Mass spectrometric detection method for simultaneously determining oxalic acid, citric acid and cystine in urine

Abstract

The invention provides a mass spectrometric detection method for simultaneously determining oxalic acid, citric acid and cystine in urine, and relates to the technical field of analytical chemistry, according to the method, degradation of a to-be-detected substance can be reduced through a simple pretreatment process, effective separation is realized in a chromatographic separation system, and the method has extremely high sensitivity and specificity and can be applied to mass spectrometric detection of oxalic acid, citric acid and cystine in urine. The method has the advantages that extremely low detection limits and lower quantitative limits of oxalic acid, citric acid and cystine can be realized, substances to be detected do not interfere with one another, the detection cost is reduced, and clinical application of the method can be effectively promoted. Metabolism assessment is carried out regularly through the method, the risk of calculus occurrence can be early warned, calculus pathogenesis can be found, metabolic abnormality can be monitored and corrected regularly, and therefore the purpose of preventing calculus is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry and relates to a mass spectrometry detection method for simultaneously determining oxalic acid, citric acid and cystine in urine. Background Art

[0002] Urinary stones are one of the most common urinary diseases, with an incidence rate of 6% to 10% in my country. Studies have found that more than 90% of stone patients have abnormal urine metabolism, among which urine oxalic acid, urine citrate, and urine cystine have the main warning value for the risk of stone occurrence, and their metabolic abnormalities can lead to primary hyperoxaluria (rare disease), secondary hyperoxaluria, hypocitrateuria, and cystinuria (rare disease). Therefore, urine metabolic assessment is very necessary for both people at high risk of stones and patients with urinary stones. It can not only provide early warning of the risk of stone occurrence, but also help to discover the cause of stones, regularly monitor and correct metabolic abnormalities, thereby achieving the purpose of preventing stones.

[0003] The prior art discloses a method for simultaneously detecting citric acid, oxalic acid and cystine in urine. For example, CN115389684A discloses a method for simultaneously detecting oxalic acid, citric acid and cystine in 24h urine. However, the derivatization step in the detection process requires the use of multiple reagents such as benzyl alcohol, trimethylsilyl chloride, tetramethylammonium fluoride and acetonitrile, and the derivatization reaction temperature is as high as 80°C, and the derivatization reaction time requires 75min. CN117330684B discloses a detection method and a kit for simultaneously detecting citric acid, oxalic acid and cystine in urine, which improves the temperature and time of the derivatization reaction, but the entire pretreatment process still takes a long time. For example, CN116381104A discloses a detection method and application of an organic acid. Although no derivatization treatment is performed in the pretreatment, the separation degree and peak shape of each substance after chromatographic separation are poor, which is greatly affected by the environmental pH and cannot be applied to the preparation of a kit for detection.

[0004] Therefore, how to provide a detection method that is convenient and fast in sample pretreatment, does not require a derivatization step, has high detection accuracy, and has better separation and peak shape and is suitable for large-scale clinical applications has become a technical problem that needs to be solved urgently in this field. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide a detection method for simultaneously determining oxalic acid, citric acid and cystine in urine with high sensitivity, high specificity, good separation, convenience and rapidity.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical scheme: The first aspect of the present invention provides a detection method for simultaneously determining oxalic acid, citric acid and cystine in urine, and the detection method comprises the following steps: pretreatment of the sample to be tested, preparation of the working solution, preparation of the injection solution, high performance liquid chromatography tandem mass spectrometry detection, substituting the detection value into the standard curve, and calculating the content of oxalic acid, citric acid and cystine in the sample to be tested.

[0007] Furthermore, the conditions for the high performance liquid chromatography tandem mass spectrometry detection include: chromatographic column: CSH chromatographic column; mobile phase A: 0.4% ammonia water + 10 mM ammonium acetate; mobile phase B: acetonitrile.

[0008] Furthermore, the conditions for the HPLC-MS / MS detection also include: flow rate: 0.2 mL / min; injection volume: 5 μL; column temperature: 40°C; initial mobile phase: 97% mobile phase A.

