Sodium oleate bacterial endotoxin inspection method

Abstract

The invention discloses a sodium oleate bacterial endotoxin detection method, which comprises: S1, adding a sodium oleate sample to be detected into BET water, mixing, and placing in a water bath to dissolve; s2, after dissolving, adjusting the PH to be neutral by using hydrochloric acid to obtain a test sample solution with the initial concentration of 100-200mg / ml, and diluting the test sample solution by using a diluent to obtain a series of test sample solutions with dilution concentrations; the diluent is diluted by the steps of a magnesium ion buffer solution, a Tween-containing BET aqueous solution and BET water; s3, detecting the diluted sample solution to be tested; and S4, setting the positive control solution, the positive control group and the negative control group of the test sample, performing the same detection with the test sample solution, and performing comparison. The method can be used for quantitatively, efficiently and quickly detecting the bacterial endotoxin in the sodium oleate sample to be detected.

Description

Technical Field

[0001] The present invention relates to an inspection method, in particular to a method for inspecting sodium oleate bacterial endotoxins. Background Art

[0002] Bacterial endotoxin testing is an important means to ensure the safety of drugs, biological products and medical supplies, but there are still some shortcomings in practical applications.

[0003] Traditional bacterial endotoxin detection methods, such as gel-based and 96-well plate-based assays, rely on extensive manual labor, including standard curve preparation and pipetting steps. This is not only time-consuming and labor-intensive, but can also lead to high retesting rates. Furthermore, frequent manual operations pose risks to the health and safety of analysts.

[0004] Traditional testing methods use large volumes of horseshoe crab reagents, with a complete sample run potentially requiring up to 10 ml of reagent. This high consumption not only increases costs, but also raises ecological concerns as horseshoe crabs are a finite resource. Summary of the Invention

[0005] In order to solve the problem of low efficiency in bacterial endotoxin detection, the present invention provides a sodium oleate bacterial endotoxin detection method.

[0006] The present invention provides the following technical solutions:

[0007] The method for testing bacterial endotoxins of sodium oleate is characterized by comprising the following steps:

[0008] S1. Add the sodium oleate sample to be tested into BET water and mix, then place in a water bath to dissolve;

[0009] S2. After dissolution, adjust the pH to 7.0 with hydrochloric acid, and the solution becomes milky white to obtain a test solution with an initial concentration of 50-200 mg / ml. Then dilute the test solution with a diluent to obtain a series of dilution concentrations of the test solution; the diluent dilution steps are magnesium ion buffer, Tween-containing BET aqueous solution, and BET water;

[0010] S3. Detect the diluted test solution;

[0011] S4. Set up the test sample positive control solution, positive control group and negative control group, and perform the same test as the test sample solution for comparison.

[0012] Furthermore, the test solution was diluted to a multiple not exceeding the maximum effective dilution multiple MVD, and the dilution multiples were 20 times and 40 times, respectively.

[0013] Furthermore, the mass concentration of the hydrochloric acid is 36-38%.

[0014] Furthermore, the test sample positive control solution is prepared: the sodium oleate sample to be tested is added to BET water and mixed, and dissolved in a water bath. After dissolution, the pH is adjusted to a neutral pH of 7.0 with hydrochloric acid, mixed with a bacterial endotoxin standard, and then diluted with a diluent to obtain the test sample positive control group.

[0015] Furthermore, the positive control group was prepared by mixing BET water with a bacterial endotoxin standard to obtain the positive control group.

[0016] Furthermore, the negative control group is BET water.

[0017] Furthermore, the positive control group also contains Limulus amebocyte lysate.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] By adding BET water and using a water bath, the sodium oleate sample to be tested can be effectively and fully dissolved, allowing the endotoxin in the sample to fully enter the aqueous phase. The pH value is then adjusted to neutral with hydrochloric acid. Within the appropriate range for bacterial endotoxin testing, the aqueous phase is taken for subsequent operations, which will not affect the recovery of endotoxins and avoid interference with the test results. The diluent is water for bacterial endotoxin testing and a magnesium ion buffer solution. Adding a certain proportion of magnesium ion buffer ensures that the solution remains within the appropriate pH range, and an appropriate amount of magnesium ions can supplement divalent cations (calcium and magnesium ions) to eliminate interference caused by the decreased activation ability of Factor C. Detection using photometry can quantitatively detect the endotoxin content in the sample.

