A method for determining ribose content in transfer factor for injection and its application

Abstract

The present invention belongs to the technical field of drug analysis, and specifically relates to a method and application for determining the ribose content in transfer factor for injection. The determination method comprises the following steps: (1) preparation of a test solution: weighing sterile powder of transfer factor for injection, dissolving it in water, then adding solution A for dilution, centrifuging, taking the supernatant to evaporate, adding trichloroacetic acid solution to dissolve, and obtaining a test solution; (2) preparation of a reference solution; (3) determination: taking the test solution, and determining the absorbance at 645-655nm using the orcinol colorimetric method; (4) drawing a standard curve; (5) calculation of the ribose content; wherein the solution A described in step (1) is a mixed solution of methanol, ethanol and acetonitrile. The present invention eliminates the interference problem of dextran 40 in the determination of ribose in transfer factor for injection by optimizing the sample pretreatment process, thereby improving the accuracy of the determination method.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and specifically relates to a method for determining the ribose content in transfer factor for injection and its application. Background Art

[0002] Transfer factor is a small molecule polypeptide and nucleotide complex made from pig or cattle spleen through homogenization and dialysis. Transfer factor for injection is a sterile lyophilized powder made by adding appropriate excipients to transfer factor solution. After entering the body, transfer factor causes the hematopoietic system to produce a large number of white blood cells in a short period of time, while activating lymphocytes, increasing the production of auxiliary cells and releasing more lymphocytes. This variety is widely used in clinical practice as a cellular immune enhancer and immunomodulator.

[0003] In the current standard ribose content determination, the orcinol colorimetric method is used to calculate the content using D-ribose. The principle of the method is to use orcinol reagent to react with ribose produced by nucleotide degradation to generate furfural for color development, and calculate the ribose content from the D-ribose regression curve. Dextran 40 in transfer factor for injection reacts with orcinol reagent in this colorimetric method to generate a precipitate, which interferes with the determination of ribose in the sample.

[0004] Therefore, further research is needed on the ribose content determination method to meet the quality control requirements of transfer factor for injection. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a method for determining the ribose content in transfer factor for injection and its application.

[0006] In order to achieve the above-mentioned purpose of the present invention, the specific technical solution adopted by the present invention is:

[0007] A method for determining the ribose content in transfer factor for injection comprises the following steps:

[0008] (1) Preparation of test solution: Weigh the sterile powder of transfer factor for injection, dissolve it in water, then add solution A to dilute it, centrifuge it, evaporate the supernatant, and dissolve it in trichloroacetic acid solution to obtain the test solution;

[0009] (2) Preparation of reference solution: Weigh D-ribose reference and dissolve it in trichloroacetic acid solution to obtain reference solution;

[0010] (3) Determination: Take the test sample solution and measure the absorbance at 645-655 nm using the orcinol colorimetric method;

[0011] (4) Drawing of standard curve: Take the reference solution and inject it sequentially according to the method in step (3) by using the sequential injection method, and draw a standard curve with the injection concentration as the horizontal axis and the absorbance as the vertical axis;

[0012] (5) Calculation of ribose content: Calculate the ribose content of the test sample in step (3) based on the standard curve obtained in step (4);

[0013] Wherein, the solution A in step (1) is a mixed solution of methanol, ethanol and acetonitrile.

[0014] Preferably, the volume ratio of ethanol, methanol and acetonitrile is 6-8:2-4:1.

[0015] Preferably, the mass volume ratio of the sterile transfer factor powder for injection to water in step (1) is 1 g:30-40 mL, and the volume ratio of solution A to water is 3-5:1.

[0016] Preferably, the centrifugal speed in step (1) is 9000-10000 r / min, and the centrifugal time is 8-12 min.

[0017] Preferably, the mass concentration of the trichloroacetic acid solution in step (1) and step (2) is 4%-6%.

