A method for detecting oxalic acid by isotopic dilution high performance liquid chromatography tandem mass spectrometry

Abstract

The present application relates to oxalic acid detection technical field, specifically to a kind of method for detecting the oxalic acid content in human blood and urine by isotope dilution high performance liquid chromatography tandem mass spectrometry, the present application is with 4-methoxy o-phenylenediamine dihydrochloride as derivatization reagent, and oxalic acid is reacted to generate 2,3-dihydroxy-6-methoxy quinoline, it is well retained on ordinary reversed-phase chromatographic column, and isotope dilution high performance liquid chromatography tandem mass spectrometry is used to detect.The method provided in the present application, sample need not be pretreated, derivatization reaction process is simple to operate, condition is mild and well retained on ordinary reversed-phase chromatographic column, peak type is good, repeatability is good, anti-interference ability is strong, flux is high, suitable for the detection of large quantities of samples in clinic.

Description

TECHNICAL FIELD

[0001] The application relates to an oxalic acid detection technology field, in particular to a method for detecting the oxalic acid content in human blood and urine by isotopic dilution high performance liquid chromatography tandem mass spectrometry. BACKGROUND

[0002] Urinary calculi is a common and frequently-occurring disease in clinic, which seriously affects the health of the human body. Calcium oxalate calculi are most common in urinary calculi, and high oxalate urine caused by abnormal oxalate metabolism has an important influence on the formation of calculi. However, the oxalic acid content in urine, serum and plasma is very small, and the detection is difficult.

[0003] The methods for determining the concentration of oxalic acid in blood mainly include enzyme analysis, capillary electrophoresis and liquid chromatography. However, oxalic acid has large polarity and has no retention on an ordinary reversed-phase column. The existing processing mode is to perform derivatization on the sample, and to perform sample pretreatment on the urine and blood, so that the oxalic acid in the urine and blood reacts with o-phenylenediamine as a derivatizing agent to generate a strong ultraviolet absorption compound, 2,3-dihydroxyquinoxaline. However, the pretreatment mode has the following disadvantages: 1. The derivatization reaction degree of oxalic acid in biological samples of different individuals is different, the matrix is also different, impurity components are extremely easy to be generated, the peak is complex, and the oxalic acid peak is not obvious, but is only mixed in the peaks, and the very accurate requirement cannot be met. 2. The pretreatment needs to go through complicated experimental steps, and the time consumption is long. 3. The temperature is high during the derivatization treatment, needs to be heated to 140 DEG C, and concentrated hydrochloric acid needs to be added, so that there is a great safety hazard to the operator. Therefore, a method for determining the oxalic acid content in human blood and urine needs to be researched and developed, which is efficient, safe and simple to operate. SUMMARY

[0004] In the application, 4-methoxy o-phenylenediamine dihydrochloride is used as a derivatizing reagent to react with oxalic acid to generate 2,3-dihydroxy-6-methoxyquinoxaline, which has good retention on an ordinary reversed-phase chromatographic column, and isotopic dilution high performance liquid chromatography tandem mass spectrometry is used for detection, so that the problems in the background technology are solved.

[0005] To achieve the above object, the application provides the following technical scheme.

[0006] A method for detecting oxalic acid by isotopic dilution high performance liquid chromatography tandem mass spectrometry, comprising the following steps:

[0007] S1: preparing an oxalic acid standard solution, adding an internal standard solution and 4-methoxy o-phenylenediamine dihydrochloride solution to the standard solution and the sample to be detected to perform a derivatization reaction; after the reaction is completed, centrifuging to obtain supernatant;

[0008] S2: The supernatant is diluted for injection, and high performance liquid chromatography tandem mass spectrometry detection is carried out; a standard curve is drawn according to the concentration of the oxalic acid standard solution, the peak area ratio of the detection peak to the internal standard peak; the peak area ratio of the oxalic acid detection peak in the sample to be measured to the internal standard peak is substituted into the standard curve for quantification, so that the content of oxalic acid in the sample to be measured can be calculated.