[0020] The present invention can perform quantitative, efficient and rapid detection of bacterial endotoxins in a sodium oleate test sample, and reduces the use of horseshoe crab resources. DETAILED DESCRIPTION

[0021] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] Determination of the maximum effective dilution factor:

[0023] The samples are tested for bacterial endotoxins at a concentration of the test solution that does not exceed the maximum effective dilution factor or is not less than the minimum effective dilution concentration. The maximum effective dilution factor refers to the maximum dilution factor allowed for the test solution in the test, and the endotoxin limit test is performed at a concentration that does not exceed this dilution factor. The following formula is used to determine the MVD (maximum effective dilution factor): MVD = cL / λ

[0024] Photometric method: where c is the concentration of the test solution, L is the bacterial endotoxin limit of the test sample, and λ is the sensitivity of the limulus amebocyte lysate (LAS). To calculate the test sample concentration at the MVD, the minimum effective dilution concentration, use the formula c = λ / L.

[0025] The maximum effective dilution factor MVD is

[0026]

[0027] Sample preparation:

[0028]

[0029] Example 1 (interference test):

[0030] Purpose: To formally determine that the sample has no interference with the bacterial endotoxin detection test at this concentration and can be used for bacterial endotoxin detection. The interference test is performed at a positive control solution concentration of the test sample with a recovery rate of 50%-200% in the pre-interference test.

[0031] By pre-adding a certain concentration of bacterial endotoxin (λ Δ ), after being processed according to the test solution preparation method, a pre-endotoxin recovery test is performed. When the recovery rate is between 50% and 200%, it can be considered that the test solution preparation process does not destroy the background bacterial endotoxins of the test sample or does not affect the detection of background bacterial endotoxins.

[0032] Preparation of test solution (solution A and impact test solution A r ):

[0033] Solution A: (Batch No. 20220801K) Weigh 224.82 mg, add 2.25 ml of BET water, mix well, and dissolve in a 50°C water bath. Adjust the pH to approximately 7.0 with 0.02 ml of hydrochloric acid to prepare a 100 mg / ml test solution. Then dilute 40-fold as follows:

[0034] Dilution steps:

[0035]

[0036] Solution Ar: Weigh 221.92 mg of the test product and add 1.903 ml of BET water, mix thoroughly, and dissolve in a 50°C water bath. Adjust the pH to approximately 7.0 with 0.02 ml of hydrochloric acid. Add 0.317 ml of a 70 EU / ml endotoxin solution to make a 100 mg / ml test solution containing 10 EU / ml. Dilute 40-fold as in Solution A.

[0037] Preparation of standard curve solution C:

[0038] Bacterial endotoxin standard dilution

[0039] Take one tube of bacterial endotoxin working standard, add 1.0 ml of BET water, place on a vortex mixer and mix for 15 minutes, then gradually dilute with BET water to a series of bacterial endotoxin standard solutions with several concentrations, vortex for 30 seconds at each dilution step, and establish a standard curve with 4 concentrations of endotoxin standard solutions of 5 EU / ml, 0.5 EU / ml, 0.05 EU / ml, and 0.005 EU / ml.

[0040] Preparation of test sample positive control solution (Solution B):

[0041] Take 2 times the concentration of the test solution and 2 times the concentration of m The endotoxin standard solution was mixed in equal proportions and vortexed for at least 30 seconds to prepare a sample with the same concentration as the test solution and containing λ m Endotoxin standard test sample positive control solution. m =0.25EU / ml.

[0042] Take the auxiliary reagent solution with the same dilution ratio as the test solution and 2 times the m Endotoxin standard solution of 100mg / ml was mixed in equal proportions and used as auxiliary reagent solution for positive control of the test sample.

[0043] Negative control: BET water.

