Method for detecting residual dimethyl sulfoxide in polysaccharide gel

Abstract

The invention discloses a method for detecting residual dimethyl sulfoxide in polysaccharide gel, and belongs to the technical field of analysis and detection of pharmaceutic adjuvants. The detection method comprises the following steps: adding a polysaccharide gel sample into a solvent, ultrasonically mixing uniformly, and filtering to obtain a test solution; then performing high performance liquid chromatography determination on the test solution to obtain a dimethyl sulfoxide peak area; and substituting the peak area into a dimethyl sulfoxide standard curve to obtain the residual quantity of dimethyl sulfoxide in the polysaccharide gel sample. According to the method, the high performance liquid chromatography is optimized, methanol is used as a sample diluent, and water and methanol are used as mobile phases, so that the problems of overlong detection time and poor separation effect are solved, the detection time is greatly shortened, the separation degree, sensitivity and accuracy of dimethyl sulfoxide detection are improved, and the experimental result is more reliable.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection of pharmaceutical excipients, and particularly relates to a method for detecting residual dimethyl sulfoxide in a polysaccharide gel. Background Art

[0002] Dimethyl sulfoxide is a very important non-protonic polar solvent with low toxicity, high boiling point, good thermal stability and miscibility with water. In the pharmaceutical industry, pharmaceutical excipient-grade dimethyl sulfoxide is often used as an organic solvent, inducer and permeability medium. Based on its ability to effectively eliminate the phase interface in the aqueous solution of epichlorohydrin and increase the activation reaction rate, it is applied to the cross-linking process of cross-linked polysaccharide gel as a facial filler. As an organic solvent in the preparation process of agarose gel, dimethyl sulfoxide will remain in the agarose gel and have a harmful effect on the human body. Therefore, the method of detecting residual dimethyl sulfoxide in agarose gel is of great significance for the safety of product use. At present, there are various ways to detect dimethyl sulfoxide, such as gas chromatography, high performance liquid chromatography and ultraviolet detection.

[0003] The detection of dimethyl sulfoxide in polysaccharide gel is mainly based on gas chromatography, but dimethyl sulfoxide has the characteristics of high boiling point, which leads to the headspace sampling method can only detect the high concentration of the solvent residue in the medicine, resulting in low detection sensitivity. High performance liquid chromatography has a better detection effect on high boiling point substances, and the operation process is simpler than that of gas chromatography. High performance liquid chromatography detection of dimethyl sulfoxide is widely used in the field of medicine, and has not yet been studied in polysaccharide gel. For example, Chinese patent CN111323516A provides a detection method for dimethyl sulfoxide in sodium glucose, which adopts high performance liquid chromatography, water as diluent, and gradient elution to detect dimethyl sulfoxide in sodium glucose. Although the sample processing process of this patent is simple, it is not suitable for water-insoluble polysaccharide gel sample processing. The patent adopts gradient elution but the detection time is long. Chinese patent CN108802254A also provides a method for detecting dimethyl sulfoxide in raw materials. The method adopts high performance liquid chromatography, acetonitrile or methanol acidic aqueous solution as diluent, and adopts isocratic elution. Although the detection time is relatively shortened, the detection and separation effect of this product is poor.

[0004] Therefore, optimizing the HPLC method to overcome the problems of long detection time and poor separation effect in the prior art is of great significance for achieving efficient detection of residual dimethyl sulfoxide in polysaccharide gel. Summary of the invention

[0005] The present invention provides a method for detecting residual dimethyl sulfoxide in a polysaccharide gel, comprising the following steps:

[0006] The polysaccharide gel sample is added to a solvent, ultrasonically mixed, and filtered to obtain a test solution; the test solution is then subjected to high performance liquid chromatography to obtain a dimethyl sulfoxide peak area; the peak area is substituted into a dimethyl sulfoxide standard curve to obtain the residual amount of dimethyl sulfoxide in the polysaccharide gel sample.

[0007] In the above detection method, the polysaccharide gel is selected from one or more of agarose gel, cross-linked sodium hyaluronate gel, sodium alginate gel and chitosan derivative gel.

[0008] In the above detection method, the solvent is methanol.

[0009] In the above detection method, the amount of the solvent used is: 10 mL of solvent is used for every 0.5 g of polysaccharide gel sample.

[0010] In the above detection method, the filtration refers to passing the mixed solution through a 0.22 μm filter membrane.

[0011] In the above detection method, the chromatographic conditions of the high performance liquid chromatography are: chromatographic column: (Diamonsil Plus 5μmC18, 250×4.6mm); column temperature: 32~38℃; wavelength: 200~245nm; flow rate: 0.8~1.2mL / min; injection volume 25μL; mobile phase: a mixed solution of methanol and water; elution time: 15min.

[0012] In the above mobile phase, the volume ratio of water to methanol is 70:30 to 90:10.

[0013] In a specific embodiment, the chromatographic conditions of the HPLC are: chromatographic column: (DiamonsilPlus 5μmC18, 250×4.6mm); column temperature: 35°C; wavelength: 215nm; flow rate: 1ml / min; injection volume 25μL; mobile phase: water: methanol = 80:20; elution time: 15min.

[0014] In the above detection method, the dimethyl sulfoxide standard curve is drawn by the following method:

[0015] Methanol was used to prepare dimethyl sulfoxide standard solutions with concentrations of 0.2 μg / mL, 0.4 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL and 8.0 μg / mL, respectively. The dimethyl sulfoxide standard solutions were subjected to high performance liquid chromatography to obtain the peak area of ​​dimethyl sulfoxide. A working curve was then plotted with the peak area of ​​dimethyl sulfoxide as the X-axis and the concentration of dimethyl sulfoxide as the Y-axis to prepare a linear regression equation.

[0016] The invention provides application of the method in detecting residual dimethyl sulfoxide in polysaccharide gel.

[0017] The beneficial effects of the present invention are:

[0018] The invention optimizes the high performance liquid chromatography method, adopts methanol as the sample diluent, and uses water and methanol as the mobile phase, thereby overcoming the problems of long detection time and poor separation effect, greatly shortening the detection time, and improving the separation degree, sensitivity and accuracy of dimethyl sulfoxide detection, and the experimental result is more reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the high performance liquid chromatogram of dimethyl sulfoxide described in Example 1;

[0020] Figure 2 is the high performance liquid chromatogram of dimethyl sulfoxide described in Example 2;

[0021] Figure 3 is the high performance liquid chromatogram of dimethyl sulfoxide described in Example 3;

[0022] Figure 4 is the high performance liquid chromatogram of dimethyl sulfoxide described in Comparative Example 1;

[0023] Figure 5 is the high performance liquid chromatogram of dimethyl sulfoxide described in Comparative Example 2;

[0024] Figure 6 is the high performance liquid chromatogram of dimethyl sulfoxide described in Comparative Example 3;

[0025] Figure 7 HPLC chromatograms of blank solution and dimethyl sulfoxide standard solution. DETAILED DESCRIPTION

[0026] The instruments and reagents used in the present invention are as follows:

[0027] Instruments: Shimadzu high performance liquid chromatograph, vortex oscillator, digitally controlled ultrasonic cleaner. Reagents: dimethyl sulfoxide, methanol, purified water, sodium hydroxide, epichlorohydrin, agarose.

[0028] To draw a dimethyl sulfoxide standard curve, the steps are as follows:

[0029] (1) Preparation of standard stock solution

[0030] Take a 100mL volumetric flask, add a little methanol first, accurately transfer 100mg of dimethyl sulfoxide standard into the volumetric flask, add methanol to make up to volume, shake well, and prepare a standard solution with a concentration of 1mg / mL. Then take 2mL of the standard solution into a 50mL volumetric flask to prepare a standard stock solution with a concentration of 40μg / mL, and refrigerate at 4℃ for later use.

[0031] Take 6 10mL volumetric flasks, add a little methanol first, add the corresponding volume of dimethyl sulfoxide external standard stock solution according to the concentration requirements in Table 1 below, add methanol to make up to volume, shake well, filter with a 0.22μm filter membrane and test. The required volume of standard stock solution is shown in Table 1 below.

[0032] (2) Chromatographic conditions

[0033] Chromatographic column: (Diamonsil Plus 5μm C18, 250×4.6mm); column temperature: 35℃; wavelength: 215nm; flow rate: 1ml / min; injection volume: 25μL; mobile phase: water:methanol=80:20; elution time: 15min.

[0034] (3) Draw a standard curve

[0035] The test solution was taken and measured according to the above chromatographic conditions. The test results are shown in Table 1.

[0036] Table 1 Peak areas of dimethyl sulfoxide standards at different concentrations

[0037]

[0038] According to the data in Table 1, the working curve was drawn with the peak area of ​​dimethyl sulfoxide as the X-axis and the concentration of the dimethyl sulfoxide standard solution as the Y-axis. The linear regression equation was Y=14172x-3127.6, R 2 =0.9969.

[0039] The agarose gel used in the present invention is homemade, and its preparation method is as follows:

[0040] (1) Microsphere preparation

[0041] Weigh 5g of agarose powder in 155g of water, place in a pressure steam sterilizer, sterilize at 121℃ for 15min, dissolve the agarose, and place in a water bath to keep warm. Weigh 12g of Span 85 in 300g of liquid paraffin, stir for 30min in a 60℃ water bath stirrer. Pour the mixture of Span 85 and liquid paraffin into the agarose solution, and emulsify it at 2000r / min for 30min under 60℃ water bath conditions. After emulsification, place the beaker in a room temperature water bath (26℃) and stir to cool down, with a speed of 200r / min, stir for 30min, and solidify. After solidification, use 700mL of anhydrous ethanol (40℃) to wash at 250r / min for 30min, for a total of 4 times; then centrifuge at 4000r / min for 10min. Then, 700 mL of RO water was used to wash at 250 r / min for 30 min, for a total of 3 times; then, the microspheres were centrifuged at 4000 r / min for 10 min, and before the last centrifugation, the microspheres were sieved and filtered with a 100-mesh screen to obtain agarose microspheres.

[0042] (2) Gel preparation

[0043] Take 30mL NaOH (1mol / L), add 0.2g epichlorohydrin and 0.4mL dimethyl sulfoxide to it, and mix well. Weigh 30g agarose microspheres, add the mixture of NaOH, epichlorohydrin and dimethyl sulfoxide to the agarose microspheres, mix again to completely disperse the microspheres in the centrifuge tube. Pour the mixed microspheres into a three-necked flask, water bath at 60℃, 250r / min, stir and crosslink for 4h. After crosslinking, terminate the crosslinking with RO water and wash 5 times with 250mL PBS (stirring conditions: 250r / min, 15min). Separate the agarose gel by centrifugation at 10000r / min for 10min, pour out the water after centrifugation, add clean PBS to rinse the gel in the centrifuge cup, repeat 5 times to obtain agarose gel.

[0044] Other materials used in the present invention, if not otherwise stated, can be obtained through commercial channels. Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way.

[0045] Example 1

[0046] The determination of residual dimethyl sulfoxide in agarose gel is as follows:

[0047] Weigh 0.5 g of agarose gel sample, add 10 mL of methanol, mix by ultrasonication for 30 min, and filter through a 0.22 μm filter membrane to prepare the test solution.

[0048] Chromatographic conditions:

[0049] Chromatographic column: (Diamonsil Plus 5μm C18, 250×4.6mm); column temperature: 35℃; wavelength: 215nm; flow rate: 1ml / min; injection volume: 25μL; mobile phase: water:methanol=80:20; elution time: 15min.

[0050] The test results are as follows Figure 1 As shown:

[0051] This method can be used to detect dimethyl sulfoxide in the test solution, and its dimethyl sulfoxide retention time is 3.5min, and the peak shape and separation (1.853) are good. The peak area is measured to be 1809, and the data is substituted into the linear regression equation to obtain the concentration of dimethyl sulfoxide in the test solution to be 0.35μg / mL.

[0052] Example 2

[0053] This example adopts the chromatographic conditions in the above-mentioned Example 1, and uses an acidic aqueous solution (phosphoric acid adjusted to pH 2.2) and methanol as the test sample diluent for detection.

[0054] The determination of residual dimethyl sulfoxide in agarose gel is as follows:

[0055] Weigh 0.5 g of agarose gel sample, add 10 mL of a mixture of phosphoric acid aqueous solution (pH 2.2) and methanol (90:10), mix by ultrasonication for 30 min, and filter through a 0.22 μm filter membrane to prepare the test solution.

[0056] Chromatographic conditions:

[0057] Chromatographic column: (Diamonsil Plus 5μm C18, 250×4.6mm); column temperature: 35℃; wavelength: 215nm; flow rate: 1ml / min; injection volume: 25μL; mobile phase: water:methanol=80:20; elution time: 15min.

[0058] The test results are as follows Figure 2 As shown:

[0059] Depend on Figure 2 It can be seen that when phosphoric acid aqueous solution and methanol are used as diluents for the test sample, the peak shape of the target peak (RT-3.052) is abnormal.

[0060] Example 3

[0061] This example adopts the chromatographic conditions in the above example 1, and uses an acidic aqueous solution (phosphoric acid adjusted to pH 2.2) and methanol as the mobile phase for detection.

[0062] The determination of residual dimethyl sulfoxide in agarose gel is as follows:

[0063] Weigh 0.5 g of agarose gel sample, add 10 mL of methanol, mix by ultrasonication for 30 min, and filter through a 0.22 μm filter membrane to prepare the test solution.

[0064] Chromatographic conditions:

[0065] Chromatographic column: (Diamonsil Plus 5μm C18, 250×4.6mm); column temperature: 35℃; wavelength: 215nm; flow rate: 1ml / min; injection volume: 25μL; mobile phase: phosphoric acid aqueous solution (pH 2.2): methanol = 90:10; elution time: 15min.

[0066] The test results are as follows Figure 3 As shown:

[0067] Depend on Figure 3 It can be seen that the retention time of dimethyl sulfoxide is 3.236 min, and its separation degree (≥1.5) is 0.523. It can be seen that the separation effect is poor when using phosphoric acid aqueous solution and methanol as mobile phases for detection.

[0068] Comparative Example 1

[0069] The dimethyl sulfoxide residue in the agarose gel test sample was detected by the method provided in patent CN 111323516 A; the test sample used purified water as the diluent.

[0070] The test results are shown in Table 2 and Figure 4 As shown: The residual amount of dimethyl sulfoxide in agarose gel could not be detected, indicating that this method is not suitable for detecting the residual amount of dimethyl sulfoxide in agarose gel.

[0071] Comparative Example 2

[0072] The method provided in patent CN 108802254 A was used to detect the residual amount of dimethyl sulfoxide in the agarose gel test sample.

[0073] The test results are shown in Table 2 and Figure 5 As shown:

[0074] The retention time of dimethyl sulfoxide is 2.98 min, and the resolution is 0.495. The separation effect of this method is poor and cannot achieve good detection of dimethyl sulfoxide.

[0075] Table 2 Test results of each test solution

[0076] Serial number Group Retention time (min) Separation (≥1.5) 1 Test solution - Example 1 3.500 1.853 2 Standard solution-Comparative Example 1 3.166 1.103 3 Test solution - comparative example 1 — — 4 Test solution - Comparative Example 2 2.980 0.495

[0077] Comparative Example 3

[0078] The method in Example 1 and patent CN 108802254 A was used to simultaneously determine 8 μg / mL dimethyl sulfoxide standard, and the injection volume was 25 μL.

[0079] The test results are shown in Table 3 and Figure 6 As shown:

[0080] Table 3 Determination results under different test methods

[0081]

[0082] From the above test data, it can be seen that the detection sensitivity of the detection method of Example 1 of the present invention is much higher than that of the patent CN

[0083] The method in 108802254A is more reliable for detecting products with lower dimethyl sulfoxide content.

[0084] Verification Example 1 Detection Limit and Quantification Limit Test

[0085] The standard solution was gradually diluted and detected using the method in Example 1, with a signal-to-noise ratio of 3:1 as the detection limit of dimethyl sulfoxide and a signal-to-noise ratio of 10:1 as the quantification limit of dimethyl sulfoxide. The test results are as follows: the detection limit concentration of dimethyl sulfoxide is 0.28 μg / mL, and the quantification limit concentration of dimethyl sulfoxide is 1.0 μg / mL.

[0086] Verification Example 2: Specificity Test

[0087] Blank solution: methanol.

[0088] Standard solution: The same as the standard solution with a concentration of 4.0 μg / mL under the standard curve in Example 1.

[0089] Test solution: the same as the test solution in Example 1.

[0090] Test sample-standard solution: Mix 1.5 mL of test sample solution with 1.5 mL of standard solution to obtain the test sample-standard solution.

[0091] The dimethyl sulfoxide in the above solution was detected using the method in Example 1.

[0092] The test results are shown in Table 4:

[0093] Table 4 Specificity test

[0094]

[0095]

[0096] As shown in Table 4, the blank solution (chromatogram Figure 7 ) does not interfere with the detection of dimethyl sulfoxide. In addition, the resolution between each peak is greater than 1.5, and the separation effect is good. It meets the requirements of specificity test.

[0097] Verification Example 3: Precision Test

[0098] Take the 4 μg / mL standard solution in Example 1 and divide it into 6 parts for testing. The testing method is the same as that in Example 1 above.

[0099] The test results are shown in Table 5:

[0100] Table 5 Precision test

[0101]

[0102] As shown in Table 5, the target peak area RSD is 1.50%, and the lower the RSD, the better the precision result, which meets the requirements of the precision test.

[0103] Verification Example 4 Accuracy Test

[0104] 80% spiked test solution: Take 0.5 mL of the test solution of Example 1, accurately add 0.5 mL of the standard solution (4 μg / mL), then add 9 ml of methanol, mix well, and filter through a 0.22 μm filter membrane; prepare 3 portions in parallel.

[0105] 100% spiked test solution: Take 0.5 ml of the test solution of Example 1, accurately add 0.5 ml of the standard solution (5 μg / mL), then add 9 ml of methanol, mix well, and filter through a 0.22 μm filter membrane; prepare 3 portions in parallel.

[0106] 120% spiked test solution: Take 0.5 ml of the test solution of Example 1, accurately add 0.5 ml of the standard solution (6 μg / mL), then add 9 ml of methanol, mix well, and filter through a 0.22 μm filter membrane; prepare 3 portions in parallel.

[0107] The dimethyl sulfoxide in the above solution was detected using the method in Example 1.

[0108] The test results are shown in Table 6:

[0109] Table 6 Accuracy test

[0110]

[0111]

[0112] As shown in Table 6, the recovery rate of dimethyl sulfoxide in the test sample is about 100.5%, which meets the requirement of accuracy limit of 80-115%. The accuracy of this method is good.

[0113] Verification Example 5

[0114] In order to further verify that the detection method of the present invention can be used for the detection of polysaccharide gel, cross-linked sodium hyaluronate gel was selected as the object for the detection of dimethyl sulfoxide.

[0115] The method in Example 1 was used to test the cross-linked sodium hyaluronate gel as a test sample.

[0116] Determination of dimethyl sulfoxide content in the test sample:

[0117] The same test solution (3) was taken and tested according to the above test conditions. The test results are shown in Table 7.

