A method for detecting the content of glycolide based on liquid chromatography

Abstract

This application relates to the technical field of chromatographic analysis, and specifically relates to a method for detecting the content of glycolide based on liquid chromatography. The method includes: preparing a sample solution to be tested and standard stock solutions diluted to different concentrations, and respectively obtaining a chromatogram to be tested and standard chromatograms of different concentrations through a liquid chromatograph; determining the baseline disturbance degree of the chromatogram to be tested; obtaining the target chromatographic peak and its corresponding chromatographic peak curve in the chromatogram to be tested; calculating the disturbance evaluation value, asymmetry coefficient, residual interference degree, and discrimination coefficient of the chromatogram to be tested to evaluate the chromatogram to be tested; according to the change relationship between different concentrations and the corresponding peak areas of the chromatographic peaks in the standard chromatograms, obtaining a fitting function, and combining the peak area corresponding to the target chromatographic peak to determine the content of glycolide. This application can improve the detection accuracy of the glycolide content and more accurately determine the content of glycolide.

Description

Technical Field

[0001] This application relates to the field of chromatographic analysis technology, and specifically relates to a method for detecting the content of glycolide based on liquid chromatography. Background Art

[0002] Glycolide, as an important monomer of high molecular materials such as polyglycolic acid (PGA) and poly(lactic acid-glycolic acid) copolymer (PLGA), has an irreplaceable position in the field of biodegradable materials. Its purity will affect the mechanical properties, degradation rate and biocompatibility of high molecular materials, and residual impurities will affect the decline of the mechanical strength of high molecular materials, out-of-control degradation or the risk of biotoxicity.

[0003] Liquid chromatography can efficiently detect the content of glycolide. The poor condition of liquid chromatography detection equipment, as well as impurities such as residual organic solvents, acids, and glycolic acid oligomers in glycolide, will interfere with liquid chromatography determination, resulting in abnormal chromatographic peaks and baseline drift in the liquid chromatogram, making it difficult to determine the content of glycolide in the liquid chromatogram and affecting the accuracy of the detection results. Summary of the Invention

[0004] In order to solve the above technical problems, a method for detecting the content of glycolide based on liquid chromatography is provided to solve the existing problems.

[0005] The solution of this application to solve the technical problem is to provide a method for detecting the content of glycolide based on liquid chromatography, including the following steps:

[0006] Prepare a sample solution to be measured and standard stock solutions diluted to different concentrations, and respectively obtain a chromatogram to be measured and standard chromatograms of different concentrations through a liquid chromatograph;

[0007] Analyze the deviation of the low-frequency components of the absorbance at all times on the baseline in the frequency domain and the discreteness of the absorbance on the baseline in the chromatogram to be measured, and determine the baseline disturbance degree of the chromatogram to be measured;

[0008] Obtain the target chromatographic peak and its corresponding chromatographic peak curve in the chromatogram to be measured through the moments corresponding to the chromatographic peaks in all standard chromatograms; analyze the tailing situation of the chromatographic peak curve, and combine the baseline disturbance degree to calculate the disturbance evaluation value of the chromatogram to be measured;

[0009] Record the remaining peaks on the chromatographic peak curve except the target chromatographic peak as impurity peaks; analyze the normal distribution characteristics of the chromatographic peak curve and the interval between different impurity peaks and the target chromatographic peak to obtain the asymmetry coefficient of the chromatogram to be measured, and combine the autocorrelation characteristics of the absorbance in the neighborhood range corresponding to the moments of different impurity peaks to determine the residual interference degree of the chromatogram to be measured;

[0010] Based on the perturbed evaluation value and the residual interference degree, a discrimination coefficient of the chromatogram to be measured is obtained to evaluate the chromatogram to be measured; according to the change relationship between different concentrations and the corresponding peak areas of the chromatographic peaks in the standard chromatograms, a fitting function is obtained, and in combination with the peak area corresponding to the target chromatographic peak, the content of glycolide is determined.

[0011] Preferably, the determination of the baseline perturbation degree of the chromatogram to be measured includes:

[0012] All the moments corresponding to the baseline in the chromatogram to be measured are recorded as baseline moments; the absorbances at all baseline moments are subjected to smoothing processing and then frequency-domain analysis to obtain a spectrogram;

[0013] Low-frequency components are screened from all the frequency components in the spectrogram, and the relative deviation is determined based on the offset of the energies corresponding to different low-frequency components;

[0014] The dispersion degree of the absorbances at all baseline moments after smoothing processing is calculated;

[0015] The baseline perturbation degree is the product of the relative deviation and the dispersion degree.

