Method for detecting concentration of organic matter in urine

Abstract

The present application relates to the technical field of analytical detection, and particularly relates to a detection method of organic matter concentration in urine. The detection method comprises mixing a urine sample with sulfuric acid for primary acidification, mixing the supernatant after acidification with a mixture of aniline and dimethyl sulfoxide, then adding hydrochloric acid for secondary acidification, adding an aqueous solution of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride for derivatization after acidification, mixing the supernatant after derivatization with a sample diluent, centrifuging, and then detecting by liquid chromatography-tandem mass spectrometry. The detection method is simple and accurate, can simultaneously detect creatinine, citric acid, oxalic acid and cystine in human urine, can meet the requirements of relevant regulations on linearity, repeatability, matrix effect, accuracy and the like of creatinine, citric acid, oxalic acid and cystine, and has important reference significance for evaluating kidney stones.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, and in particular to a method for detecting the concentration of organic matter in urine. Background Technology

[0002] Organic substances in urine (such as creatinine, citric acid, oxalic acid, and cystine) are often used to assess the metabolism of patients with urinary stones.

[0003] Calcium oxalate is the most common type of urinary stone, mainly caused by an imbalance of calcium and oxalate concentrations in the body, leading to hypercalciuria and hyperoxaluria. Hyperoxaluria can be classified into primary (rare) and secondary (common) hyperoxaluria based on its etiology. Methods for detecting oxalate mainly include liquid chromatography, liquid chromatography-mass spectrometry, colorimetric test strips, and ion chromatography.

[0004] Citric acid is a crystal inhibitor in urine, reducing the risk of kidney stones by chelating metal ions. Hypocitric aciduria is considered a cause of kidney stone formation. The main methods for detecting citric acid include liquid chromatography and liquid chromatography-mass spectrometry.

[0005] Cystinuria is an autosomal recessive or incompletely dominant genetic disorder. This disease disrupts the transport of cystine and other diamino acids in the proximal tubules of the kidney, leading to recurrent kidney stone formation. Cystine has low solubility in urine, increasing the predisposition of patients to cystine stones. Methods for detecting cystine mainly include liquid chromatography-mass spectrometry (LC-MS) and gas chromatography (GC).

[0006] Creatinine is a metabolic byproduct of muscle metabolism in the human body, primarily excreted through glomerular filtration. Creatinine is an important indicator of kidney function; elevated creatinine levels suggest kidney disease. Common methods for detecting creatinine include high-performance liquid chromatography (HPLC), spectrophotometry, and the picric acid method.

[0007] Currently, there are many articles or patent documents related to the detection of creatinine, citric acid, oxalic acid and cystine. For example, CN117330684A discloses a detection method and kit for the simultaneous detection of citric acid, oxalic acid and cystine in urine. However, there are few articles or patent documents that report the simultaneous determination of these four organic compounds in urine. Summary of the Invention

[0008] To address the shortcomings of the existing technology, this invention provides a method for detecting the concentration of organic matter in urine, comprising: mixing a urine sample with sulfuric acid for a first acidification; mixing the acidified supernatant with derivatizing agent A; then adding hydrochloric acid for a second acidification; and adding derivatizing agent B for derivatization after acidification; mixing the derivatized supernatant with a sample diluent; centrifuging; and detecting the sample using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The derivatizing agent A is a mixture of aniline and dimethyl sulfoxide; The derivatizing agent B is an aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; The sample diluent is an aqueous methanol solution containing ammonium formate; The organic compounds include creatinine, citric acid, oxalic acid, and cystine.

[0009] Through extensive experimentation with pretreatment steps, this invention has found that two acidification treatments, using sulfuric acid and hydrochloric acid respectively, are required to ensure that the recovery rates of four organic compounds—creatinine, citric acid, oxalic acid, and cystine—are within an acceptable range, thereby enabling accurate simultaneous detection of the concentrations of these four organic compounds in urine.

[0010] Preferably, the sulfuric acid is a 2M sulfuric acid solution; each 1 mL urine sample is mixed with (20~80) μL of the 2M sulfuric acid solution. More preferably, each 1 mL urine sample is mixed with (40~60) μL of the 2M sulfuric acid solution.

[0011] Preferably, the volume ratio of the acidified supernatant to the derivatizing agent A is 1:(1~5).

[0012] More preferably, the volume ratio of the acidified supernatant to the derivatizing agent A is 1:(2~3).

[0013] Preferably, the hydrochloric acid is a 6M hydrochloric acid solution, and the volume ratio of the acidified supernatant to the 6M hydrochloric acid solution is 1:(0.5~1.5), more preferably 1:(0.8~1.2).

