercise and the eating condition of a subject, and the time point of collecting blood, saliva, and urine. In Reference Example 8, it is a figure which showed the time-dependent change of the concentration of 3-HIB and 3-HB in the serum of a subject. In Reference Example 8, it is a figure which showed the time-dependent change of the urine excretion amount (μmol / h) per unit time at each collection time of 3-HIB and 3-HB of a subject. In Reference Example 8, it is a figure which showed the time-dependent change of the concentration of 3-HIB and 3-HB in the saliva of a subject. In Reference Example 9, it is the figure which showed the measurement result of 3-HIB concentration and 3-HMB concentration in the serum of the liver cirrhosis patient group and the healthy person group. In Reference Example 9, it is a figure which showed the measurement result of 3-HIB concentration and 3-HMB concentration in saliva of a liver cirrhosis patient group and a healthy person group. It is a schematic diagram of the Schirmer filter paper 1 used in Example 1. In Example 1, three Schirmer filter papers were prepared by immersing a region up to 10 mm from the tip of the Schirmer filter paper (the end on the region side in contact with saliva) in the saliva in the container and sucking up saliva by capillarity. Variations in concentration (coefficient of variation, 3-HMB) of four types of organic acids (3-HB, 3-HIB, 2-HB and 3-HMB) measured from each fragment divided into fragments (collection sites (1) to (3)) It is a figure which showed the result of having calculated% CV). In Example 2, 10 Schirmer filter papers in which saliva was sucked up by capillarity by contacting only the region up to 10 mm from the tip of the Schirmer filter paper (the end on the region side to be in contact with saliva) under the tongue of the subject. Variations (variations) in the concentration of four types of organic acids (3-HB, 3-HIB, 2-HB and 3-HMB) measured from each fragment divided into three fragments (collection sites (1) to (3)) It is a figure which showed the result of having calculated the coefficient (% CV). In Example 3, the concentration of DL-3-HB- ¹³ C ₄ sodium salt in saliva soaked in Sylmar filter paper is a diagram showing each drying time of the Schirmer paper. In Example 3, the concentration of 3-HB in saliva impregnated with Schirmer filter paper is shown for each drying time of Schirmer filter paper. In Example 3, the concentration of 3-HMB in saliva impregnated in Schirmer filter paper is shown for each drying time of Schirmer filter paper. In Example 4, a diagram illustrating saliva, and the concentration of 20 minutes is absorbed naturally dried DL-3-HB- ¹³ C ₄ sodium salt measured from the filter paper was saliva. It is a figure which showed the saliva and the concentration of 3-HB measured from each filter paper which absorbed saliva and air-dried for 20 minutes in Example 4. FIG. It is a figure which showed the saliva and the concentration of 3-HIB measured from each filter paper which absorbed saliva and air-dried for 20 minutes in Example 4. FIG. It is a figure which showed the saliva and the concentration of 3-HMB measured from each filter paper which absorbed saliva and air-dried for 20 minutes in Example 4. FIG. It is a figure which showed the saliva and the concentration of 2-HB measured from each filter paper which absorbed saliva and air-dried for 20 minutes in Example 4. FIG. In Reference Example 10, it is a figure which showed the product ion chromatogram when m / z 182.1 of the sample containing lactic acid derivatized with 2-piperidine ethanol and its deuterium isotope was used as a precursor ion. It is a figure which showed the MS / MS spectrum of the lactate peak derivatized with 2-piperidine ethanol of FIG. 26A. In Reference Example 10, it is a figure which showed the typical SRM chromatogram of D-lactic acid (standard article) derivatized with 2-pyridinemethanol and its deuterium isotope. In Reference Example 10, a typical SRM chromatogram of saliva-derived lactic acid derivatized with 2-pyridinemethanol and its deuterium isotope is shown. In Reference Example 11, it is a figure which showed the time-dependent change of the organic acid concentration in saliva at room temperature. In Reference Example 12, it is a figure which showed the measurement result of the organic acid concentration in saliva which added various additives and left at room temperature for 24 hours. In Reference Example 13, it is a figure which showed the result of having investigated the correlation between the serum concentration of lactic acid and the concentration in saliva (saliva).

<First method of preparing saliva sample>
The method for preparing a saliva sample according to the present invention is characterized in that a saliva sample in which a predetermined amount of solids derived from saliva is held in a saliva absorbing member in a dry state is prepared. Since the saliva sample prepared by the preparation method is in a dry state, various biomolecules in saliva are stable even when stored at room temperature or outside air temperature until it is subjected to clinical examination or the like. And be maintained. In addition, it is easier to store and transfer than the case where liquid saliva is collected and stored as it is in a container or the like, and it is also excellent in handleability. Further, since the saliva sample prepared by the preparation method contains a predetermined amount of solids derived from saliva, the same inspection method can be applied to a plurality of saliva samples prepared by the preparation method. By providing this, it is possible to suppress variations in measurement for each sample, and it is possible to directly compare the contents of biomolecules contained in each saliva sample without performing normalization treatment.

The solid content derived from saliva means the remaining components obtained by removing water from saliva. That is, the solid content includes organic acids, nucleic acids, proteins, sugars, polysaccharides, sugar proteins, lipids and the like.

In order to prepare a saliva sample containing a solid content derived from a predetermined amount of saliva, the method for preparing a saliva sample according to the present invention specifically comprises an absorption step of absorbing saliva into a saliva absorbing member and the above-mentioned. It has a drying step of drying a saliva absorbing member that has absorbed saliva in the absorption step, and in the absorbing step, the saliva absorbing member absorbs a predetermined amount of saliva, or after the drying step, the saliva absorbing member. Then, the region where a predetermined amount of saliva-derived solids are adsorbed is cut and recovered.

In the absorption step, the saliva to be absorbed by the saliva absorbing member may be directly collected from the oral cavity of the subject by bringing the saliva absorbing member into contact with the saliva in the oral cavity of the subject, and is stored in a container. It may be saliva (saliva) in a liquid state. The hypersalivation may be collected by the subject directly dripping into the container, or may be collected by other means and contained in the container. For example, the saliva absorbing member can absorb saliva by including the saliva absorbing member in the oral cavity, or by holding at least a part of the saliva absorbing member in the mouth and sucking up saliva by a capillary phenomenon. In particular, when the saliva absorbing member is strip-shaped or rod-shaped, saliva can be easily collected from the subject even when the subject is in a lying posture or is continuing exercise. .. Further, for example, by immersing the saliva absorbing member in the saliva collected in the container, or by bringing a part of the saliva absorbing member into contact and sucking saliva by the capillary phenomenon, the saliva absorbing member absorbs saliva. be able to.

In the absorption step, the amount of saliva absorbed by the saliva absorbing member is not particularly limited, but it is preferably an amount that can be collected by moistening the saliva absorbing member in the oral cavity. Specifically, 100 μL or less is preferable, 1 to 50 μL is more preferable, and 10 to 50 μL is further preferable. In the method for preparing a saliva sample according to the present invention, since saliva is collected by absorbing a very small amount of saliva into a saliva absorbing member, a subject having a small amount of saliva such as a child, an elderly person, a patient, etc. The required amount of saliva can be safely and quickly collected from a subject who is exercising.

The saliva absorbing member used in the method for preparing a saliva sample according to the present invention may be a porous member capable of absorbing a liquid component, and is used for collecting a biological sample containing a liquid component such as saliva or tears. It can be appropriately selected and used from the porous members. Above all, it is preferable to use filter paper or a swab. The shape of the saliva absorbing member is not particularly limited, and may be lump-shaped, strip-shaped, or rod-shaped. Further, for example, it may be a composite member of a saliva absorbing member and a member that does not absorb saliva, such as a member in which a swab, which is a saliva absorbing member, is bonded to the tip of a rod-shaped member that does not absorb saliva. It is also preferable to dry the saliva absorbing member by preserving it with a desiccant such as silica gel before using it in the absorption step.

When the saliva absorbing member is strip-shaped or rod-shaped, and the subject holds one end in the mouth and sucks saliva into the saliva absorbing member by capillarity, the saliva absorbed by the saliva absorbing member The amount depends on the distance from the end of the subject's mouth to the tip of the saliva sucked up. Therefore, in order to visualize that the saliva absorbing member has absorbed the required amount of saliva, it is visualized in advance when the subject of the saliva absorbing member comes into contact with saliva at a predetermined distance from the end of the mouth. It is also preferable to retain the dye to be used.

The material of the saliva absorbing member used in the method for preparing a saliva sample according to the present invention may be, for example, a natural material such as cotton, wool, silk, linen, lignocellulose, starch, dextran, or acrylic or polyethylene terephthalate. Polyester such as polyester, polyolefin such as polyethylene and polypropylene, polyurethane, polyamide, polyvinyl alcohol, rayon, nitrocellulose, cellulose acetate, ABS (acrylonitrile, butadiene, styrene) resin, or synthetic resin fiber such as a mixture thereof as appropriate. It may be an inorganic material such as glass fiber, silica gel, active white clay, polyester clay, carbon powder, active alumina and the like. As the material of the saliva absorbing member used in the present invention, synthetic resin fibers and inorganic materials are preferable because they are easily dried after absorbing saliva, and synthetic resin fibers are more preferable because they have high saliva absorption. preferable.

After the absorption step, as a drying step, the saliva absorbing member that has absorbed saliva in the absorption step is dried. The drying method is not particularly limited as long as it can remove water without damaging biological components such as organic acids in saliva absorbed by the saliva absorbing member, and may be naturally dried and warm air. It may be dried, dried under reduced pressure, or freeze-dried. In the present invention, it is preferable to dry naturally because it can be easily dried without requiring a special drying device.

In the drying step, when a paper or the like is brought into contact with the surface of the saliva absorbing member that has absorbed saliva, it is preferable that the paper is sufficiently dried so that moisture does not bleed. Conditions such as the drying temperature and the drying time can be appropriately adjusted according to the material of the saliva absorbing member and the drying method. For example, when the material of the saliva absorbing member is cotton cellulose and it is naturally dried in an environment of 20 to 25 ° C., the drying time is preferably 24 hours or more, more preferably 48 hours or more. When the material of the saliva absorbing member is a synthetic resin fiber and it is naturally dried in an environment of 20 to 25 ° C., the drying time is preferably 20 minutes or more, more preferably 1 hour or more.

The saliva sample obtained in the method for preparing a saliva sample according to the present invention is used for analysis of biomolecules contained in saliva. Depending on the analysis method, the biomolecule to be analyzed is extracted from the saliva sample with an organic solvent, but when the water content of the saliva absorbing member is high, the polar organic solvent does not sufficiently permeate and the organic acid derived from saliva. May be difficult to extract. Therefore, in the drying step, the water content of the saliva absorbing member after drying is such that the polar organic solvent extends to the center of the saliva absorbing member when the saliva absorbing member is immersed in the polar organic solvent. Is preferably dried until it is low enough to penetrate.

The saliva absorbing member after the drying step can be stored in an environment where no special temperature control such as room temperature is performed until it is subjected to analysis. It is also preferable to store the dried saliva absorbing member with a desiccant. When the drying step is carried out by natural drying, it is preferable that the saliva absorbing member that has absorbed saliva is left to stand in a room temperature environment together with a desiccant such as silica gel to be naturally dried.

In the method for preparing a saliva sample according to the present invention, a saliva sample in which a predetermined fixed amount of saliva-derived solid content is retained by allowing a saliva absorbing member to absorb a predetermined amount of saliva in the absorption step. Can be prepared. For example, the maximum absorption amount of the saliva absorbing member is adjusted to the predetermined amount, and the saliva absorbing member is allowed to absorb saliva to the limit (that is, until no more saliva can be absorbed) in the absorption step. By using a saliva absorbing member having a certain size, a certain amount of saliva can always be collected.

In the method for preparing a saliva sample according to the present invention, a region in which a predetermined amount of saliva-derived solids are adsorbed is cut and recovered from the saliva absorbing member after the drying step. A saliva sample can be prepared in which a certain amount of saliva-derived solids is retained. For example, when the saliva absorbing member is strip-shaped or rod-shaped, one end of the saliva absorbing member is brought into contact with saliva, and saliva is sucked up by a capillary phenomenon, so that the saliva absorbing member can absorb saliva ( Absorption process). Since the capillary phenomenon is used, it is difficult for the saliva absorbing member to absorb only a predetermined amount of saliva in the absorption process, but in principle, the amount of saliva absorbed by the saliva absorbing member is the saliva absorbing member. The saliva inside is proportional to the volume sucked up. Further, in a saliva absorbing member having a constant thickness such as a strip shape or a rod shape, the amount of saliva absorbed is, in principle, proportional to the area in which saliva is sucked up in the saliva absorbing member. Therefore, by cutting out a region of a predetermined surface integral or volume from the region where saliva of the saliva absorbing member after the drying step is sucked up, a saliva absorbing member always holding a predetermined amount of saliva-derived solid content can be obtained. Be done. The saliva absorbing member cut and recovered is the target saliva sample.

