Method for simultaneously detecting mannitol and trehalose in acellular matrix
By simultaneously detecting mannitol and trehalose in decellularized matrix under the same conditions using high-performance liquid chromatography, the problems of cumbersome detection and high cost in traditional methods are solved, achieving rapid and accurate detection results and improving the level of product quality control.
Patent Information
- Application Number
- CN202511511320.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-22
- Publication Date
- 2026-01-27
AI Technical Summary
In existing technologies, the methods for detecting mannitol and trehalose in decellularized matrices are cumbersome, inefficient, and difficult to monitor the residual amounts of both simultaneously, affecting the accuracy of the results and the detection cost.
High-performance liquid chromatography (HPLC) was used to simultaneously detect mannitol and trehalose under the same chromatographic conditions. By preparing standard solutions and sample solutions of different concentrations, and combining them with HPLC analysis, specificity, linearity, and accuracy were ensured.
This technology enables rapid and simultaneous separation and detection of mannitol and trehalose, improving detection efficiency, reducing costs, ensuring the accuracy and reliability of results, and meeting the needs of modern medical device quality control.
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Figure CN121410136A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of decellularization detection technology, specifically a detection method for simultaneously detecting mannitol and trehalose in decellularized matrix. Background Technology
[0002] Decellularized matrix is one of the key materials in the field of regenerative medicine. By completely removing the cellular components of allogeneic or xenogeneic tissues, it retains the natural extracellular matrix structure and bioactive components. As an ideal tissue engineering scaffold, it is widely used in the repair and regeneration of various tissues such as skin, cartilage, bone, and cardiovascular system.
[0003] In the freeze-drying process of decellularized matrices, sugars or sugar alcohols are often added as freeze-drying protectants to stabilize their three-dimensional porous structure and protect their bioactivity. Mannitol, as an excellent excipient and freeze-drying protectant, provides good product shapeability; while trehalose, due to its superior "water substitution" and "vitrification" effects, is recognized as a leading biomolecular protectant, effectively preventing protein denaturation and inactivation during freeze-drying and storage. The synergistic effect of both ensures the stability of the final product during storage and its biofunctionality after implantation. Therefore, precise monitoring of the residual content of mannitol and trehalose in the decellularized matrix is a key quality control step for evaluating the consistency of the production process and ensuring stable and controllable product quality between batches.
[0004] However, traditional methods are mainly for quantitative analysis of single components. To detect mannitol and trehalose separately, two different chromatographic conditions and two independent sample pretreatments and analyses are required. This traditional method has significant drawbacks: first, the operation is cumbersome, the detection cycle is long, and the efficiency is low, greatly increasing labor and time costs; second, multiple sample processing may introduce larger systematic errors, affecting the accuracy and reliability of the results; finally, it is difficult to efficiently and quickly monitor the dosage ratio and residual amount of the two excipients simultaneously, failing to fully meet the urgent needs of modern medical device quality systems for efficient and precise quality control.
[0005] Therefore, there is an urgent need in this field to develop an analytical method that can simultaneously, rapidly, and accurately detect the content of mannitol and trehalose in decellularized matrices, in order to overcome the shortcomings of existing technologies, improve product quality control, and provide a solid technical guarantee for the safe production and widespread application of such advanced regenerative medicine materials. Summary of the Invention
[0006] The purpose of this invention is to provide a method for simultaneously detecting mannitol and trehalose in decellularized matrix in order to solve the problems mentioned above.
[0007] The technical solution adopted in this invention is as follows: A method for simultaneous detection of mannitol and trehalose content in decellularized matrix, comprising the following steps:
[0008] Step S1: Instrument preparation: High performance liquid chromatograph;
[0009] Step S2: Solution preparation
[0010] Test solution: Weigh the sample (20-30 mg), add water to make up to 10 mL, sonicate in an ultrasonic machine for 30 min, and after the sample is dissolved, centrifuge at 10000 r / min for 10 min to prepare 3 parallel samples.
[0011] Blank sample solution: Prepared without adding any sample, following the same procedure as the test sample solution.
[0012] Mannitol standard stock solution: Take mannitol standard and add primary water to prepare stock solutions with concentrations of 1.1834 mg / mL and 3.5970 mg / mL.
[0013] Trehalose standard stock solution: Take trehalose standard and add primary water to prepare stock solutions with concentrations of 1.1985 mg / mL and 3.4137 mg / mL.
