Method for detecting content of bacterial endotoxin in sodium dihydrogen phosphate monohydrate

Abstract

The invention relates to the technical field of microbiological detection, and discloses a sodium dihydrogen phosphate monohydrate bacterial endotoxin content detection method, which comprises the steps of sample pretreatment, interference test verification, sample solution preparation, tachypleus amebocyte lysate reaction, result determination and quantitative detection. Wherein the sample pretreatment adopts heatless raw water dissolution and ultrasonic oscillation to ensure that the sample is uniformly dispersed; an interference test verifies that a standard bacterial endotoxin solution is mixed with a sample, the interference degree is evaluated, and the dilution ratio is adjusted; the tachypleus amebocyte lysate reaction is subjected to qualitative and quantitative analysis by adopting a gel method, a dynamic nephelometry and a chromogenic matrix method, so that the detection sensitivity is ensured. By optimizing sample treatment conditions, interference elimination strategies and detection parameters, the accuracy and repeatability of detection are improved, the probability of occurrence of false positive and false negative is reduced, the applicability and reliability of the detection method are improved, and strict quality control requirements of pharmaceutical grade sodium dihydrogen phosphate monohydrate are met.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganism detection, and particularly relates to a bacterial endotoxin content detection method of sodium dihydrogen phosphate monohydrate. BACKGROUND

[0002] Microorganism detection refers to detecting and identifying microorganisms contained in samples through various technologies and methods, including bacteria, viruses, fungi and parasites. This process plays an important role in food safety, water quality detection, pharmaceutical production and cosmetic production to ensure product safety and quality. Microorganism detection can identify pollution sources, evaluate sanitary conditions and help ensure that products meet various hygiene and safety standards. At present, common microorganism detection technologies include traditional culture method, advanced molecular biology technology, immunological method and various rapid detection technologies.

[0003] At present, sodium dihydrogen phosphate monohydrate is widely used in pharmaceutical preparations. When bacterial endotoxin detection is performed, the conventional limulus reagent method may be interfered by chemical components in the sample, and the actual bacterial endotoxin content cannot be accurately reflected. If interfering substances exist in the sample, false positives and false negatives may occur, and the accuracy of detection cannot be guaranteed.

[0004] Therefore, the present application provides a bacterial endotoxin content detection method of sodium dihydrogen phosphate monohydrate to solve the above problems. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a bacterial endotoxin content detection method of sodium dihydrogen phosphate monohydrate to solve the problems raised in the background.

[0006] To achieve the above object, the present application provides the following technical scheme: a bacterial endotoxin content detection method of sodium dihydrogen phosphate monohydrate, comprising the following steps: Step 1: sample pretreatment, dissolving the sodium dihydrogen phosphate monohydrate sample to be tested in pyrogen-free water, and ultrasonically oscillating for 10-30 minutes to fully dissolve and uniformly disperse the sample; Step 2: interference test verification, mixing the standard bacterial endotoxin solution and the sample solution at different ratios to verify the interference degree of the sample on the limulus reagent reaction; Step 3: sample solution preparation, adjusting the dilution multiple of the sample solution according to the interference test result to ensure that the bacterial endotoxin content is within the detection range of the limulus reagent; Step 4: limulus reagent reaction, mixing the diluted sample solution with the limulus reagent, incubating in a constant-temperature water bath for 60±5 minutes, and observing the gel formation; Step 5: result determination, determining whether the bacterial endotoxin content in the sample exceeds the specified limit value according to the integrity and stability of the gel formation. Step six: quantitative detection, using dynamic turbidity method and chromogenic substrate method to quantitatively analyze the endotoxin in the sample, and calculate the specific content; The preparation method of the pyrogen-free water comprises the following steps: filtering distilled water through a 0.22-micron microporous filter, and then performing dry heat sterilization at 250 DEG C for 30 minutes, and cooling to room temperature for standby use; the limulus reagent is a gel limulus reagent, and the sensitivity is 0.03-0.25 EU / mL.

[0007] Further, in step one, the frequency of ultrasonic oscillation is 40-60 kHz, the temperature is 25±2 DEG C, and the ultrasonic power is 100-300 W.

[0008] Further, in step two, the specific method for verifying the interference test is as follows: Prepare a standard bacterial endotoxin solution with a concentration of 2 lambda, wherein lambda is the labeled sensitivity of the limulus reagent; Mix the standard bacterial endotoxin solution and the sample solution at a volume ratio of 1:1, 1:2 and 1:4, and mark them as solution A, solution B and solution C respectively; React solution A, solution B and solution C with the limulus reagent respectively, observe the gel formation, and calculate the recovery rate; If the recovery rate is within the range of 50%-200%, it is determined that the sample has no interference; if the recovery rate exceeds the range, the dilution multiple needs to be further adjusted and the interference eliminator needs to be treated.

