Simultaneous determination of oxalate, uric acid, citrate, creatinine and cystine in urine

Abstract

The application provides a method for simultaneously detecting oxalic acid, uric acid, citric acid, creatinine and cystine in urine, and relates to the technical field of analytical chemistry. The method comprises the following steps: using high performance liquid chromatography tandem mass spectrometry to detect a pretreated sample to be measured, bringing a detection value into a standard curve, and calculating the content of oxalic acid, uric acid, citric acid, creatinine and cystine in urine. The method alleviates the technical problem that oxalic acid, uric acid, citric acid, creatinine and cystine cannot be simultaneously detected.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, and in particular to a method for the simultaneous detection of oxalic acid, uric acid, citric acid, creatinine and cystine in urine. Background Technology

[0002] The most common type of kidney stone, calcium oxalate, is mainly formed due to an imbalance between calcium and oxalate levels in the body and / or a lack of sufficient crystallization inhibitors. Hyperoxaluria, or excessive urinary oxalate, can be classified as primary (rare) or secondary (common) hyperoxaluria based on etiology and the severity of clinical manifestations. Oxalate detection methods include liquid chromatography, liquid chromatography-mass spectrometry, colorimetric test strips, and ion chromatography.

[0003] Citrate is a potent inhibitor of calcium salt crystallization. Potassium citrate treatment is effective for patients with primary or secondary hypocituria and acidification disorders. Hypocituria is considered a cause of urinary stone formation. Three properties of citrate are believed to help prevent urinary stone formation: urinary citrate acts as an inhibitor of the growth and aggregation of calcium oxalate and calcium phosphate crystals; citrate alters the ionic reactivity of calcium oxalate and phosphate; and the formation of intestinal complexes between calcium and citrate may reduce calcium excretion. Citrate is primarily detected by liquid chromatography and liquid chromatography-mass spectrometry.

[0004] Uric acid exists in the human body mainly in the form of uric acid and urate. When uric acid levels rise, urate levels also rise accordingly, leading to urate supersaturation. Urate crystals then precipitate, initiating a series of processes including crystal aggregation, retention, nucleation, and adhesion, ultimately forming stones. Patients with uric acid stones all have high blood uric acid or high uric acid in urine due to various causes, resulting in acidic urine. The pH value of urine has a significant impact on the formation of uric acid stones. Uric acid detection methods mainly include enzymatic methods, uric acid sensor detection methods, voltammetry, high-performance liquid chromatography (HPLC), and the tungsten-molybdenum-phosphate method. Creatinine is a metabolic byproduct of muscle metabolism in the body, primarily filtered and excreted by the glomeruli. Creatinine is an important indicator of kidney function; elevated creatinine levels indicate kidney disease. Normally, kidney stones do not cause elevated creatinine levels. However, severe urinary stones place a significant burden on the kidneys, leading to elevated creatinine levels. If your creatinine level is too high, please consult a doctor for a thorough examination and comprehensive analysis of your kidneys to determine kidney function. Common methods for detecting creatinine include high-performance liquid chromatography (HPLC), spectrophotometry, and the picric acid method.

[0005] Cystinuria is an autosomal recessive or incompletely inherited dominant genetic disorder. This disease disrupts the transport of cystine and other diamino acids in the proximal tubules of the kidney, leading to recurrent urinary stone formation. A defective reabsorption of cystine by proximal tubular cells results in excessive excretion of cystine into the urine, promoting its precipitation in the distal tubules. The low solubility of cystine in urine further contributes to the high predisposition of patients to cystine stones. Methods for detecting cystine mainly include liquid chromatography-mass spectrometry (LC-MS) and gas chromatography (GC).

[0006] There are many articles on the detection of oxalic acid, citric acid, uric acid, cystine, and creatinine, but few report the simultaneous determination of all five substances. Therefore, how to simultaneously detect oxalic acid, citric acid, uric acid, cystine, and creatinine remains an unsolved problem.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] The purpose of this invention is to provide a method for the simultaneous detection of oxalic acid, uric acid, citric acid, creatinine and cystine in urine, so as to alleviate the technical problem that oxalic acid, uric acid, citric acid, creatinine and cystine cannot be detected simultaneously in the prior art.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: According to one aspect of the present invention, the present invention provides a method for simultaneous detection of oxalic acid, uric acid, citric acid, creatinine and cystine in urine, the method comprising detecting a pretreated sample using high performance liquid chromatography-tandem mass spectrometry, substituting the detected values ​​into a standard curve, and calculating the content of oxalic acid, uric acid, citric acid, creatinine and cystine in the sample.

[0010] The cystine is L-cystine; Preferably, the pretreatment is liquid-liquid extraction; Preferably, after mixing the extract with the sample, the mixture is shaken at 1000-2000 rpm for 5-15 min, centrifuged at 3000-5000 rpm for 5-15 min, and then detected by high performance liquid chromatography-tandem mass spectrometry. Preferably, after mixing the extract with the sample, the mixture is shaken at 1500 rpm for 10 min, centrifuged at 4000 rpm for 10 min at 4°C, and then detected by high performance liquid chromatography-tandem mass spectrometry. Preferably, the extractant for the liquid-liquid extraction is an ammonia solution; Preferably, the concentration of the ammonia solution is 1-20% v / v; more preferably 10% v / v, and the 10% ammonia solution also contains an internal standard. Preferably, the volume ratio of the sample to the extract is 1:(5~50); more preferably, it is 1:10. Preferably, the extract also contains an internal standard.

