CN112154325B - Method for analyzing sugar chain distribution of low-molecular heparin and application thereof - Google Patents
Method for analyzing sugar chain distribution of low-molecular heparin and application thereof Download PDFInfo
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Abstract
A method for analyzing sugar chain distribution of low-molecular heparin, comprising the steps of: separating the low molecular weight heparin by a chromatographic column of High Performance Liquid Chromatography (HPLC); and then detected by an electric fog type detector (CAD). The method has short detection time and low detection cost, and can simultaneously realize accurate qualitative and quantitative analysis of sugar chains with different lengths in the low molecular heparin sample.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an analysis method for sugar chain distribution of low-molecular heparin and application thereof.
Background
The polydispersity of the chain length is a factor of the molecular diversity of low-molecular heparin, one index for measuring the polydispersity of the chain length is molecular weight distribution, oligosaccharide chains with different lengths are detected by separation of chromatographic columns, most laboratories use chromatographic columns at present and analyze the oligosaccharide chains by UPLC (quantitative trait locus) mass spectrometry, the method can solve the problem that the heparin sodium has no ultraviolet characteristic absorption, and meanwhile, polysaccharide chains are separated out by the UPLC high-efficiency separation capability, but the method cannot accurately quantify the separated oligosaccharide chains with different lengths.
The prior art document (Joseph Zaia et al, "Complete molecular micro arm Weight profiling of Low-molecular micro arm Weight Using Size extrusion Chromatography-Ion supressor-High-Resolution Mass Spectrometry". Anal. Chemi. (2016) 88, 10654-10660) uses an UPLC instrument, BEH SEC 125.6 x 150mm and BEH SEC 125.6 x 300mm in series, in combination with a salt removal Suppressor ACRS500 mm before the Mass spectrometer (QTOF), to qualitatively analyze them by TIC map, without quantitative conditions at all. The method still has a running time of 90min under the condition of using UPLC.
It is clear that the prior art solutions have the following drawbacks: 1) UPLC combined QTOF is needed, so the cost is higher; 2) The separation effect can only separate dp20, and accurate quantitative analysis cannot be carried out; 3) The running time is long and reaches 90min.
Therefore, at present, there is a need for an analysis method that has a short detection time and a low detection cost and can simultaneously realize accurate qualitative and quantitative analysis of sugar chains with different lengths in a low-molecular heparin sample.
Disclosure of Invention
In a first aspect, the present invention relates to a method for analyzing sugar chain distribution of low-molecular heparin, the method comprising the steps of: (1) Separating the low molecular weight heparin by a chromatographic column of High Performance Liquid Chromatography (HPLC); (2) And then detected by an electric fog type detector (CAD).
In some embodiments, the chromatography column used in step (1) is a Size Exclusion (SEC) chromatography column.
In some embodiments, in step (1), the conditions of the high performance liquid chromatography are a flow rate of 0.1 to 2mL/min, isocratic elution, a column temperature of 20 to 30 ℃, and wherein the mobile phase is an ammonium salt solution.
In some embodiments, the ammonium salt is ammonium acetate or ammonium formate at a concentration of 50 to 250mM and the flow rate is 0.1 to 1mL/min. Further, the concentration is 100-200mM, and the flow rate is 0.3-0.8mL/min. Still further, the concentration is about 100mM or about 150mM, and the flow rate is about 0.7mL/min.
In some embodiments, the method further comprises the steps of: comparing the detection result obtained in the step (2) with the detection results of oligosaccharide standard products with different sugar chain lengths, and/or comparing the detection result obtained in the step (2) with the detection result of the ultra performance liquid chromatography-mass spectrometry of the low molecular heparin; thereby obtaining the qualitative and quantitative results of the chain distribution of the low molecular heparin in the step (1).
In some embodiments, the low molecular heparin is dalteparin sodium, enoxaparin sodium, or nadroparin calcium.
In a first aspect the invention relates to the use of said analytical method for the quality detection of low molecular weight heparins.
In some embodiments, the method comprises performing multiple tests on the low molecular heparin standard and/or the primary sample using the analysis method, and establishing a quality standard according to the test results.
Drawings
FIG. 1 shows the SEC-CAD integration plot of the sodium dalteparin European pharmacopoeia reference standard with a mobile phase concentration of 100mM ammonium acetate, a sample size of 16 μ l, and a flow rate of 0.7mL/min.
FIG. 2 shows a SEC-CAD integration plot of the DaGansu sodium European pharmacopoeia reference standard with a mobile phase concentration of 150mM ammonium acetate, a sample size of 16 μ l, and a flow rate of 0.5mL/min.
FIG. 3 shows a SEC-CAD integration plot of the DaGansu sodium European pharmacopoeia reference standard with a mobile phase concentration of 50mM ammonium acetate, a sample size of 8 μ l, and a flow rate of 0.7mL/min.
