Method for detecting content of liquid paraffin in polysaccharide gel

Abstract

The invention discloses a method for detecting the content of liquid paraffin in polysaccharide gel, and belongs to the technical field of analysis and detection of pharmaceutic adjuvants. The detection method comprises the following steps: introducing a polysaccharide gel test solution into a gas chromatograph, measuring the peak area of liquid paraffin, selecting a most representative peak, and substituting the peak area of the peak into a liquid paraffin standard curve to obtain the concentration of the liquid paraffin in the polysaccharide gel test solution, and finally substituting the concentration of the liquid paraffin into a calculation formula to obtain the content of the liquid paraffin in the test solution. According to the method, the gas chromatograph is used for detecting the polysaccharide gel containing the liquid paraffin, the liquid paraffin is directly used as a reference substance, the n-hexane is used as a solvent, and the content of the liquid paraffin is calculated by selecting the most representative peak area, so that the sensitivity and the accuracy of liquid paraffin detection are improved, the detection time is shortened, and the experimental result is more reliable. And meanwhile, quantitative detection of the content of the liquid paraffin can be realized.

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 the content of liquid paraffin in a polysaccharide gel. Background Art

[0002] Liquid paraffin is a typical industrial mineral oil, which is divided into industrial grade, food grade, cosmetic grade and pharmaceutical grade, and can be mixed with most fatty oils except castor oil. In food, liquid paraffin is often used for food glazing, anti-sticking, moisture-proof, defoaming, sealing, demoulding, etc., and can extend the storage and shelf life of fruits, vegetables, eggs, canned food, etc., but it is not absorbed by the stomach and intestines. If it is ingested for a long time, it can cause digestive system disorders and fat-soluble vitamin absorption disorders. In medicine, pharmaceutical-grade liquid paraffin can be used as an organic solvent and mixed with Span 85 (emulsifier) ​​to form an oil phase for the preparation of small-particle polysaccharide microspheres, and further prepare polysaccharide gels for facial filling. According to the current Chinese Pharmacopoeia and new drug approval regulations, residual solvents in drug synthesis need to be controlled, otherwise the product will cause harm to the human body. Therefore, it is necessary to detect the content of liquid paraffin in the polysaccharide gel to ensure that most of the liquid paraffin has been removed in the subsequent cleaning steps.

[0003] There are many detection methods for liquid paraffin at present, but there is no method for detecting the content of liquid paraffin in gel samples. Most of them are for detecting liquid paraffin in food or hemostatic powder. For example, Chinese patent CN107478650A provides a detection method for residual liquid paraffin in hemostatic powder. The method utilizes a Soxhlet extractor and a petroleum ether (anhydrous ether) water bath heating method to extract the liquid paraffin in the hemostatic powder. By comparing the turbidity of the sample solution with that of a 2% liquid paraffin reference substance, it is determined whether the liquid paraffin in the hemostatic powder is lower than or higher than 2%. However, the patent method can only qualitatively detect the content of liquid paraffin in the hemostatic powder, and there are many interfering factors, so the result is inaccurate and cannot be quantitatively determined. Chinese patent CN110221005A also provides a detection method for liquid paraffin in hemostatic powder. The method utilizes a gas phase-mass spectrometer, takes biphenyl as an internal standard, and performs constant volume detection with chloroform. Although the sample processing process is simple, it has high requirements for instruments and equipment and a long detection time. Summary of the invention

[0004] In order to overcome the problems of many interference factors and long detection time in the prior art, the present invention provides a method for detecting the liquid paraffin content in a polysaccharide gel, which utilizes the GC-FID method to detect a test sample containing liquid paraffin, directly using liquid paraffin as a reference substance and n-hexane as a solvent. Due to the isocarbon response characteristics of a hydrogen flame ionization detector (FID), the problem of lack of a mineral oil standard is avoided, the sensitivity and accuracy of liquid paraffin detection are improved, and the detection result is more reliable.

[0005] The technical solution of the present invention is as follows:

[0006] A method for detecting the content of liquid paraffin in a polysaccharide gel comprises the following steps:

[0007] The polysaccharide gel test solution is introduced into a gas chromatograph, the peak area of ​​the liquid paraffin is measured, the most representative peak is selected, and then the peak area of ​​the peak is substituted into the liquid paraffin standard curve to obtain the liquid paraffin concentration in the polysaccharide gel test solution; finally, the liquid paraffin concentration is substituted into the calculation formula to obtain the liquid paraffin content in the test solution.

[0008] In the above detection method, the polysaccharide gel test solution is prepared by the following method:

[0009] Take 15g of polysaccharide gel sample, add petroleum ether, place in a Soxhlet extraction apparatus, reflux extraction for 1-2h, the reflux temperature is 40-60℃; recover the solvent and evaporate the solvent; dissolve the petroleum ether extract with 5mL of n-hexane and transfer it to a 50mL centrifuge tube, add 4mL of 0.4mol / L potassium hydroxide-methanol solution, shake, let stand for 10min, add 10mL of water, and let stand until the upper solution is transparent; take the upper transparent solution and pass it through a 0.22μm filter membrane to obtain a polysaccharide gel test solution.

