Detection method and application of 3-nitropropionic acid

Abstract

The present invention provides a detection method and application of 3-nitropropionic acid, and relates to the field of analytical detection technology. The detection method includes using high performance liquid chromatography tandem mass spectrometry to detect the pretreated sample, and then bringing the detection value into the standard curve to calculate the content of 3-nitropropionic acid in the sample; wherein the sample includes a blood sample; the standard curve is constructed using an external standard method; the pretreatment includes mixing the sample with a protein precipitation reagent containing methanol and / or acetonitrile, and performing high performance liquid chromatography tandem mass spectrometry detection after separating the supernatant; the high performance liquid chromatography conditions include: the chromatographic column uses a C18 chromatographic column; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is an aqueous solution of formic acid, and mobile phase B is acetonitrile. The detection method alleviates the problem of the lack of detection methods for 3-nitropropionic acid in blood samples in the prior art, and the cumbersome detection operation and high detection cost.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a detection method for 3-nitropropionic acid and application thereof. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] 3-nitropropionic acid (3-NPA) is a neurotoxin and the main toxic substance that causes moldy sugarcane poisoning. It can cause damage to the central nervous system, interfere with the metabolism of intracellular enzymes, and severely lead to cerebral ischemia and necrosis, causing serious damage to the central nervous system. At present, the detection method of 3-nitropropionic acid in sugarcane is mainly based on the thin layer chromatography method determined by the health industry standard (WS / T10-1996). There are also reports on quantitative determination using gas chromatography, high performance liquid chromatography, gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry.

[0004] Monitoring the 3-nitropropionic acid content in the blood of the test sample is helpful for the development of drugs for treating 3-nitropropionic acid poisoning, but the current detection of 3-nitropropionic acid is mainly carried out on sugarcane samples. The physical and chemical properties of sugarcane samples and blood samples are quite different, and the interfering components for the separation and detection of 3-nitropropionic acid are also different. Therefore, the detection method in the prior art is not suitable for the monitoring of 3-nitropropionic acid in blood samples. In addition, the solid phase extraction method is often used in the prior art to pretreat the sample. The pretreatment method is cumbersome, takes a long time to process, and the cost of the solid phase extraction column is high. Therefore, how to simplify the detection method of 3-nitropropionic acid and apply it to the detection of blood samples is a problem that needs to be solved at present.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a method for detecting 3-nitropropionic acid, so as to alleviate the problems in the prior art that the detection operation of 3-nitropropionic acid is complicated, the detection cost is high, and there is a lack of a method for detecting 3-nitropropionic acid in a blood sample.

[0007] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0008] In a first aspect, a method for detecting 3-nitropropionic acid is provided, the method comprising detecting a pretreated sample using high performance liquid chromatography tandem mass spectrometry, inserting the detected value into a standard curve, and calculating the content of 3-nitropropionic acid in the sample;

[0009] The sample includes a blood sample;

[0010] The standard curve is constructed using the external standard method;

[0011] The pretreatment includes mixing the sample with a protein precipitation reagent, separating the supernatant and performing high performance liquid chromatography tandem mass spectrometry detection, wherein the protein precipitation reagent contains methanol and / or acetonitrile;

[0012] HPLC conditions included:

[0013] The chromatographic column used is C18 chromatographic column;

[0014] The mobile phase includes a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution of formic acid, and the mobile phase B is acetonitrile.

[0015] In the second aspect, the application of the detection method of the first aspect in the preparation of a 3-nitropropionic acid detection product is also provided.

