CN117805277A - Detection method of enoxaparin sodium injection macromolecular impurities - Google Patents
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- 238000001514 detection method Methods 0.000 title claims abstract description 23
- 229960005153 enoxaparin sodium Drugs 0.000 title claims abstract description 22
- CIJQTPFWFXOSEO-NDMITSJXSA-J tetrasodium;(2r,3r,4s)-2-[(2r,3s,4r,5r,6s)-5-acetamido-6-[(1r,2r,3r,4r)-4-[(2r,3s,4r,5r,6r)-5-acetamido-6-[(4r,5r,6r)-2-carboxylato-4,5-dihydroxy-6-[[(1r,3r,4r,5r)-3-hydroxy-4-(sulfonatoamino)-6,8-dioxabicyclo[3.2.1]octan-2-yl]oxy]oxan-3-yl]oxy-2-(hydroxy Chemical compound [Na+].[Na+].[Na+].[Na+].O([C@@H]1[C@@H](COS(O)(=O)=O)O[C@@H]([C@@H]([C@H]1O)NC(C)=O)O[C@@H]1C(C[C@H]([C@@H]([C@H]1O)O)O[C@@H]1[C@@H](CO)O[C@H](OC2C(O[C@@H](OC3[C@@H]([C@@H](NS([O-])(=O)=O)[C@@H]4OC[C@H]3O4)O)[C@H](O)[C@H]2O)C([O-])=O)[C@H](NC(C)=O)[C@H]1C)C([O-])=O)[C@@H]1OC(C([O-])=O)=C[C@H](O)[C@H]1O CIJQTPFWFXOSEO-NDMITSJXSA-J 0.000 title claims abstract description 22
- 239000012535 impurity Substances 0.000 title claims abstract description 21
- 238000002347 injection Methods 0.000 title claims abstract description 21
- 239000007924 injection Substances 0.000 title claims abstract description 21
- 239000000243 solution Substances 0.000 claims abstract description 56
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 12
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 8
- 238000000926 separation method Methods 0.000 claims abstract description 6
- RAQYHWNDOFAXAS-UHFFFAOYSA-M sodium dihydrogen phosphate perchloric acid Chemical compound P(=O)([O-])(O)O.Cl(=O)(=O)(=O)O.[Na+] RAQYHWNDOFAXAS-UHFFFAOYSA-M 0.000 claims abstract description 6
- SFRUVBWDAJNXSB-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;but-1-enylbenzene Chemical compound CCC=CC1=CC=CC=C1.C=CC1=CC=CC=C1C=C SFRUVBWDAJNXSB-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000000945 filler Substances 0.000 claims abstract description 4
- 125000000524 functional group Chemical group 0.000 claims abstract description 4
- 239000002952 polymeric resin Substances 0.000 claims abstract description 4
- 125000001453 quaternary ammonium group Chemical group 0.000 claims abstract description 4
- 229920003002 synthetic resin Polymers 0.000 claims abstract description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 27
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 18
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 claims description 18
- 229920000045 Dermatan sulfate Polymers 0.000 claims description 17
- AVJBPWGFOQAPRH-FWMKGIEWSA-L dermatan sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@H](OS([O-])(=O)=O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](C([O-])=O)O1 AVJBPWGFOQAPRH-FWMKGIEWSA-L 0.000 claims description 17
- 229940051593 dermatan sulfate Drugs 0.000 claims description 17
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 claims description 15
- 229920001287 Chondroitin sulfate Polymers 0.000 claims description 15
- 229940059329 chondroitin sulfate Drugs 0.000 claims description 15
- 239000013558 reference substance Substances 0.000 claims description 15
- 229920002567 Chondroitin Polymers 0.000 claims description 11
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 11
- DLGJWSVWTWEWBJ-HGGSSLSASA-N chondroitin Chemical compound CC(O)=N[C@@H]1[C@H](O)O[C@H](CO)[C@H](O)[C@@H]1OC1[C@H](O)[C@H](O)C=C(C(O)=O)O1 DLGJWSVWTWEWBJ-HGGSSLSASA-N 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 235000010288 sodium nitrite Nutrition 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 229920000669 heparin Polymers 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 7
- 239000012088 reference solution Substances 0.000 claims description 7
- 238000005303 weighing Methods 0.000 claims description 7
- 239000012488 sample solution Substances 0.000 claims description 6
- 229960002897 heparin Drugs 0.000 claims description 5
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 5
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 5
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 5
- BAZAXWOYCMUHIX-UHFFFAOYSA-M sodium perchlorate Chemical compound [Na+].[O-]Cl(=O)(=O)=O BAZAXWOYCMUHIX-UHFFFAOYSA-M 0.000 claims description 4
- 229910001488 sodium perchlorate Inorganic materials 0.000 claims description 4
- 239000012085 test solution Substances 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 3
- 239000000523 sample Substances 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 2
- 238000010812 external standard method Methods 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 238000003908 quality control method Methods 0.000 abstract description 4
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
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- 108010074860 Factor Xa Proteins 0.000 description 3