[0016] Preferably, the determination process of the relative deviation is:

[0017] The mean value of the energies corresponding to all the frequency components in the spectrogram is calculated and recorded as the average energy;

[0018] The relative deviation is the sum of the differences between the energies corresponding to all the low-frequency components and the average energy.

[0019] Preferably, the obtaining of the target chromatographic peak and its corresponding chromatographic peak curve in the chromatogram to be measured includes:

[0020] The mode of the moments corresponding to the chromatographic peaks in all the standard chromatograms is obtained and recorded as the standard moment;

[0021] The peaks of the absorbances at all moments in the chromatogram to be measured are obtained; the peak with the smallest time interval between the moments corresponding to all the peaks in the chromatogram to be measured and the standard moment is recorded as the target chromatographic peak;

[0022] The curve between the two nearest baseline moments on the left and right sides of the moment corresponding to the target chromatographic peak in the chromatogram to be measured is recorded as the chromatographic peak curve.

[0023] Preferably, the calculation of the perturbed evaluation value of the chromatogram to be measured includes:

[0024] The tailing factor of the chromatographic peak curve is calculated;

[0025] The perturbed evaluation value is the product of the tailing factor and the baseline perturbation degree.

[0026] Preferably, obtaining the asymmetry coefficient of the chromatogram to be measured includes:

[0027] Calculating the test statistic of the absorbance at all times on the chromatographic peak curve of the chromatogram to be measured;

[0028] Denoting the difference between the corresponding time of the target chromatographic peak and the corresponding times of each impurity peak as the time difference;

[0029] Calculating the absolute value of the sum of the ratios of the time differences of all impurity peaks to their peak widths;

[0030] The asymmetry coefficient is the ratio of the absolute value to the test statistic.

[0031] Preferably, the further measurement process of the autocorrelation feature is as follows:

[0032] Taking the corresponding time of each impurity peak as the center, setting a time window with a preset size;

[0033] Based on the autocorrelation function of the absorbance at all times within the time window, calculating the average value of the autocorrelation coefficients corresponding to multiple preset lags; taking the sum value of the average values of all impurity peaks as the autocorrelation degree of the chromatogram to be measured.

[0034] Preferably, the residual interference degree is the ratio of the asymmetry coefficient to the autocorrelation degree.

[0035] Preferably, the discrimination coefficient is the normalized result of the product of the perturbed evaluation value and the residual interference degree.

[0036] Preferably, evaluating the chromatogram to be measured includes: if the discrimination coefficient is greater than or equal to a preset threshold, the test result of the chromatogram to be measured is unqualified; otherwise, the test result of the chromatogram to be measured is qualified.

[0037] This application has at least the following beneficial effects:

[0038] This application performs frequency-domain analysis on the absorbance at the baseline of the chromatogram to be measured, analyzes the deviation of the low-frequency components and the fluctuation of the absorbance at the baseline, and calculates the baseline disturbance degree of the chromatogram to be measured. The beneficial effect is that it takes into account the baseline drift phenomenon and the degree of noise interference in the chromatogram to be measured to evaluate the impact of the detection equipment in an abnormal condition; calculates the disturbance evaluation value of the chromatogram to be measured through the tailing situation of the chromatographic peak corresponding to glycolide in the chromatogram to be measured. The beneficial effect is that it takes into account the tailing degree of the chromatographic peak corresponding to the glycolide component caused by the abnormal condition of the detection equipment to reflect the more likely impact of the chromatogram to be measured by the abnormal condition of the detection equipment, and further reflects the interference impact on the detection of the glycolide content in the chromatogram to be measured; secondly, analyzes the situation of impurity peaks such as shoulder peaks and peak bifurcations in the chromatographic peak curve where the chromatographic peak corresponding to glycolide is located, analyzes the normal distribution characteristics of the chromatographic peak curve, and the degree of asymmetry of the chromatographic peak curve caused by the impurity peaks, obtains the asymmetry coefficient of the chromatogram to be measured, and combines the autocorrelation characteristics of the impurity peaks to calculate the residual interference degree of the chromatogram to be measured. The beneficial effect is to evaluate the separation effect of liquid chromatography to reflect the interference impact of the residual organic solvents and impurities on the chromatographic peak curve, and further reflect the interference situation on the detection of the glycolide content in the chromatogram to be measured; obtains the discrimination coefficient of the chromatogram to be measured and evaluates the chromatogram to be measured; according to the change relationship between different concentrations and the corresponding peak areas of the chromatographic peaks in the standard chromatogram, obtains the fitting function, and combines the peak area corresponding to the target chromatographic peak to determine the glycolide content. The beneficial effect is that it takes into account the interference of the chromatogram to be measured by the abnormal condition of the detection equipment and the residual organic solvents and impurities to evaluate the inaccuracy of the detection result, so as to adjust the detection equipment subsequently to reduce the interference of the residual organic solvents and impurities on the detection result, improve the separation effect of glycolide in the liquid chromatography detection process, thereby improving the detection accuracy of the glycolide content and more accurately determining the glycolide content. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The following further elaborates in detail a method for detecting glycolide content based on liquid chromatography according to this application with reference to the accompanying drawings.

