RCF determination method based on the integration of qNMR and chromatography technology

Abstract

The present invention discloses a method for determining RCF based on the fusion of qNMR and chromatography technology. In the same qNMR measurement, the spectral constants of all excited atoms are the same and the NMR signal intensity is proportional to the number of atoms. A stable compound or other compound in the system is used as an internal standard. The molar ratio of the internal standard compound to the test compound is calculated from the nuclear magnetic signal. The chromatographic peak area ratio of the internal standard compound to the test compound is determined using chromatography. The two results are fused to calculate the chromatographic RCF of the test compound. Thus, even in the absence of a standard sample of the test compound, the RCF of an unstable, difficult to obtain, and expensive compound can be determined using a readily available, stable, and inexpensive compound.

Description

Technical Field

[0001] The present invention relates to the technical field of organic compound analysis and testing, and in particular to a method for determining a relative molar correction factor (RCF) based on the fusion of quantitative nuclear magnetic resonance (qNMR) technology and chromatography technology. Background Art

[0002] There are three commonly used methods for calculating relative correction factors: the slope ratio method, the multi-point method, and the absorbance ratio method. These methods all require the use of standards, but three types of standards are difficult to purchase and expensive: (1) rare compounds, such as low-content plant active ingredients and drug impurities; (2) unstable compounds, such as cis- and trans-isomers that are susceptible to configurational transformations under the influence of light and heat; and (3) special compounds with personalized needs, such as special catalysts and fluorescent sensors.

[0003] The analysis object of quantitative nuclear magnetic resonance (qNMR) is the atoms contained in the compound molecules (such as 1 H. 13 C. 19 F. 31 qNMR does not require a standard substance identical to the analyte; instead, a compound containing the same atoms as the analyte can be used as an internal standard to achieve absolute quantification, eliminating the need for a standard curve. It has been widely used in pharmaceutical analysis, natural product characterization, and reference material quality control. According to the International Committee for Weights and Measures' definition of benchmark analytical methods, qNMR meets the requirements of benchmark quantitative methods (Fardus-Reid, F., Warren, J., Le Gresley, A., Validating heteronuclear 2D quantitative NMR [J], Analytical methods, 2016, 8(9): 2016-2019).

[0004] In the same qNMR measurement, the spectral constants of all excited atoms are the same and the NMR signal intensity is proportional to the number of atoms. Stable and inexpensive compounds or other compounds in the system can be used as internal standards. The amount of substance ratio of the internal standard to the compound to be measured can be calculated from the nuclear magnetic resonance signal. The ratio of the chromatographic peak area of ​​the internal standard to the compound to be measured is determined using chromatography technology. The results of the two are then combined to calculate the chromatographic RCF of the organic compound to be measured, thereby achieving the purpose of measuring the RCF of unstable, difficult to obtain, and expensive compounds using readily available, stable, and inexpensive compounds.

[0005] In the prior art, Chinese patent publication CN111579663B discloses a method and application for determining the relative correction factor of methanol in methoxymethanol without a standard sample. This method utilizes the reaction of formaldehyde in methoxymethanol with a reagent to release methanol from the methoxymethanol. The ratio of the increase in methanol content to the decrease in methoxymethanol content is the correction factor of methanol in the methoxymethanol relative to free methanol. Chinese patent publication CN110687239A discloses a method for determining the relative mass correction factor of the chromatographic response of impurity derivatives in low-purity samples. This method requires derivatizing the impurities in the sample and then performing gas or liquid chromatography response correction.

[0006] The above-mentioned methods for determining the correction factor are not only cumbersome, requiring weighing, dilution, or derivatization, but also have limited applicability and high operational requirements. Therefore, it is necessary to develop a simple, convenient, and rapid method for determining the relative correction factor. Summary of the Invention

[0007] This invention provides a method for determining RCF based on the fusion of qNMR and chromatography. This method can be used to determine relative molar correction factors (RCFs) in the absence of a standard sample. This method aims to address the difficulty in determining RCFs for compounds for which standards are difficult to purchase. This method is particularly suitable for determining RCFs for compounds for which standards are expensive, unstable, or difficult to obtain.

