CN115656067A - Method for detecting bacterial endotoxin of sodium dihydrogen phosphate monohydrate - Google Patents
Method for detecting bacterial endotoxin of sodium dihydrogen phosphate monohydrate Download PDFInfo
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- 239000002158 endotoxin Substances 0.000 title claims abstract description 68
- 238000000034 method Methods 0.000 title claims abstract description 64
- BBMHARZCALWXSL-UHFFFAOYSA-M sodium dihydrogenphosphate monohydrate Chemical compound O.[Na+].OP(O)([O-])=O BBMHARZCALWXSL-UHFFFAOYSA-M 0.000 title claims abstract description 29
- 238000012360 testing method Methods 0.000 claims abstract description 67
- 239000012085 test solution Substances 0.000 claims abstract description 41
- 239000000523 sample Substances 0.000 claims abstract description 39
- 239000000243 solution Substances 0.000 claims abstract description 39
- 238000001514 detection method Methods 0.000 claims abstract description 32
- 239000012488 sample solution Substances 0.000 claims abstract description 10
- 238000002156 mixing Methods 0.000 claims abstract description 9
- 230000002452 interceptive effect Effects 0.000 claims abstract description 8
- 150000002500 ions Chemical class 0.000 claims abstract description 8
- 238000007865 diluting Methods 0.000 claims abstract description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 162
- 239000003153 chemical reaction reagent Substances 0.000 claims description 35
- 241000239218 Limulus Species 0.000 claims description 30
- 239000012895 dilution Substances 0.000 claims description 30
- 238000010790 dilution Methods 0.000 claims description 30
- 238000003556 assay Methods 0.000 claims description 16
- 230000035484 reaction time Effects 0.000 claims description 14
- 230000035945 sensitivity Effects 0.000 claims description 13
- 239000003002 pH adjusting agent Substances 0.000 claims description 6
- 239000011159 matrix material Substances 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 abstract description 16
- 229910000403 monosodium phosphate Inorganic materials 0.000 abstract description 10
- 235000019799 monosodium phosphate Nutrition 0.000 abstract description 10
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 abstract description 10
- 238000005220 pharmaceutical analysis Methods 0.000 abstract description 2
- 230000001105 regulatory effect Effects 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 description 10
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- 239000013642 negative control Substances 0.000 description 7
- 238000011084 recovery Methods 0.000 description 7
- 239000011734 sodium Substances 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 5
- 238000002834 transmittance Methods 0.000 description 5
- 230000008859 change Effects 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
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- 238000002965 ELISA Methods 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000012417 linear regression Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000004848 nephelometry Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910001424 calcium ion Inorganic materials 0.000 description 2
- 235000020061 kirsch Nutrition 0.000 description 2
- 229910001425 magnesium ion Inorganic materials 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 235000009161 Espostoa lanata Nutrition 0.000 description 1
- 240000001624 Espostoa lanata Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000001450 anions Chemical group 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 108010055222 clotting enzyme Proteins 0.000 description 1
- 238000011981 development test Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-M dihydrogenphosphate Chemical compound OP(O)([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-M 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- GVALZJMUIHGIMD-UHFFFAOYSA-H magnesium phosphate Chemical compound [Mg+2].[Mg+2].[Mg+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O GVALZJMUIHGIMD-UHFFFAOYSA-H 0.000 description 1
- 239000004137 magnesium phosphate Substances 0.000 description 1
- 229910000157 magnesium phosphate Inorganic materials 0.000 description 1
- 229960002261 magnesium phosphate Drugs 0.000 description 1
- 235000010994 magnesium phosphates Nutrition 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000003541 multi-stage reaction Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002510 pyrogen Substances 0.000 description 1
- 238000006748 scratching Methods 0.000 description 1
- 230000002393 scratching effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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Abstract
A detection method of bacterial endotoxin of sodium dihydrogen phosphate monohydrate belongs to the technical field of pharmaceutical analysis; the method comprises the following steps: dissolving a test sample, and then mixing and reacting the test sample with a pH regulator to eliminate interfering ions in the test sample and obtain a test solution meeting a set pH value; diluting the test solution to obtain a test solution; performing photometric determination on the test sample solution to obtain bacterial endotoxin concentration; the pH regulator is adopted to regulate the test solution to a set pH value, the pH value of the solution can be regulated, and the reaction with sodium dihydrogen phosphate can be carried out, so that interference ions are reduced.
