Method for detecting related substances in sapropterin hydrochloride tablets

Abstract

The invention relates to a method for detecting related substances in sapropterin hydrochloride tablets. The related substances are selected from biological pterin, pterin, neopterin, pteridine and S-isomer. High performance liquid chromatography is adopted for detection, and a filling material of a stationary phase of a chromatographic column is octadecylsilane chemically bonded silica; a ghost peak trapping small column is arranged between the position behind the converging outlet of the pump and the sample injector; the mobile phase A is a phosphate buffer solution with the concentration of 10-30 mmol / L, and the pH value of the mobile phase A is 2.75-2.80; a mobile phase B is methanol; dissolving a to-be-detected sample with a diluent, and fixing the volume to form a sample injection solution; the diluent is a phosphate buffer solution containing ascorbic acid, the concentration of the phosphate buffer solution is 10-30 mmol / L, the pH value is 2.0-2.5, and the concentration of the ascorbic acid is 1-2 g / L.

Description

Technical Field

[0001] The present invention belongs to the field of detection of drug impurities, and particularly relates to a method for detecting related substances in sapropterin dihydrochloride tablets. Background Art

[0002] Phenylketonuria (PKU) is an autosomal recessive genetic disease. Patients are called "children who do not eat the fireworks of the world" (because of the lack of phenylalanine hydroxylase in the liver or the defects of tetrahydrobiopterin synthetase and dihydrobiopterin reductase, children cannot metabolize phenylalanine normally. If they eat ordinary protein foods, a large amount will accumulate in the body, resulting in symptoms of hyperphenylalaninemia). High concentrations of phenylalanine in the blood can cause irreversible damage to the central nervous system, resulting in clinical symptoms such as mental retardation, autism, epilepsy, and movement disorders in patients. If the disease is not treated in time, it will cause serious damage to the central nervous system of patients.

[0003] Sapropterin Dihydrochloride, also known as tetrahydrobiopterin (BH4), is currently the only treatment drug for PKU. It is a synthetic tetrahydrobiopterin and is a coenzyme of phenylalanine, tyrosine, and tryptophan hydroxylase. It can be used to replace the lacking BH4, thereby restoring the activity of phenylalanine hydroxylase. Therefore, the treatment of PKU with sapropterin dihydrochloride is called BH4 replacement therapy.

[0004] Sapropterin dihydrochloride tablets were developed by BioMarin International Limited and are applicable to hyperphenylalaninemia (HPA) caused by tetrahydrobiopterin deficiency that responds to the treatment of this product. As a rare disease drug, sapropterin dihydrochloride was first marketed in the United States as an orphan drug in 2007 (trade name: Kuvan), was rapidly approved for marketing in the European Union in 2008, and was approved for entry into China in September 2010.

[0005] The chemical name of sapropterin dihydrochloride is (6R)-5,6,7,8-tetrahydrobiopterin dihydrochloride, and its structural formula is as follows:

[0006]

[0007] At present, this drug is not included in the pharmacopoeia. According to the related substance information provided by the API manufacturer, there are 2 degradation impurities (biopterin and pterin) and 3 process impurities (S-isomer, pteridine, neopterin) in the sapropterin hydrochloride API. The 2 degradation impurities need to be accurately quantified. At the same time, the inventor also hopes that the 3 process impurities can be effectively separated without interfering with the quantification of the degradation impurities. However, the existing technical methods require multiple methods to effectively separate all the above impurities, which is time-consuming and complex in operation. At the same time, due to the poor stability of sapropterin hydrochloride, it is easily oxidized, and unknown impurities are easily degraded during the detection by the existing methods, which is not sufficient to support the detection of batch samples. Therefore, a method capable of accurately and stably detecting the related substances in sapropterin hydrochloride tablets simultaneously is needed. Summary of the Invention

[0008] Aiming at the above problems existing in the prior art, the purpose of the present invention is to provide a method capable of accurately and stably detecting as many related substances as possible in sapropterin hydrochloride tablets, so as to better monitor the quality of sapropterin hydrochloride tablets and improve the clinical medication safety of this drug.

[0009] The present invention discloses a method for detecting related substances in sapropterin hydrochloride tablets, and the related substances are selected from Table 1:

[0010] Table 1. Related substance names and structural formulas

[0011]

[0012] The detection method uses high performance liquid chromatography for detection. The packing material of the stationary phase of the chromatographic column is octadecylsilyl bonded silica gel; a ghost peak trapping column is installed between the confluence outlet of the pump and the injector; Mobile phase A: phosphate buffer solution with a concentration of 10 - 30 mmol / L, pH 2.75 - 2.80; Mobile phase B: methanol; The mobile phase gradient is as shown in the following table:

[0013]

[0014]

[0015] Mobile phase flow rate: 0.5 - 0.7 mL / min; Chromatographic column temperature: 25°C - 35°C; Injector temperature: 2°C - 8°C; Detection wavelength: 275 nm; Injection volume: 10 μl - 20 μl;

[0016] The sample to be tested is dissolved and fixed volume with a diluent to form an injection solution; the diluent is a phosphate buffer solution containing ascorbic acid, the concentration of the phosphate buffer solution is 10 - 30 mmol / L, pH 2.0 - 2.5, and the concentration of ascorbic acid is 1 - 2 g / L.

[0017] In some embodiments, the diluent is a potassium dihydrogen phosphate buffer solution containing ascorbic acid. The concentration of the potassium dihydrogen phosphate buffer solution is 20 mmol / L, the pH is adjusted to 2.5 with phosphoric acid, and the concentration of ascorbic acid is 1 g / L.

[0018] In some embodiments, the sample to be tested is dissolved and made up to volume with the diluent to form an injection solution as follows: the sample to be tested is a sapropterin hydrochloride tablet; the powder obtained by grinding the sapropterin hydrochloride tablet is weighed and dissolved with the diluent to prepare a sapropterin hydrochloride solution containing 0.4 - 0.6 mg (preferably 0.5 mg) in 1 mL.

[0019] In some embodiments, an impurity reference substance is used to determine the peak emergence time of the impurities in the related substances in the high - performance liquid chromatography (HPLC) chromatogram; preparation of the impurity reference substance solution: it is ultrasonically dissolved with 0.1 N hydrochloric acid solution and then made up to 1 mL with the diluent to prepare a solution containing 1 - 2 μg (preferably 1 μg) of the corresponding impurity.

[0020] In some embodiments, the chromatographic system is a high - performance liquid chromatograph equipped with a PDA / DAD / VWD detector.

[0021] In some embodiments, the high - performance liquid chromatograph is an Agilent 1260Ⅱ or a Waters Arc or a Waters e2695.

[0022] In some embodiments, the chromatographic column is a YMC - Pack ODS - AQ, with a specification of 4.6 mm×250 mm, 3.0 μm.

[0023] In some embodiments, the ghost peak trapping column is a Yuexu Ghost - Buster Column, or a Ghost - Buster HP Column, or a Ghost - Buster Column II.

[0024] In some embodiments, the ghost peak trapping column is a Yuexu Ghost - Buster Column, with a specification of 4.6×50 mm.

[0025] In some embodiments, the chromatographic column is a YMC - Pack ODS - AQ, with a specification of 4.6 mm×250 mm, 3.0 μm; the ghost peak trapping column is a Yuexu Ghost - Buster Column, with a specification of 4.6×50 mm; mobile phase A is a 20 mmol / L potassium dihydrogen phosphate buffer solution with a pH of 2.75 - 2.80; mobile phase flow rate: 0.6 ml / min; chromatographic column temperature: 30℃; auto - sampler temperature: 5℃.

