Method for determining ethambutol hydrochloride content based on surface enhanced Raman spectroscopy

Abstract

The invention discloses a method for determining the content of ethambutol hydrochloride based on a surface enhanced Raman spectrum technology, through enhancement of nano-silver particles, the intensity of a characteristic peak in a Raman spectrum is remarkably increased, the detection sensitivity is greatly improved, a coagulant is added to enable the nano-silver particles to be in a certain aggregation state, and the detection sensitivity is greatly improved. The characteristic peak in the Raman spectrum is further enhanced; compared with traditional high performance liquid chromatography (HPLC) detection, the method has the advantages that the analysis and detection time is greatly shortened, the detection operation is simplified, the use of an organic solvent is reduced, and the economic benefit is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, specifically to a method for determining the content of ethambutol hydrochloride based on surface-enhanced Raman spectroscopy. Background Technology

[0002] Ethylaminobutanol hydrochloride (chemical formula C) 10 H 26 Cl2N2O2 is a synthetic antibacterial drug, belonging to the class of bacteriostatic agents used in the treatment of tuberculosis. It is often used in combination with rifampin and isoniazid for various types of active tuberculosis and atypical Mycobacterium tuberculosis infections. This drug exerts its antibacterial effect by inhibiting bacterial RNA synthesis and metabolism. Common dosage forms include tablets and capsules. Its structural formula is as follows:

[0003] Since ethambutol hydrochloride lacks UV-absorbing functional groups, it cannot be detected by conventional high-performance liquid chromatography-UV detection. The 2025 edition of the Chinese Pharmacopoeia proposes a method based on the strong UV absorption of the complex formed by ethambutol hydrochloride and copper ions. This method uses ammonium acetate / copper acetate solution (50g ammonium acetate and 0.2g copper acetate dissolved in water and diluted to 1000ml, pH adjusted to 5.0 with glacial acetic acid)-methanol (88:12) as the mobile phase for high-performance liquid chromatography-UV detection. However, this method has several drawbacks, such as: 1) the generation of bubbles that affect baseline stability: residual bubbles may remain during solution preparation or use, easily causing baseline fluctuations when passing through the chromatographic column; 2) solubility and compatibility issues: mixing with some organic solvents may lead to miscibility due to polarity differences, or separation may be affected by salting-out. 3) Limited buffering capacity and affected by the environment: The buffering range is fixed (approximately pH 4.6-5.6), and changes in temperature and ionic strength may affect the buffering effect; 4) Potential toxicity and corrosion risk: Copper acetate has a certain degree of toxicity, and high concentration solutions may corrode the chromatographic column material.

[0004] Therefore, it is necessary to provide a new determination method to overcome the shortcomings of the existing technical standards for content detection by high performance liquid chromatography. Summary of the Invention

[0005] This invention provides a method for determining the content of ethylamine hydrochloride based on surface-enhanced Raman spectroscopy, overcoming the shortcomings of the reliability of content detection in existing technologies.

[0006] In view of this, the solution of the present invention is as follows: A method for determining the content of ethylamine butanol hydrochloride based on surface-enhanced Raman spectroscopy, comprising the following steps: S1. Prepare an aqueous solution of the test sample using ethambutol hydrochloride; S2. Take the aqueous solution of the test sample, add silver colloid solution and sodium chloride solution in sequence, mix well, and scan with a Raman spectrometer to obtain surface-enhanced Raman spectra. Record the Raman characteristic peak of ethylamine butanol hydrochloride at 3311 cm⁻¹. -1 Strength at the location; S3. Calculate the content of ethambutol hydrochloride in the test sample solution based on the standard linear regression equation and the intensity of the characteristic peak of the test sample, and then calculate the content of ethambutol hydrochloride in the drug.

[0007] Further, in step S2, the silver colloid solution is prepared by dissolving 45 mg of silver nitrate in 250 mL of deionized water, heating to boiling, adding 5 mL of 1% sodium citrate solution dropwise while stirring, continuing to stir while boiling, cooling, and then centrifuging to concentrate to a concentration of 1 to 5 times.

[0008] Preferably, in step S2, the volume ratio of the test sample aqueous solution, silver paste solution, and sodium chloride solution is (3~8):(0.1~0.3):(0.03~0.08), the concentration of the test sample aqueous solution is 0.15~0.25 mg / ml, and the concentration of the sodium chloride solution is 0.05~0.2 mol / L.

[0009] Further, in step S2, the Raman spectrometer laser source is 1064 nm; and / or, the scanning process has an exposure time of 20 seconds, 5 exposures, and a displacement range of 3500–50 cm. -1 .