[0010] In the above detection method, the reflux extraction time is 2 hours; the reflux temperature is 50°C.

[0011] In the above detection method, the chromatographic conditions of the gas chromatograph are as follows:

[0012] Chromatographic column: Agilent HP-5 (30m*0.32mm*0.25μm); programmed temperature: starting temperature 100℃, maintained for 5min, heated to 250℃ at 20℃ / min, maintained for 30min; injection port temperature: 230~260℃; FID detector temperature: 250~270℃; carrier gas flow rate 40mL / min; hydrogen flow rate 55mL / min; non-split injection, injection volume: 1μL; carrier gas is high-purity nitrogen.

[0013] In the above detection method, the most representative peak is selected from the characteristic peak of liquid paraffin with a retention time in the range of 9.102 to 9.109 min; preferably, the characteristic peak of liquid paraffin with a retention time of 9.107 min.

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

[0015] (1) Preparation of standard stock solution

[0016] Weigh 0.1 g of liquid paraffin into a 50 mL volumetric flask, dissolve it in n-hexane and dilute to the mark, shake well, and use this as paraffin standard stock solution 1, with a concentration of 2 mg / mL; take 25 mL of standard stock solution 1 and put it into a 50 mL volumetric flask, dissolve it in n-hexane and dilute to the mark, shake well, and use this as paraffin standard stock solution 2, with a concentration of 1 mg / mL;

[0017] (2) Preparation of standard curve solution

[0018] The liquid paraffin standard stock solution 2 was gradiently diluted to a series of different standard solutions of 0.0025, 0.005, 0.025, and 0.5 mg / mL, and filtered with a 0.22 μm filter membrane to obtain a standard curve solution;

[0019] (3) Draw a standard curve

[0020] Inject the standard curve solution into the gas chromatograph, determine the peak area of ​​liquid paraffin, record the corresponding chromatogram, draw the standard curve with the concentration of liquid paraffin standard as the abscissa x and the peak area as the ordinate y, and calculate the regression equation.

[0021] In the above detection method, the regression equation can be: y=45.039x+6.3613, R 2 =0.9978.

[0022] In the above detection method, the calculation formula is: C = (C 0 ×5) / M; where C represents the content of liquid paraffin in the test sample, in mg / g; C 0 It represents the concentration of liquid paraffin in the test sample, in mg / mL; M represents the weight of the sample weighed, in g.

[0023] The present invention provides application of the above method in detecting the content of liquid paraffin in polysaccharide gel.

[0024] In the present invention, the polysaccharide gel is selected from one of emulsified cross-linked sodium hyaluronate gel, konjac glucomannan gel and agarose gel; preferably agarose gel.

[0025] The beneficial effects of the present invention are:

[0026] The present invention uses a gas chromatograph to detect polysaccharide gel containing liquid paraffin, directly uses liquid paraffin as a reference substance (using liquid paraffin as a standard substance, preparing liquid paraffin standard solutions of different concentrations, and making a standard curve according to the concentration and peak area of ​​the liquid paraffin standard solution), and n-hexane as a solvent. The content of liquid paraffin is obtained by converting the most representative peak area, thereby improving the sensitivity and accuracy of liquid paraffin detection, shortening the detection time, and making the experimental results more reliable. At the same time, the detection method can quantitatively detect the content of liquid paraffin, has good linearity, and can accurately and sensitively detect polysaccharide gel containing liquid paraffin residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the standard curve of liquid paraffin;

[0028] Figure 2 is the gas chromatogram of the test solution;

[0029] Figure 3 is the gas chromatogram of n-hexane (blank solution);

[0030] Figure 4 It is the gas chromatogram of liquid paraffin standard solution (0.5 mg / mL). DETAILED DESCRIPTION

[0031] The instruments and reagents used in the present invention are as follows:

[0032] Instruments: Soxhlet extractor; Agilent 7890 gas chromatograph; chromatographic analysis column (HP-5, Agilent Technologies Co., Ltd.); vortex oscillator; constant temperature water bath.

[0033] Reagents: liquid paraffin (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); n-hexane; petroleum ether; potassium hydroxide; methanol; purified water; sodium hydroxide; epichlorohydrin; agarose.

[0034] In the present invention, an Rxi-50 capillary column (30m×0.25mm×0.25μm) can also be used to detect the liquid paraffin content in the sample.

