A method for detecting residual polydimethylsiloxane content in cytidine sodium from engineering bacteria by ultraviolet-visible spectrophotometry

Abstract

The application belongs to the technical field of analysis and detection, and provides a method for detecting residual polydimethylsiloxane content in engineering bacteria-derived cytidine 5'-diphosphocholine sodium by using ultraviolet-visible spectrophotometry. The detection method provided by the application is mainly based on the coordination reaction of polydimethylsiloxane and chromogenic solution, and uses ultraviolet-visible spectrophotometry to detect the absorbance value of the blue complex product at 621 nm. The detection method solves the problem of high cost of high-end instrument detection, and has the characteristics of high sensitivity, good repeatability and accuracy. Relevant data shows that the relative standard deviation is less than 2.0%, the recovery rate is in the range of 80% to 120%, and RSD is less than or equal to 2%.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, and specifically relates to a method for detecting the content of residual polydimethylsiloxane in sodium citicoline derived from engineered bacteria using ultraviolet-visible spectrophotometry. Background Technology

[0002] Currently, the synthesis of citicoline sodium mainly involves two methods: chemical synthesis and biosynthesis. In the biosynthesis of citicoline sodium, polydimethylsiloxane (PDMS) is typically added to the fermentation broth to inhibit or reduce foam formation, inevitably resulting in PMS residue in the citicoline sodium product. Citicoline sodium is currently available in injection, tablet, and capsule forms. Especially when used as an injection, residual PMS can decrease the physical stability of the product, such as leading to excessive levels of insoluble particles, thus posing significant safety risks. Furthermore, residual PMS can be recognized as a foreign substance by the human immune system, causing allergic reactions or inflammation. In addition, this substance can accelerate the degradation of citicoline sodium or its excipients. Therefore, research on PMS residue is of great significance for the safety of citicoline sodium derived from microorganisms.

[0003] Currently, the main component of polydimethylsiloxane used in the fermentation of sodium citicoline, a source of engineered bacteria, is (CH3)3Si[(CH3)2OSi). n SiO(CH3)3. This component has a large molecular weight and is a mixture, making it difficult to perform effective quantitative analysis using conventional laboratory chromatographic methods such as liquid chromatography and gas chromatography. It is typically analyzed using expensive equipment such as inductively coupled plasma atomic emission spectrometry (ICP-AES) or gas chromatography-mass spectrometry (GC-MS), resulting in high detection costs and complex procedures. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a method for detecting the residual polydimethylsiloxane content in sodium cytidine diphosphate choline derived from engineered bacteria using ultraviolet-visible spectrophotometry. This method is based on the coordination reaction of polydimethylsiloxane with a chromogenic solution. The absorbance value of the resulting colored complex is measured to reflect the residual polydimethylsiloxane content in the sodium cytidine diphosphate choline. The advantages of this method include high detection sensitivity, accurate and reliable results, and good repeatability.

[0005] The technical solution of this invention is:

[0006] A method for detecting residual polydimethylsiloxane content in sodium cytidine diphosphate choline derived from engineered bacteria using ultraviolet-visible spectrophotometry includes the following steps:

[0007] (1) Preparation of colorimetric solution: Accurately weigh cobalt nitrate hexahydrate, ammonium chloride, and potassium thiocyanate, dissolve them in ultrapure water to obtain the colorimetric solution, and store it away from light;

[0008] (2) Plotting the standard curve: Accurately weigh polydimethylsiloxane, dissolve it in dichloromethane to obtain standard solutions with different concentration gradients, and plot the standard curve.

[0009] (3) Preparation of test solution: Accurately weigh the sodium citrate produced by engineered bacteria, dissolve it in ultrapure water to obtain test solution;

[0010] (4) Preparation of reaction solution: Take dichloromethane, colorimetric solution and test solution, mix and shake well, separate the layers, take the lower layer solution, add isopropanol, shake well to obtain reaction solution;

[0011] (5) Detection: Take the reaction solution from (4) to prepare the test sample solution, measure the absorbance at a wavelength of 621 nm using a UV-Vis spectrophotometer, and then obtain the concentration of the test sample solution through a standard curve.

