HPLC Detection Method for Minocycline Hydrochloride and Its Impurities

Abstract

The present invention relates to a high performance liquid chromatography detection method for minocycline hydrochloride and its impurities. The method for determining related substances in minocycline hydrochloride comprises the following operations: according to the specifications of the high performance liquid chromatography method, a test solution, a reference solution, a system suitability solution, and a sensitivity solution are prepared, and a chromatographic column with octadecylsilane bonded silica gel as a filler is used; a mixture of ammonium acetate solution-methanol containing disodium ethylenediaminetetraacetic acid is used as mobile phase A, and acetonitrile-methanol is used as mobile phase B, and linear gradient elution is performed, and the separation degree of the minocycline peak and the RS12 peak should be not less than 1.5; if there is an impurity peak corresponding to the relative retention time in the chromatogram of the test solution, the percentage of the impurity is calculated according to the main component reference substance external standard method with a correction factor. The present invention also provides a minocycline hydrochloride. The method of the present invention has the excellent technical effect as described in the specification.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to a quality analysis method for drugs, in particular to minocycline hydrochloride and its impurities, and more particularly to a method for detecting related substances in minocycline hydrochloride using high performance liquid chromatography. The related substance detection method of the present invention can simultaneously detect more than ten related substances in minocycline hydrochloride. Background Art

[0002] Minocycline (minocycline), whose hydrochloride is commonly used clinically. The molecular formula of minocycline hydrochloride is C23H27N3O7·HCl, with a molecular weight of 493.94. Its chemical name is: [4S-(4α,4aα,5aα,12aα)]-4,7-bis(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,10,12,12a-tetrahydroxy-1,11-dioxo-2-naphthacenecarboxamide hydrochloride. Its chemical structure is as follows:

[0003]

[0004] Minocycline hydrochloride is a yellow crystalline powder, odorless, and hygroscopic; it is soluble in methanol, slightly soluble in water, slightly soluble in ethanol, and almost insoluble in ether.

[0005] Minocycline is a second-generation tetracycline antibiotic, a long-acting, highly effective, semi-synthetic tetracycline. Its antimicrobial spectrum is similar to that of tetracycline, and its antimicrobial activity is the strongest among tetracyclines. Its activity against Gram-positive bacteria is 2-4 times stronger than that of tetracycline, and it has strong activity against both Gram-positive and Gram-negative bacteria. While cross-resistance exists among tetracycline antibiotics, minocycline exhibits strong antimicrobial activity against clinically isolated tetracycline-resistant Staphylococcus aureus, Staphylococcus epidermidis, and Streptococci. Its effectiveness against resistant bacteria makes it clinically favored. Adverse reactions are largely similar to those of other tetracyclines, including reversible vestibular reactions, including nausea, vomiting, and ataxia.

[0006] Taking minocycline hydrochloride API as an example, minocycline API is included in the 2020 edition of the Chinese Pharmacopoeia, the United States Pharmacopoeia USP (effective date is May 1, 2020), the European Pharmacopoeia (EP11.0) and the Japanese Pharmacopoeia (JP18).

[0007] The 2020 edition of the Chinese Pharmacopoeia uses an ammonium acetate-dimethylformamide-tetrahydrofuran-disodium ethylenediaminetetraacetic acid mobile phase system and a C8 chromatography system to control related substances. The main controlled impurities are diastereoisomers (<1.2%), other single impurities (<1.2%), and the total amount of other impurities (<2.0%). In the current pharmacopoeias of USP, EP, and JP, the mobile phase system for the detection of related substances of minocycline hydrochloride raw materials is: oxalate-dimethylformamide-tetrahydrofuran-disodium ethylenediaminetetraacetic acid system, which controls diastereoisomers, other single impurities, and the total amount of impurities.

[0008] However, facing increasingly stringent requirements for controlling impurities related to substances, most companies' internal control of specific impurities is not limited to diastereomers. They often require the separation and detection of several or even dozens of specific impurities to better control product quality. However, using the analytical methods in current standards, the diastereomer minocycline peak, other impurities, and the minocycline peak, as well as some impurity peaks, cannot be effectively separated. Therefore, it is necessary to develop more specific and selective analytical detection methods that can effectively separate complex specific impurities and unknown impurities.

[0009] With the development of society and the increasing emphasis on human health and safety, the application of green analytical chemistry concepts to reduce the use of toxic and hazardous reagents in experiments has become an inevitable trend. Currently, mainstream detection methods for the detection of related substances in tetracycline antibiotics still use toxic and hazardous reagents such as tetrahydrofuran and dimethylformamide in the preparation of mobile phases, causing chronic damage to the health of laboratory personnel and exacerbating environmental pollution through the discharge of wastewater. To protect the health of analysts and reduce environmental pollution, the use of these reagents should be avoided as much as possible. If the use of organic reagents cannot be avoided, less toxic alternatives should be used.