[0035] In the present invention, the agarose gel used is prepared by the following method:

[0036] (1) Microsphere preparation

[0037] Weigh 4.80g of agarose powder in 160g of water, place in a pressure steam sterilizer, solid mode 121℃, sterilize for 15min, dissolve the agarose, and place in a water bath to keep warm. Weigh 20g 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 with a stainless steel stirring paddle 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 and stir for 30min. After solidification, wash 4 times with 700mL of 40℃ anhydrous ethanol: 250r / min, 30min; centrifuge: 4000r / min, 10min. Wash with 700 mL of RO water three times: 250 r / min, 30 min; centrifuge: 4000 r / min, 10 min, and filter the microspheres with a 100-mesh screen for the last time.

[0038] (2) Gel preparation

[0039] Measure 30mL 1mol / L NaOH, add 0.5g epichlorohydrin to it, and mix well. Weigh 30g agarose microspheres, add the mixture of NaOH and epichlorohydrin 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 with PBS 5 times (stirring conditions are: 250r / min, 15min, 250mL). 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, and repeat 5 times.

[0040] The present invention provides a method for drawing a liquid paraffin standard curve, the steps of which are as follows:

[0041] (1) Preparation of standard stock solution

[0042] Weigh 0.1 g of liquid paraffin into a 50 mL volumetric flask, dissolve it in n-hexane and dilute to the mark, shake well, and use it as paraffin standard stock solution 1 (2 mg / mL). Take 25 mL of standard stock solution 1 into a 50 mL volumetric flask, dissolve it in n-hexane and dilute to the mark, shake well, and use it as paraffin standard stock solution 2 (1 mg / mL).

[0043] (2) Preparation of standard curve solution

[0044] A 1 mg / mL liquid paraffin standard stock solution 2 was gradiently diluted to a series of different standard solutions of 0.0025, 0.005, 0.025, and 0.5 mg / mL, and the solutions were filtered through a 0.22 μm filter membrane and then tested.

[0045] (3) Chromatographic conditions

[0046] Chromatographic column: Agilent HP-5 (30m*0.32mm*0.25μm);

[0047] Program temperature rise: starting temperature 100℃, maintain for 5 min, increase to 250℃ at 20℃ / min, maintain for 30 min; injection port temperature: 230-260℃; FID detector temperature: 250-270℃; carrier gas flow rate 40mL / min; hydrogen flow rate 55mL / min; splitless injection, injection volume: 1μL; carrier gas is high-purity nitrogen.

[0048] (4) Draw a standard curve

[0049] Inject the standard solution into the gas chromatograph and perform the test according to the above chromatographic conditions. The blank solution is n-hexane. Record the corresponding chromatogram, draw the standard curve with the concentration of the liquid paraffin standard as the abscissa x and the peak area as the ordinate y, and calculate the regression equation.

[0050] like Figure 1 As shown in Table 1:

[0051] Table 1 Peak areas of standards and samples

[0052] Serial number Concentration (mg / mL) Retention time (min) Peak area B1 0.025 9.107 7.021 B2 0.05 9.106 8.892 B3 0.25 9.105 18.494 B4 0.5 9.108 29.236

[0053] According to the peak conditions of the standard solutions (B1-B4) with different concentration gradients, the most representative peak was selected for quantification, that is, the peak with a peak time of 9.107 min was selected as the control, and the standard curve formula was prepared. The regression equation was y=45.039x+6.3613, R 2 =0.9978.

[0054] (5) Calculation formula for liquid paraffin content

[0055] C=(C 0 ×5) / M

[0056] In the above formula, C: liquid paraffin content in the test sample, unit: mg / g; C 0 : The concentration of liquid paraffin in the test sample, in mg / mL; M: The weight of the sample weighed, in g.

[0057] The liquid paraffin content detection method described in the present invention is to introduce a test solution into a gas chromatograph to measure the liquid paraffin peak area (selecting the most representative peak); then the liquid paraffin peak area is substituted into a liquid paraffin standard curve to obtain the liquid paraffin concentration in the test solution; finally, the liquid paraffin concentration is substituted into a calculation formula to obtain the liquid paraffin content in the test solution.

[0058] 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.

[0059] Example 1

[0060] The content of liquid paraffin in agarose gel is detected as follows:

[0061] (1) Preparation of test solution

[0062] Weigh 15g of agarose gel sample, add petroleum ether, place in a Soxhlet extraction apparatus, reflux extraction for 2h, reflux temperature is 50℃. Recover the solvent and evaporate the solvent. Dissolve the petroleum ether extract with 5mL of n-hexane and transfer to a 50mL centrifuge tube, add 4mL of 0.4mol / L potassium hydroxide-methanol solution, shake, let stand for 10min, add 10mL of water, and let stand until the upper layer solution is transparent. Take the upper transparent solution and filter it through a 0.22μm filter membrane to obtain the test solution.