[0014] Linear solutions: Mannitol and trehalose standard stock solutions were diluted with primary water to prepare standard curve solutions with mannitol concentrations of 10.00 μg / mL, 107.91 μg / mL, 359.70 μg / mL, 719.40 μg / mL, 1079.10 μg / mL, 1438.80 μg / mL, and 1798.50 μg / mL, and linear solutions with trehalose concentrations of 35.00 μg / mL, 102.41 μg / mL, 341.37 μg / mL, 682.74 μg / mL, 1024.11 μg / mL, 1365.48 μg / mL, and 1706.85 μg / mL, respectively. These were designated as STD-1, STD-2, STD-3, STD-4, STD-5, STD-6, and STD-7, respectively.
[0015] Detection limit solution: Take an appropriate amount of mannitol and trehalose standard solution to make the signal-to-noise ratio of the corresponding concentrations of mannitol and trehalose in the solution ≥3:1.
[0016] Limit of Quantification Solution: Take an appropriate amount of mannitol and trehalose standard solution to ensure that the signal-to-noise ratio of the corresponding concentrations of mannitol and trehalose in the solution is ≥10:1.
[0017] Repeatability and accuracy solutions: Weigh 9 samples respectively. Add 0.5 mL of mannitol solution with a concentration of 3.5970 mg / mL and 0.4 mL of trehalose standard solution with a concentration of 3.4137 mg / mL to samples 1-3, and dilute with water to 10 mL. Add 1.5 mL of mannitol solution with a concentration of 3.5970 mg / mL to samples 4-6, and dilute with water to 10 mL. Add 1.4 mL of trehalose standard solution with a concentration of 3.4137 mg / mL to samples 7-9. Add 2.5 mL of mannitol solution with a concentration of 3.5970 mg / mL and 2.4 mL of trehalose standard solution with a concentration of 3.4137 mg / mL to samples 9, and dilute with water to 10 mL. The corresponding repeatability and accuracy solutions are obtained.
[0018] Step S3: Validate the detection method using high performance liquid chromatography;
[0019] Step S4: The contents of mannitol and trehalose in the sample were determined by high performance liquid chromatography.
[0020] In a preferred embodiment, in step S1, the instrument preparation involves adjusting various parameters to the working state using a high-performance liquid chromatograph (HPLC) paired with a refractive index detector (RID).
[0021] In a preferred embodiment, the specific chromatographic conditions in step S1 are as follows: an amino-bonded silica column (Agilent Polaris 5 NH2, 250 × 4.6 mm) is used as the chromatographic column.
[0022] In a preferred embodiment, in step S1, an acetonitrile-water solution with a volume ratio of 80:20 is used as the mobile phase.
[0023] In a preferred embodiment, in step S1, the flow rate is 1.0 mL / min, the column temperature is 40°C, the injection volume is 20 µL, and the run time is 15 minutes.
[0024] In a preferred embodiment, step S3 includes verification of specificity, limit of detection (LOD), limit of quantitation (LOQ), linearity and range, repeatability, and accuracy. For specificity verification, blank sample solution, standard solution, and spiked solution of the test sample are injected for analysis to confirm that the blank solution has no interfering peaks at the retention time of the target peak, that the mannitol and trehalose peaks in the standard solution are well separated, and that the target peak in the spiked solution of the test sample does not overlap with other component peaks. For linearity verification, standard solutions with concentration gradients are used, with mannitol concentrations ranging from 10.00 μg / mL to 1798.50 μg / mL and trehalose concentrations ranging from 35.00 μg / mL to 1706.85 μg / mL. A standard curve is plotted with concentration on the x-axis and peak area on the y-axis, requiring a correlation coefficient of not less than 0.9999. The LOD and LOQ are determined by stepwise dilution of the standard solution, with signal-to-noise ratios of 3:1 and 10:1, respectively, and the relative standard deviation of the peak area at low concentrations must meet the specified requirements.
[0025] In a preferred embodiment, in step S3, repeatability verification involves injecting the same test solution six times consecutively and calculating the relative standard deviation of the peak area. The relative standard deviations for mannitol and trehalose should not exceed 2.7% and 2.6%, respectively. Accuracy verification employs a spiked recovery experiment, with spikes added at low, medium, and high concentration levels. Three parallel samples are prepared for each level. The recoveries of mannitol and trehalose must be controlled between 90.0% and 99.0%, and the relative standard deviation of the average recovery rate should not exceed 3%. The entire verification process must strictly adhere to the set chromatographic conditions, including an amino-bonded silica column, an acetonitrile-water mobile phase, a flow rate of 1.0 mL / min, a column temperature of 40°C, an injection volume of 20 μL, and a run time of 15 minutes, to ensure that all verification indicators meet the detection requirements.
