CN112326839B - Method for detecting residual quantity of lactic acid - Google Patents
Method for detecting residual quantity of lactic acid Download PDFInfo
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Abstract
The application provides a detection method of residual quantity of lactic acid, which comprises the following steps: extracting lactic acid in a sample to be detected by using absolute ethyl alcohol to prepare a solution to be detected; detecting the solution to be detected by using high performance liquid chromatography; and calculating the residual quantity of the lactic acid in the sample to be detected based on the detection result. The chromatographic column in the method has high column efficiency and good durability, and an organic phase is not required to be added in the mobile phase, so that the lactic acid residue in the hyaluronic acid can be effectively detected.
Description
Technical Field
The application relates to the field of detection of hyaluronic acid, and more specifically relates to a method for detecting lactic acid residues.
Background
Hyaluronic Acid (HA), also known as Hyaluronic acid, is a high molecular weight linear mucopolysaccharide composed of (1-3) -2-acetamido-2-deoxy-D-glucose (1-4) -D-glucuronic acid disaccharide repeating units. First extracted from bovine glass eyeball in 1934 by Meyer et al. Hyaluronic acid exhibits various important physiological functions in the body with its unique molecular structure and physicochemical properties, such as lubricating joints, regulating permeability of blood vessel walls, regulating proteins, regulating diffusion and operation of aqueous electrolytes, promoting wound healing, and the like. More importantly, hyaluronic acid HAs a special water retention effect, is a substance which is found to have the best moisture retention in nature at present, is called an ideal natural moisture retention factor, and is widely applied to the industrial fields of cosmetics, foods, medicines, clinical treatment and the like because HA HAs good moisture retention, viscoelasticity, permeability and ductility and does not have any immunogenicity and toxicity.
HA was originally extracted from animal tissues such as cockscomb, eyeball, etc., and due to limited sources and high cost of these raw materials, the production of HA by extraction HAs not been able to meet the increasing demands of HA in the pharmaceutical and cosmetic production. In order to expand the production scale and reduce the production cost, the technology for producing HA by a fermentation method is rapidly developed. The strain used in HA fermentation is streptococcus, and the total amount of small-molecule organic acid produced in the fermentation process is far larger than that of HA. Under the existing production process conditions, the yield of HA in the fermentation liquor reaches 6-10 g/L, and the content of lactic acid is 30-40 g/L. After fermentation is finished, the HA is generally precipitated and further purified by using ethanol, lactic acid is used as the organic acid with the highest content in the fermentation liquid, and the detection of the lactic acid residue in the finished product HA can reflect the purification process effect and the product impurity control degree.
At present, RP-C18 chromatographic columns are generally adopted for detection and analysis of small-molecular organic acids, a mobile phase is mainly methanol/acetonitrile-dilute acid solution with the pH of 2-3, and chromatographic packing is used in a low-pH environment for a long time and is easy to cause hydrolysis of a bonding phase, so that the column efficiency is continuously reduced.
Disclosure of Invention
In order to solve the above problems, the applicant of the present application has discovered, by chance in daily analytical work, a cation exchange chromatography column which has a good analytical effect on lactic acid at a high column temperature, a high column efficiency, and a high durability, and does not require the addition of an organic phase to a mobile phase, thereby establishing the above method for the detection of lactic acid residues in hyaluronic acid-related products.
In order to solve the technical problem, the technical scheme adopted by the application is as follows:
1. a method for detecting residual quantity of lactic acid is characterized by comprising the following steps:
extracting lactic acid in a sample to be detected by using absolute ethyl alcohol to prepare a solution to be detected;
detecting the solution to be detected by using high performance liquid chromatography;
and calculating the residual quantity of the lactic acid in the sample to be detected based on the detection result.
2. The method according to item 1, wherein the sample to be tested contains hyaluronic acid.
3. The method according to item 1, wherein the solution to be tested is prepared by adding 40 to 100ml of the absolute ethanol to 10g of the sample to be tested.