The region cut and recovered from the strip-shaped or rod-shaped saliva absorbing member after the drying step is separated from the tip of the sucked saliva in the region where the saliva-derived solid content of the saliva absorbing member is adsorbed. It is preferably a region. In the vicinity of the tip of saliva sucked up in the saliva absorbing member due to the capillary phenomenon, the amount of saliva absorbed by the saliva absorbing member per unit area or unit volume tends to vary, and the saliva sample including the vicinity of the tip is dried. This is because the amount of saliva that was previously absorbed tends to vary. In the present invention, the region cut and collected from the strip-shaped or rod-shaped saliva absorbing member after the drying step is preferably a region separated from the tip of the sucked saliva by 8 mm or more, and the sucked saliva. It is more preferable that the region is separated from the tip of the above by 10 mm or more.

When one end of a strip-shaped or rod-shaped saliva absorbing member is immersed in saliva contained in another container to absorb saliva by the capillary phenomenon, the unit is other than the vicinity of the tip of the sucked saliva. Since the amount of saliva absorbed by the saliva absorbing member per area or unit volume is almost uniform, the area other than the vicinity of the tip of the sucked saliva can be cut and collected as a saliva sample. It may include the area that has been immersed, or may be only the area that has not been immersed in saliva. On the other hand, when one end of the saliva absorbing member is put into the mouth of the subject and the saliva is sucked up by the capillary phenomenon to cause the saliva absorbing member to absorb saliva, the saliva absorbing member after the drying step is performed. Of the regions where solids derived from saliva are adsorbed, the region is the region that was not in direct contact with saliva in the oral cavity of the subject, and is 8 mm or more, preferably 10 mm or more away from the tip of the sucked saliva. A predetermined area or volume of the area is cut from the area and collected as a saliva sample. In the region of the saliva absorbing member that was in direct contact with saliva in the subject's mouth, the amount of saliva absorbed by the saliva absorbing member per unit area or unit volume tends to vary, and the saliva is in direct contact with saliva. This is because it is more likely that the amount of saliva absorbed per unit area or unit volume is more uniform in the region that was not used. The reason why the amount of saliva absorbed per unit area or unit volume in the area that was in direct contact with saliva varies widely is the amount of saliva that directly touches the saliva absorbing member in the oral cavity and the amount of saliva that directly touches the saliva in the saliva absorbing member. It is considered that the proportion occupied by the contacted area varies, and that the saliva absorbing member is deformed by the physical contact with the oral surface of the subject.

When one end of the saliva absorbing member is put into the subject's mouth and saliva is sucked up by the capillary phenomenon to cause the saliva absorbing member to absorb saliva, the surface area in direct contact with saliva in the oral cavity is always constant. By covering a part of the surface of the saliva absorbing member with a water-repellent member in advance so that saliva does not come into direct contact with the saliva absorbing member even when it is placed in the oral cavity (non-contact area). ) Is also preferable. Examples of the water-repellent member include metal foil such as aluminum foil, paraffin paper, and synthetic resin film.

The saliva sample obtained by the method for preparing a saliva sample according to the present invention can be used for inspection of biological components in saliva and the like. Therefore, the saliva absorbing member used in the present invention may contain in advance a substance that can serve as an internal standard substance in the test in which the saliva sample is provided. Examples of the internal standard substance include radioactive isotopes of the substance to be inspected. For example, the internal standard substance can be retained in the saliva absorbing member by impregnating the saliva absorbing member with a solution in which the internal standard substance is dissolved and then sufficiently drying the saliva absorbing member. The internal standard substance is preferably held in a non-contact region of the saliva absorbing member that does not come into direct contact with saliva in the oral cavity of the subject.

By using the method for preparing a saliva sample according to the present invention, a saliva sample in which a small amount of saliva is always retained at all times can be relatively easily prepared from a subject. Furthermore, since the prepared saliva sample is in a dry state and can be easily stored for a long period of time or mailed, when the saliva collection time or collection place is separated from the saliva sample analysis time or analysis place. It is also preferable as a sample. The method for preparing a saliva sample according to the present invention is for preparing a saliva sample for analyzing the biological components in saliva of a large number of subjects and for investigating (monitoring) changes in biological components in saliva over time. It is particularly suitable for preparing saliva samples from the same subject over time.

The saliva sample obtained by the method for preparing a saliva sample according to the present invention is subjected to a highly sensitive analysis method capable of analyzing a very small amount of biomolecules in saliva of 100 μL or less, preferably 1 to 50 μL. Suitable as a sample. In particular, it is suitable as a sample for analyzing organic acids in saliva contained in 1 to 50 μL of saliva, and among them, 3-hydroxybutyric acid (3-HB) and 3-hydroxyisobutyric acid (3-HIB). , 3-Hydroxyisovaleric acid (3-HMB), 2-hydroxybutyric acid (2-HB), and lactic acid are suitable as samples for analysis of one or more selected from the group.

In the method for preparing a saliva sample according to the present invention, it is preferable that the saliva-derived solid content in the saliva sample coexists with the antibacterial agent. 2-HB and lactic acid in saliva have lower storage stability than 3-HB and the like, but by coexisting with an antibacterial agent, 2-HB and lactic acid in saliva samples can be stored more stably. it can. The antibacterial agent is not particularly limited as long as it is a substance having an action of suppressing growth or growth or an action of killing bacteria such as Gram-positive bacteria and Gram-negative bacteria and yeast-like fungi, and is antibacterial. It can be appropriately selected and used from various antibacterial agents used as agents and disinfectants. Examples of the antibacterial agent include a quaternary ammonium salt-based antibacterial agent such as benzalkonium chloride and benzethonium chloride, chlorine such as sodium hypochlorite, an iodine agent such as hydrogen peroxide solution and povidone iodine, ethanol and isopropanol. Alcohols and the like can be mentioned. In the present invention, quaternary ammonium salts such as benzalkonium chloride and benzethonium chloride are generally high in activity of altering and damaging cell membranes of bacteria and the like contained in saliva and high in antibacterial activity. Antibacterial agents are preferred. In addition, since many bacteria die in an acidic environment and their growth and growth are suppressed, a solution containing an acid having a concentration capable of lowering the pH of saliva to 4.0 or less is also available. It can be used as an antibacterial agent in the invention. The acids may be inorganic acids such as hydrochloric acid or organic acids such as formic acid.

For example, before the drying step, the saliva absorbing member that has absorbed saliva in the absorbing step contains an antibacterial agent so that the saliva-derived solid content is retained in the saliva absorbing member in a state of coexisting with the antibacterial agent. Can be done. As a method of incorporating an antibacterial agent into a saliva absorbing member that has absorbed saliva before the drying step, for example, a method of immersing the saliva absorbing member in an antibacterial agent solution (a solution in which the antibacterial agent is dissolved in an appropriate solvent) or , A method of spraying the antibacterial agent solution on the surface of the saliva absorbing member, a method of dropping the antibacterial agent solution on the surface of the saliva absorbing member using a dropper or the like, and the like. The solvent for preparing the antibacterial agent solution may be water, alcohols such as ethanol and isopropanol, a mixed solvent of water and alcohols, and various surfactants. It may contain a dissolving aid of an antibacterial agent such as.

Further, by mixing an antibacterial agent with saliva before being absorbed by the saliva absorbing member and allowing the saliva absorbing member to absorb a mixture of saliva and the antibacterial agent in the absorption step, the solid content derived from saliva is also antibacterial. It can be held by the saliva absorbing member in a state of coexisting with the agent. For example, the antibacterial agent is added to the saliva contained in the container and mixed so that the concentration of the antibacterial agent in saliva becomes a concentration necessary and sufficient to exhibit sufficient antibacterial activity. Alternatively, the hypersalivation may be collected in a container containing an antibacterial agent in advance, and the hypersalivation and the antibacterial agent may be mixed in the container. The antibacterial agent to be added to the saliva collected in the container and the antibacterial agent to be placed in the container for collection in advance may be an antibacterial agent solution dissolved in a solvent such as water, or a solid antibacterial agent such as powder. It may be an agent. Since it is relatively easy to adjust the concentration of the desired antibacterial agent, it is preferable that the antibacterial agent to be placed in the container for collection in advance is a solid antibacterial agent, and it is easy to mix more uniformly with the saliva. From the point of view, powdered antibacterial agents are more preferable. When collecting hypersalivation directly in the collection container, it is often difficult to strictly control the amount of hypersalivation collected, but a relatively large amount of solid antibacterial agent is contained in the collection container. By doing so, it is possible to mix with a sufficient amount of antibacterial agent necessary for enhancing storage stability without strictly adjusting the amount of saliva collected.

The antibacterial agent may be held in advance in the saliva absorbing member before the saliva is absorbed. By allowing the saliva absorbing member holding the antibacterial agent in advance to absorb saliva, the saliva absorbing member can hold the solid content derived from saliva in a state of coexisting with the antibacterial agent. For example, a method of immersing the saliva absorbing member in an antibacterial agent solution and then drying it, a method of uniformly spraying the antibacterial agent solution on the surface of the saliva absorbing member and then drying it, or a method of uniformly applying the antibacterial agent solution to the surface of the saliva absorbing member. The antibacterial agent can be retained in the saliva absorbing member by a method of drying later. Further, the antibacterial agent may be retained only in the non-contact region in the saliva absorbing member that does not come into direct contact with saliva and in which saliva is sucked up by the capillary phenomenon. In particular, when one end of a saliva absorbing member holding an antibacterial agent in advance is put into the subject's mouth and saliva is sucked up by capillarity, the antibacterial agent is put into saliva in the saliva absorbing member or in the oral cavity. It is preferable to keep it in a non-contact area where it does not come into direct contact.

<Method of preparing the second saliva sample>
Of the organic acids in saliva, 3-HB, 3-HIB, 3-HMB, 2-HB, and lactic acid can all be stably stored for a relatively long time in the presence of the antibacterial agent. it can. Therefore, a saliva sample that stably contains a saliva-derived organic acid can also be prepared by immersing the saliva absorbing member that has absorbed saliva in a solution containing an antibacterial agent without drying it. Specifically, it is carried out by an absorption step of absorbing saliva into a saliva absorbing member and an antibacterial agent treatment step of immersing the saliva absorbing member having absorbed saliva in the absorption step in a solution containing an antibacterial agent. .. As the saliva absorbing member and the antibacterial agent used, the same ones as those used in the above-mentioned method for preparing the first saliva sample can be used, and the absorption step is the absorption in the above-mentioned method for preparing the first saliva sample. It can be carried out in the same manner as the process. Further, the antibacterial agent solution can be used as the solution containing the antibacterial agent for immersing the saliva absorbing member in the antibacterial agent treatment step.

In order to prepare a saliva sample containing a predetermined amount of saliva-derived solids, only the portion of the saliva absorbing member holding the predetermined amount of saliva-derived solids is immersed in the antibacterial agent solution. There is a need. Therefore, in the absorption step, a predetermined amount of saliva is absorbed by the saliva absorbing member, or a region in which a predetermined amount of saliva-derived solid content is adsorbed is cut and recovered from the saliva absorbing member after the absorption step. Is immersed in an antibacterial agent solution. In the absorption step, the method of causing the saliva absorbing member to absorb a predetermined amount of saliva and the method of cutting and recovering the region where a predetermined amount of saliva-derived solids are adsorbed from the saliva absorbing member are described in the first method. It can be carried out in the same manner as the method for preparing a saliva sample.

<Method of detecting organic acids in saliva>
The method for detecting an organic acid in saliva according to the present invention (hereinafter, may be referred to as “detection method according to the present invention”) is an organic acid in a saliva sample obtained by the method for preparing a saliva sample according to the present invention. Is derivatized with a specific derivatizing reagent and then detected by LC-P-ESI-MS / MS. In the detection method according to the present invention, since the organic acid is derivatized with a specific derivatizing reagent and detected, even if the provided saliva sample is prepared from a very small amount of saliva of 1 to 50 μL, 3 It is possible to detect organic acids such as −HB, 3-HIB, 3-HMB, 2-HB, and lactic acid (hereinafter, collectively referred to as “3-HB and the like”).

Specifically, first, the saliva sample obtained by the method for preparing a saliva sample according to the present invention is immersed in a polar organic solvent to extract a saliva-derived organic acid from the saliva sample (extraction step). , The polar organic solvent from which the organic acid has been extracted is separated from the saliva sample and recovered (recovery step). The recovered polar organic solvent can be used as a sample for detecting saliva-derived organic acids.

The polar organic solvent is not particularly limited as long as it can dissolve an organic acid, and examples thereof include acetonitrile, acetone, ethyl methyl ketone, 2-pentanone, 3-pentanone, ethanol, methanol and the like. Be done. As the polar organic solvent, acetonitrile is preferable because it has good solubility of 3-HB and the like.

In order to quantitatively detect organic acids in saliva, it is preferable to add a certain amount of an internal standard substance to the saliva-derived organic acid detection sample. Examples of the internal standard substance include stable isotopes of organic acids to be detected. The internal standard substance may be added to the polar organic solvent (sample for detecting saliva-derived organic acid) recovered in the recovery step, or may be added to the polar organic solvent before being added to the saliva sample.

The organic acid in the saliva-derived organic acid detection sample has the following general formulas (1) -1 to (1) -3 (in the formulas (1) -1 to 3, R ¹ and R ² have one carbon number. A derivative is synthesized by ester-bonding with a derivatizing reagent represented by either a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, which represents 1 to 6 hydroxyalkyl groups, and the other represents the derivative. Can be detected by the electrospray ionized LC-MS / MS (liquid chromatography-tandem mass analysis) method in the positive ion mode (detection step).

