Glycolide content detection method based on liquid chromatography

Abstract

The invention relates to the technical field of chromatographic analysis, in particular to a glycolide content detection method based on liquid chromatography, which comprises the following steps: preparing a to-be-detected sample solution and diluting the to-be-detected sample solution into standard stock solutions with different concentrations, and respectively obtaining a to-be-detected chromatogram and standard chromatograms with different concentrations through a liquid chromatograph; determining the baseline deflection of the to-be-detected chromatogram; obtaining a target chromatographic peak in the to-be-detected chromatogram and a corresponding chromatographic peak curve; calculating a disturbed evaluation value, an asymmetry coefficient, a residual interference degree and a discrimination coefficient of the to-be-detected chromatogram, and evaluating the to-be-detected chromatogram; obtaining a fitting function according to different concentrations and the change relation between the peak areas corresponding to the chromatographic peaks in the corresponding standard chromatograms, and determining the glycolide content by combining the peak area corresponding to the target chromatographic peak. According to the method, the detection precision of the glycolide content can be improved, and the glycolide content can be measured more accurately.

Description

Technical Field

[0001] The present application relates to the technical field of chromatography analysis, and in particular to a method for detecting glycolide content based on liquid chromatography. Background Art

[0002] Glycolide, as an important monomer of polymer materials such as polyglycolic acid (PGA) and polylactic-co-glycolic acid (PLGA), has an irreplaceable position in the field of biodegradable materials. Its purity will affect the mechanical properties, degradation rate and biocompatibility of polymer materials, and residual impurities will affect the mechanical strength of polymer materials, uncontrolled degradation or biotoxicity risks.

[0003] Liquid chromatography can efficiently detect the content of glycolide. The poor condition of liquid chromatography detection equipment and impurities such as residual organic solvents, acids, and glycolide oligomers in glycolide will interfere with the 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, affecting the accuracy of the test results. Summary of the invention

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

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

[0006] 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;

[0007] 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;

[0008] 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;

[0009] The remaining peaks on the chromatographic peak curve except the target chromatographic peak are recorded as impurity peaks; the normal distribution characteristics of the chromatographic peak curve and the intervals between different impurity peaks and the target chromatographic peak are analyzed to obtain the asymmetry coefficient of the chromatogram to be tested, and the residual interference degree of the chromatogram to be tested is determined by combining the autocorrelation characteristics of the absorbance in the neighborhood range at the corresponding time of different impurity peaks;

[0010] 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.

[0011] Preferably, the determining of the baseline disturbance degree of the chromatogram to be measured comprises:

[0012] 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;

[0013] 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;

[0014] Calculate the discrete degree of absorbance at all baseline moments after smoothing;

[0015] The baseline disturbance degree is the product of the relative deviation and the discrete degree.

[0016] Preferably, the relative deviation is determined as follows:

[0017] Calculate the average value of energy corresponding to all frequency components in the spectrum diagram, and record it as 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 step of obtaining a target chromatographic peak in the chromatogram to be measured and its corresponding chromatographic peak curve comprises:

[0020] Obtain the mode of the time corresponding to the chromatographic peak in all standard chromatograms, and record it as the standard time;

[0021] 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;

[0022] 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.

[0023] Preferably, the calculation of the disturbance assessment value of the chromatogram to be tested comprises:

[0024] Calculating the tailing factor of the chromatographic peak curve;

[0025] The disturbance assessment value is the product of the tailing factor and the baseline disturbance.

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

[0027] Calculate the test statistic of the absorbance at all times on the chromatographic peak curve in the chromatogram to be tested;

[0028] The difference between the time corresponding to the target chromatographic peak and the time corresponding to each impurity peak is recorded as the time difference;

[0029] Calculate the absolute value of the cumulative 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:

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

[0033] 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.

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

[0035] Preferably, the discrimination coefficient is a normalized result of the product of the disturbance assessment value and the residual interference degree.

[0036] Preferably, the evaluating 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.

