CN115436542B - Method for identifying sheep-derived heparin doping proportion in pig intestinal mucosa heparin - Google Patents

Abstract

The invention provides a method for identifying sheep-derived heparin doping proportion in pig intestinal mucosa heparin, belonging to the technical field of heparin source identificationThe method comprises the following steps: 1) Carrying out enzymolysis on a heparin sample to be detected to obtain heparin enzymolysis liquid to be detected; 2) Detecting the delta IS and delta IIIA content of heparin enzymolysis liquid to be detected by adopting liquid chromatography; 3) Calculating the ratio of Δis/- Δiiia, y= Δis/- Δiiia; 4) Calculating the proportion X of the heparan in the heparin to be detected: y= [ (Δis/Δiiia) _{Sheep (sheep)} ‑(△IS/△IIIA) _Pig ]X+(△IS/△IIIA) _Pig . The method can rapidly identify whether the pig intestinal mucosa heparin is mixed with sheep-derived heparin. The method is simple and convenient to operate, easy to analyze and convenient to popularize and use.

Description

Method for identifying sheep-derived heparin doping proportion in pig intestinal mucosa heparin

Technical Field

The invention belongs to the technical field of heparin source identification, and particularly relates to a method for identifying a sheep-derived heparin doping proportion in pig intestinal mucosa heparin.

Background

Heparin is used clinically as an anticoagulant and antithrombotic drug. Heparin is widely distributed in mammalian tissues and can be extracted from small intestinal mucosa of pigs, cattle, sheep and the like, bovine lung and the like. Heparin sodium of different sources has different chemical structures and biological activities, and the adverse reaction degree is also different, for example, the adverse reaction of bovine-derived heparin sodium for causing thrombocytopenia is about 2 times that of porcine-derived heparin sodium. Heparin sodium and low molecular heparin approved by the European Union and the United states at present are only sourced from pig intestinal mucosa, and heparin sodium mixed with sources of cattle, sheep and the like is regarded as an unqualified product. Heparin is a resource-starving product based on this. Under the high profit temptation, the merchant illegally adulterates heparin from sources such as cattle, sheep and the like in the pig intestinal mucosa heparin, thereby bringing serious hidden danger to clinical application. Therefore, distinguishing species sources has become an important analytical indicator for assessing heparin sodium quality. The anticoagulation potency of bovine heparin and the potency of anti-IIa factor are about 1/2 of that of pig farm mucosa heparin, and the anticoagulation potency and the anti-IIa factor are obviously different. The anticoagulation potency and anti-IIa factor potency of the heparins are similar to those of the porcine intestinal mucosa heparin, the molecular weight of the heparins is similar, the heparins are easier to be adulterated into the porcine intestinal mucosa heparin, and the heparins need to be identified by a finer method.

The current method for detecting and analyzing heparin sodium from different species comprises the following steps: fluorescent quantitative PCR method, nuclear magnetic resonance method, electrospray mass spectrometry (ESI-MS), immunological detection method, etc. The fluorescent quantitative PCR method does not directly detect heparin, but indirectly proves whether the sample is polluted by heparin of other sources by detecting whether the sample has DNA of other ruminants or not, but the DNA is easy to be processed and destroyed under the conditions of oxidization, nuclease treatment and the like, so that the actual condition of the sample with the DNA processed is difficult to be effectively reflected by the fluorescent quantitative PCR method; the methods such as nuclear magnetic resonance method, electrospray mass spectrometry and immunological detection are expensive in required instruments, high in use cost, complex in detection and analysis steps and difficult to master quickly.

Disclosure of Invention

The invention provides a method for identifying the doping proportion of sheep-derived heparin in pig intestinal mucosa heparin, which can directly represent the composition difference of heparin sugar chains, identify whether a sample to be tested contains sheep-derived heparin or not, and has simple and rapid detection method.

