CN110907571B - Method for detecting and analyzing heparan sulfate impurities in heparin sodium - Google Patents
Method for detecting and analyzing heparan sulfate impurities in heparin sodium Download PDFInfo
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Abstract
The invention discloses a method for detecting and analyzing heparan sulfate impurities in heparin sodium, which comprises the steps of adopting a liquid chromatograph and an SAX strong anion exchange chromatographic column, taking sodium dihydrogen phosphate solution as a mobile phase A, taking a mobile phase B as a mobile phase A containing sodium perchlorate, and carrying out detection and analysis by gradient elution. The invention can accurately analyze the relative content of the heparin sodium and the heparan sulfate by simple liquid chromatography measurement, and can quickly and accurately perform qualitative and content analysis on the heparan sulfate impurities in the heparin sodium.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a method for detecting and analyzing heparan sulfate impurities in heparin sodium.
Background
Heparin is an important polysaccharide drug, is mainly used as an anticoagulant drug clinically for treating thromboembolic diseases caused by formation of deep venous thrombosis, and also has multiple biological effects of regulating blood fat concentration, resisting inflammation, resisting allergy and the like.
Modern heparin is mainly extracted from porcine small intestinal mucosa, and because heparin does not exist alone, the heparin and other glycosaminoglycans coexist, substances such as Dermatan Sulfate (DS), Heparan Sulfate (HS) and the like exist together in the production process, and the substances are linear long-chain macromolecular polysaccharides consisting of a type of repeated disaccharide structural units, and the analysis and detection of the substances are very important for the production and quality detection of heparin.
Heparin and heparan sulfate are mainly composed of repeated uronic acid and glucosamine through 1,4 glycosidic bond, except that heparin is composed of iduronic acid and heparan sulfate is composed of glucuronic acid, and the sulfation degree of heparan sulfate is lower than that of heparin, as shown in the above formula R and R2May be substituted by H or SO3-substituted, R3Can be H, acetyl (acetyl) or SO3-substitution; as for dermatan sulfate, it is mainly composed of repetitive uronic acids and galactosamine linked by 1,3 glycosidic bonds.
These glycosaminoglycans have very similar and complex structures, and most of them coexist in the same organism and even on the same sugar chain, so that the separation is very difficult, and it is very difficult to obtain a completely single kind of glycosaminoglycans. In order to ensure the safety of heparin administration, the purity of heparin must be improved, and effective confirmation of whether the removal of these structurally similar glycosaminoglycan impurities is complete is required.
At present, for separation and analysis of glycosaminoglycan polysaccharide, methods such as Nuclear Magnetic Resonance (NMR), Capillary Electrophoresis (CE), High Performance Liquid Chromatography (HPLC), mass spectrum combination and the like are combined, and the high performance liquid chromatography has the most research value due to economic factors such as industrial production cost and the like and the consideration of laboratory feasibility. In 2009, the company dean, usa, after improving and optimizing the anion exchange HPLC method, introduced a new strong anion exchange HPLC detection method (Thermo Fisher Scientific inc. Determination of over-surface chromatography and chromatography in the presence of sodium using-exchange chromatography with uv detection [ EB/OL ]. 2010 [ 2012-05-11 ]) and adopted by the USP33-NF 28; the european pharmacopoeia commission also introduced an improved strong anion exchange method in EP 7.0, but required sample pretreatment for nitrite degradation of sodium heparin. The chinese pharmacopoeia 2010 edition introduced a strong anion exchange method consistent with USP33-NF 28. However, the above-mentioned strong anion exchange chromatography detection method is not perfect, and has the defect of serious baseline drift, even the weak anion exchange method reported in Hashii NHashii N, Kawasaki N, Itoh S, et al, Heparin identification and purity test for OSCS in Heparin sodium and Heparin calcium by way of exchange-exchange high-performance chromatographic, Biologicals 539, 2010, 38 (5) has the same problems. In addition, although the above methods can separate and measure the relative contents of HP and DS and the relative content of contaminant chondroitin persulfate (OSCS), none of them can separate and accurately measure HS which may coexist in the same way.
Therefore, there is a need for a liquid phase method capable of simultaneously and effectively separating and detecting various glycosaminoglycans, and confirming the composition of a glycosaminoglycan drug, so as to achieve the purpose of guiding industrial production and drug composition analysis and ensuring drug safety.