[0040] Figure 1 It is a flowchart of the steps of a method for detecting glycolide content based on liquid chromatography provided by an embodiment of this application;

[0041] Figure 2 It is a flowchart of the steps of a method for obtaining the baseline disturbance degree of the chromatogram to be measured provided by an embodiment of this application;

[0042] Figure 3 It is a flowchart of a method for obtaining the discrimination coefficient provided by an embodiment of this application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0043] In order to make the objectives, technical solutions and advantages of this application more clear and understandable, the following further elaborates in detail a method for detecting the content of glycolide based on liquid chromatography in combination with the accompanying drawings and implementation examples. It should be understood that the specific implementation examples described herein are only used to explain this application and are not used to limit this application.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs.

[0045] Please refer to Figure 1 , which shows a step flow chart of a method for detecting the content of glycolide based on liquid chromatography provided by an embodiment of this application. The method includes the following steps:

[0046] Step 1, prepare a sample solution to be measured and standard stock solutions diluted to different concentrations. Through a liquid chromatograph, obtain the chromatogram to be measured and standard chromatograms of different concentrations respectively.

[0047] Poly(glycolide) (PGA) is widely used in fields such as surgical sutures, controlled drug release, bone marrow fixation and repair due to its excellent biodegradability and biocompatibility. High-purity glycolide is an important monomer for producing high-performance polymer materials such as PGA. Its content directly affects the molecular weight, crystallinity, degradation rate and mechanical properties of the final polymer. To ensure whether the production quality of glycolide meets the process standards, it is necessary to accurately measure the content of glycolide to control the conditions of the polymerization reaction and ensure stable product performance.

[0048] Based on the above analysis, accurately weigh 25 mg of glycolide sample, place it in a 25 ml volumetric flask, add the mobile phase to dissolve, and make up to the scale line. After shaking well, prepare a sample solution to be measured with a concentration of 1.0 mg / ml, filter it with a 0.22 μm nylon filter, and perform on-machine analysis through a liquid chromatograph. Before detection, the liquid chromatograph flushes the chromatographic column with the mobile phase for at least 30 minutes until the baseline is stable; obtain the chromatogram to be measured;

[0049] In this embodiment, mobile phase A is 30% acetonitrile, mobile phase B is 90% acetonitrile, and an EClassical3200 liquid chromatograph is used, equipped with a reversed-phase C18 chromatographic column to detect the sample solution to be measured. Among them, the flow rate of the liquid chromatograph is 1.0 mL / min, the column temperature is 30 °C, the detection wavelength is 215 nm, and the injection volume is set to 20 μL; as other implementation methods, the implementer can set it according to the actual situation.

[0050] Accurately weigh 25 mg of glycolide standard, place it in a 25-ml volumetric flask, add mobile phase to dissolve it, and make up to the calibration line. After shaking well, prepare a standard stock solution with a concentration of 2.0 mg / ml. Then, serially dilute the standard stock solution to prepare standard solutions with concentrations of 0.1, 0.2, 0.4, 0.6, 1.0, and 2.0 mg / ml. Inject and detect them through a liquid chromatograph respectively to obtain the standard chromatograms corresponding to different concentrations.

[0051] It should be noted that the abscissa in the chromatogram represents time, indicating the time for different components to pass through the chromatographic column, and the ordinate represents absorbance, indicating the intensity of the detector response.

[0052] Thus, the chromatogram to be measured and the standard chromatograms corresponding to different concentrations are obtained.