In the general formulas (1) -1 to 3, R ¹ and R ² represent one of them represents a hydroxyalkyl group having 1 to 6 carbon atoms and the other represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. That is, when R ¹ is a hydroxyalkyl group having 1 to 6 carbon atoms, R ² is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and when R ² is a hydroxyalkyl group having 1 to 6 carbon atoms, R ¹ is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. By using a compound having a 6- or 5-membered heterocycle containing a nitrogen atom and a hydroxyl group linked by an alkyl group near the nitrogen atom as a derivatization reagent, the organic acid can be efficiently ionized. Furthermore, since the LC retention time of the obtained derivative (esterified product) is relatively long, it can be clearly separated from the reagent used for derivatization.

The alkyl group having 1 to 6 carbon atoms may be a linear alkyl group or a branched-chain alkyl group. Specifically, methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, i-butyl group, s-butyl group, t-butyl group, n-pentyl group, n-hexyl. Group etc. can be mentioned. As R ¹ or R ² in the general formula (1) -1 to 3, a methyl group, an ethyl group, an n-propyl group, or an i-propyl group is preferable.

The hydroxyalkyl group having 1 to 6 carbon atoms may be a group in which one hydrogen atom in a linear alkyl group having 1 to 6 carbon atoms is substituted with a hydroxyl group, and the hydroxyalkyl group has 1 to 6 carbon atoms. It may be a group in which one hydrogen atom in the branched alkyl group of is substituted with a hydroxyl group. Specific examples thereof include a 2-hydroxyethyl group, a 3-hydroxypropyl group, a 4-hydroxybutyl group, a 5-hydroxypentyl group, a 6-hydroxyhexyl group and the like.

The derivatization reagent represented by the general formula (1) is one or more selected from the group consisting of 2-pyridinemethanol, 1-piperidineethanol, and 2- (2-hydroxyethyl) -1-methylpyrrolidine. It is preferable to have. By esterifying the carboxyl group with these compounds, organic acids having similar structures such as 3-HIB, 3-HB, 3-HMB and 2-HB can be well separated by LC. As a result, simultaneous detection of both (detection by one LC-P-ESI-MS / MS for one sample) becomes possible. Among the derivatizing reagents represented by the general formula (1), 2-pyridine is used because the retention time is slow and the effect of the derivatizing reagent on the peak of the derivatized organic acid ester to be detected is small. Methanol is particularly preferred.

In the detection method according to the present invention, the LC-P-ESI-MS / MS method can be carried out by a conventional method except that the compound represented by the general formula (1) is used as the derivatization reagent. Specifically, HPLC or MS in LC-MS / MS can be performed in the same manner as known conditions for detecting a carboxylic acid ester, or under appropriately modified conditions.

In the detection method according to the present invention, only one kind of organic acid may be detected in one LC-P-ESI-MS / MS, or two or more kinds of organic acids may be detected at the same time. The organic acid to be detected in the detection method according to the present invention is not particularly limited as long as it is a compound having a carboxyl group, and the detection method is applicable to the detection of many organic acids having a carboxyl group. it can. The detection method according to the present invention is preferably used for the detection and quantification of organic acids which are intermediate metabolites such as glycolysis system, TCA cycle and fatty acid synthesis system. Above all, the detection method according to the present invention is preferably used for detecting an organic acid represented by the following general formula (2).

In the general formula (2), R ³ represents a hydroxyalkyl group having 2 to 6 carbon atoms. The hydroxyalkyl group having 2 to 6 carbon atoms may be a branched-chain hydroxyalkyl group or a linear hydroxyalkyl group. Examples of the hydroxyalkyl group include 2-hydroxyethyl group, 3-hydroxypropyl group, 2-hydroxypropyl group, 4-hydroxybutyl group, 2-hydroxybutyl group, 2- (hydroxymethyl) propyl group and 2-. Examples thereof include a hydroxypentyl group, 3-hydroxy-2, 2-dimethylpropyl group, 3-hydroxypentyl group, 5-hydroxypentyl group, 6-hydroxyhexyl group, 2-hydroxyhexyl group and the like.

As the organic acid represented by the general formula (2), R ³ is preferably a hydroxyalkyl group having 2 to 3 carbon atoms. In particular, the organic acid represented by the general formula (2) is preferably one or more selected from the group consisting of 3-HIB, 3-HB, 3-HMB, and 2-HB, and 3-. It is more preferable to detect HB and 3-HIB, or 3-HIB, 3-HB and 3-HMB at the same time. 3-HB is a marker of fatty acid catabolism in the liver, and 3-HIB and 3-HMB can be markers of BCAA catabolism in skeletal muscle. That is, according to the detection method according to the present invention, a marker for fatty acid catabolism in the liver and a marker for BCAA catabolism in skeletal muscle can be detected at the same time.

In the detection method according to the present invention, as in the case of other detection methods using LC-MS / MS, the organic acid to be detected is quantitatively detected based on a calibration curve prepared using an organic acid having a known concentration. can do. When preparing the calibration curve, it is also preferable to add a certain amount of stable isotope to all the dilution series of the organic acid having a known concentration in order to improve the quantitativeness.

Further, in the detection of an organic acid in a biological sample, the conventional analysis method often requires more biological samples due to insufficient sensitivity, or the content is too small to detect in the first place. On the other hand, since the detection method according to the present invention has extremely high detection sensitivity, it is possible to detect the target organic acid from a smaller amount of biological sample than before. In addition, it is possible to detect organic acids in biological samples that could not be detected in the past because the content is extremely small.

For example, the detection sensitivity when 3-HIB is detected by the detection method according to the present invention is 100 times or more as compared with the case where 3-HIB is detected by LC-N-ESI-MS / MS without derivatization. .. In addition, the sensitivity is 10 times or more higher than that when a derivative obtained by derivatizing 3-HIB with HCl-butanol is detected by positive ESI-LC-MS / MS. Therefore, the detection method according to the present invention includes simultaneous quantification of 3-HIB and 3-HB, simultaneous quantification of 3-HIB, 3-HB, 3-HMB and 2-HB, and 3-HB even in saliva of 5 μL or less. Simultaneous quantification of HIB, 3-HB, 3-HMB, 2-HB and lactic acid is possible.

In muscle, metabolism of leucine produces 3-HMB, and metabolism of valine produces 3-HIB. That is, the 3-HMB concentration and 3-HIB concentration in the living body reflect BCAA catabolism in muscle, and 3-HMB and 3-HIB function as amino acid metabolism markers reflecting BCAA catabolism in muscle. .. In addition, when threonine, methionine, and fatty acids are metabolized in the liver, 2-HB and 3-HB are produced. That is, 2-HB and 3-HB function as metabolic pathway markers or fat metabolism markers for threonine and methionine. In addition, when glucose is metabolized, lactic acid is produced. That is, lactic acid functions as a glucose metabolism marker.

The degree of BCAA catabolism in skeletal muscle, that is, the 3-HMB concentration and 3-HIB concentration in vivo depends on, for example, liver cirrhosis, hepatic encephalopathy, myitis, sarcopenia, heart failure, and the use of anticancer agents. Patients suffering from diseases such as plague, rhabdomyolysis, disused muscle atrophy, hyperthyroidism, chronic fatigue syndrome, sarcopenia (age-related muscle weakness), mitochondrial fatty acid dysoxidation, dialysis An index (biomarker) of hyponutrition and muscle atrophy for patients, surgically invasive patients, elderly people, etc. who have decreased or may have decreased blood albumin concentration or blood BCAA concentration. can do. It can also be used as an index of undernutrition and muscle protein degradation for athletes such as marathons and triathlons, who are prone to promote muscle protein catabolism.

Since the 3-HIB concentration in saliva can be used as an index of nutritional status, the 3-HIB concentration in saliva is a marker indicating, for example, drug discovery screening, particularly the pharmacological effect of a therapeutic agent candidate for various diseases described above. It is also expected to be used as a marker for toxicity evaluation in safety tests of active ingredients.

As energy expenditure increases due to exercise, fatty acid catabolism in the liver increases, and the amount of 3-HB in body fluids (blood, saliva) increases. In addition, in a state where BCAA catabolism of skeletal muscle is enhanced, the amount of 3-HIB and the amount of 3-HMB increase. Therefore, the 3-HIB concentration, 3-HB concentration, and 3-HMB concentration in saliva evaluate the energy supply source balance (dependence of energy metabolic tissue and nutrient supply) of the liver and skeletal muscle during or after exercise. It can be an index (biomarker) to be used. If the 3-HB concentration and 3-HIB concentration in saliva do not change much from rest during or after exercise, it can be evaluated that sugar is used as an energy supply source. When the 3-HB concentration or 3-HIB concentration in saliva is significantly higher than that at rest during or after exercise, it is said that energy is produced by fatty acid catabolism in the liver or BCAA catabolism in skeletal muscle. Can be evaluated. As a biomarker for evaluating the use of skeletal muscle BCAA as an energy supply source during or after exercise, the concentration of 3-HIB in saliva is preferable to 3-HMB. Both 3-HIB and 3-HB concentrations in saliva need to be measured as biomarkers for assessing energy production by different tissues and different nutrient sources in the liver and skeletal muscle during or after exercise. ..

Specifically, according to the method for preparing a saliva sample according to the present invention, 3-HIB and / or 3-HB is detected from a saliva sample prepared from saliva collected from a subject during exercise by the detection method according to the present invention. The energy supply source during exercise in the subject can be evaluated by detecting and measuring the 3-HIB concentration and / or 3-HB concentration and comparing the concentration with a preset predetermined threshold. The threshold value is preferably set for each subject, but it is set from the result of measuring the 3-HIB concentration and the like after performing exercise that is expected to have the same exercise load for a large number of subjects. May be good.

In addition, saliva samples are prepared from the subject over time from immediately before the start of exercise to after a certain period of time after the end of exercise, and the 3-HIB concentration and / or 3-HB concentration in each saliva sample is determined in the present invention. By measuring by such a detection method, it is possible to monitor the 3-HIB concentration and / or the 3-HB concentration in saliva (examine the change with time). Based on the time course of 3-HIB concentration and / or 3-HB concentration obtained as a result of monitoring, the energy supply source during exercise in the subject can be evaluated. If the patient is not at rest (normal state) immediately before the start of exercise, a saliva sample is prepared in advance from the resting subject, and the 3-HIB concentration and / or 3-HB concentration in each saliva sample is determined in the present invention. It is preferable to measure by the detection method according to the above, and to use the obtained measured value as a reference for evaluation of the energy supply source during exercise.

Insufficient rest and nutrition, in addition to excessive exercise, results in an excessive relative exercise load, a so-called overwork condition. Undernutrition, such as during starvation, activates the increased ketone body production obtained by fatty acid catabolism in the liver and the increased energy production by BCAA catabolism obtained by protein degradation in skeletal muscle, resulting in 3-HB in saliva. Concentration and 3-HIB concentration increase. In particular, endurance athletes and athletes and enthusiasts who are losing weight or restricting their diet are more dependent on using amino acids in skeletal muscle as an energy source because the amount of lipid stored as an energy source after sugar consumption is small. It gets higher. By increasing or sustaining the utilization of such skeletal muscle amino acids, muscle protein decomposition is promoted, resulting in a systemic energy metabolism deficiency state accompanied by symptoms such as skeletal muscle atrophy. In many of these cases, we are in a so-called overwork state. In such an overwork state, by measuring the 3-HB concentration and 3-HIB concentration in saliva, the energy supply source balance and dependence due to the ketone body derived from fatty acid catabolism in the liver and BCAA catabolism derived from skeletal muscle protein Since it can be evaluated, it can be an index of overwork status. For example, the 3-HIB concentration and 3-HB concentration at rest (normal state) when not exercising, and the 3-HIB concentration and 3-HB concentration in saliva after exercising to the extent that it does not cause overwork. Was measured by the detection method according to the present invention, and from these measured values, the 3-HIB concentration and 3-HB concentration in the overwork state and the 3-HIB concentration and 3-HB concentration in the non-overwork state were obtained. The threshold for dividing (overwork threshold) is set in advance. The 3-HIB and 3-HB concentrations in the saliva sample prepared from the subject after excessive repeated exercise that causes overwork are detected by the detection method according to the present invention, and the 3-HIB concentration in the saliva is detected. And 3-HB concentrations are measured and the concentration is compared to a preset overwork threshold to assess the energy supply source balance in the subject. Specifically, when the 3-HIB concentration and 3-HB concentration of the subject are lower than the overwork threshold, the exercise load of the subject is appropriate, and it is highly possible that the subject is not overwork. When the 3-HIB concentration of the subject is equal to or higher than the overwork threshold value, it can be evaluated that the exercise load of the subject is excessive and the subject is likely to be overworked. Further, when only the 3-HB concentration of the subject is equal to or higher than the threshold value, it can be evaluated that there is a high possibility that the subject has not yet reached overwork even if the subject is in an energy deficient state. The overwork threshold is preferably set for each subject, but is set from the result of measuring the 3-HIB concentration or 3-HB concentration after exercising to the extent that it does not cause overwork for a large number of healthy subjects. May be good. By preparing saliva samples from exercising subjects over time and measuring and monitoring 3-HIB and 3-HB concentrations over time, exercise can be terminated before a dangerous overwork condition occurs. it can. In this case, sampling from the subject does not necessarily have to be during exercise, and the results obtained from the sample taken from the rest of the subject undergoing a long-term exercise program or training are the same before the exercise program and before training. It can also be used to evaluate the overwork state by comparing it with the results obtained from the samples collected from the subjects. That is, by monitoring the 3-HIB concentration and 3-HB concentration during or for a long period of time by the method according to the present invention, an appropriate amount of exercise can be evaluated and an overwork state can be prevented.