4. The method according to item 1, characterized in that in the preparation of the solution to be measured, the sample to be measured is added into the absolute ethyl alcohol, and is uniformly mixed to form a mixed solution, then the mixed solution is sealed and is subjected to ultrasonic treatment, then the mixed solution is transferred into a volumetric flask, the absolute ethyl alcohol is used for constant volume, then the supernatant is taken out and volatilized to obtain a solid mixture, then the solid mixture is dissolved by a weak acid solution, the constant volume is obtained, and then the filtrate is filtered to obtain the solution to be measured.
5. The method according to item 4, wherein the sonication time is 15 to 40 min.
6. The method according to item 4, wherein the weak acid solution is a phosphoric acid solution having a concentration of 0.5 wt% to 1.2 wt%.
7. The method according to item 1, wherein the column of high performance liquid chromatography is a cation exchange chromatography column.
8. The method of item 7, wherein the cation exchange chromatography column is a strong cation H packed with sulfonated crosslinked styrene divinylbenzene copolymer + A type exchange chromatography column.
9. The method of any one of claims 7-8, wherein the cation exchange chromatography column is an MCI GEL CK08EH chromatography column.
10. The method according to item 1, wherein the mobile phase of the high performance liquid chromatography is a weak acid solution.
11. The method according to item 10, wherein the mobile phase is a phosphoric acid solution having a concentration of 0.5 to 1.2wt%, preferably 0.8 to 1.0 wt%.
12. The method of any of items 10 to 11, wherein the flow rate of the mobile phase is 0.3 to 1.0ml/min, preferably 0.4 to 0.8 ml/min.
13. The method according to item 1, wherein the column temperature of the column used in the high performance liquid chromatography is 70 to 95 ℃, preferably 75 to 90 ℃.
14. The method according to item 1, wherein the detection wavelength in the high performance liquid chromatography is 190 to 220nm, and the amount of sample is 5 to 100. mu.L.
15. The method according to any one of items 1 to 14, wherein the residual amount of lactic acid in the sample to be tested is calculated by using an external standard method.
16. The method according to any one of items 1 to 15, wherein the detection limit of the residual amount of lactic acid is 5 to 8 ppm.
According to the method, the sample to be detected is pretreated by using absolute ethyl alcohol, and the impurity residual quantity is obtained by combining a liquid chromatography separation technology, so that the method is high in sensitivity. Because of adopting cation exchange chromatographic column as the analytical column, the analytical effect to lactic acid is good at higher column temperature, and the column is high-efficient, and the durability is strong, and the mobile phase only adopts diluted acid solution, need not use organic phase such as methyl alcohol acetonitrile of certain proportion, can increase the life of chromatographic column, and the experimentation security is higher. By applying the chromatographic condition, the obtained detection object has better peak shape, can improve the detection sensitivity of the lactic acid and can detect the content of the lactic acid with extremely low content.
Drawings
FIG. 1 is a liquid chromatogram of example 1.
Fig. 2 is a liquid chromatogram of comparative example 1.
Detailed Description
In the context of the present specification, chromatography (also known as "chromatography", or "chromatography") is a method of separation and analysis and is widely used in the fields of analytical chemistry, organic chemistry, biochemistry, and the like. The chromatography uses the selective distribution of different substances in different phase states to elute a mixture in a mobile phase and a stationary phase, and different substances in the mixture can move along the stationary phase at different speeds, so that the separation effect is finally achieved.
High Performance Liquid Chromatography (HPLC) was developed on the basis of gas chromatography and classical chromatography. Modern liquid chromatography is not substantially different from classical liquid chromatography. The difference is only that the modern liquid chromatography has higher efficiency and realizes automatic operation than the classical liquid chromatography. In the classical liquid chromatography, a mobile phase is conveyed under normal pressure, and the used stationary phase has low column efficiency and long analysis period. Modern liquid chromatography introduces the theory of gas chromatography, and the mobile phase is changed into high-pressure delivery (the highest delivery pressure can reach 4.9X 10) 7 Pa); the chromatographic column is formed by filling the filler with small particle size by a special method, so that the column efficiency is greatly higher than that of a classical liquid chromatogram (the number of tower plates per meter can reach tens of thousands or hundreds of thousands); meanwhile, a high-sensitivity detector is connected behind the column, and the effluent can be continuously detected. Therefore, the high performance liquid chromatography has the characteristics of high analysis speed, high separation efficiency, automation and the like. It is called high pressure, high speed, high performance or modern liquid chromatography.
