Dual-signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance digital measurement method

Abstract

The invention relates to the technical field of medicine, food and chemistry, and particularly discloses a double-signal inhibition-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance digital measurement method which comprises the following steps: separating and collecting an eluent of a target analyte through high performance liquid chromatography by using a non-deuterated solvent as a mobile phase; adding an internal standard into the eluent to form a solution B, and performing quantitative nuclear magnetic resonance analysis and double-signal inhibition on the solution B to obtain nuclear magnetic resonance quantitative peak areas of the target analyte and the internal standard; preparing a solution A by using a to-be-detected sample and an internal standard; respectively carrying out high performance liquid chromatography analysis on the solution A and the solution B to obtain respective high performance liquid chromatography peak areas of the target analyte and the internal standard, and further obtaining the purity value of the sample. Solvent signal interference in the nuclear magnetic resonance spectrum can be effectively eliminated through double-signal suppression, variables in the sample preparation and analysis process can be reduced to the minimum through an internal standard correction strategy, and therefore the time-consuming freeze-drying step in the prior art is omitted.

Description

Technical Field

[0001] The invention relates to the technical fields of medicine, food and chemistry, and in particular to a dual-signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance digital measurement method. Background Art

[0002] Purity assessment of compounds is a critical step in the biomedical and chemical fields. Accurate purity analysis methods are the cornerstone of ensuring that products meet the requirements of regulatory agencies such as the FDA and EMA, which highlights the importance of accurate quantitative analysis. The use of advanced quantitative purity analysis methods will continue to drive innovation and excellence in the field of chemistry. These methods include mass balance, quantitative nuclear magnetic resonance (qNMR), and differential scanning calorimetry (DSC). Mass balance methods use various instruments to determine various types of impurities, which is cumbersome, time-consuming, and requires a large amount of sample. For the determination of low-purity compounds, the mass balance method should be used to characterize the main impurities until the total amount of unknown impurities does not exceed 1%. However, many impurities are difficult to identify, obtain, or synthesize, and the associated measurement costs may be too high. Therefore, a lot of research work may be required to identify these impurities, and the analysis work may fail if some major impurities cannot be identified or cannot be obtained in pure form. DSC is mainly applicable to high-purity compounds, while simple detection methods for low-purity compounds are relatively limited.

[0003] Among these analytical methods for determining purity, qNMR is recognized as one of the most promising methods. Using high performance liquid chromatography (HPLC) to collect a small amount of eluate of the target analyte from a low-purity organic compound can separate most of the impurities in the low-purity compound without the need to characterize the impurities, while solving the problem of impurity interference. Compared with traditional methods, the use of HPLC purification can greatly reduce the cost of detection. Therefore, many new HPLC-qNMR methods have emerged.

[0004] Saito et al. used two tritiated reagents as mobile phases to directly perform HPLC-qNMR online coupled analysis, but this required a large amount of tritiated reagents and was extremely costly. Huang et al. used two non-tritiated solvents (CH3CN and H2O) as mobile phases for HPLC and developed a HPLC-qNMR method that supports TSS (double signal suppression). This method uses common solvents to reduce the detection cost; however, compared with traditional methods, the result deviation of this method is about 0.93%, and the accuracy is low. Zhang et al. developed an internal standard recovery correction HPLC-qNMR method (ISR-HPLC-qNMR, ISR is standard correction), which uses non-tritiated solvents as mobile phases for HPLC separation and collects the eluent containing the analyte to be tested and the internal standard in the same test tube. However, this method requires a long freeze-drying process. Sun et al. improved the ISC-HPLC-qNMR method based on the ISC-HPLC-qNMR method and shortened the freeze-drying time by half. The use of internal standard calibration reduces the deviation and improves the accuracy. However, in ISC-HPLC-qNMR, the time for HPLC acquisition, HPLC of solution A, HPLC of solution B, lyophilization and qNMR were 1 hour, 1 hour, 1 hour, 24 hours and 1 hour, respectively, which took a total of 28 hours. It can be seen that the lyophilization step is still time-consuming and is more than 24 hours.

[0005] In order to speed up the analysis of sample purity, effectively solve the problem of over-evaluation of purity due to the presence of impurities in quantitative nuclear magnetic resonance, and thus improve the accuracy and reliability of quantitative results, the inventors conducted a lot of analysis and research on relevant theories and technologies.

