Method for determining chiral isomers of pilocarpine hydrochloride by high performance liquid chromatography and application thereof
By using a chromatographic column packed with amylose-tris(3,5-dimethylphenylcarbamate) silica gel and a specific mobile phase, the problem of insufficient separation of chiral isomers of pilocarpine hydrochloride was solved, and efficient and accurate quality control was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHINA OTSUKA PHARM CO LTD
- Filing Date
- 2026-03-03
- Publication Date
- 2026-05-29
AI Technical Summary
Existing technologies struggle to achieve baseline separation of pilocarpine hydrochloride from its chiral isomers, resulting in insufficient separation (Rs < 1.5), and lack specific and reproducible analytical methods.
A high-performance liquid chromatography (HPLC) method was used to determine the chiral isomers of pilocarpine hydrochloride using a column packed with amylose-tris(3,5-dimethylphenylcarbamate) silica gel and a ternary mobile phase consisting of hexane, methanol, and an ethanol solution containing an organic base.
Baseline separation of pilocarpine hydrochloride and its chiral isomer was achieved (resolution Rs ≥ 1.5). The method is highly specific, reproducible, and accurate in quantification, with a limit of quantification as low as 0.38 μg/mL, meeting the quality control requirements.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for determining chiral isomers of pilocarpine hydrochloride by high performance liquid chromatography and its application. Background Technology
[0002] Pilocarpine hydrochloride is a cholinergic muscarinic agonist that activates muscarinic receptors located on smooth muscles such as the iris sphincter and ciliary muscle. Clinically, it is used to treat presbyopia. Its molecular structure contains a chiral center, thus resulting in two enantiomers. Different chiral isomers may differ in pharmacological activity, metabolic behavior, and toxicity; therefore, effective isomer separation and quality control of chiral drugs are of great significance. The molecular formula of pilocarpine hydrochloride is C0. 11 H 16 N2O2 · HCl, with the following structural formula:
[0003] Current techniques for separating the chiral isomers of pilocarpine hydrochloride suffer from the following problems: Common chiral chromatographic columns (such as cellulose-tris(3,5-dimethylphenylcarbamate) silica gel columns (Chiralcel OD-H) and cellulose-tris(4-methylbenzoate) silica gel columns (Chiralcel OJ-H)) often fail to achieve baseline separation of pilocarpine hydrochloride from its chiral isomers, with resolution (Rs) typically below 1.5. Changes in flow rate can also lead to peak broadening that makes elution difficult or baseline separation (Rs < 1.5) between pilocarpine hydrochloride and its chiral isomers.
[0004] Currently, there is a lack of a reliable analytical method that is highly specific, reproducible, and suitable for the quality control of chiral isomers of pilocarpine hydrochloride.
[0005] In view of this, the present invention is proposed. Summary of the Invention
[0006] One of the objectives of this invention is to provide a method for determining the chiral isomers of pilocarpine hydrochloride by high performance liquid chromatography, thereby achieving quality control of pilocarpine hydrochloride.
[0007] The second objective of this invention is to provide a method for determining the chiral isomers of pilocarpine hydrochloride using high performance liquid chromatography, and its application in the preparation of products for detecting the chiral isomers of pilocarpine hydrochloride.
[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a method for determining chiral isomers of pilocarpine hydrochloride by high performance liquid chromatography, comprising: detecting and analyzing a pilocarpine hydrochloride sample solution by liquid chromatography; wherein the chromatographic column of the liquid chromatography is packed with amylose-tris(3,5-dimethylphenylcarbamate) silica gel; and the mobile phase of the liquid chromatography comprises n-hexane, methanol, and an ethanol solution containing an organic base.
[0009] Furthermore, the organic base includes at least one of diethylamine, triethylamine, and diisopropylamine.
[0010] Furthermore, the volume ratio of the hexane, the methanol, and the ethanol solution containing the organic base is 750-850:80-120:80-120.
[0011] Furthermore, the volume ratio of the hexane, the methanol, and the diethylamine-containing ethanol solution is 800:100:100.
[0012] Furthermore, in the ethanol solution containing the organic base, the volume percentage concentration of the organic base is 0.05%-0.2%.
[0013] Furthermore, in the ethanol solution containing the organic base, the volume percentage concentration of the organic base is 0.1%.
