Raman determination of boric acid and berberine hydrochloride in compound zinc sulfate eye drops

Abstract

The application discloses a kind of Raman determination methods of boric acid and berberine hydrochloride in compound zinc sulfate eye drops, belong to the technical field of drug quality detection, the method determines the total boron content of sample using ion chromatography, determines the borax content of sample using potentiometric titration, boracic acid chemical reference value is obtained according to formula, and the chemical reference value of berberine hydrochloride is determined using high performance liquid chromatography method;Collect series sample Raman spectrum and carry out pretreatment;Using partial least square method, the quantitative model of boric acid and berberine hydrochloride is established respectively;The Raman spectrum of sample to be measured is substituted into the model, and the content of boric acid and berberine hydrochloride is output simultaneously.Compared with the current legal standard determination method, the application overcomes the problem of component interference determination in compound system, has higher specificity and accuracy, realizes the rapid, non-destructive, synchronous detection of two main components, and can be used for online monitoring in production process and daily supervision of drug supervision department.

Description

Technical Field

[0001] The present invention relates to the technical field of drug quality inspection, and particularly relates to a method for simultaneously determining the contents of boric acid and berberine hydrochloride in compound zinc sulfate eye drops based on Raman spectroscopy combined with a chemometric model. Background Art

[0002] Compound zinc sulfate eye drops are a compound preparation, and its main components are berberine hydrochloride, zinc sulfate and boric acid, with a specification of containing 1 mg of berberine hydrochloride, 3 mg of zinc sulfate and 20 mg of boric acid per milliliter. This variety has certain application value in ophthalmic preparations.

[0003] In the current quality standard, the content of boric acid is determined by the volumetric method. That is, after adding water and glycerol to dilute the sample, phenolphthalein is used as an indicator solution, and the sodium hydroxide titrant is titrated to the end point with an orange-red color. However, when this method is used for compound zinc sulfate eye drops, the following problems exist: First, the specificity is insufficient. Components such as berberine hydrochloride and zinc sulfate in the prescription can react with sodium hydroxide, resulting in systematic deviation in the determination result of the boric acid content, and the method specificity is not strong. After verification, when other components are mixed according to the prescription ratio and determined by the current volumetric method, the result calculated as boric acid is about 8.4% of the labeled amount, and the interference of zinc sulfate is the most significant; there is a statistically significant difference between the determination results of the volumetric method and the ion chromatography method, which may lead to misjudgment of unqualified products as qualified, posing a quality safety hazard.

[0004] Second, it is difficult to judge the end point. Berberine hydrochloride itself is yellow, making the background color of compound zinc sulfate eye drops yellow. In the volumetric method, the color change at the end point is not easily accurately judged under the interference of the yellow background, resulting in poor repeatability and accuracy of the determination result.

[0005] Third, the detection efficiency is low. The volumetric method requires adding chemical reagents and manual operation, and it is difficult to achieve rapid, accurate and simultaneous determination of multiple components in a compound system, and it is difficult to meet the requirements of rapid drug inspection, regulatory screening and on-line monitoring during the production process.

[0006] In addition, in the current quality standard, the content of berberine hydrochloride is determined by the ultraviolet reference substance method. This method can achieve the conventional content control of berberine hydrochloride, but it needs to be detected separately from boric acid, and it is difficult to meet the requirement of synchronous rapid analysis of multiple components in a compound preparation; at the same time, the resolution ability of ultraviolet detection for coexisting components and related substances in a complex system is relatively limited, which is not conducive to the evaluation of specificity.

[0007] Raman spectroscopy is an analytical technique based on the inelastic scattering of molecular vibrations. When a laser beam illuminates a sample, the scattered light contains not only an elastic component with the same frequency as the excitation light, but also an inelastic scattering component with a changed frequency; this latter component is Raman scattering. Different molecules exhibit different vibrational modes, therefore Raman spectroscopy can serve as a molecular fingerprint for substance identification and quantitative analysis. Because water exhibits extremely weak Raman scattering, Raman spectroscopy is particularly suitable for the detection of aqueous solutions and has significant application value in the quality analysis of liquid preparations such as eye drops.

