Method for detecting content of glycerol in biological product

Abstract

The invention provides a method for detecting the content of glycerol in a biological product. Specifically, glycerol and NAD < + > react under the action of glycerol dehydrogenase, so that NAD < + > is reduced into NADH, and the content of NADH is detected by a colorimetric method, so that the content of glycerol in a sample is indirectly obtained. The method overcomes the defects of high cost, long time consumption and large error of the traditional glycerol determination method, and has the application prospect in the field of biological product detection.

Description

Technical Field

[0001] The present invention relates to the field of biological product detection, and in particular to a method for determining the glycerol content in biological products. Background Art

[0002] Glycerol, also known as propylene glycol, has the chemical formula C3H8O3. It is colorless, odorless, sweet, and appears as a clear, viscous liquid. Biological products are often added to biological products for protection due to their properties, but the amount of glycerol added is subject to certain restrictions. The 2020 edition of the Chinese Pharmacopoeia, Part II, stipulates that 1.4-1.8g of glycerol and 0.25g of phenol can be added to every 100ml of insulin injection and protamine zinc insulin injection. Glycerol fructose sodium chloride injection: 90-110g of glycerol can be added to every 1000ml. Therefore, the amount of glycerol finally added to biological products needs to be tested and monitored.

[0003] At present, the main methods for determining glycerol in biological product samples include: 1) Chemical method: that is, glycerol is reacted with potassium periodate in an aqueous solution to generate formic acid, and the generated formic acid is then neutralized with standard sodium hydroxide to obtain the content of free glycerol; although this method has low cost, it is cumbersome to operate, time-consuming, and sometimes has large accidental errors; 2) Enzyme assay: It only reacts specifically with glycerol in the solution and has strong specificity, but the reagents required are expensive, which is not conducive to industrial application and detection of large quantities of samples; 3) Gas chromatography: It has high accuracy and is the most sensitive, but the operation steps are complicated and the detection cost is high.

[0004] Therefore, there is a need in the art to develop a low-cost, rapid, accurate and safe detection method for the glycerol content in biological products to ensure process quality control of biological products. Summary of the invention

[0005] The purpose of the present invention is to overcome some defects of the existing methods for detecting glycerol in biological products and to provide a method for determining the glycerol content in biological products which is low-cost, easy to operate, highly accurate and safe.

[0006] In a first aspect of the present invention, there is provided a method for determining the glycerol content in a sample, comprising the steps of:

[0007] 1) reacting the sample to be tested with NAD+ in the presence of glycerol dehydrogenase to produce NADH;

[0008] 2) Detecting the NADH produced in step 1), thereby calculating the glycerol content or concentration in the sample.

[0009] In another preferred embodiment, the concentration of glycerol dehydrogenase is 1-20 mM, preferably 5-15 mM, and more preferably 10-12 mM.

[0010] In another preferred embodiment, the NAD+ concentration is 1-200 mM, preferably 50-150 mM, and more preferably 100-120 mM.

[0011] In another preferred embodiment, the molar ratio of glycerol dehydrogenase to NAD+ is 100:1 to 1:100, preferably 50:1 to 1:50, and more preferably 10:1 to 1:10.

[0012] In another preferred embodiment, the reaction time of step 1) is 10-60 min, preferably 20-45 min, and more preferably 30-40 min.

[0013] In another preferred embodiment, the reaction temperature of step 1) is 25-45°C, preferably 30-40°C, and more preferably 35-37°C.

[0014] In another preferred embodiment, the detection in step 2) is colorimetric detection.

[0015] In another preferred embodiment, the detection in step 2) is performed using an ELISA reader.

[0016] In another preferred embodiment, in step 2), the detection of NADH is performed by detecting the absorbance of the sample at 340 nm.

[0017] In another preferred embodiment, step 2) includes the step of converting the NADH detection result into the content or concentration of glycerol.

[0018] In another preferred embodiment, in step 2), the NADH detection result (eg, absorbance measured by colorimetry) is compared with a standard substance or a standard curve to calculate the content or concentration of glycerol in the sample.