[0044] Turn on the dynamic cuvette reader and preheat to 37°C ± 1°C. Dissolve the limulus amebocyte lysate (LA) according to the instructions. Combine the reconstituted LA with BET water (if necessary) and dispense into the pyrogen-free tubes supplied with the dynamic cuvette reader. Add 0.1ml of each of the solutions listed in the table above, in parallel, to each set of two tubes. After addition, gently mix to avoid bubbles. Place the sample into the dynamic cuvette reader for data collection. Incubate for 90 minutes.

[0045] Table 1 Preparation of interference test solution and impact test solution

[0046]

[0047] a. Solution A r The test solution is pre-added with endotoxin and the dilution factor does not exceed MVD.

[0048] b. Solution B r To add λ m Concentration of endotoxin, and with the test solution A r There are test solution with the same dilution factor.

[0049] The test must meet the following conditions to be valid

[0050] 1. The results of series C solution must meet the requirements of the "Reliability test of the standard curve".

[0051] 2. The reaction time of solution D (negative control) is greater than the reaction time of the lowest point of the standard curve.

[0052] 3. Solution B (Solution B r ) minus the endotoxin concentration in solution A (solution A r ) after the endotoxin concentration in the sample was determined, the calculated endotoxin recovery rate should be in the range of 50%-200%.

[0053] 4. The coefficient of variation of all replicate tubes does not exceed 10%. If, during the test, the reaction time of all reaction tubes in the negative control replicate tubes or the test sample replicate tubes is greater than the reaction time of the lowest point of the standard curve, there is no need to consider the coefficient of variation between the negative control or test sample replicate tubes.

[0054] Formal interference test and impact test results

[0055] Table 2 Results of formal interference test and impact test of sodium oleate (for injection)

[0056]

[0057] Table 3 Sodium oleate test results

[0058]

[0059] Conclusion: In the formal interference test, the correlation coefficient of the standard curve |r| ≥ 0.980, the reaction time of the negative control is greater than the reaction time of the lowest concentration of the standard curve, and the coefficient of variation of the parallel groups is less than 10%, indicating that the test is valid. The test sample was dissolved in a 50°C water bath with BET water, and the pH was adjusted to about 7.0 with hydrochloric acid to prepare a 100 mg / ml test solution. The recovery rate when it was diluted 2 times with magnesium ion buffer and then diluted 20 times with BET water was 110%, which is within the range of 50%-200% and meets the regulations. The auxiliary reagent solution does not affect the calculation of the test sample recovery rate. The recovery rate of the test sample (pre-added with endotoxin) was 22%, which is not within the range of 50%-200% and does not meet the regulations. This indicates that the solution preparation process cannot effectively extract endotoxins from the test sample. The pre-interference test was subsequently repeated.

[0060] Example 2 (interference test):

[0061] Purpose: To formally determine that the sample has no interference with the bacterial endotoxin detection test at this concentration and can be used for bacterial endotoxin detection. The interference test is performed at a positive control solution concentration of the test sample with a recovery rate of 50%-200% in the pre-interference test.

[0062] By pre-adding a certain concentration of bacterial endotoxin (λ Δ ), after being processed according to the test solution preparation method, a pre-endotoxin recovery test is performed. When the recovery rate is between 50% and 200%, it can be considered that the test solution preparation process does not destroy the background bacterial endotoxins of the test sample or does not affect the detection of background bacterial endotoxins.

[0063] Preparation of test solution (solution A and impact test solution A r ):

[0064] Solution A: (Batch No. 20230201K) Weigh 204.90 mg, add 2.05 ml of BET water, mix well, and dissolve in a 50°C water bath. Adjust the pH to approximately 7.0 with 0.02 ml of hydrochloric acid to prepare a 100 mg / ml test solution. Then dilute 40-fold as follows:

[0065] Dilution steps:

[0066]

[0067] Solution Ar: Weigh 224.43 mg of the test product and add 1.92 ml of BET water, mix thoroughly, and dissolve in a 50°C water bath. Adjust the pH to approximately 7.0 with 0.02 ml of hydrochloric acid. Add 0.32 ml of a 70 EU / ml endotoxin solution to make a 100 mg / ml test solution containing 10 EU / ml. Dilute 40-fold as in Solution A.

