Pretreatment method for detecting endotoxin in sodium alginate sample and application thereof

Abstract

The invention relates to a pretreatment method for detecting endotoxin in a sodium alginate sample and application of the pretreatment method, and the pretreatment method comprises the following steps: mixing the sodium alginate sample, calcium salt and a solvent to obtain a pretreatment solution; according to the method disclosed by the invention, the detection interference is reduced by utilizing the specific binding of Ca < 2 + > and sodium alginate, the detection recovery rate and precision are improved, and reliable technical support is provided for the quality control of sodium alginate and similar polysaccharide materials.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a pretreatment method for detecting endotoxin in a sodium alginate sample and an application thereof. Background Art

[0002] Sodium alginate, also known as sodium alginate, is a natural polysaccharide extracted from the cell wall of brown algae such as kelp and giant kelp. It is essentially a linear natural polymer and a byproduct of extracting iodine and mannitol from kelp or sargassum of brown algae. Its molecule is composed of β-D-mannuronic acid (β-D-mannuronic, M) and α-L-guluronic acid (α-L-guluronic, G) connected by (1→4) bonds. The aqueous solution of sodium alginate has a high viscosity and has been used as a thickener, stabilizer, emulsifier, etc. for food. It has good biocompatibility and shows broad application prospects as a biomaterial for tissue regeneration. However, endotoxins in alginate materials will seriously affect their biocompatibility during application, so it is necessary to accurately detect the endotoxins contained therein.

[0003] The commonly used method for endotoxin detection is the Limulus amebocyte lysate (LAL) method, including the gel method, turbidity method and colorimetric method. Because sodium alginate itself has an adsorption effect on endotoxin, it hinders the reaction between endotoxin and the enzyme in the LAL reagent, resulting in a low recovery rate and reduced detection accuracy.

[0004] Therefore, it is necessary to provide a pretreatment method for endotoxin detection in sodium alginate to improve the accuracy and recovery rate of detection. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a pretreatment method for detecting endotoxin in sodium alginate samples and its application. 2+ The specific binding with sodium alginate reduces the interference of detection, improves the recovery rate and precision of detection, and provides reliable technical support for the quality control of sodium alginate and similar polysaccharide materials.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a pretreatment method for detecting endotoxin in a sodium alginate sample, the pretreatment method comprising: mixing a sodium alginate sample, a calcium salt and a solvent to obtain a pretreatment solution.

[0008] The present invention introduces calcium salt and utilizes Ca 2+The high affinity for the carboxyl group (-COO-) in the sodium alginate molecule forms a spatially stable calcium alginate gel microstructure, which effectively shields its competitive chelation of key divalent metal ions in the LAL reaction system, thereby eliminating interference with endotoxin detection and improving the accuracy and reliability of endotoxin detection.

[0009] The present invention introduces Ca in the pretreatment 2+ Compared with other metal ions (Mg 2+ 、Zn 2+ , Ba 2+ ) has better detection accuracy, which can be analyzed from the following three aspects:

[0010] (1) Ion selectivity effect: Through the analysis of ion radius and charge density, Ca 2+ The stability of the combination with the carboxyl group of sodium alginate is stronger.

[0011] (2) Electronic structure and coordination mechanism: Ca 2+ The superiority of Ca can be attributed to its unique electronic structure and coordination geometry. 2+ The formed "egg-box structure" exhibited the best gel properties, while the complexes formed by other metal ions presented different micromorphologies and structural stabilities.

[0012] (3) Compatibility of LAL enzyme system: In addition to coordination ability, different metal ions also have different degrees of influence on the LAL enzyme system itself, especially Zn 2+ , which has strong enzyme inhibitory activity, while Ca 2+ It shows the best compatibility with the LAL enzyme system and reduces additional interference to the detection system.

[0013] Preferably, the calcium salt includes any one of calcium chloride, calcium acetate, calcium nitrate or calcium lactate, or a combination of at least two thereof.

[0014] Preferably, the calcium salt is a combination of calcium chloride and calcium acetate.

[0015] Preferably, the molar ratio of calcium chloride to calcium acetate is 3:1 to 1:3 (for example, it can be 3:1, 2:1, 1:1, 1:2, 1:3, etc.).

