Method for detecting content of 4-boron-L-phenylalanine in animal blood

Abstract

The invention provides a method for detecting the content of 4-boron-L-phenylalanine in animal blood, which is characterized in that the content of 4-boron-L-phenylalanine (L-BPA) is detected through LC-MS / MS. The detection method comprises the following steps: S1, pretreating the animal blood to obtain a computer analysis solution; s2, carrying out on-machine separation on the on-machine analysis solution through LC-MS / MS and carrying out mass spectrometry; and S3, analyzing and processing the data to obtain the content of the 4-boron-L-phenylalanine (L-BPA). The 4-boron-L-phenylalanine (L-BPA) detection method provided by the invention is simple in pretreatment step and accurate in detection result, and has the advantages of high precision and easiness in operation.

Description

Technical Field

[0001] This invention relates to the field of 4-boron-L-phenylalanine content detection technology, specifically to a method for detecting 4-boron-L-phenylalanine content in animal blood. Background Technology

[0002] L-BPA, chemically named 4-boron-L-phenylalanine, was first synthesized by Snyder et al. in 1958. After years of research and clinical trials, L-BPA, as a boron drug, can specifically enter and accumulate in tumor cells through a highly expressed amino acid transporter pathway, thereby enabling boron neutron capture therapy (BNCT) under neutron radiation. It is noteworthy that although L-BPA shares a chemical structural similarity with phenylalanine, the active optical isomer of the α-amino acid in vivo, the introduction of a boron atom at the 4-position of the benzene ring in L-BPA leads to significant differences in its biosynthesis and transformation, properties and applications, as well as detection and analysis. In recent years, although domestic and international research institutions and companies have been continuously exploring the application of L-BPA in BNCT, limitations in its detection methods still hinder the assurance of safety and efficacy in BNCT treatment, thus limiting its widespread application and in-depth exploration in BNCT therapy.

[0003] Currently, the main method for detecting L-BPA content is inductively coupled plasma (ICP). While ICP can detect boron concentration, it suffers from drawbacks such as complex operation, difficult sample preparation, high operating costs, expensive instruments, and interference from biological sample matrices. Furthermore, it can only measure elemental concentration and cannot distinguish between the isotopes boron-11 and boron-10. However, for BNCT therapy, accurately measuring the distribution of boron drugs in tumor tissue is crucial. Therefore, a convenient, rapid, and accurate method for detecting L-BPA is urgently needed. Summary of the Invention

[0004] The purpose of this invention is to solve the technical problems of cumbersome pretreatment and poor detection accuracy in existing methods for detecting 4-boron-L-phenylalanine in animal blood, and to provide the following technical solution:

[0005] A method for detecting the content of 4-boron-L-phenylalanine in animal blood, wherein the content of 4-boron-L-phenylalanine (L-BPA) is detected by LC-MS / MS.

[0006] Includes the following steps:

[0007] S1: Pre-process the animal blood to obtain an analytical solution;

[0008] S2: The analytical solution was separated by LC-MS / MS and analyzed by mass spectrometry.

[0009] S3: Data analysis and processing to obtain the content of 4-boron-L-phenylalanine (L-BPA).

[0010] The specific operation of step S1 is as follows:

[0011] S11: Take animal blood and let it stand in a centrifuge tube. Centrifuge at the first temperature and separate the plasma sample into another centrifuge tube under ice bath conditions. Add sodium bisulfite, lysis working solution, and sodium hydroxide, then add n-butanol. Vortex centrifuge and take the upper organic phase as the first intermediate sample.

[0012] S12: Add internal standard working solution to the first intermediate sample in step S11, vortex and centrifuge at the second temperature, and take the supernatant into another centrifuge tube to obtain the second intermediate sample.

[0013] S13: Add ultrapure water to the second intermediate sample, vortex, centrifuge at the third temperature, sample, filter, dilute, and filter through an aqueous PES membrane to obtain the upper analytical solution.

[0014] In step S11, the pyrolysis working solution is an aqueous solution of any one of formic acid, hydrochloric acid, and phosphoric acid.