In order to achieve the above purpose, the invention provides a method for identifying porcine intestinal mucosa heparin and sheep-derived heparin, comprising the following steps:

1) Carrying out enzymolysis on a heparin sample to be detected to obtain heparin enzymolysis liquid to be detected;

2) Detecting the delta IS and delta IIIA content of heparin enzymolysis liquid to be detected by adopting liquid chromatography; wherein Δis IS 2-sulfo- Δ4, 5-glucuronic acid- (β1→4) -3-sulfo-N-sulfonic acid glucosamine; delta IIIA is 2-sulfo-Delta 4, 5-glucuronic acid- (beta 1- > 4) -3-sulfo-N-acetylglucosamine;

3) Calculating the ratio of Δis/- Δiiia, y= Δis/- Δiiia;

4) Calculating the proportion X of the heparan in the heparin to be detected: y= [ (Δis/Δiiia) _{Sheep (sheep)} -(△IS/△IIIA) _Pig ]X+(△IS/△IIIA) _Pig 。

Preferably, said (. DELTA.IS/. DELTA.IIIA) _{Sheep (sheep)} And (. DELTA.IS/. DELTA.IIIA) _Pig Average of 3 batches of pure sheep-derived heparin and 3 batches of pure pig intestinal mucosa-derived heparin, respectivelyValues.

Preferably, y=75.18x+36.25.

Preferably, the silica gel chemical bonding strong-alkalinity quaternary ammonium salt anion exchange stationary phase is used as a chromatographic column filling agent when liquid chromatography is adopted for detection.

Preferably, when detection is performed by liquid chromatography, gradient elution is performed using a mobile phase a and a mobile phase B; the mobile phase A is 2mmol/L sodium dihydrogen phosphate aqueous solution, and the pH=3.0; the mobile phase B is an aqueous solution of 1mol/L sodium perchlorate and 2mmol/L sodium dihydrogen phosphate, and the pH=3.0.

Preferably, the gradient elution is performed as shown in the following table:

preferably, the detection conditions when detection is performed by liquid chromatography are as shown in the following table:

preferably, ΔIS and ΔIIIA are calculated using area normalization.

Preferably, the enzymolysis method in step 1) includes the following steps: weighing 20mg of heparin sample to be detected, adding 1ml of purified water for dissolution, and filtering with a 0.22 mu m filter membrane to obtain sample solution to be subjected to enzymolysis detection;

taking 20 mu L of a sample solution to be subjected to enzymolysis, and carrying out enzymolysis by using 100 mu L of a mixed solution of heparanase I, heparanase II and heparanase III to obtain heparin enzymolysis solution to be subjected to enzymolysis;

the heparanase I, II and III mixed solution is prepared by mixing heparanase I, II and III with the concentration of 0.4 IU/mu L in equal volume;

the heparanase I is CAS NO:9025-39-2; heparanase II is CAS NO:149371-12-0; heparanase III is CAS NO:37290-86-1.

Compared with the prior art, the invention has the advantages and positive effects that:

1. the method is a method for directly, but not indirectly, characterizing the composition difference of heparin sugar chains, and directly reflects the difference of species sources.

2. The instruments and equipment related in the invention are conventional instruments and equipment, so that the method can be widely applied to daily detection of enterprises, institutions and the like.

3. The detection method is easy to learn, simple and convenient to operate, easy to analyze and convenient to popularize and use.

4. The parameters related in the invention are sensitive along with different changes of the addition amount of the heparosan, and can correctly reflect the actual situation.