[0014] Preferably, the aniline in the derivative A accounts for 10% to 30% of the volume percentage of the mixture, more preferably 15% to 25%.

[0015] Preferably, the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in derivatizer B is 100-500 mg / mL, more preferably 100-300 mg / mL, and even more preferably 150-250 mg / mL.

[0016] Preferably, the volume ratio of derivatizing agent A to derivatizing agent B is (1~3):1, more preferably (1.5~2.5):1.

[0017] Preferably, the sample diluent is an aqueous methanol solution containing 15-25 mM ammonium formate.

[0018] Preferably, the volume ratio of methanol to water in the sample diluent is (2~10):1, more preferably (3~5):1.

[0019] Preferably, the volume ratio of the derivatized supernatant to the sample diluent is 1:(5~15), more preferably 1:(8~12).

[0020] Preferably, the volume ratio of the acidified supernatant to the derivatizing agent A is 1:(2~3), the hydrochloric acid is a 6M hydrochloric acid solution, the volume ratio of the acidified supernatant to the 6M hydrochloric acid solution is 1:(0.8~1.2), and the volume ratio of derivatizing agent A to derivatizing agent B is (1.5~2.5):1.

[0021] The present invention further discovers that, under the above-mentioned reagent dosage conditions, the chromatographic peak areas of four organic compounds—creatinine, citric acid, oxalic acid, and cystine—can be maximized.

[0022] Preferably, the derivatized supernatant is mixed with the sample diluent, shaken, and then centrifuged in a refrigerated centrifuge at 3000-5000 rpm. The supernatant is then analyzed by liquid chromatography-tandem mass spectrometry.

[0023] Preferably, the acidified supernatant is first added with an internal standard before being mixed with derivatizing agent A; the internal standard includes creatinine internal standard, citrate internal standard, oxalate internal standard and cystine internal standard.

[0024] Preferably, the volume ratio of the acidified supernatant to the total volume of the internal standard is 1:(0.5~1.5), more preferably 1:(0.8~1.2).

[0025] Preferably, the instrument model used in the liquid chromatography-tandem mass spectrometry method includes, but is not limited to, the AB SCIEX TripleQuard. TM 4500, AB SCIEX Triple Quard TM 5500 or Waters Xevo TQ-S.

[0026] Preferably, in the liquid chromatography-tandem mass spectrometry method, the chromatographic column is an Eclipse Plus C18 with dimensions of 3.5 μm and 3.0 × 100 mm; mobile phase A is a mixture of ammonium formate solution, formic acid, and water in a volume ratio of (1~3):(0.5~1.5):1000; mobile phase B is a mixture of ammonium formate solution, formic acid, and methanol in a volume ratio of (1~3):(0.5~1.5):1000; the chromatographic elution program is as follows:

[0027] During the elution process, the sum of the volume percentages of mobile phase A and mobile phase B is 100%.

[0028] Preferably, the mobile phase A is a mixture of ammonium formate solution, formic acid and water in a volume ratio of (1~3):(0.5~1.5):1000.

[0029] More preferably, the mobile phase A is a mixture of ammonium formate solution, formic acid and water in a volume ratio of (1.5~2.5):1:1000.

[0030] Most preferably, the mobile phase A is a mixture of ammonium formate solution, formic acid and water in a volume ratio of 2:1:1000.

[0031] Preferably, the mobile phase B is a mixture of ammonium formate solution, formic acid and methanol in a volume ratio of (1~3):(0.5~1.5):1000.

[0032] More preferably, the mobile phase B is a mixture of ammonium formate solution, formic acid and methanol in a volume ratio of (1.5~2.5):1:1000.

[0033] Most preferably, the mobile phase B is a mixture of ammonium formate solution, formic acid and methanol in a volume ratio of 2:1:1000.

[0034] Preferably, the concentration of the ammonium formate solution is 4-6M, more preferably 5M.

[0035] The present invention also optimized and screened the mobile phase system, and finally found that when the above-mentioned mobile phase A and mobile phase B are composed, the chromatographic peak shapes of the four organic compounds, creatinine, citric acid, oxalic acid and cystine, are better.

[0036] Preferably, the column temperature of the chromatographic column is 30~40℃; and / or, the flow rates of mobile phase A and mobile phase B are 0.4~0.5mL / min.