[0008] However, when using Raman spectroscopy for quantitative analysis of compound preparations, the establishment of the model depends on accurate chemical reference values. Since boric acid has no significant ultraviolet absorption and no fluorescence properties, it is difficult to directly and accurately determine it using conventional high-performance liquid chromatography (HPLC) with ultraviolet detection. Therefore, the accurate acquisition of its chemical reference values ​​is a key issue restricting the establishment of Raman quantitative models.

[0009] Therefore, it is necessary to establish a specific, rapid, accurate analytical method suitable for aqueous solutions to simultaneously determine the contents of boric acid and berberine hydrochloride in compound zinc sulfate eye drops. Summary of the Invention

[0010] The purpose of this invention is to overcome the problems of poor specificity, inaccurate endpoint judgment, and low detection efficiency in the existing methods for determining the content of compound zinc sulfate eye drops, and to provide a rapid analytical method based on Raman spectroscopy combined with partial least squares method to simultaneously determine the content of boric acid and berberine hydrochloride in compound zinc sulfate eye drops.

[0011] To achieve the above objectives, the present invention adopts the following technical solution.

[0012] A Raman spectroscopy method for determining boric acid and berberine hydrochloride in compound zinc sulfate eye drops, comprising the following steps: Step (1), obtain chemical reference values: Obtaining the chemical reference value of boric acid: The total boron content (Ct) in the sample was determined by ion chromatography, and the borax content (Cs) was determined by potentiometric titration. The chemical reference value of boric acid (Ca) was calculated according to the following formula: Ca = Ct - K × Cs In the formula, Ca is the boric acid content in the sample, in mg / mL; Ct is the total boron content in the sample determined by ion chromatography, in mg / mL; Cs is the borax content in the sample determined by potentiometric titration, in mg / mL; K is the conversion factor for borax to boric acid, K=4×M(H3BO3)÷M(Na2B4O7·10H2O)≈0.6488, where M(H3BO3) is the molar mass of boric acid and M(Na2B4O7·10H2O) is the molar mass of borax.

[0013] Obtaining the chemical reference value for berberine hydrochloride: The content of berberine hydrochloride in the sample was determined by high performance liquid chromatography and used as the chemical reference value for berberine hydrochloride.

[0014] Step (2): Collect Raman spectra of a series of samples: A series of concentrations of compound zinc sulfate eye drops were sampled using a Raman spectrometer, and the obtained Raman spectra were used for subsequent model building.

[0015] Furthermore, a quartz cuvette was used to load the sample for collection in order to obtain a higher intensity of scattered light.

[0016] Furthermore, the acquisition conditions were: 785nm laser, -50℃ DDC detector, fiber optic probe, 5mm focal length, and 4cm resolution. -1 Scanning time: 100 seconds.

[0017] Furthermore, three bottles were selected from each batch of samples, and each bottle was measured once. The average spectrum of the three original spectra was used for modeling or prediction.

[0018] Step (3), establish a quantitative correction model: The Raman spectra obtained in step (2) were preprocessed, and the partial least squares method was used to establish quantitative correction models for boric acid and berberine hydrochloride, respectively, based on the chemical reference values ​​obtained in step (1).

[0019] Furthermore, the preprocessing method for the boric acid quantitative correction model was maximum-minimum normalization, and the modeling spectral range was 1667.7–1498.7 cm⁻¹. -1 1416.8~1247.8cm -1 1165.9~1080.1cm -1 and 998.2~829.2cm -1 The number of smoothed points is 17, and the number of principal components is 7.

[0020] Furthermore, the preprocessing method for the berberine hydrochloride quantitative correction model was to eliminate constant offset, and the modeling spectral range was 2901.4–2299.5 cm⁻¹. -1 2000.5~799.3cm -1 and 500.3~200cm -1 The number of smoothed points is 17, and the number of principal components is 8.