[0026] In some embodiments, the content of each impurity is calculated according to the following formula:

[0027]

[0028] Wherein: A: the peak area of a single impurity in the test solution; W STD : the mass of the control substance of sapropterin hydrochloride, in mg; P: the content of the control substance of sapropterin hydrochloride; V SAP : the dilution volume of the test substance, in mL; W AVE : the average mass of the test substance, in mg; RRF: relative response factor, wherein the RRF of biopterin is 2.50, the RRF of pterin is 2.56, and the RRF of the remaining unknown single impurities is calculated as 1.0; A STD : the average peak area of sapropterin hydrochloride in the first 6 control substance solutions; V STD : the dilution volume of the control substance of sapropterin hydrochloride, in mL; W SAP : the mass of the test substance, in mg; LC: the labeled amount of sapropterin hydrochloride in the test substance, in 100 mg.

[0029] In some embodiments, the specification of the sapropterin hydrochloride tablets is 100 mg / tablet.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The present invention can achieve excellent resolution for the 5 impurities in Table 1, and the method has good specificity. Moreover, this method has the advantages of excellent accuracy, precision and durability for the determination of 2 degradation impurities.

[0032] (2) The method for detecting related substances in sapropterin hydrochloride tablets of the present invention can meet the requirements of system suitability.

[0033] (3) The method for detecting related substances in sapropterin hydrochloride tablets of the present invention can accurately quantify degradation impurities biopterin and pterin above 0.25 μg / ml (0.05%).

[0034] (4) The method for detecting related substances in sapropterin hydrochloride tablets of the present invention can detect degradation impurities biopterin and pterin above 0.075 μg / ml (0.015%).

[0035] (5) The method for detecting related substances in sapropterin hydrochloride tablets of the present invention not only simplifies the preparation process of mobile phase A and diluent, but also the test solution is very stable within 3 days, which can support the detection of a batch of test substances.

[0036] The following will further illustrate the concept, specific structure and technical effects of the present invention with reference to the accompanying drawings, so as to fully understand the purpose, features and effects of the present invention. Description of the Drawings

[0037] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0038] Figure 1 It is the chromatogram obtained by detecting the 1% known impurity spiked sample solution using the related substance method of Manufacturer 1 in Example 1.

[0039] Figure 2 It is the chromatogram obtained by detecting the 0.2% known impurity spiked sample solution using the related substance method of Manufacturer 2 in Example 1.

[0040] Figure 3 It is the superimposed chromatogram obtained by detecting the 0.2% known impurity spiked sample solution and the pteridine positioning solution using Method 1 of the related substances of Manufacturer 3 in Example 1.

[0041] Figure 4 It is the superimposed chromatogram obtained by detecting the 0.2% impurity positioning solution and the test solution using Method 2 of the related substances of Manufacturer 3 in Example 1.

[0042] Figure 5 It is the chromatogram of the 0.2% known impurity spiked sample solution under the specificity item in Example 3 of the present invention.

[0043] Figure 6 It is the chromatogram of the blank solution under the specificity item in Example 3 of the present invention.

[0044] Figure 7 It is the chromatogram of the blank excipient under the specificity item in Example 3 of the present invention.

[0045] Figure 8 It is the chromatogram of the sample solution damaged by high temperature under the specificity item in Example 3 of the present invention;

[0046] Figure 9 It is the chromatogram of the sample solution damaged by acid under the specificity item in Example 3 of the present invention;

[0047] Figure 10 It is the chromatogram of the sample solution damaged by alkali under the specificity item in Example 3 of the present invention;

[0048] Figure 11 It is the chromatogram of the sample solution damaged by oxidation under the specificity item in Example 3 of the present invention;

[0049] Figure 12It is the chromatogram of the sample solution damaged by light under the specificity item in Example 3 of the present invention;

[0050] Figure 13 It is the linear relationship diagram of sapropterin hydrochloride, biopterin and pterin under the linearity item in Example 5 of the present invention.

[0051] Figure 14 It is the chromatogram overlay for investigating the solution stability of the test solution prepared with diluent 1 in Comparative Example 1.

[0052] Figure 15 It is the chromatogram overlay for investigating the solution stability of the test solution prepared with diluent 2 in Comparative Example 1.

[0053] Figure 16 It is the chromatogram overlay for investigating the solution stability of the test solution prepared with diluent 3 in Comparative Example 1.

[0054] Figure 17 It is the chromatogram overlay for investigating the solution stability of the test solution prepared with the diluent of the present invention in Comparative Example 1. Detailed implementation manners

[0055] In order to make the technical means, creative features, achieved purposes and effects of the invention easy to understand, the present invention will be further described below with reference to specific illustrations. However, the present invention is not limited to the following implemented cases.

[0056] Definitions

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Although any methods and materials similar to or equivalent to those described herein may be used in the practice of testing the present invention, specific test methods and materials are listed for further illustrating the technical solution of the present invention. The following terms will be used in the description and claim of the present invention.

[0058] The term "S-isomer" refers to the S-isomer of sapropterin hydrochloride. Unless otherwise specified or a recognized measurement unit (such as concentration units μg / ml, mg / ml, etc.), words connected by " / " generally mean "or".

[0059] Process impurities: refer to those generated during the drug synthesis process. Chemical drugs are usually obtained through multiple chemical reactions. In the process, various starting materials, intermediates and reagents / solvents may be used, and various by-products may be generated in these reactions, and these by-products are process impurities.

[0060] Degradation impurities: refer to those generated during the storage of drugs due to external conditions (such as temperature, humidity, light) or microbial action. During the storage and transportation of drugs, hydrolysis, oxidation, ring-opening, polymerization and other reactions may occur, thus forming degradation impurities.

[0061] Reference substance: a standard substance used for identification, inspection and content determination.

[0062] Purity angle: refers to the average value of the angles between the spectra at various positions of the peak and the spectrum at the peak apex.

[0063] Purity threshold: the critical angle used to judge the purity of a peak. If the purity angle of a peak is less than this threshold, the peak is considered pure; if the purity angle is greater than or equal to the threshold, the peak may contain impurities.

[0064] Sapropterin hydrochloride tablets: a self-developed product of Shanghai Aokeda Pharmaceutical Technology Co., Ltd., with a specification of 100 mg and a tablet weight of 300 mg.

[0065] 0.1N hydrochloric acid solution: Pipette 8.5 mL of concentrated hydrochloric acid into a 1 L volumetric flask containing an appropriate amount of water, dilute to the mark with water, and shake well.

[0066] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5.

[0067] The following examples are only used to further illustrate the present invention but do not limit the scope of the present invention to these examples.

[0068] Example 1: Detect process impurities (S-isomer, pteridine, neopterin) and degradation impurities (biopterin and pterin) in sapropterin hydrochloride tablets by the related substance detection methods of three API manufacturers.

[0069] 1. Related substance detection method of manufacturer 1:

[0070] - High performance liquid chromatography, strong cation exchange column based on benzenesulfonic acid group (Partisil 10SCX chromatographic column, with a specification of 4.6 mm × 250 mm, 10 μm);

[0071] - Mobile phase: a mixed solution of 30 mmol / L ammonium dihydrogen phosphate and 3 mmol / L ammonium sulfate (adjust the pH value to 3.00 ± 0.02 with phosphoric acid);

[0072] - Mobile phase elution mode: isocratic elution;

[0073] - Diluent: Take 0.2 g of dithiopropanol and 1.0 g of L-cysteine hydrochloride monohydrate, dissolve with an appropriate amount of water and dilute to 1000 ml, and shake well;

[0074] - Flow rate: 1.5 ml / min; Column temperature: 40 °C; Detection wavelength: 265 nm; Injection volume: 50 μl; Run time: 20 min.

