Method for detecting thiamine mixed phosphate components and thiamine monophosphate impurities

Abstract

The application discloses a thiamine mixed phosphate ester component and thiamine monophosphate impurity detection method, and belongs to the technical field of compound detection. The method comprises the following steps: (1) preparing a test sample solution of the thiamine mixed phosphate ester or the thiamine monophosphate to be detected; (2) taking control sample thiamine monophosphate, thiamine pyrophosphate, thiamine triphosphate, thiamine tetraphosphate, disulfide thiamine monophosphate, disulfide thiamine diphosphate, disulfide thiamine triphosphate, disulfide thiamine tetraphosphate and thiamine, and preparing a system suitability solution containing each control sample; (3) taking the system suitability solution and the corresponding test sample solution, and detecting by means of a high performance liquid chromatograph, and adopting a peak area normalization method to calculate the content of each substance. The method can realize effective separation of compounds with similar structures, has a short analysis cycle and high sensitivity, and is suitable for separation and detection of each component in the thiamine mixed phosphate ester and impurities in thiamine monophosphate synthesis.

Description

Technical Field

[0001] This invention relates to the field of compound detection technology, and more specifically, to a method for detecting the components of thiamine mixed phosphate esters and thiamine monophosphate ester impurities. Background Technology

[0002] In the industrial synthesis of thiamine mixed phosphate esters, the phosphorylation reaction is a stepwise reversible process, and the products naturally form a multi-component mixed system (including thiamine monophosphate, thiamine pyrophosphate, thiamine triphosphate, dithiamine monophosphate, etc.). The composition and content of these components directly determine the product's efficacy and safety. Vitamin B1 (thiamine), after entering cells, also forms thiamine mixed phosphate esters through phosphorylation, of which 80% is thiamine pyrophosphate, 10% is thiamine triphosphate, and the remainder is thiamine monophosphate, etc. However, existing detection technologies have poor selectivity for thiamine phosphate esters and derivatives with similar structures and polarities, making baseline separation difficult. They can only detect single or a few types of phosphate esters, resulting in limited coverage. Furthermore, they suffer from long analysis cycles, low sensitivity, and poor reproducibility, failing to meet the needs of monitoring the synthesis process and detecting the quality of the finished product.

[0003] Thiamine monophosphate is an important active metabolite of vitamin B1 (also known as thiamine or thiamine hydrochloride) in the body and a key intermediate in vitamin B1 supplements and the therapeutic drug phenylphosphine sulfate. Its synthesis process easily generates various byproducts and degradation impurities, which directly affect reaction process control, product purity and yield, and the quality and safety of the downstream drug phenylphosphine sulfate. Existing impurity detection technologies in thiamine monophosphate production have significant limitations: conventional detection methods struggle to effectively separate structurally similar impurities, leading to peak overlap; and simultaneous quantitative detection of trace impurities and the main component is difficult, with sensitivity failing to meet quality control requirements.

[0004] Therefore, developing a method that can separate and detect the components in thiamine mixed phosphate esters and the impurities in the synthesis of thiamine monophosphate esters would be of great practical significance. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for detecting the components of thiamine mixed phosphate and thiamine monophosphate impurities.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for detecting the components of thiamine mixed phosphate esters and thiamine monophosphate ester impurities includes the following steps:

[0008] (1) Prepare a test solution of the mixed phosphate ester or monophosphate ester of thiamine to be tested;

[0009] (2) Take the reference standards thiamine monophosphate, thiamine pyrophosphate, thiamine triphosphate, thiamine tetraphosphate, dithiamine monophosphate, dithiamine diphosphate, dithiamine triphosphate, dithiamine tetraphosphate and thiamine, and prepare a system suitability solution containing each reference standard.

[0010] (3) Take the system suitability solution and the test solution of the thiamine mixed phosphate ester or the thiamine monophosphate ester to be tested, and detect them by high performance liquid chromatography. Calculate the contents of thiamine monophosphate, thiamine pyrophosphate, thiamine triphosphate, thiamine tetraphosphate, dithiamine monophosphate, dithiamine diphosphate, dithiamine triphosphate, dithiamine tetraphosphate and thiamine in the thiamine mixed phosphate ester or the thiamine monophosphate ester to be tested, respectively, using the peak area normalization method.

