A method for detecting the purity and specific impurity L-thioproline in a semi-leafy vegetable

Abstract

The application discloses a detection method for detecting the purity and specific impurity L-thio-proline in Banxia, which detects Banxia products by using HPLC. The liquid chromatography method of the application selects YMC-Pack ODS-AQ type octadecylsilane bonded silica gel reversed-phase chromatographic column (USP L1); the chromatographic column filler is monomolecular bonded C18 silica gel, which is hydrophilic modified and capped, and is suitable for a high aqueous mobile phase system. The mobile phase is a reverse solvent: phosphoric acid aqueous solution-acetonitrile solution. The liquid chromatography method adopts an ultraviolet detector. The application has a high response value, can effectively separate products and impurities, has simple detection operation, high accuracy, and meets the requirements of specificity, linearity and range, and solves the problems of purity detection and raw material L-thio-proline control. The specificity is strong, the sensitivity meets the requirements of pharmacopoeia and chemical industry quality control, and the application is suitable for industrialized popularization and application.

Description

Technical Field

[0001] This invention discloses a liquid phase detection method for the purity of hemifolin and specific impurities, belonging to the field of analytical detection technology. Background Technology

[0002] Hemifolin, chemically known as N-acetylthioproline or 3-acetylthiazolidin-4-carboxylic acid, with the molecular formula C6H9NO3S, is a white crystalline powder at room temperature with a slightly acidic odor and is readily soluble in various organic solvents. Hemifolin possesses excellent plant growth regulating activity, capable of regulating plant cell osmotic pressure, maintaining the balance of water and nutrient transport within the plant, promoting seed germination and plant cell division and growth, reducing chlorophyll loss, and significantly improving crop fruit setting rate and fruit yield. It is now widely used in various agricultural plant growth regulator products.

[0003] The effective content of hemifolin directly determines the field application effect of agricultural inputs, and accurate quantitative detection is the core foundation for raw material quality control and finished product quality standard management. The molecular structure of hemifolin contains carboxyl and amide groups, and its overall polarity is extremely strong. Conventional ordinary C18 reversed-phase chromatography columns have very weak retention capacity for it, which easily leads to problems such as peak tailing and co-elution of the main component and matrix impurities, making it difficult to achieve effective separation and resulting in insufficient quantitative accuracy and specificity.

[0004] Chinese patent application CN116482273A (application date 2023.05.19, invention title: A liquid chromatography method for determining hemifolinin content) discloses a hemifolinin content detection scheme. This scheme uses a HILIC hydrophilic interaction mode chromatographic column, coupled with a diode array detector for detection. The mobile phase system consists of an organic phase and a sodium dihydrogen phosphate buffer solution, with the volume ratio of organic phase to buffer solution controlled to be greater than 60:40 and less than 90:10. Hemifolinin is separated from other components in the sample by relying on hydrophilic interaction, which to some extent improves the defect of insufficient retention of hemifolinin by ordinary C18 chromatographic columns. The detection method has good reproducibility and accuracy.

[0005] However, the previously disclosed HILIC liquid chromatography detection system still has significant technical shortcomings: First, the system requires a continuous use of more than 60% organic phase as the mobile phase. For hemifolin samples such as aqueous solutions and water-soluble fertilizers that use aqueous phase as the extraction medium, complex solvent replacement operations are required before instrument detection, making the sample pretreatment process cumbersome and the detection cycle long. Second, the HILIC hydrophilic column is highly sensitive to the concentration of mobile phase buffer salts, pH value, and ambient temperature and humidity. Column equilibration takes a long time, and the reproducibility of detection results varies greatly between different batches and different laboratories. Third, this method can only quantify the main components of hemifolin and has poor selectivity for separating trace polar process impurities in the sample. It cannot simultaneously complete the determination of the main component content and the monitoring of related impurities in the same chromatographic program, making it difficult to meet the industry needs for comprehensive quality control of raw material purity and rapid screening of samples from large-scale production.