[0021] Step (4), Sample detection: Raman spectra of the sample to be tested are collected and substituted into the boric acid quantitative correction model and berberine hydrochloride quantitative correction model established in step (3) respectively, so as to obtain the contents of boric acid and berberine hydrochloride in the sample to be tested.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention solves the problem of accurately obtaining the chemical reference value of boric acid in compound systems. It determines the total boron content using ion chromatography, combines this with potentiometric titration to determine the borax content, and indirectly calculates the chemical reference value of boric acid using the formula Ca=Ct-K×Cs. This effectively solves the problem that boric acid, due to its lack of significant ultraviolet absorption, is difficult to determine directly using conventional chromatographic methods, and overcomes the issue of poor specificity of volumetric methods in compound systems.

[0023] 2. This invention overcomes the difficulty in determining the endpoint color using volumetric methods. Existing volumetric methods are affected by the yellow background of compound zinc sulfate eye drops, leading to large errors in endpoint determination. This invention uses Raman spectroscopy for detection, which is independent of color changes and fundamentally avoids the problem of color interference.

[0024] 3. This invention enables rapid and simultaneous determination of two components in a compound system. Through a single Raman spectroscopy acquisition combined with two independent quantitative calibration models, it simultaneously outputs the contents of boric acid and berberine hydrochloride, significantly improving detection efficiency.

[0025] 4. Rapid and non-destructive testing, suitable for aqueous solutions. This invention requires no complex sample pretreatment, does not use chemical colorimetric reagents, and does not damage the sample. It can be used for routine supervision by drug regulatory authorities and online monitoring of the production process.

[0026] 5. The models exhibit high accuracy and robustness. The internal cross-validation R² of the boric acid quantitative calibration model is 97.90, and the test set validation R² is 98.25, with relative deviations of less than 3% for the vast majority of samples. The internal cross-validation R² of the berberine hydrochloride quantitative calibration model is 97.63, demonstrating high predictive accuracy within the actual sample concentration range. Both models have been validated with samples at different temperatures and time points, exhibiting good robustness. Attached Figure Description

[0027] Figure 1 A comparison of Raman spectra for the three sampling methods; Figure 2 Raman spectra of the raw materials and excipients for compound zinc sulfate eye drops; Figure 3 This is a comparative spectral diagram of the characteristic segments of the three main components of compound zinc sulfate eye drops; Figure 4 A graph showing the fitting of predicted values ​​to chemical reference values ​​for the boric acid model test set; Figure 5 A comparison chart of the chemical reference values ​​and predicted values ​​for boric acid content model; Figure 6 A comparison chart of the chemical reference values ​​and predicted values ​​for berberine hydrochloride content in the model; Figure 7This is a comparison chart of the chemical reference values ​​and predicted values ​​for zinc sulfate content in the model. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Example

[0029] In this embodiment, a total of 31 batches of compound zinc sulfate eye drops samples were collected from Heilongjiang Tianlong Pharmaceutical Co., Ltd., with a specification of 1 mg berberine hydrochloride, 3 mg zinc sulfate, and 20 mg boric acid per milliliter. In addition to the above 31 batches, the modeling samples also included a series of concentration samples of each component prepared according to the prescription and stability test samples, totaling 64 batches.

[0030] Raman spectroscopy acquisition conditions Spectroscopic data were acquired using a Metageopal 3000 Raman spectrometer, with the following parameters: Detector: -50℃ DDC detector; Laser: 785nm; Probe: Fiber optic probe; Focal length: 5mm; Resolution: 4cm -1 ; Scan time: 100s.

[0031] Three bottles were selected from each batch of samples, and each bottle was measured once to obtain three raw spectra. The average spectrum of the three spectra was used for modeling or prediction.

[0032] Sampling method filtering Samples were loaded into quartz cuvettes, glass vials, and original eye drop bottles (made of low-density polyethylene), respectively, and Raman spectra were collected under the same conditions. The results are as follows: Figure 1 As shown.

