Method for detecting intermediate acetyl glucose of tetraacetyl by HPLC-CAD (High Performance Liquid Chromatography-Computer Aided Design) method

Abstract

The invention discloses a method for detecting an intermediate acetylglucose of tetraacetyl by an HPLC-CAD (High Performance Liquid Chromatography-Computer Aided Design) method. The method comprises the following steps: (1) preparing a reference substance solution, namely dissolving acetylglucose by adding a diluent, and diluting to prepare the reference substance solution; (2) preparation of a test solution: taking tetraacetyl, adding a diluent solvent, and diluting to prepare the test solution; and (3) detecting the content of acetyl glucose in the test sample by adopting an HPLC-CAD (High Performance Liquid Chromatography-Computer Aided Design) method, and taking octadecylsilane chemically bonded silica as a filler in an adopted chromatographic column. According to the method disclosed by the invention, the intermediate acetyl glucose of the tetraacetyl substance is detected based on an HPLC-CAD (High Performance Liquid Chromatography-Computer Aided Design) method, and CAD parameters, a chromatographic column, a mobile phase, a diluent and a sample concentration are selected, so that the aims of effectively separating and accurately detecting the acetyl glucose in the sample can be fulfilled; the method has the advantages of simplicity, sensitivity, high accuracy, high repeatability, high specificity, good durability, high precision and the like, and has certain guiding significance on carbohydrate compound detection.

Description

Technical Field

[0001] The present invention belongs to the field of chemical drug analysis and detection, and specifically relates to a method for detecting acetylglucose, an intermediate of tetraacetate, by HPLC-CAD method. Background Art

[0002] In the late 1970s, Zhou Jun et al. isolated a new compound from the traditional Chinese medicine Gastrodia elata, and through structural identification, it was determined to be 4-(hydroxymethyl)phenyl-β-D-glucopyranoside. Pharmacological studies have shown that this compound has sedative and hypnotic effects and is considered to be the active ingredient of the traditional Chinese medicine Gastrodia elata, named gastrodin.

[0003] In 1980, Zhou Jun et al. completed the chemical synthesis of gastrodin. Subsequently, researchers improved the process based on its synthetic route, especially for the key intermediate: 2-Formylphenyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside. Specifically, it is usually prepared by reacting bromoacetylglucose with p-hydroxybenzaldehyde. Among them, acetylglucose is brominated to form bromoacetylglucose, and bromoacetylglucose reacts with p-hydroxybenzaldehyde under catalysis to form tetraacetate. The chemical structural formula of acetylglucose is as follows:

[0004]

[0005] As the starting material / intermediate for synthesizing tetraacetate, monitoring the acetylglucose that may be generated during the synthesis process of tetraacetate can better control the quality of tetraacetate. Since acetylglucose only has end absorption (see appendix Figure 3 ), when detected by HPLC-UV method, there is a large interference, and there was once no effective method to detect it, that is, it was impossible to monitor the acetylglucose in tetraacetate.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for detecting acetylglucose, an intermediate of tetraacetate, by HPLC-CAD method, so that its specificity, quantitative limit, detection limit, linear range, repeatability, accuracy, etc. meet the standards and have high sensitivity and high precision.

[0008] To solve the above technical problems, the basic concept of the technical solution adopted by the present invention is:

[0009] A method for detecting acetylglucose, an intermediate of tetra-acetate, by HPLC-CAD method, comprising the following steps:

[0010] (1) Preparation of reference solution: Dissolve and dilute acetylglucose with a diluent to prepare a reference solution;

[0011] (2) Preparation of test solution: Dissolve and dilute tetra-acetate with a diluent to prepare a test solution;

[0012] (3) Using the HPLC-CAD method, detect the content of acetylglucose in the test sample. Among them, the chromatographic column used is packed with octadecylsilyl silica gel.

[0013] The present invention adopts the HPLC-CAD method. Through a large number of experimental explorations, it is found that by using a chromatographic column packed with octadecylsilyl silica gel and a mobile phase with a simple composition, effective separation can be achieved and it can accurately evaluate whether there is acetylglucose in the sample. The detection method is simple, sensitive and highly accurate.

[0014] Further, the diluent is selected from a mixed solution of acetonitrile and water, or a mixed solution of methanol and water;

[0015] Preferably, the volume ratio of acetonitrile to water is 6-4:4-6; the volume ratio of methanol to water is 6-4:4-6;

[0016] Preferably, the volume ratio of acetonitrile to water is 5:5.