[0009] Preferably, the volume ratio of the oxalic acid standard solution or the sample to be measured to the internal standard solution and the 4-methoxy-ortho-phenylenediamine dihydrochloride solution is 2-5:1:1; the supernatant needs to be diluted by 5-15 times with water before injection.

[0010] Preferably, the sample to be measured includes urine, serum or plasma.

[0011] Preferably, the internal standard solution is oxalic acid- 13 C2 solution, the solvent is methanol, and the concentration of the internal standard solution is 5-15 μg / mL.

[0012] Preferably, the concentration of the 4-methoxy-ortho-phenylenediamine dihydrochloride solution is 3-8 mg / mL.

[0013] Preferably, in the derivatization reaction, the temperature of vortex oscillation is 50-75°C, and the time is 0.5-2 h.

[0014] Preferably, the temperature of centrifugation is 4-10°C, the rotation speed is 12000-15000 rpm, and the time is 5-20 min.

[0015] Preferably, the chromatographic column of the high performance liquid chromatography is Acquity UPLC® BEH C18 (2.1*50 mm, 1.7 μm); the mobile phase A is 0.1%-0.5% formic acid-water, and the mobile phase B is 0.1%-0.5% formic acid-methanol; the elution program is gradient elution; and the flow rate is 0.2-0.5 mL / min.

[0016] Preferably, the ion source of the mass spectrometry is an electrospray ion source in positive ion mode; the scanning mode is multiple reaction monitoring, 2,3-dihydroxy-6-methoxy-quinoxaline (DMNA) 193.03>147.03 is selected as a quantitative ion pair, 193.03>120.04 is selected as a qualitative ion pair, and 2,3-dihydroxy-6-methoxy-quinoxaline- 13 C2 195.10>148.13 (DMNA-IS) is selected as an internal standard quantitative ion pair.

[0017] Preferably, the method for detecting oxalic acid by isotopic dilution high performance liquid chromatography tandem mass spectrometry is applied to an oxalic acid detection kit.

[0018] Compared with the prior art, the method has the following beneficial effects:

[0019] (1) Select 2, 3-dihydroxy-6-methoxyquinoxaline (DMNA) 193.03 > 147.03 as the quantitative ion pair, 193.03 > 120.04 as the qualitative ion pair, and 2, 3-dihydroxy-6-methoxyquinoxaline- 13 C2 (DMNA-IS) 195.10 > 148.13 as the internal standard quantitative ion pair, and use MRM mode to determine the content of oxalic acid, which can effectively overcome the problem of different degrees of oxalic acid derivatization in different individual biological samples, overcome the matrix effect, and accurately determine the content of oxalic acid in different biological matrix samples.

[0020] (2) The sample does not need to be pretreated, and the urine / serum / plasma sample can be directly used for detection reaction, which simplifies the operation steps and improves the detection efficiency.

[0021] (3) The derivatization reagent 4-methoxy o-phenylenediamine dihydrochloride itself releases hydrochloric acid in the reaction process, and there is no need to add concentrated hydrochloric acid during operation, which ensures the safety of the operator; at the same time, the methoxy group is an electron-donating group, which makes the derivatization reaction easier to proceed, and the reaction temperature is 65℃, and the reaction condition is mild.

[0022] (4) The derivatization product is well retained on the ordinary reversed-phase chromatographic column, has good peak shape, good repeatability, strong anti-interference ability, and only needs 5 min for chromatographic time; the liquid before the peak time of the derivatization product is flowed into the waste liquid, which removes the excess unreacted derivatization reagent, and can protect the mass spectrometry ion source from pollution; high flux, suitable for detection of large number of clinical samples. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is the standard curve graph of oxalic acid;

[0024] Figure 2 is the peak out graph of the derivatization product of oxalic acid and its isotopic internal standard in standard solution 2;

[0025] Figure 3 is the peak out graph of the derivatization product of oxalic acid and its isotopic internal standard in urine sample 2;