[0006] Based on this technical background, the present invention studies a dual signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance digital measurement method. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a dual signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance digital measurement method. The dual signal suppression can effectively eliminate the solvent signal interference in the nuclear magnetic resonance spectrum, and the internal standard correction strategy can reduce the variables in the sample preparation and analysis process to a minimum, thereby eliminating the time-consuming freeze-drying step in the prior art, greatly accelerating the speed of sample purity analysis, and effectively solving the problem of over-evaluation of purity due to the presence of impurities in quantitative nuclear magnetic resonance.

[0008] In order to achieve the above object, the present invention provides a dual signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance digital measurement method, comprising:

[0009] Using a non-deuterated solvent as a mobile phase, the target analyte is separated from low-purity organic compounds by high performance liquid chromatography and the eluate is collected;

[0010] Adding an internal standard substance to the eluent to form a solution B, and performing quantitative nuclear magnetic resonance analysis and double signal suppression on the solution B to obtain the nuclear magnetic resonance quantitative peak areas of the target analyte and the internal standard;

[0011] Prepare solution A using the sample to be tested and the internal standard substance;

[0012] Performing high performance liquid chromatography-diode array detector analysis on the solution A and the solution B, respectively, to obtain high performance liquid chromatography peak areas of the respective target analytes and internal standards;

[0013] The sample purity value is calculated based on the quantitative peak area and the HPLC peak area.

[0014] The beneficial effects of the present invention include:

[0015] (1) The present invention proposes a dual signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance digital measurement method, which can effectively eliminate the solvent signal interference in the nuclear magnetic resonance spectrum through dual signal suppression, and the internal standard correction strategy can minimize the variables in the sample preparation and analysis process, thereby eliminating the time-consuming freeze-drying step in the prior art, greatly speeding up the speed of sample purity analysis, and effectively solving the problem of over-evaluation of purity due to the presence of impurities in quantitative nuclear magnetic resonance.

[0016] (2) The present invention proposes a dual signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance digital measurement method, dual signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance (TSS-ISC-HPLC-qNMR), which applies the internal standard correction mechanism of ISC-HPLC-qNMR to TSS-HPLC-qNMR analysis, thereby effectively reducing the relative standard deviation and improving the accuracy and reliability of the quantitative results.

[0017] (3) The dual signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance digital measurement method proposed in the present invention adopts a TSS-ISC-HPLC-qNMR combined measurement method compared with the ISC-HPLC-qNMR quantitative method. The time for HPLC acquisition, HPLC of solution A, HPLC and qNMR of solution B are 1 hour, 1 hour, 1 hour and 1 hour respectively, which takes a total of 4 hours. Since the freeze-drying step is eliminated, the measurement time is greatly saved.

[0018] (4) The present invention proposes a dual signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance digital measurement method. Compared with HPLC-qNMR of TSS, TSS-ISC-HPLC-qNMR does not need to collect 100% recovery rate of target analytes, and the deviation of recovery rate can be corrected by internal standard correction method. The deviation of HPLC-qNMR result of TSS is about 0.93%, while the deviation of TSS-ISC-HPLC-qNMR result is only 0.01%. In addition, this method does not need to collect internal standard substances, and the operation is simple and fast.

[0019] (5) The present invention proposes a dual signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance digital measurement method, TSS-ISC-HPLC-qNMR, which uses dual signal suppression to suppress excessive solvent signals in the NMR spectrum and enhance the signal intensity of the internal standard quantitative signal in the target sample, thereby greatly improving the signal-to-noise ratio. Consistent results show that the TSS suppression procedure in this method will neither produce signal deviation nor obvious signal distortion, and no additional deviation correction is required.

[0020] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0022] Figure 1 The present invention provides a schematic flow chart of a dual signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance digital measurement method.

[0023] Figure 2 The present invention provides a schematic flow chart of a specific implementation of a dual signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance digital measurement method. DETAILED DESCRIPTION

[0024] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0025] The present invention provides a dual signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance digital measurement method, such as Figure 1 As shown, including:

[0026] Using a non-deuterated solvent as a mobile phase, the target analyte is separated from low-purity organic compounds by high performance liquid chromatography and the eluate is collected;

[0027] An internal standard substance is added to the eluent to form a solution B, and the solution B is subjected to quantitative nuclear magnetic resonance analysis and double signal suppression to obtain the nuclear magnetic resonance quantitative peak areas of the target analyte and the internal standard;

[0028] Prepare solution A using the sample to be tested and the internal standard substance;

[0029] Solution A and solution B are analyzed by high performance liquid chromatography-diode array detector, respectively, to obtain the high performance liquid chromatography peak areas of the respective target analytes and internal standards;

[0030] The sample purity value was calculated based on the quantitative peak area and the HPLC peak area.