[0019] In another preferred embodiment, the standard curve is obtained by the following method:

[0020] (a) preparing 0-10 parts of a glycerol solution with a concentration of 0-2 mM (preferably 0.1-2 mM), reacting with NAD+ in the presence of glycerol dehydrogenase to produce NADH;

[0021] (b) measuring the absorbance of the solution obtained in step (a) by colorimetry;

[0022] (c) Draw a standard curve of absorbance and glycerol concentration.

[0023] In another preferred embodiment, after step 2), the method further comprises the following steps:

[0024] Determine whether the glycerol concentration exceeds the linear detection range. If so, dilute or concentrate the sample to be tested and repeat steps 1)-2).

[0025] In another preferred embodiment, the linear detection range is 0.01 mM-5.0 mM, preferably 0.1 mM-2.0 mM.

[0026] In another preferred embodiment, the detection sensitivity of the method for determining the glycerol content in the sample is 0.1 mM; preferably 0.08 mM; most preferably 0.05 mM.

[0027] In another preferred embodiment, the sample to be tested is a biological product.

[0028] In another preferred embodiment, the sample to be tested is an injection solution.

[0029] In another preferred embodiment, the samples to be tested include: insulin injection and glycerol fructose sodium chloride injection.

[0030] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The following drawings are used to illustrate specific embodiments of the present invention and are not used to limit the scope of the present invention defined by the claims.

[0032] Figure 1 A schematic diagram of the preparation process of 6Mol / L glycerol is shown.

[0033] Figure 2 A schematic diagram of the basic operation flow of the present invention is shown.

[0034] Figure 3 The glycerol standard curve is shown with A on the ordinate 340 The average absorbance value is shown in Figure 2, and the horizontal axis is the concentration of the glycerol standard in mMol. DETAILED DESCRIPTION

[0035] After extensive and in-depth research, the inventors have developed a method for determining the glycerol content in a sample for the first time. Specifically, the present invention allows glycerol and NAD+ to react under the action of glycerol dehydrogenase, so that NAD+ is reduced to NADH, and the NADH content is detected by colorimetry, thereby indirectly obtaining the glycerol content in the sample. The present invention overcomes the shortcomings of the traditional glycerol determination method, which is high cost, time-consuming, and has large errors. On this basis, the present invention was completed.

[0036] Method of the present invention

[0037] The inventors creatively react glycerol and NAD+ under the action of glycerol dehydrogenase to reduce NAD+ to NADH, and detect the NADH content by colorimetry, thereby being able to simply and accurately detect the glycerol content in the sample.

[0038] In addition, the method of the present invention also has a wide linear detection range (0.1mM-2.0mM). Those skilled in the art know that a wide linear detection range, especially a higher upper limit, means that the method of the present invention has a greater tolerance to the glycerol content in the sample; that is, the method of the present invention can be directly applied to samples with a high glycerol content without diluting the sample to be tested before detection, or only a small amount of dilution operation is required. In fact, the glycerol content in biological products is often high, so the method of the present invention can be directly applied to samples with a high glycerol content, which further increases the convenience of the method of the present invention.

[0039] Detection principle

[0040] Glycerol dehydrogenase is an oxidoreductase that uses NAD+ or NADP+ as an acceptor and acts on the donor CH-OH group. This enzyme can catalyze the following enzymatic reactions:

[0041]

[0042] The present invention utilizes glycerol and NAD+ under the action of glycerol dehydrogenase, NAD+ is reduced to NADH, and the amount of NADH is positively correlated with the glycerol concentration; NADH has corresponding absorption light at 340nm, and the glycerol content in the sample is calculated by detecting NADH at 340nm.

[0043] Sample pretreatment

[0044] The detection method of the present invention may include a sample pretreatment step. Depending on the sample conditions, clear liquid biological product samples such as injections and vaccines can be directly measured; if the initial measurement result exceeds the linear range of the kit, it can be concentrated or diluted with ddH2O and then measured again.

