Method for detecting heavy metal residues in medical grade sodium hyaluronate

Abstract

The application belongs to the technical field of sodium hyaluronate heavy metal detection, and specifically discloses a medical-grade sodium hyaluronate heavy metal residue detection method. After homogenization of a medical-grade sodium hyaluronate sample, the sample is equally divided into two parts. A preliminary heavy metal determination result is obtained by non-digestion pretreatment of one sub-sample. A simplified standard working solution series is prepared based on the preliminary result, so that subsequent digestion determination can be efficiently completed in a significantly narrowed calibration range. Under the premise of ensuring detection accuracy, the analysis throughput and resource utilization efficiency are significantly improved. After completing the preliminary heavy metal determination and digestion determination, range compliance verification is first performed on the digestion determination result. If the verification is passed, consistency verification is performed by quantifying the deviation between the digestion determination value and the preliminary determination value. The progressive two-level verification system not only realizes active risk control, but also greatly improves the reliability of the detection result.

Description

Technical Field

[0001] This invention belongs to the field of heavy metal detection technology in sodium hyaluronate, and specifically discloses a method for detecting heavy metal residues in medical-grade sodium hyaluronate. Background Technology

[0002] Sodium hyaluronate, as a biomedical material, is widely used in pharmaceutical preparations, medical devices, and cosmetics. Its molecular structure contains functional groups such as carboxyl groups, which readily complex or adsorb heavy metal ions, leading to the risk of heavy metal residues. Therefore, medical-grade sodium hyaluronate must undergo heavy metal residue testing before the final product is released to ensure it meets the limits for elemental impurities set by pharmacopoeias or relevant regulations, thus guaranteeing the safety of clinical use.

[0003] Currently, the detection of heavy metal residues mainly employs a method combining complete digestion with instrumental analysis. Specifically, strong acids and high temperatures are used to thoroughly destroy the carbon skeleton structure of organic molecules, degrading them into smaller molecules, thereby breaking all chemical bonds and releasing heavy metal ions completely into a free inorganic ion state for quantitative analysis. While this method boasts high accuracy and sensitivity, it still has significant limitations in practical applications. First, the digestion method involves high-temperature, high-pressure digestion, sample volume adjustment, instrumental analysis, and sample solution determination. During the instrumental analysis stage, a calibration curve needs to be established using a series of standard solutions covering the full concentration range. Regardless of the actual sample concentration, a complete injection process from low to high concentrations is required. This fixed calibration method leads to prolonged detection time, high consumption of standards and reagents, and low detection efficiency, especially when screening large batches of samples, where resource waste is particularly prominent.

[0004] Secondly, existing detection methods rely on a single digestion assay result and lack an effective verification mechanism. When operational errors, sample inhomogeneity, or instrument drift occur, the obtained results may deviate from the true value, but this is difficult to identify in a timely manner, affecting the reliability of the test results. Although some laboratories conduct quality control through parallel sample determination or spiked recovery, these measures occur after the test is completed, making real-time process monitoring impossible and creating potential quality blind spots. Summary of the Invention

[0005] In order to solve the above-mentioned technical problems, or at least partially solve the above-mentioned technical problems, the present invention provides a method for detecting heavy metal residues in medical-grade sodium hyaluronate.

[0006] The objective of this invention can be achieved through the following technical solution: a method for detecting heavy metal residues in medical-grade sodium hyaluronate, comprising the following steps: S1, after homogenizing the sodium hyaluronate sample, two equal quantum samples are taken, one of the samples is subjected to non-digestion treatment, and the preliminary determination result of heavy metals in the sample is measured using an electrochemical analyzer.

[0007] S2. Based on the preliminary heavy metal determination results, perform bidirectional concentration expansion to obtain the upper and lower limits of the calibration concentration. Within the calibration concentration range determined by these upper and lower limits, prepare a series of simplified standard working solutions containing multiple gradient concentrations.

[0008] S3. Using the simplified standard working solution series, establish a local calibration curve, completely digest another sample, and then perform instrument measurement to obtain the digestion measurement results.

[0009] S4. Determine whether the digestion measurement result is within the calibration concentration range. If not, the rationality verification is deemed unsuccessful and a retesting process is triggered. If yes, then a consistency verification is performed.

[0010] S5. Compare the digestion test results with the preliminary heavy metal test results, and determine the consistency based on the relationship between the deviation between the two and the preset deviation limit. If they are consistent, output the digestion test results as the final test conclusion; if they are inconsistent, trigger the retesting process.

