CN111855852A

CN111855852A - Method for detecting 21 organic acids in urine by ultra-high performance liquid chromatography tandem mass spectrometry technology

Info

Publication number: CN111855852A
Application number: CN202010704566.8A
Authority: CN
Inventors: 成晓亮; 李美娟
Original assignee: Nanjing Pinsheng Medical Laboratory Co ltd
Current assignee: Nanjing Pinsheng Medical Laboratory Co ltd
Priority date: 2020-07-21
Filing date: 2020-07-21
Publication date: 2020-10-30

Abstract

The invention discloses a method for detecting 21 organic acids in urine by an ultra-high performance liquid chromatography tandem mass spectrometry technology, wherein the organic acids are respectively as follows: HA. HBA, HPA, IAA, TCA, XA, beta-HIVA, SA, EMA, PYA, CA, SCA, HMGA, MDA, PGA, GA, alpha-HB, HVA, VMA, HIA, and KA; detecting the content of organic acids in the pretreated urine by adopting an ultra-high performance liquid chromatography tandem mass spectrometry, quantifying by utilizing a mass spectrometry isotope internal standard method, establishing a calibration curve by 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, and calculating the content of 21 organic acids in the urine; the urine sample is mixed with the mixed internal standard solution of all the objects to be detected, the sample does not need derivatization treatment, the pretreatment is simple, the sample dosage is small, the sensitivity is high, the specificity is strong, the detection types are more, 21 organic acids can be simultaneously detected within 5.0 minutes, and the method can be used for clinical diagnosis and health assessment of the urine organic acids.

Description

Method for detecting 21 organic acids in urine by ultra-high performance liquid chromatography tandem mass spectrometry technology

Technical Field

The invention belongs to the technical field of urine detection, and particularly relates to a method for detecting 21 organic acids in urine by using an ultra-high performance liquid chromatography tandem mass spectrometry technology.

Background

Organic acids refer to some organic compounds that are acidic and are a large class of compounds produced by the body's metabolism. They come from dietary proteins, fats and carbohydrates, and most of the organic acids are directly involved in biochemical reactions, which are used by the body to produce cellular energy and provide nutrients needed for cellular function. Meanwhile, the organic acid has the effects of bacteriostasis, inflammation diminishing, virus resistance, mutation resistance, cancer resistance and the like, some organic acids can increase the blood flow of filling arteries, inhibit the generation of lipid peroxides of brain tissues, soften blood vessels, promote the absorption of calcium and iron, and some organic acids can help stomach digest fat and protein; some have the functions of preventing diseases and promoting metabolism in new castle, thus being beneficial to the health of human body. Therefore, the organic acid test in urine can be used as an important diagnosis index of metabolic abnormality. 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.

Protein, fat and carbohydrate taken in by human body are used for generating cell energy and providing nutrition required by cell function, but other necessary nutrients are also included in the food, and the food can be converted into energy and the nutrition can be absorbed by human body only with the help of the necessary nutrients. 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.

At present, the method for detecting the concentration of organic acid in urine resistance mainly comprises an ultra-high performance liquid chromatography-tandem mass spectrometry or a gas chromatography-tandem mass spectrometry, and most of the methods reported in documents are used for detecting 1-3 organic acids at one time, for example, Chinese patent application (publication number: CN103837624A) discloses a method for detecting liquid chromatography-tandem mass spectrometry of phenylglyoxylic acid and phenylglycolic acid in urine, only two organic acids are detected at one time, the variety of the organic acids in the urine is various, and the detection process is complicated.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for detecting 21 organic acids in urine by using an ultra-high performance liquid chromatography tandem mass spectrometry technology, wherein the 21 organic acids are respectively as follows: hippuric Acid (HA), 4-hydroxybenzoic acid (HBA), 4-hydroxyphenylacetic acid (HPA), indoleacetic acid (IAA), tricarballylic acid (TCA), Xanthylic Acid (XA), beta-hydroxyisovaleric acid (beta-HIVA), Suberic Acid (SA), ethylmalonic acid (EMA), pyruvic acid (PYA), Citric Acid (CA), succinic acid (SCA), hydroxymethylglutaric acid (HMGA), mandelic acid (MDA), phenylglyoxylic acid (PGA), Gluconic Acid (GA), alpha-hydroxybutyric acid (alpha-HB), homovanillic acid (HVA), vanillylmandelic acid (VMA), 5-hydroxyindoleacetic acid (HIA) and Kynurenic Acid (KA);