[0031] In the present invention, the solvent signal interference in the nuclear magnetic resonance spectrum can be effectively eliminated by double signal suppression, and the internal standard correction strategy can minimize the variables in the sample preparation and analysis process, thereby eliminating the time-consuming freeze-drying step in the prior art, greatly speeding up the sample purity analysis, and effectively solving the problem of over-evaluation of purity due to the presence of impurities in quantitative nuclear magnetic resonance.

[0032] According to the present invention, the formula used to calculate the purity value to be measured based on the quantitative peak area and the high performance liquid chromatography peak area is:

[0033]

[0034] Wherein, P is the purity value, sam represents the sample, samA is the sample in solution A, i.e., the target analyte in solution A, samB is the target analyte in solution B, std represents the internal standard, stdA is the internal standard in solution A, stdB is the internal standard in solution B, A represents the HPLC peak area, N represents the number of protons in the quantitative signal, M represents the molar mass, m represents the weighed mass, and I represents the nuclear magnetic resonance quantitative peak area.

[0035] According to the present invention, separating and collecting the eluate of the target analyte from the low-purity organic compound by high performance liquid chromatography comprises:

[0036] Set the HPLC injection volume to 30-70 μL;

[0037] During the elution process, when the eluate containing the target analyte is detected, the collection tube made of polyetheretherketone material is manually operated and introduced into the collection bottle to ensure effective collection of the sample.

[0038] According to the present invention, adding an internal standard substance to the eluent to form solution B comprises:

[0039] The internal standard substance is added to the eluent to dissolve and form a solution B with uniform concentration.

[0040] According to the present invention, the solution is subjected to quantitative nuclear magnetic resonance analysis and dual signal suppression to obtain the nuclear magnetic resonance quantitative peak areas of the target analyte and the internal standard, comprising:

[0041] Adding a tritiated solvent to solution B is used to lock the magnetic field during quantitative NMR analysis;

[0042] Configuring specific quantitative nuclear magnetic resonance analysis parameters, performing quantitative nuclear magnetic resonance analysis on solution B to which the tritiated solvent is added to obtain a nuclear magnetic spectrum;

[0043] First suppression: Based on the NMR spectrum, select the center of the non-deuterated solvent signal as the reference point for the irradiation frequency shift, adjust the experimental parameters, and fine-tune the value of the irradiation frequency shift until the amplitude of the non-deuterated solvent signal is minimized;

[0044] A suppression macro with appropriate parameters was created and applied, and preliminary scans were performed to measure the signal heights of nondeuterated and tritiated solvents;

[0045] Second suppression: If the tritiated solvent signal height is higher, repeatedly adjust the offset of the irradiation frequency offset until both solvent heights are reduced to the minimum;

[0046] After increasing the automatic receiver gain, the NMR quantitative peak areas of the target analytes and internal standards were obtained after manual phase correction, automatic baseline correction and manual integration.

[0047] In the present invention, compared with the ISC-HPLC-qNMR quantitative method, the TSS-ISC-HPLC-qNMR combined measurement method is adopted, and the time for HPLC acquisition, HPLC of solution A, HPLC and qNMR of solution B are 1 hour, 1 hour, 1 hour and 1 hour respectively, which takes a total of 4 hours. Since the freeze-drying step is eliminated, the measurement time is greatly saved.

[0048] Preferably, configuring specific quantitative NMR analysis parameters includes

[0049] Set the pulse width to 30°, the number of scans to 32, and the relaxation delay to 60s;

[0050] 65536 data points were used for data collection to obtain the collected data;

[0051] After Fourier transforming the acquired data, exponential filtering was applied and the data was expanded to 131,072 time domain points to improve signal quality;

[0052] Before each data measurement, automatic receiver gain is performed to prevent signal overflow.

[0053] According to the present invention, the experimental parameters include a cycle counter, a scan number, and a relaxation delay;

[0054] The frequency of the nondeuterated solvent signal center is 1980-2180 Hz;

[0055] The volume concentration of the deuterated solvent in solution B is 2-10%.