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

[0038] The present application performs frequency domain analysis on the absorbance on the baseline in the chromatogram to be tested, analyzes the deviation of the low-frequency component and the fluctuation of the absorbance on the baseline, and calculates the baseline disturbance of the chromatogram to be tested. The beneficial effect is that the drift phenomenon of the baseline in the chromatogram to be tested and the degree of noise interference are taken into account to evaluate the impact of the poor condition of the detection equipment; the disturbance evaluation value of the chromatogram to be tested is calculated by the tailing of the chromatographic peak corresponding to glycolide in the chromatogram to be tested, and the beneficial effect is that the tailing degree of the chromatographic peak corresponding to the glycolide component caused by the poor condition of the detection equipment is taken into account to reflect that the chromatogram to be tested is more likely to be affected by the poor condition of the detection equipment, and then reflects the interference effect on the glycolide content of the chromatogram to be tested; secondly, 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 is analyzed, and the normal distribution characteristics of the chromatographic peak curve and the degree of asymmetry of the chromatographic peak curve caused by the impurity peak are analyzed to obtain the chromatogram to be tested. The asymmetry coefficient is combined with the autocorrelation characteristics of the impurity peak to calculate the residual interference of the chromatogram to be tested. Its beneficial effect is to evaluate the separation effect of liquid chromatography to reflect the interference of residual organic solvents and impurities on the chromatographic peak curve, and further reflect the interference of the chromatogram to be tested on the detection of glycolide content; the discrimination coefficient of the chromatogram to be tested is obtained to evaluate the chromatogram to be tested; according to the change relationship between the corresponding peak area of ​​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. Its beneficial effect is that it takes into account the interference of the chromatogram to be tested by the poor condition of the detection equipment and the residual organic solvents and impurities, so as to evaluate the inaccuracy of the detection result, so as to adjust the detection equipment later to reduce the interference of residual organic solvents and impurities on the detection result, so that the liquid chromatography detection process can improve the separation effect of glycolide, thereby improving the detection accuracy of glycolide content and more accurately determining the glycolide content. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] A method for detecting glycolide content based on liquid chromatography of the present application is further described in detail below in conjunction with the accompanying drawings.

[0040] Figure 1 A flow chart of the steps of a method for detecting glycolide content based on liquid chromatography provided in an embodiment of the present application;

[0041] Figure 2 A flowchart of the steps of a method for obtaining the baseline disturbance degree of a chromatogram to be measured provided in an embodiment of the present application;

[0042] Figure 3 A flowchart of a method for obtaining a discrimination coefficient provided in an embodiment of the present application. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the following is a further detailed description of a method for detecting glycolide content based on liquid chromatography proposed in the present application in conjunction with the accompanying drawings and implementation examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application.

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0045] See also Figure 1 , which shows a flow chart of a method for detecting glycolide content based on liquid chromatography provided in one embodiment of the present application, the method comprising the following steps:

[0046] Step 1: 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.

[0047] Polyglycolide (PGA) is widely used in 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 the production of high-performance PGA and other polymer materials. Its content directly affects the molecular weight, crystallinity, degradation rate and mechanical properties of the final polymer. In order to ensure that the production quality of glycolide meets the process standards, it is necessary to accurately measure the glycolide content to control the conditions of the polymerization reaction and ensure stable product performance.

[0048] Based on the above analysis, 25 mg of glycolide sample was accurately weighed, placed in a 25 ml volumetric flask, dissolved with mobile phase, and fixed to the scale line, shaken to prepare a 1.0 mg / ml sample solution to be tested, filtered with a 0.22 μm nylon filter, and analyzed on a liquid chromatograph, wherein the liquid chromatograph flushed the chromatographic column with mobile phase for at least 30 minutes before detection until the baseline was stable; the chromatogram to be tested was obtained;

[0049] In this embodiment, mobile phase A is 30% acetonitrile, mobile phase B is 90% acetonitrile, and an EClassical 3200 liquid chromatograph is used in combination with a reversed-phase C18 chromatographic column to detect the sample solution. 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 them according to actual conditions.