Further, for example, by measuring the concentration of 3-HIB or 3-HB in a saliva sample prepared from one animal individual over time by the detection method according to the present invention, BCAA catabolism of skeletal muscle and fatty acids in the liver can be measured. It is also possible to monitor the state of catabolism. 3-HIB concentration monitoring and 3-HB concentration monitoring are, for example, the administration of BCAA preparations and the start of nutrition therapy for the purpose of preventing skeletal muscle atrophy and improving malnutrition in patients with liver cirrhosis, malnutrition or bedridden. It can be used to determine the presence or absence of necessity and to determine the treatment of them. For example, if there is no change in 3-HB concentration but only 3-HIB concentration tends to increase, the ability to produce low energy in the liver due to decreased liver function is skeletal muscle protein catabolism. It can be evaluated that there is a high possibility that the liver is undernourished by compensating for the production of energy derived from amino acids.

In addition, by measuring the concentrations of 3-HIB and 3-HB by the detection method according to the present invention, the energy source-dependent balance (energy metabolism state) of lipids and amino acids in the liver and skeletal muscle in the field of sports medicine can be determined. , It is also possible to monitor using saliva. Furthermore, in the fields of weight management and cosmetology by anti-aging and dieting, the metabolic state of sugars, amino acids and fatty acids is measured by analyzing saliva by the detection method according to the present invention and measuring the concentrations of 3-HIB and 3-HB. It is also possible to effectively manage nutrition, exercise, and physical condition while evaluating. Further, the detection method according to the present invention can also be used for evaluation of nutritional / ingestion effect when a supplement containing nutrients such as BCAA or a nutritional drink is ingested.

In addition, in patients with liver cirrhosis, the supply of sugar from the liver to skeletal muscle decreases due to the decrease in liver function, resulting in starvation and increased amino acid catabolism. Therefore, the concentration of 3-HIB, 3-HMB, and 2-HB in the body fluid tends to be higher in the liver cirrhosis patient group than in the healthy subject group. Therefore, 3-HIB, 3-HMB, and 2-HB can be used as biomarkers for liver cirrhosis. Specifically, a threshold value for separating a cirrhosis patient and a healthy person is set in advance, and the concentrations of 3-HIB, 3-HMB, and 2-HB in the saliva of the subject are measured by the detection method according to the present invention. , The possibility of developing liver cirrhosis can be evaluated by comparing the concentration with a predetermined threshold value. Specifically, when the concentration of 3-HIB or the like in the saliva of the subject is equal to or higher than a predetermined threshold value, it can be evaluated that the subject is highly likely to have cirrhosis. .. The threshold for separating cirrhosis patients and healthy subjects can be set experimentally. For example, the detection method according to the present invention is used for saliva collected from a population known not to develop cirrhosis and saliva collected from a population known to develop cirrhosis. It can be appropriately set by measuring the concentration of 3-HIB or the like in these saliva and comparing the measured values of both populations. As a biomarker for liver cirrhosis, 3-HIB or 3-HMB is preferable, and since the difference between the liver cirrhosis patient group and the healthy subject group is clearer, 3-HIB in saliva or 3-HMB in saliva is more preferable. preferable.

When cirrhosis progresses and the urea cycle, which is one of the liver functions, declines, ammonia is compensatoryly metabolized to glutamine and alanine in skeletal muscle and detoxified. At that time, glutamic acid produced as a by-product in the process of catabolizing BCAA to branched chain α-keto acid by BCAT in skeletal muscle is used for ammonia detoxification. Therefore, it is necessary to enhance skeletal muscle BCAA catabolism during ammonia detoxification, which is compensated by skeletal muscle during liver cirrhosis, and as a result, the 3-HIB concentration in body fluid increases. It is thought that encephalopathy is more likely to be induced when ammonia detoxification is insufficient and the concentration of ammonia in body fluids is high. Therefore, in addition to the decrease in ammonia detoxification ability in the liver of patients with liver cirrhosis, the decrease in ammonia detoxification compensation ability mediated by skeletal muscle BCAA catabolism is considered to cause hepatic encephalopathy. For this reason, 3-HIB can be used as a biomarker for hepatic encephalopathy or its potential to develop, and can provide useful information for its prevention and early treatment. For example, for patients with liver cirrhosis who are evaluated to have a high risk of developing hepatic encephalopathy based on 3-HIB concentration, a protein-restricted diet is recommended or BCAA intake is recommended even if encephalopathy is not manifested. It may be possible to prevent the onset of hepatic encephalopathy by making recommendations.

For example, when the 3-HIB concentration in saliva collected from a liver cirrhosis patient over time is monitored, if the 3-HIB concentration tends to decrease, the liver cirrhosis patient is likely to develop hepatic encephalopathy. .. Therefore, the 3-HIB concentration in saliva collected from a subject suffering from hepatic cirrhosis was measured by the detection method according to the present invention, and the obtained 3-HIB concentration was measured before the time when the saliva was collected. The possibility of developing hepatic encephalopathy can be evaluated by comparing with the 3-HIB concentration in saliva collected from the subject. Specifically, when the obtained 3-HIB concentration is significantly lower than the 3-HIB concentration of previously collected saliva, or as a result of monitoring, it is higher than the 3-HIB concentration of previously collected saliva. If the tendency to decrease continues to some extent, it is evaluated that the subject is likely to develop hepatic encephalopathy.

Whether or not the 3-HIB concentration in a liver cirrhosis patient is decreased can also be evaluated by using a threshold value that separates a liver cirrhosis patient and a healthy person. Specifically, the 3-HIB concentration in saliva collected from a subject developing liver cirrhosis is measured by the detection method according to the present invention, and the obtained 3-HIB concentration is set to a predetermined threshold value (for example, The possibility of developing hepatic encephalopathy can be evaluated by comparing with the above-mentioned threshold value for separating a patient with liver cirrhosis and a healthy person). Specifically, the subject develops hepatic encephalopathy when the 3-HIB concentration of the subject is less than a predetermined threshold value or when the state of being below the predetermined threshold value continues to some extent. It can be evaluated that there is a high possibility of doing so.

In addition, as described above, 3-HIB concentration in biological samples such as saliva and serum can be a predictor of sarcopenia, but since sarcopenia is due to long-term changes, an increase in 3-HIB concentration is observed. It may be difficult to do. In addition, since the 3-HIB concentration is also affected by the nutritional status and metabolic status at the time of measurement, long-term changes may be overlooked because of short-term effects. By preloading BCAA (oral administration) before measurement, the difference in 3-HIB concentration between healthy subjects and sarcopenia patients or their reserve group can be clarified more clearly. For example, in healthy subjects, even when BCAA preparations are taken orally three times a day for two weeks, the 3-HIB concentration in blood does not change much compared to before ingestion, but the energy dependence of BCAA in skeletal muscle is high. In the subject, the 3-HIB concentration in blood or saliva increases after ingesting BCAA as compared with before ingesting BCAA. Therefore, the 3-HIB concentration in saliva or blood before and after the BCAA load is measured, and when the 3-HIB concentration is increased by the BCAA load, BCAA catabolism in skeletal muscle is enhanced, which is sarcopenia. It can be predicted that there is a high probability or that there is a high risk of developing sarcopenia.

When BCAA loading is performed, the valine concentration should be used as a reference, and the ratio of 3-HIB concentration to valine concentration should be used as a biomarker in consideration of the effects of intestinal absorption of ingested BCAA and changes in blood concentration. Is preferable. By evaluating the degree of increase in 3-HIB concentration due to BCAA intake based on the degree of increase in valine concentration, more reliable results can be expected.

3-HMB can be used as a marker for protein synthesis in liver and skeletal muscle derived from BCAA and a marker for skeletal muscle hypertrophy, and 2-HB is a marker for early insulin resistance and glucose intolerance. It is also useful as a marker for colorectal cancer. Lactate is used to determine metabolic acidosis (lactic acidosis) and is elevated during hypoxia, circulatory failure, poorly managed diabetes, liver failure, and skeletal muscle spasm, resulting in glycogen storage disease and lactate dehydrogenase deficiency. It becomes low at the time of illness. Lactic acid is also used as a marker for exercise load (exercise intensity). By using the detection method according to the present invention, these biomarkers can be detected with high sensitivity.

Since NAD is used as a coenzyme for alcohol degradation in the liver, the NAD / NADH ratio in the liver is low. This decrease in the NAD / NADH ratio reduces fatty acid catabolism (β-oxidation) in the liver, so that alcohol intake causes fatty liver and obesity. Since NAD is also used for metabolism of α-ketobutyric acid into propionyl-CoA in mitochondria, when the NAD / NADH ratio decreases, α-ketobutyric acid is not metabolized in mitochondria, and α-ketobutyric acid is 2 in the cytoplasm. -It is thought that the rate of metabolism to HB is high. Therefore, the amount of 2-HB is higher in alcoholic fatty liver (ASH) patients than in non-alcoholic fatty liver (NASH) patients, and 2-HB is a biomarker for distinguishing between NASH and ASH. Can be used.

The detection method according to the present invention can simultaneously quantify 3-HIB, 3-HMB, 2-HB, 3-HB, and lactic acid, which are energy metabolism markers of amino acids, fats, or sugars, from a trace amount of saliva. Therefore, by using the detection method according to the present invention, the state of energy metabolism of the subject can be analyzed in more detail. By analyzing the amount of these organic acids, the metabolic balance of sugars, fats, and amino acids in the elderly and the like can be investigated.

The detection method according to the present invention is also useful for evaluating the degree of fatigue accumulation. For example, humans and other mammals use sugar as their main energy source at rest, but become more dependent on lipids and amino acids during exercise. Resting after exercise restores the pre-exercise energy source balance, which uses sugar as the main energy source, and recovers from fatigue. By applying an exercise load before the energy supply source balance returns to the resting state, the energy supply source balance cannot return from the state of high dependence on amino acids and fat to the resting state before exercise. By simultaneously detecting 3-HIB, 3-HMB, 2-HB, 3-HB, and lactic acid by the detection method according to the present invention, the degree of energy metabolism of amino acids, fats, and sugars is evaluated to balance the energy supply source. Can be examined, and the degree of accumulation of fatigue can be examined.

Next, the present invention will be described in more detail with reference to Examples and the like, but the present invention is not limited thereto.

<Reagents>
The reagents used in the following examples and reference examples are as follows. As the D-3-HB, L-3-HB, and 3-HIB sodium salts, those manufactured by Sigma-Aldrich Chemical Co., Ltd. were used. ^DL-3-HB- 13 _{C 4} sodium salt was used made of Taiyo Nippon Sanso Corporation. 2-Pyridinemethanol, 2-methyl-6-nitrobenzoic acid anhydride, 1-piperidinethanol, 2- (2-hydroxyethyl) -1-methylpyrrolidin, 3-pyridinemethanol, 2-diethylaminoethanol, 3-hydroxy- As 1-methylpiperidine and 4-pyridinemethanol, those manufactured by Tokyo Kasei Kogyo Co., Ltd. were used, and as 4-dimethylaminopyridine, those manufactured by Wako Pure Chemical Industries, Ltd. were used. As the 3-HB dehydrogenase, one manufactured by Roche Diagnostics was used.

<Preparation of serum sample>
The serum samples used in the following reference examples were prepared as follows.
Serum was collected from blood collected from a healthy person and stored at −20 ° C. until the time of use for measurement. Serum (10 [mu] L), placed in a 1.5mL centrifuge plastic tube, further acetonitrile / water (1/19 containing ^DL-3-HB- 13 _{C 4} sodium salt of 769pmol (100ng) as an internal standard, volume ratio ) Solution (100 μL) was added and stirred for 1 minute, and then centrifuged at 2000 × g for 1 minute. The supernatant was placed in a water / acetonitrile (1/19, volume ratio) solution, deproteinized, and then the liquid phase was recovered and evaporated to dryness at 80 ° C. under a nitrogen gas spray.

<Preparation of 3-HB-free serum>
The 3-HB-free serum used in the following reference examples was prepared as follows.
3-HB-free serum is prepared by adding normal serum (10 μL) to 150 mM Tris-HCl (pH 8.6) supplemented with 1.8 mM NAD and 0.075 U 3-hydroxybutyric acid dehydrogenase at 37 ° C. Prepared by incubating for 30 minutes in.