[0014] Furthermore, the chromatographic column has a length of 250 mm and an inner diameter of 4.6 mm, and the packing material has a particle size of 5 μm.
[0015] Furthermore, the detection conditions for the liquid chromatography include one or more of the following (a) to (c): (a) The flow rate of the mobile phase is 0.8-1.2 ml / min; (b) Column temperature is 25-35℃; (c) The detection wavelength is 215 nm; (d) The injection volume is 2-10 μL.
[0016] Furthermore, the preparation process of the pilocarpine hydrochloride sample solution includes: taking pilocarpine hydrochloride raw material, dissolving it in ethanol, and quantitatively diluting it to prepare a solution containing 1-5 mg per 1 mL.
[0017] Secondly, the present invention provides an application of the method described herein in the preparation of a product for detecting chiral isomers of pilocarpine hydrochloride.
[0018] Compared with the prior art, the present invention has the following beneficial effects: The high-performance liquid chromatography (HPLC) method for determining the chiral isomers of pilocarpine hydrochloride provided by this invention utilizes a column packed with amylose-tris(3,5-dimethylphenylcarbamate) silica gel and a ternary mobile phase consisting of hexane, methanol, and an ethanol solution containing an organic base. This method achieves baseline separation of pilocarpine hydrochloride and its chiral isomers (resolution Rs ≥ 1.5), significantly superior to the insufficient resolution (Rs < 1.5) of existing technologies using cellulose-based chiral columns (such as Chiralcel OD-H and OJ-H). This method is highly specific, reproducible, and accurate in quantification, with a limit of quantitation (LOQ) as low as 0.38 μg / mL (equivalent to a sample concentration of 0.019%), fully meeting the quality control requirements for chiral impurities in pilocarpine hydrochloride raw materials and preparations. Attached Figure Description
[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0020] Figure 1 This is a high-performance liquid chromatogram of the system suitability solution obtained in Example 1 of the present invention; Figure 2 High performance liquid chromatograms of the system suitability solutions obtained from Comparative Example 1 and Comparative Example 2; Figure 3 High performance liquid chromatograms of the system suitability solutions obtained in Comparative Examples 3 and 4; Figure 4 High-performance liquid chromatography (HPLC) chromatograms of the system suitability solutions obtained in Examples 2 and 3; Figure 5 This is a high-performance liquid chromatogram used to verify the specificity of the method in Example 1 of this invention; Figure 6 This is a linear relationship diagram for verifying the linear range of the method in Embodiment 1 of the present invention; Figure 7 The structural formulas are those of chiral isomers I and II of pilocarpine hydrochloride. Detailed Implementation
[0021] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.
[0022] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.
[0023] The first aspect of this invention provides a method for determining chiral isomers of pilocarpine hydrochloride by high performance liquid chromatography, comprising: detecting and analyzing a pilocarpine hydrochloride sample solution by liquid chromatography; wherein the chromatographic column of the liquid chromatography is packed with amylose-tris(3,5-dimethylphenylcarbamate) silica gel; and the mobile phase of the liquid chromatography comprises n-hexane, methanol, and an ethanol solution containing an organic base.
[0024] This invention provides a method for the analysis and determination of pilocarpine hydrochloride and its chiral isomers. A linear starch-tris(3,5-dimethylphenylcarbamate) silica gel column (250×4.6mm, 5µm) is used as the packed column, and a mobile phase of a certain proportion of n-hexane-ethanol solution (0.1% diethylamine)-methanol is used to separate and analyze the chiral isomers of pilocarpine hydrochloride, thereby enabling accurate control of the quality of pilocarpine hydrochloride and its preparations.
[0025] In some preferred embodiments, the organic base includes at least one of diethylamine, triethylamine, and diisopropylamine.
[0026] In some preferred embodiments, the volume ratio of the hexane, the methanol, and the ethanol solution containing the organic base is 750-850:80-120:80-120; Among them, "750-850" can be, for example, 750, 800, 850, etc.; "80-120" can be, for example, 80, 90, 100, 110, 120, etc.
[0027] More preferably, the volume ratio of the hexane, the methanol, and the ethanol solution containing diethylamine is 800:100:100.