[0012] Preferably, in (1), the concentration of cobalt nitrate hexahydrate in the colorimetric solution is 30 g / L, the concentration of ammonium chloride is 143 g / L, and the concentration of potassium thiocyanate is 256 g / L.

[0013] Preferably, in (2), the concentration range of the standard solution is 2.0-15.0 μg / mL.

[0014] Preferably, in (3), the concentration of sodium citicoline in the test solution is not less than 100 mg / mL.

[0015] Preferably, in (4), the volume ratio of dichloromethane, colorimetric solution and test solution is 1:1:1.

[0016] Preferably, in (5), the preparation process of the test sample solution is as follows: take the reaction solution, dilute it with isopropanol, and shake it well; the concentration of sodium cytidine diphosphate choline in the test sample solution is 45-90 mg / mL.

[0017] In developing a method for determining polydimethylsiloxane residues, the inventors discovered that this substance has a large molecular weight, stable structure, and no ultraviolet absorption, and high-performance liquid chromatography (HPLC) did not achieve ideal results. However, through experimental exploration and research, it was found that polydimethylsiloxane can undergo a complexation reaction with cobalt ions and thiocyanate ions to generate a blue substance. Therefore, this invention considers establishing a linear regression relationship between the ultraviolet absorbance of this blue complex and the concentration of polydimethylsiloxane, and based on this, establishes a method for determining polydimethylsiloxane residues. Regarding solvents, polydimethylsiloxane has good lipid solubility; therefore, organic reagents such as dichloromethane, trichloromethane, and petroleum ether can be used as solvents in this method. Considering that sodium cytidine diphosphate choline is a quaternary ammonium salt compound, readily soluble in water but not readily soluble in lipid solvents such as petroleum ether, an extraction method is considered to enrich and separate polydimethylsiloxane for determining its residue, ensuring the sensitivity of the analytical method.

[0018] Experiments revealed that the blue complex formed by the coordination reaction between the colorimetric solution and the polydimethylsiloxane in the test sample solution is water-soluble and readily distributed at the interface between the aqueous and organic phases, making it difficult to dissolve. Therefore, isopropanol was used to dilute the reaction solution, which promoted the dissolution of the complex and also improved the color and clarity of the reaction solution. A full scan of the solution after the reaction of polydimethylsiloxane and the colorimetric solution was performed in the range of 190–800 nm. The maximum absorption was found at 621 nm; therefore, 621 nm was selected as the measurement wavelength for this invention.

[0019] The present invention has the following advantages and effects compared with the prior art:

[0020] (1) The present invention uses ultraviolet-visible spectrophotometry to determine the residual polydimethylsiloxane in sodium citicoline (polydimethylsiloxane added during the fermentation of engineered strains), which solves the problem of high detection costs caused by using high-end and expensive instruments such as inductively coupled plasma atomic emission spectrometry or gas chromatography-mass spectrometry to detect polydimethylsiloxane residues.

[0021] (2) The present invention has high sensitivity, good repeatability and good accuracy, ensuring the accuracy and stability of data results. Data in the embodiments show that the detection method provided by the present invention has a relative standard deviation of less than 2.0% and a recovery rate in the range of 80%~120%, with an RSD of recovery rate of ≤2%. Attached Figure Description

[0022] Figure 1 This is the standard curve in Embodiment 1 of the present invention;

[0023] Figure 2 This is the standard curve in Embodiment 2 of the present invention;

[0024] Figure 3 This is the standard curve in Embodiment 3 of the present invention. Detailed Implementation

[0025] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0026] 1.1 Instruments

[0027] Shimadzu UV1900i UV-Vis spectrophotometer, quartz cuvette (751, 10mm).