[0006] Therefore, there is an urgent need in this field to develop a reversed-phase liquid chromatography detection method that is compatible with highly polar hemifolin, has simple sample pretreatment, rapid chromatographic equilibration, can simultaneously determine the content of main components and trace impurities, and has excellent stability, so as to improve the whole-process quality control system for hemifolin-based agricultural products. Summary of the Invention

[0007] This invention addresses the shortcomings of existing central control detection methods and technologies by providing a method for detecting the purity and specific impurity L-thioproline in hemifolin. This method features high separation, good stability, high precision, high accuracy, and simple operation, meeting the needs of daily quality testing and standard establishment for hemifolin raw materials and finished products. It has significant practical value and application prospects.

[0008] Therefore, the technical solution adopted in this invention is as follows:

[0009] A method for detecting the purity and specific impurity L-thioproline in hemifolin, comprising the following steps:

[0010] The test sample was dissolved in a diluent and detected by liquid chromatography. The chromatographic column used in the liquid chromatography was an octadecylsilane-bonded silica reversed-phase column, which was hydrophilically modified and end-capped, and adapted to high-aqueous mobile phase systems. Mobile phase A was an aqueous solution containing 0.1% phosphoric acid, and mobile phase B was an acetonitrile solution. The liquid chromatography was performed using an ultraviolet detector with a detection wavelength of 210 nm.

[0011] The gradient elution procedure for liquid chromatography is shown in Table 1 below:

[0012] The column temperature is 20℃-40℃, the mobile phase flow rate is 0.8-1.2 ml / min, and the injection volume is 5-15 μl.

[0013] The purity of hemifolin and the content of the specific impurity L-thioproline were calculated using the area normalization method.

[0014] Preferably, the chromatographic column is a YMC-Pack ODS-AQ type octadecylsilane-bonded silica reversed-phase chromatographic column with dimensions of 4.6×250mm×5μm.

[0015] Preferably, the column temperature is 30°C, the mobile phase flow rate is 1.0 ml / min, and the injection volume is 10 μl.

[0016] Preferably, the concentration of the test solution is 0.8-1.2 mg / ml.

[0017] Preferably, the diluent is an acetonitrile-water mixed solvent, wherein the volume ratio of acetonitrile to water is 2:8.

[0018] Furthermore, the detection method also includes a step of determining a standard curve, specifically including:

[0019] S01) Accurately weigh 10 mg of L-thioproline into a 10 ml volumetric flask, dissolve and dilute to the mark with diluent to prepare L-thioproline stock solution; then accurately weigh 1 g of hemifolin into a 100 ml volumetric flask, transfer 1 ml of the above L-thioproline stock solution, dilute to the mark with diluent to prepare a standard stock solution containing 10 mg / ml hemifolin and 10 μg / ml L-thioproline;

[0020] S02) Accurately measure 1.0 ml, 2.5 ml, 4.0 ml, 5.0 ml, 6.0 ml, and 7.5 ml of the standard stock solution into 50 ml volumetric flasks, and dilute to the mark with diluent to obtain standard curve solutions containing 0.2 mg / ml, 0.5 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml, and 1.5 mg / ml of hemifolin and 0.2 μg / ml, 0.5 μg / ml, 0.8 μg / ml, 1.0 μg / ml, 1.2 μg / ml, and 1.5 μg / ml of L-thioproline;

[0021] S03) Take the standard curve solution and perform high performance liquid chromatography analysis according to the above chromatographic conditions, and plot the standard curve with concentration as the abscissa and chromatographic peak area as the ordinate.

[0022] Furthermore, the limit of quantification (LOQ) for the specific impurity L-thioproline is 0.20 μg / ml, and the limit of detection (LOD) is 0.06 μg / ml.

