CN119198705A - A method for determining ribose content in transfer factor for injection and its application - Google Patents
A method for determining ribose content in transfer factor for injection and its application Download PDFInfo
- Publication number
- CN119198705A CN119198705A CN202411720227.3A CN202411720227A CN119198705A CN 119198705 A CN119198705 A CN 119198705A CN 202411720227 A CN202411720227 A CN 202411720227A CN 119198705 A CN119198705 A CN 119198705A
- Authority
- CN
- China
- Prior art keywords
- solution
- injection
- ribose
- sample
- measuring
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 title claims abstract description 71
- 238000000034 method Methods 0.000 title claims abstract description 47
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 title claims abstract description 39
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 title claims abstract description 39
- 238000002347 injection Methods 0.000 title claims abstract description 38
- 239000007924 injection Substances 0.000 title claims abstract description 38
- 108010074506 Transfer Factor Proteins 0.000 title claims description 29
- 239000000243 solution Substances 0.000 claims abstract description 91
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 45
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 45
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 42
- 239000012488 sample solution Substances 0.000 claims abstract description 36
- 239000000843 powder Substances 0.000 claims abstract description 28
- 238000002835 absorbance Methods 0.000 claims abstract description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000013558 reference substance Substances 0.000 claims abstract description 12
- 238000005303 weighing Methods 0.000 claims abstract description 11
- 239000006228 supernatant Substances 0.000 claims abstract description 10
- 239000011259 mixed solution Substances 0.000 claims abstract description 7
- 238000012546 transfer Methods 0.000 claims abstract description 6
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000011161 development Methods 0.000 claims abstract description 4
- 238000010790 dilution Methods 0.000 claims abstract description 3
- 239000012895 dilution Substances 0.000 claims abstract description 3
- OIPPWFOQEKKFEE-UHFFFAOYSA-N orcinol Chemical compound CC1=CC(O)=CC(O)=C1 OIPPWFOQEKKFEE-UHFFFAOYSA-N 0.000 claims description 28
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 16
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 16
- 238000010438 heat treatment Methods 0.000 claims description 13
- 239000012085 test solution Substances 0.000 claims description 12
- 238000009835 boiling Methods 0.000 claims description 10
- 239000000523 sample Substances 0.000 claims description 10
- 238000001816 cooling Methods 0.000 claims description 7
- 238000005259 measurement Methods 0.000 claims description 6
- 239000012088 reference solution Substances 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims 1
- 238000001303 quality assessment method Methods 0.000 claims 1
- 229940119744 dextran 40 Drugs 0.000 abstract description 8
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 abstract description 8
- 238000004090 dissolution Methods 0.000 abstract description 5
- 239000003814 drug Substances 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 description 13
- 230000000052 comparative effect Effects 0.000 description 11
- 238000011835 investigation Methods 0.000 description 9
- 238000007865 diluting Methods 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 4
- 238000001704 evaporation Methods 0.000 description 4
- 230000008020 evaporation Effects 0.000 description 4
- 238000009210 therapy by ultrasound Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 229920002097 Lichenin Polymers 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- HYBBIBNJHNGZAN-UHFFFAOYSA-N furfural Chemical compound O=CC1=CC=CO1 HYBBIBNJHNGZAN-UHFFFAOYSA-N 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 210000000777 hematopoietic system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000013441 quality evaluation Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 150000003384 small molecules Chemical group 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
Landscapes
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the technical field of medicine analysis, and particularly relates to a method for measuring ribose content in transfer factors for injection and application thereof. The determination method comprises the following steps of (1) preparing a sample solution, namely weighing sterile powder of transfer factors for injection, adding water for dissolution, then adding solution A for dilution, centrifuging, taking supernatant for volatilizing, adding trichloroacetic acid solution for dissolution, and obtaining the sample solution, (2) preparing a reference substance solution, (3) determining, namely taking the sample solution, adopting a lichen phenol color development method to determine absorbance at 645-655nm, (4) drawing a standard curve, and (5) calculating ribose content, wherein the solution A in the step (1) is a mixed solution of methanol, ethanol and acetonitrile. The invention eliminates the problem of interference of dextran 40 on ribose determination in transfer factors for injection by optimizing the pretreatment process of samples, and improves the accuracy of the determination method.