[0075] - The solutions were prepared as follows:

[0076] Stock solution of impurity reference standards (0.1 mg / mL): Accurately weigh about 5 mg of each of the biopterin, pterin, pteridine, S-isomer, and neopterin reference standards into different 50 mL volumetric flasks, add 25 mL of 0.1 N hydrochloric acid, sonicate to dissolve completely, and make up the volume to the mark with diluent and mix well.

[0077] 1% localization solution for each impurity: Pipette 10.0 mL of the stock solution of impurity reference standards of biopterin, pterin, pteridine, S-isomer, and neopterin into different 50 mL volumetric flasks respectively, make up the volume to the mark with diluent, and shake well.

[0078] 1% spiked sample solution of known impurities (2 mg / mL of sapropterin hydrochloride, 20 μg / mL of biopterin, pterin, pteridine, S-isomer, and neopterin respectively): Accurately weigh about 300 mg of the test sample powder (containing about 100 mg of sapropterin hydrochloride) into a 50 mL volumetric flask, add an appropriate amount of diluent to dissolve, then add 10.0 mL of the stock solution of impurity reference standards of biopterin, pterin, pteridine, S-isomer, and neopterin respectively into the above 50 mL volumetric flask, make up the volume to the mark with diluent and mix well. Filter through a 0.22 μm nylon membrane filter, discard at least 1.0 mL, and the subsequent filtrate is the 1% spiked sample solution of known impurities. Note: In this article, the percentage before the "spiked sample solution of known impurities" is obtained by the concentration of the impurity / the concentration of sapropterin hydrochloride. That is, when calculating the percentage of each impurity, the denominator is the concentration of the active ingredient (API). Therefore, the percentage of each impurity in this related substances method refers to the percentage of the concentration of the impurity to the concentration of the API. For example, since the 1% spiked sample solution of known impurities contains 2 mg / mL of sapropterin hydrochloride and 20 μg / mL of biopterin, pterin, pteridine, S-isomer, and neopterin respectively, 20 μg / mL is 1% of 2 mg / mL, so it is the 1% spiked sample solution of known impurities. Although there is no sapropterin hydrochloride in the localization solution of impurities, the percentage before the localization solution of impurities is given with reference to the concentration of the impurity / the concentration of sapropterin hydrochloride in the spiked sample solution of known impurities. In addition, the sensitivity solution is also calculated in this way. For example, the 0.05% sensitivity solution is obtained by diluting the 0.2% reference solution by 4 times, and the 0.2% reference solution is the percentage of the reference substance concentration to the test sample concentration.

[0079] Inject the above solutions for analysis respectively, record the chromatograms, and the chromatogram of the 1% spiked sample solution of known impurities obtained is as Figure 1 .

[0080] 2. Substance detection method for Manufacturer 2:

[0081] - High performance liquid chromatography, strong cation exchange column based on benzenesulfonic acid group (Luna SCX chromatographic column, specification: 4.6mm×250mm, 5μm);

[0082] - Mobile phase A: 12g sodium dihydrogen phosphate monohydrate to 2L water, adjust pH to 2.5±0.05 with phosphoric acid, mix well, filter.

[0083] - Buffer salt: 12g sodium dihydrogen phosphate monohydrate to 2L water, adjust pH to 2.8±0.05 with phosphoric acid, mix well, filter.

[0084] - Mobile phase B: Buffer salt: Acetonitrile = 90:10 (V / V)

[0085] - Mobile phase gradient

[0086] Time, minutes Mobile phase A, % Mobile phase B, % 0 100 0 15 100 0 45 40 60 55 40 60 57 100 0 65 100 0

[0087] - Diluent: Pipette 45mL concentrated hydrochloric acid into a 1L volumetric flask containing 750mL water, add 1.0g ascorbic acid, dissolve by ultrasound, add water to the scale, shake well.

[0088] - Flow rate: 0.6ml / min; Column temperature: 45℃; Detection wavelength: 220nm; Injection volume: 20μl; Running time: 65min, Sample tray temperature: 5℃.

[0089] - Solution preparation is as follows:

[0090] Impurity reference stock solution (0.1mg / mL): Accurately weigh about 5mg of each of the biopterin, pterin, pteridine, S-isomer, and neopterin impurity reference standards into different 50mL volumetric flasks, add 25mL 0.1N hydrochloric acid, dissolve completely by ultrasound, and make up to the mark with diluent and mix well.

[0091] 0.2% positioning solution for each impurity: Pipette 2.0mL of the impurity reference stock solution of biopterin, pterin, pteridine, S-isomer, and neopterin into different 50mL volumetric flasks respectively, make up to the mark with diluent, and shake well.

[0092] 0.2% Known Impurity Spiked Sample Solution (2 mg / mL of sapropterin hydrochloride, 4 μg / mL each of biopterin, pterin, pteridine, S-isomer, and neopterin): Accurately weigh about 300 mg of the test sample powder (about 100 mg of sapropterin hydrochloride) into a 50 mL volumetric flask, add an appropriate amount of diluent to dissolve, and then add 2.0 mL each of the impurity reference stock solutions of biopterin, pterin, pteridine, S-isomer, and neopterin to the above 50 mL volumetric flask. Dilute to volume with diluent and mix well. Filter through a 0.22 μm nylon membrane filter, discard at least 1.0 mL, and the subsequent filtrate is the 0.2% known impurity spiked sample solution.

[0093] Inject the above solutions for analysis respectively, record the chromatogram, and the chromatogram of the 0.2% known impurity spiked sample solution obtained is as Figure 2 .

[0094] 3. Related Substances Detection Method of Manufacturer 3 (Manufacturer 3 jointly detects 5 impurities with two methods):

[0095] Method 1:

[0096] - High performance liquid chromatography, octadecylsilyl silica gel (YMC-Pack ODS-AQ, specification 4.6 mm × 250 mm, 3.0 μm);

[0097] - Mobile phase A: Accurately weigh and dissolve 2.72 g of potassium dihydrogen phosphate in 1 L of water, adjust the pH value to 3.0 with phosphoric acid, mix well, filter through a 3M activated carbon filter membrane (Cat No 66896-U) and degas;

[0098] - Mobile phase B: Methanol: Acetonitrile = 900:100 (V / V);

[0099] - Mobile phase gradient

[0100] Time, minutes Mobile phase A, % Mobile phase B, % 0.01 98 2 5 90 10 10 50 50 15 20 80 25 20 80 30 98 2 45 98 2

[0101] - Diluent: Accurately weigh and dissolve 2.72 g of potassium dihydrogen phosphate in 1 L of water, adjust the pH value to 3.0 with phosphoric acid, mix well, filter through a 3M activated carbon filter membrane (Cat No 66896-U) and degas, then add 10 mg of ascorbic acid to dissolve and mix well;

[0102] - Flow rate: 0.6 ml / min; Column temperature: 30 °C; Detection wavelength: 220 nm / 275 nm; Injection volume: 5 μl; Sample tray temperature: 5 °C, Running time: 45 min.

