A detection method and preparation method of nitisinone impurities

Abstract

The invention discloses a method for detecting nitisinone impurities. Compared with the prior art, the detection method has the advantages of reliability, strong specificity and strong practicability. Each impurity can be effectively detected, and nitisinone and each impurity are well separated. The detection method can detect impurities A, impurity B, impurity C, impurity D, impurity E and impurity F. The invention also discloses a preparation method thereof. A substrate is first prepared into an acyl chloride derivative, and then condensed with 1,3-cyclohexanedione under an inorganic base condition to obtain the impurity. The method is efficient, simple and highly selective. The preparation of impurity C using a cyclization reaction promoted by ultraviolet light (>300 nm) has not been reported. The photocatalytic method has the characteristics of being environmentally friendly. The preparation of impurities involved in the invention has not been reported.

Description

Technical Field

[0001] The invention relates to the technical field of drug synthesis and analysis, and in particular to a detection method and a preparation method of nitisinone impurities. Background Art

[0002] Nitisinone is a synthetic reversible inhibitor of 4-hydroxyphenylpyruvate dioxygenase, mainly used for the treatment of rare pediatric hereditary tyrosinemia type I (HT-1). By inhibiting the normal metabolism of tyrosine in HT-1 patients, it can prevent the accumulation of highly toxic maleylacetoacetate, fumarylacetoacetate and its bypass metabolite succinylacetone, thereby alleviating liver and kidney damage, relieving symptoms, significantly improving long-term prognosis, increasing survival rate, and reducing the incidence of hepatocellular carcinoma. It is suitable for hereditary tyrosinemia type I (HT-1) and can assist in dietary restriction of tyrosine and phenylalanine.

[0003] Any substance that affects the purity of a drug is called an impurity. Drugs may produce different impurity spectra under different process conditions. The control of impurities in drugs is an important aspect of drug research and development, and is also an important indicator for clinical toxicity research. By detecting impurities, we can find out the source, nature, detection method and limit of impurities in drugs, optimize the drug manufacturing process, and then avoid the generation of impurities or reduce impurities to a minimum, thus ensuring and improving drug quality from many aspects.

[0004] Nitisinone APIs will produce byproducts or other degradation products during the synthesis process, storage and transportation. The types and contents of impurities have an important impact on the quality of Nitisinone. Therefore, the research on the preparation and detection methods of impurities in Nitisinone is of great significance. The impurity products prepared by the preparation method can be used for the qualitative and quantitative analysis of impurities in the production of Nitisinone APIs and preparations. The detection method can be used to clarify the source, nature, and limit of impurities in Nitisinone, thereby improving the quality standards of Nitisinone APIs and preparations, and providing guidance for people's drug safety.

[0005] At present, there are very few reports on the detection of impurities in nitisinone. The pharmacopoeias of various countries do not include the standard of nitisinone, and the detection method of impurities in nitisinone needs to be established by ourselves. Therefore, providing a preparation and detection method of nitisinone impurities is of great significance for the quality control of nitisinone. Summary of the invention

[0006] The object of the present invention is to provide a method for detecting and preparing nitisinone impurities, so as to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for detecting impurities of nitisinone is provided, wherein the content of impurities in nitisinone is determined by high performance liquid chromatography, wherein the high performance liquid chromatography is a reverse phase chromatography, and the chromatographic conditions are as follows: chromatographic column: OSAKA SODA SUPERIOREXODS 4.6 mm×250 mm, 5 μm, 0.05%-0.15% (V / V) phosphoric acid solution is used as mobile phase A, and acetonitrile is used as mobile phase B. The detection method comprises the following steps:

[0009] 1) Take an appropriate amount of nitisinone, dissolve it in acetonitrile and quantitatively dilute it to make a solution containing about 1.5 mg to 3 mg per 1 ml;

[0010] 2) Set the mobile phase flow rate to 1.3ml-1.9ml per minute, the column temperature to 25℃-35℃, and the detection wavelength to 225nm-250nm; the elution procedure is as shown in the table below;

[0011]

[0012] 3) Take 5 μl-15 μl of the sample solution in step 1), inject it into a liquid chromatograph, record the chromatogram, and complete the sample detection.

[0013] As a further embodiment of the present invention: the detection method can determine the content of impurity A, impurity B, impurity C, impurity D, impurity E and impurity F in nitisinone.

[0014] As a further solution of the present invention: the concentration of phosphoric acid in the mobile phase A is 0.1%.

[0015] As a further solution of the present invention: in step 1), each 1 ml of the sample solution contains 2 mg of the sample to be tested.