[0011] Combining all the above technical solutions, the positive effects of this invention are as follows: 1. This invention homogenizes a medical-grade sodium hyaluronate sample and divides it into two equal parts. One part of the sample is processed without digestion to quickly obtain preliminary heavy metal determination results. Based on these preliminary results, a local calibration concentration range covering a reasonable fluctuation range is dynamically constructed. Within this range, a series of simplified standard working solutions are prepared. This allows the instrument to efficiently complete quantitative analysis within a significantly narrowed calibration range after the other part of the sample is completely digested. This significantly improves detection efficiency and resource utilization while ensuring detection accuracy.

[0012] 2. After completing the preliminary determination and digestion determination of heavy metals in two sub-samples, this invention first verifies the rationality of the digestion determination results. If the result passes, the consistency verification is performed by quantifying the deviation between the digestion determination value and the preliminary determination value. This progressive two-level verification system, on the one hand, places quality judgment in the data generation stage, realizing proactive risk control, and on the other hand, realizes the intrinsic self-consistency verification of the test results, greatly improving the reliability of the test results. Attached Figure Description

[0013] The present invention will be further described with reference to the accompanying drawings, but the embodiments in the drawings do not constitute any limitation on the present invention. For those skilled in the art, other drawings can be obtained based on the following drawings without creative effort.

[0014] Figure 1 This is a diagram illustrating the implementation steps of the method of the present invention.

[0015] Figure 2This is a flowchart illustrating the determination of the expansion factor and reduction factor in this invention.

[0016] Figure 3 This is a flowchart of the consistency verification process in this invention. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] See Figure 1 As shown, the present invention proposes a method for detecting heavy metal residues in medical-grade sodium hyaluronate, comprising the following steps: S1, after homogenizing the sodium hyaluronate sample, two equal quantum samples are taken, one of the samples is subjected to non-digestion treatment, and the preliminary determination result of heavy metals in the sample is measured using an electrochemical analyzer.

[0019] Considering that medical-grade sodium hyaluronate is usually in the form of a high-viscosity gel, its physical state can easily lead to uneven distribution of heavy metals at the microscale, such as adsorption on local particles, aggregation on container walls, or formation of complex micro-regions. Homogenization treatment, such as thorough dissolution and mixing, can break spatial heterogeneity and make heavy metals achieve statistically significant uniform distribution in the entire sample system. Only on this basis can two sub-samples truly represent the overall heavy metal level of the same batch of products.

[0020] Furthermore, the core logic of this invention lies in using the preliminary non-digestion test results to guide the calibration and verification of the digestion test. This requires that the two sub-samples be highly consistent in the heavy metal occurrence state. If they are not homogenized or the samples are not evenly separated, the two samples may show significant deviations due to local concentration differences, resulting in a mismatch between the preliminary and precise test results, and thus failing to guide the calibration and verification of the digestion test. Therefore, the purpose of homogenization and equal quantum fractionation is to ensure that the two sub-samples used for different processing paths are completely equivalent in terms of heavy metal content and distribution, thereby providing a reliable data foundation for subsequent calibration and verification based on the correlation between the two.

[0021] Based on the above considerations, the advantages of using non-digestion treatment combined with electrochemical methods for preliminary heavy metal determination of one sample are as follows: electrochemical technology features high analytical throughput and a simplified pretreatment process, eliminating the need for strong acid digestion, high temperature and pressure, or complex purification steps, thereby significantly shortening the detection cycle and reducing operational complexity. This characteristic aligns perfectly with the core objective of preliminary determination: to rapidly obtain representative apparent heavy metal concentrations, providing efficient and reliable prior information for subsequent precise analysis.

[0022] More importantly, under non-digestion conditions, electrochemical methods can selectively respond to heavy metal species in the sample existing in free ionic or weakly complexed states. These forms, due to their high electrochemical activity and migration capacity, can directly undergo redox reactions on the working electrode surface and be quantitatively detected. In medical-grade sodium hyaluronate, due to its strict production process and pure matrix, the vast majority of residual heavy metals exist primarily as soluble inorganic ions or dynamically reversible complexes formed with carboxyl groups. Therefore, the results obtained from non-digestion electrochemical determination can accurately reflect the main heavy metal content of the sample and have good reference value.

[0023] In a specific implementation of the present invention, the process of preliminary determination of heavy metals using non-digestion treatment includes the following steps: mixing the subsample with a specific complexing reagent solution in a certain proportion, and performing continuous shaking treatment under constant temperature conditions to allow the heavy metal ions to fully react with the complexing reagent.