[0015] In one embodiment, in step S11, the values ​​of plasma, sodium bisulfite, formic acid or phosphoric acid, and sodium hydroxide are in the following relationship: 1 mL : (0.1-0.2) mg : (0.2-0.5) μL : (0.25-0.5) mmol.

[0016] In one embodiment, in step S11, the values ​​of plasma, sodium bisulfite, hydrochloric acid, and sodium hydroxide are in the following relationship: 1 mL : (0.1-0.2) mg : (0.1-0.2) μL : (0.25-0.5) mmol.

[0017] Preferably, the volume fraction of the formic acid aqueous solution and the volume fraction of the phosphoric acid aqueous solution are both (2-5)%, and the volume fraction of the hydrochloric acid aqueous solution is (0.5-1)%.

[0018] Preferably, the first temperature, the second temperature, and the third temperature are all between 2°C and 8°C.

[0019] In step S2, the chromatographic separation conditions for LC-MS / MS are as follows:

[0020] Chromatographic column: C18 column (2.1×50mm, 1.6μm),

[0021] Mobile phase: Mobile phase A: 2 mmol / L ammonium acetate aqueous solution containing 0.1% formic acid (volume fraction);

[0022] Mobile phase B: a 2 mmol / L ammonium acetate to acetonitrile solution containing 0.1% formic acid (volume fraction) (2 mmol / L ammonium acetate to acetonitrile volume ratio of 5:95);

[0023] Flow rate: 0.5 mL / min;

[0024] Injection volume: 2 μL;

[0025] Column temperature: 40℃;

[0026] Elution method: gradient elution.

[0027] The specific conditions for gradient elution are as follows:

[0028]

[0029]

[0030] In step S2, the LC-MS / MS mass spectrometry analysis uses an ESI ion source, positive ion mode, and multiple reaction monitoring.

[0031] In step S2, the mass spectrometry analysis conditions for LC-MS / MS are as follows:

[0032] Spray voltage: 5000V;

[0033] Vortex ion spray temperature: 500℃

[0034] Air curtain gas: 35 psi;

[0035] Collision gas: 10 psi;

[0036] Nebulizer gas: 45 psi;

[0037] Auxiliary gas: 45 psi;

[0038] Input voltage: 10V;

[0039] Data acquisition time: 3.00 min.

[0040] The present invention has the following advantages:

[0041] (1) This invention is the first to propose a method using LC-MS / MS to detect the content of 4-boron-L-phenylalanine (L-BPA) in animal blood. This method not only allows for the complete extraction and release of 4-boron-L-phenylalanine (L-BPA) from animal blood through convenient and simple sample pretreatment, improving the accuracy of content detection, but also accurately detects and analyzes the content of isotopes boron-11 and boron-10. Furthermore, this method is simple to operate, rapid and efficient, requires simple sample preparation and small sample volumes, and the chromatographic and mass spectrometric conditions are easy to optimize and control, resulting in fast analysis speed, making it suitable for widespread application.

[0042] (2) In the sample pretreatment method provided by this invention, by adding sodium bisulfite, lysis working solution, and sodium hydroxide under ice bath conditions and during the extraction process, followed by the addition of n-butanol, the extraction rate of 4-boron-L-phenylalanine during pretreatment is improved, thus increasing the accuracy of detecting 4-boron-L-phenylalanine (L-BPA) in animal blood. Simultaneously, the chromatographic separation of 4-boron-L-phenylalanine (L-BPA) is simplified, resulting in very clear mass spectrometry peaks without the influence of impurity peaks. Furthermore, the detection method provided by this invention, combined with the C18 column used in LC-MS / MS, although simple in operation, results in fewer impurities in the analyzed sample and a good peak shape for the target analyte (L-BPA), leading to high overall detection accuracy.

[0043] The pretreatment working solution, combined with the LC-MS / MS technology used, can detect compounds at extremely low concentrations. Through simple pretreatment, the combination of chromatographic separation and mass spectrometry detection eliminates interference from endogenous substances, isomers and other metabolites, providing accurate qualitative and quantitative results. It can detect L-BPA in blood samples and determine the elemental valence state, effectively improving the detection rate of boron 10 concentration.

