阅读说明：本技术 一种检测人体干尿滤纸片中多种有机酸的定量检测方法 (Quantitative detection method for detecting multiple organic acids in human body dry urine filter paper sheet ) 是由 邬智刚 朱文漓 叶尚宇 于 2020-06-11 设计创作，主要内容包括：本发明涉及一种同步定量检测人体干尿滤纸片中多种有机酸含量的方法。该方法使用滤纸片收集尿液,经过洗脱、纯化、采用液相色谱与质谱联用方法对目标有机酸进行同步定性定量分析。本发明方法具有如下优点：1.本发明采用滤纸片收集尿液标本,降低样本的保存和运输条件,对样本的运输、保存无特殊需求,样本保存时间长,可以很好的扩大该检测方法的适用范围。通过邮寄的方式收集样本进行检测,利于大规模商业化使用该检测方法；2.本发明提供的人尿液中有机酸代谢产物检测方法具有灵敏度高、精确度高、重复性好以及检测速度快的优点,适用于功能性评估特定营养素的需求,在此基础上创建个性化治疗方案是改善患者症状的最佳选择。(The invention relates to a method for synchronously and quantitatively detecting the contents of various organic acids in a human body dry urine filter paper sheet. The method uses a filter paper sheet to collect urine, and performs synchronous qualitative and quantitative analysis on target organic acid by eluting, purifying and adopting a liquid chromatography-mass spectrometry combined method. The method of the invention has the following advantages: 1. the invention adopts the filter paper sheet to collect the urine specimen, reduces the storage and transportation conditions of the specimen, has no special requirements on the transportation and storage of the specimen, has long storage time of the specimen, and can well expand the application range of the detection method. Samples are collected by mailing for detection, so that the detection method is beneficial to large-scale commercial use; 2. the method for detecting the organic acid metabolites in the human urine has the advantages of high sensitivity, high accuracy, good repeatability and high detection speed, is suitable for functionally evaluating the requirements of specific nutrients, and is the best choice for improving the symptoms of patients by establishing a personalized treatment scheme on the basis.)

1. A quantitative detection method for detecting a plurality of organic acids in a human body dry urine filter paper sheet is characterized by comprising the following steps: firstly, collecting a urine sample by adopting a filter paper sheet; secondly, carrying out elution treatment on the dry urine filter paper sheet, and measuring the creatinine content; mixing the eluted urine with an internal standard solution, extracting and purifying the target organic acid by using an organic solvent, drying by using nitrogen, dissolving in water, purifying by using a strong anion exchange column, drying by using nitrogen after eluting the organic solvent, and dissolving in water to obtain a sample to be detected; and fourthly, the sample to be detected is detected by the combination of liquid chromatography and mass spectrometry, and qualitative and quantitative batch detection is synchronously carried out on various organic acids.

2. The method of claim 1, further comprising: the filter paper sheets used to collect urine are filter papers cut to specific sizes, including but not limited to, Xinhua No. 1, Xinhua No. 3, and Whatman corporation No. 903 filter papers.

3. The method of claim 1, further comprising: the sample collected was human morning urine.

4. The method of claim 1, further comprising: the dry diaper is prepared by dripping a certain amount of fresh morning urine of human body onto a quantitative filter paper sheet to make the urine uniformly distributed, and air-drying the filter paper sheet and then sealing and storing for later use.

5. The method of claim 1, further comprising: the solvent used in the dry diaper elution process includes, but is not limited to, water, PBS, physiological saline, and preferably the elution solvent is physiological saline.

6. The detection method according to claims 1 to 5, wherein the conditions of the liquid chromatography in the LC-MS detection in the step (iv) are as follows: the chromatographic column is a bonded phase chromatographic column, the sample injection amount is 5-50 mu L, the mobile phase A is a pure water containing 1-10mmol/L ammonium acetate water solution and 0.1-1% formic acid, the mobile phase B is a mixed solution of acetonitrile and methanol, and the mass ratio of the acetonitrile to the methanol is 9-1: 1-9; the flow rate of the mobile phase is 0.1-2.0 mL/min; gradient of mobile phase (in volume percent):

0-1min,A:30-100％,B:70-0％；

1.1-5min,A:10-60％,B:90-60％；

5.1-9min,A:0-50％,B:100-50％；

9.1-11min,A:50-100％,B:50-0％；

11.1min,A:30-100％,B:70-0％。

7. the detection method according to claim 6, wherein the bonded phase chromatographic column is a bonded phase chromatographic column such as C18 or C8, and has a specification of 50-150mm x 2.1-4.6mm,1.8-5 μm.

