Analysis method of sub-nitrous acid degradation product of dalteparin sodium and application thereof

Abstract

This invention relates to an analytical method for the degradation products of dalteparin sodium in nitrite. The method includes: completely degrading the low molecular weight heparin with nitrite to obtain degradation products, and detecting the degradation products by high performance liquid chromatography (HPLC) coupled with an electro-fogging detector (CAD). This method eliminates the need for derivatization and deacetylation steps, resulting in shorter preparation time, lower analytical costs, and higher detection accuracy.

Description

[0001] This application is a divisional application of the invention entitled “An analytical method for the degradation products of sodium dalteparin nitrite and its application”, filed on July 23, 2019, with application number CN201980027376.2. Technical Field

[0002] This invention relates to an analytical method for the degradation products of dalteparin sodium nitrite and its application. Background Technology

[0003] Dalteparin sodium is a low-molecular-weight heparin sodium composed of polysaccharide chains of varying molecular weights, and it itself does not exhibit an absorption peak in ultraviolet light. Heparinases, whether heparinase I, II, or III, specifically cleave the glycosidic bonds linking glucosamine (1-4) hexuronic acids, forming a double bond between C4 and C5 of the hexuronic acid. The enzymatic hydrolysis products of dalteparin sodium obtained by heparinase exhibit absorption peaks in ultraviolet light, and can be quantitatively analyzed using an ultraviolet detector.

[0004] To determine the quality of dalteparin sodium, it is commonly used to enzymatically hydrolyze the sodium with heparinase, then separate the degradation products using a liquid chromatography system. Qualitative analysis is performed by comparing retention times with commercially available disaccharide standards, and quantitative analysis is then conducted using standard curves of detector response values ​​and molar amounts for each disaccharide standard. Because disaccharide standards are relatively expensive, a subsequent development involved using a high-performance liquid chromatography (HPLC) system to separate the degradation products. The separated products are then further characterized using mass spectrometry (MS), enabling qualitative analysis of the dalteparin sodium degradation products. Quantitative analysis of the separated components is then performed using an ultraviolet (UV) detector, ultimately achieving the analysis of the dalteparin sodium's quality.

[0005] To further improve the qualitative / quantitative analysis methods for heparinase hydrolysates, existing techniques often involve derivatization reactions before or after the heparin sodium product is loaded onto the column. For example, existing technology (CN201310711455.X) discloses derivatizing the hydrolysate after heparinase hydrolysis to heparin sodium and before column separation in a liquid chromatography system, and then using reversed-phase chromatography coupled with high-resolution mass spectrometry to detect the derivatized product, ensuring that all hydrolysate products can be qualitatively analyzed. Existing technology (CN201410123609.8) discloses performing a fluorescence derivatization reaction after the heparin sodium hydrolysate is separated by a liquid chromatography column, followed by detection. This provides detectable fluorescent groups for low molecular weight heparin components without UV absorption, achieves highly sensitive fluorescence detection with higher sample response values ​​in the spectrum, and avoids interference from derivatization byproducts, improving the accuracy and repeatability of the analysis.

[0006] In addition, to avoid the limitations of heparinase degradation, existing technology (CN201310695114.8) also includes a method for detecting the composition of heparin disaccharide. This method mainly involves first deacetylifying heparin to obtain deacetylated heparin, then degrading the deacetylated heparin with nitrous acid to obtain heparin disaccharide; concentrating the heparin disaccharide to dryness and dissolving it in water, adjusting the pH to alkaline, adding pyrazolone derivatizing reagents, and after the reaction is complete, extracting three times with chloroform to remove unreacted derivatizing reagents; and then detecting the derivatized disaccharide using liquid chromatography or liquid chromatography-mass spectrometry.

[0007] The existing technology using deacetylation followed by deacetylated heparin nitrite degradation can degrade glucosamine containing N-sulfate groups in dalteparin sodium, but it cannot degrade glucosamine containing N-acetyl groups in dalteparin sodium. In other words, the deacetylation step eliminates the N-acetyl glucosamine information present in dalteparin sodium, and this method also requires a derivatization step, making it cumbersome.

[0008] Regarding the existing heparinase degradation method, firstly, heparinase is easily inactivated and unstable; secondly, the enzymatic hydrolysis of dalteparin sodium requires a long time (48 hours for a single enzymatic hydrolysis experiment), is cumbersome, and has high detection costs (requiring mass spectrometry). Furthermore, during enzymatic degradation of dalteparin sodium, glucuronic acid (GlcA) and iduronic acid (IdoA) dehydrate to form structurally identical dehydrated uronic acids, thus losing the content and structural information of glucuronic acid and iduronic acid in dalteparin sodium. Moreover, heparinase cannot completely degrade dalteparin sodium into disaccharides.

[0009] In existing technologies, analytical methods for dalteparin sodium degradation products are inefficient, costly, and do not provide complete information about the dalteparin sodium being analyzed. Therefore, there is a need for analytical methods that offer shorter analysis times, lower costs, higher accuracy, and a more comprehensive representation of the dalteparin sodium being analyzed. Summary of the Invention

[0010] In a first aspect of the present invention, there is a method for analyzing dalteparin sodium nitrite degradation products, the method comprising the following steps: (1) mixing the dalteparin sodium with nitrite to achieve complete degradation; and (2) detecting the degradation products by high performance liquid chromatography coupled with an electro-fogging detector.

[0011] In some embodiments, step (1) further includes adding a pH adjuster to terminate the reaction after the sodium dalaridin and nitrite mixture has been completely degraded, then adding sodium borohydride solution for reduction, and further adding one or more pH adjusters to terminate the reaction and adjust the pH to neutral to obtain the final degradation product.

[0012] In some embodiments, in step (2), the conditions for the high performance liquid chromatography and electro-fogging detector are: flow rate 0.1-2 mL / min; elution gradient 0-45 min; 10%-60% mobile phase A; 90%-40% mobile phase B, wherein mobile phase A is an ammonium salt solution and mobile phase B is acetonitrile.

[0013] In some embodiments, the ammonium salt is ammonium acetate or ammonium formate, with a concentration of 20-200 mM, and the flow rate is 0.1-1 mL / min, and the elution gradient is 0-45 min, with 10%-45% mobile phase A and 90%-55% mobile phase B.