Disclosure of Invention
The invention aims to provide a method for detecting and analyzing heparan sulfate impurities in heparin sodium, which adopts a high performance liquid chromatograph to carry out detection and analysis, can accurately separate and measure the relative component contents of the heparin sodium and the heparan sulfate, and improves the sensitivity and the accuracy of detecting the heparan sulfate impurities in a heparin sodium product.
The purpose of the invention is realized by the following technical scheme:
a method for detecting and analyzing heparan sulfate impurities in heparin sodium comprises the following steps of detecting and analyzing by using a high performance liquid chromatograph under the following liquid chromatography conditions:
adopting SAX strong anion exchange chromatographic column; the flow rate of the mobile phase is 0.1-0.3ml/min, the detection wavelength is 200-215 nm, and the sample introduction amount of the sample to be detected is 2-20 mul; the mobile phase A is 2-3mM sodium dihydrogen phosphate solution, the mobile phase B is mobile phase A containing 1-2M sodium perchlorate, and the pH of the mobile phase A, B is adjusted to 2.0-4.0 by phosphoric acid; and (3) isocratic elution of the initial mobile phase A: the mobile phase B = 80: 20 for 2-5 minutes, then the continuous increase of the mobile phase B is gradually increased from 20% to 50%, the continuous decrease of the mobile phase A is gradually decreased from 80% to 50% for 20-30 minutes, then the continuous increase of the mobile phase B is gradually increased from 50% to 80%, the continuous decrease of the mobile phase A is gradually decreased from 50% to 20% for 15-20 minutes, after the ratio is kept for continuous elution for 5-10 minutes, the mobile phase A is rapidly adjusted to be increased from 20% to 80% in 1-2 minutes, the mobile phase B is decreased from 80% to 20%, the elution is continuously performed for 30-50 minutes, and the detection is finished.
The liquid chromatography detection method provided by the invention can be used for accurately separating and detecting the relative content of heparin sodium (HP) and Heparan Sulfate (HS).
Drawings
FIG. 1 shows a liquid chromatogram of water (blank) in example 1;
FIG. 2 is a liquid chromatogram of heparan sulfate in example 1;
FIG. 3 is a liquid chromatogram of heparin sodium in example 1;
FIG. 4 is a liquid chromatogram of dermatan sulfate of example 1;
FIG. 5 is a liquid chromatogram of the mixed solution in example 1;
FIG. 6 is a liquid chromatogram of solution 1 in example 2;
FIG. 7 is a liquid chromatogram of solution 2 of example 2;
FIG. 8 is a liquid chromatogram of solution 3 of example 2;
FIG. 9 shows a liquid chromatogram of solution 4 in example 2;
FIG. 10 is a liquid chromatogram of solution 5 of example 2;
FIG. 11 is a liquid chromatogram of solution 6 of example 2;
FIG. 12 is a liquid chromatogram of solution 7 of example 2;
FIG. 13 shows a liquid chromatogram of a solution T1-T6 in example 3;
FIG. 14 is a high performance liquid chromatogram obtained in comparative example 1;
FIG. 15 is a high performance liquid chromatogram obtained in comparative example 2;
FIG. 16 is a high performance liquid chromatogram obtained in comparative example 3;
FIG. 17 is a high performance liquid chromatogram obtained in comparative example 4.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
The instruments used in the examples include: SHIMADZU (Shimadzu)®LC-20AD liquid chromatograph;
heparan sulfate standard (GLYCOSCI batch: ZZS 18040905);
heparin sodium standard (China food and drug testing research institute batch No. 140787-;
dermatan sulfate standard (China food and drug testing research institute batch No. 140788-;
chondroitin sulfate standard (China food and drug testing research institute batch number: 140792-2017);
a oversulfated chondroitin sulfate standard substance (China food and drug testing research institute batch No. 140789-;
sodium dihydrogen phosphate (national pharmaceutical group chemical Co., Ltd., batch No. 20150228);
sodium perchlorate (national drug group chemical Co., Ltd., batch No. 20170306);
phosphoric acid (Jiangsu Qiangsheng functional chemistry GmbH, batch No. 20140203).
Example 1 verification of separation Effect
1. Respectively and precisely weighing 10mg of each of heparan sulfate standard (HS), heparin sodium standard (HP) and dermatan sulfate standard (DS) in 3 centrifuge tubes of 5.0ml (or 4.0 ml), placing in the centrifuge tubes, respectively adding 2.0ml of water, dissolving and mixing uniformly, and marking as HS, HP and DS; the solution was filtered through a 0.22 μm filter.