The absolute ethyl alcohol is colorless clear liquid, has special fragrance, is easy to flow, is easy to absorb water from air, and can be mutually dissolved with various organic solvents such as water, chloroform, ether and the like in any proportion. Can form an azeotropic mixture (containing 4.43 percent of water), the azeotropic point is 78.15 ℃, the melting point is-114.1 ℃ and the boiling point is 78.5 ℃.
The chemical name of lactic acid: 2-hydroxypropionic acid, a compound that plays a role in a variety of biochemical processes. It is a carboxylic acid of formula C 3 H 6 O 3 . It is a carboxylic acid containing a hydroxyl group and is therefore an alpha-hydroxy acid (AHA). In aqueous solution, its carboxyl group releases a proton to produce the lactate ion CH3CHOHCOO - . Lactate dehydrogenase converts pyruvate to L-lactate during fermentation. Lactic acid is produced continuously during normal metabolism and exercise, but its concentration does not generally rise.
The external standard method is one of the commonly used methods for instrument analysis, and is one of the comparison methods. Compared with the internal standard method, the external standard method does not add a standard substance into a sample to be measured, but independently measures under the same chromatographic condition with the sample to be measured, and compares the obtained chromatographic peak area with the chromatographic peak area of the component to be measured to obtain the content of the component to be measured. The external standard substance and the component to be measured are both a substance but require certain purity, and the concentration of the external standard substance is close to that of the component to be measured during analysis, so that the accuracy of quantitative analysis is facilitated.
The present application will be described in detail below.
According to the application, a method for measuring the residual quantity of lactic acid is provided, which comprises the following steps: firstly, extracting and concentrating lactic acid in a hyaluronic acid sample by using absolute ethyl alcohol to obtain liquid to be detected, wherein the liquid to be detected does not contain hyaluronic acid; and then detecting the content of the lactic acid in the liquid to be detected by using an ion exchange chromatographic column for analyzing the organic acid and a mobile phase without an organic phase. Simultaneously preparing a reference solution of lactic acid (the preparation method of the reference solution comprises precisely weighing about 50mg of lactic acid reference in a 50mL volumetric flask, dissolving the mobile phase, fixing the volume to scale, mixing, and filtering with a 0.22 μm filter membrane to obtain the reference solution); and then detecting the obtained solution to be detected and the reference substance solution by using high performance liquid chromatography, and finally determining the residual amount of the lactic acid in the sample to be detected by using an external standard method based on a chromatographic detection result.
According to the method, the ion exchange chromatographic column for analyzing the organic acid is used for detecting the lactic acid in the hyaluronic acid extracted by the absolute ethyl alcohol, so that the adverse effect of the dissolution of the hyaluronic acid on the subsequent detection is avoided, the service life of the chromatographic column is prolonged, and the use of harmful reagents such as methanol and the like in the analysis process is avoided.
According to the method, the sample is pretreated by using absolute ethyl alcohol, and the content of the sample is obtained by combining a liquid chromatography separation technology, so that the method is high in specificity.
In the present application, the sample to be tested contains hyaluronic acid. The main component of the sample to be detected is hyaluronic acid, and the invention aims to detect the residual quantity of lactic acid in the hyaluronic acid.
In the application, in the preparation of the solution to be tested, 40-100 ml of the absolute ethyl alcohol is added into every 10g of the sample to be tested. The term "absolute ethanol" means ethanol (alcohol) containing 99.5% or more of ethanol. By extracting the lactic acid in the hyaluronic acid by using the absolute ethyl alcohol instead of other organic solvents, the lactic acid in the hyaluronic acid sample can be more sufficiently extracted, so that the detection is more accurate. But also avoids the influence on the hyaluronic acid itself.
