WO2020020152A1 - 一种达肝素钠亚硝酸降解产物的分析方法及其应用 - Google Patents

一种达肝素钠亚硝酸降解产物的分析方法及其应用 Download PDF

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WO2020020152A1
WO2020020152A1 PCT/CN2019/097283 CN2019097283W WO2020020152A1 WO 2020020152 A1 WO2020020152 A1 WO 2020020152A1 CN 2019097283 W CN2019097283 W CN 2019097283W WO 2020020152 A1 WO2020020152 A1 WO 2020020152A1
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sodium
dalteparin sodium
usp
solution
dalteparin
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PCT/CN2019/097283
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English (en)
French (fr)
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罗君君
石瑛
林森茂
陈永杰
李锂
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深圳市海普瑞药业集团股份有限公司
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Priority to CN202310030098.4A priority Critical patent/CN116879411A/zh
Priority to CN201980027376.2A priority patent/CN112005110B/zh
Publication of WO2020020152A1 publication Critical patent/WO2020020152A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph

Definitions

  • the invention relates to an analysis method and application of dalteparin sodium nitrite degradation products.
  • Dalparin sodium is a low-molecular-weight heparin sodium, composed of polysaccharide chains of different molecular weights, which itself has no absorption peaks in the ultraviolet.
  • Heparinase whether heparinase I, II or III, they all specifically cleave the glycosidic bond between glucosamine (1-4) hexuronic acid and form between C4 and C5 of hexuronic acid A double bond.
  • the enzymolysis product obtained after heparinase hydrolyzes daparin sodium can have an absorption peak in ultraviolet light, and the enzymolysis product of daparin sodium can be quantitatively analyzed by a UV detector.
  • heparinase is usually used to hydrolyze dalteparin sodium, and then the degradation products are separated by a liquid phase system, and the retention time is qualitatively compared with commercially available disaccharide standard products, and then the two The sugar standard quantifies the standard curve of the detector response and mass. Since the disaccharide standard is relatively expensive, the subsequent development of the separation of degradation products by a high-performance liquid phase system, and then the structural characterization of the separated products by mass spectrometry, so as to achieve the qualitative analysis of the degradation products of sodium heparin, using UV The detector performs quantitative analysis on the separated components, and finally realizes the analysis of the quality of dalteparin sodium.
  • the prior art In order to further improve the method of qualitative / quantitative analysis of the enzymatic hydrolyzed components of the heparin, the prior art often performs a derivatization reaction before or after the heparin enzymatic hydrolysis reaches the heparin sodium product, for example: the prior art (CN201310711455 .X) discloses that after the heparinase is hydrolyzed to heparin sodium and before the upper liquid phase column is separated, the enzymatic product is derivatized, and the derivative products are detected by reversed-phase chromatography and high-resolution mass spectrometry to ensure that all Enzymatic hydrolysis products can be qualitatively analyzed.
  • CN201410123609.8 discloses that after a heparinase hydrolyzes a heparin sodium liquid phase system column to separate an enzymatic product, a fluorescence derivatization reaction is performed, and then detection is performed. This not only provides detectable fluorophores for low-molecular-weight heparin components without UV absorption, but also enables high-sensitivity fluorescence detection with higher sample response values in the spectrum; it avoids chromatographic separation caused by the by-products of the derivatization reaction. Interference, improving the accuracy and repeatability of the analysis.
  • heparinase degradation a method for detecting heparin disaccharide composition exists in the prior art (CN201310695114.8), which mainly deacetylates heparin first to obtain deacetylated heparin, and then deacetylates Heparin is degraded with nitrous acid to obtain heparin disaccharide; heparin disaccharide is concentrated to dryness and then dissolved in water, the pH value is adjusted to alkaline, and pyrazolinone derivative reagents are added. After the reaction is completed, extraction is performed three times with chloroform to remove unreacted Derivatization reagent; detection of derivatized disaccharides by liquid chromatography or liquid chromatography-mass spectrometry.
  • deacetylation followed by deacetylated nitrite degradation method which can degrade glucosamine with N-sulfate group in daparin sodium, but cannot degrade N-acetyl in daparin sodium Glucosamine. That is to say, the deacetylation step makes the information of glucosamine containing N-acetyl group present in dalteparin sodium disappear, and the method must also include a derivatization step, which is complicated.
  • heparinase degradation method in the prior art, first, heparinase is easily inactivated and its properties are unstable; second, heparinase enzymolysis takes a long time to reach heparin sodium (the time required for a single enzymolysis experiment is 48h), and the steps are complicated High detection cost (requires mass spectrometry).
  • the glucuronic acid (GlcA) and iduronic acid (IdoA) are dehydrated to form the same structure of dehydrated uronic acid, and the glucuronic acid and idurol in dalteparin sodium are lost.
  • the content and structure of uronic acid, and heparinase cannot completely degrade dalteparin sodium into disaccharides.
  • the analysis method for the degradation product of sodium dalteparin sodium is inefficient and costly, and the results obtained by the analysis method for the degradation product of sodium dalteparin sodium cannot completely reflect the situation of the analysis of dalteparin sodium. Therefore, there is currently a need for an analysis method that has a short analysis time, low analysis cost, high detection accuracy rate, and fully reflects the situation of the dalteparin sodium being analyzed.
  • a method for analyzing a product of degradation of nitroheparin sodium nitrite comprising the following steps: (1) mixing said dalteparin sodium with nitrous acid to be completely degraded; (2) The degradation products were detected by a high-performance liquid chromatography coupled with an electric aerosol detector.
  • the step (1) further comprises after the dalteparin sodium and nitrous acid are completely degraded, adding a pH adjuster to stop the reaction, and then adding a sodium borohydride solution to reduce, further adding one or more A pH adjuster stops the reaction and adjusts the pH to neutral to obtain the final degradation product.
  • the conditions of the high-performance liquid chromatography and the electric aerosol detector are a flow rate of 0.1-2 mL / min; and an elution gradient of 0-45 min, 10% -60%.
  • the ammonium salt is ammonium acetate or ammonium formate
  • the concentration is 20-200 mM
  • the flow rate is 0.1-1 mL / min
  • the elution gradient is 0-45 min, 10% -45 % Mobile phase A, 90% -55% mobile phase B.
  • the ratio (g / mol) of the mass of dalteparin sodium to the number of moles of nitrous acid is 17.5-405: 1, preferably 25-250: 1, 35-175: 1, 50- 130: 1.
  • the concentration of said dalteparin sodium after mixing with nitrous acid is between 4 mg / mL and 150 mg / mL.
  • the pH adjusting agent is selected from one or more of the following: sodium carbonate, sodium bicarbonate, sodium hydroxide, hydrochloric acid, sulfuric acid, and glacial acetic acid.
  • the method optionally includes the step of: performing the detection result obtained in step (2) and the result of the complete degradation of dalteparin sodium by nitrous acid by ultra-high performance liquid chromatography coupled with mass spectrometry.
  • the qualitative and quantitative information of the product of dalteparin sodium degraded by nitrous acid in step (1) is finally obtained.
  • the present invention relates to the application of the analysis method in the quality detection of dalteparin sodium.
  • the application includes using the analysis 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.
  • dalteparin sodium standards including but not limited to European or American standards
  • Figure 1 shows a stack of blank solution and Dal-USP-RED-02.
  • the top image is Dal-USP-RED-02 and the bottom image is BLANK-RED.
  • Figure 2 shows the overlay of Dal-USP-RED-01 and Dal-USP-RED-02.
  • the upper picture is Dal-USP-RED-02 and the lower picture is Dal-USP-RED-01.
  • Figure 3 shows the overlay of Dal-USP-RED-03 and Dal-USP-RED-04.
  • the upper picture is Dal-USP-RED-04 and the lower picture is Dal-USP-RED-03.
  • Figure 4 shows Dal-USP-RED-04 to Dal-USP-RED-08 overlays.
  • FIG. 5 is a graph showing the results of sample detection.
  • Figure 6 shows the 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, Specificity-Original sample-RED-01.
  • Figure 7 shows the 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.
  • room temperature means 25 ° C ⁇ 5 ° C. At the same time, if the experimental temperature is not specified, it is room temperature.
  • the term "about” refers to ⁇ 20% of the value modified by the term, preferably ⁇ 10%, and more preferably ⁇ 5%, so those skilled in the art can clearly understand The numerical value determines the scope of the term "about.”
  • sodium heparin sodium standard refers to the sodium heparin standard including but not limited to the United States Pharmacopeia and European Pharmacopoeia.
  • original sample of dalteparin sodium refers to the marketed products of dalteparin sodium from the original research manufacturer Pfizer, including but not limited to the marketed products of dalteparin sodium in the United States and Europe.
  • peak area percentage% refers to individual peaks (e.g., 11 peaks) obtained by detection of degradation products of heparin sodium by high performance liquid chromatography in combination with an electric aerosol detector or high efficiency liquid The percentage of the peak area of each peak obtained by detecting the degradation product of sodium heparin with phase chromatography combined with mass spectrometry as a percentage of the peak area of all peaks.
