Method and kit for simultaneously detecting uridine and uridine 5 '-monophosphate in serum by LC / MS-MS (Liquid Chromatography / Mass Spectrometry-Mass Spectrometry)

Abstract

The invention relates to the technical field of analysis and detection, in particular to a method and a kit for simultaneously detecting uridine and uridine 5 '-monophosphate in serum by LC / MS-MS (Liquid Chromatography / Mass Spectrometry-Mass Spectrometry). The method comprises the following steps: S1, mixing a detection sample with an internal standard, and then adding a protein precipitant with the pH value of 2.7-3.5 to obtain a first mixed solution; the protein precipitant is an acetonitrile solution with the pH value of 2.7 to 3.5; s2, sequentially carrying out vortex oscillation and centrifugation on the first mixed solution, and then sequentially carrying out nitrogen blowing and redissolving treatment on supernate obtained by centrifugation to obtain a second mixed solution; and S3, sequentially carrying out vortex oscillation and centrifugation on the second mixed solution, and carrying out on-machine detection on a supernatant obtained by centrifugation. According to the method, the uridine and the uridine 5 '-monophosphate in the human serum can be accurately, efficiently and simultaneously detected, and the requirements of related laws and regulations on linearity, repeatability, accuracy, stability, specificity and the like of uridine and uridine 5'-monophosphate detection in the human serum are met.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical detection, and particularly relates to a method and a kit for simultaneously detecting uridine and uridine 5'-monophosphate in serum by LC / MS-MS. Background Art

[0002] Pyrimidine metabolism is crucial for healthy somatic functions. Its intermediate metabolites are involved in the synthesis and decomposition of DNA and RNA, and are also important components of cellular energy metabolism and signal transduction. Uridine is a key precursor of pyrimidine metabolism and is a pyrimidine nucleoside essential for RNA synthesis. Uridine is involved in various physiological processes, including RNA synthesis, glycogen synthesis, and lipid deposition, etc. These factors act together to regulate the concentration of uridine and maintain a relatively stable state. Uridine is closely related to the homeostasis of the body and is involved in regulating the metabolism of glucose, lipids, amino acids, and other substances. Uridine 5'-monophosphate (UMP) is the central metabolite of pyrimidine metabolism and is also the precursor of all pyrimidine nucleotides. Pyrimidine nucleotides are essential substances for DNA and RNA synthesis and play a key role in protein glycosylation, lipid metabolism, polysaccharide biosynthesis, signal transduction, and proliferation, etc. Abnormalities in the uridine metabolic pathway can lead to various diseases, such as developmental epileptic encephalopathy type 50, Miller syndrome, and orotic aciduria. The main clinical features of developmental epileptic encephalopathy type 50 are global developmental delay, loss of skills, refractory epilepsy, brain atrophy, and dyserythropoietic anemia. Miller syndrome is a rare autosomal recessive postaxial acrofacial dysostosis syndrome, mainly characterized by micrognathia, orofacial cleft, cup-shaped ears, and hypoplasia of the zygomatic bone, accompanied by postaxial limb deformities, such as the absence of the fifth finger. The main clinical features of orotic aciduria are megaloblastic anemia, accompanied by a significant increase in the excretion of orotic acid in urine, and some patients are accompanied by immunodeficiency, developmental delay, and growth retardation. Since there is still no authoritative and clear method for measuring uridine and uridine 5'-monophosphate in serum, the diagnosis of related metabolic diseases still has great difficulties.

[0003] To better understand how pyrimidine metabolism changes are involved in the complex pathogenesis of metabolic diseases, there is an urgent need to accurately measure these metabolites to provide biochemical metabolic markers for related diseases. Considering the wide concentration range of different pyrimidine metabolites in the body, the sensitivity of the detector is required to reach the nanogram level. Therefore, mass spectrometry detectors have advantages over ultraviolet-visible detectors, evaporative light scattering detectors, and electrochemical detectors. Currently, high-performance liquid chromatography or gas chromatography equipped with an MS detector (including Q-TOF and triple quadrupole detectors) has good separation and sensitivity in monitoring purine metabolites in the body. However, whether the method is based on GC-MS, LC-MS / MS, or LC-Q-TOF / MS, most of these detection methods are relatively quantitative for both untargeted and targeted metabolomics. Although these relatively quantitative metabolomics methods are suitable for high-throughput preliminary screening, they do not reflect the actual concentrations of metabolites in the body. Summary of the Invention

[0004] To solve the above problems in the prior art, the present invention provides a pretreatment method, a detection method, and a kit for simultaneously detecting uridine and uridine 5'-monophosphate in serum by LC / MS-MS.