[0017] Further, the chromatographic column is a Waters Atlantis T3 column, with a length of 150×4.6 mm and a particle size of 3 μm.

[0018] Further, when performing HPLC detection, a mixed solution of acetonitrile and water is used as the mobile phase;

[0019] Preferably, the volume ratio of acetonitrile to water is 55-45:45-55;

[0020] Preferably, the volume ratio of acetonitrile to water is 50:50.

[0021] Further, the conditions for CAD electrospray detection include: the vaporization temperature is 30-40 °C, and the acquisition frequency is 10 Hz;

[0022] Preferably, the vaporization temperature is 35 °C.

[0023] The charged aerosol detector (CAD) is a universal detector, which is characterized by high sensitivity, good reproducibility, and the detection being independent of the molecular structure of the compound. It is suitable for detecting components with no or weak ultraviolet absorption. Compared with using an ultraviolet detector to detect end-absorbing components, it has less baseline interference and can provide a better choice for the inspection of acetylglucose.

[0024] Further, in the step (1), each 1 ml of the reference solution contains 1 μg of acetylglucose.

[0025] Further, in the step (2), each 1 ml of the test solution contains 0.9 - 1.1 mg of tetra-acetyl compound;

[0026] Preferably, each 1 ml of the test solution contains 1 mg of tetra-acetyl compound.

[0027] Further, when performing HPLC detection, the column temperature used is 33°C - 37°C; preferably 35°C.

[0028] Further, when performing HPLC detection, the elution mode is isocratic elution;

[0029] Preferably, the flow rate is 0.9 ml / min - 1.1 ml / min; preferably 1.0 ml / min.

[0030] Further, in the step (3), the test solution and the reference solution are respectively measured and injected into the liquid chromatograph, the chromatogram is recorded, and the external standard method is used to calculate the content of acetylglucose in the test solution based on the peak area;

[0031] The calculation formula is as follows:

[0032]

[0033] Where:

[0034] A spl is the peak area of acetylglucose in the test solution;

[0035] A std is the peak area of acetylglucose in the reference solution;

[0036] V std is the dilution volume (ml) of the reference solution;

[0037] V spl is the dilution volume (ml) of the test solution;

[0038] W std is the weighed amount (mg) of the reference substance;

[0039] W spl is the weighed amount (mg) of the test sample;

[0040] P is the purity of the reference substance.

[0041] After adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art.

[0042] The present invention is based on the HPLC-CAD method to detect the intermediate acetylglucose of tetra-acetate. By selecting CAD parameters, chromatographic column, mobile phase, diluent and sample concentration, the purpose of effectively separating and accurately detecting acetylglucose in the sample can be achieved. This detection method has the advantages of simplicity, sensitivity, high accuracy, strong repeatability, strong specificity, good durability and high precision, and has guiding significance for the detection of carbohydrate compounds.

[0043] The following further describes in detail the specific embodiments of the present invention with reference to the accompanying drawings. Description of the Drawings

[0044] The accompanying drawings, as part of the present invention, are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention, but do not constitute an improper limitation of the present invention. Obviously, the accompanying drawings in the following description are only some embodiments, and those of ordinary skill in the art can obtain other drawings based on these drawings without creative efforts. In the drawings:

[0045] Figure 1 are the chromatograms of the reference substance solution, test sample solution, and mixed solution of other impurities; among them, 1 refers to the blank solvent; 2 refers to the mixed solution of other impurities; 3 refers to the reference substance solution; 4 refers to the test sample solution;

[0046] Figure 2 are the linear relationship diagrams of the peak area and concentration of acetylglucose;

[0047] Figure 3 are the ultraviolet absorption diagrams of acetylglucose.

[0048] It should be noted that these drawings and textual descriptions are not intended to limit the scope of the concept of the present invention in any way, but to illustrate the concept of the present invention to those skilled in the art by referring to specific embodiments. Specific Embodiments

[0049] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0050] 1. The information of instruments and reagents is shown in Table 1 below:

[0051] Table 1:

[0052]

[0053] Example 1

[0054] (1) Preparation of diluent: Measure 500 ml of acetonitrile and 500 ml of water, mix well to obtain it.

[0055] (2) Preparation of reference solution: Take an appropriate amount of acetylglucose reference substance, dissolve it with diluent and quantitatively dilute to prepare a solution containing about 1 μg per 1 ml.