  • ratio of the relative retention time of each peak refers to the retention time of each peak obtained by detecting the degradation products of daparin sodium by high performance liquid chromatography in combination with an electric aerosol detector, The ratio of the retention time of the peak with the largest peak area (for example, the fourth peak), or the retention time of each peak obtained by detecting the degradation product of sodium heparin by high performance liquid chromatography and mass spectrometry, as described therein. The ratio of the retention time of the peak with the largest peak area among the peaks.
  • UPLC Ultra High Performance Liquid Chromatograph
  • HPLC High Performance Liquid Chromatograph
  • QTOF Time-of-Flight Mass Spectrometer
  • CAD Electrospray Detector
  • dp Degree of Polymerization of Sugar Chains
  • MS Mass Spectrometer
  • TIC Total ion current map
  • RRT relative retention time
  • the method used in the present invention does not require derivatization and deacetylation steps, and the degradation product has a short preparation time (about 5 hours), low analysis cost, high detection accuracy, and fully reflects the situation of the analyzed dalteparin sodium.
  • the principle of CAD detection is that the solute (analyte) droplets are dried to form solute particles, which collide with positively charged nitrogen particles to make the solute particles positively charged. Then, the charged particles transfer their charge to the collector, and finally use high sensitivity
  • the electrostatic detection meter measures the signal current of the charged solute. The resulting signal current is directly proportional to the mass content of the solute (analytical substance) and has nothing to do with the chemical structure of the solute (analytical substance) itself. Since the components degraded by dalteparin sodium nitrite have no ultraviolet characteristic absorption, combined detection with CAD can solve this problem.
  • dalteparin sodium nitrite degradation components including 4 disaccharide peaks, 5 tetrasaccharide peaks and 2 hexasaccharide peaks, and 8 component peaks above the limit of quantification (Corresponding to 2-9 in Table 25).
  • the method of the present invention is completed by using an HPLC combined with a detector CAD.
  • the degradation product of dalteparin sodium nitrite is first separated by a chromatography column (HILIC column (hydrophilic interaction chromatography)), and then detected by a CAD detector, so that according to the CAD detection result Composition information of dalteparin sodium nitrite degradation products was obtained.
  • HILIC column hydrophilic interaction chromatography
  • the method of the present invention can also perform the detection result of the degradation product detected by ultra high performance liquid chromatography coupled with mass spectrometry and the detection result of the degradation product detected by high performance liquid chromatography coupled with an electric aerosol detector.
  • the comparison makes it possible to realize the quasi-determinism of the degradation products only by detecting the degradation products of dalteparin sodium nitrite in the future by performing high-performance liquid chromatography combined with an electrospray detector.
  • the present invention relates to a method for analyzing the degradation products of nitroheparin sodium nitrite, the method includes the following steps or consists of the following steps: (1) mixing the dalteparin sodium and nitrous acid to be completely degraded; (2) ) Degradation products were detected by high performance liquid chromatography (HPLC) combined with an electric aerosol detector (CAD).
  • HPLC high performance liquid chromatography
  • CAD electric aerosol detector
  • the step (1) further comprises after the dalteparin sodium and nitrous acid are completely degraded, adding a pH adjuster to stop the reaction, and then adding a sodium borohydride solution to reduce, further adding one or more A pH adjuster stops the reaction and adjusts the pH to neutral (about 7.0) to obtain the final degradation product.
  • the step (1) further comprises mixing the dalteparin sodium and nitrite (the ratio (g / mol) of the mass of the dalteparin sodium to the number of moles of the nitrite is 17.5-405: 1).
  • dalteparin sodium and nitrite the ratio (g / mol) of the mass of the dalteparin sodium to the number of moles of the nitrite is 17.5-405: 1).
  • sodium carbonate to adjust the pH to 7-9 (preferably about 8.5)
  • sodium borohydride solution to reduce.
  • 3-5 hours preferably about 4 hours
  • add glacial acetic acid to adjust the pH to 3- 5 (preferably about 4) to stop the reaction, and then add sodium hydroxide to adjust the pH to 6.5-7.5 (preferably about 7) to obtain a final degradation product.
  • a 105 ⁇ L sample of 100 mg / mL dalteparin sodium was mixed with 200 ⁇ L of a nitric acid solution of pH 1.5, and the reaction was allowed to stand for more than 30 minutes, and then shaken on the mixer for 2 minutes, so that the dalteparin sodium was completely degraded.
  • Add 120 ⁇ L of glacial acetic acid to adjust the pH to 4.0 to stop the reaction.
  • the condition of the high-performance liquid chromatography and the electric aerosol detector is a flow rate of 0.1-2 mL / min, preferably 0.1-1 mL / min, and more preferably 0.3 mL. / min; elution gradient is 0-45min, 10% -60% mobile phase A, 90% -40% mobile phase B; preferably, the elution gradient is 0-45min, 10% -45% mobile phase A, 90 % -55% mobile phase B.
  • the elution gradient is 0-10min, 15% mobile phase A, 85% mobile phase B; 10-25min, 15% -33% mobile phase A, 85% -67% mobile phase B; The elution gradient was 25-45 min, 35% -45% mobile phase A, and 67% -55% mobile phase B.
  • the conditions of the high performance liquid chromatography optionally further include: an elution gradient of 45-50min, 45% of mobile phase A, 55% of mobile phase B; an elution gradient of 50-60min, 45% -15% Mobile phase A, 55% -85% mobile phase B, wherein the mobile phase A is an ammonium salt solution and the mobile phase B is acetonitrile.
  • the column temperature is 20-30 ° C, preferably 25 ° C, and the injection volume is 1-10 ⁇ L, preferably 3 ⁇ L.
  • the conditions of the high-performance liquid chromatography are shown in Table 1, and the conditions of the electric aerosol detector are that the atomization temperature is about 35 ° C, and other parameters (such as power function, Frequency, filtering value, etc.), preferably as shown in Table 2.
  • the ammonium salt is ammonium acetate or ammonium formate (in the analysis method of the present invention, ammonium acetate or ammonium formate can be replaced with each other without affecting the experimental results), and its concentration is 20-200mM, preferably 100-150mM , More preferably 100 mM and 150 mM, and the flow rate is 0.1-1 mL / min, preferably 0.3 mL / min.
  • the ratio (g / mol) of the mass of dalteparin sodium to the number of moles of nitrous acid is 17.5-405: 1, preferably 25-250: 1, 35-175: 1, 50- 130: 1.
  • the concentration of the dalteparin sodium is 4 mg / mL-150 mg / mL, preferably 4.76 mg / mL, 20 mg / mL, 30 mg / mL, 34.4 mg / mL, 51.6 mg / mL, 55mg / mL, 68.9mg / mL, 70mg / mL, 100mg / mL, 103.3mg / mL, 137.7mg / mL and the range between the numerical points, including but not limited to 20mg / mL-100mg / mL, 20mg / mL-70mg / mL, 30mg / mL-55mg / mL, etc.
  • the pH adjusting agent is selected from one or more of the following: sodium carbonate, sodium bicarbonate, sodium hydroxide, hydrochloric acid, sulfuric acid, and glacial acetic acid.
  • the dalteparin sodium solution nitrite degradation product solution is stable at room temperature for about one month, preferably about one week, and more preferably about three days.
  • the method optionally includes the step of: performing the detection result obtained in step (2) and the result of the complete degradation of dalteparin sodium by nitrous acid by ultra-high performance liquid chromatography coupled with mass spectrometry. By comparison, the product information of the dalteparin sodium described in step (1) after nitrous acid degradation is finally obtained.
  • the comparison of the detection results is to compare the ratio of the relative retention time of each peak obtained in step (2) with each peak of the degradation products detected by a high-performance liquid chromatography coupled with an electric mist detector. The relative retention time ratios are compared.
  • the present invention relates to the application of the analysis method in the quality detection of dalteparin sodium.
  • the application includes performing multiple times using the analytical method on a daparin sodium standard (e.g., European or American standard) and / or original sample (e.g., Pfizer products marketed in the United States or Europe) Testing, establishing quality standards based on testing results.
  • a daparin sodium standard e.g., European or American standard
  • original sample e.g., Pfizer products marketed in the United States or Europe
  • the quality standards established according to the detection results described in the present invention may be changed according to the general knowledge of those skilled in the art, and these changed technical solutions fall within the scope of the application claimed in the present invention.
  • the quality standard can be established by using the range of the peak area percentage% of each peak in Table 30 in the example of the present application. Further, the quality standard is to convert the peak area percentage% of each peak in Table 30 to the quality standards 1, 2 and 3 in Table 32 (which respectively correspond to high quality standard intensity, neutrality, and neutrality) through the rules in Table 31. low). Further, the quality standards are described in the following table.