[0005] Based on this, the present invention has the following technical solutions: In the first aspect, the present invention provides a method for simultaneously detecting uridine and uridine 5'-monophosphate in serum by LC / MS-MS, including: S1: Mix the test sample with an internal standard, and then add a protein precipitant with a pH value of 2.7 to 3.5 to obtain a first mixed solution; the protein precipitant is an acetonitrile solution with a pH value of 2.7 to 3.5; S2: Vortex and centrifuge the first mixed solution in sequence, and then subject the supernatant obtained by centrifugation to nitrogen blowing and reconstitution treatments to obtain a second mixed solution; S3: Vortex and centrifuge the second mixed solution in sequence, and the supernatant obtained by centrifugation is the test sample.

[0006] The present invention discovers that when detecting uridine and uridine 5'-monophosphate in human serum by liquid chromatography-tandem mass spectrometry, adding acetonitrile within the above pH range to precipitate the protein in the serum, and concentrating the supernatant by nitrogen blowing can achieve the effect of efficiently extracting uridine and uridine 5'-monophosphate in the serum, thereby improving the detection sensitivity. Specifically, using conventional protein precipitants such as methanol or a mixture of methanol and acetonitrile cannot completely precipitate the protein, and the supernatant after centrifugation is still turbid to varying degrees.

[0007] In the present invention, the pH value of the protein precipitant is 2.7 to 3.5, specifically it can be any value among 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, etc. or a value range with any two of the above values as endpoints.

[0008] In the present invention, the internal standards for uridine and 5'-monophosphoric acid uridine can use the internal standards already disclosed in the art, preferably uridine-5,6-d2.

[0009] Preferably, the protein precipitant is an acetonitrile solution containing formic acid, that is, the pH value of the protein precipitant is adjusted to 2.7 to 3.5 using formic acid.

[0010] In the specific implementation process, those skilled in the art can adjust the appropriate formic acid concentration according to needs to obtain the pH value within the above range.

[0011] The present invention finds that when using formic acid to adjust the pH value of the protein precipitant, the precipitation effect of serum protein is better; as the content of formic acid increases and the pH value of the protein precipitant exceeds the above-defined range, the protein precipitation effect gradually becomes worse, and when the formic acid concentration exceeds 1 wt%, the protein does not precipitate at all.

[0012] Preferably, the volume ratio of the test sample to the protein precipitant is 1:(8 - 12).

[0013] Preferably, during liquid chromatography detection, the sample to be tested is gradient eluted using mobile phase A and mobile phase B; in mobile phase A, the solvent is water and the solutes are formic acid and ammonium formate; in mobile phase B, the solvents are acetonitrile and water and the solutes are formic acid and ammonium formate; wherein, the ammonium formate concentrations in mobile phase A and mobile phase B are the same.

[0014] The present invention finds that adding ammonium formate with the same concentration to mobile phase A and B makes the concentration of ammonium formate remain unchanged after the mixing of mobile phase A and B during gradient elution, which can better adjust the gradient setting and make the peak time of the target substance more stable.

[0015] Preferably, the concentration of ammonium formate in mobile phase A and mobile phase B is 5 - 15 mM, which can be any value among 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM or a value range with any two of the above values as endpoints.

[0016] Preferably, when the mass ratio of acetonitrile to water is (8 - 10):1, it helps to well match the properties of the test sample and the chromatographic column stationary phase, and at the same time helps to improve the detection efficiency.

[0017] Preferably, the concentration of formic acid in mobile phase A and mobile phase B is independently the same or different and is 0.01 wt% to 0.05 wt%. For example, it can be values such as 0.01 wt%, 0.02 wt%, 0.03 wt%, 0.04 wt%, or 0.05 wt%, or a numerical range with any two of these values as endpoints.

[0018] Preferably, the gradient elution conditions include:

[0019] where %, represents volume percentage, and the sum of the volume percentage of mobile phase A and the volume percentage of mobile phase B is 1.

[0020] More preferably, the gradient elution conditions include:

[0021] where %, represents volume percentage, and the sum of the volume percentage of mobile phase A and the volume percentage of mobile phase B is 1.

