High performance liquid detection method for glycoluril tetramer

Abstract

The invention discloses a high performance liquid detection method for glycoluril tetramer. The invention provides a high performance liquid chromatography detection method for glycoluril tetramer, which comprises the following steps: carrying out gradient elution on a glycoluril tetramer sample solution in a chromatographic column by adopting high performance liquid chromatography; the filler of the chromatographic column is octadecyl silane bonded silica gel; the mobile phase A is prepared from the following components in percentage by volume: 5 percent to 50 percent of hexafluoroisopropanol and 50 percent to 95 percent of water; the mobile phase B is prepared from the following components in percentage by volume: 20 percent to 100 percent of hexafluoroisopropanol and 0 percent to 80 percent of water. The detection method can sensitively, accurately and quickly detect the related substances and purity of the glycoluril tetramer with good reproducibility, and has outstanding technical advantages.

Description

Technical Field

[0001] This invention relates to the field of detection and analysis technology, specifically to a high-performance liquid chromatography method for the detection of glycourea tetramer. Background Technology

[0002] Cucurbituril, due to its strong structural rigidity, high binding strength with guest molecules, and high selectivity, has become a research hotspot in materials science and medicine. Glycourea tetramer is a crucial raw material for the synthesis of cucurbituril and its derivatives, and its purity plays a vital role in the synthesis of cucurbituril. However, due to its extremely poor solubility, analytical methods for glycourea tetramer are limited. To date, no high-performance liquid chromatography (HPLC) method for the detection of glycourea tetramer has been published. Therefore, providing an HPLC method for the detection of glycourea tetramer is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0003] The technical problem this invention aims to solve is the lack of a high-performance liquid chromatography (HPLC) method for the detection of glycourea tetramer in the prior art. Therefore, this invention provides an HPLC method for the detection of glycourea tetramer. This method can sensitively, accurately, rapidly, and with good reproducibility detect related substances and purity of glycourea tetramer, exhibiting significant technical advantages.

[0004] This invention provides a high-performance liquid chromatography (HPLC) method for detecting glycourea tetramer, comprising the following steps: using HPLC to perform gradient elution of the glycourea tetramer sample solution in a chromatographic column;

[0005] The chromatographic column is packed with octadecylsilane-bonded silica gel.

[0006] The gradient elution mobile phase consists of mobile phase A and mobile phase B;

[0007] The mobile phase A comprises the following components by volume percentage: 5% to 50% hexafluoroisopropanol and 50% to 95% water; the volume percentage is the percentage of the volume of each component relative to the volume of the mobile phase A.

[0008] The mobile phase B comprises the following components by volume percentage: 20% to 100% hexafluoroisopropanol and 0% to 80% water; the volume percentage is the percentage of the volume of each component relative to the volume of the mobile phase B.

[0009] During the gradient elution process, the volume percentage of mobile phase A in the mobile phase decreases from 90%~100% to 0~75%; the volume percentage is the percentage of the volume of mobile phase A to the total volume of the mobile phase.

[0010] The structure of the glycouretetramer is shown in the following formula:

[0011] .

[0012] In one embodiment, the mobile phase A comprises the following components by volume percentage: 5% to 25% hexafluoroisopropanol and 75% to 95% water; for example, 5% to 15% hexafluoroisopropanol and 85% to 95% water; or, for example, 10% hexafluoroisopropanol and 90% water.

[0013] In one embodiment, the mobile phase A consists of the following components by volume percentage: 5% to 50% hexafluoroisopropanol and 50% to 95% water; for example, 5% to 15% hexafluoroisopropanol and 85% to 95% water; or, for example, 10% hexafluoroisopropanol and 90% water.

[0014] In one embodiment, the mobile phase B comprises the following components by volume percentage: 30% to 100% hexafluoroisopropanol and 0% to 70% water; for example, 35% to 100% hexafluoroisopropanol and 0% to 70% water; or, for example, 40% hexafluoroisopropanol and 60% water, 70% hexafluoroisopropanol and 30% water or hexafluoroisopropanol.

[0015] In one embodiment, the mobile phase B comprises the following components by volume percentage: 20% to 100% hexafluoroisopropanol and 0% to 80% water; for example, 35% to 100% hexafluoroisopropanol and 0% to 70% water; or, for example, 40% hexafluoroisopropanol and 60% water, 70% hexafluoroisopropanol and 30% water or hexafluoroisopropanol.