[0033] The comparison results show that the Raman scattering intensity of all three principal components is highest when the sample is loaded in a quartz cuvette. Therefore, a quartz cuvette was selected as the final sampling carrier in this invention.

[0034] Spectral analysis of raw and auxiliary materials Using berberine hydrochloride, zinc sulfate, and boric acid as raw materials and excipients provided by the manufacturer, aqueous solutions were prepared according to the prescribed amounts. Raman spectra of each component were then collected. Figure 2 As shown.

[0035] After comparison with the sample spectrum ( Figure 3 It can be concluded that: Approximately 880cm -1 The region represents the characteristic spectral band of boric acid. Approximately 980cm -1 This is the characteristic spectral band of zinc sulfate; 1700~1100cm -1 The segment represents the characteristic spectral band of berberine hydrochloride.

[0036] The characteristic spectral bands of the three principal components have a certain degree of distinguishability, which provides a basis for establishing quantitative correction models for each component.

[0037] Establishment and validation of a quantitative correction model for boric acid Obtaining Chemical Reference Values: Since boric acid has no significant ultraviolet absorption and no fluorescence, it is difficult to directly and accurately determine its chemical reference values ​​using conventional high-performance liquid chromatography (HPLC) with ultraviolet detection. This invention employs the following method to indirectly obtain the chemical reference values ​​for boric acid.

[0038] (1) Determination of total boron content by ion chromatography The total boron content (Ct) in compound zinc sulfate eye drops was determined by ion chromatography. The total boron content includes all boron elements contained in boric acid and borax in the sample.

[0039] The specific measurement conditions are as follows: The instrument used was a Dionex ICS-5000 ion chromatograph; the column was an IonPac ICE Borate ion exclusion analysis column (9 mm × 250 mm); the eluent was a mixture of 2.5 mmol / L methanesulfonic acid and 60 mmol / L mannitol; the chemical inhibition regeneration buffer was a mixture of 25 mmol / L tetramethylammonium hydroxide and 15 mmol / L mannitol; the flow rate was 1.0 mL / min; the column temperature was 30 °C; the injection volume was 25 μL; the detection method was a suppression conductivity detector using an exclusion anion micromembrane suppressor (ACRS-ICE500 9 mm).

[0040] Preparation of test solution: Accurately measure 1 mL of this product and place it in a 50 mL volumetric flask. Dilute with water to the mark and shake well. Accurately measure 1 mL of this product and place it in a 25 mL volumetric flask. Dilute with water to the mark and shake well.

[0041] Preparation of reference solution: Weigh approximately 20 mg of boric acid reference standard accurately, place it in a 50 mL volumetric flask, add an appropriate amount of water, shake to dissolve, dilute with water to the mark, shake well, accurately measure 1 mL, place it in a 25 mL volumetric flask, dilute with water to the mark, shake well, and the solution is ready.

[0042] Method validation showed that water and excipients did not interfere with the determination of boric acid, and the theoretical plate number of boric acid was not less than 8000. The linear relationship of boric acid concentration was good in the range of 3.41 μg / mL to 34.10 μg / mL, with r = 0.9997. The repeatability RSD was 0.6%. The average recovery rate was 100.8%. Both the reference solution and the test solution were stable within 22 hours.

[0043] (2) Determination of borax content by potentiometric titration The borax content (Cs) in compound zinc sulfate eye drops was determined by potentiometric titration.

[0044] The specific measurement conditions are as follows: The instrument used was a Wantong fully automatic potentiometric titrator 809; the determination was performed according to the potentiometric titration method (Chinese Pharmacopoeia 2020 edition, Part IV, General Chapter 0701); 50 mL of this product was accurately measured and titrated with hydrochloric acid titrant (0.01 mol / L); each 1 mL of hydrochloric acid titrant (0.01 mol / L) is equivalent to 1.907 mg of Na2B4O7·10H2O.

