Normal-phase chromatographic analysis and detection method for tiagonium bromide intermediate and enantiomer thereof

Abstract

The invention discloses a normal-phase chromatographic analysis and detection method for a tiagonium bromide intermediate and an enantiomer of the tiagonium bromide intermediate, and belongs to the technical field of analytical chemistry. The method comprises the following steps: preparing a system applicability solution, a test solution and a contrast solution; setting liquid chromatography detection conditions, and adopting a normal phase chromatographic column with cellulose-tri (3, 5-dimethyl phenyl carbamate) coated silica gel as a filling agent; finally, the system applicability solution, the test solution and the contrast solution are taken and injected into a liquid chromatograph, and chromatograms are recorded. The detection method can be used for rapidly detecting the content of enantiomer impurities in the tiagradionium bromide intermediate, is high in specificity and separation degree, and can be used for well separating an R-configuration tiagradionium bromide intermediate and an S-configuration enantiomer impurity; the method has the advantages of high sensitivity and good repeatability, and can accurately quantify the content of enantiomer impurities in the tiagradionium bromide intermediate.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to a normal-phase chromatographic analysis and detection method for thioglucopyranoammonium intermediates and their enantiomers. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD) is a common and frequently occurring progressive respiratory disease, including chronic bronchitis, asthma, bronchiectasis, cystic fibrosis, etc. Drug treatment for COPD mainly includes the use of anti-inflammatory drugs such as glucocorticoids and leukotriene inhibitors, as well as bronchodilators such as anticholinergic drugs and adrenal β2 receptor agonists.

[0003] Tioglitazone is a newly discovered, highly selective anticholinergic drug targeting M3 cholinergics, classified as a Class 1 innovative drug under the new chemical drug registration classification. Its chemical name is 3R-1,1-dimethyl-3-(2-hydroxy-2,2-dithiophene-2-ylacetoxy)pyrrolidine bromide, with the molecular formula C1. 16 H 20 BrNO3S2, the structural formula is shown in Formula I. As can be seen from Formula I, thioglucopyranoammonium contains a chiral carbon atom, so it has an S-type enantiomer impurity 3S-1,1-dimethyl-3-(2-hydroxy-2,2-dithiophene-2-ylacetoxy)pyrrolidine bromide, the structural formula of which is shown in Formula II.

[0004]

[0005] (R)-1-methylpyrrolidone-3-yl-2-hydroxy-2,2-di(thiophene-2-yl)acetate is an important intermediate in the synthesis of thioglucopyranoammonium, and its molecular formula is C1. 15 H 17 NO3S2, with the structural formula shown in Formula III. As can be seen from Formula III, this thioglitazone intermediate also contains the S-enantiomer impurity (S)-1-methylpyrrolidone-3-yl-2-hydroxy-2,2-di(thiophene-2-yl)acetate, with the structural formula shown in Formula IV. The S-enantiomer impurity in this thioglitazone intermediate is the main source of the S-enantiomer impurities in the aforementioned thioglitazone, therefore, the content of the S-enantiomer impurity in the thioglitazone intermediate directly affects the quality of the final product, thioglitazone.

[0006]

[0007] Given that enantiomers often possess drastically different pharmacological activities, it is necessary to control the content of S-type enantiomer impurities in thioglucopyranoside intermediates at the source to ensure drug quality. Since thioglucopyranoside is a newly developed innovative COPD drug, there are currently no reported methods for detecting enantiomer impurities in thioglucopyranoside and its intermediates. Therefore, this patent aims to provide a highly accurate and well-resolution forward chromatographic analysis method to monitor the content of enantiomer impurities in thioglucopyranoside intermediates, providing technical assurance for the control of enantiomer impurities in thioglucopyranoside and its formulations at the source. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a normal phase chromatographic analysis and detection method for thiogluconium intermediates and their enantiomers. The method is rapid, simple, effective, specific, has good separation, high sensitivity and good repeatability.