[0010] Further, in step S3, the method for obtaining the linear regression equation of the ethambutol hydrochloride standard solution is as follows: the sodium chloride solution, silver colloid solution, and ethambutol hydrochloride standard solution with increasing concentration gradient are vortexed and mixed, transferred to a cuvette, and measured using a Raman spectrometer to obtain surface-enhanced Raman spectra. The linear regression equation is obtained by fitting the relationship between the concentration of ethambutol hydrochloride and the intensity of the characteristic peaks in the obtained spectrum. To ensure detection accuracy, the amounts of sodium chloride and silver colloid added to the ethambutol hydrochloride standard solution are consistent with the amounts of sodium chloride and silver colloid added to the test sample aqueous solution in step S3, so that the content conditions of sodium chloride and silver colloid in the two solutions are parallel during comparison.

[0011] Preferably, the concentration gradient of the ethambutol hydrochloride standard solution with increasing concentration gradient is 0.05, 0.10, 0.20, 0.30, and 0.40 mg / mL.

[0012] Furthermore, the preparation process of the test sample aqueous solution is as follows: take ethambutol hydrochloride and grind it into a fine powder, mix it evenly, weigh an appropriate amount according to the theoretical content, add water and shake to dissolve the main drug, and filter to obtain the test sample solution.

[0013] Preferably, the ethambutol hydrochloride drug includes, but is not limited to, one of tablets or capsules; when the ethambutol hydrochloride drug is a capsule, the capsule shell is removed before grinding, and then the contents are collected.

[0014] Preferably, the shaking time during the preparation of the test sample aqueous solution is ≥10 min.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method that significantly increases the intensity of characteristic peaks in Raman spectra through the enhancement of silver nanoparticles, greatly improving the detection sensitivity. Furthermore, the addition of a coagulant in the present invention causes the silver nanoparticles to exhibit a certain aggregation state, further enhancing the characteristic peaks in the Raman spectrum. Compared with the traditional method of detecting content using high performance liquid chromatography (HPLC), the present invention greatly shortens the analysis and detection time, simplifies the detection operation, reduces the use of organic solvents, and significantly improves economic efficiency.

[0016] Meanwhile, since ethambutol hydrochloride tablets are in solid form, they contain various excipients that can interfere with the accuracy of detection. The pretreatment method provided by this invention can effectively remove some of the excipients. Raman spectroscopy was performed on the remaining excipients, and it was found that the Raman characteristic peaks of ethambutol hydrochloride and the Raman spectral peaks of the excipients did not overlap. By analyzing the Raman characteristic peaks of ethambutol hydrochloride, the specificity of detection was further improved. Attached Figure Description

[0017] Figure 1 This is the surface-enhanced Raman spectrum of ethylamine butanol hydrochloride described in Example 1 of the present invention.

[0018] Figure 2 This is a schematic diagram of linear regression fitting of the ethylamine butanol hydrochloride standard solution in Example 1 of the present invention. Detailed Implementation

[0019] The technical solution of the present invention will now be clearly and completely described in conjunction with preferred embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] In one embodiment, a method for determining the content of ethylamine butanol hydrochloride based on surface-enhanced Raman spectroscopy is provided, comprising the following steps: S1. Prepare an aqueous solution of the test sample using ethambutol hydrochloride; S2. Take the aqueous solution of the test sample, add silver colloid solution and sodium chloride solution in sequence, mix well, and scan with a Raman spectrometer to obtain surface-enhanced Raman spectra. Record the Raman characteristic peak of ethylamine butanol hydrochloride at 3311 cm⁻¹.-1 The strength at the point; the silver paste solution is prepared from silver nitrate and citrate as raw materials, and consists of nano-silver particles; S3. Calculate the content of ethambutol hydrochloride in the test sample solution based on the standard linear regression equation and the intensity of the characteristic peak of the test sample, and then calculate the content of ethambutol hydrochloride in the drug.

[0021] In the above embodiments, the intensity of characteristic peaks in the Raman spectrum is significantly increased by the enhancement of silver nanoparticles, which greatly improves the detection sensitivity. Furthermore, the addition of a coagulant in this invention causes the silver nanoparticles to exhibit a certain aggregation state, further enhancing the characteristic peaks in the Raman spectrum.