[0103] Method 2:

[0104] - High performance liquid chromatography, octadecylsilyl silica gel (YMC-Pack ODS-AQ, size: 4.6 mm × 250 mm, 3.0 μm);

[0105] - Mobile phase A: Accurately weigh and dissolve 2.72 g of potassium dihydrogen phosphate in 1 L of water, then add 100 mg of sodium sulfite and dissolve. Adjust the pH value to 2.5 with phosphoric acid, mix well, and filter;

[0106] - Mobile phase B: Methanol;

[0107] - Mobile phase gradient:

[0108] Time, minutes Mobile phase A, % Mobile phase B, % 0.01 98 2 15 90 10 30 50 50 35 50 50 37 98 2 55 98 2

[0109] - Diluent: Accurately weigh and dissolve 2.72 g of potassium dihydrogen phosphate in 1 L of water, then add 10 mg of ascorbic acid and dissolve. Adjust the pH value to 2.5 with phosphoric acid, mix well, then add 100 mg of sodium sulfite and dissolve and mix well, and filter;

[0110] - Flow rate: 0.5 ml / min; Column temperature: 30 °C; Detection wavelength: 220 nm; Injection volume: 5 μl; Sample tray temperature: 5 °C, Running time: 55 min.

[0111] - Solution preparation is as follows:

[0112] Impurity reference stock solution (0.1 mg / mL): Consistent with the manufacturer's method 2.

[0113] 0.2% known impurity spiked sample solution (0.5 mg / mL of sapropterin hydrochloride, 1 μg / mL of biopterin, pterin, pteridine, S-isomer, neopterin): Accurately weigh about 75 mg of the test sample powder (about 25 mg of sapropterin hydrochloride) into a 50 mL volumetric flask, add an appropriate amount of diluent to dissolve, then add 0.5 mL of the impurity reference stock solution of biopterin / pterin / pteridine / S-isomer / neopterin to the above 50 mL volumetric flask respectively, and make up the volume with diluent and mix well. Filter through a 0.22 μm nylon filter membrane, discard at least 1.0 mL, and the subsequent filtrate is the 0.2% known impurity spiked sample solution.

[0114] Test sample solution: Accurately weigh about 75 mg of the test sample powder (about 25 mg of sapropterin hydrochloride) into a 50 mL volumetric flask, add an appropriate amount of diluent to completely dissolve, make up the volume with diluent and mix well, discard at least 1.0 mL of the filtrate through a nylon filter membrane (0.22 μm, diameter 25 mm), and the subsequent filtrate is the test sample solution.

[0115] 0.2% impurity localization solution: Pipette 0.5 mL of the impurity reference stock solutions of biopterin, pterin, pteridine, S - isomer, and neopterin into different 50 - mL volumetric flasks respectively, dilute to the mark with diluent, and mix well.

[0116] Under the conditions of Method 1, inject the impurity localization solutions and the spiked sample solutions respectively, record the chromatograms, and the chromatogram overlay of the 0.2% known impurity spiked sample solution and pteridine is as Figure 3 .

[0117] Under the conditions of Method 2, inject the impurity localization solutions and the test sample solutions respectively, record the chromatograms, and the chromatogram overlay of the 0.2% impurity localization solutions and the test sample solution is as Figure 4 .

[0118] 4. The method of the present invention

[0119] Chromatographic conditions:

[0120] - Chromatographic system: High - performance liquid chromatograph with a PDA / DAD / VWD detector; such as Agilent 1260Ⅱ or Waters Arc or Waters e2695 or similar instruments;

[0121] - Chromatographic column: Octadecylsilyl - bonded silica gel (YMC - Pack ODS - AQ, specifications: 4.6 mm × 250 mm, 3.0 μm);

[0122] - Ghost peak trapping column: Yuexu, 4.6 × 50 mm;

[0123] - Mobile phase A: 20 mmol / L potassium dihydrogen phosphate buffer solution, pH 2.75 - 2.80 (preparation process: accurately weigh and dissolve 2.72 g of potassium dihydrogen phosphate in 1 L of water, adjust the pH value to 2.75 - 2.80 with phosphoric acid, mix well, and filter);

[0124] - Mobile phase B: Methanol;

[0125] - Mobile phase gradient

[0126] Time, minutes Mobile phase A, % Mobile phase B, % 0 98 2 5 90 10 20 50 50 25 20 80 30 20 80 35 98 2 50 98 2

[0127] - Mobile phase flow rate: 0.6 ml / min;

[0128] - Chromatographic column temperature: 30℃;

[0129] - Autosampler temperature: 5℃;

[0130] - Detection wavelength: 275 nm;

[0131] - Injection volume: 10 μl.

[0132] The present invention prepares the following solutions:

[0133] 1) 20 mmol potassium dihydrogen phosphate buffer: Weigh 2.72 g of potassium dihydrogen phosphate into 1 L of water, adjust the pH to 2.80 (2.75 - 2.80) with phosphoric acid, mix well, and filter through a membrane filter.

[0134] 2) Diluent: Weigh 2.72 g of potassium dihydrogen phosphate into 1 L of water, adjust the pH to 2.5 with phosphoric acid, add 1 g of ascorbic acid and mix well, then filter through a membrane filter.

[0135] 3) 0.1 N hydrochloric acid solution: Pipette 8.5 mL of concentrated hydrochloric acid into a 1 L volumetric flask containing an appropriate amount of water, dilute to the mark with water, and shake well.

[0136] 4) Stock solution of reference substance: Accurately weigh 5 mg of sapropterin hydrochloride reference substance into a 100 mL volumetric flask, add 50 mL of 0.1 N hydrochloric acid solution, dissolve by ultrasonic treatment, dilute with the diluent to the mark, and mix well.

[0137] 5) Reference substance solution (1 μg / mL): Accurately pipette 1.0 mL of the stock solution of reference substance into a 50 mL volumetric flask, dilute to the mark with the diluent, and mix well.

[0138] 6) Rechecked stock solution of reference substance: Accurately weigh 5 mg of sapropterin hydrochloride reference substance into a 100 mL volumetric flask, add 50 mL of 0.1 N hydrochloric acid solution, dissolve by ultrasonic treatment, dilute to the mark with the diluent, and mix well.

[0139] 7) Rechecked reference substance solution (1 μg / mL): Accurately pipette 1.0 mL of the rechecked stock solution of reference substance into a 50 mL volumetric flask, dilute to the mark with the diluent, and shake well.

[0140] 8) System suitability stock solution: Accurately weigh 5 mg of biopterin and pterin reference substances into a 100 mL volumetric flask, add 50 mL of 0.1 N hydrochloric acid solution, dissolve by ultrasonic treatment, dilute to the mark with the diluent, and mix well.

[0141] 9) System suitability solution: Pipette 1.0 mL of the system suitability stock solution into a 50 mL volumetric flask containing an appropriate amount of diluent, dilute to the mark with the diluent, and mix well.

[0142] 10) Sensitivity solution (0.05%): Accurately pipette 2.5 mL of the reference substance solution into a 10 mL volumetric flask, dilute to the mark with the diluent, and mix well.

[0143] 11) Test sample powder: Take not less than 10 sapropterin hydrochloride tablets, weigh and transfer them to an agate mortar and grind evenly to obtain.

[0144] 12) Test sample solution: Accurately weigh about 75 mg of the test sample powder (containing about 25 mg of sapropterin hydrochloride), place it in a 50 mL volumetric flask, add an appropriate amount of diluent to completely dissolve it, make up the volume with the diluent and mix well. Discard at least 1.0 mL of the filtrate through a nylon filter membrane (0.22 μm, 25 mm in diameter), and the subsequent filtrate is the test sample solution.