[0053] Step 2: Analyze the deviation of the low-frequency components of the absorbance at all times on the baseline in the frequency domain in the chromatogram to be measured, as well as the discreteness of the absorbance on the baseline, and determine the baseline disturbance degree of the chromatogram to be measured; obtain the target chromatographic peak and its corresponding chromatographic peak curve in the chromatogram to be measured through the times corresponding to the chromatographic peaks in all standard chromatograms; analyze the tailing situation of the chromatographic peak curve, and combine the baseline disturbance degree to calculate the disturbance evaluation value of the chromatogram to be measured.

[0054] During the detection of glycolide content by a liquid chromatograph, poor equipment conditions will cause abnormal peak shape characteristics in the chromatogram, making it impossible to accurately measure the glycolide content. For example, the blockage of the sieve plate will hinder the passage of the mobile phase, resulting in a time delay and causing errors in the detection of the chromatogram to be measured; the collapse of the chromatographic column will make the substances unable to be effectively retained, ultimately resulting in obvious peak tailing in the liquid chromatogram; the bubbles in the detection cell and the unstable pressure of the injection pump will affect the delivery of the mobile phase, resulting in serious interference of the baseline in the chromatogram to be measured by low-frequency noise.

[0055] Secondly, since the main component of the glycolide sample is glycolide, and there will also be impurities such as residual organic solvents, acids, and glycolic acid oligomers, therefore, during the liquid chromatographic detection process, it will interfere with the chromatographic peak, resulting in inaccurate detection of the glycolide content.

[0056] Based on the above analysis, by analyzing the fluctuation change and frequency distribution of the baseline in the chromatogram to be measured, evaluate the noise situation of the baseline, and obtain the baseline disturbance degree. The step flow chart of the method for obtaining the baseline disturbance degree of the chromatogram to be measured provided by the embodiment of the present application is as Figure 2 shown, and specifically includes:

[0057] Record all the times corresponding to the baseline in the chromatogram to be measured as baseline times;

[0058] It should be noted that the determination of the baseline in the chromatogram to be measured is a well-known technique and will not be described in detail here. In this embodiment, the baseline is manually marked by a technician.

[0059] The absorbance at all baseline moments is smoothed and then analyzed in the frequency domain to obtain a spectrum.

[0060] In this embodiment, a wavelet transform algorithm is used for smoothing, wherein the wavelet transform algorithm is a well-known technology and will not be described in detail here; secondly, a fast Fourier transform is used for frequency domain analysis to obtain a spectrum diagram, wherein the fast Fourier transform is a well-known technology and will not be described in detail here. As other implementation methods, the implementer can adopt other methods of the prior art, such as discrete Fourier transform, etc., and this embodiment does not impose any special restrictions on this.

[0061] It should be noted that the high-frequency components corresponding to the noise are removed by smoothing the absorbance at the baseline moment.

[0062] Arrange all the frequency components in the spectrum in descending order according to their corresponding energies, and select a plurality of frequency components that are arranged at the front and record them as low-frequency components;

[0063] In this embodiment, 10% of the number of all frequency components is selected as another implementation method, and the implementer can set it according to the actual situation; that is, the number of all frequency components in the spectrum diagram is , then select the one that is ranked first frequency components.

[0064] Calculate the discrete degree of absorbance at all baseline moments after smoothing;

[0065] In this embodiment, the degree of dispersion is measured by calculating the variance of the absorbance at all baseline moments after smoothing. As other implementations, the implementer may adopt other methods of the prior art, such as standard deviation, coefficient of variation, etc. This embodiment does not impose any special restrictions on this.

[0066] Calculate the average value of energy corresponding to all frequency components in the spectrum diagram, and record it as average energy;

[0067] Calculate the sum of the differences between the energies corresponding to all the low-frequency components and the average energy, and record it as a relative deviation;

[0068] In this embodiment, the sum of the absolute values ​​of the differences between the energies corresponding to all the low-frequency components and the average energy is calculated and recorded as the relative deviation.

[0069] The product of the relative deviation and the discrete degree is used as the baseline disturbance of the chromatogram to be measured;

[0070] It should be noted that the greater the degree of dispersion, the greater the fluctuation of the baseline in the chromatogram to be measured, indicating the worse the stability of the baseline. The greater the relative deviation, the more serious the baseline drift in the chromatogram to be measured, and the greater the degree of baseline disturbance obtained, indicating that the baseline in the chromatogram to be measured is more seriously affected by drift and noise, which reflects that in the process of liquid chromatography detection, it is more affected by the poor condition of the detection equipment and the lower the accuracy of the detection result.