[0023] Furthermore, the test sample is hemifolin technical, hemifolin aqueous solution, or hemifolin water-soluble fertilizer.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. Excellent retention and peak shape of highly polar target substances, resulting in stronger separation selectivity: The YMC-Pack ODS-AQ column stationary phase has a hydrophilically modified surface, providing sufficient reversed-phase retention for highly polar hemifolin containing carboxyl and amide structures. This avoids the elution of target substances with dead volume without requiring a high organic phase environment. The addition of 0.1% phosphoric acid to the mobile phase inhibits the dissociation of hemifolin carboxyl groups, effectively eliminating peak tailing. At the same time, it can achieve baseline separation of various polar impurities generated in the hemifolin synthesis process. One chromatographic program can simultaneously complete the quantification of the main component content and the limit check of trace impurities, making up for the deficiency of the HILIC system in that it cannot simultaneously monitor impurities.

[0026] 2. Suitable for water-based samples, simple and efficient pretreatment. The mobile phase of this invention uses an aqueous buffer solution as mobile phase A, which can directly inject water-containing matrix samples such as aqueous agents, water-soluble fertilizers, and water-extracted raw materials without complicated pretreatment operations, greatly simplifying the sample preparation steps and meeting the needs of rapid detection for quality control in large-scale production in factories.

[0027] 3. The chromatographic system is stable, the equilibration is fast, and the detection reproducibility is excellent. This invention adopts a conventional reversed-phase binary gradient system, and the chromatographic column does not need to be equilibrated for a long time, which is durable. Compared with the HILIC mode, which is extremely sensitive to salt concentration, this method has a stable chromatographic baseline, a smaller precision RSD, and stronger method durability.

[0028] 4. Lower reagent costs and greater applicability: This invention uses only phosphoric acid aqueous solution and acetonitrile as the mobile phase; the YMC-Pack ODS-AQ is a conventional reversed-phase chromatographic column, eliminating the need to purchase special chromatographic consumables, making it easy for various agricultural and chemical enterprises in the industry to directly implement and use, and facilitating the establishment of unified hemifolin quality testing standards.

[0029] 5. Wide linear range, suitable for detection of all types of raw materials and formulations. This system can cover both the low concentration linear range of trace impurities and the high content linear range of the main component of hemifolin through gradient elution. It can be used for the purity determination of high-purity hemifolin raw materials, as well as the detection of active ingredients in low-content foliar fertilizers and plant growth regulators, and has a wider range of applicable samples. Attached Figure Description

[0030] Figure 1 Linear graph of L-thioproline.

[0031] Figure 2 This is a linear graph of hemifolin elements.

[0032] Figure 3 The chromatogram is of the hemifolin test solution.

[0033] Figure 4This is a chromatogram for the suitability test of the hemifolin system. Detailed Implementation

[0034] The following will provide further details with reference to embodiments of the present invention.

[0035] Example 1

[0036] 1. Instruments and reagents

[0037] High performance liquid chromatograph (equipped with ultraviolet detector), electronic analytical balance (1 / 100,000), acetonitrile (chromatographic grade), phosphoric acid (85%-90%, chromatographic grade).

[0038] 2. High-performance liquid chromatography (HPLC) analysis conditions:

[0039] Chromatographic column: YMC-Pack ODS-AQ hydrophilic reversed-phase C18 column (4.6×250mm×5μm, Agilent)

[0040] Mobile phase A: 0.1% aqueous solution of phosphoric acid (V / V)

[0041] Mobile phase B: Acetonitrile;

[0042] The detection wavelength for liquid chromatography is 210 nm;

[0043] The column temperature for liquid chromatography is 30℃.

[0044] The mobile phase flow rate was 1.0 ml / min, and the elution method was gradient elution. The gradient elution conditions are shown in Table 2 below:

[0045] The injection volume for liquid chromatography is 10 μl.

[0046] 3. Solution preparation:

[0047] Preparation of mobile phase:

[0048] Mobile phase A (aqueous solution containing 0.1% phosphoric acid): Take 1.0 ml of phosphoric acid, add water to 1000 ml, and mix well;

[0049] Mobile phase B: Acetonitrile;

[0050] Preparation of test solution: Weigh 10 mg of hemifolin test sample accurately into a 10 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well to obtain a test solution with a concentration of 1 mg / ml.