Description
Technical Field
The application relates to the technical field of pharmaceutical analysis, in particular to a method for detecting bacterial endotoxin of sodium dihydrogen phosphate monohydrate.
Background
Sodium Dihydrogen PHospHate Monohydrate (Sodium Dihydrogen PHospHate Monohydrate), also known as Sodium Dihydrogen PHospHate Monohydrate, has the molecular formula NaH 2 PO 4 ·H 2 O, molecular weight 137.99, and the medicinal grade is mainly used as acid-base regulator and phosphorus-supplementing medicine.
At present, the detection method for detecting bacterial endotoxin of sodium dihydrogen phosphate is mainly a gel method, and the sample limit value is as follows: 25EU/g (i.e., 0.025 EU/mg); sensitivity of limulus reagent: 0.06EU/ml; minimum effective dilution concentration of sample: 2.5mg/ml.
Sodium dihydrogen phosphate monohydrate is generally considered to eliminate interference at a minimum effective dilution concentration of 2.5mg/ml, however the applicant found that in the face of sodium dihydrogen phosphate monohydrate as produced by Kirsch PHarma GmbH [ endotoxin limit criteria: 5EU/g (i.e. 0.005 EU/mg) and the like are not applicable to the interference enhancement of different manufacturers in the production process, and the interference still exists when the solution is diluted to 2.5mg/ml and 0.25 mg/ml.
Disclosure of Invention
The application provides a method for detecting bacterial endotoxin of sodium dihydrogen phosphate monohydrate, which aims to solve the problem that the interference-enhanced sodium dihydrogen phosphate monohydrate cannot be detected at present.
The embodiment of the application is realized as follows:
the application example provides a method for detecting bacterial endotoxin of sodium dihydrogen phosphate monohydrate, which comprises the following steps:
dissolving a test sample, and then mixing and reacting the test sample with a pH regulator to eliminate interfering ions in the test sample and obtain a test solution meeting a set pH value;
diluting the test solution to obtain a test solution;
and performing photometric determination on the test sample solution to obtain the bacterial endotoxin concentration.
According to the method provided by the embodiment of the application, the pH value of the solution can be adjusted by adjusting the pH value of the test solution to the set pH value by using the pH adjusting agent, and the reaction with sodium dihydrogen phosphate can be realized while the pH value of the solution is adjusted, so that interfering ions are reduced.
In addition, compared with a gel method, the method has the advantages that the detection sensitivity is improved by adopting photometric determination, the dilution factor is increased, the anti-interference capability of the test solution is improved, and the problem that the interference-enhanced sodium dihydrogen phosphate cannot be detected at present is solved.
As an alternative embodiment, the set pH is 7.5 to 8.
As an alternative embodiment, the pH adjusting agent comprises sodium hydroxide;
in an alternative embodiment, the sodium hydroxide is a sodium hydroxide solution, and the molar concentration of the sodium hydroxide solution is 0.05-0.2mol/L.
More preferably, the sodium hydroxide is sodium hydroxide solution, and the molar concentration of the sodium hydroxide solution is 0.1mol/L
As an alternative embodiment, the reaction time of the sodium hydroxide and the test sample does not exceed 1 hour.
As an alternative embodiment, the reaction time of the sodium hydroxide and the test sample does not exceed 0.5h.
In an alternative embodiment, the test article has a minimum endotoxin limit of 5EU/g.
As an alternative embodiment, the minimum effective dilution concentration of the test solution is 1.0mg/ml.