[0028] In some preferred embodiments, the volume percentage concentration of the organic base in the ethanol solution containing the organic base is 0.05%-0.2%, for example, it can be 0.05%, 0.1%, 0.15%, 0.2%, etc.
[0029] More preferably, the volume percentage concentration of the organic base in the ethanol solution containing the organic base is 0.1%.
[0030] In some preferred embodiments, the chromatographic column has a length of 250 mm and an inner diameter of 4.6 mm, and the packing material has a particle size of 5 μm.
[0031] Specifically, the liquid chromatography method in this invention is normal phase liquid chromatography, and the chromatographic column in this invention is a chiral chromatographic column with silica gel as the matrix and a surface coated with amylose-tris(3,5-dimethylphenylcarbamate).
[0032] In some preferred embodiments, the detection conditions of the liquid chromatography include one or more of the following (a) to (c): (a) The flow rate of the mobile phase is 0.8-1.2 ml / min, for example, it can be 0.8 ml / min, 0.9 ml / min, 1 ml / min, 1.1 ml / min, 1.2 ml / min, etc.; (b) The column temperature is 25-35℃, for example, it can be 25℃, 30℃, 35℃, etc.; (c) The detection wavelength is 215 nm; (d) The injection volume is 2-10 μL, for example, it can be 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, etc.
[0033] In some preferred embodiments, the preparation process of the pilocarpine hydrochloride sample solution includes: taking pilocarpine hydrochloride raw material, dissolving it in ethanol, and quantitatively diluting it to prepare a solution containing 1-5 mg per 1 mL.
[0034] In some preferred embodiments, the method includes at least the following steps: (1) Take an appropriate amount of pilocarpine hydrochloride, weigh it accurately, dissolve it in ethanol and prepare a solution containing about 2 mg per ml.
[0035] (2) Set the mobile phase flow rate to 0.8~1.2 ml / min; column temperature to 25~35℃; and detection wavelength to 215 nm.
[0036] (3) Take 5 μl of the sample solution from step (1) and inject it into the liquid chromatograph to complete the separation and analysis of pilocarpine hydrochloride and its chiral isomer.
[0037] This invention provides an efficient, rapid, and accurate HPLC method for separating and determining the chiral isomers of pilocarpine hydrochloride, meeting the practical needs in its synthesis, active pharmaceutical ingredient (API), and formulation quality control. This method effectively analyzes the chiral isomers of pilocarpine hydrochloride. The experiment used a linear starch-tris(3,5-dimethylphenylcarbamate) silica gel column (250 × 4.6 mm, 5 µm) as the packed column, and a hexane-ethanol solution (0.1% diethylamine)-methanol (volume ratio 800:100:100) as the mobile phase to rapidly and accurately determine the chiral isomers of pilocarpine hydrochloride. This method effectively controls impurities in pilocarpine hydrochloride, ensuring its quality control and is of significant importance in the quality control of synthesis and formulation processes.
[0038] The second aspect of this invention provides a method for determining the chiral isomers of pilocarpine hydrochloride by high performance liquid chromatography, and its application in the preparation of products for detecting the chiral isomers of pilocarpine hydrochloride.
[0039] This invention effectively determines the chiral isomers of pilocarpine hydrochloride. The method is simple, rapid, highly sensitive, and yields accurate and reliable results. It can be used for the quality control of pilocarpine hydrochloride, ensuring the quality control of both the active pharmaceutical ingredient and the finished product.
[0040] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.
[0041] Example 1 This embodiment provides a method for determining the chiral isomers of pilocarpine hydrochloride using high performance liquid chromatography, including: (1) Instruments, reagents and conditions Experimental materials: ethanol (chromatographic grade), n-hexane (chromatographic grade), methanol (chromatographic grade), diethylamine (analytical grade); pilocarpine hydrochloride, pilocarpine hydrochloride chiral isomer I, and pilocarpine hydrochloride chiral isomer II (the structural formulas of pilocarpine hydrochloride chiral isomer I and pilocarpine hydrochloride chiral isomer II are shown below). Figure 7 As shown); High Performance Liquid Chromatograph: Shimadzu: LC-20AT; Column: A linear starch-tris(3,5-dimethylphenylcarbamate) silica gel column (250×4.6mm, 5µm); Mobile phase: n-hexane-ethanol solution (0.1% diethylamine)-methanol (volume ratio of the three is 800:100:100); Chromatographic conditions: Flow rate: 1.0 ml / min; Detection wavelength: 215 nm; Column temperature: 30 ℃; Injection volume: 5 μl.