[0018] Preferably, the concentration of the test solution in step (1) is 2-3 μg / mL; the concentration of the reference solution in step (2) is 18-22 μg / mL.

[0019] Preferably, the orcinol colorimetric method in step (3) is to add 3,5-dihydroxytoluene solution to the test solution, shake well, heat, cool, accurately pipette the test solution, and perform the determination.

[0020] 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 test solution is 1:0.8-1.2.

[0021] Further preferably, the heating condition is boiling water bath heating, and the heating time is 25-35 minutes.

[0022] The invention also relates to the application of the above determination method in the quality evaluation of transfer factor for injection.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention optimizes the sample pretreatment process and selects a mixed solution of ethanol, methanol and acetonitrile as a precipitant to eliminate the interference of dextran 40 on the determination of ribose in transfer factor for injection, thereby improving the accuracy of the determination method.

[0025] (2) The method for preparing the test solution of the present invention has simple steps and has good specificity as confirmed by experiments. It can preliminarily meet the detection requirements of ribose in transfer factor for injection and provide a reference for the quality control of transfer factor for injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the standard curve drawn in the linear range investigation of the present invention. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0028] The reagent purchase information of each embodiment and comparative example of the present invention is shown in Table 1.

[0029] Table 1 Reagent information

[0030]

[0031] Example 1

[0032] A method for determining the ribose content in transfer factor for injection, comprising the following steps:

[0033] (1) Preparation of test solution: Accurately weigh 28.8 mg of sterile powder of transfer factor for injection and place it in a 5 ml volumetric flask, add 1 ml of water and sonicate to completely dissolve it, dilute to scale with solution A (ethanol, methanol and acetonitrile in a volume ratio of 7:3:1) and shake well. Centrifuge the solution at 9500 r / min for 10 min, take 2.5 ml of the supernatant and place it in an evaporating dish to evaporate, add 5% trichloroacetic acid solution (mass concentration) and sonicate to dissolve it, wash it 3 times and transfer it to a 10 ml volumetric flask, dilute it with 5% trichloroacetic acid solution to make a solution containing 2.5 μg of ribose per 1 ml, shake well to obtain the test solution.

[0034] (2) Preparation of reference solution: Accurately weigh D-ribose reference substance, dissolve it in 5% trichloroacetic acid solution and make a solution containing 20 μg per 1 ml to obtain the reference solution.

[0035] (3) Determination: Take 2.0 ml of the test sample solution, add 2.0 ml of 1% 3,5-dihydroxytoluene solution, shake well, heat in a boiling water bath for 30 minutes, quickly cool to room temperature, and measure the absorbance at a wavelength of 650 nm.

[0036] (4) Drawing of standard curve: Take the reference solution and inject it sequentially according to the method in step (3) by using the sequential injection method, and draw a standard curve with the injection concentration as the horizontal axis and the absorbance as the vertical axis;

[0037] Accurately measure 0.0ml, 0.2ml, 0.6ml, 1.0ml, 1.5ml, and 2.0ml of the reference solution and place them in stoppered test tubes respectively. Add an appropriate amount of 5% trichloroacetic acid solution to 2.0ml, and then add 2.0ml of 1% 3,5-dihydroxytoluene solution to each. Heat in a boiling water bath for 30 minutes, quickly cool to room temperature, measure the absorbance at a wavelength of 650nm, and draw a standard curve with concentration as the horizontal axis and absorbance as the vertical axis.

[0038] (5) Calculation of ribose content: Substitute the absorbance of the test solution into the standard curve obtained in step (4) to obtain the concentration of ribose in the test solution. According to m=cv, calculate the mass of ribose (i.e., the measured amount), and then calculate the ribose content in transfer factor for injection according to the following formula:

[0039] The calculation formula is: Ribose (%) = ×100%.

