Preparation method of compound sodium nitrophenolate stable isotope internal standard reagent and application of compound sodium nitrophenolate stable isotope internal standard reagent in quantitative detection of compound sodium nitrophenolate

Abstract

The invention discloses a preparation method of a compound sodium nitrophenolate stable isotope internal standard reagent and application of the compound sodium nitrophenolate stable isotope internal standard reagent in quantitative detection of compound sodium nitrophenolate, and belongs to the technical field of chemical analysis. The internal standard reagent is deuterated compound sodium nitrophenolate; the deuterated compound sodium nitrophenolate comprises deuterated sodium o-nitrophenolate, deuterated sodium p-nitrophenolate and deuterated 5-nitro o-methoxy sodium phenolate. The reagent can eliminate the interference influence caused by a matrix effect and a pretreatment process in a liquid chromatography-tandem mass spectrometry method, can be used as a standard reagent for quantitatively detecting the compound sodium nitrophenolate, can accurately correct ionization efficiency deviation and the matrix effect, and remarkably improves the recovery rate and repeatability of the method. In addition, according to the synthetic method of the deuterated compound sodium nitrophenolate, raw materials are cheap and easy to obtain, reaction conditions are mild, the yield and the atom utilization rate are high, and the synthetic method is suitable for stable isotope labeled compound sodium nitrophenolate.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and particularly relates to a preparation method of a sodium nitrophenolate stable isotope internal standard reagent and application of the reagent in the quantitative detection of sodium nitrophenolate. Background Art

[0002] Sodium Compound Nitrophenolate (Sodium Compound Nitrophenolate) is a compound plant growth regulator composed of three sodium nitrophenol salts: sodium o-nitrophenolate (ONP), sodium p-nitrophenolate (PNP), and sodium 5-nitro-o-methoxyphenol (5NG, also known as sodium 5-nitroguaiacol). Its chemical structure is as follows: Among them, A is sodium o-nitrophenolate, B is sodium p-nitrophenolate, and C is sodium 5-nitro-o-methoxyphenolate.

[0003] Sodium nitrophenolate exhibits significant cross-species bioactivity, possessing plant and animal physiological regulation properties and broad-spectrum applications. Recent studies have confirmed that sodium nitrophenolate accumulates in the human body through the food chain, inducing methemoglobinemia, eye irritation, and multi-organ toxicity. The synergistic effect of its phenolic and nitrophenyl groups can damage the human endocrine, circulatory, and reproductive systems. Although this chemical component has been included in the "List of Prohibited Drugs and Other Compounds for Use in Food Animals" through Announcement No. 250 and has a maximum residue limit (MRL) in plant-derived foods set by GB 2763-2021, no systematic technical specifications for its detection have been established, resulting in a "no-standard" residue monitoring environment. Currently, the main testing methods for sodium nitrophenolate include liquid chromatography, liquid chromatography-tandem mass spectrometry, and gas chromatography. Liquid chromatography-tandem mass spectrometry and gas chromatography offer higher sensitivity than liquid chromatography. Gas chromatography requires a derivatization reagent to determine sodium nitrophenolate. While liquid chromatography-tandem mass spectrometry is the gold standard for trace residue quantification, its detection efficiency is limited by complex matrix interferences. The introduction of a stable isotope internal standard method can accurately correct for ionization efficiency deviations and matrix effects, significantly improving method recovery (>90%) and reproducibility (RSD <5%).

[0004] Currently, there is no corresponding stable isotope-labeled sodium nitrophenolate internal standard compound in the existing technology. Therefore, the development of stable isotope-labeled sodium nitrophenolate internal standard reagent is of great significance to food production companies, third-party testing agencies, food safety regulatory departments and other related fields. Summary of the Invention

[0005] The present invention aims to provide a stable isotope-labeled deuterated sodium nitrophenolate, which can eliminate the interference caused by matrix effects and pretreatment processes in liquid chromatography-tandem mass spectrometry and serve as a standard reagent for the quantitative detection of sodium nitrophenolate.

