Method for detecting tetrahydrocurcumin and impurities thereof by using high performance liquid chromatography

Abstract

The invention belongs to the technical field of analysis and detection, and provides a method for detecting tetrahydrocurcumin and impurities thereof by using high performance liquid chromatography. According to the detection method provided by the invention, the main component tetrahydrocurcumin and the impurities curcumin, dihydrocurcumin, hexahydrocurcumin and octahydrocurcumin are detected mainly by utilizing a high performance liquid chromatography. Specific chromatographic conditions are as follows: a chromatographic column is an octadecyl silane bonded silica gel chromatographic column; the detection wavelength is 280 nm; a mobile phase is an organic solvent and an alkaline ammonium salt solution. The method is specially used for detecting the process and degradation impurities in tetrahydrocurcumin production, solves the problem of abnormal peak pattern under the acidic condition, ensures the accuracy, can simultaneously identify and quantitatively analyze curcumin, dihydrocurcumin, hexahydrocurcumin, octahydrocurcumin and other impurities, realizes comprehensive and clear separation, improves the analysis efficiency and impurity identification comprehensiveness, and has wide application prospects. The method shows excellent separation degree, high sensitivity, good repeatability and durability.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and particularly relates to a method for detecting tetrahydrocurcumin and its impurities by utilizing high performance liquid chromatography. Background Art

[0002] Tetrahydrocurcumin, with the chemical formula of 1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione, is derived from the plant Curcuma longa L. of the Zingiberaceae family. It is one of the main active ingredients of the plant and also an important metabolite of curcumin in the human body. It has multiple functions such as anti-oxidation, whitening and skin care, anti-tumor, anti-diabetes and anti-inflammatory, especially showing significant activity in anti-oxidation, and has attracted much attention in recent years.

[0003] Since tetrahydrocurcumin compounds contain a diketone structure, there is tautomerism between the keto form and the enol form, and these two forms can be mutually converted under specific conditions. Therefore, when tetrahydrocurcumin is detected by high performance liquid chromatography, the coexistence of boat-shaped peaks of the keto form and the enol form is often observed in the relevant literature. This phenomenon not only hinders the accurate determination of the content of tetrahydrocurcumin, but also increases the difficulty of detecting other process impurities (such as curcumin, dihydrocurcumin, hexahydrocurcumin and octahydrocurcumin, which also have tautomerism, and the conditions of tautomerism are different), thereby causing an adverse effect on the quality control of tetrahydrocurcumin related products. Summary of the invention

[0004] The present invention is to solve the above technical problems, and proposes a method for detecting tetrahydrocurcumin and its impurities using high performance liquid chromatography. This method mainly establishes a detection and analysis means for tetrahydrocurcumin and its impurities (including curcumin, dihydrocurcumin, hexahydrocurcumin, octahydrocurcumin) by high performance liquid chromatography. The characteristics of the inventive method are that the detection result is accurate and reliable, with high sensitivity, excellent repeatability, strong accuracy and stability, and can effectively evaluate the quality level of tetrahydrocurcumin-related products, providing a solid basis for the quality control of tetrahydrocurcumin products.

[0005] In the process of exploring the quality control test of tetrahydrocurcumin products, the inventors found that the unique properties of tautomerism between keto and enol forms can be used to cleverly promote the conversion of keto to enol form by carefully controlling temperature and pH, and by optimizing liquid phase conditions, it is ensured that tetrahydrocurcumin is stably maintained in the single structural form of enol form. However, tetrahydrocurcumin compounds exhibit weak acidic characteristics due to the phenolic hydroxyl functional groups contained in their structure. When the pH value of the mobile phase is increased, the compounds will be converted into ionic states and easily react with NH4OH to form salts, which in turn leads to a significant increase in the polarity of the compounds, thereby weakening their retention effect in the test (seeFigure 2 ).

[0006] Faced with this problem, the inventor further proposed a solution of introducing ion pair reagents into the mobile phase. This measure not only effectively increases the surface tension of the mobile phase while maintaining the local pH steady state of the mobile phase, but also significantly improves the retention factor of tetrahydrocurcumin. This design not only ensures that tetrahydrocurcumin and its various impurities can be accurately detected, but also further improves the quality level of the product on the basis of ensuring that degradation impurities and process impurities in tetrahydrocurcumin products are effectively controlled.