[0008] The specific technical solutions are as follows:

[0009] See also Figure 1 A method for determining RCF based on the fusion of qNMR and chromatography techniques. In a single qNMR measurement, the spectral constants of all excited atoms are the same, and the NMR signal intensity is proportional to the number of atoms. A stable compound or other compound in the system is used as an internal standard. The molar ratio of the internal standard compound to the test compound is calculated from the NMR signal. The chromatographic peak area ratio of the internal standard compound to the test compound is determined using chromatography. The two results are combined to calculate the chromatographic RCF of the test compound. This allows the RCF of unstable, difficult-to-obtain, and expensive compounds to be determined using readily available, stable, and inexpensive compounds, even in the absence of a standard analyte.

[0010] The chromatogram RCF is calculated as follows:

[0011]

[0012] Where:

[0013] f represents the relative molar correction factor of the test compound relative to the internal standard compound;

[0014] I A Represents the integrated area of ​​the specified peak of the test compound in the NMR spectrum;

[0015] I B Represents the integrated area of ​​the specified peak of the internal standard compound in the NMR spectrum;

[0016] N A Indicates the number of atoms represented by the specified peak in the NMR spectrum of the test compound;

[0017] N B Indicates the number of atoms represented by the specified peak of the internal standard compound in the NMR spectrum;

[0018] A A It represents the peak area of ​​the test compound in the chromatogram;

[0019] A B Represents the peak area of ​​the internal standard compound in the chromatogram.

[0020] The innovation of the determination method of the present invention lies in: making full use of the characteristic that the spectral constants of all excited atoms in qNMR determination are the same, and for the first time integrating nuclear magnetic resonance quantitative technology with chromatography technology to determine the relative molar correction factor of a compound. This can realize the determination of the relative correction factor of a compound in the absence of a standard sample, overcoming the limitation of traditional methods that must use a standard sample that is exactly the same as the analyte.

[0021] The nuclear magnetic spectrum can be a hydrogen spectrum, a fluorine spectrum, a phosphorus spectrum or a carbon spectrum.

[0022] The designated peak is preferably a non-overlapping signal or a single peak signal. If there is no single peak, a split signal peak is selected for calculation.

[0023] Because each atom has a different spatial environment in a magnetic field, resulting in different relaxation times, insufficient relaxation time can lead to errors in the integration ratio. Preferably, the relaxation delay time (D1) in qNMR measurements should be set to at least 5 times the longitudinal relaxation time (T1) of the atom representing the designated peak.

[0024] The number of scans in the qNMR measurement is preferably such that the signal-to-noise ratio of the atomic signal representing the designated peak is greater than 250.

[0025] The chromatographic technique can be gas chromatography or liquid chromatography. The chromatographic conditions are optimized to the optimal conditions so that each peak can achieve baseline separation, and the ratio of the chromatographic peak areas of the two compounds is measured.

[0026] Since this method was proposed for the first time, in order to ensure the accuracy and reliability of the method, two common compounds were randomly selected. The conventional methods for calculating relative correction factors (slope ratio method, multi-point method) and the method of the present invention were used to calculate the RCF values, respectively, and the results were compared to verify the accuracy of the method.

[0027] In one embodiment of the present invention, the test compound is dimethyl terephthalate; the internal standard compound is coumarin; the NMR spectrum is a proton spectrum; the qNMR measurement conditions are: pulse angle 90°, relaxation delay time 40s, 16 scans; the designated peaks are H-6 and H-6' of dimethyl terephthalate, δ 3.89, and H-3 of coumarin, δ 6.50; the chromatography technique uses a liquid chromatograph, and the chromatographic conditions are: Shimadzu InertSustain C18 column, 250mm×4.6mm, particle size 5μm, mobile phase 80vol% acetonitrile in water, flow rate 1.0mL / min, column temperature 40°C, detection wavelength 254nm, injection volume 10μL. The results of this embodiment show that the RCF values ​​of dimethyl terephthalate relative to coumarin determined by the traditional slope ratio method, multi-point method, and the fusion technique proposed in this study are consistent, indicating that the method of the present invention is feasible and the measurement results are accurate, thus verifying the accuracy of the method.