[0056] In the present invention, compared with HPLC-qNMR of TSS, TSS-ISC-HPLC-qNMR does not need to collect 100% recovery rate of target analyte, and the deviation of recovery rate can be corrected by internal standard correction method. The deviation of HPLC-qNMR result of TSS is about 0.93%, while the deviation of TSS-ISC-HPLC-qNMR result is only 0.01%. In addition, this method does not need to collect internal standard, and the operation is simple and quick.

[0057] According to the present invention, the commands used to create and apply a suppression macro with corresponding parameters are:

[0058] "st generate Rectangle 50000 100filename=H2O_ACN_SHAPE1; stmanipulate H2O_ACN_SHAPE1 offs bs 1000000 2 0 100 1672.8 100".

[0059] According to the present invention, the last five digits of the command are represented from front to back as follows: (a) the total number of suppressions; (b) the offset of the first suppression relative to the irradiation frequency offset; (c) the intensity of the first suppression; (d) the offset of the second suppression relative to the irradiation frequency offset; (e) the intensity of the second suppression.

[0060] According to the present invention, the non-deuterated solvent is water or acetonitrile;

[0061] If the nondeuterated solvent is acetonitrile, the value of the offset relative to the irradiation frequency shift in the first suppression is 0;

[0062] If the non-deuterated solvent is water, the value of the offset of the irradiation frequency shift in the second suppression is the offset of water minus the offset of acetonitrile.

[0063] The present invention, dual signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance (TSS-ISC-HPLC-qNMR), applies the internal standard method correction mechanism of ISC-HPLC-qNMR to TSS-HPLC-qNMR analysis, thereby effectively reducing the relative standard deviation (no more than 1%) and improving the accuracy and reliability of the quantitative results.

[0064] In the present invention, TSS-ISC-HPLC-qNMR adopts dual signal suppression to suppress excessive solvent signals in the NMR spectrum and simultaneously enhance the signal intensity of the internal standard quantitative signal in the target sample, thereby greatly improving the signal-to-noise ratio. Consistent results show that the TSS suppression procedure in this method produces neither signal deviation nor obvious signal distortion, and no additional deviation correction is required.

[0065] The present invention will be described in more detail below by way of examples.

[0066] Embodiment 1:

[0067] like Figure 2 As shown, this embodiment proposes a dual signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance digital measurement method, comprising:

[0068] 1. Select internal standard and quantitative signal: dissolve the sample and candidate internal standard substance in solvent and conduct HPLC and 1HqNMR preliminary test;

[0069] 2. Sample collection: accurately measure the weight of the sample and dissolve it in the predetermined solvent for HPLC analysis, with the injection volume set to 50 μL; during the elution process, when the eluent containing the target analyte is detected, manually operate the collection tube made of polyetheretherketone material and introduce it into a 2mL collection bottle to ensure effective sample collection;

[0070] 3. TSS-qNMR; Use a high-precision balance to accurately weigh the required internal standard substance and dissolve it in 1 mL of the eluent prepared in the collection step to form a solution B with uniform and controllable concentration;

[0071] About 0.6 mL of solution B was transferred to the NMR tube, and an additional 0.06 mL of tritiated solvent was added specifically to lock the magnetic field during NMR analysis. Specific qNMR parameters were configured to achieve double signal suppression, including the use of 30° RF pulses and 65,536 data points for data acquisition. After Fourier transformation, exponential filtering was applied and the data were expanded to 131,072 time domain points to improve signal quality. Before each measurement, automatic receiver gain (RG) was performed to prevent signal overflow. To determine the chemical shift of the two solvents, the number of scans (NS) was set to 1 and the relaxation delay (D1) was set to 1 second to obtain basic 1H-NMR spectra. In this configuration, due to the automatic The receiver gain was set very low (RG about 1.00), and only the signals of acetonitrile and water were observed, so no analyte signal could be detected; the pulse program was set to noesygpps1d.comp1.d1 (Bruker Corporation, Rheinstetten, Germany), and the center of the acetonitrile signal (about 2080 Hz) was chosen as the reference point for the irradiation frequency shift (O1); the experimental parameters included setting the cycle counter (L6) to 3, D1 to 5 s, and NS to 1; in order to adjust O1 precisely to the center of the acetonitrile signal, this step was repeated several times and the O1 value was fine-tuned until the acetonitrile signal amplitude was minimized; subsequently, the "stgenerate rectangle 50000 100