[0063] (2) Determination of liquid paraffin content

[0064] The test solution was injected into an Agilent 7890 gas chromatograph for determination. The instrument chromatographic conditions were consistent with those in the above standard curve drawing method. The peak area of ​​the test solution was measured to be 7.952, the retention time was 9.107 min, and the gas chromatogram was as follows: Figure 2 As shown, the peak area is substituted into the linear regression equation of the standard curve, and the content of liquid paraffin in the test sample is obtained to be 0.012 mg / g.

[0065] Example 2

[0066] Repeatability test of test products:

[0067] Six samples were tested for repeatability using the same method as in Example 1. The test results are shown in Table 2:

[0068] Table 2 Repeatability test of test solution

[0069]

[0070] As shown in Table 2, the relative standard deviation of the calculated content of liquid paraffin in each test sample is 4.76%, and the measured content is basically consistent with that in Example 1, indicating that the repeatability of this method is good.

[0071] Comparative Example 1

[0072] Use clarity to test liquid paraffin, the steps are as follows:

[0073] (1) Preparation of test solution

[0074] Accurately weigh 1g of agarose gel sample and put it into a defatted filter paper bag. Place the filter paper bag into the extraction cylinder of the Soxhlet extractor, connect it to a fat flask that has been dried to constant weight, add anhydrous ether from the upper end of the extractor condenser to 2 / 3 of the bottle volume, pass condensed water, immerse the bottom bottle in a water bath to heat, gently plug a small ball of absorbent cotton into the upper end of the condenser, control the water bath temperature at 45°C, and extract for 8 hours.

[0075] After the extraction is completed, remove the fat flask and recover the anhydrous ether. When there is only 1-2 mL of ether left in the flask, evaporate the residual solvent on a water bath, add 5 mL of anhydrous ethanol to the fat flask, rinse the flask thoroughly in an 80°C water bath (to avoid spilling droplets), add 5 drops of 0.5 mol / L sodium hydroxide solution, continue to react in an 80°C water bath for 10 min, remove and add 5 mL of purified water, shake well and transfer to a cuvette.

[0076] (2) Preparation of 2% liquid paraffin reference substance

[0077] Dissolve 2 mL of liquid paraffin in 98 mL of anhydrous ethanol in a water bath at 80°C. Take 1 mL of the hot anhydrous ethanol solution of liquid paraffin and place it in a colorimetric tube. Then add 5 mL of anhydrous ethanol and 5 drops of 0.5 mol / L sodium hydroxide. Heat at 80°C for 10 min. Add 5 mL of purified water and observe the clarity.

[0078] (3) Clarity comparison

[0079] By comparing the clarity of the test solution with that of the 2% liquid paraffin reference, it is determined whether the liquid paraffin is lower or higher than 2%. The comparison results are shown in Table 3.

[0080] Table 3 Clarity comparison results

[0081] name Test solution 2% liquid paraffin solution Clarity Slightly turbid turbid

[0082] According to the clarity comparison results, it was determined that the liquid paraffin content in the test solution was less than 2%, but this method can only be used to roughly examine the liquid paraffin content in the test solution and cannot be used for accurate quantification.

[0083] Comparative Example 2

[0084] The content of liquid paraffin in agarose gel is detected as follows:

[0085] Weigh 15g of agarose gel sample, add petroleum ether, place in a Soxhlet extraction apparatus, reflux extraction for 2h, reflux temperature is 70℃. Recover the solvent and evaporate the solvent. Dissolve the petroleum ether extract with 5mL of n-hexane and transfer to a 50mL centrifuge tube, add 4mL of 0.4mol / L potassium hydroxide-methanol solution, shake, let stand for 10min, add 10mL of water, and let stand until the upper solution is transparent. After taking the upper transparent solution through a 0.22μm filter membrane, inject the test solution into an Agilent 7890 gas chromatograph for determination.

[0086] According to the test results, the reason for the lower extraction efficiency of petroleum ether with a higher boiling point in the oil bath may be that the sample peak area is small and the separation is low, which is not conducive to calculation. Therefore, under the same reflux extraction time conditions, the lower the reflux temperature (50°C), the higher the extraction efficiency.

[0087] Verification Example 1

[0088] Specificity test:

[0089] Weigh 15g of agarose gel sample, add petroleum ether, place in a Soxhlet extraction apparatus, reflux extraction for 2h, reflux temperature is 50℃. Recover the solvent and evaporate the solvent. Dissolve the petroleum ether extract with 5mL of n-hexane and transfer to a 50mL centrifuge tube, add 4mL of 0.4mol / L potassium hydroxide-methanol solution, shake, let stand for 10min, add 5mL of 0.025mg / mL liquid paraffin standard solution, add 5mL of water, let stand until the upper solution is transparent, and obtain the test sample-standard solution. Inject 1μL for GC analysis.

