A method for detecting 55 kinds of organic acids and sugar alcohols in urine by gas chromatography tandem mass spectrometry

Abstract

The application provides a method for detecting 55 kinds of urine organic acids and sugar alcohols by using gas chromatography tandem mass spectrometry. The internal standard method is used for quantitative detection of 55 kinds of organic acids and sugar alcohols in urine samples, and the quantitative results are used for interpretation of human metabolism, and the quantitative lower limit can reach 0.5 ug / mL. The pretreatment method of enzymolysis-oximation-liquid-liquid extraction-silane derivatization is used, the pH is adjusted by using sodium hydroxide solution, the response of the measured substance is high, the peak type is good, and the treated sample has good stability, and can be stored at 4 DEG C and-20 DEG C for 10 days. The application innovatively uses glass consumables for the whole pretreatment process, improves the accuracy of quantification. The method has been tested on 300 random urine samples of human population, the standard curve range can meet the detection requirements of most clinical urine samples, and the secondary retest caused by too high concentration of the measured substance is reduced.

Description

[0001] This application is a divisional application of parent application 202311008873.2, filed on August 11, 2023. Technical Field

[0002] This invention belongs to the field of chemical analysis technology, specifically relating to a method for detecting 55 organic acids and sugar alcohols in urine using gas chromatography-tandem mass spectrometry. Background Technology

[0003] The human body is constantly undergoing thousands of biochemical reactions to maintain its growth, development, and energy metabolism. Under normal circumstances, these biochemical reactions proceed smoothly through various cycles. Each step of the reaction requires specific enzymes to catalyze it. Currently, it is not possible to directly detect the activity of enzymes in the human body. However, each enzyme needs specific coenzymes to function, which usually refer to nutrients such as B vitamins and trace elements. When these nutrients are lacking, enzymes cannot function properly, which can lead to the obstruction of metabolism of some substances in the body, resulting in their accumulation in urine.

[0004] Organic acid and sugar alcohol metabolism analysis involves detecting organic acids and sugar alcohols in urine to analyze whether there are physiological imbalances in the intestinal absorption and assimilation, energy metabolism, biotransformation, and information exchange systems. If these physiological imbalances can be detected in time and corrected through effective intervention, the root cause of the disease can be eliminated, thereby preventing the occurrence and development of the disease.

[0005] Currently, the main methods for detecting the concentration of organic acids and sugar alcohols in urine are ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) or gas chromatography-tandem mass spectrometry (GC-MS / MS). However, the detection results of related methods in existing literature or patents are not ideal. For example, patent CN113777189A detects 3-methylcrotonylglycine and 3-hydroxyisovaleric acid in urine, which involves the addition of hydroxylamine hydrochloride for oximation. However, neither of these methods provides the reaction conditions for oximation. Under acidic conditions, the oximation reaction is incomplete, and hydroxylamine hydrochloride is acidic. Therefore, the above oximation method is not suitable for detection. The results were not ideal. For example, patent CN113777188A detects glutaric acid, 3-hydroxyglutaric acid, and pentenic acid in urine, which also involves oxime reaction. However, none of these three substances contain carbonyl groups. Oxime reaction converts carbonyl groups (C=O) into oximes (C=NO). Therefore, this patent does not require oxime reaction at all. Adding oxime reaction does not improve the detection but increases the processing steps. The detection effect of the method mentioned in this patent is not ideal. Moreover, the above-mentioned method detects a small number of organic acids and sugar alcohols.

[0006] Urine contains a wide variety of organic acids and sugar alcohols, including 3-hydroxybutyric acid, methylmalonic acid, benzoic acid, 3-methyl-2-oxovalerate, 4-methyl-2-oxovalerate, α-ketoisovalerate, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citric acid, malic acid, adipic acid, pyroglutamic acid, α-ketoglutarate, tartaric acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, orotic acid, isocitrate, 2-methylhippuric acid, 2-hydroxyhippuric acid, and 3-hydroxypropionic acid. Organic acids such as 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, glutaric acid, malonic acid, 4-hydroxyphenylpyruvic acid, homogentisic acid, N-acetyl-L-aspartic acid, phenylpyruvic acid, phenylacetic acid, sebacic acid, and 3,4-dihydroxyphenylpropionic acid are metabolic products of nutrients, liver decomposition markers, and intestinal flora markers. Their content in urine can help determine the level of nutrient metabolism in the human body, the liver's metabolic capacity, and whether there is an imbalance in the intestinal flora.

[0007] Therefore, it is urgent to develop a detection method that can accurately quantify and simultaneously analyze multiple organic acids and sugar alcohols in urine. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides a method for detecting 55 organic acids and sugar alcohols in urine using gas chromatography-tandem mass spectrometry (GC-MS). This method enables a single, fully quantitative analysis of 55 organic acids and sugar alcohols in urine. A pretreatment process involving enzymatic digestion, oxime conversion, liquid-liquid extraction, and silanization derivatization is employed to improve analyte response and stability. Based on the quantitative results, insights into human metabolism can be gained. The limit of quantification reaches 0.5 μg / mL. The treated samples are stable for 10 days at room temperature and for 10 days under storage conditions at 4°C and -20°C. Inter-batch and intra-batch precision is less than 15%, ensuring accurate quantification using GC-MS.

[0009] The 55 organic acids and sugar alcohols mentioned in this invention are: lactic acid, 2-hydroxybutyric acid, pyruvic acid, 3-hydroxybutyric acid, β-hydroxyisovaleric acid, methylmalonic acid, benzoic acid, 3-methyl-2-oxovaleric acid, 4-methyl-2-oxovaleric acid, α-ketoisovaleric acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citrate, malic acid, adipic acid, pyroglutamic acid, phenylacetic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, α-ketoglutaric acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3- Pyridine dicarboxylic acid, arabinitol, cis-aconitine, orotic acid, homovanillic acid, citric acid, isocitrate, hippuric acid, vanillylmandelic acid, 2-methylhippuric acid, 3-indoleacetic acid, 5-hydroxyindole-3-acetic acid, 2-hydroxyhippuric acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, 4-hydroxyphenylpyruvic acid, homohydric acid, N-acetyl-L-aspartic acid, phenylpyruvic acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid.

[0010] Common gas chromatography methods for detecting organic acids and sugar alcohols often employ butyl ester derivatization, but this method cannot detect most of the aforementioned organic acids and sugar alcohols. This invention first uses hydroxylamine hydrochloride to oxime-enzyme-enzyme in urine, followed by silane derivatization. This amplifies the signals of organic acids and sugar alcohols for easier detection, while also staggering the chromatographic peaks to differentiate them. This allows for the detection of 55 organic acids and sugar alcohols in urine, while simultaneously improving the precision and accuracy of the detection, with a limit of quantitation reaching 0.5 μg / mL.

[0011] In the above-mentioned oxime process, we specified that the oxime reaction was carried out under alkaline conditions, particularly at pH 8.2 to 9. Under these conditions, all 55 organic acids and sugar alcohols that the technical solution provided by this invention can be detected. Furthermore, after pretreatment under these conditions and storage at 4°C for 10 days, all 55 organic acids and sugar alcohols can still be detected. This has a significant beneficial effect on the practical application of urine organic acid and sugar alcohol detection.

[0012] This invention provides a pretreatment method for the detection of organic acids and sugar alcohols in urine. The pretreatment process is as follows: enzymatic hydrolysis-oximeization-liquid extraction-silanization. After this pretreatment method, the urine sample has good stability and can remain stable for 10 days under storage conditions of 4℃ and -20℃, which has practical application value.

[0013] The present invention also provides an optimized instrument detection temperature program under which the chromatographic peaks of organic acids and sugar alcohols exhibit better peak shapes and higher resolution between peaks. This temperature program enables the detection of 55 organic acids and sugar alcohols and further improves the accuracy and sensitivity of detection.

[0014] Plastic consumables introduce uncontrollable errors into the 55 organic acids and sugar alcohols tested in this invention. The amount of error introduced varies each time and for each detection index. This invention uses glass consumables (e.g., glass centrifuge tubes and glass droppers used for sample pretreatment of urine samples). This reduces background noise in gas chromatography for the 55 organic acids and sugar alcohols, improving detection accuracy. Furthermore, it prevents potential interactions between substances in the plastic and trace amounts of organic acids and sugar alcohols in the urine sample, further improving precision, accuracy, and sensitivity. Simultaneously, pretreatment with glass consumables ensures the stability of the treated urine samples, maintaining stability for up to 10 days under storage conditions at 4°C and -20°C.

[0015] Therefore, in one aspect, the present invention provides a method for detecting organic acids and sugar alcohols in urine using gas chromatography-tandem mass spectrometry, comprising the following steps:

[0016] (1) Enzymatic hydrolysis: Add urease to a glass centrifuge tube, add the sample, and perform enzymatic hydrolysis;

[0017] (2) Oximeification: Add oxime reagent and NaOH solution to the glass centrifuge tube to carry out the oximeification reaction. After the reaction is completed, add HCl solution to neutralize.

[0018] (3) Liquid-liquid extraction: Add ethyl acetate, shake and centrifuge, and transfer the supernatant to a glass centrifuge tube using a glass pipette;

[0019] (4) Silane derivatization: Dry with nitrogen, add silane derivatization reagent, and incubate;

[0020] (5) On-machine testing.

