Method for detecting urinary oxalic acid by adopting HPLC-ESI-MS / MS technology

Abstract

The invention provides a method for detecting oxalic acid in urine by adopting an HPLC-ESI-MS / MS. The method comprises the following steps: S1, taking a collected sample, adding oxalic acid-1, 2-13C as an internal standard of oxalic acid, mixing an internal standard solution and the collected sample according to a ratio of 2: 1, performing vortex for 5 minutes, and performing centrifugation for 5 minutes at 4000 rpm to obtain a urine sample; s2, detecting the sample by using a liquid chromatography tandem mass spectrometer; s3, adopting stable isotope labeled oxalic acid-1, 2-13C as an internal standard of oxalic acid, preparing a series of standard substances with different concentrations, making a standard curve, taking the concentration of an oxalic acid working standard solution as an X axis, taking a peak area ratio of the standard to the internal standard of a read result as a Y axis, carrying out linear regression analysis, and obtaining a regression equation through '1 / x2' weight; and substituting the area ratio of the oxalic acid in the sample to the internal standard peak into the standard curve equation, and calculating the concentration of the oxalic acid in the urine sample. According to the method, derivatization, solid-phase extraction, acidification and other operations are not needed, the analysis technology is greatly simplified, the detection time is shortened, and the sample stability is improved.

Description

Technical Field

[0001] The invention relates to the technical field of biochemistry, and in particular to a method for detecting urine oxalic acid by adopting HPLC-ESI-MS / MS technology. Background Art

[0002] Oxalic acid is a highly polar small molecule acidic substance. From a technical point of view, the determination of urine oxalic acid (UOx) is challenging. Internationally, the analysis technology of urine oxalic acid concentration has always been one of the hot topics in the field of clinical chemistry. A reliable analytical method is a necessary condition for implementing valuable urine oxalic acid concentration monitoring.

[0003] In recent years, various methods for detecting urine oxalic acid have been reported, including enzymatic methods, HPLC, LC-MS / MS, GC-MS, ion chromatography, colorimetry, fluorescence, electrochemical methods, etc. However, these methods have some disadvantages, such as low specificity of enzyme immunoassay and expensive kits; poor practicality of gas chromatography, long time consumption, and the need for complex pre-column derivatization; and some other methods have the problems of requiring tedious sample pretreatment (such as acidification), low efficiency of liquid-liquid extraction and solid-phase extraction, large consumption of biological samples (≥100 μL), and low accuracy. In addition, the urine samples measured by these methods are mostly 24-hour urine samples, which are not suitable for patients who are inconvenient or difficult to collect 24-hour urine samples. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a method for detecting urine oxalic acid using HPLC-ESI-MS / MS technology.

[0005] The technical solution adopted by the present invention to solve its technical problem is: A method for detecting urine oxalic acid using HPLC-ESI-MS / MS technology comprises the following steps: S1. Take the collected sample and add 1,2-oxalic acid 13 C was used as the internal standard of oxalic acid. The internal standard solution was mixed with the collected sample at a ratio of 2:1, vortexed for 5 min, and centrifuged at 4000 rpm for 5 min to obtain the urine sample; S2. Sample detection is performed using a liquid chromatography tandem mass spectrometer; the detection conditions of the liquid chromatography are: Liquid chromatography system high pressure pump A mobile phase: 0.01% by volume ammonia-1mM ammonium acetate-ultrapure water solution; Liquid chromatography system high pressure pump B mobile phase: 0.01% by volume ammonia-1mM ammonium acetate-acetonitrile solution; The chromatographic conditions were as follows: the column was a Waters Symmetry C18 column, 3.5 µm, 4.6 mm × 75 mm, column temperature 40 °C; detection mode MRM, ion source temperature 500 °C; flow rate 0.4 ml / min; gradient elution program: 70% by volume of mobile phase A + 30% by volume of mobile phase B, isocratic run for 3 min; injection volume 5 µL; needle wash mode: needle wash for 5 s before and after injection; The mass spectrometry conditions were: curtain gas 35 psi, impingement gas 4 psi, ionization voltage 4000 V, ion source temperature 500 °C, nebulizer gas 50 psi, auxiliary heating gas 50 psi; S3, using stable isotope labeled oxalic acid-1,2- 13 C was used as the internal standard of oxalic acid. A series of standards with different concentrations were prepared to make a standard curve. The concentration of the oxalic acid working standard solution was taken as the X-axis, and the peak area ratio of the standard and internal standard was taken as the Y-axis. Linear regression analysis was performed. 2 The regression equation was obtained by substituted the peak area ratio of oxalic acid in the sample to its internal standard into the standard curve equation to calculate the oxalic acid concentration in the urine sample.