[0068] Preparation of standard curve solution C:

[0069] Bacterial endotoxin standard dilution

[0070] Take one tube of bacterial endotoxin working standard, add 1.0 ml of BET water, place on a vortex mixer and mix for 15 minutes, then gradually dilute with BET water to a series of bacterial endotoxin standard solutions with several concentrations, vortex for 30 seconds at each dilution step, and establish a standard curve with 4 concentrations of endotoxin standard solutions of 5 EU / ml, 0.5 EU / ml, 0.05 EU / ml, and 0.005 EU / ml.

[0071] Preparation of test sample positive control solution (Solution B):

[0072] Take 2 times the concentration of the test solution and 2 times the concentration of m The endotoxin standard solution was mixed in equal proportions and vortexed for at least 30 seconds to prepare a sample with the same concentration as the test solution and containing λ m Endotoxin standard test sample positive control solution. m =0.25EU / ml.

[0073] Take the auxiliary reagent solution with the same dilution ratio as the test solution and 2 times the m Endotoxin standard solution of 100mg / ml was mixed in equal proportions and used as auxiliary reagent solution for positive control of the test sample.

[0074] Negative control: BET water.

[0075] Turn on the dynamic cuvette reader and preheat to 37°C ± 1°C. Dissolve the limulus amebocyte lysate (LA) according to the instructions. Combine the reconstituted LA with BET water (if necessary) and dispense into the pyrogen-free tubes supplied with the dynamic cuvette reader. Add 0.1ml of each of the solutions listed in the table above, in parallel, to each set of two tubes. After addition, gently mix to avoid bubbles. Place the sample into the dynamic cuvette reader for data collection. Incubate for 90 minutes.

[0076] Table 4 Preparation of interference test solution and impact test solution

[0077]

[0078] a. Solution A r The test solution is pre-added with endotoxin and the dilution factor does not exceed MVD.

[0079] b. Solution B r To add λ m Concentration of endotoxin, and with the test solution A r There are test solution with the same dilution factor.

[0080] The test must meet the following conditions to be valid

[0081] 1. The results of series C solution must meet the requirements of the "Reliability test of the standard curve".

[0082] 2. The reaction time of solution D (negative control) is greater than the reaction time of the lowest point of the standard curve.

[0083] 3. Solution B (Solution B r ) minus the endotoxin concentration in solution A (solution A r ) after the endotoxin concentration in the sample was determined, the calculated endotoxin recovery rate should be in the range of 50%-200%.

[0084] 4. The coefficient of variation of all replicate tubes does not exceed 10%. If, during the test, the reaction time of all reaction tubes in the negative control replicate tubes or the test sample replicate tubes is greater than the reaction time of the lowest point of the standard curve, there is no need to consider the coefficient of variation between the negative control or test sample replicate tubes.

[0085] Table 5 Results of formal interference test and impact test of sodium oleate (for injection)

[0086]

[0087] Conclusion: In the formal interference test, the standard curve correlation coefficient |r| ≥ 0.980, the negative control reaction time was greater than the reaction time of the lowest concentration of the standard curve, and the coefficient of variation of the parallel groups was less than 10%, indicating that the test was valid. The test sample was dissolved in BET water at 50°C in a water bath, and the pH was adjusted to approximately 7.0 with hydrochloric acid to prepare a 100 mg / ml test solution. The solution was diluted 2-fold with magnesium ion buffer, 2.5-fold with BET aqueous solution containing 3% Tween 80, and then diluted 8-fold with BET water. The recovery rate was 103%, which is within the range of 50%-200% and meets the requirements. The auxiliary reagent solution did not affect the calculation of the test sample recovery rate. The test sample (pre-added with endotoxin) had a recovery rate of 47%, which is not within the range of 50%-200% and does not meet the requirements. This indicates that the solution preparation process cannot effectively extract endotoxin from the test sample. The pre-interference test was subsequently repeated.

[0088] Example 3 (formal interference test):

[0089] Purpose: To formally determine that the sample has no interference with the bacterial endotoxin detection test at this concentration and can be used for bacterial endotoxin detection. The interference test is performed at a positive control solution concentration of the test sample with a recovery rate of 50%-200% in the pre-interference test.