In this application, in the preparation of solution to be measured, will the sample that awaits measuring adds in among the absolute ethyl alcohol, the misce bene forms mixed solution, then seals mixed solution, and carries out ultrasonic treatment, later with the mixed solution transfer to the volumetric flask in to with the absolute ethyl alcohol constant volume, then take the supernatant to volatilize futilely, obtain solid mixture, later dissolve with weak acid solution solid mixture to the constant volume, then filter, the filtrate that obtains is the solution that awaits measuring.
The time for the ultrasonic treatment is, for example, 15 to 40min, preferably 20 to 35min, and particularly preferably 30 min.
Ultrasonic treatment is ultrasonic extraction in this application, and ultrasonic extraction utilizes multistage effects such as strong cavitation response effect, mechanical vibration, disturbance effect, high acceleration, emulsification, diffusion, crushing and stirring effect that ultrasonic radiation pressure produced, increases material molecule motion frequency and speed, increases solvent penetrating power to the going on of target component entering solvent is accelerated, the promotion extraction.
The time of the ultrasonic treatment affects the extraction effect of the lactic acid, and when the time of the ultrasonic treatment is short, the extraction of the lactic acid is incomplete. Determination of ultrasound time: the results were obtained by performing comparative tests for 5min, 10min, 30min, and 60min of ultrasound treatment: the lactic acid concentration rises obviously 10min before ultrasonic treatment, the lactic acid concentration does not change greatly when the ultrasonic time is 10 min-60 min, and the lactic acid concentration reaches a peak value when the ultrasonic time is about 30 min.
In the present application, the weak acid solution is a phosphoric acid solution, preferably a phosphoric acid solution having a concentration of 0.5 wt% to 1.2 wt%. The concentration of the phosphoric acid solution may be 0.5 wt%, 0.55 wt%, 0.6 wt%, 0.65 wt%, 0.7 wt%, 0.75 wt%, 0.8 wt%, 0.85 wt%, 0.9 wt%, 0.95 wt%, 1 wt%, 1.05 wt%, 1.1 wt%, 1.15 wt%, 1.2 wt%.
In the present application, the ion exchange chromatography column for analyzing organic acids may be a cation exchange chromatography column, and particularly, may be a strong cationic hydrogen type exchange chromatography column in which a sulfonated crosslinked styrene divinylbenzene copolymer is used as a filler. For example, MCI GEL CK08EH column (8X 300mm, 5 μm). The MCI GEL CK08EH chromatographic column is a cation exchange chromatographic column belonging to CK08E series and produced by Mitsubishi chemistry, and is a strong cation hydrogen type exchange chromatographic column taking sulfonated and crosslinked styrene divinyl benzene copolymer as a filler.
In the present application, the mobile phase of the high performance liquid chromatography is a weak acid solution, and the concentration of the phosphoric acid is 0.5 wt% to 1.2wt%, preferably 0.8 wt% to 1.0 wt%.
The concentration of the phosphoric acid may be 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, 1.1 wt%, 1.2 wt%.
Because the mobile phase of the high performance liquid chromatography does not add any organic phase such as methanol and acetonitrile, the potential harm to experimenters caused by volatilization of organic reagents in use can be effectively prevented. Hydrogen cation exchange chromatography columns lose surface bound H during ion exchange with the test substance in the mobile phase + Therefore, the general ion exchange chromatographic column needs to be regenerated by an acid solution after being used for a period of time, the phosphoric acid with higher concentration does not need to be added with a regeneration step, and the protective effect is provided for the chromatographic column, the service life of the chromatographic column using the high-concentration phosphoric acid as a mobile phase is generally more than 3600h, and is improved by about 30 times compared with the service life of the chromatographic column using the low-concentration phosphoric acid as a mobile phase.