As an alternative embodiment, the minimal sensitivity value of the limulus reagent of the photometric assay is 0.01EU/ml or less.
As an alternative embodiment, the sensitivity of the limulus reagent is 0.005 to 50EU/ml.
As an alternative embodiment, the photometric determination includes a chromogenic matrix method and a nephelometric method.
In an alternative embodiment, the detection wavelength of the photometric assay is matched to the limulus reagent of the photometric assay; and/or
The OD value of the photometric assay is matched to the limulus reagent of the photometric assay.
Drawings
To more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and those skilled in the art can also obtain other related drawings based on the drawings without inventive efforts.
Fig. 1 is a flowchart of a method provided in an embodiment of the present application.
Detailed Description
Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The applicant finds in the course of the invention that: when meeting sodium dihydrogen phosphate monohydrate produced by different manufacturers in the production process, such as Kirsch PHarma GmbH (endotoxin limit standard: 5EU/g (i.e., 0.005 EU/mg) interference enhancement, the currently used gel method for detecting bacterial endotoxin of sodium dihydrogen phosphate monohydrate is not applicable even when diluted to the minimum effective dilution concentration of 2.5mg/ml which is currently considered to eliminate interference, and interference still exists when the sodium dihydrogen phosphate monohydrate is diluted to 2.5mg/ml and 0.25 mg/ml.
In addition, compared with a gel method, the sensitivity of detection is improved by adopting photometric determination, the dilution factor is increased, the anti-interference capability of the test solution is improved, and the problem that the interference-enhanced sodium dihydrogen phosphate cannot be detected at present is solved.
As shown in fig. 1, the present application provides a method for detecting bacterial endotoxin of sodium dihydrogen phosphate monohydrate, which comprises:
s1, dissolving a test sample, and then mixing and reacting the test sample with a pH regulator to obtain a test solution meeting a set pH value;
in some embodiments, the pH adjuster comprises sodium hydroxide;
it should be noted that in some embodiments, a single sodium hydroxide may be used as the pH adjusting agent, and in other embodiments, sodium hydroxide and other agents may be used in combination as the pH adjusting agent, and in general, a tris buffer and sodium hydroxide may be selected for use in combination. Typically sodium hydroxide and other reagents are used in the order: sodium hydroxide first, followed by the other reagents.
In some embodiments, the set pH is 7.5-8, including but not limited to 7.5, 7.6, 7.7, 7.8, 7.9, and 8, and the like.
The following mechanism is explained by using sodium hydroxide as a pH regulator:
the reaction equation of sodium dihydrogen phosphate and sodium hydroxide is as follows:
formula 1: naH (sodium hydroxide) 2 PO 4 +NaOH＝Na 2 HPO 4 +H 2 O
Formula 2: na (Na) 2 HPO 4 +NaOH＝Na 3 PO 4 +H 2 O
The above is a stepwise reaction, and the overall reaction equation is as follows:
formula 3: naH 2 PO 4 +2NaOH＝Na 3 PO 4 +2H 2 O
As can be seen from the above reaction, the extent of the reaction requires consideration of the ratio between sodium dihydrogen phosphate and sodium hydroxide, and if the sodium dihydrogen phosphate is excessive, the reaction of formula 1 occurs, and if the sodium hydroxide is excessive, the reaction of formula 3, i.e., the total reaction of formula 1 and formula 2 occurs, and if the ratio between them is in an intermediate state, the reaction of formula 1 and formula 2 occurs, and the product will have Na 2 HPO 4 、Na 3 PO 4 、H 2 O。
And the applicant finds that: in the process of producing the limulus reagent, calcium and magnesium ions are required to be used for regulating the activity of the limulus reagent, and if a test sample contains phosphate ions, hydrogen phosphate ions, dihydrogen phosphate ions and the like, the calcium and magnesium ions in the limulus reagent and the anions form slightly soluble substances such as calcium phosphate, magnesium phosphate and the like, so that a white precipitation phenomenon is caused.