[0026] In a preferred embodiment, in step S4, the test solution needs to be pretreated before detection. The sample is weighed and diluted to 10 mL with primary water. It is then sonicated for 30 minutes to ensure complete dissolution, followed by centrifugation at 10,000 rpm for 10 minutes. The supernatant is used as the test solution. During detection, the test solution is injected into the high-performance liquid chromatograph (HPLC), along with a blank sample solution as a control. Separation and detection are performed according to the set chromatographic conditions. Throughout the process, the injection volume and instrument operating status must be strictly controlled to avoid air bubbles or impurities interfering with the separation of chromatographic peaks.
[0027] In a preferred embodiment, in step S4, the concentrations of mannitol and trehalose in the sample are calculated according to the standard curve equations. The standard curve equation for mannitol is y = 181.01813x - 1208.54476, and the standard curve equation for trehalose is y = 183.37463x - 464.63383, where y is the peak area and x is the concentration. Substituting the corresponding peak area from the sample chromatogram into the equations yields the concentration value, which is then calculated using the formula. The concentration unit in the formula is micrograms per milliliter, the sample solution volume is 10 milliliters, and the sample mass unit is milligrams. The result is converted to milligrams per milligram using a unit conversion factor of 1000. The final result is the average of the parallel samples.
[0028] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:
[0029] This invention achieves rapid and simultaneous separation and detection of mannitol and trehalose in decellularized matrix under the same chromatographic conditions. Methodological validation shows that this method has high specificity, no blank interference, good linearity (R = 0.9999), and meets the detection requirements for repeatability (RSD ≤ 2.7%) and accuracy (recovery rate 90.0%–99.0%). This method significantly improves detection efficiency, reduces detection costs, and provides a reliable guarantee for the quality control and process stability evaluation of decellularized matrix products. Attached Figure Description
[0030] Figure 1 This is a schematic diagram illustrating the process principle of the present invention;
[0031] Figure 2 The spectra of blank sample solutions of mannitol and trehalose are shown.
[0032] Figure 3 The spectrum of mannitol and trehalose standard solutions;
[0033] Figure 4 Spectra of spiked solutions of mannitol and trehalose test samples;
[0034] Figure 5 This is a linear graph of the test results for the linear solution in this invention. Detailed Implementation
[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0036] Example:
[0037] Reference Figure 1-5A method for simultaneously detecting mannitol and trehalose in decellularized matrix, specifically comprising:
[0038] Instruments and reagents:
[0039] High performance liquid chromatograph (1260 Infinity II, Agilent Technologies), electronic balance (accuracy 0.00001g, METTLER), D-mannitol standard (98.7%, Tanmo Quality Inspection Technology Co., Ltd.), trehalose (98.4%, Tanmo Quality Inspection Technology Co., Ltd.), acetonitrile (HPLC) (chromatographic grade, PorMee).
[0040] Instrument conditions:
[0041] The instrument parameter settings for mannitol and trehalose liquid chromatography are shown in Table 1.
[0042] Table 1 Instrument conditions for mannitol and trehalose
[0043] Parameter name Specific parameters chromatographic column <![CDATA[Agilent Polaris 5 NH2250×4.6mm]]> mobile phase 80% acetonitrile + 20% water Time (min) 15.0 mobile phase velocity 1.0 mL / min detector RID detector Injection volume 20 µL Column temperature 40 ℃
[0044] Solution preparation
[0045] Test solution: Weigh the sample (20-30 mg), add water to make up to 10 mL, sonicate in an ultrasonic machine for 30 min, and after the sample is dissolved, centrifuge at 10000 r / min for 10 min to prepare 3 parallel samples.
[0046] Blank sample solution: Prepared without adding any sample, following the same procedure as the test sample solution.
[0047] Mannitol standard stock solution: Take mannitol standard and add primary water to prepare stock solutions with concentrations of 1.1834 mg / mL and 3.5970 mg / mL.
[0048] Trehalose standard stock solution: Take trehalose standard and add primary water to prepare stock solutions with concentrations of 1.1985 mg / mL and 3.4137 mg / mL.