[0016] In the present invention, when the calcium salt is a combination of calcium chloride and calcium acetate, a synergistic effect can be produced. Calcium chloride provides a rapid binding ability, while calcium acetate provides a more stable and long-lasting coordination effect, which jointly improves the detection recovery rate, so that the recovery rate is 81.67%, which is better than the detection effect of a single calcium salt.

[0017] Preferably, the solvent comprises water.

[0018] Preferably, the concentration of sodium alginate in the pretreatment solution is 8-13 ng / mL (for example, it can be 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, etc.), and the concentration of calcium salt is 0.4-0.65 mmol / L (for example, it can be 0.4 mmol / L, 0.45 mmol / L, 0.5 mmol / L, 0.55 mmol / L, 0.6 mmol / L, 0.65 mmol / L, etc.).

[0019] In the present invention, by analyzing the net fluorescence difference (ΔRFU) pattern under different dilution conditions, it is found that as the dilution increases, the interference pattern of the sample on the LAL enzymatic luminescence system changes from enhanced interference (the ΔRFU of the unspiked sample is abnormally high) to a controllable response (the ΔRFU of the spiked sample is in a good linear relationship with the standard curve). This change in interference pattern is a key indicator for evaluating the effectiveness of the method. The detection data show that there is an ideal "detection window", that is, while ensuring sufficient sensitivity (endotoxin can be detected), the sodium alginate concentration must be diluted to a sufficiently low level to eliminate interference. Therefore, within the concentration range defined by the present invention, higher detection accuracy and precision can be achieved.

[0020] Preferably, the pretreatment method specifically comprises: mixing and dissolving sodium alginate and a solvent to obtain a sodium alginate solution; and mixing the sodium alginate solution and a calcium salt solution to obtain a pretreatment solution.

[0021] Preferably, the dissolution temperature is 40-60°C (for example, 40°C, 45°C, 50°C, 55°C, 60°C, etc.), and the dissolution time is 25-35min (for example, 25min, 26min, 28min, 30min, 32min, 34min, 35min, etc.).

[0022] In the present invention, if the dissolution temperature is too low, the sodium alginate will not be fully dissolved, thereby reducing the recovery rate; if the dissolution temperature is too high, the thermal degradation products may interfere with the LAL reaction, thereby reducing the reliability of the detection.

[0023] In a second aspect, the present invention provides an application of the pretreatment method for endotoxin detection in a sodium alginate sample according to the first aspect in endotoxin detection.

[0024] In a third aspect, the present invention provides a method for detecting endotoxins in a sodium alginate sample, the method comprising: performing endotoxin detection on a pretreatment solution prepared by the pretreatment method described in the first aspect.

[0025] Preferably, the detection is performed using a Factor C endotoxin detection kit.

[0026] In the present invention, the mechanism of sodium alginate interfering with detection and introducing calcium ions is analyzed as follows:

[0027] Molecular mechanism of sodium alginate chelation interference:

[0028] (1) Multi-site chelation theory: Sodium alginate is a linear copolymer composed of β-D-mannuronic acid (M unit) and α-L-guluronic acid (G unit) in different proportions and sequences, with a carboxyl density of up to one per monosaccharide unit. This high-density anionic functional group can form a multi-site chelation structure, competitively binding to key divalent metal ions (mainly Mg) in the LAL reaction system. 2+ ), resulting in decreased enzyme activity.

[0029] (2) Endotoxin masking effect: Sodium alginate can directly form a complex with endotoxin lipopolysaccharide molecules, masking the key phosphate groups and acyl chain structures therein, thereby reducing its recognition efficiency with lipopolysaccharide binding protein (LBP) in the LAL system.

[0030] (3) Concentration dependence of interference pattern: As the concentration of sodium alginate increases, the interference pattern changes from a simple effect on enzyme activity to a combined effect of endotoxin molecule masking and enzyme inhibition, which explains why a more serious negative recovery phenomenon is observed at low dilution factors.