[0006] Preferably, the mass spectrometry conditions in step S1 include oxalic acid and its isotopes oxalic acid-1,2- 13 The electrical parameter conditions of C are as follows: Oxalic acid: parent ion 88.9Da, daughter ion 60.8Da, scan time 200 msec, declustering voltage 40V, entrance voltage 7V, collision energy 10V, collision cell exit voltage 15V; Oxalic acid-1,2- 13 C: parent ion 90.8Da, product ion 61.8Da, scan time 200 msec, declustering voltage 14V, inlet voltage 2V, collision energy 10V, collision cell outlet voltage 4V.

[0007] Preferably, the components and contents of the relevant reagents in step S2 are: Needle washing solution: 40% by volume methanol, 40% by volume acetonitrile, 10% by volume isopropanol, and 10% by volume ultrapure water; Internal standard solution: dissolve 1,2-oxalic acid in methanol 13 C, prepare a 1.00 mg / mL internal standard stock solution, and then dilute it to 200 ng / mL with acetonitrile containing 0.3% ammonia by volume; Calibration curve solution: dissolve oxalic acid in methanol to prepare a 1.00 mg / mL oxalic acid stock solution; dilute the stock solution with methanol: water at a volume ratio of 1:1 to obtain oxalic acid working standard solutions with concentrations of 0.2, 0.5, 1, 5, 50, 50, 100, 250 and 500 μg / mL, respectively; use ultrapure water to dilute the oxalic acid working standard solution to prepare standard curve solutions with concentrations of 0.020, 0.050, 0.100, 0.500, 10.0, 5.0, 10.0, 25.0, 50.0 μg / mL, respectively; Quality control solutions QCs: First, dilute the 1.00 mg / mL oxalic acid stock solution with methanol water at a volume ratio of 1:1 to obtain oxalic acid quality control working standard solutions with concentrations of 0.2, 1, 50, and 400 µg / mL, respectively. Use ultrapure water to dilute the oxalic acid quality control working standard solution to obtain quality control samples with concentrations of 0.020, 0.100, 5, and 40.0 µg / mL, respectively; Calibration curve samples and quality control samples: 200 μL of precipitant was mixed with 100 μL of calibration curve solution and quality control solution QCs, respectively. After vortexing for 5 min, the mixture was centrifuged at 4000 rpm for 5 min to obtain calibration curve samples and quality control samples, respectively.

[0008] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention does not require derivatization, solid phase extraction, and acidification operations, which greatly simplifies the analysis technology, shortens the detection time, and improves the stability of the sample; 2. The present invention can detect urine samples including but not limited to 24h. When it is inconvenient or difficult to collect 24h urine samples, the oxalic acid content in the sample can be calculated by combining the detection data of urine creatinine with the detection method of the present invention, which increases the application scenarios of the present invention; 3. 0.01% NH3∙H2O and 1mM NH4AC were used as additives to facilitate mass spectrometry ionization of oxalic acid; 4. Choose oxalic acid-1,2- 13 C is the internal standard, which has exactly the same structure as the analyte, thus eliminating the matrix effect and making the results more accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The graphs are related to the optimization of mass spectrometry conditions.

[0010] Figure 2 The chromatograms involved in screening columns and mobile phases.

[0011] Figure 3 Figure 1 is a graph involving screening of internal standards, flow rate, column temperature, alternative matrices, and optimization of sample preparation methods.

[0012] Figure 4 Representative chromatograms of oxalic acid and its internal standard in this study.

[0013] Figure 5 For supplementary atlas. DETAILED DESCRIPTION

[0014] The technical scheme of the present invention is described below in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The experimental methods and detection methods described in the embodiments are conventional methods unless otherwise specified. The reagents and materials, unless otherwise specified, can be purchased on the market.

