Method for detecting content of hydroxyapatite in sodium hyaluronate composite solution for injection by using ICP-MS (Inductively Coupled Plasma Mass Spectrometry)

Abstract

The invention provides a method for detecting the content of hydroxyapatite in a sodium hyaluronate composite solution for injection by using ICP-MS. The method comprises the following steps: heating and digesting a digestion solution in a specific proportion, taking out the digestion solution, diluting the digestion solution with ultrapure water, shaking uniformly, and filtering with a filter membrane to obtain a test solution; a series of calcium element standard solutions are accurately prepared by using a dilute nitric acid solution as a blank solvent, the content of the calcium element in the standard substance and a test substance is detected by adopting an internal standard method and utilizing ICP-MS (Inductively Coupled Plasma Mass Spectrometry), and then the content of hydroxyapatite is converted and calculated. The method has good performance in the aspects of linearity, accuracy, specificity, quantitation limit, range, solution stability and durability, and is high in detection sensitivity and accurate in result. The quality of the sodium hyaluronate composite solution for injection can be effectively controlled, so that the clinical use safety of the sodium hyaluronate composite solution for injection is ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and in particular, relates to a method for detecting the content of hydroxyapatite in a sodium hyaluronate composite solution for injection by using ICP-MS. Background Art

[0002] The chemical formula of hydroxyapatite is Ca 10 (PO 4 ) 6 (OH) 2 , is currently widely used in the fields of medicine, cosmetology, dentistry, industry, etc., and is one of the most popular biomaterials in research. Hydroxyapatite has good biocompatibility and osteoconductivity, and can form a good combination with human bones and teeth. However, excessive use may cause problems such as tooth sensitivity, bone pain, and impaired kidney function.

[0003] Currently, there is no detection method for hydroxyapatite content in the Chinese Pharmacopoeia and related industry standards. Therefore, it is of great significance to develop a method that can accurately test the hydroxyapatite content in sodium hyaluronate composite solution for injection.

[0004] The prior art has the following technical problems:

[0005] 1) There are many types of digestion solutions, but the digestion effects vary greatly;

[0006] 2) The digestion conditions include higher temperature and longer digestion time;

[0007] 3) The choice of internal standard elements can affect the reliability and accuracy of detection. Summary of the invention

[0008] The technical problem to be solved by the present invention is to determine the hydroxyapatite content in a sample by ICP-MS. A method for detecting the hydroxyapatite content in a sodium hyaluronate composite solution for injection by using ICP-MS is disclosed. The method completes the detection by liquid sampling, and the detection amount can reach an ultra-trace level (1 ppb). The method has the characteristics of high sensitivity, good repeatability, and good accuracy. The present invention is achieved by the following technical solutions:

[0009] The present invention discloses a method for detecting the content of hydroxyapatite in a sodium hyaluronate composite solution for injection by using ICP-MS, and the specific steps are:

[0010] 1) Preparation of test solution: weigh the sodium hyaluronate composite solution for injection into a digestion tube, add a digestion solution composed of one or more of nitric acid, hydrochloric acid, and hydrofluoric acid and ultrapure water, so that the volume ratio of sample to digestion solution is about 1:10 to 1:20, and shake evenly; the digestion solution ratio is usually ultrapure water: nitric acid volume ratio of about 1:1 to 1:4 or ultrapure water: nitric acid: hydrochloric acid volume ratio of about 2:2:1 to 2:4:1 or ultrapure water: nitric acid: hydrofluoric acid volume ratio of about 1:2:1 to 1:5:1; the sample and digestion solution are heated and digested at 80 to 150°C for 50 to 120 minutes, then taken out and diluted with ultrapure water, shaken evenly, and filtered with a filter membrane as the test solution;

[0011] 2) Preparation of blank sample: Take a blank digestion tube, add digestion solution, heat and digest, dilute with ultrapure water, shake well, filter with a filter membrane, and use it as a blank sample;

[0012] 3) Preparation of standard solution: Take the calcium standard stock solution and use dilute nitric acid solution as solvent to accurately prepare a series of standard solutions;