[0016] In one embodiment, during the gradient elution process, the volume percentage of mobile phase A in the mobile phase decreases from 95%~100% to 0~70%; for example, from 100% to 0~70%; or for example, from 100% to 0, from 100% to 50%, or from 100% to 67%.

[0017] In one implementation scheme, the decrease is a uniform decrease.

[0018] In one embodiment, the gradient elution duration is 12-20 minutes, for example, 12 minutes or 15 minutes, where the duration is the total time from the start point to the end point of the gradient elution process.

[0019] In one embodiment, the packing material of the chromatographic column is octadecylsilane-bonded ethylene-bridged hybrid particles.

[0020] In one embodiment, the length of the chromatographic column is 100 mm to 300 mm; for example, 140 mm to 160 mm; or for example, 150 mm.

[0021] In one embodiment, the inner diameter of the chromatographic column is 1 mm to 10 mm; for example, 4 mm to 5 mm; or for example, 4.6 mm.

[0022] In one embodiment, the particle size of the filler is 1 μm to 10 μm; for example, 3 μm to 4 μm; or for example, 3.5 μm.

[0023] In one embodiment, the chromatographic column has the following specifications: a column length of 150 mm, an inner diameter of 4.6 mm, and a packing particle size of 3.5 μm; for example, Waters Xbridge C18 150*4.6 mm 3.5 μm.

[0024] In one embodiment, the flow rate of the high-performance liquid chromatography is 0.5 to 3 mL / min; for example, 0.5 to 1.5 mL / min; or for example, 1 mL / min.

[0025] In one embodiment, the solvent of the glycourea tetramer sample solution comprises the following components by volume percentage: 10% to 100% hexafluoroisopropanol and 0% to 90% water; for example, 35% to 100% hexafluoroisopropanol and 0% to 65% water, or for example, 40% hexafluoroisopropanol and 60% water, 70% hexafluoroisopropanol and 30% water or hexafluoroisopropanol.

[0026] In one embodiment, the solvent of the glycourea tetramer sample solution is composed of the following components by volume percentage: 10% to 100% hexafluoroisopropanol and 0% to 90% water; for example, 35% to 100% hexafluoroisopropanol and 0% to 65% water, or for example, 40% hexafluoroisopropanol and 60% water, 70% hexafluoroisopropanol and 30% water or hexafluoroisopropanol.

[0027] In one embodiment, the concentration of the glycourea tetramer sample in the glycourea tetramer sample solution is (0.002-3) mg / mL, for example (0.002-2.3) mg / mL, or for example 2 mg / mL.

[0028] In one embodiment, the glycourea tetramer sample is obtained by the following preparation method, which includes the following steps: in a solvent, compound 3 and compound 4 react as shown in the following formula to obtain the glycourea tetramer;

[0029] .

[0030] In one embodiment, the solvent is a sulfonic acid solvent, such as methanesulfonic acid.

[0031] In one embodiment, the ratio of the number of moles of compound 3 to the volume of the solvent is (0.1-1) mol / L, for example, 0.45 mol / L.

[0032] In one embodiment, the molar ratio of compound 4 to compound 3 is (1-5):1, for example, 4.4:1.

[0033] In one embodiment, the reaction temperature is 40°C-80°C, for example, 50°C.

[0034] In one implementation, the reaction time is 2h-6h, for example 3h.

[0035] In one embodiment, the reaction is followed by a post-processing step, which is filtration and recrystallization. The solvent for recrystallization is trifluoroacetic acid and water, for example, the volume ratio of trifluoroacetic acid to water is 1:4.

[0036] In one embodiment, compound 3 is prepared by a method comprising the following steps: reacting glycourea and paraformaldehyde in a solvent to obtain compound 3.

[0037] In one embodiment, in the preparation method of compound 3, the solvent is an aqueous hydrochloric acid solution, such as an 8M aqueous hydrochloric acid solution. The amount of solvent is not limited, as long as it does not affect the reaction.

[0038] In one embodiment, in the preparation method of compound 3, the molar ratio of paraformaldehyde and glycourea is (1-3):1, for example 1:1.

[0039] In one embodiment, in the preparation method of compound 3, the reaction temperature is 40°C-80°C, for example, 50°C.

[0040] In one embodiment, the preparation method of compound 3 further includes post-treatment steps of filtration, washing (e.g., with water) and crystallization after the reaction is completed, wherein the solvent for recrystallization is trifluoroacetic acid.