the isotope internal standard substances corresponding to the 21 kinds of organic acids are respectively as follows: hippuric acid-d 5(HA-d5), 4-hydroxybenzoic acid-d 4(HBA-d4), indoleacetic acid-d 3(IAA-d3), xanthuric acid-d 4(XA-d4), citric acid-d 4(CA-d4), succinic acid-d 4(SCA-d4), phenylglyoxylic acid-d 4(PGA-d4), alpha-hydroxybutyric acid-d 3 (alpha-HB-d 3), homovanillic acid-d 3(HVA-d3), vanillylmandelic acid-d 3(VMA-d3), 5-hydroxyindoleacetic acid-d 5(HIA-d5) and kynurenic acid-d 5(KA-d 5);

in order to solve the technical problems, the technical scheme adopted by the invention is as follows: a method for detecting 21 organic acids in urine by using an ultra-high performance liquid chromatography tandem mass spectrometry technology comprises the steps of detecting the content of the organic acids in the pretreated urine by using the ultra-high performance liquid chromatography tandem mass spectrometry, quantifying by using a mass spectrometry isotope internal standard method, establishing a calibration curve by using 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, and calculating the content of the 21 organic acids in the urine;

(1) Chromatographic conditions are as follows:

mobile phase A: water containing 0.01 to 0.5% formic acid;

mobile phase B: acetonitrile;

a chromatographic column: ACQUITYUPLC BEH C18 (2.1X 100mm,1.7 μm);

the gradient elution mode is adopted, and is shown in the table 1;

the flow rate is 0.2-0.5 mL/min, the column temperature is 35-45 ℃, and the sample injection volume is 1-10 mu L;

TABLE 1 mobile phase gradient elution parameters

(2) Mass spectrum conditions: in an electrospray ionization (ESI) mode, negative ion scanning is performed by adopting multi-reaction monitoring (MRM); the spray voltage was 2.5kV (ESI-); source temperature: 120 ℃; temperature of atomized gas: 400 ℃, atomizing gas flow rate: 800L/h, taper hole air flow rate: 150L/h; simultaneously monitoring a standard substance and internal standard parent ions, ionic ions, cluster removing voltage and collision voltage corresponding to a target object, wherein the parameters are shown in a table 2;

TABLE 2 Mass spectrometric parameters

Wherein the urine is human or animal urine.

Wherein the pretreated urine is prepared according to the following method: centrifuging the urine sample, taking supernatant, filtering, adding mixed internal standard working solution into filtrate, mixing uniformly by vortex, adding diluent for dilution, and carrying out sample injection.

The standard substance is prepared according to the following steps:

preparing the standard substance of each substance to be detected into standard substance mother liquor by using methanol, wherein the concentrations are respectively as follows: HA 10mg/mL, HBA5mg/mL, HPA 10mg/mL, IAA 5mg/mL, TCA 5mg/mL, XA 10mg/mL, beta-HIVA 5mg/mL, SA 4mg/mL, EMA 5mg/mL, PYA 5mg/mL, CA 10mg/mL, SCA 5mg/mL, HMGA 5mg/mL, MDA 5mg/mL, PGA 5mg/mL, GA 10mg/mL, alpha-HB 5mg/mL, HVA 2mg/mL, VMA 4mg/mL, HIA 4mg/mL and KA 5mg/mL, and then formulated with methanol in water to contain HA 1000. mu.g/mL, HBA 50. mu.g/mL, HPA 250. mu.g/mL, IAA 250. mu.g/mL, TCA 5. mu.g/mL, mixed standard stock solutions of XA 25. mu.g/mL, β -HIVA 100. mu.g/mL, SA 100. mu.g/mL, EMA 250. mu.g/mL, PYA 250. mu.g/mL, CA 2500. mu.g/mL, SCA 100. mu.g/mL, HMGA 100. mu.g/mL, MDA 50. mu.g/mL, PGA 5. mu.g/mL, GA 1000. mu.g/mL, α -HB 100. mu.g/mL, HVA 100. mu.g/mL, VMA 100. mu.g/mL, HIA 100. mu.g/mL, and KA 50. mu.g/mL;