[0037] Preferably, in the liquid chromatography-tandem mass spectrometry method, the ion source parameters of the mass spectrometer include: ion source ESI+, curtain gas 35~45psi, collision gas 5~10psi, spray voltage 5300~5800 volts, temperature 430~480℃, and spray gas 50~60 psi.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a simple and highly accurate method for simultaneously detecting creatinine, citric acid, oxalic acid, and cystine in human urine. This method meets the relevant regulatory requirements for linearity, repeatability, matrix effect, and accuracy of creatinine, citric acid, oxalic acid, and cystine, and has important reference value for assessing kidney stones. Attached Figure Description

[0039] Figure 1 This is a liquid chromatogram of the detection method in Example 1. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0041] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels. Since human samples without background data are difficult to obtain, the following embodiments use artificial urine as a substitute matrix for human urine to prepare the concentration points of the standard curve and for quality control.

[0042] Example 1 This embodiment provides a method for detecting the concentration of organic matter in urine, the steps of which are as follows: 1. Reagent preparation The preparation of the compound stock solution is shown in Table 1.

[0043] Table 1

[0044] The preparation of calibrator C6 is shown in Table 2.

[0045] Table 2

[0046] Preparation of calibrator C0: Take 0.32 mL of 1M hydrochloric acid and 7.68 mL of artificial urine, mix them thoroughly, and that is C0.

[0047] The preparation of calibrators C1 to C5 and the low-, medium-, and high-concentration quality control samples LQC, MQC, and HQC are shown in Table 3.

[0048] Table 3

[0049] In the final calibrators C1–C6, the concentrations are as follows: oxalic acid (OX): 5–250 μg / mL; citric acid (CA): 20–1000 μg / mL; cystine (CYS): 4–200 μg / mL; creatinine (CR): 50–2500 μg / mL. In the quality control LQC, the concentrations are as follows: oxalic acid (OX): 15 μg / mL; citric acid (CA): 60 μg / mL; cystine (CYS): 12 μg / mL; creatinine (CR): 150 μg / mL. In the quality control MQC, the concentrations are as follows: oxalic acid (OX): 125 μg / mL; citric acid (CA): 500 μg / mL; cystine (CYS): 100 μg / mL; creatinine (CR): 1250 μg / mL. In the quality control HQC, the concentrations are as follows: oxalic acid (OX): 175 μg / mL; citric acid (CA): 700 μg / mL; cystine (CYS): 140 μg / mL. μg / mL; Creatinine (CR): 1750 μg / mL.

[0050] Internal standard solutions: Oxalic acid internal standard (OX-IS): 100 μg / mL; Citric acid internal standard (CA-IS): 50 μg / mL; Cystine internal standard (CYS-IS): 50 μg / mL; Creatinine (CR-IS): 100 μg / mL.

[0051] Derivatizer A: A mixture of aniline and dimethyl sulfoxide, wherein aniline accounts for 20% by volume and dimethyl sulfoxide accounts for 80% by volume. Derivatizer B: An aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride at a concentration of 200 mg / mL; Preparation of 5M ammonium formate solution as mobile phase additive: Accurately weigh 12.6 g of ammonium formate using an electronic balance, add 40 mL of pure water to prepare 40 mL of 5M ammonium formate solution, and place it in a refrigerator at 4℃ for later use.

[0052] Preparation of mobile phase A: Accurately transfer 1000 mL of water using a graduated cylinder, add 2 mL of 5M ammonium formate solution and 1 mL of formic acid, mix well, filter and sonicate to degas, and set aside for use.

[0053] Preparation of mobile phase B: Accurately transfer 1000 mL of methanol using a graduated cylinder, add 2 mL of 5M ammonium formate solution and 1 mL of formic acid, mix well, filter and sonicate to degas, and set aside for use.

[0054] Preparation of injection washing solution: Accurately transfer 500 mL of methanol and 500 mL of water using a graduated cylinder, mix well, filter, and degas by sonication, then set aside for use.

[0055] Sample diluent: A methanol-water solution containing 20 mM ammonium formate, with a methanol to water volume ratio of 4:1.

[0056] 2. Pretreatment methods (1) Urine acidification: Take 1 mL of urine and add 50 μL of acidifying agent 1 (2M sulfuric acid solution); (2) Urine centrifugation: The urine was centrifuged using a high-speed centrifuge at 10,000 rpm for 2 min; (3) Derivatization: Accurately transfer 40 μL of the sample to be tested (urine, calibrator, quality control) using a pipette, add 40 μL of internal standard solution, 100 μL of derivatizing agent A, 40 μL of acidifying agent 2 (6 M hydrochloric acid solution), and 50 μL of derivatizing agent B, and place them together in a 2 mL centrifuge tube, cover it and place it on a multi-tube vortex mixer and shake for 1 min; (4) Transfer: Take 20 μL of supernatant, add 200 μL of sample diluent, shake for 5 min at 1500 rpm in a microplate constant temperature shaker, then centrifuge for 5 min at 4000 rpm in a large capacity refrigerated centrifuge, and take the supernatant for LC-MS / MS detection.