Description
Technical Field
The invention belongs to the technical field of medicine analysis, and particularly relates to a method for measuring ribose content in transfer factors for injection and application thereof.
Background
The transfer factor is a small molecule polypeptide and nucleotide complex obtained by taking pig or cattle spleen as a raw material and carrying out homogenate dialysis. The transfer factor for injection is sterile freeze-dried powder prepared by adding proper excipient into transfer factor solution. After the transfer factor enters the body, the hematopoietic system generates a large amount of white blood cells in a short period, activates lymphocytes, increases the production of auxiliary cells and releases more lymphocytes. The variety is used as a cell immunity enhancer and an immunity regulator and is widely applied clinically.
In the current standard ribose content determination, the content is calculated by using the D-ribose by using a lichenin color development method. The principle of the method is that the furfural generated by the reaction of the lichenin reagent and the nucleotide degradation generates ribose is developed, and the ribose content is calculated by a D-ribose regression curve. Dextran 40 in the transfer factor for injection reacts with the lichenin reagent in the chromogenic method to form a precipitate, interfering with the ribose determination in the sample.
Therefore, the ribose content determination method needs to be further researched to meet the quality control requirement of the transfer factor for injection.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for measuring ribose content in transfer factors for injection and application thereof.
In order to achieve the above purpose of the present invention, the present invention adopts the following specific technical scheme:
The method for measuring ribose content in transfer factor for injection comprises the following steps:
(1) Preparing a test solution by weighing transfer factor sterile powder for injection, adding water for dissolving, then adding solution A for dilution, centrifuging, taking supernatant for volatilizing, and adding trichloroacetic acid solution for dissolving to obtain the test solution;
(2) Preparing reference substance solution by weighing D-ribose reference substance, dissolving with trichloroacetic acid solution to obtain reference substance solution;
(3) Measuring by taking sample solution, and measuring absorbance at 645-655nm by using a lichen phenol color development method;
(4) Drawing a standard curve, namely taking a reference substance solution, sequentially injecting samples by adopting a sequence injection method according to the method of the step (3), and drawing the standard curve by taking the injection concentration as an abscissa and the absorbance as an ordinate;
(5) Calculating the ribose content according to the standard curve obtained in the step (4), and calculating the ribose content of the sample in the step (3);
Wherein, the solution A in the step (1) is a mixed solution of methanol, ethanol and acetonitrile.
Preferably, the volume ratio of the methanol to the ethanol to the acetonitrile is 6-8:2-4:1.
Preferably, the mass volume ratio of the transfer factor sterile powder for injection to water in the step (1) is 1g:30-40mL, and the volume ratio of the solution A to the water is 3-5:1.
Preferably, the rotational speed of the centrifugation in the step (1) is 9000-10000r/min, and the centrifugation time is 8-12min.
Preferably, the mass concentration of the trichloroacetic acid solution in the step (1) and the step (2) is 4% -6%.
Preferably, the concentration of the sample solution in the step (1) is 2-3 mug/mL, and the concentration of the reference solution in the step (2) is 18-22 mug/mL.
Preferably, in the step (3), the lichen phenol chromogenic method is to add 3, 5-dihydroxytoluene solution into the test solution, shake the solution evenly, heat the solution, cool the solution, suck the test solution precisely, and measure the solution.
Further preferably, the mass concentration of the 3, 5-dihydroxytoluene solution is 0.8% -1.2%, and the volume ratio of the 3, 5-dihydroxytoluene solution to the sample solution is 1:0.8-1.2.
Further preferably, the heating condition is boiling water bath heating, and the heating time is 25-35min.
The invention also relates to application of the measuring method in quality evaluation of the transfer factor for injection.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the invention, through optimizing the pretreatment process of the sample and selecting the mixed solution of ethanol, methanol and acetonitrile as the precipitant, the problem of interference of dextran 40 on the determination of ribose in the transfer factor for injection is eliminated, and the accuracy of the determination method is improved.
(2) The preparation method of the sample solution has simple steps, and experiments prove that the method has good specificity, can preliminarily meet the detection requirement of ribose in the transfer factor for injection, and provides reference for quality control of the transfer factor for injection.