[0040] Example 2

[0041] A method for determining the ribose content in transfer factor for injection, comprising the following steps:

[0042] (1) Preparation of test solution: Accurately weigh 30 mg of sterile powder of transfer factor for injection and place it in a 5 ml volumetric flask. Add 1 ml of water and sonicate to completely dissolve it. Dilute to scale with solution A (ethanol, methanol and acetonitrile in a volume ratio of 6:4:1) and shake well. Centrifuge the solution at a speed of 10000 r / min for 8 min, take 2.5 ml of the supernatant and place it in an evaporating dish to evaporate, add 5% trichloroacetic acid solution (mass concentration) and sonicate to dissolve it, wash it 3 times and transfer it to a 10 ml volumetric flask. Dilute it with 5% trichloroacetic acid solution to make a solution containing 2.5 μg of ribose per 1 ml, shake well, and obtain the test solution.

[0043] The remaining steps are consistent with Example 1.

[0044] Example 3

[0045] A method for determining the ribose content in transfer factor for injection, comprising the following steps:

[0046] (1) Preparation of test solution: Accurately weigh 30 mg of sterile powder of transfer factor for injection and place it in a 5 ml volumetric flask, add 1 ml of water and sonicate to completely dissolve it, dilute to scale with solution A (ethanol, methanol and acetonitrile in a volume ratio of 8:2:1) and shake well. Centrifuge the solution at 9000 r / min for 12 min, take 2.5 ml of the supernatant and place it in an evaporating dish to evaporate, add 5% trichloroacetic acid solution (mass concentration) and sonicate to dissolve it, wash it 3 times and transfer it to a 10 ml volumetric flask, dilute it with 5% trichloroacetic acid solution to make a solution containing 2.5 μg of ribose per 1 ml, shake well to obtain the test solution.

[0047] The remaining steps are consistent with Example 1.

[0048] Comparative Example 1

[0049] The difference between this comparative example and Example 1 is that in the preparation of the test solution in step (1), solution A is only ethanol.

[0050] Comparative Example 2

[0051] The difference between this comparative example and Example 1 is that in the preparation process of the test solution in step (1), solution A is a mixed solution of methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 3:1.

[0052] Comparative Example 3

[0053] The difference between this comparative example and Example 1 is that, in the preparation process of the test solution in step (1), solution A is a mixed solution of ethanol and methanol, and the volume ratio of ethanol to methanol is 7:4.

[0054] Comparative Example 4

[0055] The difference between this comparative example and Example 1 is that in the preparation process of the test solution in step (1), solution A is a mixed solution of ethanol and acetonitrile, and the volume ratio of ethanol to acetonitrile is 7:4.

[0056] The test methods of Examples 1-3 and Comparative Examples 1-4 were used to detect the ribose content in the sterile powder of transfer factor for injection from the same batch. The test results are shown in Table 2.

[0057] Table 2 Ribose content test results

[0058]

[0059] In Example 1-3, ethanol, methanol and acetonitrile are mixed in a certain proportion, and dextran 40 can be completely precipitated. Dextran 40 does not interfere with the determination of ribose in the test sample at 650nm, and the absorbance relative to the test sample is 0.0485%-0.0759%. However, in Comparative Examples 1-4, there is still interference from dextran 40, and the absorbance of dextran 40 relative to the test sample is relatively high.

[0060] Methodological investigation

[0061] 1. Reagent Preparation

[0062] Preparation of reference solution: Accurately weigh D-ribose reference, dissolve it in 5% trichloroacetic acid solution and make a solution containing 20 μg per 1 ml to obtain the reference solution.

[0063] Preparation of test solution: accurately weigh 28.8 mg of sterile powder of transfer factor for injection into a 5 ml volumetric flask, add 1 ml of water and ultrasonically dissolve it completely, dilute it to the mark with solution A (ethanol, methanol and acetonitrile in a volume ratio of 7:3:1) and shake well. Centrifuge the solution at a speed of 9500 r / min for 10 min, take 2.5 ml of the supernatant and evaporate it in an evaporating dish, add 5% trichloroacetic acid solution (mass concentration) and ultrasonically dissolve it, wash it 3 times and transfer it to a 10 ml volumetric flask, dilute it with 5% trichloroacetic acid solution to make a solution containing 2.5 μg of ribose per 1 ml, shake well, and obtain the test solution.