[0006] The technical solutions of the present invention are as follows: The present invention provides a sodium nitrophenolate stable isotope internal standard reagent, wherein the internal standard reagent is deuterated sodium nitrophenolate; the deuterated sodium nitrophenolate comprises deuterated sodium o-nitrophenolate, deuterated sodium p-nitrophenolate and deuterated sodium 5-nitro-o-methoxyphenolate; specifically, the deuterated sodium o-nitrophenolate is sodium o-nitrophenolate-D4, the deuterated sodium p-nitrophenolate is sodium p-nitrophenolate-D4, and the deuterated sodium 5-nitro-o-methoxyphenolate is sodium 5-nitro-o-methoxyphenolate-D3.

[0007] The present invention provides application of the sodium nitrophenolate stable isotope internal standard reagent in the quantitative detection of sodium nitrophenolate in a matrix.

[0008] In the above application, the matrix is ​​selected from one of the animal foods of pig, cattle, chicken meat, liver, kidney and fat.

[0009] The preparation method of the above-mentioned sodium o-nitrophenolate-D4 or sodium p-nitrophenolate-D4 comprises the following steps: Phenol-2,3,4,5,6-D5 and ferric nitrate nonahydrate are added to a solvent to react; after the reaction is completed, extraction is performed; the organic phase is subjected to gradient elution to obtain o-nitrophenol-D4 and p-nitrophenol-D4, respectively; then o-nitrophenol-D4 or p-nitrophenol-D4 and sodium hydroxide are added to a solvent to react; after the reaction is completed, reduced pressure distillation is performed to obtain sodium o-nitrophenol-D4 or sodium p-nitrophenol-D4.

[0010] In the above-mentioned preparation method of sodium o-nitrophenolate-D4 or sodium p-nitrophenolate-D4, the molar ratio of phenol-2,3,4,5,6-D5 and ferric nitrate nonahydrate is selected from 1 to 3:1, preferably 2:1.

[0011] In the above-mentioned preparation method of sodium o-nitrophenolate-D4 or sodium p-nitrophenolate-D4, the reaction conditions are selected from: reacting at room temperature for 0.5 to 20 hours.

[0012] In the above-mentioned preparation method of sodium o-nitrophenolate-D4 or sodium p-nitrophenolate-D4, the elution solvent is a mixed solution of EA and PE, and the volume ratio of the two is set in a gradient from 1:20 to 1:5.

[0013] In the above-mentioned preparation method of sodium o-nitrophenolate-D4 or sodium p-nitrophenolate-D4, the molar ratio of o-nitrophenol-D4 or p-nitrophenol-D4 to sodium hydroxide is selected from 0.5 to 2:1; preferably 1:1.

[0014] The preparation method of the above-mentioned 5-nitro-o-methoxyphenol sodium-D3 comprises the following steps: Add o-phenylenedimethyl ether-D4 and ferric nitrate nonahydrate to a solvent and react; after the reaction is completed, extract; elute the organic phase to obtain 3,4-dimethoxynitrobenzene-D3; then add 3,4-dimethoxynitrobenzene-D3 and sodium hydroxide to a solvent and react under light; after the reaction is completed, extract, elute the organic phase to obtain 5-nitro-o-methoxyphenol-D3; then add 5-nitro-o-methoxyphenol-D3 and sodium hydroxide to a solvent and react; after the reaction is completed, distill under reduced pressure to obtain sodium 5-nitro-o-methoxyphenol-D3.

[0015] In the above-mentioned preparation method of sodium 5-nitro-o-methoxyphenolate-D3, the molar ratio of the o-phenylenedimethyl ether-D4 and ferric nitrate nonahydrate is selected from 1 to 3:1, preferably 1:1.

[0016] In the above-mentioned preparation method of sodium 5-nitro-o-methoxyphenol-D3, the molar ratio of the 3,4-dimethoxynitrobenzene-D3 and sodium hydroxide is selected from 0.5 to 2:3; preferably 1:3.

[0017] In the above-mentioned preparation method of sodium 5-nitro-o-methoxyphenol-D3, the molar ratio of 5-nitro-o-methoxyphenol-D3 and sodium hydroxide is selected from 1 to 3:1; preferably 1:1.