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- 230000000694 effects Effects 0.000 description 3
- 229960000610 enoxaparin Drugs 0.000 description 3
- 108010079356 FIIa Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 210000001772 blood platelet Anatomy 0.000 description 2
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 2
- 229960001008 heparin sodium Drugs 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000003055 low molecular weight heparin Substances 0.000 description 2
- 229940127215 low-molecular weight heparin Drugs 0.000 description 2
- 230000003285 pharmacodynamic effect Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 200000000007 Arterial disease Diseases 0.000 description 1
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000002262 Thromboplastin Human genes 0.000 description 1
- 108010000499 Thromboplastin Proteins 0.000 description 1
- 102100030951 Tissue factor pathway inhibitor Human genes 0.000 description 1
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- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 229940018872 dalteparin sodium Drugs 0.000 description 1
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- 238000002474 experimental method Methods 0.000 description 1
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- 108010013555 lipoprotein-associated coagulation inhibitor Proteins 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
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- 230000011164 ossification Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 208000004043 venous thromboembolism Diseases 0.000 description 1
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- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a method for determining macromolecular impurities of enoxaparin sodium injection. The detection method is high performance liquid chromatography, and the chromatographic conditions are as follows: adopting ethylvinylbenzene-divinylbenzene polymer resin with alkanol quaternary ammonium as a functional group as a filler; mobile phase a: adjusting the pH value of the solution to 2.0-4.0 by phosphoric acid; mobile phase B: sodium perchlorate-phosphate solution; the flow rate is 0.2-0.3ml per minute; the detection wavelength is 200-220nm; column temperature is 30-45 ℃; the sample injection volume is 10-50 μl. The detection method has the advantages of good separation degree, strong specificity, high sensitivity and high accuracy, and can be used for quality control in the production of enoxaparin sodium injection.
Description
Technical Field
The invention belongs to the technical field of drug impurity detection, and particularly relates to a method for determining macromolecular impurities of enoxaparin sodium injection.
Background
Low Molecular Weight Heparin (LMWHs) is derived from plain heparin (UFH) by chemical or enzymatic depolymerization reactions. LMWHs has reduced inhibition activity on FIIa compared to factor Xa (FXa), has a higher benefit/risk ratio than heparin, and has significant Pharmacokinetic (PK)/Pharmacodynamic (PD) properties. Enoxaparin is one of the widely used LMWHs, which is obtained by alkaline depolymerization of heparin benzyl esters of porcine intestinal mucosa. Enoxaparin has a higher FXa/FIIa activity ratio than UFH, more consistent tissue factor inhibitor (TFPI) release, weaker interaction with platelets, and less inhibition of bone formation. Enoxaparin has a higher and more consistent bioavailability after Subcutaneous (SC) administration, and a longer plasma half-life and weaker binding to plasma proteins than UFH. These properties can be converted to more reliable anticoagulation without laboratory monitoring, and safety and efficacy have been established in a wide range of arterial and venous thromboembolic diseases [7] . Enoxaparin sodium injection 10 months 1990 was marketed by Sanofi in the United kingdom under the trade nameThe current market specifications are: 2000AxaIU (20 mg)/0.2 ml, 4000AxaIU (40 mg)/0.4 ml, 6000AxaIU (60 mg)/0.6 ml, 8000AxaIU (80 mg)/0.8 ml, 10000AxaIU (100 mg)/1 ml; FDA approval was obtained at 3 months of 1993 and marketed in the United states by Sanofi-aventis U.S.LLC under the trade nameApproved for sale in japan, 4 months in 2008, trade name +.>The commercial specification was 2000AxaIU/0.2ml. In 2005, shenzhen Tiandao medical Co., 2006, hangzhou Jiuyuan Gene engineering Co., ltd, imitates the national market. At present, low molecular weight heparin represented by enoxaparin sodium, nadroparin sodium and dalteparin sodium has strong antithrombotic effect, small side effect on blood platelets and little bleedingThe characteristics of the medicine are gradually becoming the dominant variety of anticoagulants, and the medicine is being widely used by clinical departments.