[0007] The technical solution of the present invention is:

[0008] A method for detecting tetrahydrocurcumin and its impurities using high performance liquid chromatography mainly comprises the following steps:

[0009] (1) Preparation of reference substance mixed standard solution: accurately weigh each impurity, dissolve it completely with an aqueous solution of an organic solvent, transfer it to a volumetric flask, add an aqueous solution of an organic solvent to dilute to the mark, and shake well to prepare an impurity stock solution;

[0010] Accurately weigh tetrahydrocurcumin, dissolve it completely in an aqueous solution of an organic solvent, and transfer it to a volumetric flask. Accurately measure the impurity stock solution in the volumetric flask, add the aqueous solution of the organic solvent to dilute to the scale, and shake well to obtain a mixed standard solution of the reference substance.

[0011] (2) Prepare the test solution: accurately weigh the product, dissolve it completely in an aqueous solution of an organic solvent, transfer it to a volumetric flask, add an aqueous solution of an organic solvent to dilute to the mark, shake well and filter, and the filtrate is used as the test solution;

[0012] (3) Accurately pipette the reference substance mixed standard solution in (1) and the test substance solution in (2) respectively, inject them into the high performance liquid chromatograph for analysis and detection.

[0013] Preferably, the instrument parameters of the high performance liquid chromatograph are as follows:

[0014] Chromatographic column: Octadecylsilane bonded silica gel chromatographic column, specification: 4.6mm×250mm, 5μm;

[0015] Column temperature: 30-40°C;

[0016] Mobile phase: Phase A is a mixed solution of alkaline ammonium salt buffer and ion pair reagent, and phase B is acetonitrile;

[0017] Flow rate: 0.9-1.1 mL / min;

[0018] Gradient elution: 0.01-2.00min, 20% B phase; 2.00-20.00min, 20-40% B phase; 20.00-21.00min, 40-50% B phase; 21.00-23.00min, 50% B phase; 23.00-23.10min, 50-20% B phase; 23.10-35.00min, 20% B phase;

[0019] Detection wavelength: 275-285nm;

[0020] Injection volume: 20 μL;

[0021] The detector is a diode array detector.

[0022] Preferably, in (1), the impurity is selected from at least one of curcumin, dihydrocurcumin, hexahydrocurcumin, and octahydrocurcumin; and the concentration of the impurity in the reference mixed standard solution is 0.5 μg / mL;

[0023] The concentration of tetrahydrocurcumin in the reference mixed standard solution was 500 μg / mL.

[0024] Preferably, the aqueous solution of the organic solvent in (1) is a solution of acetonitrile and water mixed in a volume ratio of 1:1.

[0025] Preferably, the aqueous solution of the organic solvent in (2) is a solution of acetonitrile and water mixed in a volume ratio of 1:1.

[0026] Preferably, the product in (2) is a tetrahydrocurcumin product, which is obtained by catalyzing curcumin by curcumin reductase.

[0027] Preferably, the alkaline ammonium salt buffer in the mobile phase is ammonium bicarbonate buffer, and the ion pair reagent is triethylamine.

[0028] Preferably, the concentration of the ammonium bicarbonate buffer is 0.02-0.04 mol / L, and the concentration of the triethylamine is 0.6-0.7% by volume.

[0029] Compared with the prior art, the present invention has the following advantages and effects:

[0030] (1) The present invention adopts high performance liquid chromatography technology, which is specifically used to detect process impurities and possible degradation impurities generated in the production process of tetrahydrocurcumin (obtained by fermentation of engineered strains with curcumin reductase catalyzing the substrate curcumin). This method successfully solves the problem of abnormal chromatographic peak shape of tetrahydrocurcumin and its related impurities under acidic conditions, ensuring the accuracy of the test results;

[0031] (2) In addition, this method can also simultaneously identify and quantitatively analyze curcumin, dihydrocurcumin, hexahydrocurcumin, and octahydrocurcumin in impurities, achieving comprehensive and clear separation of curcumin-related impurities. This ability to simultaneously detect multiple components greatly improves the analysis efficiency and the comprehensiveness of impurity identification.

[0032] (3) This method exhibits excellent separation, high sensitivity response, good repeatability and durability, ensuring the stability and accuracy of the data results. Its wide range of applications provides an efficient and reliable method for the quality control of tetrahydrocurcumin products, which is of great significance for improving product quality and ensuring consumer safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the liquid chromatogram of tetrahydrocurcumin under acidic mobile phase;

[0034] Figure 2 It is the completely ionized form of tetrahydrocurcumin in alkaline mobile phase;

[0035] Figure 3 The chromatogram of tetrahydrocurcumin with the mobile phase being acetonitrile-0.03 mol / L ammonium bicarbonate solution (0.65% triethylamine) (20:80);

[0036] Figure 4 The chromatogram is obtained from the mixed positioning solution of tetrahydrocurcumin and impurities in the present invention. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0038] Instruments and reagents

[0039] 1. Instrument

[0040] Thermo U3000 DAD high performance liquid chromatograph, chromatographic column: octadecylsilane bonded silica gel chromatographic column, Waters XBridge C18, 4.6 mm × 250 mm, 5 μm.