[0051] Blank solution: acetonitrile-water (2:8, v / v).

[0052] 3. Measurement

[0053] HPLC was used for detection, and the detection conditions were as described above.

[0054] The blank, system suitability solution, and test solution were injected into the liquid chromatograph under the chromatographic conditions described in section 2 above, and the chromatograms were recorded. In the system suitability solution, the resolution between hemifolin, the impurity L-thioproline, and surrounding impurities was 3.76, greater than 1.5, meeting the purity requirements for hemifolin. The results are as follows. Figure 2 As shown.

[0055] Example 2

[0056] 1. Exclusivity

[0057] 1.1 Solution preparation:

[0058] Blank / Diluent: Acetonitrile-water (2:8, v / v)

[0059] L-Thioproline stock solution: Take 10 mg of L-thioproline and place it in a 100 ml volumetric flask. Dissolve it with diluent and then dilute to the mark.

[0060] System suitability solution: Take 100 mg of hemifolin and place it in a 100 ml volumetric flask. Then, transfer 1 ml of L-thioproline stock solution to the same volumetric flask, add diluent to dissolve, and dilute to the mark.

[0061] Test solution: Weigh 10 mg of hemifolin accurately, place it in a 10 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well to obtain a hemifolin solution with a concentration of 1 mg / ml.

[0062] 1.2 Determination Method

[0063] Inject blank, system suitability solution, and test solution in the following order and record the chromatogram.

[0064] The injection sequence is shown in Table 3 below:

[0065] 1.3 Test Results: No interference was observed in the blank; the resolution between the peaks of each component in the system suitability solution was greater than 1.5, meeting the detection requirements; the relative deviation between the retention time of the main peak in the test solution and the retention time of the main peak in the system suitability solution was 0.78%, indicating that the retention times were consistent.

[0066] The experimental results are shown in Table 4 below:

[0067] 2. Linearity and Range

[0068] 2.1 Solution preparation:

[0069] Blank / Diluent: Acetonitrile-water (2:8, v / v)

[0070] L-Thioproline stock solution: Accurately weigh 10 mg of L-thioproline into a 10 ml volumetric flask, dissolve and dilute to the mark with diluent;

[0071] Linear stock solution: Take 1g of hemifolin and place it in a 100ml volumetric flask. Then transfer 1ml of the above L-thioproline stock solution to the same 100ml volumetric flask, dissolve and dilute to the mark with diluent to prepare a standard solution containing 10mg / ml hemifolin and 10μg / ml L-thioproline.

[0072] Accurately pipette 1.0 ml, 2.5 ml, 4.0 ml, 5.0 ml, 6.0 ml, and 7.5 ml into 50 ml volumetric flasks, and then dilute to the mark with diluent to obtain standard curve solutions containing 0.2 mg / ml, 0.5 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml, and 1.5 mg / ml of hemifolin and 0.2 μg / ml, 0.5 μg / ml, 0.8 μg / ml, 1.0 μg / ml, 1.2 μg / ml, and 1.5 μg / ml of L-thioproline.

[0073] 20% linear solution ① Preparation: Take 1.0 ml of linear stock solution and place it in a 50 ml volumetric flask. Add diluent to the mark and shake well.

[0074] 50% linear solution ② Preparation: Take 2.5 ml of linear stock solution and place it in a 50 ml volumetric flask. Add diluent to the mark and shake well.

[0075] 80% linear solution ③ Preparation: Take 4.0 ml of linear stock solution and place it in a 50 ml volumetric flask. Add diluent to the mark and shake well.

[0076] 100% linear solution ④ Preparation: Take 5.0 ml of linear stock solution and place it in a 50 ml volumetric flask. Add diluent to the mark and shake well.

[0077] 120% linear solution ⑤ Preparation: Take 6.0 ml of linear stock solution and place it in a 50 ml volumetric flask. Add diluent to the mark and shake well.