<Esterification reaction of organic acids using 2-pyridinemethanol as a derivatization reagent>
In the following reference example, the esterification reaction of an organic acid using 2-pyridinemethanol as a derivatization reagent was prepared as follows.
The esterification reaction of an organic acid using 2-pyridinemethanol as a derivatization reagent was carried out by improving the method for synthesizing a carboxy ester by Siina et al. (See Non-Patent Document 13). Specifically, a mixed reagent for 2PM derivatization consisting of 2-methyl-6-nitrobenzoic anhydride (67 mg), 4-dimethylaminopyridine (20 mg), pyridine (900 μL), and 2-pyridinemethanol (100 μL). Was used. First, the reaction solution prepared by adding the 2PM derivatization mixed reagent (50 μL) prepared immediately before to a dried sample containing an organic acid was allowed to stand at room temperature for 30 minutes. Then, n-hexane (1 mL) was added to the reaction solution, and the mixture was stirred for 30 seconds, centrifuged at 700 × g for 1 minute, and the recovered supernatant was evaporated at 80 ° C. under a nitrogen gas spray. The residue was redissolved in a 1 volume% aqueous solution of formic acid (150 μL), centrifuged again at 7000 × g for 1 minute, and the recovered supernatant was subjected to ESI-LC-MS / MS as a measurement sample.

<LC-P-ESI-MS / MS>
The LC-MS / MS system is a triple quadrupole mass spectrometer TSQ Vantage (Thermo Fisher Scientific) equipped with a HESI-II probe and a Prominence Ultra Fast Liquid Chromatography (UFLC) system (manufactured by Shimadzu Corporation). .. Separation in chromatography was performed at 40 ° C. using a Hypersil GOLD aQ column (2.1 × 150 mm, 3 μm, manufactured by Thermo Fisher Scientific). First, the mobile phase was flowed as acetonitrile / water (1/19, volume ratio) containing 0.2% by volume of formic acid at a flow rate of 300 μL / min for 5 minutes. After 5 minutes, the mobile phase was changed to acetonitrile containing 0.2% by volume of formic acid and allowed to flow at a flow rate of 300 μL / min for 7 minutes. General MS / MS conditions are as follows;
Spray voltage: 3000V,
Atomizer temperature: 450 ° C,
Sheath gas (nitrogen) pressure: 50 psi,
Auxiliary gas (nitrogen) flow rate: 15 arbitrary units,
Ion transfer capillary temperature: 220 ° C,
Collision gas (argon) pressure: 1.0 mTorr,
Collision energy: 15V,
Ion polarity: Positive mode.

FIG. 1B shows a typical P-ESIMS spectrum of 3-HB (2PM-3-HB) derivatized with 2-pyridinemethanol. 2PM-3-HB is found at m / z 196 with [M + H] ⁺ ions as the base peak. In the product ion MS spectrum (FIG. 1A) when this m / z 196 is used as the precursor ion, the fragment ion of [C ₅ H ₄ NCH ₂ OH + H] ⁺ is observed at m / z 110 as the most prominent peak. SRM is performed using m / z 196 → m / z 110 for the detection of 2PM-3-HB, and m / z 200 → m / z 110 for the detection of the [ ¹³ C ₄ ] isotope (internal standard). Was performed using.

<Creation of calibration curve>
To create the calibration curve, add a standard storage solution (200 ng / μL) in which D-3-HB sodium salt is dissolved in acetonitrile / water (1/19, volume ratio), and then acetonitrile / water (1/19). A standard solution of D-3-HB sodium (1 to 200 ng / 100 μL) having a known concentration was used, which was prepared by appropriately diluting with (volume ratio). Each standard solution as an internal standard, were added ^DL-3-HB- 13 _{C 4} sodium salt (100 ng), after the mixture was evaporated to dryness and the obtained by the derivatization (esterification with derivatives) , ESI-LC-MS / MS. To examine the matrix effect of the calibration curve, 3-HB-free serum was added to each standard solution as a blank matrix.

<LC-N-ESI-MS / MS>
LC-N-ESI-MS / MS of 3-HB was carried out using the same LC-MS / MS system used in LC-P-ESI-MS / MS without derivatization. Separation in chromatography was performed at 40 ° C. using a Hypersil GOLD aQ column (2.1 × 150 mm, 3 μm, manufactured by Thermo Fisher Scientific). For the mobile phase, methanol / water (1/9, volume ratio) containing 0.1% by volume of formic acid was flowed at a flow rate of 200 μL / min. General MS / MS conditions are as follows;
Spray voltage: 2500V,
Atomizer temperature: 450 ° C,
Sheath gas (nitrogen) pressure: 50 psi,
Auxiliary gas (nitrogen) flow rate: 15 arbitrary units,
Ion transfer capillary temperature: 220 ° C,
Collision gas (argon) pressure: 1.0 mTorr,
Collision energy: 15V,
Ion polarity: Negative mode,
3-HB SRM (selective reaction monitoring): m / z 103 → m / z 59,
[ ¹³ C ₄ ] SRM: m / z 107 → m / z 61.

<Statistical processing>
The numerical values in the following examples and reference examples are mean ± SD (standard deviation). The linearity of the calibration curve was analyzed by simple regression analysis. Reproducibility was analyzed by one-way analysis of variance (one-way ANOVA) (JMP software, manufactured by SAS Institute). An estimated ± 95% confidence limit was obtained as an accuracy index (see Non-Patent Document 14). In order to calculate the value, orthogonal regression analysis was performed in the reproducibility test using JMP software. The statistical significance of the differences between the different groups was assessed by Student's two-tailed t-test. In all analyzes, P <0.05 was considered significant.

[Reference example 1]
3-HB and 3-HIB derivatized with 2-pyridinemethanol were detected by LC-P-ESI-MS / MS. Specifically, a sample solution prepared by dissolving only D-3-HB sodium salt (standard, 1 μg) in acetonitrile / water (1/19, volume ratio) (100 μL), D-3-HIB sodium salt (standard). A sample solution in which only the product (1 μg) was dissolved, and a sample solution in which D-3-HB sodium salt (standard product, 1 μg) and D-3-HIB sodium salt (standard product, 1 μg) were dissolved were prepared. After each was evaporated and dried, it was derivatized (esterified with a derivative) with the above-mentioned mixed reagent for 2PM derivatization. Finally, it is redissolved in 1% by volume of formic acid aqueous solution (1 mL), and 1 μL (equivalent to 1 ng each of D-3-HB sodium salt and D-3-HIB sodium salt) is added to LC-P-ESI- as described above. Measured by MS / MS.

The results of detecting 3-HB and 3-HIB derivatized with 2-pyridinemethanol by LC-P-ESI-MS / MS are shown in FIG. FIG. 2A shows the total ion chromatogram (upper) and MS chromatogram (lower) of the sample containing only the derivatized 3-HB, FIG. 2B shows the MS spectrum of FIG. 2A, and FIG. 2C shows the derivatized 3 The total ion chromatogram (upper) and MS chromatogram (lower) of the sample containing only −HIB, the MS spectrum of FIG. 2C in FIG. 2D, and 3-HB and 3-HIB derivatized in FIG. 2E are included. The product ion chromatogram when m / z 196 of the sample is used as the precursor ion is shown, and FIG. 2F shows the MS / MS spectrum of the 3-HIB peak of FIG. 2E.

As a result, although the sample used for the measurement contained only 1 ng, both the 3-HB and 3-HIB derivatives could be spectrally analyzed in the low-sensitivity and high-information scan mode. It was. Moreover, in the LC chromatogram, the separation of the 3-HB derivative and the 3-HIB derivative was good. In addition, since the LC retention time is slow, the effect on the peak of the 3-HB derivative or 3-HIB derivative of the derivatization reagent or the like (for example, 4-dimethylaminopyridine) that first comes out from the column is very large. It was small. That is, from these results, it was found that organic acids such as 3-HB and 3-HIB can be detected with high accuracy and high sensitivity by derivatization with 2-pyridinemethanol.

[Reference example 2]
3-HB and 3-HIB derivatized with 1-piperidine ethanol were detected by LC-P-ESI-MS / MS. Specifically, as the derivatization reagent, instead of the mixed reagent for 2PM derivatization, 2-methyl-6-nitrobenzoic acid anhydride (67 mg), 4-dimethylaminopyridine (20 mg), pyridine (900 μL), and Derivatization was carried out in the same manner as in Reference Example 1 except that a 1PE derivatization mixed reagent consisting of 1-piperidin ethanol (100 μL) was used, and the measurement was performed by LC-P-ESI-MS / MS.

FIG. 3 shows the results of detecting 3-HB and 3-HIB derivatized with 1-piperidine ethanol by LC-P-ESI-MS / MS. 3A shows the total ion chromatogram (upper) and MS chromatogram (lower) of the sample containing only the derivatized 3-HB, FIG. 3B shows the MS spectrum of FIG. 3A, and FIG. 3C shows the derivatized 3 The total ion chromatogram (upper) and MS chromatogram (lower) of the sample containing only −HIB, the MS spectrum of FIG. 3C in FIG. 3D, and 3-HB and 3-HIB derivatized in FIG. 3E are included. The product ion chromatogram when m / z 216 of the sample is used as the precursor ion is shown, and FIG. 3F shows the MS / MS spectrum of the 3-HIB peak of FIG. 3E.

As a result, although the sample used for the measurement contained only 1 ng, both the 3-HB and 3-HIB derivatives could be spectrally analyzed in the low-sensitivity and high-information scan mode. It was. Moreover, in the LC chromatogram, the separation of the 3-HB derivative and the 3-HIB derivative was good. In addition, the peaks of both the 3-HB derivative and the 3-HIB derivative had good S / N in the product ion chromatogram, and the sensitivity was particularly good. That is, from these results, it was found that organic acids such as 3-HB and 3-HIB can be detected with high accuracy and high sensitivity by derivatization with 1-pyridineethanol.

[Reference example 3]
3-HB and 3-HIB derivatized with 2- (2-hydroxyethyl) -1-methylpyrrolidine were detected by LC-P-ESI-MS / MS. Specifically, as the derivatization reagent, instead of the mixed reagent for 2PM derivatization, 2-methyl-6-nitrobenzoic acid anhydride (67 mg), 4-dimethylaminopyridine (20 mg), pyridine (900 μL), and Derivatized in the same manner as in Reference Example 1 except that a mixed reagent for 1PE derivatization consisting of 2- (2-hydroxyethyl) -1-methylpyrrolidin (100 μL) was used, and by LC-P-ESI-MS / MS. It was measured.

The results of detecting 3-HB and 3-HIB derivatized with 2- (2-hydroxyethyl) -1-methylpyrrolidine by LC-P-ESI-MS / MS are shown in FIG. FIG. 4A shows the total ion chromatogram (upper) and MS chromatogram (lower) of the sample containing only the derivatized 3-HB, FIG. 4B shows the MS spectrum of FIG. 4A, and FIG. 4C shows the derivatized 3 The total ion chromatogram (upper) and MS chromatogram (lower) of the sample containing only −HIB, the MS spectrum of FIG. 4C in FIG. 4D, and 3-HB and 3-HIB derivatized in FIG. 4E are included. The product ion chromatogram when m / z 216 of the sample is used as the precursor ion is shown, and FIG. 4F shows the MS / MS spectrum of the 3-HIB peak of FIG. 4E.

As a result, although the sample used for the measurement contained only 1 ng, both the 3-HB and 3-HIB derivatives could be spectrally analyzed in the low-sensitivity and high-information scan mode. It was. Moreover, in the LC chromatogram, the separation of the 3-HB derivative and the 3-HIB derivative was good. In addition, the peaks of both the 3-HB derivative and the 3-HIB derivative had good S / N in the product ion chromatogram, and the sensitivity was particularly good. That is, from these results, it was found that by derivatizing with 2- (2-hydroxyethyl) -1-methylpyrrolidine, organic acids such as 3-HB and 3-HIB can be detected with high accuracy and high sensitivity. ..

[Reference example 4]
The sample added ^DL-3-HB- 13 _{C 4} sodium salt as an internal standard serum harvested 10μL from healthy subjects, in the same manner as in Reference Example 1 was derivatized with 2-pyridinemethanol, LC-P-ESI -Measured by MS / MS. FIG. 5 shows typical SRM chromatograms of 3-HB (2PM-3-HB) and [ ¹³ C ₄ ] isotopes derivatized with 2-pyridinemethanol. From the chromatogram, the peak area area ratio of 2PM-3-HB to the [ ¹³ C ₄ ] isotope was calculated, and the ratio was applied to the calibration curve to determine the 3-HB concentration in serum. The peak of 2PM-3-HB in the chromatogram corresponded to 5.1 pmol (76 μM). The peak seen at 4.53 minutes of chromatogram A was identified as a 2PM ester derivative of 3-HIB by comparison with the 2PM-3HIB standard.