[0021] In some embodiments, the organic acid and sugar alcohol are lactic acid, 2-hydroxybutyric acid, pyruvic acid, 3-hydroxybutyric acid, β-hydroxyisovaleric acid, methylmalonic acid, benzoic acid, 3-methyl-2-oxovaleric acid, 4-methyl-2-oxovaleric acid, α-ketoisovaleric acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citrate, malic acid, adipic acid, pyroglutamic acid, phenylacetic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, α-ketoglutaric acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3- Pyridine dicarboxylic acid, arabinitol, cis-aconitine, orotic acid, homovanillic acid, citric acid, isocitrate, hippuric acid, vanillylmandelic acid, 2-methylhippuric acid, 3-indoleacetic acid, 5-hydroxyindole-3-acetic acid, 2-hydroxyhippuric acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, 4-hydroxyphenylpyruvic acid, homohydric acid, N-acetyl-L-aspartic acid, phenylpyruvic acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid.

[0022] Furthermore, the centrifuge tubes and straws are made of glass.

[0023] Furthermore, the oxime reaction is carried out under alkaline conditions, and then the alkaline conditions are changed to acidic conditions after the reaction is completed.

[0024] Furthermore, in step (2), NaOH solution is added to adjust the pH to pH = 7-9.

[0025] Furthermore, in step (2), NaOH solution is added to adjust the pH to pH = 8.2-9.

[0026] Furthermore, in step (2), the oxime reagent includes one of hydroxylamine ethanol, hydroxylamine hydrochloride, and hydroxylamine sulfate.

[0027] Hydroxylamine hydrochloride and hydroxylamine sulfate are used in oxime reactions using aqueous solutions; hydroxylamine ethanol is an ethanol solution of hydroxylamine hydrochloride.

[0028] All of the above-mentioned oxime reagents can achieve comprehensive detection of 55 organic acids and sugar alcohols in urine. Among them, hydroxylamine hydrochloride and hydroxylamine ethanol have similar effects and are superior to hydroxylamine sulfate. Urine samples treated with hydroxylamine hydrochloride and hydroxylamine ethanol have high stability and can remain stable for 10 days under storage conditions of 4℃ and -20℃. The 55 organic acids and sugar alcohols are basically not degraded.

[0029] Furthermore, in step (2), the concentration of the oxime reagent is 1%-10%; the volume ratio of the oxime reagent to the enzymatically digested sample is 1:1-4.

[0030] In some methods, the concentration of hydroxylamine hydrochloride is 5%.

[0031] In some methods, the ratio of hydroxylamine hydrochloride to the volume of the enzymatically digested sample is 1:2.

[0032] Furthermore, in step (1), the enzymatic hydrolysis process is carried out in a water bath at 37°C for 30 minutes.

[0033] Furthermore, in step (4), the silanization derivatization reagent is BSTFA-TMCS.

[0034] In BSTFA-TMCS, BSTFA refers to bis(trimethylsilyl)trifluoroacetamide, TMCS refers to trimethylchlorosilane, and BSTFA-TMCS is a derivatizing reagent with a ratio of BSTFA:TMCS = 99:1.

[0035] Furthermore, in step (5), a heating program is set during the on-machine testing: 100℃ for 4 minutes, then heated to 280℃ at a rate of 4℃ / min and held for 11 minutes.

[0036] On the other hand, the present invention provides a method for detecting organic acids and sugar alcohols in urine using gas chromatography-tandem mass spectrometry, including sample pretreatment and on-machine detection. The container used for holding urine in the sample pretreatment is a glass container, such as a glass centrifuge tube or a glass pipette.

[0037] On the other hand, the present invention provides the use of a combination of an oxime reagent and a silanization derivatization reagent for preparing a formulation that improves the stability of organic acids and sugar alcohols in urine samples.

[0038] In some respects, improved stability refers to reducing the degradation rate of organic acids and sugar alcohols in urine samples. After the treated urine samples have been stored for a period of time, the organic acids and sugar alcohols can still be detected by gas chromatography-tandem mass spectrometry.

[0039] In some respects, the oxime reagent includes hydroxylamine ethanol, hydroxylamine hydrochloride, and hydroxylamine sulfate.

[0040] In some respects, the silanizing derivatizing agents include BSTFA-TMCS, MSTFA-TMCS, HDMS-TMCS, trimethylchlorosilane, and triethylchlorosilane.

[0041] In some respects, methods for improving the stability of organic acids and sugar alcohols in urine samples include the following steps:

[0042] (1) Enzymatic hydrolysis: Add urease to a glass centrifuge tube, add the sample, and perform enzymatic hydrolysis;

[0043] (2) Oximeification: Add oxime reagent and NaOH solution to the glass centrifuge tube to carry out the oximeification reaction. After the reaction is completed, adjust the pH with HCl solution.

[0044] (3) Liquid-liquid extraction: Add ethyl acetate, shake and centrifuge, and transfer the supernatant to a glass centrifuge tube using a glass pipette;

[0045] (4) Silane derivatization: Dry with nitrogen, add silane derivatization reagent, and incubate.

[0046] In MSTFA-TMCS, MSTFA stands for N-methyltrimethylsilyltrifluoroacetamide, and TMCS stands for trimethylchlorosilane. MSTFA-TMCS is a silanizing agent with a ratio of MSTFA:TMCS = 99:1. In HDMS-TMCS, HDMS stands for 1,1,1,3,3,3-hexamethyldisilazane, and TMCS stands for trimethylchlorosilane. HDMS-TMCS is a silanizing agent with a ratio of HDMS:TMCS = 99:1.

[0047] The beneficial effects of this invention are as follows:

[0048] 1. The internal standard method can be used to quantitatively detect 55 organic acids and sugar alcohols in urine samples. The quantitative results can be interpreted to understand human metabolism. The lower limit of quantification for all 55 organic acids and sugar alcohols in urine samples can reach 0.5 μg / mL.

[0049] 2. The pretreatment method of enzymatic hydrolysis-oxime-liquid extraction-silanization derivatization results in high analyte response and good peak shape. The treated sample also exhibits good stability, remaining stable for 10 days under storage conditions of 4℃ and -20℃.

[0050] 3. Commonly used plastic consumables increase the background noise of 55 organic acids and sugar alcohols in gas chromatography. They also interact with some organic acids and sugar alcohols, affecting quantification and introducing errors that are difficult to measure, significantly impacting the detection quantity of organic acids and sugar alcohols. Therefore, this invention innovatively uses glass consumables for the entire pretreatment process, improving the detection quantity of organic acids and sugar alcohols while also ensuring accurate quantification.

[0051] 4. This method has been tested on 300 randomized population urine samples. The standard curve range can meet the detection needs of most clinical urine samples, reducing the need for secondary testing due to excessively high analyte concentrations. Attached Figure Description

[0052] Figure 1 The chromatogram is for 3-phenylpropionic acid;

[0053] Figure 2 The chromatogram is for adipic acid.

[0054] Figure 3 Chromatograms of benzoic acid, mandelic acid, and 4-hydroxyphenyllactic acid;

[0055] Figure 4Chromatograms of 3-methyl-2-oxovaleric acid and 4-methyl-2-oxovaleric acid;

[0056] Figure 5 The chromatograms are of fumaric acid and isocitric acid.

[0057] Figure 6 Chromatograms of succinic acid, citrate, and 3-hydroxy-3-methylglutaric acid;

[0058] Figure 7 Chromatograms of α-ketoisovaleric acid and α-ketoglutaric acid;

[0059] Figure 8 The chromatograms are of 4-hydroxybenzoic acid and 2,3-pyridinedicarboxylic acid.

[0060] Figure 9 The chromatogram is for 2-hydroxyisohexanoic acid;

[0061] Figure 10 The chromatogram is for phenylacetic acid.

[0062] Figure 11 The chromatogram is for pyridine-2-carboxylic acid.

[0063] Figure 12 The chromatogram is for phenylpyruvic acid.

[0064] Figure 13 The chromatogram is for tricarboxylic acid.

[0065] Figure 14 The chromatogram is for orotic acid.

[0066] Figure 15 The chromatogram of homovanillic acid is shown.

[0067] Figure 16 The chromatogram of vanillylmandelic acid is shown.

[0068] Figure 17 The chromatogram is for 2-methylhippuric acid.

[0069] Figure 18 The chromatogram is for 3-indoleacetic acid;

[0070] Figure 19 Chromatograms of lactic acid, 2-hydroxybutyric acid, 3-hydroxypropionic acid, pyruvic acid, 3-hydroxybutyric acid, 2-hydroxyisovaleric acid, malonic acid, β-hydroxyisovaleric acid, methylmalonic acid, and ethylmalonic acid;

[0071] Figure 20 Chromatograms of glutaric acid, malic acid, pyroglutamic acid, phenylethyl ketone acid, phenyllactic acid, 4-hydroxybenzoic acid, N-acetyl-L-aspartic acid, succinic acid, and cis-aconitic acid;

[0072] Figure 21 Chromatograms of hippuric acid, citric acid, homogentisic acid, 4-hydroxybenzoic acid, 3,4-dihydroxyphenylpropionic acid, and 2-hydroxyhippuric acid;

[0073] Figure 22 The peak shape of the butylated malate chromatogram;

[0074] Figure 23 The peak shape of the butyl citrate chromatogram;

[0075] Figure 24 The peak shape of the chromatogram of malic acid silanization derivatization;

[0076] Figure 25 The peak shape diagram of citric silanization derivatization;

[0077] Figure 26 The chromatogram of benzoic acid before instrument temperature program optimization;

[0078] Figure 27 Chromatogram of 3-hydroxypropionic acid before instrument temperature program optimization;

[0079] Figure 28 Chromatogram of 2-hydroxyisovaleric acid before instrument temperature program optimization;

[0080] Figure 29 The chromatogram of benzoic acid after optimizing the instrument's temperature program;

[0081] Figure 30 Chromatogram of 3-hydroxypropionic acid after optimization of the instrument's temperature program;

[0082] Figure 31 The chromatogram of 2-hydroxyisovaleric acid after optimization of the instrument's temperature program;

[0083] Figure 32 The chromatograms are obtained from the gas chromatography detection of 55 organic acids in urine. Detailed Implementation

[0084] The preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and are not intended to limit it in any way. All features disclosed in the embodiments of the present invention, or all steps in all methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any way.

