CN111830111A - A kind of CS capillary electrophoresis analysis method and application - Google Patents

Abstract

The invention belongs to the technical field of capillary electrophoresis detection, and relates to a CS capillary electrophoresis analysis method and application, wherein CS is enriched by using an off-line enrichment method, and then is enriched on line by using a method of combining field amplification with large-volume electrokinetic sample injection stacking; mixing the CS water solution with the quaternary ammonium salt cationic surfactant solution, adding a sulfuric acid solution, discarding the supernatant, adding NaCl into the precipitate, carrying out alcohol precipitation, separating the precipitate and the supernatant, carrying out multiple alcohol precipitations on CS, combining the CS precipitates, and dissolving with ultrapure water to obtain the enrichment solution. Preparing the enriched solution after the offline enrichment to obtain a CS alkali solution, and carrying out large-volume electrokinetic sample injection, wherein BGE is 20mmol/LNaH₂PO₄-90mmol/L EDA-phosphate buffer, BGE plusNaCl, pH3.0 was added. Can accurately detect the macromolecular substance CS, and has better enrichment effect.

Description

A kind of CS capillary electrophoresis analysis method and application

technical field

The invention belongs to the technical field of capillary electrophoresis detection, in particular to a CS capillary electrophoresis analysis method and application.

Background technique

The disclosure of information in this Background section is only for enhancement of understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

Chondroitin Sulfate (CS) is a glycosaminoglycan ubiquitously present in animal cartilage and connective tissue. Its sugar structure and composition are complex and diverse. Chondroitin sulfate has certain applications in the field of food additives and cosmetics.

Capillary Electrophoresis (Capillary Electrophoresis, CE for short). Capillary electrophoresis is a type of liquid phase separation technology that uses a high-voltage electric field as the driving force and the capillary as the separation channel to achieve separation according to the differences in the mobility and distribution behavior of each component in the sample. The basic instrumentation of CE consists of capillary, buffer tank, sampling system, high voltage power supply and detector.

The inventors found that in the process of detecting CS by the existing capillary electrophoresis analysis method, due to the large molecular weight of CS and the lack of ultraviolet chromophore, the detection sensitivity of the UV detection by the capillary electrophoresis analysis method is low.

SUMMARY OF THE INVENTION

In view of the above problems in the prior art, the purpose of the present invention is to provide a CS capillary electrophoresis analysis method and application.

In order to solve the above technical problems, the technical scheme of the present invention is:

The first aspect is a CS capillary electrophoresis analysis method, which uses an offline enrichment method to enrich CS, and then utilizes field amplification combined with a large-volume electrokinetic injection stacking method for online enrichment;

The off-line enrichment method is as follows: CS aqueous solution is mixed with quaternary ammonium salt cationic surfactant solution, then sulfuric acid solution is added, supernatant is discarded, NaCl solution is added to the precipitate, alcohol precipitation, separation of precipitate and supernatant, CS through alcohol Precipitate, combine CS precipitation and dissolve with ultrapure water to obtain enrichment solution.

The enrichment solution after offline enrichment is prepared to obtain CS alkali solution, and the online enrichment adopts the method of field amplification combined with large-volume electric injection, BGE is 20mmol/LNaH ₂ PO ₄ -90mmol/L EDA-NaCl-phosphate buffer, pH3.0.

Due to the large molecular weight and weak acidity of CS, the enrichment effect of dynamic pH modulation stacking and micellar scanning technology is not obvious.

In the online enrichment method: adding a certain concentration of NaCl solution to BGE (buffer solution in capillary) buffer solution of 20mmol/L NaH ₂ PO ₄ -90mmol/LEDA-phosphoric acid (pH 3.0) can improve the conductivity of BGE, When the CS sample moved to the junction of the sample zone and BGE, the electric field strength decreased, so that the migration speed was slowed to achieve the enrichment effect.

In the offline enrichment method: CS is a polyanion, and CS reacts with quaternary ammonium salt cationic surfactants to form stable associate particles. Offline enrichment can be achieved through large-volume extraction.

The combination of offline enrichment and online enrichment for enrichment, compared with the conventional injection method, the enrichment multiple is 130 times. The conventional analysis method is: no NaCl is added to BGE, the injection pressure is 0.5psi, and the injection time is 0.5 psi. for 10s.