[0056] (3) Preparation of test solution: Take about 20 mg of tetra-acetyl compound (batch number: C0621905-31), accurately weigh it, place it in a 20-ml volumetric flask, dissolve it with diluent, dilute to the mark with diluent, and shake well to obtain it.

[0057] (4) Accurately measure the test solution and the reference solution respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of acetylglucose in the test solution by the external standard method based on the peak area;

[0058] Chromatographic conditions:

[0059] Chromatographic column: Waters Atlantis T3, 4.6×150 mm, 3 μm;

[0060] Mobile phase: Acetonitrile: water (volume ratio is 50 ml:50 ml);

[0061] Elution mode: Isocratic elution;

[0062] Flow rate: 1.0 ml / min;

[0063] Column temperature: 35 °C;

[0064] CAD parameters: Data Collection Rate: 10, Filter: 3.6, Peak width: 0.02, Controlevaporater temperature: Low (35 °C);

[0065] Injection volume: 50 μl;

[0066] Formula for calculating the content of acetylglucose:

[0067]

[0068] Where:

[0069] A spl is the peak area of acetylglucose in the test solution;

[0070] A stdis the peak area of acetylglucose in the reference solution;

[0071] V std is the dilution volume (ml) of the reference solution;

[0072] V spl is the dilution volume (ml) of the test solution;

[0073] W std is the weighed amount of the reference substance (mg);

[0074] W spl is the weighed amount of the test sample (mg);

[0075] P is the purity of the reference substance.

[0076] Results: The chromatograms of the reference solution and the test solution in this example are as Figure 1 shown. Among them, peak 1 - acetylglucose; 1 refers to the blank solvent; 3 refers to the reference solution; 4 refers to the test solution. It can be seen from the figure that there is no acetylglucose in the test solution of this example.

[0077] Example 2

[0078] (1) Preparation of the diluent: Measure 600 ml of acetonitrile and 400 ml of water, mix well to obtain.

[0079] (2) Preparation of the reference solution: Take an appropriate amount of acetylglucose reference substance, dissolve it with the diluent and quantitatively dilute it to prepare a solution containing about 1 μg per 1 ml.

[0080] (3) Preparation of the test solution: Take 18 mg of tetra-acetyl compound (batch number: C0621905 - 31), weigh it accurately, place it in a 20 ml volumetric flask, dissolve it with the diluent, and dilute it to the mark with the diluent, shake well to obtain.

[0081] (4) Accurately measure the test solution and the reference solution respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of acetylglucose in the test solution by the external standard method with the peak area;

[0082] Chromatographic conditions:

[0083] Chromatographic column: Waters Atlantis T3, 4.6×150 mm, 3 μm;

[0084] Mobile phase: Acetonitrile: water (volume ratio is 55 ml:45 ml);

[0085] Elution mode: Isocratic elution;

[0086] Flow rate: 0.9 ml / min;

[0087] Column temperature: 33 °C;

[0088] CAD parameters: Data Collection Rate: 10, Filter: 3.6, Peak width: 0.02, Controlevaporater temperature: Low (30 °C);

[0089] Sample injection volume: 50 μl;

[0090] Calculation formula for acetylglucose content:

[0091]

[0092] In the formula:

[0093] A spl is the peak area of acetylglucose in the test solution;

[0094] A std is the peak area of acetylglucose in the reference solution;

[0095] V std is the dilution volume (ml) of the reference solution;

[0096] V spl is the dilution volume (ml) of the test solution;

[0097] W std is the weighed amount of the reference substance (mg);

[0098] W spl is the weighed amount of the test sample (mg);

[0099] P is the purity of the reference substance.

[0100] Example 3

[0101] (1) Preparation of diluent: Measure 400 ml of acetonitrile and 600 ml of water, mix well to obtain.

[0102] (2) Preparation of reference solution: Take an appropriate amount of acetylglucose reference substance, dissolve it with diluent and quantitatively dilute it to a solution containing about 1 μg per 1 ml.

[0103] (3) Preparation of test solution: Take 22 mg of tetra-acetyl compound (batch number: C0621905 - 31), weigh it accurately, place it in a 20 ml volumetric flask, dissolve it with diluent, dilute it to the scale with diluent, and shake well to obtain.