[0145] Record the chromatogram and analyze the obtained chromatographic peaks. Calculate the content of each impurity according to the following formula:

[0146]

[0147] Where: A: Peak area of a single impurity in the test sample solution; A STD : Average peak area of sapropterin hydrochloride in the first 6 injections of the reference solution; W STD : Weight (mg) of the sapropterin hydrochloride reference substance; P: Content of the sapropterin hydrochloride reference substance; V SAP : Dilution volume (mL) of the test sample; V STD : Dilution volume (mL) of the sapropterin hydrochloride reference substance; W SAP : Weight of the test sample (mg); W AVE : Average weight of the test sample (mg); LC: Labeled amount of sapropterin hydrochloride in the test sample (100 mg); RRF: Relative response factor, where the RRF of biopterin is 2.50, the RRF of pterin is 2.56, and the RRF of the remaining unknown single impurities is calculated as 1.0. The content of each impurity was calculated using this formula in Example 7.

[0148] The detection results of each method in Example 1 are shown in Table 2:

[0149] Table 2. Detection results

[0150]

[0151]

[0152] The following comprehensively examines the method of the present invention in terms of system suitability, specificity, limit of quantitation, limit of detection, precision, accuracy, and durability.

[0153] Example 2. System suitability test

[0154] The chromatographic conditions and solution preparation in this experiment are the same as those of the method of the present invention in Example 1.

[0155] The injection process in this experiment is as follows:

[0156] Inject the blank solution (diluent), sensitivity solution, system suitability solution, reference solution, and recheck reference solution respectively, and record the chromatograms. The injection process is as follows: (1) Blank solution (number of injections ≥ 1 needle), (2) Sensitivity solution (number of injections is 1 needle), (3) System suitability solution (number of injections is 1 needle), (4) Reference solution (number of injections is 6 needles), (5) Recheck reference solution (number of injections is 2 needles). (4) Inject the reference solution 6 times to check the system precision, and (5) Inject the recheck reference solution 2 times to calculate the recovery rate for evaluating the preparation accuracy of the (4) reference solution.

[0157] System suitability requirements: Except for the solvent peak, the blank solution should have no interference or the interference should be less than 0.05%; the signal-to-noise ratio of sapropterin hydrochloride in the sensitivity solution should not be less than 10; the resolution between biopterin and pterin in the system suitability solution should not be less than 1.5; the relative standard deviation (RSD) of the peak area of sapropterin hydrochloride in the first 6 injections of the reference solution should not be greater than 5.0%; the recovery rate of sapropterin hydrochloride in the recheck reference solution should be between 90.0 - 110.0%. The results are shown in Table 3 as follows:

[0158] Table 3. System suitability results

[0159] Item Result Acceptable standard Resolution of biopterin and pterin ＞3.0 Resolution is not less than 1.5 Sensitivity: Signal-to-noise ratio of sapropterin hydrochloride 148～200 Signal-to-noise ratio is not less than 10 RSD% of the peak areas of the first 6 injections of the reference substance 0.5 ≥5.0 Recovery rate of the rechecked reference substance % 100.6 Recovery rate is between 90.0 - 110.0%

[0160] The results in Table 3 show that: the blank solution has no interference; the resolution between biopterin and pterin in the system suitability solution is greater than 1.5; the signal-to-noise ratio of sapropterin hydrochloride in the sensitivity solution is 148 - 200, far greater than 10; the RSD of the peak area of the first 6 injections of the reference solution is 0.5%, far less than 5.0%; the recovery rate of the recheck reference is between 90.0 - 110.0%. These results indicate that the system suitability of the method in Example 2 meets the requirements.

[0161] Example 3. Specificity test

[0162] The chromatographic conditions in this experiment are the same as those of the method of the present invention in Example 1.

[0163] Prepare the blank solution (diluent), blank excipient solution, reference solution, 0.2% solution for each impurity location, 0.2% known impurity spiked sample solution, and test solution. Among them, the blank solution (diluent), reference solution, and test solution are the same as those of the method of the present invention in Example 1. The preparation methods of other solutions are as follows:

[0164] Blank excipient solution: Accurately weigh about 20 mg of blank excipients (excipients in the sapropterin hydrochloride tablets of Shanghai Aokeda Pharmaceutical Technology Co., Ltd.) into a 20 mL volumetric flask, add an appropriate amount of diluent to completely dissolve, and make up the volume to the mark with diluent and mix well. Filter through a 0.22 μm nylon filter membrane, discard at least 1.0 mL, and the subsequent filtrate is the blank excipient solution.

[0165] Impurity reference substance stock solution (0.05 mg / mL): Accurately weigh about 2.5 mg of each of the biopterin, pterin, pteridine, S-isomer, and neopterin reference substances into different 50 mL volumetric flasks, add 25 mL of 0.1 N hydrochloric acid, and ultrasonically dissolve until completely dissolved. Dilute to volume with diluent and mix well.

[0166] 0.2% positioning solution for each impurity: Pipette 0.5 mL of the impurity reference substance stock solution of biopterin / pterin / pteridine / S-isomer / neopterin into different 25 mL volumetric flasks respectively, dilute to volume with diluent, and shake well.

[0167] 0.2% spiked sample solution of known impurities (0.5 mg / mL of sapropterin hydrochloride, 1 μg / mL of biopterin, pterin, pteridine, S-isomer, and neopterin): Accurately weigh about 75 mg of the test sample powder (containing about 25 mg of sapropterin hydrochloride) into a 50 mL volumetric flask, add an appropriate amount of diluent to dissolve, and then add 1.0 mL of the impurity reference substance stock solution of biopterin, pterin, pteridine, S-isomer, and neopterin to the above 50 mL volumetric flask respectively. Dilute to volume with diluent and mix well. Filter through a 0.22 μm nylon filter membrane, discard at least 1.0 mL, and the subsequent filtrate is the 0.2% spiked sample solution of known impurities.

[0168] Take the blank solution (diluent), blank excipient solution, reference solution, 0.2% positioning solution for each impurity, 0.2% spiked sample solution of known impurities, and test sample solution, inject and analyze according to the above chromatographic conditions, and record the chromatogram. The 0.2% spiked sample solution of known impurities, blank solution, and blank excipient solution are as Figure 5 、 Figure 6 、 Figure 7 。From Figures 5 - 7 it can be seen that the blank solvent and blank excipients have no interference at the peak positions of sapropterin hydrochloride or other known impurities. Table 4 shows the detection results obtained by detecting the 0.2% spiked sample solution of known impurities using the method of the present invention.

[0169] Table 4. Detection results of 0.2% spiked sample solution of known impurities

[0170] Name Retention time / min Relative retention time Resolution Pteridine 5.203 0.83 / Sapropterin hydrochloride 6.288 1.00 ＞1.5 S-isomer 7.146 1.14 ＞1.5 Neopterin 10.179 1.62 ＞1.5 Biopterin 13.984 2.22 ＞1.5 Pterin 14.491 2.30 3.6

[0171] From the retention times, resolution results of each peak in Table 4 and Figure 5 it can be seen that each related substance can be effectively separated, indicating that the method of the present invention has good specificity.

[0172] Conduct forced degradation tests on an appropriate amount of sapropterin hydrochloride tablets under high temperature, acid, base, oxidation, and light.