[0041] 2. Reagents and test drugs

[0042] Acetonitrile (chromatographically pure, ACS), triethylamine (chromatographically pure, purchased from Fuchen Chemical Reagent Co., Ltd.), tetrahydrocurcumin reference substance (purchased from McLean, purity 98%), curcumin reference substance (purchased from McLean, purity 98%), dihydrocurcumin reference substance (purchased from McLean, purity 98%), hexahydrocurcumin reference substance (purchased from Aladdin, purity 99%), octahydrocurcumin reference substance (purchased from Shanghai Yuanye Biological Co., Ltd., purity 96%);

[0043] The tetrahydrocurcumin product involved in the present invention is a self-made product, which is obtained by catalyzing the substrate curcumin by curcumin reductase obtained by fermentation of an engineered strain, and has a purity of greater than 98%. The preparation steps are: culturing the engineered strain of curcumin reductase at 37°C and 200rpm for 5h, adding 0.1mM inducer to the above fermentation medium for induced expression of enzyme production, continuing to culture at 30°C and 200rpm for 10h, centrifuging the fermentation broth to obtain wet cells, and resuspending the wet cells with Tris-HCl buffer to obtain a whole-cell catalyst; adding an appropriate amount of the whole-cell catalyst so that its final OD in the reaction system is 50, adding curcumin with a final concentration of 20g / L, 0.5g / L NADP + , 5% isopropanol, 100mM Tris-HCl buffer, and heat-retaining reaction at 30°C for 12h to obtain a reaction solution of a reduction product. The reaction solution is further separated and purified by extraction, nanofiltration, ultrafiltration, resin column and other means to obtain a tetrahydrocurcumin product.

[0044] Analytical method in the present invention is developed and designed according to the idea of ​​ICHQ14 guiding principles using the concept of quality from design. By analyzing tetrahydrocurcumin to be measured and its impurity structure, it is confirmed that such compounds are β-dicarbonyl compounds, and such compounds are very prone to keto-enol tautomerism in chromatographic system, and there are differences in tetrahydrocurcumin and its impurity structure, and the distribution of electron clouds is different, and the acid-base of α-H is different, and the conditions for tautomerism are also different (tetrahydrocurcumin or part of impurities undergo structural transformation under the same conditions and other do not undergo transformation). Analytical method in the present invention is that impurity analysis method takes into account the assay of main component (tetrahydrocurcumin), and it is also necessary to consider the sensitivity and specificity of tetrahydrocurcumin impurity (content is one thousandth of the main component) detection, so analytical method development difficulty is very great. Based on the above-mentioned theoretical analysis, the preliminary design mobile phase is alkaline conditions, and tetrahydrocurcumin and its impurity isomerization is enol structure and adopts hybrid silica gel chromatographic column to carry out the development of analytical method. After screening different buffer salts, mobile phase pH and organic phase types of mobile phase, it was found that tetrahydrocurcumin and its impurities showed different performances under the same mobile phase conditions (some impurities had good peak shapes, and some impurities showed boat-shaped peaks). Only under extreme conditions of mobile phase pH>11 and column temperature>50°C, tetrahydrocurcumin and its impurities had good peak shapes and existed in a single form. However, under such conditions, the chromatographic column filler was seriously lost, and the life was greatly affected, which had a huge impact on the quality control cost of the product, so triethylamine was introduced into the mobile phase. On the one hand, tetrahydrocurcumin and its impurities were promoted to form an enol configuration, and on the other hand, as an ion pair reagent, the adsorption force generated by the hydroxyl group in the compound promoted the separation of tetrahydrocurcumin and its impurities. Finally, the concentration of the buffer, the concentration of triethylamine and the optimization of the chromatographic condition parameters were optimized to realize the detection and analysis of this product under normal conditions. The analytical method in the present invention can simultaneously monitor the quality of impurities and main components in tetrahydrocurcumin products, and has the advantages of simple operation, time saving, environmental protection, economy, etc. in the production industry, and has good innovation.