[0015] Embodiment 1: This embodiment provides a method for detecting urine oxalic acid using HPLC-ESI-MS / MS technology, comprising the following steps: S1. Take the collected sample and add 1,2-oxalic acid 13 C was used as the internal standard of oxalic acid. The internal standard solution was mixed with the collected sample at a ratio of 2:1, vortexed for 5 min, and centrifuged at 4000 rpm for 5 min to obtain the urine sample; S2. Sample detection is performed using a liquid chromatography tandem mass spectrometer; the detection conditions of the liquid chromatography are: Liquid chromatography system high pressure pump A mobile phase: 0.01% by volume ammonia-1mM ammonium acetate-ultrapure water solution; Liquid chromatography system high pressure pump B mobile phase: 0.01% by volume ammonia-1mM ammonium acetate-acetonitrile solution; The chromatographic conditions were as follows: the column was a Waters Symmetry C18 column, 3.5 µm, 4.6 mm × 75 mm, column temperature 40 °C; detection mode MRM, ion source temperature 500 °C; flow rate 0.4 ml / min; gradient elution program: 70% by volume of mobile phase A + 30% by volume of mobile phase B, isocratic run for 3 min; injection volume 5 µL; needle wash mode: needle wash for 5 s before and after injection; The mass spectrometry conditions were as follows: curtain gas 35 psi, impingement gas 4 psi, ionization voltage 4000 V, ion source temperature 500 °C, spray gas 50 psi, and auxiliary heating gas 50 psi.

[0016] S3, using stable isotope labeled oxalic acid-1,2- 13 C was used as the internal standard of oxalic acid. A series of standards with different concentrations were prepared to make a standard curve. The concentration of the oxalic acid working standard solution was taken as the X-axis, and the peak area ratio of the standard and internal standard was taken as the Y-axis. Linear regression analysis was performed. 2The regression equation was obtained by substituted the peak area ratio of oxalic acid in the sample to its internal standard into the standard curve equation to calculate the oxalic acid concentration in the urine sample.

[0017] The mass spectrometry conditions in step S1 include oxalic acid and its isotopes oxalic acid-1,2- 13 The electrical parameter conditions of C are as follows: Oxalic acid: parent ion 88.9Da, daughter ion 60.8Da, scan time 200 msec, declustering voltage 40V, entrance voltage 7V, collision energy 10V, collision cell exit voltage 15V; Oxalic acid-1,2- 13 C: parent ion 90.8Da, product ion 61.8Da, scan time 200 msec, declustering voltage 14V, inlet voltage 2V, collision energy 10V, collision cell outlet voltage 4V.

[0018] The components and contents of the relevant reagents in step S2 are: Needle washing solution: Pipette 400 mL of methanol, 400 mL of acetonitrile, 100 mL of isopropanol and 100 mL of water, mix well and set aside.

[0019] Internal standard solution: dissolve 1,2-oxalic acid in methanol 13 C, prepare a 1.00 mg / mL internal standard stock solution, and then dilute it to 200 ng / mL with acetonitrile containing 0.3% ammonia by volume.

[0020] Calibration curve solution: dissolve oxalic acid in methanol to prepare a 1.00 mg / mL oxalic acid stock solution; dilute the stock solution with methanol: water at a volume ratio of 1:1 to obtain oxalic acid working standard solutions with concentrations of 0.2, 0.5, 1, 5, 50, 50, 100, 250 and 500 μg / mL, respectively; use ultrapure water to dilute the oxalic acid working standard solution to prepare standard curve solutions with concentrations of 0.020, 0.050, 0.100, 0.500, 10.0, 5.0, 10.0, 25.0, 50.0 μg / mL, respectively; Quality control solutions QCs: First, dilute the 1.00 mg / mL oxalic acid stock solution with methanol water at a volume ratio of 1:1 to obtain oxalic acid quality control working standard solutions with concentrations of 0.2, 1, 50, and 400 µg / mL, respectively. Use ultrapure water to dilute the oxalic acid quality control working standard solution to obtain quality control samples with concentrations of 0.020, 0.100, 5, and 40.0 µg / mL, respectively.

[0021] Calibration curve samples and quality control samples: 200 μL of precipitant was mixed with 100 μL of calibration curve solution and quality control solution QCs, respectively. After vortexing for 5 min, the mixture was centrifuged at 4000 rpm for 5 min to obtain calibration curve samples and quality control samples, respectively.

[0022] Example 2: Optimization of mass spectrometry conditions The present invention tests and compares the two modes of ESI (+) and ESI (-) respectively to obtain satisfactory mass spectrometry conditions for the analyte and the internal standard. Due to the lack of product ions in the ESI positive mode, the MRM scan in the negative ion mode was finally adopted. According to the mass-to-charge ratio m / z of the parent ion and the fragment information of the daughter ion, the present invention analyzes two MRM transitions of oxalic acid, namely 88.9 / 60.8 and 88.9 / 43.0. At the same time, oxalic acid-1,2- 13 C (stable isotope internal standard) through two MRM transitions, namely 90.8 / 61.8 and 90.8 / 45.0. Then, the compound-related ionization parameters (DP, EP, CE and CXP) and source-related parameters (CAD, CUR, GS1, GS2, IS, TEM) were optimized to obtain better response of the target analytes. The mass spectral information of oxalic acid and its stable isotope internal standard, Figure 1 The fragment intensity information of the specific protonated molecular ion [M+H]+ as the precursor ion and the major product ion acquired by MRM is shown. Figure 1 A shows the fragment ions of oxalic acid. Figure 1 B shows oxalic acid-1,2- 13 Fragment ions of C (internal standard, IS). Figure 1 C shows the chromatographic peaks for each ion transition of oxalic acid (tR = 1.12 min). Figure 1 D shows oxalic acid-1,2- 13 Chromatographic peaks for each ion transition in C (internal standard, IS) (tR = 1.12 min). Figure 1 E shows the MRM transitions and mass spectrometry parameters used for quantification of oxalic acid and its internal standard.