[0028] 1.2 Reagents and Test Chemicals

[0029] Cobalt nitrate hexahydrate (AR, Fuchen Tianjin Chemical Reagent Co., Ltd.), potassium thiocyanate (AR, Fuchen Tianjin Chemical Reagent Co., Ltd.), ammonium chloride (AR, Yantai Yuandong Fine Chemical Co., Ltd.), dichloromethane (AR, Tianjin Damao Chemical Reagent Factory), isopropanol (AR, Tianjin Fuyu Fine Chemical Co., Ltd.), chloroform (chromatographic grade, Fuchen Tianjin Chemical Reagent Co., Ltd.), polydimethylsiloxane (food grade), sodium citicoline (Suzhou Zhengji), sodium citicoline (self-made).

[0030] The sodium citicoline test sample involved in this invention is a self-made product, and the self-made process is as follows:

[0031] First, fermentation broth is obtained by fermentation with engineered strains of Bacillus subtilis. Then, the fermentation broth is separated and purified by methods such as cell wall disruption, nanofiltration, ultrafiltration, and resin column to obtain the finished product.

[0032] This invention uses an ultraviolet spectrophotometer to measure the absorbance of polydimethylsiloxane complexes for quantitative analysis. The operation is simple and economical, and it is suitable for the determination of polydimethylsiloxane residues in the industrial production of sodium citicoline.

[0033] Example 1

[0034] A method for detecting the residual amount of polydimethylsiloxane in sodium citicoline includes the following steps:

[0035] (1) Preparation of colorimetric solution: Accurately weigh 15g of cobalt nitrate, 71.5g of ammonium chloride and 128g of potassium thiocyanate, dissolve them completely in ultrapure water, transfer them to a 500mL volumetric flask, dilute to the mark with ultrapure water, shake well and store in the dark.

[0036] (2) Draw the standard curve:

[0037] Polydimethylsiloxane stock solution: Accurately weigh 105.2 mg of polydimethylsiloxane into a 100 mL volumetric flask, dissolve it in dichloromethane and dilute to the mark, shake well to obtain the polydimethylsiloxane stock solution with a concentration of 1.052 mg / mL.

[0038] Polydimethylsiloxane standard solution: Accurately transfer 10 mL of the stock solution into a 100 mL volumetric flask, dilute to the mark with dichloromethane, and shake well to obtain the polydimethylsiloxane standard solution with a concentration of 0.1052 mg / mL.

[0039] Take six 250mL separatory funnels and add 20, 19, 18, 17, 16, 15, and 14mL of dichloromethane, and 0, 1, 2, 3, 4, 5, and 6mL of polydimethylsiloxane standard solution, respectively. Add 20mL of colorimetric reagent to each funnel and 20mL of ultrapure water. Stopper the funnels and shake horizontally for 1 minute to release the gas. Discard 1mL of the lower layer solution and begin collecting the lower layer solution. Transfer 15mL of the collected lower layer solution to a centrifuge tube, add 5mL of isopropanol, and mix well to obtain the standard reaction solution.

[0040] Take six 25mL volumetric flasks, add 10mL of isopropanol to each, and then dilute to the mark with the above reaction solution. Shake well to obtain the standard solutions to be tested. Except for the blank test solution, the concentrations of the other standard solutions to be tested are 2.367μg / mL, 4.734μg / mL, 7.101μg / mL, 9.468μg / mL, 11.835μg / mL, and 14.202μg / mL, respectively.

[0041] The absorbance was measured at 621 nm using a UV-Vis spectrophotometer, and a standard curve (C-Abs) was plotted. The results are shown in Table 1. The standard curve is shown in... Figure 1 As shown.

[0042] Table 1. Linearity Results of Standard Curve

[0043]

[0044] (3) Preparation of test solution: Weigh 10g of sodium citicoline sample, dissolve it completely in ultrapure water, transfer it to a 50mL volumetric flask, dilute to the mark with ultrapure water, shake well, and the test solution with a concentration of 200mg / mL is obtained.