[0016] In a third aspect, a 3-nitropropionic acid detection product is also provided, and the 3-nitropropionic acid detection product includes the protein precipitation reagent and mobile phase described in the first aspect.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] In the detection method of the present invention, sample pretreatment uses a protein precipitation reagent containing methanol and / or acetonitrile to precipitate proteins in blood samples, which is simpler to operate than solid phase extraction, and significantly reduces the detection cost. The mobile phase of the high performance liquid chromatography of the present invention does not contain ion pair reagents of metal salts, is not easy to contaminate the mass spectrometer, and is friendly to the long-term stability of the instrument. One of the disadvantages of mass spectrometry is that expensive internal standard substances are required, and the detection method of the present invention uses an external standard method to construct a standard curve, without the need for internal standard substances to calibrate the matrix interference. The present invention achieves accurate quantification of 3-nitropropionic acid in plasma using an external standard method by optimizing the pretreatment method, chromatographic column and chromatographic conditions. The detection method provided by the present invention has been verified by methodology, and the limit of quantification can be as low as 1 ng / mL, and the limit of detection can be as low as 0.1 ng / mL. In summary, the detection method of 3-nitropropionic acid provided by the present invention simplifies the detection steps, reduces the detection cost, and can maintain an excellent detection effect.

[0019] Based on the detection method provided by the present invention, the 3-nitropropionic acid detection product provided by the present invention is used to detect 3-nitropropionic acid in blood samples, which can provide a reference for the diagnosis of 3-nitropropionic acid poisoning and the clinical treatment of 3-nitropropionic acid poisoning. In addition, the product has the advantages of simple pretreatment steps and significantly compressed detection time when used, which shortens the time for poison identification, and gains more treatment time for clinical treatment of sugarcane poisoning patients, and can significantly reduce the cost of pretreatment consumables and thus reduce the burden on patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 is the spectrum of 3-NPA in the surrogate matrix in Example 2;

[0022] Figure 2 is the spectrum of 3-NPA in the standard solution in Example 2;

[0023] Figure 3 is the spectrum of 3-NPA in blank plasma in Example 2;

[0024] Figure 4 This is the spectrum of the plasma spiked sample when the protein is precipitated using methanol-acetonitrile solution in Example 3;

[0025] Figure 5 is the spectrum of the blank plasma sample when the protein is precipitated using methanol-acetonitrile solution in Example 3;

[0026] Figure 6 This is the spectrum of the plasma spiked sample when the protein is precipitated with acetonitrile solution in Example 3;

[0027] Figure 7 is the spectrum of the blank plasma sample when the protein is precipitated with acetonitrile solution in Example 3;

[0028] Figure 8 This is a typical spectrum of 3-NPA obtained by the detection method using elution procedure 1 in Example 5;

[0029] Fig. 9 This is the minimum quantification limit spectrum of 3-NPA in plasma matrix samples detected using Waters Acquity BEH C18 (2.1 mm×150 mm×1.7 μm) in Example 6;

[0030] Fig.10 This is the minimum quantification limit spectrum of 3-NPA in plasma matrix samples detected by using Phenomenex Kinetex C18 (2.6 μm, 3 mm×100 mm) in Example 6;

[0031] Fig.11 This is the minimum quantification limit spectrum of 3-NPA in plasma matrix samples detected using Phenomenex Kinetex EVO C18 (2.6μm, 3mm×100mm) in Example 6. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] In a first aspect, a method for detecting 3-nitropropionic acid is provided, which comprises detecting a pretreated sample using high performance liquid chromatography tandem mass spectrometry, substituting the detection value into a standard curve, and calculating the content of 3-nitropropionic acid in the sample.

[0034] The detection sample of the detection method includes a blood sample, and the blood sample includes but is not limited to whole blood, plasma or serum.

[0035] The pretreatment includes mixing the sample with a protein precipitation reagent to precipitate the protein in the blood sample, separating the supernatant and performing high performance liquid chromatography tandem mass spectrometry detection, wherein the protein precipitation reagent contains methanol and / or acetonitrile.

[0036] In an optional embodiment, the protein precipitation reagent is methanol or acetonitrile.

[0037] In an optional embodiment, the protein precipitation reagent contains methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is (0-2): (0-2), and the volumes of methanol and acetonitrile are not 0. The volume ratio of methanol to acetonitrile can be, for example, but not limited to, 0.1:2, 0.5:2, 1:2, 1.5:2, 2:2, 2:1.5, 2:1, 2:0.5 or 2:0.1, preferably 1:1.