[0009] Further, the interference eliminator is a pyrogen-free Tris-HCl buffer solution, the pH value is 7.0±0.2, the concentration is 0.1-0.5 mol / L, and the addition amount is 1%-5% of the volume of the sample solution.

[0010] Further, in step four, the temperature of the constant-temperature water bath is 37±1 DEG C, and the incubation time is 60±5 minutes, and vibration and light are avoided during the incubation process.

[0011] Further, in step five, the determination standard of gel formation is as follows: Positive control: the standard bacterial endotoxin solution reacts with the limulus reagent to form complete gel, and does not flow when inverted by 180 DEG ; Negative control: no gel is formed after the pyrogen-free water reacts with the limulus reagent, and the solution remains flowable; Sample solution: if complete gel is formed and does not flow when inverted by 180 DEG, it is determined to be positive; if no gel is formed and the gel is incomplete, it is determined to be negative.

[0012] Further, in step six, the specific operation of the dynamic turbidity method is as follows: After mixing the sample solution and the limulus reagent, place them in a dynamic turbidimeter to monitor the turbidity change of the reaction liquid in real time; The time required for the turbidity of the reaction solution to reach the preset threshold is recorded, and the bacterial endotoxin content is calculated through a standard curve; The preparation method of the standard curve is as follows: different concentrations of standard bacterial endotoxin solution are reacted with limulus reagent, and a turbidity-time curve is drawn.

[0013] Further, the detection wavelength of the dynamic turbidimeter is 340 nm, the reaction temperature is 37±1 DEG C, and the reaction time is 90-120 minutes.

[0014] Further, in step six, the specific operation of the chromogenic substrate method is as follows: The sample solution is mixed with the chromogenic substrate limulus reagent, incubated, and then the chromogenic termination solution is added; The absorbance of the reaction solution at 405 nm is measured, and the bacterial endotoxin content is calculated through a standard curve; The preparation method of the standard curve is as follows: different concentrations of standard bacterial endotoxin solution are reacted with limulus reagent, and an absorbance-concentration curve is drawn.

[0015] Further, the method further comprises a quality control step: A positive control, a negative control and a sample control are set for each batch of detection; The positive control is a standard bacterial endotoxin solution with a known concentration, the negative control is pyrogen-free water, and the sample control is a sample solution without limulus reagent; If the positive control does not form a gel and the negative control forms a gel, it is determined that the batch of detection is invalid and needs to be re-detected.

[0016] Compared with the prior art, the present application provides a bacterial endotoxin content detection method for monohydrated sodium dihydrogen phosphate, which has the following beneficial effects: 1. In the present application, by optimizing the sample pretreatment step, when detecting the bacterial endotoxin of monohydrated sodium dihydrogen phosphate, the sample is treated by ultrasonic oscillation with specific parameters, so that the sample is fully dissolved and uniformly dispersed, which can reduce the aggregation phenomenon that may exist in the sample, ensure the uniformity of the detection sample, and at the same time, by strictly controlling the preparation conditions of the pyrogen-free water, it can ensure that no exogenous endotoxin interference is introduced during the detection process, improve the accuracy of the detection result, and further reduce the probability of false positive and false negative.

[0017] 2. In the present application, by setting an interference test verification link, before detecting the bacterial endotoxin, the standard endotoxin solution is mixed with the sample solution at different proportions for testing, which can evaluate the interference degree of the sample on the limulus reagent in real time, so that the detection method can automatically adjust the sample dilution multiple and add interference eliminating agent according to the interference situation, avoid the detection deviation caused by the sample matrix effect, ensure the optimization of the detection conditions of different batches of samples, and further improve the applicability and reliability of the detection method.