[0045] (4) Preparation of reaction solution: Take a 250mL separatory funnel, add 20mL of dichloromethane, 20mL of colorimetric solution, and 20mL of test solution. Stopper the funnel, shake horizontally for 1min, release the gas, discard 1mL of the lower layer solution, and then start collecting the lower layer solution. Take 15mL of the collected lower layer solution into a centrifuge tube, add 5mL of isopropanol, and shake well to obtain the reaction solution.

[0046] (5) Detection: Take one 25 mL volumetric flask, add 10 mL of isopropanol, dilute to the mark with the above reaction solution, shake well, and the test solution is obtained with a concentration of 90 mg / mL. Measure its absorbance at 621 nm using a UV-Vis spectrophotometer and calculate its concentration as 4.12 μg / mL by substituting it into the above standard curve.

[0047] Example 2

[0048] The detection method proposed in Example 1 was validated by performing five tests: specificity test, linearity and range test, limit of quantitation test, accuracy test, and repeatability test.

[0049] 2.1 Specificity test

[0050] Instruments and conditions: Same as in Example 1.

[0051] Test solution: Weigh 10g of sodium citicoline sample, dissolve it completely in ultrapure water, transfer it to a 50mL volumetric flask, dilute to the mark with ultrapure water, and shake well to obtain the test solution.

[0052] Blank solution: Take two 250mL separatory funnels, add 20mL of dichloromethane, 20mL of colorimetric solution, 20mL of test solution, and 20mL of blank solution to each funnel. Stopper the funnels and shake horizontally for 1 minute to release the gas. Discard 1mL of the lower layer solution and begin collecting the lower layer solution. Take 15mL of the collected lower layer solution into a centrifuge tube, add 5mL of isopropanol, and shake well to obtain the blank solution.

[0053] Take two 25 mL volumetric flasks, add 10 mL of isopropanol, dilute to the mark with the above reaction solution, and shake well to obtain the test solution with a concentration of 90 mg / mL. Measure the absorbance at 621 nm using a UV-Vis spectrophotometer. The concentration is calculated from the standard curve in the linearity test. Repeat the above experiment three times, and the specificity results are shown in Table 2.

[0054] Table 2 Specificity Test Results

[0055] name Absorbance value (Abs) Corresponding polydimethylsiloxane concentration (μg / mL) Blank solution ① 0.000 N. D. Blank solution② 0.000 N. D. Blank solution ③ 0.000 N. D. Test solution ① 0.002 4.00 Test solution ② 0.002 4.00 Test solution ③ 0.002 4.00

[0056] In our laboratory, polydimethylsiloxane was added to the self-made cytidine diphosphate choline sodium during fermentation. Therefore, as shown in Table 2, this method can determine the residual amount of polydimethylsiloxane in engineered bacterial cytidine diphosphate choline sodium, and cytidine diphosphate choline sodium does not affect the determination of polydimethylsiloxane.

[0057] 2.2 Linearity and Range Tests

[0058] Instruments and conditions: Same as in Example 1.

[0059] Experimental steps:

[0060] Polydimethylsiloxane standard solution: Accurately transfer 10 mL of the above polydimethylsiloxane stock solution into a 100 mL volumetric flask, dilute to the mark with dichloromethane, and shake well to obtain the polydimethylsiloxane standard solution with a concentration of 0.1052 mg / mL.

[0061] Solution preparation: Take eight 250mL separatory funnels and add 20, 19, 18.5, 18, 17, 16, 15, and 14mL of dichloromethane sequentially, followed by 0, 1, 1.5, 2, 3, 4, 5, and 6mL of polydimethylsiloxane standard solution sequentially; add 20mL of colorimetric reagent to each funnel; and add 20mL of ultrapure water. Stopper the funnels and shake horizontally for 1 minute to release the gas. Discard 1mL of the lower layer solution and begin collecting the lower layer solution. Transfer 15mL of the collected lower layer solution to a centrifuge tube, add 5mL of isopropanol, and mix well to obtain the standard reaction solution.