The enoxaparin sodium injection mainly comprises enoxaparin sodium, the enoxaparin sodium is degraded and produced by taking the heparin sodium as a raw material, and macromolecular impurities contained in the heparin sodium can be transferred into the enoxaparin sodium injection, so that the macromolecular impurities in the enoxaparin sodium injection are studied.
The structural formulas of macromolecular impurities are respectively as follows:
the quality control of macromolecular impurities generated in the process is needed in raw materials and preparations, so that separation and analysis of enoxaparin sodium and macromolecular impurities are realized, and the method has important practical significance for quality control of enoxaparin sodium injection.
Disclosure of Invention
The invention aims to provide a method for separating and measuring macromolecular impurities in enoxaparin sodium injection by using a liquid chromatography, which can be used for quality control in the production of enoxaparin sodium injection.
The detection method of the enoxaparin sodium injection macromolecular impurities is high performance liquid chromatography, and the chromatographic conditions are as follows: adopting ethylvinylbenzene-divinylbenzene polymer resin with alkanol quaternary ammonium as a functional group as a filler; mobile phase a: adjusting the pH value of the solution to 2.0-4.0 by phosphoric acid; mobile phase B: sodium perchlorate-phosphate solution; the flow rate is 0.2-0.3ml per minute; the detection wavelength is 200-220nm; column temperature is 30-45 ℃; the sample injection volume is 10-50 μl; linear gradient elution was performed as follows:
| time (minutes) | Mobile phase a (%) | Mobile phase B (%) | Remarks |
| 0～10 | 75 | 25 | Isocratic of |
| 10～35 | 75→0 | 25→100 | Gradient of |
| 35～40 | 0 | 100 | Isocratic of |
| 40～55 | 75 | 25 | Rebalancing |
。
The preparation method of the sodium perchlorate-phosphate solution comprises the following steps: 100-180g of sodium perchlorate is taken, dissolved and diluted to 1000ml by 0.05 percent of sodium dihydrogen phosphate solution, and the pH value is regulated to 2.0-4.0 by phosphoric acid.
The detection method comprises the following specific steps:
(1) Preparing a test solution: precisely measuring 0.5ml of enoxaparin sodium injection, adding 0.25ml of 1mol/L hydrochloric acid solution and 0.05ml of 25% sodium nitrite solution, shaking and mixing uniformly, reacting for 40 minutes, and adding 0.2ml of 1mol/L sodium hydroxide solution to terminate the reaction;
(2) Preparing dermatan sulfate reference substance solution: precisely weighing 0.15ml of 2% dermatan sulfate reference substance, adding 1.35ml of water, and mixing; precisely weighing 0.5ml, adding 0.25ml of 1mol/L hydrochloric acid solution and 0.05ml of 25% sodium nitrite solution, shaking and mixing uniformly, reacting for 40 minutes, and adding 0.2ml of 1mol/L sodium hydroxide solution to terminate the reaction;
(3) Preparing chondroitin sulfate reference substance solution: precisely measuring 0.15ml of 2% chondroitin sulfate reference substance, adding 1.35ml of water, uniformly mixing, precisely measuring 0.5ml, adding 0.25ml of 1mol/L hydrochloric acid solution and 0.05ml of 25% sodium nitrite solution, shaking, uniformly mixing, reacting for 40 minutes, and adding 0.2ml of 1mol/L sodium hydroxide solution to terminate the reaction;
(4) Preparing a chondroitin polysulfate reference substance solution: precisely measuring 0.15ml of 2% chondroitin polysulfate reference substance, adding 1.35ml of water, uniformly mixing, precisely measuring 0.5ml, adding 0.25ml of 1mol/L hydrochloric acid solution and 0.05ml of 25% sodium nitrite solution, shaking, uniformly mixing, reacting for 40 minutes, and adding 0.2ml of 1mol/L sodium hydroxide solution to terminate the reaction;
(5) Precisely measuring the sample solution, the dermatan sulfate reference solution, the chondroitin sulfate reference solution and the chondroitin polysulfate reference solution, respectively injecting into a liquid chromatograph, and recording a chromatogram; the chromatogram of the test solution includes dermatan sulfate/chondroitin sulfate and chondroitin sulfate peaks, which are calculated by external standard method.
No heparin peak appears in the chromatogram of the sample solution, and the separation degree of dermatan sulfate/chondroitin sulfate and chondroitin polysulfate chromatographic peaks is not less than 3.0.