[0090] By pre-adding a certain concentration of bacterial endotoxin (λ Δ ), after being processed according to the test solution preparation method, a pre-endotoxin recovery test is performed. When the recovery rate is between 50% and 200%, it can be considered that the test solution preparation process does not destroy the background bacterial endotoxins of the test sample or does not affect the detection of background bacterial endotoxins.

[0091] Preparation of test solution (solution A and impact test solution A r ):

[0092] Solution A:

[0093] ① (Batch number: 20230201K) Weigh 286.42 mg, add 2.86 ml of BET water, mix evenly, place in a 50°C water bath to dissolve, and adjust the pH to about 7.0 with 0.02 ml of hydrochloric acid to prepare a 100 mg / ml test solution; ② (Batch number: 20220801K) Weigh 291.13 mg, add 2.91 ml of BET water, mix evenly, place in a 50°C water bath to dissolve, and adjust the pH to about 7.0 with 0.02 ml of hydrochloric acid to prepare a 100 mg / ml test solution; ③ (Batch number: 20230402K) Weigh 242.51 mg, add 2.43 ml of BET water, mix evenly, place in a 50°C water bath to dissolve, and adjust the pH to about 7.0 with 0.02 ml of hydrochloric acid to prepare a 100 mg / ml test solution; then dilute 40 times according to the following steps:

[0094] Dilution steps:

[0095]

[0096] Solution Ar: ① (Batch number: 20230201K) Weigh 258.77 mg, add 2.22 ml of BET water, mix well, and dissolve in a 50°C water bath. Adjust the pH to approximately 7.0 with 0.02 ml of hydrochloric acid, and then add 0.37 ml of 70 EU / ml endotoxin solution to prepare a 100 mg / ml test solution containing 10 EU / ml.

[0097] ② (Batch number: 20220801K) Weigh 235.06 mg, add 2.014 ml of BET water, mix well, place in a 50°C water bath to dissolve, adjust the pH to about 7.0 with 0.02 ml of hydrochloric acid, and add 0.336 ml of 70 EU / ml endotoxin solution to prepare a 100 mg / ml test solution containing 10 EU / ml;

[0098] ③ (Batch number: 20230402K) Weigh 256.96 mg, add 2.203 ml of BET water, mix well, place in a 50°C water bath to dissolve, adjust the pH to about 7.0 with 0.02 ml of hydrochloric acid, and then add 0.367 ml of 70 EU / ml endotoxin solution to make a 100 mg / ml test solution containing 10 EU / ml; dilute 40-fold as in solution A.

[0099] Preparation of standard curve solution C:

[0100] Bacterial endotoxin standard dilution

[0101] Take one tube of bacterial endotoxin working standard, add 1.0 ml of BET water, place on a vortex mixer and mix for 15 minutes, then gradually dilute with BET water to a series of bacterial endotoxin standard solutions with several concentrations, vortex for 30 seconds at each dilution step, and establish a standard curve with 4 concentrations of endotoxin standard solutions of 5 EU / ml, 0.5 EU / ml, 0.05 EU / ml, and 0.005 EU / ml.

[0102] Preparation of test sample positive control solution (Solution B):

[0103] Take 2 times the concentration of the test solution and 2 times the concentration of m The endotoxin standard solution was mixed in equal proportions and vortexed for at least 30 seconds to prepare a sample with the same concentration as the test solution and containing λ m Endotoxin standard test sample positive control solution. m =0.25EU / ml.

[0104] Take the auxiliary reagent solution with the same dilution ratio as the test solution and 2 times the m Endotoxin standard solution of 100mg / ml was mixed in equal proportions and used as auxiliary reagent solution for positive control of the test sample.

[0105] Negative control: BET water.

[0106] Turn on the dynamic cuvette reader and preheat to 37°C ± 1°C. Dissolve the limulus amebocyte lysate (LA) according to the instructions. Combine the reconstituted LA with BET water (if necessary) and dispense into the pyrogen-free tubes supplied with the dynamic cuvette reader. Add 0.1ml of each of the solutions listed in the table above, in parallel, to each set of two tubes. After addition, gently mix to avoid bubbles. Place the sample into the dynamic cuvette reader for data collection. Incubate for 90 minutes.