[0050] Accurately weigh 25 mg of glycolide standard, place it in a 25 ml volumetric flask, add mobile phase to dissolve, and dilute to the scale line. Shake well to prepare a 2.0 mg / ml standard stock solution, and then dilute the standard stock solution step by step to prepare 0.1, 0.2, 0.4, 0.6, 1.0, and 2.0 mg / ml standard solutions, respectively, and perform injection detection through liquid chromatograph to obtain standard chromatograms corresponding to different concentrations.

[0051] It should be noted that the horizontal axis in the chromatogram is time, indicating the time it takes for different components to pass through the chromatographic column, and the vertical axis is absorbance, indicating the intensity of the detector response.

[0052] At this point, the chromatogram to be tested and the standard chromatograms corresponding to different concentrations are obtained.

[0053] Step 2, analyzing 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 of the chromatogram to be tested; obtaining the target chromatographic peak in the chromatogram to be tested and its corresponding chromatographic peak curve through the moments corresponding to the chromatographic peaks in all standard chromatograms; analyzing the tailing of the chromatographic peak curve, and calculating the disturbance evaluation value of the chromatogram to be tested in combination with the baseline disturbance.

[0054] In the process of detecting the glycolide content by liquid chromatography, poor equipment condition will cause abnormal peak shape characteristics of the chromatogram, and it is impossible to accurately determine the glycolide content. For example, 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 tested; the collapse of the chromatographic column will make it impossible to effectively retain the substance formed, and ultimately lead to obvious peak tailing in the liquid chromatogram; bubbles in the detection pool and unstable pressure of the injection pump will affect the delivery of the mobile phase, causing the baseline in the chromatogram to be tested to be seriously disturbed by low-frequency noise.

[0055] Secondly, since the main content of glycolide samples is glycolide, there will also be impurities such as residual organic solvents, acids and hydroxyacetic acid oligomers. Therefore, during the liquid chromatography 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 and frequency distribution of the baseline in the chromatogram to be tested, the noise of the baseline is evaluated, and the baseline disturbance is obtained. The step flow chart of the method for obtaining the baseline disturbance of the chromatogram to be tested provided in the embodiment of the present application is as follows: Figure 2 As shown, specifically including:

[0057] All the moments corresponding to the baseline in the chromatogram to be measured are recorded as baseline moments;

[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] Calculating the sum of the differences between the energies corresponding to all the low-frequency components and the average energy, and recording 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 discreteness, the greater the fluctuation of the baseline in the chromatogram to be tested, which reflects that the stability of the baseline is worse; the greater the relative deviation, the more serious the baseline drift in the chromatogram to be tested; the greater the obtained baseline disturbance, the more serious the baseline in the chromatogram to be tested is disturbed by drift and noise, which is reflected in the liquid chromatography detection process. The greater the impact of the poor condition of the detection equipment, the lower the accuracy of the detection result.

[0071] Furthermore, the tailing of the chromatographic peaks in the chromatogram to be tested is analyzed, and the disturbance evaluation value is calculated in combination with the baseline disturbance degree to evaluate the interference of the chromatogram to be tested during the liquid chromatography detection process, specifically:

[0072] Obtain the mode of the time corresponding to the chromatographic peak in all standard chromatograms, and record it as the standard time;

[0073] It should be noted that in the chromatogram, the retention time is the moment corresponding to the peak in the chromatogram. 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 of different concentrations. Therefore, the retention time of the chromatographic peak corresponding to glycolide in the chromatogram to be tested should be the same as that of the standard chromatogram.

[0074] Obtain the peak of absorbance at all times in the chromatogram to be measured;

[0075] In this embodiment, an AMPD (Automatic Multiscale-based Peak Detection) peak detection algorithm is used to obtain the wave peak, wherein the AMPD peak detection algorithm is a well-known technology and will not be described in detail here.

[0076] 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 is recorded as the target chromatographic peak;

[0077] The curve between the baseline moments on the left and right sides of the time corresponding to the target chromatographic peak in the chromatogram to be tested is recorded as the chromatographic peak curve;

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

[0079] It should be noted that the calculation of the tailing factor is a well-known technique, and 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, which will not be described in detail here.