[0041] In one embodiment, compound 4 is prepared by a method comprising the following steps: dimethylglyoxal ( Compound 4 is obtained by reacting compound 4 and paraformaldehyde in a solvent.

[0042] In one embodiment, in the preparation method of compound 4, the solvent is an aqueous solution of hydrochloric acid, such as an 8M aqueous solution of hydrochloric acid. The amount of solvent is not limited, as long as it does not affect the reaction.

[0043] In one embodiment, in the preparation method of compound 3, the molar ratio of paraformaldehyde and glycourea is (1-10):1, for example 5:1.

[0044] In one embodiment, in the preparation method of compound 3, the reaction temperature is 40°C-80°C, for example, 50°C.

[0045] In one embodiment, the preparation method of compound 3 further includes post-treatment steps of filtration, washing (e.g., water and ethanol) and crystallization after the reaction is completed, wherein the solvent for recrystallization is trifluoroacetic acid.

[0046] In one embodiment, the dimethylglycourea is prepared by reacting 2,3-butanedione and urea in an aqueous hydrochloric acid solution.

[0047] In one embodiment, the injection volume of the high-performance liquid chromatography is 5~30 μl; for example, 10~20 μl; or for example, 15 μl.

[0048] In one embodiment, the column temperature of the high-performance liquid chromatography is 30~50℃, for example 40~50℃, or for example 45℃.

[0049] In one embodiment, the detector for the high-performance liquid chromatography is an ultraviolet detector.

[0050] In one embodiment, the detection wavelength of the high-performance liquid chromatography is 150-250 nm, for example, 195 nm.

[0051] In one embodiment, the mobile phase A consists of the following components by volume percentage: 5% to 15% hexafluoroisopropanol and 85% to 95% water; the mobile phase B consists of the following components by volume percentage: 35% to 100% hexafluoroisopropanol and 0% to 70% water.

[0052] In one embodiment, the high-performance liquid chromatography method is selected from any of the following:

[0053] Option 1: The packing material for the chromatographic column is octadecylsilane-bonded silica gel;

[0054] The mobile phase A consists of the following components by volume percentage: 10% hexafluoroisopropanol and 90% water;

[0055] The mobile phase B consists of the following components by volume percentage: 40% hexafluoroisopropanol and 60% water;

[0056] During the gradient elution process, the volume percentage of mobile phase A in the mobile phase decreases from 100% to 0 at a constant rate.

[0057] The flow rate of the high-performance liquid chromatography method is 1 mL / min;

[0058] Option 2: The packing material for the chromatographic column is octadecylsilane-bonded silica gel;

[0059] The mobile phase A consists of the following components by volume percentage: 10% hexafluoroisopropanol and 90% water;

[0060] The mobile phase B consists of the following components by volume percentage: 70% hexafluoroisopropanol and 30% water;

[0061] During the gradient elution process, the volume percentage of mobile phase A in the mobile phase decreases at a constant rate from 100% to 50%.

[0062] The flow rate of the high-performance liquid chromatography method is 1 mL / min;

[0063] Option 3: The packing material for the chromatographic column is octadecylsilane-bonded silica gel;

[0064] The mobile phase A consists of the following components by volume percentage: 10% hexafluoroisopropanol and 90% water;

[0065] The mobile phase B is hexafluoroisopropanol;

[0066] During the gradient elution process, the volume percentage of mobile phase A in the mobile phase decreases at a constant rate from 100% to 67%.

[0067] The flow rate of the high-performance liquid chromatography method is 1 mL / min.

[0068] In one implementation scheme, in schemes 1, 2, and 3:

[0069] The chromatographic column is packed with octadecylsilane-bonded silica gel; the column specifications are a length of 150 mm, an inner diameter of 4.6 mm, and a packing particle size of 3.5 μm.

[0070] The column temperature is 45℃;

[0071] The injection volume was 15 μl;

[0072] The detector is an ultraviolet detector;

[0073] The detection wavelength is 195nm.

[0074] In this invention, the glycourea tetramer sample solution has chromatographic peaks within the corresponding retention time range, and the content of glycourea tetramer can be calculated by peak area.

[0075] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0076] The reagents and raw materials used in this invention are all commercially available.