[0011] Preferably, the mobile phase for high-performance liquid chromatography (HPLC) detection includes mobile phase A and mobile phase B; mobile phase A is an aqueous phase, and mobile phase B is an organic phase; the mobile phase contains ammonium fluoride. Preferably, the concentrations of ammonium fluoride in mobile phase A and mobile phase B are each independently preferably 0.1~10 mM; Preferably, the concentrations of ammonium fluoride in mobile phase A and mobile phase B are each independently preferably 0.1~2 mM; Preferably, the concentrations of ammonium fluoride in mobile phase A and mobile phase B are each independently preferably 0.5 mM; Preferably, mobile phase B is acetonitrile containing ammonium fluoride; Preferably, the high-performance liquid chromatography detection uses a pentafluorophenyl column.

[0012] Preferably, the elution program for chromatography is as follows: 0.00 min, mobile phase A 85%, mobile phase B 15%; 1.50 min, mobile phase A 75%, mobile phase B 25%; 1.51 min, mobile phase A 25%, mobile phase B 75%; 2.00 min, mobile phase A 25%, mobile phase B 75%; 2.01 min, mobile phase A 85%, mobile phase B 15%; 3.00 min, mobile phase A 85%, mobile phase B 15%.

[0013] Preferably, the ion source parameters for mass spectrometry detection are as follows: Positive ion mode: curtain gas: 40 psi; collision gas: 8 psi; ion source spray voltage: 5500V; ion source temperature: 500℃; ion source gas 1: 55 psi; and ion source gas 2: 60 psi; Negative ion mode: curtain gas: 40psi; collision gas: 9psi; ion source spray voltage: -4500V; ion source temperature: 500℃; ion source gas 1: 55 psi; and ion source gas 2: 60 psi.

[0014] Preferably, the standard curve is the correspondence between the content of the analyte in the standard and the detection value of the standard; the detection value of the standard is the peak value ratio of the standard and the internal standard, and the peak value includes peak height or peak area; Preferably, the standard is composed of several standards with a series of concentrations, wherein the concentrations of oxalic acid, uric acid, citric acid, creatinine, and L-cysteine ​​are independently distributed in the series of standards, ranging from 10 to 250 μg / mL, 60 to 1500 μg / mL, 60 to 1500 μg / mL, 120 to 3000 μg / mL, and 8 to 200 μg / mL, respectively. Preferably, the internal standard is oxalic acid, uric acid, citric acid, creatinine, and cystine labeled with isotopes.

[0015] Preferably, the determination method further includes testing quality control samples; Preferably, the quality control material includes at least two of low-value quality control material, medium-value quality control material, and high-value quality control material; the low-value quality control material is 2 to 4 times the linear minimum limit concentration, the medium-value quality control material is 40 to 50% of the linear maximum limit concentration, and the high-value quality control material is 60 to 80% of the linear maximum limit concentration.

[0016] Preferably, the source of the test sample includes mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, dairy cows, pigs, horses, monkeys, or humans; Preferably, the sample is a urine sample; Preferably, the matrix of the standard and quality control products includes artificial urine.

[0017] According to another aspect of the present invention, the present invention also provides the application of the above-described method for simultaneous detection of oxalic acid, uric acid, citric acid, creatinine and cystine in urine in the preparation of products for detecting oxalic acid, uric acid, citric acid, creatinine and cystine. The cystine in question is L-cysteine.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This method employs high-throughput liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) for the high-throughput detection of oxalic acid, uric acid, citric acid, creatinine, and cystine in human urine. Five compounds can be detected simultaneously in a single assay. After sample pretreatment, the sample is injected into the HPLC-MS / MS instrument for detection. The optimal testing time for all five compounds can be completed within three minutes, making it convenient and rapid. The detection method provided by this invention features high sensitivity, good repeatability, high accuracy, and good specificity. The method exhibits linearity in the following ranges: oxalic acid 10-250 μg / mL, citric acid 60-1500 μg / mL, uric acid 60-1500 μg / mL, cystine 8-200 μg / mL, and creatinine 120-3000 μg / mL, with correlation coefficients (r) ≥ 0.990 for all samples. The coefficient of variation (CV) for repeatability of low-value control samples is ≤ 20%, for medium-value control samples ≤ 15%, and for high-value control samples ≤ 15%. The relative deviation (B) of accuracy is ≤ ±15%, and the spiked recovery rate is 85%~115%. The simultaneous detection method for oxalic acid, uric acid, citric acid, creatinine, and cystine in urine provided by this invention meets the relevant regulatory requirements for linearity, repeatability, batch-to-batch variation, and accuracy of these components, and plays an important role in the study of the pathological mechanisms of urinary calculi and the development of related drugs. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 The citric acid chromatogram of the detection method provided in Example 1; Figure 2 The chromatogram of uric acid provided in Example 1; Figure 3 The oxalic acid chromatogram of the detection method provided in Example 1; Figure 4 The chromatogram of creatinine provided in Example 1; Figure 5 The chromatogram of cystine is provided by the detection method in Example 1. Detailed Implementation

[0021] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] According to one aspect of the present invention, a method for simultaneous detection of oxalic acid, uric acid, citric acid, creatinine, and cystine in urine is provided. The detection method includes detecting a pretreated sample using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), inputting the detected values ​​into a standard curve, and calculating the content of oxalic acid, uric acid, citric acid, creatinine, and cystine in the sample.