[0062] Take six 25mL volumetric flasks, add 10mL of isopropanol to each, and then dilute to the mark with the above reaction solution. Shake well to obtain the standard solutions to be tested. Except for the blank test solution, the concentrations of the other standard solutions to be tested are 2.367μg / mL, 3.5505μg / mL, 4.734μg / mL, 7.101μg / mL, 9.468μg / mL, 11.835μg / mL, and 14.202μg / mL, respectively.

[0063] Solution determination: The absorbance was measured at 621 nm using a UV-Vis spectrophotometer, and a standard curve (C-Abs) was plotted. The linearity results are shown in Table 3. Figure 2 As shown.

[0064] Table 3. Linearity test results of concentration C and absorbance Abs

[0065]

[0066] As shown in Table 3, the correlation coefficient of polydimethylsiloxane is greater than 0.99 in the range of 2.5~15.0 μg / mL, and the correlation coefficient is greater than 0.999 when the concentration is ≥10 μg / mL. The absorbance Abs shows a good linear relationship with the concentration C.

[0067] 2.3 Limit of Quantitation Test

[0068] Instruments and conditions: Same as in Example 1.

[0069] Polydimethylsiloxane standard solution: Take the polydimethylsiloxane standard solution from section 2.2.

[0070] Solution preparation: Take six 250mL separatory funnels, add 19mL of dichloromethane and 1mL of polydimethylsiloxane standard solution to each funnel; add 20mL of colorimetric reagent to each funnel; and add 20mL of ultrapure water. Stopper the funnels, shake horizontally for 1 minute to release gas, discard 1mL of the lower layer solution, and begin collecting the lower layer solution. Take 15mL of the collected lower layer solution into a centrifuge tube, add 5mL of isopropanol, and shake well to obtain the standard reaction solution.

[0071] Take six 25mL volumetric flasks, add 10mL of isopropanol to each, and then dilute each flask to the mark with the above reaction solution. Shake well to obtain the standard solution to be tested. The concentration of the standard solution to be tested is 2.367μg / mL.

[0072] Solution determination: The absorbance was measured at 621 nm using a UV-Vis spectrophotometer, and the results are shown in Table 4.

[0073] Table 4 Results of Limit of Quantitation Test

[0074] Concentration C (μg / mL) 2.367 2.367 2.367 2.367 2.367 2.367 RSD (%) Absorbance (Abs) 0.001 0.001 0.001 0.001 0.001 0.001 0.00

[0075] As shown in Table 4, when the absorbance value is 0.001, the detectable concentration is 2.367 μg / mL. Therefore, the limit of quantitation for this method of detecting polydimethylsiloxane content is 2.4 μg / mL.

[0076] 2.4 Accuracy Test

[0077] Instruments and conditions: Same as in Example 1.

[0078] Homemade solution of cytidine diphosphate choline sodium: Weigh approximately 20g of cytidine diphosphate choline sodium sample, dissolve it completely in ultrapure water, transfer it to a 100mL volumetric flask, dilute to the mark with ultrapure water, and shake well to obtain the test solution. Prepare two portions.

[0079] Polydimethylsiloxane standard solution: Take the polydimethylsiloxane standard solution from section 2.2.

[0080] Solution preparation: Take seven 250 mL separatory funnels and add 20, 18, 18, 18, 15, 15, and 15 mL of dichloromethane sequentially, followed by 0, 2, 2, 2, 5, 5, and 5 mL of polydimethylsiloxane standard solution sequentially; add 20 mL of colorimetric reagent to each funnel; and add 20 mL of the test solution. Stopper the separatory funnels, shake horizontally for 1 min, release the gas, discard 1 mL of the lower layer solution, and begin collecting the lower layer solution. Take 15 mL of the collected lower layer solution into a centrifuge tube, add 5 mL of isopropanol, and shake well to obtain the spiked reaction solution.