Drawings
FIG. 1 is a standard curve plotted in a linear range study of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
The reagent purchase information for each example of the present invention and comparative example is shown in Table 1.
TABLE 1 reagent information
Example 1
The method for measuring ribose content in transfer factor for injection comprises the following steps:
(1) Preparation of sample solution, namely precisely weighing 28.8mg of transfer factor sterile powder for injection, placing the powder into a 5ml measuring flask, adding 1ml of water, carrying out ultrasonic treatment to completely dissolve the powder, diluting the powder to a scale by using solution A (ethanol, methanol and acetonitrile with the volume ratio of 7:3:1), and shaking the powder uniformly. Taking the solution, centrifuging for 10min at the rotating speed of 9500r/min, taking 2.5ml of supernatant, placing the supernatant in an evaporation dish for volatilizing, adding 5% trichloroacetic acid solution (mass concentration) for ultrasonic dissolution, washing for 3 times, transferring the solution into a 10ml measuring flask, diluting the solution with 5% trichloroacetic acid solution to prepare a solution with 2.5 mug of nuclear sugar per 1ml, and shaking the solution uniformly to obtain a sample solution.
(2) Preparation of reference solution by precisely weighing D-ribose reference, adding 5% trichloroacetic acid solution for dissolving, and making into solution containing 20 μg per 1ml to obtain reference solution.
(3) Measuring, namely taking 2.0ml of a sample solution, adding 2.0ml of 1% 3, 5-dihydroxytoluene solution, shaking uniformly, heating in a boiling water bath for 30 minutes, rapidly cooling to room temperature, and measuring absorbance at the wavelength of 650 nm.
(4) Drawing a standard curve, namely taking a reference substance solution, sequentially injecting samples by adopting a sequence injection method according to the method of the step (3), and drawing the standard curve by taking the injection concentration as an abscissa and the absorbance as an ordinate;
Precisely measuring 0.0ml, 0.2ml, 0.6ml, 1.0ml, 1.5ml and 2.0ml of reference substance solution, respectively placing into test tubes with plugs, respectively adding a proper amount of 5% trichloroacetic acid solution to 2.0ml, respectively adding 2.0ml of 1% 3, 5-dihydroxytoluene solution, heating in boiling water bath for 30min, rapidly cooling to room temperature, measuring absorbance at 650nm wavelength, taking concentration as horizontal coordinate and absorbance as vertical coordinate, and drawing a standard curve.
(5) Calculating ribose content, namely substituting absorbance of the test solution into the standard curve obtained in the step (4) to obtain ribose concentration in the test solution, calculating ribose quality (measured quantity) according to m=cv, and then calculating ribose content in the transfer factor for injection according to the following formula;
Ribose (%) = ×100%。
Example 2
The method for measuring ribose content in transfer factor for injection comprises the following steps:
(1) Preparation of sample solution, namely, precisely weighing 30mg of transfer factor sterile powder for injection, placing the powder into a 5ml measuring flask, adding 1ml of water, carrying out ultrasonic treatment to completely dissolve the powder, diluting the powder to a scale by using solution A (ethanol, methanol and acetonitrile with the volume ratio of 6:4:1), and shaking the powder uniformly. Taking the solution, centrifuging for 8min at the rotating speed of 10000r/min, taking 2.5ml of supernatant, placing the supernatant in an evaporation dish for volatilizing, adding 5% trichloroacetic acid solution (mass concentration) for ultrasonic dissolution, washing for 3 times, transferring the solution into a 10ml measuring flask, diluting the solution with 5% trichloroacetic acid solution to prepare a solution with 2.5 mug of nuclear sugar per 1ml, and shaking the solution uniformly to obtain a sample solution.
The remaining steps are identical to those of example 1.