[0071] Furthermore, analyze the tailing situation of the chromatographic peaks in the chromatogram to be measured, and combine it with the degree of baseline disturbance to calculate the disturbance evaluation value to evaluate the interference situation of the chromatogram to be measured in the process of liquid chromatography detection. Specifically:

[0072] Obtain the mode of the corresponding moments of the chromatographic peaks in all standard chromatograms, denoted as the standard moment;

[0073] It should be noted that in the chromatogram, the retention time is the moment corresponding to the peak value in the chromatogram, and the retention time of the same substance in the chromatogram is the same. Therefore, for the standard chromatogram, there is only one chromatographic peak. Therefore, the retention time of the glycolide corresponding to its chromatographic peak is consistent in the standard chromatograms with different concentrations, and the retention time of the chromatographic peak corresponding to glycolide in the chromatogram to be measured should be the same as that of the standard chromatogram.

[0074] Obtain the peaks of the absorbance at all moments in the chromatogram to be measured;

[0075] In this embodiment, the AMPD (Automatic Multiscale-based Peak Detection) peak detection algorithm is used to obtain the peaks. Among them, the AMPD peak detection algorithm is a well-known technology and will not be elaborated here.

[0076] Denote the peak with the smallest time interval between the moments corresponding to all the peaks in the chromatogram to be measured and the standard moment as the target chromatographic peak;

[0077] Denote the curve between the left and right adjacent baseline moments corresponding to the moment of the target chromatographic peak in the chromatogram to be measured as the chromatographic peak curve;

[0078] Calculate the tailing factor of the chromatographic peak curve;

[0079] It should be noted that the calculation of the tailing factor is a well-known technology. The tailing factor is measured by calculating the ratio of the peak width at 5% of the peak height to the distance from the peak apex to the peak front edge and will not be elaborated here.

[0080] Take the product of the tailing factor and the degree of baseline disturbance as the disturbance evaluation value of the chromatogram to be measured;

[0081] It should be noted that the tailing factor reflects the degree of tailing of the chromatographic peaks in the chromatogram to be measured caused by the poor condition of the detection equipment. The larger the obtained tailing factor, the more serious the tailing phenomenon of the chromatographic peaks in the chromatogram to be measured, and the greater the degree of interference. The larger the interference evaluation value, the more likely the chromatogram to be measured is affected by the poor conditions of the detection equipment such as sieve plate blockage and column collapse during the liquid chromatography detection process.

[0082] Thus, the interference evaluation value of the chromatogram to be measured is obtained.

[0083] Step 3, mark the remaining peaks on the chromatographic peak curve except the target chromatographic peak as impurity peaks; analyze the normal distribution characteristics of the chromatographic peak curve and the time intervals between different impurity peaks and the target chromatographic peak to obtain the asymmetry coefficient of the chromatogram to be measured, and combine the autocorrelation characteristics of the absorbance in the neighborhood range corresponding to the moments of different impurity peaks to determine the residual interference degree of the chromatogram to be measured.

[0084] During the liquid chromatography detection process, in addition to the poor conditions of the detection equipment, impurities such as residual organic solvents, acids, and glycolic acid oligomers in the glycolide sample may co-elute with the glycolide, resulting in the appearance of unclear shoulder peaks or peak splitting phenomena. Secondly, the presence of acid impurities in the glycolide may also cause fluctuations in the pH of the mobile phase, leading to more obvious retention time shifts of the chromatographic peaks in the liquid chromatogram.

[0085] Secondly, when the residual organic solvents, acids, and glycolic acid oligomers in the glycolide sample interfere more severely with the liquid chromatography detection results, the chromatographic separation effect is worse, the chromatographic peaks deviate more from the normal distribution; and the more obvious the shoulder peaks and peak splitting phenomena appear in the chromatogram to be measured, the more blurred the symmetry of the chromatographic peaks, and thus the greater the measurement error of the glycolide content.

[0086] Based on the above analysis, first analyze the normal distribution of the chromatographic peak curve, specifically:

[0087] Calculate the test statistic of the absorbance at all moments on the chromatographic peak curve of the chromatogram to be measured;

[0088] In this embodiment, the Shapiro-Wilk test algorithm is used to calculate the test statistic of the absorbance at all moments on the chromatographic peak curve. Among them, the Shapiro-Wilk test algorithm is a well-known technology and will not be elaborated here. As other implementation manners, the implementer can adopt other methods of the existing technology, for example, the Kolmogorov-Smirnov test, etc. This embodiment does not make special restrictions on this.