[0064] 2. Exclusive inspection

[0065] The absorbance of the excipient (dextran 40) solution was measured at a wavelength of 650 nm. The absorbance (Abs) of the excipient solution was -0.0002;

[0066] Accurately measure 2.0 ml of the test solution, add 2.0 ml of 1% 3,5-dihydroxytoluene solution, shake well, heat in a boiling water bath for 30 minutes, quickly cool to room temperature, and measure the absorbance at a wavelength of 650 nm. The absorbance (Abs) of the test solution is 0.4139;

[0067] The absorbance of the excipient relative to the test sample was <0.1%, indicating that the excipient solution did not interfere with the ribose determination of the test sample at 650nm.

[0068] 3. Accuracy test

[0069] Prepare relevant solutions for accuracy assessment according to the above method, accurately measure 2.0 ml of the test solution, add 2.0 ml of 1% 3,5-dihydroxytoluene solution, shake well, heat in a boiling water bath for 30 minutes, quickly cool to room temperature, measure the absorbance at a wavelength of 650 nm, calculate the RSD value, and the results are shown in Table 3.

[0070] Table 3 Accuracy experimental results

[0071]

[0072] The results showed that within the range of 80%-250% of the limit concentration, the average recovery rate of D-ribose in the sample recovery solution was 99.32%, with RSD=6.17% (n=9).

[0073] 4. Precision inspection

[0074] Accurately measure 2.0 ml of the test solution, add 2.0 ml of 1% 3,5-dihydroxytoluene solution, shake well, heat in a boiling water bath for 30 minutes, quickly cool to room temperature, measure the absorbance at a wavelength of 650 nm, repeat the measurement 6 times, calculate the RSD value, and the results are shown in Table 4.

[0075] Table 4 Precision investigation results

[0076]

[0077] The results showed that the RSD value of the method for determining the ribose content in the transfer factor for injection of the present invention was 0.13%, indicating that the system precision of the method of the present invention was good.

[0078] 5. Repeatability test

[0079] Accurately weigh 6 portions of sterile transfer factor powder for injection from the same batch, prepare test solutions according to the preparation method of the test solution in Example 1, accurately measure 2.0 ml of the test solution, add 2.0 ml of 1% 3,5-dihydroxytoluene solution, shake well, heat in a boiling water bath for 30 minutes, quickly cool to room temperature, measure the absorbance at a wavelength of 650 nm, calculate the RSD value, and the results are shown in Table 5.

[0080] Table 5 Repeatability test results

[0081]

[0082] The results showed that the RSD value of the ribose content determination method of the present invention was 1.32%, indicating that the method had good repeatability.

[0083] 6. Intermediate precision

[0084] The intermediate precision evaluation of the reference solution and the test solution was performed by different experimenters using different equipment on the same batch of samples. The RSD of D-ribose content in the 6 test solutions was 1.94%; the RSD of D-ribose content in the 12 test solutions was 1.79%. See Table 6-7.

[0085] Table 6 Repeatability test results

[0086]

[0087] Table 7 Intermediate precision test results

[0088]

[0089] 7. Linear range investigation

[0090] Take the reference solution (20 μg / ml) and inject it sequentially using the sequential injection method. Draw a standard curve with the injection concentration (mg / ml) as the horizontal axis and the absorbance as the vertical axis. The results are shown in Table 8 and Figure 1 shown.

[0091] Table 8 D-ribose linearity test results

[0092]

[0093] The results showed that the linear relationship was good within the range of 2.205-16.54 μg / ml, which was approximately equivalent to 80-600% limit concentration. The linear equation of absorbance and concentration of D-ribose at 650 nm was y=0.06883x-0.04019 (r=0.9993, n=5).