The invention prepares the mixed standard stock solution into seven standard solutions with different concentration points by a blank urine substrate, and the specific method comprises the following steps: adding 40 μ L of the mixed standard solution to 160 μ L of 0.3% formic acid aqueous solution as a first high concentration point (S7); diluting the first high-value concentration point (S7) with an equal volume of 0.3% formic acid aqueous solution to obtain a second high-value concentration point (S6); diluting the first high-value concentration point (S7) with 4 times of 0.3% formic acid aqueous solution to obtain a third high-value concentration point (S5); diluting the second high-value concentration point (S6) with 4 times of 0.3% formic acid aqueous solution to obtain a fourth high-value concentration point (S4); diluting the third high-value concentration point (S5) with 4 times of 0.3% formic acid aqueous solution to obtain a fifth high-value concentration point (S3); diluting the fourth high-value concentration point (S4) with 4 times of 0.3% formic acid aqueous solution to obtain a sixth high-value concentration point (S2); diluting the fifth high-value concentration point (S3) with 4 times of 0.3% formic acid aqueous solution to obtain a seventh high-value concentration point (S1);

the seven concentration points of the standard solution are:

the concentrations of HA and GA are the same in the order: 1.6. mu.g/mL, 4. mu.g/mL, 8. mu.g/mL, 20. mu.g/mL, 40. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL;

the concentrations of HBA, MDA and KA are the same in sequence: 0.08. mu.g/mL, 0.2. mu.g/mL, 0.4. mu.g/mL, 1. mu.g/mL, 2. mu.g/mL, 5. mu.g/mL, 10. mu.g/mL;

The concentrations of HPA, IAA, EMA and PYA are the same in order: 0.4. mu.g/mL, 1. mu.g/mL, 2. mu.g/mL, 5. mu.g/mL, 10. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL;

the concentrations of TCA and PGA are the same in the order: 0.008. mu.g/mL, 0.02. mu.g/mL, 0.04. mu.g/mL, 0.1. mu.g/mL, 0.2. mu.g/mL, 0.5. mu.g/mL, 1. mu.g/mL;

the concentration of XA is as follows: 0.04. mu.g/mL, 0.1. mu.g/mL, 0.2. mu.g/mL, 0.5. mu.g/mL, 1. mu.g/mL, 2.5. mu.g/mL, 5. mu.g/mL;

the concentrations of beta-HIVA, SA, SCA, HMGA, alpha-HB, HVA, VMA and HIA are the same, and the following are sequentially adopted: 0.16. mu.g/mL, 0.4. mu.g/mL, 0.8. mu.g/mL, 2. mu.g/mL, 4. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL;

the concentrations of CA are, in order: 4. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 250. mu.g/mL, 500. mu.g/mL.

The mixed internal standard working solution is prepared by the following method: preparing isotope internal standard mother liquor with the concentrations of HA-d 51 mg/mL, HBA-d 41 mg/mL, IAA-d 31 mg/mL, XA-d 41 mg/mL, CA-d 41 mg/mL, SCA-d 41 mg/mL, PGA-d 41 mg/mL, alpha-HB-d 31 mg/mL, HVA-d 31 mg/mL, VMA-d 32 mg/mL, HIA-d 52 mg/mL and KA-d 51 mg/mL by using methanol, and then preparing the isotope internal standard mother liquor with aqueous methanol solution to contain HA-d 5100 mu g/mL, HBA-d 45 mu g/mL, IAA-d 325 mu g/mL, XA-d 42.5 mu g/mL, CA-d 4250 mu g/mL, SCA-d 410 mu g/mL, PGA-d 40.5 mu g/mL, alpha-HB-d 310 mu g/mL and SCA, Mixed internal standard solutions of HVA-d 310. mu.g/mL, VMA-d 310. mu.g/mL, HIA-d 510. mu.g/mL and KA-d 55. mu.g/mL; and adding 900 mu L of formic acid aqueous solution into 100 mu L of the mixed internal standard solution, and uniformly mixing to obtain the mixed internal standard working solution.