[0022] Preferably, the reconstitution solution is a mixed solution of mobile phase A and mobile phase B; the volume ratio of mobile phase A to mobile phase B is (2 - 4):1.

[0023] Preferably, the chromatographic column is a HILIC chromatographic column; preferably, the chromatographic column includes: Waters Atlantis Premier BEH Z - HILIC Column 2.5 µm 2.1x100 mm.

[0024] Preferably, the flow rate of mobile phase A and mobile phase B is 0.4 - 0.6 mL / min.

[0025] Preferably, the needle - washing solution during injection is an acetonitrile aqueous solution, preferably an acetonitrile aqueous solution of 45 - 55 wt%.

[0026] Preferably, the column temperature is 38 - 42 °C.

[0027] Preferably, the injector temperature is 8 - 12 °C.

[0028] Preferably, the injection volume is 3 - 7 µL.

[0029] Preferably, the mass spectrometry detection conditions include:

[0031] In a second aspect, the present invention provides a kit for implementing the above - mentioned detection method, including a sample to be tested, a protein precipitant, mobile phase A, and mobile phase B; The sample to be tested includes at least one of human serum, calibration product, and quality control product; The pH value of the protein precipitant is 2.7 - 3.5; preferably, the protein precipitant is an acetonitrile solution with a pH value of 2.7 - 3.5; more preferably, the protein precipitant is an acetonitrile solution containing formic acid; In mobile phase A, the solvent is water, and the solutes are formic acid and ammonium formate; in mobile phase B, the solvent is acetonitrile and water, and the solutes are formic acid and ammonium formate; wherein, the ammonium formate concentrations in mobile phase A and mobile phase B are the same; preferably, the ammonium formate concentration in mobile phase A and mobile phase B is 5 - 15 mM; preferably, the mass ratio of acetonitrile to water is (8 - 10):1; preferably, the formic acid concentrations in mobile phase A and mobile phase B are independently, identically or differently 0.01 wt% - 0.05 wt%.

[0032] Preferably, the concentration range of uridine in the calibration product is 20 - 10000 ng / mL; the concentration of uridine in the quality control product is 100 - 5000 ng / mL; And / or, the concentration range of uridine 5'-monophosphate in the standard product is 4 - 2000 ng / mL; the concentration range of uridine 5'-monophosphate in the quality control product is 20 - 1000 ng / mL.

[0033] In the present invention, since it is difficult to obtain human-derived samples, phosphate buffered saline (PBS) added with about 5% bovine serum albumin (BSA) is used as a substitute matrix to prepare each concentration point of the standard curve and quality control.

[0034] A method and kit for simultaneously detecting uridine and uridine 5'-monophosphate in serum by LC / MS-MS provided by the present invention can accurately and efficiently detect uridine and uridine 5'-monophosphate in human serum simultaneously. This method can meet the requirements of relevant regulations for the linearity, repeatability, accuracy, stability, specificity, etc. of the detection of uridine and uridine 5'-monophosphate in human serum, and plays an important role in monitoring uridine and uridine 5'-monophosphate in human serum. Description of the Drawings

[0035] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0036] Figure 1It is the test result of the calibration product containing alternative matrix under the Waters Atlantis Premier BEH Z-HILIC Column 2.5µm 2.1x100mm provided by the present invention; among them, the upper figure is the test result diagram of uridine 5′-monophosphate, the middle figure is the test result diagram of the isotopic internal standard of uridine, and the lower figure is the test result diagram of uridine.

[0037] Figure 2 It is the test result of the calibration product containing alternative matrix under the Waters XBridge BEH Amide 2.5um 100×2.1mm chromatographic column provided by the present invention; among them, the upper figure is the test result diagram of uridine 5′-monophosphate, the middle figure is the test result diagram of the isotopic internal standard of uridine, and the lower figure is the test result diagram of uridine. Detailed implementation manners

[0038] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0039] Unless otherwise specified, all kinds of raw materials used in the examples and comparative examples are commercially available conventional raw materials, and the technical means used are conventional means well known to those skilled in the art.