[0104] (4) Accurately measure the test solution and the reference solution respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of acetylglucose in the test solution by the external standard method based on the peak area;

[0105] Chromatographic conditions:

[0106] Chromatographic column: Waters Atlantis T3, 4.6×150 mm, 3 μm;

[0107] Mobile phase: acetonitrile: water (volume ratio 45 ml: 55 ml);

[0108] Elution mode: isocratic elution;

[0109] Flow rate: 1.1 ml / min;

[0110] Column temperature: 37 °C;

[0111] CAD parameters: Data Collection Rate: 10, Filter: 3.6, Peak width: 0.02, Controlevaporater temperature: Low (40 °C);

[0112] Sample injection volume: 50 μl;

[0113] Calculation formula for acetylglucose content:

[0114]

[0115] Where:

[0116] A spl is the peak area of acetylglucose in the test solution;

[0117] A std is the peak area of acetylglucose in the reference solution;

[0118] V std is the dilution volume (ml) of the reference solution;

[0119] V spl is the dilution volume (ml) of the test solution;

[0120] W std is the weighed amount (mg) of the reference substance;

[0121] W spl is the weighed amount (mg) of the test sample;

[0122] P is the purity of the reference substance.

[0123] Example 4

[0124] (1) Preparation of diluent: Measure 500 ml of methanol and 500 ml of water, mix well to obtain.

[0125] (2) Preparation of reference solution: Take an appropriate amount of acetylglucosamine reference substance, dissolve it in the diluent and quantitatively dilute to prepare a solution containing about 1 μg per 1 ml.

[0126] (3) Preparation of test solution: Take 22 mg of tetra-acetyl compound (batch number: C0621905-31), accurately weigh it, place it in a 20-ml volumetric flask, dissolve it with the diluent, dilute to the mark with the diluent, and shake well to obtain the solution.

[0127] (4) Accurately measure appropriate volumes of the test solution and the reference solution respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of acetylglucosamine in the test solution by the external standard method based on the peak area;

[0128] Chromatographic conditions:

[0129] Chromatographic column: Waters Atlantis T3, 4.6×150 mm, 3 μm;

[0130] Mobile phase: acetonitrile: water (volume ratio is 45 ml: 55 ml);

[0131] Elution mode: isocratic elution;

[0132] Flow rate: 1.1 ml / min;

[0133] Column temperature: 37 °C;

[0134] CAD parameters: Data Collection Rate: 10, Filter: 3.6, Peak width: 0.02, Controlevaporater temperature: Low (40 °C);

[0135] Injection volume: 50 μl;

[0136] Formula for calculating the content of acetylglucosamine:

[0137]

[0138] In the formula:

[0139] A spl is the peak area of acetylglucosamine in the test solution;

[0140] A std is the peak area of acetylglucosamine in the reference solution;

[0141] V std is the dilution volume (ml) of the reference solution;

[0142] V spl is the dilution volume (ml) of the test solution;

[0143] W std is the weighing amount of the reference substance (mg);

[0144] W spl is the weighing amount of the test sample (mg);

[0145] P is the purity of the reference substance.

[0146] Example Five

[0147] Selection of diluent

[0148] Referring to the preparation methods of the reference substance solution and the test sample solution in Example One, compare the detection effects after dilution with two diluents, the methanol-water mixed solution and the acetonitrile-water mixed solution:

[0149] Due to the poor water solubility of the tetra-acetate, common reagents such as the mixed solution of methanol and water or the mixed solution of acetonitrile and water are selected for dilution. When the volume ratio of acetonitrile to water is 6 - 4:4 - 6, or the volume ratio of methanol to water is 6 - 4:4 - 6, acetylglucose shows a single symmetric peak and can be used as a diluent. However, since the mobile phase uses an acetonitrile system, from the perspective of preparing the sample solution with the mobile phase as much as possible, the mixed solution of acetonitrile and water is selected as the diluent and has a better detection effect.

[0150] Example Six

[0151] Selection of chromatographic conditions

[0152] (1) Selection of chromatographic column

[0153] On the basis of Example One, adjust some chromatographic conditions. Replace the chromatographic column with Waters XBridge C18 (4.6 mm × 150 mm, 5 μm), the flow rate is 0.5 ml / min, and the mobile phase is acetonitrile-water (volume ratio 50 ml:50 ml) for detection. The detection results show that acetylglucose and the tetra-acetate without impurities can be effectively separated. However, other process impurities usually present in the tetra-acetate, such as p-hydroxybenzaldehyde, cannot be effectively separated from acetylglucose; by adjusting the flow rate, changing the mobile phase ratio and setting a gradient mobile phase, it is found that acetylglucose still cannot be effectively separated from other process impurities present in the tetra-acetate.