[0072] filename=H2O_ACN_SHAPE1;st manipulate H2O_ACN_SHAPE1 offs b s10000002 0 100 1672.8 The "100" command creates and applies a suppression macro with the corresponding parameters; the last five digits of the command represent: (a) the total number of suppressions; (b) the offset of the first suppression relative to O1 (always 0 for acetonitrile); (c) the intensity of the first suppression (the recommended value is 100); (d) the offset of the second suppression relative to O1 (for water suppression, the value is the offset of water minus the offset of acetonitrile); (e) the intensity of the second suppression (the recommended value is 100); Perform a preliminary scan to measure the water and acetonitrile signal heights; if the water level is still high, adjust the offset of the second suppression from O1 in the previous step and repeat this process until both solvent heights are minimized; subsequently, increase the automatic receiver gain (e.g. RG: 12.7) so that the analyte signal is detected; after manual phase correction, automatic baseline correction and manual integration, the signal areas of the sample quantitative signal and the internal standard quantitative signal are obtained;

[0073] 4. Determination of peak area: Use a high-precision analytical balance and an aluminum weighing boat to accurately weigh the test sample and internal standard, and dissolve them in the solvent; To ensure the repeatability and accuracy of the experimental data, prepare solution A repeatedly for each experiment; Use HPLC-DAD to determine the peak area of ​​the analyte and internal standard in solution A and solution B, with an injection volume of 10 μL;

[0074] The purity was calculated based on the ratio of qNMR and theoretical derivation of the Lambert-Beer law for HPLC-DAD, and was corrected by the ratio of the HPLC-DAD signal areas of the two solutions:

[0075]

[0076] Wherein, P is purity; sam represents sample; samA is the sample in solution A; samB is the sample in solution B; std represents internal standard; stdA is the internal standard in solution A; stdB is the internal standard in solution B; A represents the peak area of ​​HPLC; N represents the number of protons of the quantitative signal; M represents molar mass; m represents weighed mass; and I represents the peak area of ​​qNMR.

[0077] In this embodiment, a non-deuterated solvent is first used as a mobile phase to separate and collect the target analyte from a low-purity organic compound by HPLC; then an internal standard is added to the collected solution and named solution B, and a dual signal suppression pulse sequence is used to selectively suppress the proton signals in the two non-deuterated solvents to replace the traditional time-consuming steps of freeze-drying and re-dissolving with a deuterated solvent. This method greatly reduces the analysis time and cost. Finally, a mixed solution containing the sample to be tested and the internal standard substance is prepared as the solution before solution B treatment, named solution A; the purity value is calculated by a formula including conditions such as nuclear magnetic resonance signal, chromatographic peak area and mass, thereby further improving the accuracy of the determination.

[0078] Embodiment 2:

[0079] This embodiment provides a dual signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance digital measurement method for determining the value of a standard substance acesulfame potassium (AF), comprising the following steps:

[0080] S1: Potassium hydrogen phthalate (KHP) was selected as the internal standard substance of AF. The H-2 signal (δ5.91ppm) and KHP signal (δ7.76ppm) of AF were selected as quantitative signals;

[0081] S2: The acesulfame potassium aqueous solution (20 mg / mL) was subjected to HPLC analysis (ODS-SP column, 0.1% formic acid aqueous solution: acetonitrile = 85:15 as the mobile phase composition, wavelength of 265 nm), and the injection volume was set to 50 μL; during the elution process, when the eluent containing the target analyte was detected, the collection tube made of polyetheretherketone material was manually operated and introduced into a 2 mL collection bottle to ensure effective sample collection;

[0082] S3: TSS-qNMR; KHP was weighed on a high-precision balance and dissolved in 1 mL of the collected solution, which was solution B; 0.06 mL of heavy water was added to 0.6 mL of solution B; qNMR measurement was performed (pulse width was 30°, scan number was 32, relaxation delay was 60 s), the NMR spectrum of the sample was obtained, and a double signal suppression operation was performed;

[0083] S4: Determination of HPLC peak area; AF (0.6 mg) and KHP (0.6 mg) were accurately weighed using a high-precision analytical balance and an aluminum weighing boat, and dissolved in water to form solution A; the peak areas of the analyte and internal standard in solution A and solution B were determined using HPLC-DAD, with an injection volume of 10 μL;