[0077] The positive and progressive effects of the present invention are as follows: the detection method of the present invention has one or more of the following advantages: high sensitivity, high accuracy, speed, and good reproducibility. Attached Figure Description

[0078] Figure 1 This is the blank solvent chromatogram of Example 1 without interference.

[0079] Figure 2 This is the HPLC resolution chromatogram of the sample solution from Example 1.

[0080] Figure 3 This is the blank solvent chromatogram without interference from Example 2.

[0081] Figure 4 This is the HPLC resolution chromatogram of the sample solution from Example 2.

[0082] Figure 5 This is the blank solvent chromatogram without interference for Example 3.

[0083] Figure 6 This is the HPLC resolution chromatogram of the sample solution in Example 3.

[0084] Figure 7 To verify the repeatability of the chromatogram in Example 1.

[0085] Figure 8 To verify the detection limit chromatogram of Example 2.

[0086] Figure 9 To verify the limit of quantitation chromatogram of Example 2.

[0087] Figure 10 To verify the linearity and range chromatograms of Example 3.

[0088] Figure 11 To verify the linear relationship of glycourea tetramer in Example 3.

[0089] Figure 12 The HPLC resolution chromatogram is shown for the sample solution of Comparative Example 1.

[0090] Figure 13 The HPLC resolution chromatogram is shown for the sample solution of Comparative Example 2. Detailed Implementation

[0091] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0092] Instrument: Waters high performance liquid chromatograph.

[0093] Chromatographic column: Waters Xbridge C18 150*4.6mm 3.5μm (octadecylsilane bonded silica gel)

[0094] Preparation Example 1: Preparation of Glycourea Tetramer Sample

[0095] The preparation of the glycourea tetramer sample was carried out according to the method in the reference "Acyclic Cucurbit[n]uril Molecular Containers Enhance the Solubility and Bioactivity of Poorly Soluble Pharmaceuticals" (Da Ma et al., NATURE CHEMISTRY, 2012. DOI: 10.1038 / NCHEM.1326).

[0096] The specific steps are as follows:

[0097] Step 1:

[0098]

[0099] A mixture of 500 g (3.51 mol) of glycourea and 105 g (3.51 mol) of paraformaldehyde in an aqueous HCl solution (8 M, 700 mL) was heated at 50 °C for 48 hours. The reaction mixture was cooled and filtered. The solid was washed with water (500 mL) and then recrystallized with TFA (1.5 L) to give compound 3 as a white solid (334 g, 62%).

[0100] Step 2:

[0101]

[0102] A solution of urea (1140 g, 19.0 mol) in hydrochloric acid (0.3 mol, 2.8 L) was treated with 2,3-butanedione (500 g, 5.8 mol). The solution was stirred at room temperature for 12 hours. The reaction mixture was filtered, and the solid was washed with water (2.0 L × 2) and then with ethanol (2.0 L) to give a white solid dimethylglyoxal (749 g, 76%).

[0103] Step 3:

[0104]

[0105] A solution of dimethylglyoxal (749 g, 4.4 mol) and paraformaldehyde (650 g, 21.7 mol) in hydrochloric acid-water mixture (9 mol, 3.8 L) was stirred for 24 hours. Water (14.0 L) was added, and the mixture was stirred for another 12 hours. The mixture was then filtered and washed with water (2 L) and ethanol (2 L) to give compound 4 as a white solid (719 g, 65%).

[0106] Step 4:

[0107]

[0108] A solution of compound 3 (84 g, 0.27 mol) in anhydrous MeSO₃H (600 mL) was treated with compound 4 (304 g, 1.20 mol). The mixture was stirred and heated at 50 °C for 3 hours. The reaction mixture was poured into water (6.0 L). After filtration, the crude solid was dried under high vacuum. The crude solid was recrystallized from a mixture of TFA (350 mL) and water (1.4 L) to give a glycourea tetramer as a white solid (76 g, 36%).

[0109] Example 1:

[0110] Mobile phase A: 10% hexafluoroisopropanol aqueous solution;

[0111] Mobile phase B: 40% hexafluoroisopropanol aqueous solution;

[0112] Gradient elution procedure:

[0113] Table 1

[0114]

[0115] Detector: Ultraviolet detector;

[0116] Column temperature: 45℃;

[0117] Detection wavelength: 195nm;

[0118] Flow rate: 1.0 ml / min;

[0119] Injection volume: 15 μl;

[0120] Diluent: 40% hexafluoroisopropanol aqueous solution.