[0078] Preparation of 150% linear solution ⑥: Take 7.5 ml of linear stock solution and place it in a 50 ml volumetric flask, add diluent to the mark, and shake well.

[0079] 2.2 Determination Method:

[0080] Take linear solutions ① through ⑥, inject each twice, and record the chromatograms. Perform linear regression using the concentrations and peak areas of the linear solutions, and plot a standard curve.

[0081] 2.3 Experimental Results:

[0082] Table 5: Linearity data for L-thioproline

[0083] Table 6: Linear Data of Hemifolin

[0084] Linear regression was performed with concentration as the abscissa (x) and peak area as the ordinate (y). The results showed that, under the chromatographic conditions described in Example 1, L-thioproline exhibited good linearity in the concentration range of 0.20 μg / ml to 1.52 μg / ml, with the linear equation y = 13066.4492x + 32.9223 and R = 0.9990; hemifolin also showed good linearity in the concentration range of 0.20 mg / ml to 1.53 mg / ml, with the linear equation y = 13066.4492x + 32.9223 and R = 0.9999, both meeting the detection requirements.

[0085] The above content is a further detailed description of the technical solution provided in conjunction with the preferred embodiments of the present invention. It should not be considered that the specific implementation of the present invention is limited to the above description. For those skilled in the art, any simple deductions or substitutions made without departing from the concept of the present invention should be considered as necessary to fall within the protection scope of the present invention.

[0086] 3. Limit of Detection and Limit of Quantification

[0087] L-Thioproline Limit of Quantitation (LOQ) Solution: Take 1.0 ml of 20% linear solution ① to obtain the solution.

[0088] L-Thioproline detection limit (LOD) solution: Take 3.0 ml of the quantitation limit solution and pour it into a 10 ml volumetric flask. Add diluent to the mark and shake well.

[0089] Take 10 μL of the limit of quantitation (LOQ) solution and the limit of detection (LOD) solution, and inject them into the liquid chromatograph. Inject the LOD solution twice consecutively and the LOD solution six times consecutively. Record the chromatograms and calculate the signal-to-noise ratio (S / N). The results are shown in Tables 7 and 8.

[0090] Table 7. Detection results of specific impurity L-thioproline at the limit of quantitation

[0091] Table 8 Detection Limit Results for Specific Impurity L-Thioproline

[0092] As shown in Tables 7 and 8, the method of the present invention has high detection sensitivity and can accurately determine the content of the specific impurity L-thioproline, thus meeting the detection requirements.

[0093] Example 3

[0094] A liquid chromatography method for detecting the purity of hemifolin products and the specific impurity L-thioproline, specifically including the following steps:

[0095] Take different batches of hemifolin test samples, accurately weigh appropriate amounts of each sample, dissolve and dilute them with acetonitrile-water (2:8, V / V, V / V) mixed solvent, and prepare a test sample solution with a concentration of 1 mg / mL.

[0096] Referring to the liquid chromatography conditions specified in Example 1, blank solvent and each batch of test solution were injected sequentially, and chromatograms were collected and recorded. The purity of the main peak of hemifolin was calculated using the area normalization method, and the percentage content of the characteristic impurity L-thioproline was also calculated. The test data results for each batch are shown in Table 9.

[0097] Table 9 Results of hemifolin detection for each batch

[0098] As shown in the table above, the differences between batches are small. This method can accurately detect the purity of hemifolin and the content of the specific impurity L-thioproline, meeting the experimental requirements.

[0099] The results show that this high-performance liquid chromatography method can accurately quantify the purity of the main peak of hemifolin and the content of the specific impurity L-thioproline. After methodological validation, the area normalization method was used to control the quality of the main components and key impurities of the product. The detection results are accurate and the data are reliable. It can be adapted to the daily quality inspection of hemifolin products and can meet the quality control requirements of the whole process of the product. It has strong practical application value and promotion prospects.