[0081] Take seven 25mL volumetric flasks, add 10mL of isopropanol to each, and then dilute to the mark with the above reaction solution. Shake well to obtain the spiking reaction solution to be tested. After conversion, the added concentrations are 7.101μg / mL and 11.835μg / mL, respectively.

[0082] The absorbance of the reaction solution was measured at 621 nm using a UV-Vis spectrophotometer. The concentration of polydimethylsiloxane was calculated using the standard curve described in section 2.2, and the results are shown in Table 5.

[0083] Table 5. Accuracy Test Results

[0084]

[0085] As shown in Table 5, the recovery rate of this method for detecting polydimethylsiloxane residue is in the range of 80% to 120%, and the RSD of the recovery rate is ≤2%, indicating that the accuracy of this detection method is good.

[0086] 2.5 Repeatability Test

[0087] Instruments and conditions: Same as in Example 1.

[0088] Homemade solution of sodium citicoline: Weigh about 20g of sodium citicoline sample, dissolve it completely in ultrapure water, transfer it to a 100mL volumetric flask, dilute to the mark with ultrapure water, shake well, and the test solution is obtained. Prepare two portions.

[0089] Solution preparation: Take six 250mL separatory funnels, add 20mL of dichloromethane to each, then add 20mL of colorimetric reagent to each, and finally add 20mL of the test solution. Stopper the separatory funnels, shake horizontally for 1 minute to release the gas, discard 1mL of the lower layer solution, and begin collecting the lower layer solution. Take 15mL of the collected lower layer solution into a centrifuge tube, add 5mL of isopropanol, and shake well to obtain the standard reaction solution.

[0090] Take six 25mL volumetric flasks, add 10mL of isopropanol to each, then dilute each flask to the mark with the above reaction solution, shake well, and the test solution with a concentration of 90 mg / mL is obtained.

[0091] Solution determination: The absorbance was measured at 621 nm using a UV-Vis spectrophotometer, and the concentration was calculated based on the standard curve in section 2.2. The results are shown in Table 6.

[0092] Table 6 Results of Repeatability Tests

[0093] Absorbance (Abs) 0.002 0.002 0.002 0.002 0.002 0.002 RSD (%) Concentration C (μg / mL) 4.00 4.00 4.00 4.00 4.00 4.00 0

[0094] As shown in Table 6, there was no significant difference in the concentration of polydimethylsiloxane measured in the six test solutions, and the relative standard deviation was less than 2.0%. Therefore, the repeatability of the above detection method is good.

[0095] Example 3

[0096] 1. Polydimethylsiloxane standard solution: Accurately transfer 10 mL of the above polydimethylsiloxane stock solution into a 100 mL volumetric flask, dilute to the mark with dichloromethane, and shake well to obtain the polydimethylsiloxane standard solution with a concentration of 0.1052 mg / mL.

[0097] 2. Standard Curve: Take six 250mL separatory funnels and add 20, 19, 18, 17, 16, 15, and 14mL of dichloromethane, and 0, 1, 2, 3, 4, 5, and 6mL of polydimethylsiloxane standard solution, respectively. Add 20mL of colorimetric reagent to each funnel and 20mL of ultrapure water. Stopper the funnels and shake horizontally for 1 minute to release the gas. Discard 1mL of the lower layer solution and begin collecting the lower layer solution. Transfer 15mL of the collected lower layer solution to a centrifuge tube, add 5mL of isopropanol, and mix well to obtain the standard reaction solution.

[0098] Take six 25mL volumetric flasks, add 10mL of isopropanol to each, and then dilute to the mark with the above reaction solution. Shake well to obtain the standard solutions to be tested. Except for the blank test solution, the concentrations of the other standard solutions to be tested are 2.367μg / mL, 4.734μg / mL, 7.101μg / mL, 9.468μg / mL, 11.835μg / mL, and 14.202μg / mL, respectively.