[0085] Example 1: A method for detecting 55 organic acids and sugar alcohols in urine by gas chromatography-tandem mass spectrometry

[0086] I. Preparation of Standard Curve and Related Solutions:

[0087] 1. Preparation of standard single-standard stock solution:

[0088] Table 1: Preparation Table of Standard Stock Solutions

[0089]

[0090]

[0091] 2. Prepare the mixed standard solution required for the experiment using the prepared single standard solution:

[0092] Table 2: Preparation Table of Mixed Standard Solutions

[0093] 3. Prepare standard curves and quality control samples using the prepared mixed standard solutions:

[0094] Table 3: Standard Curve for Mixed Solutions

[0095] STD 6 mix1 4000 8000 100 STD 5 mix1 2000 8000 50 STD 4 mix1 400 8000 10 STD 3 STD5 800 8000 5 STD2 STD4 800 8000 1 STD 1 STD3 800 8000 0.5

[0096] Table 4: Quality control samples for the preparation of mixed solutions

[0097] LQC 400 19600 4 HQC 2000 18000 20

[0098] II. Preparation of Internal Standard and Experimental Reagents:

[0099] 1. Preparation of internal standard working solution:

[0100] Accurately dissolve 50 mg of troponic acid in 50 mL of ethyl acetate, and store the prepared internal standard working solution at 4 °C.

[0101] 2. Preparation of experimental reagents:

[0102] Urease preparation: Add 12KU urease to 12mL of water and mix well. Aliquot according to sample volume, avoiding repeated freeze-thaw cycles. Store the prepared urease at -20℃.

[0103] 5% hydroxylamine hydrochloride: Accurately dissolve 10g of hydroxylamine hydrochloride in 200mL of water.

[0104] 5% Hydroxylamine Ethanol: Accurately dissolve 10g of hydroxylamine hydrochloride in 200mL of ethanol.

[0105] 5% hydroxylamine sulfate: Accurately dissolve 10g of hydroxylamine sulfate in 200mL of water.

[0106] Preparation of 2.5N NaOH: Accurately dissolve 20g of NaOH in 200mL of water.

[0107] Preparation of 6N HCl: Transfer 30mL of 37% HCl and add 20mL of water and mix well.

[0108] III. Pretreatment methods:

[0109] 1. Sample pretreatment methods:

[0110] Add 20 μL of urease to a 10 mL glass centrifuge tube, then add 500 μL of sample (urine sample, from morning urine of a randomized population), shake for 5 min, incubate at 37 °C for 30 min, then add 40 μL of internal standard and 500 μL of water; then add 500 μL of 5% hydroxylamine hydrochloride, and adjust the pH to 8.2 with 400 μL of 2.5N NaOH, shake for 10 min, and let stand for 1 h. Add 350 μL of 6N HCl to neutralize and bring the pH to 7, shake for 5 min. Add 5 mL of ethyl acetate, shake for 10 min, centrifuge for 5 min, and transfer the supernatant to a 10 mL glass centrifuge tube using a glass pipette. Repeat the above extraction steps (add 5 mL of ethyl acetate, shake for 10 min, centrifuge for 5 min), and combine the supernatant with the first supernatant. Dry the supernatant with liquid nitrogen, add 100 μL of BSTFA-TMCS, shake for 5 min, incubate at 80 °C for 30 min, and take 90 μL for analysis.

[0111] 2. The quality control sample processing method is as described above.

[0112] 3. Standard curve preprocessing methods:

[0113] Add 500 μL of the standard curve (the solution prepared in Table 3 for standard curve determination) to a 10 mL glass centrifuge tube, add 500 μL of water, add 500 μL of 5% hydroxylamine hydrochloride, and add 400 μL of 2.5N NaOH to adjust the pH to 8.2. Shake for 10 min and let stand for 1 h. Add 350 μL of 6N HCl to neutralize the pH to 7 and shake for 5 min. Add 5 mL of ethyl acetate, shake for 10 min, centrifuge for 5 min, and transfer the supernatant to a 10 mL glass centrifuge tube using a glass pipette. Repeat the above extraction steps (add 5 mL of ethyl acetate, shake for 10 min, centrifuge for 5 min). Combine the supernatant with the first supernatant, dry the supernatant with liquid nitrogen, add 100 μL of BSTFA-TMCS, shake for 5 min, incubate at 80 °C for 30 min, and take 90 μL for analysis.

[0114] IV. Instrument Requirements:

[0115] Column: DB-5 (30m × 0.25mm × 1μm); Carrier gas: Helium; Injection volume: 1μL; Injector temperature: 280℃; Septum purge flow rate: 5mL / min; Purge time: 0.75min; Injector mode: Split injection, split ratio 10:1; Flow rate: 1.0mL / min, constant flow rate; Transfer line: 280℃. Temperature program: 100℃ held for 4min, then ramped to 280℃ at a rate of 4℃ / min and held for 11min; Acquisition mode: SIM; Ion source temperature: 250℃; Quadrupole temperature: 150℃; Solvent delay: 8min.

[0116] Table 5: Quantitative ions, qualitative ions, and retention times

[0117]

[0118]

[0119] Table 6: Linear equations and R² of the standard curve

[0120]

[0121]

[0122] V. Methodological Performance:

[0123] Table 7: Inter-batch precision of lactic acid

[0124]

[0125] Table 8: Lactic Acid Standard Curve

[0126]

[0127] Table 9: Inter-batch precision of 2-hydroxybutyric acid

[0128]

[0129] Table 10: Standard Curve for 2-Hydroxybutyric Acid

[0130]

[0131] Table 11: Inter-batch precision of pyruvate

[0132]

[0133] Table 12: Pyruvate Standard Curve

[0134]

[0135] Table 13: Inter-batch precision of 3-hydroxybutyric acid

[0136]

[0137] Table 14: Standard Curve for 3-Hydroxybutyric Acid

[0138]

[0139] Table 15: Inter-batch precision of methylmalonic acid

[0140]

[0141] Table 16: Standard Curve for Methylmalonic Acid

[0142]

[0143] Table 17: Inter-batch precision of benzoic acid

[0144]

[0145] Table 18: Benzoic Acid Standard Curve

[0146]

[0147] Table 19: Inter-batch precision of 3-methyl-2-oxovalerate

[0148]

[0149] Table 20: Standard Curve for 3-Methyl-2-oxovalerate

[0150]

[0151] Table 21: Inter-batch precision of 4-methyl-2-oxovalerate

[0152]

[0153] Table 22: Standard Curve for 4-Methyl-2-oxovalerate

[0154]

[0155] Table 23: Inter-batch precision of succinic acid

[0156]

[0157] Table 24: Succinic Acid Standard Curve

[0158]

[0159] Table 25: Inter-batch precision of fumarate

[0160]

[0161] Table 26: Inter-batch standard curves of fumaric acid

[0162]

[0163] Table 27: Inter-batch precision of 3-phenylpropionic acid

[0164]

[0165] Table 28: Standard Curve for 3-Phenylacetic Acid

[0166]

[0167] Table 29: Inter-batch precision of citric acid

[0168]

[0169] Table 30: Standard Curve for Limonic Acid

[0170]

[0171] Table 31: Inter-batch precision of malic acid

[0172]

[0173] Table 32: Standard Curve for Malic Acid

[0174]

[0175] Table 33: Adipic Acid Batch Precision

[0176]

[0177] Table 34: Adipic Acid Standard Curve

[0178]

[0179] Table 35: Inter-batch precision of pyroglutamic acid

[0180]

[0181] Table 36: Standard Curve for Pyroglutamic Acid

[0182]

[0183] Table 37: Inter-batch precision of phenacetinic acid

[0184]

[0185] Table 38: Standard Curve for Phenylacetic Acid

[0186]

[0187] Table 39: Inter-batch precision of 4-hydroxybenzoic acid

[0188]

[0189] Table 40: Standard Curve for 4-Hydroxybenzoic Acid

[0190]

[0191] Table 41: Inter-batch precision of α-ketoglutaric acid

[0192]

[0193] Table 42: Standard Curve for α-Ketoglutarate

[0194]

[0195] Table 43: Inter-batch precision of 4-hydroxyphenylacetic acid

[0196]

[0197] Table 44: Standard Curve for 4-Hydroxyphenylacetic Acid

[0198]

[0199] Table 45: Inter-batch precision of tartaric acid

[0200]

[0201] Table 46: Tartaric Acid Standard Curve

[0202]

[0203] Table 47: Inter-batch Precision of Octadiic Acid

[0204]

[0205] Table 48: Standard Curve for Octadionic Acid

[0206]

[0207] Table 49: Inter-batch precision of tricarboxylic acid

[0208]

[0209] Table 50: Standard Curve for Tricarboxylic Acid

[0210]

[0211] Table 51: Inter-batch precision of 2,3-pyridinedicarboxylic acid

[0212]

[0213] Table 52: Standard Curve for 2,3-Pyridinedicarboxylic Acid

[0214]

[0215] Table 53: Inter-batch precision of cis-aconic acid

[0216]

[0217] Table 54: Standard Curve for Cis-Aconitic Acid

[0218]

[0219] Table 55: Inter-batch precision of whey acid

[0220]

[0221] Table 56: Orytic Acid Standard Curve

[0222]