[0014] In some embodiments, the ratio (g / mol) of the mass of the dalteparin sodium to the molar number of the nitrite is 17.5-405:1, preferably 25-250:1, 35-175:1, or 50-130:1.

[0015] In some embodiments, the concentration of dalteparin sodium after mixing with nitrite is between 4 mg / mL and 150 mg / mL.

[0016] In some embodiments, the pH adjuster is selected from one or more of the following: sodium carbonate, sodium bicarbonate, sodium hydroxide, hydrochloric acid, sulfuric acid, and glacial acetic acid.

[0017] In some embodiments, the method optionally includes the following steps: comparing the detection results obtained in step (2) with the results of detection by ultra-high performance liquid chromatography coupled with mass spectrometry of the product after complete degradation of dalteparin sodium by nitrite, and finally obtaining the qualitative and quantitative information of the product after degradation of dalteparin sodium by nitrite in step (1).

[0018] In a second aspect of the invention, the analytical method is used in the quality detection of dalteparin sodium.

[0019] In some embodiments, the application includes using the analytical method to perform multiple tests on dalteparin sodium standards (including but not limited to European or American standards) and / or original samples, and establishing quality standards based on the test results. Attached Figure Description

[0020] Figure 1 The image shows an overlay of the blank solution and Dal-USP-RED-02, with the top image showing Dal-USP-RED-02 and the bottom image showing BLANK-RED.

[0021] Figure 2 This shows an overlay of Dal-USP-RED-01 and Dal-USP-RED-02, with the top image being Dal-USP-RED-02 and the bottom image being Dal-USP-RED-01.

[0022] Figure 3 This shows an overlay of Dal-USP-RED-03 and Dal-USP-RED-04 images, with the top image being Dal-USP-RED-04 and the bottom image being Dal-USP-RED-03.

[0023] Figure 4 Display the overlay images from Dal-USP-RED-04 to Dal-USP-RED-08.

[0024] Figure 5 The image shows the sample test results.

[0025] Figure 6 The image shows an overlay of blank solution, dalteparin sodium standard, and original dalteparin sodium sample, from bottom to top: specificity-BLANK-RED, specificity-Dal-USP-RED-01, specificity-Dal-EP-RED-01, and specificity-original sample-RED-01.

[0026] Figure 7 The image shows an overlay of blank solution, dalteparin sodium standard, and original dalteparin sodium sample, from bottom to top: BLANK-RED, Dal-USP-RED-01, Dal-EP-RED-01, and original sample-RED-01.

[0027] definition

[0028] As used in this article, the term "room temperature" refers to 25℃±5℃. Unless otherwise specified, all experimental temperatures are room temperature.

[0029] As used herein, the term “about” means ±20%, preferably ±10%, and more preferably ±5% of the value modified by the term, so that those skilled in the art can clearly determine the range of the term “about” based on the modified value.

[0030] As used herein, the term "dalteparin sodium standard" refers to dalteparin sodium standards, including but not limited to those in the United States Pharmacopeia and the European Pharmacopeia.

[0031] As used herein, the term "dalteparin sodium original sample" refers to the dalteparin sodium marketed by the original manufacturer Pfizer, including but not limited to dalteparin sodium marketed in the United States and Europe.

[0032] As used herein, the term "peak area percentage %" refers to the percentage of the peak area of ​​each individual peak (e.g., 11 peaks) obtained by detection of dalteparin sodium degradation products using high performance liquid chromatography coupled with an electro-fogging detector or by detection of dalteparin sodium degradation products using high performance liquid chromatography coupled with mass spectrometry, relative to the total peak area.

[0033] As used herein, the term "relative retention time ratio of each peak" refers to the ratio of the retention time of each peak obtained by detecting dalteparin sodium degradation products using high performance liquid chromatography coupled with an electro-fogging detector to the retention time of the peak with the largest peak area (e.g., the fourth peak) among said peaks, or the ratio of the retention time of each peak obtained by detecting dalteparin sodium degradation products using high performance liquid chromatography coupled with mass spectrometry to the retention time of the peak with the largest peak area among said peaks.

[0034] As used herein, the following abbreviations have meanings commonly known to those skilled in the art. Specifically, UPLC: Ultra-high performance liquid chromatography; HPLC: High performance liquid chromatography; QTOF: Time-of-flight mass spectrometry; CAD: Electro-fogging detector; dp: Degree of polymerization of sugar chains; MS: Mass spectrometer; TIC: Total ion chromatogram; RRT: Relative retention time. Invention Details

[0036] The method used in this invention does not require derivatization and deacetylation steps, has a short preparation time for degradation products (about 5 hours), low analysis cost, high detection accuracy, and fully reflects the condition of the analyzed dalteparin sodium.

[0037] The principle of CAD detection is that after the solute (analyte) droplets dry, they form solute particles. These particles collide with positively charged nitrogen particles, causing the solute particles to become positively charged. The charged particles then transfer their charge to a collector, and finally, a highly sensitive electrostatic detector measures the signal current of the charged solute. The resulting signal current is proportional to the mass content of the solute (analyte) and is independent of the chemical structure of the solute (analyte) itself. Since the components of dalteparin sodium after nitrite degradation do not exhibit characteristic ultraviolet absorption, CAD detection can overcome this problem. Therefore, it can be used for the qualitative and quantitative analysis of the components of dalteparin sodium after nitrite degradation, including 4 disaccharide peaks, 5 tetrasaccharide peaks, and 2 hexasaccharide peaks. Quantitative analysis was also performed on 8 component peaks exceeding the limit of quantitation (corresponding to 2-9 in Table 25).

[0038] The method of this invention is performed using HPLC coupled with a CAD detector. The dalteparin sodium nitrite degradation products are first separated by a HILIC column (hydrophilic interaction chromatography) and then detected by a CAD detector, thereby obtaining the composition information of the dalteparin sodium nitrite degradation products based on the CAD detection results.