Wherein the pretreated urine is prepared according to the following method: taking 200 mu L urine, placing in a 1.5mL EP tube, centrifuging at 12000-15000 r/min at 10-20 ℃ for 2-5 min to obtain supernatant, sucking 150 mu L supernatant, and filtering with a 0.22 mu m nylon filter membrane to obtain filtrate; and (3) adding 20 mu L of filtrate into 20 mu L of mixed internal standard working solution, uniformly mixing for 5s in a vortex manner, adding 760 mu L of formic acid aqueous solution for dilution, uniformly mixing, taking 80 mu L of diluent into a plastic lining tube, and carrying out sample injection.

Wherein the volume concentration of the methanol aqueous solution is 10-90%, and the volume concentration of the formic acid aqueous solution is 0.1-0.5%.

The method comprises the steps of mixing a urine sample with mixed internal standard solutions of all objects to be detected, separating a target object to be detected from interfering components in a urine matrix by using ultra-high performance liquid chromatography, detecting the mass-to-charge ratio (m/z) of the target object and the corresponding isotope internal standard thereof by using mass spectrometry, quantifying by using an isotope internal standard method, and accurately calculating the content of 21 organic acids.

Has the advantages that: the urine sample is mixed with the mixed internal standard solution of all the objects to be detected, the sample does not need derivatization treatment, the pretreatment is simple, the sample dosage is small, the sensitivity is high, the specificity is strong, the detection types are more, 21 organic acids can be simultaneously detected within 5.0 minutes, and the method can be used for clinical diagnosis and health assessment of the urine organic acids.

Drawings

FIG. 1 is a selective ion flow chromatogram of 21 organic acid standards;

FIG. 2 is a selective ion flow chromatogram of 21 organic acids in urine.

Detailed Description

For the purpose of enhancing the understanding of the present invention, the present invention will be further described in detail with reference to the following examples and the accompanying drawings, which are only used for explaining the present invention and are not to be construed as limiting the scope of the present invention.

Example 1

1. Material

(1) The instrument comprises the following steps: xevo TQ-S triple quadrupole mass spectrometer (Waters Corporation); UPLC I-Class ultra high performance liquid chromatography system (with autosampler, Waters Corporation); SCILOGEX D2012 high speed bench top centrifuge (usa); ultra pure water meter (ELGA LabWater, uk); multi-tube Vortex mixer (Vortex genie2, usa); an adjustable pipettor (Eppendorf0.5-10 muL, 10-100 muL, 100-1000 muL); glassware, graduated cylinders, etc.;

(2) reagent consumables: MS grade acetonitrile (Fisher, usa); 0.22 μm nylon filter (Millipore, usa); column model ACQUITYUPLC BEH C18 (2.1X 100mm,1.7 μm) (Waters, USA).

(3) And (3) standard substance: HA. HBA, HPA, IAA, TCA, XA, beta-HIVA, SA, EMA, PYA, CA, SCA, HMGA, MDA, PGA, GA, alpha-HB, HVA, VMA, HIA, and KA; HA-d5, HBA-d4, IAA-d3, XA-d4, CA-d4, SCA-d4, PGA-d4, alpha-HB-d 3, HVA-d3, VMA-d3, HIA-d5 and KA-d 5; TCA was purchased from Sigma, and the remainder from TRC.

(4) Quality control product: the blank urine matrix solution containing 21 organic acids has low, medium and high concentrations of QC (L), QC (M) and QC (H), respectively, and the specific concentrations are shown in Table 3 (unit: μ g/mL)

TABLE 3 corresponding concentrations of quality control substances

2. Method of producing a composite material

(1) Chromatographic conditions

Mobile phase A: water containing 0.01 to 0.5% formic acid; mobile phase B: acetonitrile; a chromatographic column: ACQUITY UPLC BEHC18 (2.1X 100mm,1.7 μm); the flow rate is 0.2-0.5 mL/min, the column temperature is 35-45 ℃, and the sample injection volume is 1-10 mu L; the gradient elution mode is adopted, and is shown in the table 1;