Drawings

FIG. 1 is a disaccharide profile of sheep 1-derived heparin;

FIG. 2 is a disaccharide profile of sheep 2-derived heparin;

FIG. 3 is a disaccharide profile of sheep 3-derived heparin;

FIG. 4 is a disaccharide profile of porcine 1 intestinal mucosa derived heparin;

FIG. 5 is a disaccharide profile of porcine 2 intestinal mucosa derived heparin;

FIG. 6 is a disaccharide profile of porcine 3 intestinal mucosa derived heparin;

FIG. 7 is a disaccharide profile of a sample to be tested;

FIG. 8 IS an enlarged partial schematic view of the ΔIS spectrum of FIG. 7;

FIG. 9 is an enlarged partial schematic view of the ΔIIIA spectrum of FIG. 7;

fig. 10 is a graph comparing sheep 1 and sheep 2.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The invention provides a method for identifying sheep-derived heparin doping proportion in pig intestinal mucosa heparin, which comprises the following steps:

1) Carrying out enzymolysis on a heparin sample to be detected to obtain heparin enzymolysis liquid to be detected;

2) Detecting the delta IS and delta IIIA content of heparin enzymolysis liquid to be detected by adopting liquid chromatography; wherein Δis IS 2-sulfo- Δ4, 5-glucuronic acid- (β1→4) -3-sulfo-N-sulfonic acid glucosamine; delta IIIA is 2-sulfo-Delta 4, 5-glucuronic acid- (beta 1- > 4) -3-sulfo-N-acetylglucosamine;

3) Calculating the ratio of Δis/- Δiiia, y= Δis/- Δiiia;

4) Calculating the proportion X of the heparan in the heparin to be detected: y= [ (Δis/Δiiia) _{Sheep (sheep)} -(△IS/△IIIA) _Pig ]X+(△IS/△IIIA) _Pig 。

The heparin sample to be detected is subjected to enzymolysis to obtain heparin enzymolysis liquid to be detected. In the present invention, the enzymolysis method of the heparin sample to be detected preferably comprises the following steps: weighing 20mg of heparin sample to be detected, adding 1ml of purified water for dissolution, and filtering with a 0.22 mu m filter membrane to obtain sample solution to be subjected to enzymolysis detection; taking 20 mu L of a sample solution to be subjected to enzymolysis, and carrying out enzymolysis by using 100 mu L of a mixed solution of heparanase I, heparanase II and heparanase III to obtain heparin enzymolysis solution to be subjected to enzymolysis; the heparanase I, II and III mixed solution is prepared by mixing heparanase I, II and III with the concentration of 0.4 IU/mu L in equal volume; the heparanase I is CAS NO:9025-39-2; heparanase II is CAS NO:149371-12-0; heparanase III is CAS NO:37290-86-1. The heparanase I, II and III belong to heparan polysaccharide lyase.

After heparin enzymolysis liquid to be detected IS obtained, the delta IS and delta IIIA content of the heparin enzymolysis liquid to be detected IS detected by adopting liquid chromatography; wherein Δis IS 2-sulfo- Δ4, 5-glucuronic acid- (β1→4) -3-sulfo-N-sulfonic acid glucosamine; delta IIIA is 2-sulfo-Delta 4, 5-glucuronic acid- (beta 1- & gt 4) -3-sulfo-N-acetylglucosamine.

In the invention, the silica gel chemical bonding strong alkaline quaternary ammonium salt anion exchange stationary phase is preferably used as a chromatographic column filling agent when liquid chromatography is adopted for detection.

In the present invention, when detection is performed by liquid chromatography, gradient elution is preferably performed by using a mobile phase a and a mobile phase B; the mobile phase A is 2mmol/L sodium dihydrogen phosphate aqueous solution, and the pH=3.0; the mobile phase B is an aqueous solution of 1mol/L sodium perchlorate and 2mmol/L sodium dihydrogen phosphate, and the pH=3.0.

In the present invention, the procedure at the time of the gradient elution is preferably as shown in the following table 1:

TABLE 1

In the present invention, the detection conditions when detection is performed by liquid chromatography are shown in the following table 2:

TABLE 2

In the present invention, Δis and Δiiia are preferably calculated by an area normalization method.

After obtaining Δis and Δiiia, the invention calculates the ratio of Δis/Δiiia, y= Δis/Δiiia;

the delta IS and delta IIIA belong to heparin core disaccharides, and the applicant finds that the delta IS and delta IIIA are greatly different for pig intestine mucosa source and sheep source heparin, so that the delta IS and delta IIIA are adopted as parameters for detecting the doping proportion of sheep source heparin in the invention, and when delta IS/delta IIIA are adopted, the difference IS amplified more, so that the method IS more suitable for identifying pig intestine mucosa source and sheep source heparin.