[0039] The method of the present invention can also compare the detection results of the degradation products detected by ultra-high performance liquid chromatography coupled with mass spectrometry with the detection results of the degradation products detected by high performance liquid chromatography coupled with an electro-fogging detector, so that in the future, only the degradation products of dalteparin sodium nitrite can be detected by high performance liquid chromatography coupled with an electro-fogging detector to achieve accurate characterization of the degradation products.

[0040] This invention relates to an analytical method for the degradation products of dalteparin sodium with nitrite, the method comprising or consisting of the following steps: (1) mixing the dalteparin sodium with nitrite to achieve complete degradation; (2) detecting the degradation products by high performance liquid chromatography (HPLC) coupled with an electro-fogging detector (CAD).

[0041] In some embodiments, step (1) further includes adding a pH adjuster to terminate the reaction after the sodium dalteparin and nitrite mixture has been completely degraded, then adding sodium borohydride solution for reduction, and further adding one or more pH adjusters to terminate the reaction and adjust the pH to neutral (about 7.0) to obtain the final degradation product.

[0042] Further, step (1) also includes, after the dalteparin sodium and nitrite (the ratio of the mass of the dalteparin sodium to the moles of the nitrite (g / mol) is 17.5-405:1) are completely degraded, adding sodium carbonate to adjust the pH to 7-9 (preferably about 8.5) to terminate the reaction, then adding sodium borohydride solution for reduction, and after 3-5 hours (preferably about 4 hours), adding glacial acetic acid to adjust the pH to 3-5 (preferably about 4) to terminate the reaction, and then adding sodium hydroxide to adjust the pH to 6.5-7.5 (preferably about 7) to obtain the final degradation product.

[0043] Furthermore, 105 μL of 100 mg / mL dalteparin sodium sample was mixed with 200 μL of pH 1.5 nitrite solution, and allowed to stand for at least 30 min. Then, the mixture was shaken on a mixer for 2 min to ensure complete degradation of the dalteparin sodium. 80 μL of 1M sodium carbonate solution was added to adjust the pH to 8.5 to terminate the reaction. 145 μL of freshly prepared 30 mg / mL sodium borohydride solution was added, and the reaction was reduced at room temperature for at least 4 hours. 120 μL of glacial acetic acid was added to adjust the pH to 4.0 to terminate the reaction. 150 μL of 4M NaOH was added to neutralize and adjust the pH to approximately 7.0, with a total volume of approximately 800 μL, yielding the final degradation product. 0.3 mL of the degradation product liquid was placed in a centrifuge tube, 0.7 mL of ultrapure water was added and mixed well, and the mixture was filtered through a 0.22 μm filter membrane for later use.

[0044] In some embodiments, in step (2), the conditions for the high performance liquid chromatography and the electro-fogging detector are: a flow rate of 0.1-2 mL / min, preferably 0.1-1 mL / min, more preferably 0.3 mL / min; an elution gradient of 0-45 min, 10%-60% mobile phase A, and 90%-40% mobile phase B; preferably, the elution gradient is 0-45 min, 10%-45% mobile phase A, and 90%-55% mobile phase B.

[0045] In some embodiments, the elution gradient is 0-10 min, 15% mobile phase A, 85% mobile phase B; 10-25 min, 15%-33% mobile phase A, 85%-67% mobile phase B; or 25-45 min, 33%-45% mobile phase A, 67%-55% mobile phase B. Further, the high-performance liquid chromatography (HPLC) conditions optionally include: an elution gradient of 45-50 min, 45% mobile phase A, 55% mobile phase B; or an elution gradient of 50-60 min, 45%-15% mobile phase A, 55%-85% mobile phase B, wherein mobile phase A is an ammonium salt solution and mobile phase B is acetonitrile. The column temperature is 20-30°C, preferably 25°C, and the injection volume is 1-10 μL, preferably 3 μL. Preferably, the conditions for the high-performance liquid chromatography are shown in Table 1, and the conditions for the electro-nebulizer detector are that the nebulization temperature is about 35°C, and other parameters (e.g., power function, acquisition frequency, filtration value, etc.) are adjusted according to the nebulization temperature, preferably as shown in Table 2.

[0046] 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 analytical method of the present invention without affecting the experimental results), and its concentration is 20-200 mM, preferably 100-150 mM, more preferably 100 mM and 150 mM, and the flow rate is 0.1-1 mL / min, preferably 0.3 mL / min.

[0047] In some embodiments, the ratio (g / mol) of the mass of the dalteparin sodium to the molar number of the nitrite is 17.5-405:1, preferably 25-250:1, 35-175:1, or 50-130:1.

[0048] In some embodiments, after mixing with nitrous acid, the concentration of dalteparin sodium is 4 mg / mL to 150 mg / mL, preferably 4.76 mg / mL, 20 mg / mL, 30 mg / mL, 34.4 mg / mL, 51.6 mg / mL, 55 mg / mL, 68.9 mg / mL, 70 mg / mL, 100 mg / mL, 103.3 mg / mL, 137.7 mg / mL, and the range between these values, including but not limited to 20 mg / mL to 100 mg / mL, 20 mg / mL to 70 mg / mL, 30 mg / mL to 55 mg / mL, etc.

[0049] In some embodiments, the pH adjuster is selected from one or more of the following: sodium carbonate, sodium bicarbonate, sodium hydroxide, hydrochloric acid, sulfuric acid, and glacial acetic acid.

[0050] In some embodiments, the dalteparin sodium solution of nitrite degradation products remains stable at room temperature for about one month, preferably about one week, and more preferably about three days.

[0051] In some embodiments, the method optionally includes the following steps: comparing the detection results obtained in step (2) with the results of detection by ultra-high performance liquid chromatography coupled with mass spectrometry of the product after complete degradation of dalteparin sodium by nitrite, and finally obtaining the product information of dalteparin sodium after degradation by nitrite in step (1).

[0052] In some embodiments, the comparison of the detection results is to compare the ratio of the relative retention times of each peak obtained in step (2) with the ratio of the relative retention times of each peak of the degradation product detected by high performance liquid chromatography coupled with an electro-fogging detector.

[0053] In a second aspect of the invention, the analytical method is used in the quality determination of dalteparin sodium.