[0223] Table 57: Inter-batch precision of high vanillic acid

[0224]

[0225] Table 58: Standard Curve for Pervanillic Acid

[0226]

[0227] Table 59: Inter-batch precision of isocitric acid

[0228]

[0229] Table 60: Standard Curve for Isocitrate

[0230]

[0231] Table 61: Inter-batch precision of citric acid

[0232]

[0233] Table 62: Citric Acid Standard Curve

[0234]

[0235] Table 63: Inter-batch precision of hippuric acid

[0236]

[0237] Table 64: Standard Curve for Hippuric Acid

[0238]

[0239] Table 65: Vanilla Almond Acid Inter-bit Precision

[0240]

[0241] Table 66: Standard Curve for Vanillyl Mandelic Acid

[0242]

[0243] Table 67: Inter-batch precision of 2-methylhippuric acid

[0244]

[0245] Table 68: Standard Curve for 2-Methylhippuric Acid

[0246]

[0247] Table 69: Inter-batch precision of pyridine-2-carboxylic acid

[0248]

[0249] Table 70: Standard Curve for Pyridine-2-Carboxylic Acid

[0250]

[0251] Table 71: Inter-batch precision of arabinitol

[0252]

[0253] Table 72: Standard Curve of Arabinol

[0254]

[0255] Table 73: Inter-batch precision of 3-hydroxy-3-methylglutaric acid

[0256]

[0257] Table 74: Standard Curve for 3-Hydroxy-3-methylglutaric acid

[0258]

[0259] Table 75: Inter-batch precision of β-hydroxyisovaleric acid

[0260]

[0261] Table 76: Standard Curve for β-Hydroxyisovalerate

[0262]

[0263] Table 77: Inter-batch precision of 3-indoleacetic acid

[0264]

[0265] Table 78: Standard Curve for 3-Indoleacetic Acid

[0266]

[0267] Table 79: Inter-batch precision of 5-hydroxy-indole-3-acetic acid

[0268]

[0269] Table 80: Standard Curve for 5-Hydroxy-Indole-3-Acetic Acid

[0270]

[0271] Table 81: Inter-batch precision of 2-hydroxyhippuric acid

[0272]

[0273] Table 82: Standard Curve for 2-Hydroxyhippuric Acid

[0274]

[0275] Table 83: Inter-batch precision of 3-hydroxypropionic acid

[0276]

[0277] Table 84: Standard Curve for 3-Hydroxypropionic Acid

[0278]

[0279] Table 85: Inter-batch precision of 2-hydroxyisovaleric acid

[0280]

[0281] Table 86: Standard Curve for 2-Hydroxyisovalerate

[0282]

[0283] Table 87: Inter-batch precision of 2-hydroxyisohexanoic acid

[0284]

[0285] Table 88: Standard Curve for 2-Hydroxyisohexanoic Acid

[0286]

[0287] Table 89: Inter-batch precision of 4-hydroxyphenyllactic acid

[0288]

[0289] Table 90: Standard Curve for 4-Hydroxyphenyllactic Acid

[0290]

[0291] Table 91: Inter-batch precision of phenyllactic acid

[0292]

[0293] Table 92: Standard Curve for Phenolic Acid

[0294]

[0295] Table 93: Inter-batch precision of ethylmalonic acid

[0296]

[0297] Table 94: Standard Curve for Ethylmalonic Acid

[0298]

[0299] Table 95: Inter-batch precision of mandelic acid

[0300]

[0301] Table 96: Standard Curve for Mandelic Acid

[0302]

[0303] Table 97: Inter-batch precision of glutaric acid

[0304]

[0305] Table 98: Standard Curve for Glutaric Acid

[0306]

[0307] Table 99: Inter-batch precision of malonic acid

[0308]

[0309] Table 100: Standard Curve for Malonic Acid

[0310]

[0311] Table 101: Inter-batch precision of 4-hydroxyphenylpyruvic acid

[0312]

[0313] Table 102: Standard Curve for 4-Hydroxyphenylpyruvic Acid

[0314]

[0315] Table 103: Inter-batch precision of homogentisic acid

[0316]

[0317] Table 104: Standard Curve for Alkali Acid

[0318]

[0319] Table 105: Inter-batch precision of N-acetyl-L-aspartic acid

[0320]

[0321] Table 106: Standard Curve for N-acetyl-L-aspartic acid

[0322]

[0323] Table 107: Inter-batch precision of phenylpyruvic acid

[0324]

[0325] Table 108: Standard Curve for Phenylacetic Acid

[0326]

[0327] Table 109: Inter-batch precision of phenylacetic acid

[0328]

[0329] Table 110: Standard Curve for Phenylacetic Acid

[0330]

[0331] Table 111: Inter-batch precision of sebacic acid

[0332]

[0333] Table 112: Standard Curve for Sebacic Acid

[0334]

[0335] Table 113: Inter-batch precision of 3,4-dihydroxyphenylpropionic acid

[0336]

[0337] Table 114: Standard Curve for 3,4-Dihydroxyphenylpropionic Acid

[0338]

[0339] Table 115: Inter-batch precision of α-ketoisovaleric acid

[0340]

[0341] Table 116: Standard Curve for α-Ketoisovaleric Acid

[0342]

[0343] The urine sample, after being processed and detected using the method of this invention, yields the following overall chromatogram: Figure 32 As shown, 55 organic acids and sugar alcohols can be accurately quantified. Chromatograms of some organic acids and sugar alcohols are shown below:

[0344] Figure 1 The chromatogram is for 3-phenylpropionic acid; Figure 2 The chromatogram is for adipic acid. Figure 3 Chromatograms of benzoic acid, mandelic acid, and 4-hydroxyphenyllactic acid; Figure 4 Chromatograms of 3-methyl-2-oxovaleric acid and 4-methyl-2-oxovaleric acid; Figure 5 The chromatograms are of fumaric acid and isocitric acid. Figure 6 Chromatograms of succinic acid, citrate, and 3-hydroxy-3-methylglutaric acid; Figure 7 Chromatograms of α-ketoisovaleric acid and α-ketoglutaric acid; Figure 8 The chromatograms are of 4-hydroxybenzoic acid and 2,3-pyridinedicarboxylic acid. Figure 9 The chromatogram is for 2-hydroxyisohexanoic acid; Figure 10 The chromatogram is of phenylacetic acid. Figure 11 The chromatogram is for pyridine-2-carboxylic acid. Figure 12 The chromatogram is for phenylpyruvic acid. Figure 13 The chromatogram is for tricarboxylic acid; Figure 14 The chromatogram is for orotic acid. Figure 15 The chromatogram of homovanillic acid is shown. Figure 16 The chromatogram of vanillylmandelic acid is shown. Figure 17 The chromatogram is for 2-methylhippuric acid. Figure 18 The chromatogram is for 3-indoleacetic acid; Figure 19 Chromatograms of lactic acid, 2-hydroxybutyric acid, 3-hydroxypropionic acid, pyruvic acid, 3-hydroxybutyric acid, 2-hydroxyisovaleric acid, malonic acid, β-hydroxyisovaleric acid, methylmalonic acid, and ethylmalonic acid; Figure 20 Chromatograms of glutaric acid, malic acid, pyroglutamic acid, phenylethyl ketone acid, phenyllactic acid, 4-hydroxybenzoic acid, N-acetyl-L-aspartic acid, succinic acid, and cis-aconitic acid; Figure 21 Chromatograms of hippuric acid, citric acid, homogentisic acid, 4-hydroxybenzoic acid, 3,4-dihydroxyphenylpropionic acid, and 2-hydroxyhippuric acid.

[0345] The pretreatment and detection method of this invention enables the detection of all 55 organic acids and sugar alcohols in urine, and the peaks of the 55 organic acids and sugar alcohols are highly distinguishable. When viewed individually, the peaks of the 55 organic acids and sugar alcohols have good peak shapes, which is conducive to accurate quantification.

[0346] Six: Stability

[0347] When detecting organic acids and sugar alcohols in urine samples, a complex pretreatment process is required before instrumental analysis. If the pretreated samples are unstable and difficult to store, each pretreatment step must be freshly prepared and used immediately, causing significant inconvenience to the operation. For example, after receiving the samples, gas chromatography analysis may not be possible immediately due to instrument occupancy. In practice, the situation is quite complex; the samples may deteriorate while waiting in line for injection after pretreatment, making analysis impossible. Another important scenario is when the samples are critical and require multiple analyses. In such cases, the pretreated samples need to be stored for a period of time. If the pretreated samples deteriorate or the organic acids and sugar alcohols decompose during this period, it is tantamount to losing important samples, which is extremely detrimental to the research. Alternatively, after one analysis, the samples need to be sent to other departments for secondary analysis. If the pretreated samples deteriorate during the transfer process after pretreatment, it will severely affect the research.

[0348] The pretreatment method provided in this application results in samples with excellent stability, which can be stored for a long time at 4℃ and -20℃. The samples can be taken out and used directly for instrument testing when needed, and the test results can still maintain extremely high accuracy.

[0349] During the research process of this invention, we found that urine samples, after being pretreated by this invention, exhibit outstanding stability and can be stored for 10 days at 4°C and -20°C. During this period, samples can be taken at any time for instrument testing, and 55 kinds of organic acids and sugar alcohols can be accurately detected. It is evident that the method provided by this invention has very beneficial value for practical work.

[0350] We set the standard storage conditions as follows: 4℃ and -20℃. After performing the same pretreatment as described above on the LQC quality control products, we stored them at the above temperatures. Every other day, we took a portion of the pretreated LQC quality control products for testing using the above method. We compared the results with those from the first day to calculate the peak area ratio. The results are shown in Table 117.