After the Y value is obtained, the invention calculates the proportion X of the heparins in the heparin to be detected: y= [ (Δis/Δiiia) _{Sheep (sheep)} -(△IS/△IIIA) _Pig ]X+(△IS/△IIIA) _Pig . Since the enzymatic hydrolysis and the analysis of disaccharides by a strong anion exchange chromatography column are slightly different for each detection, in the present invention, the method (. DELTA.IS/. DELTA.IIIA) IS used to improve the accuracy of the detection _{Sheep (sheep)} And (. DELTA.IS/. DELTA.IIIA) _Pig Preferably, the average value of 3 batches of pure sheep-derived heparin and 3 batches of pure pig intestinal mucosa-derived heparin is respectively obtained.

Although there was a difference in Δis/. DELTA.IIIA between the pure sheep-derived heparin and the pure porcine intestinal mucosa-derived heparin for each batch, the actual variation was not too great. Therefore, when the purpose of the detection is to only approximately understand whether the sample to be tested contains heparin of sheep origin and the approximate proportion, in the present invention, it is preferable to directly calculate the formula y=75.18x+36.25. By the calculation of the formula, the delta IS/deltaIIIA of the pure sheep-derived heparin and the pure pig intestinal mucosa-derived heparin are not required to be additionally measured, and the use IS more convenient.

It should be noted that: since the area normalization method is a rough calculation method, the method provided by the application is more used for identifying whether the pig intestinal mucosa heparin is mixed with sheep-derived heparin and the approximate proportion thereof.

The technical solutions provided by the present invention are described in detail below in conjunction with examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.

Example 1

1. Instrument for measuring and controlling the intensity of light

High performance liquid chromatograph, electronic balance and acidimeter

2. Reagent and test solution

TABLE 3 reagents and test solutions

3. Sample processing

And preparing a mixed product of the pure sheep-derived heparin and the pure pig intestinal mucosa-derived heparin, wherein the ratio (mass ratio) of the sheep heparin is 5%, and the mixed product is used as a sample to be detected.

20mg of the sample was weighed, dissolved in 1ml of purified water, and filtered through a 0.22 μm filter membrane to obtain a sample to be subjected to enzymolysis.

4. Sample enzymolysis

4.1 sodium calcium acetate pH7.0 solution preparation: calcium acetate (32 mg) and bovine serum albumin (10 mg) were weighed into 60mL of purified water, 580. Mu.L of glacial acetic acid was added, pH7.0 was adjusted with 2mol/L sodium hydroxide, transferred into a 100mL volumetric flask, diluted to scale with purified water, and filtered through a 0.22 μm filter.

4.2 preparation of Potassium dihydrogen phosphate 7.0 buffer: weighing KH ₂ PO ₄ After complete dissolution, 68mg and 10mg of bovine serum albumin in 30mL of purified water were transferred to a 50mL volumetric flask, pH7.0 was adjusted with 2mol/L KOH, diluted to scale with purified water and filtered through a 0.22 μm filter.

4.3 heparin enzyme I solution preparation: heparanase I was dissolved in potassium dihydrogen phosphate 7.0 buffer to 0.4IU/mL, mixed in a vortex mixer and stored at-20℃before use.

4.4 heparin enzyme II solution preparation: heparanase II was dissolved in potassium dihydrogen phosphate 7.0 buffer to 0.4IU/mL, mixed in a vortex mixer and stored at-20℃before use.

4.5 heparinase III solution preparation: heparanase III was dissolved in potassium dihydrogen phosphate 7.0 buffer to 0.4IU/mL, mixed in a vortex mixer and stored at-20℃before use.

4.6 heparanase I, II, III mixed solution: the heparanase solutions I, II and III are taken according to the following ratio of 1:1: mixing in proportion of 1.