[0038] In an optional embodiment, the volume ratio of the sample to the protein precipitant is 1: (3-5), preferably 1: 4. For example, 100 μL of sample is mixed with 300, 400 or 500 μL of protein precipitant and the supernatant is separated.

[0039] In an optional embodiment, the pretreatment includes vortex mixing the sample and the protein precipitation reagent, and collecting the supernatant after centrifugation for high performance liquid chromatography tandem mass spectrometry detection.

[0040] The HPLC conditions in the HPLC-MS / MS detection step include:

[0041] The chromatographic column uses a C18 chromatographic column. In an optional embodiment, the chromatographic column is a Waters Acquity BEHC18 chromatographic column with a specification of 2.1 mm×150 mm×1.7 μm.

[0042] In an optional embodiment, the column temperature of the chromatographic column is 20-50 °C, preferably 40 °C.

[0043] The mobile phase includes a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution of formic acid, and the mobile phase B is acetonitrile.

[0044] In an optional embodiment, the mobile phase A contains 0.05-0.2% v / v of formic acid, preferably 0.1% v / v.

[0045] In an optional embodiment, according to gradient elution, the elution procedure is:

[0046] At 0-1.5 min, the volume ratio of the mobile phase A to the mobile phase B was maintained at 85:15;

[0047] At 1.5-1.6 min, the volume ratio of the mobile phase A to the mobile phase B changed from 85:15 to 10:90;

[0048] At 1.6-2.2 min, the volume ratio of the mobile phase A to the mobile phase B was maintained at 10:90;

[0049] At 2.2-2.3 min, the volume ratio of the mobile phase A to the mobile phase B changed from 10:90 to 5:95;

[0050] At 2.3-3.7 min, the volume ratio of the mobile phase A to the mobile phase B was maintained at 5:95;

[0051] At 3.7-3.8 min, the volume ratio of the mobile phase A to the mobile phase B changed from 5:95 to 85:15;

[0052] From 3.8 to 5.0 min, the volume ratio of the mobile phase A to the mobile phase B was maintained at 85:15.

[0053] In an optional embodiment, the flow rate of the mobile phase is 0.2~0.5 mL / min.

[0054] In an optional embodiment, the flow rate is 0.2 mL / min at 0-1.5 min; the flow rate changes from 0.2 mL / min to 0.5 mL / min at 1.5-1.6 min; the flow rate is 0.5 mL / min at 1.6-2.2 min; the flow rate changes from 0.5 mL / min to 0.2 mL / min at 2.2-2.3 min; and the flow rate is 0.2 mL / min at 2.3-5.0 min.

[0055] In an optional embodiment, the mass spectrometry conditions include:

[0056] Scanning mode: electrospray ionization positive ion mode;

[0057] Acquisition mode: multiple reaction monitoring mode;

[0058] Electrospray voltage: -3000 V; curtain gas: 30 psi; nebulizer gas: 40 psi; auxiliary gas: 50 psi; ion source temperature: 650 °C.

[0059] In an optional embodiment, high performance liquid chromatography tandem mass spectrometry detection is implemented using a SCIEX Triple Quad 4500MD system, which has a high market share and is more conducive to the promotion of the detection method.

[0060] The detection method of 3-nitropropionic acid provided by the invention adopts an external standard method to construct a standard curve.

[0061] In an optional embodiment, the standard curve is the correspondence between the content of 3-nitropropionic acid in the standard and the detection value of the standard; the detection value of the standard is the peak value of the standard, and the peak value includes but is not limited to the peak height or peak area, preferably the peak area.

[0062] In an optional embodiment, the concentrations of 3-nitropropionic acid in several standards used to construct a standard curve are 50 ng / mL, 25 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, 2 ng / mL and 1 ng / mL, respectively.

[0063] In an optional embodiment, the detection method also includes detecting a quality control product.

