CN115902031A - Method for detecting energy substances in cell culture solution and application thereof - Google Patents
Method for detecting energy substances in cell culture solution and application thereof Download PDFInfo
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Abstract
The invention provides a detection method of energy substances in a cell culture solution and application thereof, wherein the detection method comprises the following steps: (1) Filtering a sample to be detected by using a filter membrane to obtain a test sample solution; (2) Performing liquid phase detection on the test solution, and calculating the content of energy substances in the sample to be detected according to the detection result and the standard curve; the mobile phase of the liquid phase detection in the step (2) is a mixed solution of an acid solution and an organic solvent; the organic solvent comprises any one or the combination of at least two of isopropanol, acetonitrile or ethanol; the energy substance comprises sodium lactate and/or sodium pyruvate. The detection method provided by the invention has high precision and high accuracy, and can be used for effectively and accurately quantifying sodium pyruvate and sodium lactate in a cell culture solution.
Description
Technical Field
The invention belongs to the field of biological material detection, particularly relates to a method for detecting energy substances in a cell culture solution and application thereof, and particularly relates to a method for detecting energy substances in a cell culture solution with high accuracy and application thereof.
Background
Pyruvate and lactate are primarily used as the primary energy sources in the early vegetative stages of development of certain cells, such as embryonic cells. The deficiency of the pyruvic acid can lead the development to be blocked when reaching the 8 cell stage and even before reaching the 8 cell stage, and is also involved in the antioxidant stress of the cells; lactic acid, as an energy substance, has a synergistic effect with pyruvic acid, mainly affecting cell viability. Therefore, in the in vitro cell culture process, various cell culture fluids used as direct contact environments for cell survival generally include sodium pyruvate and/or sodium lactate as an energy supply source in place of pyruvic acid and/or lactic acid. There are two reasons why pyruvic acid and/or lactic acid are not used directly as an energy source: 1. pyruvic acid and lactic acid are both acidic, and if the pyruvic acid and the lactic acid are used as preparation components, the pH of the culture solution is easily reduced seriously, so that the material balance is difficult to realize; 2. lactic acid readily enters the cell membrane before other nutrient components, lowering the intracellular pH and thus negatively affecting viability.
The cell culture solution provided by the prior art is mostly used as a commercial reagent, and the main components of the cell culture solution, including sodium pyruvate and/or sodium lactate, need to be subjected to content determination before the cell culture solution leaves a factory according to the requirements of corresponding regulations so as to ensure the safety and effectiveness of the cell culture solution in the actual use process.
In the prior art, a chemical method is conventionally adopted for detection and analysis of sodium pyruvate and/or sodium lactate, but the cell culture solution often contains interfering substances formed by various amino acids and saccharides, so that the detection result is not high in accuracy. In addition, the detection result is also easily affected by human subjective factors such as different operators or different understandings of the operators to the operation specification, so that the detection result has errors or even mistakes, and further results such as unavailable detection data or resource waste are caused. Therefore, how to provide a high-accuracy method for detecting and analyzing sodium pyruvate and/or sodium lactate is a problem to be solved urgently.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a method for detecting energy substances in a cell culture solution and application thereof, in particular to a method for detecting energy substances in a cell culture solution with high accuracy and application thereof. The detection method provided by the invention has high precision and high accuracy, and can effectively and accurately quantify sodium pyruvate and sodium lactate in the cell culture solution.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the invention provides a method for detecting an energy substance in a cell culture solution, the method comprising the steps of:
(1) Filtering a sample to be detected by using a filter membrane to obtain a test solution;
(2) Performing liquid phase detection on the test solution, and calculating the content of energy substances in the sample to be detected according to the detection result and the standard curve;
and (3) the mobile phase in the liquid phase detection in the step (2) is a mixed solution of an acid solution and an organic solvent.
The organic solvent includes any one or a combination of at least two of isopropyl alcohol, acetonitrile or ethanol, such as a combination of isopropyl alcohol and acetonitrile, a combination of acetonitrile and ethanol, or a combination of isopropyl alcohol and ethanol, but is not limited to the above-listed combinations, and other combinations not listed within the above-mentioned combinations are also applicable.