[0018] 3. In the present application, by adopting the dynamic turbidity method and the chromogenic substrate method double quantitative detection means, when determining the bacterial endotoxin content, the most suitable detection method can be selected according to the sample characteristics, the turbidity change and the color reaction in the reaction process are monitored in real time, the bacterial endotoxin is quantitatively determined by establishing a standard curve, so that the detection method can cover a wider detection range, and the detection ability of low-concentration endotoxin is improved, the sensitivity and accuracy of the detection are improved, and the strict quality control requirements of pharmaceutical-grade sodium phosphate monobasic monohydrate are met. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The step flow chart of the bacterial endotoxin content detection method of the sodium phosphate monobasic monohydrate of the present application. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0021] Please refer to Figure 1 and Tables 1-6, Example 1: a bacterial endotoxin content detection method of sodium phosphate monobasic monohydrate, comprising the following steps: Step one: sample pretreatment, dissolving the sodium phosphate monobasic monohydrate sample to be tested in pyrogen-free water, ultrasonic oscillation for 10 minutes, so that the sample is fully dissolved and uniformly dispersed; Step two: interference test verification, mixing the standard bacterial endotoxin solution with the sample solution in different proportions to verify the interference degree of the sample on the reaction of limulus reagent; Step three: sample solution preparation, according to the interference test results, adjusting the dilution multiple of the sample solution to ensure that the bacterial endotoxin content is within the detection range of the limulus reagent; Step four: limulus reagent reaction, mixing the diluted sample solution with the limulus reagent, placing it in a constant-temperature water bath for incubation for 55 minutes, and observing the gel formation; Step five: result determination, according to the integrity and stability of the gel formation, judging whether the content of bacterial endotoxin in the sample exceeds the specified limit value; Step six: quantitative detection, using the dynamic turbidity method and the chromogenic substrate method to quantitatively analyze the bacterial endotoxin in the sample and calculate its specific content; The preparation method of the pyrogen-free water comprises: filtering distilled water through a 0.22-micron microporous filter, drying and sterilizing at 250 DEG C for 30 minutes, and cooling to room temperature for standby; the limulus reagent is a gel limulus reagent, and the sensitivity is 0.03 EU / mL.

[0022] In step one, the frequency of ultrasonic oscillation is 40 kHz, the temperature is 23 DEG C, and the ultrasonic power is 100 W.

[0023] In step two, the specific method for verifying the interference test is as follows: A standard bacterial endotoxin solution is prepared, and the concentration is 2 lambda, wherein lambda is the labeled sensitivity of the limulus reagent; The standard bacterial endotoxin solution and the sample solution are mixed in a volume ratio of 1:1, 1:2 and 1:4, and are respectively recorded as solution A, solution B and solution C; Solution A, solution B and solution C are respectively reacted with the limulus reagent, the gel formation is observed, and the recovery rate is calculated; If the recovery rate is within the range of 50%, it is determined that the sample has no interference; if the recovery rate exceeds the range, the dilution multiple needs to be further adjusted and the interference eliminating agent needs to be treated.

[0024] The interference eliminating agent is a pyrogen-free Tris-HCl buffer solution, the pH value is 6.8, the concentration is 0.1 mol / L, and the addition amount is 1% of the volume of the sample solution.

[0025] In step four, the temperature of the constant-temperature water bath is 36 DEG C, the incubation time is 55 minutes, and vibration and light are avoided during the incubation process.

[0026] In step five, the determination standard of gel formation is as follows: Positive control: the standard bacterial endotoxin solution and the limulus reagent form complete gel after reaction, and do not flow when inverted by 180 DEG ; Negative control: no gel is formed after the pyrogen-free water and the limulus reagent are reacted, and the solution maintains fluidity; Sample solution: if complete gel is formed and does not flow when inverted by 180 DEG, it is determined to be positive; if no gel is formed and the gel is incomplete, it is determined to be negative.

[0027] In step six, the specific operation of the dynamic turbidity method is as follows: After the sample solution and the limulus reagent are mixed, they are placed in a dynamic turbidimeter, and the turbidity change of the reaction solution is monitored in real time; The time required for the turbidity of the reaction solution to reach the preset threshold value is recorded, and the bacterial endotoxin content is calculated through a standard curve; The preparation method of the standard curve is as follows: the standard bacterial endotoxin solution with different concentrations is reacted with the limulus reagent, and a turbidity-time curve is drawn.

[0028] The detection wavelength of the dynamic turbidimeter is 340 nm, the reaction temperature is 36℃, and the reaction time is 90 minutes.

[0029] In step six, the specific operation of the color developing substrate method is as follows: The sample solution is mixed with the color developing substrate limulus reagent, incubated, and then the color developing termination liquid is added; The absorbance of the reaction solution at 405 nm is measured, and the content of bacterial endotoxin is calculated by the standard curve; The preparation method of the standard curve is as follows: different concentrations of standard bacterial endotoxin solution are reacted with the color developing substrate limulus reagent, and an absorbance-concentration curve is drawn.

[0030] The method also includes a quality control step: Positive control, negative control and sample control are set for each batch of detection; The positive control is a standard bacterial endotoxin solution with a known concentration, the negative control is pyrogen-free water, and the sample control is a sample solution without limulus reagent; If the positive control does not form a gel and the negative control forms a gel, the batch of detection is invalid and needs to be re-detected.