[0090] The analysis results are as follows Figure 3 As shown in Table 4:

[0091] Table 4 Specificity test

[0092] Serial number name Retention time (min) Separation (≥1.5) 1 n-Hexane(blank) ——— —— 2 Liquid paraffin standard solution (0.025 mg / mL) 9.105 54.04 3 Test sample-liquid paraffin standard solution 9.106 51.69

[0093] As shown in Table 4, the blank solution has no interference at the peak position of liquid paraffin. According to the measured separation degree of the most representative peak of liquid paraffin in the test-standard solution and the adjacent peaks is 51.69, the specificity of this method is good.

[0094] Verification Example 2

[0095] Repeatability test:

[0096] Take a 0.5 mg / mL standard solution and divide it into 5 samples. Inject 1 μL for GC analysis.

[0097] The analysis results are as follows Figure 4 As shown in Table 5:

[0098] Table 5 Repeatability test of liquid paraffin standard solution

[0099]

[0100] It can be seen from Table 5 that the relative standard deviation of the peak area was calculated based on the measured peak area of ​​the standard solution, and the result was 1.65%, which meets the requirement of not exceeding 4% stipulated in the General Rules 9101 of Part IV of the 2020 edition of the Chinese Pharmacopoeia. This method has good repeatability.

[0101] Verification Example 3

[0102] Detection limit and quantification limit test:

[0103] Take standard solutions of different concentrations (1.2 μg / mL, 1.8 μg / mL, 2.7 μg / mL, 5.5 μg / mL, 8.2 μg / mL, 10 μg / mL) and inject 1 μL for GC analysis.

[0104] According to the General Rules 9101 of Part IV of the 2020 edition of the "Chinese Pharmacopoeia", the detection limit signal-to-noise ratio method: the detection limit is generally determined by the corresponding concentration when the signal-to-noise ratio is 3:1 or the amount injected into the instrument. The measured standard concentration corresponding to S / N=3.05 is 2.7μg / mL; the quantification limit signal-to-noise ratio method: the quantification limit is generally determined by the corresponding concentration when the signal-to-noise ratio is 10:1 or the amount injected into the instrument. The measured standard concentration corresponding to S / N=10.08 is 8.2μg / mL.

[0105] Verification Example 4

[0106] Prepare the test solution according to Example 1 until the step before adding water:

[0107] Weigh 15g of agarose gel sample, add petroleum ether, place in a Soxhlet extraction apparatus, reflux for 2h, reflux temperature is 50℃. Recover the solvent and evaporate it. Dissolve the petroleum ether extract with 5mL of n-hexane and transfer to a 50mL centrifuge tube, add 4mL of 0.4mol / L potassium hydroxide-methanol solution, shake, and let stand for 10min.

[0108] To the above test solution, accurately add 5 mL of liquid paraffin standard solution (0.04 mg / mL), then add 5 mL of water, mix well, let stand and take the supernatant; prepare 3 portions in parallel to obtain 80% sampled test solutions 1 to 3.

[0109] To the above test solution, accurately add 5 mL of liquid paraffin standard solution (0.05 mg / mL), then add 5 mL of water, mix well, let stand and take the supernatant; prepare 3 portions in parallel to obtain 100% sampled test solutions 1 to 3.

[0110] To the above test solution, accurately add 5 mL of liquid paraffin standard solution (0.06 mg / mL), then add 5 mL of water, mix well, let stand and take the supernatant; prepare 3 portions in parallel to obtain 120% sampled test solutions 1 to 3.

[0111] 1 μL of the above solution was injected for GC analysis.

[0112] The analysis results are shown in Table 6:

[0113] Table 6 Spiked recovery results

[0114]

[0115]

[0116] It can be seen from Table 6 that the accuracy was calculated based on the measured content of liquid paraffin added to the test sample. The results showed that the recovery rate of liquid paraffin in the test sample was 92.5-108%, which meets the accuracy limit of 85-115% specified in General Rules 9101 of Part IV of the 2020 edition of the Chinese Pharmacopoeia. The accuracy of this method is good.

[0117] Example 3

[0118] The technical solution of the present invention can also be used for quantitative detection of liquid paraffin content in other polysaccharide gels, such as emulsified cross-linked sodium hyaluronate gel for injection (Chinese patent CN 111686664 A). The preparation method of the liquid paraffin standard curve and the gas chromatography conditions are consistent with those of the above-mentioned agarose gel detection. The sample processing steps are as follows:

[0119] (1) Preparation of test solution

[0120] Weigh 15g of emulsified cross-linked sodium hyaluronate gel sample for injection, add petroleum ether, place in a Soxhlet extraction apparatus, reflux extraction for 2h, reflux temperature is 50℃. Recover the solvent and evaporate the solvent. Dissolve the petroleum ether extract with 5mL of n-hexane and transfer to a 50mL centrifuge tube, add 4mL of 0.4mol / L potassium hydroxide-methanol solution, shake, stand for 10min, add 10mL of water, and stand until the upper solution is transparent. Take the upper transparent solution and filter it through a 0.22μm filter membrane to obtain the test solution.