[0040] Example 1 A method and kit for simultaneously detecting uridine and uridine 5′-monophosphate in serum by LC / MS-MS provided by the present invention include: 1. Main reagents: Table 1

[0041] 2. Main equipment Table 2

[0042] 3. Preparation of uridine, uridine 5′-monophosphate standards and quality control products: 3.1 Preparation of concentrated stock solution (1) Accurately weigh 10 mg of purchased uridine (BePure, 26458-5g), add 2 mL (accurately pipetted) of 50% methanol aqueous solution, shake well to ensure complete dissolution, obtain a 5 mg / mL uridine concentrated stock solution, transfer it to a 1.5 mL brown vial, label it, and store it at -20 °C; (2) Accurately weigh 5 mg of purchased uridine 5′-monophosphate (BePure, 26442 - 100 mg), add 4 mL (accurately pipetted) of 50% methanol aqueous solution, and shake well to ensure complete dissolution to obtain a concentrated stock solution of uridine 5′-monophosphate at 1.25 mg / mL. Transfer it to a 1.5 mL brown vial, label it, and store it at -20 °C.

[0043] (3) The purchased uridine 5,6-[d2] (BePure, MD - 8140 - 10 mg) has an accurate mass. Without weighing, directly add 10 mL (accurately pipetted) of 50% methanol aqueous solution, shake well to ensure complete dissolution to obtain a concentrated stock solution of uridine 5,6-[d2] at 1 mg / mL. Transfer it to a 1.5 mL brown vial, label it, and store it at -20 °C; Finally, the following stock solutions are obtained: Table 3

[0044] 3.2 Preparation of alternative matrix: (1) Accurately pipette 1 pack of PBS particles into 2 L of ultrapure water and mix well to obtain a 10 mM phosphate buffer solution, and store it in a refrigerator at 2 - 8 °C; (2) Accurately pipette 100 mL of the 10 mM phosphate buffer solution into a 500 mL glass bottle, add 5 g of bovine serum albumin, and dissolve it thoroughly by ultrasound to finally obtain 100 mL of the alternative matrix H0 solution.

[0045] 3.3 Preparation of calibrators and quality control samples Table 4

[0046] The 10X calibrators C1 - C7, quality control samples QCL, QCH, and alternative matrix H0 are mixed in a ratio of 1:9 to obtain the final calibrators C1 - C7 and quality control samples QCL, QCH. The concentrations after preparation are as shown in the following table. After preparation, label them and store them at -20 °C: Table 5

[0047] 3.4 Preparation of mobile phase (1) Preparation of mobile phase A: Accurately weigh 0.3125 mg of ammonium formate, 0.25 mL of formic acid, and 500 mL of deionized water into a 500 mL reagent bottle, and ultrasonicate for 5 minutes, then set aside.

[0048] (2) Preparation of mobile phase B: Accurately weigh 0.63 mg of ammonium formate, 0.5 mL of formic acid, and 100 mL of water, transfer all to a 1000 mL reagent bottle, ultrasonicate for 5 minutes, then continue to add 900 mL of acetonitrile and ultrasonicate for 5 minutes before setting aside.

[0049] 4. This embodiment provides a method for detecting uridine and 5'-uridine monophosphate in human serum by liquid chromatography-tandem mass spectrometry, including the following pretreatment processes: 4.1 Preparation of pretreatment reagents: Taking the amount for processing 100 samples as an example, the following reagents need to be prepared: 1) 100 mL of 50% methanol-water: Measure 50 mL of methanol and 50 mL of ultrapure water with a graduated cylinder and pour them into a suitable container and mix evenly.

[0050] 2) 100 mL of acetonitrile: Measure 100 mL of HPLC-grade acetonitrile.

[0051] 3) 10 mL of reconstitution solution: Use a pipette to measure 7.6 mL of mobile phase B and 2.4 mL of mobile phase A respectively, and put them into a suitable container and mix evenly. (The ratio of the reconstitution solution is based on the actual ratio of the mobile phase).

[0052] 4.2 Preparation of test samples: 1) Protein precipitation: Add 50 μL of calibrator, quality control product, sample and 20 μL of internal standard to a 1 mL 96-well plate respectively, vortex for 1 min, and then add 500 μL of acetonitrile solution with a pH of 3 (adjust the pH to 3 using formic acid); vortex at 1500 rpm for 10 min, centrifuge at 3000 rpm for 10 min, and take 450 μL of the supernatant to another new 1 mL 96-well plate.

[0053] 2) Nitrogen blowing / reconstitution: Place a 1 mL 96-well U-shaped plate on a nitrogen evaporator, dry it with nitrogen at 45 °C, and then reconstitute it with 80 μL of the reconstitution solution.