[0009] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0010] A normal-phase chromatographic method for the detection of thiogluconium intermediates and their enantiomers, using liquid chromatography, includes the following steps:

[0011] 1) Prepare system suitability solution, test solution and control solution for later use;

[0012] 2) Setting up liquid chromatography detection conditions: A normal phase column coated with cellulose-tris(3,5-dimethylphenylcarbamate) silica gel was used as the packing material; the mobile phase was n-alkane solvent-alcohol solvent-amine tailing inhibitor;

[0013] 3) Take the system suitability solution, test solution and control solution respectively, inject them into the liquid chromatograph and record the chromatogram.

[0014] Further, in step 1), the preparation of the system suitability solution is as follows: Take appropriate amounts of thioglucopyranoammonium intermediate and enantiomer reference standard, dissolve and dilute them in solvent to prepare a mixed solution containing approximately 0.5 mg of thioglucopyranoammonium intermediate and 10 μg of enantiomer per 1 ml, and shake well.

[0015] Further, in step 1), the preparation of the test solution is as follows: take an appropriate amount of thioglucopyranoammonium intermediate, accurately weigh it, dissolve and dilute it with solvent to prepare a solution containing about 0.5 mg per ml, and shake well.

[0016] Further, in step 1), the preparation of the control solution is as follows: accurately measure an appropriate amount of the test solution, quantitatively dilute it with a solvent to prepare a solution containing approximately 5 μg per 1 ml, and shake well.

[0017] Further, in step 2), the chromatographic column is selected from either YMC CHIRAL ART Cellulose-C, 250 mm × 4.6 mm, 5 μm or DAICEL CHIRALCEL OD-H, 250 mm × 4.6 mm, 5 μm.

[0018] Further, in step 2), the volume ratio of n-alkane solvent, alcohol solvent and amine tailing inhibitor is 88-98:12-2:0.1-0.5; the n-alkane solvent is selected from n-hexane or n-heptane, the alcohol solvent is selected from one, two or three of methanol, ethanol or isopropanol, and the amine tailing inhibitor is selected from diethylamine, dibutylamine or triethylamine.

[0019] Furthermore, in step 2), the flow rate is 0.6–1.5 ml / min.

[0020] Furthermore, in step 2), the column temperature is 20–40°C; the sample pan temperature is 4–10°C.

[0021] Furthermore, in step 2), the detection wavelength is 230–245 nm; the running time is 15 min.

[0022] Furthermore, in step 2), the injection volume is 10 μl.

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

[0024] This invention provides a normal-phase chromatographic method for the detection of thioglucopyranoammonium intermediates and their enantiomers, employing liquid chromatography. This method can rapidly detect the content of enantiomeric impurities in thioglucopyranoammonium intermediates, exhibiting high specificity and good resolution, effectively separating R-configuration thioglucopyranoammonium intermediates and S-configuration enantiomers. It also demonstrates high sensitivity and good repeatability, accurately quantifying the content of enantiomeric impurities in thioglucopyranoammonium intermediates. This ensures accurate and reliable analytical results, providing technical support for the quality control of thioglucopyranoammonium raw materials and their formulations. Attached Figure Description

[0025] Figure 1 This is a chromatogram of the solution used to detect the suitability of the system in Example 1 of this application;

[0026] Figure 2 This is the chromatogram of the test solution in Example 1 of this application;

[0027] Figure 3 This is the chromatogram of the control solution used in Example 1 of this application;

[0028] Figure 4 This is a standard curve diagram of the thioglucopyranoium intermediate in Example 2 of this application;

[0029] Figure 5 This is a standard curve diagram of the enantiomers in Embodiment 2 of this application. Detailed Implementation

[0030] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are implemented based on the technical solutions of the present invention, and it should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0031] Example 1

[0032] A normal-phase chromatographic analysis method for the detection of thiogluconium intermediates and their enantiomers includes the following steps:

[0033] 1) Instrument and sample selection: The liquid chromatograph is a Thermo Fisher U3000; the batch number of the thiogluconium bromide intermediate is SA-a202206003, and the manufacturer is Nanjing Lianzhi Pharmaceutical Technology Co., Ltd.