[0022] In a preferred embodiment, the preparation process of the test sample aqueous solution is as follows: ethambutol hydrochloride is ground into a fine powder, mixed evenly, and an appropriate amount is weighed according to the theoretical content. Water is added and the mixture is shaken for ≥10 minutes to dissolve the active ingredient. The solution is then filtered to obtain the test sample solution. The ethambutol hydrochloride can be in tablet or capsule form. When the ethambutol hydrochloride is in capsule form, the capsule shell is removed before grinding, and the contents are collected. The above methods can effectively detect the ethambutol hydrochloride content in tablets or capsules, and the detection results are consistent with the pharmacopoeia method. The treatment of the test sample aqueous solution can effectively remove some excipients. Raman spectroscopy of the remaining excipients reveals that the Raman characteristic peaks of ethambutol hydrochloride and the excipients do not overlap. Analysis of the Raman characteristic peaks of ethambutol hydrochloride further improves the specificity of the detection.

[0023] In a preferred embodiment, the Raman spectrometer laser source is 1064 nm; the scanning process has an exposure time of 20 seconds, 5 exposures, and a displacement range of 3500–50 cm. -1 .

[0024] In the above embodiments, experiments showed that the order in which the test sample solution, silver colloid, and sodium chloride solution were added significantly affected the intensity of the characteristic peak. The strongest characteristic peak of ethambutol hydrochloride was observed when the addition order was solution-silver colloid-sodium chloride solution, while the weakest was observed when the order was silver colloid-sodium chloride solution-solution solution. Both silver nanoparticles and ethambutol hydrochloride molecules carry charges, and these charges are opposite. When the silver colloid and solution were mixed first, the positively charged ethambutol hydrochloride molecules and the negatively charged silver nanoparticles exhibited adsorption. The subsequent addition of sodium chloride solution as a flocculant caused the silver nanoparticles adsorbed with ethambutol hydrochloride molecules to aggregate, generating more active sites. This increased the surface area of ​​ethambutol hydrochloride, making its characteristic peak more prominent.

[0025] In the following examples, the excipients of the ethambutol hydrochloride tablets used are starch, lactose, and magnesium stearate.

[0026] Example 1

[0027] A method for detecting the content of ethambutol hydrochloride tablets based on surface-enhanced Raman spectroscopy includes the following steps: 1) Preparation of silver colloid solution: Using a three-necked flask as the reaction vessel, prepare 10 -3 After adding 200 ml of silver nitrate solution M, the mixture was stirred continuously in an oil bath at 108°C until it boiled. Then, 5 mL of sodium citrate solution (1%) was added in four equal time intervals. After maintaining boiling and stirring for 1 hour, the power was turned off to stop the reaction. The prepared silver sol was then allowed to cool naturally to room temperature and placed in a 4°C constant temperature refrigerator for later use. Before use, an appropriate amount of the prepared silver sol solution was centrifuged and concentrated to a 5-fold concentration.

[0028] 2) Preparation of standard solutions of ethambutol hydrochloride at different concentrations: Weigh an appropriate amount of ethambutol hydrochloride, dissolve it in water, and then dilute it with water to prepare standard solutions of different concentrations. The concentration gradient of the standard solutions is 0.05, 0.10, 0.20, 0.30, and 0.40 mg / ml.

[0029] 3) Obtaining the linear regression equation for the ethambutol hydrochloride standard solution: 200 μL of silver colloid solution was added to 5 mL of ethambutol hydrochloride standard solution with an increasing concentration gradient and vortexed for 30 s. Then, 50 μL of sodium chloride solution was added and vortexed for 30 s. The solution was then transferred to a cuvette and measured with a Raman spectrometer to obtain the surface-enhanced Raman spectrum. The linear regression equation was obtained by fitting the relationship between the concentration of ethambutol hydrochloride and the intensity of the characteristic peaks in the obtained spectrum.

[0030] Raman spectroscopy tests for all samples were performed at room temperature using an RFS-100 Raman spectrometer with the following parameters: laser source 1064 nm, exposure time 20 seconds, 5 exposures, and displacement range 3500–50 cm. -1 .

[0031] The measured surface-enhanced Raman spectrum of ethylamine hydrochloride is as follows: Figure 1 As shown, 3311cm was selected. -1 Characteristic peaks are used for content calculation; the concentration, corresponding peak intensity, and the fitted straight line are shown below. Figure 2 As shown, the offline regression equation for concentration and peak intensity is obtained as follows: y = 0.7291x + 0.0033, R² = 0.9989.

[0032] 4) Preparation of test solution: Take 10 tablets of this product, grind them into fine powder, mix them evenly, accurately weigh an appropriate amount (approximately equivalent to 20 mg of ethambutol hydrochloride) and place it in a 100 ml volumetric flask, add an appropriate amount of water, shake for 20 min to fully dissolve the active ingredient, then dilute with water to the mark, shake well, filter, discard 5 ml, and take the filtrate to obtain the test solution.