[0042] (2) Experimental procedure: Take 2 mg each of pilocarpine hydrochloride isomer I and pilocarpine hydrochloride isomer II, place them in the same 100 ml volumetric flask, dissolve and dilute to the mark with ethanol, and shake well. Accurately weigh 20 mg of pilocarpine hydrochloride, place it in a 10 ml volumetric flask, add 1 ml of the above isomer solution, dissolve and dilute to the mark with ethanol, and shake well. This is the system suitability solution. Perform high-performance liquid chromatography (HPLC) analysis on the system suitability solution under the above chromatographic conditions, record the chromatogram, and the results are shown in the figure. Figure 1 .
[0043] Figure 1 Peak 1 represents isomer I of pilocarpine hydrochloride; peak 2 represents pilocarpine hydrochloride; and peak 3 represents isomer II of pilocarpine hydrochloride. This three-peak separation indicates that, under the conditions specified in this invention (using a column packed with amylose-tris(3,5-dimethylphenylcarbamate) silica gel and a mobile phase (n-hexane, methanol, and an ethanol solution containing an organic base), effective separation of pilocarpine hydrochloride and its chiral isomers is achieved.
[0044] Example 2 This embodiment provides a method for determining the chiral isomer of pilocarpine hydrochloride by high performance liquid chromatography, which differs from Example 1 in that the flow rate is 0.5 ml / min.
[0045] Example 3 This embodiment provides a method for determining the chiral isomer of pilocarpine hydrochloride by high performance liquid chromatography, which differs from Example 1 in that the flow rate is 1.5 ml / min.
[0046] The results of Examples 2 and 3 are attached. Figure 4 . Figure 4 This is a high-performance liquid chromatography (HPLC) chromatogram of a solution suitable for system application when the mobile phase flow rate deviates from the preferred range (0.8-1.2 mL / min) of this invention. The results indicate that at mobile phase flow rates of 0.5 mL / min or 1.5 mL / min (i.e., below the lower limit or above the upper limit), independent identification and accurate quantification of the main component and each isomer are impossible. This experiment further confirms that the mobile phase flow rate range (0.8-1.2 mL / min) defined in this invention is a key operating parameter ensuring effective separation of chiral isomers.
[0047] Comparative Example 1 This comparative example provides a high-performance liquid chromatography method for determining the chiral isomers of pilocarpine hydrochloride. The difference between this method and Example 1 is that the chromatographic column is a cellulose-tris(3,5-dimethylphenylcarbamate) silica column (Chiralcel OD-H, 250×4.6mm, 5µm).
[0048] Comparative Example 2 This comparative example provides a high-performance liquid chromatography method for determining the chiral isomers of pilocarpine hydrochloride. The difference between this method and Example 1 is that the chromatographic column is a cellulose-tris(4-methylbenzoate) silica column (Chiralcel OJ-H, 250×4.6mm, 5µm).
[0049] The results of Comparative Example 1 and Comparative Example 2 are shown in Figure 2 . Figure 2 Peaks 1 and 2 are chromatographic peaks of the pilocarpine hydrochloride isomers. This result indicates that, under the same sample, mobile phase, and instrument conditions, the use of a cellulose derivative-based chiral column cannot achieve effective separation of the main component of pilocarpine hydrochloride and its chiral isomers. Comparative Examples 1 and 2 further confirm the necessity and irreplaceability of using amylose-tris(3,5-dimethylphenylcarbamate) silica gel as the packing material in this invention.
[0050] Comparative Example 3 This comparative example provides a method for determining the chiral isomer of pilocarpine hydrochloride by high performance liquid chromatography. The difference from Example 1 is that the mobile phase is n-hexane-ethanol (800:200).
[0051] Comparative Example 4 This comparative example provides a method for determining the chiral isomer of pilocarpine hydrochloride by high performance liquid chromatography. The difference from Example 1 is that the mobile phase is n-hexane-methanol (800:200).