[0009] Furthermore, the mobile phase A is prepared by preparing 400 mL of water + 800 μL of 5M ammonium acetate + 1600 μL of aqueous ammonia, which is prepared immediately before use.

[0010] Furthermore, the instrument used for the high performance liquid chromatography tandem mass spectrometry detection is a liquid chromatography tandem mass spectrometer.

[0011] Furthermore, the sample pretreatment is centrifugation to obtain a supernatant.

[0012] Furthermore, the pretreatment of the sample to be tested is to take 1 ml of the sample to be tested, centrifuge it at 12,000 rpm for 15 minutes, and obtain the supernatant.

[0013] Furthermore, the preparation of the working solution includes: 1) preparation of a standard solution; 2) preparation of a mixed standard solution; 3) preparation of an internal standard working solution; 4) preparation of a diluent containing an internal standard; and 5) preparation of a standard curve sample.

[0014] 1) The preparation of the standard solution includes: oxalic acid internal standard stock solution: oxalic acid (oxalic acid)-13C2 is configured with pure water to a final concentration of 100PPM; citric acid internal standard stock solution: citric acid-D4 is configured with pure water to a final concentration of 100PPM; cystine internal standard stock solution: L-cystine-3,3,3',3'-D4 is configured with pure water to a final concentration of 1000PPM.

[0015] 2) The preparation of the mixed standard solution includes: Mix A (concentration of 200 PPM): oxalic acid internal standard mother solution, citric acid internal standard mother solution, cystine internal standard mother solution and mobile phase A in a ratio of 1:1:1:2. Mix A is diluted with mobile phase A to obtain Mix B to Mix F, whose concentrations are 100 PPM, 50 PPM, 20 PPM, 10 PPM and 5 PPM respectively.

[0016] Furthermore, the preparation of the mixed A includes: 40 μL oxalic acid internal standard stock solution + 40 μL citric acid internal standard stock solution + 40 μL cysteine ​​internal standard stock solution + 80 μL mobile phase A; the preparation of the mixed B (100PPM) includes: 20 μL mixed A + 20 μL mobile phase A; the preparation of the mixed C (50PPM) includes: 10 μL mixed A + 30 μL mobile phase A; the preparation of the mixed D (20PPM) includes: 10 μL mixed A + 90 μL mobile phase A; the preparation of the mixed E (10PPM) includes: 10 μL mixed A + 190 μL mobile phase A; the preparation of the mixed F (5PPM) includes: 5 μL mixed A + 195 μL mobile phase A.

[0017] 3) The preparation of the internal standard working solution includes: diluting the oxalic acid or citric acid internal standard stock solution 10 times with mobile phase A, diluting the cystine internal standard stock solution 10 times with water and then diluting it 10 times with mobile phase A to prepare an internal standard working solution with a concentration of 10 PPM.

[0018] 4) The preparation of the internal standard diluent includes: mixing oxalic acid internal standard working solution, citric acid internal standard working solution, cystine internal standard working solution and mobile phase A in a ratio of 2:2:1:14, and mixing to obtain the internal standard diluent.

[0019] 5) The preparation of the standard curve samples includes: mixing a mixed standard solution containing an internal standard diluent and mobile phase A, and mixed A to mixed F in a ratio of 19:1 to obtain standard curve samples with concentrations of blank, 250 ppb, 500 ppb, 1000 ppb, 2000 ppb, 5000 ppb, and 10000 ppb.

[0020] Further, the blank concentration sample refers to a sample containing an internal standard diluent and mobile phase A in a ratio of 19:1; the 250ppb concentration sample refers to a sample containing an internal standard diluent and mixed F in a ratio of 19:1; the 5000ppb concentration sample refers to a sample containing an internal standard diluent and mixed E in a ratio of 19:1; the 1000ppb concentration sample refers to a sample containing an internal standard diluent and mixed D in a ratio of 19:1; the 2000ppb concentration sample refers to a sample containing an internal standard diluent and mixed C in a ratio of 19:1; the 5000ppb concentration sample refers to a sample containing an internal standard diluent and mixed B in a ratio of 19:1; the 10000ppb concentration sample refers to a sample containing an internal standard diluent and mixed A in a ratio of 19:1.