[0099] The absorbance was measured at 621 nm using a UV-Vis spectrophotometer, and a standard curve (C-Abs) was plotted. The obtained standard curve is shown in Table 7. Figure 3 As shown.

[0100] Table 7. Linearity Results of the Standard Curve

[0101]

[0102] 3. Test solution: Weigh about 10g of the self-made sodium citicoline, dissolve it completely in ultrapure water, transfer it to a 50mL volumetric flask, dilute to the mark with ultrapure water, and shake well to obtain the test solution.

[0103] Sodium citicoline (Suzhou Zhengji) solution: Weigh about 5g of sodium citicoline sample, dissolve it completely in ultrapure water, transfer it to a 50mL volumetric flask, dilute to the mark with ultrapure water, and shake well to obtain the solution.

[0104] Solution processing procedure: Take two 250mL separatory funnels, add 20mL of dichloromethane and 20mL of colorimetric solution, then add 20mL of the test solution and 20mL of the Suzhou Zhengji sample solution, respectively. Stopper the funnels, shake horizontally for 1 minute to release gas, discard 1mL of the lower layer solution, and begin collecting the lower layer solution. Take 15mL of the collected lower layer solution into a centrifuge tube, add 5mL of isopropanol, and shake well to obtain the reaction solution with a concentration of 90 mg / mL.

[0105] Solution determination: The absorbance was measured at 621 nm using a UV-Vis spectrophotometer, and the concentration was calculated based on the standard curve mentioned above. The results are shown in Table 8.

[0106] Table 8 Results of Sample Concentration Detection

[0107]

[0108] Suzhou Zhengji's sodium citicoline product is known to be produced via chemical synthesis, without the use of polydimethylsiloxane (PDMS). However, our company's sodium citicoline product incorporates PDMS during fermentation. As shown in Table 8, this PDMS detection method can distinguish between sodium citicoline from chemical synthesis and sodium citicoline from fermentation microorganisms.

[0109] In summary, the analytical method for determining residual polydimethylsiloxane in sodium cytidine diphosphate choline from engineered bacteria using a UV-Vis spectrophotometer provided by this invention solves the problems of difficult-to-obtain analytical equipment, complex operation, and high detection costs in the industrial production of sodium cytidine diphosphate choline. It achieves accurate determination of its residual amount, improves detection efficiency, and provides an efficient and reliable method for quality control of fermentation-derived sodium cytidine diphosphate choline products.

[0110] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A method for detecting the content of residual polydimethylsiloxane in sodium cytidine diphosphate choline derived from engineered bacteria using ultraviolet-visible spectrophotometry, characterized in that, The steps include the following: (1) Preparation of colorimetric solution: Accurately weigh cobalt nitrate hexahydrate, ammonium chloride and potassium thiocyanate, dissolve them in ultrapure water to obtain colorimetric solution, and store it in the dark. The mass ratio of cobalt nitrate hexahydrate: ammonium chloride: potassium thiocyanate is 30:143:

256. (2) Plotting the standard curve: Accurately weigh polydimethylsiloxane, dissolve it in dichloromethane, dilute it to obtain standard solutions with different concentration gradients, and plot the standard curve; (3) Preparation of test solution: Accurately weigh the sodium citicoline sample produced by engineered bacteria, dissolve it in ultrapure water to obtain the test solution; (4) Preparation of reaction solution: Take dichloromethane, colorimetric solution and test solution, mix and shake well, separate the layers, take the lower layer solution, add isopropanol, shake well, and obtain the reaction solution; (5) Detection: Take the reaction solution in (4) and dilute it with isopropanol, shake it well, prepare the test solution, measure the absorbance at a wavelength of 621 nm with a UV-Vis spectrophotometer, and then obtain the concentration of the test solution through the standard curve.

2. The method as described in claim 1, characterized in that, (2) The concentration range of the standard solution is 2.0-15.0 μg / mL.

3. The method as described in claim 1, characterized in that, (3) The concentration of sodium cytidine diphosphate choline in the test solution is not less than 100 mg / mL.

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