[Reference example 5]
3-HB, 3-HIB, 3-HMB, and 2-HB derivatized with 2-pyridinemethanol were detected by LC-P-ESI-MS / MS. Specifically, D-3-HB sodium salt (standard, 30 ng), D-3-HIB sodium salt (standard, 30 ng) and D in acetonitrile / water (1/19, volume ratio) (100 μL). -3-HMB sodium salt (preparation, 30 ng) and D-2-HB sodium salt (preparation, 30 ng) and ^DL-3-HB- 13 _{C 4} sodium salt (preparation, 80 ng) sample solution obtained by dissolving Was prepared, evaporated and dried, and then derivatized (esterified with a derivative) with the above-mentioned mixed reagent for 2PM derivatization. Finally, it is redissolved in 1% by volume of formic acid aqueous solution (1 mL), and 1 μL (D-3-HB sodium salt, D-3-HIB sodium salt, D-3-HMB sodium salt, D-2-HB sodium salt) is dissolved. There corresponds 3ng _{^{respectively, DL-3-HB- 13 C}} 4 sodium salt of 8ng equivalent), it was measured by LC-P-ESI-MS / MS as described previously.

The results of detecting 3-HB, 3-HIB, 3-HMB, and 2-HB derivatized with 2-pyridinemethanol by LC-P-ESI-MS / MS are shown in FIG. The total ion chromatogram of the sample is in the upper part of FIG. 6, the product ion chromatogram when m / z 196 is used as the precursor ion in the second stage from the top, and m / z 200 is used as the precursor ion in the third stage from the top. The product ion chromatogram at the time of preparation is shown, and the product ion chromatogram when m / z 210 is used as a precursor ion is shown at the bottom.

As a result, although the sample used for the measurement contained only 3 ng, 2 PM of 3-HB, 3-HIB, 3-HMB, and 2-HB in the scan mode with low sensitivity and high information amount. All of the ester derivatives could be spectrally analyzed. Moreover, in the LC chromatogram, the separation of the 3-HB derivative, the 3-HIB derivative, the 3-HMB derivative and the 2-HB derivative was good. That is, from these results, it was found that by derivatizing with 2-pyridinemethanol, structurally similar 3-HB, 3-HIB, 3-HMB, and 2-HB can be simultaneously detected with high accuracy and high sensitivity. ..

[Reference example 6]
The stability of 3-HIB and 3-HB in serum and saliva was examined when the serum and saliva collected from the subject were left at room temperature for a certain period of time.

<Serum>
Blood was collected from healthy subjects in 6 blood collection tubes. One of the six blood collection tubes was centrifuged immediately after blood collection, and the collected serum was cryopreserved at −20 ° C. The remaining 5 sera were left at room temperature for 1, 2, 4, 6 or 24 hours, respectively, and then centrifuged, and the collected sera were stored at −20 ° C.
The concentration of 3-HIB and 3-HB in each serum after storage at -20 ° C., using a ^DL-3-HB- 13 _{C 4} sodium salt as an internal standard, using 2-pyridinemethanol as a derivatizing reagent , Measured by LC-P-ESI-MS / MS in the same manner as in Reference Example 4. From the chromatogram, calculate the peak area area ratio of 2PM-3-HB to the [ ¹³ C ₄ ] isotope, apply the ratio to the calibration curve, and determine the 3-HB concentration and 3-HIB concentration in serum. did. The measurement results are shown in FIG. As a result, the 3-HB concentration and 3-HIB concentration in the serum decreased until 6 hours after the blood was collected and left at room temperature, but did not change until 24 hours thereafter.

<Saliva>
Saliva collected (saliva) from a healthy subject into one tube was dispensed into six tubes. One of the six tubes was cryopreserved at −20 ° C. immediately after collection. The remaining 5 bottles were left at room temperature for 1, 2, 4, 6 or 24 hours, respectively, and then stored at −20 ° C. The cryopreserved saliva sample was naturally thawed at room temperature at the time of analysis. After mixing using a vortex mixer, the mixture was centrifuged (3000 rpm, 15 minutes) to decompose the viscous protein (mucin), precipitated together with food debris contained in saliva, and the supernatant was used for analysis.
The concentration of 3-HIB and 3-HB in the saliva after storage at -20 ° C., using a ^DL-3-HB- 13 _{C 4} sodium salt as an internal standard, using 2-pyridinemethanol as a derivatizing reagent , Measured by LC-P-ESI-MS / MS in the same manner as in Reference Example 4. From the chromatogram, calculate the peak area area ratio of 2PM-3-HB to the [ ¹³ C ₄ ] isotope, apply the ratio to the calibration curve, and determine the 3-HB concentration and 3-HIB concentration in saliva. did. The measurement results are shown in FIG. As a result, the 3-HB concentration and 3-HIB concentration in saliva were almost stable regardless of the time left at room temperature after collection.

[Reference Example 7]
The correlation between the serum collected from the subject and the concentrations of 3-HIB, 3-HB, and 2-HB in saliva (saliva) was examined. Specifically, serum and saliva were collected from 3 healthy subjects (subjects α, β, γ), 5 samples each, and the concentrations of 3-HIB, 3-HB, and 2-HB were measured. It was measured and the correlation between serum concentration and saliva concentration was investigated. The concentrations of 3-HIB, 3-HB, and 2-HB in serum and saliva were measured in the same manner as in Reference Example 6. The measurement result of 3-HB concentration is shown in FIG. 9A, the measurement result of 3-HIB concentration is shown in FIG. 9B, and the measurement result of 2-HB concentration is shown in FIG. 9C. As a result, it was confirmed that the saliva concentration correlates with the serum concentration in all of 3-HIB, 3-HB, and 2-HB.

[Reference Example 8]
The 3-HIB and 3-HB concentrations in serum, saliva, and urine before and after running exercise were measured, and changes over time were observed.
Specifically, a subject (healthy male subject) was jogging for 60 minutes (mileage: about 9 km, speed: about 6.7) after collecting urine for 24 hours from 11:00 am on the previous day to 11:00 am on the day of running exercise. Minutes / km), and then 24 hours (12:00 noon on the day after the running exercise day) was used as the experimental period. The subjects ingested lunch and supper on the previous day, lunch and supper on the day of running exercise, and breakfast on the next day from 11:00 am on the previous day to the end of the experimental period. FIG. 10 shows the subject's exercise and dietary status, and the time points for collecting blood, saliva, and urine. The 60-minute jogging performed was a slightly higher load exercise than the exercise routinely performed for the subject.

The 3-HIB concentration and 3-HB concentration in serum, saliva, and urine were measured in the same manner as in Reference Example 4. The measurement results of 3-HIB and 3-HB in each collected sample are shown in FIGS. 11 to 13. FIG. 11 shows the time course of the concentrations of 3-HIB and 3-HB in serum. FIG. 12 shows the time course of the urine excretion amount ([urine excretion amount] / [elapsed time from the previous collection]) (μmol / h) per unit time at each collection time of 3-HIB and 3-HB. Each is shown. FIG. 13 shows changes over time in the concentrations of 3-HIB and 3-HB in saliva. The lower part of FIG. 13 is an enlarged view of the vertical axis scale showing the measurement result during the running motion of the upper part.
As a result, by derivatizing with 2-pyridinemethanol and performing LC-P-ESI-MS / MS, both 3-HIB and 3-HB were obtained in all of serum, saliva and urine collected from the subjects. It was confirmed that it could be detected.

The concentration of 3-HB increased in both serum and saliva due to running exercise, but the serum 3-HB concentration increased as it was after running exercise, and the saliva 3-HB concentration was increased. Although it decreased immediately after the running exercise, it increased again after that (Fig. 11 and FIG. 13, upper row). From these results, it was confirmed that the 3-HB concentration in saliva is increased by exercise as well as the 3-HB concentration in serum which is increased by exercise. In addition, after exercise, the 3-HB concentration in both serum and saliva continues to rise until food is ingested, so post-exercise hepatic fatty acid catabolism (fat burning) is applied to both blood and saliva samples. I was also able to confirm that it can be monitored.

On the other hand, the concentration of 3-HIB in both serum and saliva increases due to running exercise, and after a certain period of time has passed after the end of exercise, the concentration rapidly decreases and returns to the normal state before exercise. Was confirmed (FIG. 11 and 13 upper row). The skeletal muscle BCAA catabolism does not persist after exercise compared to during exercise, and in healthy individuals, only transient exercise does not result in catabolism of skeletal muscle proteolytic BCAAs or free BCAAs. confirmed. In particular, it was found that the 3-HIB concentration in saliva hardly fluctuated from the time when 30 minutes passed after the end of exercise to the time when the end of the experimental period, and was not easily affected by food and the like. On the other hand, the serum 3-HIB concentration decreased to a normal state 1 hour after the end of exercise, but slightly increased 4 hours after the end of exercise. Since the diet was consumed 2.25 hours after the end of the exercise, it is possible that the increase in serum 3-HIB concentration 4 hours after the end of the exercise was slightly affected by the diet. Was done.

From these results, since 3-HB concentration and 3-HIB concentration in serum and saliva are increased by exercise, they are simultaneously evaluated as liver fatty acid catabolism (fat burning) marker and skeletal muscle BCAA catabolism marker by exercise. It is possible, useful as a marker for evaluating the energy metabolism state (and thus the balance of energy supply sources) by tissue (liver and skeletal muscle) during or after exercise, and is not easily affected by diet, etc. From this, it was found that for 3-HIB, the concentration in saliva is preferable to the concentration in serum as a marker.

As shown in the lower part of FIG. 12, the 3-HIB concentration in saliva during running exercise does not change much from the start of exercise (normal state) until 15 minutes after the start of exercise, but tends to increase gradually thereafter. Was observed. The 3-HB concentration in saliva during running exercise does not change much from the start of exercise (normal state) until 35 minutes after the start of exercise, but after that, it rises more rapidly than the 3-HIB concentration. A tendency was observed. Sugar is preferentially used as an energy source during exercise, but fat and amino acids are used as an energy source as exercise time and intensity increase. From these results, the increase in 3-HB concentration and 3-HIB concentration indicates that fat and amino acids were used as energy sources instead of sugar, and when both increased, the dependence on energy production Is considered to indicate the point of transition from glucose metabolism to lipid and amino acid metabolism. In recent years, it has been suggested that amino acids are used as an energy source in addition to sugars and lipids from the early stage of exercise, but the result that 3-HIB starts to rise earlier than 3-HB is that amino acids It is considered that metabolism is also activated from the early stage of exercise. In this reference example, the 3-HIB concentration in saliva dropped to a steady state immediately after the end of running exercise, but when the exercise load became too excessive, the so-called overwork state became a steady state even after the end of exercise. It is presumed that it will be difficult to return to.

FIG. 12 shows the amount of 3-HB excreted in urine and the amount of 3-HIB excreted per unit time before and after exercise. Unlike serum and saliva, exercise did not increase urinary 3-HB excretion and 3-HIB excretion, and urinary 3-HIB excretion tended to decrease (1 hour after exercise). ). The amount of 3-HB excreted in urine then tended to increase until 3 hours of exercise, then decreased once, but then increased again until 12.5 hours of exercise. The amount of 3-HIB excreted in urine increased up to 9.5 hours of exercise with repeated slight increases and decreases. Neither urinary 3-HB nor 3-HIB excretion was associated with changes in serum or saliva concentrations, suggesting that reabsorption in renal tissue had an effect. When urine is used as a sample, considering the need for correction with urine, livestock urine, or creatinine at any time, both 3-HB and 3-HIB are markers for evaluating the energy metabolism status of each tissue during and after exercise. It was found that evaluation by serum or saliva is preferable to the amount of excretion in urine.

[Reference example 9]
Blood and saliva were collected from 15 healthy subjects and 20 patients with liver cirrhosis, and 3-HIB and 3-HMB concentrations in serum and saliva were measured and compared. The 3-HIB concentration and 3-HMB concentration in serum and saliva were measured in the same manner as in Reference Example 6.
The measurement results of 3-HIB concentration and 3-HMB concentration in serum are shown in FIG. 14A, and the measurement results of 3-HIB concentration and 3-HMB concentration in saliva are shown in FIG. 14B, respectively. As a result, it was observed that the 3-HIB concentration and 3-HMB concentration in the liver cirrhosis patient group tended to be higher than those in the healthy subject group in both saliva and serum, indicating that they are useful as liver cirrhosis markers. Was done. In particular, the 3-HIB concentration and 3-HMB concentration in saliva were significantly higher in the liver cirrhosis patient group than in the healthy subject group.

[Example 1]
After immersing one end of a strip-shaped filter paper in the saliva collected in a container to absorb saliva by capillarity, and then using a dried saliva sample, 3-HIB, 3-HB, 2- The contents (concentrations) of HB and 3-HMB were measured, and the variation between saliva samples was examined.

Specifically, first, the hypersalivation collected from one subject was divided into 10 tubes. Each tube is immersed in a region up to 10 mm from the tip of Schirmer filter paper (manufactured by Alcon Laboratories) (the end on the region side where it comes into contact with saliva), and saliva is sucked up to about 25 mm from the tip by capillary action. After that, the Schirmer filter paper was allowed to dry at room temperature (20 to 25 ° C.) for 20 minutes. The dried Schirmer filter paper is cut at three points, 10 mm, 15 mm, and 20 mm from the tip, and a fragment from the tip to 10 mm (collection site (1)) and a distance from the tip. Obtained a fragment from 10 mm to 15 mm (collection site (2)) and a fragment from the tip portion from 15 mm to 20 mm (collection site (3)). That is, the collection site (1) is a site that is directly contacted with saliva and absorbed, and the collection sites (2) and (3) are not in direct contact with saliva, and the capillary phenomenon from the collection site (1). It is the part that sucked up saliva.