[0064] The detection method of 3-nitropropionic acid provided by the present invention, the source of the sample can be, for example, but not limited to, mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys or humans. It should be noted that the detection method of 3-nitropropionic acid provided by the present invention is for non-diagnostic and therapeutic purposes. Specifically, for example, the sample derived from the above-mentioned mammal is an experimental animal sample, and the determination method of the present invention is used to provide experimental results, which are further used to alleviate the fields of drug mechanism of action or pharmacokinetic research of 3-nitropropionic acid poisoning; and, knowing the content of 3-nitropropionic acid in the sample to be tested does not mean that the result can directly point to the diagnosis result of the disease, so the detection method of 3-nitropropionic acid provided by the present invention is for non-diagnostic and therapeutic purposes.

[0065] In the second aspect, the application of the detection method of 3-nitropropionic acid in the first aspect in the preparation of a 3-nitropropionic acid detection product is also provided. The above-mentioned determination method can be used as an evaluation standard to evaluate the detection effect of other 3-nitropropionic acid detection methods, or the above-mentioned determination method is preset in the detection product as an operation module, and the reagent used in the above-mentioned determination method is used as a supporting reagent in the determination product; or the above-mentioned determination method is preset in the detection product as an operation module to support the diagnosis or auxiliary diagnosis product of 3-nitropropionic acid poisoning.

[0066] In the third aspect, a 3-nitropropionic acid detection product is also provided, and the 3-nitropropionic acid detection product may include the protein precipitation reagent and mobile phase in the first aspect; the protein precipitation reagent contains methanol and / or acetonitrile; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is an aqueous solution of formic acid, and the mobile phase B is acetonitrile.

[0067] In an optional embodiment, the protein precipitation reagent is methanol or acetonitrile.

[0068] In an optional embodiment, the protein precipitation reagent contains methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is (0-2): (0-2), and the volumes of methanol and acetonitrile are not 0. The volume ratio of methanol to acetonitrile can be, for example, but not limited to, 0.1:2, 0.5:2, 1:2, 1.5:2, 2:2, 2:1.5, 2:1, 2:0.5 or 2:0.1, preferably 1:1.

[0069] In an optional embodiment, the mobile phase A contains 0.05-0.2% v / v of formic acid, preferably 0.1% v / v.

[0070] In an optional embodiment, the 3-nitropropionic acid detection product also includes standard samples, and the standard samples include standard samples S1~S7. The working concentrations of 3-nitropropionic acid in the standard samples S1~S7 are 50ng / mL, 25ng / mL, 20ng / mL, 10ng / mL, 5ng / mL, 2ng / mL and 1ng / mL respectively.

[0071] In an optional embodiment, the 3-nitropropionic acid detection product also includes a quality control product.

[0072] In an optional embodiment, the 3-nitropropionic acid detection product further comprises a C18 chromatographic column, preferably a Waters Acquity BEH C18 chromatographic column.

[0073] In an optional embodiment, the 3-nitropropionic acid detection product also includes an operation module, which records an elution program and mass spectrometry parameters; when the operation module is run, the high performance liquid chromatography tandem mass spectrometry detection system is operated according to the elution program and mass spectrometry parameters recorded in the operation module.

[0074] In an optional embodiment, the operation module records the elution program and mass spectrometry conditions and parameters described in the first aspect.

[0075] In an optional embodiment, the 3-nitropropionic acid detection product also includes an analysis module, in which a standard curve is recorded. The analysis module obtains the detection value of high performance liquid chromatography tandem mass spectrometry and obtains the content of 3-nitropropionic acid in the sample based on the standard curve.

[0076] In an optional embodiment, the 3-nitropropionic acid detection product also includes a blood sample separation module for collecting samples.

[0077] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0078] Example 1

[0079] Provided is a method for detecting 3-nitropropionic acid:

[0080] 1. Reagent preparation:

[0081] The 3-nitropropionic acid (3-NPA) standard diluent is a pure aqueous solution; the surrogate matrix is ​​a pure aqueous solution; the precipitated protein solution is a mixed solution of methanol and acetonitrile in a volume ratio of 1:1; and the plasma sample is human plasma without 3-NPA.

[0082] 1. Preparation of 3-NPA linear working solution: Dilute the secondary stock solution containing 1 μg / mL 3-NPA to 500 ng / mL, 250 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 20 ng / mL and 10 ng / mL using standard diluent.