[0054] In some embodiments, the application includes using the analytical method to perform multiple tests on dalteparin sodium standards (e.g., European or US standards) and / or original samples (e.g., Pfizer products marketed in the US or Europe), and establishing quality standards based on the test results.

[0055] In some embodiments, the quality standards established based on the test results according to the present invention can be varied according to the general knowledge of those skilled in the art, and these varied technical solutions all fall within the scope of the application claimed by the present invention. Further, the quality standards can be established by the range of peak area percentages (%) of each peak as shown in Table 30 of the embodiments of this application. Further, the quality standards are converted from the peak area percentages (%) of each peak in Table 30 to quality standards 1, 2, and 3 in Table 32 (which correspond to high, medium, and low quality standard intensities, respectively) according to the rules in Table 31. Further still, the quality standards are as shown in the following table.

[0056] Detailed Implementation

[0057] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope covered by the claims of the present invention.

[0058] 1. Source of reagents used in this invention:

[0059]

[0060] 2. Preparation of reagent samples used in this invention:

[0061] (1) Preparation of mobile phase: The volume of the solution can be adjusted according to the actual amount used and the solution ratio.

[0062] Preparation of mobile phase A (20-200 mM ammonium acetate or ammonium formate solution, preferably 150 mM ammonium acetate): Weigh an appropriate amount of ammonium acetate or ammonium formate into a 1000 mL volumetric flask, dilute to the mark with ultrapure water, shake well, transfer to a blue-capped bottle, and sonicate for 10 minutes before use.

[0063] Mobile phase B: Take an appropriate amount of acetonitrile into a clean blue-capped bottle, sonicate for 10 minutes and set aside.

[0064] (2) Preparation of heparin-based nitrite degradation product solution: The volume of the solution can be adjusted according to the actual amount used and the solution ratio.

[0065] (3) Preparation of 0.5M H2SO4 solution: Take 0.3mL of concentrated sulfuric acid and slowly add it to a small amount of water. Make up to 10mL with ultrapure water. After cooling, store in the refrigerator for later use.

[0066] (4) 0.5M Ba(NO2)2 (barium nitrite): Weigh 1.2303g of Ba(NO2)2 monohydrate, dissolve it in ultrapure water and make up to 10mL, then store it under cold.

[0067] (5) Preparation of pH 1.5 nitrous acid (HONO) solution: In a fume hood, take equal volumes of refrigerated 0.5M H2SO4 and refrigerated 0.5M Ba(NO2)2 and mix them directly. Centrifuge at 11000rpm for two minutes to remove BaSO4 precipitate. Take the supernatant, which is the pH 1.5 nitrous acid solution. Prepare it fresh before use.

[0068] (6) Preparation of 1M sodium carbonate solution: Dissolve 1.06g of sodium carbonate in ultrapure water and make up to 10mL. Shake well.

[0069] (7) Preparation of 30mg / mL NaBH4 solution: Take 30mg NaBH4 into a clean centrifuge tube, add 1mL of ultrapure water to dissolve, shake well, and prepare fresh before use.

[0070] (8) Preparation of 4M sodium hydroxide solution: Take 1.6g of sodium hydroxide, add 10mL of ultrapure water to dissolve, and shake well.

[0071] (9) Preparation of 100 mg / mL dalteparin sodium sample solution: Weigh 0.1 g dalteparin sodium sample (original sample or European Pharmacopoeia Chemical Reference or United States Pharmacopoeia Chemical Reference) into a clean centrifuge tube and add 1 mL of ultrapure water until completely dissolved.

[0072] (10) Preparation of dalteparin sodium nitrite degradation product solution and blank solution:

[0073] Exemplary preparation of dalteparin sodium nitrite degradation product solution: Take a certain volume (e.g., 105 μL) of dalteparin sodium sample (e.g., 100 mg / mL) solution into a centrifuge tube, add 200 μL of pH 1.5 HONO solution, shake well on a mixer, let stand for at least 30 min, shake on a mixer for 2 min, add 80 μL of 1M sodium carbonate solution to adjust the pH to 8.5 to terminate the reaction, add 145 μL of freshly prepared 30 mg / mL sodium borohydride solution, reduce at room temperature for at least 4 hours, add 120 μL of glacial acetic acid to adjust the pH to 4.0 to terminate the reaction, then add 150 μL of 4M NaOH to neutralize and adjust the pH to approximately 7.0, the total volume is approximately 800 μL. Take 0.3 mL of the degradation solution into a centrifuge tube, add 0.7 mL of ultrapure water and mix well, filter through a 0.22 μm filter membrane and set aside for use.

[0074] Preparation of blank solution: Replace 105 μL of dalteparin sodium sample solution in the preparation of dalteparin sodium sample solution with 105 μL of ultrapure water. The solvent used and the entire preparation process are the same as those for the preparation of dalteparin sodium sample solution.

[0075] Example 1: Chromatographic separation and analysis of nitrite degradation products of heparin sodium at different concentrations

[0076] 1. High performance liquid chromatography conditions are shown in Table 1.

[0077] Table 1

[0078]

[0079]

[0080] 2. The CAD requirements are shown in Table 2.

[0081] Table 2

[0082] Condition content Name / Indicator CAD <![CDATA[ Thermo Corona Veo RS ]]> power function 1.0 sampling frequency 10Hz Filter value 5.0 atomization temperature 35℃ Collection time 60min

[0083] 3. The preparation methods of nitrite degradation samples of different concentrations of heparin sodium are shown in Table 3.

[0084]

[0085]

[0086] Sample naming rules: USP and EP standards are named starting with Dal-USP / EP-RED-XX, where the batch number starting with -RED indicates the sample after degradation and reduction, and XX indicates the sample number; blank samples are named starting with Blank-RED, where Blank-RED indicates the blank sample after degradation and reduction.

[0087] In Table 3, Dal-USP-RED-01 represents the concentration at which complete degradation is achieved in the prior art. If the result of Dal-USP-RED-02 is the same as that of Dal-USP-RED-01, it indicates that a 5-fold increase in the initial concentration of the sample can also achieve complete degradation. Dal-USP-RED-04 represents an initial concentration that is 2.1 times higher than that of Dal-USP-RED-02. Since the initial concentrations of Dal-USP-RED-03 and Dal-USP-RED-02 are the same, comparing Dal-USP-RED-03 with Dal-USP-RED-04 can prove whether Dal-USP-RED-04 is completely degraded.