[0107] Table 6 Preparation of interference test solution and impact test solution

[0108]

[0109] a. Solution A r The test solution is pre-added with endotoxin and the dilution factor does not exceed MVD.

[0110] b. Solution B r To add λ m Concentration of endotoxin, and with the test solution A r There are test solution with the same dilution factor.

[0111] To be valid, the test must meet the following conditions:

[0112] 1. The results of series C solution must meet the requirements of the "Reliability test of the standard curve".

[0113] 2. The reaction time of solution D (negative control) is greater than the reaction time of the lowest point of the standard curve.

[0114] 3. Solution B (Solution B r ) minus the endotoxin concentration in solution A (solution A r ) after the endotoxin concentration in the sample was determined, the calculated endotoxin recovery rate should be in the range of 50%-200%.

[0115] 4. The coefficient of variation of all replicate tubes does not exceed 10%. If, during the test, the reaction time of all reaction tubes in the negative control replicate tubes or the test sample replicate tubes is greater than the reaction time of the lowest point of the standard curve, there is no need to consider the coefficient of variation between the negative control or test sample replicate tubes.

[0116] Formal interference test results

[0117] The test results of the first batch are shown in Table 18. If the test results of the first batch meet the validity requirements, the second and third batches of formal interference tests are carried out in the same manner as in Example 3. The test results are shown in Table 19.

[0118] Table 7 Results of formal interference test and impact test of sodium oleate (for injection)

[0119]

[0120] Table 8 Results of formal interference test and impact test of sodium oleate (for injection)

[0121]

[0122] Conclusion: In the three formal interference tests in Example 3, the standard curve correlation coefficient |r| ≥ 0.980, the negative control reaction time is greater than the reaction time of the lowest concentration of the standard curve, and the coefficient of variation of the parallel groups is less than 10%, indicating that the test is valid. The test sample was dissolved in a 50°C water bath with BET water, and the pH was adjusted to about 7.0 with hydrochloric acid to prepare a 100 mg / ml test solution. The recovery rates when diluted 2 times with magnesium ion buffer, 2.5 times with a BET aqueous solution containing 1% Tween 80, and then diluted 8 times with BET water were 121%, 98%, and 92%, respectively, all within the range of 50%-200%, and all met the regulations. The auxiliary reagent solution does not affect the calculation of the test sample recovery rate. The test sample (pre-added with endotoxin) recovery rates were 85%, 54%, and 55%, respectively, all within the range of 50%-200%, and all met the regulations. This shows that the solution preparation process can effectively extract endotoxins from the test sample.

[0123] Note: The manufacturer of the Limulus amebocyte lysate reagent used in this verification is Zhanjiang Andus Biological Co., Ltd.

[0124] Comparative Example:

[0125] Table 9 Differences between the present invention and comparative examples

[0126]

[0127] in conclusion:

[0128] Sodium oleate (for injection) (326829) was validated according to Section 1143 of the Chinese Pharmacopoeia (2020 edition), Part IV, General Rules, to determine the bacterial endotoxin test method for this product. The specific procedure is as follows: The standard stipulates that the bacterial endotoxin content in 1g of sodium oleate should be less than 2.0EU.

[0129] 1. Turn on the computer and bacterial endotoxin detector in sequence, and preheat the bacterial endotoxin detector. When the display on the instrument interface changes from "PLEASE WAIT..." to "37.0℃" or to "405nm" or "660nm", the preheating is completed.

[0130] 2. Preparation of bacterial endotoxin standard solution: Take one bacterial endotoxin standard, break the bottle at the upper part of the neck, add 1.0 ml of BET water to reconstitute, place on a vortex mixer and mix for at least 15 minutes, then dilute with BET water to 4 concentrations of 5 EU / ml, 0.5 EU / ml, 0.05 EU / ml, and 0.005 EU / ml (mix on a vortex mixer for at least 30 seconds for each dilution step).

[0131]

[0132] 3. Preparation of test solution: Weigh an appropriate amount of test sample (M should be in the range of 200 mg-400 mg), mix with BET water V (V = M / 100 mg / ml), and dissolve in a 50°C water bath (the solution is completely clear). After taking out, adjust the pH to about 7.0 with hydrochloric acid (about 0.02 ml) (the solution becomes milky white) to make a 100 mg / ml test solution. When diluted to 50 mg / ml with magnesium ion buffer, stratification occurs. Remove the lower aqueous phase and further dilute.