FIG. 4 shows the SEC-CAD integration plot of the sodium dalteparin European pharmacopoeia reference standard with a mobile phase concentration of 200mM ammonium acetate, a sample size of 8 μ l, and a flow rate of 0.7mL/min.
FIG. 5 shows the SEC-CAD integration plot of the sodium dalteparin European pharmacopoeia reference standard with a mobile phase concentration of 250mM ammonium acetate, a sample size of 8 μ l, and a flow rate of 0.7mL/min.
FIG. 6 shows a SEC-CAD integration plot of the daparinux sodium European pharmacopoeia reference standard with a mobile phase concentration of 200mM ammonium formate, a sample size of 8 μ l, and a flow rate of 0.7mL/min.
FIG. 7 shows a SEC-CAD integration plot of the daparinux sodium European pharmacopoeia reference standard with a mobile phase concentration of 50mM ammonium formate, a sample size of 8 μ l, and a flow rate of 0.7mL/min.
Figure 8 shows a overlay of blank solution (bottom) and dalteparin sodium usp reference standard solution (top).
Figure 9 shows a overlay of dalteparin sodium usp reference standard with oligosaccharide standard.
Figure 10 shows a overlay of darabin sodium european pharmacopoeia reference standard and oligosaccharide standard.
Figure 11 shows a overlay of darabin sodium usp reference standard with Dermatan Sulfate (DS), naCl, methanol and ethanol (from bottom to top darabin sodium usp reference standard, dermatan Sulfate (DS), naCl, methanol and ethanol, respectively).
Definition of
As used herein, the term "low molecular heparin" refers to a generic name for a class of lower molecular weight heparins prepared by depolymerization of normal heparin, including but not limited to: enoxaparin sodium, nadroparin calcium, dalteparin sodium, etc.
As used herein, the term "room temperature" refers to 25 ℃ ± 5 ℃. Meanwhile, if the experimental temperature is not specified, the temperature is room temperature.
As used herein, the term "about" refers to ± 20%, preferably ± 10%, more preferably ± 5% of the numerical value modified by the term, and thus the range of the term "about" can be clearly determined according to the modified numerical value by one of ordinary skill in the art.
As used herein, the term "low molecular heparin standard" refers to usp reference standards and european pharmacopoeia reference standards including, but not limited to, low molecular heparin.
As used herein, the term "low molecular heparin raw research sample" refers to the low molecular heparin marketed product of the raw research manufacturer, including but not limited to low molecular heparin marketed products in the united states and europe.
As used herein, the term "dalteparin sodium standard" refers to us pharmacopoeia reference standards and european pharmacopoeia reference standards including, but not limited to, dalteparin sodium.
As used herein, the term "heparanan sodium raw research sample" refers to the marketed product of heparanan sodium from the raw research manufacturer, pfeiri, including but not limited to the marketed product of heparanan sodium in the united states and europe.
As used herein, the following english abbreviations have the meaning commonly known to those skilled in the art. Specifically, UPLC: ultra-high performance liquid chromatography; QTOF: a time-of-flight mass spectrometer; CAD: an electrospray detector (electrospray detector); dp: represents the degree of polymerization of the sugar chain; and (2) MS: a mass spectrometer; and (3) TIC: a total ion flow pattern; SEC: size exclusion.
Detailed Description
The invention uses High Performance Liquid Chromatography (HPLC) combined with a spray detector (CAD) detector, solves the problem of difficult quantification, can carry out quantitative analysis to dp26, does not need to use a UPLC combined mass spectrum detector for each detection, reduces the detection cost and remarkably reduces the detection time (to 35 min).
The CAD detection principle is that solute (analyte) droplets are dried to form solute particles, the solute particles are positively charged by collision with positively charged nitrogen particles, the charged particles transfer their charge to a collector, and the signal current of the charged solute is measured by a high-sensitivity electrostatic detector. The resulting signal current is proportional to the mass content of the solute (analyte substance) regardless of the chemical structure of the solute (analyte substance) itself.
The analysis method is completed by adopting HPLC combined with a novel detector CAD, the low molecular heparin sample is firstly separated by a chromatographic column (SEC column) and then enters a CAD detector for detection, and thus, the sugar chain distribution information of the low molecular heparin sample is obtained according to the CAD detection result. This problem is solved by the combination of CAD detection, since low molecular heparin (e.g., dalteparin sodium) does not have characteristic uv absorption. Therefore, the method can be used for qualitative and quantitative analysis of the sugar chain distribution of the low molecular weight heparin. The method can achieve the same effect as UPLC by HPLC separation, and can quantitatively analyze the peak area percentage of the components dp8-dp26 and more than or equal to dp28 which are absorbed by the low molecular heparin without ultraviolet characteristic after being combined with a novel detector CAD.