[0083] 2. Preparation of 3-NPA quality control working solution: Dilute the secondary stock solution containing 1 μg / mL 3-NPA with standard diluent to 350 ng / mL (H), 150 ng / mL (M) and 25 ng / mL (L).

[0084] 2. Sample preprocessing:

[0085] Sample to be tested: Add 100 μL of the plasma sample to be tested to a clean 2 mL EP tube, add 400 μL of the precipitated protein solution, and vortex evenly on an oscillator. Centrifuge at 12,000 rpm for 10 minutes, and transfer the supernatant for injection.

[0086] Standard curve: Add 90 μL of surrogate matrix to a clean 2 mL EP tube, add 10 μL of working solution at 7 linear concentrations, add 400 μL of precipitated protein solution, place on a shaker and vortex evenly. Centrifuge at 12,000 rpm for 10 minutes, transfer the supernatant for injection.

[0087] 3. Instrument testing conditions:

[0088] 1. High performance liquid chromatography (HPLC) detection conditions:

[0089] LC-MS system: SCIEX Triple Quad 4500MD system;

[0090] Chromatographic column: Waters Acquity BEH C18 (2.1 mm × 150 mm × 1.7 μm);

[0091] Mass spectrometer: 4500MD liquid-mass spectrometer;

[0092] Mobile phase A: 0.1% v / v formic acid in water;

[0093] Mobile phase B: acetonitrile solution;

[0094] Column temperature: 40 °C;

[0095] Injection volume: 2 μL.

[0096] Table 1 Elution procedure

[0097]

[0098] 2. Mass spectrometry conditions

[0099] Scanning mode: electrospray ionization positive ion mode;

[0100] Acquisition mode: multiple reaction monitoring mode (MRM);

[0101] The mass spectrometry parameters were automatically optimized using FIA, and the following parameters were obtained: electrospray voltage: -3000 V; curtain gas (curtain gas): 30 psi; nebulizer gas (gas 1): 40 psi; auxiliary gas (gas 2): 50 psi; ion source temperature (TEM): 650 °C.

[0102] Table 2 Mass spectrometry parameter information

[0103]

[0104] Example 2

[0105] Methodological verification of the detection method of 3-nitropropionic acid provided in Example 1:

[0106] 1. Intra-batch and inter-batch precision and accuracy assessment:

[0107] Multiple human plasma samples without 3-NPA were selected and mixed as blank matrices for methodological validation. 3-NPA quality control solutions of corresponding concentrations were added to the plasma blank matrices to prepare plasma quality control samples of low, medium and high concentrations, and the procedures were performed according to the "Sample Pretreatment" in Example 1. Within the same batch, 6 samples were prepared and measured at each concentration to examine the intra-batch variation.

[0108] Three different batches of plasma samples with low, medium and high concentrations were prepared according to the above method, and the standard curve prepared on the same day was used for analysis and determination to examine the batch variation. The accuracy of the method was examined based on the degree of proximity between the sample measured concentration and the theoretical true concentration.

[0109] The results are shown in Table 3. The test results show that the accuracy of all quality control samples is within the error range, and all samples are included in the statistics. The accuracy of all three quality control samples with different concentrations is between 92.00% and 103.44%, and the RSD% of the quality control samples is <9.11%. This shows that this quantitative method is stable and reliable, and meets the requirements for human plasma sample detection.

[0110] Table 3 Intra-batch and inter-batch precision and accuracy data of 3-NPA

[0111]

[0112] 2. Selective inspection:

[0113] The chromatographic separation of 3-NPA and the interfering peaks in the matrix was tested. The results showed that the separation was greater than 1.5, and the interference around the 3-NPA peak time would not affect the quantitative analysis. The chromatograms of 3-NPA in the surrogate matrix, standard solution, and blank plasma were compared. Figure 1~Figure 3 shown.