[0049] Linear solutions: Mannitol and trehalose standard stock solutions were diluted with primary water to prepare standard curve solutions with mannitol concentrations of 10.00 μg / mL, 107.91 μg / mL, 359.70 μg / mL, 719.40 μg / mL, 1079.10 μg / mL, 1438.80 μg / mL, and 1798.50 μg / mL, and linear solutions with trehalose concentrations of 35.00 μg / mL, 102.41 μg / mL, 341.37 μg / mL, 682.74 μg / mL, 1024.11 μg / mL, 1365.48 μg / mL, and 1706.85 μg / mL, respectively. These were designated as STD-1, STD-2, STD-3, STD-4, STD-5, STD-6, and STD-7, respectively.
[0050] Detection limit solution: Take an appropriate amount of mannitol and trehalose standard solution to make the signal-to-noise ratio of the corresponding concentrations of mannitol and trehalose in the solution ≥3:1.
[0051] Limit of Quantification Solution: Take an appropriate amount of mannitol and trehalose standard solution to ensure that the signal-to-noise ratio of the corresponding concentrations of mannitol and trehalose in the solution is ≥10:1.
[0052] Repeatability and accuracy solutions: Weigh 9 samples respectively. Add 0.5 mL of mannitol solution with a concentration of 3.5970 mg / mL and 0.4 mL of trehalose standard solution with a concentration of 3.4137 mg / mL to samples 1-3, and dilute with water to 10 mL. Add 1.5 mL of mannitol solution with a concentration of 3.5970 mg / mL to samples 4-6, and dilute with water to 10 mL. Add 1.4 mL of trehalose standard solution with a concentration of 3.4137 mg / mL to samples 7-9. Add 2.5 mL of mannitol solution with a concentration of 3.5970 mg / mL and 2.4 mL of trehalose standard solution with a concentration of 3.4137 mg / mL to samples 9, and dilute with water to 10 mL. The corresponding repeatability and accuracy solutions are obtained.
[0053] The methodological validation results of a test method for mannitol and trehalose content in decellularized matrix are as follows:
[0054] Exclusivity:
[0055] Take the STD-4 linear standard solution, spiked test solution, and blank control solution, and perform the test according to the conditions in Table 5. No impurity peaks were observed near the mannitol and trehalose peaks in the STD-4 linear standard solution, spiked test solution, and blank control solution, indicating that this method has good specificity and linearity.
[0056] Linear:
[0057] Linear solutions were tested according to the conditions in Table 1, and chromatograms were recorded. A standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The linear correlation coefficient R should be ≥0.99. The test results are shown in Table 2. As can be seen from the results in Table 2, this method has good linearity.
[0058] Table 2 Linear Results
[0059]
[0060] Limit of detection and limit of quantitation:
[0061] Take the solutions with the limit of detection (LOD) and limit of quantitation (LOQ) and inject them according to the conditions in Table 1. Record the chromatograms. The solution concentration with a signal-to-noise ratio (SNR) greater than 3:1 is used as the LOD, and the solution concentration with a SNR greater than 10:1 is used as the LOQ. For mannitol and trehalose, the RSD of the peak area for solutions with LOD and LOQ concentrations less than or equal to 10 μg / mL should be ≤ 6%, and the RSD of the peak area for solutions with concentrations less than 100 μg / mL should be ≤ 4%. The test results are shown in Table 3. The results show that the LOD and LOQ meet the relevant requirements.
[0062] Table 3 Results of Limit of Detection and Limit of Quantification for Mannitol
[0063]
[0064] Accuracy and repeatability:
[0065] Nine accuracy solutions (low, medium, and high accuracy) were taken and tested according to the conditions in Table 1, and the chromatograms were recorded. According to Guideline 9101 of Part IV of the 2020 Edition of the Chinese Pharmacopoeia, the acceptable recovery rate of the target analyte in the solution is 90%–108%, and the acceptable RSD of the target analyte in the solution should be ≤3%. The results for mannitol are shown in Table 4, and the results for trehalose are shown in Table 5. The results in the tables show that this method has good accuracy.
[0066] Table 4. Accuracy Results of Mannitol
[0067]
[0068] Table 5. Trehalose accuracy results
[0069]
[0070] Summary of validation results for mannitol and trehalose methods:
[0071] The results of the mannitol and trehalose method validation are summarized in Table 6. As can be seen from the results, the validation results of each indicator meet the requirements of the evaluation criteria. This method can be used to determine the concentration of mannitol in micronized decellularized matrix.