[0045] Methodological validation showed that neither water nor excipients interfered with the determination of borax; the average recovery rate was 100.4%, and the RSD was 0.7%.

[0046] (3) Calculation of the chemical reference value of boric acid Borax reacts with boric acid as follows: Na₂B₄O₇ + 2H⁺ + 5H₂O → 4H₃BO₃ The chemical reference value of boric acid in the sample is calculated by subtracting the boric acid content (converted from borax) from the total boron content, using the following formula: Ca = Ct - K × Cs In the formula: Ca represents the boric acid content in the sample, expressed in mg / mL. Ct represents the total boron content of the sample determined by ion chromatography, in mg / mL. Cs represents the borax content in the sample determined by potentiometric titration, in mg / mL. K is the conversion factor for borax to boric acid, K=4×M(H3BO3)÷M(Na2B4O7·10H2O)=4×61.83÷381.37≈0.6488, where M(H3BO3) is the molar mass of boric acid (61.83g / mol) and M(Na2B4O7·10H2O) is the molar mass of borax (381.37g / mol).

[0047] The above-mentioned indirect quantitative method effectively solves the problem of the difficulty in accurately obtaining the chemical reference value of boric acid in compound systems.

[0048] Modeling parameters: A quantitative calibration model for boric acid was established using 63 batches of samples, including 54 batches for calibration and 9 batches for validation. OPUS software was used to process the spectra, and partial least squares modeling was employed. The coefficient of determination (R²) and root mean square error of prediction (RMSEV) were used as indicators to optimize the spectral segments, preprocessing methods, and modeling batches. The final model parameters are shown in Table 1.

[0049] Table 1 Parameters of the boric acid quantitative correction model

[0050] Model validation results: The model quality was evaluated using internal cross-validation and test set validation. The degree of agreement between the model's predicted values ​​and the chemical reference values ​​is represented by the relative deviation Rp, calculated as follows: Rp = |Cp - Cr| / Cr × 100% In the formula: Rp represents the relative deviation; Cp is the model's predicted value; Cr is a chemical reference value.

[0051] During external validation, an anomaly was found in the boric acid linear 2 solution. After checking the spectral and chemical reference values ​​of the sample, no abnormalities were found. It was analyzed that the anomaly was likely caused by accidental factors. Therefore, the sample was removed from the modeling process.

[0052] The results of internal cross-validation and test set validation are shown in Table 2. Figure 4 , Figure 5 As can be seen, among the 54 internal cross-validation samples, except for YP2023CJ1561 (Rp=3.8%) and sample 5 (Rp=3.4%), the relative deviations of the remaining samples are all less than 3%; among the 9 test set validation samples, except for sample 1 (concentration 5.221mg / mL, close to the lower end of the linear range, Rp=6.4%), the relative deviations of the remaining 8 samples are all less than 3%.

[0053] The prediction deviations of the stability test samples (at different temperatures of 20℃, 30℃, 40℃, and 60℃, and at different time points of 5 days, 10 days, 20 days, and 30 days) were all within 3%, indicating that the model has good robustness.

[0054] Table 2. Model Predicted Values ​​and Chemical Reference Values ​​for Boric Acid Content

[0055] Establishment and validation of a quantitative correction model for berberine hydrochloride Obtaining chemical reference values: The content of berberine hydrochloride in compound zinc sulfate eye drops samples was determined by high performance liquid chromatography and used as the chemical reference value for berberine hydrochloride.

[0056] The specific measurement conditions are as follows: The instrument was an Agilent 1260 high-performance liquid chromatograph equipped with a diode array detector; the chromatographic column was a Kromasil C18 (5 μm, 4.6 × 250 mm); the column temperature was 35 ℃; the detection wavelength was 347 nm; the injection volume was 20 μL; the flow rate was 1 mL / min; 0.02 mol / L ammonium acetate solution (adjusted to pH 3.5 with glacial acetic acid) was used as mobile phase A, and acetonitrile was used as mobile phase B, with gradient elution performed according to Table 3.