[0024] In an example of the above implementation, a specific complexing reagent solution comprises an aminocarboxylic acid complexing agent and a buffer salt component, wherein the aminocarboxylic acid complexing agent is selected from at least one of ethylenediaminetetraacetic acid and diethyltriaminepentaacetic acid, and the buffer salt component is used to maintain the pH stability of the reaction system.

[0025] It is important to understand that the purpose of first mixing the sample with a specific complexing reagent during non-digestion treatment is that the specific complexing reagent can form a stable, electrochemically active complex with the free or weakly bound heavy metal ions in the sample. This process effectively preserves the apparent form of heavy metals without damaging the sodium hyaluronate polymer matrix, while improving its response sensitivity in electrochemical detection, thus providing a high-fidelity detection signal basis for subsequent determination.

[0026] The mixed solution after shaking was centrifuged, and the supernatant was used as the sample to be tested.

[0027] It is further important to understand that the purpose of non-digestion assay in this invention is to determine heavy metals in free or weakly complexed states. Centrifugation is essentially a physical separation method that can retain soluble heavy metal-complexing reagent complexes in the supernatant. The supernatant more accurately represents the form of heavy metals that can be released and detected in the sample.

[0028] The three-electrode system of an electrochemical analyzer is used to scan the sample and record its current response signal at the characteristic oxidation / reduction potential.

[0029] The recorded current response signal is substituted into the pre-established standard response curve of the heavy metal-complexation system to obtain the apparent concentration of heavy metals in the subsample, which is the preliminary determination result of heavy metals.

[0030] Further understanding is needed regarding the process of enrichment and dissolution of heavy metal complexes on the working electrode surface using differential pulse anodic stripping voltammetry in a three-electrode system. This generates a current signal proportional to the concentration. According to Faraday's law, the current response intensity is linearly related to the concentration of heavy metal ions in the solution. By fitting this linear relationship and translating the recorded current response signal into this equation, a preliminary determination of heavy metals in medical sodium hyaluronate samples can be achieved.

[0031] The process of establishing the standard response curve of the above heavy metal-complexation system is as follows: First, a series of heavy metal standard working solutions of different concentrations are prepared. Each standard solution is constructed in a matrix environment that is completely consistent with the sample to be tested, including the same buffer system, sodium hyaluronate background and an equal amount of specific complexing reagent, to ensure that the heavy metals exist in the same form as in the sample.

[0032] Subsequently, each standard working solution was introduced into a three-electrode electrochemical detection system identical to the sample to be tested for measurement, and the peak current response value generated at the corresponding characteristic dissolution potential for each concentration was recorded.

[0033] Finally, using the concentration of heavy metal ions as the independent variable and the measured characteristic peak current intensity as the dependent variable, a linear regression analysis was performed using the least squares method to establish a quantitative relationship model between the two, yielding a model in the form of... The standard response curve equation, where The sensitivity of the calibration curve is represented by the slope of the regression line. The intercept of the regression line represents the background current or system offset at theoretical zero concentration. Indicates heavy metal concentration. This indicates the characteristic peak current intensity.

[0034] S2. Based on the preliminary heavy metal determination results, perform bidirectional concentration expansion to obtain upper and lower limits of calibration concentration. Within the calibration concentration range determined by these upper and lower limits, prepare a series of simplified standard working solutions containing multiple gradient concentrations.

[0035] Given that preliminary measurement results can accurately and stably reflect the background level of heavy metals in the sample to be digested, while traditional full digestion detection usually relies on a series of standard solutions covering a wide concentration range to construct a global calibration curve during the instrument analysis stage, regardless of the actual content of the sample, a complete multi-point injection process must be performed, resulting in high consumption of standard products, long analysis cycle and low resource utilization.

[0036] This invention uses preliminary measurement results as the core and constructs a calibration concentration range through bidirectional expansion. Only standard working solutions of gradient concentrations need to be prepared within this range to meet high-precision quantitative requirements. This design significantly reduces the number of standard solution systems and avoids redundant measurements at irrelevant concentration points, thereby greatly improving detection efficiency while ensuring analytical accuracy.

[0037] In the preferred implementation of the above scheme, the two-way concentration expansion includes the following: multiplying the preliminary heavy metal measurement results by a preset expansion factor to obtain the upper limit of the calibration concentration.