[0121] Sample solution: Take an appropriate amount of glycouretetramer, accurately weigh it, add diluent and quantitatively dilute it to a solution containing 2 mg of glycouretetramer per 1 ml.

[0122] The peak area was calculated using the area normalization method.

[0123] The results are as follows Figure 1 , Figure 2 The blank solvent shown has no interference, and there is good separation between the main peak and adjacent impurity peaks (as shown in Table 2, with a minimum separation of 1.4). The analysis time is short, which has good analytical guidance significance for the production of glycourea tetramer.

[0124] Table 2

[0125]

[0126] Example 2

[0127] Mobile phase A: 10% hexafluoroisopropanol aqueous solution;

[0128] Mobile phase B: 70% hexafluoroisopropanol aqueous solution;

[0129] Gradient elution procedure:

[0130] Table 3

[0131]

[0132] Detector: Ultraviolet detector;

[0133] Column temperature: 45℃;

[0134] Detection wavelength: 195nm;

[0135] Flow rate: 1.0 ml / min;

[0136] Injection volume: 15 μl;

[0137] Diluent: 70% hexafluoroisopropanol aqueous solution.

[0138] Sample solution: Take an appropriate amount of glycouretetramer, accurately weigh it, add diluent and quantitatively dilute it to a solution containing 2 mg of glycouretetramer per 1 ml.

[0139] The peak area was calculated using the area normalization method.

[0140] The results are as follows Figure 3 and Figure 4 The blank solvent shown has no interference, and there is good separation between the main peak and adjacent impurity peaks (as shown in Table 4, the minimum separation is 1.3). The analysis time is short, which has good analytical guidance significance for the production of glycourea tetramer.

[0141] Table 4

[0142]

[0143] Example 3

[0144] Mobile phase A: 10% hexafluoroisopropanol aqueous solution;

[0145] Mobile phase B: hexafluoroisopropanol;

[0146] Gradient elution procedure:

[0147] Table 5

[0148]

[0149] Detector: Ultraviolet detector;

[0150] Column temperature: 45℃;

[0151] Detection wavelength: 195nm;

[0152] Flow rate: 1.0 ml / min;

[0153] Injection volume: 15 μl;

[0154] Diluent: Hexafluoroisopropanol.

[0155] Sample solution: Take an appropriate amount of glycouretetramer, accurately weigh it, add diluent and quantitatively dilute it to a solution containing 2 mg of glycouretetramer per 1 ml.

[0156] The peak area was calculated using the area normalization method.

[0157] The results are as follows Figure 5 , Figure 6 The blank solvent shown has no interference, and there is good separation between the main peak and adjacent impurity peaks (as shown in Table 6, with a minimum separation of 1.3). The analysis time is short, which has good analytical guidance significance for the production of glycourea tetramer.

[0158] Table 6

[0159]

[0160] Verification Example 1: Repeatability

[0161] Sample solution: Accurately weigh an appropriate amount of glycourea tetramer, add 40% hexafluoroisopropanol aqueous solution and dilute quantitatively to a solution containing approximately 2 mg of glycourea tetramer per ml. Prepare 6 parallel solutions.

[0162] Precisely measure 15 μl of sample according to the chromatographic analysis conditions of Example 1, and inject 6 samples consecutively, one injection per sample. Record the chromatograms, and calculate the maximum single impurity and total impurities in each sample using the area normalization method. The results are shown in Table 7 and... Figure 7 As shown, the absolute values ​​of the absolute deviations of the six largest single impurities are all less than 0.2%, and the absolute values ​​of the absolute deviations of the total impurities are all less than 0.5%.

[0163] Table 7 Statistical table of sample repeatability injection data for Validation Example 1

[0164]

[0165] Validation Example 2: Limit of Detection and Limit of Quantification

[0166] Detection limit and quantitation limit solutions: Take an appropriate amount of glycouretetramer, accurately weigh it, dissolve it in 40% hexafluoroisopropanol aqueous solution and gradually dilute it to prepare glycouretetramer solutions of different concentrations.

[0167] Precisely measure 15 μl according to the chromatographic analysis conditions of Example 1, inject it into the liquid chromatograph, and record the chromatogram.

[0168] Using a concentration with a S / N ratio of approximately 10 as the limit of quantitation (LOQ) and a concentration with an S / N ratio of approximately 3 as the LQ, the results showed that the LQ was 2.068 μg / ml and the LQ was 0.827 μg / ml. See the results below. Figure 8 , Figure 9 As shown in Table 8.