[0052] The results of Comparative Examples 3 and 4 are attached. Figure 3 . Figure 3 Peaks 1 and 2 are chromatographic peaks of the pilocarpine hydrochloride isomers (peak 2 coincides with the main component pilocarpine hydrochloride peak under hexane-methanol (800:200) conditions; under hexane-ethanol (800:200) conditions, the resolution (Rs) between peak 1 and the main component pilocarpine hydrochloride peak is less than 1.5). This result indicates that in the absence of the ethanol solution component containing an organic base, using only a binary mobile phase system of hexane and methanol, or in the absence of the methanol solution component using only a binary mobile phase system of hexane and ethanol, the main component of pilocarpine hydrochloride and its chiral isomers cannot be separated (separation requirement is Rs not less than 1.5). This comparative experiment further confirms that the ternary mobile phase system (hexane, methanol, and an ethanol solution containing an organic base) defined in this invention is an important condition for achieving effective separation of chiral isomers.
[0053] Example 4: Methodological Validation In this embodiment, the chromatographic conditions and other parameters were performed according to the method in Example 1.
[0054] (a) Specificity test of Example 1 Isomer I positioning solution: Take an appropriate amount of pilocarpine hydrochloride isomer I, dissolve it in ethanol and dilute it quantitatively to prepare a solution containing about 4 μg per 1 ml.
[0055] Isomer II positioning solution: Take an appropriate amount of pilocarpine hydrochloride isomer II, dissolve it in ethanol and dilute it quantitatively to prepare a solution containing about 4 μg per 1 ml.
[0056] System suitability solution: Take 2 mg each of pilocarpine hydrochloride isomer I and pilocarpine hydrochloride isomer II, place them in the same 100 ml volumetric flask, dissolve and dilute to the mark with ethanol, and shake well; take 20 mg of pilocarpine hydrochloride, accurately weigh it, place it in a 10 ml volumetric flask, add 1 ml of the above isomer solution, dissolve and dilute to the mark with ethanol, and shake well.
[0057] Test solution: Weigh 20 mg of pilocarpine hydrochloride accurately, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with ethanol, and shake well.
[0058] Mixed localization (related substances): Take appropriate amounts of EP / USP impurity A, EP / USP impurity B, and USP impurity C reference standards, dissolve and dilute them in solvent to prepare a solution containing approximately 5 μg per 1 ml. It should be noted that impurities A, B, and C are impurities under the related substances section of pilocarpine hydrochloride, used to demonstrate that related substances do not interfere with the isomers.
[0059] Accurately measure 5 μl of each of the above solutions, inject them into the liquid chromatograph, record the chromatograms, and see the results below. Figure 5 The results showed that the blank solvent and other impurities under the related substances section did not interfere with the determination of isomers in the test solution, and the resolution between each chromatographic peak in the system suitability solution was greater than 1.5; it met the acceptable standard for method validation, indicating that the method has good specificity.
[0060] (II) Linear Range Test of Example 1 Linear solutions of pilocarpine hydrochloride, isomer impurity I, and isomer impurity II were prepared with concentrations ranging from the limit of quantitation to 10 μg / ml (equivalent to 0.5% of the sample detection concentration). The concentrations were as follows: isomer impurity I: 0.42 μg / ml, 1.04 μg / ml, 2.08 μg / ml, 4.15 μg / ml, 10.39 μg / ml; main component: 0.40 μg / ml, 0.99 μg / ml, 1.98 μg / ml, 3.96 μg / ml, 9.91 μg / ml; isomer impurity II: 0.38 μg / ml, 0.96 μg / ml, 1.92 μg / ml, 3.83 μg / ml, 9.59 μg / ml.
[0061] Accurately measure 5 μl of each of the above solutions, inject them into the liquid chromatograph, record the chromatograms, and see the results below. Figure 6 See Table 1. The results show that within the above concentration range, the concentration and peak area of each component exhibit a good linear relationship, meeting the acceptable criteria for method validation.
[0062] Table 1
[0063] (III) Limit of Quantitation and Limit of Detection Test in Example 1 Prepare solutions with a peak height signal-to-noise ratio of approximately 10 for pilocarpine hydrochloride, isomer impurity I, and isomer impurity II, and use these solutions as the limit of quantitation solutions. Also prepare solutions with a peak height signal-to-noise ratio of approximately 3, and use these solutions as the limit of detection solutions.