[0038] Multiply the preliminary heavy metal measurement results by a preset reduction factor to obtain the lower limit of the calibration concentration.

[0039] The expansion factor is a real number greater than 1, and the reduction factor is a positive real number less than 1.

[0040] The calibration concentration range is determined based on the upper and lower limits of the calibration concentration.

[0041] See Figure 2 As shown, in a further optimized implementation, the process for determining the expansion factor and reduction factor is as follows: obtain the legally permissible limits for heavy metals in medical-grade sodium hyaluronate, compare them with the preliminary determination results of heavy metals, and calculate the proportion of the difference between the legally permissible limits for heavy metals and the preliminary determination results of heavy metals to the legally permissible limits, which is defined as the safety margin.

[0042] It should be noted that the safety margin calculation is only performed if the preliminary test results of heavy metals are below the legally permissible limits. If the preliminary test results have exceeded the legally permissible limits, it indicates that the sample has obvious compliance risks. In this case, the sample should be directly subjected to full-range digestion and testing to confirm whether the total content of heavy metals has indeed exceeded the regulatory limits.

[0043] It should be noted that the legally permissible limits for heavy metals in medical-grade sodium hyaluronate are the highest acceptable concentration limits of heavy metals in the final product as stipulated by current pharmacopoeia standards or relevant medical device / drug regulatory regulations. These limits are not only used for compliance determination of the final results, but also serve as the boundary for expanding the preliminary heavy metal determination results.

[0044] Specifically, by comparing the preliminary heavy metal test results with the legally permissible limits, a safety margin characterizing the risk level of heavy metal residues in the sample can be calculated. When the safety margin is high, it indicates that the heavy metal content in the sample is significantly lower than the regulatory limit, and the risk is low. In this case, the upper limit of the calibration concentration range can be reasonably set as the legally permissible limit itself to avoid over-expansion. However, when the safety margin is low, it suggests that the sample is close to the regulatory limit. In order to fully cover the heavy metal content that may be released during the complete digestion process, the upper limit of the calibration concentration needs to be appropriately extended above the legally permissible limit.

[0045] Compare the calculated safety margin with the critical threshold: the aforementioned critical threshold is used to determine whether the safety margin is at a sufficient level.

[0046] Given that the range of values ​​for the safety margin is Between these values, more than half can be taken. For example, 0.7 can be used as the safety margin. When the safety margin is greater than or equal to the critical threshold, it is considered that the current initial test result has sufficient buffer to be far from the regulatory limit. Otherwise, it is considered that the current test result has insufficient buffer.

[0047] If the safety margin is greater than or equal to the critical threshold, the ratio of the legally permissible limit for heavy metals to the preliminary determination result of heavy metals is calculated, and this ratio is used as the expansion factor, and the reciprocal of the expansion factor is used as the reduction factor.

[0048] With sufficient safety margin, since the legally permissible limit for heavy metals is greater than the preliminary measurement result, the ratio between the two is greater than 1, which meets the mathematical requirement that the expansion factor must be greater than 1. This also aligns the upper limit of the calibration concentration with the regulatory limit, thereby ensuring that in low-risk scenarios, the calibration range fully covers the potential true concentration while avoiding unnecessary over-expansion.

[0049] Accordingly, the reduction factor is defined as the reciprocal of this ratio, which meets the mathematical requirement that the reduction factor should be less than 1, and is used to compress the calibration concentration range downward.

[0050] If the safety margin is less than the critical threshold, the ratio of the legally permissible limit for heavy metals to the preliminary determination result of heavy metals is calculated, and the multiple of this ratio is used as the expansion factor. For example, this multiple can be 2 or 3 times, and the reciprocal of this ratio is used as the reduction factor.

[0051] When the safety margin is insufficient, by using multiples of the ratio as expansion factors, the upper limit of the calibration concentration can be extended to above the legally permissible limit, effectively covering the concentration increase caused by speciation during digestion and avoiding false negative results due to insufficient calibration range.

[0052] The reduction factor was not the reciprocal of the expansion factor, but rather the reciprocal of the original ratio. The reason for this is that if the reciprocal of the expansion factor were used as the reduction factor, the lower limit of calibration concentration would be excessively compressed, significantly lower than the actual possible concentration level. This would not only be unnecessary, but also, given insufficient safety margin, the focus would not be on accurate resolution at the low concentration end, but rather on reliable coverage and accurate quantification of potential exceedances at the high concentration end. Using the reciprocal of the original ratio as the reduction factor would keep the lower limit of calibration at a reasonable level and prevent it from being excessively lowered.