[0016] As a further solution of the present invention: the flow rate of the mobile phase in step 2) is 1.6 ml per minute; the column temperature of the chromatographic column is 30° C.; and the detection wavelength is 240 nm.

[0017] As a further solution of the present invention: the injection volume of the sample solution in step 3) is 10 μl.

[0018] As a further scheme of the present invention: The present invention also discloses a method for preparing nitisinone impurities, including the preparation of impurities C, impurity D and impurity E. The preparation method comprises first preparing substrates 2-chloro-4-trifluoromethylbenzoic acid, 2-nitro-4-trifluoromethylbenzoic acid and 4-nitro-2-trifluoromethylbenzoic acid into acyl chloride derivatives respectively, and then condensing them with 1,3-cyclohexanedione under inorganic base conditions to obtain impurities C, impurity D and impurity E respectively.

[0019] As a further solution of the present invention: the preparation method of the impurity C is: the substrate 2-chloro-4-trifluoromethylbenzoic acid is subjected to a chlorination reaction with thionyl chloride under the catalysis of DMF, the solvent is evaporated, and condensed with 1,3-cyclohexanedione under an alkaline environment, and then irradiated under ultraviolet light to obtain impurity C, and the reaction formula is as follows:

[0020]

[0021] As a further solution of the present invention: the preparation method of the impurity D is: the substrate 2-nitro-4-trifluoromethylbenzoic acid is subjected to a chlorination reaction with thionyl chloride under the catalysis of DMF, the solvent is evaporated, and the impurity D is obtained by condensation with 1,3-cyclohexanedione under an alkaline environment. The reaction formula is as follows:

[0022]

[0023] As a further solution of the present invention: the preparation method of the impurity E is: the substrate 4-nitro-2-trifluoromethylbenzoic acid is subjected to a chlorination reaction with thionyl chloride under the catalysis of DMF, the solvent is evaporated, and the impurity E is obtained by condensation with 1,3-cyclohexanedione under an alkaline environment. The reaction formula is as follows:

[0024]

[0025] Compared with the prior art, the present invention has the following beneficial effects: the detection method of nitisinone-related substances provided by the present invention has the advantages of being reliable, highly specific, and highly practical; each impurity can be effectively detected; and nitisinone and each impurity are well separated; the system can be used to detect a variety of impurities (impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F).

[0026] The invention also discloses a preparation method of nitisinone impurity C, impurity D and impurity E. Compared with the prior art, the preparation method of the invention is to first prepare a substrate into an acyl chloride derivative, and then condense the acyl chloride derivative with 1,3-cyclohexanedione under inorganic base conditions. The method is efficient, simple and highly selective. The preparation of impurity C uses a cyclization reaction promoted by ultraviolet light (>300nm), which has not been reported, and the photocatalytic method has the characteristic of being environmentally friendly. The impurities C, impurity D and impurity E prepared by the preparation method of the invention are easy to purify, and the product purity is above 98%. The preparation of impurities involved in the invention has not been reported.

[0027] The structural formula and chemical name of the Nitisinone impurity described in the present invention are as follows:

[0028] Impurity A: 2-nitro-4-trifluoromethylbenzoic acid;

[0029]

[0030] Impurity B: 1,3-cyclohexanedione;

[0031]

[0032] Impurity C: 6-(trifluoromethyl)-3,4-dihydro-1H-xanthine-1,9(2H)-dione;

[0033]

[0034] Impurity D: 2-nitro-4-(trifluoromethyl)-3-oxo-1-cyclohexen-1-yl benzoate;

[0035]

[0036] Impurity E: 2-(4-nitro-2-(trifluoromethyl)benzoyl)cyclohexane-1,3-dione;

[0037]

[0038] Impurity F: 2-hydroxy-4-trifluoromethylbenzoic acid;

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of impurity C;

[0041] Figure 2 is the carbon NMR spectrum of impurity C;

[0042] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of impurity D;

[0043] Figure 4 is the carbon NMR spectrum of impurity D;

[0044] Figure 5 is the hydrogen nuclear magnetic resonance spectrum of impurity E;

[0045] Figure 6 is the carbon NMR spectrum of impurity E;

[0046] Figure 7 It is the HPLC chromatogram of the test solution at different flow rates;

[0047] Figure 8 It is the HPLC chromatogram of the test solution at different column temperatures;

[0048] Fig. 9 The HPLC chromatograms of the test solution at different injection volumes;

[0049] Fig.10The HPLC chromatograms of the test solution under different mobile phase gradient elution methods are as follows: A is the chromatogram of the first mobile phase gradient elution method; B is the chromatogram of the second mobile phase gradient elution method; C is the chromatogram of the third mobile phase gradient elution method; D is the chromatogram of the fourth mobile phase gradient elution method; E is the chromatogram of the fifth mobile phase gradient elution method;

[0050] Fig.11 It is the high performance liquid chromatogram of separation impurity A, impurity B, impurity C, impurity D, impurity E and impurity F in the solution. DETAILED DESCRIPTION

[0051] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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.