[0010] At present, relevant research teams are exploring and studying low-toxic solvent replacements and have made certain progress. For example, the literature of Gao Yanxia's team [Gao Yanxia, Jiang Jianguo, Du Zenghui. Determination of the content of minocycline hydrochloride for injection and related substances by HPLC [J]. Chinese Journal of Antibiotics, 2005, 30 (12): 744-746] reported that using a Kromasil C8 (4.6 mm × 250 mm, 5 μm) column, 0.2 mol / L ammonium acetate: N, N-dimethylformamide: tetrahydrofuran (600: 398: 2, containing 0.01 mol / L disodium ethylenediaminetetraacetic acid) as the mobile phase, and a detection wavelength of 280 nm, when detecting the related substances of minocycline hydrochloride, it was possible to detect 5 special impurities: demethylchlortetracycline, 6-demethyltetracycline (DMTC), 6-demethyl-6-deoxytetracycline (DMDOTC), 11a-chloro-6-demethyl-6-deoxytetracycline (11a-Cl This method effectively separates and controls 2-amino-6-demethyl-6-deoxytetracycline (DMDOTC) and 7-amino-6-demethyl-6-deoxytetracycline (ADMDOTC). This method uses acetate, which is more water-soluble, to reduce salt concentration and avoid the use of oxalate (which crystallizes at low temperatures and can easily damage the instrument and chromatographic column), offering improved applicability. However, this literature method only controls five specific impurities and cannot meet the requirements for controlling multiple complex specific impurities in the relevant substances.

[0011] The literature of Wang Long's team [Wang Long, Wang Hongmei. Determination of related substances of minocycline hydrochloride by high performance liquid chromatography [J]. Strait Pharmacy, 2006, 18(12006):85-86], using Kromasil A C8 chromatographic column (250 mm × 4.6 mm, 5 μm) was used with ammonium acetate buffer-acetonitrile-methanol (830:170:5) adjusted to pH 6.55 with concentrated ammonia or acetic acid as the mobile phase. The detection wavelength was 280 nm. Eight impurities that may be present in minocycline hydrochloride, namely, ①7-bis-demethylminocycline, ②demethyltetracycline, ③demeclocycline, ④7-monodemethylminocycline, ⑤pyrazol-minocycline, ⑥demethyldeoxytetracycline, ⑦4-di-minocycline, and ⑧hydroxymethylminocycline, were separated and determined to meet the detection requirements for the control of the above eight impurities. Methanol and acetonitrile were used instead of tetrahydrofuran as the organic phase to reduce the harm of tetrahydrofuran to the human body.

[0012] The literature of Mei Qian's team [Mei Qian, Li Tonghui, Li Shaojie, et al. Quality evaluation of minocycline hydrochloride capsules [J]. Chinese Journal of Antibiotics, 2020, 45(3): 224-233], using a chromatographic column of Dikma Platisil ODS (250mm×4.6mm, 5μm); mobile phase A was 0.05mol / L ammonium acetate solution (adjusted to pH 8.0 with ammonia water), mobile phase B was methanol, mobile phase C was acetonitrile, and a gradient elution program was performed with a flow rate of 1.0mL / min; UV detector, detection wavelength 280nm; column temperature 30℃; injection volume 10μL, to detect related substances in minocycline. Compared with the current pharmacopoeia method, this method has lower column pressure, better separation effect between impurities, detection of 3 more impurities, and a more stable system. In addition to detecting differential minocycline impurities, 10 unknown impurities were also detected, all of which were effectively separated.

[0013] However, with the continuous improvement of separation technology, more specific impurities have been separated and purified, and companies have become increasingly stringent in their requirements for impurity control. The number of specific impurities has increased to more than ten or even twenty, and the limits have been tightened. It is difficult to completely separate impurities with existing detection methods. Continuous improvement and optimization are needed to develop new analytical methods to improve the separation and detection capabilities of impurities in related substance detection. Currently, most chromatographic columns used for the detection of minocycline hydrochloride raw materials in China are octadecylsilane bonded silica gel columns (C18) or octylsilane bonded silica gel columns (C8), which are prone to peak broadening and tailing.

[0014] However, there is still a need in the art for new methods for detecting related substances in minocycline hydrochloride. For example, it is expected that this method can improve column efficiency, improve the separation effect between impurities, and have one or more beneficial effects such as higher flux and higher column efficiency of the chromatographic column, thereby providing a new and beneficial option for the development of methods for detecting related substances of antibiotics. Summary of the Invention

[0015] The object of the present invention is to provide a method for determining related substances in minocycline hydrochloride, and it is expected that the method can present one or more beneficial effects, such as accurate and reliable determination method, low labor protection cost, etc.