FIG. 15 shows a schematic view of the Schirmer filter paper 1 used. In FIG. 15, the tip end portion (the end portion on the region side in contact with saliva) of the Schirmer filter paper 1 is the left end portion. Further, in the figure, the area of "(1)" is the collection site (1) (the area from the tip of Schirmer filter paper 1 to 10 mm), and the area of "(2)" is the collection site (2) (Schirmer filter paper). The distance from the tip of 1 is 10 mm to 15 mm), and the area of "(3)" is the collection site (3) (the distance from the tip of Schirmer filter paper 1 is 15 mm to 20 mm). is there. In addition, the tip of saliva sucked up by Schirmer filter paper 1 is indicated by an arrowhead.

For the collection site (1), collection site (2), and collection site (3) cut from 10 Sylmer filter papers, each fragment was added to 100 μL of an internal standard-containing acetonitrile solution (100 μL of acetonitrile in 150 ng of DL-. 3-HB- ¹³ C ₄ sodium salt solution prepared by dissolving (preparation)) to about 1 minute, by immersing while mixing with a vortex mixer, the organic acid of saliva from that had been present in each fragment Extracted. The acetonitrile solution from which the organic acid was extracted was recovered as a sample for detecting the organic acid.

Each organic acid detection sample is evaporated and dried, and then derivatized (esterified with a derivative) with the above-mentioned 2PM derivatization mixed reagent in the same manner as in Reference Example 4, and finally 1 volume is used. Resolve in% aqueous formic acid solution (1 mL) and measure 1 μL by LC-P-ESI-MS / MS to determine the content (concentration) of 3-HIB, 3-HB, 2-HB, and 3-HMB. I asked.

The concentrations of each organic acid in the 10 collection sites (1) were compared, and the variation (coefficient of variation,% CV) was calculated. Similarly, the variation in the concentration of each organic acid in the 10 collection sites (2) and the variation in the concentration of each organic acid in the 10 collection sites (3) were calculated, respectively. The calculation result is shown in FIG. As a result, among all four types of organic acids, the collection site (3) had the largest variation, and the collection site (1) and the collection site (2) had the same variation. From these results, when a strip-shaped or stick-shaped filter paper such as Schirmer filter paper is used as a saliva absorbing member and saliva is sucked up from one end by a capillary phenomenon, a fragment containing a region near the tip of the sucked saliva. It was found that using a fragment containing a region farther from the tip of the sucked saliva suppresses the variation between the samples and gives more reliable detection results.

[Example 2]
After absorbing saliva by capillarity by the subject holding one end of the Sylmer filter paper in the mouth, using a dried saliva sample, 3-HIB, 3-HB, 2-HB, and 3 -The content (concentration) of HMB was measured and the variation between saliva samples was examined.
Specifically, first, the other areas (collection site (2) and collection site (3)) so that the area in the oral cavity that comes into direct contact with saliva is only the area from the tip to 10 mm (collection site (1)). ))) Was covered with silver paper, and 10 Schirmer filter papers (manufactured by Alcon Laboratories) were prepared. For these 10 Schirmer filter papers, one by one, the area from the tip not covered with silver paper to 10 mm is brought into contact with the same subject's tongue for about 5 seconds, and saliva is sucked up and absorbed by capillary action. After that, it was dried by leaving it at room temperature (20 to 25 ° C.) for 20 minutes.
The silver paper is removed from the 10 Schirmer filter papers after drying, and in the same manner as in Example 1, the pieces are cut at 3 points of 10 mm, 15 mm and 20 mm from the tip, and fragments from the tip to 10 mm. (Collection site (1)), fragments with a distance from the tip of 10 mm to 15 mm (collection site (2)), fragments with a distance from the tip of 15 mm to 20 mm (collection site (3)) The contents (concentrations) of 3-HIB, 3-HB, 2-HB, and 3-HMB were determined from the obtained, collected site (1), collected site (2), and collected site (3).

Variations in the concentration of each organic acid in the 10 collection sites (1) (coefficient of variation,% CV), variations in the concentration of each organic acid in the 10 collection sites (2), and 10 collection sites (3) ), The variation in the concentration of each organic acid was calculated. The calculation result is shown in FIG. As a result, among all four types of organic acids, the collection site (3) having the shortest distance from the tip of the sucked saliva had the largest variation, and the collection site (2) had the smallest variation. In addition, there was a tendency for the variation to be larger than in Example 1. The collection site (2) that was not in direct contact with saliva had less variation than the collection site (1) that was in direct contact with saliva because the amount of contact with saliva in the mouth was sparse. Therefore, although saliva permeates the inside of the filter paper toward the site (3) after collection, it is considered that the influence of the amount of direct contact with saliva may remain. The collection site (2) was more likely to contain more uniform saliva than the collection site (1).

[Example 3]
The organic acid concentration of the saliva sample dried by impregnating the tip of the Sylmer filter paper with 5 μL of saliva collected in a container was measured, and the effect of the drying time on the measured value of the organic acid concentration was investigated.
Specifically, 5 μL of each of the salivation was dropped into a range of 10 mm from the tip of nine Schirmer filter papers (manufactured by Alcon Laboratories), and then naturally dried by leaving at room temperature (20 to 25 ° C.). .. The drying time was 20 minutes for 5 of the 9 Schirmer filter papers, 1 day for 2 sheets, and 2 days for 2 sheets.

From the dried Sylmer filter paper, 10 mm fragments (areas impregnated with saliva) were cut out from the tip, and each fragment was divided into 100 μL of an internal standard-containing acetonitrile solution (100 μL of acetonitrile and 150 ng of DL in the same manner as in Example 1. -3-HB- ¹³ C ₄ is immersed in a sodium salt solution obtained by dissolving (preparation)), the acetonitrile solution was extracted and the organic acid is recovered as a sample for detection organic acids, DL of the detection in a sample each organic acid ^-3-HB- 13 _{C 4} sodium salt, 3-HB, and detects a 3-HMB, was determined and their content (ng / 5μL). The content of ^DL-3-HB- 13 _{C 4} sodium salt 18, 19 the content of 3-HB, in FIG. 20 the content of 3-HMB, respectively. In FIGS. 18 to 20, the concentration shown by the dotted line is DL-3-HB- ¹³ C ₄ measured in the same manner as a mixed solution obtained by mixing 5 μL of the same salivation with 100 μL of the acetonitrile solution containing the internal standard as a sample for detecting organic acids. The contents (ng / 5 μL) (actual concentration) (n = 4) of sodium salt, 3-HB, and 3-HMB are shown.

As a result, the concentration of DL-3-HB- ¹³ C ₄ sodium salt is much less than the actual concentration in the case the drying time is 20 minutes, close to the actual concentration as the drying time becomes longer (Fig. 17 ). It was presumed that this was because a large amount of water remained in the filter paper when the standing (drying) time was short, and the acetonitrile solution containing the internal standard did not sufficiently soak into the filter paper. In addition, the concentrations of 3-HB and 3-HMB were much higher than the actual concentrations when the drying time was 20 minutes, but were close to the actual concentrations in the samples having a drying time of 1 to 2 days. It became the concentration. It is presumed that the reason why the concentrations of 3-HB and 3-HMB were calculated higher than the directly measured values when the drying time was 20 minutes was that the internal standard values were measured low. From these results, the saliva sample collected by absorbing it in a filter paper is measured after it has been sufficiently dried and then subjected to measurement, which is almost the same as the value obtained by directly measuring organic acids such as 3-HB in saliva. It was confirmed that the results were obtained.

[Example 4]
Using filter papers made of three different materials as saliva absorbing members, the effect of the materials of the saliva absorbing members on the measured values of organic acids was investigated.
Specifically, as a saliva absorbing member, Schirmer filter paper (manufactured by Alcon Laboratories), GF / B filter paper made of glass fiber (manufactured by Whatman), and SOS (SALIMETRICS ORAL SWAB) filter paper made of synthetic resin fiber (manufactured by SALIMETRICS). ) Was used. After dropping 5 μL of the saliva collected from one subject into a container on each filter paper, the mixture was allowed to air dry at room temperature (20 to 25 ° C.) for 20 minutes. In the case of Schirmer filter paper, the area 10 mm from the tip is cut out (n = 5), in the case of GF / B filter paper, it is divided into 8 equal parts (n = 5), and in the case of SOS filter paper, the height. The cylinder cut so that the size was 5 mm was cut in half (n = 5), and the saliva was added dropwise to each of the fragments.

The solution in the same manner as in Example 1, in which each filter paper fragments after drying 100 [mu] L of internal standard-containing acetonitrile solution (100 [mu] L of acetonitrile to dissolve the ^DL-3-HB- 13 _{C 4} sodium salt of 150 ng (preparation) ) the soaked, the acetonitrile solution was extracted and the organic acid is recovered as a sample for detection organic _{^{acid, DL-3-HB- 13 C}} 4 sodium salt of the organic acid for detection in a sample, 3-HB, 3-HIB , 3-HMB and 2-HB were detected, and their contents (ng / 5 μL) were determined. Further, the concentration (actual concentration) of each organic acid was measured in the same manner using a mixed solution prepared by mixing 5 μL of the same saliva with 100 μL of the acetonitrile solution containing the internal standard as a sample for detecting organic acids (n = 4) (saliva direct). Measurement). The content of ^DL-3-HB- 13 _{C 4} sodium salt 21, 22 the content of 3-HB, in Figure 23 the content of 3-HIB, the content of 3-HMB 24 The content of 2-HB is shown in FIG. 25, respectively. As a result, of the three types of filter paper, the SOS filter paper has the same internal standard measurement value as the direct saliva measurement value even if the natural drying time is 20 minutes, and any of the four types of organic acids in saliva Was able to be measured almost accurately.

[Reference Example 10]
The deuterium isotopes of lactic acid and D-lactic acid in the saliva of healthy subjects were derivatized with 2-pyridinemethanol and then detected by LC-P-ESI-MS / MS.
First, the saliva collected from a healthy person is frozen at -20 ° C, then thawed, and then centrifuged (3000 rpm, 15 minutes) to decompose the viscous protein (mucin) and contain it in saliva. It was precipitated together with the food waste and the like to obtain a supernatant. The obtained saliva supernatant was used for analysis.
Then, in acetonitrile / water (1/19, volume ratio) (100 μL), D-lactate (standard, 100 ng) or the saliva supernatant (5 μL) and the deuterium isotope of D-lactate (standard, D) A sample solution in which -lactic acid-d ₃ , 500 ng) was dissolved was prepared, evaporated and dried, and then derivatized (esterified with a derivative) with the above-mentioned mixed reagent for 2PM derivatization. Finally, it is redissolved in a 1 volume% aqueous solution of formic acid (100 μL), and 5 μL (equivalent to solid content in 250 nL of saliva, equivalent to 5 ng of D-lactic acid, equivalent to 25 ng of D-lactic acid-d ₃ ) is added as described above. Measured by LC-P-ESI-MS / MS.

The results detected by LC-P-ESI-MS / MS are shown in FIG. FIG. 26A shows a product using m / z 182.1 as a precursor ion of a sample containing lactic acid (2PM-Lactate) derivatized with 2-pyridinemethanol and a deuterium isotope (2PM-Lactate-d ₃ ). Ion chromatograms are shown in FIG. 26B and the MS / MS spectrum of the 2PM-Lactate peak in FIG. 26A, respectively. Further, in FIG. 26C, the SRM chromatograms of 2 PM-Lactate and 2 PM-Lactate-d ₃ in the case of using the preparation D- lactic acid, in FIG. 26D, and 2 PM-Lactate in the case of using a saliva supernatant 2PM The SRM chromatogram of -Lactate-d ₃ is shown. The lactic acid concentration in saliva can be determined by calculating the peak area area ratio with respect to 2PM-Lactate-d ₃ from the chromatogram and applying the ratio to the calibration curve. As a result, although the sample used for the measurement contained only lactic acid derived from 5 μL of saliva, lactic acid was derivatized with 2-pyridinemethanol in a scan mode with low sensitivity and high information content. Was able to be spectrally analyzed. Moreover, in the LC chromatogram, the 2PM ester derivative of lactic acid could be clearly separated and analyzed from the 2PM ester derivatives of 3-HB, 3-HIB, 3-HMB, and 2-HB. From these results, by derivatizing with 2-pyridinemethanol and performing LC-P-ESI-MS / MS, trace amounts of lactic acid in saliva can be removed with structurally similar 3-HB, 3-HIB, 3-HMB, And 2-HB, it was found that simultaneous detection can be performed with high accuracy and high sensitivity.

[Reference Example 11]
The storage stability of 3-HB, 3-HIB, 3-HMB, 2-HB, and lactic acid in saliva (saliva) collected from the subject was examined at room temperature.
Specifically, saliva collected (saliva) from a healthy subject into one tube was dispensed into seven tubes. One of the seven tubes was cryopreserved at −20 ° C. immediately after collection. The remaining 6 bottles were left at room temperature for 1, 2, 4, 6, 10 or 24 hours, respectively, and then stored at −20 ° C. The cryopreserved saliva sample was naturally thawed at room temperature at the time of analysis. After mixing using a vortex mixer, the mixture was centrifuged (3000 rpm, 15 minutes) to decompose the viscous protein (mucin), precipitated together with food debris contained in saliva, and the supernatant was used for analysis.