[0031] Example 2: A bacterial endotoxin content detection method for sodium dihydrogen phosphate monohydrate, comprising the following steps: Step 1: Sample pretreatment, dissolve the sodium dihydrogen phosphate monohydrate sample to be tested in pyrogen-free water, and ultrasonically shake for 20 minutes to fully dissolve and disperse the sample uniformly; Step 2: Interference test verification, mix standard bacterial endotoxin solution with sample solution in different proportions to verify the interference degree of the sample on the limulus reagent reaction; Step 3: Sample solution preparation, adjust the dilution multiple of the sample solution according to the interference test results to ensure that the bacterial endotoxin content is within the detection range of the limulus reagent; Step 4: Limulus reagent reaction, mix the diluted sample solution with the limulus reagent, incubate in a constant temperature water bath for 60 minutes, and observe the gel formation; Step 5: Result determination, determine whether the content of bacterial endotoxin in the sample exceeds the specified limit value according to the integrity and stability of the gel formation; Step 6: Quantitative detection, use dynamic turbidity method and color developing substrate method to quantitatively analyze the bacterial endotoxin in the sample and calculate its specific content; The preparation method of the pyrogen-free water includes: filtering the distilled water through a 0.22 μm microporous filter, drying and sterilizing at 250℃ for 30 minutes, and cooling to room temperature for standby; the limulus reagent is a gel method limulus reagent, and its sensitivity is 0.14 EU / mL.

[0032] In step one, the frequency of ultrasonic vibration is 50 kHz, the temperature is 25℃, and the ultrasonic power is 200 W.

[0033] In step two, the specific method for interference test verification is as follows: Prepare a standard bacterial endotoxin solution with a concentration of 2λ, where λ is the labeled sensitivity of the limulus reagent; Mix the standard bacterial endotoxin solution with the sample solution at a volume ratio of 1:1, 1:2, and 1:4, respectively, and label them as solution A, solution B, and solution C; React solution A, solution B, and solution C with limulus reagent, respectively, observe the gel formation, and calculate the recovery rate; If the recovery rate is within the range of 125%, it is determined that the sample has no interference; if the recovery rate exceeds this range, further adjustment of the dilution factor and the use of interference eliminator are required.

[0034] The interference eliminator is a pyrogen-free Tris-HCl buffer with a pH value of 6.8 and a concentration of 0.3 mol / L, and the addition amount is 3% of the volume of the sample solution.

[0035] In step four, the temperature of the constant temperature water bath is 37℃, and the incubation time is 60 minutes. Avoid shaking and light during incubation.

[0036] In step five, the judgment standard for gel formation is as follows: Positive control: After the standard bacterial endotoxin solution reacts with the limulus reagent, complete gel is formed, and it does not flow when inverted 180°; Negative control: No gel is formed after the pyrogen-free water reacts with the limulus reagent, and the solution remains fluid; Sample solution: If complete gel is formed and does not flow when inverted 180°, it is determined to be positive; if no gel is formed and the gel is incomplete, it is determined to be negative.

[0037] In step six, the specific operation of dynamic turbidity method is as follows: After mixing the sample solution with the limulus reagent, place it in the dynamic turbidimeter to monitor the turbidity change of the reaction solution in real time; Record the time required for the turbidity of the reaction solution to reach the preset threshold, and calculate the bacterial endotoxin content through the standard curve; The preparation method of the standard curve is as follows: react the limulus reagent with standard bacterial endotoxin solutions of different concentrations to draw the turbidity-time curve.

[0038] The detection wavelength of the dynamic turbidimeter is 340 nm, the reaction temperature is 37℃, and the reaction time is 105 minutes.

[0039] In step six, the specific operation of the color developing substrate method is as follows: Mix the sample solution with the color developing substrate limulus reagent, incubate, and then add the color developing stop solution; The absorbance of the reaction solution at 405 nm was measured, and the bacterial endotoxin content was calculated by a standard curve; The preparation method of the standard curve was as follows: different concentrations of standard bacterial endotoxin solution were reacted with the chromogenic substrate limulus reagent, and an absorbance-concentration curve was drawn.

[0040] The method further comprises a quality control step: A positive control, a negative control and a sample control are set for each batch of detection; The positive control is a standard bacterial endotoxin solution with a known concentration, the negative control is pyrogen-free water, and the sample control is a sample solution without limulus reagent; If the positive control does not form a gel and the negative control forms a gel, the batch of detection is determined to be invalid and needs to be re-detected.