[0052] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0053] Example 1 Preparation of Nitisinone Impurity C

[0054] Under nitrogen protection, 100 ml of toluene and 20 drops of DMF dissolved 11.54 g of 2-chloro-4-trifluoromethylbenzoic acid (50 mmol, 1.0 eqv.) were added to a 250 ml three-necked flask, and 30 ml of toluene dissolved 10.71 g of thionyl chloride (90 mmol, 1.8 eqv.) solution was slowly added dropwise under ice bath, and the temperature was raised to 110°C and refluxed after the addition was completed. After the reaction was completed, the solvent was evaporated under reduced pressure, and the residual thionyl chloride was dried with 60 ml of n-heptane 3 times, and after evaporation, 100 ml of acetonitrile was added to dissolve. Under nitrogen protection, 6.21 g 1,3-cyclohexanedione (55 mmol, 1.1 eqv.), 13.56 g dried potassium fluoride powder (231 mmol, 4.6 eqv.) and 20 ml acetonitrile were added to a 500 ml three-necked flask and dissolved. The mixture was stirred at 25 °C for 3 h. The acetonitrile solution after evaporation was slowly added dropwise under an ice bath. After the addition was completed, the mixture was heated to 25 °C and continued to react for 38.5 hours. 80 ml of water was taken to quench the reaction solution. The solution was separated and the acetonitrile layer was concentrated to dryness. 400 ml of ethyl acetate was added to dissolve the solution. The solution was washed twice with 80 ml of 10% sodium chloride solution, and then the organic phase was washed twice with 300 ml of 2 mol / L hydrochloric acid solution and once with 300 ml of saturated brine. The solution was dried and concentrated to obtain a crude chloro-NTBC analog. Take the crude product and add 205 ml of acetone at 45°C to dissolve it, slowly add 153 ml of water to squeeze out crystals, keep warm and stir for 1 hour, cool to 0°C, stir for 2 hours, filter, and dry the filter cake to obtain the pure chloro-NTBC analog. Take the pure chloro-NTBC analog and dissolve it in 270 ml of dichloromethane, place it under ultraviolet light (365nm) for 13 days, concentrate it, use ethyl acetate: formic acid (20:1) as eluent, use 300 mesh silica gel as stationary phase, separate by column chromatography, concentrate it, recrystallize it with ethyl acetate: n-heptane (5:1), filter it, and dry it to obtain 0.86 g of pure light yellow crystalline impurity C, with a yield of 50%. Its nuclear magnetic resonance hydrogen spectrum is shown in Figure 1 , its carbon NMR spectrum can be found at Figure 2 , quantitative NMR purity: 99.57%.

[0055] Example 2 Preparation of Nitisinone Impurity D

[0056] Under nitrogen protection, 3.53g 2-nitro-4-trifluoromethylbenzoic acid (15mmol, 1eqv.), 3 drops of DMF and 30ml toluene were added to a three-necked flask in sequence. After stirring in an ice bath for 0.5h, a solution of 2.68g thionyl chloride (22.5mmol, 1.5eqv.) dissolved in 10ml toluene was slowly added dropwise. After the addition was complete, the temperature was raised to 110°C and refluxed. After the reaction of 2-nitro-4-trifluoromethylbenzoic acid was complete, the solvent was evaporated under reduced pressure. After evaporation, the residual thionyl chloride was removed three times with 15ml dry n-heptane. After evaporation, it was dissolved in 30ml dichloromethane for later use. Under nitrogen protection, 1.85g 1,3-cyclohexanedione (16.5mmol, 1.1eqv.), 2.61g pyridine (33mmol, 2.2eqv.) and 60ml dichloromethane were stirred in an ice bath for 1.5h, and then the dichloromethane solution prepared in the previous step was slowly added dropwise. After the addition was completed, the temperature was raised to room temperature for reaction. After the reaction was completed, 30ml of water was added to quench the reaction, and the liquid was separated. The dichloromethane layer was washed three times with 50ml 1mol / L hydrochloric acid solution, and then washed with 50ml 5% sodium bicarbonate solution, 50ml saturated brine, and 25ml 10% sodium chloride solution in sequence, and the organic phase was dried over anhydrous sodium sulfate. Filtration and concentration gave 4.45 g of a yellow solid. n-heptane / ethyl acetate (2:1) was used as a recrystallization solvent. 15 ml of a mixed solvent was added under reflux, and the mixture was cooled naturally to precipitate. The mixture was filtered and dried to give 4.0 g of a solid. The mixture was recrystallized with 30 ml of n-heptane / ethyl acetate (4:1) under reflux, and the solid was cooled naturally to precipitate. The solid was filtered and dried to give 3.9 g of a pure impurity D with a yield of 79%. The H NMR spectrum of the impurity D is shown in Figure 3 , its carbon NMR spectrum can be found at Figure 4 , quantitative NMR purity: 98.93%.