[0016] To this end, the first aspect of the invention provides a method for determining related substances in minocycline hydrochloride, comprising the following operations:

[0017] (1) Follow the HPLC method according to the specifications, and prepare each test solution freshly before use;

[0018] (2) Preparation of test solution:

[0019] Test solution: Take an appropriate amount of the test sample, dissolve it in water and dilute it to make a solution containing approximately 1 mg of minocycline per 1 ml;

[0020] Reference solution: Take an appropriate amount of minocycline hydrochloride reference substance, accurately weigh it, dissolve it in water and quantitatively dilute it to make a solution containing about 5 μg per 1 ml;

[0021] System suitability solution: Take an appropriate amount of minocycline hydrochloride system suitability reference substance, dissolve it in water and quantitatively dilute it to make a solution containing approximately 1 mg per 1 ml;

[0022] Sensitivity solution: Accurately measure an appropriate amount of reference solution and quantitatively dilute with water to make a solution containing approximately 0.5 μg per 1 ml;

[0023] (3) Chromatographic conditions:

[0024] A chromatographic column was filled with octadecylsilane bonded silica gel; mobile phase A was a mixture of 0.25 mol / L ammonium acetate solution and methanol (90:10) containing 0.002 mol / L disodium ethylenediaminetetraacetic acid (pH adjusted to 6.75±0.05 with aqueous ammonia or glacial acetic acid), and mobile phase B was acetonitrile-methanol (90:10). Elution was performed using a linear gradient according to the table below, with a flow rate of 1.0 ml / min. The column temperature was 35°C; the detection wavelength was 280 nm; the sample tray temperature was controlled at 2-8°C, and the injection volume was 10 μl.

[0025] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 12 92 8 22 92 8 42 85 15 50 75 25 60 75 25 60.1 95 5 70 95 5

[0026] System suitability requirements: In the system suitability solution chromatogram, the separation between the minocycline peak and the RS12 peak should be no less than 1.5; in the sensitivity solution chromatogram, the signal-to-noise ratio of the main peak should be no less than 10; after six consecutive injections of the reference solution, the relative standard deviation of the minocycline peak area should be no greater than 5.0%;

[0027] (4) Determine and calculate the percentage of impurities: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram. If there is an impurity peak corresponding to the relative retention time (RRT) listed in the table below in the chromatogram of the test solution, calculate the percentage of the impurity by the main component reference substance external standard method with the correction factor added.

[0028]

[0029]

[0030] According to the method described in the first aspect of the present invention, the specifications for the high performance liquid chromatography method are the specifications for the high performance liquid chromatography method included in previous editions of the Chinese Pharmacopoeia, the United States Pharmacopoeia, the European Pharmacopoeia, or the Japanese Pharmacopoeia, for example, the specifications for high performance liquid chromatography method in Part Four General Chapter 0512 of the 2020 edition of the Chinese Pharmacopoeia.

[0031] According to the method described in the first aspect of the present invention, the minocycline hydrochloride system suitability reference substance contains impurities RS02, RS16, RS32, RS03, RS12, RS15, RS11 and minocycline.

[0032] According to the method described in the first aspect of the present invention, the minocycline hydrochloride system suitability reference substance contains impurities RS02, RS16, RS32, RS03, RS12, RS15, RS11 and minocycline, and further contains impurities RS17, impurity RS28, impurity RS30, impurity RS33, and impurity RS40, whose structural formulas and / or chemical names are:

[0033] Impurity RS17:

[0034] (4S,4aS,5aR,12aS)-9-amino-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (9-aminosancycline),

[0035] Impurity RS28 or 9-aminominocycline:

[0036]

[0037] Impurity RS30 or monomethylethyl minocycline:

[0038]

[0039] Impurity RS33:

[0040] (4S,4aS,5aR,12aS)-7-amino-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (7-aminosancycline),

[0041] Impurity RS40 or Demeclocycline:

[0042]

[0043] According to the method of the first aspect of the present invention, the chromatographic column filled with octadecylsilane bonded silica gel is an Agilent InfinityLab Poroshell HPH-C18 column with a specification of 4.6 mm×150 mm and a diameter of 2.7 μm, or other chromatographic columns with equivalent performance.

[0044] According to the method described in the first aspect of the present invention, minocycline hydrochloride is considered qualified when it is detected that the minocycline hydrochloride contains impurities that meet the following limit requirements: RS03 shall not exceed 1.2%, RS32 shall not exceed 0.8%, RS02 shall not exceed 0.6%, RS11 shall not exceed 0.6%, RS12 shall not exceed 0.5%, RS15 shall not exceed 0.5%, RS16 shall not exceed 0.3%, other individual impurities shall not exceed 0.10%, the total amount of impurities (excluding RS03) shall not exceed 2.0%, and peaks smaller than the main peak area of the sensitivity solution (0.05%) shall be ignored.

[0045] According to the method of the first aspect of the present invention, the theoretical plate number of the minocycline peak is greater than 10,000, such as greater than 20,000, such as greater than 50,000.

[0046] According to the method described in the first aspect of the present invention, the minocycline hydrochloride system suitability reference substance contains impurities RS02, RS16, RS32, RS03, RS12, RS15, RS11 and minocycline, and the resolution between adjacent peaks is greater than 1.5.

[0047] According to the method described in the first aspect of the present invention, the minocycline hydrochloride system suitability reference substance contains impurities RS02, RS16, RS32, RS03, RS12, RS15, RS11 and minocycline, and also contains impurities RS17, impurity RS28, impurity RS30, impurity RS33, and impurity RS40, and the separation between adjacent peaks is greater than 1.5.