(2) conditions of Mass Spectrometry

In an electrospray ionization (ESI) mode, negative ion scanning is performed by adopting multi-reaction monitoring (MRM); the spray voltage was 2.5kV (ESI-); source temperature: 120 ℃; temperature of atomized gas: 400 ℃, atomizing gas flow rate: 800L/h, taper hole air flow rate: 150L/h; simultaneously monitoring a standard product and internal standard parent ions, ionic ions, cluster removing voltage and collision voltage corresponding to the target object, wherein the mass spectrum parameters of each target object are shown in a table 2;

(3) preparation of standard substance

Preparing the standard substance of each substance to be detected into standard substance mother liquor by using methanol, wherein the concentrations are respectively as follows: HA 10mg/mL, HBA5mg/mL, HPA 10mg/mL, IAA 5mg/mL, TCA 5mg/mL, XA 10mg/mL, beta-HIVA 5mg/mL, SA 4mg/mL, EMA 5mg/mL, PYA 5mg/mL, CA 10mg/mL, SCA 5mg/mL, HMGA 5mg/mL, MDA 5mg/mL, PGA 5mg/mL, GA 10mg/mL, alpha-HB 5mg/mL, HVA 2mg/mL, VMA 4mg/mL, HIA 4mg/mL and KA 5mg/mL, and then prepared into a solution containing HA 1000. mu.g/mL, HBA 50. mu.g/mL, HPA 250. mu.g/mL, IAA 5mg/mL, and, Mixed standard stock solutions of TCA 5. mu.g/mL, XA 25. mu.g/mL, beta-HIVA 100. mu.g/mL, SA 100. mu.g/mL, EMA 250. mu.g/mL, PYA 250. mu.g/mL, CA 2500. mu.g/mL, SCA 100. mu.g/mL, HMGA 100. mu.g/mL, MDA 50. mu.g/mL, PGA 5. mu.g/mL, GA 1000. mu.g/mL, alpha-HB 100. mu.g/mL, HVA 100. mu.g/mL, VMA 100. mu.g/mL, HIA 100. mu.g/mL and KA 50. mu.g/mL, the preparation methods are shown in Table 4;

Table 4 preparation of stock solutions for mixed standards

(4) Preparation of mixed internal standard working solution

Weighing each isotope internal standard substance into a 5mL centrifuge tube, preparing isotope internal standard mother liquor by using methanol, wherein the concentrations are respectively HA-d 51 mg/mL, HBA-d 41mg/mL, IAA-d 31 mg/mL, XA-d 41mg/mL, CA-d41mg/mL, SCA-d41mg/mL, PGA-d 41mg/mL, alpha-HB-d 31 mg/mL, HVA-d 31 mg/mL, VMA-d 32 mg/mL, HIA-d 52 mg/mL, KA-d 51 mg/mL, and then preparing the isotope internal standard substance into a methanol water solution with the volume concentration of 50 percent, wherein the methanol water solution comprises HA-d 5100 mu g/mL, HBA-d 45 mu g/mL, IAA-d 325 mu g/mL, IAA-d 42.5 mu g/mL, CA-d 4250 mu g/mL, SCA-d 410 mu g/mL, PGA-d 40.5 mu g/mL and PGA-d 40.5 mu g/mL, The specific preparation process of the mixed internal standard solution of alpha-HB-d 310. mu.g/mL, HVA-d 310. mu.g/mL, VMA-d 310. mu.g/mL, HIA-d 510. mu.g/mL and KA-d 55. mu.g/mL is shown in Table 5; adding 100 mu L of mixed internal standard solution into 900 mu L of formic acid aqueous solution, and uniformly mixing to obtain mixed internal standard working solution;

TABLE 5 preparation of mixed internal standard solutions

(5) Preparation of quality control product

QC (L) is that the mixed standard stock solution is diluted to 250 times by blank urine matrix solution;

QC (M) is that the mixed standard stock solution is diluted to 50 times by blank urine matrix solution;

Qc (h) diluted 10-fold for the mixed standard stock with blank urine matrix solution.