[0057] Example 3 Preparation of Nitisinone Impurity E

[0058] Under nitrogen protection, add 1.00g 4-nitro-2-trifluoromethylbenzoic acid (4.25mmol, 1.0eqv.), 2 drops of DMF and 7ml dry toluene to a three-necked flask, stir in an ice bath for 20min, slowly dropwise add 0.91g thionyl chloride (7.65mmol, 1.8eqv.) solution dissolved in 3ml dry toluene, and after the dropwise addition is complete, heat to 110℃ and reflux. After the reaction is completed by TLC monitoring, cool to room temperature and evaporate the solvent under reduced pressure, remove the remaining thionyl chloride 3 times with 5ml toluene each time, evaporate to dryness, and add 8.5ml acetonitrile for use. Under nitrogen protection, 0.56 g of 1,3-cyclohexanedione (4.67 mmol, 1.1 eqv.), 1.20 g of dry potassium fluoride powder (19.55 mmol, 4.6 eqv.) and 17 ml of acetonitrile were added to a three-necked flask and stirred for reaction at room temperature for 3 h. After stirring for 0.5 h in an ice bath, the acetonitrile solution was slowly added dropwise. After the addition was completed, the mixture was stirred for 0.5 h, and the temperature was raised to room temperature for reaction. The reaction was tracked by HPLC for 46 h. After the reaction was completed, 6 ml of water was added to quench the reaction, the liquid was separated, the organic phase was concentrated below 45°C, 34 ml of ethyl acetate was added to dissolve, 7 ml of 10% sodium chloride solution was washed twice, and then 32 ml of 1 mol / L hydrochloric acid was washed 3 times, and finally 32 ml of saturated sodium chloride was washed once, the liquid was separated, and the organic phase was concentrated under reduced pressure below 45°C; dissolved with 130 ml of 1 mol / L sodium hydroxide solution, stirred at room temperature for 30 min, and then filtered, the insoluble matter was discarded, the filtrate was washed twice with 51 ml of dichloromethane, the aqueous phase was adjusted to pH 2 with hydrochloric acid, the aqueous phase was extracted with ethyl acetate twice, 100 ml was used each time, the ethyl acetate phases were combined, and washed once with 190 ml of saturated sodium chloride and 25 ml of 10% sodium chloride solution, respectively, and the organic phase was dried over anhydrous sodium sulfate. Filter and concentrate to obtain the crude impurity E, which was separated by column chromatography using dichloromethane as the eluent and 300 mesh silica gel as the stationary phase. Filter and concentrate to obtain 0.599 g of the pure impurity E with a yield of 43%. The H NMR spectrum is shown in Figure 5 , its carbon NMR spectrum can be found at Figure 6 , quantitative NMR purity: 99.19%.

[0059] Example 4 Optimization of liquid phase detection conditions and methodological verification of Nitisinone and its impurities

[0060] 1. Preparation of test solution

[0061] Take an appropriate amount of this product, weigh it accurately, add solvent to dissolve it and quantitatively dilute it to make a solution containing about 2 mg per 1 ml.

[0062] 2. Preparation of control solution

[0063] Accurately measure an appropriate amount of the test sample solution and quantitatively dilute it with solvent to make a solution containing approximately 2 μg per 1 ml.

[0064] 3. Optimization of liquid chromatography conditions

[0065] Mixed control solutions were prepared for all impurities in the impurity spectrum and the chromatographic conditions were optimized.