[0057] 3. Detection by LC-MS / MS method (1) Chromatographic conditions: The instrument model for liquid chromatography-tandem mass spectrometry was AB SCIEX Triple Quard. TM 4500; The chromatographic column was an Eclipse Plus C18 with dimensions of 3.5 μm and 3.0 × 100 mm; Column temperature: 40℃; Injection volume: 1 μL; Sample manager temperature: 10℃; The chromatographic elution program is shown in Table 4.

[0058] Table 4

[0059] (2) Mass spectrometry conditions: Ion source parameters are shown in Table 5.

[0060] Table 5

[0061] Ion information is shown in Table 6.

[0062] Table 6

[0063] * indicates quantitative ions.

[0064] 4. Linear Range Experiment The above pretreatment method and LC-MS / MS method were used to detect calibrators C1~C6. The correlation coefficient r of linear regression was calculated with reference to the formula. The correlation coefficient r of linear regression for creatinine, citrate, oxalate and cystine should be ≥0.990.

[0065] x i : Concentration of calibrators C1~C6.

[0066] y i : The average peak area ratio of the calibrator to its internal standard in the corresponding concentration solution.

[0067] The linear regression data for oxalic acid, citric acid, cystine, and creatinine are shown in Tables 7 to 10, respectively.

[0068] Table 7

[0069] Table 8

[0070] Table 9

[0071] Table 10

[0072] Conclusion: The linear range for oxalic acid was 5–250 μg / mL, for citric acid 20–1000 μg / mL, for cystine 4–200 μg / mL, and for creatinine 50–2500 μg / mL. The correlation coefficients (r) for the linear regression of citric acid, oxalic acid, creatinine, and cystine were all ≥0.990, meeting the acceptance criteria.

[0073] 5. Repeatability test Using actual human samples as background levels as low-concentration samples, and adding high-concentration standards to the samples as high-concentration samples (oxalic acid concentration: 175 μg / mL, citric acid concentration: 700 μg / mL, cystine concentration: 140 μg / mL, creatinine concentration: 1750 μg / mL), the samples were analyzed using the above pretreatment method and LC-MS / MS. Each sample was measured five times, and the tests were conducted in three batches. The coefficient of variation (CV) for repeatability was calculated using the formula. The CV for low-value quality control samples should be ≤15%, and the CV for high-value quality control samples should be ≤15%.

[0074] : The average of 5 measurements; S: The standard deviation of 5 measurements.

[0075] The intra-batch and inter-batch precision data for oxalic acid, citric acid, cystine, and creatinine are shown in Tables 11 to 14, respectively.

[0076] Table 11

[0077] Table 12

[0078] Table 13

[0079] Table 14

[0080] Conclusion: The coefficient of variation (CV) for low-concentration samples was ≤15%, and the coefficient of variation (CV) for high-concentration samples was ≤15%, which met the acceptance criteria.

[0081] 6. Matrix effect experiment Six independent human matrix samples were collected, along with a 50% methanol-water solvent sample, a matrix sample, and a 1:1 volume ratio (solvent and matrix) mixture. In the low-concentration sample, the concentrations were: oxalic acid 15 μg / mL, citric acid 60 μg / mL, cysteine ​​12 μg / mL, and creatinine 150 μg / mL. In the high-concentration sample, the concentrations were: oxalic acid 175 μg / mL, citric acid 700 μg / mL, cysteine ​​140 μg / mL, and creatinine 1750 μg / mL. Peak areas were measured for all samples. The mean peak area ratio of analyte to internal standard in the solvent sample was A; in the matrix sample, it was BP; and in the 1:1 volume ratio (solvent and matrix), it was CP. Calculate the matrix deviation, matrix deviation (%) = (CP - (A + BP) / 2) / ((A + BP) / 2), the matrix deviation should be ≤ ±20%.

[0082] The intra-batch and inter-batch precision data for oxalic acid, citric acid, cystine, and creatinine are shown in Tables 15 to 18, respectively.

[0083] Table 15

[0084] Table 16

[0085] Table 17

[0086] Table 18

[0087] Conclusion: The relative matrix effect deviations are all ≤ ±20%, which meets the requirements.

[0088] The liquid chromatogram of the detection method in this embodiment is as follows: Figure 1 As shown.

[0089] Example 2 This embodiment provides a method for detecting the concentration of organic matter in urine. The only difference from Embodiment 1 is that the pretreatment method is set with different reagent dosages according to the different schemes in Table 19.