[0044] (4) Compared with the existing technology of using ICP emission spectroscopy to detect L-BPA, which requires a large amount of inert gas (such as argon) to be consumed during operation and has high operating costs, the LC-MS / MS method can not only reduce the cost of analysis and detection, but its highly automated sample processing and analysis system can reduce human operation errors and interference, and improve the accuracy and reliability of analysis. Attached Figure Description

[0045] Figure 1 The LC-MS / MS spectrum of the internal standard (Acetaminophen-IS1);

[0046] Figure 2 The LC-MS / MS spectrum of L-BPA;

[0047] Figure 3 The image shows the LC-MS / MS spectra of L-BPA and internal standard in the animal blood sample from Example 1.

[0048] Figure 4 The image shows the LC-MS / MS spectrum of L-BPA in the animal blood sample from Example 10. Detailed Implementation

[0049] The following detailed description of embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely illustrates some embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0050] It should be noted that, unless otherwise specified, the embodiments and features and technical solutions in the present invention can be combined with each other.

[0051] Example 1:

[0052] The content of 4-boron-L-phenylalanine (L-BPA) in animal blood was detected by LC-MS / MS.

[0053] Includes the following steps:

[0054] S1: Pre-process the animal blood to obtain an analytical solution;

[0055] S2: The analytical solution was separated by LC-MS / MS and analyzed by mass spectrometry.

[0056] S3: Data analysis and processing to obtain the content of 4-boron-L-phenylalanine (L-BPA).

[0057] The specific operation of step S1 is as follows:

[0058] S11: Take 1 mL of animal blood (in this example, the animal blood is from a beagle dog) and let it stand for 2 hours in a centrifuge tube containing EDTA-K2 anticoagulant. Centrifuge at 2-8°C at a speed of 2000g for 8 minutes. After centrifugation, separate the plasma sample into another blank centrifuge tube under ice bath conditions. Add 10 μL of 2% formic acid aqueous solution (volume fraction) (i.e., 0.2 μL of formic acid) to the centrifuge tube, 0.1 mg of sodium bisulfite and 100 μL of 2.5 mol / L sodium hydroxide solution (i.e., 0.25 mmol of sodium hydroxide) to the centrifuge tube. After shaking and mixing, add 250 μL of n-butanol and centrifuge by vortexing. Take 100 μL of the upper organic phase into another blank centrifuge tube to obtain the first intermediate sample.

[0059] S12: Add 300 μL of Acetaminophen-IS1 internal standard solution with a concentration of 1 μg / mL to the first intermediate sample in step S11, vortex at 2500 rpm for 3 min, centrifuge at (2-8)℃ and 3200 g for 10 min, and take the supernatant into another blank centrifuge tube (in some embodiments, this step can be done by using a 96-well plate instead of a centrifuge tube) to obtain the second intermediate sample;

[0060] S13: Add 250 μL of ultrapure water to the second intermediate sample, vortex at 2500 rpm for 3 min, centrifuge at 3200 g for 5 min at (2-8) °C, take a sample, filter through an aqueous PES membrane, and obtain the analytical solution. As described in the above embodiments, the first, second, and third temperatures are (2-8) °C.

[0061] In step S2, the chromatographic separation conditions for LC-MS / MS are set as follows:

[0062] Chromatographic column: C18 column (2.1×50mm, 1.6μm),

[0063] Mobile phase: Mobile phase A: 2 mmol / L ammonium acetate aqueous solution containing 0.1% formic acid (volume fraction);

[0064] Mobile phase B: a 2 mmol / L ammonium acetate to acetonitrile solution containing 0.1% formic acid (volume fraction) (2 mmol / L ammonium acetate to acetonitrile volume ratio of 5:95); in this embodiment, mobile phase A is prepared by adding 1 mL of formic acid to 1 L of 2 mmol / L ammonium acetate aqueous solution; mobile phase B is prepared by mixing 50 mL of 2 mmol / L ammonium acetate with 950 mL of acetonitrile, and then adding 1 mL of formic acid.