By studying the large stacking sampling method, field amplification combined with large-volume pressure sampling and stacking method, or field amplification combined with large-volume electric sampling and stacking method, the field amplification combined with large-volume electric sampling and stacking method is selected to improve the detection sensitivity.

In some embodiments of the present invention, in the online enrichment, the concentration of NaCl in BGE is 10-30 mmol/L; preferably 10-20 mmol/L.

There is a difference between large volume pressure injection and electrodynamic injection. Pressure injection is not selective and will lead to the entry of a large amount of sample matrix, which may interfere with the separation and enrichment effect. Motorized injection can selectively enter only the analytical sample and expel the sample matrix to obtain better peak shape and sensitivity.

Selecting the above-mentioned concentration range of NaCl is beneficial to improve the enrichment effect.

In some embodiments of the present invention, in the field amplification combined with the large-volume electric sampling stacking, the injection time is 60-420s; preferably, the injection time is 300-420s. With the extension of injection time, the peak area gradually increased, and after 300 s, the peak area gradually decreased.

In some embodiments of the present invention, in field amplification combined with large-volume electrokinetic injection stacking, the alkali concentration is 27-108 mmol/L; preferably, the alkali concentration is 27-54 mmol/L. The alkali concentration is the concentration of the alkali in the solution obtained by mixing the CS aqueous solution and the alkali solution. When the alkali concentration is 0, the peak shape is poor. After adding the alkali, it is in the above range, which helps to improve the peak shape. In the range of 27-54mmol/L, it has a good peak shape.

In the field amplification combined with the large-volume electrodynamic injection stacking method, the enrichment effect is increased by 46.4 times compared with the conventional injection method. The conventional analysis method is that no NaCl is added to the BGE, the injection pressure is 0.5psi, and the injection time is 10s. .

In some embodiments of the present invention, the quaternary ammonium salt type cationic surfactant is cetylpyridinium chloride, cetylpyridinium bromide, cetyltrimethylammonium chloride (CTAC), Hexacyltrimethylammonium bromide (CTAB), etc.; preferably cetyltrimethylammonium chloride (CTAC).

In some embodiments of the present invention, in the offline enrichment method, the concentration of the CTAC solution is 4 g/mL, and the volume ratio of the CTAC solution to the CS aqueous solution is 1-1.5:10. The volume ratio of CTAC solution to CS aqueous solution, that is, the amount of CTAC, affects the enrichment effect. Preferably, the volume ratio of CTAC solution to CS aqueous solution is 1:10

In some embodiments of the present invention, the sulfuric acid solution is a solution of 98% sulfuric acid and water, the volume ratio of 98% sulfuric acid and water is 1:9, and the volume ratio of CS aqueous solution to sulfuric acid solution is 4-6:1; preferably 5 :1.

In some embodiments of the present invention, in the offline enrichment method, the concentration of the NaCl solution is 4-6 mol/L, and the volume ratio of the CS aqueous solution to the NaCl solution is 9-11:1; preferably, the volume ratio is 10:1 , the concentration of NaCl solution is 5mol/L.

In some embodiments of the present invention, the volume ratio of ethanol to the sample used in the alcohol precipitation is 5-10:1; preferably 8-10:1; more preferably 8:1. The amount of ethanol used in alcohol precipitation affects the effect of enrichment, and when the volume ratio is 5:2, there is no enrichment effect.

In some embodiments of the present invention, the number of times of alcohol precipitation is 1-5 times; preferably, it is 2-5 times. When the number of alcohol precipitation increases, the enrichment effect is better.

In some embodiments of the present invention, the injection voltage of the online enrichment electrokinetic injection is -8 to -12Kv; preferably -10Kv.

In some embodiments of the present invention, the electrophoresis conditions are: detection wavelength 200nm, electrophoresis temperature 20-30°C, separation voltage -10--20Kv; preferably, electrophoresis conditions are: electrophoresis temperature 25°C, separation voltage -15Kv.

In some embodiments of the present invention, the capillary is activated with 0.1 mol/L NaOH before use, rinsed under 20 psi pressure, and then sequentially rinsed with ultrapure water and BGE under 20 psi pressure, and finally added to both ends of the capillary. High voltage operation, the voltage value of the high voltage is 20kV, and the BGE is 20mmol/ _{LNaH2PO4-90mmol} /L EDA _- NaCl-phosphate buffer solution (pH 3.0).