3-HB of the saliva after storage at -20 ℃, 3-HIB, 3 -HMB, 2-HB, and the concentration of lactic _{^{acid, DL-3-HB- 13 C}} 4 sodium salt and as an internal standard D- It was measured by LC-P-ESI-MS / MS in the same manner as in Reference Example 4 using lactic acid-d ₃ and 2-pyridinemethanol as a derivatization reagent. From the chromatogram, calculate the peak area area ratio of 2PM-3-HB to the [ ¹³ C ₄ ] isotope and D-lactic acid-d ₃ , apply the ratio to the calibration curve, and apply the 3-HB concentration in saliva. , 3-HIB concentration, 3-HMB concentration, 2-HB concentration, and lactic acid concentration were determined. The measurement result is shown in FIG. As a result, 3-HB, 3-HIB, 3-HMB, 2-HB, and lactic acid could be separated and quantitatively detected from the saliva sample. In addition, the 3-HB concentration and 3-HIB concentration in saliva are almost stable regardless of the time left at room temperature after collection, and the 3-HMB concentration is stable up to 6 hours of storage at room temperature. A gradual decreasing tendency was observed after 10 hours of storage at room temperature. On the other hand, the 2-HB concentration and the lactic acid concentration in saliva decreased sharply after 1 hour of storage at room temperature, and it was confirmed that the storage stability at room temperature was low.

[Reference Example 12]
To saliva collected by the saliva method, sodium oxamate, which is an LDH (lactate dehydrogenase) inhibitor, was added to a final concentration of 200 μM, and the same as in Reference Example 11 for 1 to 24 hours at room temperature. The effect of the LDH inhibitor on the decrease of 2-HB concentration and lactic acid concentration in saliva when left at room temperature was investigated. As a result, although the decreasing tendency of the lactic acid concentration in saliva was slightly slower than that in the case where sodium oxamate was not added, the lactic acid concentration was still decreasing with time. In addition, the 2-HB concentration in saliva was not different between the case where sodium oxamate was added and the case where sodium oxamate was not added (data not shown).
Further, even when the final concentration of sodium oxamate to be added is increased to 500 μM, 1 mM, 5 mM, or 10 mM and the sample is examined after being left for 24 hours, the 2-HB concentration in saliva and The decrease in lactic acid concentration could not be suppressed (data not shown).

Antibacterial agent "Clear Bath®" (manufactured by Specrum Laboratories) containing formic acid, sodium hydroxide, hydrochloric acid, or benzalkonium chloride as an active ingredient in saliva (salting) collected from a subject, composition: 50 Mass% benzalkonium chloride, 10 mass% ethanol, 40 mass% water) was added, and the storage stability of 3-HB, 3-HIB, 3-HMB, 2-HB, and lactic acid at room temperature was examined.
Specifically, saliva collected (saliva) from a healthy subject into one tube was dispensed into eight tubes by 500 μL per tube. One of the eight tubes was cryopreserved at −20 ° C. immediately after collection. Nothing was added to one of the remaining seven, and the remaining six were 0.001% by volume caustic aqueous solution, 0.01% by volume caustic aqueous solution, 1% by volume caustic aqueous solution, and 0.001N water, respectively. A 1/100 times amount (5 μL) of each of an aqueous sodium oxide solution, 0.001N hydrochloric acid, or a 0.1% by volume Clear Bath aqueous solution was immediately mixed, left at room temperature for 24 hours, and then stored at −20 ° C. When the pH of each saliva was measured using pH test paper, the pH of saliva to which a 1% by volume aqueous solution of formic acid was added was about 3 to 4, and the pH of other saliva was about 6 to 8. It was sex. The cryopreserved saliva sample was naturally thawed at room temperature at the time of analysis. After mixing using a vortex mixer, centrifugation (3000 rpm, 15 minutes) is performed to decompose the viscous protein (mucin) and precipitate it together with food debris contained in saliva, and the obtained saliva supernatant is analyzed. Used for.

The concentrations of 3-HB, 3-HIB, 3-HMB, 2-HB, and lactic acid in each saliva after storage at −20 ° C. were measured in the same manner as in Reference Example 11. The measurement result is shown in FIG. As a result, in saliva left at room temperature for 24 hours, the concentrations of 3-HMB, 2-HB, and lactic acid were remarkably decreased, and the concentration of 3-HB was increased, but 0.1% by volume Clear. In saliva to which Bath was added, the concentrations of 2-HB, lactic acid, and 3-HMB hardly decreased, and the concentration of 3-HB did not increase even after being left at room temperature for 24 hours, and the saliva was left at room temperature. The concentration was about the same as that of no saliva. It was also found that saliva to which a 1% by volume formic acid aqueous solution having an acidic pH was added also suppressed a decrease in the concentration of 3-HMB, 2-HB, and lactic acid and an increase in the concentration of 3-HMB. In saliva to which other additives were added, a decrease in the concentration of 3-HMB, 2-HB, and lactic acid and an increase in the concentration of 3-HMB were observed when left at room temperature, as in the case where no additive was added. From these results, it can be said that the storage stability of 3-HB, 3-HMB, 2-HB, and lactic acid in saliva can be improved by adding an antibacterial agent to saliva or adjusting the pH of saliva to be acidic. all right. Moreover, since the decrease in the concentration of 2-HB and lactic acid was suppressed by the addition of the antibacterial agent, it was presumed that the decrease in the amount of 2-HB and the amount of lactic acid due to leaving saliva at room temperature was due to the decomposition by oral bacteria.

Further, when acetonitrile / water (1/19, volume ratio) was mixed with the saliva supernatant as a sample to be derivatized with the above-mentioned 2PM derivatization mixed reagent, the saliva supernatant to which no additive was added became turbid. However, in the saliva supernatant to which 0.1% by volume Clear Bath aqueous solution was added, coagulation and precipitation occurred, and the liquid part was transparent. It was speculated that the turbidity of saliva was mainly bacteria, and that the saliva became transparent as a result of being killed by the addition of an antibacterial agent, agglutinating and precipitating.

[Reference Example 13]
The correlation between the serum collected from the subject and the concentration of lactic acid in saliva (saliva) was investigated. Specifically, the concentrations of these lactic acids were measured for blood and saliva collected simultaneously from one healthy person on different days, and the correlation between the serum concentration and the saliva concentration was investigated.
Blood was collected in a blood collection tube containing a serum separating agent, and after coagulation, serum was collected by centrifugation and stored at −20 ° C. until analysis. After collecting saliva by the saliva method, 5 μL of 0.1% Clear Bath aqueous solution was immediately mixed with 500 μL, and the saliva was stored at −20 ° C. until analysis. The concentrations of lactic acid in serum and saliva were measured in the same manner as in Reference Example 11. The measurement result is shown in FIG. As a result, it was confirmed that the salivary concentration of lactic acid correlates with the serum concentration.

1 ... Schirmer filter paper

Claims (19)

An absorption process that allows saliva to be absorbed by the saliva absorbing member,
In the absorption step, a drying step of drying the saliva absorbing member that has absorbed saliva, and a drying step.
Have,
In the absorption step, the saliva absorbing member absorbs a predetermined amount of saliva, and after the drying step, a region in which a predetermined amount of saliva-derived solid content is adsorbed from the saliva absorbing member is formed. By cutting and collecting, a saliva sample in which a predetermined amount of solids derived from saliva is held in a saliva absorbing member in a dry state is prepared.
The saliva absorbing member is in the shape of a strip or a rod,
In the absorption step, one end of the saliva absorbing member is brought into contact with saliva, and saliva is sucked up by a capillary phenomenon so that the saliva absorbing member absorbs saliva.
After the drying step, in the region where the saliva-derived solid content of the saliva absorbing member is adsorbed, from a portion 8 mm or more away from the tip of the sucked saliva toward the end side in contact with saliva. A method for preparing a saliva sample, which comprises collecting a saliva sample obtained by cutting a region of a predetermined area or volume. The method for preparing a saliva sample according to claim 1 , wherein the saliva is stored in a saliva container in a liquid state. The method for preparing a saliva sample according to claim 1 or 2, wherein the absorption step is performed by bringing the saliva absorbing member into contact with saliva in the oral cavity of a subject. The saliva absorbing member is in the shape of a strip or a rod,
In the absorption step, one end of the saliva absorbing member is put into the mouth of the subject, and saliva is sucked up by the capillary phenomenon so that the saliva absorbing member absorbs saliva.
After the drying step, among the regions where saliva-derived solids of the saliva absorbing member are adsorbed, the regions that are not in direct contact with saliva in the oral cavity of the subject and the tip of the sucked saliva. The saliva sample according to claim 3, wherein a saliva sample obtained by cutting a region of a predetermined surface integral or volume from a portion separated from the saliva by 8 mm or more toward the end side in contact with saliva is collected. Preparation method. The method for preparing a saliva sample according to any one of claims 1 to 4 , wherein an antibacterial agent is contained in a saliva absorbing member that has absorbed saliva in the absorbing step before the drying step. The method for preparing a saliva sample according to claim 1 or 2 , wherein in the absorption step, a mixture of saliva and an antibacterial agent is absorbed by the saliva absorbing member. The method for preparing a saliva sample according to any one of claims 1 to 4 , wherein the saliva absorbing member holds an antibacterial agent in advance. An absorption process that allows saliva to be absorbed by the saliva absorbing member,
An antibacterial agent treatment step of immersing a saliva absorbing member that has absorbed saliva in the absorption step in a solution containing an antibacterial agent,
Have,
In the absorption step, the saliva absorbing member absorbs a predetermined amount of saliva, and after the absorption step, a region in which a predetermined amount of saliva-derived solid content is adsorbed is cut from the saliva absorbing member. By immersing the recovered product in a solution containing the antibacterial agent, a saliva sample, which is a solution containing a predetermined amount of saliva-derived solids and a saliva absorbing member, is prepared.
The saliva absorbing member is in the shape of a strip or a rod,
In the absorption step, one end of the saliva absorbing member is brought into contact with saliva, and saliva is sucked up by a capillary phenomenon so that the saliva absorbing member absorbs saliva.
After the drying step, in the region where the saliva-derived solid content of the saliva absorbing member is adsorbed, from a portion 8 mm or more away from the tip of the sucked saliva toward the end side in contact with saliva. A method for preparing a saliva sample, which comprises collecting a saliva sample obtained by cutting a region of a predetermined area or volume. The method for preparing a saliva sample according to any one of claims 5 to 8 , wherein the antibacterial agent is a quaternary ammonium salt-based antibacterial agent. The method for preparing a saliva sample according to any one of claims 1 to 9 , wherein the amount of saliva absorbed by the saliva absorbing member in the absorption step is the maximum amount of saliva absorbed by the saliva absorbing member. The method for preparing a saliva sample according to any one of claims 1 to 10 , wherein the predetermined amount is 1 to 50 μL. The method for preparing a saliva sample according to any one of claims 1 to 11 , wherein the saliva absorbing member is made of a synthetic resin fiber. The method for preparing a saliva sample according to any one of claims 1 to 7 , wherein the drying in the drying step is natural drying. A saliva sample is prepared by the method for preparing a saliva sample according to any one of claims 1 to 13 .
An extraction step of extracting the organic acid derived from saliva from the saliva sample by immersing the obtained saliva sample in a polar organic solvent.
After the extraction step, a recovery step of separating and recovering the polar organic solvent from which the organic acid has been extracted from the saliva sample as a saliva-derived organic acid detection sample,
The organic acid in the saliva-derived organic acid detection sample recovered by the recovery step is represented by the following general formula (1) -1 to (1) -3.

(In formulas (1) -1 to 3, R ¹ and R ² represent one of them represents a hydroxyalkyl group having 1 to 6 carbon atoms and the other represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.)
A derivative is synthesized by ester-bonding with a derivatization reagent represented by any of the above, and the derivative is detected by the electrospray ionization LC-MS / MS (liquid chromatography-tandem mass spectrometry) method in the positive ion mode. Detection process and
A method for detecting an organic acid in saliva, which comprises having. The method for detecting an organic acid in saliva according to claim 14 , wherein the polar organic solvent is acetonitrile. The method for detecting an organic acid in saliva according to claim 14 or 15 , wherein in the extraction step, the saliva sample is immersed in a polar organic solvent containing an internal standard substance in advance. Any of claims 14 to 16 , wherein the derivatization reagent is at least one selected from the group consisting of 2-pyridinemethanol, 1-piperidineethanol, and 2- (2-hydroxyethyl) -1-methylpyrrolidine. The method for detecting an organic acid in saliva according to item 1. Any of claims 14 to 17 , wherein the organic acid is at least one selected from the group consisting of 3-hydroxybutyric acid, 3-hydroxyisobutyric acid, 3-hydroxyisovaleric acid, 2-hydroxybutyric acid, and lactic acid. The method for detecting an organic acid in saliva according to item 1. The method for detecting an organic acid in saliva according to any one of claims 14 to 18 is performed on a plurality of saliva samples prepared over time from the same subject, and the organic acid in the saliva of the subject is performed. A method for measuring changes in organic acid concentration over time, which comprises examining changes in concentration over time.

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