[0072] Table 6 Summary of validation results for mannitol and trehalose methods
[0073] Method validation metrics Evaluation indicators Method validation results Does it meet the requirements? Exclusivity The blank solution and the spiked test solution should not interfere with the target peak. No impurity peaks were observed near the mannitol peak in the spiked test solution and blank solution. yes linear The correlation coefficient R of the standard curve is ≥ 0.99. Mannitol R = 0.999980, Trehalose R = 0.999994 yes Detection limit S / N ≥ 3:1 The detection limits (S / N) for both mannitol and trehalose were >3:1. yes Limit of Quantification S / N ≥ 10:1 The limits of quantitation for mannitol and trehalose are S / N > 10:1 yes System Applicability Peak area RSD ≤ 3% Mannitol peak area RSD: 0.33%; Trehalose peak area RSD: 0.44% yes Repeatability RSD ≤ 3% Mannitol RSD: 2.7%; Trehalose RSD: 2.6% yes Accuracy Recovery rate is between 90% and 108%. Minimum recovery of mannitol: 91.7%; Maximum recovery of mannitol: 99.0%; Minimum recovery of trehalose: 90.0%; Maximum recovery of trehalose: 99.0%. yes
[0074] Mannitol and trehalose content test:
[0075] Take the test solution and blank solution, and test them according to the conditions in Table 1. Calculate the contents of mannitol and trehalose in the sample extract according to the standard curve equation. The contents of mannitol and trehalose are calculated according to formula (1), and the test results are shown in Table 7.
[0076]
[0077] In formula (1):
[0078] A – Content of the target compound, in mg / mg;
[0079] C – The test concentration of the target compound, in µg / mL;
[0080] V—Volume of the test solution, in mL;
[0081] m—Sample mass used for extraction, in mg;
[0082] 1000 – Unit conversion factor.
[0083] Table 7. Content of mannitol and trehalose
[0084]
[0085] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.
[0086] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.
[0087] From the above, we can conclude that:
[0088] This invention achieves rapid and simultaneous separation and detection of mannitol and trehalose in decellularized matrix under the same chromatographic conditions. Methodological validation shows that this method has high specificity, no blank interference, good linearity (R = 0.9999), and meets the detection requirements for repeatability (RSD ≤ 2.7%) and accuracy (recovery rate 90.0%–99.0%). This method significantly improves detection efficiency, reduces detection costs, and provides a reliable guarantee for the quality control and process stability evaluation of decellularized matrix products.
[0089] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the term "comprising" or any other variations thereof is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.
[0090] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for simultaneous detection of mannitol and trehalose content in decellularized matrix, characterized in that: The methods and steps include the following: Step S1: Instrument preparation: High performance liquid chromatograph; Step S2: Solution preparation: Test solution: After weighing the sample, add water to make up to 10 mL, sonicate in an ultrasonic machine for 30 min, and after the sample is dissolved, centrifuge at 10000 r / min for 10 min to prepare 3 parallel samples. Blank sample solution: Prepared without adding any sample, following the same procedure as the test sample solution. Mannitol standard stock solution: Take mannitol standard and add primary water to prepare stock solutions with concentrations of 1.1834 mg / mL and 3.5970 mg / mL; Trehalose standard stock solution: Take trehalose standard and add primary water to prepare stock solutions with concentrations of 1.1985 mg / mL and 3.4137 mg / mL; Linear solutions: Mannitol and trehalose standard stock solutions were diluted with primary water to prepare standard curve solutions with mannitol concentrations of 10.00 μg / mL, 107.91 μg / mL, 359.70 μg / mL, 719.40 μg / mL, 1079.10 μg / mL, 1438.80 μg / mL, and 1798.50 μg / mL, respectively, and linear solutions with trehalose concentrations of 35.00 μg / mL, 102.41 μg / mL, 341.37 μg / mL, 682.74 μg / mL, 1024.11 μg / mL, 1365.48 μg / mL, and 1706.85 μg / mL, respectively. These solutions were designated as STD-1, STD-2, STD-3, STD-4, STD-5, STD-6, and STD-7, respectively. Detection limit solution: Take an appropriate amount of mannitol and trehalose standard solution to make the signal-to-noise ratio of the corresponding concentrations of mannitol and trehalose in the solution ≥3:1; Limit of Quantification Solution: Take an appropriate amount of mannitol and trehalose standard solution to make the signal-to-noise ratio of the corresponding concentrations of mannitol and trehalose in the solution ≥10:1; Repeatability and accuracy solutions: Weigh 9 samples respectively. Add 0.5 mL of 3.5970 mg / mL mannitol solution and 0.4 mL of 3.4137 mg / mL trehalose standard solution to samples 1-3, and dilute with water to 10 mL. Add 1.5 mL of 3.5970 mg / mL mannitol solution to samples 4-6, and dilute with water to 10 mL. Add 1.4 mL of 3.4137 mg / mL trehalose standard solution. Add 2.5 mL of 3.5970 mg / mL mannitol solution and 2.4 mL of 3.4137 mg / mL trehalose standard solution to samples 7-9, and dilute with water to 10 mL. The corresponding repeatability and accuracy solutions are obtained. Step S3: Validate the detection method using high performance liquid chromatography; Step S4: The contents of mannitol and trehalose in the sample were determined by high performance liquid chromatography.