The method can be further combined with the detection result of the mass spectrum for use, and the result of identifying the product separated by the same chromatographic column by a mass spectrometer is compared, so that the accurate qualification of the substance corresponding to the CAD detected peak is realized.
The first aspect of the present invention relates to a method for analyzing sugar chain distribution of low-molecular heparin, comprising the steps of: (1) Separating the low molecular weight heparin by a chromatographic column of High Performance Liquid Chromatography (HPLC); (2) And then detected by an electric fog type detector (CAD).
In some embodiments, the chromatography column used in step (1) is a Size Exclusion (SEC) chromatography column.
In some embodiments, the chromatography columns used in step (1) are two Size Exclusion (SEC) chromatography columns in series.
In some embodiments, in the step (1), the conditions of the high performance liquid chromatography are a flow rate of 0.1 to 5mL/min, isocratic elution, a column temperature of 20 to 30 ℃ (preferably 25 ℃), a sample volume of 5 to 20 μ L (preferably 8 μ L), wherein the mobile phase is an ammonium salt solution. Preferably, the conditions of the high performance liquid chromatography are as shown in table 2, and the conditions of the electrospray detector are that the atomization temperature is about 35 ℃, and other parameters (e.g., power function, acquisition frequency, filtering value, etc.) are adjusted according to the atomization temperature, preferably as shown in table 3.
In some embodiments, the ammonium salt is ammonium acetate or ammonium formate (ammonium acetate or ammonium formate can be substituted for each other in the assay of the invention without affecting the results of the experiment), the concentration is 50-250mM, and the flow rate is 0.1-1mL/min. Further, the concentration is 100-200mM, and the flow rate is 0.3-0.8mL/min. Still further, the concentration is about 100mM or about 150mM, and the flow rate is about 0.7mL/min.
In some embodiments, the method further comprises the steps of: and (3) comparing the detection result obtained in the step (2) with the detection results of oligosaccharide standard products with different sugar chain lengths, and/or comparing the detection result obtained in the step (2) with the detection result of low molecular heparin (original ground sample or standard product) by ultra high performance liquid chromatography-mass spectrometry, so as to obtain the qualitative and quantitative results of the chain distribution of the low molecular heparin in the step (1).
In some embodiments, when comparing the detection result obtained in step (2) with the detection result of oligosaccharide standards with different sugar chain lengths, the comparison is that the retention time of each peak of the low molecular heparin sugar chain obtained in step (2) is compared with the retention time of each peak of the oligosaccharide standards with different sugar chain lengths of the low molecular heparin. Through the comparison, the sugar chain length corresponding to each peak of the low molecular heparin can be determined, qualitative analysis of each peak in a peak shape graph obtained by detecting the low molecular heparin is realized, and quantitative analysis of each peak in the peak shape graph obtained by detecting the low molecular heparin through peak area integration and ratio calculation in the peak shape graph obtained by a CAD detector is further realized.
In some embodiments, when the detection result obtained in step (2) is compared with the detection result of low molecular heparin by hplc, the comparison refers to comparing the peak shape of the low molecular heparin sugar chain obtained in step (2) with the peak shape of the low molecular heparin sugar chain obtained by hplc under the same or substantially the same chromatographic conditions. By the comparison, the qualitative of each peak in the peak shape diagram obtained by detecting the low molecular heparin can be determined, and the quantitative analysis of each peak in the peak shape diagram obtained by detecting the low molecular heparin can be realized by further calculating the peak area integral and the ratio of the low molecular heparin in the peak shape diagram obtained by a CAD detector.
In some embodiments, when the detection result obtained in step (2) is compared with the detection result of oligosaccharide standard products with different sugar chain lengths and the detection result obtained in step (2) is compared with the detection result of low molecular heparin by ultra high performance liquid chromatography mass spectrometry, the comparison means that the retention time of each peak of the oligosaccharide standard products with different sugar chain lengths of low molecular heparin is respectively compared with the retention time of each peak of the low molecular heparin sugar chain obtained in step (2) and the retention time of each peak of the low molecular heparin detected by ultra high performance liquid chromatography mass spectrometry under the same or basically the same chromatographic conditions. And (3) correlating the detection result of the low molecular heparin sugar chain obtained in the step (2) with the detection result of the low molecular heparin oligosaccharide standard product with different sugar chain lengths with the detection result of the low molecular heparin by ultra performance liquid chromatography-mass spectrometry under the same or basically the same chromatographic conditions. By the comparison, the qualitative of each peak in the peak shape chart obtained by detecting the low molecular heparin can be determined, and the quantitative analysis of each peak in the peak shape chart of detecting the low molecular heparin can be realized by further calculating the peak area integral and the ratio of the low molecular heparin in the peak shape chart obtained by a CAD detector.
In some embodiments, the low molecular heparin is dalteparin sodium, enoxaparin sodium, or nadroparin calcium.