Example 3
The method for measuring ribose content in transfer factor for injection comprises the following steps:
(1) Preparation of sample solution, namely, precisely weighing 30mg of transfer factor sterile powder for injection, placing the powder into a 5ml measuring flask, adding 1ml of water, carrying out ultrasonic treatment to completely dissolve the powder, diluting the powder to a scale by using solution A (ethanol, methanol and acetonitrile with the volume ratio of 8:2:1), and shaking the powder uniformly. Taking the solution, centrifuging for 12min at the rotating speed of 9000r/min, taking 2.5ml of supernatant, volatilizing in an evaporation dish, adding 5% trichloroacetic acid solution (mass concentration), ultrasonically dissolving, washing for 3 times, transferring to a 10ml measuring flask, diluting with 5% trichloroacetic acid solution to prepare a solution with 2.5 mug of nuclear sugar per 1ml, and shaking uniformly to obtain a sample solution.
The remaining steps are identical to those of example 1.
Comparative example 1
The comparative example differs from example 1 only in that in the preparation of the test sample solution of step (1), the solution a is only ethanol.
Comparative example 2
The comparative example differs from example 1 only in that in the preparation of the sample solution in step (1), the solution A is a mixed solution of methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 3:1.
Comparative example 3
The comparative example differs from example 1 only in that in the preparation of the sample solution in step (1), the solution A is a mixed solution of ethanol and methanol, and the volume ratio of ethanol to methanol is 7:4.
Comparative example 4
The comparative example differs from example 1 only in that in the preparation of the sample solution in step (1), the solution A is a mixed solution of ethanol and acetonitrile, and the volume ratio of ethanol to acetonitrile is 7:4.
The ribose content of the transfer factor sterile powder for injection of the same batch was measured by the test methods of examples 1 to 3 and comparative examples 1 to 4, and the measurement results are shown in Table 2.
TABLE 2 ribose content detection results
Examples 1-3 the dextran 40 was completely precipitated by mixing ethanol, methanol and acetonitrile in a ratio such that the dextran 40 did not interfere with the ribose measurement of the test sample at 650nm and the absorbance was 0.0485% -0.0759% relative to the test sample. In comparative examples 1-4, however, there was still interference of the dextran 40, and the absorbance of the dextran 40 relative to the sample was relatively high.
Methodology investigation
1. Reagent preparation
Preparation of reference solution by precisely weighing D-ribose reference, adding 5% trichloroacetic acid solution for dissolving, and making into solution containing 20 μg per 1ml to obtain reference solution.
Preparation of sample solution, namely precisely weighing 28.8mg of transfer factor sterile powder for injection, placing the powder into a 5ml measuring flask, adding 1ml of water, carrying out ultrasonic treatment to completely dissolve the powder, diluting the powder to a scale by using solution A (ethanol, methanol and acetonitrile with the volume ratio of 7:3:1), and shaking the powder uniformly. Taking the solution, centrifuging for 10min at the rotating speed of 9500r/min, taking 2.5ml of supernatant, placing the supernatant in an evaporation dish for volatilizing, adding 5% trichloroacetic acid solution (mass concentration) for ultrasonic dissolution, washing for 3 times, transferring the solution into a 10ml measuring flask, diluting the solution with 5% trichloroacetic acid solution to prepare a solution with 2.5 mug of nuclear sugar per 1ml, and shaking the solution uniformly to obtain a sample solution.
2. Investigation of specificity
Measuring absorbance of the adjuvant (dextran 40) solution at 650nm, wherein the absorbance (Abs) of the adjuvant solution is-0.0002;
precisely measuring 2.0ml of a sample solution, adding 2.0ml of 1% 3, 5-dihydroxytoluene solution, shaking uniformly, heating in a boiling water bath for 30 minutes, rapidly cooling to room temperature, and measuring absorbance at 650nm, wherein the absorbance (Abs) of the sample solution is 0.4139;
the absorbance of the auxiliary materials relative to the test sample is less than 0.1%, which indicates that the auxiliary material solution does not interfere with the ribose determination of the test sample under 650 nm.
3. Accuracy investigation
Preparing relevant solution according to the method for accuracy evaluation, precisely measuring 2.0ml of sample solution, adding 2.0ml of 1% 3, 5-dihydroxytoluene solution, shaking uniformly, heating in boiling water bath for 30 min, rapidly cooling to room temperature, measuring absorbance at 650nm wavelength, and calculating RSD value, wherein the result is shown in Table 3.
TABLE 3 accuracy test results
The results showed that the average recovery of D-ribose in the sample recovery solution was 99.32% with rsd=6.17% (n=9) in the range of 80% -250% of the limit concentration.