[0173] Note: Synchronously putting the blank excipient and the sarpogrelate hydrochloride raw material drug is used to determine the impurity attribution of sample degradation.

[0174] Thermal degradation (105 °C): Accurately weigh about 30 mg of the test sample powder, about 20 mg of the blank excipient, and about 10 mg of the sarpogrelate hydrochloride raw material drug into different 20-mL volumetric flasks, place them in an oven at 105 °C and let them stand for degradation for 2 h. After cooling, add an appropriate amount of diluent, make up the volume, and vortex for about 30 s to mix evenly. Filter through a 0.22-μm nylon membrane filter, discard at least 1.0 mL, and the subsequent filtrate is used as the thermally degraded test sample solution.

[0175] Acid degradation (1N hydrochloric acid): Accurately weigh about 30 mg of the test sample powder, about 20 mg of the blank excipient, and about 10 mg of the sarpogrelate hydrochloride raw material drug into different 20-mL volumetric flasks, add 5 mL of 1N hydrochloric acid, let them stand for degradation for 4 h, make up the volume with diluent, and vortex for about 30 s to mix evenly. Filter through a 0.22-μm nylon membrane filter, discard at least 1.0 mL, and the subsequent filtrate is used as the acid-degraded test sample solution.

[0176] Alkali degradation (0.1N sodium hydroxide): Accurately weigh about 30 mg of the test sample powder, about 20 mg of the blank excipient, and about 10 mg of the sarpogrelate hydrochloride raw material drug into different 20-mL volumetric flasks, add 1 mL of 0.1N sodium hydroxide, immediately terminate the degradation by adding 1 mL of 0.1N hydrochloric acid, make up the volume with diluent, and vortex for about 30 s to mix evenly. Filter through a 0.22-μm nylon membrane filter, discard at least 1.0 mL, and the subsequent filtrate is used as the alkali-degraded test sample solution.

[0177] Oxidation degradation (0.3% hydrogen peroxide): Accurately weigh about 30 mg of the test sample powder, about 20 mg of the blank excipient, and about 10 mg of the sarpogrelate hydrochloride raw material drug into different 20-mL volumetric flasks, add 5 mL of 0.3% hydrogen peroxide, let them stand for degradation for 25 minutes, make up the volume with diluent, and vortex for about 30 s to mix evenly. Filter through a 0.22-μm nylon membrane filter, discard at least 1.0 mL, and the subsequent filtrate is used as the oxidation-degraded test sample solution (inject immediately).

[0178] Light degradation (9000 lx ± 500 lx): Put 20 tablets of sarpogrelate hydrochloride tablets, an appropriate amount of excipient, and the sarpogrelate hydrochloride raw material drug into different plastic-sealed bags respectively. Place two copies in parallel, wrap one of them with aluminum foil. And place them in a drug strong light irradiation test chamber and let them stand for degradation for 6 days. At the specified time to be measured, take out the exposed and unexposed light-exposed samples and accurately weigh about 30 mg of the test sample powder, about 20 mg of the blank excipient, and about 10 mg of the sarpogrelate hydrochloride raw material drug into different 20-mL volumetric flasks, make up the volume with diluent, and vortex for about 30 s to mix evenly. Filter through a 0.22-μm nylon membrane filter, discard at least 1.0 mL, and the subsequent filtrate is used as the light-degraded test sample solution.

[0179] For each solution prepared after undergoing high temperature, acid, base, oxidation, and light destruction experiments, injection analysis was carried out to determine the content of sapropterin hydrochloride and the total impurity content. Among them Figures 8 - 12 are the chromatograms obtained after high temperature, acid, base, oxidation, and light destruction experiments respectively. The statistical results of the content of sapropterin hydrochloride, total impurity content, material balance, and peak purity after forced degradation of the test substance are shown in Table 5.

[0180] Table 5. Content of sapropterin hydrochloride, total impurity content, material balance, and peak purity after forced degradation of the test substance

[0181] Degradation conditions Purity angle Purity threshold Main peak content % Total impurities % Material balance % Not destroyed 7.738 9.218 100.0 0.14 100 Acid-destroyed for 4 h 6.459 9.221 96.4 0.20 97 Base-destroyed for 0 h 7.226 9.135 96.0 1.11 97 Oxidation-destroyed for 25 min 5.348 8.961 87.6 7.09 95 High temperature-destroyed for 2 h 6.262 9.191 97.9 0.44 98 Light-destroyed for 6 days 6.775 9.166 99.8 0.14 100 Light-protected-destroyed for 6 days 6.887 9.130 97.5 0.13 98

[0182] Note: Material balance: Main peak content + total impurities. The difference in material balance between the destroyed sample and the non-destroyed sample is within the range of ±5%.

[0183] As can be seen from Table 5, under the conditions of high temperature, acid, base, oxidation, and light destruction, mass conservation is achieved, and at the same time, the peak purity also meets the requirements.

[0184] Through Figures 8 - 12 it can be seen that the unknown impurities generated by degradation are well separated from the main peak and two degradation impurities (biopterin and pterin), and the specificity of the present invention can meet the requirements.

[0185] Example 4. Quantitative limit and detection limit tests

[0186] The chromatographic conditions for this experiment are the same as those of the method of the present invention in Example 1.

[0187] Take the solutions of the limit of quantitation (LOQ; the lowest concentration solution that satisfies the linear relationship, approximately 0.25 μg / mL) and the detection limit (dilute the LOQ solution by 3 times) and inject them for analysis according to the chromatographic conditions in the method of the present invention in Example 1. The signal-to-noise ratio (S / N) of 6 parallel injections of the LOQ solution should be greater than 10, and the RSD of the peak areas should not be greater than 5.0%; the signal-to-noise ratio (S / N) of 3 parallel injections of the detection limit solution should not be less than 3. The results are shown in Table 6 and Table 7.

[0188] Table 6. Results of the investigation of the limit of quantitation

[0189]

[0190]

[0191] Table 7. Results of the investigation of the detection limit

[0192]

[0193] The results in Table 6 and Table 7 show that: at the quantitation limit concentration level of the test article at 0.05% (the reporting limit of this product is 0.05%), the signal-to-noise ratios of sapropterin hydrochloride, biopterin, and pterin are all greater than 10, and at the detection limit level of the test article at 0.015%, the signal-to-noise ratios of sapropterin hydrochloride, biopterin, and pterin are all greater than 3, indicating that this method can accurately quantify the contents of known and unknown degradation impurities (substituting the main component sapropterin hydrochloride for verification) in sapropterin hydrochloride tablets.

[0194] Example 5, Linearity and Range

[0195] The purpose of this experiment is to examine the ability of the high performance liquid chromatography method to have a proportional relationship between the peak area of the analyte and the concentration within the designed range, as well as the accuracy of calculating the contents of various impurities by the external standard method.

[0196] The chromatographic conditions for this experiment are the same as those of the method of the present invention in Example 1.

[0197] Prepare standard solutions at 5 concentration levels, which are 0.05% (LOQ), 0.1%, 0.2%, 0.3%, and 0.4% of the sample concentration (0.5 mg / mL) respectively.

[0198] Inject one injection of each of the above solutions and record the chromatograms. Using the concentration as the abscissa and the peak area as the ordinate, make a linear regression equation, and require that the correlation coefficient r should not be less than 0.990, and the y-axis intercept should be within 20% of the target concentration. The results are shown in Table 8.