In a first aspect the invention relates to the use of said analytical method for the quality detection of low molecular weight heparins.
In some embodiments, comprising using the assay method to perform multiple tests on a low molecular heparin standard (e.g., a european or usp reference standard) and/or a primary research sample (e.g., a product marketed by feverre in the us or europe), a quality standard is established based on the test results.
In some embodiments, the quality criteria established according to the test results of the present invention may vary according to the general knowledge of a person skilled in the art, and such varying solutions fall within the scope of the application claimed by the present invention. Further, the quality standard can be established by the range of peak area percentages of the respective peaks as in table 23 of examples herein. Further, the quality standard is to convert the peak area percentage (%) of each peak in table 23 into preferred 1, 2 and 3 in table 25 (which correspond to low, medium and high intensity of the quality standard, respectively) by the rule in table 24. Still further, the quality criteria are set forth in the following table.
| Component name | Mass standard range (%) (preferably 1) | Mass standard range (%) (preferably 2) | Mass standard range (%) (preferably 3) |
| ≥dp24 | 28.0-39.7 | 29.6-38.0 | 31.3-36.2 |
| dp22 | 4.1-11.6 | 4.7-10.8 | 5.4-9.9 |
| dp20 | 4.7-11.6 | 5.4-10.8 | 6.1-9.9 |
| dp18 | 5.5-12.4 | 6.3-11.5 | 7.1-10.6 |
| dp16 | 5.9-13.8 | 6.8-12.8 | 7.7-11.7 |
| dp14 | 6.2-13.8 | 7.1-12.8 | 8.1-11.7 |
| dp12 | 6.0-13.6 | 6.9-12.6 | 7.8-11.6 |
| dp10 | 5.1-12.0 | 5.9-11.1 | 6.6-10.2 |
| dp8 | 2.5-6.3 | 2.9-5.9 | 3.2-5.4 |
| ≤dp6 | ≤0.95 | ≤0.87 | Less than or equal to the limit of quantitation (0.85) |
Detailed Description
The invention is further described below with reference to the following figures and specific examples. It is to be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the invention covered by the claims.
Example 1 Experimental conditions and reagents
1. Sources of reagents used in the present invention:
TABLE 1
2. Preparation of the reagent samples used in the present invention:
(1) Preparation of mobile phase: the preparation volume of the solution can be adjusted according to the solution proportion according to the actual dosage.
Preparation of the mobile phase (50-250 mM ammonium acetate or ammonium formate solution, preferably 100-200mM, most preferably 150 mM): weighing a proper amount of ammonium acetate or ammonium formate, adding a proper amount of ultrapure water for dissolving, transferring to a 1000mL volumetric flask, fixing the volume to a scale with the ultrapure water, shaking up, transferring to a blue-cap bottle, and performing ultrasonic treatment for 10 minutes before use.
(2) Preparation of system suitability solution (dalteparin sodium united states pharmacopoeia reference standard solution): the following is described in detail by taking 100mg/mL as an example, and the specific configuration can be appropriately adjusted as necessary: weighing 100mg of a reference standard substance of the United states Pharmacopeia of dalteparin sodium in a clean 5mL centrifuge tube, adding 1mL of ultrapure water to prepare a sample into a solution of 100mg/mL (adjusting the volume of the added ultrapure water according to the sample weighing amount), fully mixing uniformly, and filtering with a filter membrane of 0.22 mu m for later use.
(3) Preparing a blank solution: taking a proper amount of ultrapure water for standby.
(4) Preparing a sample solution: reference system suitability test sample preparation.
3. High performance liquid chromatography conditions:
TABLE 2
| Conditional content | Name/index |
| High performance liquid chromatograph | Thermo Ultimate 3000 |
| Detector | Corona Veo RS(CAD) |
| Chromatographic column | Agilent advanced Bio SEC 130A (2.7um, 7.8X 300 mm), two in series |
| Flow rate of flow | 0.3-0.8mL/min |
| Gradient of mobile phase | Isocratic elution |
| Sample volume | 5-20μL |
| Column temperature | 20-30℃ |
| Work station | Chromeleon 7.2 |
CAD conditions are shown in Table 3:
TABLE 3
Example 2 Effect of Experimental conditions on chromatographic separation of daparinux sodium
(1) See example 1 for experimental conditions wherein the mobile phase is 100mM or 150mM ammonium acetate at a flow rate of 0.7mL/min or 0.5mL/min.
Data processing: appropriate integration parameters are set, for example, in Table 4, and the CAD drawing of the sample is integrated by area normalization to calculate the percentage area of each component dp8, dp10, dp12, dp14, dp16, dp18, dp20, dp22, dp24, dp26, dp 28. Because of the characteristics of the dalteparin sodium product, the content of oligosaccharide fragments less than or equal to dp6 is less than the limit of quantitation and can be ignored, so the peak area percentage of components less than or equal to dp6 is not calculated.