4. Precision investigation
2.0Ml of the sample solution was precisely measured, 2.0ml of 1% 3, 5-dihydroxytoluene solution was added thereto, the mixture was shaken well, heated in a boiling water bath for 30 minutes, rapidly cooled to room temperature, absorbance was measured at a wavelength of 650nm, the measurement was repeated 6 times, and the RSD value was calculated, and the results were shown in Table 4.
Table 4 results of precision investigation
The result shows that the RSD value of the ribose content determination method in the transfer factor for injection is 0.13%, which indicates that the system precision of the method is good.
5. Repeatability investigation
6 Parts of transfer factor sterile powder for injection in the same batch are precisely weighed, the sample solutions are prepared according to the preparation method of the sample solutions in the example 1, 2.0ml of the sample solutions are precisely weighed, 2.0ml of 1% 3, 5-dihydroxytoluene solution is added, shaking is carried out, the mixture is heated in a boiling water bath for 30 minutes, the mixture is rapidly cooled to room temperature, absorbance is measured at a wavelength of 650nm, and RSD values are calculated, and the results are shown in Table 5.
Table 5 results of repeatability investigation
The results show that the RSD value of the ribose content determination method is 1.32%, which shows that the method has good repeatability.
6. Intermediate precision
The intermediate precision evaluation is carried out on the reference substance solution and the sample solution, and different experimenters carry out on the same batch of samples by different equipment. 6 test solutions were tested for D-ribose content result RSD=1.94%, and 12 test solutions were tested for D-ribose content result RSD=1.79%. As shown in tables 6-7.
TABLE 6 results of repeatability experiments
TABLE 7 results of intermediate precision experiments
7. Linear range investigation
The control solution (20. Mu.g/ml) was sampled sequentially by a sequential sampling method, the sampling concentration (mg/ml) was taken as the abscissa, the absorbance was taken as the ordinate, and a standard curve was drawn, and the results are shown in Table 8 and FIG. 1.
TABLE 8D results of ribose linearity experiments
The results show that the D-ribose has good linear relation in the range of 2.205-16.54 mug/ml, which is approximately equivalent to 80-600% of limit concentration, and the linear equation of the absorbance and concentration of the D-ribose at the wavelength of 650nm is y= 0.06883x-0.04019 (r=0.9993, n=5).
8. Durability inspection
And (5) examining whether the water bath time and the small change of the detection wavelength influence the measurement of the D-ribose content.
Precisely measuring 2.0ml of the sample solution, adding 2.0ml of 1% 3, 5-dihydroxytoluene solution, shaking, heating in boiling water bath for 25, 30 and 35 min, rapidly cooling to room temperature, measuring absorbance at 650nm, calculating ribose content and RSD value, precisely measuring 2.0ml of the sample solution, adding 2.0ml of 1% 3, 5-dihydroxytoluene solution, shaking, heating in boiling water bath for 30 min, rapidly cooling to room temperature, measuring absorbance at 645nm, 650nm and 655nm, and calculating ribose content and RSD value, and the results are shown in Table 9.
TABLE 9D results of ribose durability experiments
The results show that the small changes of the water bath time and the detection wavelength do not affect the measurement of the D-ribose content (the acceptable standard RSD is less than or equal to 6.0%).
9. Solution stability investigation
The control solution and the test solution were allowed to stand at room temperature for 1 hour, and the absorbance at 650nm of D-ribose in the control solution and the test solution was measured, and the results are shown in Table 10.
TABLE 10D results of ribose stability experiments
The results show that the RSD value of the D-ribose in the test sample solution measured at different times is 1.20%, and the D-ribose is stable under the room temperature condition for 1 hour (the acceptable standard RSD is less than or equal to 6.0%).
The foregoing detailed description is directed to one of the possible embodiments of the present invention, which is not intended to limit the scope of the invention, but is to be accorded the full scope of all such equivalents and modifications so as not to depart from the scope of the invention.