[0053] In another preferred implementation of the above scheme, the preparation of a series of simplified standard working solutions containing multiple gradient concentrations includes the following: the determined calibration concentration range is set differently based on the comparison results of the safety margin and the critical threshold: (1) when the safety margin is greater than or equal to the critical threshold, the concentration gradient points are uniformly distributed in linear segments with equal intervals in the calibration concentration range.

[0054] Understandably, when there is sufficient safety margin, the calibration concentration range is symmetrical at both ends with the initial measurement results as the center. Within this range, uniformly distributing concentration gradient points in a linear, equally spaced manner can more effectively and evenly cover the entire concentration range.

[0055] In a specific example, the uniformly distributed concentration gradient points in linear segments at equal intervals are implemented as follows: First, determine the number of concentration points to be distributed. Then, calculate the concentration interval based on the upper and lower limits of the calibration concentration interval using the number of concentration points. Finally, starting with the lower limit of the calibration concentration, the concentration intervals are accumulated sequentially to generate a concentration gradient sequence.

[0056] (2) When the safety margin is less than the critical threshold, the low concentration sub-interval formed by the lower limit of the calibration concentration and the preliminary determination result of heavy metals in the calibration concentration interval is also uniformly distributed by linear segmentation with equal spacing. In the high concentration sub-interval formed by the preliminary determination result of heavy metals and the upper limit of the calibration concentration, the concentration gradient points are set according to the reduction ratio of the concentration interval in the low concentration sub-interval.

[0057] Understandably, when the safety margin is insufficient, the preliminary measurement results of the sample are close to the legal limit, indicating that it is in a high-risk state. At this time, the focus of the analysis is on the reliable coverage and accurate quantification of potential exceedances at the high concentration end. Therefore, for the low concentration end, the same equal-interval linear segmentation method is used to set up concentration gradient points as when the safety margin is sufficient to retain the basic resolution. For the high concentration end, the density of the points is increased. Specifically, the concentration interval of the low concentration sub-interval is reduced by a preset ratio, such as 1 / 2 or 1 / 3, to set up a denser concentration gradient point, thereby achieving higher resolution at the high concentration end, while avoiding the waste of resources caused by uniform point distribution throughout the entire interval.

[0058] For each concentration gradient point, a standard working solution of the corresponding concentration is prepared using heavy metal standard substances.

[0059] A simplified standard working solution series was created by combining standard working solutions at all concentration gradient points.

[0060] S3. Using the simplified standard working solution series, establish a local calibration curve, completely digest another sample, and then perform instrument measurement to obtain the digestion measurement results.

[0061] In the optional implementation of the above scheme, the specific operation process of the above steps is as follows: the series of simplified standard working solutions are sequentially injected into the inductively coupled plasma mass spectrometer in order of increasing concentration.

[0062] Record the mass spectrometry response signal value corresponding to each concentration of standard working solution, wherein the mass spectrometry response signal value includes the ion count intensity of heavy metal elements.

[0063] A local calibration curve was established by fitting the standard working solution concentration on the x-axis and the mass spectrometry response signal value on the y-axis through linear regression.

[0064] Another sample was mixed with high-purity nitric acid in a certain proportion and then microwave-assisted digested in a sealed container.

[0065] Cool the digested sample solution to room temperature and bring it to the standard volume with ultrapure water.

[0066] Suspended particles are removed by centrifugation or filtration to obtain a clear digested sample solution.

[0067] The digested sample solution was introduced into an inductively coupled plasma mass spectrometer for measurement to obtain the mass spectrometric response signal of heavy metal elements.

[0068] The digestion measurement results are obtained by substituting the mass spectrometry response signal into the local calibration curve.

[0069] The above method for determining heavy metals by digestion follows the following principle: by completely digesting, heavy metals of various forms in the sample are completely converted into free inorganic ions, and then detected by inductively coupled plasma mass spectrometry: (i) the digestion solution is atomized and enters high-temperature argon plasma, where the elements are efficiently atomized and ionized into single-charged positive ions.

[0070] (ii) The ion beam is introduced into the high vacuum system through the interface. After being separated by the mass-to-charge ratio by the quadrupole, the ion count rate of the target heavy metal isotope, i.e. the mass spectrometry response signal, is detected by the electron multiplier.