TABLE 4
(2) Results of the experiment
When the mobile phase concentration was 100mM ammonium acetate, the sample was taken in an amount of 16. Mu.l at a flow rate of 0.7mL/min, see (FIG. 1). When the mobile phase concentration was 150mM ammonium acetate, the sample was taken in an amount of 16. Mu.l at a flow rate of 0.5mL/min, see (FIG. 2). As shown in FIGS. 1 and 2, the method of the present invention using 100mM or 150mM ammonium acetate as the mobile phase and a flow rate of 0.7mL/min or 0.5mL/min provides good separation at dp8, dp10, dp12, dp14, dp16, dp18, dp20, dp22, dp24, dp26, and/or dp 28.
In addition, the inventor further adjusts the concentration of ammonium acetate or ammonium formate solution, and proves that 50-250mM ammonium acetate or ammonium sulfate solution can achieve the invention and achieve the corresponding technical effect, and the related experimental results are shown in figures 3-7 (figures 3-7 use Dagansu sodium European pharmacopoeia reference standard with flow rate of 0.7mL/min, except that the mobile phase is 50mM ammonium acetate, 200mM ammonium acetate, 250mM ammonium acetate, 200mM ammonium formate, and 50mM ammonium formate respectively). The inventor also further verifies that the flow rate is 0.1-1mL/min to achieve the invention and achieve the corresponding technical effect, and preferably 0.3-0.8mL/min. Therefore, the method of the invention can detect the chain distribution of the dalteparin sodium, and has better separation effect on oligosaccharides with different sugar chain lengths.
Example 3 verification of the Experimental methods of the invention
The authentication items include: specificity, quantitation limit, precision (repeatability and intermediate precision), and durability. The method adopts an area normalization method to carry out quantitative analysis on each component, so that accuracy verification is not required.
Sample and standard information is shown in table 1.
(1) The specificity is as follows:
and (4) analyzing a series of oligosaccharide standard products by sample injection, and determining the attribution of each peak in the dalteparin sodium standard product. And (4) sequentially injecting and analyzing methanol, ethanol, DS and NaCl, and inspecting whether the interference of the methanol, the ethanol, the DS and the NaCl on sample detection is caused. And simultaneously carrying out sample injection analysis on the dalteparin sodium United states Pharmacopeia reference standard substance, the European Pharmacopeia reference standard substance and a sample of a former research company, and aiming at investigating whether the method is simultaneously suitable for the United states Pharmacopeia reference standard substance, the European Pharmacopeia reference standard substance and the sample of the former research company. The oligosaccharide reference used in this verification was a commercially available heparin oligosaccharide, each oligosaccharide obtained by degrading heparin with heparinase and then performing gel separation. According to the principle of enzymolysis, the non-reducing ends of the heparin oligosaccharides contain double bonds. The dalteparin sodium is obtained by degrading heparin through nitrous acid, and is structurally characterized in that the reducing end of a modified chain is a 2,5-anhydromannitol structure, and the rest of the structure still remains the structure brought by the heparin, such as glucosamine. The non-reducing end is consistent with heparin and is mostly in a saturated uronic acid structure. The dalteparin sodium oligosaccharide chain has similar or identical structural characteristics with the used oligosaccharide reference standard product, and the molecular weight difference is 13. The method in this verification uses a SEC column, which separates the same species according to their molecular weights, and the difference in molecular weights of the two species is 13, which is indistinguishable on this SEC column.
Solution preparation: mobile phase (150 mM ammonium acetate solution): weighing 11.5624g of ammonium acetate in a clean beaker, adding a proper amount of ultrapure water for dissolving, transferring to a 1000ml volumetric flask, fixing the volume to the scale by the ultrapure water, shaking up, transferring to a blue-covered flask, and carrying out ultrasonic treatment for 10 minutes before use.
Preparation of single standard solution: the specific procedure is shown in Table 5.
TABLE 5
| Name of solution | Name of reagent | Amount of reagent added | Amount of ultrapure water added | Concentration of solution |
| Oligosaccharide standard (dp 6) | Oligosaccharide standard (dp 6) | 2.0mg | 2.00ml | 1mg/ml |
| Oligosaccharide standard (dp 8) | Oligosaccharide standard (dp 8) | 2.0mg | 2.00ml | 1mg/ml |
| Oligosaccharide standard (dp 10) | Oligosaccharide standard (dp 10) | 2.0mg | 2.00ml | 1mg/ml |
| Oligosaccharide standard (dp 12) | Oligosaccharide standard (dp 12) | 2.0mg | 2.00ml | 1mg/ml |
| Oligosaccharide standard (dp 14) | Oligosaccharide standard (dp 14) | 2.0mg | 2.00ml | 1mg/ml |
| Oligosaccharide standard (dp 16) | Oligosaccharide standard (dp 16) | 2.0mg | 2.00ml | 1mg/ml |
| Oligosaccharide standard (dp 18) | Oligosaccharide standard (dp 18) | 2.0mg | 2.00ml | 1mg/ml |
| Oligosaccharide standard (dp 20) | Oligosaccharide standard (dp 20) | 2.0mg | 2.00ml | 1mg/ml |
| 10mg/ml DS | DS United states Pharmacopeia reference standard | 0.0200g | 2.00ml | 10mg/ml |
| 10mg/ml NaCl | NaCl | 0.0401g | 4.01ml | 10mg/ml |
| 10mg/ml methanol | Methanol | 0.0430g | 4.30ml | 10mg/ml |
| 10mg/ml ethanol | Ethanol | 0.0412g | 4.12ml | 10mg/ml |
Remarking: the standard oligosaccharide is purchased with a label amount of 2.0 mg/bottle, and is directly dissolved in 2.00ml of ultrapure water.