[0023] Example 3: Screening of chromatographic columns Nine different chromatography columns were screened for evaluation in the present invention. Includes: Kinetex C8 100 Å (2.1 mm × 50 mm, 5 µm), Kinetex C18 100 Å (2.1 mm × 50 mm, 1.7 µm), Kinetex C18 100 Å (2.1 mm × 50 mm, 2.6 µm), Kinetex C18 100 Å (2.1 mm × 50 mm, 5 µm), Kinetex C18 100 Å (2.1 mm × 100 mm, 2.6 µm), Luna C18(2) 100 Å (2 mm × 50 mm, 5 µm), Gemini C18110 Å (2 mm × 50 mm, 3 µm), SynergiTM Fusion-RP 80 Å (2 mm × 50 mm, 4 µm), Waters Symmetry C18 (4.6mm × 75 mm, 3.5 µm). Figure 2 A shows the chromatograms of oxalic acid on different chromatographic columns. The results show that oxalic acid can be retained on the chromatographic column Waters Symmetry C18 (4.6mm × 75 mm, 3.5 µm), but is not retained on other chromatographic columns and all peaks are eluted in the dead volume.

[0024] Example 4: Optimization of mobile phase and additives The properties and proportions of mobile phase and additives are important factors affecting chromatographic separation. The types of additives screened in this study include formic acid (FA), acetic acid (AA), ammonium acetate (NH4AC) and ammonia (NH3∙H2O), and the types of mobile phase systems screened include methanol and acetonitrile.

[0025] When acidic additives and any concentration of NH4AC are present in the mobile phase MeOH or ACN system: Figure 2 B represents the MRM chromatogram of the double blank sample. Figure 2 C represents the MRM chromatogram of oxalic acid when the concentration of NH4AC is lower than 5mM. Figure 2 D represents the MRM chromatogram of oxalic acid when the concentration of NH4AC is 5 mM.

[0026] When the mobile phase of the acetonitrile system contains only NH4AC as an additive: Figure 2 E represents the MRM chromatogram of the double blank sample (a sample containing blank matrix without oxalic acid or internal standard). Figure 2 F represents the MRM chromatogram of oxalic acid, Figure 2 G represents the MRM chromatogram of a series of oxalic acid concentrations when the concentration of NH4AC is 1 mM. Figure 2 H represents the MRM chromatogram of different concentrations of NH4AC in the mobile phase of acetonitrile system and the same concentration of oxalic acid.

[0027] When the mobile phase of the acetonitrile system contains only NH3∙H2O as an additive: Figure 2 I represents the MRM chromatogram of the double blank sample, Figure 2 J represents the MRM chromatogram of oxalic acid concentration of 40 μg / mL. Figure 2 K represents the MRM chromatogram at an oxalic acid concentration of 50 μg / mL.

[0028] When NH3∙H2O and NH4AC are used as additives in the mobile phase of the acetonitrile system: Figure 2 L represents the MRM chromatogram of the double blank sample, Figure 2 M represents the MRM chromatogram with an oxalic acid concentration of 40 μg / mL. Figure 2 N represents the MRM chromatogram at an oxalic acid concentration of 50 μg / mL.

[0029] Figure 2 O represents the line graph of the oxalic acid chromatographic peak height (blue) and retention time (orange) when the proportion of organic phase B is changed.

[0030] Firstly, methanol was used as the mobile phase system, and different ratios of FA (0.008, 0.1, and 5 mM) and / or NH4AC (0, 0.05, 1, and 5 mM) were selected as mobile phase additives. Figure 2 The results of C-2D showed that in the methanol system, as long as the acidic additive FA was present, the MRM channel 88.9 / 60.8 of oxalic acid had no chromatographic peak (when the concentration of ammonium acetate was lower than 5 mM) ( Figure 2 C), or the peak is not formed (when the concentration of ammonium acetate is 5mM) ( Figure 2 D). Figure 2 The results of B-2D showed that in the 88.9 / 43 channel, there were higher peaks in both the oxalic acid standard sample and the double blank sample. In order to eliminate the possibility of contamination or residue in the double blank sample, the double blank sample was replaced with solvents such as water / methanol / ACN / 50% methanol / 50% ACN (v / v), and the needle wash procedure was changed to 5s before and after injection. The results showed that the above phenomenon did not change. Therefore, ACN was considered as the mobile phase system for further optimization.