[0169] Table 8. Statistical table of detection limit and quantitation limit data for Validation Example 2

[0170]

[0171] Verification Example 3: Linearity and Range

[0172] Linear solutions: Accurately weigh an appropriate amount of glycouretetramer, dissolve and gradually dilute it in 40% hexafluoroisopropanol aqueous solution to prepare a series of solutions containing glycouretetramer concentrations of 4.137 μg / ml, 10.342 μg / ml, 20.684 μg / ml, 103.420 μg / ml, 206.840 μg / ml, 1241.040 μg / ml, 1654.720 μg / ml, 2068.400 μg / ml, and 2275.240 μg / ml.

[0173] Precisely measure 15 μl according to the chromatographic analysis conditions of Example 1, inject it into the liquid chromatograph, and record the chromatogram.

[0174] A linear regression was performed with concentration (μg / ml) as the x-axis and average peak area as the y-axis. The linear equation is y = 8243.1184x + 187915.3526, and the linear correlation coefficient R0 is [value missing]. 2 =0.9985 (>0.99). The results show that when the concentration of glycouretetramer is in the range of 2.068 μg / ml to 2275.240 μg / ml, the linear correlation is good. The experimental results are shown in Table 9. Figure 10 and Figure 11 .

[0175] Table 9 Statistical Table of Linear Data in Verification Example 3

[0176]

[0177] Comparative Example 1:

[0178] Instrument: Agilent high performance liquid chromatograph.

[0179] Column: Agilent ZORBAX SB-Aq 250*4.6mm 5μm (organosilane-bonded silica gel)

[0180] Mobile phase A: 10% hexafluoroisopropanol aqueous solution (containing 1 mol / L sodium trifluoroacetate);

[0181] Mobile phase B: hexafluoroisopropanol;

[0182] Gradient elution procedure:

[0183] Table 10

[0184]

[0185] Detector: Ultraviolet detector;

[0186] Column temperature: 45℃;

[0187] Detection wavelength: 195nm;

[0188] Flow rate: 1.0 ml / min;

[0189] Injection volume: 10 μl;

[0190] Diluent: 50% hexafluoroisopropanol aqueous solution.

[0191] Sample solution: Take an appropriate amount of glycouretetramer, accurately weigh it, add diluent and quantitatively dilute it to a solution containing 2 mg of glycouretetramer per 1 ml.

[0192] The results are as follows Figure 12 As shown, the main peak cannot be effectively separated from the adjacent impurity peaks.

[0193] Comparative Example 2

[0194] Instrument: Agilent high performance liquid chromatograph.

[0195] Chromatographic column: Waters Xbridge C18 150*4.6mm 3.5μm

[0196] Mobile phase A: 10% hexafluoroisopropanol aqueous solution

[0197] Mobile phase B: hexafluoroisopropanol;

[0198] Isocratic elution: Mobile phase A: Mobile phase B = 75:25

[0199] Detector: Ultraviolet detector;

[0200] Column temperature: 45℃;

[0201] Detection wavelength: 195nm;

[0202] Flow rate: 1.0 ml / min;

[0203] Injection volume: 10 μl;

[0204] Diluent: 50% hexafluoroisopropanol aqueous solution.

[0205] Sample solution: Take an appropriate amount of glycouretetramer, accurately weigh it, add diluent and quantitatively dilute it to a solution containing 2 mg of glycouretetramer per 1 ml.

[0206] The results are as follows Figure 13 As shown, the main peak cannot be effectively separated from the adjacent impurity peaks.