[0023] The testing principle of the determination method provided by this invention is as follows: A sample to be tested, such as a standard, quality control sample, or actual sample, is taken, pretreated, and then analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). When using the internal standard method for quantification, the sample to be tested also contains an internal standard. After chromatographic separation, the analyte is introduced into a mass spectrometer, where it is ionized in an ion source to form charged ions. Under the influence of an electric field, these ions are focused into a triple quadrupole mass analyzer. The precursor ion and daughter ions of the analyte are separated in the mass spectrometer analyzer, and finally enter the detector, generating a signal. The detection value of the sample is recorded, and then the content of the target substance in the sample is obtained through further analysis of the detection value.

[0024] In some optional embodiments, the assay method includes pretreatment of the sample to remove impurities and / or enrich the target analyte. Pretreatment can be performed using any acceptable method in the art, including but not limited to one or more of precipitation, liquid-liquid extraction, solid-phase extraction, and filtration; the present invention does not limit this. It is understood that those skilled in the art will select appropriate pretreatment methods based on general knowledge of the art, depending on the sample type.

[0025] In some optional embodiments, pretreatment employs liquid-liquid extraction. Liquid-liquid extraction can be performed in a 96-well plate using a multi-channel pipette for batch pretreatment. Preferred parameters for liquid-liquid extraction are: mixing the extract with the sample, shaking at 1000-2000 rpm for 5-15 min, centrifuging at 3000-5000 rpm for 5-15 min, and then detecting by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS); more preferably, mixing the extract with the sample, shaking at 1500 rpm for 10 min, centrifuging at 4°C and 4000 rpm for 10 min, and then detecting by HPLC-MS / MS. The extractant for liquid-liquid extraction is preferably an ammonia solution, with a preferred concentration of 1-20% v / v, for example, but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% v / v, preferably 10% v / v. The volume ratio of sample to extractant is preferably 1:(5~50), for example, but not limited to, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50, with 1:10 being the most preferred. For example, 20 μL of the sample to be tested is mixed thoroughly with 100 μL, 200 μL, or 1000 μL of extractant, preferably with 200 μL of extractant. The extractant preferably also contains an internal standard to facilitate its introduction into the system during the extraction process.

[0026] In some optional embodiments, internal standard quantification is employed, wherein the internal standard is an isotope of the analyte. Quantification using the corresponding isotope internal standard method can not only greatly eliminate matrix interference, but also ensure accurate quantification as the results are unaffected by pretreatment processes, instrument response fluctuations, and other conditions. Preferred internal standards include oxalic acid-[13C2], citrate-2,2,4,4-D4, creatinine-[D3], DL-cysteine-2,2',3,3,3',3'-D6, and uric acid-[1,3-15N2].

[0027] In some optional embodiments, the chromatographic conditions in the high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) detection are also optimized. The optimized mobile phase is as follows: the mobile phase comprises mobile phase A and mobile phase B; mobile phase A is an aqueous phase, and mobile phase B is an organic phase; the mobile phase contains ammonium fluoride. The concentration of ammonium fluoride in mobile phase A is preferably 0.1~10 mM, for example, but not limited to 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM, preferably 0.1~2 mM, more preferably 0.5 mM; the concentration of ammonium fluoride in mobile phase B is preferably 0.1~10 mM, for example, but not limited to 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM, preferably 0.1~2 mM, more preferably 0.5 mM. Mobile phase B is preferably acetonitrile containing ammonium fluoride. The chromatographic column is preferably a pentafluorophenyl column.

[0028] In some optional implementations, the elution program for high-performance liquid chromatography (HPLC) detection is as follows: 0.00 min, mobile phase A 85%, mobile phase B 15%; 1.50 min, mobile phase A 75%, mobile phase B 25%; 1.51 min, mobile phase A 25%, mobile phase B 75%; 2.00 min, mobile phase A 25%, mobile phase B 75%; 2.01 min, mobile phase A 85%, mobile phase B 15%; 3.00 min, mobile phase A 85%, mobile phase B 15%.

[0029] In some optional implementations, the ion source parameters for mass spectrometry detection are as follows: Positive ion mode: curtain gas: 40 psi; collision gas: 8 psi; ion source spray voltage: 5500V; ion source temperature: 500℃; ion source gas 1: 55 psi; and ion source gas 2: 60 psi; Negative ion mode: curtain gas: 40psi; collision gas: 9psi; ion source spray voltage: -4500V; ion source temperature: 500℃; ion source gas 1: 55 psi; and ion source gas 2: 60 psi.