[0041] Example 3: A bacterial endotoxin content detection method for monohydrated sodium dihydrogen phosphate, comprising the following steps: Step one: sample pretreatment, dissolving the monohydrated sodium dihydrogen phosphate sample to be tested in pyrogen-free water, and ultrasonic oscillation for 30 minutes to make the sample fully dissolved and uniformly dispersed; Step two: interference test verification, mixing standard bacterial endotoxin solution and sample solution in different proportions to verify the interference degree of the sample on the limulus reagent; Step three: sample solution preparation, adjusting the dilution multiple of the sample solution according to the interference test results to ensure that the bacterial endotoxin content is within the detection range of the limulus reagent; Step four: limulus reagent reaction, mixing the diluted sample solution with the limulus reagent, incubating in a constant temperature water bath for 65 minutes, and observing the gel formation; Step five: result determination, determining whether the content of bacterial endotoxin in the sample exceeds the specified limit value according to the integrity and stability of the gel formation; Step six: quantitative detection, quantitatively analyzing the bacterial endotoxin in the sample by dynamic turbidity method and chromogenic substrate method, and calculating the specific content; The preparation method of the pyrogen-free water comprises: filtering distilled water through a 0.22 μm microporous filter, drying and sterilizing at 250°C for 30 minutes, and cooling to room temperature for standby; the limulus reagent is a gel limulus reagent, and its sensitivity is 0.25 EU / mL.

[0042] In step one, the frequency of ultrasonic oscillation is 60 kHz, the temperature is 27°C, and the ultrasonic power is 300 W.

[0043] In step two, the specific method of interference test verification is as follows: Prepare a standard bacterial endotoxin solution with a concentration of 2λ, where λ is the labeled sensitivity of the limulus reagent; Mix the standard bacterial endotoxin solution with the sample solution at a volume ratio of 1:1, 1:2, and 1:4, respectively, and label them as solution A, solution B, and solution C; React solution A, solution B, and solution C with limulus reagent, observe gel formation, and calculate the recovery rate; If the recovery rate is within the range of 200%, it is determined that the sample has no interference; if the recovery rate exceeds this range, the dilution factor needs to be further adjusted and the interference eliminator needs to be treated.

[0044] The interference eliminator is a pyrogen-free Tris-HCl buffer with a pH of 7.2 and a concentration of 0.5 mol / L, and the addition amount is 5% of the volume of the sample solution.

[0045] In step four, the temperature of the constant temperature water bath is 38℃, and the incubation time is 65 minutes. Avoid shaking and light during incubation.

[0046] In step five, the judgment standard for gel formation is: Positive control: the standard bacterial endotoxin solution reacts with limulus reagent to form complete gel, and does not flow when inverted 180°; Negative control: no gel is formed after the pyrogen-free water reacts with the limulus reagent, and the solution remains fluid; Sample solution: if complete gel is formed and does not flow when inverted 180°, it is determined to be positive; if no gel is formed and the gel is incomplete, it is determined to be negative.

[0047] In step six, the specific operation of dynamic turbidity method is: After mixing the sample solution with the limulus reagent, place it in the dynamic turbidimeter to monitor the turbidity change of the reaction liquid in real time; Record the time required for the turbidity of the reaction liquid to reach the preset threshold, and calculate the bacterial endotoxin content through the standard curve; The preparation method of the standard curve is: react different concentrations of standard bacterial endotoxin solution with limulus reagent to draw the turbidity-time curve.

[0048] The detection wavelength of the dynamic turbidimeter is 340 nm, the reaction temperature is 38℃, and the reaction time is 120 minutes.

[0049] In step six, the specific operation of the color developing substrate method is: Mix the sample solution with the color developing substrate limulus reagent, incubate, and then add the color developing termination liquid; Determine the absorbance of the reaction liquid at 405 nm, and calculate the bacterial endotoxin content through the standard curve; The preparation method of the standard curve is: react different concentrations of standard bacterial endotoxin solution with color developing substrate limulus reagent to draw the absorbance-concentration curve.

[0050] The method further comprises a quality control step: A positive control, a negative control and a sample control are set for each batch of detection; The positive control is a standard bacterial endotoxin solution with a known concentration, the negative control is pyrogen-free water, and the sample control is a sample solution without limulus reagent; If the positive control does not form a gel and the negative control forms a gel, it is determined that the batch of detection is invalid and needs to be re-detected.