[0065] Injection washing solution: A mixed solution of methanol-acetonitrile-isopropanol-water (volume ratio of methanol-acetonitrile-isopropanol-water is 1:1:1:1) containing 0.1% formic acid (volume fraction);

[0066] Needle washing method:

[0067] Value Clean Time Solvent 2(s): 2;

[0068] Post Clean Time Solvent 2(s): 2;

[0069] Value Clean Time Solvent 1(s): 3;

[0070] Post Clean Time Solvent 1(s): 2;

[0071] StatorWash Time Solvent 2(s): 5;

[0072] StatorWash Time Solvent 1(s): 5;

[0073] Flow rate: 0.5 mL / min;

[0074] Injection volume: 2 μL;

[0075] Column temperature: 40℃;

[0076] Elution method: gradient elution.

[0077] The specific conditions for gradient elution are as follows:

[0078] Retention time (min) Mobile phase A (volume fraction, %) Mobile phase B (volume fraction, %) 0.00 99.0 1.0 1.10 99.0 1.0 1.11 10.0 90.0 2.00 10.0 90.0 2.01 99.0 1.0 3.00 99.0 1.0

[0079] In step S2, the LC-MS / MS mass spectrometry analysis is set to use an ESI ion source, positive ion mode, and multiple reaction monitoring (MRM).

[0080] In step S2, the mass spectrometry analysis conditions for LC-MS / MS are as follows:

[0081] Spray voltage: 5000V;

[0082] Vortex ion spray temperature: 500℃

[0083] Air curtain gas: 35 psi;

[0084] Collision gas: 10 psi;

[0085] Nebulizer gas: 45 psi;

[0086] Auxiliary gas: 45 psi;

[0087] Input voltage: 10V;

[0088] Data acquisition time: 3.00 min.

[0089] In step S3, the internal standard method is used to perform quantitative analysis on the mass spectrometry data detected in step S2. The specific operation is as follows:

[0090] The specific operation is as follows: Step S31: Prepare the standard curve. Weigh 100.01 mg of L-BPA standard into a volumetric flask, dilute and dissolve it with 25% formic acid solution (volume fraction) (wherein, the volume of preparation = weighed amount * content (99.9%) / concentration of preparation), to prepare a standard solution with a concentration of 48.0 mg / mL, and then dilute it with ultrapure water to prepare a standard stock solution with a concentration of 12.0 mg / mL. Then, after dilution with 25% formic acid aqueous solution (volume fraction), a series of standard curve working solutions are obtained.

[0091] Preparation of standard curve samples: Add 47.5 μL of 2% formic acid aqueous solution to blank plasma (volume ratio 1:15) to an EP tube. Then add 2.5 μL of standard curve working solution to each EP tube to prepare dilutions of 0.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 600 μg / mL. Nine standard solutions of different concentrations were prepared, and 300 μL of Acetaminophen-IS1 internal standard solution with a concentration of 1 μg / mL was added to each standard solution. Each standard solution was vortexed at 2500 rpm for 3 min and centrifuged at 3200 g for 10 min at (2-8) °C. The supernatant was then transferred to another blank centrifuge tube (in some embodiments, this step can be done using a 96-well plate instead of a centrifuge tube). 250 μL of ultrapure water was added, and the solution was vortexed at 2500 rpm for 3 min and centrifuged at 3200 g for 5 min at 2-8 °C. The sample was then filtered through an aqueous PES filter membrane to obtain the standard sample analytical solution to be analyzed.

[0092] Each standard sample was analyzed by LC-MS / MS. The ratio of the peak area of ​​the standard sample to the peak area of ​​the internal standard (Acetaminophen-IS1) was used as the y-axis value, and the concentration of the standard sample was used as the x-axis. The linear regression equation y = 0.0154x ± 0.000932 was calculated, and the standard curve was obtained. The linear correlation coefficient r in the linear regression equation is... 2 >0.9904.