[0013] 4) Preparation of internal standard solution: Take the standard substance stock solution, use dilute nitric acid solution as solvent, prepare 400-1000 μg / kg concentration of scandium, germanium, rhodium, indium, rhenium, bismuth, iridium standard solution, and select one of the elements as the internal standard solution;

[0014] 5) Detection conditions: Using ICP-MS, calcium standard solutions of different solubility, blank samples, test solutions and internal standard solutions were absorbed and injected, a standard curve of calcium content was drawn, and the hydroxyapatite content was calculated.

[0015] As a further improvement, the calculation formula for the hydroxyapatite content in the sodium hyaluronate composite solution for injection of the present invention is:

[0016]

[0017] Where: c—hydroxyapatite content, %;

[0018] c 1 —Calcium content in the sample tube, μg / kg;

[0019] m 1 —Sample volume mass, g;

[0020] m—sample weight, g;

[0021] M—Hydroxyapatite Ca 10 (PO 4 ) 6 (OH) 2 molecular weight, 1004.62;

[0022] M 1 —The total molecular weight of calcium in hydroxyapatite is 400.78.

[0023] As a further improvement, the volume ratio of the sample to the digestion solution in the preparation of the test solution described in the present invention is about 1:10 to 1:15.

[0024] As a further improvement, the digestion solution described in the present invention is composed of a mixture of one or more of nitric acid, hydrochloric acid, and hydrofluoric acid and ultrapure water; the digestion solution ratio is ultrapure water: nitric acid volume ratio of about 1:1; ultrapure water: nitric acid: hydrochloric acid volume ratio of about 2:3:1; ultrapure water: nitric acid: hydrofluoric acid volume ratio of about 1:3:1.

[0025] As a further improvement, the mass fraction of the nitric acid solution of the present invention is 67-70%, the mass fraction of the hydrochloric acid solution is 30-40%, and the mass fraction of the hydrofluoric acid solution is 35-45%.

[0026] As a further improvement, the preparation of the test solution of the present invention requires heating and digestion at 100-120° C. for 50-70 min.

[0027] As a further improvement, the dilute nitric acid solution in the preparation of the standard solution and the internal standard solution of the present invention is a 2% nitric acid solution.

[0028] As a further improvement, a micron-grade filter membrane is used in the preparation of the test solution described in the present invention.

[0029] As a further improvement, the internal standard solution of the present invention uses a scandium element standard substance.

[0030] As a further improvement, the concentration of the internal standard solution of the present invention is about 400-800 μg / kg.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1) The present invention provides a method for detecting the content of hydroxyapatite in a composite solution of sodium hyaluronate for injection, using a digestion solution with a ratio of ultrapure water: nitric acid of about 1:1 or ultrapure water: nitric acid: hydrochloric acid of about 2:3:1 or ultrapure water: nitric acid: hydrofluoric acid of about 1:3:1, heating and digesting for 50 to 70 minutes at 100 to 120 ° C, taking out and diluting with ultrapure water, shaking and shaking, filtering with a filter membrane, as a test solution. By using a dilute nitric acid solution as a blank solvent, a series of calcium standard solutions are accurately prepared, and the internal standard method is used to detect the content of calcium in the standard and the test sample by ICP-MS, and then the hydroxyapatite content is converted and calculated. The method of the present invention has good performance in linearity, accuracy, specificity, quantitative limit, range, solution stability, and durability, high detection sensitivity, and accurate results. It can effectively control the quality of the composite solution of sodium hyaluronate for injection, thereby ensuring its safety in clinical use.