[0114] 3. Linear range

[0115] Five samples of 7 relative concentrations of standard solutions of 3-NPA (3-NPA linear working solution in Example 1) of the surrogate matrix were prepared and analyzed in the same run. The allowable deviation of linearity is ±15% of the true value. With the concentration of 3-NPA as the abscissa and the peak area as the ordinate, the regression equations are shown in the following table.

[0116] Table 4 3-NPA linear range data

[0117]

[0118] 4. Recovery and Matrix Effects

[0119] According to CLSI guidelines, linear working solutions were used to calculate the recovery and matrix effect. Seven different concentrations of 3-NPA linear working solutions were added to different matrices to prepare analytical samples in triplicate. Group A contained pure water solution, and groups B and C contained blank plasma.

[0120] The working solution was added to groups A and C. Groups B and C were prepared according to the sample pretreatment steps of Example 1. After extracting the supernatant, group B was reconstituted with the linear working solution.

[0121] The recovery of the analyte was obtained by calculating the ratio of the peak area of ​​the analyte in group C to the peak area at the corresponding concentration in group B. The ratio of the linear slope of the analyte in group B to the linear slope of group A was calculated to obtain the matrix effect of the analyte. The results are shown in the following table.

[0122] Table 5 3-NPA recovery data

[0123]

[0124] Table 6 3-NPA matrix effect data

[0125]

[0126] The ratio of the linear slope of group B analytes to the linear slope of group A was calculated to obtain the matrix effect of the analytes. The matrix effect of the method in Example 1 was 87.63%, indicating that after the blood sample was pre-treated and chromatographically separated, the interference of the matrix on 3-NPA was very low.

[0127] 5. Carryover effect

[0128] The residual was evaluated by injecting the sample containing the highest linear concentration point of 3-NPA and the blank sample. The residual was required to be less than 20% of the LLOQ of the analyte. The results showed that the method of Example 1 had no carryover interference.

[0129] 6. Stability

[0130] The three concentrations of quality control solutions L, M, and H configured in Table 6 were selected and added to blank plasma samples to prepare quality control samples. The stability of the plasma quality control samples at room temperature for 6 hours and the stability of the pretreated samples refrigerated in an automatic sampler for 6 hours were investigated. The results are shown in the following table.

[0131] Table 7 3-NPA stability data

[0132]

[0133] 7. Limit of Quantitation

[0134] Add 3-NPA linear working solution at the minimum quantification limit concentration to blank plasma to prepare plasma quality control samples and follow the sample pretreatment procedure. Prepare and measure 6 samples at the minimum quantification limit concentration to examine the fluctuation of the minimum quantification limit. The experimental results are shown in the table below.

[0135] Table 8 3-NPA minimum quantification limit data

[0136]

[0137] 8. Detection Limit

[0138] According to the signal-to-noise ratio of 30 with a minimum quantification limit of 1 ng / mL, the detection limit of this method was calculated to be 0.1 ng / mL.

[0139] Example 3

[0140] Different pretreatment methods were compared to investigate the extraction efficiency of 3-NPA in plasma and the complexity of the pretreatment operation. Methods 1 to 5 were all performed according to the sample pretreatment method in step 2 of Example 1.

[0141] Method 1: Use methanol to precipitate protein.

[0142] Method 2: Use acetonitrile to precipitate protein.

[0143] Method 3: Use methanol-acetonitrile (V / V, 1:1) solution to precipitate protein.

[0144] Method 4: Use methanol-acetonitrile (V / V, 1:2) solution to precipitate protein.

[0145] Method 5: Use methanol-acetonitrile (V / V, 2:1) solution to precipitate protein.

[0146] Method 6: Use 10% trichloroacetic acid solution to precipitate protein.

[0147] Method 7: Use 5% perchloric acid solution to precipitate protein.

[0148] Method 8: Use saturated ammonium sulfate solution to precipitate protein.

[0149] Method 9: Use a saturated sodium chloride solution-acetonitrile (V / V, 1:1) solution to precipitate the protein.

[0150] Method 10: Use solid phase extraction method to precipitate protein.

[0151] Solid phase extraction column model: BondElut-PSA solid phase extraction column (3 mL / 500 mg) (Agilent, USA).