[0034] 2) Liquid chromatography conditions: The column was a normal phase column (YMC CHIRAL ART Cellulose-C, 250 mm × 4.6 mm, 5 μm) packed with cellulose-tris(3,5-dimethylphenylcarbamate) silica gel; the mobile phase was n-hexane-ethanol-diethylamine (95:5:0.1); the flow rate was 1.0 ml / min; the column temperature was 30 ℃; the sample tray temperature was 4 ℃; the detection wavelength was 237 nm; the injection volume was 10 μl; and the run time was 15 min.

[0035] 3) Preparation of sample solution

[0036] Solvent: Methanol-ethanol (5:95);

[0037] System suitability solution: Take appropriate amounts of thioglucopyranoammonium intermediate and enantiomer reference standard, dissolve and dilute with solvent to prepare a mixed solution containing approximately 0.5 mg of thioglucopyranoammonium intermediate and 10 μg of enantiomer per 1 ml, and shake well.

[0038] Test solution: Take an appropriate amount of thiogluconium bromide intermediate, accurately weigh it, dissolve and dilute it with solvent to prepare a solution containing about 0.5 mg per ml, and shake well.

[0039] Control solution: Accurately measure an appropriate amount of the test solution, quantitatively dilute it with solvent to prepare a solution containing approximately 5 μg per 1 ml, and shake well.

[0040] 4) Sample testing methods

[0041] Inject the system suitability solution, test solution, and control solution into the liquid chromatograph under the chromatographic conditions described in the method, and record the chromatograms. In the chromatogram of the system suitability solution, the elution order should be thioglucopyranocyanate intermediate and enantiomer, with a resolution greater than 1.5 between the thioglucopyranocyanate intermediate peak and the enantiomer peak, and the theoretical plate number calculated based on the thioglucopyranocyanate intermediate peak should be no less than 5000. In the chromatogram of the test solution, only the thioglucopyranocyanate intermediate and enantiomer should be integrated, calculated using the self-comparison method of the principal component.

[0042] Depend on Figure 1 The chromatogram for system suitability shows that the thioglucopyranoammonium intermediate and its enantiomers are well separated, indicating that the method has good specificity.

[0043] Depend on Figure 2 As can be seen from the chromatogram of the test solution, the main peak of the thioglucopyranoammonium intermediate is well separated from the enantiomer impurities. Other impurities in the sample do not interfere with the detection of enantiomers, and even low concentrations of enantiomers can be effectively detected.

[0044] Depend on Figure 3 As can be seen from the chromatogram of the control solution, this method has high sensitivity, and the main peak at the limit concentration is sharp and has a high response.

[0045] Example 2

[0046] The detection method of Example 1 was validated in terms of specificity, detection limit and quantitation limit, linearity and range, system precision, repeatability, recovery rate and robustness.

[0047] 1. Exclusivity

[0048] Enantiomer reference standard stock solution: Weigh approximately 1 mg of enantiomer reference standard accurately, place it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0049] System suitability solution: Accurately weigh approximately 2.5 mg of thiogluconium bromide intermediate reference standard, place it in a 5 ml volumetric flask, add 0.5 ml of the enantiomer reference standard stock solution, dissolve and dilute to the mark with solvent, and shake well to obtain the solution.

[0050] Test solution: Weigh approximately 10 mg of thioglucopyranoammonium intermediate accurately, place it in a 20 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well to obtain the test solution.

[0051] Control solution: Accurately measure 200 μl of the test solution, place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0052] Accurately measure 10 μl each of the above-mentioned enantiomer reference standard stock solution, test solution and reference solution, operate according to the chromatographic conditions of Example 1, record the chromatograms, and the results are shown in Table 1.

[0053] Table 1 Results of specificity test

[0054]

[0055]

[0056] As shown in Table 1, the elution order of each component is thioglucopyranoammonium intermediate and enantiomer. In the system suitability solution, the resolution between the enantiomer and the main peak is greater than 1.5, and the theoretical plate number of the main peak is greater than 5000, which meets the requirements. The enantiomer peak detected in the test solution has a high theoretical plate number and good resolution, indicating that the specificity of this method is good.