In the present application, the chromatographic conditions employed are: the flow rate of the mobile phase is 0.3 to 1.0ml/min, and more preferably 0.4 to 0.8 ml/min. The column temperature used in the high performance liquid chromatography is 70 ℃ to 95 ℃, preferably 75 ℃ to 90 ℃. When the column temperature is higher, the ionic thermal motion in the mobile phase is accelerated, and the exchange frequency with resin groups in unit time is increased, so that the column efficiency is improved, the lactic acid peak width is narrowed, the peak type is more symmetrical, and the chromatographic peak area is more accurately calculated.
The detection wavelength is 190-220 nm, such as 210nm, and the sample injection amount is 5-100 μ L, such as 20 μ L. Under this condition, lactic acid can be measured more accurately.
The lactic acid in the hyaluronic acid can be detected more accurately under the chromatographic condition, and the detection precision can be as low as 5 ppm. Because the hyaluronic acid solution is very viscous and is a gelatinous substance, even a 1% hyaluronic acid solution is very viscous and cannot be filtered and extracted, if the concentration of hyaluronic acid is reduced to obtain a solution with proper viscosity, the detection limit can be greatly improved, and the detection requirement cannot be met. The sample preparation method for extracting and concentrating lactic acid in hyaluronic acid by using absolute ethyl alcohol can solve the problems and ensure that the detection limit is lower.
In the embodiment of the present invention, the residual amount of lactic acid in the sample to be tested is calculated by using an external standard method.
By utilizing the method for determining the residual amount of the lactic acid in the hyaluronic acid, the ion exchange chromatographic column for analyzing the organic acid is used, the analysis effect on the lactic acid at a higher column temperature is good, the column efficiency is high, the durability is strong, the lactic acid can be eluted by using a weak acid solution without an organic phase, and the using amount of an organic reagent is reduced. By using the method, the used chromatographic column can run for a long time, and the situations of column efficiency reduction and the like can not occur.
The present application will be described in detail with reference to examples. It should be understood, however, that the present application may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. The recitation of numerical ranges in this application includes both numerical values for both endpoints of the numerical range and also includes each numerical value specifically within that numerical range, and any combination of such numerical values with the endpoints provides a new subrange.
The lifetime of a chromatography column is generally determined by the theoretical plate number and column pressure: the liquid chromatographic column generally has column efficiency lower than 2000, and the column efficiency cannot be recovered after regeneration, and then the liquid chromatographic column is judged to be discarded; in addition, the column pressure of the chromatographic column is obviously increased under the same using condition, and the chromatographic column is judged to be scrapped when the regenerated effect is not obvious.
Examples
The experimental methods used in the following examples are all conventional methods unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
1. Reagents and materials
Absolute alcohol (Tianjin Kemi Ou), phosphoric acid (national drug group chemical reagent Co., Ltd.)
2. Chromatographic conditions
A chromatographic column: MCI GEL CK08EH chromatography column (Mitsubishi chemical, 8X 300mm, 5 μm);
mobile phase: 1% phosphoric acid solution;
flow rate: 0.6 ml/min;
sample introduction amount: 20 mu L of the solution; column temperature: 80 ℃;
detection wavelength: 210 nm.
3. Solution preparation
Control solution: precisely weighing about 50mg of lactic acid reference substance in a 50mL volumetric flask, dissolving the mobile phase, fixing the volume to a scale, mixing uniformly, and filtering with a 0.22 mu m filter membrane to obtain the reference substance solution.
Test article (may also be referred to as a test sample) solution: precisely weighing a sample 10g to 50mL in a volumetric flask, adding 40mL of absolute ethyl alcohol, uniformly mixing and sealing, performing ultrasonic extraction for 30min, and fixing the volume of the absolute ethyl alcohol to the scale. Collecting supernatant 20mL, volatilizing, dissolving with mobile phase to 2 mL. Filtering with 0.22 μm filter membrane to obtain test solution.
4. Measurement of
And respectively taking 20 mu L of reference substance and sample solution, injecting, detecting according to the chromatographic conditions, and calculating the residual quantity of the lactic acid in the sample by the external standard method peak area.