[0018] In the above-mentioned preparation method of sodium 5-nitro-o-methoxyphenolate-D3, the reaction conditions are selected from: reacting at room temperature for 0.5 to 20 hours.

[0019] In the above-mentioned preparation method of sodium 5-nitro-o-methoxyphenol-D3, the eluent for the first elution is a mixed solution of EA and PE, with a volume ratio of 1:20; the eluent for the second elution is a mixed solution of EA and PE, with a volume ratio of 1:5.

[0020] The beneficial effects of the present invention are: The present invention provides a sodium nitrophenolate stable isotope internal standard reagent. This internal standard reagent is deuterated sodium nitrophenolate, including deuterated sodium o-nitrophenolate, deuterated sodium p-nitrophenolate, and deuterated sodium 5-nitro-o-methoxyphenolate. This internal standard reagent can eliminate interference caused by matrix effects and pretreatment processes in liquid chromatography-tandem mass spectrometry. As a standard reagent for the quantitative detection of sodium nitrophenolate, it can accurately correct ionization efficiency deviation and matrix effects, significantly improving the method's recovery rate (>90%) and repeatability (RSD <5%). Furthermore, the synthesis method of deuterated sodium nitrophenolate of the present invention utilizes inexpensive and readily available raw materials, mild reaction conditions, and high yield and atom utilization, making it suitable for the synthesis of stable isotope-labeled sodium nitrophenolate. The deuterated sodium nitrophenolate provided by the present invention exhibits promising application prospects and value in research fields such as food safety and environmental monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the primary mass spectrum of o-nitrophenol-D4; Figure 2 is the primary mass spectrum of p-nitrophenol-D4; Figure 3 is the primary mass spectrum of 5-nitro-o-methoxyphenol-D3; Figure 4 is the secondary mass spectrum of o-nitrophenol-D4; Figure 5 is the secondary mass spectrum of p-nitrophenol-D4; Figure 6 is the secondary mass spectrum of 5-nitro-o-methoxyphenol-D3; Figure 7 is a liquid chromatography tandem mass spectrometry; Figure 8 The quantitative limit of sodium nitrophenolate in pork; where: 1: p-nitrophenol; 2: o-nitrophenol; 3: p-nitrophenol-D4; 4: o-nitrophenol-D4; 5: 5-nitro-o-methoxyphenol; 6: 5-nitro-o-methoxyphenol-D3. DETAILED DESCRIPTION

[0022] The sodium nitrophenolate stable isotope internal standard reagent prepared by the present invention is deuterated sodium nitrophenolate, and its structural formula is as follows: Among them, A is sodium o-nitrophenolate-D4, B is sodium p-nitrophenolate-D4, and C is sodium 5-nitro-o-methoxyphenolate-D3.

[0023] The above synthesized compounds were used as internal standards for the detection of sodium nitrophenolate residues in animal foods for the first time. At the same time, the existing technology has not reported that other isotope labels can be used for the detection of sodium nitrophenolate residues.

[0024] In the present invention, hexafluoroisopropanol (HFIP), dichloroethane (DCE), ethyl acetate (EA), petroleum ether (PE), tetrahydrofuran (THF).

[0025] In the present invention, the final product synthesized is sodium phenolate. After being dissolved in a solvent, this type of compound will become phenol in subsequent measurements. Therefore, the measurement results are calculated in terms of phenol, representing sodium phenolate.

[0026] The other materials used in the present invention, unless otherwise stated, can be obtained through commercial channels. Unless otherwise specified, other terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below with reference to specific examples and data. The following examples are merely for illustration of the present invention and are not intended to limit the scope of the present invention in any way.