8. The detection method according to claims 1 to 7, wherein the mass spectrometry conditions of the LC-MS detection in step 3) are as follows: an electrospray ionization (ESI) ion source and a negative ion MRM scanning mode are adopted, the flow rate of atomizing gas is 5-10L/min, the flow rate of a gas curtain is 5-20L/min, the voltage of the ion source is 4500V of minus 2000-.

9. The assay of any one of claims 1-8, wherein the organic acid compound comprises a major organic acid product produced metabolically in human urine; the mass spectrum scanning mode of multi-reaction monitoring is adopted, and the ion pair of the target object is monitored as follows: oxalic acid (m/z 145.2-83.1), suberic acid (m/z 173.2-111.2), ethylmalonic acid (m/z 131.1-87.1), lactic acid (m/z 89.1-43.1), pyruvic acid (m/z 87.1-43.1), beta-hydroxybutyric acid (m/z 103.1-59.1), citric acid (m/z 191.1-87.1), cis-aconitic acid (m/z 173.1-85.1), isocitric acid (m/z 191.1-73.1), alpha-ketoglutaric acid (m/z 145.1-101.1), alpha-ketoisovaleric acid (m/z 115.1-115.1), alpha-ketoisocaproic acid (m/z 129.2-129.2), alpha-keto-beta-methylvaleric acid (m/z 129.2-129.2), succinic acid (m/z 115.1-73.0), fumaric acid (m/z 115.1-71.1-0), malic acid (m/z 1-71.1-0.1), malic acid (m/z) and malic acid (m/z) 1-59.1-0, Hydroxymethylglutaric acid (m/z 117.1-59.0), xanthuric acid (m/z 204.2-160.1), beta-hydroxyisovaleric acid (m/z 117.1-73.1) and methylmalonic acid (m/z 117.1-73.1); internal standard: d3-methyl malonic acid (m/z 120.1-73.1), D3-ethyl malonic acid (m/z 134.1-87.1), and D4-citric acid (m/z 195.1-87.1).

Technical Field

The invention relates to the technical field of biochemical detection, in particular to a method for synchronously and quantitatively detecting the content of various organic acids in a human body dry urine filter paper sheet.

Background

Organic acids are a large class of compounds produced during the basic metabolic processes of the body. They are derived from dietary proteins, fats and carbohydrates, which are used by the body to produce cellular energy and provide nutrients necessary for cellular function. The metabolites selected by the urine organic acid test can be used as important diagnosis indexes of abnormal metabolism. Metabolic imbalance is a common and widespread disease that may underlie many chronic diseases, such as fatigue, gastrointestinal dysfunction, muscle/joint problems, mood disorders and headaches. These diseases are often resistant to long-term treatment and sustained improvement. Organic acid analysis can be traditionally used for early detection/elimination or monitoring of metabolic disorders. Urine samples can be used to assess gut, liver and nervous system health as well as energy metabolism and nutritional deficiencies.

Proteins, fats and carbohydrates, which are the main foods of the human body, are used to produce cellular energy and provide nutrients necessary for cellular functions. However, the food should also contain other essential nutrients, and only with the help of these essential nutrients can the food be converted into energy and nutrients to be absorbed by human body. When food is metabolized, the deficiency of certain essential nutrients can lead to the blockage of pathways for energy production and nutrient absorption, and the resulting organic acid metabolites are excreted into the urine. Thus, the energy metabolism and the deficiency of specific nutrients in an individual can be assessed by the determination of organic acids in urine. For many organic acids measured, abnormally high levels in urine often indicate that a particular metabolic pathway is blocked, with low levels of nutrients necessary for that metabolic pathway. Urine organic acid testing is helpful in understanding how nutrient metabolism is performed and in determining where imbalances in the metabolic cycle may exist. By way of example, the B vitamins, in particular B1 (thiamine), B3 (niacin) and B5 (pantothenic acid), provide the necessary cofactors for all somatic energy pathways. When food is metabolized, specific compounds are formed in steps that require vitamin B to be involved. These steps occur in carbohydrate metabolism, where pyruvate and lactate are formed. Elevated levels of these compounds indicate a low helper enzyme activity for this reaction and thus an increase in the B vitamins, particularly thiamine and pantothenic acid, is desirable. After increased intake of these essential nutrients, the pathways of the reaction are opened again and the energy production efficiency of the cells is restored. Almost every element of the body system requires enzyme cofactors, and deficiencies in such cofactors as vitamins or minerals affect a wide range of body functions including the immune, endocrine, musculoskeletal and metabolic systems.