[0066] 3.1 Selection of flow rate

[0067] Chromatographic conditions: OSAKA SODA SUPERIOREX ODS 4.6mm×250mm, 5μm, Welch Ghost-Buster Column 4.6mm×50mm as trapping column, column temperature of 30°C, detection wavelength of 274nm, injection volume of 10μl, 0.1% phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B, gradient elution according to Table 1, and the overall chromatographic behavior of the test solution at flow rates of 1.4ml / min, 1.6ml / min, and 1.8ml / min were investigated respectively. The separation results are shown in Table 2.

[0068] Table 1 Mobile phase gradient elution ratio

[0069]

[0070] Table 2 Separation of various substances in the test solution at different flow rates

[0071]

[0072] From the results in Table 2, it can be seen that at a flow rate of 1.8 ml / min, the separation degree of NTBC is 1.45, which does not meet the requirements; at a flow rate of 1.6 ml / min, the separation degree of each impurity is good; at a flow rate of 1.4 ml / min, the separation degree of impurity C is 1.11, which does not meet the requirements; see the spectrum Figure 7 .

[0073] 3.2 Selection of column temperature

[0074] Chromatographic conditions: OSAKA SODA SUPERIOREX ODS 4.6mm×250mm, 5μm, Welch Ghost-Buster Column 4.6mm×50mm as trapping column, detection wavelength of 274nm, injection volume of 10μl, flow rate of 1.6ml / min, 0.1% phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B, gradient elution according to the mobile phase elution ratio of 3.1, the overall chromatographic behavior of the test solution was investigated at column temperatures of 25℃, 30℃, 35℃, 40℃, and 45℃, respectively, and the separation results are shown in Table 3.

[0075] Table 3 Separation of various substances in the test solution at different column temperatures

[0076]

[0077]

[0078] From the results in Table 3, it can be seen that at a column temperature of 25°C, impurities A and C overlap and the separation is poor; at a column temperature of 30°C, the separation of each impurity is good; at a column temperature of 35°C, the separation of NTBC is 1.45, which does not meet the requirements; at a column temperature of 40°C, the separation of impurity F is 1.29, which does not meet the requirements, and the separation of NTBC is 1.36, which also does not meet the requirements; at a column temperature of 45°C, the separation of NTBC is 1.21, which does not meet the requirements, see the spectrum Figure 8 .

[0079] 3.3 Selection of injection volume

[0080] The system suitability solution was prepared with impurity 4 (impurity D), impurity 7 (impurity F) and NTBC. Based on the optimization of 3.2, the overall chromatographic behavior of the test solution was investigated when the injection volume was 10μl, 20μl, and 30μl, and the separation results are shown in Table 4.

[0081] Table 4 System suitability under different injection volumes

[0082]

[0083] As shown in Table 4, the separation of each impurity is good at 10 μl injection volume. Fig. 9 .

[0084] 3.4 Selection of the optimal detection wavelength

[0085] Based on the poor separation of impurities No. 1 (impurity A) and No. 5 (impurity C) in the impurity spectrum, the chromatographic gradient conditions were optimized again. Considering that each impurity in the impurity spectrum has a large absorption at 240nm, the detection wavelength was determined to be 240nm.

[0086] 3.5 Selection of gradient elution method

[0087] Chromatographic conditions: OSAKA SODA SUPERIOREX ODS 4.6mm×250mm, 5μm, Welch Ghost-Buster Column 4.6mm×50mm as trapping column, detection wavelength of 240nm, injection volume of 10μl, flow rate of 1.6ml / min, column temperature of 30℃, 0.1% phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B, according to the following different mobile phase gradient elution ratios:

[0088] The gradient elution ratio of the first mobile phase is shown in Table 5:

[0089] Table 5 Gradient elution ratio of the first mobile phase

[0090]

[0091] The system suitability results are shown in Table 6:

[0092] Table 6 System suitability results for the first mobile phase gradient elution ratio

[0093]

[0094]

[0095] As shown in Table 6, the separation degree between impurity A and impurity C is 2.13, the separation degree between impurity F and impurity D is 3.00, and the separation degree between impurity D and NTBC is 1.59. Fig.10 A.

[0096] The gradient elution ratio of the second mobile phase is shown in Table 7:

[0097] Table 7 Gradient elution ratio of the second mobile phase

[0098]

[0099] The system suitability results are shown in Table 8:

[0100] Table 8 System suitability results of the second mobile phase gradient elution ratio

[0101]

[0102] As shown in Table 8, the separation degree between impurity A and impurity C is 2.33, the separation degree between impurity F and impurity D is 3.09, and the separation degree between impurity D and NTBC is 1.56. Fig.10 B.