[0030] In some optional implementations, the mass spectrometry parameters are as follows:

[0031] * indicates a quantitative ion.

[0032] In some optional embodiments, the standard curve is the correspondence between the content of the analyte in the standard and the detection value of the standard; the detection value of the standard is the peak ratio of the standard and the internal standard, and the peak includes peak height or peak area; preferably, the standard concentration is used as the independent variable xi, and the peak area ratio of the corresponding concentration standard and the internal standard is used as the dependent variable yi, and the linear regression equation y = ax + b and the correlation coefficient r are calculated.

[0033] The standard is preferably composed of several standards with a series of concentrations. The concentrations of oxalic acid in these standards are independently distributed from 10 to 250 μg / mL, with preferred concentration points being 10, 20, 40, 75, 145, and 250 μg / mL; the concentrations of uric acid are independently distributed from 60 to 1500 μg / mL, with preferred concentration points being 60, 120, 240, 450, 870, and 1500 μg / mL; and the concentrations of citric acid are independently distributed from 60 to 1... The concentrations are 500 μg / mL, with preferred concentration points set at 60, 120, 240, 450, 870, and 1500 μg / mL; the concentrations of creatinine are independently distributed in the range of 120–3000 μg / mL, with preferred concentration points set at 120, 240, 480, 900, 1740, and 3000 μg / mL; and the concentrations of cystine are independently distributed in the range of 8–200 μg / mL, with preferred concentration points set at 8, 16, 32, 60, 116, and 200 μg / mL.

[0034] In some optional embodiments, the assay method further includes the detection of quality control samples. The quality control samples preferably include at least two of low-value, medium-value, and high-value quality control samples; the low-value quality control sample is 2 to 4 times the linear minimum limit concentration, and the preferred concentrations of oxalic acid, uric acid, citric acid, creatinine, and cystine in the low-value quality control sample are: oxalic acid 30 μg / mL, citric acid 180 μg / mL, uric acid 180 μg / mL, cystine 24 μg / mL, and creatinine 360 ​​μg / mL; the medium-value quality control sample is 40 to 50% of the linear maximum limit concentration, and the preferred concentrations of oxalic acid, uric acid, citric acid, creatinine, and cystine in the medium-value quality control sample are: oxalic acid 30 μg / mL, citric acid 180 μg / mL, uric acid 180 μg / mL, cystine 24 μg / mL, and creatinine 360 ​​μg / mL. The preferred concentrations of cystine are: oxalic acid 115 μg / mL, citric acid 690 μg / mL, uric acid 690 μg / mL, cystine 92 μg / mL, and creatinine 1380 μg / mL; the high-value quality control material is 60-80% of the linear limit concentration, and the preferred concentrations of oxalic acid, uric acid, citric acid, creatinine, and cystine in the high-value quality control material are: oxalic acid 180 μg / mL, citric acid 1080 μg / mL, uric acid 1080 μg / mL, cystine 144 μg / mL, and creatinine 2160 μg / mL.

[0035] The present invention provides a method for the simultaneous detection of oxalic acid, uric acid, citric acid, creatinine, and cystine in urine. The sample source can be, for example, but not limited to, mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys, or humans. It should be noted that the method for the simultaneous detection of oxalic acid, uric acid, citric acid, creatinine, and cystine in urine provided by the present invention is not for diagnostic or therapeutic purposes. Specifically, samples from the aforementioned mammals are experimental animal samples, and the method of the present invention is used to provide experimental results for further application in fields such as the study of the mechanism of action of drugs for urinary stones or pharmacokinetic studies. Furthermore, knowing the content of oxalic acid, uric acid, citric acid, creatinine, and cystine in the sample does not mean that the result directly points to a diagnostic result for urinary stones. Therefore, the method for the simultaneous detection of oxalic acid, uric acid, citric acid, creatinine, and cystine in urine provided by the present invention is not for diagnostic or therapeutic purposes. The detection method provided by this invention does not limit the types of samples that can be tested; conventional sample types in the art can be selected, such as, but not limited to, extracts of serum, plasma, tissues or cells, and body fluids. In some optional embodiments, the sample is urine. When the sample is urine, since it is difficult to obtain human samples without background data, the matrix of the standards and quality control samples preferably includes artificial urine.

[0036] According to another aspect of the present invention, the present invention also provides a method for the simultaneous detection of oxalic acid, uric acid, citric acid, creatinine, and cystine in urine, in the preparation of products for detecting oxalic acid, uric acid, citric acid, creatinine, and cystine. The above-described determination method can be used as an evaluation standard to evaluate the detection effectiveness of other methods for detecting oxalic acid, uric acid, citric acid, creatinine, and cystine; or the above-described determination method can be pre-installed in the detection product as an operating module, and the reagents used in the above-described determination method can be used as complementary reagents in the detection product; or the above-described determination method can be pre-installed in the detection product as an operating module for use in conjunction with diagnostic or auxiliary diagnostic detection products for urinary stones.

[0037] The technical solution and technical effects of the present invention will be further described below with reference to preferred embodiments.

[0038] The main reagents and instruments are as follows: Table 1 Standards and Reagents

[0039] Table 2 Instruments and Equipment

[0040] Example 1 (a) Reagent preparation: 1.1 Preparation of stock solutions for oxalic acid, uric acid, citric acid, creatinine, and cystine: Sodium hydroxide and sodium carbonate aqueous solution: Using a 1 / 100,000 electronic balance, accurately weigh 4g of sodium hydroxide and 66.6mg of sodium carbonate into a 30mL plastic bottle, add 20mL of water, dissolve, label, and store at room temperature.

[0041] Sodium hydroxide aqueous solution: Using a 1 / 100,000 electronic balance, accurately weigh 4g of sodium hydroxide, add 20 mL of water, dissolve, label, and store at room temperature.

[0042] Preparation of oxalic acid stock solution: Using a 1 / 100,000 electronic balance, accurately weigh 10 mg of oxalic acid, place it in a 1 mL volumetric flask, dissolve it in water and dilute to the mark to prepare a stock solution with a concentration of 10,000 μg / mL. Transfer the solution to a 1.5 mL PP plastic bottle, label it, and store it at -20℃.

[0043] Preparation of citric acid monohydrate stock solution: Using a 1 / 100,000 electronic balance, accurately weigh 54.69 mg of citric acid monohydrate, place it in a 1 mL volumetric flask, dissolve it in water and dilute to the mark to prepare a stock solution with a concentration of 50,000 μg / mL. Transfer the solution to a 1.5 mL PP plastic bottle, label it, and store it at -20 ℃.

[0044] Preparation of creatinine stock solution: Using a 1 / 100,000 electronic balance, accurately weigh 50 mg of creatinine, place it in a 1 mL volumetric flask, dissolve it in water and dilute to the mark to prepare a stock solution with a concentration of 50,000 μg / mL. Transfer the solution to a 1.5 mL PP plastic bottle, label it, and store it at -20 ℃.

[0045] Preparation of cystine stock solution: Using a 1 / 100,000 electronic balance, accurately weigh 10 mg of L-cystine, place it in a 1 mL volumetric flask, dissolve it in sodium hydroxide aqueous solution and dilute to the mark to prepare a stock solution with a concentration of 10,000 μg / mL. Transfer the solution to a 1.5 mL PP plastic bottle, label it, and store it at -20℃.

[0046] Preparation of uric acid stock solution: Using a 1 / 100,000 electronic balance, accurately weigh 10 mg of uric acid, place it in a 1 mL volumetric flask, dissolve it in sodium hydroxide and sodium carbonate aqueous solution, and dilute to the mark to prepare a stock solution with a concentration of 10,000 μg / mL. Transfer the solution to a 1.5 mL PP plastic bottle, label it, and store it at -20℃.

[0047] 1.2 Preparation of standard products S1 to S6: Preparation of S0 diluent: Using a suitable pipette, mix 1.7 mL of 2% sodium hydroxide aqueous solution, 1.15 mL of water, and 7.15 mL of artificial urine thoroughly, label the mixture, and store at 2-8 ℃.

[0048] The standard products are prepared according to the high-low preparation mode, that is, after preparing S0 and S6, they are mixed in proportion to prepare S1 to S5 and LQC, MQC and HQC.

[0049] Preparation of Standard S6: Using a suitable pipette, take 50 μL of oxalic acid stock solution, 60 μL of citric acid stock solution, 40 μL of cystine stock solution, 300 μL of uric acid stock solution, 120 μL of creatinine stock solution, and 1.43 mL of artificial urine, place them in a suitable PP tube, mix thoroughly, label, and store at -20℃.

[0050] Preparation of standard S1: Using a pipette of appropriate size, take 20 μL of S6 solution and 0.48 mL of blank S0 solution, mix thoroughly, and prepare 0.5 mL of S1 standard solution. Label the solution and store at 2-8℃. Prepare and use immediately.

[0051] Preparation of standard S2: Using a pipette of appropriate size, take 40 μL of S6 solution and 0.46 mL of blank S0 solution, mix them thoroughly, and prepare 0.5 mL of S2 standard solution. Label the solution and store at 2-8℃. Prepare and use immediately.

[0052] Preparation of standard S3: Using a pipette of appropriate size, take 80 μL of S6 solution and 0.42 mL of blank S0 solution, mix thoroughly, and prepare 0.5 mL of S3 standard solution. Label the solution and store at 2-8℃. Prepare and use immediately.

[0053] Preparation of standard S4: Using a pipette of appropriate size, take 150 μL of S6 solution and 0.35 mL of blank S0 solution, mix them thoroughly, and prepare 0.5 mL of S4 standard solution. Label the solution and store at 2-8℃. Prepare and use immediately.

[0054] Preparation of standard S5: Using a pipette of appropriate size, take 290 μL of S6 solution and 0.21 mL of blank S0 solution, mix them thoroughly, and prepare 0.5 mL of S5 standard solution. Label the solution and store at 2-8℃. Prepare and use immediately.

[0055] 1.3 Preparation of quality control materials: Preparation of low-concentration quality control (LQC) solution: Using a suitable pipette, take 60 μL of S6 solution and 0.44 mL of blank S0 solution, mix thoroughly, and prepare 0.5 mL of low-concentration LQC solution. Label the solution and store at 2-8℃. Prepare and use immediately.

[0056] Preparation of medium-concentration quality control solution (MQC): Using a suitable pipette, take 230 μL of S6 solution and 0.27 mL of blank S0 solution, mix thoroughly, and prepare 0.5 mL of medium-concentration MQC solution. Label the solution and store at 2-8℃. Prepare and use immediately.

[0057] Preparation of high-concentration quality control (HQC) solution: Using a suitable pipette, take 360 ​​μL of S6 solution and 0.14 mL of blank S0 solution, mix thoroughly, and prepare 0.5 mL of high-concentration HQC solution. Label the solution and store at 2-8℃. Prepare and use immediately.

[0058] Table 3 Concentrations of various substances in standard and quality control samples

[0059] 1.4 Preparation of internal standard working solution: Preparation of 10% ammonia solution: Measure 90 mL of water using a graduated cylinder, add 10 mL of ammonia solution, mix, label, and store at room temperature.

[0060] Preparation of oxalic acid-[13C2] stock solution: Using a 1 / 100,000 electronic balance, accurately weigh 1 mg of oxalic acid-[13C2], place it in a 1 mL volumetric flask, dissolve it in water and dilute to the mark to prepare a stock solution with a concentration of 1000 μg / mL. Transfer the solution to a 1.5 mL PP plastic bottle, label it, and store it at -20℃.

[0061] Preparation of citric acid-2,2,4,4-D4 stock solution: Using a 1 / 100,000 electronic balance, accurately weigh 1 mg of citric acid-2,2,4,4-D4 monohydrate, place it in a 1 mL volumetric flask, dissolve it in water, and dilute to the mark to prepare a stock solution with a concentration of 1000 μg / mL. Transfer the solution to a 1.5 mL PP plastic bottle, label it, and store it at -20℃.

[0062] Preparation of creatinine-[D3] stock solution: Using a 1 / 100,000 electronic balance, accurately weigh 1 mg of creatinine-[D3], place it in a 1 mL volumetric flask, dissolve it in water and dilute to the mark to prepare a stock solution with a concentration of 1000 μg / mL. Transfer the solution to a 1.5 mL PP plastic bottle, label it, and store it at -20℃.

[0063] Preparation of DL-cystine 2,2',3,3,3',3'-D6 stock solution: Using a 0.0001 g electronic balance, accurately weigh 1 mg of DL-cystine 2,2',3,3,3',3'-D6, place it in a 1 mL volumetric flask, dissolve it in sodium hydroxide aqueous solution, and dilute to the mark to prepare a stock solution with a concentration of 1000 μg / mL. Transfer the solution to a 1.5 mL PP plastic bottle, label it, and store at -20℃.

[0064] Preparation of uric acid-[1,3-15N2] stock solution: Using a 1 / 100,000 electronic balance, accurately weigh 1 mg of uric acid-[1,3-15N2], place it in a 1 mL volumetric flask, dissolve it in sodium hydroxide and sodium carbonate aqueous solution, and dilute to the mark to prepare a stock solution with a concentration of 1000 μg / mL. Transfer the solution to a 1.5 mL PP plastic bottle, label it, and store it at -20℃.

[0065] Internal standard working solution (sample extraction solution): Measure 23.75 mL of 10% ammonia solution, add 0.1 mL of oxalic acid-[13C2] internal standard stock solution, 0.05 mL of citric acid-2,2,4,4-D4 internal standard stock solution, 1 mL of uric acid-[1,3-15N2] internal standard stock solution, 0.05 mL of DL-cystine-2,2',3,3,3',3'-D6 internal standard stock solution, and 0.05 mL of creatinine-[D3] internal standard stock solution, mix well, label, and store at -20℃.

[0066] 1.5 Preparation of mobile phase: Preparation of 0.5M ammonium fluoride solution: Accurately weigh 0.926 g of ammonium fluoride using an electronic balance, add 50 mL of pure water to prepare 50 mL of 0.5 mol / L ammonium fluoride solution, and store in a refrigerator at 2-8℃ until use.

[0067] Preparation of mobile phase A: Accurately transfer 1000 mL of water using a graduated cylinder, add 1000 μL of ammonium fluoride solution, mix well, filter, and degas by sonication, and set aside for use.

[0068] Preparation of mobile phase B: Accurately transfer 1000 mL of acetonitrile using a graduated cylinder, add 1000 μL of ammonium fluoride solution, mix well, filter, and degas by sonication, then set aside for use.

[0069] Preparation of injection washing solution: Accurately transfer 100 mL of acetonitrile and 900 mL of water using a graduated cylinder, add 10 mL of ammonia solution, mix well, filter and degas by sonication, and set aside for use.

[0070] (II) Pre-processing: (a) Shaking centrifugation: Take the sample solution and shake it at 1500 rpm for 5 min. After shaking, transfer 1 mL of sample into a 1.5 mL centrifuge tube and centrifuge at 10000 rpm for 10 min.

[0071] (b) Add standard and quality control solutions: Accurately transfer 20 μL of standard and quality control solutions into 1 mL of 96-well plate.

[0072] (c) Add sample extraction solution: Accurately transfer 200 μL of sample extraction solution and add it to the corresponding 96-well plates above.

[0073] (d) Add urine sample: Accurately transfer 20 μL of urine sample into 1 mL of 96-well plate.

[0074] (e) Add sample extraction solution: Accurately transfer 200 μL of sample extraction solution and add it to the corresponding 96-well plate.

[0075] (f) Oscillation: Cover the 96-well plate with the cover, place it on the 96-well plate oscillator, and oscillate fully at 1500 rpm for 10 min.

[0076] (g) Centrifugation: Centrifuge at 4 ℃ and 4000 rpm for 10 min in a refrigerated centrifuge; take the supernatant after centrifugation for LC-MS / MS separation.

[0077] (III) Chromatographic conditions: Table 4 Chromatographic conditions

[0078] (iv) Mass spectrometry parameters 4.1 Ion source parameters Table 5 Ion source parameters

[0079] 4.2 Ion Information Table 6 Ion Information

[0080] In Table 6, * indicates quantitative ions.

[0081] The chromatograms of citric acid, uric acid, oxalic acid, creatinine, and cystine detected using the method described in this embodiment are shown below. Figures 1-5 As shown.

[0082] Example 1: Linear Range 1.1 Validation Method: The standard solutions S1-S6 of the test products were processed according to the sample processing method described for the detection of oxalic acid, uric acid, citric acid, creatinine, and cystine in human urine by high performance liquid chromatography-tandem mass spectrometry. Each concentration was tested three times. The correlation coefficient r for linear regression can be calculated using the formula; the correlation coefficient r should be ≥0.990.

[0083]

[0084] r: linear regression correlation coefficient; xi: concentration of S1~S6; yi: mean ratio of peak area of ​​standard and internal standard in the corresponding concentration solution.

[0085] 1.2 Acceptance criteria: The correlation coefficients r of the regressions of citric acid, uric acid, oxalic acid, creatinine, and cystine were all ≥0.990.

[0086] 1.3 Experimental Results: Linear range: Oxalic acid 10-250 μg / mL, citric acid 60-1500 μg / mL, uric acid 60-1500 μg / mL, cystine 8-200 μg / mL, creatinine 120-3000 μg / mL.

[0087] Table 7 Linear data for citric acid

[0088] Table 8. Linear data of uric acid

[0089] Table 9. Linear data for oxalic acid

[0090] Table 10 Linear data of creatinine

[0091] Table 11 Linear data for cystine

[0092] 1.4 Conclusion: The regression correlation coefficients r of citric acid, uric acid, oxalic acid, creatinine and cystine were all ≥0.990, which met the acceptance criteria.

[0093] Example 2: Repeatability 2.1 Validation Method: The standard solutions S1-S6 and quality control samples were processed according to the sample preparation method described in Example 1 for the detection of oxalic acid, uric acid, citric acid, creatinine, and cystine in human urine using high-performance liquid chromatography-tandem mass spectrometry. Each sample was measured five times, and the tests were conducted in three batches. The coefficient of variation (CV) for repeatability can be calculated using the formula; the CV for the quality control samples was ≤15%.

[0094]

[0095] CV: Coefficient of variation for repeatability; : The average of 5 measurements; S: The standard deviation of 5 measurements.

[0096] 2.2 Acceptance criteria: The coefficient of variation (CV) for low-value quality control products is ≤15%, the coefficient of variation (CV) for medium-value quality control products is ≤15%, and the coefficient of variation (CV) for high-value quality control products is ≤15%.

[0097] 2.3 Experimental Results: Table 12. Intra-batch and inter-batch precision of citric acid

[0098] Table 13. Intra-batch and inter-batch precision of uric acid

[0099] Table 14. Intra-batch and inter-batch precision of oxalic acid

[0100] Table 15. Intra- and inter-batch precision of creatinine

[0101] Table 16 Intra- and inter-batch precision of cystine

[0102] 2.4 Conclusion: The coefficient of variation (CV) of low-value quality control products is ≤20%, and the coefficient of variation (CV) of high-value quality control products is ≤15%, which meets the acceptance criteria.

[0103] Example 3: Evaluation of matrix effect 3.1 Experimental Procedure: Six independent human matrix samples, solvent samples, matrix samples, and a 1:1 mixture of solvent and matrix samples were collected, and the peak areas of all samples were measured. The mean ratio of analyte to internal standard peak area for solvent samples was A, the mean ratio for matrix samples was BP, and the mean ratio for the 1:1 mixture of solvent and matrix samples was CP.

[0104] 3.2 Acceptance criteria: Matrix deviation (%) = (CP - (A + BP) / 2) / ((A + BP) / 2) (%), relative matrix deviation ≤ 20%.

[0105] 3.3 Experimental Results: Table 17. Results of Citric Acid Matrix Effect

[0106] Table 18. Results of uric acid matrix effect

[0107] Table 19. Oxalic acid matrix effect

[0108] Table 20. Results of creatinine matrix effect

[0109] Table 21. Cystine matrix effect

[0110] 3.4 Experimental Summary: The relative matrix deviations were all ≤20%, which meets the requirements.

[0111] Example 4: Accuracy 4.1 Experimental procedure: The spiked recovery method was used. 20 μL of accuracy working solution samples with low, medium and high concentrations (concentrations of: background value + LQC, background value + MQC, background value + HQC, respectively) were taken for pretreatment; 3 samples were replicated for each concentration level.

[0112] 4.2 Acceptance criteria: Recovery rate: 85%-115%.

[0113] Table 22 Citric Acid Accuracy

[0114] Table 23 Uric Acid Accuracy

[0115] Table 24 Oxalic Acid Accuracy

[0116] Table 25 Creatinine Accuracy

[0117] Table 26 Accuracy of Cystine

[0118] 4.3 Experimental Summary: The accuracy is within ±15% of the indicated value, which meets the requirements.

[0119] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for simultaneous detection of oxalic acid, uric acid, citric acid, creatinine, and cystine in urine, characterized in that, The detection method includes detecting the pretreated sample using high-performance liquid chromatography-tandem mass spectrometry, inputting the detected values ​​into a standard curve, and calculating the contents of oxalic acid, uric acid, citric acid, creatinine, and cystine in the sample; The cystine is L-cystine; The pretreatment includes: taking urine, adding 10% ammonia water by volume for liquid-liquid extraction, centrifuging and taking the supernatant; The 10% ammonia solution also contains an internal standard; In the high-performance liquid chromatography-tandem mass spectrometry detection, a pentafluorophenyl column is used, wherein: Mobile phase A is an aqueous solution of ammonium fluoride, and the concentration of ammonium fluoride in mobile phase A is 0.5 mM; Mobile phase B is an ammonium fluoride acetonitrile solution, and the concentration of ammonium fluoride in mobile phase B is 0.5 mM; Elute using the following gradient: 0.00 min, mobile phase A 85%, mobile phase B 15%; 1.50 min, mobile phase A 75%, mobile phase B 25%; 1.51 min, mobile phase A 25%, mobile phase B 75%; 2.00 min, mobile phase A 25%, mobile phase B 75%; 2.01 min, mobile phase A 85%, mobile phase B 15%; 3.00 min, mobile phase A 85%, mobile phase B 15%.

2. The detection method according to claim 1, characterized in that, The liquid-liquid extraction is performed by mixing the extract with the sample, shaking at 1000-2000 rpm for 5-15 min, centrifuging at 3000-5000 rpm for 5-15 min, and then performing high performance liquid chromatography-tandem mass spectrometry detection. The volume ratio of urine sample to extract is 1:(5~50).

3. The detection method according to claim 1, characterized in that, The liquid-liquid extraction was performed by mixing the extract with the sample, shaking at 1500 rpm for 10 min, centrifuging at 4000 rpm for 10 min at 4°C, and then performing high performance liquid chromatography-tandem mass spectrometry detection. The volume ratio of urine sample to extract was 1:

10.

4. The detection method according to claim 1, characterized in that, The ion source parameters for mass spectrometry detection are as follows: Positive ion mode: curtain gas: 40 psi; collision gas: 8 psi; ion source spray voltage: 5500V; ion source temperature: 500℃; ion source gas ratio: 1:55 psi; And, ion source gas 2: 60 psi; Negative ion mode: Air curtain gas: 40psi; Collision gas: 9 psi; Ion source spray voltage: -4500V; Ion source temperature: 500℃; Ion source gas 1: 55 psi; and, ion source gas 2: 60 psi.

5. The detection method according to claim 4, characterized in that, The mass spectrometry parameters are as follows: * indicates a quantitative ion.

6. The detection method according to any one of claims 1 to 5, characterized in that, The standard curve is the correspondence between the content of the analyte in the standard and the detection value of the standard; the detection value of the standard is the ratio of the peak value of the standard to the internal standard, and the peak value includes peak height or peak area. The standard consists of several standards with a series of concentrations, in which the concentrations of oxalic acid, uric acid, citric acid, creatinine, and L-cysteine ​​are independently distributed from 10 to 250 μg / mL, 60 to 1500 μg / mL, 60 to 1500 μg / mL, 120 to 3000 μg / mL, and 8 to 200 μg / mL, respectively.

7. The detection method according to any one of claims 1 to 5, characterized in that, The detection method also includes detecting quality control samples; The quality control materials include at least two of the following: low-value quality control materials, medium-value quality control materials, and high-value quality control materials; the low-value quality control materials are 2 to 4 times the linear minimum limit concentration, the medium-value quality control materials are 40 to 50% of the linear maximum limit concentration, and the high-value quality control materials are 60 to 80% of the linear maximum limit concentration.

8. The detection method according to any one of claims 1 to 5, characterized in that, The test samples may be from mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys, or humans. The matrix for standards and quality control products includes artificial urine.

9. The simultaneous detection method for oxalic acid, uric acid, citric acid, creatinine and cystine in urine according to any one of claims 1 to 5, used in the preparation of products for detecting oxalic acid, uric acid, citric acid, creatinine and cystine; The cystine in question is L-cysteine.

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