[0121] (2) Determination of liquid paraffin content

[0122] The test solution was injected into an Agilent 7890 gas chromatograph for determination. The chromatographic conditions of the instrument were consistent with those in the above-mentioned standard curve drawing method. The peak area of ​​the test solution was measured to be 10.370, and the retention time was 9.109 min. The peak area was substituted into the linear regression equation of the standard curve, and the content of liquid paraffin in the test sample was obtained to be 0.03 mg / g.

[0123] (3) Preparation of emulsified cross-linked sodium hyaluronate gel for injection

[0124] Preparation of emulsion: Sodium hyaluronate was prepared into an alkaline solution of sodium hyaluronate with a concentration of 8% using 1M NaOH solution, and the oil phase was prepared using Span 85 and liquid paraffin in a ratio of 1:20.

[0125] Preparation of microsphere emulsion: Add the cross-linking agent to the above emulsion in a certain mass ratio with sodium hyaluronate, first stir and mix in an ice bath, then conduct a preliminary reaction under stirring at room temperature, and finally react under stirring in a 40°C water bath. After the reaction, a microsphere emulsion is obtained, first cool down, and then adjust the pH to 4-6.

[0126] Cleaning: First, use n-hexane to adjust the pH of the microsphere emulsion to 4-6 and stir and clean it several times, let it stand and separate, and discard the upper oil phase. After completion, use ethyl acetate to stir and clean it several times, let it stand and separate, and discard the upper oil phase; after completion, use anhydrous ethanol to stir and clean it several times, let it stand and separate, and discard the upper oil phase.

[0127] Swelling: Place the washed microspheres in a buffer solution and allow to swell overnight, then pour out the supernatant and sieve out the microspheres that do not meet the particle size range requirements to obtain the emulsified cross-linked sodium hyaluronate gel for injection.

[0128] Example 4

[0129] The technical solution of the present invention can also be used for quantitative detection of the content of liquid paraffin in hemostatic powder. The preparation method of the liquid paraffin standard curve and the gas chromatography conditions are consistent with the detection of the agarose gel mentioned above. The sample processing steps are as follows:

[0130] (1) Preparation of test solution

[0131] Weigh 1g of hemostatic powder sample, add petroleum ether, place in a Soxhlet extraction apparatus, reflux extraction for 5h, reflux temperature is 80℃. Recover the solvent and evaporate the solvent. Dissolve the petroleum ether extract with 5mL of n-hexane and transfer to a 50mL centrifuge tube, add 4mL of 0.4mol / L potassium hydroxide-methanol solution, shake, let stand for 10min, add 10mL of water, let stand until the upper solution is transparent. Take the upper transparent solution and filter it through a 0.22μm filter membrane to obtain the test solution.

[0132] (2) Determination of liquid paraffin content

[0133] The test solution was injected into an Agilent 7890 gas chromatograph for determination. The chromatographic conditions of the instrument were consistent with those in the above-mentioned standard curve drawing method. The peak area of ​​the test solution was measured to be 7.397, and the retention time was 9.104 min. The peak area was substituted into the linear regression equation of the standard curve, and the content of liquid paraffin in the test sample was obtained to be 0.023 mg / g.

[0134] Although the method of the present invention can be used to detect the content of liquid paraffin in hemostatic powder, the hemostatic powder is in powder form, and the time required for reflux extraction of the liquid paraffin therein is longer than that of sugar gel. Therefore, it takes a long time to prepare the test solution, and the overall operation time is long.

[0135] The present invention studies the influence of injection volume and sample processing time on the quantitative detection of liquid paraffin, as follows:

[0136] Test Example 1

[0137] (1) Preparation of test solution

[0138] Weigh 1g of agarose gel sample, add petroleum ether, place in a Soxhlet extraction apparatus, reflux extraction for 8h, reflux temperature is 50℃. Recover the solvent and evaporate the solvent. Dissolve the petroleum ether extract with 5mL of n-hexane and transfer to a 50mL centrifuge tube, add 4mL of 0.4mol / L potassium hydroxide-methanol solution, shake, let stand for 10min, add 10mL of water, and let stand until the upper layer solution is transparent. Take the upper transparent solution and filter it through a 0.22μm filter membrane to obtain the test solution.

[0139] (2) Determination of liquid paraffin content

[0140] The test solution was injected into an Agilent 7890 gas chromatograph for measurement. The chromatographic conditions of the instrument were consistent with those in the above-mentioned standard curve drawing method. The results showed that no peak of the test solution was detected, which was presumably caused by the too low concentration of the test solution.