For many organic acids measured, abnormally high levels in urine generally indicate lower levels of nutrients required to break down the compound. On this basis, the need for specific nutrients is functionally assessed and creating a personalized treatment regime is the best choice to improve the patient's symptoms.

The inventor has developed a quantitative detection method for a plurality of organic acid metabolites in human urine samples in the past. In order to solve the problem, the inventor further develops a quantitative detection method using a dry urine filter paper sheet for storing the sample, so that the storage and transportation conditions of the sample are reduced, the special requirements on the transportation and storage of the sample are avoided, and the application range of the detection method can be well expanded. By comparison, the sample stored by the dry urine filter paper still has detection sensitivity and repeatability equivalent to those of a urine sample, and the method has the advantages of high detection speed, large detection batch and the like, can efficiently and quantitatively detect the organic acid metabolites, and has good scientific research and commercial application prospects.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for detecting various organic acid metabolites in human urine by using a high performance liquid chromatography-tandem mass spectrometry technology in which a dry diaper is used as a sample collection, storage and transportation mode.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

step 1, collecting urine specimens by using a filter paper sheet, airing the urine specimens, and placing the urine specimens in a special drying bag for storage and transportation;

step 2, carrying out elution treatment on the dry diaper, and determining creatinine content of the obtained eluted urine sample to determine urine concentration and obtain a urine sample for determining organic acid;

step 3, mixing the urine sample with the internal standard solution, extracting and purifying the target organic acid by using an organic solvent, drying by using nitrogen, dissolving in water, purifying by using a strong anion exchange column, eluting by using the organic solvent, drying by using nitrogen, and dissolving in water to obtain a sample to be detected;

and 4, detecting the sample by combining liquid chromatography and mass spectrometry to obtain the type and content of the organic acid.

Further, the filter paper sheets described in step 1 above include, but are not limited to, Xinhua No. 1, Xinhua No. 3 and Whatman No. 903 filter papers;

further, the urine sample in step 1 is human morning urine;

further, the dry diaper in the step 1 is prepared by dropping a certain amount of fresh human morning urine on a quantitative filter paper sheet to make the urine uniformly distributed, and the filter paper sheet is dried and then sealed for storage.

Further, the solvent used in the elution treatment of the dry diaper in the step 2 includes water, PBS, and physiological saline, and preferably, the elution solvent is physiological saline.

Further, the organic solvent used for the extraction in the step 3 comprises ethyl acetate, n-hexane, dichloromethane, chloroform and tert-butyl methyl ether, and the preferred organic extraction solvent is ethyl acetate. Furthermore, the organic extraction solvent is ethyl acetate;

further, the elution solvent of the strong anion exchange column in the step 3 is methanol or acetone (with 2% formic acid added).

Further, the step 4 specifically includes the following steps:

step A: weighing an organic acid standard substance, and dissolving the organic acid standard substance by using methanol to obtain standard solutions with different concentrations;

b, measuring the internal standard liquid after the methanol constant volume, respectively adding the internal standard liquid into the standard liquid obtained in the step A, carrying out constant volume by using ultrapure water, centrifuging, and taking supernatant as standard liquid to be measured;

and C: and C, detecting the standard solution to be detected obtained in the step B and the sample to be detected obtained in the step 3 by liquid chromatography-mass spectrometry to obtain the type and content of the organic acid metabolite.

Further, the organic acid metabolites include oxalic acid, suberic acid, ethylmalonic acid, lactic acid, pyruvic acid, β -hydroxybutyric acid, citric acid, cis-aconitic acid, isocitric acid, α -ketoglutaric acid, α -ketoisovaleric acid, α -ketoisocaproic acid, α -keto- β -methylvaleric acid, succinic acid, fumaric acid, malic acid, hydroxymethylglutaric acid, xanthuric acid, β -hydroxyisovaleric acid, and methylmalonic acid.

