Quantitative Analysis Method of 2,3,4,6-Tetrabenzyl-D-pyranoglucopyranoside-1,5-lactone

Abstract

The invention discloses a quantitative analysis method for 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone. The method adopts a high performance liquid chromatography method, takes octaalkylsilane bonded silica gel as a stationary phase, takes a methanol aqueous solution containing triethylamine and trifluoroacetic acid as a mobile phase A, and takes a methanol acetonitrile solution containing triethylamine and trifluoroacetic acid as a mobile phase B for gradient elution. After elution, an area normalization method is used to integrate a spectrum to obtain a quantitative result of 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone and impurities. The method has accurate analysis results, can effectively detect the purity and impurity content of 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone in a sample, and the detection error can be controlled within 1.54%, even within 0.64%, and can be as low as 0.07%.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis, and in particular to a quantitative analysis method for 2,3,4,6-tetrabenzyl-D-pyranoglucopyranoside-1,5-lactone. Background Art

[0002] 2,3,4,6-Tetrabenzyl-D-pyranoglucono-1,5-lactone (Formula I) is a light yellow to wine red liquid, easily soluble in organic solvents such as butanone and toluene, but insoluble in water. It is a common glucose derivative and is widely used in the synthesis of drugs, drug intermediates, and natural products.

[0003]

[0004] 2,3,4,6-Tetrabenzyl-D-pyranoglucono-1,5-lactone is one of the key raw materials for the synthesis of voglibose and sodium-sugar cotransporter (SGLT1 and SGLT2) inhibitors, and is used to treat diabetes. 2,3,4,6-Tetrabenzyl-D-pyranoglucono-1,5-lactone can be used as an intermediate for the synthesis of puerarin morphine. The use of puerarin morphine can reduce the dosage of morphine, reduce the addiction and tolerance of morphine, and can be used as a substitute for morphine for pain treatment. It can also be used to prepare miglitol, formyl C-glycosides, compounds for the treatment of high cholesterol, spirocyclic orthoester sugars, etc.

[0005] As a key intermediate, 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone, the research on its preparation and detection methods has become the primary task for R&D and analysis personnel. Currently, it has been reported that thin layer chromatography (TLC), nuclear magnetic resonance (NMR) or mass spectrometry (MS) can detect the structure and purity. However, there is no relevant literature involving the quantitative analysis method of 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone and its related substances.

[0006] Tetrabenzyl glucose is the raw material for producing 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone. The company's enterprise standard method for tetrabenzyl glucose registered on the enterprise standard information service platform uses liquid chromatography to detect the content. This method uses methanol as the solvent and the mobile phase is acidic. When 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone is detected using this method, it will hydrolyze under acidic conditions, which can easily result in a low content result.

[0007] Therefore, there is an urgent need in the art to develop a quantitative analysis method for 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone that is simple to operate and has accurate results. Summary of the invention

[0008] In view of the deficiencies of the prior art, the present invention provides a quantitative analysis method for 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone. The quantitative analysis method is easy to operate, has accurate analysis results, and can effectively detect the purity and impurity content of 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone in a sample.

[0009] The technical solution of the present invention is as follows:

[0010] A quantitative analysis method for 2,3,4,6-tetrabenzyl-D-pyranoglucopyranoside-1,5-lactone, the method being as follows:

[0011] High performance liquid chromatography was used to quantitatively analyze 2,3,4,6-tetrabenzyl-D-glucopyranose-1,5-lactone, with octaalkylsilane bonded silica gel as the stationary phase, methanol aqueous solution containing triethylamine and trifluoroacetic acid as the mobile phase A, and methanol acetonitrile solution containing triethylamine and trifluoroacetic acid as the mobile phase B for gradient elution. After elution, the spectrum was integrated by area normalization method to obtain the quantitative results of 2,3,4,6-tetrabenzyl-D-glucopyranose-1,5-lactone and impurities.

[0012] In mobile phase A, the volume ratio of solvent methanol and water is 10-30:90-70, such as 10:90, 15:85, 20:80, 25:75, 30:70, etc., preferably 20:80;

[0013] In mobile phase B, the volume ratio of solvent methanol and acetonitrile is 10-30:90-70, such as 10:90, 15:85, 20:80, 25:75, 30:70, etc., preferably 20:80;

[0014] In mobile phase A, the volume fractions of triethylamine and trifluoroacetic acid are both 0.03-0.07%, such as 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, etc., preferably 0.05%;

[0015] In mobile phase B, the volume fractions of triethylamine and trifluoroacetic acid are both 0.03-0.07%, such as 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, etc., preferably 0.05%;

[0016] The gradient elution program is as follows: based on the total volume of mobile phase A and mobile phase B, in terms of volume fraction of mobile phase A: 0-18 min: 55-65% (e.g., 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, etc.);

[0017] 18-19min: 0; 19-25min: 55-65% (e.g. 56%, 57%, 58%, 59%, 60%, 61%, 62%,

[0018] 63%, 64%, etc.);

[0019] In the gradient elution program, the injection time is recorded as 0 min, and the volume fraction of mobile phase A is adjusted, for example, the volume fraction of mobile phase A is 55-65% at 0.1 min, adjusted to 0 at 18 min, and adjusted to 55-65% at 19 min;

[0020] The preferred gradient elution program is as follows: based on the total volume of mobile phase A and mobile phase B, in terms of the volume fraction of mobile phase A: 0-18 min: 60%; 18 min: 0; 19-25 min: 60%; under this elution gradient, the degree of separation of 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone and impurities can be further improved, thereby further improving the accuracy of the quantitative analysis results;

[0021] The flow rate of the mobile phase (mobile phase A and mobile phase B combined) is 0.6 to 1.0 ml / min, for example, 0.6 ml / min, 0.7 ml / min, 0.8 ml / min, 0.9 ml / min, 1.0 ml / min, etc., preferably 0.8 ml / min;

[0022] The column temperature is 25-35°C, for example, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, etc., preferably 30°C;

[0023] The detection wavelength is 205-215 nm, for example, 206 nm, 207 nm, 208 nm, 209 nm, 210 nm, 211 nm, 212 nm, 213 nm, 214 nm, etc., preferably 210 nm;

[0024] The injection volume is 8 to 12 μl, for example, 8 μl, 9 μl, 10 μl, 11 μl, 12 μl, etc., preferably 10 μl.

[0025] Particularly preferably, the quantitative analysis method of 2,3,4,6-tetrabenzyl-D-pyranoglucopyranoside-1,5-lactone of the present invention is as follows:

[0026] 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone was quantitatively analyzed by high performance liquid chromatography, with octaalkylsilane bonded silica gel as the stationary phase, methanol aqueous solution containing triethylamine and trifluoroacetic acid as the mobile phase A, and methanol acetonitrile solution containing triethylamine and trifluoroacetic acid as the mobile phase B for gradient elution. After elution, the spectrum was integrated by area normalization method to obtain the quantitative results of 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone and impurities.

[0027] In mobile phase A, the volume ratio of solvent methanol and water was 20:80, and the volume fractions of triethylamine and trifluoroacetic acid were both 0.05%;

[0028] In mobile phase B, the volume ratio of solvent methanol and acetonitrile was 20:80, and the volume fractions of triethylamine and trifluoroacetic acid were both 0.05%;

[0029] The gradient elution program was as follows: based on the total volume of mobile phase A and mobile phase B, in terms of volume fraction of mobile phase A: 0-18 min: 60%; 18 min: 0; 19-25 min: 60%;

[0030] The flow rate of the mobile phase was 0.8 ml / min, the column temperature was 30°C, the detection wavelength was 210 nm, and the injection volume was 10 μl.

[0031] In a preferred embodiment of the present invention, a specific stationary phase and mobile phase are selected, and the elution gradient, column temperature, flow rate, detection wavelength and injection volume are set to further improve the separation effect of 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone and impurities, thereby further improving the accuracy of the quantitative analysis results.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The invention provides a method for quantitatively analyzing 2,3,4,6-tetrabenzyl-D-glucopyranose-1,5-lactone by adopting high performance liquid chromatography. A methanol aqueous solution containing triethylamine and trifluoroacetic acid and a methanol acetonitrile solution containing triethylamine and trifluoroacetic acid are used as mobile phases to elute a sample to be tested. After elution, the separation degree of 2,3,4,6-tetrabenzyl-D-glucopyranose-1,5-lactone and impurities is high. The spectrum is integrated by adopting an area normalization method to obtain quantitative results of 2,3,4,6-tetrabenzyl-D-glucopyranose-1,5-lactone and impurities.

[0034] The analysis result of the present invention is accurate, and the purity and impurity content of 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone in the sample can be effectively detected. The detection error can be controlled within 1.54%, or even within 0.64%, and the lowest can reach 0.07%. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 : The spectrum obtained by high performance liquid chromatography detection in Example 1.

[0036] Figure 2 : The spectrum obtained by high performance liquid chromatography detection in Example 2. DETAILED DESCRIPTION

[0037] The present invention is further described below by means of specific embodiments, but the protection scope of the present invention is not limited thereto.

[0038] Example 1

[0039] This embodiment provides a method for quantitative analysis of 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone using high performance liquid chromatography, as follows:

[0040] (1) Prepare sample solution:

[0041] Weigh 25 mg of 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone, dissolve it in acetonitrile to make up to 50 ml, mix well, and filter with a 0.45 μm filter membrane.

[0042] (2) Detection instrument: Agilent 1260 series liquid chromatograph.

[0043] (3) Stationary phase: octadecylsilane bonded silica gel (Waters XTerra RP85 μm, 4.6×150 mm); mobile phase A: 0.05% by volume solution of triethylamine and trifluoroacetic acid in methanol and water (volume ratio 20:80); mobile phase B: 0.05% by volume solution of triethylamine and trifluoroacetic acid in methanol and acetonitrile (volume ratio 20:80).

[0044] (4) The elution gradient is shown in the following table (the time in the table is the time point when the volume ratio is changed):

[0045] Volume ratio of mobile phase A and mobile phase B 0min 60:40 0.1min 60:40 18min 0:100 19min 60:40 25min 60:40

[0046] (5) Elution parameter setting:

[0047] The flow rate was 0.8 ml / min, the column temperature was 30 °C, the detection wavelength was 210 nm, and the injection volume was 10 μl.

[0048] (6) Data analysis:

[0049] The above test results are as follows Figure 1As shown in the figure, the peak with the largest peak area is the peak of 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone. It can be seen from the spectrum that the separation between 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone and impurities is high, the peak position is moderate, and the peak shape is good.

[0050] Example 2

[0051] The difference from Example 1 is that the elution gradient is as shown in the following table (the time in the table is the time node for changing the volume ratio):

[0052] Volume ratio of mobile phase A and mobile phase B 0min 45:55 0.1min 45:55 18min 0:100 19min 45:55 25min 45:55

[0053] The test results of Example 2 are as follows Figure 2 As shown in the figure, the peak with the largest peak area is the peak of 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone, and the peak shape is good. However, compared with Example 1, the degree of separation between 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone and impurities is relatively low, and the peak position is forward.

[0054] Example 3

[0055] The difference from Example 1 is that the elution gradient is as shown in the following table (the time in the table is the time node for changing the volume ratio):

[0056] Volume ratio of mobile phase A and mobile phase B 0min 65:35 0.1min 65:35 18min 5:95 19min 65:35 25min 65:35

[0057] Example 4

[0058] The difference from Example 1 is that the elution gradient is as shown in the following table (the time in the table is the time node for changing the volume ratio):

[0059] Volume ratio of mobile phase A and mobile phase B 0min 55:45 0.1min 55:45 18min 0:100 19min 55:45 25min 55:45

[0060] Example 5

[0061] The difference from Example 1 is that the flow rate is 0.6 ml / min.

[0062] Example 6

[0063] The difference from Example 1 is that the flow rate is 1.0 ml / min.

[0064] Example 7

[0065] The difference from Example 1 is that the flow rate is 0.4 ml / min.

[0066] Example 8

[0067] The difference from Example 1 is that the flow rate is 1.2 ml / min.

[0068] Example 9

[0069] The difference from Example 1 is that the column temperature is 25°C.

[0070] Example 10

[0071] The difference from Example 1 is that the column temperature is 35°C.

[0072] Embodiment 11

[0073] The difference from Example 1 is that the column temperature is 23°C.

[0074] Example 12

[0075] The difference from Example 1 is that the column temperature is 37°C.

[0076] Embodiment 13

[0077] The difference from Example 1 is that the detection wavelength is 205 nm.

[0078] Embodiment 14

[0079] The difference from Example 1 is that the detection wavelength is 215 nm.

[0080] Embodiment 15

[0081] The difference from Example 1 is that the detection wavelength is 202 nm.

[0082] Example 16

[0083] The difference from Example 1 is that the detection wavelength is 218 nm.

[0084] Embodiment 17

[0085] The difference from Example 1 is that the injection volume is 8 μl.

[0086] Embodiment 18

[0087] The difference from Example 1 is that the injection volume is 12 μl.

[0088] Embodiment 19

[0089] The difference from Example 1 is that the injection volume is 7 μl.

[0090] Embodiment 20

[0091] The difference from Example 1 is that the injection volume is 13 μl.

[0092] Embodiment 21

[0093] The difference from Example 1 is that the volume fraction of triethylamine and trifluoroacetic acid in mobile phase A is 0.03%, and the volume fraction of triethylamine and trifluoroacetic acid in mobile phase B is 0.03%.

[0094] Embodiment 22

[0095] The difference from Example 1 is that the volume fraction of triethylamine and trifluoroacetic acid in mobile phase A is 0.07%, and the volume fraction of triethylamine and trifluoroacetic acid in mobile phase B is 0.07%.

[0096] Embodiment 23

[0097] The difference from Example 1 is that the volume fraction of triethylamine and trifluoroacetic acid in mobile phase A is 0.02%, and the volume fraction of triethylamine and trifluoroacetic acid in mobile phase B is 0.02%.

[0098] Embodiment 24

[0099] The difference from Example 1 is that the volume fraction of triethylamine and trifluoroacetic acid in mobile phase A is 0.08%, and the volume fraction of triethylamine and trifluoroacetic acid in mobile phase B is 0.08%.

[0100] Embodiment 25

[0101] The difference from Example 1 is that the volume ratio of the solvent methanol to water in the mobile phase A is 15:85, and the volume ratio of the solvent methanol to acetonitrile in the mobile phase B is 15:85.

[0102] Embodiment 26

[0103] The difference from Example 1 is that the volume ratio of the solvent methanol to water in the mobile phase A is 25:75, and the volume ratio of the solvent methanol to acetonitrile in the mobile phase B is 25:75.

[0104] Embodiment 27

[0105] The difference from Example 1 is that the volume ratio of the solvent methanol to water in the mobile phase A is 10:90, and the volume ratio of the solvent methanol to acetonitrile in the mobile phase B is 10:90.

[0106] Embodiment 28

[0107] The difference from Example 1 is that the volume ratio of the solvent methanol to water in the mobile phase A is 30:70, and the volume ratio of the solvent methanol to acetonitrile in the mobile phase B is 30:70.

[0108] Comparative Example 1

[0109] The difference from Example 1 is that the addition of triethylamine and trifluoroacetic acid to mobile phase A and mobile phase B is omitted.

[0110] Comparative Example 2

[0111] The difference from Example 1 is that the mobile phase A remains unchanged, and the mobile phase B replaces acetonitrile with methanol.

[0112] Data Analysis:

[0113] The spectra obtained in the above examples and comparative examples were integrated by area normalization method to calculate the concentration of 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone in the tested samples, and the error (%) from the actual concentration was calculated, as shown in the following table:

[0114]

[0115]

[0116] It can be seen from the table that the quantitative analysis method provided by the present invention has high accuracy, with an error within 1.54%, even within 0.64%, and the lowest can reach 0.07%.

[0117] Compared with Example 1, the mobile phases in Comparative Examples 1 and 2 were replaced respectively, and their errors were increased and the accuracy was reduced compared with Example 1. This proves that the present invention achieves effective separation by adopting a specific mobile phase to elute the sample to be tested and obtains accurate test results. Changing any one of the two mobile phases cannot obtain the technical achievements of the present invention.

[0118] By comparing Examples 1 to 4, it can be seen that within the preferred elution gradient range of the present invention (Examples 1, 3 and 4), the test accuracy is higher, and in particular, the elution effect of Example 1 is the best.

[0119] Comparative Examples 1, 5 to 20 show that the flow rate, column temperature, detection wavelength and volume will affect the accuracy of the final test results. When the above four items all meet the preferred conditions of the present invention (flow rate 0.6 to 1.0 ml / min, column temperature 25 to 35°C, detection wavelength 205 to 215 nm, injection volume 8 to 12 μl) (Examples 1, 5, 6, 9, 10, 13, 14, 17, 18), the various parameters cooperate with the elution gradient to further improve the accuracy. Adjusting any one of the parameters of flow rate, column temperature, detection wavelength and injection volume to outside the preferred range (Examples 7, 8, 11, 12, 15, 16, 19, 20) will reduce the accuracy.

[0120] Comparative Examples 1, 21 to 24 show that the use of 0.03 to 0.07% triethylamine and trifluoroacetic acid in methanol aqueous solution and 0.03 to 0.07% triethylamine and trifluoroacetic acid in methanol acetonitrile solution as mobile phases (Examples 1, 21 and 22) can further improve the accuracy of the test results. Too low (Example 23) or too high (Example 24) concentrations will reduce the accuracy.

[0121] By comparing Examples 1 and 25 to 28, it can be seen that the use of a methanol-water solution with a volume ratio of 15 to 25: 85 to 75 and a methanol-acetonitrile solution with a volume ratio of 15 to 25: 85 to 75 as the mobile phase solvent (Examples 1, 25 and 26) can further improve the accuracy of the detection results. A methanol ratio that is too low (Example 27) or too high (Example 28) will reduce the accuracy.

Claims

1. A quantitative analysis method for 2,3,4,6-tetrabenzyl-D-pyranoglucopyranoside-1,5-lactone, characterized in that: The method is as follows: High performance liquid chromatography was used to quantitatively analyze 2,3,4,6-tetrabenzyl-D-glucopyranose-1,5-lactone, with octaalkylsilane bonded silica gel as the stationary phase, methanol aqueous solution containing triethylamine and trifluoroacetic acid as the mobile phase A, and methanol acetonitrile solution containing triethylamine and trifluoroacetic acid as the mobile phase B for gradient elution. After elution, the spectrum was integrated by area normalization method to obtain the quantitative results of 2,3,4,6-tetrabenzyl-D-glucopyranose-1,5-lactone and impurities. In mobile phase A, the volume ratio of solvent methanol to water is 10-30:90-70, and the volume fractions of triethylamine and trifluoroacetic acid are both 0.03-0.07%; in mobile phase B, the volume ratio of solvent methanol to acetonitrile is 10-30:90-70, and the volume fractions of triethylamine and trifluoroacetic acid are both 0.03-0.07%; The gradient elution program was as follows: based on the total volume of mobile phase A and mobile phase B, in terms of volume fraction of mobile phase A: 0-18 min: 55-65%; 18-19 min: 0; 19-25 min: 55-65%; The flow rate of the mobile phase is 0.6-1.0 ml / min; Column temperature is 25-35°C; The detection wavelength is 205-215nm; The injection volume is 8-12 μl.

2. The quantitative analysis method of 2,3,4,6-tetrabenzyl-D-pyranoglucopyranoside-1,5-lactone as claimed in claim 1, characterized in that: The method is as follows: 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone was quantitatively analyzed by high performance liquid chromatography, with octaalkylsilane bonded silica gel as the stationary phase, methanol aqueous solution containing triethylamine and trifluoroacetic acid as the mobile phase A, and methanol acetonitrile solution containing triethylamine and trifluoroacetic acid as the mobile phase B for gradient elution. After elution, the spectrum was integrated by area normalization method to obtain the quantitative results of 2,3,4,6-tetrabenzyl-D-pyranoglucono-1,5-lactone and impurities. In mobile phase A, the volume ratio of solvent methanol and water was 20:80, and the volume fractions of triethylamine and trifluoroacetic acid were both 0.05%; In mobile phase B, the volume ratio of solvent methanol and acetonitrile was 20:80, and the volume fractions of triethylamine and trifluoroacetic acid were both 0.05%; The gradient elution program was as follows: based on the total volume of mobile phase A and mobile phase B, in terms of volume fraction of mobile phase A: 0-18 min: 60%; 18 min: 0; 19~25min: 60%; The flow rate of the mobile phase was 0.8 ml / min, the column temperature was 30°C, the detection wavelength was 210 nm, and the injection volume was 10 μl.

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