4.7 preparation of sample solution: taking a pre-prepared 20mg/ml water solution of a sample to be detected, adding 70 mu L of a solution of sodium calcium acetate pH7.0, adding 100 mu L of a mixed solution of heparinase I, heparinase II and heparinase III, mixing by a vortex mixer, and placing in a water bath at 25 ℃ for 48 hours to obtain a solution of the sample to be detected.

5. Detection of

The sample solution was tested by liquid chromatography, the specific chromatographic conditions are shown in table 4, and the elution gradient is shown in table 5.

TABLE 4 chromatographic conditions

Table 5 detection of linear elution gradient

Time (minutes)	Mobile phase a (%)	Mobile phase B (%)
			0	97	3
20	65	35
			50	0	100
60	0	100
			61	97	3
79	97	3

Detecting by liquid chromatography to obtain 3 batches of pure sheep-derived heparin disaccharide spectrograms shown in figures 1-3; the disaccharide spectrograms of 3 batches of pure pig intestinal mucosa source heparin are shown in figures 4-6, and the disaccharide spectrograms of sheep 1 and pig 1 are shown in figure 10 (note: three batches of heparin adopt different equipment in detection, chromatographic columns are different, and peak time is different);

the DeltaIS spectrogram of the sample to be detected IS shown in fig. 7 and 8, and the DeltaIIIA spectrogram IS shown in fig. 7 and 9. The spectrograms were integrated by area normalization, and the peak area percentages ΔIS and ΔIIIA were 62.86 and 1.58, respectively, and further calculated as the ratio of ΔIS/. DELTA.IIIA was 39.78,3 for the pure sheep-derived heparin and 3 for the pure pig intestinal mucosa-derived heparin, as shown in Table 6, respectively.

Table 63 batches of heparan and 3 batches of porcine intestinal mucosa heparin disaccharides

From the data in table 6, it can be seen that the Δis/. DELTA.IIIA of 3 batches of pure sheep-derived heparin was 111.53, 112.35, 110.38, respectively, with an average value of (111.53+112.35+110.38)/3=111.43.

The DeltaIS/DeltaIIIAof 3 batches of pure porcine intestinal mucosa-derived heparin were 37.27, 35.52, 35.98, respectively, with an average value of (37.27+35.52+35.98)/3=36.25. According to formula y= [ (Δis/Δiiia) _{Sheep (sheep)} -(△IS/△IIIA) _Pig ]X+(△IS/△IIIA) _Pig . X= (39.78-36.25)/(111.43-36.25) =0.0469 was calculated, i.e. the proportion of heparan in the sample was 4.69%.

The calculation result shows that: the calculated value obtained by the method basically accords with the actual proportion of the heparins in the sample, which shows that the method has good applicability and can be used for identifying whether the heparin sodium is doped with the heparins and the doping proportion.

Example 2

The difference from example 1 is that the other procedure is exactly the same as example 1, except that the sample to be tested is a sample.

Wherein: and preparing a mixed product with the ratio (mass ratio) of the sheep heparin of 15% by adopting the pure sheep-derived heparin and the pure pig intestinal mucosa-derived heparin, and taking the mixed product as a sample to be detected.

The liquid chromatography IS adopted for detection to obtain the DeltaIS spectrograms of the sample to be detected as shown in fig. 7 and 8, and the DeltaIIIA spectrograms are shown in fig. 7 and 9. The spectrogram IS integrated by adopting an area normalization method, the area percentages of peaks DeltaIS and DeltaIIIA are 63.77 and 1.36 respectively, the ratio of DeltaIS to DeltaIIIA IS 46.89, and X=0.1415 IS calculated according to the formula Y=75.18X+36.25, namely the proportion of the heparin in the sample IS 14.15%.

The calculation result shows that: the calculated value obtained by the method basically accords with the actual proportion of the heparins in the sample, which shows that the method has good applicability and can be used for identifying whether the heparin sodium is doped with the heparins and the doping proportion.