[0027] Example 1 Preparation of sodium o-nitrophenolate-D4 and sodium p-nitrophenolate-D4, the steps are as follows: Phenol-2,3,4,5,6-D5 (2 mmol, 1 equivalent) was placed in a round-bottom flask, followed by the addition of ferric nitrate nonahydrate (1 mmol, 0.5 equivalent) and 10 mL of a mixture of HFIP and DCE (volume ratio 1:19). The reaction was allowed to react at room temperature for 12 h. After completion of the reaction, the reaction was quenched with saturated brine and filtered. The filtrate was extracted three times with 50 mL of EA. The organic phase was washed once with saturated aqueous NaCl and dried over anhydrous Na2SO4. The volatile solvent was evaporated under reduced pressure, and the remaining organic phase was mixed with silica gel powder. Elution was performed using a gradient of EA:PE (1:20 to 1:5) to obtain o-nitrophenol-D4 and p-nitrophenol-D4 (o-nitrophenol-D4 was eluted at 1:20, and p-nitrophenol-D4 was eluted with increasing the polarity to 1:5). The yields of o-nitrophenol-D4 and p-nitrophenol-D4 were 20% and 35%, respectively.

[0028] Add o-nitrophenol-D4 or p-nitrophenol-D4 (0.2 mmol, 1 equivalent) and sodium hydroxide (0.2 mmol, 1 equivalent) to a round-bottom flask with 5 mL of methanol as solvent. Stir at room temperature for 30 min. After the reaction is complete as detected by spot plate detection, vacuum distillation is performed to obtain sodium o-nitrophenol-D4 or sodium p-nitrophenol-D4.

[0029] The above reaction process is as follows: The nuclear magnetic resonance data of sodium o-nitrophenolate-D4 are as follows: 1 H NMR (500 MHz, Methanol-d4) 0 H; 13C NMR (126 MHz, Methanol-d4) δ170.44, 141.55, 137.82, 129.42, 114.79.

[0030] The nuclear magnetic resonance data of sodium p-nitrophenolate-D4 are as follows: 1 H NMR (500 MHz, Methanol-d4) 0 H; 13 C NMR (126 MHz, Methanol-d4) δ163.69, 140.24, 125.31, 114.94.

[0031] Example 2 Preparation of 5-nitro-o-methoxyphenol sodium-D3, the steps are as follows: 3 mmol, 1 equivalent of o-phenylenedimethyl ether (D4) was placed in a round-bottom flask, followed by the addition of ferric nitrate nonahydrate (3 mmol, 1 equivalent) and 20 mL of HFIP solvent. The reaction was allowed to proceed at room temperature for 12 h. After completion of the reaction, the reaction was quenched with water and filtered. The filtrate was extracted three times with 50 mL of EA. The organic phase was washed once with a saturated aqueous NaCl solution and dried over anhydrous Na2SO4. The volatile solvent was evaporated under reduced pressure, and the remaining organic phase was mixed with silica gel powder. 3,4-dimethoxynitrobenzene (D3) was obtained by column chromatography using an EA:PE ratio of 1:20 as the eluent. The product was purified by column chromatography to yield 3,4-dimethoxynitrobenzene (D3) in a 65% yield.

[0032] 3,4-Dimethoxynitrobenzene-D3 (0.3 mmol, 1 equivalent), NaOH (1 mmol, 3 equivalents), 0.1 mL H2O, and 0.9 mL THF were added to a photoreaction tube. The reaction was allowed to proceed under illumination for 4 hours. After the reaction was complete, the reaction was quenched with 1 mol / L HCl and extracted three times with 50 mL EA. The organic phase was washed once with a saturated aqueous NaCl solution and dried over anhydrous Na2SO4. The volatile solvent was evaporated under reduced pressure, and the remaining organic phase was mixed with silica gel powder. Elution with EA:PE = 1:5 gave 5-nitro-o-methoxyphenol-D3 in a 10% yield.

[0033] 5-Nitro-o-methoxyphenol-D3 (0.2 mmol, 1 equivalent) and sodium hydroxide (0.2 mmol, 1 equivalent) were added to a round-bottom flask with 5 mL of methanol as solvent. The mixture was stirred at room temperature for 30 min. After the reaction was complete as detected by spot plate detection, sodium 5-nitro-o-methoxyphenol-D3 was obtained by vacuum distillation.