[0057] Table 3 Gradient elution program

[0058] Preparation of test solution: Take 5 bottles of this product, mix the contents, accurately measure 1 mL, place it in a 50 mL volumetric flask, dilute with water to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0059] Preparation of reference solution: Accurately weigh 32 mg of berberine hydrochloride reference standard, place it in a 25 mL volumetric flask, add an appropriate amount of water, heat in an 80 °C water bath to dissolve, cool to room temperature, dilute to the mark with water, and shake well to obtain the solution.

[0060] Methodological validation showed that zinc sulfate, boric acid, and borax in the formulation had no UV absorption, the solvent did not interfere, and berberine hydrochloride could be baseline separated from specific impurities (berberine hydrochloride and palmatine hydrochloride). The linear relationship of berberine hydrochloride concentration was good in the range of 9.707 μg / mL to 29.12 μg / mL, with r = 0.9991; the repeatability RSD was 0.9%; the average recovery rate was 98.6%; the solution was stable within 24 hours, with an RSD of 0.2%.

[0061] Modeling parameters: A quantitative calibration model for berberine hydrochloride was established using 64 batches of samples, all of which were used for internal cross-validation. The spectra were processed using OPUS software, and the model was built using partial least squares method. The final model parameters are shown in Table 4.

[0062] Table 4. Parameters of the quantitative calibration model for berberine hydrochloride.

[0063] Model validation results: Internal cross-validation was used to evaluate model quality. The relative bias Rp was calculated using the following formula: Rp = |Cp - Cr| / Cr × 100% In the formula: Rp represents the relative deviation; Cp is the model's predicted value; Cr is a chemical reference value.

[0064] Four samples with significant biases were identified in the model: berberine hydrochloride linear 1 (0.1800 mg / mL, Rp=11.0%), berberine hydrochloride linear 2 (0.4579 mg / mL, Rp=5.6%), sample 1 (0.2658 mg / mL, Rp=5.0%), and sample 2 (0.3491 mg / mL, Rp=7.9%). Analysis revealed that the berberine hydrochloride concentrations in these samples were all near the detection limit, resulting in weak Raman signals and thus larger prediction biases. Since the spectra of these samples were normal, they were not removed.

[0065] Except for the low-concentration limit samples mentioned above, the relative deviations of the other samples were all small, indicating that the model has high prediction accuracy within the actual sample concentration range. The prediction deviations for the stability test samples were all within 3%, indicating that the model has good robustness. Detailed validation data are shown in Table 5 and... Figure 6 .

[0066] Table 5. Model Predicted Values ​​and Chemical Reference Values ​​for Berberine Hydrochloride Content

[0067] Comparative analysis: To further investigate the impact of the accuracy of chemical reference values ​​on the quality of the Raman quantitative model, the inventors also established a zinc sulfate content model.

[0068] Because the background color of compound zinc sulfate eye drops is yellow, it is difficult to determine the endpoint color when determining the zinc sulfate content by volumetric method. The chemical reference value has a large error, resulting in the R² of the zinc sulfate model in internal cross-validation being only 94.46%, with some samples having a relative deviation of more than 10%, indicating that the model quality is not high.

[0069] The inventors also attempted to obtain chemical reference values ​​for zinc sulfate using atomic absorption spectrometry, but due to instrument factors such as flame stability and lamp current, the measurement results had poor precision and insufficient data, and were not used for modeling.

[0070] In contrast, the boric acid chemical reference value obtained using the indirect quantitative method described in this invention achieved a model R² of 97.90%, with the relative deviation of the vast majority of samples being less than 3%, a stark contrast to the previous method. These results demonstrate that the accuracy of the chemical reference value is a key factor affecting the quality of Raman quantitative models and also verify the effectiveness of the boric acid chemical reference value acquisition method proposed in this invention.