[0093] Step S32: Substitute the L-BPA peak area obtained in step S2 into the linear regression equation to calculate the concentration of L-BPA, and then calculate the content of L-BPA in the animal blood. The content of L-BPA is 411 μg / mL.

[0094] Example 2:

[0095] Example 2 used the same animal blood and the same pretreatment and detection methods as Example 1. The difference was that in step S11, formic acid was replaced with phosphoric acid in different amounts, and the amounts of sodium bisulfite and sodium hydroxide were also different. In Example 2, 10 μL of 5% phosphoric acid aqueous solution (volume fraction) (i.e., 0.5 μL of phosphoric acid) and 0.2 mg of sodium bisulfite and 100 μL of 5 mol / L sodium hydroxide solution (i.e., 0.5 mmol of sodium hydroxide) were added in step S11. The L-BPA content was found to be 421 μg / mL.

[0096] Example 3:

[0097] Example 3 used the same animal blood and the same pretreatment and detection methods as Example 1. The difference was that in step S11, hydrochloric acid was used instead of formic acid and the amount added was different. The amounts of sodium bisulfite and sodium hydroxide added were also different. In Example 2, in step S11, 10 μL of 0.15% hydrochloric acid aqueous solution (volume fraction) (i.e., the amount of hydrochloric acid added was 0.35 μL), 0.15 mg of sodium bisulfite and 100 μL of 4 mol / L sodium hydroxide solution (i.e., 0.4 mmol of sodium hydroxide added) were added; the L-BPA content was detected to be 407 μg / mL.

[0098] Example 4:

[0099] Example 4 used the same animal blood and the same pretreatment and detection methods as Example 1. The difference was that the amounts of formic acid, sodium bisulfite, and sodium hydroxide added in step S11 were different. In Example 2, 10 μL of 3% formic acid aqueous solution (volume fraction) (i.e., the formic acid content was 0.3 μL) and 0.12 mg of sodium bisulfite and 100 μL of 3 mol / L sodium hydroxide solution (i.e., 0.3 mmol of sodium hydroxide) were added in step S11; the L-BPA content was detected to be 410 μg / mL.

[0100] Example 5:

[0101] Example 5 used the same animal blood and the same pretreatment and detection methods as Example 1. The difference was that the amounts of formic acid, sodium bisulfite, and sodium hydroxide added in step S11 were different. In Example 2, 10 μL of 7% formic acid aqueous solution (volume fraction) (i.e., the formic acid content was 0.7 μL) and 0.3 mg of sodium bisulfite and 100 μL of 7 mol / L sodium hydroxide solution (i.e., 0.3 mmol of sodium hydroxide) were added in step S11; the L-BPA content was detected to be 394 μg / mL.

[0102] Example 6:

[0103] Example 6 used the same animal blood and the same pretreatment and detection methods as Example 1. The difference was that the amounts of formic acid, sodium bisulfite, and sodium hydroxide added in step S11 were different. In Example 2, 10 μL of 1% formic acid aqueous solution (volume fraction) (i.e., the formic acid content was 0.1 μL) and 0.1 mg of sodium bisulfite and 100 μL of 1 mol / L sodium hydroxide solution (i.e., 0.1 mmol of sodium hydroxide) were added in step S11; the L-BPA content was detected to be 384 μg / mL.

[0104] Comparing Examples 1-6, the optimal and most accurate detection of L-BPA in plasma was achieved when the ratio of plasma, sodium bisulfite, formic acid or phosphoric acid, and sodium hydroxide was 1 mL : (0.1-0.2) mg : (0.2-0.5) μL : (0.25-0.5) mmol; or when the ratio of plasma, sodium bisulfite, hydrochloric acid, and sodium hydroxide was 1 mL : (0.1-0.2) mg : (0.1-0.2) μL : (0.25-0.5) mmol.

[0105] Example 7:

[0106] Example 7 used the same animal blood and the same pretreatment and detection methods as Example 1. The difference was that sodium bisulfite was not added in step S11. Everything else was the same as in Example 1. The L-BPA content was detected by LC-MS / MS to be 201 μg / mL.