The energy substance comprises sodium lactate and/or sodium pyruvate.
The method adopts a liquid phase mode, can effectively avoid the problem that the enzymatic method and the chemical method are influenced by other substances in a cell culture solution, and has higher detection accuracy; in addition, the application also can effectively improve the detection accuracy and reduce errors by selecting a specific mobile phase and adding a specific organic solvent into the mobile phase.
Preferably, the acid solution is any one of or a combination of at least two of a sulfuric acid solution, a hydrochloric acid solution or a phosphoric acid solution, such as a combination of a sulfuric acid solution and a hydrochloric acid solution, a combination of a hydrochloric acid solution and a phosphoric acid solution or a combination of a phosphoric acid solution and a sulfuric acid solution, and the like, but is not limited to the above-listed combinations, and other combinations not listed within the above-listed combinations are also applicable, and a sulfuric acid solution is preferred.
The sodium lactate and the sodium pyruvate can be effectively replaced by the specific acid solution, and then the sodium lactate and the sodium pyruvate can be effectively separated and detected by the specific chromatographic column, so that the detection accuracy is improved; and the preferable acid solution can further improve the detection accuracy.
Preferably, the acid solution has a concentration of 4-6mM, such as 4mM, 4.5mM, 5mM, 5.5mM or 6mM, but not limited to the values recited above, and other values not recited within the above range of values are equally applicable.
Preferably, the organic solvent is a combination of acetonitrile and isopropanol.
The specific solvent combination can effectively improve the detection accuracy by compounding isopropanol and acetonitrile and combining specific chromatographic conditions
Preferably, the volume fraction of the organic solvent in the acid solution is 2-5%, such as 2%, 3%, 4%, or 5%, but not limited to the above-listed values, and other values not listed within the above-mentioned range of values are also applicable.
Preferably, the stationary phase of the chromatographic column comprises a sulfonated polystyrene divinylbenzene resin or a sulfonated styrene-divinylbenzene resin, preferably a sulfonated polystyrene divinylbenzene resin.
The selection of the specific stationary phase can effectively separate sodium lactate from sodium pyruvate in the cell culture solution, reduce the influence of other components and improve the detection accuracy.
Preferably, the column temperature of the liquid phase detection is 30-42 ℃.
Preferably, the elution mode of the liquid phase detection is isocratic elution;
preferably, in the liquid phase detection, the flow rate of the mobile phase is 0.4-1mL/min.
Preferably, the detection wavelength of the liquid phase detection is 210-320nm.
Wherein the column temperature may be 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃ or 42 ℃, the flow rate may be 0.4mL/min, 0.5mL/min, 0.6mL/min, 0.7mL/min, 0.8mL/min, 0.9mL/min or 1mL/min, etc., and the detection wavelength may be 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm or 320nm, etc., but is not limited to the above-mentioned values, and other values not listed in the above-mentioned range of values are also applicable.
In another aspect, the invention also provides the use of the detection method as described above for quality control of cell culture fluids.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for detecting energy substances in a cell culture solution, which can effectively avoid the problem that other substances in the cell culture solution are influenced in an enzymatic method and a chemical method by adopting a liquid phase mode, and has higher detection accuracy; in addition, the application can effectively improve the detection accuracy and reduce errors by selecting a specific mobile phase and adding a specific organic solvent into the mobile phase.
Detailed Description
In order to further illustrate the technical means and effects of the present invention, the technical solutions of the present invention are further described below with reference to the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.
In the following examples, the test cell culture medium was purchased from Vitroffe and contained 0.66mg/mL of sodium lactate and 0.008mg/mL of sodium pyruvate.
Example 1
This example provides a method for detecting energy substances in a cell culture solution, and the detection targets are sodium lactate and sodium pyruvate.
The preparation of the reference substance stock solution and the linear test solution comprises the following specific steps:
appropriate amounts of sodium lactate and sodium pyruvate were taken and added to a 10mL volumetric flask, and then diluted with ultrapure water to 8.52mg/mL and 1.02mg/mL respectively to obtain stock solutions as controls, and the concentrations of the obtained linear test solutions were as shown in the following table.