[0034] The nuclear magnetic resonance data of 5-nitro-o-methoxyphenol sodium-D3 are as follows: 1 H NMR (500 MHz, Methanol-d4) δ 3.81 (s, 3H); 13 C NMR (126 MHz, Methanol-d4) δ 157.97, 142.10, 111.03, 109.05, 107.83, 54.59.

[0035] Using the above-mentioned deuterated sodium nitrophenolate as an internal standard reagent for the detection of sodium nitrophenolate in the liquid chromatography-mass spectrometry method can calibrate the sample pretreatment loss and the corresponding instrument deviation, significantly improving the accuracy and repeatability of the detection.

[0036] 1. UPLC-MS / MS Analysis Method Stock solutions of sodium o-nitrophenolate, sodium p-nitrophenolate, sodium 5-nitro-o-methoxyphenolate, sodium o-nitrophenolate-D4, sodium p-nitrophenolate-D4, and sodium 5-nitro-o-methoxyphenolate-D3 (calculated as o-nitrophenol, p-nitrophenol, 5-nitro-o-methoxyphenol, o-nitrophenol-D4, p-nitrophenol-D4, and 5-nitro-o-methoxyphenol-D3, respectively) at a concentration of 1 mg / mL were prepared in methanol, and then diluted to 1 μg / mL with methanol. The solutions were filtered through a 0.22 μm membrane and analyzed by UPLC-MS / MS.

[0037] The liquid chromatograph model was Agilent 1290Ⅱ, and the mass spectrometer model was AB SCIEX 5500+.

[0038] Liquid phase conditions: The chromatographic column was InfinityLab Poroshell 120 EC-C18 (1.9 μm, 2.1 × 50 mm), the column temperature was 40°C, the injection volume was 5 μL, the mobile phase A was water, the mobile phase B was acetonitrile, and the gradient elution method was adopted. The time was 6 min and the flow rate was 0.4 mL / min. The mobile phase composition was 95% A and 5% B from 0 to 1 min, 5% A and 95% B from 4 to 5 min, and 95% A and 5% B from 5.1 to 6 min.

[0039] Mass spectrometry conditions were as follows: atmospheric pressure chemical ionization source, declustering voltage of -80 V, collision energy of -20 V, curtain gas of 35 psi, collision gas of 6 psi, corona needle current of -3 mA, heating temperature of 400 °C, spray gas of 65 psi, collision cell outlet voltage of -14 V, collision cell inlet voltage of -13 V, and dwell time of 50 ms.

[0040] The test results are as follows Figures 1 to 7 As shown: Depend on Figure 1 and Figure 2 It can be seen that the parent ions of o-nitrophenol-D4 and p-nitrophenol-D4 are 142. Figure 3 It can be seen that the parent ion of 5-nitro-o-methoxyphenol-D3 is 171, and the detected value is consistent with the theoretical value. The experimental data further verified its structure. Figure 4 and Figure 5 It can be seen that the daughter ions of o-nitrophenol-D4 and p-nitrophenol-D4 are 112 and 96; Figure 6 It can be seen that the daughter ions of 5-nitro-o-methoxyphenol-D3 are 156 and 126. Therefore, o-nitrophenol-D4 and p-nitrophenol-D4 have exactly the same parent ions and daughter ions, so the established UPLC-MS / MS analysis method needs to completely separate them for differentiation. Figure 7 It can be seen that the retention times of o-nitrophenol, p-nitrophenol and 5-nitro-o-methoxyphenol are 2.83, 2.54 and 2.62 min, respectively, and the retention times of o-nitrophenol-D4, p-nitrophenol-D4 and 5-nitro-o-methoxyphenol-D3 are 2.83, 2.54 and 2.62 min, respectively. It can be seen that the UPLC-MS / MS analysis method of sodium nitrophenolate and deuterated sodium nitrophenolate is established through the above liquid phase and mass spectrometry conditions.

[0041] 2. Application of Deuterated Sodium Nitrophenolate in the Detection of Sodium Nitrophenolate in Animal Food Pork liver was selected as animal food, and the accuracy and sensitivity of the test method when deuterated sodium nitrophenolate was used as the internal standard reagent were determined through a spiked recovery experiment.