[0199] Table 8, Linear Results of Sapropterin Hydrochloride, Biopterin, and Pterin

[0200]

[0201]

[0202] Figure 13 The linear relationships of sapropterin hydrochloride, biopterin, and pterin detected by the method of the present invention are shown. Figure 13 And the results in Table 8 show that sapropterin hydrochloride has a good linear relationship in the concentration range of 0.244 μg / mL (LOQ) to 1.949 μg / mL, biopterin has a good linear relationship in the concentration range of 0.233 μg / mL (LOQ) to 1.862 μg / mL, and pterin has a good linear relationship in the concentration range of 0.245 μg / mL (LOQ) to 1.961 μg / mL.

[0203] Example 6, Accuracy Test

[0204] The chromatographic conditions for this experiment are the same as those of the method of the present invention in Example 1.

[0205] Prepare the unspiked sample solution (i.e., the test solution in the method of Example 1 of the present invention), the LOQ limit accuracy solution, the 100% limit accuracy solution, and the 150% limit accuracy solution. Other solutions not specifically stated are prepared in the same manner as the solutions in the method of the present invention in Example 1.

[0206] Impurity stock solution (0.05 mg / mL): Accurately weigh approximately 5 mg of biopterin and pterin reference standards into a 100 mL volumetric flask, add 50 mL of 0.1 N hydrochloric acid, and ultrasonically dissolve completely. Dilute to volume and mix well with the diluent. Pipette 12.5 mL of the above solution into a 50 mL volumetric flask, and dilute to volume and mix well with the diluent.

[0207] LOQ limit accuracy solution: Accurately weigh approximately 75 mg (containing approximately 25 mg of sapropterin hydrochloride) of the test sample powder and 1.0 mL of the impurity stock solution into a 50 mL volumetric flask, add an appropriate amount of diluent to dissolve completely, and dilute to volume and mix well with the diluent. Filter through a 0.22 μm nylon membrane filter, discard at least 1.0 mL, and take the subsequent filtrate for detection. Prepare three replicates in parallel.

[0208] 100% limit accuracy solution: Accurately weigh approximately 75 mg of the sample powder and 4.0 mL of the impurity stock solution into a 50 mL volumetric flask, add an appropriate amount of diluent to dissolve completely, and dilute to volume and mix well with the diluent. Filter through a 0.22 μm nylon membrane filter, discard at least 1.0 mL, and take the subsequent filtrate for detection. Prepare three replicates in parallel.

[0209] 150% limit accuracy solution: Accurately weigh approximately 75 mg of the sample powder and 6.0 mL of the impurity stock solution into a 50 mL volumetric flask, add an appropriate amount of diluent to dissolve completely, and dilute to volume and mix well with the diluent. Filter through a 0.22 μm nylon membrane filter, discard at least 1.0 mL, and take the subsequent filtrate for detection. Prepare three replicates in parallel.

[0210] Inject each of the above solutions for analysis and record the chromatograms. Calculate the recovery rates of each impurity by the external standard method. The results are shown in Table 9.

[0211] Table 9. Accuracy results of biopterin and pterin

[0212]

[0213] The results show that for the known degradation impurities biopterin and pterin, the recovery rates and average recovery rates of each sample at each concentration level are between 90.0% and 110.0%, and the RSD of the 9 recovery rates is less than 10%, indicating that the accuracy of this method is good.

[0214] Example 7. Precision test

[0215] The chromatographic conditions for this experiment are the same as those in the method of the present invention in Example 1.

[0216] Repeated sample solution: Prepared in the same manner as the 100% limit accuracy solution, and six parallel samples were prepared for injection analysis.

[0217] Inject each of the above solutions for analysis and record the chromatogram. Calculate the content of each impurity by the external standard method. The results are shown in Table 10.

[0218] Table 10. Repeatability results

[0219]

[0220]

[0221] The results showed that the RSDs of the contents of known degradation impurities and unknown impurities in the six samples were all less than 15%, indicating that the method had good repeatability in quantifying known degradation impurities and unknown impurities.

[0222] Reproducibility test

[0223] Another analyst independently established the system in the factory laboratory. Samples of the same batch as the repeated samples were taken, and six sample solutions were prepared in the same manner as the repeatability method. The reproducibility and precision results are shown in Table 11.

[0224] Table 11. Precision results

[0225]

[0226]

[0227] The results showed that among the 12 sample solutions including the repeatability ones, the RSDs of the contents of each known degradation impurity and unknown impurity were all less than 20%, indicating that the method had good precision in quantifying known impurities and unknown impurities.

[0228] Example 8. Stability test of test solution and reference solution

[0229] The chromatographic conditions and solution preparation in this experiment were the same as those of the method of the present invention in Example 1.

[0230] The reference solution and the test solution were placed at 2 - 8 °C and sampled on day 0, day 1, day 2, day 3, or day 4 respectively to investigate the solution stability. The stability results of the reference solution are shown in Table 12, and the stability results of the test solution are shown in Table 13.

[0231] Table 12. Stability results of reference solution

[0232]

[0233] Table 13. Stability results of test solution

[0234]

[0235]

[0236] Note: *The actual measurement result is 0.04%, LOQ = 0.05%.

[0237] The results show that when the reference solution is stored at 2 - 8 °C, the difference in content from the zero point is ≤ 5.0%, indicating that the reference solution is stable for 4 days under the condition of 2 - 8 °C; for the test solution samples stored at 2 - 8 °C for 3 days, compared with the zero point, no new impurities greater than the reporting limit (0.05%) appeared; the changes in the contents of each known impurity and unknown impurity were also within the reporting limit, indicating that the test solution is stable for 3 days under the condition of 2 - 8 °C.

[0238] Sapropterin hydrochloride itself is easily oxidized and is generally very unstable when stored in the presence of air. The stability results in Table 12 and Table 13 further illustrate that the diluent in the present invention has strong antioxidant ability and good protection for sapropterin hydrochloride.

[0239] Example 9, Robustness Test

[0240] Unless otherwise specified, the chromatographic conditions and solution preparation in this experiment are the same as those of the method of the present invention in Example 1.

[0241] Table 14, Items for Robustness Investigation

[0242] Investigated item Specified parameter Range of variation pH of mobile phase pH 2.80 pH 2.80 ± 0.05 Chromatographic column YMC-Pack ODS-AQ 4.6 * 250 mm 3 μm One chromatographic column of different batches

[0243] The blank solution (diluent), sensitivity solution, system suitability solution, reference solution, and recheck reference solution were respectively injected for analysis in sequence to investigate whether the system suitability meets the requirements under different chromatographic parameter conditions (Table 14); the test solution was injected for analysis to investigate the results obtained by changing different chromatographic parameter conditions and the results without changing the conditions. The investigation results are shown in Table 15 and Table 16.

[0244] Acceptable criteria for system suitability: Except for the solvent peak, the blank solution should have no interference or the interference should be less than 0.05%; the signal - to - noise ratio of sapropterin hydrochloride in the sensitivity solution should be not less than 10; the resolution between biopterin and pterin in the system suitability solution should be not less than 1.5; the RSD of the peak area of sapropterin hydrochloride in the first 6 injections of the reference solution should be not greater than 5.0%; the recovery rate of sapropterin hydrochloride in the recheck reference solution should be between 90.0 - 110.0%.

[0245] Table 15, Investigation Results of Robustness - System Suitability

[0246]

[0247]

[0248] Table 16, Durability - Test Results of Test Samples

[0249]

[0250] Note: ND - Not Detected / Not detected; LOQ = 0.05%.