The second aspect is the application of the above-mentioned CS capillary electrophoresis analysis method in the detection of chondroitin sulfate.

One or more technical solutions of the present invention have the following beneficial effects:

The CS capillary electrophoresis analysis method can accurately detect the macromolecular substance CS, and the enrichment effect is good, which solves the problem that the macromolecular substance is not enriched by the capillary electrophoresis analysis method, resulting in poor detection effect;

In the CS capillary electrophoresis analysis method, adding a certain concentration of NaCl solution to BGE can improve the conductivity of BGE. When the CS sample moves to the junction of the sample zone and BGE, the electric field intensity decreases, so that the migration speed is slowed down to achieve enrichment. Effect;

When the sample is dissolved in a low-concentration alkali solution, the ionization degree of CS becomes larger and the charge is more, so that more samples enter the capillary tube, which helps to improve the detection sensitivity;

In the off-line enrichment method, quaternary ammonium salt cationic surfactant is used to react with CS to form stable associate particles, and off-line enrichment can be achieved by large-volume extraction;

The CS capillary electrophoresis analysis method conforms to the linear regression equation for the peak area of CS in the range of 1μg/mL to 100μg/mL, the detection limit of CS is 50ng/ml, and the quantification limit is 200ng/ml;

The CS capillary electrophoresis analysis method was compared with the method in the Chinese Pharmacopoeia, and it was verified that the labeled amount of CS determined by the capillary electrophoresis analysis method was close to the method in the Pharmacopoeia.

Description of drawings

The accompanying drawings forming a part of the present invention are used to provide further understanding of the present application, and the exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

Fig. 1 is a comparison diagram of the enrichment effect of Example 1 and Comparative Example 1, (1) conventional sample injection (2) offline enrichment;

Fig. 2 is a graph showing the influence of NaCl concentration on enrichment effect under the electrokinetic injection conditions of Experimental Example 1-Experiment Example 4, (1) 0mmol/L (2) 10mmol/L (3) 20mmol/L (4) 30mmol/L ;

Fig. 3 is the contrast diagram of the enrichment effect of experimental example 5-experimental example 7, (1) 0mmol/L NaCl (2) 10mmol/LNaCl (3) 30mmol/L NaCl;

Fig. 4 is a graph showing the influence of injection time on enrichment effect;

Figure 5 is a graph of the influence of the concentration of the base of Experimental Example 14-Experiment Example 18 on the CS peak shape, (1) 0 mmol/L (2) 30 mmol/L (3) 60 mmol/L (4) 90 mmol/L (5) 120 mmol /L;

Fig. 6 is the influence figure of the concentration of the alkali of Experimental Example 14-Experiment Example 18 on the CS peak area;

Figure 7 is a comparison diagram of field amplification combined with large-volume electric injection and conventional injection;

Figure 8 is a comparison diagram of offline enrichment and conventional injection, (1) offline enrichment (2) conventional injection;

Figure 9 is the standard curve of the regression equation.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It should be noted that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural as well, furthermore, it is to be understood that when the terms "comprising" and/or "including" are used in this specification, it indicates that There are features, steps, operations, devices, components and/or combinations thereof.

Preparation of experimental solutions:

Preparation of 4% CTAC: Weigh 2.0 g of CTAC, transfer it to a 50 mL volumetric flask, add ultrapure water to dissolve, make up to the mark, and store in a 4°C refrigerator.

Preparation of 5mol/L NaCl solution: Weigh 2.9g of NaCl, transfer it to a 10mL volumetric flask, add ultrapure water to dissolve, and adjust the volume to the mark.

Preparation of 0.1mol/L NaOH solution: Weigh 0.4g of NaOH in a 25mL beaker, add an appropriate amount of ultrapure water to dissolve, transfer it to a 100mL volumetric flask, add ultrapure water to the mark, and store in a 4°C refrigerator. 30, 60, 90, and 120 mmol/L NaOH solutions were all obtained by diluting the stock solution.

