Method for measuring isotope abundance of p-acetamino anisole (methoxy-13C) in kit

Abstract

The invention relates to a method for determining isotopic abundance of p-acetamino anisole (methoxy-13C) in a kit, which comprises the following steps: grinding p-acetamino anisole (methoxy-13C) particles in the kit into powder, placing the powder in a centrifuge tube, adding an ether solvent, uniformly mixing by vortex, carrying out centrifugal extraction, filtering the obtained supernatant, drying, and grinding to obtain the isotopic abundance of p-acetamino anisole (methoxy-13C). Finally, a nuclear magnetic instrument is adopted for detection, a nuclear magnetic spectrogram is obtained, analysis and calculation are conducted, and the < 13 > C isotope abundance is obtained. Compared with the prior art, the method has the advantages that the isotope abundance of the p-acetamino anisole (methoxy-13C) in the kit subjected to specific pretreatment is measured by utilizing a nuclear magnetic method, the method is good in repeatability and high in accuracy, and the method is complementary with a mass spectrometry method to meet the requirements of the medicine field and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of stable isotope abundance and relates to a method for preparing acetaminophen (methoxy- 13 C) Methods for determining isotope abundance. Background Art

[0002] Acetaminophenoxyethyl 13 C), also known as methacetin (methoxy- 13 C), mainly used as medical diagnostic reagents, liver function and liver disease detection 13 CO2 breath reagent. In the detection of liver function and its degree of damage, acetaminophen (methoxy- 13 C) has the advantages of non-invasiveness, safety, high sensitivity, and high specificity, and is currently one of the main technical means for detecting liver function damage. 13 C) also contains excipients such as sweeteners, 4-acetaminophen (methoxy- 13 C) After oral administration of the kit, it enters the liver through the blood circulation, and the liver cells metabolize 4-acetaminophen (methoxy- 13 C) Methoxy- 13 C decomposition generates 13 C labels carbon dioxide and excretes it from the body. When the liver is damaged, it breaks down acetaminophen (methoxy- 13 C) Methoxy- 13 C weakens, resulting in a decrease in the isotope ratio of exhaled air. Therefore, acetaminophen (methoxy- 13 C) isotopic abundance is a key indicator for medicinal use.

[0003] Acetaminophenoxyethyl ether (methoxy- 13 C) The existing detection technology for isotope abundance is only liquid chromatography-mass spectrometry, which uses chromatography to separate the acetaminophen (methoxy- 13 C) is separated and then detected by mass spectrometry, and the isotope abundance is determined by the mass-to-charge ratio. According to the "9101 Analytical Method Validation Guidelines" in the 2020 edition of the "Chinese Pharmacopoeia", the key indicator test method should compare the results of the two methods. However, at present, methacetin (methoxy- 13 C) The only isotope detection method is mass spectrometry, which cannot meet the requirements for drug marketing. Therefore, a second method, methacetin (methoxy- 13 C) The detection method of isotope abundance has become a technical problem that needs to be solved urgently.

[0004] For example, Chinese patent application CN202310907680.4 provides a method for measuring the13 The method of isotope abundance of C-labeled compounds is only applicable to high-purity reagents and is not suitable for detection in kits because the acetaminophen (methoxy- 13 C) The drug is not a high-purity reagent, 4-acetaminophen (methoxy- 13 C) accounts for less than 5% of the test kit, which contains a large number of excipients, such as polyethylene glycol, organic sweeteners, inorganic salts and other excipients, among which polyethylene glycol and acetaminophen (methoxy- 13 C) have overlapping chemical shifts, which makes it impossible to distinguish the H NMR spectra, and the signal peak of polyethylene glycol covers the acetaminophenether (methoxy- 13 C) signal peak, the acetaminophen (methoxy- 13 The effective separation and purification of C) is the key to accurate NMR determination. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing acetaminophen (methoxy- 13 C) The method of isotope abundance determination is complementary to mass spectrometry to meet the requirements of diverse drug isotope detection in the medical field, and the method has good repeatability and high accuracy.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] A kind of acetaminophen (methoxy- 13 C) Method for isotope abundance determination: first, take 4-acetaminophen (methoxy- 13 C) The particles are ground into powder, placed in a centrifuge tube, and then an ether solvent is added to vortex and mixed for centrifugal extraction. The supernatant obtained is filtered and dried to obtain a nuclear magnetic resonance sample. Finally, a nuclear magnetic resonance instrument is used for detection to obtain a nuclear magnetic resonance spectrum, which is analyzed and calculated to obtain 13 C isotope abundance.