[0045] Standard curve

[0046] The detection method of the present invention may include the step of drawing a standard curve, or use an existing standard curve.

[0047] In one embodiment of the present invention, the drawing of the standard curve comprises the following steps:

[0048] Prepare glycerol solutions of different concentrations, add glycerol dehydrogenase and NAD+ to each solution of different concentrations, measure the absorbance at a specific wavelength (such as 340nm) with an enzyme reader after the reaction, use the glycerol solution concentration as the X-axis and the corresponding absorbance as the Y-axis, draw a standard curve, and fit and obtain the formula.

[0049] In one embodiment, the dilution scheme is as follows:

[0050] Provide a 100 mM glycerol standard, dilute the 100 mM glycerol standard to 2.0 mM, 1.6 mM, 1.2 mM, 0.8 mM, 0.4 mM, and 0.1 mM with ddH2O, and set up a 0 mM concentration control reaction tube.

[0051] The main advantages of the present invention include:

[0052] 1) The present invention utilizes glycerol and NAD+ under the action of glycerol dehydrogenase, NAD+ is reduced to NADH, and the amount of NADH is positively correlated with the glycerol concentration; the glycerol content in the sample is calculated by detecting NADH at 340nm. The present invention is the first to apply the above method to the determination of glycerol content in biological products, and can be used to detect finished biological products and raw materials, etc.;

[0053] 2) This kit can be used to determine the glycerol content in samples of injections, vaccines and other biological products. The kit is easy to operate, with a minimum detection limit of 0.05mM, a linear range of 0.1mM to 2.0mM, and excellent reliability and repeatability. It is suitable for biomedical, biological product, and food laboratory testing.

[0054] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0055] The equipment and materials used in this embodiment are as follows:

[0056] Equipment: 1 / 100,000 analytical balance, 100mL conical flask, Sartorius deionized water dispenser, sonicator, 2mL ball pipette, 15mL stoppered volumetric flask, MD SpectraMax M2 microplate reader, constant temperature incubator, 1000μL, 100μL, 10μL pipettes.

[0057] Materials: Glycerol dehydrogenase, NAD+, glycerol standard solution, insulin injection.

[0058] Example 1 Sample pretreatment

[0059] Since the initial determination result of insulin injection exceeds the linear range of this kit, it needs to be diluted with ddH2O and then measured again. To ensure the accuracy of high-concentration glycerol dilution, a high-concentration standard is used to verify the dilution accuracy and spike recovery rate. The national glycerol standard can be purchased, item number: 56-81-5.

[0060] 1.1 Preparation of high concentration glycerol reference

[0061] Place the 15mL volumetric flask on the weighing balance and reset to zero;

[0062] Slowly add national glycerol standard and weigh 8.2881g glycerol;

[0063] Add double distilled water to make up to 15 mL, shake to mix, the final concentration is 6 Mol / L, and then transfer to a 15 mL centrifuge tube for storage.

[0064] Example 2 Standard Dilution

[0065] Take a glycerol standard with a concentration of 100mM. The dilution scheme is as follows:

[0066] Use ddH2O to dilute the 100mM glycerol standard to 2.0mM, 1.6mM, 1.2mM, 0.8mM, 0.4mM, and 0.1mM, and set up a 0mM concentration control reaction tube. The dilution scheme is shown in Table 1. The operation diagram is shown in Figure 2 .

[0067] Table 1. Glycerol standard curve dilution gradient

[0068]

[0069] Example 3 Control Sample Treatment

[0070] Parallel sample processing: To ensure the accuracy of the results, it is recommended that each sample be tested three times in parallel, with one set of experiments being performed with and without spiked treatments.

[0071] Spike recovery (ERC): ERC is used to evaluate the authenticity and accuracy of glycerol detection, and can be used to evaluate and verify analytical methods and system performance. Spike recovery calculation formula = (detection value - background value) / added glycerol amount * 100%.