[0089] It should be noted that the larger the test statistic is, the more the chromatographic peak curve conforms to the normal distribution. That is, during the liquid chromatography detection of the glycolide sample, the better the chromatographic separation effect is, and the higher the detection accuracy of the glycolide content is.

[0090] Secondly, by combining the peak bifurcation phenomenon in the chromatographic peak curve with the test statistic, the residual interference degree is calculated as follows:

[0091] The remaining peaks on the chromatographic peak curve in the chromatogram to be measured, except for the target chromatographic peak, are recorded as impurity peaks.

[0092] It should be noted that the peaks on the chromatographic peak curve include the target chromatographic peak corresponding to glycolide. Since glycolide is the main component and its content is higher than that of impurities, its peak value is the largest, and the remaining peaks are caused by impurity residues, resulting in the peak bifurcation phenomenon on the chromatographic peak curve.

[0093] Taking the corresponding moments of each impurity peak as the center, a time window with a preset size is set.

[0094] In this embodiment, the duration of the time window is 1 min. As other implementation manners, the implementer can set it according to the actual situation.

[0095] Based on the autocorrelation function of the absorbance at all moments within the time window, the average value of the autocorrelation coefficients corresponding to multiple preset lags is calculated, and the sum of the average values of all impurity peaks is used as the autocorrelation degree of the chromatogram to be measured.

[0096] It should be noted that the determination of the autocorrelation function is a well-known technology and will not be elaborated here. The autocorrelation coefficients when the lag terms are 1, 2, 3, 4, and 5 are calculated. As other implementation manners, the implementer can set it according to the actual situation. Among them, the larger the autocorrelation degree is, the more stable or periodic the impurity peaks are, and the smaller the interference of the impurity peaks is.

[0097] Furthermore, the symmetry of the impurity peaks on both sides of the target chromatographic peak is evaluated, and by combining the test statistic, the asymmetry coefficient is calculated as follows:

[0098] The difference between the corresponding moment of the target chromatographic peak and the corresponding moments of each impurity peak is recorded as the time difference.

[0099] It should be noted that if the time difference is positive, it means that the impurity peak is on the left side of the target chromatographic peak; otherwise, it is on the right side of the target chromatographic peak.

[0100] Calculate the absolute value of the sum of the ratios of the time differences of all impurity peaks to their peak widths, and use the ratio of the absolute value to the test statistic as the asymmetry coefficient of the chromatogram to be measured.

[0101] In this embodiment, the calculation formula of the asymmetry coefficient is as follows:

[0102]

[0103] where is the asymmetry coefficient of the chromatogram to be measured, is the time corresponding to the th impurity peak, is the time corresponding to the target chromatographic peak, is the peak width of the th impurity peak, is the number of all impurity peaks, is the test statistic.

[0104] It should be noted that the calculation of the peak width is a well-known technique. In this embodiment, taking the th impurity peak as an example, two adjacent troughs corresponding to the th impurity peak are obtained, and the time interval between the corresponding times of the two troughs is recorded as the peak width. Secondly, the larger the absolute value, the more serious the peak splitting phenomenon is, the less symmetric the chromatographic peak curve is caused by the impurity peak, and the smaller the test statistic is, the more deviated the chromatographic peak curve is from the normal distribution. Then, the larger the obtained asymmetry coefficient is, the more asymmetric the chromatographic peak curve is, which reflects that the liquid chromatography separation effect is poor, the peak splitting condition of the target chromatographic peak is more serious, and the interference of impurity residues is greater.

[0105] Furthermore, based on the asymmetry coefficient and the autocorrelation degree, the residual interference degree is calculated as follows:

[0106] Calculate the ratio of the asymmetry coefficient to the autocorrelation degree as the residual interference degree of the chromatogram to be measured;

[0107] It should be noted that the larger the residual interference degree is, the greater the interference of the organic solvent and impurity residues on the curve where the chromatographic peak is located during the liquid chromatography detection process.

[0108] Thus, the residual interference degree of the chromatogram to be measured is obtained.

[0109] Step 4: Based on the perturbed evaluation value and the residual interference degree, obtain the discrimination coefficient of the chromatogram to be measured, and evaluate the chromatogram to be measured; according to the change relationship between different concentrations and the corresponding peak areas of the chromatographic peaks in the standard chromatogram, obtain the fitting function, and combine the peak area corresponding to the target chromatographic peak to determine the glycolide content.