[0103] The gradient elution ratio of the third mobile phase is shown in Table 9:

[0104] Table 9 Gradient elution ratio of the third mobile phase

[0105]

[0106] The system suitability results are shown in Table 10:

[0107] Table 10 System suitability results of the third mobile phase gradient elution ratio

[0108]

[0109] As shown in Table 10, the separation degree between impurity A and impurity C is 4.10, the separation degree between impurity F and impurity D is 3.05, and the separation degree between impurity D and NTBC is 1.50. Fig.10 C.

[0110] The gradient elution ratio of the fourth mobile phase is shown in Table 11:

[0111] Table 11 Gradient elution ratio of the fourth mobile phase

[0112]

[0113]

[0114] The system suitability results are shown in Table 12:

[0115] Table 12 System suitability results of the fourth mobile phase gradient elution ratio

[0116]

[0117] As shown in Table 12, the separation degree between impurity A and impurity C is 4.13, the separation degree between impurity F and impurity D is 3.17, and the separation degree between impurity D and NTBC is 1.49. Fig.10 D.

[0118] The gradient elution ratio of the fifth mobile phase is shown in Table 13:

[0119] Table 13 Gradient elution ratio of the fifth mobile phase

[0120]

[0121]

[0122] The system suitability results are shown in Table 14:

[0123] Table 14 System suitability results for the fifth mobile phase gradient elution ratio

[0124]

[0125] As shown in Table 14, the separation degree between impurity A and impurity C is 3.18, the separation degree between impurity F and impurity D is 3.22, and the separation degree between impurity D and NTBC is 1.54. Fig.10 E.

[0126] In summary, the chromatography control method for the final product of Nitisinone is as follows:

[0127] The method for detecting the related substances of Nitisinone is to use liquid chromatography, and the HPLC detection conditions are as follows:

[0128] Chromatographic column: OSAKA SODA SUPERIOREX ODS 4.6mm×250mm, 5μm, Welch Ghost-Buster Column 4.6mm×50mm as trapping column, column temperature 30℃, flow rate 1.6ml per minute, detection wavelength 240nm, injection volume 10μl, 0.1% phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B, gradient elution according to the following table:

[0129]

[0130]

[0131] Experimental operation

[0132] Solvent Acetonitrile

[0133] Take an appropriate amount of the product for test solution, weigh it accurately, dissolve it in solvent and quantitatively dilute it to make a solution containing about 2 mg per 1 ml.

[0134] For the control solution, accurately measure an appropriate amount of the test solution and quantitatively dilute it with solvent to make a solution containing approximately 2 μg per 1 ml.

[0135] Reference solution: Take appropriate amounts of impurity A reference substance, impurity B reference substance, impurity C reference substance, impurity D reference substance, impurity E reference substance and impurity F reference substance, weigh them accurately, dissolve them in solvent and quantitatively dilute them to make solutions containing approximately 4 μg of impurity A and impurity C and 2 μg of other impurities per 1 ml.

[0136] System suitability solution: Weigh appropriate amounts of impurity A reference substance, impurity C reference substance, impurity D reference substance, impurity E reference substance and nitisinone reference substance respectively, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing approximately 4 μg of impurity A, 4 μg of impurity C, 2 μg of impurity D, 2 μg of impurity E and 2 mg of nitisinone per 1 ml.

[0137] Sensitivity solution: Accurately measure an appropriate amount of the control solution and quantitatively dilute it with a solvent to make a solution containing approximately 1 μg per 1 ml.

[0138] System suitability requirements In the system suitability solution chromatogram, impurity A, impurity C, impurity E, impurity D and nitisinone appear in sequence, and the separation between each chromatographic peak should meet the requirements.

[0139] Determination method

[0140] Accurately measure 10 μl of the test solution, control solution, reference substance solution and sensitivity solution, respectively, and inject them into the liquid chromatograph to record the chromatogram.

[0141] limit

[0142] If there are chromatographic peaks in the chromatogram of the test solution that have the same retention time as the peaks of impurity A, impurity B, impurity C, impurity D, impurity E and impurity F in the reference solution, the peak area calculated by the external standard method shall be as follows: impurity A and impurity C shall not exceed 0.2%, impurity B, impurity D, impurity E and impurity F shall not exceed 0.1%, and the peak area of ​​other individual impurities shall not be greater than the main peak area of ​​the reference solution (0.1%), and the sum of all impurities shall not be greater than 0.5%. Peaks that are less than the main peak area of ​​the sensitivity solution shall be ignored. (Note: Impurity B may be a split peak. If it is displayed as a split peak, it shall be calculated by the sum of peak areas.) For the HPLC chromatogram of the separation solution, see Fig.11 The system suitability results are shown in Table 15.