(6) Sample processing

1) Preparation of standard curve

The standard curve is prepared by adopting a gradient dilution method, and the preparation process is as follows:

adding 40 μ L of the mixed standard stock solution into 160 μ L of 0.3% formic acid aqueous solution as a first high-value concentration point (S7); diluting the first high-value concentration point (S1) with an equal volume of 0.3% formic acid aqueous solution to obtain a second high-value concentration point (S6); diluting the first high-value concentration point (S1) with 4 times of 0.3% formic acid aqueous solution to obtain a third high-value concentration point (S5); diluting the second high-value concentration point (S2) with 4 times of 0.3% formic acid aqueous solution to obtain a fourth high-value concentration point (S4); diluting the third high-value concentration point (S3) with 4 times of 0.3% formic acid aqueous solution to obtain a fifth high-value concentration point (S3); diluting the fourth high-value concentration point (S4) with 4 times of 0.3% formic acid aqueous solution to obtain a sixth high-value concentration point (S2); diluting the fifth high-value concentration point (S5) with 4 times of 0.3% formic acid aqueous solution to obtain a seventh high-value concentration point (S1); the specific procedure is as follows in Table 6 (unit: μ g/mL)

TABLE 6 Standard Curve formulations and concentrations

2) Pretreatment of a standard product: and (3) taking 20 mu L of each concentration point sample, putting the 20 mu L of internal standard working solution into a 1.5mL EP tube, adding the internal standard working solution into the EP tube, mixing the internal standard working solution uniformly for 5s in a vortex manner, adding 760 mu L of formic acid aqueous solution with the volume concentration of 0.3% into the internal standard working solution for dilution, mixing the internal standard working solution uniformly, and filling 80 mu L of the diluted solution into a sample injection bottle with an internal insertion tube for detection.

3) Preparation of pretreated urine: taking 200 mu L urine, placing in a 1.5mL EP tube, centrifuging at 12000-15000 r/min at 10-20 ℃ for 2-5 min to obtain supernatant, sucking 150 mu L supernatant, and filtering with a 0.22 mu m nylon filter membrane to obtain filtrate; and adding 20 mu L of filtrate into 20 mu L of mixed internal standard working solution, uniformly mixing for 5s in a vortex manner, adding 760 mu L of formic acid aqueous solution with the volume concentration of 0.3% for dilution, uniformly mixing, and filling 80 mu L of diluent into a sample injection bottle with an inner inserting tube for detection.

4) Pretreatment of quality control products: respectively taking 20 mu L of quality control product solutions QC (L), QC (M) and QC (H) into a 1.5mL EP tube, adding 20uL of internal standard working solution, uniformly mixing for 5s in a vortex manner, adding 760 mu L of formic acid aqueous solution with the volume concentration of 0.3% for dilution, uniformly mixing, and filling 80 mu L of diluent into a sample injection bottle containing an inner intubation for detection.

3. Method verification

1) Selecting an ion flow spectrogram: as can be seen from FIGS. 1-2, the peak shapes of the 21 organic acids are symmetrical and there is no interference of the miscellaneous peak, which indicates that the good detection can be obtained under the conditions.

2) Calibration curve: establishing a calibration curve by adopting an isotope internal standard quantitative method and utilizing TargetLynx software to calculate the concentration of the organic acid to-be-detected substance in the urine by taking the concentration ratio of the standard substance to the 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; the linear fitting equation of the 21 organic acids in the respective concentration ranges has good linearity, the correlation coefficient is more than 0.99, and the quantitative requirements are met, which is shown in Table 7.

Linear regression equation and linear correlation coefficient for organic acids in Table 721

3) Minimum limit of quantitation

The lowest limit of quantitation (LLOQ), which is the lowest point of the standard curvilinear range, also reflects the sensitivity of the method. Part of organic acids are low in human body content, the requirements on the sensitivity and the specificity of the method are high, the method is optimized and investigated, the current minimum quantitative limit (LLOQ) basically meets the sensitivity requirement of simultaneous detection of 21 organic acids, and the concentration of the LLOQ is specifically shown in Table 8.

TABLE 8 quantitative lower limit data table

4) And (4) inspecting the standard recovery rate: randomly selecting one sample of human urine, adding 1 sample without standard substance, adding 3 other samples with mixed standard substance stock solutions with low, medium and high concentrations, respectively, processing and measuring for 5 times by the same steps, and calculating the recovery rate result, see Table 9. The results show that the result of the normalized recovery rate of the 21 organic acids in the urine is between 85.4 and 114.0 percent, and the RSD of 5 times of repeated tests is in the range of 0.66 to 14.83 percent, and the results all meet the requirements.