[0110] Further, during the liquid chromatography detection process, the better the condition of the detection equipment and the less affected it is by organic solvents and residual impurities, the more accurate the detection of glycolide content; therefore, based on the disturbance evaluation value and the residual interference degree, a discrimination coefficient is determined to evaluate the separation effect during the liquid chromatography detection process and the accuracy of glycolide content detection. Specifically:

[0111] The normalized result of the product of the disturbance evaluation value and the residual interference degree is used as the discrimination coefficient of the chromatogram to be measured;

[0112] In this embodiment, the sigmoid function is used for normalization processing. The sigmoid function is a well-known technology and will not be elaborated here. As other implementation manners, implementers can adopt other methods of the existing technology, such as the tanh function, etc. This embodiment does not make special restrictions on this.

[0113] It should be noted that the larger the discrimination coefficient, the greater the influence of the poor condition of the detection equipment and the interference of organic solvents and residual impurities, and the lower the detection accuracy of glycolide. It is necessary to further check the working state of the liquid chromatograph, eliminate the interference of equipment failures and instabilities, and timely adjust the relevant parameters of the liquid chromatograph to improve the separation effect of glycolide and impurities, thereby improving the detection accuracy of glycolide content. Among them, the flow chart of the method for obtaining the discrimination coefficient provided by the embodiment of the present application is as Figure 3 shown.

[0114] Calculate the peak area of the standard chromatogram corresponding to each concentration, and form a two-dimensional array with each concentration and its corresponding peak area;

[0115] Perform linear fitting on all two-dimensional arrays to obtain a fitting equation;

[0116] It should be noted that the calculation of the peak area is a well-known technology and will not be elaborated here.

[0117] When the discrimination coefficient is greater than or equal to the preset threshold, check the working state of the liquid chromatograph; when the discrimination coefficient is less than the preset threshold, substitute the peak area corresponding to the target chromatographic peak in the chromatogram to be measured into the fitting equation to obtain the concentration of glycolide in the sample solution to be measured; and determine the content of glycolide according to the dilution factor and injection volume of the sample solution to be measured.

[0118] In this embodiment, the preset threshold is set to 0.7. As other implementation manners, implementers can set it according to actual situations.

[0119] It should be noted that the process of determining the content of glycolide by the concentration of glycolide is a well-known technology and will not be elaborated here. Secondly, when the discrimination coefficient is greater than or equal to the preset threshold, the processing process for the equipment is as follows: Ultrasonic treatment of the sieve plate with pure water for 10 - 15 minutes can be adopted to remove blockage, reverse the operation of the chromatographic column, and repeatedly rinse with the mobile phase to repair the collapse of the column head; when the chromatographic column is severely collapsed, the chromatographic column needs to be replaced; Connect a thin-inner-diameter PEEK tube (3 - 5 cm) at the detector outlet to increase the back pressure to suppress bubbles in the detection cell, and at the same time keep the temperature of the detection cell consistent with that of the column oven to avoid local overheating; Detect the seal of the pump head and replace the worn sealing ring, and debug whether there is still an unstable injection pump pressure condition for the injection pump pressure.

[0120] It should be understood that although Figure 1 the steps in the flowchart of Figure 1 are shown in sequence according to the indication of the arrows, these steps are not necessarily executed in the order indicated by the arrows. Unless there is a clear description in this article, there is no strict order limit for the execution of these steps, and these steps can be executed in other orders. Moreover,

[0121] The technical features of the above embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0122] The above-described embodiments only represent several implementation manners of the present application, and their descriptions are relatively specific and detailed, but should not be construed as a limitation to the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several deformations and improvements can still be made. Therefore, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present application without departing from the content of the technical solution of the present application all belong to the protection scope of the technical solution of the present application.