[0057] 2. Limit of detection and limit of quantitation

[0058] Accurately weigh appropriate amounts of thiogluconium intermediate and enantiomer reference standard, add solvent and dilute to prepare a test solution of a certain concentration, and gradually dilute until the signal-to-noise ratio (S / N) is 10, which is the limit of quantitation; when the signal-to-noise ratio (S / N) is 3, it is the limit of detection. The results are shown in Table 2-3.

[0059] Table 2 Results of Limit of Quantitation Test

[0060] name thiogluconium intermediate Enantiomers Concentration (μg / ml) 0.2768 0.2452 Equivalent to the concentration (%) of the test sample 0.055 0.049 Signal-to-noise ratio (S / N) 18.3 13.7

[0061] Table 3 Results of the detection limit test

[0062] name thiogluconium intermediate Enantiomers Concentration (μg / ml) 0.1575 0.1270 Equivalent to the concentration (%) of the test sample 0.031 0.025 Signal-to-noise ratio (S / N) 8.3 3.1

[0063] As shown in Table 2-3, the limits of quantification for the thiogluconium intermediate and its enantiomer were 0.2768 μg / ml and 0.2452 μg / ml, respectively, and the limits of detection were 0.1564 μg / ml and 0.1279 μg / ml, respectively, with good sensitivity for both.

[0064] 3. Linearity and Range

[0065] Linear stock solution: Take appropriate amounts of thioglucopyranoammonium intermediate and enantiomer reference standard, accurately weigh them, dissolve them in solvent and dilute them to a solution containing approximately 100 μg of thioglucopyranoammonium intermediate and enantiomer per 1 ml.

[0066] Linear solution 1: Accurately measure 0.2 mL of the linear mother liquor, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0067] Linear solution 2: Accurately measure 0.5 mL of the linear mother liquor, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0068] Linear solution 3: Accurately measure 0.8 mL of the linear mother liquor, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0069] Linear solution 4: Accurately measure 1.0 mL of the linear mother liquor, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0070] Linear solution 5: Accurately measure 0.6 mL of the linear mother liquor, place it in a 5 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0071] Linear solution 6: Solution with limit of quantitation.

[0072] Accurately measure 10 μl of each of the above linear solutions and inject them into the liquid chromatograph. Record the chromatograms and perform linear regression with concentration on the x-axis and peak area on the y-axis. The results are shown in Tables 4-5 and 4-5. Figure 4-5 .

[0073] Table 4 Standard Curve for Thiogluconium Bromide Intermediate

[0074]

[0075] As shown in Table 4, the standard curve equation for the thioglucopyran intermediate in the range of 0.2768 μg / ml to 13.29 μg / ml is y = 0.4463x - 0.0009, with a regression coefficient r = 0.9999. The intercept is less than 20% of the y value at the 100% limit concentration, indicating a good linear relationship.

[0076] Table 5 Standard curves for enantiomers

[0077]

[0078]

[0079] As shown in Table 5, the standard curve equation for the enantiomers in the range of 0.2452 μg / ml to 11.77 μg / ml is y = 0.4510x - 0.0104, with a regression coefficient r = 0.9999. The intercept is less than 20% of the y value at the 100% limit concentration, indicating a good linear relationship.

[0080] 4. System precision

[0081] System suitability solution: Take appropriate amounts of thioglucopyranoammonium intermediate and enantiomer reference standard, dissolve and dilute them with solvent to prepare a mixed solution containing approximately 0.5 mg of thioglucopyranoammonium intermediate and 10 μg of enantiomer per 1 ml, and shake well.

[0082] Accurately measure 10 μl of the above system suitability solution, operate under the chromatographic conditions of Example 1, inject the sample 6 times consecutively, record the chromatogram, and the results are shown in Table 6.

[0083] Table 6. Results of System Precision Test

[0084]

[0085]

[0086] As shown in Table 6, the RSD values ​​of the retention times of the thiogluconium intermediate and its enantiomer were all less than 1.0% and the RSD values ​​of the peak areas were all less than 2.0% after six consecutive injections of the system suitability solution, indicating that the system has good precision.