The formulation of the dalteparin sodium solution is shown in table 6.
TABLE 6
Ultrapure water was used as a blank sample. Under the conditions of the above-mentioned apparatus (please refer to tables 1-3), after injecting sample and analyzing blank sample and oligosaccharide standard solution for 1 needle each, injecting sample and analyzing European and American dalteparin sodium standard solution for one needle each.
The experimental results are as follows:
as shown in FIG. 8 (FIG. 8 shows the blank sample and the standard solution of dalteparin sodium in USA) were analyzed by injection, the blank solution showed no chromatographic peak affecting the detection of the sample except the solvent peak.
The peak retention time of each component of the dalteparin sodium standard solution and the oligosaccharide standard solution is shown in table 7.
As can be seen from the table, the retention time of each component peak of the dalteparin sodium standard solution is basically consistent with that of each component peak of the oligosaccharide standard solution (within +/-5%), and the detection result (retention time of each peak) of the dalteparin sodium standard solution or the detection result (retention time of each peak) of each oligosaccharide standard solution can be used for judging the qualitative analysis of each peak of the dalteparin sodium sample under the same chromatographic conditions. The results of the related experiments are shown in fig. 9 and 10.
As shown in FIG. 11, the retention time of 10mg/ml DS is 13.8min, that of 10mg/ml NaCl is 28.6min, and the retention time of each component of the dalteparin sodium standard solution is 15 min-24 min, and no influence on the detection chromatographic peak of the sample by 10mg/ml methanol or ethanol occurs. Therefore, naCl, methanol and ethanol do not influence the peak appearance of the standard solution of the dalteparin sodium, and DS is expressed as the front peak of the dalteparin sodium and does not influence the peak appearance of the standard solution of the dalteparin sodium.
The results show that:
(2) Precision (repeatability and intermediate precision):
repeatability: daparinux sodium standard solutions were prepared as per table 8.
TABLE 8
| Name of solution | Adding amount of dalteparin sodium standard substance | Amount of ultrapure water added | Concentration of solution |
| Dagansu sodium standard substance solution 1 | 0.0201g | 201μl | 100mg/ml |
| Dagansu sodium standard substance solution 2 | 0.0201g | 201μl | 100mg/ml |
| Dagansu sodium standard substance solution 3 | 0.0202g | 202μl | 100mg/ml |
| Dagansu sodium standard substance solution 4 | 0.0201g | 201μl | 100mg/ml |
| Dagansu sodium |
0.0202g | 202μl | 100mg/ml |
| Dagansu sodium standard substance solution 6 | 0.0202g | 202μl | 100mg/ml |
The apparatus conditions were as described above (see tables 1 to 3).
One needle of the same inspector is used for blank, and then 1 needle of each of 6 dalteparin sodium standard solutions is injected and analyzed. Then, the relative standard deviation (RSD%) of the area percentage of each component peak such as dp8, dp10, dp12, dp14, dp16, dp18, dp20, dp22, dp24, dp26 and dp28 in the 6-pin heparin sodium standard solution CAD picture is calculated (quantitative analysis). The peak area percentages of the components of the 6-pin heparin sodium standard solution are shown in Table 9.
The maximum relative standard deviation of the peak area percentage of each component of the two experimenters 12 aiming at the heparin sodium standard is 8.1 percent, and the minimum separation degree between dp10 and dp8 is 1.2, so the intermediate precision experimental result meets the acceptable standard.
(3) And (4) quantitative limit:
the method is used for comparing and researching the percentage contents of the components by calculating the percentage contents of the components by using an area percentage method through CAD graph integration. Therefore, the minimum quantitative range of each component needs to be confirmed. According to the requirements of ICHQ2, the acceptable standard of the quantitative limit not only meets the condition that S/N is more than or equal to 10: 1, but also meets the condition that the percentage RSD percent of the peak area of the minimum peak is less than or equal to 10.0 percent.