[0033] 2) The present invention studies the digestion of hydroxyapatite in the sodium hyaluronate composite solution for injection by using different digestion solutions composed of a mixture of one or more of nitric acid, hydrochloric acid, and hydrofluoric acid and ultrapure water, which can fully dissolve and release the calcium element in the hydroxyapatite;

[0034] 3) By optimizing the digestion conditions, it was determined that the preparation of the test solution was heated and digested at 100-120°C for 50-70 minutes, which could completely digest the hydroxyapatite particles and improve the accuracy and effectiveness of the test results;

[0035] 4) The present invention uses 400-800 μg / kg scandium standard solution as the internal standard solution, which can better correct the response of calcium element and reduce non-spectral line interference and changes over time;

[0036] 5) The present invention measures the calcium content in the sample by ICP-MS to calculate the hydroxyapatite content, and the method has high sensitivity, good repeatability and good accuracy. The inventive method provides a guarantee for effectively controlling the quality of the sodium hyaluronate composite solution for injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a flow chart of the inspection process;

[0038] Figure 2 The concentrations of the calcium standard in Example 2 are Linear relationship diagram between . DETAILED DESCRIPTION

[0039] In order to better illustrate the process and scheme of the present invention, the invention is further explained in conjunction with the embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. The protection scope of the present invention shall be determined by the appended claims. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0040] The samples used in the present invention are all sodium hyaluronate composite solutions for injection, which are composed of sodium hyaluronate, hydroxyapatite, sodium chloride, phosphate buffer and water for injection and are produced by Hangzhou Xiehe Medical Supplies Co., Ltd., wherein the hydroxyapatite content is 0.005% to 0.020%.

[0041] Embodiment 1:

[0042] 1. Preparation of test solution: Weigh about 0.5 g of sample into a 50 mL digestion tube, add 3 mL of ultrapure water and 3 mL of nitric acid solution, shake well, heat at 110 ° C for 60 min, cool, add ultrapure water to make the total mass of the liquid in the digestion tube about 20 g, shake well, and filter with a 0.45 μm filter membrane.

[0043] 2. Preparation of blank sample: Prepare in the same way as the test solution.

[0044] 3. Preparation of internal standard solution: Take the scandium element standard stock solution and use 2% nitric acid solution as solvent to prepare a scandium element standard solution with a mass concentration of about 500 μg / kg.

[0045] 4. Preparation of standard solution: Take an appropriate amount of calcium standard solution and use 2% nitric acid solution as blank solvent to accurately prepare a series of standard solutions so that the mass concentration of calcium is approximately 0μg / kg, 100μg / kg, 200μg / kg, 500μg / kg, 1000μg / kg, and 2000μg / kg, respectively.

[0046] 5. Detection conditions: Use ICP-MS, with CPS as the ordinate and concentration as the abscissa, draw a standard curve, absorb and inject the standard solution and internal standard solution to draw a standard curve; absorb and inject the blank sample, test solution and internal standard solution, read the calcium content in the test solution from the standard curve and calculate the hydroxyapatite content.

[0047] 6. Calculation of hydroxyapatite content in sodium hyaluronate composite solution for injection:

[0048]

[0049] Where: c—hydroxyapatite content, %;

[0050] c 1—Calcium content in the sample tube, μg / kg;

[0051] m 1 —Sample volume mass, g;

[0052] m—sample weight, g;

[0053] M—Hydroxyapatite Ca 10 (PO 4 ) 6 (OH) 2 molecular weight, 1004.62;

[0054] M 1 —The total molecular weight of calcium in hydroxyapatite is 400.78.

[0055] Comparative Example 1:

[0056] On the basis of Example 1, the digestion solution was changed to 2 mL ultrapure water + 3 mL nitric acid + 1 mL hydrochloric acid to explore the digestion of hydroxyapatite by different digestion solutions.

[0057] Comparative Example 2:

[0058] On the basis of Example 1, the digestion solution was changed to 1 mL ultrapure water + 3 mL nitric acid + 1 mL hydrofluoric acid to explore the digestion of hydroxyapatite by different digestion solutions.