[0054] 3) Vibration: Cover the 96-well plate pad, place it in a 96-well plate mixer, vibrate fully at 1500 rpm for 5 min, centrifuge at 3000 rpm for 3 min, and take 60 - 70 μL of the supernatant to another new 450 μL 96-well plate.

[0055] 4) Detection: Put the 96-well plate on the instrument for detection.

[0056] The liquid chromatography and mass spectrometry detection conditions include: Table 6

[0057] Table 7

[0058] 5. Linear range test data 5.1. Verification method: Process the calibration solutions C1 - C7 of the product to be tested according to the sample treatment method described for the determination of the contents of uridine and 5'-uridine monophosphate in human serum by high-throughput detection, and repeat the test 3 times for each concentration. The correlation coefficient r of linear regression can be calculated with reference to the formula, and the coefficient of determination R^2 should be ≥ 0.990.

[0059] r: Correlation coefficient of linear regression x i : Concentrations of C1 - C7 y i : Mean value of the peak area ratio of the calibrator to its internal standard in the solution of the corresponding concentration.

[0060] 5.2. Acceptance criteria: The correlation coefficient r of linear regression for both uridine and 5'-uridine monophosphate should be ≥ 0.990.

[0061] 5.3. Experimental results Table 8

[0062] 5.4. Conclusion: The correlation coefficient r of linear regression for both uridine and 5'-uridine monophosphate is ≥ 0.990, meeting the acceptance criteria.

[0063] 6. Repeatability test data 6.1. Verification method: Use the prepared quality control samples, and according to the liquid chromatography-tandem mass spectrometry method for the determination of the contents of uridine and 5'-uridine monophosphate in human serum, repeat the determination 10 times for each sample. The coefficient of variation (CV) of repeatability can be calculated with reference to the formula, with CV ≤ 20% for the low-value quality control sample and CV ≤ 15% for the high-value quality control sample.

[0064] CV = S / × 100% CV: Coefficient of variation of repeatability : Mean value of the 10 measurement results S: Standard deviation of the 10 measurement results.

[0065] 6.2. Acceptance criteria: The coefficient of variation CV of the low-value quality control sample is ≤ 20%, and the coefficient of variation CV of the high-value quality control sample is ≤ 15%.

[0066] 6.3. Experimental results Table 9

[0067] 6.4. Conclusion: The coefficient of variation CV of the low-value quality control sample is ≤ 20%, and the coefficient of variation CV of the high-value quality control sample is ≤ 15%, meeting the acceptance criteria.

[0068] 7. Precision Result Evaluation 7.1 Experimental Procedure Blood samples with three different concentrations of low, medium, and high were prepared by diluting / adding standard substances with actual human serum, such that the uridine contents in the low, medium, and high concentration mixed blood samples were 100 ng / mL, 1000 ng / mL, and 8000 ng / mL respectively; the 5′-uridine monophosphate contents were 20 ng / mL, 200 ng / mL, and 1600 ng / mL respectively. Continuously test for 5 days, with 5 replicates for each concentration each day, and calculate the average value, within-batch difference, and between-batch difference of the test contents for 5 days respectively.

[0069] 7.2 Experimental Results Table 10 Evaluation Results of Intra-batch and Inter-batch Precision of Uridine

[0070] Table 11 Evaluation Results of Intra-batch and Inter-batch Precision of 5′-Uridine Monophosphate

[0071] 7.3 Summary of the Experiment The above test results show that the RSDs of intra-batch and inter-batch precision of uridine and 5′-uridine monophosphate in blood samples are both ≤ 15%, and the evaluation of intra-batch and inter-batch precision meets the methodological requirements.

[0072] 8. Matrix Effect Result Evaluation 8.1 Experimental Procedure 1) Verification method: The ratio A of the peak area measured after pretreatment of 50 μL of blood sample to the peak area of the internal standard; the ratio B of the peak area measured after pretreatment of 50 μL of matrix-containing standard sample to the peak area of the internal standard; the ratio C of the peak area measured after pretreatment of the mixture of 25 μL of blood sample and 25 μL of matrix-containing standard sample.

[0073] 2) Acceptable standard: Matrix deviation (%) = (A + B) / 2C (%), matrix deviation ≤ ±20%.

[0074] 8.2 Experimental Results Table 12 Matrix Effect Results of Uridine

[0075] Table 13 Matrix Effect Results of 5′-Uridine Monophosphate

[0076] 8.3 Summary of the Experiment The above test results show that the matrix effects of uridine and 5′-uridine monophosphate in blood samples are both ≤ ±20%, and the evaluation of matrix effect meets the methodological requirements.