[0071] (iii) Under optimized conditions, the signal intensity has a good linear relationship with the element concentration in the solution. Combined with the local calibration curve, the total content of heavy metals in the sample can be accurately inverted.

[0072] S4. Determine whether the digestion measurement result is within the calibration concentration range. If not, the rationality verification is deemed unsuccessful and a retesting process is triggered. If yes, then a consistency verification is performed.

[0073] The rationality verification using the calibration concentration range after digestion and determination is based on the fact that the local calibration curve used in digestion and determination only has a verified linear response within the calibration concentration range. If the digestion and determination result exceeds this range, it indicates that the result is in the extrapolation region of the calibration, and its concentration value is obtained by inversion through an unverified response relationship, which lacks methodological basis and metrological reliability. In this case, it is unnecessary to compare and verify the digestion and determination with the preliminary determination. The verification is directly judged as failing, avoiding invalid analysis based on unreliable data.

[0074] Therefore, the rationality verification is essentially to confirm that the quantitative results are within the legal range covered by the method verification, which is a prerequisite for data credibility.

[0075] In one possible implementation of the above scheme, determining whether the digestion measurement result is within the calibration concentration range involves comparing the digestion measurement result with the calibration concentration range.

[0076] When the digestion test results exceed the calibration concentration range, the rationality verification is deemed unsuccessful, triggering a retesting process.

[0077] When the digestion test results are within the calibration concentration range, the rationality verification is deemed successful.

[0078] S5. Compare the digestion test results with the preliminary heavy metal test results, and determine the consistency based on the relationship between the deviation between the two and the preset deviation limit. If they are consistent, output the digestion test results as the final test conclusion; if they are inconsistent, trigger the retesting process.

[0079] See Figure 3 As shown, in a further feasible way of the above scheme, the implementation process of consistency verification is as follows: the digestion test results are compared with the preliminary heavy metal test results. If the digestion test results are lower than the preliminary heavy metal test results, the consistency verification is directly judged to be unsuccessful.

[0080] It should be noted that when verifying the consistency between the digestion determination results and the preliminary heavy metal determination results, the two values ​​should be directly compared first. This is because the digestion determination completely releases all existing heavy metals in the sample into detectable inorganic ions, and the measured value is the total amount of heavy metals. The preliminary determination, on the other hand, is carried out under non-digestion conditions and can only respond to electrochemically active free or weakly bound heavy metals. Therefore, the total amount measured value should theoretically not be lower than the preliminary result that only reflects the determination of free heavy metals.

[0081] If the digestion measurement result is smaller than the preliminary measurement result, it is likely due to incomplete digestion. Therefore, prioritizing logical screening based on numerical magnitude can quickly identify anomalies and prevent subsequent deviation calculations from being based on physically infeasible data.

[0082] If the digestion test result is not lower than the preliminary heavy metal test result, the ratio of the difference between the two results to the reference standard is calculated as the normalized deviation.

[0083] The normalized deviation is compared with the configured deviation limit.

[0084] When the normalized deviation is less than or equal to the deviation limit, the consistency verification is considered to have passed.

[0085] When the normalization deviation exceeds the deviation limit, the consistency verification is deemed to have failed.

[0086] Understandably, although the preliminary determination only targets free or weakly complexed heavy metals, it usually covers the main part of the heavy metals in the sample. Under this premise, the total amount obtained from digestion determination may be slightly higher than the preliminary determination value, but the deviation between the two is theoretically small. By subtracting the two results and dividing by the reference standard, the deviation can be normalized and quantified. Then, by comparing with the deviation limit, consistency verification can be achieved.

[0087] Given the varying risk profiles of samples, a risk-adaptive strategy is adopted for setting the reference baseline and deviation limits, specifically as follows: When the safety margin is greater than or equal to the critical threshold, it indicates sufficient safety margin and a low-risk state. In this case, the legally permissible limit is used as the reference baseline. The rationale for this is that, in low-risk scenarios, compliance boundaries are the core concern, and there is a sufficient safety buffer between the preliminary measurement results and the permissible limit. Therefore, the remaining margin of the preliminary measurement results relative to the legally permissible limit is defined as the deviation limit. ,in Indicates the deviation limit. Indicates the legally permitted limit. This indicates preliminary measurement results. This setting reflects the tolerance margin of the current sample from the regulatory limit and is used as the maximum acceptable relative deviation limit.