[0031] Ca 2+ Molecular mechanism of neutralization:

[0032] (1) “Egg box model” and selective binding: Ca 2+ It forms a classic "egg-box structure" with sodium alginate, especially with the sequence segment rich in G units, forming a highly regular coordination structure. Molecular dynamics simulation shows that each Ca 2+ It can form coordination bonds with four carboxyl oxygen atoms at the same time. This structural specificity is Ca 2+ The key reason why it is superior to other metal ions.

[0033] (2) Synergistic effect mechanism: Ca 2+ The synergistic effect of neutralization and high dilution can be explained by the “critical chelation threshold” theory. Only when the sodium alginate concentration is diluted to a low enough level, the added Ca 2+ In order to form a stable coordination with most of the carboxyl sites and achieve effective neutralization; under high concentration conditions, limited Ca 2+ Failure to cover a sufficient proportion of chelation sites results in insufficient neutralization effect.

[0034] Compared with the prior art, the present invention has at least the following beneficial effects:

[0035] (1) Solving the key technical bottleneck: The detection method provided by the present invention solves the problem of recovery rate of endotoxin detection of chelating polysaccharides such as sodium alginate for the first time, and improves the recovery rate from seriously unqualified (negative value) to within the industry standard range, providing key technical support for the quality control of related products.

[0036] (2) Excellent methodological performance: The pretreatment method provided by the present invention has high recovery rate and precision, good linear relationship within the working range, and high data reliability.

[0037] (3) Easy to operate and cost-effective: The entire pretreatment process does not require special equipment. Only conventional, low-cost calcium salt reagents are added. The operation steps are simple and easy to implement in a conventional laboratory environment, which greatly reduces the detection cost and technical threshold.

[0038] (4) Wide range of applications: This method is not only applicable to sodium alginate, but can also be extended to other polysaccharides with similar chelating properties (such as carboxymethyl cellulose, pectin, etc.), greatly expanding the application prospects of the method. DETAILED DESCRIPTION

[0039] For the convenience of understanding the present invention, the present invention lists the following embodiments. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0040] Example 1

[0041] This embodiment provides a pre-treatment method for endotoxin detection in sodium alginate, comprising the following steps:

[0042] (1) Sample dissolution: Accurately weigh 0.1 g of sodium alginate sample, add 10 mL of pyrogen-free water (LAL water for endotoxin detection), and place in a 60°C constant temperature water bath to dissolve for 30 min. During this period, gently shake once every 5 min to ensure that the sample is completely dissolved into a uniform and transparent solution;

[0043] (2) Primary dilution: Use a pyrogen-free pipette to accurately take 0.1 mL of the above solution, add it to 9.9 mL of pyrogen-free water, and mix thoroughly to achieve a 100-fold primary dilution;

[0044] (3) Calcium ion neutralization: Use a pyrogen-free pipette to accurately take 1900 μL of the diluted sample solution, add 100 μL of 0.1 M calcium chloride (CaCl2) aqueous solution, and vortex mix for 15 seconds in a pyrogen-free environment;

[0045] (4) Final dilution: The solution obtained in step (3) was further diluted 10 times with pyrogen-free water, mixed and allowed to stand for 5 minutes to obtain a pretreatment solution, in which the concentration of sodium alginate was 10 ng / mL and the concentration of calcium salt was 0.5 mmol / L.

[0046] Example 2

[0047] This embodiment provides a pre-treatment method for endotoxin detection in sodium alginate, comprising the following steps:

[0048] (1) Sample dissolution: Accurately weigh 0.1 g of sodium alginate sample, add 10 mL of pyrogen-free water (LAL water for endotoxin detection), and place in a 40°C constant temperature water bath to dissolve for 35 min. During this period, gently shake once every 5 min to ensure that the sample is completely dissolved into a uniform and transparent solution;

[0049] (2) Primary dilution: Use a pyrogen-free pipette to accurately take 0.1 mL of the above solution, add it to 9.9 mL of pyrogen-free water, and mix thoroughly to achieve a 100-fold primary dilution;

[0050] (3) Calcium ion neutralization: Use a pyrogen-free pipette to accurately take 1900 μL of the diluted sample solution, add 100 μL of 0.1 M calcium acetate (Ca(CH3COO)2) aqueous solution, and vortex mix for 15 seconds in a pyrogen-free environment;

[0051] (4) Final dilution: The solution obtained in step (3) was further diluted 8 times with pyrogen-free water, mixed and allowed to stand for 5 minutes to obtain a pretreatment solution, in which the concentration of sodium alginate was 12.5 ng / mL and the concentration of calcium salt was 0.625 mmol / L.