[0154] Considering that the acetylglucose structure does not contain phenyl and hydrophilic groups, attempts were made by replacing the phenyl column and the hydrophilic column. The separation results of the phenyl column Agilent ZORBAX SB-phenyl (5μm, 4.6×150mm) and the hydrophilic column Waters BEH SheildRP18 (5μm, 4.6×150mm) were similar, and acetylglucose still could not be effectively separated from other process impurities of tetraacetate.

[0155] When using the chromatographic column Waters Atlantis T3 (3μm, 4.6×150mm) under gradient conditions, there were signs of separation between acetylglucose and other process impurities of tetraacetate, but the separation effect was still not good. Therefore, on this basis, the chromatographic conditions were further adjusted. A mixture of acetonitrile - water (volume ratio 55~45:45~55) was selected as the mobile phase for elution, and it was found that acetylglucose could be effectively separated. Further, a mixture of acetonitrile - water (volume ratio 50:50) was selected as the mobile phase, and separation was carried out under the chromatographic conditions with a flow rate of 1ml / min. It was found that acetylglucose and tetraacetate could be effectively separated without interference from impurities, and the resolution met the requirements. Therefore, the chromatographic column selected in the present invention is Waters Atlantis T3 column (length 150×4.6mm, particle size 3μm); the selected mobile phase is a mixture of acetonitrile - water (volume ratio 55~45:45~55, preferably 50:50); the selected flow rate is 1ml / min.

[0156] (2) Selection of mobile phase

[0157] On the basis of Example 1, the type of mobile phase in the chromatographic conditions was adjusted. When mobile phase A was water and mobile phase B was acetonitrile, the resolution was greater than 1.5; when mobile phase A was trifluoroacetic acid with a mass fraction of 0.1% and mobile phase B was methanol, it was found that the baseline noise increased after adding trifluoroacetic acid with a mass fraction of 0.1%, and the chromatographic peak showed fronting after adding methanol. Therefore, a water - acetonitrile system was used as the mobile phase, and a mixture of acetonitrile - water (volume ratio of acetonitrile to water is 50:50) could obtain a better resolution.

[0158] Example 7

[0159] (1) Specificity and system suitability

[0160] Examine whether the determination of acetylglucose in the test sample is interfered when there are other impurities in the blank solution or tetraacetate (such as: the process impurity p-hydroxybenzaldehyde of tetraacetate).

[0161] Reference solution: Take an appropriate amount of acetylglucose reference substance, dissolve it with a diluent and quantitatively dilute it to prepare a solution containing about 1μg per 1ml.

[0162] Preparation of test solution: Take about 20 mg of tetra-acetyl compound (batch number: C0621905-31), accurately weigh it, place it in a 20-ml volumetric flask, add diluent to dissolve it, dilute it to the mark with diluent, shake well, and you will get it.

[0163] Mixed solution of other impurities: Prepare a mixed solution of other impurities according to the limit concentration, and examine whether other impurities interfere with acetylglucose.

[0164] Accurately measure the test solution, reference solution, and mixed solution of other impurities respectively, inject them into the liquid chromatograph, and use the chromatographic conditions in Example 1 for detection. The obtained chromatograms are as Figure 1 shown. 1 refers to the blank solution; 2 refers to the mixed solution of other impurities; 3 refers to the reference solution; 4 refers to the test solution; as can be Figure 1 seen, there are no interfering peaks at the retention time of acetylglucose in the chromatograms of the blank solution and the mixed solution of other impurities, that is, the detection method of the present invention has strong specificity.

[0165] (2) Injection precision test

[0166] Prepare a reference solution according to the method in Example 1, inject 50 μl, inject continuously for 6 times, and calculate the RSD of the peak area of acetylglucose. The RSD of the 6 determination results of the reference solution is 3.2% (n = 6), as shown in Table 2 in detail.

[0167] Table 2:

[0168] N-acetylglucosamine 1 2 3 4 5 6 RSD % Peak area 0.1347 0.1395 0.1308 0.1331 0.1267 0.1341 3.2

[0169] The RSD of the peak area of acetylglucose in the chromatogram of the reference solution is 3.2% (n = 6), and the injection precision is good.

[0170] Example 8

[0171] Quantitation limit and detection limit

[0172] The quantitation limit and detection limit are determined according to the signal-to-noise ratio method. The reference solution is diluted step by step, the measured signal is compared with the baseline noise, and the lowest concentration that can be reliably detected is calculated. The signal-to-noise ratio of the peak of acetylglucose in the chromatogram of the quantitation limit solution is not less than 10, and the RSD of the peak area ≤ 10% (n = 5). The signal-to-noise ratio of the peak of acetylglucose in the chromatogram of the detection limit solution is not less than 3. The results are shown in Table 3:

[0173] Table 3:

[0174]

[0175] The quality control limit of acetylglucose in the tetra-acetate is 1.0%, the quantitation limit is 20% of the limit concentration, and the detection limit is 6% of the limit concentration. This method has high sensitivity.

[0176] Example Nine

[0177] Linearity

[0178] Examine whether there is a good linear relationship between the response value and the concentration of acetylglucose in the range of LOQ to 200% of the limit concentration (1 μg / ml). The results are shown in Table 4 and Appendix Figure 2 .

[0179] Table 4:

[0180]

[0181] From Table 4 and Figure 2 it can be seen that in the range of LOQ to 200%, the linear equation of acetylglucose is y = 0.1423x - 0.0058, and its linear correlation coefficient r = 0.9997. The Y-axis intercept is -0.0058, which is less than ±20% (±0.0279) of the response value of the solution at the limit concentration; the linear relationship of acetylglucose is good.

[0182] Example Ten

[0183] Accuracy

[0184] Determined by the method of recovery of added samples, the ratio (recovery rate) between the actual measured value and the theoretical amount of acetylglucose in the spiked test sample is expressed as a percentage. Requirements: for 3 concentrations of low, medium and high, the average recovery rate of 9 samples should be between 80% and 120%, and the RSD of the recovery rate of 9 samples should not be greater than 10%. The results are shown in Table 5.

[0185] Table 5:

[0186]

[0187]

[0188] Example Eleven

[0189] Repeatability

[0190] Take the tetra-acetate sample and prepare 6 portions of 100% spiked test sample solutions to verify the precision of the method. The results are shown in Table 6.

[0191] Table 6:

[0192]

[0193] In the 6 test solution samples prepared by a single analyst, the RSD of the measured amount of acetylglucose was 3.8% (n = 6), less than 8%; the method had good repeatability.

[0194] Example XII

[0195] Intermediate precision

[0196] Another analyst changed an instrument on another test date and prepared 6 spiked test solution samples in parallel. The content of acetylglucose was calculated based on the peak area of the reference solution, and its RSD and the RSD of the content of 12 samples prepared by the two analysts were calculated. The results are shown in Table 7.

[0197] Table 7:

[0198]

[0199]

[0200] In the 6 spiked test solution samples prepared by Analyst A, the RSD of the measured amount of acetylglucose was 3.8% (n = 6); in the 6 test solution samples prepared by Analyst B, the RSD of the measured amount of acetylglucose was 0.1% (n = 6); in the 12 test solution samples prepared by the two analysts, the RSD of the measured amount of acetylglucose was 5.0% (n = 12); less than 16%, the method had good intermediate precision.

[0201] Example XIII

[0202] Solution stability

[0203] The reference solution and the 100% spiked test solution were injected at different times to investigate the solution stability. Compared with 0 hour, taking the main peak area of the reference solution at 0 hour as 100%, within each time point, the relative amount of the main component of the reference solution should be between 90% and 110%; in the 100% spiked test solution, the absolute difference in the content of acetylglucose should not exceed 0.05%. The results are shown in Table 8 (Solution stability results - reference solution) and Table 9 (Solution stability results - 100% spiked test solution).

[0204] The absolute difference in the content of acetylglucose = |the content of acetylglucose at the investigated time point - the content of acetylglucose at 0 hour|.

[0205] Table 8:

[0206] Time Peak area Relative quantity % 0h 0.1347 / 4h 0.1377 102 8h 0.1308 97 12h 0.1332 99 18h 0.1381 103 26h 0.1409 105 35h 0.1296 96 45h 0.1317 98

[0207] Table 9:

[0208] Time Peak area Content % Content difference % 0h 0.1456 0.11 / 3h 0.1499 0.11 0.00 6h 0.1504 0.11 0.00 22h 0.1399 0.10 0.01 28h 0.1413 0.10 0.01

[0209] As can be seen from the above table, the reference solution is stable after being placed for 45 h; the test solution is stable after being placed for 28 h.

[0210] Example XIV

[0211] Robustness

[0212] Select different chromatographic conditions to test the reference solution and the 100% spiked test solution respectively, and calculate the amount of acetylglucose according to the specified method. Examine the robustness of the method when there are minor fluctuations in the test conditions. The results are shown in Table 10.

[0213] Table 10:

[0214]

[0215] Compared with the specified method, the difference in the content of acetylglucose measured in the spiked test solution under different conditions does not exceed 0.05%, and the robustness of the method is good.

[0216] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art of this patent, without departing from the scope of the technical solution of the present invention, can make some changes or modifications using the technical content prompted above into equivalent embodiments with equivalent changes. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the present invention.

Claims

1. A method for detecting acetylglucose, an intermediate of tetra-acetate, by HPLC-CAD, characterized in that: It includes the following steps: (1) Preparation of reference solution: Dissolve and dilute acetylglucose with a diluent to prepare a reference solution; (2) Preparation of test solution: Dissolve and dilute tetra-acetyl compound with a diluent to prepare a test solution; (3) Use HPLC-CAD method to detect the content of acetylglucose in the reference and test solutions. Among them, the chromatographic column used is packed with octadecylsilane-bonded silica gel.

2. The method for detecting intermediate acetylglucose of tetraacetate by HPLC-CAD according to claim 1, characterized in that: The diluent is selected from the mixed solution of acetonitrile and water, and the mixed solution of methanol and water; Preferably, the volume ratio of acetonitrile to water is 6-4:4-6; the volume ratio of methanol to water is 6-4:4-6; Preferably, the volume ratio of acetonitrile to water is 5:

5.

3. The method for detecting intermediate acetylglucose of tetra-acetate by HPLC-CAD method according to claim 1 or 2, characterized in that: The chromatographic column is Waters Atlantis T3 column, with a length of 150×4.6mm and a particle size of 3μm.

4. A method for detecting acetylglucose, an intermediate of tetra-acetate, by HPLC-CAD according to any one of claims 1-3, characterized in that: When performing HPLC detection, use the mixed solution of acetonitrile and water as the mobile phase; Preferably, the volume ratio of acetonitrile to water is 55-45:45-55; Preferably, the volume ratio of acetonitrile to water is 50:

50.

5. A method for detecting acetylglucose, an intermediate of tetraacetate, by HPLC-CAD according to any one of claims 1-4, characterized in that: The conditions for CAD electrospray detection include: the vaporization temperature is 30-40°C, and the acquisition frequency is 10Hz; Preferably, the vaporization temperature is 35°C.

6. A method for detecting acetylglucose, an intermediate of tetraacetate, by HPLC-CAD method according to any one of claims 1-5, characterized in that: In the step (1), each 1 ml of the reference solution contains 1 μg of acetylglucose.

7. A method for detecting intermediate acetylglucose of tetra-acetyl compound by HPLC-CAD method according to any one of claims 1-6, characterized in that: In the step (2), each 1 ml of the test solution contains 0.9-1.1 mg of tetra-acetyl compound; Preferably, each 1 ml of the test solution contains 1 mg of tetra-acetyl compound.

8. A method for detecting intermediate acetylglucose of tetra-acetate by HPLC-CAD method according to any one of claims 1-7, characterized in that: When performing HPLC detection, the column temperature used is 33°C - 37°C; preferably 35°C.

9. A method for detecting acetylglucose, an intermediate of tetra-acetate, by HPLC-CAD according to any one of claims 1-8, characterized in that: When performing HPLC detection, the elution method is isocratic elution; Preferably, the flow rate is 0.9 ml / min - 1.1 ml / min; preferably 1.0 ml / min.

10. A method for detecting intermediate acetylglucose of tetra-acetate by HPLC-CAD method according to any one of claims 1-9, characterized in that: In the step (3), respectively measure the test solution and the reference solution, inject them into the liquid chromatograph, record the chromatogram, and use the external standard method to calculate the content of acetylglucose in the test solution based on the peak area; The calculation formula is as follows: In the formula: A spl is the peak area of acetylglucose in the test solution; A std is the peak area of acetylglucose in the reference solution; V std is the dilution volume (ml) of the reference solution; V spl is the dilution volume (ml) of the test solution; W std is the weighing quantity of the reference substance (mg); W spl is the weighed sample amount of the test article (mg); P is the purity of the reference substance.

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