Claims

1. A high-performance liquid chromatography method for the detection of glycourea tetramer, characterized in that, It includes the following steps: High performance liquid chromatography was used to perform gradient elution of the glycourea tetramer sample solution in the chromatographic column; The chromatographic column is packed with octadecylsilane-bonded silica gel. The gradient elution mobile phase consists of mobile phase A and mobile phase B; The mobile phase A comprises the following components by volume percentage: 5% to 50% hexafluoroisopropanol and 50% to 95% water; the volume percentage is the percentage of the volume of each component relative to the volume of the mobile phase A. The mobile phase B comprises the following components by volume percentage: 20% to 100% hexafluoroisopropanol and 0% to 80% water; the volume percentage is the percentage of the volume of each component relative to the volume of the mobile phase B. During the gradient elution process, the volume percentage of mobile phase A in the mobile phase decreases from 90%~100% to 0~75%; the volume percentage is the percentage of the volume of mobile phase A to the total volume of the mobile phase. The structure of the glycouretetramer is shown in the following formula: 。 2. The high-performance liquid chromatography method for detecting glycourea tetramer as described in claim 1, characterized in that, It meets one or more of the following conditions: (1) The mobile phase A comprises the following components by volume percentage: 5% to 25% hexafluoroisopropanol and 75% to 95% water; (2) The mobile phase B comprises the following components by volume percentage: 30% to 100% hexafluoroisopropanol and 0% to 70% water; (3) During the gradient elution process, the volume percentage of mobile phase A in the mobile phase decreases from 95%~100% to 0~70%; (4) The decrease is a uniform decrease; (5) The gradient elution time is 12-20 min; (6) The length of the chromatographic column is 100 mm to 300 mm; (7) The inner diameter of the chromatographic column is 1 mm to 10 mm; (8) The particle size of the filler is 1 μm to 10 μm; (9) The flow rate of the high performance liquid chromatography method is 0.5~3 mL / min; (10) The solvent of the glycourea tetramer sample solution comprises the following components by volume percentage: 10% to 100% hexafluoroisopropanol and 0% to 90% water; (11) The concentration of the glycourea tetramer sample in the glycourea tetramer sample solution is (0.002-3) mg / mL; (12) The injection volume for the high performance liquid chromatography method is 5~30 μl; (13) The column temperature of the high performance liquid chromatography method is 30~50℃; (14) The detector used in the high performance liquid chromatography method is an ultraviolet detector; (15) The detection wavelength of the high performance liquid chromatography method is 150-250 nm; (16) The packing material of the chromatographic column is octadecylsilane-bonded ethylidene bridged hybrid particles.

3. The high-performance liquid chromatography method for detecting glycourea tetramer as described in claim 2, characterized in that, It meets one or more of the following conditions: (1) The mobile phase A comprises the following components by volume percentage: 5% to 15% hexafluoroisopropanol and 85% to 95% water; (2) The mobile phase B comprises the following components by volume percentage: 35% to 100% hexafluoroisopropanol and 0% to 70% water; (3) During the gradient elution process, the volume percentage of mobile phase A in the mobile phase decreases from 100% to 0-70%; (4) The gradient elution time is 12 min or 15 min; (5) The length of the chromatographic column is 140 mm to 160 mm; (6) The inner diameter of the chromatographic column is 4 mm to 5 mm; (7) The particle size of the filler is 3 μm to 4 μm; (8) The flow rate of the high performance liquid chromatography method is 0.5~1.5 mL / min; (9) The solvent of the glycourea tetramer sample solution includes the following components by volume percentage: 35% to 100% hexafluoroisopropanol and 0% to 65% water; (10) The concentration of the glycourea tetramer sample in the glycourea tetramer sample solution is (0.002-2.3) mg / mL; (11) The injection volume for the high performance liquid chromatography method is 10~20 μl; (12) The column temperature of the high performance liquid chromatography method is 40~50℃; (13) The detection wavelength of the high performance liquid chromatography method is 195 nm.

4. The high-performance liquid chromatography method for detecting glycourea tetramer as described in claim 3, characterized in that, It meets one or more of the following conditions: (1) The mobile phase A comprises the following components by volume percentage: 10% hexafluoroisopropanol and 90% water; (2) The mobile phase B comprises the following components by volume percentage: 40% hexafluoroisopropanol and 60% water, 70% hexafluoroisopropanol and 30% water or hexafluoroisopropanol; (3) During the gradient elution process, the volume percentage of mobile phase A in the mobile phase decreases from 100% to 0, from 100% to 50%, or from 100% to 67%. (4) The length of the chromatographic column is 150 mm; (5) The inner diameter of the chromatographic column is 4.6 mm; (6) The particle size of the filler is 3.5 μm; (7) The flow rate of the high performance liquid chromatography method is 1 mL / min; (8) The solvent of the glycourea tetramer sample solution comprises the following components by volume percentage: 40% hexafluoroisopropanol and 60% water, 70% hexafluoroisopropanol and 30% water or hexafluoroisopropanol; (9) The concentration of the glycourea tetramer sample in the glycourea tetramer sample solution is 2 mg / mL; (10) The injection volume for the high performance liquid chromatography method is 15 μl; (11) The column temperature of the high performance liquid chromatography method is 45℃.