[0052] Example 3

[0053] This embodiment provides a pre-treatment method for endotoxin detection in sodium alginate, comprising the following steps:

[0054] (1) Sample dissolution: Accurately weigh 0.1 g of sodium alginate sample, add 10 mL of pyrogen-free water (LAL water for endotoxin detection), and place in a 50°C constant temperature water bath to dissolve for 35 min. During this period, gently shake once every 5 min to ensure that the sample is completely dissolved into a uniform and transparent solution;

[0055] (2) Primary dilution: Use a pyrogen-free pipette to accurately take 0.1 mL of the above solution, add it to 9.9 mL of pyrogen-free water, and mix thoroughly to achieve a 100-fold primary dilution;

[0056] (3) Calcium ion neutralization: Use a pyrogen-free pipette to accurately take 1900 μL of the diluted sample solution and add 100 μL of 0.1 M calcium lactate (C6H 10 CaO6) aqueous solution, vortex mix for 15 seconds in a pyrogen-free environment;

[0057] (4) Final dilution: The solution obtained in step (3) was further diluted 12.5 times with pyrogen-free water, mixed and allowed to stand for 5 minutes to obtain a pretreatment solution, in which the concentration of sodium alginate was 8 ng / mL and the concentration of calcium salt was 0.4 mmol / L.

[0058] Example 4

[0059] This embodiment provides a pretreatment method for endotoxin detection in sodium alginate, which differs from Embodiment 1 only in that a combination of calcium chloride and calcium acetate is used in step (3) to neutralize calcium ions: 1900 μL of the diluted sample solution is accurately taken using a pyrogen-free pipette, 50 μL of 0.1M calcium chloride (CaCl2) and 50 μL of 0.1M calcium acetate (Ca(CH3COO)2) aqueous solution (molar ratio 1:1) are added, and vortex mixed for 15 seconds in a pyrogen-free environment. For other references, refer to Embodiment 1.

[0060] Example 5

[0061] This embodiment provides a pretreatment method for detecting endotoxin in sodium alginate, which differs from Embodiment 1 only in that, in step (4), the solution obtained in step (3) is further diluted 5 times with pyrogen-free water, mixed and allowed to stand for 5 minutes to obtain a pretreatment solution, wherein the concentration of sodium alginate in the pretreatment solution is 20 ng / mL, and the concentration of calcium salt is 1 mmol / L.

[0062] Example 6

[0063] This embodiment provides a pretreatment method for detecting endotoxin in sodium alginate, which differs from Embodiment 1 only in that the dissolution temperature is adjusted to 25° C. in step (1). For other matters, refer to Embodiment 1.

[0064] Example 7

[0065] This embodiment provides a pretreatment method for detecting endotoxin in sodium alginate, which differs from Embodiment 1 only in that the dissolution temperature is adjusted to 70° C. in step (1). For other matters, refer to Embodiment 1.

[0066] Comparative Example 1

[0067] This embodiment provides a pretreatment method for detecting endotoxin in sodium alginate, which differs from Embodiment 1 only in that calcium chloride is replaced by an equimolar amount of magnesium chloride in step (3). For other matters, refer to Embodiment 1.

[0068] Comparative Example 2

[0069] This embodiment provides a pretreatment method for detecting endotoxin in sodium alginate, which differs from Embodiment 1 only in that calcium chloride is replaced by an equimolar amount of zinc sulfate in step (3). For other matters, refer to Embodiment 1.

[0070] Comparative Example 3

[0071] This embodiment provides a pretreatment method for detecting endotoxin in sodium alginate, which differs from Embodiment 1 only in that calcium chloride is replaced by an equimolar amount of barium chloride in step (3). For other matters, refer to Embodiment 1.

[0072] Comparative Example 4

[0073] This comparative example provides a pretreatment method for detecting endotoxin in sodium alginate, which differs from Example 1 only in that calcium salt neutralization is not performed, and the solution obtained in step (1) is directly diluted to obtain a pretreatment solution with a sodium alginate concentration of 10 ng / mL.