[0059] Table 1 Comparison results of different digestion solutions

[0060] Example Name Hydroxyapatite content (%) RSD(%) Example 1 0.009 2 Comparative Example 1 0.009 7 Comparative Example 2 0.009 6

[0061] As can be seen from Table 1, under the same digestion conditions, the digestion ability of the three different digestion solutions on hydroxyapatite in the composite solution of sodium hyaluronate for injection is basically the same, but the preparation of the digestion solution in Example 1 is easier, the operation is simpler, and the experimental results show that the RSD of the hydroxyapatite content is lower. Taking all factors into consideration, the digestion solution with a volume ratio of ultrapure water: nitric acid of about 1:1 is more preferred.

[0062] The following will be combined with Example 1 of the present invention to carry out a methodological verification experiment to clearly and completely describe the technical solution of the present invention.

[0063] Embodiment 2:

[0064] Linearity verification of calcium standard curve:

[0065] 1. Preparation of standard solution: Take an appropriate amount of calcium standard solution and use 2% nitric acid solution as blank solvent to accurately prepare a series of standard solutions so that the mass concentration of calcium is approximately 0μg / kg, 100μg / kg, 200μg / kg, 500μg / kg, 1000μg / kg, and 2000μg / kg, respectively.

[0066] 2. Preparation of internal standard solution: same as in Example 1.

[0067] 3. Detection conditions: Use ICP-MS to absorb and inject the standard solution and internal standard solution. Draw a graph, list the regression equation and correlation coefficient, and draw the linear curve of calcium.

[0068] Table 2 Linearity verification results

[0069] Serial number Calcium concentration (μg / kg) CPS (Calcium) CPS(scandium) 1 0.000 490.03 53307.05 2 100.330 1217.88 52910.00 3 205.775 1985.77 52878.73 4 519.679 4285.25 53030.60 5 1027.207 7908.04 52449.46 6 1980.883 14524.60 51482.78 Regression equation <![CDATA[y=1.378×10 -4 x+9.19×10 -3 ]]> Correlation coefficient R＝0.9998

[0070] As can be seen from Table 2, the detection method of the present invention is Plotting, the linear regression equation is y = 1.378 × 10 -4 x+9.19×10 -3 , linear correlation coefficient ≥ 0.995, actual R = 0.9998, indicating that the invented method is reliable.

[0071] Embodiment 3:

[0072] Durability verification: except that the digestion time was changed from 60 min to 50 min, the rest was the same as Example 1.

[0073] Embodiment 4:

[0074] Durability verification: except that the digestion time was changed from 60 min to 70 min, the rest was the same as Example 1.

[0075] Embodiment 5:

[0076] Durability verification: except that the digestion temperature was changed from 110°C to 105°C, the rest was the same as in Example 1.

[0077] Embodiment 6:

[0078] Durability verification: except that the digestion temperature was changed from 110°C to 115°C, the rest was the same as Example 1.

[0079] Table 3 Durability verification results

[0080] Example Name Hydroxyapatite content (%) Change value (%) RSD(%) Example 1 0.009 / 2 Example 3 0.008 11.1 5 Example 4 0.009 0.0 6 Example 5 0.009 0.0 8 Example 6 0.008 11.1 4

[0081] As can be seen from Table 3, the digestion time of this method is in the range of 50 to 70 minutes, and the digestion temperature is in the range of 105°C to 115°C, and the measurement results are almost unchanged, indicating that the method has good durability.

[0082] Embodiment 7:

[0083] Exclusiveness verification:

[0084] 1. Preparation of test solution: same as in Example 1.

[0085] 2. 100% concentration level calcium reference solution: dilute with 2000 μg / kg calcium standard stock solution.

[0086] 3. Blank sample preparation: same as Example 1.

[0087] 4. Preparation of standard solution: same as in Example 1.

[0088] 5. Preparation of internal standard solution: same as in Example 1.

[0089] 6. Detection conditions: Use ICP-MS to absorb and inject the standard solution, blank sample, test solution, 100% concentration level calcium reference solution and internal standard solution respectively, calculate the concentration of calcium in the sample, and analyze whether calcium has a specific mass-to-charge ratio.

[0090] Table 4 Specificity verification results

[0091]

[0092] Note: Selecting calcium 44 with higher isotopic abundance for analysis can improve the accuracy and precision of the measurement.