[0107] Example 8:

[0108] Example 8 used the same animal blood and the same pretreatment and detection methods as Example 1. The difference was that in step S11, no lysis working solution (i.e., no formic acid, hydrochloric acid, or phosphoric acid) was added. Everything else was the same as in Example 1. The L-BPA content was 124 μg / mL as detected by LC-MS / MS.

[0109] Example 9:

[0110] Example 9 used the same animal blood and the same pretreatment and detection methods as Example 1. The difference was that sodium hydroxide was not added in step S11. Everything else was the same as in Example 1. The L-BPA content was detected by LC-MS / MS to be 214 μg / mL.

[0111] Example 10:

[0112] Example 10 used the same animal blood and pretreatment and detection methods as Example 1, except that n-butanol was not added in step S11. After vortex centrifugation, 100 μL of the supernatant was transferred to another blank centrifuge tube to obtain the first intermediate sample. Everything else was consistent with Example 1. LC-MS / MS detection and calculation showed that the L-BPA content was 398 μg / mL, but the chromatogram showed a high level of impurities (see Appendix). Figure 4 ).

[0113] Comparative analysis of Examples 1-4 and Examples 7-10 revealed that the absence of any one of the extraction agents—sodium bisulfite, lysis working solution, or sodium hydroxide—during plasma pretreatment affected the extraction rate of L-BPA in plasma, thus impacting the accuracy of L-BPA detection results. When preparing L-BPA samples for analysis, the addition of sodium bisulfite, lysis working solution, and sodium hydroxide resulted in the highest extraction rate of L-BPA from blood and the most accurate detection results. Furthermore, the addition of n-butanol facilitated sample purification. This step not only did not reduce the detection concentration of L-BPA but also improved the accuracy of the L-BPA detection results to some extent.

[0114] Example 11:

[0115] Example 11 used the same animal blood and the same pretreatment and detection methods as Example 1, except that the first, second and third temperatures were all at room temperature. After LC-MS / MS detection and analysis, the content of L-BPA was measured to be 322 μg / mL.

[0116] Example 12:

[0117] Example 12 used the same animal blood and the same pretreatment and detection methods as Example 1. The difference was that the first, second and third temperatures were all (9-15)℃. After LC-MS / MS detection and analysis, the content of L-BPA was measured to be 336 μg / mL.

[0118] Example 13:

[0119] Example 13 used the same animal blood and the same pretreatment and detection methods as Example 1, except that the first, second and third temperatures were all (-5-1)℃. After LC-MS / MS detection and analysis, the content of L-BPA was measured to be 401 μg / mL.

[0120] Comparing Examples 1, 6, and 11-13, the detection results of L-BPA were most accurate and precise only when various extraction additives were used in combination under the condition of low temperature.

[0121] Example 14: Recovery Rate Verification Experiment

[0122] S11: Take 1 mL of animal blood (in this example, the animal blood is from a beagle dog) and let it stand for 2 hours in a centrifuge tube containing EDTA-K2 anticoagulant. Centrifuge at 2-8°C at a speed of 2000g for 8 minutes. After centrifugation, separate the plasma sample into another blank centrifuge tube under ice bath conditions. Add 10 μL of 2% formic acid aqueous solution (volume fraction) (i.e., 0.2 μL of formic acid) to the centrifuge tube, 0.1 mg of sodium bisulfite and 100 μL of 2.5 mol / L sodium hydroxide solution (i.e., 0.25 mmol of sodium hydroxide) to the centrifuge tube. After shaking and mixing, add 250 μL of n-butanol and centrifuge by vortexing. Take 100 μL of the upper organic phase into another blank centrifuge tube to obtain the first intermediate sample.