[0021] Furthermore, the injection solution is prepared by mixing the supernatant and mobile phase A at a ratio of 1:1 to obtain a 2-fold diluted sample to be tested; the 2-fold diluted sample to be tested is mixed with the internal standard diluent at a ratio of 1:19 to obtain a 40-fold diluted injection solution.

[0022] The preparation of the injection solution is preferably to aspirate 100 μL of supernatant, add 100 μL of mobile phase A, blow evenly, and obtain a 2-fold diluted sample to be tested; take 10 μL of the 2-fold diluted sample to be tested, add 190 μL of internal standard diluent, blow evenly, and obtain a 40-fold diluted injection solution.

[0023] Furthermore, the high performance liquid chromatography tandem mass spectrometry detection is to perform liquid chromatography separation on the injection solution, and perform mass spectrometry analysis on the sample after liquid chromatography separation; analyze the blank, 250ppb, 500ppb, 1000ppb, 2000ppb, 5000ppb, and 10000ppb concentration standard curve samples, and calculate the standard curve according to the detection value; analyze the injection solution, substitute the detection value into the standard curve, and calculate the content of oxalic acid, citric acid, and cystine in the sample to be tested.

[0024] Furthermore, the mass spectrometry conditions are ESI ion source, positive and negative ion switching mode.

[0025] Furthermore, the sources of the samples to be tested include humans, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, pigs, monkeys, sheep, and cattle.

[0026] Furthermore, the sample to be tested is a urine sample.

[0027] Furthermore, the sample to be tested is a 24-hour urine sample.

[0028] Another aspect of the present invention provides an application of the detection method described in the first aspect of the present invention in preparing products, wherein the products include products for detecting oxalic acid, citric acid and cystine, or products for diagnosing urinary stones.

[0029] The products include, but are not limited to, kits.

[0030] Advantages and beneficial effects of the present invention: The method adopted by the present invention can directly load urine samples onto the machine through a one-step dilution method, eliminating complex pre-treatment steps such as derivatization. Compared with the existing patented method, the pre-treatment time is shortened from more than 75 minutes to less than 1 minute, greatly improving the detection efficiency and reducing the detection cost. It also has high sensitivity and high specificity, which can effectively promote the clinical application of this method. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the liquid chromatogram of oxalic acid in the sample. Figure 1 A and B in the figure are the MRM diagrams of oxalic acid in the sample. Figure 1 C in the figure is the internal standard of oxalic acid. Figure 1 D in is the standard curve of oxalic acid; Figure 2 is the liquid chromatogram of citric acid in the sample. Figure 2 A and B in the figure are the MRM diagrams of citric acid in the sample. Figure 2 The C in the figure is the internal standard of citric acid. Figure 2 D in is the standard curve of citric acid; Figure 3 is the liquid chromatogram of cystine in the sample. Figure 3 A and B in the figure are the MRM diagrams of cystine in the sample. Figure 3 The C in is the internal standard of cystine. Figure 3 D in is the standard curve of cystine; Figure 4 The chromatogram of cystine-oxalic acid-citric acid in the standard solution shows the peaks and interferences; Figure 5 This is the chromatogram peak and interference diagram of cystine-oxalate-citrate in the patient's urine. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0033] In the context of the present invention, the term "urinary calculi" refers to a common disease of the urinary system. Stones can be found in any part of the urinary system, including the kidney, bladder, ureter and urethra, with kidney and ureteral stones being more common. According to the cause of disease, urinary calculi are divided into primary urinary calculi, metabolic urinary calculi and secondary or infectious calculi. Primary urinary calculi refer to urinary calculi with unknown causes and unclear mechanisms. Metabolic urinary calculi are the most common, and are caused by metabolic disorders in the body or kidney, such as hyperthyroidism, idiopathic calciuria causing increased calcium in urine, increased uric acid excretion in gout, and a large increase in phosphate during renal tubular acidosis, etc., and the stones formed are mostly urate, carbonate, cystine xanthine stones. Secondary or infectious calculi are mainly bacterial infections of the urinary system, especially bacteria and Proteus that can decompose urea can decompose urea into free ammonia to alkalinize urine, prompting phosphate and carbonate to form stones with bacterial masses or pus masses as the core. In addition, the formation of stones is related to race, genetics (cystine stones have a genetic tendency), gender, age, geographical environment, eating habits, nutritional status, and urinary tract diseases such as urinary tract stenosis and prostatic hyperplasia.