TABLE 9 results of normalized recovery of 21 organic acids in urine (unit: μ g/mL)

5) And (3) precision test: repeatedly treating 6 batches of urine quality control samples within one day for 3 days, quantitatively determining the concentrations of the 15 organic acids by an isotope internal standard method, continuously counting the internal precision of each day for three days, and calculating the internal precision to be 2.49-14.76%; the batch precision was calculated to be 4.24-14.77% within 3 batches of three days, and the results are shown in Table 10.

TABLE 10 results of precision test within and between batches (unit: μ g/mL)

4. Discussion of the related Art

The invention adopts UPLC-MS/MS method to measure 21 organic acids in human urine, and detects the peak-out time and ion pair of the target object, with high sensitivity and strong specificity. Meanwhile, the isotope internal standard method is adopted for quantification, so that the matrix interference can be greatly eliminated, the influence of the conditions such as a pretreatment process, a sample loading volume and flow is avoided, and accurate quantification can be achieved.

The standard addition recovery rate of 21 organic acids in urine is considered to be 85-115%, and the requirements are met. The reproducibility result of the method shows that the internal precision of 21 organic acids in urine is 2.49-14.76%, the inter-batch precision is 4.24-14.77%, and the reproducibility of the method is good.

Compared with other LC-MS/MS methods, the method has higher sensitivity, simple pretreatment, only one-step centrifugal filtration treatment, small sample dosage, more detection types, capability of simultaneously detecting 21 organic acids within 5.0 minutes, and capability of being used for clinical diagnosis and health assessment of the organic acids in urine.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: modifications of the technical solutions described in the foregoing embodiments are still possible, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting 21 organic acids in urine by an ultra performance liquid chromatography tandem mass spectrometry technology is characterized in that the organic acids are respectively: HA. HBA, HPA, IAA, TCA, XA, beta-HIVA, SA, EMA, PYA, CA, SCA, HMGA, MDA, PGA, GA, alpha-HB, HVA, VMA, HIA, and KA;

detecting the content of organic acids in the pretreated urine by adopting an ultra-high performance liquid chromatography tandem mass spectrometry, firstly separating a target object to be detected from interfering components in a urine matrix by utilizing the ultra-high performance liquid chromatography, then quantifying by utilizing a mass spectrometry isotope internal standard method, establishing a calibration curve by 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, and calculating the content of 21 organic acids in the urine;

(1) chromatographic conditions are as follows:

mobile phase A: water containing 0.01 to 0.5% formic acid; mobile phase B: acetonitrile;

a chromatographic column: ACQUITYUPLC BEH C18 (2.1X 100mm,1.7 μm);

gradient elution is carried out by adopting a mobile phase A and a mobile phase B as a mixed mobile phase, and the gradient elution is shown in a table 1;

TABLE 1 mobile phase gradient elution parameters

TABLE 2 Mass spectrometric parameters

2. The method for detecting 21 organic acids in urine according to claim 1, wherein the urine is human or animal urine.

3. The method for detecting 21 organic acids in urine by using the ultra performance liquid chromatography-tandem mass spectrometry technology according to claim 1, wherein the pretreated urine is prepared according to the following method: centrifuging the urine sample, taking supernatant, filtering, adding mixed internal standard working solution into filtrate, mixing uniformly by vortex, adding diluent for dilution, and carrying out sample injection.

4. The method for detecting 21 organic acids in urine by using the ultra performance liquid chromatography-tandem mass spectrometry technology according to claim 1, wherein the standard substance is prepared by the following steps:

preparing the standard substance of each substance to be detected into standard substance mother liquor by using methanol, wherein the concentrations are respectively as follows: HA 10mg/mL, HBA 5mg/mL, HPA 10mg/mL, IAA 5mg/mL, TCA 5mg/mL, XA 10mg/mL, beta-HIVA 5mg/mL, SA 4mg/mL, EMA5mg/mL, PYA 5mg/mL, CA 10mg/mL, SCA 5mg/mL, HMGA 5mg/mL, MDA 5mg/mL, PGA 5mg/mL, GA10mg/mL, alpha-HB 5mg/mL, HVA 2mg/mL, VMA 4mg/mL, HIA 4mg/mL, and KA 5mg/mL, and then formulated with methanol aqueous solution to contain HA 1000. mu.g/mL, HBA 50. mu.g/mL, HPA 250. mu.g/mL, IAA 250. mu.g/mL, TCA 5. mu.g/mL, XA 25. mu.g/mL, and, Mixed standard stock solutions of beta-HIVA 100. mu.g/mL, SA 100. mu.g/mL, EMA 250. mu.g/mL, PYA 250. mu.g/mL, CA 2500. mu.g/mL, SCA 100. mu.g/mL, HMGA 100. mu.g/mL, MDA 50. mu.g/mL, PGA 5. mu.g/mL, GA 1000. mu.g/mL, alpha-HB 100. mu.g/mL, HVA 100. mu.g/mL, VMA 100. mu.g/mL, HIA 100. mu.g/mL, and KA 50. mu.g/mL;

Adding 40 mu L of mixed standard solution into 160 mu L of 0.3% formic acid aqueous solution to serve as a first high-value concentration point; diluting the first high-value concentration point with an equal volume of 0.3% formic acid aqueous solution to obtain a second high-value concentration point; diluting the first high-value concentration point with 4 times of 0.3% formic acid aqueous solution to obtain a third high-value concentration point; diluting the second high-value concentration point with 4 times of 0.3% formic acid aqueous solution to obtain a fourth high-value concentration point; diluting the third high-value concentration point with 4 times of 0.3% formic acid aqueous solution to obtain a fifth high-value concentration point; diluting the fourth high-value concentration point with 4 times of 0.3% formic acid aqueous solution to obtain a sixth high-value concentration point; and (4) diluting the fifth high-value concentration point with 0.3% formic acid aqueous solution with 4 times volume to obtain a seventh high-value concentration point.

5. The method for detecting 21 organic acids in urine by using the ultra performance liquid chromatography-tandem mass spectrometry technology according to claim 1, wherein the pretreated urine is prepared according to the following method: taking 200 mu L urine, placing in a 1.5mL EP tube, centrifuging at 12000-15000 r/min at 10-20 ℃ for 2-5 min to obtain supernatant, sucking 150 mu L supernatant, and filtering with a 0.22 mu m nylon filter membrane to obtain filtrate; and (3) adding 20 mu L of filtrate into 20 mu L of mixed internal standard working solution, uniformly mixing for 5s in a vortex manner, adding 760 mu L of formic acid aqueous solution for dilution, uniformly mixing, taking 80 mu L of diluent into a plastic lining tube, and carrying out sample injection.

6. The method for detecting 21 organic acids in urine by using the ultra performance liquid chromatography-tandem mass spectrometry technology according to claim 3, wherein the mixed internal standard working solution is prepared by the following method: preparing isotope internal standard mother liquor with the concentrations of HA-d 51 mg/mL, HBA-d 41mg/mL, IAA-d 31 mg/mL, XA-d 41mg/mL, CA-d41mg/mL, SCA-d41mg/mL, PGA-d 41mg/mL, alpha-HB-d 31 mg/mL, HVA-d 31 mg/mL, VMA-d 32 mg/mL, HIA-d 52 mg/mL and KA-d 51 mg/mL by using methanol, and then preparing the isotope internal standard mother liquor with aqueous methanol solution to contain HA-d 5100 mu g/mL, HBA-d 45 mu g/mL, IAA-d 325 mu g/mL, XA-d 42.5 mu g/mL, CA-d 4250 mu g/mL, SCA-d 410 mu g/mL, PGA-d 40.5 mu g/mL, alpha-HB-d 310 mu g/mL and SCA, Mixed internal standard solutions of HVA-d 310. mu.g/mL, VMA-d 310. mu.g/mL, HIA-d 510. mu.g/mL and KA-d 55. mu.g/mL; and adding 900 mu L of formic acid aqueous solution into 100 mu L of the mixed internal standard solution, and uniformly mixing to obtain the mixed internal standard working solution.

7. The method for detecting 21 organic acids in urine according to claim 4 or 5, wherein the volume concentration of the methanol aqueous solution is 10-90% and the volume concentration of the formic acid aqueous solution is 0.1-0.5%.