[0028]

[0029] Since the original intention of the present invention is to solve the problem of difficult RCF determination of compounds in the absence of standard samples, in another embodiment, an unstable vitamin D3 precursor that cannot be purchased as a standard is selected as the test compound. The test compound is a vitamin D3 precursor (pre-Vit D3); the internal standard compound is vitamin D3. The nuclear magnetic spectrum is a hydrogen spectrum; the qNMR measurement conditions are: pulse angle 90°, relaxation delay time 10s, and number of scans 64 times; the designated peaks are H-19 of vitamin D3, δ5.02, and H-9 of vitamin D3 precursor pre-Vit D3, δ5.45; the chromatographic technology uses a liquid chromatograph, and the chromatographic conditions are: YMCCarotenoid column, 250mm×4.6mm, particle size 5μm, mobile phase is 98vol% methanol aqueous solution, flow rate 1.0mL / min, column temperature 40°C, detection wavelength 265nm, injection volume 10μL;

[0030]

[0031]

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1) The present invention cleverly integrates nuclear magnetic resonance quantification technology with chromatography technology, thereby achieving the purpose of measuring the RCF of unstable, difficult to obtain, and expensive compounds using readily available, stable, and inexpensive compounds.

[0034] 2) The method proposed in the present invention does not require derivatization, multiple dilutions or weighing operations, the process is simple, and the sources of error are reduced.

[0035] 3) The present invention solves the problem of difficulty in determining the RCF of unstable, hard-to-obtain and expensive compounds.

[0036] 4) The present invention can realize the RCF determination of compounds without standard samples.

[0037] 5) The principle of the RCF determination method based on the fusion of qNMR and chromatography technology outlined in the method of the present invention is not only applicable to the compounds mentioned in the examples, but is also applicable to the determination of RCF of compounds that can be separated and detected by chromatography and whose hydrogen, fluorine, phosphorus or carbon spectra can be measured using nuclear magnetic resonance technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The flow chart of the relative molar correction factor determination method based on the fusion of nuclear magnetic resonance quantitative technology and chromatography technology of the present invention is shown in FIG. 1 , wherein Class A compounds represent test compounds, Class B compounds represent internal standard compounds, and Class C compounds represent internal standard compounds. A 、C B represent the molar concentrations of compound A and compound B respectively; f, f A 、f B Represent the molar correction factor of compound A relative to compound B, the correction factor of compound A, and the correction factor of compound B respectively; A A 、A B Respectively represent the peak areas of compound A and compound B in the chromatogram; n A 、n B Represent the amount of compound A and compound B respectively; I A , I B Respectively represent the integrated areas of the designated peaks of compound A and compound B in the NMR spectra; N A 、N B Represent the number of atoms represented by the specified peaks of compound A and compound B in the NMR spectra, respectively.

[0039] Figure 2 Schematic diagram of the structure of dimethyl terephthalate and coumarin.

[0040] Figure 3 The hydrogen spectra of dimethyl terephthalate and coumarin.

[0041] Figure 4The chromatograms are those of dimethyl terephthalate and coumarin.

[0042] Figure 5 Schematic diagram of the prototype and precursor structure of vitamin D3.

[0043] Figure 6 This is a partial magnification of the hydrogen spectrum of vitamin D3 and vitamin D3 precursor.

[0044] Figure 7 These are the chromatograms of vitamin D3 solution before and after heating at 80°C in the dark and air-proof conditions. DETAILED DESCRIPTION

[0045] The present invention will be further described below with reference to the accompanying drawings and specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0046] In the following examples, the operating methods without specifying specific conditions are generally carried out under conventional conditions or conditions recommended by the manufacturer.