[0045] Example 1

[0046] A method for detecting tetrahydrocurcumin and its impurities using high performance liquid chromatography mainly comprises the following steps:

[0047] (1) Preparation of reference substance mixed standard solution: accurately weigh 10 mg of each impurity (referring to curcumin, dihydrocurcumin, hexahydrocurcumin, octahydrocurcumin), dissolve it completely in 50% acetonitrile aqueous solution, transfer it to a 100 mL volumetric flask, add the above acetonitrile aqueous solution to dilute to the scale, shake well, and prepare an impurity stock solution with a concentration of 0.1 mg / mL;

[0048] Accurately weigh 50 mg of tetrahydrocurcumin, dissolve it completely in 50% acetonitrile aqueous solution, transfer it to a 100 mL volumetric flask, accurately measure 0.5 mL of impurity stock solution in the volumetric flask, add the above acetonitrile aqueous solution to dilute to the scale, shake well, and obtain a reference substance mixed standard solution, wherein the concentration of tetrahydrocurcumin is 500 μg / mL, and the concentration of each impurity is 0.5 μg / mL;

[0049] (2) Preparation of test solution: accurately weigh 12 mg of homemade tetrahydrocurcumin product, dissolve it completely in 50% acetonitrile aqueous solution, transfer it to a 25 mL volumetric flask, add the above acetonitrile aqueous solution to dilute to the scale, shake well and filter, the filtrate is used as the test solution, the concentration is 0.48 mg / mL;

[0050] (3) Accurately pipette 20 μL of the reference mixed standard solution in (1) and the test solution in (2) respectively, inject them into the Thermo U3000 high performance liquid chromatograph, and conduct analytical determination experiments;

[0051] Among them, the instrument parameters of Thermo U3000 high performance liquid chromatograph are as follows:

[0052] Chromatographic column: Octadecylsilane bonded silica gel column, model is Waters XBridge C18, specification is 4.6mm×250mm, 5μm;

[0053] Column temperature: 35°C;

[0054] Mobile phase: Phase A is a mixed solution of 0.03 mol / L ammonium bicarbonate buffer and 0.65% triethylamine, and phase B is acetonitrile;

[0055] Flow rate: 1.0 mL / min;

[0056] Gradient elution: 0.01-2.00min, 20% B phase; 2.00-20.00min, 20-40% B phase; 20.00-21.00min, 40-50% B phase; 21.00-23.00min, 50% B phase; 23.00-23.10min, 50-20% B phase; 23.10-35.00min, 20% B phase;

[0057] Detection wavelength: 280nm;

[0058] Injection volume: 20 μL;

[0059] Detector:DAD.

[0060] The test results obtained are as follows Figure 3As shown, under this condition, the retention time of tetrahydrocurcumin is 23.693min, the peak shape is good (single existence form), and no tetrahydrocurcumin impurity is found in the product.

[0061] Example 2 Methodology Verification Experiment

[0062] The detection method proposed in Example 1 was verified by conducting seven indices, namely, specificity test, linearity and range test, repeatability test, accuracy test, quantitative limit, solution stability, and durability test.

[0063] The main component, impurity a, impurity b, impurity c, and impurity d appearing in the following seven index experiments refer to tetrahydrocurcumin, curcumin, octahydrocurcumin, dihydrocurcumin, and hexahydrocurcumin, respectively.

[0064] 1. Specificity test

[0065] Instruments and conditions: Same as Example 1 (DAD detector)

[0066] Experimental steps:

[0067] Preparation of positioning solutions: Weigh appropriate amounts of tetrahydrocurcumin, curcumin, octahydrocurcumin, dihydrocurcumin, and hexahydrocurcumin as reference substances, place each in a 1.5 mL liquid phase injection bottle, add 1.5 mL of 50% acetonitrile aqueous solution to completely dissolve, and shake well to obtain a separate positioning solution for each reference substance.

[0068] Preparation of impurity stock solutions: accurately weigh 10 mg of each impurity curcumin, octahydrocurcumin, dihydrocurcumin and hexahydrocurcumin, dissolve them in 50% acetonitrile aqueous solution by volume, transfer them to a 100 mL volumetric flask, add 50% acetonitrile aqueous solution by volume to dilute to the scale, mix well, and prepare impurity stock solutions, which are respectively recorded as impurity a stock solution, impurity b stock solution, impurity c stock solution and impurity d stock solution, and the concentrations are all 100 μg / mL.

[0069] Preparation of reference substance mixed solution: accurately weigh 50 mg of tetrahydrocurcumin, accurately measure 0.5 mL of impurity stock solution, place in a 100 mL volumetric flask, add 50% by volume acetonitrile aqueous solution to dissolve, dilute to scale, shake well, and obtain reference substance mixed solution.