[0074] Test Example 1

[0075] Recovery test

[0076] The samples were pretreated according to Examples 1-7 and Comparative Examples 1-4, respectively, and the endotoxin content was determined using a recombinant factor C endotoxin detection kit (manufacturer Adamas life, model 96 Tests) in strict accordance with the instructions, and a recovery test was performed, wherein the spiked group added a known concentration of endotoxin standard (0.275-10EU / mL) to the sample to evaluate the recovery rate; the unspiked group was the original sample without the addition of endotoxin standard, which was used to determine the basic endotoxin content.

[0077] Table 1

[0078]

[0079]

[0080] Test Example 2

[0081] Precision test

[0082] The samples were pre-treated according to Examples 1-5 and Comparative Example 4, and the endotoxin content was determined using a recombinant factor C endotoxin detection kit (manufacturer Adamas life, model 96 Tests) in strict accordance with the instructions, and a precision test was performed (n=6). The results are shown in Table 2.

[0083] Table 2

[0084] Grouping Mean concentration (EU / mL) RSD(%) Example 1 0.200±0.005 2.67％ Example 2 0.195±0.007 3.59％ Example 3 0.189±0.008 4.23％ Example 4 0.222±0.005 2.25％ Example 5 0.346±0.013 3.78％ Comparative Example 4 0.357±0.021 5.88％

[0085] From the test results in Table 1 and Table 2, we can see that:

[0086] (1) The pretreatment method for endotoxin detection in sodium alginate provided by the present invention solves the problem of chelation interference in endotoxin detection in sodium alginate samples by using calcium salt for neutralization of calcium ions. In the preferred technical solution, a recovery rate that meets industry standards (50-200%) is achieved, and a high detection precision (RSD <5%) is achieved.

[0087] (2) By comparing Example 1 and Example 4, it can be seen that when the calcium salt is a combination of calcium chloride and calcium acetate, the two have a synergistic effect and show better recovery rate (81.67%) and precision (RSD = 2.25%).

[0088] By comparing Example 1 and Example 5, it can be seen that if the dilution factor of the pretreatment solution is too low, the interference mode in the detection changes from the simple effect of enzyme activity to the combined effect of endotoxin molecule masking and enzyme inhibition, resulting in a lower recovery rate and a decrease in precision.

[0089] By comparing Example 1 with Examples 6-7, it can be seen that if the dissolution temperature is too low, the sodium alginate cannot be fully dissolved; if the dissolution temperature is too high, thermal degradation products may appear to interfere with the LAL reaction, thereby reducing the reliability of the detection.

[0090] (3) By comparing Example 1 with Comparative Examples 1-3, it can be seen that compared with other divalent metal ions, Ca 2+ A higher recovery rate was achieved due to the higher stability of the complex formed with sodium alginate and the best compatibility with the LAL enzyme system.

[0091] By comparing Example 1 with Comparative Example 4, it can be seen that when Ca is not used 2+ After neutralization, the sample showed severe detection interference and accurate detection of endotoxin could not be achieved.

[0092] Test Example 3

[0093] Linear analysis and quantitative evaluation

[0094] The samples were pre-treated according to Example 1 and Example 5, respectively, and a commercial group C factor endotoxin detection kit was used for content detection and linear analysis. The results are shown in Table 3.

[0095] Table 3

[0096]

[0097] The test results show:

[0098] (1) Linear response characteristics: The method under the conditions of Example 1 showed an excellent linear relationship (R 2=0.996), the linear regression equation y=0.74x+0.017 shows a good slope (close to the ideal value 1.0) and a small intercept (close to the ideal value 0); in contrast, the linearity of Example 5 with a smaller dilution multiple is significantly poorer (R 2 =0.879).

[0099] (2) Method sensitivity analysis: The slope in Example 1 was significantly improved compared with that in Example 5, showing a higher response sensitivity to endotoxin.

[0100] (3) Optimization of limit of quantification (LOQ): The signal-to-noise ratio method (S / N=10) was used to calculate that, under the conditions of Example 1, the lower limit of quantification of the method was 0.018 EU / mL, which was much better than the traditional method (about 0.05 EU / mL), indicating that the present invention directly expanded the practical working range of the method.