[0034] Since metabolic urinary stones are the most common, stone composition analysis and metabolic assessment are of guiding significance in stone prevention. Metabolic assessment can clarify the causes of 95% of stones. Individualized prevention and treatment of the causes can reduce the recurrence rate of stones to 10%~15%. According to the complexity, metabolic assessment can be divided into simplified assessment, comprehensive assessment and specific assessment. Simplified assessment is suitable for patients with initial stones, aiming to screen and exclude some important causes of stones, including serum and urine examinations and stone composition analysis. Comprehensive assessment is to add 24-hour urine stone risk factor analysis on this basis, including calcium, oxalate, uric acid, citric acid, cystine, magnesium, phosphate, urea, sodium, potassium, creatinine, urine volume, ammonium, pH, urine specific gravity, etc. The applicable population includes patients with initial stones at high risk of recurrence, such as children with kidney stones, multiple stones, recurrent stones, and patients with urinary stones with urinary tract malformations; it also includes all patients with recurrent stones and those suspected of potential causes of stone formation after simplified assessment. The purpose of the specificity assessment is to classify certain metabolic causes of urinary stones, including 1 g calcium load test, ammonium chloride load test, sodium bicarbonate load test, blood gas analysis, etc., which is suitable for stone patients in special occupations such as public safety. In some embodiments, the detection method provided by the present invention can be used for 24-hour urine stone formation risk factor analysis.

[0035] In the present invention, the term "oxalic acid" (OA) refers to an organic substance with a chemical formula of H2C2O4 and its salts. Oxalic acid is a metabolic product of organisms and usually exists in organisms in the form of oxalate. Oxalate is a metabolic end product in humans and other mammals and is excreted into urine by the kidneys. When combined with calcium, oxalic acid forms an insoluble product, calcium oxalate, which is the most common compound found in kidney stones. Since mammals cannot synthesize enzymes that degrade oxalate, the oxalate level in an individual is usually maintained normal by excretion and low absorption of dietary oxalate.

[0036] In the present invention, the term "citric acid" (CA), also known as citric acid, refers to an organic weak acid and its salt with the chemical formula C6H8O7, which is a metabolic intermediate in the body. Citric acid is an important protective factor for preventing the formation of kidney stones. It can prevent the formation of kidney stones from multiple mechanisms and has important physiological functions. Citric acid can combine with calcium to form a soluble complex, thereby inhibiting the formation of crystals in urine and the supersaturation of calcium salts. Stone patients are usually accompanied by hypocitraturia, which is an important risk factor for urinary stone formation and can predict the risk of stone occurrence. Hypocitraturia refers to a daily urinary citrate excretion of less than 320 mg, which is usually related to poor dietary choices, such as high meat and sodium intake and low fruit (citrus) intake. Hypocitraturia is a common feature of patients with primary kidney stones, especially those with calcium stones.