[0026] Figure 4 is the peak out graph of the derivatization product of oxalic acid and its isotopic internal standard in serum sample 2;

[0027] Figure 5 is the peak out graph of the derivatization product of oxalic acid and its isotopic internal standard in plasma sample 2; DETAILED DESCRIPTION

[0028] With reference to the drawings of the embodiments of the present application, the technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0029] Prepare standard solution: 10 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 500 μmol / L, 1000 μmol / L oxalic acid solution;

[0030] Prepare quality control solution: 50 μmol / L, 150 μmol / L oxalic acid solution;

[0031] Prepare internal standard solution: 10 μg / mL oxalic acid 13 C2, solvent is methanol;

[0032] Prepare 5 mg / mL 4-methoxy o-phenylenediamine dihydrochloride solution.

[0033] Example 1

[0034] (1) precisely pipette 100 μL standard solution / quality control solution / urine / serum / plasma into a 1.5 mL centrifuge tube, respectively, add 50 μL internal standard solution, 50 μL 4-methoxy o-phenylenediamine dihydrochloride solution, vortex at 65°C for 1 h, then centrifuge at 4°C at high speed (14000 rpm) for 5 min, take 20 μL supernatant, add 180 μL water, vortex for 1 min, centrifuge at 4°C at high speed (14000 rpm) for 10 min, the obtained supernatant is the sample solution.

[0035] (2) take 10 μL sample solution and inject into high performance liquid chromatography-tandem mass spectrometer for detection;

[0036] a. The conditions of high performance liquid chromatography are as follows:

[0037] Chromatographic column: Acquity UPLC® BEH C18 (2.1×50 mm, 1.7 μm);

[0038] Mobile phase A: 0.1% formic acid-water; mobile phase B: 0.1% formic acid-methanol; flow rate: 0.3 mL / min; column temperature: 40°C;

[0039] The gradient elution program is shown in Table 1.

[0040] Table 1 Gradient elution conditions

[0041]

[0042] b. Mass spectrometry conditions are:

[0043] Ion source: electrospray ion source, positive ion mode;

[0044] Capillary voltage: 0.5kV; desolvation temperature: 500℃; desolvation gas: 800L / Hr; cone gas: 150L / hr;

[0045] Scan mode: multiple reaction monitoring (MRM), select 2,3-dihydroxy-6-methoxyquinoxaline (DMNA) 193.03>147.03 as quantitative ion pair, 193.03>120.04 as qualitative ion pair, select 2,3-dihydroxy-6-methoxyquinoxaline- 13 C2 (DMNA-IS) 195.10>148.13 as internal standard quantitative ion pair, conditions are shown in Table 2.

[0046] Table 2 MRM conditions of each compound

[0047]

[0048] (3) The results of high performance liquid chromatography-tandem mass spectrometry are shown in Table 3.

[0049] Table 3 High performance liquid chromatography-tandem mass spectrometry result data

[0050]

[0051] (4) Verification of sample addition recovery rate

[0052] Table 4 Sample addition recovery rate result data

[0053]

[0054] Result analysis:

[0055] As shown in Table 4: the recovery rates of low, medium and high standard concentrations of urine, serum and plasma matrix samples are between 93%-100.5%, which indicates that the detection results of oxalic acid in different matrix samples are accurate and reliable.

[0056] As shown in Table 4: the recovery rates of low, medium and high standard concentrations of urine, serum and plasma matrix samples are between 93%-100.5%, which indicates that the detection results of oxalic acid in different matrix samples are accurate and reliable. Figure 1 As shown in Table 4: the recovery rates of low, medium and high standard concentrations of urine, serum and plasma matrix samples are between 93%-100.5%, which indicates that the detection results of oxalic acid in different matrix samples are accurate and reliable. 2 The R of oxalic acid standard curve is 0.999, i.e. the standard curve is good in linearity at 10-1000μmol / L; the limit of quantification is 10μmol / L according to S / N=10, which indicates that the analysis method has sensitive quantitative detection capability for the derivative product.