[0101] (4) Dose dependence of method deviation: The relative deviation between the theoretical concentration and the detected concentration in Example 1 was significantly reduced compared with that in Example 5, indicating that Example 1 has a better detection effect.

[0102] (5) Residual analysis: Residual analysis was performed on the calibration curve under the conditions of Example 1. The residuals were randomly distributed and the mean was close to zero (p>0.05), which confirmed the applicability of the linear model and the reliability of the data quality.

[0103] Comprehensive linearity, quantitation limit and residual analysis confirmed that Example 1 had excellent quantitative performance under the conditions, with a reliable linear range of 0.018-0.5EU / mL (18-500EU / g in the original sample), which fully met the actual needs of endotoxin control in pharmaceutical-grade sodium alginate.

[0104] Test Example 3

[0105] Comprehensive evaluation of accuracy

[0106] Based on the above data, the accuracy of Example 1 and Example 5 was comprehensively evaluated, and the results are shown in Table 4.

[0107] Table 4

[0108] Grouping Theoretical recovery rate (%) Measured recovery rate (%) deviation(%) z-score Example 1 70-130 76.45 9.21 0.51 Example 5 70-130 27.83 -60.24 -3.35

[0109] The accuracy evaluation and z-score analysis results show:

[0110] (1) Compliance determination: Based on the industry standard (recovery rate 50-200%), the recovery rate under the conditions of Example 1 (76.45%) is within the acceptable range, and the z-score is 0.51 (|z|<2 is considered satisfactory).

[0111] (2) Deviation distribution characteristics: With the decrease of dilution factor, the recovery rate deviation shows an exponentially increasing trend. This characteristic reflects the nonlinear relationship between the chelation interference intensity of sodium alginate and its concentration, which is consistent with the multi-site chelation theory in coordination chemistry.

[0112] (3) Method bias evaluation: Under the conditions of Example 1, the method bias was -26.55%. Although there was a certain systematic bias, it could be effectively corrected by the calibration function, and the bias had good inter-batch stability (RSD = 4.3%, n = 3 batches).

[0113] This systematic evaluation strongly demonstrates that the present invention can provide accuracy that meets industry standards under the conditions of Example 1.

[0114] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention are within the protection scope and disclosure scope of the present invention.

Claims

1. A pretreatment method for endotoxin detection in sodium alginate samples, characterized in that: The pretreatment method comprises: mixing a sodium alginate sample, a calcium salt and a solvent to obtain a pretreatment solution.

2. The pre-treatment method according to claim 1, characterized in that: The calcium salt includes any one of calcium chloride, calcium acetate, calcium nitrate or calcium lactate, or a combination of at least two thereof.

3. The pre-treatment method according to claim 2, characterized in that: The calcium salt is a combination of calcium chloride and calcium acetate; Preferably, the molar ratio of calcium chloride to calcium acetate is 3:1 to 1:

3.

4. The pre-treatment method according to any one of claims 1 to 3, characterized in that: The solvent includes water.

5. The pre-treatment method according to any one of claims 1 to 4, characterized in that: The concentration of sodium alginate in the pretreatment solution is 8-13 ng / mL, and the concentration of calcium salt is 0.4-0.65 mmol / L.

6. The pre-treatment method according to any one of claims 1 to 5, characterized in that: The pretreatment method specifically comprises: mixing and dissolving sodium alginate and a solvent to obtain a sodium alginate solution; and mixing the sodium alginate solution and a calcium salt solution to obtain a pretreatment solution.

7. The pre-treatment method according to claim 6, characterized in that: The dissolving temperature is 40-60°C and the dissolving time is 25-35 minutes.

8. Use of the pretreatment method for endotoxin detection in a sodium alginate sample according to any one of claims 1 to 7 in endotoxin detection.

9. A method for detecting endotoxin in a sodium alginate sample, characterized in that: The method comprises: conducting endotoxin detection on the pretreatment liquid prepared by the pretreatment method according to any one of claims 1 to 7.

10. The method according to claim 9, characterized in that The detection is performed using a Factor C endotoxin detection kit.

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