[0070] 20 μL of the reference mixed standard solution was respectively drawn and injected into the Thermo U3000 high performance liquid chromatograph. The analysis spectra of the main components and impurities are shown in Figure 4 The specificity test results are shown in Table 1.

[0071] Table 1 Specificity test results

[0072]

[0073]

[0074] As shown in Table 1, under the chromatographic conditions, the retention time of impurities curcumin, dihydrocurcumin, hexahydrocurcumin and octahydrocurcumin in the reference mixed solution is consistent with the retention time in the positioning solution, each impurity can be effectively separated from the main peak of tetrahydrocurcumin, the separation between each adjacent chromatographic peak is greater than 1.5, and the purity of the main peak is 994, which is greater than 980, proving that the method has good specificity.

[0075] 2.2 Linearity and range

[0076] Instruments and conditions: Same as Example 1 (DAD detector)

[0077] Experimental steps:

[0078] Since the structures of various impurities are similar, the validation of analytical methods (such as linearity and range, repeatability and accuracy) only requires validation of one representative impurity (impurity a).

[0079] Prepare the reference substance mixed stock solution: accurately weigh 25.31 mg of the main component and 25.32 mg of the impurity a reference substance, dissolve them in 50% acetonitrile aqueous solution, transfer them to a 50 mL volumetric flask, add the above acetonitrile aqueous solution to dilute to the scale, shake well, and prepare the reference substance mixed stock solution.

[0080] Preparation of standard curve solution: Accurately measure 1.0mL, 2.0mL, 4.0mL, 6.0mL, and 8.0mL of the above-mentioned reference substance mixed stock solution, respectively, add 50% acetonitrile aqueous solution to dilute to 10mL, to prepare the reference substance mixed standard curve solution, aspirate 20μL and inject into Thermo U3000 high performance liquid chromatograph, determine the peak area, linearity and correlation coefficient of tetrahydrocurcumin and impurities, the experimental results are shown in Tables 2-1 and 2-2.

[0081] Table 2-1 Linear test results of main component concentration C and peak area A

[0082]

[0083] Table 2-2 Linearity test results of impurity a concentration C and peak area A

[0084]

[0085] From the above experimental results, it can be seen that the correlation coefficient between the concentration of the main component and impurity a is greater than 0.999 in the range of 50-500 μg / mL, and the peak area A and the concentration C show a good linear relationship. The correction factor of impurity a calculated according to the slope of the linear equation between the main component and impurity a is 1.3. In order to ensure the accuracy of the impurity determination, the content of impurity a is detected by the external standard method.

[0086] 2.3 Repeatability test

[0087] Instruments and conditions: Same as Example 1 (DAD detector)

[0088] Experimental steps:

[0089] Preparation of impurity a reference solution: accurately weigh 10.23 mg of impurity a, dissolve it in 50% acetonitrile aqueous solution, place it in a 100 mL volumetric flask, add it to the acetonitrile aqueous solution to dilute to the mark, shake well, and prepare the impurity a stock solution;

[0090] Accurately measure 0.5 mL of the impurity a stock solution, place it in a 10 mL volumetric flask, add 50% acetonitrile aqueous solution to dilute to the scale, shake well, and obtain the impurity a reference solution.

[0091] Preparation of test solution: Accurately weigh 12 mg of homemade tetrahydrocurcumin product, dissolve it in 50% acetonitrile aqueous solution, transfer it to a 25 mL volumetric flask, then add 50% acetonitrile aqueous solution to dilute to the scale, shake well, and use it as the test solution. Prepare 6 copies in parallel.

[0092] Preparation of control solution: Accurately measure 1 mL from each of the 6 test solutions, place in a 100 mL volumetric flask, add 50% acetonitrile aqueous solution to dilute to scale, then take 1 mL of the diluted solution, shake well, place in a 10 mL volumetric flask, add 50% acetonitrile aqueous solution to dilute to scale, and mix well to obtain 6 control solutions. Pipette 20 μL and inject into the Thermo U3000 high performance liquid chromatograph to determine the repeatability of impurity a. The experimental results are shown in Table 3.

[0093] Table 3 Reproducibility test results of impurity a for relevant substance methodology verification

[0094] serial number 1 2 3 4 5 6 RSD(%) Impurity a(%) 0.7591 0.7389 0.7744 0.7461 0.7552 0.7663 1.71

[0095] The experimental results show that there is no significant difference in the content of impurity a in the 6 test solutions, and the relative standard deviation is less than 2.0%, so the repeatability of the above analysis method is good.