[0090] Table 19

[0091] The same low-value urine sample and high-value urine sample were detected according to the LC-MS / MS detection conditions of Example 1, and the chromatographic peak areas were compared. The results are shown in Table 20.

[0092] Table 20

[0093] When option 2 is selected, each organic compound has the largest peak area.

[0094] Comparative Example This comparative example provides a method for detecting the concentration of organic matter in urine. The only difference between this method and Example 1 is that the acidifying agent 1 in the pretreatment method is replaced with 2M hydrochloric acid and 2M phosphoric acid, and the addition of acidifying agent 1 is omitted.

[0095] 20 μL of baseline sample (RA), low-concentration spiked recovery sample (recovery sample 1, RL), medium-concentration spiked recovery sample (recovery sample 2, RM), and high-concentration spiked recovery sample (recovery sample 3, RL) were taken for pretreatment. The samples were detected according to the LC-MS / MS detection conditions in Example 1. Three samples were used in parallel for each concentration level. The detection accuracy of different pretreatment methods was evaluated by the spike recovery method. The recovery rate of 85% to 115% was considered acceptable.

[0096] The recovery rates of oxalic acid, citric acid, cystine, and creatinine are shown in Tables 21 to 24.

[0097] Table 21

[0098] Table 22

[0099] Table 23

[0100] Table 24

[0101] It is evident that without acidifier 1, the recovery rates of oxalic acid and cystine are not within acceptable limits; with 2M hydrochloric acid as acidifier 1, the recovery rate of cystine is not within acceptable limits; and with 2M phosphoric acid as acidifier 1, the recovery rates of oxalic acid and cystine are not within acceptable limits.

[0102] 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 of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the concentration of organic matter in urine, characterized in that, include: The urine sample was acidified once by mixing it with sulfuric acid. The supernatant after acidification was mixed with derivatizing agent A, and then hydrochloric acid was added for a second acidification. After acidification, derivatizing agent B was added for derivatization. The derivatized supernatant was mixed with the sample dilution, centrifuged, and then detected by liquid chromatography-tandem mass spectrometry. The derivatizing agent A is a mixture of aniline and dimethyl sulfoxide; The derivatizing agent B is an aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; The sample diluent is an aqueous methanol solution containing ammonium formate; The organic compounds include creatinine, citric acid, oxalic acid, and cystine; The sulfuric acid is a 2M sulfuric acid solution; each 1 mL urine sample is mixed with (20~80) μL of 2M sulfuric acid solution; The hydrochloric acid is a 6M hydrochloric acid solution, and the volume ratio of the supernatant after acidification to the 6M hydrochloric acid solution is 1:(0.5~1.5). In the liquid chromatography-tandem mass spectrometry (LC-MS / MS) method, the chromatographic column was an Eclipse Plus C18 with dimensions of 3.5 μm and 3.0 × 100 mm. Mobile phase A consisted of a mixture of ammonium formate solution, formic acid, and water at a volume ratio of (1–3):(0.5–1.5):1000. Mobile phase B consisted of a mixture of ammonium formate solution, formic acid, and methanol at a volume ratio of (1–3):(0.5–1.5):1000. The chromatographic elution program was as follows: During the elution process, the sum of the volume percentages of mobile phase A and mobile phase B is 100%.

2. The detection method according to claim 1, characterized in that, The volume ratio of the acidified supernatant to the derivatizing agent A is 1:(1~5).

3. The detection method according to claim 1, characterized in that, In derivatizer A, aniline accounts for 10% to 30% of the volume of the mixture, and in derivatizer B, the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 100 to 500 mg / mL. And / or, the volume ratio of derivatizing agent A to derivatizing agent B is (1~3):

1.

4. The detection method according to claim 1, characterized in that, The sample diluent is a methanol-water solution containing 15-25 mM ammonium formate; And / or, the volume ratio of the derived supernatant to the sample diluent is 1:(5~15).

5. The detection method according to claim 1, characterized in that, The acidified supernatant is first mixed with an internal standard and then with derivatizing agent A; the internal standard includes creatinine internal standard, citrate internal standard, oxalate internal standard and cystine internal standard.

6. The detection method according to claim 1, characterized in that, The column temperature is 30-40℃; and / or the flow rates of mobile phase A and mobile phase B are 0.4-0.5 mL / min.

7. The detection method according to claim 1, characterized in that, In liquid chromatography-tandem mass spectrometry, the ion source parameters for mass spectrometry include: ESI+ ion source, curtain gas 35~45 psi, collision gas 5~10 psi, spray voltage 5300~5800 volts, temperature 430~480℃, and spray gas 50~60 psi.

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