The detection method of the enoxaparin sodium injection macromolecular impurities is a detection method for detecting genotoxic impurities with good separation degree, strong specificity, high sensitivity and high accuracy.
Drawings
FIG. 1 is a linear relationship diagram of dermatan sulfate.
FIG. 2 is a graph of chondroitin sulfate linearity.
FIG. 3 is a graph of the linear relationship of chondroitin polysulfate.
Detailed Description
The method for measuring macromolecular impurities in enoxaparin sodium injection according to the present invention will be described in further detail by way of examples, but the scope of the claims of the present invention should not be construed as limited to the following examples, and all techniques based on the above description of the present invention are within the scope of the present invention.
Example 1
1. Instrument for measuring and controlling the intensity of light
Electronic balance (Metrele-tolidol), acidometer (Metrele-tolidol), high performance liquid chromatograph (Simerfei), low temperature circulating water tank (Shanghai Shunyu Hengping scientific instruments Co., ltd.), vortex meter (Orhaus), volumetric flask (100 ml, 25ml, 20ml and 10 ml), pipette (10 ml and 1 ml), measuring cylinder (1000 ml), beaker (1000 ml and 5000 ml)
2. Reagent(s)
Sodium dihydrogen phosphate (national medicine), phosphoric acid (merck), sodium perchlorate (national medicine)
3. Chromatographic conditions
Adopting ethylvinylbenzene-divinylbenzene polymer resin with alkanol quaternary ammonium as functional group as filler; linear gradient elution was performed using a 0.05% sodium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid) as mobile phase a, a sodium perchlorate-phosphate solution (140 g of sodium perchlorate, 0.05% sodium dihydrogen phosphate solution dissolved and diluted to 1000ml, pH adjusted to 3.0 with phosphoric acid) as mobile phase B, as follows; the flow rate is 0.25ml per minute; the detection wavelength is 205nm; column temperature 40 ℃; the sample volume was 25. Mu.l.
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) | Remarks |
| 0～10 | 75 | 25 | Isocratic of |
| 10～35 | 75→0 | 25→100 | Gradient of |
| 35～40 | 0 | 100 | Isocratic of |
| 40～55 | 75 | 25 | Rebalancing |
4. Selection of detection wavelength
Taking a proper amount of dermatan sulfate reference substance, precisely weighing, adding water for dissolving and quantitatively diluting to prepare a solution containing about 20mg per 1ml; 0.15ml of 2% dermatan sulfate reference substance, 1.35ml of water, uniformly mixing, precisely weighing 0.5ml, adding 0.25ml of 1mol/L hydrochloric acid solution and 0.05ml of 25% sodium nitrite solution, shaking and uniformly mixing, reacting for 40 minutes, and adding 0.2ml of 1mol/L sodium hydroxide solution to terminate the reaction to obtain the reference substance solution. Precisely 25. Mu.l was measured and injected into a liquid chromatograph.
As is clear from the dermatan sulfate spectrum, dermatan sulfate has a maximum absorption at 205nm, so 205nm is taken as the detection wavelength.
5. The analysis method is verified as follows
5.1 Property and System applicability test
5.1.1 preparation of solutions
5.1.2 measuring method precisely measuring 25 μl of each of blank solvent and reference solution, respectively injecting into liquid chromatograph, and recording chromatogram.
5.1.3 experimental results
Table 1 proprietary and system applicability results
5.1.4 experimental conclusion
The blank solution has no interference to the detection of various impurities, and the separation degree of the dermatan sulfate/chondroitin sulfate and the chondroitin polysulfate chromatographic peak in the chromatogram is not less than 3.0. The method has good specificity and system applicability.
5.2 quantitative limit and detection limit
5.2.1 preparation of solutions
5.2.2 assays
Precisely measuring 25 μl of each of the blank solution, the detection limit and the quantitative limit solution, and injecting into a liquid chromatograph.
5.2.3 experimental results
Table 2 detection limit verification results
Table 3 quantitative limit verification results
5.2.4 experimental conclusion
The detection limit concentration of each impurity is 3-8 mug/ml, which is about 0.003-0.01% of the concentration of the sample solution, and the method can effectively detect each impurity.
The quantitative limiting concentration of each impurity is 8-16 mug/ml, which is about 0.01-0.02% of the concentration of the sample solution, the quantitative limiting solution is continuously used for 6 needles, and the peak area RSD of each impurity is 4.8% (RSD is less than or equal to 20.0%).