[0011] The present invention further specifies that the chromatographic conditions are as follows:

[0012] Chromatographic column: Welch Ultimate AQ-C18 aqueous column, 4.6 mm × 250 mm, packed with octadecylsilane-bonded silica gel with a particle size of 5 μm; Mobile phase: triethanolamine and ammonium salt ion-pair buffer solution as mobile phase A, and methanol or acetonitrile as mobile phase B, with gradient elution; Detection wavelength: 243 nm; Flow rate: 0.8 mL / min; Column temperature: 30 ℃; Injection volume: 10 μL.

[0013] The present invention is further configured such that triethanolamine and an ammonium salt are added to pure water, and the pH value is adjusted to 5-7 using one of hydrochloric acid, sulfuric acid, phosphoric acid, or acetic acid to obtain mobile phase A; the ratio of pure water to triethanolamine and ammonium salt is 1L:(1-3)g:(1-1.5)g. The ammonium salt is one of ammonium chloride, ammonium carbonate, ammonium bisulfate, ammonium formate, ammonium acetate, or tetrabutylammonium hydroxide.

[0014] The present invention is further configured such that the gradient elution program is as follows: 0-5 min, the volume fraction of mobile phase A is maintained at 90%, and the volume fraction of mobile phase B is maintained at 10%; 5-25 min, the volume fraction of mobile phase A decreases from 90% to 70%, and the volume fraction of mobile phase B increases from 10% to 30%; 25-40 min, the volume fraction of mobile phase A is maintained at 70%, and the volume fraction of mobile phase B is maintained at 30%; 40-40.2 min, the volume fraction of mobile phase A increases from 70% to 90%, and the volume fraction of mobile phase B decreases from 30% to 10%; 40.2-45 min, the volume fraction of mobile phase A is maintained at 90%, and the volume fraction of mobile phase B is maintained at 10%.

[0015] The present invention is further configured such that the concentration of the test sample solution is 0.5-1 mg / mL.

[0016] The present invention is further configured such that, in the system suitability solution, the concentration of thiamine monophosphate reference standard is 0.5-1 mg / mL, and the concentrations of thiamine reference standard, thiamine pyrophosphate reference standard, thiamine triphosphate reference standard, thiamine tetraphosphate reference standard, dithiamine monophosphate reference standard, dithiamine diphosphate reference standard, dithiamine triphosphate reference standard, and dithiamine tetraphosphate reference standard are all 0.009-0.011 mg / mL.

[0017] In summary, the present invention has the following beneficial effects:

[0018] The method of this invention can effectively separate compounds with similar structures, with a short analysis cycle and high sensitivity. It can be used for the separation and detection of components in thiamine mixed phosphate esters, as well as for the separation and detection of impurities in the synthesis of thiamine monophosphate esters. It plays a significant role in monitoring the product synthesis process and controlling the quality of finished products. Attached Figure Description

[0019] Figure 1 The mass spectrum of thiamine pyrophosphate prepared in Example 1;

[0020] Figure 2 The mass spectrum of thiamine triphosphate prepared in Example 1;

[0021] Figure 3 This is the mass spectrum of the dithiamine monophosphate prepared in Example 1;

[0022] Figure 4 Here is the liquid chromatogram of the blank solution from Example 1;

[0023] Figure 5 The liquid chromatogram of the thiamine mixed phosphate test solution in Example 1;

[0024] Figure 6 The mass spectrum of the crude thiamine monophosphate prepared in Example 2;

[0025] Figure 7 This is the blank liquid chromatogram for Example 2;

[0026] Figure 8 The liquid chromatogram of the thiamine monophosphate test solution in Example 2 is shown.

[0027] Figure 9 This is a liquid chromatogram of the system suitability solution from Example 2. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] The method for detecting the components of thiamine mixed phosphate and thiamine monophosphate impurities in this invention is as follows:

[0030] a1. Preparation of the test solution:

[0031] Take the thiamine mixed phosphate ester or the thiamine monophosphate ester to be tested, and use pure water as a solvent to prepare a test solution with a concentration of 0.5-1 mg / mL;

[0032] a2. Prepare system-suitable solutions:

[0033] Using pure water as a solvent, prepare a mixed solution with a concentration of 0.5-1 mg / mL for thiamine monophosphate reference standard and 0.009-0.011 mg / mL for thiamine reference standard, thiamine pyrophosphate reference standard, thiamine triphosphate reference standard, thiamine tetraphosphate reference standard, dithiamine monophosphate reference standard, dithiamine diphosphate reference standard, dithiamine triphosphate reference standard, and dithiamine tetraphosphate reference standard. This solution is used as a system suitability solution.