5. The high-performance liquid chromatography method for detecting glycourea tetramer as described in claim 2, characterized in that, It meets one or more of the following conditions: (1) The mobile phase A consists of the following components by volume percentage: 5% to 50% hexafluoroisopropanol and 50% to 95% water; (2) The mobile phase B consists of the following components by volume percentage: 20% to 100% hexafluoroisopropanol and 0% to 80% water; (3) The solvent of the glycourea tetramer sample solution is composed of the following components by volume percentage: 10% to 100% hexafluoroisopropanol and 0% to 90% water.

6. The high-performance liquid chromatography method for detecting glycourea tetramer as described in claim 2, characterized in that: The mobile phase A consists of the following components by volume percentage: 5% to 15% hexafluoroisopropanol and 85% to 95% water; the mobile phase B consists of the following components by volume percentage: 35% to 100% hexafluoroisopropanol and 0% to 70% water.

7. The high-performance liquid chromatography method for detecting glycourea tetramer as described in claim 2, characterized in that, The chromatographic column has the following specifications: a column length of 150 mm, an inner diameter of 4.6 mm, and a packing particle size of 3.5 μm; for example, Waters Xbridge C18 150*4.6 mm 3.5 μm.

8. The high-performance liquid chromatography method for detecting glycourea tetramer as described in claim 1, characterized in that, The high-performance liquid chromatography method is selected from any of the following schemes: Option 1: The packing material for the chromatographic column is octadecylsilane-bonded silica gel; The mobile phase A consists of the following components by volume percentage: 10% hexafluoroisopropanol and 90% water; The mobile phase B consists of the following components by volume percentage: 40% hexafluoroisopropanol and 60% water; During the gradient elution process, the volume percentage of mobile phase A in the mobile phase decreases from 100% to 0 at a constant rate. The flow rate of the high-performance liquid chromatography method is 1 mL / min; Option 2: The packing material for the chromatographic column is octadecylsilane-bonded silica gel; The mobile phase A consists of the following components by volume percentage: 10% hexafluoroisopropanol and 90% water; The mobile phase B consists of the following components by volume percentage: 70% hexafluoroisopropanol and 30% water; During the gradient elution process, the volume percentage of mobile phase A in the mobile phase decreases at a constant rate from 100% to 50%. The flow rate of the high-performance liquid chromatography method is 1 mL / min; Option 3: The packing material for the chromatographic column is octadecylsilane-bonded silica gel; The mobile phase A consists of the following components by volume percentage: 10% hexafluoroisopropanol and 90% water; The mobile phase B is hexafluoroisopropanol; During the gradient elution process, the volume percentage of mobile phase A in the mobile phase decreases at a constant rate from 100% to 67%. The flow rate of the high-performance liquid chromatography method is 1 mL / min; Preferably, in Scheme 1, Scheme 2 and Scheme 3: the chromatographic column packing material is octadecylsilane-bonded silica gel; the specifications of the chromatographic column are: column length of 150 mm, inner diameter of 4.6 mm, packing particle size of 3.5 μm; column temperature of 45 °C; injection volume of 15 μl; detector of ultraviolet detector; detection wavelength of 195 nm.

9. The high-performance liquid chromatography method for detecting glycourea tetramer as described in claim 1, characterized in that, The glycourea tetramer sample was obtained by the following preparation method, which includes the following steps: in a solvent, compound 3 and compound 4 react as shown in the following formula to obtain the glycourea tetramer; 。 10. The high-performance liquid chromatography method for detecting glycourea tetramer as described in claim 1, characterized in that, The method for preparing the glycouretetramer satisfies one or more of the following conditions: (1) The solvent is a sulfonic acid solvent, such as methanesulfonic acid; (2) The ratio of the number of moles of compound 3 to the volume of the solvent is (0.1-1) mol / L, for example 0.45 mol / L; (3) The molar ratio of compound 4 to compound 3 is (1-5):1, for example 4.4:1; (4) The reaction temperature is 40℃-80℃, for example 50℃; (5) The reaction is completed and includes a post-processing step, which is filtration and recrystallization. The solvent for crystallization is trifluoroacetic acid and water, for example, the volume ratio of trifluoroacetic acid and water is 1:4.

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