[0037] In the present invention, the term "cystine" (CYS) refers to an amino acid with a chemical formula of C6H12N2O4S2, one of the common amino acids in organisms, and its abnormal metabolism may lead to cystinuria. Cystinuria, also known as sulfite oxidase deficiency, is an autosomal recessive or incomplete genetic disease. It is a disease in which the metabolism of xanthine into uric acid, the conversion of sulfite into sulfate, and other metabolic processes in the organism are blocked due to the lack of sulfite oxidase. It will destroy the transport of cystine and other dibasic amino acids in the proximal tubules of the kidney, leading to the repeated formation of kidney stones. The main problem of this disease is that the reabsorption of cystine by the renal tubules is reduced, which causes an increase in the concentration of cystine in the urine. Cystine rarely dissolves in acidic urine. When its concentration is too high and exceeds its solubility, it precipitates and forms crystals or stones. The incidence of kidney stones caused by cystinuria in children is about 5-10%. Cystine stones form quickly and are prone to bilateral urinary system involvement, with a 5-year recurrence probability of ≥80%.

[0038] In the present invention, the term "mobile phase" refers to a substance that carries the components to be measured forward during the chromatography process. Another phase that is in equilibrium with the stationary phase and drives the sample forward. The substances used as mobile phases include: gas, liquid, supercritical fluid, etc. Common mobile phases mainly include: acetonitrile-water solution, acetonitrile-acetic acid aqueous solution, methanol-water solution, acetonitrile-phosphoric acid aqueous solution, formate aqueous solution, acetate aqueous solution, etc., and additives such as ammonia water are sometimes added. Adding organic amines to the mobile phase can weaken the strong interaction between alkaline solutes and residual silanol groups, and reduce or eliminate the peak tailing phenomenon. In a specific embodiment of the present invention, the mobile phase is an aqueous solution of ammonium formate and ammonia water, and acetonitrile.

[0039] In the present invention, the term "ion source" refers to a device that ionizes the molecules of the sample to be tested in mass spectrometry and converges the ions into an ion beam with a certain energy and geometric shape. In the actual experimental process, technicians can select a suitable ion source for experimentation according to the properties of the substance and the principle of its ionization. The more commonly used ion sources include electron impact source (EI), chemical ionization source (CI), electrospray ionization source (ESI), atmospheric pressure chemical ionization source (APCI) and matrix-assisted laser desorption ionization source (MALDI). In some embodiments, the ion source is an electrospray ionization source (ESI): ESI source is generally used in liquid chromatography-mass spectrometry instruments. This ionization method basically does not produce fragmentation peaks, so it is called soft ionization. The ESI source requires that the sample to be tested must be able to form ions in the solution; the type and concentration of the buffer salt in the mobile phase have a significant effect on the sensitivity, so the selection of the mobile phase is very important; the matrix suppression phenomenon is more obvious.

[0040] In the present invention, the term "sample to be tested" refers to a composition obtained from or derived from a subject (e.g., an individual of interest), which contains cells and / or other molecular entities to be characterized and / or identified based on, for example, physical, biochemical, chemical and / or physiological characteristics. For example, a sample refers to any sample derived from a subject of interest that is expected or known to contain metabolites to be characterized. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell cultures, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, sputum, tears, sweat, mucus, saliva, tissue culture fluid, tissue extracts, homogenized tissues, cell extracts, and combinations thereof. In a specific embodiment of the present invention, the sample is urine.

[0041] In the present invention, the source of the sample to be tested includes but is not limited to humans, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, pigs, monkeys, sheep, and cattle. Preferably, the sample to be tested is a urine sample. More preferably, it is a 24-hour urine sample.

[0042] It should be noted that the detection method for determining oxalic acid, citric acid and cystine in urine provided by the present invention is not for diagnostic and therapeutic purposes. The results obtained using the detection method of the present invention are only the contents of the three metabolites in the sample, which can be further used in the fields of urinary stone drug action mechanism research or pharmacokinetic research, or provide a certain reference value for the diagnosis of urinary stone disease, but does not mean that the result can directly represent the diagnosis result of urinary stone disease. Therefore, the detection method provided by the present invention is not for diagnostic and therapeutic purposes.

[0043] The present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Simple improvements to the present invention made according to the essence of the present invention all belong to the scope of protection claimed in the present invention. It should be noted that the experimental methods described in the following embodiments, unless otherwise specified, are conventional methods, and the reagents and materials, unless otherwise specified, can be obtained from commercial sources.