[0048] According to the method described in the first aspect of the present invention, minocycline hydrochloride is considered qualified when it is detected that the minocycline hydrochloride contains impurities that meet the following limit requirements: impurity RS17 shall not exceed 0.2%, for example, not exceed 0.10%, impurity RS28 shall not exceed 0.2%, for example, not exceed 0.10%, impurity RS30 shall not exceed 0.2%, for example, not exceed 0.10%, impurity RS33 shall not exceed 0.2%, for example, not exceed 0.10%, and impurity RS40 shall not exceed 0.2%, for example, not exceed 0.10%.

[0049] According to the method of the first aspect of the present invention, the separation degree between minocycline and adjacent impurities in the chromatogram of the test solution is greater than 2.0

[0050] Furthermore, the second aspect of the present invention provides minocycline hydrochloride, which is determined by the method described in any embodiment of the first aspect of the present invention for related substances, and impurities with the following limits are detected in the minocycline hydrochloride: no more than 1.2% of RS03, no more than 0.8% of RS32, no more than 0.6% of RS02, no more than 0.6% of RS11, no more than 0.5% of RS12, no more than 0.5% of RS15, no more than 0.3% of RS16, no more than 0.10% of other individual impurities, no more than 2.0% of the total amount of impurities (excluding RS03), and peaks smaller than the main peak area of the sensitivity solution (0.05%) are negligible.

[0051] According to the minocycline hydrochloride of the second aspect of the present invention, the related substances therein are determined using the method described in any embodiment of the first aspect of the present invention. Impurities with the following limits are detected in the minocycline hydrochloride: not more than 0.2%, for example, not more than 0.10% of impurity RS17, not more than 0.2%, for example, not more than 0.10% of impurity RS28, not more than 0.2%, for example, not more than 0.1% of impurity RS30, not more than 0.2%, for example, not more than 0.10% of impurity RS33, and not more than 0.2%, for example, not more than 0.10% of impurity RS40.

[0052] The method of the present invention has achieved several excellent results. For example, it can separate a large number of impurities and effectively separate and detect 12 specific impurities, with separations greater than 1.5 between each impurity and between the impurities and the main peak. It overcomes the problems of poor separation between impurities, low recovery, and poor solution stability. The method utilizes a specific liquid chromatography column to improve the separation between unknown impurities, overcoming the problem of poor separation of individual unknown impurities leading to combined integration and difficulty in tightening the limit for a single impurity. The limit for a single impurity can be tightened from 0.15% to 0.10%. The mobile phase of the method of the present invention does not contain highly toxic substances such as N,N-dimethylformamide and tetrahydrofuran, making it environmentally friendly and reducing harm to the human body. It also improves the stability of the instrument and chromatographic system, resulting in high safety, ease of operation, and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 : Typical HPLC profile of a system suitability solution containing seven typical impurities and minocycline.

[0054] Figure 2 : Typical chromatogram and integration data of an impurity mixture containing 12 impurities and minocycline.

[0055] Figure 3 : Local details of a typical chromatogram of an impurity mixture containing 12 impurities and minocycline during the period of 35 to 55 minutes.

[0056] Figure 4 : Typical chromatogram of a mixture containing 12 impurities and low concentration of minocycline.

[0057] Figure 5 : Typical need testing solution HPLC diagrams of three batches of samples measured using the method of Example 1 of the present invention are respectively Figure 5 A. Figure 5 B. Figure 5 C. DETAILED DESCRIPTION

[0058] The following provides some specific examples to further illustrate the present invention. Unless otherwise specified, the various reagents and instruments used in these examples are easily available on the market.

[0059] Example 1: Method for Determining Related Substances in Minocycline Hydrochloride

[0060] (1) The test solution was prepared freshly according to the specification of 0512 High Performance Liquid Chromatography in the 2020 edition of the Chinese Pharmacopoeia.

[0061] (2) Preparation of test solution:

[0062] Test solution: Take an appropriate amount of the test sample, dissolve it in water and dilute it to make a solution containing approximately 1 mg of minocycline per 1 ml;

[0063] Reference solution: Take an appropriate amount of minocycline hydrochloride reference substance, accurately weigh it, dissolve it in water and quantitatively dilute it to make a solution containing about 5 μg per 1 ml;

[0064] System suitability solution: Dissolve an appropriate amount of minocycline hydrochloride system suitability reference substance (compliant with EP11.0, containing RS02, RS16, RS32, RS03, RS12, RS15, RS11, and minocycline) in water and quantitatively dilute to a solution containing approximately 1 mg per 1 ml.