Preparation of 20 mmol/L NaH ₂ PO ₄ -90 mmol/L EDA-phosphoric acid (pH 3.0) buffer solution: Weigh 0.31 g of NaH ₂ PO ₄ , add 75 mL of ultrapure water to dissolve, add 600 μL of EDA, adjust pH to 3.0 with phosphoric acid , Transfer to a 100mL volumetric flask to constant volume, and store in a refrigerator at 4°C.

1:9 sulfuric acid solution: Precisely measure 1 mL of 98% concentrated sulfuric acid and add it to 9 mL of ultrapure water.

CS stock solution: Take about 0.25g of CS from shark, accurately weigh it, transfer it to a 25mL volumetric flask, add ultrapure water to dissolve, and make up to the mark. The concentration of the stock solution is 10mg/mL.

Preparation of 1 mg/mL CS base solution: take 20 μL of 10 mg/mL CS stock solution and dilute it with NaOH solutions of different concentrations.

Experimental example 34 utilizes the preparation of the enzyme solution and tablet sample involved in the HPLC method recorded in the Chinese Pharmacopoeia as follows:

Preparation of enzyme incubation buffer (50mmol/L Tris-60mmol/LCH ₃ COONa, pH 8.0): Weigh 0.30g of Tris and 0.41g of CH ₃ COONa, place in a 50mL volumetric flask, add 35mL of ultrapure water to dissolve Then adjust the pH to 8.0 with hydrochloric acid, and finally add ultrapure water to make up to the mark, shake well, and store in a 4°C refrigerator.

Preparation of enzyme solution: Dissolve 10 U of enzyme in 10 mL of incubation buffer to prepare 1 U/mL chondroitin sulfate ABC enzyme solution, and store it at -20°C after aliquoting it into 1.5 mL EP tubes.

Preparation of tablet samples: Weigh a certain number of tablets and calculate the average tablet weight, grind into fine powder, weigh a certain amount of tablet powder according to the labelled amount and dissolve it in ultrapure water. Take 50 μL of tablet sample solution, 50 μL of chondroitin sulfate ABC enzyme solution and 100 μL of incubation buffer into a 1.5 mL EP tube. After vortexing and mixing, the EP tube was placed in a constant temperature water bath at 37°C for 1 hour, and then the EP tube was heated in boiling water for 3 minutes to inactivate the enzyme, and then cooled to room temperature.

All the above solutions should be filtered through a 0.22 μm filter before entering the capillary.

Below in conjunction with embodiment, the present invention is further described

Example 1

CS is CS of shark origin. CS aqueous solution was prepared by CS and ultrapure water.

Measure 2.5ml of 5mg/mL CS aqueous solution into a 50ml volumetric flask, dilute with ultrapure water to the constant volume at the mark, 250μg/mL CS stock solution. Measure 1 mL of this stock solution and add 0.1 mL of 4% CTAC, shake gently and let stand for 1 h. Then, 0.2 mL of a 1:9 sulfuric acid solution was added, shaken and placed for 5 min, so that the CS-CTAC associates were precipitated to the bottom of the EP tube, and centrifuged at 7000 rpm for 20 min. The supernatant was discarded. In order to free CS from the CS-CTAC associate, 0.1 mL of 5 mol/L NaCl was added to the precipitate, and the precipitate was dissolved by vortexing for 30 s. Add 5.5 mL of ethanol to precipitate CS, and centrifuge at 7000 rpm for 10 min after mixing. Separate the precipitate and the supernatant, add 200 μL of ultrapure water to the precipitate, vortex to dissolve, add 2.5 mL of ethanol to the supernatant, mix well, centrifuge at 7000 rpm for 10 min, discard the supernatant, add 200 μL of ultrapure water to the precipitate, and vortex. Spin to dissolve. The dissolved solutions of the two precipitates were combined and filtered through a 0.22 μm filter to obtain an aqueous CS solution.

The CS aqueous solution obtained by off-line enrichment was taken, and NaOH solution was added, so that the final CS concentration was 1 mg/mL, and the NaOH concentration was 27 mmol/L. BGE is 20 mmol/L NaH ₂ PO ₄ -90 mmol/L EDA-NaCl-phosphate buffer (pH 3.0), and the final concentration of NaCl in BGE is 10 mmol/L. After mixing, it was filtered through a 0.22 μm filter. Electric sampling was used for detection, and the sampling conditions were: sampling voltage -10 kV, and sampling time 300 s.