Claims (10)
1. The method for measuring the ribose content in the transfer factor for injection is characterized by comprising the following steps of:
(1) Preparing a test solution by weighing transfer factor sterile powder for injection, adding water for dissolving, then adding solution A for dilution, centrifuging, taking supernatant for volatilizing, and adding trichloroacetic acid solution for dissolving to obtain the test solution;
(2) Preparing reference substance solution by weighing D-ribose reference substance, dissolving with trichloroacetic acid solution to obtain reference substance solution;
(3) Measuring by taking sample solution, and measuring absorbance at 645-655nm by using a lichen phenol color development method;
(4) Drawing a standard curve, namely taking a reference substance solution, sequentially injecting samples by adopting a sequence injection method according to the method of the step (3), and drawing the standard curve by taking the injection concentration as an abscissa and the absorbance as an ordinate;
(5) Calculating the ribose content according to the standard curve obtained in the step (4), and calculating the ribose content of the sample in the step (3);
Wherein, the solution A in the step (1) is a mixed solution of methanol, ethanol and acetonitrile.
2. The method according to claim 1, wherein the volume ratio of methanol, ethanol and acetonitrile is 6-8:2-4:1.
3. The method according to claim 1, wherein the mass-to-volume ratio of the transfer factor sterile powder for injection to water in step (1) is 1g:30-40mL, and the volume ratio of the solution a to water is 3-5:1.
4. The method according to claim 1, wherein the rotational speed of the centrifugation in the step (1) is 9000-10000r/min and the centrifugation time is 8-12min.
5. The method according to claim 1, wherein the trichloroacetic acid solution in step (1) and step (2) has a mass concentration of 4% to 6%.
6. The method according to claim 1, wherein the concentration of the sample solution in the step (1) is 2 to 3. Mu.g/mL, and the concentration of the reference solution in the step (2) is 18 to 22. Mu.g/mL.
7. The method according to claim 1, wherein the method of coloration of lichen phenol in step (3) comprises adding 3, 5-dihydroxytoluene solution to the sample solution, shaking, heating, cooling, and precisely sucking the sample solution for measurement.
8. The method according to claim 7, wherein the mass concentration of the 3, 5-dihydroxytoluene solution is 0.8% -1.2%, and the volume ratio of the 3, 5-dihydroxytoluene solution to the sample solution is 1:0.8-1.2.
9. The method according to claim 7, wherein the heating condition is boiling water bath heating, and the heating time is 25 to 35 minutes.
10. Use of an assay according to any one of claims 1 to 9 in the quality assessment of transfer factors for injection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411720227.3A CN119198705B (en) | 2024-11-28 | 2024-11-28 | A method for determining ribose content in transfer factor for injection and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411720227.3A CN119198705B (en) | 2024-11-28 | 2024-11-28 | A method for determining ribose content in transfer factor for injection and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN119198705A true CN119198705A (en) | 2024-12-27 |
| CN119198705B CN119198705B (en) | 2025-04-22 |
Family
ID=94061638
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202411720227.3A Active CN119198705B (en) | 2024-11-28 | 2024-11-28 | A method for determining ribose content in transfer factor for injection and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN119198705B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008127291A2 (en) * | 2006-10-10 | 2008-10-23 | Los Alamos National Security, Llc | Advanced drug development and manufacturing |
| CN108931592A (en) * | 2018-05-25 | 2018-12-04 | 金花企业(集团)股份有限公司西安金花制药厂 | A kind of quantitative detecting method of oral liquid capable of transferring factors nucleotide substance |
| CN109239218A (en) * | 2018-09-26 | 2019-01-18 | 金花企业(集团)股份有限公司西安金花制药厂 | A kind of ribonucleic acid quantitative detecting method in transfer factor oral solution |
| CN110514606A (en) * | 2019-08-16 | 2019-11-29 | 大连百利天华制药有限公司 | Spleen aminopeptide solution ribose measuring method |