[0065] Sensitivity solution: Accurately measure an appropriate amount of reference solution and quantitatively dilute with water to make a solution containing approximately 0.5 μg per 1 ml;

[0066] (3) Chromatographic conditions:

[0067] A chromatographic column filled with octadecylsilane bonded silica gel (an Agilent InfinityLab Poroshell HPH-C18 column, 4.6 mm × 150 mm, 2.7 μm, was used in this example; other chromatographic columns with comparable performance may also be used) was used. Mobile phase A was a mixture of 0.25 mol / L ammonium acetate solution and methanol (90:10) (pH adjusted to 6.75±0.05 with aqueous ammonia or glacial acetic acid) containing 0.002 mol / L disodium ethylenediaminetetraacetic acid, and mobile phase B was acetonitrile-methanol (90:10). A linear gradient elution was performed according to the table below (Table 1) at a flow rate of 1.0 ml / min. The column temperature was 35°C, the detection wavelength was 280 nm, the sample tray was temperature-controlled at 2-8°C, and the injection volume was 10 μl.

[0068] Table 1:

[0069] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 12 92 8 22 92 8 42 85 15 50 75 25 60 75 25 60.1 95 5 70 95 5

[0070] System suitability requirements: In the system suitability solution chromatogram, the separation between the minocycline peak and the RS12 peak should be no less than 1.5; in the sensitivity solution chromatogram, the signal-to-noise ratio of the main peak should be no less than 10; after six consecutive injections of the reference solution, the relative standard deviation of the minocycline peak area should be no greater than 5.0%;

[0071] (4) Determine and calculate the percentage of impurities: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram. If there is an impurity peak corresponding to the relative retention time (RRT) listed in the following table (Table 2) in the chromatogram of the test solution, calculate the percentage of the impurity by the main component reference substance external standard method with the correction factor added.

[0072] Table 2:

[0073]

[0074] According to the present inventors' testing of some typical minocycline hydrochloride samples, the general limits of certain impurities can be generally determined. For example, according to the test and calculation results of this embodiment, if there are impurity peaks in the chromatogram of the test solution, the main component reference substance external standard method with the addition of a correction factor is used to calculate the content:

[0075] RS03 can generally be stipulated not to exceed 1.2%.

[0076] RS32 can generally be specified to not exceed 0.8%.

[0077] RS02 can generally be specified to be no more than 0.6%.

[0078] RS11 can generally be stipulated not to exceed 0.6%.

[0079] RS12 can generally be specified to be no more than 0.5%.

[0080] RS15 can generally be stipulated not to exceed 0.5%.

[0081] RS16 can generally be specified to be no more than 0.3%.

[0082] Other individual impurities can generally be specified to not exceed 0.10%, the total amount of impurities (except RS03) can generally be specified to not exceed 2.0%, and peaks smaller than the main peak area of the sensitivity solution (0.05%) can generally be specified to be ignored. When determining the quality standard of minocycline hydrochloride, the general limit results obtained above can also be used, that is, the following regulations can be adopted: RS03 shall not exceed 1.2%, RS32 shall not exceed 0.8%, RS02 shall not exceed 0.6%, RS11 shall not exceed 0.6%, RS12 shall not exceed 0.5%, RS15 shall not exceed 0.5%, RS16 shall not exceed 0.3%, other individual impurities shall not exceed 0.10%, the total amount of impurities (except RS03) shall not exceed 2.0%, and peaks smaller than the main peak area of the sensitivity solution (0.05%) can be ignored.

[0083] In this embodiment, the chemical names and chemical structural formulas of the impurities listed in Table 2 are as follows:

[0084] EP.A: (4R,4aS,5aR,12aS)-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (4-epiminocycline),

[0085] EP.B: (4S,4aS,5aR,12aS)-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydronaphthacene-2-carboxamide (sancycline),

[0086] EP.C: (4S,4aS,5aR,12aS)-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-7-(methylamino)-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (7-monodemethylminocycline),

[0087] EP.E: (4S,4aS,5aR,12aS)-4,7-bis(dimethylamino)-3,10,12a-trihydroxy-12-imino-1,11-dioxo-1,4,4a,5,5a,6,11,11a,12,12a-decahydronaphthacene-2-carboxamide,

[0088] EP.F: (4S,4aS,5aR,12aS)-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-N-(hydroxymethyl)-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydronaphthacene-2-carboxamide,

[0089] EP.G: (4S,4aS,5aR,12aS)-4,7,9-tris(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide,

[0090] EP.H: (4S,4aS,12aS)-4,7-bis(dimethylamino)-3,10,11,12a-tetrahydroxy-1,12-dioxo-1,4,4a,5,12,12a-hexahydronaphthacene-2-carboxamide,

[0091] In this embodiment, a typical HPLC chart of the system suitability solution is as follows: Figure 1 As shown, the retention time of the main component minocycline is approximately 45.5 minutes, and the separation between each impurity and the main component minocycline peak is greater than 1.5. For example, the separation between the main component minocycline peak and the nearest impurity RS12 peak is 2.3, and the separation between the remaining impurities is also greater than 1.5. This shows that the HPLC method of this example can be effectively used to determine typical impurities in minocycline hydrochloride, and the assay conditions have the advantage of being environmentally friendly.