It can be seen that if the amount of sodium hydroxide is not excessive, there are many kinds of interfering ions (although the limulus reagent of Amidous is strong in anti-interference property and does not appear white precipitate, the anticoagulation effect of the sample still exists), so that the less the interfering ions are, the better the amount is, and the more the amount of sodium hydroxide is excessive.
At the same time, naH 2 PO 4 The solution is acidic and has certain pH value buffering capacity, and the applicant finds that: if the pH value is more than or equal to 7.5 after the sodium hydroxide is uniformly mixed, the sodium hydroxide is enough and excessive, and only hydrogen phosphate ions are left in the presence of interfering ions. And when more sodium hydroxide is added, the activity of endotoxin is reduced, and the applicant finds that: the pH value exceeding 8 will have a significant effect, and in summary, the control of the pH value of the test solution to 7.5-8 in the present application can produce a better effect.
In some embodiments, the reaction time of the sodium hydroxide and the test article does not exceed 1 hour.
The longer the time after mixing the sodium hydroxide with the test sample, the more likely the activity of endotoxin in the test sample is reduced. Therefore, the reaction time is controlled to be within 0.5 hour, preferably not more than 1 hour, and includes, but is not limited to, 5min, 10min, 15min, 20min, 25min, 30min, 40min, 50min and 60min.
In some embodiments, the sodium hydroxide is a sodium hydroxide solution having a molar concentration of 0.05 to 0.2mol/L.
Since the higher the concentration of sodium hydroxide, the activity of endotoxin is reduced, and the detection result is affected, the molar concentration of the sodium hydroxide solution is controlled to be 0.05-0.2mol/L, including but not limited to 0.05mol/L, 0.10mol/L, 0.15mol/L, 0.2mol/L, etc.
By adopting the design, the method can detect the sodium dihydrogen phosphate monohydrate with more strict endotoxin limit standard, and the endotoxin limit of the measurable sodium dihydrogen phosphate monohydrate can reach 5EU/g, generally speaking, the method is suitable for detecting the sodium dihydrogen phosphate monohydrate with the endotoxin limit of 5-25EU/g, and the method is also suitable for detecting the sodium dihydrogen phosphate monohydrate with the endotoxin limit exceeding 25EU/g, but comprehensively considering the factors of operation cost, convenience and the like, and is generally used for detecting the sodium dihydrogen phosphate monohydrate with the endotoxin limit of 5-25 EU/g.
S2, diluting the test solution to obtain a test solution;
by adopting the design, the method can effectively increase the minimum effective dilution concentration of the test solution to be tested to 1.0mg/ml, and compared with the prior art (specifically, see the minimum effective dilution concentration of 2.5mg/ml for detecting the bacterial endotoxin of sodium dihydrogen phosphate in the document 'checking the bacterial endotoxin of sodium dihydrogen phosphate' issued in the latest medical information Abstract of the world (vol.17, no. 22 in 2017)), the method obviously increases the dilution times and improves the anti-interference capability.
And S3, performing photometric determination on the test sample solution to obtain the bacterial endotoxin concentration.
In some embodiments, the minimum sensitivity value of the limulus reagent of the photometric assay is ≦ 0.01EU/ml. In this example, the sensitivity of the limulus reagent is 0.005 to 50EU/ml.
The improvement of the sensitivity of the limulus reagent realizes the improvement of the detection sensitivity.
In some embodiments, the photometric assay comprises a chromogenic matrix method and a nephelometric method.
The chromogenic matrix method is a method for measuring the endotoxin content by detecting the amount of a chromogen released from a specific substrate by a clotting enzyme produced during the reaction of a limulus reagent with endotoxin. According to the detection principle, the method is divided into an endpoint color development method and a dynamic color development method. The end-point color development method is a method for determining the endotoxin content based on the quantitative relationship existing between the endotoxin concentration in the reaction mixture and the amount of the chromogen released therefrom at the termination of the incubation. The dynamic color development method is a method of detecting a reaction time required for the absorbance or transmittance of a reaction mixture to reach a predetermined detection value, or a rate of increase in the detection value. In general, the operation of the endpoint color development method is more complicated, and a person skilled in the art will usually select a dynamic color development method in actual practice.