[0047] Example 1

[0048] The RCF of dimethyl terephthalate relative to coumarin was determined using the traditional slope ratio method, the multi-point method, and the fusion technology proposed in the present invention. The reliability of the method proposed in the present invention was verified by comparing the results.

[0049] Sample Preparation: Accurately weigh 5 mg each of dimethyl terephthalate and coumarin and dissolve them in 1 mL of DMSO-d6. The standard solutions were then diluted with DMSO-d6 to produce standard solutions of varying concentrations for quantitative H-spectrometry analysis. Simultaneously, a series of standard solutions were diluted 100-fold with acetonitrile and the peak areas of each compound were determined by liquid chromatography.

[0050] Step 1: Measure the NMR spectrum of the sample, select the atomic signal peak for quantification, and calculate the target signal peak area ratio. Quantitative H NMR spectrum measurement conditions: pulse angle 90°, relaxation delay time 40s, and 16 scans. Data were processed using JEOL Delta 6.0.0 software with a line width factor of 0.2Hz. Manual phase correction and baseline correction were performed. During integration, an integration region was selected, and each peak was integrated 3 to 5 times. The average value was taken when the relative standard deviation (RSD) was less than 1%. See Figure 2 、 Figure 3Spectral analysis shows that dimethyl terephthalate has single peaks at δ8.09 and 3.89, but the δ8.09 signal peak overlaps with the coumarin peak. Therefore, the signal at δ3.89 is selected as the designated peak for dimethyl terephthalate and included in the calculation. Coumarin has signals at δ8.08, 7.73, 7.63, 7.41, 7.36, and 6.50, but these signals are split. Therefore, the non-overlapping signal at δ6.50 is selected as the designated peak for coumarin and included in the calculation.

[0051] Step 2: Separate and analyze the sample using the set chromatographic conditions, collect and process the data using the data acquisition processor, and calculate the chromatographic peak area ratio of the compound (see Figure 4 Chromatographic conditions: Shimadzu InertSustain C18 column (250 mm × 4.6 mm, particle size 5 μm); mobile phase: 80 vol% acetonitrile in water; flow rate: 1.0 mL / min; column temperature: 40°C; detection wavelength: 254 nm; injection volume: 10 μL.

[0052] Step 3: Calculate the RCF of dimethyl terephthalate relative to coumarin according to the following formula:

[0053]

[0054] Where:

[0055] f represents the relative molar correction factor of dimethyl terephthalate relative to coumarin;

[0056] I A Represents the integrated area of ​​the designated peak of dimethyl terephthalate in the NMR spectrum;

[0057] I B represents the integrated area of ​​the designated peak of coumarin in the NMR spectrum;

[0058] N A Indicates the number of atoms represented by the designated peak of dimethyl terephthalate in the NMR spectrum;

[0059] N B Indicates the number of atoms represented by the specified peak of coumarin in the NMR spectrum;

[0060] A A represents the peak area of ​​dimethyl terephthalate in the chromatogram;

[0061] A B Represents the peak area of ​​coumarin in the chromatogram.

[0062] The measurement data and calculation results are detailed in Table 1 below.

[0063] Table 1

[0064]

[0065] The RCF of dimethyl terephthalate relative to coumarin was calculated using the traditional slope ratio method. Linear fitting was performed using the concentration as the horizontal axis and the chromatographic peak area measured in "Step 2" as the vertical axis. The linear equations for dimethyl terephthalate and coumarin were y = 3.162 × 10 9 x-1842(R 2 =1.00), y=6.042×10 9 x-1899(R 2 =1.00), and the slope ratio, i.e., the RCF of dimethyl terephthalate relative to coumarin, is 0.52.

[0066] The RCF of dimethyl terephthalate relative to coumarin was calculated using the traditional multi-point method. The RCF of dimethyl terephthalate relative to coumarin was calculated as follows:

[0067]

[0068] Where:

[0069] f represents the relative molar correction factor of dimethyl terephthalate relative to coumarin;

[0070] A A represents the peak area of ​​dimethyl terephthalate in the chromatogram;

[0071] A B represents the peak area of ​​coumarin in the chromatogram;

[0072] C A It represents the molar concentration of dimethyl terephthalate;

[0073] C B Indicates the molar concentration of coumarin.