[0351] Table 117: Peak area ratio under storage conditions of 4℃ and -20℃

[0352]

[0353]

[0354] The results showed that the stability was excellent after 10 days of storage at 4℃ and -20℃, with peak area ratios within 100% ± 15%, indicating that organic acids and sugar alcohols are not easily degraded after the pretreatment of this invention. Therefore, the pretreatment method of this invention is beneficial to improving the reliability of detection and making the detection more practically valuable.

[0355] Example 2: Screening Experiment of Pretreatment Methods

[0356] This embodiment uses the detection method of Example 1 and sets up other groups. The difference between the other groups is that the pretreatment method is changed. The pretreatment methods include enzymatic hydrolysis, oxime (optional), extraction (optional), and derivatization (optional, including methyl esterification, butyl esterification, and silanization). The same steps are performed in the same way to facilitate comparison of the impact of other steps on the overall pretreatment method, so as to study the most suitable pretreatment method for the detection of 55 organic acids and sugar alcohols in urine.

[0357] We have set up the following preprocessing methods:

[0358] Pretreatment method for methyl esterification: enzymatic hydrolysis-oxime conversion-extraction-methyl esterification

[0359] Add 20 μL of urease, 500 μL of LQC (quality control), shake for 5 min, incubate at 37℃ for 30 min, add 40 μL of internal standard, add 500 μL of water, add 500 μL of 5% hydroxylamine hydrochloride, add 400 μL of 2.5N NaOH to adjust pH to 8.2, shake for 10 min, and let stand for 1 h. Add 350 μL of 6N HCl to neutralize pH to 7, shake for 5 min. Add 5 mL of ethyl acetate, shake for 10 min, centrifuge for 5 min, transfer the supernatant, and repeat the above extraction steps (add 5 mL of ethyl acetate, shake for 10 min, centrifuge for 5 min). Combine the supernatant with the first supernatant, dry the supernatant under nitrogen, add 2 mL of 10% sulfuric acid-methanol, react at 62℃ for 2 h, cool to room temperature, add 2 mL of saturated NaCl, add 2 mL of ethyl acetate, shake for 10 min, centrifuge for 5 min, collect the supernatant, dry under nitrogen, add 100 μL of n-hexane to reconstitute, and analyze.

[0360] Pretreatment method for butylation: enzymatic hydrolysis - oxime conversion - extraction - butylation

[0361] Add 20 μL of urease and 500 μL of LQC (quality control standard), mix well, incubate at 37°C for 30 min, add 40 μL of internal standard, dilute to 1 mL with water, add 500 μL of hydroxylamine hydrochloride, adjust pH to 8.2 with 400 μL of 2.5N NaOH, mix well, let stand for 1 h, add 350 μL of 6N HCl to neutralize pH to 7, mix well, add 5 mL of ethyl acetate, mix well for 10 min, centrifuge for 5 min, transfer the supernatant to a 10 mL glass centrifuge tube, add another 5 mL of ethyl acetate, mix well for 10 min, centrifuge for 5 min, mix the supernatant with the first supernatant, dry under nitrogen at 60°C, add 500 μL of n-butanol-acetyl chloride, incubate at 80°C for 30 min, dry under nitrogen, reconstitute with 100 μL of n-hexane, and analyze.

[0362] Direct silanization pretreatment method: enzymatic hydrolysis-extraction-silanization

[0363] Add 20 μL of urease and 500 μL of LQC quality control, shake for 5 min, incubate at 37°C for 30 min, add 40 μL of internal standard, add 500 μL of water, add 5 mL of ethyl acetate, shake for 10 min, centrifuge for 5 min, transfer the supernatant to a 10 mL glass centrifuge tube using a glass pipette, repeat the above extraction steps (add 5 mL of ethyl acetate, shake for 10 min, centrifuge for 5 min), collect the supernatant and combine it with the first supernatant, blow the supernatant dry with liquid nitrogen, add 100 μL of BSTFA-TMCS, shake for 5 min, incubate at 80°C for 30 min, and take 90 μL for analysis.

[0364] Direct oxime derivatization pretreatment method: enzymatic digestion-oximeization-silanization

[0365] Add 20 μL of urease and 500 μL of LQC quality control sample, shake for 5 min, incubate at 37℃ for 30 min, add 40 μL of internal standard, add 500 μL of water, add 500 μL of hydroxylamine hydrochloride, adjust pH to 8.2 with 400 μL of 2.5N NaOH, mix well, let stand for 1 h, add 350 μL of 6N HCl to neutralize pH to 7, blow dry with nitrogen, add 100 μL of BSTFA-TMCS, shake for 5 min, incubate at 80℃ for 30 min, and take 90 μL for instrumental analysis.

[0366] Direct oxime pretreatment method: enzymatic hydrolysis-oximeization-extraction

[0367] Add 20 μL of urease and 500 μL of LQC (quality control), shake for 5 min, incubate at 37°C for 30 min, add 40 μL of internal standard, add 500 μL of water, add 500 μL of 5% hydroxylamine hydrochloride, add 400 μL of 2.5N NaOH to adjust pH to 8.2, shake for 10 min, and let stand for 1 h. Add 350 μL of 6N HCl to neutralize pH to 7, shake for 5 min. Add 5 mL of ethyl acetate, shake for 10 min, centrifuge for 5 min, transfer the supernatant, and repeat the above extraction steps (add 5 mL of ethyl acetate, shake for 10 min, centrifuge for 5 min). Combine the supernatant with the first supernatant, dry the supernatant with liquid nitrogen, add 100 μL of n-hexane to reconstitute, and analyze.

[0368] The pretreatment method for methyl esterification has a low response, and organic acids and sugar alcohols are both below the detection limit and cannot be detected. Specific experimental data are omitted.

[0369] The pretreatment method of butyl esterification was used to process the standard curves of 55 organic acids and sugar alcohols, but only 14 organic acids and sugar alcohols could be detected. Among them, some organic acids and sugar alcohols had poor peak shapes and low responses, and the standard curves had poor linearity, resulting in large detection errors.

[0370] For a detailed comparison of chromatographic peaks and responses of the butylation and silanization derivatization pretreatment methods, please refer to [link to relevant documentation]. Figures 22-25 ; Figure 22 The peak shape of the butylated malate chromatogram; Figure 23 The peak shape of the butyl citrate chromatogram; Figure 24 The peak shape is shown in the chromatogram of malic acid silanization derivatization; the peak on the left side of the figure is shown. Figure 25 The peak shape is shown in the chromatogram of citric silanization derivatization, and it is the peak on the left side of the figure.

[0371] Butyl esterification derivatization has poor peak shape, with peak overlap and shift, making detection difficult, resulting in poor detection results, low response, poor linearity of the standard curve, and difficulty in ensuring detection accuracy. Silanization derivatization, on the other hand, has good peak shape, clear distinction between peaks, high response, and good linearity of the standard curve, which is beneficial for the detection of 55 organic acids and sugar alcohols.

[0372] The direct silanization pretreatment method lacks a one-step oxime reaction. In the LQC (Low Quality Control) sample, organic acids and sugar alcohols containing carbonyl groups are unstable during subsequent detection because the oxime reaction has not occurred. Some organic acids fail to be detected, and the detected carbonyl-containing organic acids exhibit low peak areas due to partial degradation. Furthermore, the continuous decomposition of carbonyl-containing organic acids and sugar alcohols in subsequent detections leads to poor stability. After 10 days, all carbonyl-containing organic acids and sugar alcohols become undetectable.

[0373] The direct oxime derivatization pretreatment method suffers from a large number of interferences. The LQC quality control product after derivatization crystallizes from the derivatization reagent and cannot be tested on the instrument.

[0374] Table 118 shows the comparison of chromatographic peak areas for butylation and silanization derivatization pretreatment methods. Thirteen organic acids were selected for comparison of chromatographic peak areas (the following 13 organic acids are among the 55 organic acids and sugar alcohols that are more difficult to detect, and their chromatographic intensity is the lowest. If these 13 organic acids are detected well, the detection of the other organic acids and sugar alcohols will not be a problem. Therefore, the following experiments are all conducted using these 13 organic acids).

[0375] Table 118: Comparison of chromatographic peak areas for butylation and silanization pretreatment methods

[0376]

[0377] In addition, we statistically analyzed the types of organic acids and sugar alcohols that can be detected by various pretreatment methods and studied their stability (the changes in the types of organic acids and sugar alcohols that can be detected over time). The temperature was set at 4℃ (if it is stable at 4℃, it will definitely be stable at -20℃). The results are shown in Table 119.

[0378] Table 119: Pretreatment methods and number of detectable organic acid species

[0379]

[0380] The butylation derivatization (enzymatic hydrolysis-oxime-extraction-butylation) on the first day detected the following: lactic acid, pyruvic acid, benzoic acid, succinic acid, fumaric acid, malic acid, adipic acid, octanoic acid, orotic acid, citric acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, and 14 other organic acids.

[0381] On the third day, the following were detected: lactic acid, pyruvic acid, benzoic acid, succinic acid, orotic acid, citric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, and 9 other organic acids.

[0382] On the seventh day, the following were detected: orotic acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, and four organic acids.

[0383] On the tenth day, the following were detected: 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, and three organic acids.

[0384] On the first day of direct silanization (enzymatic hydrolysis-extraction-silanization), the following organic acids were detected: lactic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, β-hydroxyisovaleric acid, methylmalonic acid, benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citrate, malic acid, adipic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 3-indoleacetic acid, 5-hydroxyindole-3-acetic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, and 40 other organic acids.