The electrophoresis conditions were: detection wavelength 200 nm, electrophoresis temperature 25°C, sample storage temperature 4°C, separation voltage -15Kv.

The electrophoresis results are shown in Figure 1.

Comparative Example 1

Conventional injection method, the conventional injection method is direct pressure injection, no NaCl is added to the BGE, the injection pressure is 0.5psi, and the injection time is 10s. The electrophoresis conditions were the same as in Example 1.

Examination of experimental conditions:

Experimental example 1 field magnification accumulation

1 mg/mL CS base solution (NaOH concentration in CS base solution is 60 mmol/L), BGE is 20 mmol/L NaH ₂ PO ₄ -90 mmol/L EDA-NaCl-phosphate buffer (pH 3.0), NaCl final in BGE The concentration is 10mmol/L. After mixing, it was filtered through a 0.22 μm filter. Electric injection detection was adopted, and the injection conditions were as follows: injection voltage -10 kV, injection time 99.9 s.

The electrophoresis conditions were: detection wavelength 200 nm, electrophoresis temperature 25°C, sample storage temperature 4°C, and separation voltage -15V.

Experimental example 2

Compared with Experimental Example 1, the final concentration of NaCl in BGE was 0 mmol/L, that is, no NaCl was added.

Experimental example 3

Compared with Experimental Example 1, the final concentration of NaCl was 20 mmol/L.

Experimental example 4

Compared with Example 1, the final concentration of NaCl was 30 mmol/L.

The results of Experimental Example 1 to Experimental Example 4 are shown in FIG. 2 , when NaCl exceeds 10 mmol/L, a sharp peak appears after the peak. The higher the NaCl concentration, the more forward the sharp peak, which affects the chromatographic peak of CS. The reason for the sharp peak is not clear, and it is speculated that the addition of a large amount of NaCl changes the BGE environment.

Experimental example 5

Compared with Experimental Example 1, pressure injection was changed.

Experimental example 6

Compared with Experimental Example 2, pressure injection was changed.

Experimental example 7

Compared with Experimental Example 3, pressure injection was changed.

The enrichment effect of experimental example 5-experimental example 7 is shown in Figure 3. It can be seen from Figure 3 that the greater the concentration of NaCl in BGE, the greater the conductivity of BGE, and the greater the difference between the electric field strength of the sample zone and the sample zone. , the enrichment effect is better, but it also increases the current and Joule heat, which in turn causes serious diffusion. Among them, when the concentration of NaCl was 10 mmol/L, the peak area was the largest, and the CS peak shape was the best.

Experimental Example 8 Field Amplification Combined with Large-Volume Electric Sample Injection Stacking

1 mg/mL CS base solution (27 mmol/L NaOH concentration in CS base solution), 20 mmol/L NaH ₂ PO ₄ -90 mmol/L EDA-NaCl-phosphate buffer (pH 3.0) for BGE, NaCl final in BGE The concentration is 10mmol/L. After mixing, it was filtered through a 0.22 μm filter. Electric sampling was used for detection, and the sampling conditions were: sampling voltage -10 kV, and sampling time 60 s.

The electrophoresis conditions were: detection wavelength 200 nm, electrophoresis temperature 25°C, sample storage temperature 4°C, and separation voltage -15V.

Experimental example 9

Compared with Experimental Example 8, the injection time was 120s.

Experimental Example 10

Compared with Experimental Example 8, the injection time was 180s.

Experimental Example 11

Compared with Experimental Example 8, the injection time was 240s.

Experimental example 12

Compared with Experimental Example 8, the injection time was 300s.

Experimental Example 13

Compared with Experimental Example 8, the injection time was 420s.

The enrichment effect of experimental example 8-experimental example 13 is shown in Figure 4. It can be seen from Figure 4 that in the process of 60-300s, the peak area gradually becomes larger, and from 300-420s, the peak area changes It is not obvious and has a slight downward trend. The reason may be that with the extension of injection time, the sample diffusion is more serious, which reduces the sample ions that actually enter the capillary.

Experimental Example 14

Take 20 μL of 1 mg/mL CS aqueous solution, add 180 μL of water to make the final alkali concentration 0 mmol/L, BGE is 20 mmol/L NaH ₂ PO ₄ -90 mmol/L EDA-NaCl-phosphate buffer (pH3.0), in BGE The final concentration of NaCl was 10 mmol/L. After mixing, it was filtered through a 0.22 μm filter. Electric sampling was used for detection, and the sampling conditions were: sampling voltage -10 kV, and sampling time 300 s.

The electrophoresis conditions were: detection wavelength 200 nm, electrophoresis temperature 25°C, sample storage temperature 4°C, and separation voltage -15V.

Experimental example 15

Compared with Experimental Example 8, 20 μL of 1 mg/mL CS aqueous solution was taken and added to 30 mmol/L NaOH solution, and the final concentration of NaOH was 27 mmol/L.

Experimental Example 16

Compared with Experimental Example 8, 20 μL of 1 mg/mL CS aqueous solution was added to 60 mmol/L NaOH solution, and the final concentration of NaOH was 54 mmol/L.

Experimental Example 17

Compared with Experimental Example 8, 20 μL of 1 mg/mL CS aqueous solution was taken and added to 90 mmol/L NaOH solution, and the final concentration of NaOH was 81 mmol/L.

Experimental Example 18

Compared with Experimental Example 8, 20 μL of 1 mg/mL CS aqueous solution was taken and added to 120 mmol/L NaOH solution, and the final concentration of NaOH was 108 mmol/L.

The enrichment effect of experimental example 14-experimental example 18 is shown in Figure 5 and Figure 6. It can be seen from the figures that the peak shape of the CS aqueous solution without alkali is poor, and it is obviously improved after adding alkali, so the addition of alkali is necessary. In the case of adding alkali, the higher the concentration of alkali, the lower the peak area, and the change of the peak area according to the addition of alkali.

The comparison of the enrichment effect of Experimental Example 18 and Comparative Example 1 is shown in Figure 7. From Figure 7, it can be seen that the enrichment effect of Experimental Example 18 is 46.4 times that of Comparative Example 1BGE without NaCl.

Experimental example 19 Field amplification combined with large volume pressure injection

Compared with the experimental example 8, the electric injection is changed to the pressure injection. The comparison results are shown in Figure 8. From Figure 8, it can be seen that the field amplification combined with the large-volume electric injection and the field amplification combined with the large-volume pressure injection are rich. The enrichment times were 46.4 times and 40.8 times, respectively, and the enrichment times of the two were not much different. In the process of electric injection, the number of ions in the sample may affect the injection volume. In order to eliminate the electric injection being easily affected by the current and the reproducibility is poor, parallel sample preparation can be used to eliminate the interference. Comparing the CS peak shapes of the two enrichment methods, it can be seen that the electro-injection peak shape is better, the peak height is higher, and the signal-to-noise ratio is higher.

Experimental example 20

250 μg/mL CS solution, measure 1 mL of this CS solution and add 0.1 mL of 4% CTAC, shake gently and let stand for 1 h. Then, 0.2 mL of 1:9 sulfuric acid solution was added, shaken for 5 min, and the CS-CTAC associate was precipitated to the bottom of the EP tube, and centrifuged at 7000 rpm for 20 min. The supernatant was discarded. In order to free CS from the CS-CTAC associate, 0.1 mL of 5 mol/L NaCl was added to the precipitate, and the precipitate was dissolved by vortexing for 30 s. Add 5.5 mL of ethanol to precipitate CS, and centrifuge at 7000 rpm for 10 min after mixing. Separate the precipitate and the supernatant, add 200 μL of ultrapure water to the precipitate, vortex to dissolve, add 2.5 mL of ethanol to the supernatant, mix well, centrifuge at 7000 rpm for 10 min, discard the supernatant, add 200 μL of ultrapure water to the precipitate, and vortex. Spin to dissolve. The dissolved solutions of the two precipitates were combined and filtered through a 0.22 μm filter.

Then, electrophoresis detection was performed, and the electrophoresis conditions were: detection wavelength 200 nm, electrophoresis temperature 25°C, sample storage temperature 4°C, and separation voltage -15V.

Experimental example 21

Compared with Experimental Example 20, the addition amount of 4% CTAC was 0.05 mL.

Experimental example 22

Compared with Experimental Example 20, the addition amount of 4% CTAC was 0.15 mL.