[0044] Example 1 Detection method and performance verification of oxalic acid, citric acid and cystine 1. Detection method 1. Instrument Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS): equipped with an ion source.

[0045] The instrument used in the specific embodiment of the present invention is an AB4500MD liquid spectrometer-mass spectrometer and an ESI ion source.

[0046] 2. Preparation of working solution Unless otherwise specified, the water described in the specific embodiments of the present invention is pure water.

[0047] 1) Mobile phase Mobile phase A: 0.4% ammonia + 10 mM ammonium acetate Preparation of mobile phase A: 400 mL water + 800 μL 5M ammonium acetate + 1600 μL ammonia water, prepared before use.

[0048] Mobile phase B: acetonitrile 2) Preparation of standard solution Oxalic acid internal standard stock solution: Oxalic acid (oxalic acid)-13C2 is prepared with pure water to a final concentration of 100PPM and stored at 4 degrees; citric acid internal standard stock solution: Citric acid-D4 is prepared with pure water to a final concentration of 100PPM and stored at 4 degrees; cystine internal standard stock solution: L-cystine-3,3,3',3'-D4 is prepared with pure water to a final concentration of 1000PPM and stored at 4 degrees.

[0049] 3) Preparation of mixed standard solution Mix A (200PPM): 40 μL oxalic acid internal standard stock solution + 40 μL citric acid internal standard stock solution + 40 μL cystine internal standard stock solution + 80 μL mobile phase A; Mix B (100PPM): 20 μL Mix A + 20 μL mobile phase A; Mix C (50PPM): 10 μL Mix A + 30 μL mobile phase A; Mix D (20PPM): 10 μL Mix A + 90 μL mobile phase A; Mix E (10PPM): 10 μL Mix A + 190 μL mobile phase A; Mix F (5PPM): 5 μL Mix A + 195 μL mobile phase A.

[0050] 4) Preparation of internal standard working solution 10PPM internal standard working solution: dilute the oxalic acid / citric acid internal standard stock solution 10 times with mobile phase A, dilute the cystine internal standard stock solution 10 times with water and then dilute it 10 times with mobile phase A. Calculate the total number of samples n (including the standard curve and the samples to be tested) that need to add the internal standard, and obtain the amount of oxalic acid-IS / citric acid-IS working solution (n+2)×20 μL, and the amount of cystine-IS working solution (n+2)×10 μL.

[0051] 5) Preparation of diluent containing internal standard: Each standard curve or sample to be tested needs to be prepared with diluent containing internal standard in the following ratio: 10 μL standard curve or sample to be tested + 190 μL diluent containing internal standard (20 μL oxalic acid-IS working solution + 20 μL citric acid-IS working solution + 10 μL cystine-IS working solution + 140 μL mobile phase A). First calculate the total number of samples N, and prepare a total volume of 190 μL×(N+2).

[0052] 6) Preparation of standard curve samples: STD0 (BLANK): 10 μL mobile phase A + 190 μL internal standard diluent, blow evenly; STD1 (250ppb): 10 μL mixed F + 190 μL internal standard diluent, blow evenly; STD2 (500ppb): 10 μL mixed E + 190 μL internal standard diluent, blow evenly; STD3 (1000ppb): 10 μL mixed D + 190 μL internal standard diluent, blow evenly; STD4 (2000ppb): 10 μL mixed C + 190 μL internal standard diluent, blow evenly; STD5 (5000ppb): 10 μL mixed B + 190 μL internal standard diluent, blow evenly; STD6 (10000ppb): 10 μL mixed A + 190 μL internal standard diluent, blow evenly.

[0053] 3. Chromatographic conditions Flow rate: 0.2 mL / min; Injection volume: 5 μL; Column temperature: 40°C; Chromatographic column: waters ACQUITY UPLC CSH C18 1.7 μm.