[0034] The structural formula of thiamine is ;

[0035] The structural formula of thiamine monophosphate is: ;

[0036] The structural formula of thiamine pyrophosphate is: ;

[0037] The structural formula of thiamine triphosphate is: ;

[0038] The structural formula of thiamine tetraphosphate is: ;

[0039] The structural formula of dithioamine monophosphate is:

[0040] ;

[0041] The structural formula of dithioamine diphosphate is:

[0042] ;

[0043] The structural formula of dithioamine triphosphate is:

[0044] ;

[0045] The structural formula of dithioamine tetraphosphate is:

[0046] .

[0047] a3. Content determination: Inject the test solution, system suitability solution, and pure water blank solution into the high performance liquid chromatograph for determination. Calculate the content of thiamine, thiamine monophosphate, thiamine pyrophosphate, thiamine triphosphate, thiamine tetraphosphate, dithiamine monophosphate, dithiamine diphosphate, dithiamine triphosphate, and dithiamine tetraphosphate in the thiamine mixed phosphate or thiamine monophosphate test samples, respectively, using the peak area normalization method.

[0048] The chromatographic conditions are as follows:

[0049] Chromatographic column: Welch Ultimate AQ-C18 aqueous column (with octadecylsilane-bonded silica gel as packing material), 4.6 mm × 250 mm, packing particle size 5 μm; Mobile phase: Triethanolamine and ammonium salt ion-pair buffer solution as mobile phase A (take 1 L of pure water, add 1-3 g of triethanolamine and 1-1.5 g of ammonium salt (ammonium salt is one of ammonium chloride, ammonium carbonate, ammonium bisulfate, ammonium formate, ammonium acetate, tetrabutylammonium hydroxide), adjust the pH to 5-7 with one of hydrochloric acid, sulfuric acid, phosphoric acid, or acetic acid to obtain mobile phase A), methanol or acetonitrile as mobile phase B, elute according to the gradient specified in Table 1; Detection wavelength: 243 nm; Flow rate: 0.8 mL / min; Column temperature: 30 ℃; Injection volume: 10 μL.

[0050] Table 1

[0051]

[0052] Example 1: Detection of components in thiamine mixed phosphate ester

[0053] Thiamine monophosphate, thiamine pyrophosphate, thiamine triphosphate, dithiamine monophosphate, thiamine (purchased from Maclean's, catalog number B861479), and thiamine tetraphosphate (catalog number HY250112), dithiamine diphosphate (catalog number HY714588), dithiamine triphosphate (catalog number HY475808), and dithiamine tetraphosphate (catalog number HY24-250223), all purchased from Hangzhou Hanyao Biotechnology Co., Ltd., were used as standard references. The self-synthesized thiamine mixed phosphate was tested according to the above method.

[0054] The preparation method of the thiamine mixed phosphate ester to be tested is as follows: Take a reaction flask, add phosphoric acid (85% (v / v), 12g), dehydrate under vacuum at 100℃ for 1 hour, add phosphorus pentoxide (11g, 0.077mol), and react at 100℃ for 2 hours; after the reaction, add thiamine (Annegi Chemical, purity 99%, 8g, 0.024mol) in batches, and react at 110℃ for 2 hours. After confirming the complete reaction by TLC analysis (using a mixture of acetone, water, acetic acid, and 10wt% ammonia as the developing solvent, with a volume ratio of 6:2:1:0.5), the temperature was lowered to 70℃. The reaction solution was then added to 15mL of 0-4℃ ice water and stirred to dissolve. 300mL of isopropanol was added under 0-4℃ ice water bath, and the mixture was stirred for 10 minutes before being placed in a refrigerator overnight (4℃). After removing the supernatant, a milky white substance was obtained, which was the thiamine mixed phosphate ester. A portion of this substance was taken as the thiamine mixed phosphate ester to be tested.

[0055] Take 20 g of the crude thiamine mixed phosphate synthesized by the above method, dissolve it in 60 mL of pure water, and directly load the sample through a macroporous weakly basic anion exchange resin D302 (column diameter 4 cm × bed height 90 cm, amine type). Elute with pure water at a flow rate of 200 mL / h. Monitor the separation liquid using HPLC central control. First collect 0-140 mL of eluent (corresponding to thiamine monophosphate), then collect 140-550 mL of eluent (corresponding to thiamine pyrophosphate, its mass spectrum is shown in [reference needed]). Figure 1 Collect 550-680 mL of eluent (corresponding to dithiamine monophosphate, its mass spectrum is shown in [reference]). Figure 3 Then rinse with 3wt% ammonia (flow rate 200 mL / h) and collect 90 mL (corresponding to thiamine triphosphate, its mass spectrum is shown in [reference needed]). Figure 2 Each collected liquid was individually concentrated to dryness under vacuum and crystallized with 50wt% isopropanol aqueous solution to obtain white solids as reference standards.