5. Computing
The residual amount of lactic acid in the test sample was calculated according to the following formula:
wherein X represents the lactate residue in the sample, and μ g/g
Peak area of As-sample solution
Peak area of Ar-control solution
Wr-weight of control, μ g
Z-reference content
Ws-sample weight of sample, g
6. Results
The results of the detection of the lactic acid content in the test samples are shown in table 1. The chromatogram is shown in FIG. 1.
TABLE 1 chromatographic analysis results of the test article in example 1
Note: the detection limit was the residual amount of lactic acid at a signal-to-noise ratio (S/N) of 3.
And (4) continuously detecting different samples to be detected under the conditions, wherein the total running time of the chromatographic column exceeds 3600 hours, and the chromatographic column still runs normally.
Example 2 (column temperature 70 ℃ C.)
The residual amount of lactic acid in the sample was measured by setting the column temperature in example 1 to 70 ℃ and the other conditions in the same manner as in example 1.
TABLE 2 chromatographic analysis results of the test article in example 2
And (4) summarizing: as is clear from Table 2, when the column temperature was 70 ℃, the peak width of lactic acid was slightly wider than that of example 1, and the peak area was slightly larger, and the residual amount of lactic acid in the sample was 8.0, which was found to be within the allowable range of detection deviation.
Example 3 (column temperature 95 ℃ C.)
The residual amount of lactic acid in the sample was measured by setting the column temperature in example 1 to 95 ℃ and the other conditions in the same manner as in example 1.
TABLE 3 chromatographic analysis results of the test articles in example 3
And (3) knotting: as can be seen from table 3, when the column temperature is 95 ℃, the peak symmetry of lactic acid is slightly poor compared with example 1, the residual amount of lactic acid in the sample is detected to be 8.0, and the detection result is within the allowable deviation range, but the relative deviation of the two parallel samples is close to 2%, which may be related to the column temperature close to the boiling point of water, resulting in the deterioration of the sample injection volume accuracy.
Example 4 (column temperature 90 ℃ C.)
The residual amount of lactic acid in the test sample was measured by setting the column temperature in example 1 to 90 ℃ and the other conditions in the same manner as in example 1.
Table 4 chromatographic analysis results of the test articles in example 4
And (3) knotting: as is clear from Table 4, when the column temperature was 90 ℃, the residual amount of lactic acid in the sample was 8.1, which is the same as the detection result in example 1.
Example 5 (column temperature 75 ℃ C.)
The column temperature in example 1 was set to 75 ℃ and the residual amount of lactic acid in the test sample was measured under the same conditions as in example 1.
TABLE 5 chromatographic analysis results of the test articles in example 5
Example 6 (0.8% phosphoric acid solution)
The residual amount of lactic acid in the test sample was measured by setting the concentration of phosphoric acid in example 1 to 0.8% and the other conditions to those in example 1.
TABLE 6 chromatographic analysis results of the test article in example 6
Example 7 (0.9% phosphoric acid solution)
The residual amount of lactic acid in the test sample was measured under the same conditions as in example 1 except that the concentration of phosphoric acid in example 1 was set to 0.9%.
TABLE 7 chromatographic analysis results of the test article in example 7
And (3) knotting: as is clear from Table 7, when the mobile phase was a 0.9% phosphoric acid solution, the residual amount of lactic acid in the test sample was 8.1, which was the same as that in example 1.
Example 8 (1.2% phosphoric acid solution)
The residual amount of lactic acid in the test sample was measured by setting the concentration of phosphoric acid in example 1 to 1.2% and the other conditions to those in example 1.
TABLE 8 chromatographic analysis results of the test article in example 8
Example 9 (0.5% phosphoric acid solution)
The residual amount of lactic acid in the test sample was measured under the same conditions as in example 1 except that the concentration of phosphoric acid in example 1 was set to 0.5%.
TABLE 9 chromatographic analysis results of the test articles in example 9
Comparative example 1
The residual amount of lactic acid in the test sample was measured by setting the column temperature in example 1 to 40 ℃ and the other conditions to the same as in example 1.