[0074] The measurement data and calculation results are detailed in Table 2 below.

[0075] Table 2

[0076]

[0077] The RCF of dimethyl terephthalate relative to coumarin determined using the traditional slope ratio method, multi-point method, and the fusion technology proposed in this study were all 0.52, indicating that the scheme of integrating quantitative NMR and chromatography technology for RCF determination is feasible and the measurement results are accurate.

[0078] Example 2

[0079] Active vitamin D compounds have a conjugated triene structure and can be thermally isomerized into their precursor structure. In addition, under the conditions of light protection, oxygen-free, and heating at 80°C, some vitamin D compounds can be quantitatively converted into precursors without the generation of other impurities (Wang Yun, Shi Jieming, Zhang Liwen, et al., Study on the calculation method of the results of vitamin D content determination by HPLC [J]. Chinese Pharmaceutical Standards, 2020, 21(02): 107-113). Since it is impossible to purchase vitamin D3 precursor standards, it is impossible to use traditional methods to calculate the RCF of vitamin D3 precursor (pre-Vit D3) relative to vitamin D3 (Vit D3). Therefore, the fusion technology was used to determine the RCF of vitamin D3 precursor (pre-Vit D3) relative to vitamin D3 (Vit D3).

[0080] Sample Preparation: Accurately weigh 10 mg of VitD3 into a brown vial, add 1.0 mL of DMSO-d6, purge the vial with nitrogen, and place in an 80°C electric constant-temperature forced-air drying oven. Heat in the dark for 1 hour, then remove and rapidly cool. The resulting mixture is used for quantitative H NMR analysis. Simultaneously, dilute the mixture 100-fold with methanol and determine the peak areas of Vit D3 and pre-Vit D3 using liquid chromatography. Prepare three replicates.

[0081] Step 1: Measure the NMR spectrum of the sample, select the atomic signal peak for quantification, and calculate the target signal peak area ratio. After spectrum analysis, hydrogen atoms δ6.17, 5.95 and 5.02 were assigned to the H-6, H-7 and H-19 atomic signal peaks of Vit D3, and hydrogen atoms δ5.89, 5.63 and 5.45 were assigned to the H-6, H-7 and H-9 proton signal peaks of pre-Vit D3 (see Figure 5 and Figure 6 ). Quantitative NMR analysis prioritizes peaks with less coupled splitting and better separation from adjacent proton peaks as quantitative peaks. After comprehensive consideration, H-19 (δ5.02) of Vit D3 and H-9 (δ5.45) of pre-Vit D3 were selected as quantitative peaks. Quantitative H NMR spectrum measurement conditions: pulse angle 90°, relaxation delay time 10s, and 64 scans. The data were processed using JEOL Delta 6.0.0 software with a line width factor of 0.2Hz, and manual phase correction and baseline correction were performed. During integration, an integration region was selected, and each peak was integrated 3 to 5 times. The average value was taken when the relative standard deviation (RSD) was less than 1%.

[0082] Step 2: Separate and analyze the sample using the set chromatographic conditions, collect and process the data using the data acquisition processor, and calculate the chromatographic peak area ratio of the compound (see Figure 7Chromatographic conditions: YMC Carotenoid column (250 mm × 4.6 mm, particle size 5 μm); mobile phase: 98 vol% methanol in water; flow rate: 1.0 mL / min; column temperature: 40°C; detection wavelength: 265 nm; injection volume: 10 μL.