[0088] The final sample concentrations of Dal-USP-RED-01 and Dal-USP-RED-02 were the same. The final sample concentration of Dal-USP-RED-04 was 1.05 times that of Dal-USP-RED-03.

[0089] The initial concentrations of dal-USP-RED-01 to Dal-USP-RED-08 were 100 mg / mL, 100 mg / mL, 100 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 300 mg / mL and 400 mg / mL, respectively.

[0090] The above samples were tested using the instrument, and the experimental results are as follows:

[0091] like Figure 1 As shown, apart from the solvent peak, no chromatographic peaks affecting sample detection appeared in the blank solution.

[0092] Depend on Figure 1 It was learned that a large amount of solvents used in the degradation of nitrite (such as sodium carbonate, sodium borohydride, glacial acetic acid, etc.) will have peaks in CAD. If these reagents enter the CAD detector in large quantities, they will affect the lifespan of the detector. Therefore, data were only collected from 0 min to 60 min in the condition exploration experiment, and data were collected from 16 min in subsequent experiments.

[0093] like Figure 2 As shown, the final sample concentrations of Dal-USP-RED-01 and Dal-USP-RED-02 are the same. Due to the low sample concentration, only three chromatographic peaks appear in addition to the solvent peak.

[0094] like Figure 3 As shown, the final sample concentration of Dal-USP-RED-04 is 1.05 times that of Dal-USP-RED-03, and 11 chromatographic peaks appear in addition to the solvent peak.

[0095] like Figure 4 As shown, the chromatographic peaks from Dal-USP-RED-04 to Dal-USP-RED-08 were well separated.

[0096] in conclusion:

[0097] After mixing with nitrite, the concentrations of dalteparin sodium in each group were as follows (concentration = initial mass of dalteparin sodium (mg) / volume of added nitrite and dalteparin sodium (mL)): 4.76 mg / mL (Dal-USP-RED-01), 20 mg / mL (Dal-USP-RED-02), 20 mg / mL (Dal-USP-RED-03), 34.4 mg / mL (Dal-USP-RED-04), 51.6 mg / mL (Dal-USP-RED-05), 68.9 mg / mL (Dal-USP-RED-06), 103.3 mg / mL (Dal-USP-RED-07), and 137.7 mg / mL (Dal-USP-RED-08). In existing sample processing techniques, degradation reagents significantly interfere with sample detection. The inventors addressed this by first increasing the concentration of dal-heparin sodium (groups Dal-USP-RED-02 to Dal-USP-RED-08) after mixing with nitrous acid to approximately 4-30 times that of group Dal-USP-RED-01, and then diluting with ultrapure water (dilution step). This reduced the interference of the degradation reagent and also allowed for a suitable increase in the sample detection concentration. However, if the conditions after dal-heparin sodium sample degradation remain unchanged, as shown in the table above, an excessively high concentration of dal-heparin sodium during nitrous acid degradation leads to an excessively high salt concentration in the final sample, potentially affecting the column life in HPLC and reducing the accuracy of the detection results.

[0098] Therefore, as shown in Table 3 Figure 1-4 The corresponding results show that the detection method of the present invention can quickly, cost-effectively and accurately separate and detect the nitrite degradation products of dalteparin sodium samples, and further realize the analysis (quality dimension) of dalteparin sodium samples.

[0099] 4. The peak areas of each component in the nitrite degradation product solution of four doses of dalteparin sodium US Pharmacopeia standard and the relative errors of the peak areas between the two doses are shown in Tables 4 and 5.

[0100] Relative error formula:

[0101] Formula meaning: The relative error of A relative to B.

[0102] Table 4

[0103]

[0104] Table 5

[0105]

[0106] Note: Since the final sample concentration of Dal-USP-RED-04 is 1.05 times that of Dal-USP-RED-03, the peak area is proportional to the sample concentration. To calculate the relative error of the peak area between the two injections, the same sample concentration must be used. Therefore, the peak area of ​​the Dal-USP-RED-03 sample needs to be multiplied by 1.05.

[0107] in conclusion:

[0108] The blank solution, except for the solvent peak, did not show any factors that would affect the detection of the chromatographic peaks in the sample. The resolution between peak 4 and the solvent peak in the sample solution was 2.6, which meets the requirement that the minimum resolution between adjacent chromatographic peaks should not be less than 0.8, indicating that the two peaks are well separated and the solvent peak does not affect the detection of peak 4.

[0109] The sample concentration was high. The final sample concentration of Dal-USP-RED-04 was approximately 3.94 mg / ml, obtained by increasing the concentration of the sample solution and then increasing the dilution factor after degradation. The baseline noise was low, so it was selected as the optimal degradation condition.

[0110] In the following verification experiments, both the dalteparin sodium standard and the original dalteparin sodium sample were degraded under optimal conditions.

[0111] Example 2: Chromatographic separation and analysis of nitrite degradation products of heparin sodium at different concentrations

[0112] 1. Chromatography and CAD instrument conditions are the same as before.

[0113] A schematic diagram of the detection results of sodium dalteparin nitrite degradation in samples is shown below. Figure 5 As shown.

[0114] Data processing: Set appropriate integration parameters, such as those in Table 6, and integrate the CAD diagram of the sample using the area normalization method to calculate the peak area percentage of each component.

[0115] Table 6

[0116]

[0117] 2. Method validation: Validation items include specificity, limit of quantitation, precision (reproducibility and intermediate precision) and robustness.

[0118] (1) Exclusivity:

[0119] The blank solution of nitrite degradation, the nitrite degradation product solution of dalteparin sodium US Pharmacopeia standard, the nitrite degradation product solution of dalteparin sodium European Pharmacopeia standard, and the nitrite degradation product solution of the original dalteparin sodium were analyzed by injection on the HILIC-HPLC-CAD system to investigate whether the blank sample would affect the detection.