[0385] On the third day, the following were detected: 2-hydroxybutyric acid, 3-hydroxybutyric acid, β-hydroxyisovaleric acid, methylmalonic acid, benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citrate, malic acid, adipic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 3-indoleacetic acid, 5-hydroxyindole-3-acetic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, and 39 kinds of organic acids.

[0386] On the seventh day, the following were detected: β-hydroxyisovaleric acid, benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citrate, malic acid, adipic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 5-hydroxyindole-3-acetic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, and 35 kinds of organic acids.

[0387] On the tenth day, the following were detected: benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, citric acid, malic acid, adipic acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, and 30 kinds of organic acids.

[0388] The results showed that the pretreatment method described in Example 1 (enzymatic hydrolysis-oxime-liquid extraction-silanization derivatization) could detect the most types of organic acids and sugar alcohols compared to other pretreatment methods, and the 55 types of organic acids and sugar alcohols detected are of great value for the study of human metabolism; moreover, the method has extremely high stability, and the types of organic acids and sugar alcohols that can be detected remain unchanged over 10 days as time increases; and according to Example 1, its decomposition rate is also extremely low over 10 days, and high-precision detection can still be achieved on the 10th day.

[0389] By comparing different pretreatment methods, it can be found that derivatization is necessary; without derivatization, no organic acids or sugar alcohols can be detected. Comparing different derivatization methods, only silanization derivatization can detect all 55 organic acids and sugar alcohols, and it exhibits good stability. Secondly, oxime treatment is necessary; without oxime treatment, the number of detectable organic acids and sugar alcohols is reduced to 48, and some organic acids and sugar alcohols degrade within 10 days, further reducing the number of detectable species. Since the sample is urine, urease needs to be added to improve extraction efficiency and detection sensitivity. Comparison shows that without extraction, oxime treatment followed by silanization derivatization results in crystallization, and no organic acids or sugar alcohols can be detected in the supernatant. Therefore, the final pretreatment method is determined to be: enzymatic hydrolysis - oxime treatment - liquid-liquid extraction - silanization derivatization.

[0390] Example 3: Pretreatment Oxime

[0391] From Example 2, we determined the final pretreatment method to be: enzymatic hydrolysis-oximeization-liquid-liquid extraction-silanization derivatization; in this example, we studied the substantial benefits of oximeization for detection, as well as the conditions under which the oximeization reaction was carried out.

[0392] First, we set up two control experiments. For each control, 20 μL of urease was added to 500 μL of LQC quality control solution, mixed, and incubated at 37°C for 30 min. Then, 40 μL of internal standard was added, and water was added to bring the volume to 1 mL. For one control group, 500 μL of 5% hydroxylamine hydrochloride and 400 μL of 2.5N NaOH were added to adjust the pH to 8.2. The mixture was mixed, allowed to stand for 1 h, and then 350 μL of 6N HCl was added to neutralize the pH to 7. The mixture was then mixed. The other control group was left untreated. Subsequently, 5 mL of ethyl acetate was added to both control groups, shaken for 10 min, centrifuged for 5 min, and the supernatant was collected. The above extraction steps were repeated (adding 5 mL of ethyl acetate, shaking for 10 min, centrifuging for 5 min). The supernatant was collected and combined with the first supernatant. The supernatant was dried under liquid nitrogen, 100 μL of silanization derivatization reagent was added, shaken for 5 min, and incubated at 80°C for 30 min. 90 μL of the supernatant was then analyzed. The peak areas of some organic acids are shown in Table 120.

[0393] Table 120: Effect of oximation on the detection of organic acids

[0394] 4-Methyl-2-oxovaleric acid 1.49E+07 1.86E+06 2-Methylhippuric acid 4.56E+06 5.76E+05 Pyruvic acid 2.01E+07 1.03E+06 Phenylacetic acid 1.66E+07 2.19E+06 whey acid 9.28E+06 N / A α-Ketoglutaric acid 4.82E+06 3.42E+05 Pyroglutamic acid 5.42E+06 3.89E+05 Citric acid 2.37E+06 N / A 2-Hydroxyhippuric acid 2.14E+06 1.26E+05 4-Hydroxyphenylpyruvic acid 1.72E+06 2.93E+05 N-acetyl-L-aspartic acid 4.25E+06 N / A hippuric acid 2.69E+06 N / A Phenylacetic acid 4.54E+06 1.66E+05

[0395] Note: N / A indicates that it was not detected.

[0396] The results showed that 55 organic acids and sugar alcohols could be detected after oximation; however, among the 15 organic acids and sugar alcohols that did not undergo oximation and contained carbonyl groups, some had extremely low peak areas, making quantification difficult, and some could not even be detected. On the one hand, oximation can increase the number of organic acids and sugar alcohols that can be detected without oximation; on the other hand, as shown in Table 121, for organic acids and sugar alcohols that could be detected without oximation, the peak area increased significantly after oximation, which is beneficial for their accurate determination.

[0397] In addition, the oximating reagent can affect the oximation reaction. Therefore, we selected different oximating reagents, such as hydroxylamine ethanol, hydroxylamine sulfate, and hydroxylamine hydrochloride, all with a concentration of 5% and an addition amount of 500 μL. Only the oximating reagent was changed, and the other steps were the same as in Example 1. The detection peak areas of some organic acids and sugar alcohols are shown in Table 121.

[0398] Table 121: Effect of oxime reagents on the detection of organic acids

[0399]

[0400] The results showed that hydroxylamine hydrochloride and hydroxylamine ethanol were more effective than hydroxylamine sulfate, resulting in higher peak areas for the detection of organic acids and sugar alcohols; among them, hydroxylamine hydrochloride was the most effective.

[0401] Since the oximation reagent affects the oximation reaction, the choice of oximation reagent should also affect the stability of the pretreated sample. We selected different oximation reagents, hydroxylamine ethanol, hydroxylamine sulfate, and hydroxylamine hydrochloride, all with a concentration of 5% and an addition amount of 500 μL. All were carried out according to the method in Example 1, and the amount of organic acid and sugar alcohol detected over time was measured. The results are shown in Table 122.

[0402] Table 122: Oxime reagents and the number of organic acids and sugar alcohols that can be detected

[0403] Hydroxyethanol 55 54 52 51 Hydroxylamine sulfate 55 54 51 50 Hydroxylamine hydrochloride 55 55 55 55

[0404] The results showed that the choice of oxime reagent also affected the stability of the pretreated samples. Hydroxylamine hydrochloride was more effective than hydroxylamine sulfate and hydroxylamine ethanol, enabling all 55 organic acids and sugar alcohols to be detected within 10 days. Subsequent experiments showed that only hydroxylamine hydrochloride as the oxime reagent could maintain stability for 20 days under longer storage conditions.

[0405] Finally, we investigated the conditions for the oximation reaction. Since the method provided in this invention is used to detect organic acids and sugar alcohols in urine, and urine often exhibits different pH values, especially potentially acidic urine, this is quite unfavorable for the oximation reaction. Existing technologies rarely consider the influence of pH during urine oximation. In our exploratory research on pretreatment, we found that the oximation reaction did not proceed well. Through experimental exploration, we determined a method of adding NaOH solution during the oximation reaction.

[0406] In the oxime reaction, we found that without the addition of NaOH solution, the number of detectable organic acids and sugar alcohols was significantly reduced and the stability of the pretreated samples was poor. Both groups of experiments were carried out according to the method of Example 1, and the number of detectable organic acids and sugar alcohols was detected over time. The only difference was that one group did not add NaOH solution to adjust the pH, and because no NaOH solution was added, there was no need to add HCl solution for neutralization. The results are shown in Table 123.

[0407] Table 123: Number of Detectable Organic Acids in NaOH Solution

[0408] No addition 40 39 35 30 Add to 55 55 55 55

[0409] On the first day without adding NaOH solution, the following were detected: lactic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, β-hydroxyisovaleric acid, methylmalonic acid, benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citric acid, malic acid, adipic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 3-indoleacetic acid, 5-hydroxyindole-3-acetic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, and 40 kinds of organic acids.

[0410] On the third day, the following were detected: 2-hydroxybutyric acid, 3-hydroxybutyric acid, β-hydroxyisovaleric acid, methylmalonic acid, benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citrate, malic acid, adipic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 3-indoleacetic acid, 5-hydroxyindole-3-acetic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, and 39 kinds of organic acids.

[0411] On day 7, the following were detected: β-hydroxyisovaleric acid, benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citrate, malic acid, adipic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 5-hydroxyindole-3-acetic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, and 35 kinds of organic acids.

[0412] On day 10, the following were detected: benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, citric acid, malic acid, adipic acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, and 30 kinds of organic acids.

[0413] The results showed that the absence of NaOH led to a decrease in the number of detectable organic acids and sugar alcohols; therefore, NaOH solution needs to be added during the oxime step.

[0414] Clearly, the pH at which the oximation reaction occurs has a significant impact on the detection of organic acids and sugar alcohols in urine, so we need to investigate the pH. We set up multiple parallel experiments, all conducted according to the method in Example 1, and measured the changes in the detection quantities of organic acids and sugar alcohols over time. The difference was that the pH value was changed under the monitoring of a pH meter, and oximation was performed at different pH values. After oximation, HCl solution was added to adjust the pH to 7 before proceeding to the next step. The results are shown in Table 124.

[0415] Table 124: pH value and number of detectable organic acid types

[0416] Initial pH (approximately 4–7) 40 39 35 30 7.0 43 42 38 33 7.5 45 44 40 35 8 52 51 47 42 8.2 55 55 55 55 8.5 55 55 55 55 9 55 55 55 55

[0417] On the first day without pH adjustment, the following organic acids were detected: lactic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, β-hydroxyisovaleric acid, methylmalonic acid, benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citric acid, malic acid, adipic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 3-indoleacetic acid, 5-hydroxyindole-3-acetic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, and 40 other organic acids.