Example 3

The difference from example 1 is that the other procedure is exactly the same as example 1, except that the sample to be tested is a sample.

Wherein: and preparing a mixed product with the ratio (mass ratio) of the sheep heparin of 30% by adopting the pure sheep-derived heparin and the pure pig intestinal mucosa-derived heparin, and taking the mixed product as a sample to be detected.

The liquid chromatography IS adopted for detection to obtain the DeltaIS spectrograms of the sample to be detected as shown in fig. 7 and 8, and the DeltaIIIA spectrograms are shown in fig. 7 and 9. The spectrogram IS integrated by adopting an area normalization method, the area percentages of peaks of DeltaIS and DeltaIIIA are 64.08 and 1.03 respectively, the ratio of DeltaIS to DeltaIIIA IS 62.21, and X= 0.3453 percent IS calculated according to the formula Y=75.18X+36.25, namely the proportion of the heparan in the sample IS 34.53 percent.

The calculation result shows that: the calculated value obtained by the method basically accords with the actual proportion of the heparins in the sample, which shows that the method has good applicability and can be used for identifying whether the heparin sodium is doped with the heparins and the doping proportion.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (2)

1. The method for identifying the sheep-derived heparin doping proportion in the pig intestinal mucosa heparin is characterized by comprising the following steps of:

1) Carrying out enzymolysis on a heparin sample to be detected to obtain heparin enzymolysis liquid to be detected;

2) Detecting the content of heparin disaccharides delta IS and delta IIIA in the heparin enzymolysis liquid to be detected by adopting liquid chromatography;

3) Calculating the ratio of Δis/- Δiiia, y= Δis/- Δiiia; the ratio of DeltaIS/DeltaIIIAis the ratio of DeltaIS to DeltaIIIA peak area percent; y IS the ratio of delta IS to delta IIIA in heparin enzymolysis liquid to be detected;

4) The proportion X of the heparan in the heparin to be detected is calculated, and the calculation formula is as follows: y= [ (Δis/Δiiia) _{Sheep (sheep)} -（△IS/△IIIA） _Pig ]X+（△IS/△IIIA） _Pig The method comprises the steps of carrying out a first treatment on the surface of the The (DeltaIS/DeltaIIIA) _{Sheep (sheep)} And (. DELTA.IS/. DELTA.IIIA) _Pig Average of 3 batches of pure sheep-derived heparin and 3 batches of pure pig intestinal mucosa-derived heparin, respectively;

when liquid chromatography is adopted for detection, silica gel chemical bonding strong-alkalinity quaternary ammonium salt anion exchange stationary phase is adopted as a chromatographic column filler; the specification of the chromatographic column is 4mm multiplied by 250mm and 5 mu m;

when liquid chromatography is adopted for detection, the mobile phase A and the mobile phase B are used for gradient elution; the mobile phase A is 2mmol/L sodium dihydrogen phosphate aqueous solution, and the pH=3.0; the mobile phase B is an aqueous solution of 1mol/L sodium perchlorate and 2mmol/L sodium dihydrogen phosphate, and the pH=3.0;

the procedure for the gradient elution is shown in the following table:

the detection conditions when detection is carried out by liquid chromatography are shown in the following table:

the enzymolysis method in the step 1) comprises the following steps: weighing 20mg of heparin sample to be detected, adding 1ml of purified water for dissolution, and filtering with a 0.22 mu m filter membrane to obtain sample solution to be subjected to enzymolysis detection;

taking 20 mu L of a sample solution to be subjected to enzymolysis, and carrying out enzymolysis by using 100 mu L of a mixed solution of heparanase I, heparanase II and heparanase III to obtain heparin enzymolysis solution to be subjected to enzymolysis;

the heparanase I, II and III mixed solution is prepared by mixing heparanase I, II and III with the concentration of 0.4 IU/mu L in equal volume.

2. The method of claim 1, wherein Y = 75.18x+36.25.

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