[0056] In this test, the concentration of the test sample solution was 1 mg / mL; the concentration of thiamine monophosphate reference standard in the system suitability solution was 1 mg / mL, and the concentrations of the other 8 reference standards were all 0.001 mg / mL; the chromatographic conditions were as follows:

[0057] Chromatographic column: Welch Ultimate AQ-C18 aqueous column (with octadecylsilane-bonded silica gel as packing material), 4.6 mm × 250 mm, packing particle size 5 μm; Mobile phase: Triethanolamine and ammonium salt ion pair buffer solution as mobile phase A (take 1 L of pure water, add 1 g of triethanolamine and 1 g of ammonium acetate, adjust the pH to 6 with acetic acid to obtain mobile phase A), methanol as mobile phase B, elution according to the gradient specified in Table 1; Detection wavelength: 243 nm; Flow rate: 0.8 mL / min; Column temperature: 30 ℃; Injection volume: 10 μL.

[0058] The liquid chromatogram of the blank solution obtained in this embodiment is shown below. Figure 4 The liquid chromatogram of the thiamine mixed phosphate test solution is shown in [reference needed]. Figure 5 The retention times and contents of each component in the measured thiamine mixed phosphate ester are shown in Table 2.

[0059] Table 2

[0060]

[0061] Example 2: Detection of structurally similar impurities in the synthesis of thiamine monophosphate

[0062] Thiamine monophosphate, thiamine pyrophosphate, thiamine triphosphate, dithiamine monophosphate, thiamine (purchased from Maclean's, catalog number B861479), and thiamine tetraphosphate (catalog number HY250112), dithiamine diphosphate (catalog number HY714588), dithiamine triphosphate (catalog number HY475808), and dithiamine tetraphosphate (catalog number HY24-250223), all purchased from Hangzhou Hanyao Biotechnology Co., Ltd., were used as standard references. The self-synthesized thiamine monophosphate was tested according to the above method.

[0063] The preparation method of the thiamine monophosphate to be tested is as follows: Take a reaction flask, add phosphoric acid (85% (v / v), 12g), dehydrate under vacuum at 100°C for 1 hour, add phosphorus pentoxide (11g, 0.077mol), react at 100°C for 2 hours, and then add thiamine (Annex, 99%, 8g, 0.024mol) in batches, and react at 110°C for 2 hours. After confirming the completeness of the reaction by TLC (the developing solvent is a mixture of isopropanol, water, acetic acid, and 10wt% ammonia, with a volume ratio of 6:2:1:0.5), cool to 80°C, add 25mL of water, stir at 80°C for 2 hours, cool to room temperature, add 300mL of isopropanol, stir and filter to obtain a white solid, which is the thiamine monophosphate to be tested.

[0064] The preparation of the reference standards thiamine monophosphate, thiamine pyrophosphate, dithiamine monophosphate, and thiamine triphosphate was the same as in Example 1.

[0065] In this test, the concentration of the test sample solution was 1 mg / mL; the concentration of thiamine monophosphate reference standard in the system suitability solution was 1 mg / mL, and the concentrations of the other 8 reference standards were all 0.001 mg / mL; the chromatographic conditions were as follows:

[0066] Chromatographic column: Welch Ultimate AQ-C18 aqueous column (with octadecylsilane-bonded silica gel as packing material), 4.6 mm × 250 mm, packing particle size 5 μm; Mobile phase: Triethanolamine and ammonium salt ion pair buffer solution as mobile phase A (take 1 L of pure water, add 1 g of triethanolamine and 1 g of ammonium acetate, adjust the pH to 6 with acetic acid to obtain mobile phase A), methanol as mobile phase B, elution according to the gradient specified in Table 1; Detection wavelength: 243 nm; Flow rate: 0.8 mL / min; Column temperature: 30 ℃; Injection volume: 10 μL.

[0067] The blank liquid chromatogram for this embodiment is shown below. Figure 6 The liquid chromatogram of the thiamine monophosphate test solution is shown in [reference needed]. Figure 7 The retention times and contents of each component in the thiamine monophosphate were measured and are shown in Table 4. The results showed that thiamine and thiamine pyrophosphate impurities were detected in the thiamine monophosphate synthesized by the method of this embodiment.