[0057] Figures 2-5The out peak figures of the derivative products of oxalic acid and its isotope internal standard in standard 2, urine sample 2, serum sample 2 and plasma sample 2 are respectively shown, and it can be seen from the figures that the derivative products are well reserved on the common reversed-phase chromatographic column, the target peaks are clearly out peaked, the peak shape is good, the anti-interference ability is strong, and the repeatability is good.

[0058] The chromatographic time only needs 5 min, the out peak time of the derivative products is about 2.3 min, the liquid phase before this retention time is cut into waste liquid, the excess unreacted derivative reagent can be avoided from entering the mass spectrometer, the ion source is protected from being polluted, the flux is improved, and the detection of the clinical large batch samples is suitable.

[0059] It is apparent for those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, but can be implemented in other concrete forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be considered as exemplary and non-limiting, the scope of the present application is defined by the appended claims rather than the above description, and all changes falling within the meaning and range of the equivalent elements of the claims are intended to be embraced in the present application. Any reference signs in the claims should not be considered as limiting the involved claims.

Claims

1. A method for detecting oxalate using isotopic dilution high performance liquid chromatography tandem mass spectrometry, characterized by, Comprising the following steps: S1: preparing oxalic acid standard solution, adding internal standard solution and 4-methoxy o-phenylenediamine dihydrochloride solution to the standard solution and the sample to be tested for derivatization reaction; after the reaction is completed, centrifuging to obtain supernatant; S2: diluting the supernatant for sampling, and performing high performance liquid chromatography tandem mass spectrometry detection; according to the concentration of the oxalic acid standard solution, the ratio of the detection peak area to the internal standard peak area, a standard curve is drawn, and the ratio of the detection peak area to the internal standard peak area in the sample to be tested is substituted into the standard curve for quantification, so that the content of oxalic acid in the sample to be tested can be calculated; The sample to be tested includes urine, serum or plasma, in the derivatization reaction, the temperature of vortex oscillation is 50-75℃, and the time is 0.5-2h, the chromatographic column of the high performance liquid chromatography is Acquity BEH C18; the mobile phase A is 0.1%-0.5% formic acid-water, and the B is 0.1%-0.5% formic acid-methanol; the elution program is gradient elution, and the gradient elution program is shown in the following table: ； The ion source of the mass spectrum is an electrospray ion source, positive ion mode; the scan mode is multiple reaction monitoring, 2,3-dihydroxy-6-methoxyquinoxaline 193.03>147.03 is selected as the quantitative ion pair, 193.03>120.04 is selected as the qualitative ion pair, 2,3-dihydroxy-6-methoxyquinoxaline- 13 C2 195.10>148.13 is selected as the internal standard quantitative ion pair.

2. The method for detecting oxalic acid by isotope dilution high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that, The volume ratio of the oxalic acid standard solution or the sample to be tested to the internal standard solution and the 4-methoxy o-phenylenediamine dihydrochloride solution is 2-5:1:1; the supernatant needs to be diluted 5-15 times with water before sampling.

3. The method for detecting oxalic acid by isotope dilution high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that, The internal standard solution is oxalic acid 13 C2 solution, solvent is methanol, concentration of internal standard solution is 5-15 μg / mL.

4. The method for detecting oxalic acid by isotope dilution high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that, The concentration of the 4-methoxy o-phenylenediamine dihydrochloride solution is 3-8mg / mL.

5. The method for detecting oxalic acid by isotope dilution high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that, The temperature of centrifugation is 4-10℃, the rotation speed is 12000-15000rpm, and the time is 5-20min.

6. The method for detecting oxalic acid by isotope dilution high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that, The flow rate of the elution program is 0.2-0.5mL / min.

7. The method for detecting oxalic acid by isotopic dilution high performance liquid chromatography tandem mass spectrometry according to any one of claims 1-6 is applied to an oxalic acid detection kit.

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