[0093] As can be seen from Table 4, the method of using calcium 44 with a higher isotope abundance to analyze the calcium content in the sample blank solution, the test solution and the reference solution at a concentration level of 100% has good specificity.

[0094] Embodiment 8:

[0095] Limit of Quantitation Verification:

[0096] 1. Blank sample preparation: Prepare 6 blank sample solutions using the same preparation method as in Example 1.

[0097] 2. Preparation of standard solution: same as in Example 1.

[0098] 3. Preparation of internal standard solution: same as in Example 1.

[0099] 4. Detection conditions: Using ICP-MS, the standard solution, blank sample solution and internal standard solution were injected by absorption, and the standard deviation of the response values ​​of the 6 blank solutions was calculated.

[0100] 5. Calculate according to the following formula:

[0101]

[0102] Where:

[0103] σ——Standard deviation of response factor (response value deviation);

[0104] S——Slope of the calibration curve (slope of the standard curve).

[0105] Table 5 Quantitation limit verification results

[0106]

[0107] As can be seen from Table 5, the RSD of the CPS values ​​of the blank solutions of six consecutive samples was 15% and the limit of quantification concentration was about 28.3 μg / kg, indicating that the detection method had high sensitivity.

[0108] Embodiment 9:

[0109] Accuracy and Range Verification:

[0110] 1. Preparation of test solution: Take 9 portions of sample blank solution, add calcium standard stock solution, and prepare three different concentrations of test solution. Add low concentration standard solution (800μg / kg) to three portions, named "low recovery rate-1", "low recovery rate-2", "low recovery rate-3", add medium concentration standard solution (1000μg / kg) to the other three portions, named "medium recovery rate-1", "medium recovery rate-2", "medium recovery rate-3", and add high concentration standard solution (1200μg / kg) to the last three portions, named "high recovery rate-1", "high recovery rate-2", "high recovery rate-3" as test solution.

[0111] 2. Blank sample preparation: same as Example 1.

[0112] 3. Preparation of calcium standard stock solution: Prepare 20,000 μg / kg calcium standard stock solution using 2% nitric acid solution.

[0113] 4. Preparation of standard solution: same as in Example 1.

[0114] 5. Internal standard solution: same as in Example 1.

[0115] 6. Using ICP-MS, absorb and inject the standard solution, blank sample solution, test solution and internal standard solution respectively, and calculate the average recovery rate and relative standard deviation.

[0116] Table 6 Blank sample solution test results

[0117]

[0118]

[0119] Table 7 Accuracy verification results

[0120]

[0121] As can be seen from Tables 6 and 7, the average recovery of the 9 spiked sample solutions was 102%, the RSD value was less than 5%, and the range was 98% to 104%, which met the validation requirements, indicating that the method has high accuracy.

[0122] Embodiment 10:

[0123] Solution stability verification:

[0124] 1. Preparation of test solution: same as in Example 1.

[0125] 2. Blank sample preparation: same as Example 1.

[0126] 3. Preparation of standard solution: same as in Example 1.

[0127] 4. Preparation of internal standard solution: same as in Example 1.

[0128] 5. Detection conditions: Using ICP-MS, absorb and inject the standard solution, blank sample solution, test solution and internal standard solution at room temperature at the initial time point (within 3 hours), 24 hours and 48 hours, detect the calcium concentration and calculate the change value.

[0129] Table 8 Stability of standard solutions

[0130]

[0131] Table 9 Stability of sample solutions

[0132]

[0133] As can be seen from Tables 8 to 9, the change value of the standard solution within 48 hours is less than 20.0%, and the change value of the test solution within 48 hours is less than 5.0%, indicating that the solution stability of this method is good at room temperature.

[0134] Obviously, the described embodiments are only preferred examples of the present invention, not all embodiments, and do not limit the present invention. Based on the embodiments of the present invention, any modifications, improvements, and other embodiments made by those skilled in the art without creative work are within the scope of protection of the present invention.