[0077] 9. Spike Recovery Data 9.1. Verification Method: 9.1.1. Background Testing of Serum Samples: Take 4 of 50 μL serum samples from two different mixed blood types respectively, and use the prepared calibration products to test the background contents of uridine and 5'-uridine monophosphate in the serum according to the liquid chromatography-tandem mass spectrometry method for detecting the contents of uridine and 5'-uridine monophosphate in human serum. Since the background of uridine is relatively high, dilution treatment is carried out.

[0078] 9.1.2. Preparation of Spiked Samples: Take 45 μL of the mixed serum sample, and add uridine at three concentration levels of 100 ng / mL, 1000 ng / mL, and 8000 ng / mL and 5'-uridine monophosphate at concentrations of 20 ng / mL, 200 ng / mL, and 1600 ng / mL respectively, for use.

[0079] 9.1.3. Use the prepared calibration products to test the spiked serum samples according to the liquid chromatography-tandem mass spectrometry method for detecting the contents of uridine and 5'-uridine monophosphate in human serum.

[0080] 9.2. Acceptance Criteria: Spike recovery rate is 80% - 120%.

[0081] 9.3. The test results are shown in the table. The spike recovery rates of uridine are 93.5% - 102.3%, and the recovery rates of 5'-uridine monophosphate are 90.1% - 103.5%, both within the range and meeting the requirements.

[0082] Table 14. Accuracy Performance Evaluation of Uridine

[0083] Table 15. Accuracy Performance Evaluation of 5'-Uridine Monophosphate

[0084] 9.4. Conclusion: The spike recovery rates of uridine and 5'-uridine monophosphate are both within the range and meet the requirements.

[0085] Figure 1 The test results are for the Waters Atlantis Premier BEH Z-HILIC Column 2.5 µm 2.1x100 mm chromatographic column.

[0086] In this example, the liquid chromatography-tandem mass spectrometry method was used to determine the contents of uridine and 5'-uridine monophosphate in human serum. The total detection time was 7.5 min. The peak time of uridine was 1.02 min, and the peak time of 5'-uridine monophosphate was 3.5 min. The detection time was relatively short. A Waters Xevo TQS triple quadrupole mass spectrometer was used for detection. Although the sensitivity of the equipment was relatively low, the linear range of uridine in human serum was 20 - 10,000 ng / mL, and the linear range of 5'-uridine monophosphate was 4 - 2,000 ng / mL. The lowest linear point of uridine, 20 ng / mL, was lower than the lowest linear point of 2 ng / mL in the comparative example. Moreover, the content of uridine in human serum was generally in the range of 500 - 1,500 ng / mL, which had no effect on the quantification of uridine. At the same time, this method had the characteristics of simple operation, short nitrogen blowing time, high repeatability and accuracy. The linear range of uridine in this method was 20 - 10,000 ng / mL, and the linear range of 5'-uridine monophosphate was 4 - 2,000 ng / mL, and the correlation coefficient r > 0.990. The coefficient of variation (CV) of the repeatability of the low-value quality control product was ≤ 20%, and the coefficient of variation (CV) of the repeatability of the high-value quality control product was ≤ 15%. The relative deviation (B) of the accuracy was ≤ ±15%, and the spike recovery rate was 85% - 115%.

[0087] Example 2 This example provides a method for determining uridine and 5'-uridine monophosphate in human serum by liquid chromatography-tandem mass spectrometry. The difference from Example 1 is only that: formic acid in the protein precipitant is replaced with acetic acid, and the pH value remains unchanged. The results show that the protein precipitation effect is basically the same as that in Example 1.

[0088] Example 3 This example provides a method for determining uridine and 5'-uridine monophosphate in human serum by liquid chromatography-tandem mass spectrometry. The difference from Example 1 is only that: the pH value of the protein precipitant is adjusted to 2.88 with formic acid. The results show that the protein precipitation effect is basically the same as that in Example 1.

[0089] Example 4 This example provides a method for determining uridine and 5'-uridine monophosphate in human serum by liquid chromatography-tandem mass spectrometry. The difference from Example 1 is only that: in the protein precipitant, acetonitrile is replaced with a mixed solution of equal amounts of acetonitrile and methanol, and the volume ratio of acetonitrile to methanol is 1:1.