Ultrapure water was used as a blank sample. The apparatus conditions were as before (see tables 1 and 2).
Preparation of 25mg/ml dalteparin sodium United states pharmacopoeia reference standard solution: and sequentially diluting the standard solution by referring to the S/N of the peak with the minimum peak area percentage of the components of the special United states Pharmacopeia standard solution, wherein the S/N of the minimum peak in the diluted sample solution and the RSD of the minimum peak can meet the minimum sample concentration of an acceptable standard.
For example: when the sample concentration is 100 mg/mL; the peak area percentage of dp8 is 4.0%, and the S/N is 28; it was diluted to 50% (50 mg/mL). The results are calculated as in table 12.
TABLE 12
* Percent peak area after conversion = percent peak area by 50%.
From the analysis report of the special United states Pharmacopeia standard solution, the signal-to-noise ratio of dp6 is 5.3, which does not meet the acceptable standard of the quantitative limit, so dp8 is taken as the peak with the minimum peak area percentage, and the signal-to-noise ratio is referred for dilution. The specific dilution process is shown in Table 13.
Watch 13
The instrument conditions were as before. Quantitative analysis: and (4) analyzing the standard solution after the 3-pin dilution by injection, and respectively calculating the relative standard deviation (RSD%) of the peak area percentage of the minimum peak of the CAD graph of the standard solution after the 3-pin dilution. The minimum quantification range was determined by the S/N of each peak and the RSD% of the peak area percentage. The results are shown in Table 14.
TABLE 14
The peak signal-to-noise ratios of the components of the diluted 3-pin heparin sodium standard solution are calculated and shown in Table 15.
* Percent peak area after conversion = percent peak area = 25%
The results show that: the peak area percentage of the dp8 component peak of the dalteparin sodium standard solution diluted by 25% is minimum, the signal to noise ratio (S/N) is 17 and meets the acceptance standard (S/N is more than or equal to 10: 1), and the RSD% is 3.9 and meets the acceptance standard. Thus, the limit of quantitation was 0.85% peak area percentage (3.39% (dp 8) × 25% (dilution factor) when the sample concentration was 100 mg/ml.
(4) Durability:
two concentrations of 150. + -.5 mM ammonium acetate and two concentrations of 100. + -.2 mg/mL standard solutions were prepared as described in Table 16. Orthogonal experiments were performed to analyze 3 standard solutions for each sample, and the absolute value of the relative deviation between the average of the peak area percentage of each main component for 3 samples and the average of the standard solutions for 6 samples of normal concentration (reproducibility) was calculated.
TABLE 16
Quantitative analysis: orthogonal experimental analysis (see table 17) was performed with 3 pins for each condition, and each set of experiments met the acceptance criteria for system adaptability. The absolute value of relative deviation of the peak area percentage average value of each main component of the results dp8, dp10, dp12, dp14, dp16, dp18, dp20, dp22, dp24, dp26 and more than or equal to dp28 of each group of experiments with the average value of the result of the 6-needle standard solution with normal concentration (repeatability) is calculated respectively.
TABLE 17
| Conditions of the experiment | Mobile phase concentration mM | Sample concentration mg/mL |
| Group 1 | 145 | 98 |
| Group 2 | 145 | 102 |
| Group 3 | 155 | 98 |
| Group 4 | 155 | 102 |
The experimental results are as follows: experimental conditions group 1 system adaptation results: the blank solution has no chromatographic peak influencing the detection of the sample except the solvent peak; the separation degree between dp10 and dp8 is 1.2, and is in a preferable range. Experimental conditions the calculation of the absolute values of the relative deviations of the peak area percentage mean values of the 3-pin standard solutions of group 1 and the 6-pin standard solutions of normal conditions (reproducibility) is shown in table 18.
The absolute values of the relative deviations of the peak area percentages of the components from the normal conditions for each of the 4 experimental conditions are summarized in Table 22.
TABLE 22
The absolute value of the relative deviation of the results of the 4 groups of experimental conditions and the percentage of the peak area of each component of the standard solution under the normal conditions (repeated experiments) is 9 percent at most, which is in a better range. Thus, the method is robust to sample concentrations (98-102 mg/ml) and mobile phase ammonium acetate concentrations (145-155 mM).
Example 4
Using the above method (see examples 1-3), we finally determined the SEC-CAD chain distribution quality criteria for daparinux sodium by testing the standard for daparinux sodium and analyzing the results as shown in Table 23.
TABLE 23
Quality criteria applicable: according to the results of the American preliminary examination, three types of test results, high, medium and low (preferably 3, 2 and 1 in correspondence to Table 25), were calculated according to the following rules of Table 24. For example, the% dp6 peak area percentage in Table 23 is 0.2-0.7% (below the quantitation limit of 0.85%, refer to Table 24 for quantitation limit), then the mass standard intensity (i.e., high: 0.7X 115% =0.81; medium: 0.7X 125% =0.87%; low: 0.7X 135% = 0.95) is calculated with reference to the regular rightmost quantitation limit (0.85%) > percentage of each sugar chain peak area in Table 24, which corresponds to the preferred 3 (≦ quantitation limit), mass standard 2 (≦ 0.87%), and mass standard 3 (≦ 0.95) in Table 25. Other peaks were calculated with reference to the above method.