[0087] 5. Repeatability

[0088] System suitability solution: Take appropriate amounts of thioglucopyranoammonium intermediate and enantiomer reference standard, dissolve and dilute them with solvent to prepare a mixed solution containing approximately 0.5 mg of thioglucopyranoammonium intermediate and 10 μg of enantiomer per 1 ml, and shake well.

[0089] Test solution: Take an appropriate amount of thioglucopyranoammonium intermediate, accurately weigh it, dissolve and dilute it with solvent to prepare a solution containing about 0.5 mg per ml, and shake well.

[0090] Control solution: Accurately measure an appropriate amount of the test solution, quantitatively dilute it with solvent to prepare a solution containing approximately 5 μg per 1 ml, and shake well.

[0091] Accurately measure 10 μl each of the above system suitability solution, test solution and control solution, operate under the chromatographic conditions of Example 1, record the chromatograms, and the results are shown in Table 7.

[0092] Table 7 Results of Repeatability Tests

[0093]

[0094] As shown in Table 7, the purity of the main peak of the six test solutions was basically the same, and there was no significant difference in the enantiomer content, indicating that the repeatability of this detection method was good.

[0095] 6. Recovery rate

[0096] Enantiomer reference standard stock solution: Weigh approximately 2 mg of enantiomer reference standard accurately, place it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0097] Recovery solution (50%): Accurately weigh 10 mg of thioglucopyranoammonium intermediate, place it in a 20 ml volumetric flask, add an appropriate amount of solvent to dissolve it, add 0.1 ml of the enantiomer reference standard stock solution, dilute to the mark with solvent, and shake well. Prepare 3 parallel solutions.

[0098] Recovery solution (100%): Accurately weigh 5 mg of thioglucopyranoammonium intermediate, place it in a 10 ml volumetric flask, add an appropriate amount of solvent to dissolve it, add 0.3 ml of the enantiomer reference standard stock solution, dilute to the mark with solvent, and shake well. Prepare 3 parallel solutions.

[0099] Recovery solution (120%): Accurately weigh 10 mg of thioglucopyranoammonium intermediate, place it in a 20 ml volumetric flask, add an appropriate amount of solvent to dissolve it, add 0.8 ml of the enantiomer reference standard stock solution, dilute to the mark with solvent, and shake well. Prepare 3 parallel solutions.

[0100] Test solution: Weigh 10 mg of thioglucopyranoammonium intermediate accurately, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0101] Control solution: Accurately measure 200 μl of the above recovery solution and test solution, place them in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the control solution.

[0102] Accurately measure the above-mentioned recovery solution, test solution and reference solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the recovery rate by peak area according to the self-comparison method of the main component. The results are shown in Table 8.

[0103] Table 8 Results of enantiomer recovery tests

[0104]

[0105] As shown in Table 8, within the limit concentration range of 50% to 120%, the recoveries of enantiomers calculated by the self-comparison method were all between 80% and 120%, with RSD values ​​less than 10%, indicating that this method is suitable for the detection of enantiomers of thioglucopyranoammonium intermediates.

[0106] 7. Durability

[0107] The effects of fine-tuning chromatographic conditions, such as detection wavelength, column temperature, flow rate, type and ratio of mobile phase, and column, on the chromatographic behavior of this method were investigated.

[0108] System suitability solution: Take appropriate amounts of thioglucopyranoammonium intermediate and enantiomer reference standard, dissolve and dilute them with solvent to prepare a mixed solution containing approximately 0.5 mg of thioglucopyranoammonium intermediate and 10 μg of enantiomer per 1 ml, and shake well.

[0109] Test solution: Weigh 10 mg of thioglucopyranoammonium intermediate accurately, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0110] Control solution: Accurately measure 200 μl of the above test solution, place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the control solution.

[0111] The system suitability solution, the test solution, and the control solution were tested under the above-mentioned test conditions, and the results are shown in Table 9.