[0068] Table 4

[0069]

[0070] The retention time results for solution chromatography detection of the system in this embodiment are shown in Table 5.

[0071] Table 5

[0072]

[0073] In summary, sample analysis results show that structurally similar components in thiamine mixed phosphate esters can be effectively separated and detected. This method can also be used to detect related substances in thiamine monophosphate ester samples. The method of this invention can also be used to detect thiamine monophosphate esters and thiamine mixed phosphate esters synthesized by other methods.

[0074] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting impurities in various components of thiamine mixed phosphate esters and thiamine monophosphate esters, characterized in that, Includes the following steps: (1) Prepare a test solution of the mixed phosphate ester or monophosphate ester of thiamine to be tested; (2) Take the reference standards thiamine monophosphate, thiamine pyrophosphate, thiamine triphosphate, thiamine tetraphosphate, dithiamine monophosphate, dithiamine diphosphate, dithiamine triphosphate, dithiamine tetraphosphate and thiamine, and prepare a system suitability solution containing each reference standard. (3) Take the system suitability solution and the test solution of the thiamine mixed phosphate ester or the thiamine monophosphate ester to be tested, and detect them by high performance liquid chromatography. Calculate the contents of thiamine monophosphate, thiamine pyrophosphate, thiamine triphosphate, thiamine tetraphosphate, dithiamine monophosphate, dithiamine diphosphate, dithiamine triphosphate, dithiamine tetraphosphate and thiamine in the thiamine mixed phosphate ester or the thiamine monophosphate ester to be tested, respectively, using the peak area normalization method.

2. The method for detecting impurities in thiamine mixed phosphate esters and thiamine monophosphate esters according to claim 1, characterized in that, The chromatographic conditions are as follows: Chromatographic column: Welch Ultimate AQ-C18 aqueous column, 4.6 mm × 250 mm, packed with octadecylsilane-bonded silica gel with a particle size of 5 μm; Mobile phase: triethanolamine and ammonium salt ion-pair buffer solution as mobile phase A, and methanol or acetonitrile as mobile phase B, with gradient elution; Detection wavelength: 243 nm; Flow rate: 0.8 mL / min; Column temperature: 30 ℃; Injection volume: 10 μL.

3. The method for detecting impurities in thiamine mixed phosphate esters and thiamine monophosphate esters according to claim 2, characterized in that, Add triethanolamine and ammonium salt to pure water, and adjust the pH value to 5-7 with one of hydrochloric acid, sulfuric acid, phosphoric acid, or acetic acid to obtain mobile phase A; the ratio of pure water to triethanolamine and ammonium salt is 1L:(1-3)g:(1-1.5)g.

4. The method for detecting impurities in thiamine mixed phosphate esters and thiamine monophosphate esters according to claim 3, characterized in that, The ammonium salt is one of the following: ammonium chloride, ammonium carbonate, ammonium bisulfate, ammonium formate, ammonium acetate, and tetrabutylammonium hydroxide.

5. The method for detecting impurities in thiamine mixed phosphate esters and thiamine monophosphate esters according to claim 2, characterized in that, The gradient elution program was as follows: 0-5 min, mobile phase A volume fraction remained at 90%, and mobile phase B volume fraction remained at 10%; 5-25 min, mobile phase A volume fraction decreased from 90% to 70%, and mobile phase B volume fraction increased from 10% to 30%; 25-40 min, mobile phase A volume fraction remained at 70%, and mobile phase B volume fraction remained at 30%; 40-40.2 min, mobile phase A volume fraction increased from 70% to 90%, and mobile phase B volume fraction decreased from 30% to 10%; 40.2-45 min, mobile phase A volume fraction remained at 90%, and mobile phase B volume fraction remained at 10%.

6. The method for detecting impurities in thiamine mixed phosphate esters and thiamine monophosphate esters according to claim 1, characterized in that, The concentration of the test solution is 0.5-1 mg / mL.

7. The method for detecting impurities in thiamine mixed phosphate esters and thiamine monophosphate esters according to claim 1, characterized in that, In the system suitability solution, the concentration of thiamine monophosphate reference standard is 0.5-1 mg / mL, and the concentrations of thiamine reference standard, thiamine pyrophosphate reference standard, thiamine triphosphate reference standard, thiamine tetraphosphate reference standard, dithiamine monophosphate reference standard, dithiamine diphosphate reference standard, dithiamine triphosphate reference standard, and dithiamine tetraphosphate reference standard are all 0.009-0.011 mg / mL.

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