[0090] The results show that compared with Examples 1 - 3, the protein precipitation is incomplete, and the supernatant is turbid after shaking and centrifugation, indicating that the protein precipitation effect is worse than that in Example 1.

[0091] Example 5 This example provides a method for determining uridine and 5'-uridine monophosphate in human serum by liquid chromatography-tandem mass spectrometry. The difference from Example 1 is only that mobile phase A is replaced with an aqueous solution of 0.05% formic acid, and mobile phase B is replaced with an acetonitrile solution of 0.05% formic acid.

[0092] The results show that the elution time of the target compound fluctuates each time after the replacement of the mobile phase and is not fixed. This indicates that removing ammonium formate from the mobile phase affects the stability of the elution time of the target compound.

[0093] Example 6 This example provides a method for determining uridine and 5'-uridine monophosphate in human serum by liquid chromatography-tandem mass spectrometry. The difference from Example 1 is only that the ammonium formate concentrations in mobile phase A and mobile phase B are different. Specifically, the ammonium formate concentration in mobile phase A is 5 mM, and the ammonium formate concentration in mobile phase B is 5 mM.

[0094] It is found that reducing the content of ammonium formate in the mobile phase also affects the elution time of the target compound. Specifically, the elution time of the target compound fluctuates slightly each time and does not elute at a specific fixed time.

[0095] Example 7 This example provides a method for determining uridine and 5'-uridine monophosphate in human serum by liquid chromatography-tandem mass spectrometry. The difference from Example 1 is only that the chromatographic column is replaced with Waters XBridge BEH Amide 2.5um 100×2.1mm.

[0096] The results show that when using the Waters XBridge BEH Amide 2.5um 100×2.1mm column under gradient conditions, the peak of 5'-uridine monophosphate will split.

[0097] Figure 2 Test results for Waters XBridge BEH Amide 2.5um 100×2.1mm chromatographic column.

[0098] Comparative Example 1 This comparative example provides a method for determining uridine and 5'-uridine monophosphate in human serum by liquid chromatography-tandem mass spectrometry. The difference from Example 1 is only that in the protein precipitant, acetonitrile is replaced with an equal amount of methanol.

[0099] The results show that methanol cannot completely precipitate proteins, and the supernatant will still show a certain degree of turbidity after shaking and centrifugation.

[0100] Comparative Example 2 This comparative example provides a method for determining uridine and uridine 5'-monophosphate in human serum by liquid chromatography-tandem mass spectrometry. The difference from Example 1 is that the pH value of the protein precipitant is different.

[0101] The pH value of the protein precipitant is 2.58: It was detected that the sample to be measured changed, and uridine 5'-monophosphate was hydrolyzed with a decreased content, indicating that excessive addition of formic acid would affect the experimental results.

[0102] The pH value of the protein precipitant is 2.38: It was detected that the sample to be measured changed, and uridine 5'-monophosphate was hydrolyzed with a decreased content, and it was more obvious than when the pH value of the protein precipitant was 2.58, indicating that excessive addition of formic acid would affect the experimental results.

[0103] The pH value of the protein precipitant is 2.21: It was detected that the sample to be measured changed, and uridine 5'-monophosphate was hydrolyzed with a decreased content, and it was more obvious than when the pH value of the protein precipitant was 2.38, indicating that excessive addition of formic acid would affect the experimental results.

[0104] The pH value of the protein precipitant is 4: It was detected that the protein precipitation effect was worse than that in Example 1, and there was still a relatively light turbid state in the supernatant after shaking and centrifugation.

[0105] Comparative Example 3 This comparative example uses a method for quantitative analysis of 20 purine and pyrimidine metabolites in the serum and hippocampus of depressed mice by HILIC-MS / MS disclosed in the literature "Quantitative analysis of 20 purine and pyrimidine metabolites by HILIC-MS / MS in the serum and hippocampus of depressed mice" (Journal of Pharmaceutical and Biomedical Analysis), and the detection is carried out by liquid chromatography-tandem mass spectrometry. The total detection time is 14 min. Among them, the peak time of uridine is 6.74 min, and the peak time of uridine 5'-monophosphate is 7.54 min, and the detection time is relatively long. In addition, it uses an AB SCIEX 6500 triple quadrupole mass spectrometer for detection. This device has high sensitivity. The linear range of uridine in mouse serum is 2 - 5000 ng / mL, and the linear range of uridine 5'-monophosphate is 10 - 200 ng / mL.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for simultaneously detecting uridine and 5'-monophosphate uridine in serum by LC / MS-MS, characterized in that: include: S1: Mix the test sample and the internal standard, and then add a protein precipitant with a pH value of 2.7 to 3.5 to obtain a first mixed solution; the protein precipitant is an acetonitrile solution with a pH value of 2.7 to 3.5; S2: vortexing and centrifuging the first mixed solution in sequence, and then nitrogen blowing and re-dissolving the supernatant obtained by centrifugation in sequence to obtain a second mixed solution; S3: vortexing and centrifuging the second mixed solution in sequence, and testing the supernatant obtained by centrifugation.