[0051] Table 1: Experimental parameter setting table

[0052] Experimental procedure Parameter name Setting value or range Sample pretreatment Ultrasonic time 10-30 min Ultrasonic frequency 40-60 kHz Ultrasonic power 100–300W Temperature 25±2°C Pyrogen-free water preparation Filter membrane pore size 0.22 μm Dry heat sterilization condition 250 °C, 30 min LAL reagent reaction Incubation temperature 37±1°C Incubation time 60±5 min Interference elimination agent Tris-HCl buffer pH 7.0±0.2 Buffer concentration 0.1-0.5 mol / L Addition volume ratio 1%–5% Dynamic turbidity method Detection wavelength 340 nm Reaction time 90-120 min Color developing substrate method Detection wavelength 405 nm

[0053] Table 2: Recovery rate verification results of interference test

[0054] Sample number Addition endotoxin concentration (EU / mL) Measured endotoxin concentration (EU / mL) Recovery rate (%) Whether it meets the requirements (50%-200%) Sample A-1 0.25 0.245 98.0 Yes Sample A-2 0.50 0.510 102.0 Yes Sample B-1 0.25 0.230 92.0 Yes Sample B-2 0.50 0.480 96.0 Yes Sample C-1 0.25 0.255 102.0 Yes Sample C-2 0.50 0.495 99.0 Yes

[0055] All sample recoveries are within the range of 50%-200%, indicating no significant interference, and no further dilution or addition of interference elimination agent is required.

[0056] Table 3: Reproducibility and precision test results

[0057] Sample batch number Detection method Average value (EU / mL) Standard deviation (SD) RSD (%) Batch number 2023-001 Gel method 0.105 0.005 4.76 Dynamic turbidity method 0.108 0.004 3.70 Color developing substrate method 0.107 0.003 2.80 Batch number 2023-002 Gel method 0.092 0.004 4.35 Dynamic turbidity method 0.094 0.003 3.19 Color developing substrate method 0.093 0.002 2.15

[0058] RSD<5%, indicating that the method has good reproducibility and meets the requirements of the pharmacopoeia.

[0059] Table 4: Limit of detection and limit of quantification verification

[0060] Detection method LOD (EU / mL) LOQ (EU / mL) Remarks Gel method 0.03 0.06 Judgment of gel formation by naked eye Dynamic turbidity method 0.01 0.03 Signal-to-noise ratio S / N≥3 (LOD), ≥10 (LOQ) Color developing substrate method 0.005 0.015 Linear range: 0.015-1.0 EU / mL

[0061] Table 5: Comprehensive conclusion of method verification

[0062] Verification item Verification result Whether it meets the requirements Specificity No interference, qualified recovery rate Yes Accuracy Recovery rate 92-102% Yes Precision RSD < 5% Yes Detection limit / quantitative limit Compliance with the indicated sensitivity Yes Linear range 0.015-1.0 EU / mL Yes Durability pH, temperature slight change does not affect the results Yes

[0063] Table 6: Sample detection result example

[0064] Sample batch number Gel method result Dynamic turbidity method result (EU / mL) Color developing substrate method result (EU / mL) Conclusion 2023-001 Negative <0.03 <0.015 Qualified 2023-002 Positive 0.108 0.107 Unqualified 2023-003 Negative <0.03 <0.015 Qualified

[0065] Comparative Example 1, the difference between this comparative example and Example 1 is that this comparative example does not use ultrasonic oscillation treatment during sample pretreatment.

[0066] Comparative Example 2, the difference between this comparative example and Example 2 is that this comparative example does not use interference elimination agent during interference test verification.

[0067] Comparative Example 3, the difference between this comparative example and Example 3 is that this comparative example uses ordinary distilled water instead of pyrogen-free water in the limulus reagent reaction stage.

[0068] Comparative Example 4, the difference between this comparative example and Example 3 is that this comparative example only uses a single dynamic turbidity method for quantitative detection, without combining the chromogenic substrate method for verification.

[0069] The performance test of the bacterial endotoxin content detection method of sodium phosphate monobasic monohydrate in Examples 1-3 and Comparative Examples 1-4 was tested, and the test items and test methods are as follows: Detection sensitivity test: different concentrations of standard bacterial endotoxin solution were used for detection, and the lowest detectable concentration was recorded.

[0070] Interference test recovery rate test: known concentration of endotoxin standard was added to the sample solution, the recovery rate was calculated, and the interference degree of the sample matrix on the detection was evaluated.

[0071] Repeatability test: the same batch of sample was detected for 10 times, the coefficient of variation was calculated, and the precision of the method was evaluated.

[0072] Stability test: the sample solution was detected at different time points, and the change of bacterial endotoxin content was observed.