[0008] Furthermore, the polyethylene glycol added in the kit is soluble in many organic solvents, such as alcohols, ketones, chloroform, glycerides and aromatic hydrocarbons, but is insoluble in conventional ether reagents such as ether and methyl ether. 13 C) is also insoluble in reagents such as methyl ether and ethyl ether. Selecting a suitable extraction reagent is the key to separation and purification. Preferably, the ether solvent in the present invention is methyl tert-butyl ether.

[0009] Furthermore, the ratio of the added amount of the ether solvent to the powder is (5-30) mL: (0.5-3) g.

[0010] Furthermore, the pore size of the organic filter membrane used for filtration is 0.22 μm.

[0011] Furthermore, the drying process is carried out in a vacuum vortex oscillator, the drying temperature is 40-70° C., and the rotation speed is 1000-2000 rpm.

[0012] Furthermore, the analysis process of the NMR spectrum is as follows:

[0013] The range of 4.10ppm-3.30ppm was integrated and the peak area was set to 100, and the range of 3.78ppm-3.68ppm was integrated and the peak area was set to A.

[0014] Furthermore, calculation 13 The formula for C isotope abundance is 100-A, and the unit is atom%. 13 C.

[0015] Furthermore, the powder particle size is 100 mesh.

[0016] The present invention fully grinds and takes part of the sample to carry out experiments. After a large number of experiments, methyl tert-butyl ether is finally selected as the solvent to convert the acetaminophen (methoxy- 13 C) is extracted, and the selected methyl tert-butyl ether is insoluble in polyethylene glycol, organic sweeteners, inorganic salts and other auxiliary materials. At the same time, the boiling point of methyl tert-butyl ether is 55°C, which is lower than that of acetaminophen (methoxy- 13 C) melting point 129 ℃ easy to remove, to ensure that the extracted acetaminophenether (methoxy- 13 C) Purity greater than 99%; based on 13 The C-labeled isotope has a splitting effect on the nuclear magnetic hydrogen spectrum, which can distinguish 13 C and 12 C isotope, and the isotope abundance was calculated by integrating the peak area.

[0017] Compared with the prior art, the present invention uses a specific pre-treatment method to treat the particle raw materials in the kit, and then uses nuclear magnetic resonance to detect, analyze the spectrum and calculate 13 C isotope abundance, the method has good repeatability, high accuracy, and is complementary to mass spectrometry to meet the requirements of the medical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 For acetaminophenoxy ether (methoxy- 13 C) NMR spectrum.

[0019] Figure 2 NMR spectra after treatment with different solvents.

[0020] Figure 3It is the NMR spectrum of Comparative Example 2. DETAILED DESCRIPTION

[0021] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0022] In the following examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0023] Embodiment 1:

[0024] Acetaminophenoxyethyl 13 C) Isotope abundance detection

[0025] Take 2.5g of methacetin (methoxy- 13 C) The particles in the kit were ground using a high-throughput cryo-grinder to a particle size of 200 mesh. The ground sample was placed in a 50 mL centrifuge tube, 15 mL of methyl tert-butyl ether was added and vortexed for extraction, the supernatant was filtered through a 0.22 μm organic filter membrane, and the filtrate was dried by a vacuum vortex oscillator. The temperature was set to 50°C and the speed was set to 1500 r / min. 10 mg of the dried sample was prepared for NMR and transferred to an NMR tube. Two groups were prepared in parallel and sealed for testing. The samples were detected using a JEOL JNM-ECZ 500 MHz superconducting NMR spectrometer from JEOL Ltd. The instrument parameters were pulse angle: 90°, number of scans: 32 times, and relaxation delay time: 30 s. After the test, the peak area of ​​the spectra of the parallel samples after the test was integrated using Delta NMR software, and the spectra were analyzed ( Figure 1 ) is to integrate the 4.10ppm-3.30ppm interval and set the peak area to 100, integrate the 3.78ppm-3.68ppm interval and set the peak area to A, the first group: A is 0.79; the second group: A is 0.82; calculate 13 The formula for C isotope abundance is 100-A. 13 The unit of C isotope abundance is atom% 13 C:

[0026] The deuterium substitution rate of the parallel samples was calculated to be 99.21atom% 13 C. 99.18atom% 13 C, average value is 99.2atom% 13 C.

[0027] The measured data and mass spectrometry test results 99.1atom% 13 C, the deviation is only 0.1atom%13 C, the results are basically consistent, it can be seen that the present invention 13 The C isotope abundance determination method also has a very high measurement accuracy.