2. Taking 1 centrifuge tube of 5.0ml (or 4.0 ml), precisely weighing heparan sulfate standard, heparin sodium standard, dermatan sulfate standard, chondroitin sulfate standard, and oversulfated chondroitin sulfate standard 10mg each, placing into centrifuge tube, adding water of 2.0ml, dissolving, and mixing uniformly, and labeling as HH; the solution was filtered through a 0.22 μm filter.
3. Taking 5 newly opened clean liquid chromatography sample bottles, and respectively adding water (blank) filtered by a 0.22 mu m filter membrane, a heparan sulfate standard (HS) solution, a heparin sodium standard (HP) solution, a dermatan sulfate standard (DS) solution and a multi-component mixed solution HH; and (4) putting the packaged sample introduction bottle into a liquid chromatograph, and carrying out sample introduction and measurement.
4. The liquid chromatography conditions were:
a chromatographic column: welch Ultimate®XB-SAX（4.6×250mm，3μm）；
Flow rate: 0.15 ml/min;
column temperature: 40 ℃;
detection wavelength of the ultraviolet detector: 202 nm;
sample introduction amount: 10 mul;
mobile phase A: 2.5mM sodium dihydrogen phosphate buffer, pH 3.0; mobile phase B: 2M sodium perchlorate is dissolved in a mobile phase A;
the specific preparation method of the mobile phase comprises the following steps: phase A: accurately weighing 0.600g of sodium dihydrogen phosphate, dissolving in 2L of pure water to obtain 2.5mM sodium dihydrogen phosphate buffer solution, wherein the pH of the phosphoric acid adjusting solution is = 3.0; phase B: 224.736g of sodium perchlorate monohydrate were weighed out and dissolved in 800mL of mobile phase A.
The mobile phase A, B was run according to the following gradient program:
5. the experimental results are as follows:
the corresponding chromatograms are shown in figures 1-5, and the comparison of the chromatograms can lead to the conclusion that the heparan sulfate and the heparin sodium can be effectively separated and the interference of the dermatan sulfate can be removed when the analysis method is used for detection.
EXAMPLE 2 determination of the relative amounts of heparan sulfate and heparin sodium
1. Taking 2 10.0ml (or 8.0 ml) centrifuge tubes, respectively and precisely weighing heparan sulfate standard substance and heparin sodium standard substance 20mg, respectively placing into the centrifuge tubes, adding 4.0ml of water, dissolving and mixing uniformly.
2. Taking 7 2.0ml centrifuge tubes, marking as 1-7, adding 0.01ml, 0.05ml, 0.10ml, 0.50ml, 0.90ml, 0.95ml and 0.99ml of the heparan sulfate standard solution in the step 1 into the 7 centrifuge tubes in sequence, and adding 0.99ml, 0.95ml, 0.90ml, 0.50ml, 0.10ml, 0.05ml and 0.01ml of the heparin sodium standard solution in the step 1 into the 7 centrifuge tubes in the same sequence. Mixing the solutions in 7 centrifugal tubes, filtering with 0.22 μm filter membrane, loading into sample bottle, and detecting with liquid chromatograph.
3. The liquid chromatography conditions were:
a chromatographic column: welch Ultimate®XB-SAX（4.6×250mm，3μm）；
Flow rate: 0.15 ml/min;
column temperature: 40 ℃;
detection wavelength of the ultraviolet detector: 202 nm;
mobile phase A: 2.5mM sodium dihydrogen phosphate buffer, pH 3.0; mobile phase B: 2M sodium perchlorate is dissolved in a mobile phase A;
the mobile phase was prepared in the same manner as in example 1.
The mobile phase A, B was run according to the following gradient program:
analyzing according to the above conditions, and performing peak area integration on each chromatogram (shown in figures 6-12) to obtain peak area ratio of heparan sulfate and heparin sodium, which is the relative content ratio of heparan sulfate and heparin sodium, wherein the specific experimental data is as follows:
as can be seen from the data in the table above, the peak area ratio method can be used to accurately detect the relative content of heparan sulfate and heparin sodium.
Example 3 heparin product production intermediate detection
1. Taking heparin production intermediate products (Hubei Yinorui biological pharmacy Co., Ltd.), preparing the intermediate products into 5mg/ml respectively, filtering the intermediate products through a 0.22-micron filter membrane, wherein the labels of the filter membrane are T1-T6 respectively corresponding to crude heparin, enzymolysis precipitates, primary oxidation precipitates, secondary oxidation precipitates and tertiary oxidation precipitates in the heparin production process, and the final products are used as test solution.