[0120] The blank solution of nitrite degradation, the nitrite degradation product solution of dalteparin sodium US Pharmacopeia standard, the nitrite degradation product solution of dalteparin sodium European Pharmacopeia standard, and the nitrite degradation product solution of the original dalteparin sodium were injected into HILIC-UPLC-MS to investigate whether the blank sample would affect the detection, and to qualitatively identify the components after nitrite degradation.

[0121] Specific methods:

[0122] The preparation of the sodium dalteparin nitrite degradation product solution is shown in Tables 7 and 8.

[0123] Table 7

[0124] Solution name Dalteparin Sodium Standard / Sample Name Dalteparin sodium standard / sample addition amount Ultrapure water addition amount solution concentration Dal-USP Dalteparin Sodium United States Pharmacopeia Standard 0.0200g 0.200ml 100mg / ml Dal-EP Dalteparin Sodium European Pharmacopoeia Standard 0.0205g 0.205ml 100mg / ml Original sample Original Dalteparin Sodium 0.0208 0.208ml 100mg / ml

[0125] Table 8

[0126]

[0127]

[0128] Preparation of blank solution: Replace 100 mg / ml sample solution with ultrapure water, and keep the rest unchanged.

[0129] Under the aforementioned instrument conditions, one injection of blank solution, one injection of dalteparin sodium US Pharmacopeia standard nitrite degradation product solution, one injection of EP dalteparin sodium standard nitrite degradation product solution, and one injection of dalteparin sodium original sample nitrite degradation product solution were analyzed.

[0130] The experimental results are as follows:

[0131] like Figure 6 As shown, apart from the solvent peak, no chromatographic peaks affecting sample detection appeared in the blank solution.

[0132] Table 9 shows the resolution of adjacent chromatographic peaks in the CAD diagrams of dalteparin sodium US standard, European standard, and dalteparin sodium original sample nitrite degradation product solution.

[0133] Table 9

[0134]

[0135] Table 10 shows the minimum resolution, asymmetry of peak 4, and number of plates for nitrite degradation products of dalteparin sodium US standard, European standard, and original dalteparin sodium sample.

[0136] Table 10

[0137]

[0138]

[0139] in conclusion:

[0140] Apart from the solvent peak, no chromatographic peaks that would affect sample detection appeared in the blank.

[0141] Table 9 shows that the minimum resolution of adjacent chromatographic peaks in the USP dalteparin sodium standard, EP dalteparin sodium standard, and the nitrite degradation product solution of the original dalteparin sodium sample is the resolution between peak 2 and peak 3, which all meet the requirement of a resolution of not less than 0.8.

[0142] The USP dalteparin sodium standard, EP dalteparin sodium standard, and the nitrite degradation product solution of the original dalteparin sodium sample all showed good theoretical plate number, asymmetry, and resolution of adjacent chromatographic peaks (peaks 2 and 3). Therefore, the method of this invention meets the specificity requirements.

[0143] HILIC-UPLC-MS method

[0144] Mobile phases A and B, and the dalteparin sodium nitrite degradation product solution were prepared as described in the previous Example 1 and the preparation of the dalteparin sodium nitrite degradation product solution (HILIC-HPLC-CAD).

[0145] The instrument requirements are shown in Tables 11 and 12:

[0146] Table 11

[0147]

[0148] Table 12

[0149]

[0150]

[0151] The experimental results are as follows:

[0152] like Figure 7 As shown, apart from the solvent peak, no chromatographic peaks affecting sample detection appeared in the blank solution.

[0153] The RRT values ​​of each peak in the TIC and CAD mass spectra are basically consistent, and the results are detailed in Table 13.

[0154]

[0155]

[0156]

[0157]

[0158]

[0159] Note: The structural representation is as follows: Taking U8, 9, 1 as an example, the first letter U represents a saturated uronic acid structure, the first number 8 represents an octasaccharide structure, the second number represents 9 sulfonic acid groups, the third number represents 1 acetyl group, and -ManR represents a reduced end with anhydromannitol structure. AMol is 2,5-anhydromannitol; Rc-GlcN is intrachain cyclopentadiazonium glucosamine; ANAC is N-acetylated glycosamine; I in the sequence represents iduronic acid or glucuronic acid.

[0160] Since it is common knowledge in the art that acetyl groups only exist in glucosamine residues, the deacetylation step in CN201310695114.8 causes the glucosamine information containing N-acetyl groups in dalteparin sodium to disappear. As shown in Tables 14-15, the method of the present invention can fully reflect the situation of the analyzed dalteparin sodium degradation products.

[0161] Experimental conclusion:

[0162] Apart from the solvent peak, no chromatographic peaks that would affect sample detection appeared in the blank.

[0163] The USP dalteparin sodium standard, EP dalteparin sodium standard, and the nitrite degradation product solution of the original dalteparin sodium sample all meet the analytical requirements. Peak 4 has a minimum theoretical plate number of 149851, an asymmetry of 1.2, and a resolution of 0.9 between adjacent peaks (peaks 2 and 3). Qualitative analysis was performed on each peak in the CAD image using mass spectrometry to determine their possible structures and sequences. Subsequent qualitative and quantitative analysis of the sample can be achieved solely through CAD analysis.

[0164] (2) Precision (repeatability and intermediate precision):

[0165] Repeatability:

[0166] The preparation of the sodium dalteparin nitrite degradation product solution is shown in Tables 16 and 17.

[0167] Table 16

[0168]

[0169]

[0170] Preparation of blank solution: Replace 100 mg / ml sample solution with ultrapure water, and keep the rest unchanged.

[0171] The instrument conditions were as described above. One blank sample and six dalteparin sodium United States Pharmacopeia standard nitrite degradation product solutions were injected for analysis.

[0172] On the same day, the same inspector administered one injection each of the six injections of dalteparin sodium USP reference standard nitrite degradation product solution. Quantitative analysis: The relative standard deviation (RSD%) of the percentage area of ​​each component peak in the CAD plot of the six injections of dalteparin sodium USP reference standard nitrite degradation product solution was calculated.

[0173] Experimental results:

[0174] The percentage of peak area of ​​each component in the degradation of nitrite from 6 injections of heparin sodium (US standard) is shown in Table 18.