[0096] 2.4 Accuracy test

[0097] Instruments and conditions: Same as Example 1 (DAD detector)

[0098] Experimental steps:

[0099] Preparation of impurity a stock solution: accurately weigh 10.57 mg of the reference impurity a, dissolve it in 50% acetonitrile aqueous solution by volume, transfer it into a 100 mL volumetric flask, add the above acetonitrile aqueous solution to dilute to the scale, and shake well; then accurately measure 0.5 mL of the above impurity a stock solution in a 10 mL volumetric flask, add 50% acetonitrile aqueous solution by volume to dilute to the scale, shake well, and prepare the impurity a stock solution.

[0100] Preparation of test sample stock solution: Accurately weigh 101.13 mg of homemade tetrahydrocurcumin product, dissolve it in 50% acetonitrile aqueous solution by volume, transfer it into a 100 mL volumetric flask, add the above acetonitrile aqueous solution to dilute to the scale line, and shake well.

[0101] Preparation of test solution: Accurately measure 5 mL of the test sample stock solution and place it in a 10 mL volumetric flask. Then add 50% acetonitrile aqueous solution to dilute to the scale line and shake well.

[0102] Prepare 50% accuracy solution: Accurately measure 5 mL of the test sample stock solution and 0.5 mL of the impurity a stock solution, place them in a 10 mL volumetric flask, then add 50% acetonitrile aqueous solution to dilute to the scale line, shake well, and prepare 3 copies in parallel.

[0103] Prepare 100% accuracy solution: Accurately measure 5 mL of the test sample stock solution and 1.0 mL of the impurity a stock solution, place them in a 10 mL volumetric flask, then add 50% acetonitrile aqueous solution to dilute to the scale line, shake well, and prepare 3 copies in parallel.

[0104] Prepare 150% accuracy solution: Accurately measure 5 mL of the test sample stock solution and 1.5 mL of the impurity a stock solution, place them in a 10 mL volumetric flask, then add 50% acetonitrile aqueous solution to dilute to the scale line, shake well, and prepare 3 copies in parallel.

[0105] 20 μL of the above accuracy solutions of each concentration were respectively injected into the Thermo Fisher U3000 high performance liquid chromatograph to determine the peak area of ​​impurity a and calculate the recovery rate. The experimental results are shown in Table 4.

[0106] Table 4 Accuracy test results of impurity a

[0107]

[0108]

[0109] From the experimental results in the above table, it can be seen that the recoveries of impurity a at three concentration levels (50%, 100%, and 150% of the limit concentration) are all in the range of 80.0% to 120.0%, and the RSD of the recoveries is less than 10%, indicating that the above detection method has good accuracy.

[0110] 2.5 Limit of quantitation

[0111] Instruments and conditions: Same as Example 1 (DAD detector)

[0112] Experimental steps: Accurately weigh 10.11 mg of the main component and 10.57 mg of the impurity a reference substance, respectively, add 50% acetonitrile aqueous solution to dissolve and quantitatively dilute to make a solution with a concentration of 100 μg / mL of each component as a stock solution. Add 50% acetonitrile water to dilute step by step and inject into the Thermo Fisher U3000 high performance liquid chromatograph, and take the concentration when S / N ≥ 10 as the quantitative limit concentration. The experimental results are shown in Tables 5-1 and 5-2.

[0113] Table 5-1 Results of the quantitative limit test of impurity a for methodological verification of related substances

[0114]

[0115] Table 5-2 Results of principal component quantitative limit test for methodological verification of related substances

[0116]

[0117]

[0118] From the above test results, it can be seen that the signal-to-noise ratio of curcumin at this concentration is about 10:1, so the quantitative limit of curcumin under the present chromatographic conditions is 10.36 ng, which is equivalent to more than 0.10% of curcumin being quantitatively calculated; the signal-to-noise ratio of tetrahydrocurcumin is about 10:1, so the quantitative limit of the main component under the present chromatographic conditions is 9.91 ng, which is equivalent to more than 0.10% of tetrahydrocurcumin being quantitatively calculated.

[0119] 2.6 Solution stability

[0120] Instruments and conditions: Same as Example 1 (DAD detector)

[0121] Experimental steps:

[0122] Impurity stock solution: Accurately weigh 10 mg of each impurity reference substance, dissolve it in 50% by volume acetonitrile aqueous solution, transfer it into a 100 mL volumetric flask, add the above acetonitrile aqueous solution to dilute to the scale line, and shake well.