[0031] The ACN system was used as the mobile phase, and different proportions of FA (0, 1 mM), AA (0.01, 0.0.05 mM) and / or NH4AC (0, 0.5, 1, 2, 5 mM) were selected as mobile phase additives. Figure 2The results of B-2D showed that in the acetonitrile system, as long as the acidic additive was present, the MRM channel 88.9 / 60.8 of oxalic acid had no peak or the peak was not formed, while in the 88.9 / 43 channel, the oxalic acid standard sample ( Figure 2 C and 2D) and double blank samples ( Figure 2 B) have chromatographic peaks. The difference is that when the mobile phase of the acetonitrile system contains only NH4AC as an additive, the 88.9 / 60.8 channel has a good peak shape, while the 88.9 / 43 channel has no peak shape ( Figure 2 F). In addition, no interference peaks appeared in either channel of the double blank sample ( Figure 2 E). It can be seen that the additive NH4AC and ACN system is more conducive to the peak of oxalic acid 88.9 / 60.8 channel (convenient for quantitative analysis channel), and can avoid channel interference of double blank samples. However, when preparing the standard curve of pure oxalic acid solution (continuous concentrations of 0.040, 0.200, 1.0, 10.0, 20.0, 50.0 and 50.0 μg / mL), using ultrapure water containing 1mM NH4AC and acetonitrile containing 1mM NH4AC as the mobile phase, it was found that there was an inverted peak before the main peak, which affected the peak shape at low concentration points, and the main peak at high concentration points was tailing, and the standard curve was not linear ( Figure 2 G). Further optimization was performed by changing the concentration of NH4AC (1, 1.5, 2, 3, 3.5, 4, 4.5 and 5 mM). It was found that when the addition amount of NH4AC was 3.5 mM, the oxalic acid peak width was the narrowest, which improved the tailing problem at high concentration points ( Figure 2 H). However, this phenomenon could not be reproduced, which indicates that the method using NH4AC as an additive has poor reproducibility and is unstable, and needs further optimization.

[0032] Considering the alkalinity and volatility of aqueous ammonia, aqueous ammonia was added as an additive to the mobile phase of the ACN system for further optimization. Figure 2 The results of I-2K showed that when ultrapure water (UPW) and ACN were used as mobile phases without any additives, the oxalic acid peak was tailing. After adding ammonia, the peak width of oxalate became narrower ( Figure 2 J and 2K). There are no interfering peaks in the double blank samples ( Figure 2 I). However, at low concentrations, there is an interference peak behind the main peak of oxalic acid, while at high concentrations, the oxalic acid peak is tailed ( Figure 2 J and 2K). The noise baseline of the mobile phase additive with 0.01% NH3∙H2O (v / v) is lower than that of the mobile phase additive with 0.005% NH3∙H2O (v / v) ( Figure 2 K).

[0033] Using the ACN system as the mobile phase, different proportions of NH3∙H2O (0.005%, 0.01%) (v / v) and NH4AC (0, 0.1, 0.2, 0.5, 1, 2 and 5 mM) were selected as mobile phase additives. After adding NH4AC to the mobile phase containing ammonia water, the peak shape was improved. With the increase of NH4AC concentration, the peak height of oxalic acid chromatographic peak gradually decreased, especially after the NH4AC concentration exceeded 1mM. When 0.01% NH3∙H2O (v / v) and 1mM NH4AC were used as additives, the chromatographic peak baseline was the lowest, the oxalic acid peak response was higher, and the peak shape of oxalic acid at low and high concentrations was good, without interference or tailing ( Figure 2 M and 2N). In summary, 0.01% NH3∙H2O (v / v) and 1 mM NH4AC were selected as additives.

[0034] The present invention evaluates the proportion of mobile phase. By comparing gradient elution and isocratic elution, it is found that isocratic elution can obtain better peak shape. When the proportion of mobile phase B increases, the chromatographic peak moves forward. Finally, the present invention selects the proportion of organic phase to be 30% (v / v) ( Figure 2 O).