[0080] The product of the tailing factor and the baseline disturbance degree is used as the disturbance evaluation value of the chromatogram to be tested;

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

[0082] At this point, the disturbance evaluation value of the chromatogram to be tested is obtained.

[0083] Step 3, the remaining peaks on the chromatographic peak curve except the target chromatographic peak are recorded as impurity peaks; the normal distribution characteristics of the chromatographic peak curve and the time intervals between different impurity peaks and the target chromatographic peak are analyzed to obtain the asymmetry coefficient of the chromatogram to be measured, and the residual interference degree of the chromatogram to be measured is determined by combining the autocorrelation characteristics of the absorbance in the neighborhood range at the corresponding time of different impurity peaks.

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

[0085] Secondly, the more serious the interference of residual organic solvents, acids and glycolic acid oligomers in the glycolide sample on the liquid chromatography detection results, the worse the chromatographic separation effect, and the more the chromatographic peak deviates from the normal distribution; and the more obvious the shoulder peak and peak bifurcation phenomenon appear in the chromatogram to be tested, the more blurred the symmetry of the chromatographic peak, and thus the greater the error in the determination of the glycolide content.

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

[0087] Calculate the test statistic of the absorbance at all times on the chromatographic peak curve in the chromatogram to be tested;

[0088] In this embodiment, the Shapiro-Wilk test algorithm is used to calculate the test statistic of the absorbance at all times on the chromatographic peak curve, wherein the Shapiro-Wilk test algorithm is a well-known technology and will not be described here. As other implementation methods, the implementer can adopt other methods of the prior art, such as the Kolmogorov-Smirnov test, etc., and this embodiment does not impose any special restrictions on this.

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

[0090] Secondly, the residual interference is calculated by combining the peak bifurcation phenomenon in the chromatographic peak curve with the test statistics, specifically:

[0091] The remaining peaks on the chromatographic peak curve in the chromatographic diagram to be tested, 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 the impurity content, the peak value is the largest, and the remaining peaks are caused by residual impurities, resulting in peak bifurcation on the chromatographic peak curve.

[0093] Set a time window of preset size with the corresponding time of each impurity peak as the center;

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

[0095] 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 used as the autocorrelation of the chromatogram to be tested;

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

[0097] Further, the symmetry of the impurity peaks on both sides of the target chromatographic peak is evaluated, and the asymmetry coefficient is calculated in combination with the test statistic, specifically:

[0098] The difference between the time corresponding to the target chromatographic peak and the time corresponding to each impurity peak is recorded as the time difference;

[0099] It should be noted that if the time difference is a positive number, 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 cumulative 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 tested;

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

[0102]

[0103] in, is the asymmetry coefficient of the chromatogram to be tested, For the The time corresponding to the impurity peak is is the time corresponding to the target chromatographic peak, For the The peak width of the impurity peak, is the number of all impurity peaks, is the test statistic.

[0104] It should be noted that the calculation of peak width is a well-known technique. Take the impurity peak as an example and obtain the The time interval between the corresponding moments of the two troughs adjacent to the impurity peaks is recorded as the peak width; secondly, the larger the absolute value, the more serious the peak bifurcation phenomenon is, and the less symmetric the chromatographic peak curve is caused by the impurity peak, and the smaller the test statistic is, the more the chromatographic peak curve deviates from the normal distribution, and the larger the obtained asymmetry coefficient is, reflecting that the more asymmetric the chromatographic peak curve is, the poorer the liquid chromatography separation effect is, the more serious the peak bifurcation of the target chromatographic peak is, and the greater the interference from impurity residues.

[0105] Further, based on the asymmetry coefficient and the autocorrelation, the residual interference is calculated, specifically:

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

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

[0108] At this point, the residual interference of the chromatogram to be measured is obtained.

[0109] Step 4, based on the disturbance assessment value and the residual interference degree, obtain the discrimination coefficient of the chromatogram to be tested, and evaluate the chromatogram to be tested; 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, obtain the fitting function, and determine the glycolide content in combination with the peak area corresponding to the target chromatographic peak.