[0143] Table 15 System suitability results

[0144] name Retention time [min] Peak area Theoretical plates Tailing Factor Separation Signal-to-Noise Ratio Impurity B 4.653 59.49 160 0.93 40.4 Impurity A 41.389 32.8 649272 1.19 44.3 113.1 Impurity C 41.959 110.63 1033404 1.06 3.1 481.8 Impurity F 45.306 18.51 583862 2.07 16.6 52.4 Impurity E 48.465 25.00 1217386 1.08 15.3 102.6 Impurity D 48.976 37.01 1327595 1.02 3.0 159.9 NTBC 49.283 26097.92 1048640 1.04 1.7 94038.9

[0145] 4. Methodological validation:

[0146] The results of the validation of the impurity determination methodology are as follows:

[0147]

[0148]

[0149]

[0150] Comparative Example: Detection of related substances of Nitisinone (refer to the registration standard for imported drugs of Nitisinone capsules).

[0151] The method for detecting related substances in the registration standard for imported drugs of Nitisinone Capsules is liquid chromatography, and the HPLC detection conditions are as follows:

[0152] Chromatographic column: ACE 5C18, 4.6mm×150mm, 5μm, 0.1% phosphoric acid aqueous solution as mobile phase A, 0.1% phosphoric acid acetonitrile solution as mobile phase B, gradient elution as shown in the table below, detection wavelength of 274nm, flow rate of 1.8ml per minute, column temperature of 30℃, injector temperature of 4℃, injection volume of 10μl, gradient elution as shown in the table below:

[0153]

[0154] Experimental operation

[0155] Solvent Acetonitrile

[0156] Take an appropriate amount of nitisinone for the test solution, weigh it accurately, dissolve it in a solvent and quantitatively dilute it to make a solution containing about 2 mg per 1 ml.

[0157] For the control solution, accurately measure an appropriate amount of the test solution and quantitatively dilute it with solvent to make a solution containing approximately 2 μg per 1 ml.

[0158] Reference solution: Take appropriate amounts of impurity A reference substance, impurity B reference substance, impurity C reference substance, impurity D reference substance, impurity E reference substance and impurity F reference substance, weigh them accurately, dissolve them in solvent and quantitatively dilute them to make solutions containing approximately 4 μg of impurity A and impurity C and 2 μg of other impurities per 1 ml.

[0159] System suitability solution: Weigh appropriate amounts of impurity A reference substance, impurity B reference substance, impurity C reference substance, impurity D reference substance, impurity E reference substance, impurity F reference substance and nitisinone reference substance respectively, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing approximately 4 μg of impurity A, 4 μg of impurity C, 2 μg of impurity B, 2 μg of impurity D, 2 μg of impurity E, 2 μg of impurity F and 2 mg of nitisinone per 1 ml.

[0160] System suitability requirements: In the system suitability solution chromatogram, impurity B, impurity A, impurity C, impurity F, impurity E, nitisinone and impurity D appear in sequence, and the separation between each chromatographic peak should meet the requirements.

[0161] Determination method

[0162] Accurately measure 10 μl of the test solution and the reference solution, respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0163] limit

[0164] If there are chromatographic peaks in the chromatogram of the test solution with the same retention time as the peaks of impurity A, impurity B, impurity C, impurity D, impurity E and impurity F in the reference solution, the peak area calculated by the external standard method shall be as follows: impurity A and impurity C shall not exceed 0.2%, impurity B, impurity D, impurity E and impurity F shall not exceed 0.1%, and the peak area of ​​other individual impurities shall not be greater than the main peak area of ​​the reference solution (0.1%), and the sum of all impurities shall not be greater than 0.5%. (Note: Impurity B may be a split peak. If it is displayed as a split peak, it shall be calculated by the sum of peak areas.)

[0165] The system suitability results are shown in Table 16.