[0092] Example 2: Determination of certain specific impurities in minocycline hydrochloride

[0093] It should be noted that in the method of Example 1 above, the green chromatographic conditions were used to effectively separate and quantify common impurities in minocycline hydrochloride, such as the seven impurities shown in Table 2 above. However, it is well known that, depending on the preparation method of the API and its physical and chemical properties, minocycline hydrochloride also contains other relatively common impurities, such as the following impurities RS17, RS28, RS30, RS33, and RS40:

[0094] Impurity RS17 or impurity EP.I:

[0095] (4S,4aS,5aR,12aS)-9-amino-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (9-aminosancycline),

[0096] Impurity RS28 or 9-aminominocycline:

[0097]

[0098] Impurity RS30 or monomethylethyl minocycline:

[0099]

[0100] Impurity RS33 or impurity EP.D:

[0101] (4S,4aS,5aR,12aS)-7-amino-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (7-aminosancycline),

[0102] Impurity RS40 or Demeclocycline:

[0103]

[0104] The present inventors have discovered that, using the method of Example 1, in addition to being able to simultaneously separate and determine the seven impurities described therein, the five impurities mentioned above can also be simultaneously determined, allowing 13 substances including the main component minocycline to be separated and determined under the same chromatographic conditions. The specific experiments are as follows.

[0105] Refer to Example 1; take an appropriate amount of impurity RS17, impurity RS28, impurity RS30, impurity RS33, and impurity RS40, dissolve them in water and dilute them to prepare a solution containing 0.2 to 1 mg of each impurity per 1 ml, as a stock solution, take the stock solution and the system suitability solution described in Example 1 at a volume ratio of 5:95 to obtain an impurity mixture, take the impurity mixture and inject it into the liquid chromatograph for measurement according to the method of Example 1, and record the chromatogram; In this experiment, a typical HPLC diagram of the impurity mixture is as follows Figure 2 As shown, the local details during the period of 35min to 55min are as follows Figure 3As shown, the retention time of the main component minocycline is approximately 45.5 minutes, and the separation between each impurity and the main component minocycline peak is greater than 1.5. For example, the separation between the main component minocycline peak and the nearest impurity RS12 peak is 2.3, and the separation between the remaining impurities is also greater than 1.5. In addition, the theoretical plate number of each peak is greater than 5000, especially the theoretical plate number of the main component reaches more than 140,000. This shows that the high-performance liquid chromatography method of Example 1 of the present invention can be effectively used to determine up to 12 typical impurities in minocycline hydrochloride.

[0106] In addition, refer to Example 1; take the 7 impurities of Example 1 and impurity RS17, impurity RS28, impurity RS30, impurity RS33, impurity RS40 and minocycline hydrochloride reference substance in appropriate amounts, dissolve them in water and dilute them to prepare a solution containing 1 to 10 μg of each impurity and minocycline per 1 ml, as the impurity mixed reference solution. The concentration of minocycline in this solution is low, which is convenient for identifying each peak and separation status; take the impurity mixed reference solution and inject it into the liquid chromatograph for measurement according to the method of Example 1, and record the chromatogram; In this experiment, a typical HPLC diagram of the impurity mixed reference solution is as follows Figure 4 As shown, the retention time of the main component minocycline is approximately 45.4 minutes, and the separation between each impurity and the main component minocycline peak is greater than 1.5. For example, the separation between the main component minocycline peak and the nearest impurity RS12 peak is 2.3, and the separation between the remaining impurities is also greater than 1.5. This shows that the HPLC method of Example 1 of the present invention can be effectively used to determine up to 12 typical impurities in minocycline hydrochloride.

[0107] Example 3: Performance evaluation of the method for determining related substances of minocycline hydrochloride

[0108] Examples 1 and 2 of the present invention demonstrate that this method can be effectively used to determine related substances in minocycline hydrochloride, and exhibits excellent results in one or more aspects. This Example 3 examines the methodological performance of Examples 1 and 2 using conventional pharmaceutical analysis methods known to those skilled in the art. The main results are summarized below.

[0109] 1. Verification results of specificity and system applicability

[0110] (1) The blank solution has no interference;

[0111] (2) The system applicability meets the requirements;

[0112] (3) In the spiked test solution, the minimum resolution between minocycline and adjacent impurities was 2.2;

[0113] (4) Under all forced degradation conditions, minocycline was well separated from all impurity peaks, with peak purity factors greater than 999 and mass balances within the range of 95% to 105%. The results of these four items all met the general quality testing requirements in this field.

[0114] 2. Verification results of limit of quantification

[0115] The prepared limit of quantitation solution was injected six times. The signal-to-noise ratio for RS02, RS03, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33, RS40, and minocycline was generally required to be no less than 10, and the peak area RSD was generally required to be no greater than 10.0%. The results showed that the limits of quantitation for RS02, RS03, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33, RS40, and minocycline were all within the range of 0.4 to 0.5 μg / ml. For example, the limit of quantitation for minocycline was 0.465 μg / ml, with signal-to-noise ratios greater than 10 and peak area RSDs less than 10.0%.