[0088] When the safety margin is less than the critical threshold, it indicates insufficient safety margin and a high-risk state. In this case, the legally permissible limit is insufficient to provide a reasonable margin of error tolerance. Therefore, the upper limit of the calibration concentration range is used as a reference benchmark, and the buffer ratio of the preliminary heavy metal determination result relative to the upper limit of the calibration concentration is calculated as the deviation limit. ,in This indicates the upper limit of the calibration concentration.

[0089] This setting focuses on the usable safe range within the actual analysis window, ensuring that even if the concentration increases after digestion, the deviation from the initial value remains within the controllable range of the calibration system, thereby avoiding misjudgment of high-risk samples due to over-reliance on fixed limits.

[0090] Specifically, the test conclusion is: when the consistency verification is passed, the digestion test result is confirmed as the final test result.

[0091] When the consistency verification fails, a re-detection process is triggered.

[0092] It can be noted that when the consistency verification is passed, it indicates that the deviation between the digestion test results and the preliminary heavy metal test results is within the acceptable limit. Since the digestion test results represent the total content of heavy metals in the sample, they are used as the final test conclusion.

[0093] When the consistency verification passes, it indicates that the two have a large deviation. In such cases, the reliability of the data is questionable, and the digestion test results should not be used as the final conclusion. Retesting is required.

[0094] The above embodiments can be implemented, in whole or in part, by software, hardware, firmware, or any other combination thereof. When implemented using software, the above embodiments can be implemented, in whole or in part, in the form of a computer program product.

[0095] Those skilled in the art will recognize that the modules and algorithm steps of the various examples described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are implemented in hardware or software depends on the specific application and design constraints of the technical solution. Those skilled in the art can use different methods to implement the described functions for each specific application, but such implementation should not be considered beyond the scope of this application.

[0096] In addition, the functional modules in the various embodiments of this application can be integrated into one processing module, or each module can exist physically separately, or two or more modules can be integrated into one module.

[0097] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

[0098] Finally, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting heavy metal residues in medical grade sodium hyaluronate, characterized by, The method comprises the following steps: S1, after homogenizing the sodium hyaluronate sample, two equal sub-samples are taken, one of which is mixed with a specific complexing reagent solution in proportion, and the heavy metal ions are allowed to fully react with the complexing reagent under constant temperature and continuous oscillation treatment; The mixed solution after oscillation treatment is centrifuged, and the supernatant is taken as the sample to be tested; The three-electrode system of the electrochemical analyzer is used to scan the sample to be tested, and the current response signal at the characteristic oxidation / reduction potential is recorded; The recorded current response signal is substituted into the standard response curve of the heavy metal-complexing system to obtain the apparent concentration of heavy metals in the sub-sample, which is the preliminary determination result of heavy metals; S2, obtain the legal allowable limit of heavy metals in medical-grade sodium hyaluronate, and compare it with the preliminary determination result of heavy metals, calculate the proportion of the difference between the legal allowable limit of heavy metals and the preliminary determination result of heavy metals to the legal allowable limit, and define it as the safety margin; Compare the calculated safety margin with the critical threshold value, which is used to determine whether the safety margin is at a sufficient level; If the safety margin is greater than or equal to the critical threshold value, calculate the ratio of the legal allowable limit of heavy metals to the preliminary determination result of heavy metals, and use the ratio as the expansion factor, and the reciprocal of the expansion factor as the reduction factor; If the safety margin is less than the critical threshold value, calculate the ratio of the legal allowable limit of heavy metals to the preliminary determination result of heavy metals, and use the multiple of the ratio as the expansion factor, and the reciprocal of the ratio as the reduction factor; Multiply the preliminary determination result of heavy metals by the expansion factor to obtain the upper limit of the calibrated concentration; Multiply the preliminary determination result of heavy metals by the reduction factor to obtain the lower limit of the calibrated concentration; Determine the calibrated concentration interval based on the upper limit of the calibrated concentration and the lower limit of the calibrated concentration with the preliminary determination result of heavy metals as the center; Prepare a series of simplified standard working solutions containing multiple gradient concentrations within the determined calibrated concentration interval; S3, use the series of simplified standard working solutions to establish a local calibration curve, and perform instrument determination after complete digestion treatment of the other sub-sample to obtain a digestion determination result; S4, determine whether the digestion determination result is within the calibrated concentration interval, if not, determine that the rationality verification fails and trigger the re-detection process; If yes, perform consistency verification; S5, compare the digestion determination result with the preliminary determination result of heavy metals, and judge the consistency according to the relationship between the deviation of the two and the preset deviation limit value, if consistent, output the digestion determination result as the final detection conclusion; If not, trigger the re-detection process.