[0152] Solid phase extraction steps: The PSA solid phase extraction column was pre-activated with 1 mL of acetonitrile, 1 mL of plasma sample solution was passed through the column, eluted with 1 mL of 10% methanol aqueous solution, blown dry, and eluted with 1 mL of 10% ammonia-methanol solution. The eluate was collected, blown to near dryness with nitrogen, and fixed to 0.2 mL with 0.4% formic acid aqueous solution.

[0153] The results are shown in Table 9: It can be seen that the recovery rate can reach the level of pretreatment using solid phase extraction when using methanol and / or acetonitrile as protein precipitants. In addition, the recovery rates of the pretreatment methods of methods 2 and 3 can exceed those of solid phase extraction. When the pretreatment was methanol-acetonitrile (V / V, 1:1) solution to precipitate protein, the separation degree of 3-NPA and interference in plasma was good (see Figure 4 and Figure 5 ), and the recovery rate was the best, up to 98.02%; when the pretreatment was acetonitrile protein precipitation, the separation degree of 3-NPA and interference in plasma was not as good as that using methanol-acetonitrile solution (see Figure 6 and Figure 7 ), the recovery rate was 90.22%, but it also exceeded the solid phase extraction method. When using acid reagents and salt reagents to precipitate proteins, the recovery rate of 3-NPA was significantly lower than that of the precipitation agent system containing methanol and / or acetonitrile and the precipitation scheme using solid phase extraction. Therefore, it is preferred to precipitate proteins with a protein precipitant composed of methanol and / or acetonitrile, which is simple and rapid compared to solid phase extraction.

[0154] Table 9

[0155]

[0156] Example 4

[0157] Comparison of the effects of different mobile phase systems on 3-NPA detection results:

[0158] Mobile phase system 1: 0.05% v / v formic acid in water; mobile phase B: acetonitrile solution.

[0159] Mobile phase system 2: 0.1% v / v formic acid in water; mobile phase B: acetonitrile solution.

[0160] Mobile phase system three: 0.2% v / v formic acid in water; mobile phase B: acetonitrile solution.

[0161] Other detection steps and parameters are the same as those in Example 1.

[0162] The experimental results are shown in Table 10. The results show that when the ratio of formic acid solution in mobile phase A is 0.1%, the chromatographic separation effect is better, the baseline is stable, the peak shape is symmetrical, the separation degree is good, and the addition of formic acid can significantly enhance the retention of 3-NPA in the chromatographic column, thereby improving the separation of 3-NPA and matrix interference. Therefore, the mobile phase is preferably: phase A (aqueous solution containing 0.1% v / v formic acid)-phase B (acetonitrile solution).

[0163] Table 10

[0164]

[0165] Example 5

[0166] Comparison of the effects of different elution procedures on 3-NPA detection results:

[0167] Elution procedure 1: Follow the elution procedure in Table 1 of Example 1.

[0168] Elution procedure 2: as shown in Table 11

[0169] Table 11

[0170]

[0171] Other detection steps and parameters are the same as those in Example 1.

[0172] After adjusting the elution ratio of the mobile phase at different times, the retention time of each chromatographic peak eluted according to elution procedure 1 is moderate, and the baseline is stable and not easy to drift. At the same time, the separation of the chromatogram is improved, and the tailing phenomenon of the chromatogram is effectively avoided, which is beneficial to the detection and analysis of 3-NPA. The typical spectrum of 3-NPA eluted according to elution procedure 1 is as follows Figure 8 shown.

[0173] Example 6

[0174] Compare the effects of different chromatographic columns on 3-NPA detection results:

[0175] Chromatographic column 1: Waters Acquity BEH C18 (2.1 mm × 150 mm × 1.7 μm).

[0176] Chromatographic column 2: Phenomenex Kinetex C18 (2.6 μm, 3 mm × 100 mm).

[0177] Chromatographic column three: Phenomenex Kinetex EVO C18 (2.6 μm, 3 mm × 100 mm).

[0178] Other detection steps and parameters are the same as those in Example 1.