The turbidity method is a method for measuring the endotoxin content by detecting the turbidity change in the reaction process of limulus reagent with endotoxin. According to the detection principle, the method can be divided into an end point turbidimetric method and a dynamic turbidimetric method. The end-point nephelometry is a method for determining the endotoxin content based on the quantitative relationship existing between the endotoxin concentration in the reaction mixture and its turbidity (absorbance or transmittance) at the termination of incubation. The dynamic turbidity method is a method of detecting the reaction time required for the turbidity of a reaction mixture to reach a predetermined absorbance or transmittance, or detecting the turbidity increase rate. In general, the operation of the endpoint nephelometry is more complicated and dynamic nephelometry is often the choice of practice by those skilled in the art.
The photometric tests are carried out in specific instruments, the temperature being generally 37 ℃. + -. 1 ℃.
The amounts of the sample and the limulus reagent to be added, the ratio of the sample and the limulus reagent, the incubation time, and the like are described with reference to the relevant instructions of the instrument and the reagent used.
In some embodiments, the detection wavelength of the photometric assay matches the limulus reagent of the photometric assay; the OD value of the photometric assay is matched to the limulus reagent of the photometric assay. In this example, the detection wavelength was 405nm, and the OD value was 0.05.
Generally, in order to ensure the effectiveness of the turbidity and color development tests, the reliability test of the standard curve and the interference test of the test sample should be performed in advance, which can be referred to as "1143 bacterial endotoxin test method" in the general rules of the four departments of the 2020 edition of Chinese pharmacopoeia:
standard Curve reliability test the standard curve reliability test is carried out when a new lot of limulus reagents is used or there is any change in test conditions that may affect the test results.
Preparing solution with standard endotoxin, and preparing at least 3 dilutions (dilution multiple between adjacent concentrations is not more than 10), wherein the lowest concentration is not lower than the labeled detection limit of limulus reagent. The mixing time of each dilution step is the same as that of the gel method, and at least 3 parallel tubes are made for each concentration. And simultaneously, 2 negative controls are required to be made, and when the absorbance of the negative control is smaller than or the transmittance is greater than the detection value of the lowest point of the standard curve or the reaction time is greater than the reaction time of the lowest point of the standard curve, linear regression analysis is carried out on all data.
According to the linear regression analysis, the absolute value of the correlation coefficient (r) of the standard curve should be greater than or equal to 0.980, the experimenter being valid. Otherwise, the test should be repeated.
The interference test is as follows: the endotoxin concentration at or near the midpoint of the standard curve was selected (set as. Lambda.) m ) The concentration of endotoxin added in the test was considered as a test sample interference. Solutions A, B, C and D were prepared as follows.
Note: a is a test solution with the dilution factor not exceeding MVD.
B is a test solution to which a known endotoxin concentration at or near the midpoint of the standard curve is added and which has the same dilution factor as solution A.
C is the standard endotoxin solution used for preparing the standard curve as described under the section "reliability test of standard curve".
D is a negative control.
Respectively calculating the endotoxin content c of the test solution and the test solution containing standard endotoxin according to the obtained linear regression equation 1 And c n . The recovery rate (R) under the test conditions was calculated according to the following formula.
R＝(c n -c 1 )/λ m ×100％
When the recovery rate of endotoxin is 50% -200%, it is considered that no interference exists in the test solution under the test conditions.
When the recovery rate of endotoxin is not within the specified range, the interfering factor is removed by the method in the "gel method interference test" and the interference test is repeated to verify the effectiveness of the treatment.
When any change that may affect the test result occurs in the limulus reagent, the prescription of the test article, the production process change, the test environment, or the like, the interference test must be performed again.