[0110] Furthermore, in the process of liquid chromatography detection, the better the condition of the detection equipment and the less interference from organic solvents and impurities, the more accurate the detection of glycolide content; therefore, based on the interference evaluation value and the residual interference degree, the discrimination coefficient is determined to evaluate the separation effect in the process of liquid chromatography detection 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 tested;

[0112] In this embodiment, the sigmoid function is used for normalization processing, wherein the sigmoid function 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 the tanh function, etc. This embodiment does not impose any special restrictions on this.

[0113] It should be noted that the larger the discrimination coefficient is, the greater the impact of poor conditions of the detection equipment and interference from organic solvents and impurity residues, and the lower the detection accuracy of glycolide. It is necessary to further check the working status of the liquid chromatograph, eliminate the interference of equipment failure and instability, and adjust the relevant parameters of the liquid chromatograph in a timely manner to improve the separation effect of glycolide and impurities, thereby improving the detection accuracy of glycolide content. The flowchart of the method for obtaining the discrimination coefficient provided in the embodiment of the present application is as follows: 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 fitting equations;

[0116] It should be noted that the calculation of peak area is a well-known technique and will not be described in detail here.

[0117] When the discrimination coefficient is greater than or equal to a preset threshold, the working status of the liquid chromatograph is checked; when the discrimination coefficient is less than the preset threshold, the peak area corresponding to the target chromatographic peak in the chromatogram to be tested is substituted into the fitting equation to obtain the concentration of glycolide in the sample solution to be tested; and the content of glycolide is determined according to the dilution multiple and injection volume of the sample solution to be tested.

[0118] In this embodiment, the preset threshold value is 0.7. As for other implementation modes, the implementer can set it according to the actual situation.

[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 repeated here. Secondly, when the discrimination coefficient is greater than or equal to the preset threshold, the processing process of the equipment is as follows: the sieve plate can be treated with pure water ultrasound for 10 to 15 minutes to remove the blockage, the chromatographic column can be reversed, and the mobile phase can be used to repeatedly rinse and repair the column head collapse; when the chromatographic column collapses severely, the chromatographic column needs to be replaced; a thin inner diameter PEEK tube (3 to 5 cm) is connected to the detector outlet to increase the back pressure to suppress the bubbles in the detection pool, while keeping the detection pool and the column oven temperature consistent to avoid local overheating; the pump head sealing is tested and the wear of the replacement seal ring is replaced, and the injection pump pressure is debugged to see if there is still an unstable injection pump pressure.

[0120] It should be understood that although Figure 1 The steps in the flowchart are shown in sequence as indicated by the arrows, but these steps are not necessarily executed in the order indicated by the arrows. Unless otherwise specified in this document, there is no strict order restriction for the execution of these steps, and these steps can be executed in other orders. Moreover, Figure 1 At least part of the steps may include multiple sub-steps or multiple stages. These sub-steps or stages are not necessarily executed at the same time, but can be executed at different times. The execution order of these sub-steps or stages is not necessarily sequential, but can be executed in turn or alternately with other steps or at least part of the sub-steps or stages of other steps.

[0121] The technical features of the above embodiments may be combined arbitrarily. To make the description concise, 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, they should be considered to be within the scope of this specification.

[0122] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the present application. It should be pointed out that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present application. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application without departing from the content of the technical solution of the present application, shall fall within 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 normal distribution characteristics of the chromatographic peak curve and the intervals between different impurity peaks and the target chromatographic peak are analyzed to obtain the asymmetry coefficient of the chromatogram to be tested, and the residual interference degree of the chromatogram to be tested is determined by combining 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 step of obtaining the asymmetry coefficient of the chromatogram to be measured comprises: Calculate the test statistic of the absorbance at all times on the chromatographic peak curve in the chromatogram to be tested; The difference between the time corresponding to the target chromatographic peak and the time corresponding to each impurity peak is recorded as the time difference; Calculate the absolute value of the cumulative sum of the ratios of the time differences of all impurity peaks to their peak widths; The asymmetry coefficient is the ratio of the absolute value to the test statistic.

7. 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.

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

9. 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.

10. 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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