Preparing a test solution, comprising the following steps:
taking 1mL of cell culture solution to be detected, and filtering the cell culture solution through a 0.22 mu m microporous filter membrane to obtain the cell culture solution for later use.
Respectively carrying out liquid chromatography detection on the test solution and the linear test solutions 1-6 under the following conditions:
the column temperature is 32 ℃, the mobile phase (filtered by a 0.45 mu m water system filter membrane before use) is 5mM sulfuric acid solution, the volume fraction of the sulfuric acid solution comprises 2% of acetonitrile and 1% of isopropanol, the flow rate of the mobile phase is 0.8mL/min, the stationary phase is sulfonated polystyrene divinylbenzene hydrogen ion resin, the thickness of the stationary phase is 300X 7.8mM, the detection wavelength is 230nm, and the sample injection amount is 25 mu L.
Obtaining a standard curve according to the result of the linear test solution:
sodium lactate: y =515.98x-23.309, R2=0.9999;
sodium pyruvate: y =7501.4x +5.4224, and R2=0.9998.
And calculating the contents of sodium lactate and sodium pyruvate according to the standard curve and the detection result of the test solution, repeating the detection for 10 times, and calculating the average values to be 0.66mg/mL and 0.008mg/mL respectively.
Example 2
This example provides a method for detecting energy substances in a cell culture solution, which is the same as example 1 except that the sulfuric acid solution in the mobile phase is replaced by hydrochloric acid solution with equal volume and concentration, and the standard curve is recalculated.
Finally, the contents of sodium lactate and sodium pyruvate are respectively 0.69mg/mL and 0.004mg/mL through calculation.
Example 3
This example provides a method for detecting energy substances in a cell culture solution, which is the same as example 1 except that the sulfuric acid solution in the mobile phase is replaced with an equal volume of phosphoric acid solution, and the standard curve is recalculated.
Finally, the contents of sodium lactate and sodium pyruvate are respectively 0.58mg/mL and 0.011mg/mL.
Example 4
This example provides a method for detecting energetic species in a cell culture solution, which was identical to example 1 except that the stationary phase of the column was replaced with sulfonated styrene-divinylbenzene (8% hydrogen ion) and the standard curve was recalculated.
Finally, the contents of sodium lactate and sodium pyruvate are respectively 0.58mg/mL and 0.010mg/mL through calculation.
Example 5
This example provides a method for detecting energetic species in a cell culture fluid, which is the same as example 1 except that acetonitrile in the mobile phase is replaced with isopropanol of equal volume and the standard curve is recalculated.
The final calculation gave sodium lactate and sodium pyruvate in amounts of 0.93mg/mL and 0.007mg/mL, respectively.
Example 6
This example provides a method for detecting energetic materials in a cell culture fluid, which is the same as example 1 except that isopropanol in the mobile phase is replaced by acetonitrile with the same volume, and the standard curve is recalculated.
The final calculation gave sodium lactate and sodium pyruvate contents of 0.93mg/mL and 0.009mg/mL, respectively.
Example 7
This example provides a method for detecting energetic materials in a cell culture fluid, which is the same as example 1 except that acetonitrile in a mobile phase is replaced by ethanol with the same volume, and a standard curve is recalculated.
Finally, the contents of sodium lactate and sodium pyruvate are respectively 0.86mg/mL and 0.011mg/mL through calculation.
Comparative example 1
The comparative example provides a method for detecting energy substances in a cell culture solution, which comprises the following specific steps:
taking 0.2g of a sample, accurately weighing, adding 15mL of glacial acetic acid and 2mL of acetic anhydride into a conical flask, uniformly mixing, adding 1 drop of crystal violet indicator, titrating by using 0.1mol/L of perchloric acid titration solution until the solution is blue-green, correcting the titration result with a blank test, wherein each milliliter of perchloric acid titration solution is equivalent to 1.121mg of sodium lactate and sodium pyruvate.
The contents of sodium lactate and sodium pyruvate in the cell culture solution were 20.178mg/mL by titration calculation, and the recovery rate was 1133%. Because the components in the cell culture solution are complex, the cell culture solution contains various sodium salts of strong acid and weak acid, and the interference factors of the acid-base titration method are many, the accurate content detection result cannot be obtained.