[0141] Test Example 2

[0142] (1) Preparation of test solution

[0143] Weigh 15g of agarose gel sample, add petroleum ether, place in a Soxhlet extraction apparatus, reflux extraction for 8h, reflux temperature is 50℃. Recover the solvent and evaporate the solvent. Dissolve the petroleum ether extract with 5mL of n-hexane and transfer to a 50mL centrifuge tube, add 4mL of 0.4mol / L potassium hydroxide-methanol solution, shake, let stand for 10min, add 10mL of water, and let stand until the upper layer solution is transparent. Take the upper transparent solution and filter it through a 0.22μm filter membrane to obtain the test solution.

[0144] (2) Determination of liquid paraffin content

[0145] The test solution was injected into an Agilent 7890 gas chromatograph for determination. The chromatographic conditions of the instrument were consistent with those in the above-mentioned standard curve drawing method. The peak area of ​​the test solution was measured to be 7.944, and the retention time was 9.102 min. The peak area was substituted into the linear regression equation of the standard curve, and the content of liquid paraffin in the test sample was obtained to be 0.012 mg / g.

[0146] Test Example 3

[0147] (1) Preparation of test solution

[0148] Weigh 15g of agarose gel sample, add petroleum ether, place in a Soxhlet extraction apparatus, reflux extraction for 5h, reflux temperature is 50℃. Recover the solvent and evaporate the solvent. Dissolve the petroleum ether extract with 5mL of n-hexane and transfer to a 50mL centrifuge tube, add 4mL of 0.4mol / L potassium hydroxide-methanol solution, shake, let stand for 10min, add 10mL of water, and let stand until the upper solution is transparent. Take the upper transparent solution and filter it through a 0.22μm filter membrane to obtain the test solution.

[0149] (2) Determination of liquid paraffin content

[0150] The test solution was injected into an Agilent 7890 gas chromatograph for determination. The chromatographic conditions of the instrument were consistent with those in the above-mentioned standard curve drawing method. The peak area of ​​the test solution was measured to be 7.928, and the retention time was 9.105 min. The peak area was substituted into the linear regression equation of the standard curve, and the content of liquid paraffin in the test sample was obtained to be 0.012 mg / g.

[0151] Test Example 4

[0152] (1) Preparation of test solution

[0153] Weigh 15g of agarose gel sample, add petroleum ether, place in a Soxhlet extraction apparatus, reflux extraction for 2h, reflux temperature is 50℃. Recover the solvent and evaporate the solvent. Dissolve the petroleum ether extract with 5mL of n-hexane and transfer to a 50mL centrifuge tube, add 4mL of 0.4mol / L potassium hydroxide-methanol solution, shake, let stand for 10min, add 10mL of water, and let stand until the upper layer solution is transparent. Take the upper transparent solution and filter it through a 0.22μm filter membrane to obtain the test solution.

[0154] (2) Determination of liquid paraffin content

[0155] The test solution was injected into an Agilent 7890 gas chromatograph for determination. The chromatographic conditions of the instrument were consistent with those in the above-mentioned standard curve drawing method. The peak area of ​​the test solution was measured to be 7.955, and the retention time was 9.104 min. The peak area was substituted into the linear regression equation of the standard curve, and the content of liquid paraffin in the test sample was obtained to be 0.012 mg / g.

[0156] Test Example 5

[0157] (1) Preparation of test solution

[0158] Weigh 15g of agarose gel sample, add petroleum ether, place in a Soxhlet extraction apparatus, reflux extraction for 1h, reflux temperature is 50℃. Recover the solvent and evaporate the solvent. Dissolve the petroleum ether extract with 5mL of n-hexane and transfer to a 50mL centrifuge tube, add 4mL of 0.4mol / L potassium hydroxide-methanol solution, shake, let stand for 10min, add 10mL of water, let stand until the upper solution is transparent. Take the upper transparent solution and filter it through a 0.22μm filter membrane to obtain the test solution.

[0159] (2) Determination of liquid paraffin content

[0160] The test solution was injected into an Agilent 7890 gas chromatograph for determination. The chromatographic conditions of the instrument were consistent with those in the above-mentioned standard curve drawing method. The peak area of ​​the test solution was measured to be 7.914, and the retention time was 9.105 min. The peak area was substituted into the linear regression equation of the standard curve, and the content of liquid paraffin in the test sample was obtained to be 0.012 mg / g.

[0161] Test Example 6

[0162] (1) Preparation of test solution

[0163] Weigh 15g of agarose gel sample, add petroleum ether, place in a Soxhlet extraction apparatus, reflux extraction for 0.5h, reflux temperature is 50℃. Recover the solvent and evaporate the solvent. Dissolve the petroleum ether extract with 5mL of n-hexane and transfer to a 50mL centrifuge tube, add 4mL of 0.4mol / L potassium hydroxide-methanol solution, shake, let stand for 10min, add 10mL of water, let stand until the upper solution is transparent. Take the upper transparent solution and filter it through a 0.22μm filter membrane to obtain the test solution.