[0042] Working fluid: Take appropriate amounts of stock solutions of o-nitrophenol, p-nitrophenol, and 5-nitro-o-methoxyphenol to prepare a mixed working solution; the concentrations of o-nitrophenol, p-nitrophenol, and 5-nitro-o-methoxyphenol are 2.5 μg / mL, 1.0 μg / mL, and 0.5 μg / mL, respectively. Take appropriate amounts of stock solutions of o-nitrophenol-D4, p-nitrophenol-D4, and 5-nitro-o-methoxyphenol-D3 to prepare a mixed internal standard working solution; the concentrations of o-nitrophenol-D4, p-nitrophenol-D4, and 5-nitro-o-methoxyphenol-D3 are 0.5 μg / mL, 0.2 μg / mL, and 0.1 μg / mL, respectively.

[0043] Sample preparation: Take fresh or thawed pig liver tissue, mince it, and homogenize it. Each portion of 5 g (accurate to ±0.05 g) is divided into 50 mL stoppered centrifuge tubes.

[0044] extract: Take 5 g of sample (accurate to ±0.05 g) and place it in a 50 mL centrifuge tube. Add 100 μL of the mixed internal standard working solution and mix thoroughly. Add 2 mL of water and vortex for 30 seconds. Add 13 mL of acetonitrile and vortex for 30 seconds. Ultrasonic extraction is performed for 10 minutes. Centrifuge at 8000 rpm for 10 minutes. Transfer the supernatant to another 50 mL centrifuge tube, add 10 mL of acetonitrile to the residue, and repeat the extraction once. Combine the two extracts, add 2 mL of ammonia water, and set aside.

[0045] Purification: A mixed-mode strong anion exchange solid-phase extraction column (60 mg / 3 cc) was activated sequentially with 3 mL of methanol and 3 mL of water. The reserve solution was passed through the column at a controlled flow rate of 1 drop / s. Elute with 3 mL of 5% ammonia water and 3 mL of methanol, draining the column. Elute with 3 mL of 2% formic acid in methanol and collect the eluate. Add water to a volume of 5 mL, filter through a 0.22 µm filter, and prepare for liquid chromatography-tandem mass spectrometry analysis.

[0046] Add recovery rate: 5 g of the sample was placed in a 50 mL centrifuge tube and an appropriate amount of the mixed working solution was added to obtain the limit of quantification (LOQ), 2 times the limit of quantification (2 LOQ), and 10 times the limit of quantification (10 LOQ), respectively. The spiked concentrations of o-nitrophenol were 5 µg / kg, 10 µg / kg, and 50 µg / kg; the spiked concentrations of p-nitrophenol were 2 µg / kg, 4 µg / kg, and 20 µg / kg; and the spiked concentrations of 5-nitro-o-methoxyphenol were 1 µg / kg, 2 µg / kg, and 10 µg / kg.

[0047] Except for not adding the mixed internal standard working solution during extraction, the sample extraction and cleanup were carried out according to the above method, and 5 μL was injected for analysis to obtain the absolute spiked recovery.

[0048] During extraction, 100 μL of mixed internal standard working solution was added, and then sample extraction and cleanup were performed according to the above method. 5 μL was taken for injection and analysis to obtain the relative recovery rate.

[0049] The absolute and relative recovery results of sodium nitrophenolate in pig liver are shown in Table 1. The test results are calculated as o-nitrophenol, p-nitrophenol, and 5-nitro-o-methoxyphenol, respectively.