[0123] Example 14 uses the same detection and analysis method as Example 1, and the specific operation is as follows:

[0124] The stability of blank plasma is as follows:

[0125] Three 950 μL blank blood samples were collected and placed in centrifuge tubes containing EDTA-K2 anticoagulant. 50 μL of standard curve working solution of different concentrations was added to prepare three blank plasma samples with L-BPA concentrations of 1.5 μg / mL, 150 μg / mL, and 375 μg / mL, respectively. After standing for 2 h, the samples were centrifuged at 2–8 °C for 8 min at 2000 g. After centrifugation, the plasma samples were separated into blank centrifuge tubes under ice bath conditions. 10 μL of 2% formic acid aqueous solution (volume fraction) (i.e., 0.2 μL of formic acid) and 0.1 mg of sodium bisulfite and 100 μL of 2.5 mol / L sodium hydroxide solution (i.e., 0.25 mmol of sodium hydroxide) were added to each centrifuge tube sequentially, and the mixture was shaken to mix. Afterwards, 250 μL of n-butanol was added, and the mixture was vortexed and centrifuged. 100 μL of the upper organic phase was transferred to another blank centrifuge tube. 300 μL of Acetaminophen-IS1 internal standard solution (1 μg / mL) was added to each of the three centrifuge tubes. The mixture was vortexed at 2500 rpm for 3 min, centrifuged at 3200 g at (2–8) °C for 10 min, and the supernatant was transferred to another blank centrifuge tube (in some embodiments, this step can be done using a 96-well plate instead of a centrifuge tube). 250 μL of ultrapure water was added, and the mixture was vortexed at 2500 rpm for 3 min, centrifuged at 3200 g at 2–8 °C for 5 min, and the sample was filtered through an aqueous PES membrane to obtain the analytical solution. The solution was then analyzed by LC-MS / MS. After LC-MS / MS analysis, the L-BPA concentrations in three blank plasma samples containing L-BPA were calculated to be 1.45 μg / mL, 147 μg / mL, and 377 μg / mL, respectively. (The concentrations mentioned here are the converted L-BPA concentrations in the plasma.) The recovery rates for the three experiments were 96.7%, 98%, and 100.5%, respectively, all of which were higher than 95%, indicating good recovery performance.

[0126] The stability analysis of non-blank plasma was performed as follows: 950 μL of blood from Example 1 was taken into centrifuge tubes containing EDTA-K2 anticoagulant, and 50 μL of standard curve working solution of different concentrations was added to prepare three blank plasma samples with L-BPA concentrations of 1.5 μg / mL, 150 μg / mL, and 375 μg / mL, respectively. The samples were allowed to stand for 2 h, then centrifuged at 2–8 °C at 2000 g for 8 min. After centrifugation, the plasma samples were separated into blank centrifuge tubes under ice bath conditions. 10 μL of 2% formic acid aqueous solution (volume fraction) (i.e., 0.2 μL of formic acid) and 0.1 mg sodium bisulfite and 100 μL of 2.5 mol / L sodium hydroxide solution (i.e., 0.25 mmol of sodium hydroxide) were added to each centrifuge tube sequentially. l), after shaking and mixing, add 250 μL of n-butanol, vortex centrifuge, and take 100 μL of the upper organic phase into another blank centrifuge tube; add 300 μL of Acetaminophen-IS1 internal standard solution with a concentration of 1 μg / mL to each of the three centrifuge tubes, vortex at 2500 rpm for 3 min, centrifuge at (2-8) °C and 3200 g for 10 min, take the supernatant into another blank centrifuge tube (in some embodiments, this step can be done using a 96-well plate instead of centrifuge tubes), add 250 μL of ultrapure water, vortex at 2500 rpm for 3 min, centrifuge at 2-8 °C and 3200 g for 5 min, take the sample, filter through an aqueous PES filter membrane to obtain the analytical solution, and analyze it by LC-MS / MS. After LC-MS / MS analysis, the L-BPA content in the plasma of the three animals containing L-BPA was found to be 412.6 μg / mL, 563 μg / mL and 782 μg / mL, respectively. The recovery rates of the three experiments were 106.7%, 101.3% and 98.9%, which met the requirements.

[0127] The recovery rate verification test showed that the recovery rate of both blank plasma and other samples met expectations, indicating that the detection method had good stability and small error.