[0123] Test sample stock solution: Accurately weigh 100 mg of homemade tetrahydrocurcumin product, dissolve it in 50% acetonitrile aqueous solution, transfer it into a 100 mL volumetric flask, add the above acetonitrile aqueous solution to dilute to the scale line, and shake well.

[0124] Reference solution: Accurately weigh 50 mg of tetrahydrocurcumin, dissolve it in 50% acetonitrile aqueous solution, transfer it into a 100 mL volumetric flask, accurately measure 0.5 mL of impurity stock solution, add the above acetonitrile aqueous solution to dilute, dilute to volume after dissolution, and shake well to obtain.

[0125] Preparation of test solution: Accurately measure 5 mL of the test sample stock solution, place it in a 10 mL volumetric flask, add 50% acetonitrile aqueous solution to dissolve and dilute to the scale, and shake well.

[0126] Test sample spiked solution: Accurately measure 5 mL of test sample stock solution and 0.5 mL of impurity stock solution, add 50% acetonitrile aqueous solution to dilute, make up to volume after dissolving, and shake well.

[0127] The reference solution, test solution and test spiked solution were refrigerated for 12 hours. At 0, 2, 4, 6, 9 and 12 hours, 20 μL was injected into the Thermo U3000 high performance liquid chromatograph for determination and the peak areas were recorded. The experimental results are shown in Tables 6-1, 6-2 and 6-3.

[0128] Table 6-1 Results of stability test of reference solution for verification of relevant substance methodology

[0129]

[0130] Table 6-2 Results of stability test of test sample solution for verification of relevant substance methodology

[0131]

[0132]

[0133] Table 6-3 Results of stability test of spiked solution of test samples for verification of relevant substance methodology

[0134]

[0135] From the test results in Table 6-1, it can be seen that after being placed in a refrigerated environment for 12 hours, the RSDs of the peak areas of the main component and impurities a, b, c, and d in the reference solution were all less than 2.0%, indicating that the reference solution had good stability.

[0136] From the test results in Table 6-2, it can be seen that after being placed in a refrigerated environment for 12 hours, no impurities were detected in the test solution, and the RSD of the main component peak area was less than 2.0%, indicating that the test solution had good stability.

[0137] It can be seen from the test results in Table 6-3 that after being placed in a refrigerated environment for 12 hours, the RSDs of the peak areas of the main component and impurities b, c, and d in the test sample spiked solution were all less than 2.0%, and impurity a was not detected, indicating that the test sample spiked solution had good stability.

[0138] 2.7 Durability test

[0139] Instruments and conditions: The parameters of column temperature, flow rate and detection wavelength were changed (see Table 7). Other experimental conditions were the same as those in Example 1 (DAD detector).

[0140] Experimental steps:

[0141] Impurity a reference substance stock solution: Accurately weigh 10.57 mg of impurity a reference substance, dissolve it in 50% by volume acetonitrile aqueous solution, transfer it into a 100 mL volumetric flask, add the above acetonitrile aqueous solution to dilute to the scale line, and shake well.

[0142] Impurity a reference solution: Accurately measure 0.5 mL of the impurity a reference stock solution, place it in a 100 mL volumetric flask, add 50% acetonitrile aqueous solution to dilute, make up to volume after dissolving, and shake well.

[0143] Test solution: Accurately weigh 12.64 mg of homemade tetrahydrocurcumin product, dissolve it in 50% acetonitrile aqueous solution, transfer it into a 25 mL volumetric flask, add the above acetonitrile aqueous solution to dilute to the scale line, and shake well.

[0144] Test sample spike solution: Measure 1.5 mL of the test sample solution and place it in a liquid phase injection bottle. Weigh appropriate amounts of impurity b, impurity c, and impurity d and dissolve them completely as the test sample spike solution.

[0145] Control solution: Accurately measure 1.0 mL of the test solution, place it in a 100 mL volumetric flask, add 50% acetonitrile aqueous solution to dilute, dilute to volume after dissolving, then measure 1 mL of the fixed solution and place it in a 10 mL volumetric flask, add 50% acetonitrile aqueous solution to dilute, dilute to volume after dissolving, and shake well.

[0146] 20 μL was injected into Thermo U3000 high performance liquid chromatograph. By changing the flow rate, column temperature and detection wavelength, the applicability of the impurity system and the change of impurity a content (calculated by external standard method) before and after the changes were investigated. The experimental results are shown in Tables 7-1 and 7-2.