[0166] Table 16 System suitability results

[0167] name Retention time [min] Peak area Theoretical plates Tailing Factor Separation Signal-to-Noise Ratio Impurity B 2.063 29.66 31 1.07 -- 46.3 Impurity A 21.825 15.73 37730 0.91 21.2 35.4 Impurity C 24.769 28.58 73662 0.91 7.2 79.9 Impurity F 27.676 1.12 55258 0.79 6.9 2.0 Impurity E 39.016 31.74 84191 0.94 22.5 61.0 NTBC 41.830 29712.46 60074 0.89 4.6 45535.2 Impurity D 42.856 17.75 6559 4.26 0.7 23.7

[0168] In the system suitability spectrum of the comparative method, the separation degree between Nitisinone and impurity D is 0.7, which does not reach the baseline separation, the theoretical plate number is not high, the column efficiency is low, the response values ​​of each impurity and the main peak are low, especially the response value of impurity F is lower than the detection limit. It is very likely that it will not be detected in the actual test, resulting in a false negative result, and the sensitivity is low.

[0169] Compared with the system applicability results of the comparative example, the method for detecting related substances of nitisinone provided by the present invention has the advantages of reliability, strong specificity, strong practicability, and each impurity can be effectively detected. Moreover, nitisinone and each impurity are well separated, and a variety of impurities can be detected.

[0170] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0171] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for detecting nitisinone impurities, characterized in that: The content of impurities in nitisinone was determined by high performance liquid chromatography, wherein the high performance liquid chromatography was a reverse phase chromatography, and the chromatographic conditions were as follows: chromatographic column: OSAKA SODASUPERIOREX ODS 4.6 mm×250 mm, 5 μm, 0.05%-0.15% (V / V) phosphoric acid solution as mobile phase A, and acetonitrile as mobile phase B. The detection method comprises the following steps: 1) Take an appropriate amount of nitisinone, dissolve it in acetonitrile and quantitatively dilute it to make a solution containing about 1.5 mg to 3 mg per 1 ml; 2) Set the mobile phase flow rate to 1.3ml-1.9ml per minute, the column temperature to 25℃-35℃, and the detection wavelength to 225nm-250nm; the elution procedure is as shown in the table below; 3) Take 5 μl-15 μl of the sample solution in step 1), inject it into a liquid chromatograph, record the chromatogram, and complete the sample detection.

2. The method for detecting nitisinone impurities according to claim 1, characterized in that: The detection method can determine the contents of impurity A, impurity B, impurity C, impurity D, impurity E and impurity F in nitisinone.

3. The method for detecting nitisinone impurities according to claim 1, characterized in that: The phosphoric acid concentration in the mobile phase A was 0.1%.

4. The method for detecting nitisinone impurities according to claim 1, characterized in that: In step 1), each 1 ml of the sample solution contains 2 mg of the sample.

5. The method for detecting nitisinone impurities according to claim 1, characterized in that: In step 2), the flow rate of the mobile phase is 1.6 ml per minute; the column temperature of the chromatographic column is 30° C.; and the detection wavelength is 240 nm.

6. The method for detecting nitisinone impurities according to claim 1, characterized in that: The injection volume of the sample solution in step 3) is 10 μl.

7. A method for preparing nitisinone impurities, characterized in that: The invention includes the preparation of impurity C, impurity D and impurity E. The preparation method comprises the following steps: firstly preparing substrates 2-chloro-4-trifluoromethylbenzoic acid, 2-nitro-4-trifluoromethylbenzoic acid and 4-nitro-2-trifluoromethylbenzoic acid into acyl chloride derivatives respectively, and then condensing them with 1,3-cyclohexanedione under inorganic base conditions to obtain impurity C, impurity D and impurity E respectively.

8. The method for preparing nitisinone impurities according to claim 7, characterized in that: The preparation method of the impurity C is as follows: the substrate 2-chloro-4-trifluoromethylbenzoic acid is subjected to a chlorination reaction with thionyl chloride under the catalysis of DMF, the solvent is evaporated, and condensed with 1,3-cyclohexanedione under an alkaline environment, and then irradiated under ultraviolet light to obtain impurity C. The reaction formula is as follows:

9. The method for preparing nitisinone impurities according to claim 7, characterized in that: The preparation method of the impurity D is: subjecting the substrate 2-nitro-4-trifluoromethylbenzoic acid to a chlorination reaction with thionyl chloride under the catalysis of DMF, evaporating the solvent, and condensing with 1,3-cyclohexanedione under an alkaline environment to obtain impurity D, and the reaction formula is as follows:

10. The method for preparing nitisinone impurities according to claim 7, characterized in that: The preparation method of the impurity E is as follows: the substrate 4-nitro-2-trifluoromethylbenzoic acid is subjected to a chlorination reaction with thionyl chloride under the catalysis of DMF, the solvent is evaporated, and the impurity E is condensed with 1,3-cyclohexanedione under an alkaline environment to obtain the impurity E. The reaction formula is as follows:

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