  • Preparation of mobile phase According to the actual dosage, the preparation volume of the solution can be adjusted according to the ratio of the solution.
  • Preparation of mobile phase A (20-200mM ammonium acetate or ammonium formate solution, preferably 150mM ammonium acetate): Weigh an appropriate amount of ammonium acetate or ammonium formate into a 1000mL volumetric flask, make up to volume with ultrapure water, shake, and transfer to In a blue cap bottle, sonicate for 10 minutes before use.
  • Mobile phase B Take an appropriate amount of acetonitrile into a clean blue cap bottle and sonicate for 10 minutes before use.
  • Exemplary preparation of dalteparin sodium nitrite degradation product solution Take a certain volume (for example, 105 ⁇ L) of a dalteparin sodium sample (for example, 100 mg / mL) solution in a centrifuge tube, and add 200 ⁇ L of a HONO solution at pH 1.5, Shake uniformly on the mixer, let stand for 30 minutes or more, then shake for 2 minutes on the mixer, add 80 ⁇ L of 1M sodium carbonate solution to adjust the pH to 8.5, stop the reaction, add 145 ⁇ L of a new 30 mg / mL sodium borohydride solution, room temperature Reduce for at least 4 hours, add 120 ⁇ L of glacial acetic acid to adjust the pH to 4.0 to stop the reaction, and add 150 ⁇ L of 4M NaOH to neutralize and adjust the pH to about 7.0.
  • the total volume is about 800 ⁇ L. Take 0.3 ml of the degradation solution in a centrifuge tube, add 0.7 ml of ultrapure Mix with water
  • Preparation of blank solution Replace 105 ⁇ L of dalteparin sodium sample solution with 105 ⁇ L of ultrapure water in the preparation of dalteparin sodium sample solution.
  • the solvent and the entire preparation process are the same as those of dalteparin sodium sample solution.
  • Condition content Name / indicator Power function 1.0 Acquisition frequency 10Hz Filter value 5.0 Atomization temperature 35 °C Acquisition time 60min
  • Sample naming rules USP and EP standards (named after Dal-USP / EP-RED-XX), where the batch number -RED begins to degrade the reduced sample, XX represents the number of samples; blank samples start with Blank- RED is named at the beginning, and Blank-RED at the beginning represents the reduced sample after blank degradation.
  • Dal-USP-RED-01 is the concentration that is completely degraded in the prior art; if the result of Dal-USP-RED-02 is the same as Dal-USP-RED-01, it indicates that the initial concentration of the sample increased during degradation 5 times can also be completely degraded; Dal-USP-RED-04 is an additional 2.1 times the initial concentration based on Dal-USP-RED-02, because Dal-USP-RED-03 and Dal-USP-RED-02 are initially The same concentration, Dal-USP-RED-03 compared with Dal-USP-RED-04 can prove whether Dal-USP-RED-04 is completely degraded.
  • Dal-USP-RED-01 and Dal-USP-RED-02 have the same final sample concentration.
  • the final sample concentration of Dal-USP-RED-04 is 1.05 times that of Dal-USP-RED-03.
  • Dal-USP-RED-01 to Dal-USP-RED-08 have initial concentrations of dalteparin sodium of 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.
  • Dal-USP-RED-01 and Dal-USP-RED-02 have the same final sample concentration. Due to the low sample concentration, only three chromatographic peaks appear in addition to the solvent peak.
  • the degradation reagent interferes with the detection of the sample greatly.
  • the inventor first achieved the concentration of heparin sodium by mixing it with nitrous acid (Group Dal-USP-RED-02 to Dal-USP-RED-08). It is increased to about 4-30 times of the Dal-USP-RED-01 group, and then diluted with ultrapure water (dilution step), so as to reduce the interference of degradation reagents and also increase the sample detection concentration appropriately.
  • the detection method of the present invention can quickly, cost-effectively and accurately separate and detect the nitrite degradation products of the dalteparin sodium sample, thereby further realizing dalteparin sodium Analysis of samples (mass dimension).
  • the blank solution did not appear to affect the detection of each chromatographic peak of the sample except for the solvent peak; the resolution of peak 4 and the solvent peak in the sample solution was 2.6, which met the minimum resolution of adjacent chromatographic peaks not less than 0.8, indicating that the two peaks have good resolution.
  • the solvent peak does not affect the detection of peak 4.
  • the sample concentration is high.
  • the sample obtained by increasing the concentration of the sample solution and increasing the dilution factor after the degradation is completed (Dal-USP-RED-04 final sample concentration is about 3.94mg / ml), the baseline noise is small, so it is selected as the better Degradation conditions.
  • Chromatographic and CAD instrument conditions are the same as before.
  • Data processing Set appropriate integration parameters, such as Table 6, integrate the CAD drawing of the sample using the area normalization method to calculate the peak area percentage of each component.
  • the validation items include specificity, limit of quantification, precision (repeatability and intermediate precision), and durability.
  • a blank solution of nitrite degradation running on the HILIC-HPLC-CAD system, a solution of nitrite degradation product of the pharmacopoeia standard of sodium heparin, a solution of nitrite degradation product of the European pharmacopoeia standard of sodium heparin, and a degradation of the original research heparin sodium nitrite The product solution was injected into HILIC-UPLC-MS separately to check whether the blank sample would affect the detection. At the same time, the components after nitrous acid degradation were also characterized.
  • the mobile phases A, B, and sodium heparin sodium nitrite degradation product solution were all described in the previous Example 1 and the preparation of sodium heparin sodium nitrite degradation product solution (HILIC-HPLC-CAD).
  • the blank solution did not show chromatographic peaks that affected the detection of the sample.
  • the TIC of the mass spectrum is basically consistent with the RRT of each peak of the CAD chart. The results are shown in Table 13.
  • the structure is expressed as follows: Take U8,9,1 as an example, the first letter U represents a saturated uronic acid structure, the first digit 8 represents the octose structure, the second digit represents 9 sulfonic acid groups, and the third digit There is 1 acetyl group, and -ManR stands for anhydromannitol structure at the reducing end.
  • AMol is 2,5-anhydromannitol
  • Rc-GlcN is in-chain glucosamine
  • ANAC is N-acetylated glucosamine
  • I in the sequence is iduronic acid or glucuronic acid.
  • the peak area percentages are greater than LOQ (3.0%) (the limit of quantification determined by subsequent quantitation limit experiments, and the peak area percentage ⁇ 10% has the highest RSD It is 1.3%, the peak area percentage is greater than or equal to LOQ and the RSD of the peak of ⁇ 10.0% has a maximum RSD of 7.2%, so the method of the present invention passes the repeatability experiment.
  • the peak area percentages are greater than the LOQ (section (3) of the quantification limit described below) (3.0%), and the peak area percentage of the peaks ⁇ 10%
  • the maximum RSD is 2.2%
  • the peak area percentage ⁇ LOQ and the peak RSD of ⁇ 10.0% has a maximum RSD of 6.1%, which can be effectively analyzed.
  • the two experimenters had 12 needles of dalteparin sodium US standard nitrite degradation product solution. Except for the peak names of 1, 10 and 11, the peak area percentages were all greater than the LOQ (3.0%). With reference to the conclusion of the (3) quantitation limit section below, the peak RSD of peaks with a peak area percentage ⁇ 10% is 1.6%, and the peak RSD of a peak area percentage ⁇ LOQ and ⁇ 10.0% is 7.2%, which is effective. Analysis.
  • This method uses CAD area integration to calculate the percentage content of each component using the area percentage method, which is used for comparative research of component percentage content. Therefore, the minimum quantitative range of each component needs to be confirmed. According to the requirements of ICHQ2, the acceptable limit of quantification limit not only meets S / N ⁇ 10: 1, but also meets the peak area percentage RSD% ⁇ 10.0%.
  • the limit of quantification was determined by the S / N of each peak and the RSD% of the peak area percentage.
  • the signal-to-noise ratio S / N ⁇ 10: 1 is also satisfied, and the 6-pin repeatability peak area percentage RSD ⁇ 10.0%.
  • the smallest component peak name is 9, and the average peak area percentage of No. 9 is 3.0%, so In the case of a sample concentration of 100 mg / ml, the limit of quantification of the method is 3.0%.
  • the repetitive investigation uses a solution of dalteparin sodium solution of the US Pharmacopoeia standard nitrite degradation product as a 0-hour stability test sample, and it is left at room temperature for about 48 hours (the first injection time is counted as the first day, and 48 hours is the third Days) for testing, each time a sample is injected.
  • Quantitative analysis Calculate the relative standard deviation (RSD%) of the area percentage of each component peak in the CAD chart of the 12-pin dalteparin sodium USP standard nitrite degradation product solution, and use the first needle as the system applicability.
  • the experimental conditions were the same as before.
  • the present inventor believes that the method of the present invention can be used to determine the quality standard of heparin sodium by testing European original research and American original research samples, and the detection method is the same as that in the previous Example 2.
  • Table 30 shows the detection results of the degradation products of sodium heparin in 7 European original research and 11 American original research samples by using a high-performance liquid chromatography combined with an electric aerosol detector.
  • the other peaks are calculated with reference to the above method.
  • the analysis of degradation products by the method of the present invention using the original research of European and American dalteparin sodium can establish a set of quality standards for the analysis of dalteparin sodium degradation products and use them to judge the quality of dalteparin sodium samples.
  • the quality standards described in the present invention are merely exemplary, and those skilled in the art can make changes based on general knowledge, and these changed technical solutions fall within the scope of the application claimed in the present invention.

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Abstract

一种达肝素钠亚硝酸降解产物的分析方法，所述方法包括：将所述低分子肝素用亚硝酸完全降解得到降解产物，以及将所述降解产物经高效液相色谱(HPLC)联用电雾式检测器(CAD)进行检测。此方法无需衍生和脱乙酰步骤，降解产物制备时间短、分析成本低、检测准确率高。

Description

一种达肝素钠亚硝酸降解产物的分析方法及其应用 技术领域
本发明涉及一种达肝素钠亚硝酸降解产物的分析方法及其应用。
背景技术
达肝素钠是一种低分子肝素钠，由不同分子量的多糖链组成，其本身在紫外没有吸收峰。肝素酶，不论是肝素酶I、II或III，它们都是特异性裂解葡萄糖胺(1-4)己糖醛酸间相连的糖苷键，并在己糖醛酸的C4与C5间形成一个双键。肝素酶酶解达肝素钠后所得的酶解产物能够在紫外具有吸收峰，可通过紫外检测器对达肝素钠的酶解产物进行定量分析。
为了检测达肝素钠的质量，常用肝素酶酶解达肝素钠，然后用液相系统对降解产物进行分离，并通过与市售的二糖标准品比对保留时间进行定性，再通过各二糖标准品对检测器响应值和物质量的标准曲线进行定量。由于二糖标准品比较昂贵，所以后续发展出通过高效液相系统对降解产物进行分离，然后进一步对分离的产物通过质谱仪进行结构表征，从而实现对达肝素钠降解产物的定性分析，利用紫外检测器对分离出的各个组分进行定量分析，最终实现对达肝素钠质量的分析。
为了进一步提升对肝素酶酶解组分的定性/定量分析的方法，现有技术常在肝素酶酶解达肝素钠产物上柱前或后，进行衍生反应，例如：现有技术(CN201310711455.X)公开了在肝素酶酶解达肝素钠后，上液相系统柱子分离之前，对酶解产物进行衍生，并用反相色谱与高分辨质谱联用对衍生产物进行检测，以保证所有酶解产物均能进行定性分析。现有技术(CN201410123609.8)公开了在肝素酶酶解达肝素钠液相系统柱子分离酶解产物之后，进行荧光衍生反应，然后再进行检测。这既为无紫外吸收的低分子量肝素组分提供可检测的荧光基团，又可以实现高灵敏度的荧光检测，谱图中样品响应值更高；避免了衍生反应的副产物对色谱分离造成的干扰，提高分析的准确性和重复性。
另外，为了避免肝素酶降解的局限性，现有技术(CN201310695114.8)中还存在检测肝素二糖组成的方法，其主要是先将肝素脱乙酰化，得到脱乙酰肝素，然后将脱乙酰肝素用亚硝酸降解，得肝素二糖；将肝素二糖浓缩至干后溶于水，调pH值至碱性，加入吡唑啉酮类衍生试剂，反应完成后用氯仿萃取三次去除未反应的衍生试剂；用液相色谱法或液相色谱质谱联用法检测衍生后的二糖。
关于现有技术中使用脱乙酰化后然后进行脱乙酰肝素亚硝酸降解法，它能够降解达肝素钠中带有N-硫酸基的葡萄糖胺，但不能降解达肝素钠中带有的N-乙酰基的葡萄糖胺。也就是说，脱乙酰步骤使得达肝素钠中存在的含有N-乙酰基的葡萄糖胺信息消失，且该方法还必须含有衍生步骤，步骤繁琐。
关于现有技术中肝素酶降解法，首先肝素酶易失活且性质不稳定，其次肝素酶酶解达肝素钠需要的时间长(单酶解实验所需时间为48h)、步骤繁琐、检测成本高(需要使用质谱)。同时，用酶解降解达肝素钠时，葡萄糖醛酸(GlcA)及艾杜糖醛酸(IdoA)脱水后形成结构相同的脱水的糖醛酸，失去了达肝素钠中葡萄糖醛酸和艾杜糖醛酸的含量和结构信息，且肝素酶不能完全将达肝素钠降解为二糖。
在现有技术中，对达肝素钠降解产物的分析方法效率低下、成本高且对达肝素钠降解产物的分析方法所得结果不能完整体现被分析的达肝素钠的情况。因此，目前存在对分析时间短、分析成本低、检测准确率高且完整体现被分析的达肝素钠的情况的分析方法的需求。
发明内容
在本发明的第一方面，涉及一种达肝素钠亚硝酸降解产物的分析方法，所述方法包括下述步骤：(1)将所述达肝素钠与亚硝酸混合被完全降解；(2)将降解产物经高效液相色谱联用电雾式检测器进行检测。
在一些实施方式中，所述步骤(1)还包括在所述达肝素钠与亚硝酸混合被完全降解后，加入pH调节剂终止反应，然后加入硼氢化钠溶液还原，进一步加入一种或多种pH调节剂终止反应且调节pH至中性，得到最终降解产物。
在一些实施方式中，在所述步骤(2)中，所述高效液相色谱和电雾式检测器的条件是流速0.1-2mL/min；洗脱梯度为0-45min，10％-60％流动相A，90％-40％流动相B，其中所述流动相A是铵盐溶液且所述流动相B是乙腈。
在一些实施方式中，所述铵盐是醋酸铵或甲酸铵，其浓度是20-200mM，且所述流速为0.1-1mL/min，且所述洗脱梯度为0-45min，10％-45％流动相A，90％-55％流动相B。
在一些实施方式中，所述达肝素钠的质量与所述亚硝酸摩尔数的比值(g/mol)是17.5-405:1，优选为25-250:1、35-175:1、50-130:1。
在一些实施方式中，在与亚硝酸混合后所述达肝素钠的浓度在4mg/mL-150mg/mL。
在一些实施方式中，所述pH调节剂选自下述的一种或多种：碳酸钠、碳酸氢钠、氢氧化钠、盐酸、硫酸和冰醋酸。
在一些实施方式中，所述方法任选包括下述步骤：将步骤(2)所得检测结果与达肝素钠被亚硝酸完全降解后的产物经超高效液相色谱联用质谱进行检测的结果进行比对，最终获得步骤(1)所述达肝素钠经亚硝酸降解后的产物的定性和定量信息。
在本发明的第二方面，涉及所述分析方法在达肝素钠质量检测中的应用。
在一些实施方式中，所述应用包括使用所述分析方法对达肝素钠标准品(包括但不限于欧洲或美国标准品)和/或原研样品进行多次检测，根据检测结果建立质量标准。
附图说明
图1显示空白溶液和Dal-USP-RED-02叠图，上图为Dal-USP-RED-02，下图为BLANK-RED。
图2显示Dal-USP-RED-01和Dal-USP-RED-02叠图，上图为Dal-USP-RED-02，下图为Dal-USP-RED-01。
图3显示Dal-USP-RED-03和Dal-USP-RED-04叠图，上图为Dal-USP-RED-04，下图为Dal-USP-RED-03。
图4显示Dal-USP-RED-04至Dal-USP-RED-08叠图。
图5显示样品检测结果示图。
图6显示空白溶液、达肝素钠标准品和达肝素钠原研样品叠图，由下往上分别为专属性-BLANK-RED、专属性-Dal-USP-RED-01、专属性-Dal-EP-RED-01、专属性-原研样品-RED-01。
图7显示空白溶液、达肝素钠标准品和达肝素钠原研样品叠图，由下往上分别为BLANK-RED、Dal-USP-RED-01、Dal-EP-RED-01、原研样品-RED-01。
定义
如本文所使用的，术语“室温”是指25℃±5℃。同时，若没有具体指明实验温度，均为室温。
如本文所使用的，术语“约”是指该术语所修饰的数值的±20％，优选为±10％，更优选为±5％，因此本领域的普通技术人员能够清楚地根据所修饰的数值确定术语“约”的范围。
如本文所使用的，术语“达肝素钠标准品”是指包括但不限于美国药典和欧洲药典的达肝素钠标准品。
如本文所使用的，术语“达肝素钠原研样品”是指原研厂家辉瑞的达肝素钠上市产品，包括但不限于美国和欧洲的达肝素钠上市产品。
如本文所使用的，术语“峰面积百分比％”是指经高效液相色谱与电雾式检测器联用检测达肝素钠降解产物所获得的各个峰(例如，11个峰)或经高效液相色谱与质谱联用检测达肝素钠降解产物所获得的各个峰的峰面积占全部峰的峰面积的比值的百分比％。
如本文所使用的，术语“各个峰的相对保留时间的比值”是指经高效液相色谱与电雾式检测器联用检测达肝素钠降解产物所获得的各个峰的保留时间与与其中所述峰中峰面积最大的峰(例如，第4个峰)的保留时间的比值，或经高效液相色谱与质谱联用检测达肝素钠降解产物所获得的各个峰的保留时间与其中所述峰中峰面积最大的峰的保留时间的比值。
如本文所使用的，下述英文缩写具有本领域技术人员通常知晓的含义。具体地，UPLC:超高效液相色谱仪；HPLC:高效液相色谱仪；QTOF：飞行时间质谱仪；CAD：电雾式检测器；dp：表示糖链的聚合度；MS：质谱仪；TIC：总离子流图；RRT：相对保留时间。
发明详述
本发明所使用的方法无需衍生和脱乙酰步骤，降解产物制备时间短(约5小时)、分析成本低、检测准确率高且完整体现被分析的达肝素钠的情况。
CAD检测原理是溶质(分析物)液滴干燥后形成溶质颗粒，与带正电荷的氮气颗粒碰撞使 溶质颗粒带上正电，然后，带电颗粒将它们的电荷转移给收集器，最后用高灵敏度的静电检测计测出带电溶质的信号电流。由此产生的信号电流与溶质(分析物质)的质量含量成正比，与溶质(分析物质)本身化学结构无关。由于达肝素钠亚硝酸降解后的组分无紫外特征吸收，联用CAD检测可解决这一问题。因此，可以用于达肝素钠亚硝酸降解组分的定性、定量分析，包含了4个双糖峰、5个四糖峰和2个六糖峰，且对大于定量限的8个组分峰(对应表25中的2-9)进行了定量分析。
本发明使用方法采用HPLC联用检测器CAD完成，达肝素钠亚硝酸降解产物首先通过层析柱(HILIC柱(亲水相互作用色谱))分离后，进入CAD检测器检测，从而根据CAD检测结果获得达肝素钠亚硝酸降解物的组成信息。
本发明方法还可将经超高效液相色谱联用质谱进行检测的所述降解产物的检测结果与将经高效液相色谱联用电雾式检测器进行检测的所述降解产物的检测结果进行比对，使得以后仅对达肝素钠亚硝酸降解产物实现高效液相色谱联用电雾式检测器进行检测即可实现对所述降解产物的准确定性。
在本发明涉及一种达肝素钠亚硝酸降解产物的分析方法，所述方法包括下述步骤或由下述步骤组成：(1)将所述达肝素钠与亚硝酸混合被完全降解；(2)将降解产物经高效液相色谱(HPLC)联用电雾式检测器(CAD)进行检测。
在一些实施方式中，所述步骤(1)还包括在所述达肝素钠与亚硝酸混合被完全降解后，加入pH调节剂终止反应，然后加入硼氢化钠溶液还原，进一步加入一种或多种pH调节剂终止反应且调节pH至中性(约7.0)，得到最终降解产物。
进一步地，所述步骤(1)还包括在所述达肝素钠与亚硝酸(所述达肝素钠的质量与所述亚硝酸摩尔数的比值(g/mol)是17.5-405:1)混合被完全降解后，加入碳酸钠调节pH至7-9(优选约8.5)，终止反应，然后加入硼氢化钠溶液还原，3-5小时(优选约4小时)后加入冰醋酸调节pH至3-5(优选约4)终止反应，然后加入氢氧化钠调节pH至6.5-7.5(优选约7)，得到最终降解产物。
更进一步地，取105μL的100mg/mL的达肝素钠样品与200μL的pH1.5的亚硝酸溶液混合，静置反应30min以上，再在混合器上震荡2min，使得所述达肝素钠完全降解，加入80μL1M的碳酸钠溶液调节pH值至8.5，使反应终止，加入145μL新配的30mg/mL硼氢化钠溶液，室温还原至少4小时，加入冰醋酸120μL调pH值至4.0终止反应，再添加150μL 4M NaOH中和调pH至约7.0，总体积约为800μL，得到最终降解产物，取所述降解产物液体0.3ml于离心管中，加入0.7ml超纯水混匀，以0.22μm的滤膜过滤后待用。
在一些实施方式中，在所述步骤(2)中，所述高效液相色谱和电雾式检测器的条件是流速0.1-2mL/min，优选为0.1-1mL/min，更优选为0.3mL/min；洗脱梯度为0-45min，10％-60％流动相A，90％-40％流动相B；优选地，洗脱梯度为0-45min，10％-45％流动相A，90％-55％流动相B。
在一些实施方式中，所述洗脱梯度为0-10min，15％流动相A，85％流动相B；10-25min， 15％-33％流动相A，85％-67％流动相B；洗脱梯度为25-45min，33％-45％流动相A，67％-55％流动相B。进一步地，所述高效液相色谱的条件还任选地包括：洗脱梯度为45-50min，45％流动相A，55％流动相B；洗脱梯度为50-60min，45％-15％流动相A，55％-85％流动相B，其中所述流动相A是铵盐溶液且所述流动相B是乙腈。柱温为20-30℃，优选地25℃，且进样量1-10μL，优选地3μL。优选地，所述高效液相色谱的条件如表1所示，且所述电雾式检测器的条件是雾化温度约为35℃，根据雾化温度调节其它参数(例如，幂函数、采集频率、过滤值等)，优选地如表2所示。
在一些实施方式中，所述铵盐是醋酸铵或甲酸铵(在本发明的分析方法中醋酸铵或甲酸铵可以互相替换而不影响实验结果)，其浓度是20-200mM，优选100-150mM，更优选100mM和150mM，且所述流速为0.1-1mL/min，优选0.3mL/min。
在一些实施方式中，所述达肝素钠的质量与所述亚硝酸摩尔数的比值(g/mol)是17.5-405:1，优选为25-250:1、35-175:1、50-130:1。
在一些实施方式中，在与亚硝酸混合后，所述达肝素钠的浓度为4mg/mL-150mg/mL，优选为4.76mg/mL、20mg/mL、30mg/mL、34.4mg/mL、51.6mg/mL、55mg/mL、68.9mg/mL、70mg/mL、100mg/mL、103.3mg/mL、137.7mg/mL以及所述各数值点之间的范围，包括但不限于20mg/mL-100mg/mL、20mg/mL-70mg/mL、30mg/mL-55mg/mL等。
在一些实施方式中，所述pH调节剂选自下述的一种或多种：碳酸钠、碳酸氢钠、氢氧化钠、盐酸、硫酸和冰醋酸。
在一些实施方式中，所述达肝素钠液亚硝酸降解产物溶液在室温放置约1个月，优选约1周，更优选约3天内保持稳定。
在一些实施方式中，所述方法任选包括下述步骤：将步骤(2)所得检测结果与达肝素钠被亚硝酸完全降解后的产物经超高效液相色谱联用质谱进行检测的结果进行比对，最终获得步骤(1)所述达肝素钠经亚硝酸降解后的产物信息。
在一些实施方式中，所述检测的结果进行比对是将步骤(2)所得各个峰的相对保留时间的比值与经高效液相色谱联用电雾式检测器检测所述降解产物的各个峰的相对保留时间的比值进行对比。
在本发明的第二方面，涉及所述分析方法在达肝素钠质量检测中的应用。
在一些实施方式中，所述应用包括使用所述分析方法对达肝素钠标准品(例如，欧洲或美国标准品)和/或原研样品(例如，辉瑞公司在美国或欧洲上市产品)进行多次检测，根据检测结果建立质量标准。
在一些实施方式中，根据本发明所述检测结果建立的质量标准可以根据本领域的普通技术人员的一般知识进行变化，这些变化的技术方案均落入本发明要求保护的所述应用的范围之中。进一步地，所述质量标准可以通过如本申请实施例表30中各峰的峰面积百分比％的范围来建立。进一步地，所述质量标准是通过表31中的规则将表30中各峰的峰面积百分比％转换成表32中的质量标准1、2和3(其分别对应于质量标准强度高、中和低)。再进一 步地，所述质量标准如下表所述。
序号 1 2 3 4 5 6 7 8 9 10 11
质量标准1 ≤3.3 4.3-7.0 3.9-6.1 44.7-50.8 4.3-6.9 3.4-5.5 12.8-16.2 6.2-10.0 2.0-3.7 ≤3.0 ≤3.0
质量标准2 ≤3.6 3.8-7.6 3.5-6.6 43.5-52.1 3.8-7.5 3.0-6.0 12.2-16.9 5.5-10.9 1.8-4.0 ≤3.3 ≤3.3
质量标准3 ≤3.9 3.3-8.2 3.0-7.2 42.4-53.3 3.3-8.1 2.6-6.5 11.5-17.7 4.7-11.7 1.6-4.3 ≤3.5 ≤3.5
具体实施方式
以下结合附图和具体实施例，对本发明作进一步说明。应理解，以下实施例仅用于说明本发明而并非用于限定本发明权利要求中所涵盖的范围。
1.本发明所用的试剂的来源：
Figure PCTCN2019097283-appb-000001
2.本发明所用的试剂样品的制备：
(1)流动相的制备：根据实际用量可按溶液配比调整溶液的配制体积。
流动相A(20～200mM醋酸铵或甲酸铵溶液，优选150mM醋酸铵)的配制：称取醋酸铵或甲酸铵适量至1000mL容量瓶中，用超纯水定容至刻度，摇匀，转移至蓝盖瓶中，使用前超声10分钟。
流动相B：取适量乙腈至一洁净蓝盖瓶中，超声10分钟待用。
(2)肝素类亚硝酸降解产物溶液的制备：所用溶液根据实际用量可按溶液配比调整溶液的配制体积。
(3)0.5M H 2SO 4溶液的配制：取0.3mL浓硫酸缓慢加入少量水中，用超纯水定容到10mL，待冷却后，冷藏保存待用。
(4)0.5M Ba(NO 2) 2(亚硝酸钡)：称取1.2303g的一水合Ba(NO 2) 2，用超纯水溶解并定容到10mL，冷藏保存。
(5)pH 1.5亚硝酸(HONO)溶液的配制：在通风橱中，取冷藏的0.5M H 2SO 4和冷藏的0.5M Ba(NO 2) 2等体积直接混匀，11000rpm离心两分钟去除BaSO 4沉淀，取上清液，即为 pH 1.5的亚硝酸溶液，临用现配。
(6)1M碳酸钠溶液的配制：取1.06g碳酸钠用超纯水溶解，并定容到10mL，摇匀。
(7)30mg/mLNaBH 4溶液的配制：取30mgNaBH 4于洁净离心管中，加入1mL超纯水溶解，摇匀，临用现配。
(8)4M氢氧化钠溶液的配制：取1.6g氢氧化钠，加入10mL超纯水溶解，摇匀。
(9)100mg/mL达肝素钠样品溶液的配制：称取0.1g达肝素钠样品(原研样品或欧洲药典化学参考品或美国药典化学参考品)于洁净离心管中，加入1mL超纯水至完全溶解。
(10)达肝素钠亚硝酸降解产物溶液和空白溶液的制备：
达肝素钠亚硝酸降解产物溶液的示例性制备：分别取一定体积(例如，105μL)的达肝素钠样品(例如，100mg/mL)溶液于离心管中，加入200μL的pH1.5的HONO溶液，混合器上震荡均匀，静置反应30min以上，再在混合器上震荡2min，加入80μL1M的碳酸钠溶液调节pH值至8.5，使反应终止，加入145μL新配的30mg/mL硼氢化钠溶液，室温还原至少4小时，加入冰醋酸120μL调pH值至4.0终止反应，再添加150μL 4M NaOH中和调pH约7.0，总体积约为800μL，取降解液0.3ml于离心管中，加入0.7ml超纯水混匀，以0.22μm的滤膜过滤后待用。
空白溶液的制备：将达肝素钠样品溶液的制备中的105μL达肝素钠样品溶液更换为105μL超纯水，所用到的溶剂及整个制备过程同达肝素钠样品溶液的制备。
实施例1不同浓度达肝素钠亚硝酸降解产物的色谱分离及分析
1.高效液相色谱条件见表1
表1
Figure PCTCN2019097283-appb-000002
2.CAD的条件见表2
表2
条件内容 名称/指标
CAD Thermo Corona Veo RS
条件内容 名称/指标
幂函数 1.0
采集频率 10Hz
过滤值 5.0
雾化温度 35℃
采集时间 60min
3.不同浓度达肝素钠亚硝酸降解样品的制备方法见表3。
Figure PCTCN2019097283-appb-000003
Figure PCTCN2019097283-appb-000004
样品命名规则：USP和EP标准品(以Dal-USP/EP-RED-XX为开头命名)，其中批号-RED开头代表降解后经还原的样品，XX代表第几个样品；空白样品以Blank-RED为开头命名，其中Blank-RED开头代表空白降解后经还原的样品。
在表3中，其中Dal-USP-RED-01是现有技术中彻底降解的浓度；如果Dal-USP-RED-02的结果与Dal-USP-RED-01相同，说明降解时样品初始浓度增加5倍也能彻底降解；Dal-USP-RED-04为在Dal-USP-RED-02的基础上初始浓度再增加2.1倍，因Dal-USP-RED-03与Dal-USP-RED-02初始浓度相同，则Dal-USP-RED-03与Dal-USP-RED-04相比较可证明Dal-USP-RED-04是否彻底降解。
Dal-USP-RED-01与Dal-USP-RED-02最终样品浓度一致。Dal-USP-RED-04最终样品浓度是Dal-USP-RED-03的1.05倍。
Dal-USP-RED-01至Dal-USP-RED-08的达肝素钠的初始浓度分别为100mg/mL、100mg/mL、100mg/mL、100mg/mL、150mg/mL、200mg/mL、300mg/mL和400mg/mL。
将上述样品上机检测，实验结果如下：
如图1所示，空白溶液除溶剂峰外，未出现影响样品检测的色谱峰。
由图1得知，亚硝酸降解过程中使用的大量溶剂(比如碳酸钠、硼氢化钠、冰醋酸等)会在CAD出峰，这些试剂如果大量进入CAD检测器，会影响到检测器的寿命，因此只在条件摸索实验中采集0min～60min，以后实验均从16min开始采集。
如图2所示，Dal-USP-RED-01和Dal-USP-RED-02最终样品浓度一致，由于样品浓度低，除溶剂峰外仅出现3个色谱峰。
如图3所示，Dal-USP-RED-04最终样品浓度是Dal-USP-RED-03的1.05倍，除溶剂峰外出现11个色谱峰。
如图4所示，Dal-USP-RED-04至Dal-USP-RED-08各色谱峰分离良好。
结论：
在与亚硝酸混合后，各组达肝素钠的浓度如下(所述浓度＝达肝素钠初始质量(mg)/加入的亚硝酸与达肝素钠的体积(mL))：4.76mg/mL(Dal-USP-RED-01)、20mg/mL(Dal-USP-RED-02)、20mg/mL(Dal-USP-RED-03)、34.4mg/mL(Dal-USP-RED-04)、51.6mg/mL(Dal-USP-RED-05)、68.9mg/mL(Dal-USP-RED-06)、103.3mg/mL(Dal-USP-RED-07)、137.7mg/mL(Dal-USP-RED-08)。现有技术样品处理方面，降解试剂对样品检测干扰较大，发明人通过先将与亚硝酸混合后达肝素钠的浓度(第Dal-USP-RED-02至Dal-USP-RED-08组)提高至第Dal-USP-RED-01组的约4-30倍，再用超纯水稀释(稀释步骤)，从而降低降解试剂的干扰，还可适当提高样品检测浓度。假如通过上述实验，达肝素钠样品降解后的条件保持不变，和上表相同，亚硝酸降解时达肝素钠的浓度过高，导致最终上样时的样品中的盐浓度过高，从而可能影响HPLC中层析柱寿命，降低检测结果的准确性。
因此，如表3结合图1-4相应的结果显示，本发明的检测方法能够快速、低成本、准确的 实现对达肝素钠样品亚硝酸降解产物的分离和检测，进而可进一步实现达肝素钠样品的分析(质量维度)。
4.四针达肝素钠美国药典标准品亚硝酸降解产物溶液各组分峰面积以及两针之间各峰面积相对误差见表4、5。
相对误差公式：
Figure PCTCN2019097283-appb-000005
公式含义：A相对B的相对误差。
表4
Figure PCTCN2019097283-appb-000006
表5
Figure PCTCN2019097283-appb-000007
注：由于Dal-USP-RED-04最终样品浓度是Dal-USP-RED-03的1.05倍，峰面积与样品浓度成正比，计算两针之间峰面积相对误差需采用相同样品浓度，故Dal-USP-RED-03样品峰面积需乘与1.05。
结论：
空白溶液除溶剂峰外没有出现影响样品各色谱峰的检测；样品溶液中4号峰与溶剂峰的分离度为2.6，满足相邻色谱峰最小分离度不小于0.8要求，说明两峰分离度好，溶剂峰不影响4号峰的检测。
样品浓度高，通过提高样品溶液浓度，降解完成后增加稀释倍数的方式得到的样品(Dal-USP-RED-04最终样品浓度约为3.94mg/ml)，基线噪音小，所以选定为较优降解条件。
以下验证实验中达肝素钠标准品、达肝素钠原研样品均按较优条件降解。
实施例2不同浓度达肝素钠亚硝酸降解产物的色谱分离及分析
1.色谱和CAD仪器条件同前。
达肝素钠亚硝酸降解样品检测结果示意图如图5所示。
数据处理：设置合适的积分参数，例如表6，将样品的CAD图采用面积归一化法积分，计算各组分的峰面积百分比。
表6
Figure PCTCN2019097283-appb-000008
2.方法验证：验证项目包括专属性、定量限、精密度(重复性和中间精密度)和耐用性。
(1)专属性：
在HILIC-HPLC-CAD系统上进样分析亚硝酸降解空白溶液、达肝素钠美国药典标准品亚硝酸降解产物溶液、达肝素钠欧洲药典标准品亚硝酸降解产物溶液和原研达肝素钠亚硝酸降解产物溶液，考察空白样品是否会影响检测。
将在HILIC-HPLC-CAD系统上运行的亚硝酸降解空白溶液、达肝素钠美国药典标准品亚硝酸降解产物溶液、达肝素钠欧洲药典标准品亚硝酸降解产物溶液和原研达肝素钠亚硝酸降解产物溶液在HILIC-UPLC-MS分别进样，考察空白样品是否会影响检测，同时也对亚硝酸降解后的各组分进行定性。
具体方法：
达肝素钠亚硝酸降解产物溶液的配制见表7和表8。
表7
Figure PCTCN2019097283-appb-000009
表8
Figure PCTCN2019097283-appb-000010
Figure PCTCN2019097283-appb-000011
空白溶液的制备:将以超纯水代替100mg/ml样品溶液，其余不变。
在前述的仪器条件下，进样分析一针空白溶液、一针达肝素钠美国药典标准品亚硝酸降解产物溶液、一针EP达肝素钠标准品亚硝酸降解产物溶液、一针达肝素钠原研样品亚硝酸降解产物溶液。
实验结果如下：
如图6所示，空白溶液除溶剂峰外，未出现影响样品检测的色谱峰。
达肝素钠美国标准品、欧洲标准品和达肝素钠原研样品亚硝酸降解产物溶液CAD图相邻色谱峰的分离度见表9。
表9
Figure PCTCN2019097283-appb-000012
达肝素钠美国标准品、欧洲标准品和达肝素钠原研样品亚硝酸降解产物溶液CAD图(2号峰与3号峰)最小分离度、4号峰的不对称度和4号峰的塔板数见表10。
表10
Figure PCTCN2019097283-appb-000013
Figure PCTCN2019097283-appb-000014
结论：
空白中除溶剂峰外，未出现影响样品检测的色谱峰。
由表9得知USP达肝素钠标准品、EP达肝素钠标准品、达肝素钠原研样品亚硝酸降解产物溶液相邻色谱峰中最小分离度为2号峰与3号峰的分离度，均能满足分离度不小于0.8要求。
USP达肝素钠标准品、EP达肝素钠标准品、达肝素钠原研样品亚硝酸降解产物溶液4号峰理论塔板数、不对称度、相邻色谱峰(2号峰与3号峰)分离度均表现良好，因此本发明的方法满足专属性要求。
HILIC-UPLC-MS方法
流动相A、B、达肝素钠亚硝酸降解产物溶液均如先前的实施例1和达肝素钠亚硝酸降解产物溶液的制备所述(HILIC-HPLC-CAD)。
仪器条件见表11、12：
表11
Figure PCTCN2019097283-appb-000015
表12
条件内容 名称/指标
质谱仪 Waters Xevo G2-S QTOF
Mode Negative Resolution Mode【负离子分辨率模式】
Capillary Voltage 2.0kV
Sampling cone 25V
Source Offset Voltage 80V
Source Temperature 120℃
Desolvation Temperature 500℃
Cone Gas Flow 50L/Hr
Desolvation Gas Flow 800L/Hr
Start Mass 200
条件内容 名称/指标
End Mass 2000
Start Time 16min(可根据实际情况进行调整)
End Time 60min(可根据实际情况进行调整)
实验结果如下：
如图7所示，空白溶液除溶剂峰外，未出现影响样品检测的色谱峰。
质谱TIC图与CAD图各峰RRT基本一致，结果详见表13。
Figure PCTCN2019097283-appb-000016
Figure PCTCN2019097283-appb-000017
Figure PCTCN2019097283-appb-000018
Figure PCTCN2019097283-appb-000019
Figure PCTCN2019097283-appb-000020
注：结构表示方式如下：以U8,9,1为例，首位字母U表示饱和糖醛酸结构，首位数字8代表八糖结构，第二位数字代表有9个磺酸基，第三位数字代表有1个乙酰基，-ManR代表还原端为脱水甘露醇结构。AMol为2,5-脱水甘露醇；Rc-GlcN为链内缩环葡萄糖胺；ANAC为N乙酰化糖胺；序列中I表示为艾杜糖醛酸或葡萄糖醛酸。
由于乙酰基仅存在于葡萄糖胺残基中属于本领域的公知常识，因此如CN201310695114.8中的脱乙酰步骤使得达肝素钠中存在的含有N-乙酰基的葡萄糖胺信息消失，而如表14-15所见在本发明方法中能完整体现被分析的达肝素钠降解产物的情况。
实验结论：
空白中除溶剂峰外，未出现影响样品检测的色谱峰。
USP达肝素钠标准品、EP达肝素钠标准品、达肝素钠原研样品亚硝酸降解产物溶液4号峰理论塔板数最小值149851、不对称度1.2、相邻色谱峰(2号峰与3号峰)分离度为0.9，均满足分析要求。通过质谱解析对CAD图中各峰进行定性，得到其可能结构和可能序列。后续可仅通过CAD检测，实现对样品的定性和定量检测。
(2)精密度(重复性和中间精密度)：
重复性：
达肝素钠亚硝酸降解产物溶液的配制见表16、17。
表16
Figure PCTCN2019097283-appb-000021
Figure PCTCN2019097283-appb-000022
空白溶液的制备:将以超纯水代替100mg/ml样品溶液，其余不变。
仪器条件如前所述，进样分析1针空白样品、6针达肝素钠美国药典标准品亚硝酸降解产物溶液。
在同一天由同一个检验员进样分析6针达肝素钠美国药典参考标准品亚硝酸降解产物溶液各1针。定量分析：计算6针达肝素钠美国药典标准品亚硝酸降解产物溶液CAD图中各组分峰的面积百分比的相对标准偏差(RSD％)。
实验结果：
6针达肝素钠美国标准品亚硝酸降解各组分峰峰面积百分比见表18。
表18
Figure PCTCN2019097283-appb-000023
从上表可以看出，除峰名称为1、10、11外其余峰面积百分比均大于LOQ(3.0％)(后续定量限实验确定的定量限，且峰面积百分比≥10％的峰的RSD最大为1.3％，峰面积百分比≥LOQ且＜10.0％的峰的RSD最大为7.2％，因此本发明的方法通过了重复性实验。
中间精密度：
由不同的实验人员在不同的日期，按照重复性中的方法进行溶液配制。
达肝素钠亚硝酸降解产物溶液的配制见表19、20。
表19
溶液名称 达肝素钠标准品/样品名称 达肝素钠标准品/样品加入量 超纯水加入量 溶液浓度
Dal-USP-01 达肝素钠美国药典标准品 0.0205g 0.205ml 100mg/ml
Dal-USP-02 达肝素钠美国药典标准品 0.0204g 0.204ml 100mg/ml
Dal-USP-03 达肝素钠美国药典标准品 0.0201g 0.201ml 100mg/ml
Dal-USP-04 达肝素钠美国药典标准品 0.0206g 0.206ml 100mg/ml
Dal-USP-05 达肝素钠美国药典标准品 0.0202g 0.202ml 100mg/ml
Dal-USP-06 达肝素钠美国药典标准品 0.0201g 0.201ml 100mg/ml
Figure PCTCN2019097283-appb-000024
空白溶液的制备:将以超纯水代替100mg/ml样品溶液，其余不变。
流动相和仪器条件如前所述，进样分析1针空白样品，6针达肝素钠美国药典标准品亚硝酸降解产物溶液。定量分析：计算两个检验员检测的12针达肝素钠美国药典标准品亚硝酸降解产物溶液CAD图中各组分峰的面积百分比的相对标准偏差(RSD％)。
实验结果如下：
6针达肝素钠美国标准品亚硝酸降解各组分峰峰面积百分比见表21。
表21
Figure PCTCN2019097283-appb-000025
两名实验员12针达肝素钠美国标准品亚硝酸降解各组分峰的峰面积百分比见表22。
Figure PCTCN2019097283-appb-000026
从表21可以看出，除峰名称为1、10、11外其余峰面积百分比均大于LOQ(下述的定量限(3)部分)(3.0％)，且峰面积百分比≥10％的峰的RSD最大为2.2％，峰面积百分比≥LOQ且＜10.0％的峰的RSD最大为6.1％，可进行有效的分析。