2. Weighing dermatan sulfate, heparan sulfate and 5mg heparin sodium standard, adding 1ml pure water to dissolve and shake, respectively marking DS, HS and HP, and filtering with 0.22 μm filter membrane to obtain reference solution.
3. The liquid chromatography conditions were:
a chromatographic column: welch Ultimate®XB-SAX（4.6×250mm，3μm）；
Flow rate: 0.15 ml/min;
column temperature: 40 ℃;
detection wavelength of the ultraviolet detector: 202 nm;
the sample amount is 10 mul;
mobile phase A: 2.5mM sodium dihydrogen phosphate buffer, pH 3.0; mobile phase B: 2M sodium perchlorate monohydrate dissolved in phase A;
the mobile phase was prepared in the same manner as in example 1.
The mobile phase A, B was run according to the following gradient program:
the analysis under the above conditions, by comparing peak area fractions of peaks corresponding to the chromatogram (fig. 13), the mass percentage content of heparan sulfate and heparin sodium in each intermediate product can be obtained, and the results are shown in the following table:
therefore, the method can see the purpose and effect of impurities removed in each step in the heparin production process, can accurately see the mass percentage of the main components heparin sodium and the impurity components in each step, can better analyze products in each stage in the heparin production process, and can provide a guiding function for the industrial production of heparin.
Comparative example 1 comparison of the results of the measurement of the method of the present invention with those of the conventional methods
1. Weighing dermatan sulfate, heparan sulfate and 5mg heparin standard, adding 1ml of pure water, dissolving, shaking, marking DS, HS and HP, and filtering with 0.22 μm filter membrane to obtain control solution.
2. The sample of example 3 was used as a test solution, and the specific preparation method was the same as in example 3.
3. The method adopts the literature of anion exchange HPLC method for accurately determining chondroitin polysulfate and dermatan sulfate in heparin sodium (Wenkwell jin et al, J. Pharmacology analysis, Vol. 33 No. 4 of 2013, p. 642-647) for detection.
The specific chromatographic conditions were as follows:
a chromatographic column: TSK-GEL DEAE-5 PW;
mobile phase A: 2.5mM sodium dihydrogen phosphate, pH 3.0 adjusted with phosphoric acid;
mobile phase B: 2.5mM sodium dihydrogen phosphate and 1M sodium perchlorate, and adjusting the pH to 3.0 by phosphoric acid;
flow rate: 0.2 ml/min;
column temperature: 35 ℃;
wavelength: 202 nm;
elution gradient:
the analysis result is shown in figure 14, therefore, the method can separate HP from HS and DS, but HS and DS flow out simultaneously, and the resolution of the detection method provided by the invention is obviously higher than that of the method disclosed by the prior document after comparing with the separation map obtained in the example 1.
Comparative example 2 comparison of measurement results of the method of the present invention and conventional methods
1. Weighing dermatan sulfate, heparan sulfate and 5mg heparin standard, adding 1ml of pure water, dissolving, shaking, marking DS, HS and HP, and filtering with 0.22 μm filter membrane to obtain control solution.
2. The sample of example 3 was used as a test solution, and the specific preparation method was the same as in example 3.
3. The detection was carried out using the chromatographic conditions disclosed in the documents D.A Keire, et al, Assay of porous organic substrates or native imprints levels in helium by H NMR, SAX-HPLC, and antimicrobial aggregation time improvement, J Pharm Biomed Anal,2010, 52(2010): 656.
The specific chromatographic conditions were as follows:
a chromatographic column: dionex IonPac AS 11-HC;
mobile phase A: ultrapure water;
mobile phase B: 2.5M NaCl (Aladdin, batch: F1620006) containing 20mM Tris (VETEC, batch: WXBC 1913V) was adjusted to pH 3.0 with phosphoric acid;
flow rate: 0.3 ml/min;
column temperature: 35 ℃;
wavelength: 215 nm;
elution gradient:
the analysis result is shown in figure 15, therefore, in the method, HS and DS are eluted simultaneously in a broad peak, and the resolution of the detection method provided by the invention is obviously higher than that of the method disclosed by the prior document after being compared with the separation spectrum obtained in the example 1.
Comparative example 3 comparison of measurement results of different columns
1. Weighing dermatan sulfate, heparan sulfate and 5mg heparin standard, adding 1ml of pure water, dissolving, shaking, marking DS, HS and HP, and filtering with 0.22 μm filter membrane to obtain control solution.