[0128] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for detecting the content of 4-boron-L-phenylalanine in animal blood, characterized in that, The content of 4-boron-L-phenylalanine (L-BPA) was determined by LC-MS / MS.

2. The method for detecting 4-boron-L-phenylalanine content in animal blood as described in claim 1, characterized in that, Includes the following steps: S1: Pre-process the animal blood to obtain an analytical solution; S2: The analytical solution was separated by LC-MS / MS and analyzed by mass spectrometry. S3: Data analysis and processing to obtain the content of 4-boron-L-phenylalanine (L-BPA).

3. The method for detecting 4-boron-L-phenylalanine content in animal blood as described in claim 1, characterized in that, The specific operation of step S1 is as follows: S11: Take animal blood and let it stand in a centrifuge tube. Centrifuge at the first temperature and separate the plasma sample into another centrifuge tube under ice bath conditions. Add sodium bisulfite, lysis working solution, and sodium hydroxide, then add n-butanol. Vortex centrifuge and take the upper organic phase as the first intermediate sample. S12: Add internal standard working solution to the first intermediate sample in step S11, vortex and centrifuge at the second temperature, and take the supernatant into another centrifuge tube to obtain the second intermediate sample. S13: Add ultrapure water to the second intermediate sample, vortex, centrifuge at the third temperature, sample, filter, dilute, and filter through an aqueous PES membrane to obtain the upper analytical solution.

4. The method for detecting the content of 4-boron-L-phenylalanine in animal blood as described in claim 3, characterized in that, In step S11, the pyrolysis working solution is an aqueous solution of any one of formic acid, hydrochloric acid, and phosphoric acid.

5. The method for detecting the content of 4-boron-L-phenylalanine in animal blood as described in claim 4, characterized in that, In step S11, the values ​​of plasma, sodium bisulfite, formic acid or phosphoric acid, and sodium hydroxide are in the following relationship: 1 mL : (0.1-0.2) mg : (0.2-0.5) μL : (0.25-0.5) mmol.

6. The method for detecting 4-boron-L-phenylalanine content in animal blood as described in claim 4, characterized in that, In step S11, the values ​​of plasma, sodium bisulfite, hydrochloric acid, and sodium hydroxide are in the following relationship: 1 mL : (0.1-0.2) mg : (0.1-0.2) μL : (0.25-0.5) mmol.

7. The method for detecting 4-boron-L-phenylalanine content in animal blood as described in claim 3, characterized in that, The first, second, and third temperatures are all between 2°C and 8°C.

8. The method for detecting 4-boron-L-phenylalanine content in animal blood as described in claim 2, characterized in that, In step S2, the chromatographic separation conditions for LC-MS / MS are as follows: Chromatographic column: C18 column (2.1×50mm, 1.6μm), Mobile phase: Mobile phase A: 2 mmol / L ammonium acetate aqueous solution containing 0.1% formic acid (volume fraction); Mobile phase B: a 2 mmol / L ammonium acetate to acetonitrile solution containing 0.1% formic acid (volume fraction) (2 mmol / L ammonium acetate to acetonitrile volume ratio of 5:95); Flow rate: 0.5 mL / min; Injection volume: 2 μL; Column temperature: 40℃; Elution method: gradient elution.

9. The method for detecting the content of 4-boron-L-phenylalanine in animal blood as described in claim 7, characterized in that, The specific conditions for gradient elution are as follows: 。 10. The method for detecting 4-boron-L-phenylalanine content in animal blood as described in claim 2, characterized in that, In step S2, the LC-MS / MS mass spectrometry analysis used an ESI ion source, positive ion mode, and multiple reaction monitoring. The mass spectrometry analysis conditions were as follows: Spray voltage: 5000V; Vortex ion spray temperature: 500℃ Air curtain gas: 35 psi; Collision gas: 10 psi; Nebulizer gas: 45 psi; Auxiliary gas: 45 psi; Input voltage: 10V; Data acquisition time: 3.00 min.

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