[0133] 4. The dilution steps of the test solution are as follows:

[0134]

[0135] 5. Test sample positive control solution: Use a pipette to draw 0.5 ml of the test sample solution with a concentration of 5 mg / ml and mix it with 0.5 ml of the bacterial endotoxin standard solution with a concentration of 0.5 EU / ml.

[0136] 6. Negative control: Bacterial endotoxin test water (BET water) is used as the negative solution.

[0137] 7. Turn on the dynamic test tube analyzer and preheat to 37°C ± 1°C. Take the limulus amebocyte lysate (sensitivity 10-0.005EU / ml) and refer to the instructions for each tube to reconstitute it with 1.25ml of bacterial endotoxin test water, then combine (if necessary). Dispense the limulus amebocyte lysate into the test tubes that come with the dynamic test tube analyzer, 0.1ml per tube. Then add 0.1ml each of the four solutions mentioned above (2.5mg / ml test solution, test sample positive control solution, solutions at each point of the standard curve (5EU / ml, 0.5EU / ml, 0.05EU / ml, 0.005EU / ml), and negative solution) to the tubes. Each solution is added to two parallel tubes. After gently mixing the limulus amebocyte lysate mixture, insert the instrument and incubate for 90 minutes. The preset value is 95%.

[0138] 8. The absolute value of the correlation coefficient r of the standard curve is greater than or equal to 0.980, the reaction time of each negative tube is greater than the reaction time of the lowest concentration of the standard curve, and the coefficient of variation of all replicate tubes does not exceed 10% (if during the test, the reaction time of all reaction tubes in the negative control replicate tubes or the test sample replicate tubes is greater than the reaction time of the lowest point of the standard curve, there is no need to consider the coefficient of variation between the negative control or test sample replicate tubes), the test is valid.

[0139] 9. The endotoxin recovery rate of the parallel tubes of the test sample positive solution is between 50% and 200% (including 50% and 200%); all parallel tubes of the test sample solution are less than the prescribed endotoxin limit, and the test sample meets the regulations.

[0140] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for testing bacterial endotoxins of sodium oleate, characterized by: The following steps are involved: S1. Add the sodium oleate sample to be tested into BET water and mix, then place in a water bath to dissolve; S2. After dissolution, adjust the pH to neutral 7.0 with hydrochloric acid, and the solution becomes milky white to obtain a test solution with an initial concentration of 50-200 mg / ml. Then dilute the test solution with a diluent to obtain a series of dilution concentrations of the test solution; the diluent dilution steps are magnesium ion buffer, Tween-containing BET aqueous solution, and BET water; S3. Detect the diluted test solution; S4. Set up the test sample positive control solution, positive control group and negative control group, and perform the same test as the test sample solution for comparison.

2. The method for testing sodium oleate bacterial endotoxins according to claim 1, wherein: The test solution was diluted to a multiple not exceeding the maximum effective dilution multiple MVD, and the dilution multiples were 20 times and 40 times respectively.

3. The method for testing sodium oleate bacterial endotoxins according to claim 1, wherein: The mass concentration of the hydrochloric acid is 36-38%.

4. The method for testing sodium oleate bacterial endotoxins according to claim 1, wherein: Prepare the test sample positive control solution: add the sodium oleate sample to be tested into BET water and mix, place in a water bath to dissolve, adjust the pH to neutral 7.0 with hydrochloric acid after dissolution, mix with the bacterial endotoxin standard, and then dilute with a diluent to obtain the test sample positive control solution.

5. The method for testing sodium oleate bacterial endotoxins according to claim 1, wherein: The positive control group was prepared by mixing BET water with a bacterial endotoxin standard to obtain the positive control group.

6. The method for testing bacterial endotoxins of sodium oleate according to claim 1, wherein: The negative control group was BET water.

7. The method for testing sodium oleate bacterial endotoxins according to claim 1, wherein: The positive control group also contained Limulus amebocyte lysate.

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