Claims

1. A method for detecting glycolide content based on liquid chromatography, characterized in that: The method comprises the following steps: Prepare a sample solution to be tested and a standard stock solution diluted to different concentrations, and obtain a chromatogram to be tested and a standard chromatogram of different concentrations through a liquid chromatograph; Analyze the deviation of the low-frequency components of the absorbance at all moments on the baseline of the chromatogram to be tested in the frequency domain, as well as the discreteness of the absorbance on the baseline, to determine the baseline disturbance degree of the chromatogram to be tested; Obtain the target chromatographic peak in the chromatogram to be tested and its corresponding chromatographic peak curve through the time corresponding to the chromatographic peak in all standard chromatograms; analyze the tailing of the chromatographic peak curve, and calculate the disturbance evaluation value of the chromatogram to be tested in combination with the baseline disturbance degree; The remaining peaks on the chromatographic peak curve except the target chromatographic peak are recorded as impurity peaks; the test statistic of the absorbance at all times on the chromatographic peak curve in the chromatogram to be tested is calculated; the difference between the corresponding time of the target chromatographic peak and the corresponding time of each impurity peak is recorded as the time difference; the absolute value of the cumulative sum of the ratios of the time difference of all impurity peaks to their peak widths is calculated; the ratio of the absolute value to the test statistic is recorded as the asymmetry coefficient of the chromatogram to be tested, and the residual interference of the chromatogram to be tested is determined in combination with the autocorrelation characteristics of the absorbance in the neighborhood range at the corresponding time of different impurity peaks; Based on the disturbance assessment value and the residual interference degree, the discrimination coefficient of the chromatogram to be measured is obtained, and the chromatogram to be measured is evaluated; according to the change relationship between the peak area corresponding to the chromatographic peak in the standard chromatogram with different concentrations and its corresponding peak area, the fitting function is obtained, and the glycolide content is determined in combination with the peak area corresponding to the target chromatographic peak.

2. A method for detecting glycolide content based on liquid chromatography as claimed in claim 1, characterized in that: The method of determining the baseline disturbance of the chromatogram to be tested comprises: All the moments corresponding to the baseline in the chromatogram to be tested are recorded as baseline moments; the absorbances at all baseline moments are smoothed and then analyzed in the frequency domain to obtain a spectrum; Filter low-frequency components from all frequency components in the spectrum, and determine the relative deviation through the offset of the corresponding energy of different low-frequency components; Calculate the discrete degree of absorbance at all baseline moments after smoothing; The baseline disturbance degree is the product of the relative deviation and the discrete degree.

3. A method for detecting glycolide content based on liquid chromatography as claimed in claim 2, characterized in that: The relative deviation is determined as follows: Calculate the average value of energy corresponding to all frequency components in the spectrum diagram, and record it as average energy; The relative deviation is the sum of the differences between the energies corresponding to all the low-frequency components and the average energy.

4. A method for detecting glycolide content based on liquid chromatography as claimed in claim 2, characterized in that: The step of obtaining the target chromatographic peak in the chromatogram to be tested and its corresponding chromatographic peak curve comprises: Obtain the mode of the time corresponding to the chromatographic peak in all standard chromatograms, and record it as the standard time; Obtain the peaks of absorbance at all times in the chromatogram to be tested; record the peak with the smallest time interval between the time corresponding to all the peaks in the chromatogram to be tested and the standard time as the target chromatographic peak; The curve between the baseline moments on the left and right sides of the corresponding moment of the target chromatographic peak in the chromatogram to be tested is recorded as the chromatographic peak curve.

5. The method for detecting glycolide content based on liquid chromatography according to claim 1, characterized in that: The method of calculating the disturbance evaluation value of the chromatogram to be tested comprises: Calculating the tailing factor of the chromatographic peak curve; The disturbance assessment value is the product of the tailing factor and the baseline disturbance.

6. The method for detecting glycolide content based on liquid chromatography according to claim 1, characterized in that: The further measurement process of the autocorrelation feature is: Set a time window of preset size with the corresponding time of each impurity peak as the center; Based on the autocorrelation function of the absorbance at all moments in the time window, the average values ​​of the autocorrelation coefficients corresponding to the plurality of preset lag terms are calculated; and the sum of the average values ​​of all impurity peaks is taken as the autocorrelation of the chromatogram to be tested.

7. A method for detecting glycolide content based on liquid chromatography as claimed in claim 6, characterized in that: The residual interference degree is a ratio of the asymmetry coefficient to the autocorrelation degree.

8. The method for detecting glycolide content based on liquid chromatography according to claim 1, characterized in that: The discrimination coefficient is a normalized result of the product of the disturbance assessment value and the residual interference degree.

9. The method for detecting glycolide content based on liquid chromatography according to claim 1, characterized in that: The evaluating of the chromatogram to be tested includes: if the discrimination coefficient is greater than or equal to a preset threshold, the test result of the chromatogram to be tested is unqualified; otherwise, the test result of the chromatogram to be tested is qualified.

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