[0035] Example 5: Screening of stable isotope internal standards The similarity of the physicochemical properties and ionization process between the stable isotope internal standard and the target analyte can overcome the potential matrix effect. Therefore, two isotope internal standards were selected for comparison: oxalic acid-d2 and oxalic acid-1,2- 13 C. Figure 3 A is the mass spectrum of oxalic acid-d2. The results show that: in both positive and negative ion modes, no corresponding parent ion can be found for oxalic acid-d2. The mass-to-charge ratio of the parent ion peak with the highest response is m / z=88.9, which is the same as the parent ion mass-to-charge ratio of oxalic acid. 13 After C was used as the internal standard, everything went smoothly. According to the peak response, 90.8 / 61.8 was selected as the internal standard quantification channel, and the internal standard concentration was set to 200 ng / mL.

[0036] Example 6: Screening of flow rate and column temperature The effects of different flow rates (0.45, 0.4, 0.35, and 0.3 mL / min) and different column temperatures (45, 40, 35, and 30 °C) on peak height and retention time were evaluated. Figure 3 B shows the effect of flow rate (0.45, 0.4, 0.35, 0.3 mL / min) on the peak height (orange) and retention time (blue) of oxalic acid. Figure 3 C represents the effect of column temperature (45, 40, 35, 30 °C) on the peak height (orange) and retention time (blue) of oxalic acid. Figure 3The results of B-3C showed that the retention time of oxalic acid decreased with the increase of flow rate and column temperature. Taking the peak height into consideration, the optimal flow rate was 0.4 mL / min and the optimal column temperature was 40 ̊℃.

[0037] Example 7: Screening for alternative matrices Since oxalic acid is an endogenous substance, it is difficult to find a matrix without oxalic acid. The present invention detects plasma from different sources (rat, mouse, rabbit, beagle, human), human urine samples and fetal bovine serum albumin, and finds that they all contain oxalic acid. In mass spectrometry, if the analyte eluent is present with most matrix components, this may lead to significant matrix effects. Therefore, ultrapure water UPW is used as a substitute matrix, and a relative matrix effect evaluation is performed through a mixing experiment to prove that the existence of the matrix effect of this method does not affect the accurate quantification of the target analyte. Figure 3 D represents the double blank sample chromatograms of various types of biological matrices, including: plasma from different sources (including: rat plasma, mouse plasma, rabbit plasma, fetal bovine serum albumin FBSA, beagle dog plasma, human plasma, human urine, ultrapure water).

[0038] Mixing experiment: Three types of target analytical samples were measured - the first type was the quality control solution QCs using UPW as a surrogate matrix, divided into four concentration levels: 0.020 μg / mL (LLOQ), 0.100 μg / mL (LQC), 5 μg / mL (MQC), 40.0µg / mL (HQC). The second type was urine samples from four different sources. The third type was a mixed sample obtained by mixing the first two types of samples 1:1. Each concentration level was measured 6 times. The results of the relative matrix effect are summarized in Table 1. RSD represents the relative standard deviation of the sample, RE represents the relative error of the sample, and n represents the number of replicates of the sample. The results of the mixing experiment showed that in the relative matrix effect results of the mixed sample (1:1), RE was -13.9%~18.2%, and RSD was less than 7.9%. These data were within the acceptable standards (RSD<15%, RE within ±15%), indicating that the matrix effect did not affect the accurate quantification of the target analyte.

[0039] Table 1:

[0040] Example 8: Optimizing sample pretreatment methods According to the acidic properties of oxalic acid, the present invention studied the effect of NH3∙H2O concentration in the precipitant on the extraction efficiency, and screened different concentrations of NH3∙H2O, including 0, 0.005%, 0.05%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4% and 0.5% (v / v). Figure 3 E represents the effect of NH3∙H2O concentration in the oxalic acid precipitant on the peak height and peak shape. The results show that when the precipitant (IS solution) contains 0.3% NH3∙H2O (v / v), the peak shape and peak height of oxalic acid are optimal.

[0041] Example 9: Calculating the oxalic acid content in the sample by combining urine creatinine test data

[0042] According to the method of the literature “Groothoff JW, Metry E, Deesker L, et al. Clinical practice recommendations for primary hyperoxaluria: an expert consensus statement fromERKNet and OxalEurope. Nature Reviews Nephrology. 2023; 19(3): 194-211.”, the oxalate content in the sample was assessed based on the ratio of oxalate to creatinine.

[0043] Example 10: Method Validation Figure 4 A represents the representative chromatogram of the blank sample, Figure 4 B represents the representative chromatogram of the sample with lower limit of quantitation. Figure 4 C represents the representative chromatogram of the upper limit of quantitation sample (UPLC) without internal standard. Figure 4 D represents the representative chromatogram of a blank sample containing only the internal standard. Figure 4 E represents a representative chromatogram of a urine sample from a pediatric nephrology patient.