[0100] Example 4

[0101] Comparative Study of the Invention Method and Existing HILIC Methods

[0102] By conducting parallel comparative experiments with the closest prior art (HILIC hydrophilic interaction chromatography disclosed in CN116482273A), the method of the present invention was verified in terms of separation effect, peak shape symmetry, system equilibrium speed and adaptability to water-based samples of hemifolin and specific impurity L-thioproline.

[0103] Comparison method

[0104] The HILIC comparison group was set according to the conditions described in Example 1 of CN116482273A:

[0105] Chromatographic column: Hydrophilic interaction column (HILIC, 4.6×150mm, 5μm)

[0106] Mobile phase: Acetonitrile-25mM sodium dihydrogen phosphate buffer (pH 3.0) = 75:25 (V / V)

[0107] Column temperature: 30℃

[0108] Detection wavelength: 210nm

[0109] Flow rate: 1.0 ml / min

[0110] Isocratic elution

[0111] Test sample

[0112] Sample A: Hemifolin test solution (1.0 mg / ml, solvent: acetonitrile-water = 2:8, v / v)

[0113] Sample B: System suitability solution (hemifolin 1.0 mg / ml + L-thioproline 10 μg / ml, solvent as above)

[0114] Experimental results

[0115] Table 10 Comparison of key chromatographic parameters between the method of this invention and the HILIC method.

[0116] Table 11 Comparison of the durability of the method of the present invention and the HILIC method.

[0117] Table 12 Comparison of linear range between the method of this invention and the HILIC method.

[0118] The comparative experimental results from Tables 10 to 12 show that:

[0119] (1) Significantly superior separation selectivity: The method of this invention, using a YMC-Pack ODS-AQ hydrophilic reversed-phase column and a 0.1% phosphoric acid aqueous solution-acetonitrile gradient elution system, can achieve complete baseline separation of hemifolin and the polar impurity L-thioproline (resolution 2.81), which is far superior to the HILIC method (resolution 1.25). This is because the hydrophilic modified C18 stationary phase used in this invention retains the selective separation ability of reversed-phase chromatography for non-polar / moderately polar impurities while retaining the polar compounds, whereas in the HILIC mode, polar impurities elute prematurely and are not sufficiently separated from the main peak.

[0120] (2) Significantly improved peak shape quality: In the method of this invention, the tailing factor of hemifolin is 1.07 and the tailing factor of L-thioproline is 0.91, both within the ideal symmetrical peak range (0.9-1.2); while in the HILIC method, both show obvious tailing (1.33 and 1.68, respectively), which seriously affects the accuracy of integration. The addition of 0.1% phosphoric acid to the mobile phase effectively inhibits the dissociation of carboxyl groups in the hemifolin structure and eliminates peak broadening caused by secondary interactions.

[0121] (3) Enhanced system stability and durability: When the column temperature, pH value and organic phase ratio of the present invention fluctuate reasonably, the resolution change is less than 0.4, which is much lower than the change of the HILIC method (up to 0.7). This indicates that the present invention is less sensitive to operating parameters and is more suitable for the reproducibility requirements of different laboratories and different operators in industrial applications.

[0122] (4) Significantly improved operational efficiency and versatility: The column equilibration time of the method of this invention is only one-third that of the HILIC method, and it allows direct injection of samples with an aqueous phase as the main component without the need for solvent replacement. This has important practical significance for the rapid quality control of hemifolin preparations with high water content, such as aqueous solutions and water-soluble fertilizers.

[0123] (5) Wider linear range and more accurate quantification: The method of the present invention shows excellent linearity (correlation coefficient R≥0.9990) in the range of 0.2-1.5 mg / ml of hemifolin and 0.2-1.5 μg / ml of L-thioproline. The quantification ability at the low concentration end is better than that of the HILIC method, which is beneficial to the accurate control of trace impurities.

[0124] This invention is not limited to the above embodiments. Based on the technical solutions disclosed in this invention, those skilled in the art can make some substitutions and modifications to some of the technical features without creative effort, and all such substitutions and modifications are within the protection scope of this invention.