[0072] Example 4 Glycerol determination

[0073] 4.1 Preparation of reaction solution

[0074] 1) The kit needs to be stored at -18℃ or below when it is unopened; fresh reaction solution needs to be prepared for each reaction, and repeated freezing and thawing is not allowed. During the preparation of the reaction solution, Assay BufferⅡ and Enzyme Mix need to be operated on ice; the specific reaction system is as follows:

[0075] Table 2. Reaction solution preparation

[0076] Assay BufferⅠ Assay BufferⅡ Enzyme Mix Volume (μL) 68 2 10

[0077] The specific composition of Enzyme Mix is ​​glycerol dehydrogenase, EDTA, and glucose, including that the concentrations of glycerol dehydrogenase and NAD+ are 10 mMol and 100 mMol, respectively, with a molar ratio of 1:10.

[0078] The reaction Assay Buffer in the system can be purchased commercially.

[0079] 2) Add 20 μL of the sample to be tested and the glycerol standard working solution into the corresponding wells of a 96-well transparent flat-bottom plate. Three replicate wells should be set up for each well.

[0080] 3) Prepare a reaction mixture according to Table 3, add 80 μL of the reaction mixture to each reaction well, incubate in a 37° C. constant temperature incubator for 30 minutes, and then measure its absorbance at 340 nm using an ELISA reader.

[0081] 4.2 Result determination

[0082] 1) Calculation of A340 absorbance

[0083] First, use the absorbance value of 0 mM concentration to zero the absorbance values ​​of the remaining standard curve concentrations and the sample to be tested.

[0084] 2) Drawing and calculation of standard curve

[0085] The linear regression equation was obtained by taking the average absorbance value of A340 of each well of the standard as the ordinate (y) and the corresponding glycerol standard concentration (mMol) as the abscissa (x). Figure 3 The standard curve fitting range is ST1 to ST6, and the value of 0 is not included in the standard curve.

[0086] That is: y=A+Bx;

[0087] The linear correlation coefficient of the standard curve is required to be R2≥0.980.

[0088] Substitute the absorbance value of the sample into the standard curve, read the corresponding concentration of the sample from the standard curve, and multiply it by the corresponding dilution factor to obtain the actual residual glycerol in the sample.

[0089] Table 3. Recovery rate of diluted spiked solution of high concentration glycerol insulin injection and test results of diluted samples

[0090]

[0091] sample: insulin injection;

[0092] Spiked sample: insulin injection: 1M glycerol = 1:1;

[0093] Test sample: ①sample diluted 300 times; ②spiked sample diluted 600 times

[0094] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for determining the glycerol content in a sample, comprising the steps of: 1) reacting the sample to be tested with NAD+ in the presence of glycerol dehydrogenase to produce NADH; 2) Detecting the NADH produced in step 1), thereby calculating the glycerol content or concentration in the sample.

2. The method according to claim 1, characterized in that The concentration of glycerol dehydrogenase is 1-20 mM, preferably 5-15 mM, and more preferably 10-12 mM.

3. The method according to claim 1, characterized in that The NAD+ concentration is 1-200mM, preferably 50-150mM, and more preferably 100-120mM.

4. The method according to claim 1, characterized in that The molar ratio of glycerol dehydrogenase to NAD+ is 100:1 to 1:100, preferably 50:1 to 1:50, and more preferably 10:1 to 1:

10.

5. The method according to claim 1, characterized in that In step 2), the detection of NADH is performed by detecting the absorbance of the sample at 340 nm.

6. The method according to claim 1, characterized in that After step 2), the following steps are also included: Determine whether the glycerol concentration exceeds the linear detection range. If so, dilute or concentrate the sample to be tested and repeat steps 1)-2).

7. The method according to claim 1, characterized in that The linear detection range is 0.01mM-5.0mM, preferably 0.1mM-2.0mM.

8. The method according to claim 1, characterized in that The detection sensitivity of the method for determining the glycerol content in the sample is 0.1 mM; preferably 0.08 mM; most preferably 0.05 mM.

9. The method according to claim 1, characterized in that The sample to be tested is a biological product.

10. The method according to claim 1, characterized in that The samples to be tested include: insulin injection and glycerol fructose sodium chloride injection.

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