[0073] The test data of the bacterial endotoxin content detection method of sodium phosphate monobasic monohydrate in Examples 1-3 and Comparative Examples 1-4 were recorded in the following table:

[0074] Test item Example one Example two Example three Comparative example one Comparative example two Comparative example three Comparative example four Detection sensitivity (EU / mL) 0.03 0.14 0.25 0.10 0.20 0.50 0.30 Interference test recovery rate (%) 98-102 95-105 90-110 70-130 80-120 50-150 85-115 Repeatability (CV%) ≤5 ≤5 ≤5 ≤15 ≤10 ≤20 ≤8 Stability (change rate within 6 h) % ≤2 ≤2 ≤2 ≤10 ≤5 ≤15 ≤4

[0075] By comparing and analyzing the data in the table, it can be seen that the bacterial endotoxin content detection method of sodium dihydrogen phosphate monohydrate in examples 1-3 is obviously more excellent than that in comparative examples 1-4, which shows that by optimizing the sample pretreatment step, when detecting the bacterial endotoxin of sodium dihydrogen phosphate monohydrate, the ultrasonic oscillation treatment with specific parameters is adopted to make the sample fully dissolved and uniformly dispersed, which can reduce the aggregation phenomenon that may exist in the sample, ensure the uniformity of the detection sample, strictly control the preparation conditions of the pyrogen-free water to ensure that no exogenous endotoxin is introduced during the detection process, improve the accuracy of the detection result, further reduce the probability of false positive and false negative, set up the interference test verification link, mix the standard endotoxin solution and the sample solution in different proportions before detecting the bacterial endotoxin, which can evaluate the interference degree of the sample on the limulus reagent reaction in real time, so that the detection method can automatically adjust the sample dilution multiple and add interference eliminating agent according to the interference situation, avoid the detection deviation caused by the sample matrix effect, ensure the optimization of the detection conditions of different batches of samples, further improve the applicability and reliability of the detection method, by using the dynamic turbidity method and the chromogenic substrate method, when detecting the bacterial endotoxin content, the most suitable detection method can be selected according to the characteristics of the sample, the turbidity change and the color reaction in the reaction process are monitored in real time, the bacterial endotoxin is quantified accurately by establishing a standard curve, so that the detection method can cover a wider detection range, at the same time, the detection ability of low concentration endotoxin is improved, the sensitivity and accuracy of the detection are improved, and the strict quality control requirements of medical grade sodium dihydrogen phosphate monohydrate are met.

[0076] By comparing and analyzing the related data in the table, it can be seen that the bacterial endotoxin content detection method of sodium dihydrogen phosphate monohydrate provided by the application not only realizes the detection requirements of high sensitivity and high accuracy, but also solves the key technical problems of many interference factors and poor repeatability in the traditional method. Therefore, the bacterial endotoxin content detection method of sodium dihydrogen phosphate monohydrate provided by the application has a broader market prospect and is more suitable for promotion.

[0077] In the description of the present specification, the description of the terms "one embodiment", "example", "specific example" and the like means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are contained in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0078] The preferred embodiments of the application disclosed above are only to facilitate the elucidation of the application. The preferred embodiments do not describe all the details of the application and limit the application to the specific embodiments. Obviously, many modifications and variations can be made in light of the teachings above. The description is chosen and described in order to provide the best illustration of the application and its practical application to those skilled in the art and to enable those skilled in the art to best utilize the application. The application is limited only by the claims and their full scope and equivalents.

Claims

1. A method for detecting bacterial endotoxin content in sodium dihydrogen phosphate monohydrate, characterized in that: The following steps are involved: Step 1: Sample pretreatment: dissolve the sodium dihydrogen phosphate monohydrate sample to be tested in pyrogen-free water and ultrasonically vibrate for 10-30 minutes to fully dissolve and evenly disperse the sample; Step 2: Interference test verification, using standard bacterial endotoxin solution and sample solution mixed in different proportions to verify the degree of interference of the sample on the Limulus Amebocyte Lysate reaction; Step 3: Prepare the sample solution. According to the interference test results, adjust the dilution ratio of the sample solution to ensure that the bacterial endotoxin content is within the detection range of the Limulus amebocyte lysate. Step 4: Limulus amebocyte lysate reaction: Mix the diluted sample solution with Limulus amebocyte lysate, incubate in a constant temperature water bath for 60 ± 5 minutes, and observe the gel formation; Step 5: Result determination: Based on the integrity and stability of the gel formation, determine whether the bacterial endotoxin content in the sample exceeds the prescribed limit; Step 6: Quantitative detection: Use dynamic turbidity method and chromogenic matrix method to quantitatively analyze the bacterial endotoxins in the sample and calculate its specific content; Among them, the preparation method of pyrogen-free water includes: filtering distilled water through a 0.22μm microporous filter membrane, dry heat sterilizing at 250°C for 30 minutes, and cooling to room temperature for use; the horseshoe crab reagent is a gel-based horseshoe crab reagent with a sensitivity of 0.03-0.25EU / mL.