[0071] Method Application: The method of this invention was used to test compound zinc sulfate eye drops samples. The operation steps are as follows: (1) Place the sample to be tested in a quartz cuvette; (2) Collect the Raman spectrum of the sample to be tested according to the aforementioned parameters; (3) Substitute the obtained spectra into the boric acid quantitative correction model and the berberine hydrochloride quantitative correction model, respectively; (4) Read and record the predicted values ​​of boric acid and berberine hydrochloride content.

[0072] The entire process is simple to operate, with a single sample testing time of approximately 100 seconds. No chemical reagents are required, and the sample is not damaged.

[0073] The method of this invention can be used by drug regulatory authorities for routine supervision and rapid screening of compound zinc sulfate eye drops, and can also be used for online monitoring of the production process of manufacturing enterprises.

[0074] 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 Raman spectroscopy method for determining boric acid and berberine hydrochloride in compound zinc sulfate eye drops, characterized in that, Includes the following steps: (1) Obtaining chemical reference values: The total boron content (Ct) in the sample was determined by ion chromatography, and the borax content (Cs) was determined by potentiometric titration. The chemical reference value (Ca) of boric acid was calculated using the following formula: Ca = Ct - K × Cs Where Ca is the boric acid content in the sample, in mg / mL; Ct is the total boron content in the sample determined by ion chromatography, in mg / mL; Cs is the borax content in the sample determined by potentiometric titration, in mg / mL; and K is the conversion factor for borax to boric acid. The content of berberine hydrochloride in the sample was determined by high performance liquid chromatography, and the chemical reference value of berberine hydrochloride was obtained. (2) Collect Raman spectra of a series of samples; (3) The Raman spectra obtained in step (2) are preprocessed, and the partial least squares method is used to establish quantitative correction models for boric acid and berberine hydrochloride, respectively, based on the chemical reference values ​​obtained in step (1). (4) Collect the Raman spectrum of the sample to be tested, and substitute it into the boric acid quantitative correction model and the berberine hydrochloride quantitative correction model established in step (3) respectively, and obtain the content of boric acid and berberine hydrochloride in the sample to be tested.

2. The Raman determination method according to claim 1, characterized in that, In step (2), a quartz cuvette is used to load the sample for Raman spectroscopy acquisition.

3. The Raman determination method according to claim 1, characterized in that, The Raman spectroscopy acquisition conditions in step (2) are: 785nm laser, -50℃ DDC detector, fiber optic probe, focal length 5mm, resolution 4cm⁻¹, and scanning time 100s.

4. The Raman determination method according to claim 1, characterized in that, In step (2), three bottles are selected for each batch of samples, and each bottle is measured once. The average spectrum of the three original spectra is used for modeling or prediction.

5. The Raman determination method according to claim 1, characterized in that, The modeling parameters for the boric acid quantitative correction model in step (3) are as follows: the preprocessing method is maximum-minimum normalization, and the modeling spectral range is 1667.7–1498.7 cm⁻¹. -1 1416.8~1247.8cm -1 1165.9~1080.1cm -1 and 998.2~829.2cm -1 The number of smoothed points is 17, and the number of principal components is 7.

6. The Raman determination method according to claim 1, characterized in that, In step (3), the modeling parameters for the quantitative correction model of berberine hydrochloride are as follows: the preprocessing method is to eliminate constant offset, and the modeling spectral range is 2901.4–2299.5 cm⁻¹. -1 2000.5~799.3cm -1 and 500.3~200cm -1 The number of smoothed points is 17, and the number of principal components is 8.

7. The Raman determination method according to claim 1, characterized in that, The linear range of the boric acid quantitative correction model was 3.408–25.98 mg / mL, and the linear range of the berberine hydrochloride quantitative correction model was 0.1800–1.364 mg / mL.

8. The Raman determination method according to claim 1, characterized in that, The compound zinc sulfate eye drops contain 1 mg of berberine hydrochloride, 3 mg of zinc sulfate and 20 mg of boric acid per milliliter.

9. The Raman determination method according to any one of claims 1 to 8, characterized in that, The method is used for online monitoring or daily quality supervision and rapid screening of the production process of compound zinc sulfate eye drops.

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