[0033] 5) Surface-enhanced Raman spectroscopy determination of the test sample: Take 5 ml of the filtered test sample solution, add 200 μL of silver colloid solution and vortex mix, add 50 μL of sodium chloride solution (concentration 0.1 mol / L) and vortex mix, transfer to a cuvette, and scan with a Raman spectrometer to obtain the surface-enhanced Raman spectrum. Record the characteristic peak of ethambutol hydrochloride (3311 cm⁻¹). -1 The intensity of ).

[0034] Six samples were measured in parallel using both the content detection method listed in the Chinese Pharmacopoeia and the method provided in this embodiment. The results are as follows: , Example 2

[0035] The test solution, silver colloid, and sodium chloride of the same concentration were prepared according to the method in Example 1. Samples were prepared using three different addition sequences, with the same amounts as in Example 1, and the characteristic peak (3311 cm⁻¹) was compared. -1 The strength of ) is as follows:

[0036] The results show that the order in which the test solution, silver paste, and sodium chloride solution are added significantly affects the intensity of the characteristic peak in the Raman spectrum of ethylamine butanol hydrochloride. The addition method in sequence 1 (Example 1) shows a significantly stronger characteristic peak, which is more conducive to detection.

[0037] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for determining the content of ethylamine butanol hydrochloride based on surface-enhanced Raman spectroscopy, characterized in that the steps include... include: S1. Prepare an aqueous solution of the test sample using ethambutol hydrochloride; S2. Take the aqueous solution of the test sample, add silver colloid solution and sodium chloride solution in sequence, mix well, and scan with a Raman spectrometer to obtain surface-enhanced Raman spectra. Record the Raman characteristic peak of ethylamine butanol hydrochloride at 3311 cm⁻¹. -1 Strength at the location; S3. Calculate the content of ethambutol hydrochloride in the test sample solution based on the standard linear regression equation and the intensity of the characteristic peak of the test sample, and then calculate the content of ethambutol hydrochloride in the drug.

2. The determination method according to claim 1, characterized in that, In step S2, the silver colloid solution is prepared by dissolving 45 mg of silver nitrate in 250 mL of deionized water, heating to boiling, adding 5 mL of 1% sodium citrate solution dropwise while stirring, continuing to stir while boiling, cooling, and then centrifuging to concentrate to a concentration of 1 to 5 times.

3. The determination method according to claim 2, characterized in that, In step S2, the volume ratio of the test sample aqueous solution, silver paste solution, and sodium chloride solution is (3~8):(0.1~0.3):(0.03~0.08), the concentration of the test sample aqueous solution is 0.15~0.25 mg / ml, and the concentration of the sodium chloride solution is 0.05~0.2 mol / L.

4. The determination method according to claim 1, characterized in that, In step S2, the Raman spectrometer laser source is 1064 nm; and / or, the scanning process has an exposure time of 20 seconds, 5 exposures, and a displacement range of 3500–50 cm. -1 .

5. The determination method according to claim 1, characterized in that, In step S3, the method for obtaining the linear regression equation of the ethambutol hydrochloride standard solution is as follows: the sodium chloride solution, silver paste solution and the gradient-increased ethambutol hydrochloride standard solution are vortexed and mixed, transferred to a cuvette and measured with a Raman spectrometer to obtain a surface-enhanced Raman spectrum, and the linear regression equation is obtained by fitting the relationship between the concentration of ethambutol hydrochloride and the intensity of the characteristic peaks in the obtained spectrum.

6. The determination method according to claim 5, characterized in that, The concentration gradient of the ethambutol hydrochloride standard solution, which increases with concentration, is 0.05, 0.10, 0.20, 0.30, and 0.40 mg / mL.

7. The determination method according to claim 1, characterized in that, The preparation process of the test sample aqueous solution is as follows: take ethambutol hydrochloride, grind it into fine powder, mix it evenly, weigh an appropriate amount according to the theoretical content, add water and shake to dissolve the main drug, and filter to obtain the test sample solution.

8. The determination method according to claim 7, characterized in that, The ethambutol hydrochloride drug is in the form of tablets or capsules. When the ethambutol hydrochloride drug is in the form of capsules, the capsule shell is removed before grinding, and the contents are collected.

9. The determination method according to claim 7, characterized in that, During the preparation of the aqueous solution of the test sample, the shaking time is ≥10 min.

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