5.3 Linear sum Range
5.3.1 preparation of solutions
5.3.2 assays
Precisely weighing the blank solution, the quantitative limiting solution and 25 mu l of each linear solution, and injecting the solutions into a liquid chromatograph.
5.3.3 experimental results
TABLE 4 results of Linear and Range experiments
5.3.4 experimental conclusion
Dermatan sulfate: in the concentration range of 0.015mg/ml to 4.096mg/ml, the linear equation is: y=26386.19x+1388.69, r is 0.9990 (r. Gtoreq.0.999), Y-intercept is 2% of the 100% concentration response value;
chondroitin polysulfate: in the concentration range of 0.021 mg/ml-4.124 mg/ml, the linear equation is: y=14047.78x+899.33, r is 0.9994 (r. Gtoreq.0.999), Y-intercept is 3% of the 100% concentration response value;
chondroitin sulfate: in the concentration range of 0.010mg/ml to 4.012mg/ml, the linear equation is: y=30829.47x+1997.36, r is 0.9990 (r. Gtoreq.0.999), Y-axis intercept is 3% of the 100% concentration response value.
Claims (4)
1. The detection method of the enoxaparin sodium injection macromolecular impurities is characterized by comprising the following steps of: adopting ethylvinylbenzene-divinylbenzene polymer resin with alkanol quaternary ammonium as a functional group as a filler; mobile phase a: adjusting the pH value of the solution to 2.0-4.0 by phosphoric acid; mobile phase B: sodium perchlorate-phosphate solution; the flow rate is 0.2-0.3ml per minute; the detection wavelength is 200-220nm; column temperature is 30-45 ℃; the sample injection volume is 10-50 μl; linear gradient elution was performed as follows:
。
2. The method according to claim 1, wherein the sodium perchlorate-phosphate solution is prepared by the following steps: taking 100-180g of sodium perchlorate, dissolving and diluting to 1000ml by using 0.05% sodium dihydrogen phosphate solution, and regulating the pH value to 2.0-4.0 by using phosphoric acid.
3. The detection method according to claim 1, characterized in that the specific steps of the detection method are:
(1) Preparing a test solution: precisely measuring 0.5ml of enoxaparin sodium injection, adding 0.25ml of 1mol/L hydrochloric acid solution and 0.05ml of 25% sodium nitrite solution, shaking and mixing uniformly, reacting for 40 minutes, and adding 0.2ml of 1mol/L sodium hydroxide solution to terminate the reaction;
(2) Preparing dermatan sulfate reference substance solution: precisely weighing 0.15ml of 2% dermatan sulfate reference substance, adding 1.35ml of water, and mixing; precisely weighing 0.5ml, adding 0.25ml of 1mol/L hydrochloric acid solution and 0.05ml of 25% sodium nitrite solution, shaking and mixing uniformly, reacting for 40 minutes, and adding 0.2ml of 1mol/L sodium hydroxide solution to terminate the reaction;
(3) Preparing chondroitin sulfate reference substance solution: precisely measuring 0.15ml of 2% chondroitin sulfate reference substance, adding 1.35ml of water, uniformly mixing, precisely measuring 0.5ml, adding 0.25ml of 1mol/L hydrochloric acid solution and 0.05ml of 25% sodium nitrite solution, shaking, uniformly mixing, reacting for 40 minutes, and adding 0.2ml of 1mol/L sodium hydroxide solution to terminate the reaction;
(4) Preparing a chondroitin polysulfate reference substance solution: precisely measuring 0.15ml of 2% chondroitin polysulfate reference substance, adding 1.35ml of water, uniformly mixing, precisely measuring 0.5ml, adding 0.25ml of 1mol/L hydrochloric acid solution and 0.05ml of 25% sodium nitrite solution, shaking, uniformly mixing, reacting for 40 minutes, and adding 0.2ml of 1mol/L sodium hydroxide solution to terminate the reaction;
(5) Precisely measuring the sample solution, the dermatan sulfate reference solution, the chondroitin sulfate reference solution and the chondroitin polysulfate reference solution, respectively injecting into a liquid chromatograph, and recording a chromatogram; the chromatogram of the test solution includes dermatan sulfate/chondroitin sulfate and chondroitin sulfate peaks, which are calculated by external standard method.
4. The method according to claim 1, wherein no heparin peak appears in the chromatogram of the sample solution, and the separation degree of dermatan sulfate/chondroitin sulfate from the chondroitin polysulfate chromatographic peak is not less than 3.0.
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