[0175] Table 18

[0176]

[0177] As can be seen from the table above, except for peaks named 1, 10, and 11, the percentage of the area of ​​all other peaks is greater than LOQ (3.0%) (the limit of quantitation determined by subsequent limit of quantitation experiments). The maximum RSD of peaks with a peak area percentage ≥ 10% is 1.3%, and the maximum RSD of peaks with a peak area percentage ≥ LOQ and < 10.0% is 7.2%. Therefore, the method of this invention has passed the repeatability experiment.

[0178] Intermediate precision:

[0179] The solutions were prepared by different researchers on different dates, following the methods described in the repeatability section.

[0180] The preparation of the sodium dalteparin nitrite degradation product solution is shown in Tables 19 and 20.

[0181] Table 19

[0182] Solution name Dalteparin Sodium Standard / Sample Name Dalteparin sodium standard / sample addition amount Ultrapure water addition amount solution concentration Dal-USP-01 Dalteparin Sodium United States Pharmacopeia Standard 0.0205g 0.205m1 100mg / ml Dal-USP-02 Dalteparin Sodium United States Pharmacopeia Standard 0.0204g 0.204ml 100mg / m1 Dal-USP-03 Dalteparin Sodium United States Pharmacopeia Standard 0.0201g 0.201ml 100mg / m1 Dal-USP-04 Dalteparin Sodium United States Pharmacopeia Standard 0.0206g 0.206ml 100mg / m1 Dal-USP-05 Dalteparin Sodium United States Pharmacopeia Standard 0.0202g 0.202ml 100mg / ml Dal-USP-06 Dalteparin Sodium United States Pharmacopeia Standard 0.020lg 0.201ml 100mg / ml

[0183]

[0184] Preparation of blank solution: Replace 100 mg / ml sample solution with ultrapure water, and keep the rest unchanged.

[0185] The mobile phase and instrument conditions were as described above. One blank sample and six dalteparin sodium USP standard nitrite degradation product solutions were injected for analysis. Quantitative analysis: The relative standard deviation (RSD%) of the peak area percentage of each component in the CAD plots of the 12 dalteparin sodium USP standard nitrite degradation product solutions analyzed by two inspectors was calculated.

[0186] The experimental results are as follows:

[0187] The percentage of peak area of ​​each component in the degradation of nitrite from 6 injections of heparin sodium (US standard) is shown in Table 21.

[0188] Table 21

[0189]

[0190] The percentage of peak area of ​​each component in the degradation of nitrite by two experimenters using 12 injections of dalteparin sodium (US standard) is shown in Table 22.

[0191]

[0192] As can be seen from Table 21, except for peaks named 1, 10, and 11, the percentage of the area of ​​all other peaks is greater than the LOQ (the limit of quantitation (3) below) (3.0%), and the maximum RSD of peaks with a percentage of area ≥ 10% is 2.2%, while the maximum RSD of peaks with a percentage of area ≥ LOQ and < 10.0% is 6.1%, which allows for effective analysis.

[0193] As can be seen from Table 22, the nitrite degradation product solution of dalteparin sodium US standard for the two experimenters, except for peaks named 1, 10, and 11, all had peak area percentages greater than LOQ (3.0%). The results of the limit of quantitation are referenced in the research conclusions of the limit of quantitation section (3) below. The maximum RSD of peaks with a peak area percentage ≥10% is 1.6%, and the maximum RSD of peaks with a peak area percentage ≥LOQ and <10.0% is 7.2%, which can be effectively analyzed.

[0194] The method of the present invention has been verified by intermediate precision experiments.

[0195] (3) Limit of quantitation:

[0196] This method calculates the percentage content of each component using the area percentage method through CAD plot integration, and is used for comparative studies of component percentage content. Therefore, it is necessary to confirm the minimum quantitation range for each component. According to ICH Q2 requirements, the acceptable criteria for the limit of quantitation are that the S / N ≥ 10:1 and the peak area percentage RSD% ≤ 10.0%.

[0197] The mobile phase and instrument conditions are as described above.

[0198] The preparation of the sodium dalteparin nitrite degradation product solution is shown in Tables 23 and 24.

[0199] Table 23

[0200] Solution name Dalteparin Sodium Standard / Sample Name Dalteparin sodium standard / sample addition amount Ultrapure water addition amount solution concentration Dal-USP Dalteparin Sodium United States Pharmacopeia Standard 0.0203g 0.203m1 100mg / ml

[0201] Table 24

[0202]

[0203] Preparation of blank solution: Replace 100 mg / ml sample solution with ultrapure water, and keep the rest unchanged.

[0204] One injection of blank sample and three injections of dalteparin sodium US Pharmacopeia standard nitrite degradation product solution were used for analysis.

[0205] For repeatability tests, the RSD% and limit of quantitation for each component of the 6-dalteparin sodium US Pharmacopeia standard nitrite degradation product solution were as follows: for the 3-dalteparin sodium standard solution, the minimum peak area percentage was the acceptable standard for the limit of quantitation.

[0206] Quantitative analysis: The relative standard deviation (RSD%) of the peak area percentage of the CAD chromatograms of the 6 repeatable injections of dalteparin sodium US Pharmacopeia standard nitrite degradation product solutions was calculated.

[0207] The limit of quantitation is determined by the S / N ratio and the RSD% of the peak area percentage for each peak.

[0208] Table 25 shows the peak area percentage and signal-to-noise ratio of each component in the degradation of nitrite from 3-dalteparin sodium (US standard).

[0209] Table 25

[0210]

[0211] As can be seen from the table above, the minimum component peak name that simultaneously satisfies the requirements of signal-to-noise ratio S / N ≥ 10:1, 6-needle repeatability peak area percentage RSD ≤ 10.0% is 9, and the average peak area percentage of peak 9 is 3.0%. Therefore, when the sample concentration is 100 mg / ml, the limit of quantitation for this method is 3.0%.

[0212] (4) Durability:

[0213] The mobile phase and instrument conditions are the same as before.

[0214] The preparation of the sodium dalteparin nitrite degradation product solution is shown in Tables 26 and 27.