[0050] Table 1 Recovery rates of the three components of sodium nitrophenolate As shown in Table 1, the absolute recovery of sodium nitrophenolate is between 75.19% and 92.41%, and the RSD is between 5.27% and 9.20%, of which the absolute recovery of 5-nitro-o-methoxyphenol is only 75.19-80.92%. By adding the mixed internal standard working solution, the recovery of sodium nitrophenolate has been greatly improved and the experimental error has been reduced, especially 5-nitro-o-methoxyphenol has been greatly improved. The relative recovery rate is above 90%, and the RSD is less than 5%. It can be seen that the recovery rate and repeatability of the method are significantly improved by adding deuterated sodium nitrophenolate. At the same time, the above data can meet the requirements of quantitative limit detection. The quantitative limit results of sodium nitrophenolate detection in pork are as follows. Figure 8 shown.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. A sodium nitrophenolate stable isotope internal standard reagent, characterized in that: The internal standard reagent is deuterated sodium nitrophenolate; the deuterated sodium nitrophenolate includes deuterated sodium o-nitrophenolate, deuterated sodium p-nitrophenolate and deuterated sodium 5-nitro-o-methoxyphenolate.

2. The sodium nitrophenolate stable isotope internal standard reagent according to claim 1, wherein The preparation method of sodium o-nitrophenolate-D4 or sodium p-nitrophenolate-D4 comprises the following steps: Phenol-2,3,4,5,6-D5 and ferric nitrate nonahydrate are added to a solvent to react; after the reaction is completed, extraction is performed; the organic phase is subjected to gradient elution to obtain o-nitrophenol-D4 and p-nitrophenol-D4, respectively; then o-nitrophenol-D4 or p-nitrophenol-D4 and sodium hydroxide are added to a solvent to react; after the reaction is completed, reduced pressure distillation is performed to obtain sodium o-nitrophenol-D4 or sodium p-nitrophenol-D4.

3. The sodium nitrophenolate stable isotope internal standard reagent according to claim 2, wherein The molar ratio of the phenol-2,3,4,5,6-D5 to ferric nitrate nonahydrate is selected from 1 to 3:1; the molar ratio of the o-nitrophenol-D4 or p-nitrophenol-D4 to sodium hydroxide is selected from 0.5 to 2:

1.

4. The sodium nitrophenolate stable isotope internal standard reagent according to claim 2, wherein The reaction conditions are selected from: reacting at room temperature for 0.5 to 20 h.

5. The sodium nitrophenolate stable isotope internal standard reagent according to claim 2, wherein The eluent for elution is a mixed solution of EA and PE, with a volume ratio of the two being set in a gradient from 1:20 to 1:

5.

6. The sodium nitrophenolate stable isotope internal standard reagent according to claim 1, wherein The preparation method of 5-nitro-o-methoxyphenol sodium-D3 comprises the following steps: Add o-phenylenedimethyl ether-D4 and ferric nitrate nonahydrate to a solvent and react; after the reaction is completed, extract; elute the organic phase to obtain 3,4-dimethoxynitrobenzene-D3; then add 3,4-dimethoxynitrobenzene-D3 and sodium hydroxide to a solvent and react under light; after the reaction is completed, extract, elute the organic phase to obtain 5-nitro-o-methoxyphenol-D3; then add 5-nitro-o-methoxyphenol-D3 and sodium hydroxide to a solvent and react; after the reaction is completed, distill under reduced pressure to obtain sodium 5-nitro-o-methoxyphenol-D3.

7. The sodium nitrophenolate stable isotope internal standard reagent according to claim 6, wherein The molar ratio of the o-phenylenedimethyl ether-D4 and ferric nitrate nonahydrate is selected from 1 to 3:1; the molar ratio of the 3,4-dimethoxynitrobenzene-D3 and sodium hydroxide is selected from 0.5 to 2:3; and the molar ratio of the 5-nitro-o-methoxyphenol-D3 and sodium hydroxide is selected from 1 to 3:

1.

8. The sodium nitrophenolate stable isotope internal standard reagent according to claim 6, wherein The reaction conditions are selected from: reacting at room temperature for 0.5 to 20 h.

9. The sodium nitrophenolate stable isotope internal standard reagent according to claim 6, characterized in that The eluent for the first elution is a mixed solution of EA and PE, with a volume ratio of 1:20; the eluent for the second elution is a mixed solution of EA and PE, with a volume ratio of 1:

5.

10. Use of the sodium nitrophenolate stable isotope internal standard reagent according to claim 1 in the quantitative detection of sodium nitrophenolate in a matrix.

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