[0094] 8. Durability inspection

[0095] The aim of this study was to investigate whether slight changes in water bath time and detection wavelength would affect the determination of D-ribose content.

[0096] Accurately measure 2.0 ml of the test solution, add 2.0 ml of 1% 3,5-dihydroxytoluene solution, shake well, heat in a boiling water bath for 25, 30, and 35 minutes, respectively, cool rapidly to room temperature, measure the absorbance at a wavelength of 650 nm, and calculate the ribose content and RSD value; separately accurately measure 2.0 ml of the test solution, add 2.0 ml of 1% 3,5-dihydroxytoluene solution, shake well, heat in a boiling water bath for 30 minutes, cool rapidly to room temperature, measure the absorbance at wavelengths of 645 nm, 650 nm, and 655 nm, and calculate the ribose content and RSD value; the results are shown in Table 9.

[0097] Table 9 D-ribose durability test results

[0098]

[0099] The results showed that slight changes in water bath time and detection wavelength did not affect the determination of D-ribose content (acceptable standard RSD≤6.0%).

[0100] 9. Solution stability study

[0101] The reference solution and the test solution were placed at room temperature for 1 hour, and the absorbance of D-ribose in the reference solution and the test solution at 650 nm was detected. The results are shown in Table 10.

[0102] Table 10 D-ribose stability test results

[0103]

[0104] The results showed that the RSD value of D-ribose in the test solution measured at different times was 1.20%, and it was stable after being placed at room temperature for 1 hour (acceptable standard RSD≤6.0%).

[0105] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A method for determining the ribose content in transfer factor for injection, characterized in that: The following steps are involved: (1) Preparation of test solution: Weigh the sterile powder of transfer factor for injection, dissolve it in water, then add solution A to dilute it, centrifuge it, evaporate the supernatant, and dissolve it in trichloroacetic acid solution to obtain the test solution; (2) Preparation of reference solution: Weigh D-ribose reference and dissolve it in trichloroacetic acid solution to obtain reference solution; (3) Determination: Take the test sample solution and measure the absorbance at 645-655 nm using the orcinol colorimetric method; (4) Drawing of standard curve: Take the reference solution and inject it sequentially according to the method in step (3) by using the sequential injection method, and draw a standard curve with the injection concentration as the horizontal axis and the absorbance as the vertical axis; (5) Calculation of ribose content: Calculate the ribose content of the test sample in step (3) based on the standard curve obtained in step (4); Wherein, the solution A in step (1) is a mixed solution of methanol, ethanol and acetonitrile; the volume ratio of ethanol, methanol and acetonitrile is 7:3:1; The mass volume ratio of the sterile transfer factor powder for injection to water in step (1) is 1 g:30-40 mL, and the volume ratio of solution A to water is 3-5:

1.

2. The measuring method according to claim 1, characterized in that The centrifugal speed in step (1) is 9000-10000 r / min, and the centrifugal time is 8-12 min.

3. The measuring method according to claim 1, characterized in that The mass concentration of the trichloroacetic acid solution in step (1) and step (2) is 4%-6%.

4. The measuring method according to claim 1, characterized in that The concentration of the test solution in step (1) is 2-3 μg / mL; the concentration of the reference solution in step (2) is 18-22 μg / mL.

5. The measuring method according to claim 1, characterized in that The orcinol colorimetric method in step (3) is to add 3,5-dihydroxytoluene solution to the test solution, shake well, heat, cool, accurately pipette the test solution, and measure at a wavelength of 645-655 nm.

6. The measuring method according to claim 5, characterized in that 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 test solution is 1:0.8-1.

2.

7. The measuring method according to claim 5, characterized in that The heating condition is heating in a boiling water bath, and the heating time is 25-35 minutes.

8. Use of the determination method according to any one of claims 1 to 7 in the quality evaluation of transfer factor for injection.

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