[0118] Table 7 Cross-linked sodium hyaluronate gel results

[0119] Serial number name Retention time (min) Separation (≥1.5) 1 Cross-linked sodium hyaluronate gel 3.529 1.753 2 Cross-linked sodium hyaluronate gel 3.525 1.689 3 Cross-linked sodium hyaluronate gel 3.528 1.725

[0120] As can be seen from Table 7, this method can be used for the detection of dimethyl sulfoxide in cross-linked sodium hyaluronate gel, which further verifies the versatility and accuracy of the method of the present invention in the detection of dimethyl sulfoxide residues in polysaccharide gels.

[0121] In this verification example, the cross-linked sodium hyaluronate gel used was prepared according to patent CN110396204A, and its preparation method is as follows:

[0122] (1) Preparation of gel: First prepare 100 mL of 0.5 mol / L sodium hydroxide solution, add about 5 g of sodium hyaluronate at room temperature, mix well and stir to dissolve until no white undissolved HA is visible to the naked eye, adjust the water bath temperature to 50° C., then add 2.5 g of cross-linking agent, cross-link for 4 hours to obtain a gel.

[0123] (2) Preparation of gel microparticles: The cross-linked gel was swollen in purified water for 1 hour and then prepared into gel microparticles of appropriate particle size using a high-speed disperser.

[0124] (3) Washing of gel particles: Adjust the pH to 7.2 with dilute hydrochloric acid, add three times the volume of dimethyl sulfoxide, stir and wash for 1 hour, filter, and wash three times. Then use ten times the volume of purified water, stir and wash for 1 hour, filter, and wash five times. Obtain cross-linked sodium hyaluronate gel.

[0125] Unless otherwise stated, other materials used in the present invention can be obtained through commercial channels. Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0126] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A method for detecting residual dimethyl sulfoxide in polysaccharide gel, characterized in that: The steps include: The polysaccharide gel sample is added to a solvent, ultrasonically mixed, and filtered to obtain a test solution; the test solution is then subjected to high performance liquid chromatography to obtain a dimethyl sulfoxide peak area; the peak area is substituted into a dimethyl sulfoxide standard curve to obtain the residual amount of dimethyl sulfoxide in the polysaccharide gel sample.

2. The detection method according to claim 1, characterized in that: The polysaccharide gel is selected from one or more of agarose gel, cross-linked sodium hyaluronate gel, sodium alginate gel and chitosan derivative gel.

3. The detection method according to claim 1, characterized in that: The solvent is methanol.

4. The detection method according to claim 1, characterized in that: The amount of the solvent used is: 10 mL of solvent is used for every 0.5 g of polysaccharide gel sample.

5. The detection method according to claim 1, characterized in that: The filtering refers to passing the mixed solution through a 0.22 μm filter membrane.

6. The detection method according to claim 1, characterized in that: The chromatographic conditions of the HPLC are as follows: chromatographic column: (Diamonsil Plus 5 μm C18, 250×4.6 mm); column temperature: 32-38° C.; Wavelength: 200-245 nm; flow rate: 0.8-1.2 mL / min; injection volume: 25 μL; mobile phase: a mixed solution of methanol and water; elution time: 15 min.

7. The detection method according to claim 6, characterized in that: The volume ratio of water to methanol is 70:30 to 90:

10.

8. The detection method according to claim 7, characterized in that: The volume ratio of water to methanol is 80:

20.

9. The detection method according to claim 1, characterized in that: The dimethyl sulfoxide standard curve is drawn by the following method: Methanol was used to prepare dimethyl sulfoxide standard solutions with concentrations of 0.2 μg / mL, 0.4 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL and 8.0 μg / mL, respectively. The dimethyl sulfoxide standard solutions were subjected to high performance liquid chromatography to obtain the peak area of ​​dimethyl sulfoxide. A working curve was then plotted with the peak area of ​​dimethyl sulfoxide as the X-axis and the concentration of dimethyl sulfoxide as the Y-axis to prepare a linear regression equation.

10. Use of the method according to any one of claims 1 to 9 in detecting residual dimethyl sulfoxide in polysaccharide gel.

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