| CN114450576A (en) * | 2019-10-01 | 2022-05-06 | 生物E有限公司 | Methods of carbohydrate quantification |
| CN117562855A (en) * | 2023-09-16 | 2024-02-20 | 湖南一格制药有限公司 | Preparation process of transfer factor solution and freeze-dried powder injection |
-
2024
- 2024-11-28 CN CN202411720227.3A patent/CN119198705B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008127291A2 (en) * | 2006-10-10 | 2008-10-23 | Los Alamos National Security, Llc | Advanced drug development and manufacturing |
| CN108931592A (en) * | 2018-05-25 | 2018-12-04 | 金花企业(集团)股份有限公司西安金花制药厂 | A kind of quantitative detecting method of oral liquid capable of transferring factors nucleotide substance |
| CN109239218A (en) * | 2018-09-26 | 2019-01-18 | 金花企业(集团)股份有限公司西安金花制药厂 | A kind of ribonucleic acid quantitative detecting method in transfer factor oral solution |
| CN110514606A (en) * | 2019-08-16 | 2019-11-29 | 大连百利天华制药有限公司 | Spleen aminopeptide solution ribose measuring method |
| CN114450576A (en) * | 2019-10-01 | 2022-05-06 | 生物E有限公司 | Methods of carbohydrate quantification |
| CN117562855A (en) * | 2023-09-16 | 2024-02-20 | 湖南一格制药有限公司 | Preparation process of transfer factor solution and freeze-dried powder injection |
Non-Patent Citations (3)
| Title |
|---|
| 国家药典委员会: "《国家药品标准 化学药品地方标准上升国家标准 第十六册》", 31 December 2003, 国家药品监督管理局, pages: 103 - 104 * |
| 李皋: "冻干转移因子赋形剂的筛选研究", 西北国防医学杂志, no. 03, 25 September 1985 (1985-09-25) * |
| 黄青 等: "转移因子注射剂质量评价与研究", 《中国药学杂志》, vol. 50, no. 13, 8 July 2015 (2015-07-08), pages 1146 - 1152 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN119198705B (en) | 2025-04-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Neeld Jr et al. | Macro-and micromethods for the determination of serum vitamin A using trifluoroacetic acid | |
| CN112526134A (en) | Chitinase 3-like protein 1 determination kit | |
| CN103076455B (en) | Quantitative detection serum amyloid A protein kit and Synthesis and applications thereof | |
| CN113049699B (en) | Method for detecting biphenyl anhydride and related substances thereof and application | |
| CN113049727B (en) | Method for detecting content of polysorbate 80 | |
| CN111398490A (en) | Kit for detecting free triiodothyronine and free thyroxine by mass spectrometry | |
| CN104711279A (en) | Vascular endothelium growth factor detection kit and raw material preparation | |
| CN115839945B (en) | Photosensitive microsphere for photoexcitation chemiluminescence detection | |
| CN119198705B (en) | A method for determining ribose content in transfer factor for injection and its application | |
| CN106932347A (en) | A kind of mezlocillin and its quality index detection method | |
| CN114878825A (en) | C peptide determination kit and method for detecting content of C peptide in human serum | |
| WO2022099972A1 (en) | Relaxation nuclear magnetic resonance method for measuring glucose content in liquid biological sample | |
| US3897212A (en) | Test for systemic lupus erythematosus | |
| CN106501526A (en) | A kind of combination directing agent optimizes latex coupled antibody detection prealbumin（PA）Test kit | |
| CN111307990A (en) | Method for simultaneously determining content of cystine and methionine in artificial tiger bone powder | |
| CN114112957B (en) | Adiponectin determination kit and application thereof | |
| CN115236245A (en) | A kind of measuring method of serum homocysteine, candidate standard substance and preparation method | |
| CN113567607A (en) | Method for detecting content of penicillin G acylase in cefaclor | |
| CN108593696B (en) | Method for determining trifluoroacetic acid residue in polypeptide by using quantitative nuclear magnetism | |
| CN112684031A (en) | HPLC (high Performance liquid chromatography) determination method for content of povidone K30 | |
| CN112326967A (en) | A kit for simultaneous detection of alpha-fetoprotein and carcinoembryonic antigen | |
| Curtis-Prior et al. | A colorimetric method for the determination of deoxyribonucleic acid in adipose tissue | |
| CN115656390B (en) | Method for measuring content of paracetamol and oxycodone sustained release tablets | |
| CN108051594A (en) | A kind of latex enhancing immune turbidimetry quantitatively detects the reagent of chloramphenicol | |
| AF et al. | Simple methods for measuring serum levels of the glutamic-oxalacetic and glutamic-pyruvic transaminases in routine laboratories. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |