Method for detecting enamine content of vitamin B1 intermediate

Abstract

According to the detection method, two isomers of the intermediate enamine are separated through preparative chromatography, reference substances of two structures are prepared, then the content of a sample is measured through an HPLC external standard method, methodology verifies that specificity, linear relation, precision, repeatability and stability are good, and the content of the vitamin B1 intermediate enamine is measured through the HPLC external standard method. The method can meet accurate determination of the enamine content of the vitamin B1 intermediate, overcomes the defect of poor content determination accuracy in the prior art, and provides a detection basis for guiding vitamin B1 production.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, in particular to a method for detecting the content of vitamin B1 intermediate enamine. Background Art

[0002] Enamine, chemical name: α-(o-chlorophenyl)-aminomethylene-β-formamidopropionitrile), structural formula: It is an intermediate in the production process of vitamin B1. Its content determination can adopt common methods such as ultraviolet-visible spectrophotometry and HPLC. What is adopted now is the ultraviolet-visible spectrophotometry reference substance comparison method to determine the content of enamine sample. The enamine reference substance is a working reference substance prepared after refining the enamine sample. It is found that the sample content varies greatly during the determination process, sometimes only 89%, and sometimes it may reach more than 102%, which cannot guide production well. It is found that enamine presents bimodal in HPLC chromatogram by liquid chromatography detection, and the area percentage of the two peaks is not fixed. Due to the double bond contained in its molecular structure, there are two structures of cis and trans isomers. The ultraviolet response values ​​of the two structures at a specific wavelength have certain differences, and due to the slight changes in process parameters (such as pH value, temperature, etc.) in the production process, the ratio of the two structures can be inconsistent, and the ratio of the two peaks in the reference substance and the sample may be inconsistent, resulting in that the analysis result cannot reflect the true content of the sample; by using HPLC external standard method to determine, the result difference is also very large, which is basically consistent with ultraviolet-visible spectrophotometry, and the content of the sample cannot be determined well.

[0003] Therefore, it is necessary to propose a method for detecting the content of vitamin B1 intermediate enamine to overcome the defect of poor accuracy of the detection results of the prior art. Summary of the invention

[0004] The invention provides a method for detecting the content of vitamin B1 intermediate enamine. Two isomers in the enamine are separated by preparative chromatography, two reference substances with different structures are prepared, and the content of the sample is determined by an HPLC external standard method, thereby overcoming the defect of poor accuracy of content determination in the prior art.

[0005] In view of this, the scheme of the present invention is:

[0006] A method for detecting the content of vitamin B1 intermediate enamine, the detection method using HPLC method, the chromatographic conditions are: the chromatographic column is filled with octadecylsilane bonded silica gel, the mobile phase is a mixture of acetonitrile and water in a volume ratio of (36-44): (56-64), the flow rate is 0.9-1.1 mL / min, the detection wavelength is 285-289 nm, and the column temperature is 38-42° C.

[0007] The detection method comprises the following steps:

[0008] Preparation of enamine reference substance: inject the sample into preparative chromatography, collect the cis-isomer and trans-isomer components of the enamine in the order of peak elution, and then rotary evaporate to dryness to prepare enamine cis-isomer and enamine trans-isomer reference substance working solutions respectively;

[0009] Detection of enamine content: The sample is injected into the chromatogram, and the enamine content is calculated according to the external standard method based on the chromatogram: enamine content = enamine trans isomer peak area multiplied by correction factor + enamine cis isomer peak area;

[0010] The enamine is α-(o-chlorophenyl)-aminomethylene-β-formamidopropionitrile, and the structural formula is:

[0011]

[0012] Furthermore, during the detection process, standard curves of the trans isomer of the enamine and the cis isomer are drawn respectively, and the correction factor is the coefficient ratio of the standard curve of the cis isomer to the standard curve of the trans isomer of the enamine.

[0013] Furthermore, the correction factor is 0.82.

[0014] Furthermore, the injection volume of the enamine content detection process is 10 μL.

[0015] Furthermore, the concentration of the sample and / or reference working solution is 20-200 μg / ml; preferably, the diluent of the working reference solution is the mobile phase.

[0016] Furthermore, the chromatographic column is XB-C18 4.6mm*250mm*5um.

[0017] Furthermore, the chromatographic conditions are as follows: the mobile phase is a mixture of acetonitrile and water in a volume ratio of 40:60, the flow rate is 1.0 mL / min, the detection wavelength is 2887 nm, and the column temperature is 40°C.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The detection method provided by the present invention separates two isomers of the intermediate enamine by preparative chromatography, prepares reference substances of two structures, and then determines the content of the sample by HPLC external standard method. The methodological verification shows that the specificity, linear relationship, precision, repeatability and stability are all good, and the content of the vitamin B1 intermediate enamine can be accurately determined, and the defect of poor accuracy of content determination in the prior art is overcome, so as to provide a detection basis for guiding the production of vitamin B1. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a comparison chart of the cis-isomers of the enamine described in the present invention.

[0021] Figure 2 This is a comparison chart of the trans isomers of the enamine described in the present invention.

[0022] Figure 3 This is the specificity verification spectrum of the present invention. DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be clearly and completely described below in conjunction with the preferred embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The inventors have collected some data on the determination of enamine content using existing methods in previous years, as shown in the following table.

[0025] The enamine content result is on the low side, which means that the detection results of existing means are inaccurate and not conducive to guiding production.

[0026] batch number content batch number content batch number content 20191014 94.0％ 20201114 92.7％ 20211021 90.2％ 20191015 93.1％ 20201115 88.4％ 20211022 91.3％ 20191016 92.8％ 20201116 93.2％ 20211023 92.6％

[0027] In order to solve the above problems, a method for detecting the content of vitamin B1 intermediate enamine is proposed. The two isomers in the enamine are separated by preparative chromatography, and reference substances with two structures are prepared. Then, the content of the sample is determined by HPLC external standard method. The method is feasible. Specifically, it is determined by high performance liquid chromatography, with octadecylsilane bonded silica gel as filler (such as: XB C18 4.6*250*5um), flow rate 1.0ml / min, detection wavelength 287nm, column temperature 40℃, accurately weigh about 50mg of this product, put it in a 50ml volumetric flask, add mobile phase to dissolve and dilute to the scale, shake well, accurately measure 5ml, put it in a 50ml volumetric flask, dilute to the scale with mobile phase, shake well, as the test solution, accurately measure 10ul and inject it into the liquid chromatograph, record the chromatogram, the order of peaks is enamine cis-isomer and enamine trans-isomer, take about 50mg of enamine working standard product, put it in a 50ml volumetric flask, and determine it in the same way. According to the external standard method, the peak area of ​​enamine trans-isomer multiplied by the sum of the correction factor and the peak area of ​​enamine cis-isomer is calculated.

[0028] The specific steps are as follows:

[0029] 1. Preparation of enamine reference substance: Prepare by preparative chromatography, mobile phase (acetonitrile: water (4:6)), detection wavelength 287nm, flow rate 10ml / min, injection volume 10ml, collect two components separately, and then rotary evaporate to dryness to obtain pure products of two components. The purity of both components is above 99.5% after HPLC detection. Since the preparation amount of the two isomers is very small, in order to ensure the accuracy of the results, it is best to use a mixed reference substance (cis isomer and trans isomer are closer to the sample) as a control to determine the content of the sample.

[0030] The reference substances include reference substances 1 to 3, as shown in the following table: Among them, the HPLC detection reference spectrum of the enamine cis isomer is as follows Figure 1 As shown, the reference spectrum of the trans isomer of the enamine is as Figure 2 shown.

[0031] Reference Cis isomer content Trans isomer content Reference substance-1 99.9％ 0.1％ Reference substance-2 0.2％ 99.8％ Reference substance-3 66.0％ 34.0％

[0032] 2. Determination of enamine sample content

[0033] Weigh about 50 mg of this product accurately, place it in a 50 ml volumetric flask, add mobile phase to dissolve and dilute to scale, shake well, accurately measure 5 ml, place it in a 50 ml volumetric flask, dilute to scale with mobile phase, shake well, as the test solution, according to the following chromatographic conditions, accurately measure 10 μl and inject it into the liquid chromatograph, record the chromatogram, the peak order is enamine cis isomer and enamine trans isomer, take about 50 mg of enamine working standard product, place it in a 50 ml volumetric flask, and determine it in the same way. According to the external standard method, the enamine trans isomer peak area multiplied by the correction factor f and the sum of the enamine cis isomer peak area is calculated, that is, the enamine content is obtained. Correction factor f = 0.82.

[0034] Specifically, the above chromatographic conditions are as follows:

[0035] Detection Methods High performance liquid chromatography Chromatographic columns XB-C18 4.6mm*250mm*5um Mobile phase Acetonitrile-water (40:60) Detection wavelength 287nm Injection volume 10μl Column temperature 40℃ Control / Sample Concentration 100μg / ml Theoretical plates Both cis and trans isomers of enamine should be greater than 5000 Separation The separation degree between cis and trans isomers of enamine should be greater than 3.0

[0036] Methodological validation

[0037] 1. Exclusivity

[0038] According to the above liquid chromatography conditions, 10 μl of blank solvent (acetonitrile-water (4:6)) was accurately measured and injected into the liquid chromatograph. There were no absorption peaks at the retention times of the two enamine peaks, the solvent did not interfere with the determination, and the method had good specificity.

[0039] 2. Linear range

[0040] Accurately weigh about 50 mg each of enamine cis isomer, enamine trans isomer and enamine working reference substance, place in a 50 ml volumetric flask, add mobile phase to dissolve and dilute to the scale, shake well, as the reference substance stock solution, then take 1, 2, 3, 4, 5, 10 ml of the reference substance stock solution respectively, place in a 50 ml volumetric flask, add mobile phase to dilute to the scale, shake well, to obtain linear solution-1, linear solution-2, linear solution-3, linear solution-4, linear solution-5, linear solution-6, according to the above chromatographic conditions, accurately measure 10 μl and inject into liquid chromatograph, draw a standard curve with concentration as the horizontal axis and peak area as the vertical axis.

[0041]

[0042]

[0043] It can be seen that the response of the trans isomer peak is larger than that of the cis isomer. When calculating, the trans isomer peak area can be multiplied by the correction factor f=0.82 (f=50.016 / 60.988) for correction. The linear relationship is good in the range of 20--200ug / ml.

[0044] 3. Injection precision

[0045] Six injections were made of the enamine cis isomer, enamine trans isomer, and enamine mixed reference solution (100ug / ml) respectively, and the RSD of the peak area was calculated. The results are as follows:

[0046]

[0047]

[0048] 4. Repeatability

[0049] Take the same test sample and measure the content of 6 portions continuously according to the sample determination method. The results are as follows:

[0050] 1 2 3 4 5 6 average RSD 96.76％ 97.74％ 98.14％ 97.61％ 98.07％ 98.58％ 97.8％ 0.6％

[0051] The same sample was measured six times in succession, and the results were between 96.8% and 98.6%, with an RSD of 0.6%, indicating good repeatability.

[0052] 5. Solution stability

[0053] Take the same reference solution and test solution, and inject samples at 0h, 5h, 20h, and 24h respectively to observe the changes in peak area.

[0054]

[0055] 6. Durability

[0056] By changing the mobile phase ratio, column temperature, flow rate, etc., the separation degree and plate number of the two enamine peaks can meet the requirements, and the method has good durability, as shown in the following table.

[0057]

[0058] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting the content of vitamin B1 intermediate enamine, characterized in that: The detection method uses HPLC, and the chromatographic conditions are: the chromatographic column is filled with octadecylsilane bonded silica gel, the mobile phase is a mixture of acetonitrile and water in a volume ratio of (36-44): (56-64), the flow rate is 0.9-1.1 mL / min, the detection wavelength is 285-289 nm, and the column temperature is 38-42° C.; The detection method comprises the following steps: Preparation of enamine reference substance: inject the sample into preparative chromatography, collect the cis-isomer and trans-isomer components of the enamine in the order of peak elution, and then rotary evaporate to dryness to prepare enamine cis-isomer and enamine trans-isomer reference substance working solutions respectively; Detection of enamine content: The sample is injected into the chromatogram, and the enamine content is calculated according to the external standard method based on the chromatogram: enamine content = enamine trans isomer peak area multiplied by correction factor + enamine cis isomer peak area; The enamine is α-(o-chlorophenyl)-aminomethylene-β-formamidopropionitrile.

2. The detection method according to claim 1, characterized in that: During the detection process, standard curves of the trans isomer of the enamine and the cis isomer are drawn respectively, and the correction factor is the coefficient ratio of the standard curve of the cis isomer to the standard curve of the trans isomer of the enamine.

3. The detection method according to claim 1 or 2, characterized in that: The correction factor is 0.

82.

4. The detection method according to claim 1, characterized in that: The injection volume of the enamine content detection process is 10 μL.

5. The detection method according to claim 1, characterized in that: The concentration of the sample and / or reference substance working solution is 20-200 μg / ml.

6. The detection method according to claim 1, characterized in that: The chromatographic column is XB-C18 4.6mm*250mm*5um.

7. The detection method according to claim 1, characterized in that: The chromatographic conditions are as follows: the mobile phase is a mixture of acetonitrile and water in a volume ratio of 40:60, the flow rate is 1.0 mL / min, the detection wavelength is 2887 nm, and the column temperature is 40°C.

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