According to the data, the detection method provided by the invention has the advantages that compared with the conventional detection method (comparative example 1), the detection accuracy is effectively improved; comparing examples 1 to 3, it can be found that the present invention can further improve the accuracy of detection by selecting a specific mobile phase; comparing examples 1 and 4, it can be found that the accuracy of detection can be further improved by selecting a specific stationary phase; comparing examples 1, 5-7, it can be seen that the present invention can further improve the accuracy of detection compared to other organic solvents by selecting a specific combination of organic solvents.
And (3) specificity test:
(1) Test procedure
Filtering the blank solution (ultrapure water) with a 0.22 μm microporous membrane to obtain the final product.
Taking 0.9020mg/mL sodium lactate as a positioning solution, and filtering with a 0.22-micron microporous filter membrane to obtain the sodium lactate.
Taking 0.1005mg/mL sodium pyruvate as a positioning solution, and filtering the solution through a 0.22 mu m microporous filter membrane to obtain the product.
Taking a mixed solution of 0.9020mg/mL sodium lactate and 0.1005mg/mL sodium pyruvate, filtering with 0.22 μm microporous membrane to obtain control solution.
Weighing appropriate amount of cell culture solution sample, and filtering with 0.22 μm microporous membrane.
And taking each solution, injecting samples according to the method in the embodiment 1, and recording the retention time, peak area and separation degree of chromatographic peaks of sodium lactate and sodium pyruvate.
(2) Statistics of results
Specificity of sodium lactate and sodium pyruvate
| Name(s) | Blank solution | Sodium lactate positioning solution | Control solution | Test article |
| Retention time | / | 10.313 | 10.326 | 10.336 |
| Peak area | / | 689.5 | 188.7 | 307.2 |
| Degree of separation | / | ＞1.5 | 7.5 | 7.4 |
| Name (R) | Blank solution | Sodium pyruvate positioning solution | Control solution | Test article |
| Retention time | / | 6.752 | 6.739 | 6.776 |
| Peak area | / | 775.4 | 46.6 | 15.4 |
| Degree of separation | / | ＞1.5 | ＞1.5 | 2.2 |
(3) Analysis of results
According to GB/T27417-2017 & lt & gt guidelines for the confirmation and verification of qualification chemical analysis methods & lt/EN & gt, and the fourth part of the 2020 edition & lt/EN & gt 9101 analysis method verification guide principle & lt/EN & gt, the separation degree of chromatographic peaks of sodium lactate and sodium pyruvate is required to be more than or equal to 1.5.
In the above test, the retention time of sodium lactate and sodium pyruvate in the cell culture solution sample is consistent with that of the positioning solution and the reference solution, which indicates that the sodium lactate and sodium pyruvate in the sample can be effectively identified; the separation degree of a sodium lactate chromatographic peak is 8.2, and the separation degree of a sodium pyruvate chromatographic peak is 3.3, which all meet the requirements of relevant standards, thereby showing that the detection method provided by the invention has better specificity and can effectively identify and separate sodium lactate and sodium pyruvate
And (3) precision test:
the test steps are as follows:
weighing a cell culture solution sample of a proper amount of stock solution, evenly dividing into 6 parts of 1mL, and filtering by a 0.22 mu m microporous filter membrane to obtain the cell culture solution.
The content of sodium lactate and sodium pyruvate in the cell culture sample was measured according to the content measurement method provided in example 1, and the relative standard deviation (RSD,%) was calculated.
The results are as follows
The correlation coefficient RSD is required to be less than or equal to 2.0 percent according to GB/T27417-2017 'guide for confirming and verifying qualified chemical analysis method' and '9101 analysis method verification guiding principle' in the fourth part of the pharmacopoeia of the people's republic of China' 2020 edition.
In the cell culture solution of the embodiment 1, the precision repeatability RSD of sodium lactate is 0.54 percent, the precision repeatability RSD of sodium pyruvate is 0.45 percent, and the requirements of related standards are met; the above tests were also carried out in the same manner as in examples 2 to 7, and the RSD values were calculated as follows:
the results show that the detection method provided by the invention further improves the precision of the method by selecting the combination of the specific mobile phase, the specific stationary phase and the specific organic solvent; meanwhile, compared with the existing method, the detection method provided by the invention has the advantages of better precision and small error of the measurement result.