Watch 24
TABLE 25
As can be seen from the above table, when the method of the present invention is used for analyzing sugar chains of daparinux sodium, a set of quality standards for distribution analysis of sugar chains of daparinux sodium can be established and used for rapid detection and judgment of the quality of a sample of daparinux sodium. The quality standards described herein are exemplary only and variations can be made by one of ordinary skill in the art based on general knowledge and these variations fall within the scope of the claimed application.
Various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference (including all patents, patent applications, journal articles, books, and any other publications) cited in this application is hereby incorporated by reference in its entirety.
Claims (8)
1. A method for analyzing sugar chain distribution of low-molecular heparin, comprising the steps of:
(1) Separating the low molecular heparin by a chromatographic column of high performance liquid chromatography;
(2) Then detecting the mixture by an electric fog type detector;
(3) Comparing the detection result obtained in the step (2) with the detection results of oligosaccharide standard products with different sugar chain lengths, and/or comparing the detection result obtained in the step (2) with the detection result of the low molecular heparin through ultra performance liquid chromatography-mass spectrometry, so as to obtain the qualitative and quantitative results of the chain distribution of the low molecular heparin in the step (1); wherein, in the step (1), the chromatographic column is a size exclusion chromatographic column, and the conditions of the high performance liquid chromatography are as follows: the chromatographic column is Agilent advanced Bio SEC 130A,2.7um, 7.8X 300mm, the flow rate is 0.1-2mL/min, the elution is carried out isocratic, the column temperature is 20-30 ℃, and the mobile phase is ammonium salt solution.
2. The assay of claim 1, wherein the ammonium salt is ammonium acetate or ammonium formate, the ammonium salt solution is at a concentration of 50-250mM, and the flow rate is 0.1-1mL/min.
3. The assay of claim 2, wherein the ammonium salt solution is at a concentration of 100-200mM and the flow rate is 0.3-0.8mL/min.
4. The assay of claim 3, wherein the concentration is about 100mM or about 150mM and the flow rate is about 0.7mL/min.
5. An assay as claimed in any one of claims 1 to 4 wherein in step (1) the chromatographic columns used are two size exclusion chromatographic columns in series.
6. The assay of any one of claims 1-4, wherein the low molecular weight heparin is dalteparin sodium, enoxaparin sodium, or nadroparin calcium.
7. Use of the assay of any one of claims 1-4 for the detection of low molecular weight heparin quality.
8. Use according to claim 7, comprising performing a plurality of tests on a low molecular heparin standard and/or a sample under investigation using the assay according to any one of claims 1 to 6, establishing a quality standard based on the test results.
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| EP2642288A1 (en) * | 2012-03-21 | 2013-09-25 | Istituto di Richerche Chimiche e Biochimiche G. Ronzoni | Profiling oligosaccharides associated with biological activities of heparin or heparin derivatives |
| TW201502507A (en) * | 2013-07-11 | 2015-01-16 | Scinopharm Taiwan Ltd | Analytical method for detecting sulfated oligosaccharides |
| CN105431733A (en) * | 2013-07-11 | 2016-03-23 | 台湾神隆股份有限公司 | Analytical method for detecting sulfated oligosaccharides |
| CN106442799A (en) * | 2016-11-04 | 2017-02-22 | 深圳市天道医药有限公司 | Low-molecular-weight heparin molecular weight and molecular weight distribution detection method |
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| EP2642288A1 (en) * | 2012-03-21 | 2013-09-25 | Istituto di Richerche Chimiche e Biochimiche G. Ronzoni | Profiling oligosaccharides associated with biological activities of heparin or heparin derivatives |
| TW201502507A (en) * | 2013-07-11 | 2015-01-16 | Scinopharm Taiwan Ltd | Analytical method for detecting sulfated oligosaccharides |
| CN105431733A (en) * | 2013-07-11 | 2016-03-23 | 台湾神隆股份有限公司 | Analytical method for detecting sulfated oligosaccharides |
| CN106442799A (en) * | 2016-11-04 | 2017-02-22 | 深圳市天道医药有限公司 | Low-molecular-weight heparin molecular weight and molecular weight distribution detection method |
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| Complete Molecular Weight Profiling of Low-Molecular Weight Heparins Using Size Exclusion Chromatography-Ion Suppressor High-Resolution Mass Spectrometry;Zaia, J等;《ANALYTICAL CHEMISTRY》;20161006;第88卷;10654-10660 * |
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