[0028] Comparative Example 1:

[0029] Take 2.5g of methacetin (methoxy- 13 C) The particles in the kit were ground using a high-throughput cryo-grinder to a particle size of 200 mesh. The ground sample was placed in a 50 mL centrifuge tube, and 15 mL of an organic solvent (ethanol) was added and vortexed for extraction. The supernatant was filtered through a 0.22 μm organic filter membrane, and the filtrate was dried by a vacuum vortex oscillator with a speed set to 1500 r / min. 10 mg of the dried sample was prepared for nuclear magnetic resonance and transferred to a nuclear magnetic resonance tube. Two groups were prepared in parallel, sealed for testing, and detected using a JEOL JNM-ECZ 500 MHz superconducting nuclear magnetic resonance spectrometer from JEOL Ltd. The instrument parameters were pulse angle: 90°, number of scans: 32 times, and relaxation delay time: 30 s.

[0030] After the test, the peak area of ​​the spectrum of the parallel sample after the test was integrated using Delta NMR software, and the spectrum was analyzed ( Figure 2 ) as shown: It is not difficult to find out from the spectrum that the extraction effect is very poor, and the auxiliary materials are also extracted, interfering with the detection.

[0031] Comparative Example 2:

[0032] Using the test method of Example 1 of invention patent CN202310907680.4, a deuterated solvent (acetone-D6) was added to the sample, and the NMR test spectrum was as follows Figure 3 As shown in the test results, acetaminophen (methoxy- 13 In C), the signal is completely covered and cannot be resolved.

[0033] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for preparing a kit containing acetaminophen (methoxy- 13 C) A method for isotope abundance determination, characterized in that First take the acetaminophen ether (methoxy- 13 C) The particles are ground into powder, placed in a centrifuge tube, and then an ether solvent is added to vortex and mixed for centrifugal extraction. The supernatant obtained is filtered and dried to obtain a nuclear magnetic resonance sample. Finally, a nuclear magnetic resonance instrument is used for detection to obtain a nuclear magnetic resonance spectrum, which is analyzed and calculated to obtain 13 C isotope abundance.

2. A method for preparing a kit of claim 1 comprising preparing acetaminophen (methoxy- 13 C) A method for isotope abundance determination, characterized in that The ether solvent is methyl tert-butyl ether.

3. A method for preparing a kit of claim 1 comprising preparing acetaminophen (methoxy- 13 C) A method for isotope abundance determination, characterized in that The ratio of the added amount of the ether solvent to the powder is (5-30) mL: (0.5-3) g.

4. A method for preparing a kit of claim 1 comprising preparing acetaminophen (methoxy- 13 C) A method for isotope abundance determination, characterized in that The pore size of the organic filter membrane used for filtration is 0.22 μm.

5. A method for preparing a kit of claim 1 comprising preparing acetaminophen (methoxy- 13 C) A method for isotope abundance determination, characterized in that The drying process was carried out in a vacuum vortex shaker.

6. A method for preparing a kit of claim 1 comprising preparing acetaminophen (methoxy- 13 C) A method for isotope abundance determination, characterized in that The drying temperature is 40-70°C and the rotation speed is 1000-2000rpm.

7. A method for preparing a kit of claim 1 comprising preparing acetaminophen (methoxy- 13 C) A method for isotope abundance determination, characterized in that The process of analyzing the NMR spectrum is as follows: The range of 4.10ppm-3.30ppm was integrated and the peak area was set to 100, and the range of 3.78ppm-3.68ppm was integrated and the peak area was set to A.

8. A method for preparing the kit of claim 7 comprising preparing acetaminophen (methoxy- 13 C) A method for isotope abundance determination, characterized in that calculate 13 The formula for C isotope abundance is 100-A, and the unit is atom%. 13 C.

9. A method for preparing a kit according to claim 1 comprising preparing acetaminophen (methoxy- 13 C) A method for isotope abundance determination, characterized in that The particle size of the ground powder is 100-200 mesh.

10. A method for preparing a kit according to claim 1 comprising preparing acetaminophen (methoxy- 13 C) A method for isotope abundance determination, characterized in that The nuclear magnetic resonance instrument used was a JEOL JNM-ECZ 500 MHz superconducting nuclear magnetic resonance spectrometer produced by JEOL Ltd. The instrument parameters were set as follows: pulse angle 90°, scan number 32 times, and relaxation delay time 30 s.

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