Experimental example 23

Compared with Experimental Example 20, the amount of 4% CTAC added was 0.2 mL.

Experimental example 24

Compared with Experimental Example 20, the addition amount of 4% CTAC was 0.25 mL.

The enrichment peak areas of Experimental Example 20 to Experimental Example 24 are shown in Table 1.

Table 1 Optimization of CTAC dosage

From Table 1, it can be seen that with the increase of CTAC dosage, the enrichment effect does not increase significantly.

Experimental example 25

Compared with Experimental Example 20, the amount of ethanol used was 10 mL.

Experimental example 26

Compared with Experimental Example 20, the amount of ethanol used was 6 mL.

Experimental example 28

Compared with Experimental Example 20, the amount of ethanol used was 5 mL.

Experimental example 29

Compared with Experimental Example 20, the amount of ethanol used was 2.5 mL.

The peak area of the enrichment effect of Experimental Example 20, Experimental Example 25-Experimental Example 29 is shown in Table 2. It can be seen from Table 2 that with the increase of ethanol consumption, the enrichment effect increases, but when V _ethanol : V _sample is greater than After 8:1, the enrichment effect did not increase significantly.

Table 2 Optimization of ethanol dosage

Experimental example 30

Compared with Experimental Example 20, the number of alcohol precipitation was changed to 1 time.

Experimental example 31

Compared with Experimental Example 20, the number of alcohol precipitation was changed to 3 times.

Experimental example 32

Compared with Experimental Example 20, the number of alcohol precipitation was changed to 4 times.

Experimental example 33

Compared with Experimental Example 20, the number of alcohol precipitation was changed to 5 times.

The peak areas of the enrichment effect of Experimental Example 20, Experimental Example 30 to Experimental Example 33 are shown in Table 3. From Table 3, it can be obtained that the enrichment effect increases with the increase of the number of alcohol precipitations. However, after two alcohol precipitations, increasing the number of alcohol precipitations will make the sample pretreatment steps more complicated, while the enrichment effect is not significantly increased.

The enrichment effect comparison between offline enrichment (Experiment 20) and conventional injection (Comparative Example 1) is shown in Figure 8. It can be seen that offline enrichment can achieve 37.7 times the enrichment effect.

Establishment of standard curve

A series of concentrations of CS solutions were prepared and determined using an online enrichment method after off-line enrichment pretreatment. As shown in Figure 9, the peak area of CS has a good linear relationship with the CS concentration in the range of 1 μg/mL to 100 μg/mL, and the regression equation is y=265033x+319323, r=0.9990 (n=6).

Methodological validation

To determine the limit of quantification and limit of detection of this method, a series of low-concentration CS solutions were prepared, and the limit of quantification and detection limit of this method were determined with 10-fold and 3-fold signal-to-noise ratio (S/N), respectively. The experimental results showed that the quantification limit of this method was 200ng/ml (S/N=10.32), and the detection limit was 50ng/ml (S/N=4.34).

The precision and accuracy of the established method are also verified in the experiments. The CS concentrations were selected as 10 μg/mL, 50 μg/mL and 90 μg/mL for verification, and each concentration was measured three times in parallel. The experimental results are shown in Table 4.

Table 4 Precision and Accuracy of Methods

Experimental example 34

In order to verify the feasibility of the established CE enrichment method, the experiment verified the applicability of the method by measuring the CS content in compound chondroitin sulfate tablets. The experiment used the Chinese Pharmacopoeia (2015 edition) method to determine the same sample (the same sample as Example 1), and the HPLC method was used to determine the content of CS, and the CS was enzymatically decomposed into disaccharide units with chondroitin sulfate ABC enzyme. The linear relationship between the sum of the peak areas of the products CSA, CSB and CSC and the concentration of CS was used to quantify CS. The chromatographic column used in the experiment was a strong anion exchange column. Mobile phase A is 2mol/L NaCl-HCl solution (pH 3.5), mobile phase B is HCl solution (pH 3.5), gradient elution. For the specific steps, please refer to the Chinese Pharmacopoeia (2015 edition) on page 1340 of the second part.

The results obtained by the two methods (Example 1 and the method of the Chinese Pharmacopoeia) were compared with the labeled amount, as shown in Table 5. From Table 5, it can be seen that the enrichment method of the present invention is similar to the declared amount in the method in the Pharmacopoeia.