Claims

1. A method for detecting the purity and specific impurity L-thioproline in hemifolin, characterized in that, Includes the following steps: The test sample was dissolved in a diluent and detected by liquid chromatography. The liquid chromatography column used is an octadecylsilane-bonded silica reversed-phase column, which is a hydrophilically modified end-capped column adapted to high aqueous mobile phase systems. Mobile phase A is an aqueous solution containing 0.1% phosphoric acid, and mobile phase B is an acetonitrile solution; The liquid chromatography method uses an ultraviolet detector with a detection wavelength of 210 nm. The liquid chromatography method employs a gradient elution process. The gradient elution program is as follows: 0-5 min: mobile phase A is maintained at 100% and mobile phase B at 0%; 5-20 min: mobile phase A linearly decreases from 100% to 30% and mobile phase B linearly increases from 0% to 70%; 20-25 min: mobile phase A is maintained at 30% and mobile phase B at 70%; 25-25.1 min: mobile phase A linearly increases from 30% to 100% and mobile phase B linearly decreases from 70% to 0%; 25.1-35 min: mobile phase A is maintained at 100% and mobile phase B at 0%. The column temperature is 20℃-40℃, the mobile phase flow rate is 0.8-1.2 ml / min, and the injection volume is 5-15 μl. The purity of hemifolin and the content of the specific impurity L-thioproline were calculated using the area normalization method.

2. The method for detecting the purity and specific impurity L-thioproline in hemifolin according to claim 1, characterized in that, The chromatographic column is a YMC-Pack ODS-AQ type octadecylsilane-bonded silica reversed-phase chromatographic column with dimensions of 4.6×250mm×5μm.

3. The method for detecting the purity and specific impurity L-thioproline in hemifolin according to claim 1, characterized in that, The column temperature is 30℃, the mobile phase flow rate is 1.0 ml / min, and the injection volume is 10 μl.

4. The method for detecting the purity and specific impurity L-thioproline in hemifolin according to claim 1, characterized in that, The concentration of the test solution is 0.8-1.2 mg / ml.

5. The method for detecting the purity and specific impurity L-thioproline in hemifolin according to claim 1, characterized in that, The diluent is an acetonitrile-water mixed solvent, wherein the volume ratio of acetonitrile to water is 2:

8.

6. The method for detecting the purity and specific impurity L-thioproline in hemifolin according to claim 1, characterized in that, It also includes the steps for determining the standard curve, specifically including: S01) Accurately weigh 10 mg of L-thioproline into a 10 ml volumetric flask, dissolve and dilute to the mark with diluent to prepare L-thioproline stock solution; then accurately weigh 1 g of hemifolin into a 100 ml volumetric flask, transfer 1 ml of the above L-thioproline stock solution, dilute to the mark with diluent to prepare a standard stock solution containing 10 mg / ml hemifolin and 10 μg / ml L-thioproline; S02) Accurately measure 1.0 ml, 2.5 ml, 4.0 ml, 5.0 ml, 6.0 ml, and 7.5 ml of the standard stock solution into 50 ml volumetric flasks, and dilute to the mark with diluent to obtain standard curve solutions containing 0.2 mg / ml, 0.5 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml, and 1.5 mg / ml of hemifolin and 0.2 μg / ml, 0.5 μg / ml, 0.8 μg / ml, 1.0 μg / ml, 1.2 μg / ml, and 1.5 μg / ml of L-thioproline; S03) Take the standard curve solution and perform high performance liquid chromatography analysis according to the chromatographic conditions described in claim 1, and plot the standard curve with concentration as the abscissa and chromatographic peak area as the ordinate.

7. The method for detecting the purity and specific impurity L-thioproline in hemifolin according to claim 1, characterized in that, The limit of quantification (LOQ) for the specific impurity L-thioproline is 0.20 μg / ml, and the limit of detection (LOD) is 0.06 μg / ml.

8. The method for detecting the purity and specific impurity L-thioproline in hemifolin according to claim 1, characterized in that, The test sample is hemifolin technical, hemifolin aqueous solution, or hemifolin water-soluble fertilizer.

Images