[0112] Table 9. Durability Test Results

[0113]

[0114]

[0115] As shown in Table 9, fine-tuning the chromatographic conditions, including the detection wavelength, flow rate, column temperature, type and ratio of mobile phase, and column, has no significant impact on system suitability or sample detection. When the flow rate, column temperature, and alcohol ratio in the mobile phase are increased, the resolution between the main peak and isomers in the system suitability solution decreases slightly, and the isomer content in the sample also decreases slightly, but all remain within acceptable limits. When the flow rate, column temperature, and alcohol ratio in the mobile phase are decreased, there is no significant difference in the resolution between the main peak and isomers in the system suitability solution or the isomer content in the sample, indicating that this method has good robustness.

[0116] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A normal-phase chromatographic method for the detection of thiogluconium intermediates and their enantiomers, characterized in that: The liquid chromatography method includes the following steps: 1) Prepare system suitability solution, test solution and control solution for later use; 2) Setting up liquid chromatography detection conditions: A normal phase column coated with cellulose-tris(3,5-dimethylphenylcarbamate) silica gel was used as the packing material; the mobile phase was n-alkane solvent-alcohol solvent-amine tailing inhibitor; 3) Take the system suitability solution, test solution and control solution respectively, inject them into the liquid chromatograph and record the chromatogram.

2. The normal-phase chromatographic analysis and detection method for thiogluconium intermediates and their enantiomers according to claim 1, characterized in that: In step 1), the system suitability solution is prepared by taking appropriate amounts of thioglucopyranoammonium intermediate and enantiomer reference standard, dissolving and diluting them with solvent to prepare a mixed solution containing approximately 0.5 mg of thioglucopyranoammonium intermediate and 10 μg of enantiomer per 1 ml, and shaking well.

3. The normal-phase chromatographic analysis and detection method for the thiogluconium intermediate and its enantiomers according to claim 1, characterized in that: In step 1), the test solution is prepared by accurately weighing an appropriate amount of thioglucopyranoammonium intermediate, dissolving and diluting it with solvent to prepare a solution containing approximately 0.5 mg per ml, and then shaking well.

4. The normal-phase chromatographic analysis and detection method for the thiogluconium intermediate and its enantiomers according to claim 1, characterized in that: In step 1), the preparation of the control solution is as follows: accurately measure an appropriate amount of the test solution, quantitatively dilute it with a solvent to prepare a solution containing approximately 5 μg per 1 ml, and shake well.

5. The normal-phase chromatographic analysis method for the thiogluconium intermediate and its enantiomers according to claim 1, characterized in that: In step 2), the chromatographic column is selected from either YMC CHIRAL ART Cellulose-C, 250 mm × 4.6 mm, 5 μm or DAICEL CHIRALCEL OD-H, 250 mm × 4.6 mm, 5 μm.

6. The normal-phase chromatographic analysis and detection method for the thiogluconium intermediate and its enantiomers according to claim 1, characterized in that: In step 2), the volume ratio of n-alkane solvent, alcohol solvent and amine tailing inhibitor is 88-98:12-2:0.1-0.5; the n-alkane solvent is selected from n-hexane or n-heptane, the alcohol solvent is selected from one, two or three of methanol, ethanol or isopropanol, and the amine tailing inhibitor is selected from diethylamine, dibutylamine or triethylamine.

7. The normal-phase chromatographic analysis and detection method for the thiogluconium intermediate and its enantiomers according to claim 1, characterized in that: In step 2), the flow rate is 0.6–1.5 ml / min.

8. The normal-phase chromatographic analysis and detection method for the thiogluconium intermediate and its enantiomers according to claim 1, characterized in that: In step 2), the column temperature is 20–40°C; the sample pan temperature is 4–10°C.

9. The normal-phase chromatographic analysis method for the thiogluconium intermediate and its enantiomers according to claim 1, characterized in that: In step 2), the detection wavelength is 230–245 nm; the running time is 15 min.

10. The normal-phase chromatographic analysis and detection method for the thiogluconium intermediate and its enantiomers according to claim 1, characterized in that: In step 2), the injection volume is 10 μl.

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