2. A method for detecting heavy metal residues in medical grade sodium hyaluronate according to claim 1, characterized in that: The preparation of the series of simplified standard working solutions containing multiple gradient concentrations comprises the following contents: The determined calibrated concentration interval is differentiated to set the concentration gradient points according to the comparison result of the safety margin and the critical threshold value: (1) When the safety margin is greater than or equal to the critical threshold value, the concentration gradient points are uniformly arranged in the calibrated concentration interval by equal interval linear segmentation; (2) When the safety margin is less than the critical threshold, the low-concentration sub-interval formed by the lower limit of the calibration concentration in the calibration concentration interval and the preliminary determination result of the heavy metal is also uniformly arranged with concentration gradient points by equal interval linear segmentation, and the concentration gradient points in the high-concentration sub-interval formed by the preliminary determination result of the heavy metal and the upper limit of the calibration concentration are arranged according to the reduction ratio of the concentration interval in the low-concentration sub-interval; For each concentration gradient point, a standard working solution with a corresponding concentration is prepared using a heavy metal standard substance; The standard working solution series of all concentration gradient points is used to form a simplified standard working solution series.

3. A method for detecting heavy metal residues in medical grade sodium hyaluronate according to claim 1, characterized in that: The specific implementation process of S3 is as follows: The simplified standard working solution series is sequentially injected into the inductively coupled plasma mass spectrometer in order of concentration from low to high; Record the mass spectrum response signal value corresponding to each concentration standard working solution, which includes the ion count intensity of the heavy metal element; Take the standard working solution concentration as the abscissa and the mass spectrum response signal value as the ordinate, and establish a local calibration curve by linear regression fitting; Another subsample is mixed with high-purity nitric acid in a certain proportion, and microwave-assisted digestion is carried out in a sealed container; Cool the digested sample solution to room temperature, and dilute to the calibrated volume with ultrapure water; Remove the suspended particles by centrifugation or filtration to obtain a clear digested sample solution; The digested sample solution is introduced into the inductively coupled plasma mass spectrometer for determination to obtain the mass spectrum response signal of the heavy metal element; The mass spectrum response signal is substituted into the local calibration curve to obtain the digestion determination result.

4. A method for detecting heavy metal residues in medical grade sodium hyaluronate according to claim 1, characterized in that: The operation of determining whether the digestion determination result is within the calibration concentration interval is as follows: Compare the digestion determination result with the calibration concentration range; When the digestion determination result exceeds the calibration concentration range, it is determined that the reasonableness verification fails, triggering the re-detection process; When the digestion determination result is within the calibration concentration range, it is determined that the reasonableness verification passes.

5. A method for detecting heavy metal residues in medical grade sodium hyaluronate according to claim 1, characterized in that: The implementation process of comparing the digestion determination result with the preliminary determination result of the heavy metal and judging the consistency according to the relationship between the deviation of the two and the preset deviation limit value is as follows: Compare the digestion determination result with the preliminary determination result of the heavy metal. If the digestion determination result is lower than the preliminary determination result of the heavy metal, it is directly determined that the verification fails; If the digestion determination result is not lower than the preliminary determination result of the heavy metal, calculate the difference between the two results and the reference benchmark as the normalized deviation; Compare the normalized deviation with the configured deviation limit value; When the normalized deviation is less than or equal to the deviation limit value, it is determined that the consistency verification passes; When the normalized deviation is greater than the deviation limit value, it is determined that the consistency verification fails.

6. A method for detecting heavy metal residues in medical grade sodium hyaluronate according to claim 5, characterized in that: The deviation limit value is configured as follows: When the safety margin is greater than or equal to the critical threshold, the remaining space ratio of the preliminary determination result of the heavy metal relative to the legal limit of the heavy metal is calculated as the deviation limit value, taking the legal limit of the heavy metal as the reference benchmark; When the safety margin is less than the critical threshold, the buffer ratio of the preliminary determination result of the heavy metal relative to the upper limit of the calibration concentration is calculated as the deviation limit value, taking the upper limit of the calibration concentration as the reference benchmark.

7. A method for detecting heavy metal residues in medical grade sodium hyaluronate according to claim 1, characterized in that: The final detection conclusion includes the following contents: When it is determined that the consistency verification passes, the result of the elimination assay is confirmed as the final detection result; When it is determined that the consistency verification fails, a re-detection procedure is triggered.

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