The results of the measurements are shown in table 10 and fig. 2.
TABLE 10 chromatographic analysis results of the test articles in comparative example 1
And (3) knotting: under the chromatographic condition that the temperature is lower than 70 ℃, the lactic acid chromatographic peak has obvious tailing and broadening, and the detection result has deviation due to the reduction of the column efficiency.
Comparative example 2
The concentration of the mobile phase in example 1 was set to 0.01%, and the residual amount of lactic acid in the test sample was measured under the same conditions as in example 1.
TABLE 11 chromatographic analysis results of the test articles in comparative example 2
And (3) knotting: the peak shape is poor under the chromatographic condition that the concentration of the mobile phase is 0.01 percent, the residual quantity of the lactic acid of a test sample is low, and integration cannot be realized due to no obvious chromatographic peak.
Comparative example 3
The residual amount of lactic acid in the sample was measured under the same conditions as in example 1 except that the column temperature in example 1 was set to 40 ℃ and the mobile phase concentration was set to 0.01%.
TABLE 12 chromatographic analysis results of the test articles in comparative example 3
And (3) knotting: under the chromatographic conditions that the concentration of the mobile phase is 0.01 percent and the column temperature is 40 ℃, the lactic acid does not peak within the detection time.
Comparative example 4
The residual amount of lactic acid in the test sample was determined by substituting the lactic acid extraction solvent of example 1 with methanol (which is more toxic than ethanol) under the same conditions as in example 1.
TABLE 13 chromatographic analysis results of the test articles in comparative example 4
TABLE 14 comparison of chromatographic conditions and running effects for the examples
Indicates that the chromatographic column needs regeneration treatment after 120h of use
And (3) knotting: as can be seen from the above table, the chromatographic conditions of a column temperature of 70 ℃ to 95 ℃ and a mobile phase concentration of 0.5% to 1.2% are adopted to detect the lactic acid residue in the test sample, the detection sensitivity is high, the column life is long, and the peak type is good, especially under the chromatographic conditions of a column temperature of 75 ℃ to 90 ℃ and a mobile phase concentration of 0.8% to 1.0%, the detection limit is extremely low, the column efficiency is high, i.e., the sensitivity is high, meanwhile, the phosphoric acid with a certain concentration in the mobile phase can keep the activity of the cation exchange chromatographic column, an additional chromatographic column regeneration process is not needed, the feasibility of continuous detection is improved, and the service life of the chromatographic column is prolonged. Therefore, by the detection method of the residual lactic acid, a lower detection limit, namely higher method sensitivity can be obtained, and the service life of the chromatographic column is prolonged.
Claims (13)
1. A method for detecting residual quantity of lactic acid is characterized by comprising the following steps:
extracting lactic acid in a sample to be detected by using absolute ethyl alcohol to prepare a solution to be detected;
detecting the solution to be detected by using high performance liquid chromatography;
calculating the residual quantity of lactic acid in the sample to be detected based on the detection result;
the chromatographic column of the high performance liquid chromatography takes sulfonated cross-linked styrene divinyl benzene copolymer as a fillerStrong cation H + A type exchange chromatography column;
the mobile phase of the high performance liquid chromatography is phosphoric acid solution, and the concentration of the phosphoric acid solution is 0.5-1.2 wt%;
the column temperature of a chromatographic column used in the high performance liquid chromatography is 70-95 ℃;
the sample to be detected is hyaluronic acid.
2. The method according to claim 1, wherein the solution to be tested is prepared by adding 40-100 ml of the absolute ethanol to 10g of the sample to be tested.
3. The method according to claim 1, wherein in the preparation of the solution to be tested, the sample to be tested is added into the absolute ethyl alcohol and mixed uniformly to form a mixed solution, then the mixed solution is sealed and subjected to ultrasonic treatment, then the mixed solution is transferred into a volumetric flask and is subjected to constant volume by the absolute ethyl alcohol, then a supernatant is taken and volatilized to obtain a solid mixture, then the solid mixture is dissolved by a weak acid solution and is subjected to constant volume, and then the solid mixture is filtered, so that the obtained filtrate is the solution to be tested.