[0215] Table 26

[0216]

[0217]

[0218]

[0219] As can be seen from the table above, the test results of the 6-needle robustness and 6-needle repeatability of dalteparin sodium US standard solution, except for peaks named 1, 10, and 11, show that the percentage of the area of ​​all other peaks is greater than LOQ (3.0%). The maximum RSD of peaks with a percentage of area ≥10% is 2.0%, and the maximum RSD of peaks with a percentage of area ≥LOQ and <10.0% is 9.6%. Therefore, the method of the present invention has robustness to chromatographic columns with different serial numbers, as tested by the robustness test.

[0220] (5) Solution stability:

[0221] The dalteparin sodium solution, a solution of nitrite degradation products from the United States Pharmacopeia standard, was used as the stability test sample for the 0-hour test. The sample was placed at room temperature for approximately 48 hours (the first injection time was counted as day 1, and 48 hours as day 3) and tested. One injection was given for each sample at each time point.

[0222] Quantitative analysis: The relative standard deviation (RSD%) of the peak area percentage of each component in the CAD diagram of the nitrite degradation product solution of 12 injections of dalteparin sodium USP standard was calculated, with the first injection also used as the system suitability.

[0223] The experimental conditions were the same as before.

[0224] The percentage of peak area of ​​each component in the degradation of nitrite from 12 injections of dalteparin sodium (US standard) is shown in Table 29.

[0225]

[0226] The stability and repeatability of the 6-injection solution of dalteparin sodium US standard solution are shown in the table above. Except for peaks named 1, 10, and 11, the percentage area of ​​all other peaks is greater than the LOQ (3.0%). Furthermore, the maximum RSD for peaks with a percentage area ≥10% is 2.5%, and the maximum RSD for peaks with a percentage area ≥LOQ and <10.0% is 9.1%. Therefore, the method of this invention, through solution stability experiments, shows that the dalteparin sodium solution and the nitrite degradation product solution of the US Pharmacopeia standard are stable after 3 days and do not affect the sample detection results.

[0227] Example 3

[0228] The experimental conditions and reagents are as described in Example 2.

[0229] The inventors believe that by testing original samples from Europe and the United States, the standard for using the method of this invention to detect the quality of dalteparin sodium can be determined. The testing method is the same as that in Example 2 above. The detection results of degradation products of dalteparin sodium from 7 batches of original European and 11 batches of original US samples by high performance liquid chromatography coupled with an electrospray detector are shown in Table 30.

[0230] Table 30

[0231]

[0232] The applicable quality standards are as follows: Based on the original EU GATT test results, three levels are categorized: high, medium, and low (corresponding to quality standards 1, 2, and 3 in Table 32), calculated according to the rules in Table 31 below. For example, if the peak area percentage of the first peak in Table 30 is 2.1-2.9% (below the limit of quantitation 3.0%, please refer to Table 25 for the limit of quantitation), then the quality standard strength is calculated by referring to the rightmost column of the rules in Table 31, where the limit of quantitation is greater than the peak area percentage (i.e., high: 2.9 × 115% = 3.3; medium: 2.9 × 125% = 3.6; low: 2.9 × 135% = 3.9), which corresponds to quality standard 1 (≤3.3%), quality standard 2 (≤3.6%), and quality standard 3 (≤3.9%) in Table 32. Other peaks are calculated using the same method.

[0233] Table 31

[0234]

[0235] Table 32 (Peak Area Percentage %)

[0236]

[0237] As shown in the table above, by using the original European and American dalteparin sodium and analyzing its degradation products using the method of this invention, a set of quality standards for the analysis of dalteparin sodium degradation products can be established and used for the quality judgment of dalteparin sodium samples. The quality standards described in this invention are merely exemplary; those skilled in the art can make variations based on general knowledge, and all such variations fall within the scope of the applications claimed in this invention.

[0238] In addition to those described herein, various modifications of the invention will be apparent to those skilled in the art based on the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. All references cited in this application (including all patents, patent applications, journal articles, books, and any other disclosures) are incorporated herein by reference in their entirety.

Claims

1. A method for analyzing the degradation products of dalteparin sodium nitrite, the method comprising the following steps: (1) The sodium dalteparin is mixed with nitrite and completely degraded. A pH adjuster is added to terminate the reaction. Then, sodium borohydride solution is added for reduction. One or more pH adjusters are added to terminate the reaction and adjust the pH to neutral to obtain the final degradation product. (2) The degradation products were detected by high performance liquid chromatography coupled with mass spectrometry. Finally, qualitative information on the products of sodium dalteparin degradation by nitrite obtained in step (1) is obtained; in, In step (2), the detection conditions of the high performance liquid chromatography coupled with mass spectrometry are as follows: mobile phase A is ammonium acetate solution or ammonium formate solution, and mobile phase B is acetonitrile; 2. The analytical method according to claim 1, wherein the detection conditions of the mass spectrometry in the high performance liquid chromatography-mass spectrometry are as follows: 。 3. The analytical method according to claim 1, wherein, The concentration of the ammonium acetate solution or ammonium formate solution is 20-200 mM.

4. The analytical method according to claim 1, wherein the ratio of the mass of dalteparin sodium to the molar number of nitrites (g / mol) is 17.5-405:

1.

5. The analytical method according to claim 1, wherein the ratio of the mass of dalteparin sodium to the molar number of nitrites (g / mol) is 35-175:

1.

6. The analytical method according to claim 1, wherein the ratio of the mass of dalteparin sodium to the molar number of nitrites (g / mol) is 50-130:

1.

7. The analytical method according to claim 1, wherein the concentration of dalteparin sodium after mixing with nitrite is between 4 mg / mL and 150 mg / mL.

8. The analytical method according to claim 1, wherein the pH adjuster is selected from one or more of the following: sodium carbonate, sodium bicarbonate, sodium hydroxide, hydrochloric acid, sulfuric acid, and glacial acetic acid.

9. The application of the analytical method of any one of claims 1-8 in the quality determination of dalteparin sodium.

10. The application according to claim 9 includes performing multiple tests on dalteparin sodium standards and / or original samples using the analytical method according to any one of claims 1-8, and establishing quality standards based on the test results.

Images