[0084] S5: Purity calculation was based on the ratio of qNMR and theoretical derivation of the Lambert-Beer law of HPLC-DAD, and was corrected by the ratio of the HPLC-DAD signal areas of the two solutions:

[0085]

[0086] Wherein, P is purity; sam represents sample; samA is the sample in solution A; samB is the sample in solution B; std represents internal standard; stdA is the internal standard in solution A; stdB is the internal standard in solution B; A represents the peak area of ​​HPLC; N represents the number of protons of the quantitative signal; M represents the molar mass; m represents the weighed mass; I represents the peak area of ​​qNMR;

[0087] The average value of all measured purities was 99.89% (standard deviation was 0.021%), which was very close to the certified purity value of reference material AF of 99.88% (standard uncertainty was 0.025%); the consistency between the purity values ​​of the TSS-ISC-HPLC-qNMR method and the certified purity values ​​of reference material AF enhanced the accuracy and reliability of the adopted analytical protocol.

[0088] Embodiment three:

[0089] This embodiment provides a quantitative purity determination method based on double signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance to determine the value of digitoxin, comprising the following steps:

[0090] S1: ethyl thiocarbamide (EP) was selected as the internal standard of digitocin, and the H-8 resonance of digitocin at δ5.91 ppm and the EP signal at δ6.82 ppm were selected as the quantitative NMR signals for determining the concentration of digitocin;

[0091] S2: The methanol solution of digetoin (12 mg / mL) was subjected to HPLC analysis (Waters-XBridge chromatographic column, 0.1% formic acid aqueous solution: acetonitrile = 60:40 as the mobile phase composition, wavelength of 221 nm), and the injection volume was set to 50 μL; during the elution process, when the eluent containing the target analyte was detected, the collection tube made of polyetheretherketone material was manually operated and introduced into a 2 mL collection bottle to ensure effective collection of the sample;

[0092] S3: TSS-qNMR; KHP was weighed on a high-precision balance and dissolved in 1 mL of the collected solution, which was solution B; 0.06 mL of deuterated methanol was added to 0.6 mL of solution B; qNMR measurement was performed (pulse width of 30°, number of scans of 32, relaxation delay of 60 s), the NMR spectrum of the sample was obtained, and a double signal suppression operation was performed;

[0093] S4: Determination of HPLC peak area; Digitosine (0.6 mg) and EP (0.6 mg) were accurately weighed using a high-precision analytical balance and an aluminum weighing boat, and dissolved in water to form solution A; the peak areas of the analyte and internal standard in solution A and solution B were determined using HPLC-DAD, with an injection volume of 10 μL;

[0094] S5: Purity calculation was based on the ratio of qNMR and theoretical derivation of the Lambert-Beer law of HPLC-DAD, and was corrected by the ratio of the HPLC-DAD signal areas of the two solutions:

[0095]

[0096] Wherein, P is purity; sam represents sample; samA is the sample in solution A; samB is the sample in solution B; std represents internal standard; stdA is the internal standard in solution A; stdB is the internal standard in solution B; A represents the peak area of ​​HPLC; N represents the number of protons of the quantitative signal; M represents the molar mass; m represents the weighed mass; I represents the peak area of ​​qNMR;

[0097] The average purity of all measured data was 94.45%, which was very close to the average purity of 94.45% of the qNMR measurements with deconvolution; the agreement between the purity values ​​of the TSS-ISC-HPLC-qNMR method and the qNMR measurements with deconvolution reinforced the accuracy and reliability of the adopted analytical protocol.

[0098] The embodiment of the present invention proposes a dual signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance digital measurement method. The dual signal suppression can effectively eliminate the solvent signal interference in the nuclear magnetic resonance spectrum, and the internal standard correction strategy can minimize the variables in the sample preparation and analysis process, thereby eliminating the time-consuming freeze-drying step in the prior art, greatly speeding up the sample purity analysis, and effectively solving the problem of over-evaluation of purity due to the presence of impurities in quantitative nuclear magnetic resonance.

[0099] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A dual signal suppression-internal standard correction-high performance liquid chromatography-quantitative nuclear magnetic resonance digital measurement method, characterized in that: include: Using a non-deuterated solvent as a mobile phase, the target analyte is separated from low-purity organic compounds by high performance liquid chromatography and the eluate is collected; Adding an internal standard substance to the eluent to form a solution B, and performing quantitative nuclear magnetic resonance analysis and double signal suppression on the solution B to obtain the nuclear magnetic resonance quantitative peak areas of the target analyte and the internal standard; Prepare solution A using the sample to be tested and the internal standard substance; Performing high performance liquid chromatography-diode array detector analysis on the solution A and the solution B, respectively, to obtain high performance liquid chromatography peak areas of the respective target analytes and internal standards; The sample purity value is calculated based on the quantitative peak area and the HPLC peak area.