2. The method for simultaneously detecting mannitol and trehalose in decellularized matrix as described in claim 1, characterized in that: In step S1, the instrument preparation involves adjusting various parameters to the working state, using a high-performance liquid chromatograph paired with a differential refractive index detector.
3. The method for simultaneously detecting mannitol and trehalose in decellularized matrix as described in claim 1, characterized in that: In step S1, an amino-bonded silica gel column is used as the chromatographic column.
4. The method for simultaneously detecting mannitol and trehalose in decellularized matrix as described in claim 1, characterized in that: In step S1, an acetonitrile-water solution with a volume ratio of 80:20 is used as the mobile phase.
5. The method for simultaneously detecting mannitol and trehalose in decellularized matrix as described in claim 1, characterized in that: In step S1, the flow rate is 1.0 mL / min, the column temperature is 40℃, the injection volume is 20 µL, and the running time is 15 minutes.
6. The method for simultaneously detecting mannitol and trehalose in decellularized matrix as described in claim 1, characterized in that: In step S3, the verification items include specificity, limit of detection, limit of quantitation, linearity and range, repeatability, and accuracy. For specificity verification, blank sample solution, standard solution, and spiked solution of the test sample are injected for analysis to confirm that the blank solution has no interfering peaks at the retention time of the target peak, that the mannitol and trehalose peaks in the standard solution are well separated, and that the target peak in the spiked solution of the test sample does not overlap with other component peaks. For linearity verification, standard solutions with concentration gradients are used, with mannitol concentration ranging from 10.00 μg / mL to 1798.50 μg / mL and trehalose concentration ranging from 35.00 μg / mL to 1706.85 μg / mL. A standard curve is plotted with concentration on the x-axis and peak area on the y-axis, requiring a correlation coefficient of not less than 0.9999.
7. The method for simultaneously detecting mannitol and trehalose in decellularized matrix as described in claim 1, characterized in that: In step S3, repeatability verification involves injecting the same test solution six times consecutively and calculating the relative standard deviation of the peak area. The relative standard deviations of mannitol and trehalose should not exceed 2.7% and 2.6%, respectively. Accuracy verification uses a spiked recovery experiment, with spikes added at low, medium, and high concentration levels. Three parallel samples are prepared for each level. The recoveries of mannitol and trehalose should be controlled between 90.0% and 99.0%, and the relative standard deviation of the average recovery rate should not exceed 3%.
8. The method for simultaneously detecting mannitol and trehalose in decellularized matrix as described in claim 1, characterized in that: In step S4, the test solution needs to be pretreated before detection. After weighing the sample, add primary water to make up to 10 ml, sonicate for 30 minutes to fully dissolve the sample, and then centrifuge at 10,000 rpm for 10 minutes. Take the supernatant as the test solution. During detection, inject the test solution into the high performance liquid chromatograph, and inject a blank sample solution as a control. Separation and detection are performed according to the set chromatographic conditions.
9. The method for simultaneously detecting mannitol and trehalose in decellularized matrix as described in claim 1, characterized in that: In step S4, the concentrations of mannitol and trehalose in the sample are calculated according to the standard curve equations. The standard curve equation for mannitol is y = 181.01813x - 1208.54476, and the standard curve equation for trehalose is y = 183.37463x - 464.63383, where y is the peak area and x is the concentration. Substituting the corresponding peak area from the sample chromatogram into the equations, the concentration values are obtained, and the content is calculated according to the formula. In the formula, the concentration unit is micrograms per milliliter, the volume of the test solution is 10 milliliters, and the sample mass unit is milligrams. The result is converted to milligrams per milligram using a unit conversion factor of 1000. The final result is the average of the parallel samples.