[0054] The gradient elution program is shown in Table 1: Table 1. Gradient elution program

[0055] 4. Mass spectrometry conditions 1) Ion source parameters (positive and negative ion switching mode) Table 2. Ion source parameters

[0056] 2) Mass spectrometry analysis conditions Table 3. Mass spectrometry analysis conditions

[0057] 5. Sample processing Take 1 ml of the sample to be tested, centrifuge at 12000 rpm for 15 minutes, and obtain the supernatant. Pipette 100 μL of the supernatant and add 100 μL of mobile phase A, blow evenly, and obtain a 2-fold diluted sample to be tested. Take 10 μL of the 2-fold diluted sample to be tested and add 190 μL of the internal standard diluent, blow evenly, and obtain a 40-fold diluted injection solution.

[0058] In this embodiment, the sample to be tested is a 24-hour urine sample.

[0059] 6. Analysis results Peak graph and standard curve Figure 1-Figure 5 shown.

[0060] 2. Performance Verification 1. Regression linear equation (standard curve see Figure 1 D in Figure 2 D in Figure 3 D) Table 3. Regression linear equation

[0061] 2. Retention time, recovery rate and precision Table 4. Retention time, recovery and precision of three metabolites

[0062] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that for ordinary technicians in this field, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for simultaneously determining oxalic acid, citric acid and cystine in urine, the method comprising the following steps: Pretreatment of the sample to be tested, preparation of the working solution, preparation of the injection solution, high performance liquid chromatography tandem mass spectrometry detection, substituting the detection value into the standard curve, and calculating the content of oxalic acid, citric acid and cystine in the sample to be tested.

2. The detection method according to claim 1, characterized in that: The conditions for the high performance liquid chromatography tandem mass spectrometry detection include: Chromatographic column: CSH chromatographic column; Mobile phase A: 0.4% ammonia + 10 mM ammonium acetate; Mobile phase B: acetonitrile.

3. The detection method according to claim 2, characterized in that: The conditions for the high performance liquid chromatography tandem mass spectrometry detection also include: Flow rate: 0.2 mL / min; Injection volume: 5 μL; Column temperature: 40°C; Initial mobile phase: 97% mobile phase A; Preferably, the mobile phase A is prepared by mixing 400 mL of water + 800 μL of 5M ammonium acetate + 1600 μL of ammonia water, which is prepared on the spot. Preferably, the instrument used for the high performance liquid chromatography tandem mass spectrometry detection is a liquid chromatography tandem mass spectrometer.

4. The detection method according to claim 1, characterized in that: The sample pretreatment is centrifugation to obtain a supernatant; Preferably, the pretreatment of the sample to be tested is to take 1 ml of the sample to be tested, centrifuge it at 12,000 rpm for 15 minutes, and obtain the supernatant.