[0418] Day 3: 2-Hydroxybutyric acid, 3-Hydroxybutyric acid, β-hydroxyisovaleric acid, methylmalonic acid, benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citrate, malic acid, adipic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 3-indoleacetic acid, 5-hydroxyindole-3-acetic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, 39 kinds of organic acids;

[0419] Day 7: β-hydroxyisovalerate, benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citrate, malic acid, adipic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 5-hydroxyindole-3-acetic acid, 3-hydroxypropionic acid, 2-hydroxyisovalerate, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, 35 kinds of organic acids;

[0420] Day 10: Benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, citric acid, malic acid, adipic acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, 30 kinds of organic acids;

[0421] When pH=7:

[0422] The following were detected on the first day: lactic acid, 2-hydroxybutyric acid, methylmalonic acid, benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citric acid, malic acid, adipic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 3-indoleacetic acid, 5-hydroxyindole-3-acetic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, pyruvic acid, α-ketoglutaric acid, citric acid, and 43 other organic acids.

[0423] Day 3: 2-Hydroxybutyric acid, 3-Hydroxybutyric acid, β-hydroxyisovaleric acid, methylmalonic acid, benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citrate, malic acid, adipic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 3-indoleacetic acid, 5-hydroxyindole-3-acetic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, pyruvic acid, α-ketoglutaric acid, citric acid, 42 kinds of organic acids;

[0424] Day 7: β-hydroxyisovalerate, benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citric acid, malic acid, adipic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 5-hydroxyindole-3-acetic acid, 3-hydroxypropionic acid, 2-hydroxyisovalerate, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, pyruvic acid, α-ketoglutaric acid, citric acid, 38 kinds of organic acids;

[0425] Day 10: Benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, citric acid, malic acid, adipic acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, pyruvic acid, α-ketoglutaric acid, citric acid, 33 kinds of organic acids;

[0426] At pH = 7.5:

[0427] The following were detected on day one: lactic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, β-hydroxyisovaleric acid, methylmalonic acid, benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citric acid, malic acid, adipic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, and vanilla. Acids, isocitric acid, vanillylmandelic acid, 3-indoleacetic acid, 5-hydroxyindole-3-acetic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, pyruvic acid, α-ketoglutaric acid, citric acid, phenylpyruvic acid, 4-hydroxyphenylpyruvic acid, 45 kinds of organic acids;

[0428] Day 3: 2-Hydroxybutyric acid, 3-Hydroxybutyric acid, β-hydroxyisovaleric acid, methylmalonic acid, benzoic acid, trisuccinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citric acid, malic acid, adipic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocyanate. Citric acid, vanillylmandelic acid, 3-indoleacetic acid, 5-hydroxyindole-3-acetic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, pyruvic acid, α-ketoglutaric acid, citric acid, phenylpyruvic acid, 4-hydroxyphenylpyruvic acid, 44 kinds of organic acids;

[0429] Day 7: β-hydroxyisovalerate, benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citric acid, malic acid, adipic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 5-hydroxyindole-3-acetic acid, 3-hydroxypropionic acid, 2-hydroxyisovalerate, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, pyruvic acid, α-ketoglutaric acid, citric acid, phenylpyruvic acid, 4-hydroxyphenylpyruvic acid, 40 kinds of organic acids;

[0430] Day 10: Benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, citric acid, malic acid, adipic acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, pyruvic acid, α-ketoglutaric acid, citric acid, phenylpyruvic acid, 4-hydroxyphenylpyruvic acid, 35 kinds of organic acids; at pH=8:

[0431] The following were detected on day one: lactic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, β-hydroxyisovaleric acid, methylmalonic acid, benzoic acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citric acid, malic acid, adipic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 3-indoleacetic acid, 5-hydroxyindoleacetic acid. 3-Dole-3-acetic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, 4-methyl-2-oxovaleric acid, pyruvic acid, phenylacetic acid, orotic acid, α-ketoglutarate, pyroglutamic acid, citric acid, 4-hydroxyphenylpyruvic acid, hippuric acid, phenylpyruvic acid, 3-methyl-2-oxovaleric acid, α-ketoisovaleric acid, 52 kinds of organic acids;

[0432] Day 3: 2-Hydroxybutyric acid, 3-Hydroxybutyric acid, β-hydroxyisovaleric acid, methylmalonic acid, benzoic acid, 3-methyl-2-oxovaleric acid, α-ketoisovaleric acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citric acid, malic acid, adipic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 3-indoleacetic acid 5-Hydroxyindole-3-acetic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, 4-methyl-2-oxovaleric acid, pyruvic acid, phenylacetic acid, orotic acid, α-ketoglutarate, pyroglutamic acid, citric acid, 4-hydroxyphenylpyruvic acid, hippuric acid, phenylpyruvic acid, 3-methyl-2-oxovaleric acid, α-ketoisovaleric acid, 51 kinds of organic acids;

[0433] Day 7: β-hydroxyisovalerate, benzoic acid, 3-methyl-2-oxovalerate, α-ketoisovalerate, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citric acid, malic acid, adipic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 5-hydroxyindole-3-acetic acid. 3-Hydroxypropionic acid, 2-Hydroxyisovaleric acid, 2-Hydroxyisohexanoic acid, 4-Hydroxyphenyllactic acid, Phenylactic acid, Ethylmalonic acid, Mandelic acid, Glutaric acid, Malonic acid, Hypoglycic acid, Phenylacetic acid, Sebacic acid, 3,4-Dihydroxyphenylpropionic acid, 4-Methyl-2-oxovaleric acid, Pyruvic acid, Phenylacetic acid, Orotic acid, α-Ketoglutarate, Pyroglutamic acid, Citric acid, 4-Hydroxyphenylpyruvic acid, Hipuric acid, Phenylacetic acid, 3-Methyl-2-oxovaleric acid, α-Ketoisovaleric acid, 47 kinds of organic acids;

[0434] Day 10: Benzoic acid, 3-methyl-2-oxovaleric acid, α-ketoisovaleric acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, citric acid, malic acid, adipic acid, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, 2,3-pyridinedicarboxylic acid, arabinitol, cis-aconitine, homovanillic acid, isocitrate, vanillylmandelic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2- Hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, mandelic acid, glutaric acid, malonic acid, homohydric acid, phenylacetic acid, sebacic acid, 3,4-dihydroxyphenylpropionic acid, 4-methyl-2-oxovaleric acid, pyruvic acid, phenylacetic acid, orotic acid, α-ketoglutarate, pyroglutamic acid, citric acid, 4-hydroxyphenylpyruvic acid, hippuric acid, phenylpyruvic acid, 3-methyl-2-oxovaleric acid, α-ketoisovaleric acid, 42 kinds of organic acids;

[0435] The results showed that 55 organic acids in urine could be detected simultaneously at pH 8.2, and the stability was highest after pretreatment under this condition. All 55 organic acids could still be detected after 10 days of storage at 4°C. The effect did not change as the pH continued to increase. In principle, oxime reactions need to be carried out under appropriate alkaline conditions, but the specific pH value needs to be determined according to the specific reaction substrate. Based on a large number of experiments, this invention determined that the optimal pH value for the oxime reaction of 55 organic acids in urine suitable for this invention is 8.2.

[0436] Example 4: Pretreatment Derivatization

[0437] From Example 2, we can conclude that: the methyl esterification pretreatment method has a low response, and organic acids and sugar alcohols are all below the detection limit and cannot be detected; the butyl esterification pretreatment method processes 55 organic acids and sugar alcohol standard curves, but can only detect 14 organic acids and sugar alcohols, some of which have poor peak shapes, low response, and poor linearity of the standard curves; the silanization pretreatment method can detect all 55 organic acids and sugar alcohols, and has good peak shapes, good response, and accurate detection.

[0438] Silyl group refers to trimethylsilane Si(CH3)3, also known as TMS. Silanization refers to the introduction of a silyl group into the molecule, typically by substituting an active hydrogen atom. Substituting the active hydrogen with a silyl group reduces the compound's polarity and decreases hydrogen bond binding. Silanized compounds exhibit increased stability and enhanced detectability under gas chromatography due to the reduced number of reactive sites containing active hydrogen.

[0439] In this embodiment, we conducted a specific study on silanization to identify the most favorable silanization derivatization reagents for the detection of 55 organic acids and sugar alcohols.

[0440] BSTFA-TMCS, MSTFA-TMCS, HDMS-TMCS, trimethylchlorosilane, and triethylchlorosilane were selected as silanizing reagents to pretreat and detect the LQC quality control sample. Other steps were the same as in Example 1. The peak areas of some organic acids are shown in Table 125.