[0064] Accurately measure 5 μl of each of the above solutions and inject them into the liquid chromatograph. Record the chromatograms, and the results are shown in Table 2. The results show that the signal-to-noise ratio of the quantitation limit solutions for each component is greater than 10; the signal-to-noise ratio of the detection limit solutions is greater than 3. Both the method quantitation limit and the detection limit meet the acceptable standards, indicating that the method has good sensitivity.
[0065] Table 2
[0066] (iv) Accuracy test of Example 1 According to the analytical method, a mixed solution containing isomer impurity I at concentrations of 1 μg / ml, 2 μg / ml, and 3 μg / ml, and pilocarpine hydrochloride at 2 ml / ml was prepared and tested. The accuracy was assessed based on the comparison between the detected value and the added value.
[0067] Accurately measure 5 μl of each of the above solutions and inject them into the liquid chromatograph. Record the chromatograms, and the results are shown in Table 3. The results show that the recoveries of isomer I and isomer II in the spiked test solutions of different concentrations are between 90.0% and 108.0%, which meets the acceptable standard for method validation, indicating that the method has good accuracy.
[0068] Table 3
[0069] (v) Repeatability test of Example 1 According to the analytical method, a mixed solution containing isomer impurity I and isomer impurity II at a concentration of 2 μg / ml and pilocarpine hydrochloride at a concentration of 2 mg / ml was prepared and tested. Repeatability was assessed based on the RSD of the impurity detection in 6 spiked test solution samples.
[0070] Accurately measure 5 μl of each of the above solutions and inject them into the liquid chromatograph. Record the chromatograms, and the results are shown in Table 4. The results show that when six parallel spiked test solutions were prepared, the RSD values of isomer I and isomer II were between 1.7% and 2.7%, less than 5.0%, which meets the acceptable standard for method validation, indicating that the method has good repeatability.
[0071] Table 4
[0072] The method provided by this invention can effectively analyze the chiral isomers of pilocarpine hydrochloride. This invention utilizes a linear starch-tris(3,5-dimethylphenylcarbamate) silica gel column (250×4.6mm, 5µm) as the packed column to rapidly and accurately determine the chiral isomers of pilocarpine hydrochloride. This allows for the experimental control of impurities in pilocarpine hydrochloride, ensuring its quality control and holding significant importance for quality control in synthesis and formulation processes.
[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for determining chiral isomers of pilocarpine hydrochloride by high performance liquid chromatography, characterized in that, include: The sample solution of pilocarpine hydrochloride was analyzed by liquid chromatography; wherein the chromatographic column of the liquid chromatography was packed with amylose-tris(3,5-dimethylphenylcarbamate) silica gel; and the mobile phase of the liquid chromatography included hexane, methanol, and an ethanol solution containing an organic base.
2. The method according to claim 1, characterized in that, The organic base includes at least one of diethylamine, triethylamine, and diisopropylamine.
3. The method according to claim 1, characterized in that, The volume ratio of the n-hexane, the methanol, and the ethanol solution containing the organic base is 750-850:80-120:80-120.
4. The method according to claim 1, characterized in that, The volume ratio of the hexane, the methanol, and the diethylamine-containing ethanol solution is 800:100:
100.
5. The method according to claim 1, characterized in that, The volume percentage concentration of the organic base in the ethanol solution containing the organic base is 0.05%-0.2%.
6. The method according to claim 1, characterized in that, The volume percentage concentration of the organic base in the ethanol solution containing the organic base is 0.1%.
7. The method according to claim 1, characterized in that, The chromatographic column is 250 mm long and has an inner diameter of 4.6 mm. The particle size of the packing material is 5 μm.
8. The method according to claim 1, characterized in that, The detection conditions for the liquid chromatography include one or more of the following (a) to (c): (a) The flow rate of the mobile phase is 0.8-1.2 ml / min; (b) Column temperature is 25-35℃; (c) The detection wavelength is 215 nm; (d) The injection volume is 2-10 μL.
9. The method according to claim 1, characterized in that, The preparation process of the pilocarpine hydrochloride sample solution includes: taking pilocarpine hydrochloride raw material, dissolving it in ethanol, and quantitatively diluting it to prepare a solution containing 1-5 mg per 1 mL.
10. The application of the method according to any one of claims 1-9 in the preparation of a product for detecting chiral isomers of pilocarpine hydrochloride.