[0135] All the above specific embodiments are only part of the implementation schemes of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field within the technical scope disclosed by the present invention, and any equivalent replacement or change made by the technicians in this technical field according to the technical scheme and inventive concept of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting the hydroxyapatite content in a sodium hyaluronate composite solution for injection using ICP-MS, characterized in that: The specific steps are: 1) Preparation of test solution: weigh the sodium hyaluronate composite solution for injection into a digestion tube, add a digestion solution composed of one or more of nitric acid, hydrochloric acid, and hydrofluoric acid and ultrapure water, so that the volume ratio of sample to digestion solution is about 1:10 to 1:20, and shake evenly; the digestion solution ratio is usually ultrapure water: nitric acid volume ratio of about 1:1 to 1:4 or ultrapure water: nitric acid: hydrochloric acid volume ratio of about 2:2:1 to 2:4:1 or ultrapure water: nitric acid: hydrofluoric acid volume ratio of about 1:2:1 to 1:5:1; the sample and digestion solution are heated and digested at 80 to 150°C for 50 to 120 minutes, then taken out and diluted with ultrapure water, shaken evenly, and filtered with a filter membrane as the test solution; 2) Preparation of blank sample: Take a blank digestion tube, add digestion solution, heat and digest, dilute with ultrapure water, shake well, filter with a filter membrane, and use it as a blank sample; 3) Preparation of standard solution: Take the calcium standard stock solution and use dilute nitric acid solution as solvent to accurately prepare a series of standard solutions; 4) Preparation of internal standard solution: Take the standard substance stock solution, use dilute nitric acid solution as solvent, prepare 400-1000 μg / kg concentration of scandium, germanium, rhodium, indium, rhenium, bismuth, iridium standard solution, and select one of the elements as the internal standard solution; 5) Detection conditions: Using ICP-MS, calcium standard solutions of different solubility, blank samples, test solutions and internal standard solutions were absorbed and injected, a standard curve of calcium content was drawn, and the hydroxyapatite content was calculated.

2. The method according to claim 1, characterized in that: The calculation formula of hydroxyapatite content in sodium hyaluronate composite solution for injection is: Where: c—hydroxyapatite content, %; c1—Calcium content in the sample tube, μg / kg; m1—sample volume mass, g; m—sample weight, g; M—Hydroxyapatite Ca 10 The molecular weight of (PO4)6(OH)2 is 1004.62; M1—The total molecular weight of calcium in hydroxyapatite, 400.

78.

3. According to the method of claim 1, preferably, the volume ratio of the sample to the digestion solution in the preparation of the test solution is about 1:10 to 1:

15.

4. The method according to claim 1, 2 or 3, characterized in that: The digestion solution is composed of a mixture of one or more of nitric acid, hydrochloric acid, and hydrofluoric acid and ultrapure water; the volume ratio of the digestion solution is ultrapure water: nitric acid is about 1:1; the volume ratio of ultrapure water: nitric acid: hydrochloric acid is about 2:3:1; the volume ratio of ultrapure water: nitric acid: hydrofluoric acid is about 1:3:

1.

5. The method according to claim 4, characterized in that: The mass fraction of the nitric acid solution is 67-70%, the mass fraction of the hydrochloric acid solution is 30-40%, and the mass fraction of the hydrofluoric acid solution is 35-45%.

6. The method according to claim 5, characterized in that: The preparation of the test solution requires heating and digestion at 100-120° C. for 50-70 min.

7. The method according to claim 1 or 2 or 3 or 5 or 6, characterized in that: The dilute nitric acid solution in the preparation of the standard solution and the internal standard solution is a 2% nitric acid solution.

8. The method according to claim 7, characterized in that: A micron-sized filter membrane is used in the preparation of the test solution.

9. The method according to claim 1 or 2 or 3 or 5 or 6 or 8, characterized in that: The internal standard solution is a scandium element standard substance.

10. The method according to claim 9, characterized in that: The concentration of the internal standard solution is about 400-800 μg / kg.

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