[0164] (2) Determination of liquid paraffin content

[0165] The test solution was injected into an Agilent 7890 gas chromatograph for determination. The chromatographic conditions of the instrument were consistent with those in the above-mentioned standard curve drawing method. The peak area of ​​the test solution was measured to be 7.715, and the retention time was 9.102 min. The peak area was substituted into the linear regression equation of the standard curve, and the content of liquid paraffin in the test sample was obtained to be 0.010 mg / g.

[0166] From the above experimental results, it can be seen that when the reflux time of agarose gel sample is reduced from 8h to 0.5h, the detected liquid paraffin content is reduced from 0.012mg / g to 0.010mg / g accordingly. This shows that the reflux time will affect the extraction rate of liquid paraffin in the gel sample. Considering the extraction rate and extraction time comprehensively, the reflux time of 1-2h is selected for sample processing.

[0167] 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 the content of liquid paraffin in a polysaccharide gel, characterized in that: The steps include: The polysaccharide gel test solution is introduced into a gas chromatograph, the peak area of ​​the liquid paraffin is measured, the most representative peak is selected, and then the peak area of ​​the peak is substituted into the liquid paraffin standard curve to obtain the liquid paraffin concentration in the polysaccharide gel test solution; finally, the liquid paraffin concentration is substituted into the calculation formula to obtain the liquid paraffin content in the test solution.

2. The detection method according to claim 1, characterized in that: The polysaccharide gel test solution is prepared by the following method: Take 15g of polysaccharide gel sample, add petroleum ether, place in a Soxhlet extraction apparatus, reflux extraction for 1-2h, the reflux temperature is 40-60℃; recover the solvent and evaporate the solvent; dissolve the petroleum ether extract with 5mL of n-hexane and transfer it to a 50mL centrifuge tube, add 4mL of 0.4mol / L potassium hydroxide-methanol solution, shake, let stand for 10min, add 10mL of water, and let stand until the upper solution is transparent; take the upper transparent solution and pass it through a 0.22μm filter membrane to obtain a polysaccharide gel test solution.

3. The detection method according to claim 2, characterized in that: The reflux extraction time is 2 hours; the reflux temperature is 50°C.

4. The detection method according to claim 1, characterized in that: The chromatographic conditions of the gas chromatograph are as follows: Chromatographic column: Agilent HP-5 (30m*0.32mm*0.25μm); programmed temperature: starting temperature 100℃, maintained for 5min, heated to 250℃ at 20℃ / min, maintained for 30min; injection port temperature: 230~260℃; FID detector temperature: 250~270℃; carrier gas flow rate 40mL / min; hydrogen flow rate 55mL / min; non-split injection, injection volume: 1μL; carrier gas is high-purity nitrogen.

5. The detection method according to claim 1, characterized in that: The most representative peak is selected from the characteristic peak of liquid paraffin with a retention time in the range of 9.102 to 9.109 min.

6. The detection method according to claim 5, characterized in that: The most representative peak is a characteristic peak of liquid paraffin with a retention time of 9.107 min.

7. The detection method according to claim 1, characterized in that: The liquid paraffin standard curve is drawn by the following method: (1) Preparation of standard stock solution Weigh 0.1 g of liquid paraffin into a 50 mL volumetric flask, dissolve it in n-hexane and dilute to the mark, shake well, and use this as paraffin standard stock solution 1, with a concentration of 2 mg / mL; take 25 mL of standard stock solution 1 and put it into a 50 mL volumetric flask, dissolve it in n-hexane and dilute to the mark, shake well, and use this as paraffin standard stock solution 2, with a concentration of 1 mg / mL; (2) Preparation of standard curve solution The liquid paraffin standard stock solution 2 was gradiently diluted to a series of different standard solutions of 0.0025, 0.005, 0.025, and 0.5 mg / mL, and filtered with a 0.22 μm filter membrane to obtain a standard curve solution; (3) Draw a standard curve Inject the standard curve solution into the gas chromatograph, determine the peak area of ​​liquid paraffin, record the corresponding chromatogram, draw the standard curve with the concentration of liquid paraffin standard as the abscissa x and the peak area as the ordinate y, and calculate the regression equation.

8. The detection method according to claim 7, characterized in that: The regression equation is: y=45.039x+6.3613, R 2 =0.9978.

9. The detection method according to claim 1, characterized in that: The calculation formula is: C=(C0×5) / M; wherein C represents the content of liquid paraffin in the test sample, in mg / g; C0 represents the concentration of liquid paraffin in the test sample, in mg / mL; and M represents the weight of the weighed sample, in g.

10. Use of the method according to any one of claims 1 to 9 in detecting the content of liquid paraffin in polysaccharide gel.

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