Table 5 Comparison of CE method and Chinese Pharmacopoeia (2015 edition) method

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. A CS capillary electrophoresis analysis method is characterized in that: enriching CS by using an off-line enrichment method, and then carrying out sample injection by using a method of combining field amplification with large-volume electric sample injection accumulation;

the off-line enrichment method comprises the following steps: mixing the CS water solution with the quaternary ammonium salt cationic surfactant solution, adding a sulfuric acid solution, discarding the supernatant, adding NaCl into the precipitate, carrying out alcohol precipitation, separating the precipitate and the supernatant, carrying out alcohol precipitation on the CS, combining the CS precipitates, and dissolving the CS precipitates with ultrapure water to obtain an enrichment solution.

Preparing the enrichment solution after the off-line enrichment into a CS alkali solution, and adopting a method of combining field amplification and large-volume electrokinetic sample injection for the on-line enrichment, wherein BGE is 20mmol/LNaH₂PO₄90mmol/L EDA-NaCl-phosphate buffer, pH 3.0.

2. The CS capillary electrophoresis analysis method of claim 1, wherein: in the electric sample injection, the concentration of NaCl in BGE is 10-30 mmol/L; preferably 10-20 mmol/L.

3. The CS capillary electrophoresis analysis method of claim 1, wherein: in the field amplification and large-volume electric sample injection accumulation, the sample injection time is 60-420 s; preferably, the sample injection time is 300-420 s.

4. The CS capillary electrophoresis analysis method of claim 1, wherein: in the field amplification and large-volume electrokinetic sample accumulation, the alkali concentration is 27-108 mmol/L; preferably, the concentration of the base is 27 to 54 mmol/L.

5. The CS capillary electrophoresis analysis method of claim 1, wherein: the quaternary ammonium salt cationic surfactant is cetylpyridinium chloride, cetylpyridinium bromide, cetyltrimethylammonium chloride and cetyltrimethylammonium bromide; preferably cetyltrimethylammonium chloride.

6. The CS capillary electrophoresis analysis method of claim 1, wherein: in the off-line enrichment method, the concentration of the CTAC solution is 4g/mL, and the volume ratio of the CTAC solution to the CS aqueous solution is 1-1.5: 10.

7. The CS capillary electrophoresis analysis method of claim 1, wherein: the sulfuric acid solution is a solution of 98% sulfuric acid and water, the volume ratio of the 98% sulfuric acid to the water is 1:9, and the volume ratio of the CS aqueous solution to the sulfuric acid solution is 4-6: 1; preferably 5: 1.

8. The CS capillary electrophoresis analysis method of claim 1, wherein: in the off-line enrichment method, the concentration of the NaCl solution is 4-6mol/L, and the volume ratio of the CS aqueous solution to the NaCl solution is 9-11: 1; preferably, the volume ratio is 10:1, and the concentration of the NaCl solution is 5 mol/L;

or, the volume ratio of ethanol used in alcohol precipitation to the sample is 5-10: 1; preferably 8-10: 1; further preferred is 8: 1;

or, the times of alcohol precipitation are 1-5 times; preferably 2 to 5 times;

or the sample injection voltage of the online enriched electric sample injection is-8 to-12 Kv; preferably-10 Kv;

or, the electrophoresis conditions are as follows: the detection wavelength is 200nm, the electrophoresis temperature is 20-30 ℃, and the separation voltage is-10 to-20 Kv; preferably, the electrophoresis conditions are: the electrophoresis temperature is 25 ℃, and the separation voltage is-15 Kv.

9. The CS capillary electrophoresis analysis method of claim 8, wherein: before the capillary tube is used, 0.1mol/L NaOH is used for activation, the capillary tube is washed under the pressure of 20psi, then ultrapure water and BGE are used for washing under the pressure of 20psi, and finally high voltage is applied to two ends of the capillary tube for running, the high voltage value is 20kV, the BGE is 20mmol/LNaH₂PO₄90mmol/L EDA-NaCl-phosphate buffer, pH 3.0.

10. Use of the CS capillary electrophoresis assay of any one of claims 1 to 9 for the detection of chondroitin sulfate.

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