[0179] The results showed that 3-NPA was moderately retained on the Waters Acquity BEH C18 column (2.1 mm × 150 mm × 1.7 μm), the chromatographic peak was symmetrical and had good separation from the interferences in plasma (see Fig. 9 ), which has poor retention in Phenomenex Kinetex C18 (2.6 μm, 3 mm × 100 mm) and Phenomenex Kinetex EVO C18 (2.6 μm, 3 mm × 100 mm) ( Fig.10 and Fig.11 ) resulted in incomplete separation of 3-NPA and interference in plasma, resulting in loss of sensitivity. Therefore, Waters Acquity BEH C18 (2.1mm×150mm×1.7μm) was selected as the preferred chromatographic column.

[0180] Example 7

[0181] The effects of different column temperatures (20°C, 30°C, 40°C and 50°C) on the detection results of 3-NPA were compared. Except for the column temperature, the other detection steps and parameters were the same as those in Example 1. The results showed that when the column temperature was 40°C, the retention time of the chromatographic peak was appropriate, the baseline was stable, the separation of each chromatographic peak was good, and the peak shape was symmetrical, so the column temperature was preferably 40°C.

[0182] 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 it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting 3-nitropropionic acid, characterized in that, The detection method comprises detecting the pretreated sample using high performance liquid chromatography tandem mass spectrometry, inserting the detection value into the standard curve, and calculating the content of 3-nitropropionic acid in the sample; The sample includes a blood sample; The standard curve is constructed using the external standard method; The pretreatment is to mix the sample with a protein precipitation reagent, separate the supernatant and perform high performance liquid chromatography tandem mass spectrometry detection, wherein the protein precipitation reagent contains methanol and acetonitrile in a volume ratio of 1:1; HPLC conditions included: The chromatographic column used was a C18 column with a specification of 2.1 mm × 150 mm × 1.7 μm; The mobile phase includes mobile phase A and mobile phase B, wherein the mobile phase A is an aqueous solution of formic acid, and the mobile phase B is acetonitrile; According to the gradient elution, the elution program is: At 0-1.5 min, the volume ratio of the mobile phase A to the mobile phase B was maintained at 85:15; At 1.5-1.6 min, the volume ratio of the mobile phase A to the mobile phase B changed from 85:15 to 10:90; At 1.6-2.2 min, the volume ratio of the mobile phase A to the mobile phase B was maintained at 10:90; At 2.2-2.3 min, the volume ratio of the mobile phase A to the mobile phase B changed from 10:90 to 5:95; At 2.3-3.7 min, the volume ratio of the mobile phase A to the mobile phase B was maintained at 5:95; At 3.7-3.8 min, the volume ratio of the mobile phase A to the mobile phase B changed from 5:95 to 85:15; At 3.8-5.0 min, the volume ratio of the mobile phase A to the mobile phase B was maintained at 85:15; and, the mobile phase flow rate was 0.2~0.5mL / min.

2. The detection method according to claim 1, characterized in that: The mobile phase A contains formic acid 0.05-0.2% v / v.

3. The detection method according to claim 1, characterized in that At 0~1.5min, the flow rate was 0.2 mL / min; at 1.5~1.6min, the flow rate changed from 0.2mL / min to 0.5mL / min; at 1.6~2.2min, the flow rate was 0.5 mL / min; at 2.2~2.3min, the flow rate changed from 0.5mL / min to 0.2mL / min; at 2.3~5.0min, the flow rate was 0.2 mL / min.

4. The detection method according to claim 1, characterized in that The chromatographic column is a Waters Acquity BEHC18 chromatographic column, with specifications of 2.1 mm×150 mm×1.7 μm.

5. The detection method according to claim 1, characterized in that: Mass spectrometry conditions included: Scanning mode: electrospray ionization positive ion mode; Acquisition mode: multiple reaction monitoring mode; Electrospray voltage: -3000 V; curtain gas: 30 psi; nebulizer gas: 40 psi; auxiliary gas: 50 psi; ion source temperature: 650°C.

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