2. A method for detecting bacterial endotoxin content of sodium dihydrogen phosphate monohydrate according to claim 1, characterized in that: In step 1, the frequency of ultrasonic oscillation is 40-60 kHz, the temperature is 25±2° C., and the ultrasonic power is 100-300 W.

3. A method for detecting bacterial endotoxin content of sodium dihydrogen phosphate monohydrate according to claim 1, characterized in that: In step 2, the specific method of interference test verification is: Prepare a standard bacterial endotoxin solution with a concentration of 2λ, where λ is the labeled sensitivity of the limulus amebocyte lysate; The standard bacterial endotoxin solution was mixed with the sample solution at a volume ratio of 1:1, 1:2, and 1:4, and recorded as solution A, solution B, and solution C, respectively; Solution A, solution B, and solution C were reacted with limulus amebocyte lysate, the gel formation was observed, and the recovery rate was calculated; If the recovery rate is within the range of 50%-200%, the sample is judged to be free of interference; if the recovery rate exceeds this range, it is necessary to further adjust the dilution factor and use interference eliminator treatment.

4. A method for detecting bacterial endotoxin content of sodium dihydrogen phosphate monohydrate according to claim 3, characterized in that: The interference eliminator is a pyrogen-free Tris-HCl buffer with a pH of 7.0±0.2 and a concentration of 0.1-0.5 mol / L. The amount added is 1%-5% of the volume of the sample solution.

5. A method for detecting bacterial endotoxin content of sodium dihydrogen phosphate monohydrate according to claim 1, characterized in that: In step 4, the temperature of the constant temperature water bath is 37±1°C, and the incubation time is 60±5 minutes. Avoid vibration and light during the incubation process.

6. A method for detecting bacterial endotoxin content of sodium dihydrogen phosphate monohydrate according to claim 1, characterized in that: In step 5, the criteria for determining gel formation are: Positive control: Standard bacterial endotoxin solution reacts with limulus amebocyte lysate to form a complete gel, which does not flow when inverted 180°; Negative control: No gel is formed after the reaction between pyrogen-free water and limulus amebocyte lysate, and the solution maintains fluidity; Sample solution: If a complete gel is formed and there is no flow when inverted 180°, it is judged as positive; if no gel is formed or the gel is incomplete, it is judged as negative.

7. A method for detecting bacterial endotoxin content of sodium dihydrogen phosphate monohydrate according to claim 1, characterized in that: In step six, the specific operation of the dynamic turbidity method is: After mixing the sample solution with the limulus amebocyte lysate, place it in a dynamic turbidity meter to monitor the turbidity changes of the reaction solution in real time; Record the time required for the turbidity of the reaction solution to reach the preset threshold value, and calculate the bacterial endotoxin content using the standard curve; The preparation method of the standard curve is: using standard bacterial endotoxin solutions of different concentrations to react with Limulus amebocyte lysate to draw a turbidity-time curve.

8. A method for detecting bacterial endotoxin content of sodium dihydrogen phosphate monohydrate according to claim 7, characterized in that: The detection wavelength of the dynamic turbidity meter is 340 nm, the reaction temperature is 37±1°C, and the reaction time is 90-120 minutes.

9. A method for detecting bacterial endotoxin content of sodium dihydrogen phosphate monohydrate according to claim 1, characterized in that: In step six, the specific operation of the color development matrix method is as follows: The sample solution was mixed with the color-developing matrix Limulus amebocyte lysate, and after incubation, the color-developing stop solution was added; The absorbance of the reaction solution was measured at 405 nm, and the bacterial endotoxin content was calculated using the standard curve; The preparation method of the standard curve is: using standard bacterial endotoxin solutions of different concentrations to react with the color-developing matrix Limulus amebocyte lysate reagent to draw an absorbance-concentration curve.

10. A method for detecting bacterial endotoxin content of sodium dihydrogen phosphate monohydrate according to claim 1, characterized in that: The method also includes quality control steps: Positive control, negative control and sample control must be set up for each batch of testing; The positive control was a standard bacterial endotoxin solution of known concentration, the negative control was pyrogen-free water, and the sample control was a sample solution without limulus amebocyte lysate. If the positive control does not form a gel and the negative control does form a gel, the test batch is considered invalid and needs to be retested.

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