2. The method according to claim 1, characterized in that: The formula used to calculate the purity value to be measured based on the quantitative peak area and the HPLC peak area is: Wherein, P is the purity value, sam represents the sample, samA is the sample in solution A, i.e., the target analyte in solution A, samB is the target analyte in solution B, std represents the internal standard, stdA is the internal standard in solution A, stdB is the internal standard in solution B, A represents the HPLC peak area, N represents the number of protons in the quantitative signal, M represents the molar mass, m represents the weighed mass, and I represents the nuclear magnetic resonance quantitative peak area.

3. The method according to claim 1, characterized in that Separation of target analytes from low-purity organic compounds by HPLC and collection of eluents include: The HPLC injection volume is set to 30-70 μl; During the elution process, when the eluate containing the target analyte is detected, the collection tube made of polyetheretherketone material is manually operated and introduced into the collection bottle to ensure effective collection of the sample.

4. The method according to claim 1, characterized in that: Adding an internal standard substance to the eluent to form solution B comprises: The internal standard substance is added into the eluent to dissolve and form a solution B with uniform concentration.

5. The method according to claim 1, characterized in that The solution is subjected to quantitative nuclear magnetic resonance analysis and double signal suppression to obtain the nuclear magnetic resonance quantitative peak areas of the target analyte and the internal standard, including: Adding a tritiated solvent to the solution B to lock the magnetic field during the quantitative nuclear magnetic resonance analysis; configuring specific quantitative nuclear magnetic resonance analysis parameters, and performing a quantitative nuclear magnetic resonance analysis on the solution B to which the tritiated solvent is added to obtain a nuclear magnetic spectrum; First suppression: based on the NMR spectrum, select the center of the non-deuterated solvent signal as the reference point of the irradiation frequency shift, adjust the experimental parameters, and fine-tune the value of the irradiation frequency shift until the amplitude of the non-deuterated solvent signal is minimized; A suppression macro with corresponding parameters was created and applied to perform a preliminary scan to measure the signal heights of the non-deuterated solvent and the tritiated solvent; Second suppression: if the tritiated solvent signal height is high, repeatedly adjusting the offset of the irradiation frequency offset until both solvent heights are reduced to the minimum; After increasing the automatic receiver gain, the NMR quantitative peak areas of the target analyte and the internal standard were obtained through manual phase correction, automatic baseline correction and manual integration.

6. The method according to claim 5, characterized in that Configuration specific parameters for the quantitative NMR analysis include Set the pulse width to 30°, the number of scans to 32, and the relaxation delay to 60s; 65536 data points were used for data collection to obtain the collected data; After Fourier transforming the collected data, exponential filtering is applied and the data is expanded to 131072 time domain points to improve signal quality; Before each data measurement, automatic receiver gain is performed to prevent signal overflow.

7. The method according to claim 5, characterized in that The experimental parameters include cycle counter, scan number and relaxation delay; The frequency of the non-deuterated solvent signal center is 1980-2180 Hz; The volume concentration of the tritiated solvent in the solution B is 2-10%.

8. The method according to claim 5, characterized in that The commands used to create and apply a suppression macro with the appropriate parameters are: "st generate Rectangle 50000100filename=H2O_ACN_SHAPE1; st manipulateH2O_ACN_SHAPE1 offs bs 1000000201001672.8100".

9. The method according to claim 8, characterized in that The last five digits of the command represent, from front to back, the following: (a) the total number of suppressions; (b) the offset of the first suppression relative to the irradiation frequency offset; (c) the strength of the first suppression; (d) the offset of the second suppression relative to the irradiation frequency offset; (e) the strength of the second suppression.

10. The method according to claim 9, characterized in that The non-deuterated solvent is water or acetonitrile; If the non-deuterated solvent is acetonitrile, in the first suppression, the value of the offset relative to the irradiation frequency offset is 0; If the non-deuterated solvent is water, in the second suppression, the value of the offset of the irradiation frequency shift is the offset of water minus the offset of acetonitrile.

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