[0251] The results show that: by changing different parameters, the system suitability meets the requirements. However, under the condition of pH 2.85, the content change of the test sample solution is greater than the limit value (±0.05%). Therefore, the pH value of mobile phase A is set between 2.75 and 2.80, and the durability of the test sample solution meets the requirements under other conditions, indicating that the durability of this method is good.

[0252] The above is only the preferred embodiment of the present invention and is not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0253] Comparative Example 1, Investigating the Interference of Different Diluents on the Detection of Test Samples and the Influence on the Stability of Test Sample Solutions The chromatographic conditions of this experiment are the same as those of the method of the present invention in Example 1.

[0254] Diluent 1: Weigh 2.72 g of potassium dihydrogen phosphate into 1 L of water, adjust the pH to 3.0 with phosphoric acid, add 1 g of ascorbic acid to dissolve and mix well, and filter through a membrane filter.

[0255] Diluent 2: Accurately pipette 8.5 mL of concentrated hydrochloric acid into 1 L of water, and then add 1 g of ascorbic acid to dissolve and mix well.

[0256] Diluent 3: Weigh 0.2 g of mercaptoethanol and 1 g of L - cysteine hydrochloride monohydrate into 1 L of water, dissolve and mix well by ultrasonic treatment, and filter through a membrane filter.

[0257] Prepare 4 test sample solutions respectively using the diluent of the present invention, Diluent 1, Diluent 2, and Diluent 3 according to the method for preparing test sample solutions in Example 1 of the present invention. Then inject the zero - point samples, 12 - hour samples, and 24 - hour samples of each diluent and the above - mentioned test sample solutions respectively, and record the chromatograms. Figures 14 - 17 The chromatographic overlay map showing the stability of the test sample solutions prepared with 4 diluents is shown. It can be seen that Diluent 2 has interference at the peak position of sapropterin hydrochloride, and the stability of the test sample solutions of Diluent 1 and Diluent 3 is poor. The impurities at the retention times of about 8 min and 12 min - 14 min increase significantly at 12 h. While there is no obvious increase in the impurities in the test sample solution of the diluent of the present invention at 24 h. This further shows that the diluent of the method of the present invention in Example 1 has good protection.

[0258] In summary, by adopting the combination of the chromatographic conditions and detection conditions of the present invention, the method of the present invention can achieve a resolution greater than 1.5, a detection limit of 0.015%, and a quantification limit of 0.05%. The above excellent detection limits enable the simultaneous detection of 5 impurities including biopterin and pterin.

[0259] The use of any and all examples or exemplary language (e.g., "such as") provided herein is only intended to better illustrate the present invention and does not constitute a limitation on the scope of the present invention, unless otherwise required. The language in the specification should not be construed as indicating that any non-claimed element is necessary for the implementation of the present invention.

[0260] All publications and patent applications cited in this specification are incorporated herein by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In addition, any theory, mechanism, proof, or discovery described herein is intended to further enhance the understanding of the present invention and is not intended to limit the present invention in any way to such theory, mechanism, proof, or discovery. Although the present invention has been shown and described in detail in the drawings and the foregoing description, the present invention should be considered illustrative rather than restrictive.

Claims

1. A method for detecting related substances in sapropterin hydrochloride tablets, characterized in that: The related substances are selected from Table 1: Table 1. Names and structural formulas of relevant substances The detection method adopts high performance liquid chromatography for detection, and the filling material of the chromatographic column stationary phase is octadecylsilane bonded silica gel; a ghost peak capture column is installed between the confluence outlet of the pump and the injector; mobile phase A: phosphate buffer with a concentration of 10 to 30 mmol / L and a pH of 2.75-2.80; mobile phase B: methanol; the mobile phase gradient is shown in the following table: Mobile phase flow rate: 0.5~0.7mL / min; chromatographic column temperature: 25℃~35℃; injector temperature: 2℃~8℃; detection wavelength: 275nm; injection volume: 10μl~20μl; The sample to be tested is dissolved in a diluent and fixed to a fixed volume to form an injection solution; the diluent is a phosphate buffer containing ascorbic acid, the concentration of the phosphate buffer is 10-30 mmol / L, the pH is 2.0-2.5, and the concentration of ascorbic acid is 1-2 g / L.

2. The method for detecting related substances in sapropterin hydrochloride tablets according to claim 1, characterized in that: The diluent is potassium dihydrogen phosphate buffer containing ascorbic acid, the concentration of potassium dihydrogen phosphate buffer is 20 mmol / L, the pH is adjusted to 2.5 with phosphoric acid, and the concentration of ascorbic acid is 1 g / L.

3. The method for detecting related substances in sapropterin hydrochloride tablets according to claim 1, characterized in that: The sample to be tested is dissolved in a diluent and fixed to a certain volume to form an injection solution. Specifically, the sample to be tested is a sapropterin hydrochloride tablet; the powder obtained by grinding the sapropterin hydrochloride tablet is weighed and dissolved in the diluent to prepare a 1 mL solution containing 0.4-0.6 mg of sapropterin hydrochloride.

4. The method for detecting related substances in sapropterin hydrochloride tablets according to claim 1, characterized in that: An impurity reference substance is used to determine the peak time of the impurities in the related substances in the HPLC. The impurity reference substance solution is prepared by ultrasonically dissolving it with a 0.1N hydrochloric acid solution and then adding the diluent to prepare a 1mL solution containing 1-2μg of the corresponding impurity.

5. The method for detecting related substances in sapropterin hydrochloride tablets according to claim 1, characterized in that: The chromatographic system is a high performance liquid chromatograph with a PDA / DAD / VWD detector; the high performance liquid chromatograph is Agilent 1260Ⅱ or Waters Arc or Waters e2695.

6. The method for detecting related substances in sapropterin hydrochloride tablets according to claim 1, characterized in that: The chromatographic column is YMC-Pack ODS-AQ, with specifications of 4.6 mm×250 mm and 3.0 μm.

7. The method for detecting related substances in sapropterin hydrochloride tablets according to claim 1, characterized in that: The ghost peak capture column is Yuexu's Ghost-Buster Column, or Ghost-Buster HP Column, or Ghost-Buster Column II.

8. The method for detecting related substances in sapropterin hydrochloride tablets according to claim 7, characterized in that: The ghost peak capture column is Yuexu's Ghost-Buster Column, with a specification of 4.6×50 mm.

9. The method for detecting related substances in sapropterin hydrochloride tablets according to claim 1, characterized in that: Mobile phase A was 20 mmol / L potassium dihydrogen phosphate buffer, pH 2.75-2.80; mobile phase flow rate: 0.6 ml / min; chromatographic column temperature: 30°C; automatic sampler temperature: 5°C.

10. The method for detecting related substances in sapropterin hydrochloride tablets according to claim 1, characterized in that: The content of each impurity is calculated according to the following formula: Where: A: peak area of ​​a single impurity in the test solution; W STD : The mass of sapropterin hydrochloride reference substance, in mg; P: content of sapropterin hydrochloride reference substance; V SAP W: dilution volume of the test sample, in mL; AVE : average mass of the test sample, in mg; RRF: relative response factor, where the RRF of biopterin is 2.50, the RRF of pterin is 2.56, and the other unknown impurities are calculated as 1.0; A STD : The average peak area of ​​sapropterin hydrochloride in the first 6 samples of the reference solution; V STD W: dilution volume of sapropterin hydrochloride reference substance, unit: mL; SAP : The mass of the test sample, in mg; LC: labeled amount of sapropterin hydrochloride in the test sample, unit: 100 mg.

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