从表22可以看出，两个实验员12针达肝素钠美国标准品亚硝酸降解产物溶液，除峰名称为1、10、11外其余峰面积百分比均大于LOQ(3.0％)，定量限结果参考下述(3)定量限部分的研究结论，且峰面积百分比≥10％的峰的RSD最大为1.6％，峰面积百分比≥LOQ且＜10.0％的峰的RSD最大为7.2％，可进行有效的分析。
以上，本发明的方法通过了中间精密度实验验证。
(3)定量限：
本方法是通过CAD图积分采用面积百分比法计算各组分的百分含量百分比，用于组分百分含量对比研究。因此需对各组分的最小定量范围进行确认。根据ICHQ2要求，定量限的可接受标准既满足S/N≥10：1，同时满足峰面积百分比RSD％≤10.0％。
流动相和仪器条件如前所述。
达肝素钠亚硝酸降解产物溶液的配制见表23、24。
表23
溶液名称 达肝素钠标准品/样品名称 达肝素钠标准品/样品加入量 超纯水加入量 溶液浓度
Dal-USP 达肝素钠美国药典标准品 0.0203g 0.203ml 100mg/ml
表24
Figure PCTCN2019097283-appb-000027
空白溶液的制备:将以超纯水代替100mg/ml样品溶液，其余不变。
进样分析1针空白样品，3针达肝素钠美国药典标准品亚硝酸降解产物溶液。
重复性实验6针达肝素钠美国药典标准品亚硝酸降解产物溶液每个组分RSD％及定量限3针信噪比(S/N)均符合上述定量限的可接受标准的最小峰面积百分比为定量限。
定量分析：分别计算重复性实验6针达肝素钠美国药典标准亚硝酸降解产物溶液CAD图谱峰面积百分比的相对标准偏差(RSD％)。
通过每个峰的S/N和峰面积百分比的RSD％来确定定量限。
3针达肝素钠美国标准品亚硝酸降解各组分峰峰面积百分比、信噪比见表25。
表25
Figure PCTCN2019097283-appb-000028
从上表可以看出同时满足信噪比S/N≥10:1，6针重复性峰面积百分比RSD≤10.0％最小组分峰名称为9，9号峰峰面积百分比平均值3.0％，因此在样品浓度为100mg/ml的情况下，该方法定量限为3.0％。
(4)耐用性：
流动相和仪器条件同前。
达肝素钠亚硝酸降解产物溶液的配制见表26、27。
表26
Figure PCTCN2019097283-appb-000029
Figure PCTCN2019097283-appb-000030
Figure PCTCN2019097283-appb-000031
从上表可以看出6针耐用性与6针重复性达肝素钠美国标准品溶液的检测结果除峰名称为1、10、11外其余峰面积百分比均大于LOQ(3.0％)，且峰面积百分比≥10％的峰的RSD最大为2.0％，峰面积百分比≥LOQ且＜10.0％的峰的RSD最大为9.6％，因此，本发明的方法通过耐用性实验检测，对不同序列号的色谱柱具有耐用性。
(5)溶液稳定性：
将重复性考察用达肝素钠液美国药典标准品亚硝酸降解产物溶液作为0小时的稳定性实验样品，置于室温放置约48小时(首次进样时间计为第1天，48小时为第3天)进行检测，每个时间点样品各进一针。
定量分析：计算12针达肝素钠美国药典标准品亚硝酸降解产物溶液CAD图中各组分峰的面积百分比的相对标准偏差(RSD％)，以第一针同时作为系统适用性。
实验条件同前。
12针达肝素钠美国标准品亚硝酸降解各组分峰峰面积百分比见表29。
Figure PCTCN2019097283-appb-000032
6针溶液稳定性与6针重复性达肝素钠美国标准品溶液从上表可以看出除峰名称为1、10、11外其余峰面积百分比均大于LOQ(3.0％)，且峰面积百分比≥10％的峰的RSD最大为2.5％，峰面积百分比≥LOQ且＜10.0％的峰的RSD最大为9.1％，因此，本发明的方法通过溶液稳定性实验，达肝素钠液美国药典标准品亚硝酸降解产物溶液放置3天溶液稳定，不影响样品检测结果。
实施例3
实验条件和实验试剂如实施例2所述。
本发明人认为通过对欧洲原研和美国原研样品的检测，可确定本发明的方法用于检测达肝素钠质量的标准，检测方法同前实施例2中的方法。对7个欧洲原研、11个美国原研样品批次的达肝素钠降解产物经高效液相色谱联用电雾式检测器的检测结果如表30所示。
表30
Figure PCTCN2019097283-appb-000033
可采用的质量标准：按照欧美原研检测结果，分高、中和低(对应表32中的质量标准1、2和3)三种，按以下表31的规则计算。例如，表30中第1个峰的峰面积百分比％是2.1-2.9％(低于定量限3.0％，关于定量限请参考表25)，那么参考表31中的规则最右侧一栏定量限>峰面积百分比％来计算质量标准强度(即，高：2.9×115％＝3.3；中：2.9×125％＝3.6；低： 2.9×135％＝3.9)，其对应于表32中的质量标准1(≤3.3％)、质量标准2(≤3.6％)和质量标准3(≤3.9％)。其它峰参考上述方法进行计算。
表31
Figure PCTCN2019097283-appb-000034
表32(峰面积百分比％)
序号 1 2 3 4 5 6 7 8 9 10 11
质量标准1 ≤3.3 4.3-7.0 3.9-6.1 44.7-50.8 4.3-6.9 3.4-5.5 12.8-16.2 6.2-10.0 2.0-3.7 ≤3.0 ≤3.0
质量标准2 ≤3.6 3.8-7.6 3.5-6.6 43.5-52.1 3.8-7.5 3.0-6.0 12.2-16.9 5.5-10.9 1.8-4.0 ≤3.3 ≤3.3
质量标准3 ≤3.9 3.3-8.2 3.0-7.2 42.4-53.3 3.3-8.1 2.6-6.5 11.5-17.7 4.7-11.7 1.6-4.3 ≤3.5 ≤3.5
由上表可知，使用欧洲和美国原研达肝素钠通过本发明方法进行降解产物的分析，可建立一套达肝素钠降解产物分析的质量标准并用于达肝素钠样品的质量判断。本发明所述质量标准仅仅是示例性的，本领域的普通技术人员可以根据一般知识进行变化，这些变化的技术方案均落入本发明要求保护的所述应用的范围之中。
除本文中描述的那些外，根据前述描述，本发明的多种修改对本领域技术人员而言会是显而易见的。这样的修改也意图落入所附权利要求书的范围内。本申请中所引用的各参考文献(包括所有专利、专利申请、期刊文章、书籍及任何其它公开)均以其整体援引加入本文。

Claims (10)

  1. 一种达肝素钠亚硝酸降解产物的分析方法，所述方法包括下述步骤：
    (1)将所述达肝素钠与亚硝酸混合被完全降解；
    (2)将降解产物经高效液相色谱联用电雾式检测器进行检测。
  2. 根据权利要求1所述的分析方法，其中所述步骤(1)还包括在所述达肝素钠与亚硝酸混合被完全降解后，加入pH调节剂终止反应，然后加入硼氢化钠溶液还原，进一步加入一种或多种pH调节剂终止反应且调节pH至中性，得到最终降解产物。
  3. 根据权利要求1或2所述的分析方法，其中在所述步骤(2)中，所述高效液相色谱和电雾式检测器的条件是流速0.1-2mL/min；洗脱梯度为0-45min，10％-60％流动相A，90％-40％流动相B，其中所述流动相A是铵盐溶液且所述流动相B是乙腈。
  4. 根据权利要求3所述的分析方法，其中所述铵盐是醋酸铵或甲酸铵，其浓度是20-200mM，所述流速为0.1-1mL/min，且所述洗脱梯度为0-45min，10％-45％流动相A，90％-55％流动相B。
  5. 根据权利要求1-4中任一项所述的分析方法，其中所述达肝素钠的质量与所述亚硝酸摩尔数的比值(g/mol)是17.5-405:1，优选为25-250:1、35-175:1、50-130:1。
  6. 根据权利要求1-5中任一项所述的分析方法，其中在与亚硝酸混合后所述达肝素钠的浓度在4mg/mL-150mg/mL。
  7. 根据权利要求2-6中任一项所述的分析方法，其中所述pH调节剂选自下述的一种或多种：碳酸钠、碳酸氢钠、氢氧化钠、盐酸、硫酸和冰醋酸。
  8. 根据权利要求1-7中任一项的分析方法，所述方法任选包括下述步骤：
    将步骤(2)所得检测结果与达肝素钠被亚硝酸完全降解后的产物经超高效液相色谱联用质谱进行检测的结果进行比对，最终获得步骤(1)所述达肝素钠经亚硝酸降解后的产物的定性和定量信息。
  9. 权利要求1-8中任一项的分析方法在达肝素钠质量检测中的应用。
  10. 根据权利要求9的应用，包括使用根据权利要求1-8中任一项的分析方法对达肝素钠标准品和/或原研样品进行多次检测，根据检测结果建立质量标准。
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