4. The method according to claim 3, wherein the ultrasonic treatment time is 15-40 min.
5. The method according to claim 3, wherein the weak acid solution is a phosphoric acid solution having a concentration of 0.5 wt% to 1.2 wt%.
6. The method of claim 1, wherein the cation exchange chromatography column is an MCI GEL CK08EH chromatography column.
7. The method of claim 1, wherein the concentration of the phosphoric acid solution is 0.8 wt% to 1.0 wt%.
8. The method according to claim 1, wherein the flow rate of the mobile phase is 0.3 to 1.0 ml/min.
9. The method according to claim 7, wherein the flow rate of the mobile phase is 0.4 to 0.8 ml/min.
10. The method of claim 1, wherein the column temperature of the chromatography column used in the high performance liquid chromatography is 75 ℃ to 90 ℃.
11. The method according to claim 1, wherein the detection wavelength in the high performance liquid chromatography is 190 to 220nm, and the amount of the sample is 5 to 100. mu.L.
12. The method according to any one of claims 1 to 11, wherein the residual amount of lactic acid in the sample to be tested is calculated using an external standard method.
13. The method according to any one of claims 1 to 11, wherein the detection limit of the residual amount of lactic acid is 5 to 8 ppm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN202011278713.6A CN112326839B (en) | 2020-11-16 | 2020-11-16 | Method for detecting residual quantity of lactic acid |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106568851A (en) * | 2015-10-13 | 2017-04-19 | 中粮集团有限公司 | Detection method of lactic acid in baijiu base liquor |
| CN109212104A (en) * | 2018-11-13 | 2019-01-15 | 华熙福瑞达生物医药有限公司 | The remaining detection method of phthalic acid ester plasticiser in a kind of hyaluronic acid |
| CN109678696A (en) * | 2019-01-31 | 2019-04-26 | 华熙福瑞达生物医药有限公司 | A method of extracting lactic acid from hyaluronic acid fermentation liquor |
| CN110749679A (en) * | 2019-11-08 | 2020-02-04 | 华熙生物科技股份有限公司 | Detection method of trimethyl pyrazine residues |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPWO2015170516A1 (en) * | 2014-05-08 | 2017-04-20 | 昭和電工株式会社 | Organic acid mass spectrometry method, analytical column and analyzer |
-
2020
- 2020-11-16 CN CN202011278713.6A patent/CN112326839B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106568851A (en) * | 2015-10-13 | 2017-04-19 | 中粮集团有限公司 | Detection method of lactic acid in baijiu base liquor |
| CN109212104A (en) * | 2018-11-13 | 2019-01-15 | 华熙福瑞达生物医药有限公司 | The remaining detection method of phthalic acid ester plasticiser in a kind of hyaluronic acid |
| CN109678696A (en) * | 2019-01-31 | 2019-04-26 | 华熙福瑞达生物医药有限公司 | A method of extracting lactic acid from hyaluronic acid fermentation liquor |
| CN110749679A (en) * | 2019-11-08 | 2020-02-04 | 华熙生物科技股份有限公司 | Detection method of trimethyl pyrazine residues |
Non-Patent Citations (4)
| Title |
|---|
| 6种常用低聚糖对嗜热链球菌体外生长的影响;夏晓风 等;《食品与生物技术学报》;20161231;第35卷(第3期);第310-317页 * |
| 8种药材中6种有机酸类成分的HPLC含量测定;刘慧 等;《中药材》;20170531;第40卷(第5期);第1130-1133页 * |
| 刘慧 等.8种药材中6种有机酸类成分的HPLC含量测定.《中药材》.2017,第40卷(第5期), * |
| 大肠杆菌9种有机酸代谢产物的高效液相检测条件优化;马建刚等;《现代食品科技》;20111231;第27卷(第05期);第591-594，519页 * |
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