2. The sample of example 3 was used as a test solution, and the specific preparation method was the same as in example 3.
3. The chromatographic conditions were checked with reference to the chromatographic conditions of example 1, using the column types: dionex IonPac AS 11-HC.
The results of the analysis are shown in FIG. 16, from which it can be seen that HS and DS cannot be separated according to the chromatographic conditions provided by the present invention using the column of this comparative example.
Comparative example 4 comparison of measurement results of different chromatographic conditions
1. Heparan sulfate and heparin standard 10mg are weighed, dissolved by adding 2ml of pure water and shaken up, marked with HS and HP respectively and filtered through a 0.22 mu m filter membrane to be used as reference substance solution.
2. The sample of example 3 was used as a test solution, and the specific preparation method was the same as in example 3.
3. The chromatographic conditions were examined with reference to the chromatographic conditions of comparative example 2, using the types of chromatographic columns: welch Ultimate®XB-SAX(4.6×250mm,3μm）。
The results of the analysis are shown in FIG. 17, from which it can be seen that heparan sulfate and heparin could not be separated according to the chromatographic conditions of comparative example 2 using the column of the present invention.
Claims (3)
1. A method for detecting and analyzing impurities of heparan sulfate and dermatan sulfate in heparin sodium is characterized by comprising the following steps: the method adopts a high performance liquid chromatograph to carry out detection and analysis, and the conditions of the liquid chromatograph are as follows:
using Welch Ultimate®An XB-SAX strong anion exchange chromatography column;
the flow rate of the mobile phase is 0.1-0.3 ml/min;
the detection wavelength is 200nm-215 nm;
the sample amount of the sample to be detected is 2-20 mul;
the mobile phase A is 2-3mM sodium dihydrogen phosphate solution, the mobile phase B is mobile phase A containing 1-2M sodium perchlorate, and the pH of the mobile phase A, B is adjusted to 2.0-4.0 by phosphoric acid;
and (3) isocratic elution of the initial mobile phase A: the mobile phase B = 80: 20 for 2-5 minutes, then the continuous increase of the mobile phase B is gradually increased from 20% to 50%, the continuous decrease of the mobile phase A is gradually decreased from 80% to 50% for 20-30 minutes, then the continuous increase of the mobile phase B is gradually increased from 50% to 80%, the continuous decrease of the mobile phase A is gradually decreased from 50% to 20% for 15-20 minutes, after the ratio is kept for continuous elution for 5-10 minutes, the mobile phase A is rapidly adjusted to be increased from 20% to 80% in 1-2 minutes, the mobile phase B is decreased from 80% to 20%, and the elution is continuously carried out for 30-50 minutes.
2. The detection analysis method according to claim 1, characterized in that: the Welch Ultimate®The column length of the XB-SAX strong anion exchange chromatographic column is 150-250mm, and the column temperature is 30-50 ℃.
3. The detection analysis method according to claim 1, characterized in that: the Welch Ultimate®The matrix of the XB-SAX strong anion exchange chromatographic column is porous spherical silica gel, and the pore diameter is 120-300A; the filler is silica gel matrix bonded with quaternary ammonium group, and the particle size of the filler is 3 μm.
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| EP3004181A1 (en) * | 2013-05-27 | 2016-04-13 | Agency For Science, Technology And Research | Heparan sulphate |
| CN109596742A (en) * | 2018-12-25 | 2019-04-09 | 深圳市格利科生物科技有限公司 | A method of Contents of Main Components in detection Heparan sulfate finished product |
| CN110172107A (en) * | 2019-05-28 | 2019-08-27 | 苏州鸿洋医药科技有限公司 | The separation method of Heparan sulfate and dermatan sulfate in a kind of heparan |
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| EP3004181A1 (en) * | 2013-05-27 | 2016-04-13 | Agency For Science, Technology And Research | Heparan sulphate |
| CN109596742A (en) * | 2018-12-25 | 2019-04-09 | 深圳市格利科生物科技有限公司 | A method of Contents of Main Components in detection Heparan sulfate finished product |
| CN110172107A (en) * | 2019-05-28 | 2019-08-27 | 苏州鸿洋医药科技有限公司 | The separation method of Heparan sulfate and dermatan sulfate in a kind of heparan |
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| Assay of possible economically motivated additives or native impurities levels in heparin by 1H NMR, SAX-HPLC, and anticoagulation time approaches;David A.Keire 等;《Journal of Pharmaceutical andB iomedical Analysis》;20100220;第52卷;656-664 * |
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