2. The method for simultaneously detecting uridine and 5'-monophosphate uridine in serum by LC / MS-MS according to claim 1, characterized in that: The protein precipitant is an acetonitrile solution containing formic acid.

3. The method for simultaneously detecting uridine and 5'-monophosphate uridine in serum by LC / MS-MS according to claim 1 or 2, characterized in that: The volume ratio of the test sample to the protein precipitant is 1:(8-12).

4. The method for simultaneously detecting uridine and 5'-monophosphate uridine in serum by LC / MS-MS according to any one of claims 1 to 3, characterized in that: During liquid chromatography detection, mobile phase A and mobile phase B are used to perform gradient elution on the sample to be tested; in mobile phase A, the solvent is water, and the solutes are formic acid and ammonium formate; in mobile phase B, the solvent is acetonitrile and water, and the solutes are formic acid and ammonium formate; wherein the concentrations of ammonium formate in mobile phase A and mobile phase B are the same; Preferably, the concentration of ammonium formate in the mobile phase A and the mobile phase B is 5-15 mM; more preferably, the mass ratio of acetonitrile to water is (8-10):1; And / or, the concentration of formic acid in the mobile phase A and in the mobile phase B is independently, the same or different from 0.01 wt % to 0.05 wt %.

5. The method for simultaneously detecting uridine and 5'-monophosphate uridine in serum by LC-MS / MS according to any one of claims 1 to 4, characterized in that: The gradient elution conditions include: Wherein, % represents volume percentage, and the sum of the volume percentage of mobile phase A and the volume percentage of mobile phase B is 1.

6. The method for simultaneously detecting uridine and 5'-monophosphate uridine in serum by LC-MS / MS according to any one of claims 1 to 5, characterized in that: The reconstituted solution is a mixed solution of mobile phase A and mobile phase B; the volume ratio of mobile phase A to mobile phase B is (2-4):

1.

7. The method for simultaneously detecting uridine and 5'-monophosphate uridine in serum by LC-MS / MS according to any one of claims 1 to 6, characterized in that: The chromatographic column is a HILIC chromatographic column; preferably, the chromatographic column comprises: Waters Atlantis PremierBEH Z-HILIC Column.

8. The method for simultaneously detecting uridine and 5'-monophosphate uridine in serum by LC-MS / MS according to any one of claims 1 to 7, characterized in that: Mass spectrometry detection conditions include: 。 9. A kit for implementing the method according to any one of claims 1 to 8, characterized in that: It includes a sample to be tested, a protein precipitant, a mobile phase A and a mobile phase B; The sample to be tested includes at least one of human serum, calibrator and quality control product; The pH value of the protein precipitant is 2.7-3.5; the protein precipitant is an acetonitrile solution with a pH value of 2.7-3.5; preferably, the protein precipitant is an acetonitrile solution containing formic acid; In the mobile phase A, the solvent is water, and the solutes are formic acid and ammonium formate; in the mobile phase B, the solvent is acetonitrile and water, and the solutes are formic acid and ammonium formate; wherein the concentrations of ammonium formate in the mobile phase A and the mobile phase B are the same; preferably, the concentrations of ammonium formate in the mobile phase A and the mobile phase B are 5-15 mM; preferably, the mass ratio of acetonitrile to water is (8-10):1; preferably, the concentrations of formic acid in the mobile phase A and the mobile phase B are independently, identically or differently, 0.01 wt%-0.05 wt%.

10. The kit according to claim 9, characterized in that The concentration range of uridine in the calibration product is 20-10000 ng / mL; the concentration of uridine in the quality control product is 100-5000 ng / mL; And / or, the concentration range of 5'-uridine monophosphate in the standard is 4-2000 ng / mL; the concentration range of 5'-uridine monophosphate in the quality control is 20-1000 ng / mL.

Images