[0044] Figure 5 A represents the chromatogram of the blank sample injected after the quantitative upper limit concentration quality control sample ULOQ, Figure 5 B represents the linear graph of oxalic acid (0.020-50.0 μg / mL), Figure 5 C represents the typical MRM chromatogram of the quality control sample with the lower limit of detection of oxalic acid (LOD) (10 ng / mL). Blank test: Figure 4 The results for A show that the blank sample has no interference with the detection of the analyte and internal standard. There is no detectable interference between the analyte and internal standard.

[0045] Residue detection: Figure 5 The results for A showed that the chromatograms obtained when the blank sample was injected after the UPLC sample without internal standard showed no interfering signals beyond the acceptability criteria. Therefore, carryover was considered negligible, indicating that subsequent samples could be analyzed reliably regardless of the concentration of the previously injected sample.

[0046] Linearity, lower limit of quantitation, and detection limit verification: The method showed good linearity in the concentration range of 0.020~50.0 µg / mL. The typical regression equation of the calibration curve was y = 5.27x +0.0685 (r2 = 0.9980, n = 8) (y represents the peak area ratio of the analyte to the IS. x represents the concentration of the analyte) ( Figure 5 B). The LLOQ of the quality control sample for oxalate is 0.020 µg / mL, and the signal-to-noise ratio is much higher than 5 ( Figure 4 B). The detection limit concentration of oxalate in the quality control sample LOD is 0.010µg / mL, and the signal-to-noise ratio is between 2-3 ( Figure 5 B) The signal-to-noise ratio is used to determine whether the measured concentration is suitable for use as the lower limit of quantification or the lower limit of detection.

[0047] Accuracy and precision detection: The present invention evaluates the intra-day and inter-day precision and accuracy of oxalic acid by measuring four QC concentration levels. The results of intra-day and inter-day precision and accuracy of oxalic acid in alternative matrices are shown in Table 2, where RSD represents the relative standard deviation of the sample, RE represents the relative error of the sample, and n represents the number of replicates of the sample. The intra-day precision and accuracy are 2.5%~9.5% and 100.4%~106.1%, respectively, and the inter-day precision and accuracy are 3.0%~9.5% and 97.9%~105%, respectively. These data show good precision and accuracy, within the acceptable standards (RSD<15%, RE within ± 15%, except for LLOQ, where a 20% deviation is allowed).

[0048] Table 2:

[0049] Repeatability test: The samples of the analytical batch were stored at 4°C for 3 days and 2 hours before determination. The repeatability results of the urine oxalate method are shown in Table 3. RSD represents the relative standard deviation of the sample, and RE represents the relative error of the sample. The precision and accuracy of the LQC were 2.9% and 103.0%, respectively. The precision and accuracy of the HQC were 2.6% and 95.0%, respectively. These data were within the acceptable standard (RSD < 15%, RE within ± 15%).

[0050] Table 3:

[0051] Recovery rate test: The results are summarized in Table 4. RSD represents the relative standard deviation of the sample, and n represents the number of replicates of the sample. The average recovery rate data of QC samples at different analysis levels ranged from 101.4% to 104.4%, which met the requirements.

[0052] Table 4:

[0053] Stability test: The results of the stability test of urine oxalic acid are summarized in Table 5 (n=3). RSD represents the relative standard deviation of the sample, RE represents the relative error of the sample, and n represents the number of replicates of the sample. The RSD values ​​of the samples exposed to four different storage conditions were 1.1%~10.7%, and the RE values ​​were -12.0%~3.2%. The LOQ and HOQ of oxalic acid were stable at room temperature for 4 h and 24 h, respectively. It was stable for 5 days and 1 hour at 4°C. It was stable for 30 days at -20°C. In addition, oxalic acid remained stable after 5 freeze-thaw cycles (-20°C).

[0054] Table 5:

[0055] Dilution integrity verification: The dilution integrity results of urine oxalic acid are summarized in Table 6. RSD represents the relative standard deviation of the sample, RE represents the relative error of the sample, and n represents the number of replicates of the sample. The results show that a 10-fold dilution does not affect the concentration determination of urine oxalic acid samples and medium and high concentration quality control samples.

[0056] Table 6:

[0057] The embodiments of the present invention are only to introduce the specific implementation methods thereof, and are not intended to limit the protection scope thereof. The technicians in the industry may make some modifications inspired by the embodiments, so any equivalent changes or modifications made according to the scope of the present invention are within the scope of the patent claims of the present invention.