5. The detection method according to claim 1, characterized in that: The preparation of the working solution includes: 1) preparation of a standard solution; 2) preparation of a mixed standard solution; 3) preparation of an internal standard working solution; 4) preparation of a dilution solution containing an internal standard; 5) preparation of a standard curve sample; 1) The preparation of the standard solution includes: Oxalic acid internal standard stock solution: Oxalic acid (oxalic acid)-13C2 is prepared with pure water to a final concentration of 100 PPM; Citric acid internal standard stock solution: Citric acid-D4 is prepared with pure water to a final concentration of 100 PPM; Cystine internal standard stock solution: L-cystine-3,3,3',3'-D4 is prepared with pure water to a final concentration of 1000 PPM; 2) The preparation of the mixed standard solution includes: Mix A (concentration 200 PPM): The ratio of oxalic acid internal standard mother solution, citric acid internal standard mother solution, cystine internal standard mother solution and mobile phase A is 1:1:1:2; Dilute Mix A with mobile phase A to obtain Mix B to Mix F, whose concentrations are 100 PPM, 50 PPM, 20 PPM, 10 PPM, and 5 PPM, respectively; Preferably, the preparation of the mixed A comprises: 40 μL oxalic acid internal standard mother solution + 40 μL citric acid internal standard mother solution + 40 μL cystine internal standard mother solution + 80 μL mobile phase A; Preferably, the preparation of the mixed B (100 PPM) includes: 20 μL of mixed A + 20 μL of mobile phase A; Preferably, the preparation of the mixed C (50 PPM) includes: 10 μL mixed A + 30 μL mobile phase A; Preferably, the preparation of the mixed D (20 PPM) includes: 10 μL mixed A + 90 μL mobile phase A; Preferably, the preparation of the mixed E (10 PPM) includes: 10 μL mixed A + 190 μL mobile phase A; Preferably, the preparation of the mixed F (5 PPM) includes: 5 μL mixed A + 195 μL mobile phase A; 3) The preparation of the internal standard working solution includes: The oxalic acid or citric acid internal standard stock solution was diluted 10 times with mobile phase A, and the cystine internal standard stock solution was diluted 10 times with water and then diluted 10 times with mobile phase A to prepare an internal standard working solution with a concentration of 10 PPM; 4) The preparation of the internal standard diluent comprises: The ratio of oxalic acid internal standard working solution, citric acid internal standard working solution, cystine internal standard working solution and mobile phase A is 2:2:1:14, and they are mixed to obtain an internal standard-containing dilution solution; 5) The preparation of the standard curve sample includes: Mix the mixed standard solution containing the internal standard diluent and mobile phase A, mixed A to mixed F at a ratio of 19:1 to obtain standard curve samples with concentrations of blank, 250ppb, 500ppb, 1000ppb, 2000ppb, 5000ppb, and 10000ppb; Preferably, the blank concentration sample refers to a sample containing an internal standard diluent and mobile phase A in a ratio of 19:1; Preferably, the sample with a concentration of 250 ppb refers to a sample containing an internal standard diluent and a mixed F ratio of 19:1; Preferably, the sample with a concentration of 5000 ppb refers to a sample containing an internal standard diluent and mixed E in a ratio of 19:1; Preferably, the sample with a concentration of 1000 ppb refers to a sample containing an internal standard diluent and a mixed D ratio of 19:1; Preferably, the sample with a concentration of 2000 ppb refers to a sample containing an internal standard diluent and mixed C in a ratio of 19:1; Preferably, the sample with a concentration of 5000 ppb refers to a sample containing an internal standard diluent and mixed B in a ratio of 19:1; Preferably, the sample with a concentration of 10000 ppb refers to a sample containing an internal standard diluent and mixed A in a ratio of 19:

1.

6. The detection method according to claim 5, characterized in that: The injection solution is prepared by mixing the supernatant and mobile phase A at a ratio of 1:1 to obtain a 2-fold diluted sample to be tested; The 2-fold diluted sample to be tested was mixed with the diluent containing the internal standard at a ratio of 1:19 to obtain a 40-fold diluted injection solution.

7. The detection method according to claim 1, characterized in that: The high performance liquid chromatography tandem mass spectrometry detection is to perform liquid chromatography separation on the injected solution, and perform mass spectrometry analysis on the sample after liquid chromatography separation; Analyze blank, standard curve samples with concentrations of 250ppb, 500ppb, 1000ppb, 2000ppb, 5000ppb, and 10000ppb, and calculate the standard curve based on the test values; The injected solution was analyzed, and the detected value was substituted into the standard curve to calculate the contents of oxalic acid, citric acid and cystine in the sample to be tested.

8. The detection method according to claim 7, characterized in that: The mass spectrometry analysis conditions are ESI ion source and positive and negative ion switching mode.

9. The detection method according to claim 1, characterized in that: The sources of the samples to be tested include humans, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, pigs, monkeys, sheep, and cattle; Preferably, the sample to be tested is a urine sample; Preferably, the sample to be tested is a 24-hour urine sample.

10. Use of the detection method according to any one of claims 1 to 9 in the preparation of a product for detecting oxalic acid, citric acid and cystine, or in the preparation of a product for diagnosing urinary stones.

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