The method comprises the steps of adopting an organic solvent to extract and a strong anion exchange column to purify and process the organic acid metabolites in human urine, utilizing high performance liquid chromatography to separate 20 organic acid metabolites, utilizing a mass spectrum isotope internal standard quantitative method, taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, establishing a calibration curve, and calculating the content of the organic acid metabolites.

Further, the liquid chromatography conditions for the LC-MS detection in the step C are as follows:

the chromatographic column is a bonded phase chromatographic column, the sample injection amount is 5-50 mu L, the mobile phase comprises A and B, wherein A is an aqueous solution, B is a mixed solution, and the flow rate of the mobile phase is 0.1-2.0 mL/min; gradient of mobile phase (in volume percent): 0-1min, 30-100% of A and 70-0% of B;

1.1-5min,A:10-60％,B:90-60％；

5.1-9min,A:0-50％,B:100-50％；

9.1-11min,A:50-100％,B:50-0％；

11.1min,A:30-100％,B:70-0％。

furthermore, the bonded phase chromatographic column is C18 or C8, etc., and has a specification of 50-150mm × 2.1-4.6mm,1.8-5 μm.

Furthermore, in the condition of liquid chromatography, the mobile phase A is 1-10mmol/L ammonium acetate water solution and 0.1% -1% formic acid pure water, the mobile phase B is a mixed solution of acetonitrile and methanol, and the mass ratio of the acetonitrile to the methanol is 9-1: 1-9.

Further, the mass spectrometry conditions of the LC-MS detection are as follows: ESI ion source, negative ion MRM scanning, atomizing airflow rate of 5-10L/min, air curtain airflow rate of 5-20L/min, ion source voltage of 2000-4500V, and ion source temperature of 200-400 ℃.

Further, in an electrospray negative ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the ion pair monitoring of the target object is as follows: oxalic acid (m/z 145.2-83.1), suberic acid (m/z 173.2-111.2), ethylmalonic acid (m/z 131.1-87.1), lactic acid (m/z 89.1-43.1), pyruvic acid (m/z 87.1-43.1), beta-hydroxybutyric acid (m/z 103.1-59.1), citric acid (m/z 191.1-87.1), cis-aconitic acid (m/z 173.1-85.1), isocitric acid (m/z 191.1-73.1), alpha-ketoglutaric acid (m/z 145.1-101.1), alpha-ketoisovaleric acid (m/z 115.1-115.1), alpha-ketoisocaproic acid (m/z 129.2-129.2), alpha-keto-beta-methylvaleric acid (m/z 129.2-129.2), succinic acid (m/z 115.1-73.0), fumaric acid (m/z 115.1-71.1-0), malic acid (m/z 1-71.1-0.1), malic acid (m/z) and malic acid (m/z) 1-59.1-0, Hydroxymethylglutaric acid (m/z 117.1-59.0), xanthuric acid (m/z 204.2-160.1), beta-hydroxyisovaleric acid (m/z 117.1-73.1) and methylmalonic acid (m/z 117.1-73.1). Internal standard: d3-methyl malonic acid (m/z 120.1-73.1), D3-ethyl malonic acid (m/z 134.1-87.1), and D4-citric acid (m/z 195.1-87.1).

Compared with the prior art, the invention has the advantages that:

1. the invention adopts the filter paper sheet to collect the urine specimen, reduces the storage and transportation conditions of the specimen, has no special requirements on the transportation and storage of the specimen, has long specimen storage time, can well expand the application range of the detection method, accords with the characteristics of wide breadth of our country, numerous population and unbalanced regional development, collects the specimen by mailing for detection, and is beneficial to large-scale commercial use of the detection method.

2. The method for detecting the organic acid metabolites in the human urine, provided by the invention, has the advantages of high sensitivity, high accuracy, good repeatability and high detection speed, is suitable for functionally evaluating the requirements of specific nutrients, and the establishment of a personalized treatment scheme on the basis is the best choice for improving the symptoms of patients.

Drawings

FIG. 1: and comparing the total ion current chromatogram of the standard stock solution and the standard dry filter paper sheet.

FIG. 2: and (4) comparing the total ion current chromatogram of the same urine sample with that of the dry diaper.

Detailed Description

The present invention will be better understood from the following examples. It is easily understood by those skilled in the art that the descriptions of the embodiments are only for illustrating the present invention and should not be construed as limiting the present invention as detailed in the claims. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without inventive step, are within the scope of protection of the present invention.