[0441] Table 125: Effect of silanizing reagents on the detection of organic acids

[0442] Methylmalonic acid 1.49E+07 1.55E+07 1.38E+07 1.33E+07 1.30E+07 benzoic acid 4.56E+06 4.70E+06 4.44E+06 4.36E+06 4.40E+06 Succinic acid 2.01E+07 2.12E+07 2.00E+07 1.95E+07 1.92E+07 fumaric acid 1.66E+07 1.70E+07 1.64E+07 1.58E+07 1.56E+07 3-Phenylonpropionic acid 9.28E+06 9.41E+06 9.25E+06 9.20E+06 9.11E+06 malic acid 4.82E+06 4.80E+06 4.71E+06 4.62E+06 4.70E+06 adipic acid 5.42E+06 5.69E+06 5.39E+06 5.34E+06 5.31E+06 4-Hydroxyphenylacetic acid 2.37E+06 2.44E+06 2.20E+06 2.11E+06 2.17E+06 Oleic acid 2.14E+06 2.11E+06 2.07E+06 2.02E+06 2.03E+06 cis-aconitine 1.72E+06 1.98E+06 1.66E+06 1.70E+06 1.59E+06 High vanillic acid 4.25E+06 4.32E+06 4.04E+06 4.14E+06 4.12E+06 hippuric acid 2.69E+06 2.88E+06 2.62E+06 2.58E+06 2.59E+06 Citric acid 4.54E+06 4.44E+06 4.32E+06 4.39E+06 4.33E+06

[0443] The results showed that all five silanization derivatizing agents could detect 55 organic acids and sugar alcohols, and the detection effects were generally similar. MSTFA-TMCS had the best overall effect, followed by BSTFA-TMCS. However, MSTFA-TMCS is very expensive. Considering that BSTFA-TMCS has a lower economic cost and its detection effect is almost the same as that of MSTFA-TMCS, this invention selected BSTFA-TMCS as the silanization derivatizing agent.

[0444] Example 5: Instrument detection of heating program

[0445] When using conventional methods for detection, due to the large number of 55 organic acids and sugar alcohols, the peaks obtained by gas chromatography often overlap. This problem cannot be solved by conventional chemical changes (such as derivatization). Therefore, after extensive research, we found a suitable method that can distinguish between the peaks and optimized the heating program.

[0446] The initial heating program was as follows: hold at 60℃ for 2.5 min, increase the temperature to 220℃ at a rate of 10℃ / min, and then increase the temperature to 325℃ at a rate of 15℃ / min, and hold for 1 min.

[0447] Optimized heating program: Hold at 100℃ for 4 minutes, then increase to 280℃ at a rate of 4℃ / min and hold for 11 minutes.

[0448] Benzoic acid, 3-hydroxypropionic acid, and 2-hydroxyisovaleric acid are the three most difficult organic acids and sugar alcohols to detect among the 55 organic acids and sugar alcohols in this invention. Their peak shapes are complex, and there are certain peak shifts and peak overlaps. Therefore, we will examine whether the optimized heating program can have a beneficial effect on the detection of the above three organic acids.

[0449] Using the two heating programs described above, and with other methods the same as in Example 1, chromatograms of benzoic acid, 3-hydroxypropionic acid, and 2-hydroxyisovaleric acid were obtained.

[0450] Before optimization, some chromatographic peaks of the indicators were as follows: Figure 26 Chromatogram of benzoic acid; Figure 27Chromatogram of 3-hydroxypropionic acid; Figure 28 Chromatogram of 2-hydroxyisovaleric acid;

[0451] Optimized chromatographic peaks: Figure 29 Chromatogram of benzoic acid; Figure 30 Chromatogram of 3-hydroxypropionic acid; Figure 31 This is the spectrum of 2-hydroxyisovalerate.

[0452] When the preheating procedure is optimized, not only do the chromatographic peaks of the three organic acids mentioned above overlap and stack, but other organic acids and sugar alcohols also show a certain degree of peak shift during detection, and there is also overlap between the peaks of the analytes and between the analytes and impurities. This makes it difficult to accurately quantify all 55 organic acids and sugar alcohols.

[0453] However, after optimizing the temperature program, the chromatographic peaks were completely separated and the peak shapes were good. For example, benzoic acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, etc., which could not be distinguished and detected before, can now be detected distinguishably. The accuracy of the detection of each index compound is improved. Further experiments also show that the optimized instrument detection temperature program in this embodiment can detect 55 kinds of organic acids and sugar alcohols.

[0454] Example 6: Use of Glass Consumables

[0455] During the research process of this invention, we found that there were quantitative inaccuracies when using plastic consumables (consumables refer to centrifuge tubes and droppers), but this quantitative inaccuracy could be avoided when using glass consumables. The peak areas of 0.5 μg / mL organic acids after treatment with plastic and glass consumables are shown in Table 126.

[0456] Table 126: Peak areas of some organic acids at 0.5 μg / mL after treatment with plastic and glass consumables.

[0457]

[0458] The peak areas of plastic consumables were larger than those of glass consumables in the above results. In reality, for the 55 organic acids and sugar alcohols, the peak areas detected by plastic consumables were all larger than those detected by glass consumables (only methylmalonic acid was unaffected).

[0459] Therefore, we continued to use centrifuge tubes and droppers made of plastic to repeat the detection scheme of Example 1, tested the quality control product LQC, calculated the ratio of the test result concentration to the actual concentration, and recorded it as the accuracy. The accuracy of some organic acids is shown in Table 127.

[0460] Table 127: Accuracy of Organic Acids in Plastic and Glass Materials

[0461]

[0462] The results showed that using glass centrifuge tubes and droppers resulted in detection results closer to the actual concentrations. Beyond what is shown in the table above, for the 55 organic acids and sugar alcohols, the accuracy using glass centrifuge tubes and droppers was within 100% ± 15%, indicating minimal error and accurate quantification. However, using plastic centrifuge tubes and droppers resulted in significantly higher overall accuracy, almost all exceeding 115% (except for methylmalonic acid), severely impacting quantification accuracy. In principle, plastic consumables interfere with the quantification of individual organic acids and sugar alcohols, and the amount of interference introduced by each plastic consumable is different and uncontrollable. Therefore, using plastic consumables affects the actual sample quantification. This further illustrates that when using plastic containers for sample pretreatment, the obtained results deviate significantly from the actual organic acid and sugar alcohol content, often exceeding the actual content in the sample, leading to inaccurate test results.

[0463] Conventional testing methods often use consumables made of plastic, such as plastic droppers and plastic centrifuge tubes. This is one of the reasons why existing technologies are difficult to accurately quantify or even detect organic acids and sugar alcohols in urine. The method provided by this invention solves this problem and has a better detection effect on samples with low organic acid and sugar alcohol content. It solves the problems that organic acids and sugar alcohols in urine are difficult to detect and that trace amounts of organic acids and sugar alcohols are difficult to detect due to background impurities.

[0464] Example 7: Method Application

[0465] We randomly selected 300 morning urine samples from the surrounding population and samples submitted by the hospital, and tested them using the method provided in this invention. The pretreatment and testing methods were the same as in Example 1.

[0466] Supplement: According to the performance verification results, the accuracy of the samples is 100% ± 15%, and the precision meets the detection requirements. The lower limit of quantitation can reach 0.5 μg / mL. The detection results of 300 random population samples show that the detection rate of organic acids and sugar alcohols in each sample is 98%, and the actual concentrations of organic acids and sugar alcohols in human urine samples are all within the linear range of the standard curve.

[0467] The embodiments described above provide a detailed explanation of the technical solution of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting 55 organic acids and sugar alcohols in urine using gas chromatography-tandem mass spectrometry, characterized in that, The process includes sample pretreatment and instrumental detection. The centrifuge tubes and pipettes used for sample pretreatment are made of glass. The pretreated urine samples can still be detected with 55 organic acids and sugar alcohols after being placed at 4°C or -20°C for 10 days. The 55 organic acids and sugar alcohols are lactic acid, 2-hydroxybutyric acid, pyruvic acid, 3-hydroxybutyric acid, β-hydroxyisovaleric acid, methylmalonic acid, benzoic acid, 3-methyl-2-oxovaleric acid, 4-methyl-2-oxovaleric acid, α-ketoisovaleric acid, succinic acid, pyridine-2-carboxylic acid, fumaric acid, 3-phenylpropionic acid, citrate, malic acid, adipic acid, pyroglutamic acid, phenylacetic acid, 3-hydroxy-3-methylglutaric acid, 4-hydroxybenzoic acid, α-ketoglutaric acid, 4-hydroxyphenylacetic acid, tartaric acid, octanoic acid, tricarboxylic acid, and 2,3-pyridine. Dicarboxylic acid, arabinitol, cis-aconitine, orotic acid, homovanillic acid, citric acid, isocitrate, hippuric acid, vanillylmandelic acid, 2-methylhippuric acid, 3-indoleacetic acid, 5-hydroxyindole-3-acetic acid, 2-hydroxyhippuric acid, 3-hydroxypropionic acid, 2-hydroxyisovaleric acid, 2-hydroxyisohexanoic acid, 4-hydroxyphenyllactic acid, phenyllactic acid, ethylmalonic acid, mandelic acid, glutaric acid, malonic acid, 4-hydroxyphenylpyruvic acid, homohydric acid, N-acetyl-L-aspartic acid, phenylpyruvic acid, phenylacetic acid, sebacic acid, and 3,4-dihydroxyphenylpropionic acid; The sample pretreatment method consists of the following steps: (1) Enzymatic hydrolysis: Add urease to a glass centrifuge tube, add the sample, and perform enzymatic hydrolysis; (2) Oxime reaction: Add hydroxylamine hydrochloride and NaOH solution to a glass centrifuge tube to carry out the oxime reaction. Add NaOH solution to adjust the pH to pH=8.

2. After the reaction is completed, add HCl solution to neutralize. (3) Liquid-liquid extraction: Add ethyl acetate, shake and centrifuge, and transfer the supernatant to a glass centrifuge tube using a glass pipette; (4) Silane derivatization: Dry with nitrogen, add BSTFA-TMCS, and incubate; During the instrumental analysis, the chromatographic column was DB-5, and the temperature program was set as follows: 100℃ for 4 min, then increase to 280℃ at a rate of 4℃ / min and hold for 11 min.

2. The method as described in claim 1, characterized in that, In step (2), the concentration of hydroxylamine hydrochloride is 1%-10%; the volume ratio of hydroxylamine hydrochloride to the enzymatically digested sample is 1:1-4.

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