Recovery rate test:
test procedure
Preparation of test solution
Taking cell culture solution samples subjected to content determination, averagely dividing the cell culture solution samples into 9 parts of 1mL, and numbering and grouping the samples;
the different volumes of the linear test solution 4 (sodium lactate concentration 0.902mg/mL, sodium pyruvate 0.1005 mg/mL) in example 1 were added to each group in sequence; 0.8mL for the first group, 1mL for the second group, and 1.2mL for the third group.
The contents of sodium lactate and sodium pyruvate (sodium lactate concentration 0.66mg/mL and sodium pyruvate concentration 0.008 mg/mL) in the test solution were measured according to the content measurement method provided in example 1, and the recovery rates thereof were calculated. The detailed grouping is as follows:
the recovery rate calculation formula is as follows:
according to GB/T27417-2017 'quality assessment chemical analysis method validation and verification guide' and '9101 analysis method verification guide principle' in the fourth part of the 2020 edition of the pharmacopoeia of the people's republic of China', the required recovery rate of sodium lactate is 90-108%; the recovery rate of sodium pyruvate is required to be 80-115%.
The recovery rates of the sodium lactate and the sodium pyruvate in the embodiment 1 are respectively 100.59 percent and 100.57 percent, and the recovery rates meet the requirements of related standards; the above test was also carried out in the same manner as in examples 2 to 7, and the recovery rate was calculated as follows:
the results show that the detection method provided by the invention further improves the precision of the method by selecting the combination of the specific mobile phase, the specific stationary phase and the specific organic solvent; meanwhile, the detection method provided by the invention has good accuracy, and the loss of sodium lactate and sodium pyruvate is not caused in the determination process.
The applicant states that the present invention is described by the above examples of the method for detecting an energetic substance in a cell culture solution and the application thereof, but the present invention is not limited to the above examples, that is, the present invention is not limited to the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (10)
1. A method for detecting an energetic substance in a cell culture fluid, the method comprising the steps of:
(1) Filtering a sample to be detected by using a filter membrane to obtain a test sample solution;
(2) Performing liquid phase detection on the test solution, and calculating the content of energy substances in the sample to be detected according to the detection result and the standard curve;
the mobile phase of the liquid phase detection in the step (2) is a mixed solution of an acid solution and an organic solvent;
the organic solvent comprises any one or the combination of at least two of isopropanol, acetonitrile or ethanol;
the energy substance comprises sodium lactate and/or sodium pyruvate.
2. The detection method according to claim 1, wherein the acid solution is any one of a sulfuric acid solution, a hydrochloric acid solution, or a phosphoric acid solution, or a combination of at least two thereof, preferably a sulfuric acid solution.
3. The detection method according to claim 1 or 2, wherein the concentration of the acid solution is 4 to 6mM.
4. The detection method according to any one of claims 1 to 3, wherein the organic solvent is a combination of acetonitrile and isopropanol.
5. The detection method according to any one of claims 1 to 4, wherein the volume fraction of the organic solvent in the acid solution is 4 to 10%.
6. The detection method according to any one of claims 1 to 5, wherein the stationary phase of the chromatographic column comprises a sulfonated polystyrene divinylbenzene resin or a sulfonated styrene-divinylbenzene resin, preferably a sulfonated polystyrene divinylbenzene resin.
7. The detection method according to any one of claims 1 to 6, wherein the column temperature of the liquid phase detection is 30 to 42 ℃.
8. The detection method according to any one of claims 1 to 7, wherein the elution manner of the liquid phase detection is isocratic elution;
preferably, in the liquid phase detection, the flow rate of the mobile phase is 0.4-1mL/min.
9. The detection method according to any one of claims 1 to 8, wherein the detection wavelength of the liquid phase detection is 210 to 320nm.
10. Use of the detection method according to any one of claims 1 to 9 for quality control of a cell culture solution.
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