Claims

1. A method for detecting urine oxalic acid using HPLC-ESI-MS / MS technology, characterized in that: The following steps are involved: S1. Take the collected sample and add 1,2-oxalic acid 13 C was used as the internal standard of oxalic acid. The internal standard solution was mixed with the collected sample at a ratio of 2:1, vortexed for 5 min, and centrifuged at 4000 rpm for 5 min to obtain the urine sample; S2. Sample detection is performed using a liquid chromatography tandem mass spectrometer; the detection conditions of the liquid chromatography are: Liquid chromatography system high pressure pump A mobile phase: 0.01% by volume ammonia-1mM ammonium acetate-ultrapure water solution; Liquid chromatography system high pressure pump B mobile phase: 0.01% by volume ammonia-1mM ammonium acetate-acetonitrile solution; The chromatographic conditions were as follows: the column was a Waters Symmetry C18 column, 3.5 µm, 4.6 mm × 75 mm, column temperature 40 °C; detection mode MRM, ion source temperature 500 °C; flow rate 0.4 ml / min; gradient elution program: 70% by volume of mobile phase A + 30% by volume of mobile phase B, isocratic run for 3 min; injection volume 5 µL; needle wash mode: needle wash for 5 s before and after injection; The mass spectrometry conditions were: curtain gas 35 psi, impingement gas 4 psi, ionization voltage 4000 V, ion source temperature 500 °C, nebulizer gas 50 psi, auxiliary heating gas 50 psi; S3, using stable isotope labeled oxalic acid-1,2- 13 C was used as the internal standard of oxalic acid. A series of standards with different concentrations were prepared to make a standard curve. The concentration of the oxalic acid working standard solution was taken as the X-axis, and the peak area ratio of the standard and internal standard was taken as the Y-axis. Linear regression analysis was performed. The results were obtained by "1 / x 2 The regression equation was obtained by substituted the peak area ratio of oxalic acid in the sample to its internal standard into the standard curve equation to calculate the oxalic acid concentration in the urine sample.

2. The method for detecting urinary oxalic acid using HPLC-ESI-MS / MS technology according to claim 1, characterized in that: The mass spectrometry conditions in step S1 include oxalic acid and its isotopes oxalic acid-1,2- 13 The electrical parameter conditions of C are as follows: Oxalic acid: parent ion 88.9Da, daughter ion 60.8Da, scan time 200 msec, declustering voltage 40V, entrance voltage 7V, collision energy 10V, collision cell exit voltage 15V; Oxalic acid-1,2- 13 C: parent ion 90.8Da, product ion 61.8Da, scan time 200 msec, declustering voltage 14V, entrance voltage 2V, collision energy 10V, collision cell exit voltage 4V.

3. The method for detecting urine oxalic acid using HPLC-ESI-MS / MS technology according to claim 1, characterized in that: The components and contents of the relevant reagents in step S2 are: Needle washing solution: 40% by volume methanol, 40% by volume acetonitrile, 10% by volume isopropanol, and 10% by volume ultrapure water; Internal standard solution: dissolve 1,2-oxalic acid in methanol 13 C, prepare a 1.00 mg / mL internal standard stock solution, and then dilute it to 200 ng / mL with acetonitrile containing 0.3% ammonia by volume; Calibration curve solution: dissolve oxalic acid in methanol to prepare a 1.00 mg / mL oxalic acid stock solution; dilute the stock solution with methanol: water at a volume ratio of 1:1 to obtain oxalic acid working standard solutions with concentrations of 0.2, 0.5, 1, 5, 50, 50, 100, 250 and 500 μg / mL, respectively; use ultrapure water to dilute the oxalic acid working standard solution to prepare standard curve solutions with concentrations of 0.020, 0.050, 0.100, 0.500, 10.0, 5.0, 10.0, 25.0, 50.0 μg / mL, respectively; Quality control solutions QCs: First, dilute the 1.00 mg / mL oxalic acid stock solution with methanol water at a volume ratio of 1:1 to obtain oxalic acid quality control working standard solutions with concentrations of 0.2, 1, 50, and 400 µg / mL, respectively. Use ultrapure water to dilute the oxalic acid quality control working standard solution to obtain quality control samples with concentrations of 0.020, 0.100, 5, and 40.0 µg / mL, respectively; Calibration curve samples and quality control samples: 200 μL of precipitant was mixed with 100 μL of calibration curve solution and quality control solution QCs, respectively. After vortexing for 5 min, the mixture was centrifuged at 4000 rpm for 5 min to obtain calibration curve samples and quality control samples, respectively.

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