[0116] 3. Verification results of detection limit

[0117] The prepared detection limit solution was used for repeated injection three times, and the signal-to-noise ratio of RS02, RS03, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33, RS40 and minocycline was not less than 3. As a result, the detection limit concentrations of RS02, RS03, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33, RS40 and minocycline were all in the range of 0.21-0.26 μg / ml. For example, the quantification limit concentration of minocycline was measured to be 0.233 μg / ml, and the signal-to-noise ratios were all greater than 3.

[0118] 4. Linearity and range verification results

[0119] It was determined that the correlation coefficients for RS02, RS03, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33, and RS40 were greater than 0.9999 when they were in the range of 50% to 150% (relative to the limit concentration) and for minocycline when it was in the concentration range of 2.323 μg / ml to 6.970 μg / ml. The absolute value of the Y-axis intercept is generally required to be less than 10% of the response value of the 100% linear concentration level (the actual measured values were all less than 6.6%).

[0120] 5. Accuracy verification results

[0121] It was determined that the recoveries of RS02, RS03, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33, and RS40 within the range of 50% to 150% (relative to the limit concentration) were all between 90% and 110%, and the RSD values were all less than 5.0%; for example, the average recoveries of each impurity were all within the range of 92 to 103%.

[0122] 6. Precision verification results

[0123] In the repeatability test, analyst A prepared 6 test sample solutions in parallel and tested them according to the law: when comparing the 6 test results, the ranges of RS02, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33, RS40, and other largest single impurities were all less than 0.05%; the RSDs of RS03 and total impurities (excluding RS03) were 1.4% and 0.8%, respectively.

[0124] 6. Verification results of solution stability

[0125] The reference solution and the test solution were placed at 2-8°C for a period of time and then tested. The test results at each time point were compared with the results at 0 h. The results showed that (1) the reference solution was tested after being placed at 2-8°C for 22 h, and the RD value of the main peak area was less than 2.9% compared with that at 0 h; (2) the test solution was tested after being placed at 2-8°C for 54 h, and the absolute difference of the impurities less than 0.25% (RS02, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33, RS40, and other largest single impurities) was less than 0.05% compared with that at 0 h; the RD values of RS03 and total impurities (excluding RS03) were 9.8% and 8.3%, respectively. Unfortunately, it has been found that when ammonium acetate used in the mobile phase of the method of Example 1 is replaced with ammonium oxalate of the same concentration, the RD values of RS03 and total impurities (excluding RS03) are 21.2% and 16.8%, respectively, indicating that ammonium acetate should not be arbitrarily replaced.

[0126] 7. Durability verification results

[0127] The flow rate, column temperature and mobile phase pH value were changed respectively: (1) the system suitability met the requirements; (2) the absolute differences of the impurities less than 0.25% (RS02, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33, RS40 and other largest single impurities) under various conditions were less than 0.05%; the RD of RS03 and total impurities (excluding RS03) were 5.9% and 4.2%, respectively.

[0128] The above verification results are in full compliance with the general analytical method requirements.