[0083] Step 3. Calculate the RCF of vitamin D3 precursor (pre-Vit D3) relative to vitamin D3 (Vit D3) according to the following formula:

[0084]

[0085] Where:

[0086] f represents the relative molar correction factor of vitamin D3 precursor relative to vitamin D3;

[0087] I A It represents the integrated area of ​​the designated peak of vitamin D3 precursor in the NMR spectrum;

[0088] I B It represents the integrated area of ​​the designated peak of vitamin D3 in the NMR spectrum;

[0089] N A Indicates the number of atoms represented by the specified peak of vitamin D3 precursor in the NMR spectrum;

[0090] N B Indicates the number of atoms represented by the specified peak of vitamin D3 in the NMR spectrum;

[0091] A A It represents the peak area of ​​vitamin D3 precursor in the chromatogram;

[0092] A B Represents the peak area of ​​vitamin D3 in the chromatogram.

[0093] The measurement data and calculation results are detailed in Table 3 below.

[0094] Table 3

[0095]

[0096] In addition, it should be understood that after reading the above description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for determining RCF based on the fusion of qNMR and chromatography technology, characterized in that: In the same qNMR measurement, the spectral constants of all excited atoms are the same and the NMR signal intensity is proportional to the number of atoms. A stable compound or other compound in the system is used as an internal standard. The molar ratio of the internal standard compound to the test compound is calculated from the NMR signal. The chromatographic peak area ratio of the internal standard compound and the test compound is determined using chromatography. The two results are combined to calculate the chromatographic RCF of the test compound. The chromatogram RCF is calculated as follows: Where: f represents the relative molar correction factor of the test compound relative to the internal standard compound; I A Represents the integrated area of ​​the specified peak of the test compound in the NMR spectrum; I B Represents the integrated area of ​​the specified peak of the internal standard compound in the NMR spectrum; N A Indicates the number of atoms represented by the specified peak in the NMR spectrum of the test compound; N B Indicates the number of atoms represented by the specified peak of the internal standard compound in the NMR spectrum; A A It represents the peak area of ​​the test compound in the chromatogram; A B Represents the peak area of ​​the internal standard compound in the chromatogram.

2. The measuring method according to claim 1, wherein The nuclear magnetic spectrum is a hydrogen spectrum, a fluorine spectrum, a phosphorus spectrum or a carbon spectrum.

3. The measuring method according to claim 1, wherein The designated peaks are non-overlapping signals and single-peak signals. If there is no single peak, a split signal peak is selected for calculation.

4. The measuring method according to claim 1, wherein The relaxation delay time in qNMR measurement is set to 5 times or more the longitudinal relaxation time of the atom represented by the designated peak.

5. The measuring method according to claim 1, wherein The number of scans in qNMR measurement is set to a value that ensures that the signal-to-noise ratio of the atomic signal represented by the designated peak is greater than 250.

6. The measuring method according to claim 1, wherein The chromatography technique is gas chromatography technique or liquid chromatography technique.

7. The measuring method according to claim 1, wherein The test compound is dimethyl terephthalate; the internal standard compound is coumarin; the nuclear magnetic resonance spectrum is a hydrogen spectrum; the qNMR measurement conditions are: pulse angle 90°, relaxation delay time 40s, and number of scans 16 times; the designated peaks are H-6 and H-6' of dimethyl terephthalate, δ3.89, and H-3 of coumarin, δ6.50; the chromatography technique uses a liquid chromatograph, and the chromatographic conditions are: Shimadzu InertSustain C18 column, 250mm×4.6mm, particle size 5μm, mobile phase 80vol% acetonitrile aqueous solution, flow rate 1.0mL / min, column temperature 40°C, detection wavelength 254nm, injection volume 10μL; 8. The measuring method according to claim 1, wherein The test compound is vitamin D3 precursor; the internal standard compound is vitamin D3; the nuclear magnetic resonance spectrum is a hydrogen spectrum; the qNMR measurement conditions are: pulse angle 90°, relaxation delay time 10s, and scan number 64 times; the designated peaks are H-19 of vitamin D3, δ 5.02, and H-9 of vitamin D3 precursor, δ 5.45; the chromatography technique uses a liquid chromatograph, and the chromatographic conditions are: YMC Carotenoid column, 250mm×4.6mm, particle size 5μm, mobile phase 98vol% methanol aqueous solution, flow rate 1.0mL / min, column temperature 40°C, detection wavelength 265nm, and injection volume 10μL;

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