The photometric determination in the present application specifically includes: the detection is carried out according to the procedure in the "interference test with photometric method".
The endotoxin concentration of each of the parallel channels of solution a was calculated using a standard curve generated for the series of solutions C.
The test must meet the following three conditions:
(1) The result of the series solution C is required to meet the requirement of a reliability test of a standard curve;
(2) After the endotoxin concentration in the solution A is subtracted from the endotoxin concentration in the solution B, the calculated recovery rate of the endotoxin is within the range of 50-200 percent;
(3) The absorbance of the negative control is less than or the transmittance is greater than the detection value of the lowest point of the standard curve or the reaction time is greater than the reaction time of the lowest point of the standard curve.
If the average endotoxin concentration of all the parallel tubes of the test solution is multiplied by the dilution times and is less than the specified endotoxin limit value, the test solution is judged to be in accordance with the specification. If the endotoxin is not less than the prescribed endotoxin limit, the test article is judged to be out of specification.
By adopting the design, the method provided by the application participates in the reaction with the test sample through the sodium hydroxide, then the bacterial endotoxin is detected by photometric determination, and the interference can be eliminated by adding a proper amount of sodium hydroxide solution into the sample solution; the photometric chromogenic matrix method is suitable for samples with smaller endotoxin standard limit and larger dilution factor (maximum effective dilution factor). In summary, the method has at least the following effects: the detection sensitivity is effectively improved, the anti-interference capability of a detection sample is improved, and the kit is suitable for samples with smaller endotoxin standard limit values.
The present application is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application. The experimental methods of the following examples, which are not specified under specific conditions, are generally determined according to national standards. If there is no corresponding national standard, it is carried out according to the usual international standards, to the conventional conditions or to the conditions recommended by the manufacturer.
The apparatus used in the following examples and comparative examples is specified below:
| name(s) | Model number | Numbering |
| Enzyme-linked immunosorbent assay (ELISA) instrument | Multiskan FC/ET | 13-A023 |
| Electronic balance | XSR105 | 13-B089 |
| Liquid transfer device | 100-1000μl | 215025591 |
| Liquid transfer device | 20-200μl | F210408004B |
| Liquid transfer device | 10-100μl | F190505253B |
The reagents used in the following examples and comparative examples are as follows:
examples 1 to 6
A method for detecting bacterial endotoxin of sodium dihydrogen phosphate monohydrate, which comprises the following steps:
s1, dissolving a test sample, and mixing with a sodium hydroxide pH regulator to obtain a test solution meeting a set pH value;
s2, diluting the test solution to obtain a test solution;
and S3, performing photometric determination on the test sample solution to obtain the bacterial endotoxin concentration.
The specific parameters of the experimental procedure are shown in the following table:
in which examples 1-3 were experiments on the same day, and examples 4-6 were experiments on the same day.
The specific experimental procedures of the examples have similarities, and are only illustrated by examples 4-6, and the specific experimental procedures of examples 4-6 are as follows:
(1) Interference test:
the assay was carried out using a limulus reagent of 1 manufacturer (Zhanjiang Amidism Co., ltd.) and 3 times of assays were carried out at a concentration of 1mg/ml to confirm the reproducibility of the results.
Sodium dihydrogen phosphate monohydrate the bacterial endotoxin limit (L) should be less than 0.005EU per mg sodium dihydrogen phosphate monohydrate.
Determination of minimum effective dilution concentration of sodium dihydrogen phosphate monohydrate: the minimum effective dilution concentration c = λ/L =0.005EU/ml/0.005EU/mg =1mg/ml of the test article. Wherein lambda is limulus reagent labeling sensitivity of 50-0.005EU/ml; l is the bacterial endotoxin limit of the test article of 0.005EU/mg.
(2) Preparation of the reaction solution
Preparation of standard curve solution C: and (3) lightly flicking the bottle wall of one working standard product of bacterial endotoxin to enable powder to fall into the bottom of the bottle, then lightly scratching the upper part of the bottle neck by using a grinding wheel, wiping the bottle neck by using a 75% alcohol cotton ball and opening the bottle, wherein glass scraps are prevented from falling into the bottle in the opening process. According to the standard specification, a specified amount of bacterial endotoxin test water is added to dissolve the contents, the bottle mouth is sealed by a sealing film, and the mixture is placed on a vortex mixer to be mixed for 30 minutes. Then, dilution is carried out, and mixing is carried out on a vortex mixer for more than 30 seconds in each dilution step. The dilution times should not exceed 10 times each time. The dilution can be made with reference to the following table:
| initial concentration (EU/ml) | 90 | 10 | 1.0 | 0.5 | 0.1 | 0.05 |
| Add volume (ml) | 0.2 | 0.5 | 1.0 | 1.0 | 1.0 | 0.5 |
| Adding BET volume (ml) | 1.6 | 4.5 | 1.0 | 4.0 | 1.0 | 4.5 |
| Concentration of solution (EU/ml) | 10 | 1.0 | 0.5 | 0.1 | 0.05 | 0.005 |
Preparation of test solution A: precisely weighing 0.04g (0.0400 g-0.0404 g) of a test solution to be tested, placing the test solution into a clean test tube without pyrogen, adding 2.3ml of BET water to dissolve the test solution, adding 2.7ml of sodium hydroxide solution (0.1 mol/L) to prepare 8mg/ml of test solution (on the premise of ensuring that the concentration of the 8mg/ml test solution is not changed, the pH value of the test solution in a proper range after the sample and the sodium hydroxide solution are mixed can be obtained by micro-adjusting the amount of the BET water and the sodium hydroxide solution which are dissolved in the sample), and gradually diluting the 8mg/ml test solution to 1mg/ml to be used as the test solution. Each dilution step should be mixed on a vortex mixer for more than 30 seconds. The dilution times should not exceed 10 times each time. The dilution can be made with reference to the following table:
| initial concentration (mg/ml) | 8.0 | 4.0 | 2.0 |
| Add volume (ml) | 1.0 | 1.0 | 1.0 |
| Add BET volume (ml) | 1.0 | 1.0 | 1.0 |
| Concentration of solution (mg/ml) | 4.0 | 2.0 | 1.0 |
Preparation of a positive control solution B of the test article: s 2 +E 0.1 ＝S 1 E 0.05 Wherein S is 2 And E 0.1 Is 1:1.
negative control solution D: namely, water for bacterial endotoxin test
Sample adding: adding 100 mul of samples into corresponding holes of the ELISA plate, adding the solution D in the sample adding sequence, adding the solution C from low concentration to high concentration, adding the solution A and the solution B, and replacing the heat source-free gun heads at different concentrations.
Mu.l of reconstituted limulus reagent was rapidly added to each well with sample in the order of negative control, then the standard solution, sample solution and sample positive control, taking care not to spill or bubble the limulus reagent. The addition of the limulus reagent should be completed in 2 minutes or less.
And (3) detection: and (3) putting the well-loaded elisa plate into an elisa plate reader or an equivalent analyzer, and selecting a dynamic color development method, wherein the preset temperature is 37 +/-1 ℃, the OD limit value is 0.05, and the reaction time is 3600 second. And after the sample information in the software is confirmed to be correct, the detection is started. Reading the endotoxin detection results from the instrument print report.
Acceptance criteria: the experiment must meet the following conditions to be effective, otherwise, the experiment needs to be retested. 1) When the reaction time of the negative control is longer than that of the lowest concentration of the standard curve; 2) The correlation coefficient | r | of the standard curve is more than or equal to 0.980; 3) Coefficient of variation CV% is less than or equal to 10%; 4) When the average recovery rate of the test sample positive tube is in the range of 50-200%, the test sample solution is considered to be free of interference to the test under the test condition.
Comparative examples 1 to 3
The specific steps of the gel method for the determination of the comparative examples 1 to 3 can be found in 1143 bacterial endotoxin test method in the general rules of the four parts of the 2020 edition of Chinese pharmacopoeia.
The specific parameters of the experimental procedure are shown in the following table:
comparative examples 4 to 7
Comparative examples 4 to 7 were carried out by the following method:
a method for detecting bacterial endotoxin of sodium dihydrogen phosphate monohydrate, which comprises the following steps:
s1, dissolving a test sample, and mixing with a sodium hydroxide pH regulator to obtain a test solution meeting a set pH value;
s2, diluting the test solution to obtain a test solution;
and S3, performing photometric determination on the test sample solution to obtain the bacterial endotoxin concentration.
The specific parameters of the experimental procedure are shown in the following table:
the experimental results of examples 1-6 and comparative examples 1-7 are shown in the following table:
in the table, N/a indicates that the data was not measured.
The applicant believes that: with respect to comparative examples 1 to 7, which cause a lower recovery rate due to insufficient amount of sodium hydroxide, the reaction was incomplete, wherein comparative examples 4 and 5 did not interfere at a dilution factor of 0.5mg/ml, but at this time the sample concentration was lower than the minimum effective dilution concentration, and the test results were invalid; with respect to examples 1-3, the reaction was complete for both the sodium dihydrogen phosphate monohydrate and the sodium hydroxide in example 1, which is theoretically the best, and a slight excess of examples 2 and 3 resulted in a somewhat lower recovery; with respect to examples 4-6, both the sodium dihydrogen phosphate monohydrate and the sodium hydroxide in examples 4-6 reacted completely, theoretically best, but did not show a good degree of similarity, which was analyzed as possibly due to the timing of the addition of the sodium hydroxide (the time of addition of the sodium hydroxide in examples 4-6 was: example 4 precedes example 5 before example 6).
From the above table, it can be seen that the method provided by the embodiments of the present application achieves no interference in detection at a minimum effective dilution concentration of 1.0mg/ml.
The foregoing is illustrative of the present application and is not to be construed as limiting thereof, as numerous modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (10)
1. A method for detecting bacterial endotoxin in sodium dihydrogen phosphate monohydrate, which comprises the following steps:
dissolving a test sample, and then mixing and reacting the test sample with a pH regulator to eliminate interfering ions in the test sample and obtain a test solution meeting a set pH value;
diluting the test solution to obtain a test solution;
and performing photometric determination on the test sample solution to obtain the bacterial endotoxin concentration.
2. The detection method according to claim 1, wherein the set pH is 7.5 to 8.
3. The detection method according to claim 1, wherein the pH adjuster comprises sodium hydroxide;
more preferably, the sodium hydroxide is a sodium hydroxide solution, and the molar concentration of the sodium hydroxide solution is 0.05-0.2mol/L;
more preferably, the sodium hydroxide is a sodium hydroxide solution, and the molar concentration of the sodium hydroxide solution is 0.1mol/L.
4. The detection method according to claim 3, wherein the reaction time of the sodium hydroxide and the test sample is not more than 1h;
more preferably, the reaction time of the sodium hydroxide and the test sample is not more than 0.5h.
5. The method of any one of claims 1 to 4, wherein the test sample has a minimum endotoxin limit of 5EU/g.
6. The detection method according to any one of claims 1 to 4, wherein the minimum effective dilution concentration of the sample solution is 1.0mg/ml.
7. The detection method according to any one of claims 1 to 4, wherein the minimum sensitivity value of the limulus reagent of the photometric assay is 0.01EU/ml or less.
8. The detection method according to claim 7, wherein the sensitivity of the limulus reagent is 0.005 to 50EU/ml.
9. The detection method according to any one of claims 1 to 4, wherein the photometric determination includes a chromogenic matrix method and a nephelometric method.
10. The detection method according to any one of claims 1 to 4, wherein the detection wavelength of said photometric method matches with the limulus reagent of said photometric method; and/or
The OD value of the photometric assay is matched to the limulus reagent of the photometric assay.
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