[0147] Table 7-1 Results of the test on the suitability of the system for the durability impurities of the relevant substance methodology verification

[0148]

[0149]

[0150] Table 7-2 Test results of the durability test sample solution for relevant substance methodology verification

[0151]

[0152] It can be seen from Table 7-1 and Table 7-2 that after changing the column temperature, flow rate and detection wavelength, the separation between the main peak and each adjacent impurity is greater than 1.5, and baseline separation is achieved; the determination result of the impurity a content has no obvious change, and the detection of related substances is good. It can be seen that the durability of the analysis and detection method of the present invention is good.

[0153] In summary, the method for detecting tetrahydrocurcumin and its impurities by high performance liquid chromatography provided by the present invention is specifically used to detect the process and degradation impurities in the production of tetrahydrocurcumin products (curcumin obtained by fermentation of engineered strains and produced by reductase catalysis), solves the problem of abnormal peak shape under acidic conditions, ensures accuracy, and can simultaneously identify and quantitatively analyze impurities such as curcumin, dihydrocurcumin, hexahydrocurcumin and octahydrocurcumin, achieves comprehensive and clear separation, improves analysis efficiency and comprehensiveness of impurity identification, shows excellent separation, high sensitivity, good repeatability and durability, and provides an efficient and reliable method for quality control of tetrahydrocurcumin products.

[0154] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A method for detecting tetrahydrocurcumin and its impurities using high performance liquid chromatography, characterized in that, The main steps include: (1) Preparation of reference substance mixed standard solution: Accurately weigh the impurity, dissolve it completely with an aqueous solution of an organic solvent, transfer it to a volumetric flask, add an aqueous solution of an organic solvent to dilute to the mark, shake well, and prepare an impurity stock solution; Accurately weigh tetrahydrocurcumin, dissolve it completely in an aqueous solution of an organic solvent, and transfer it to a volumetric flask. Accurately measure the impurity stock solution in the volumetric flask, add the aqueous solution of the organic solvent to dilute to the scale, and shake well to obtain a mixed standard solution of the reference substance. (2) Preparation of test solution: Accurately weigh the product, dissolve it completely in an aqueous solution of an organic solvent, transfer it to a volumetric flask, add an aqueous solution of an organic solvent to dilute to the mark, shake well and filter. The filtrate is used as the test solution. (3) Accurately pipette the reference mixed standard solution in (1) and the test solution in (2) into a high performance liquid chromatograph for analysis.

2. The detection method as claimed in claim 1, characterized in that The instrument parameters of the high performance liquid chromatograph are as follows: Chromatographic column: octadecylsilane bonded silica gel chromatographic column, specification: 4.6 mm×250 mm, 5 μm; Column temperature: 30-40°C; Mobile phase: Phase A is a mixed solution of alkaline ammonium salt buffer and ion pair reagent, and phase B is acetonitrile; Flow rate: 0.9-1.1 mL / min; Gradient elution: 0.01-2.00min, 20% B phase; 2.00-20.00min, 20-40% B phase; 20.00-21.00min, 40-50% B phase; 21.00-23.00min, 50% B phase; 23.00-23.10min, 50-20% B phase; 23.10-35.00min, 20% B phase; Detection wavelength: 275-285nm; Injection volume: 20 μL; The detector is a diode array detector.

3. The detection method as claimed in claim 1, characterized in that In (1), the impurity is selected from at least one of curcumin, dihydrocurcumin, hexahydrocurcumin, and octahydrocurcumin; and the concentration of the impurity in the reference mixed standard solution is 0.5 μg / mL; The concentration of tetrahydrocurcumin in the reference mixed standard solution was 500 μg / mL.

4. The detection method as claimed in claim 1, characterized in that The aqueous solution of the organic solvent in (1) is a solution obtained by mixing acetonitrile and water in a volume ratio of 1:

1.

5. The detection method as claimed in claim 1, characterized in that The aqueous solution of the organic solvent in (2) is a solution obtained by mixing acetonitrile and water in a volume ratio of 1:

1.

6. The detection method as claimed in claim 1, characterized in that The product in (2) is a tetrahydrocurcumin product, which is obtained by catalyzing curcumin by curcumin reductase.

7. The detection method as claimed in claim 2, characterized in that The alkaline ammonium salt buffer in the mobile phase is ammonium bicarbonate buffer, and the ion pair reagent is triethylamine.

8. The detection method as claimed in claim 7, characterized in that The concentration of the ammonium bicarbonate buffer is 0.02-0.04 mol / L, and the concentration of triethylamine is 0.6-0.7% by volume.

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