[0129] Example 4: Selection of test methods and conditions

[0130] The methods for determining related substances in minocycline hydrochloride provided in Examples 1 and 2 were developed after tremendous creative effort. It has been found that the unique selection of chromatographic columns and mobile phases is essential for the successful separation of the 12 impurities. Some of the results that emerged were unforeseen by the prior art. The specific experiments are as follows. Referring to the method of Example 1, the only difference was that the following brands / models of chromatographic columns were used: Sunniest C18 column (4.6 mm × 250 mm, 5 μm), Inertsustain C8 column (4.6 × 250 mm, 5 μm), Waters Symmetey C18 column (4.6 × 250 mm, 5 μm), Agilent InfinityLab Poroshell 120Aq-C18 column (4.6 mm × 150 mm, 2.7 μm), Agilent InfinityLab Poroshell 120CS-C18 column (4.6 mm × 150 mm, 2.7 μm), Agilent InfinityLab Poroshell 120SB-C18 column (4.6 mm × 150 mm, 2.7 μm). As a result, all 12 impurities could not be effectively separated; specifically: Sunniest C18 column, Inertsustain C8 column and Waters Symmetey All three C18 columns could not effectively separate RS02, RS40 and RS16 (the resolution between adjacent peaks was less than 0.8), the RS12 peak was hidden inside the minocycline peak and could not be separated from the two, and the resolution between the RS15 peak and the minocycline peak was in the range of 0.9 to 1.1. For example, when using the Sunniest C18 column, the resolution between RS02 and RS40 was only 0.56, and the resolution between the RS15 peak and the minocycline peak was 1.03; all three Agilent InfinityLab Poroshell 120 columns could not separate RS02 from RS40 (the resolution was in the range of 0.7 to 1.0), and could not separate the RS12 peak from the minocycline peak (the resolution was in the range of 0.8 to 1.0). When using a 120SB-C18 column for separation, the resolution between RS02 and RS40 was 0.84, and the resolution between the RS12 peak and the minocycline peak was 0.92. It is well known that when using reversed-phase HPLC for separation and analysis of chemical substances, a resolution of 1.5 or greater between peaks is generally required. However, the present inventors found that even using an Agilent InfinityLab Poroshell column of the same brand as that used in Example 1 of the present invention, the separation effect was significantly inferior to that of the column in Example 1.Refer to the method of Example 1, except that the following four pharmacopoeia mobile phases were used and eluted at a flow rate of 1.0 ml / min: CP mobile phase: 0.2 M ammonium acetate-dimethylformamide-tetrahydrofuran (600:398:2, containing 0.01 mol / L disodium edetate), USP mobile phase: dimethylformamide-tetrahydrofuran-0.2 M ammonium oxalate-0.01 M disodium edetate (120:80:600:180, ammonium hydroxide adjusted to pH = 7.2), JP mobile phase: a mixture of ammonium oxalate monohydrate solution (7 g / 250 ml), dimethylformamide, 0.1 M disodium edetate (11:5:4) with tetrabutylammonium hydroxide adjusted to pH 6.5, EP mobile phase: dimethylformamide-tetrahydrofuran-solution A (volume ratio 12:8:78, solution A is a mixture of 18 volumes of 3.75 g / L sodium edetate solution and 60 volumes of 28.3 g / L ammonium oxalate solution and the pH is adjusted with ammonia 7.2); when the conditions of Example 1 of the present invention were used but the mobile phase was changed to the above-mentioned four isocratic elutions, the results showed that all 12 impurities could not be effectively separated; specifically: none of the four mobile phases could separate RS33 and RS17 (the two peaks overlapped), RS02, RS40, and RS16 peaks could not achieve acceptable separation (the separation between the two adjacent peaks was less than 0.7), and RS12, minocycline, and RS15 peaks could not achieve acceptable separation (the separation between the two adjacent peaks was less than 0.8 or RS12 was hidden in the minocycline peak). For example, when the CP mobile phase was used, the RS33 and RS17 peaks overlapped, the separation between the RS02 and RS40 peaks was 0.46, RS12 was hidden in the minocycline peak, and the separation between the minocycline peak and RS15 was 0.68; these results show that even if the chromatographic column of the present invention is used but the mobile phase of the prior art is used, the 12 impurities and minocycline cannot be separated. Refer to the relevant substance inspection method under the minocycline hydrochloride variety item of Part II of the Chinese Pharmacopoeia 2020 edition, but use the chromatographic column of Example 1 of the present invention to determine the present invention. Figure 2 When the test solution containing 12 impurities and minocycline is used, only 7 impurity peaks are shown that can achieve a separation degree of 1.5 or more from adjacent peaks; the method for detecting related substances under the minocycline hydrochloride variety in the Japanese Pharmacopoeia JP XVIII edition is referred to, but the chromatographic column of Example 1 of the present invention is used for determination. Figure 2 When the test solution containing 12 impurities and minocycline is used, only 5 impurity peaks are shown that can achieve a separation degree of 1.5 or more from adjacent peaks; the method for detecting related substances under the minocycline hydrochloride variety in European Pharmacopoeia 11.0 is referred to, but the chromatographic column of Example 1 of the present invention is used for determination. Figure 2When the test solution containing 12 impurities and minocycline was used, only 6 impurity peaks were shown to have a separation degree of 1.5 or more from adjacent peaks. In addition, some documents record that when determining related substances in minocycline hydrochloride, about 1% triethylamine was added to the mobile phase. The inventors also tried to add a small amount of triethylamine to the mobile phase to examine its separation effect, and found that the result was completely unacceptable. The specific experiment was carried out as follows: Referring to the methods of Example 1 and Example 2, the only difference was that the mobile phase A was a 0.25 mol / L ammonium acetate solution-methanol (90:10) mixture containing 0.002 mol / L disodium ethylenediaminetetraacetic acid and 1% triethylamine (the pH value was adjusted to 6.75±0.05 with aqueous ammonia or glacial acetic acid), and the others remained unchanged. In the test solution containing 12 impurities and minocycline, the separation degree of 1.5 or more was 1.5, and the separation degree of 1.5 was 1.5. Figure 2 When the impurity mixture was measured, both impurities R12 and R15 were hidden in the minocycline peak. The separation between impurities R02 and R40 was 0.36, and the separation between R40 and R16 was 0.53. The separation of the three impurities could not meet the general measurement requirements at all. Therefore, the addition of triethylamine in the mobile phase did not help to effectively separate and analyze the main component and the 12 impurities.

[0131] Example 5: Determination of some minocycline hydrochloride samples using the method of the present invention

[0132] In this example, the method of Example 1 of the present invention was used to determine the related substances in some minocycline hydrochloride samples (three batches, with batch numbers MN2102002B, MN2102003B, and MN2102003B). At the same time, the related substance determination methods under the same varieties of the 2020 edition of the Chinese Pharmacopoeia and the European Pharmacopoeia EP11.0 were used for determination and comparison. The results are as follows.

[0133] The results of the determination using the method of Example 1 of the present invention are shown in Table 3 below. The typical HPLC patterns of the test solutions of the three batches of samples are shown in Table 3. Figure 5 A. Figure 5 B. Figure 5 C.

[0134] Table 3:

[0135]

[0136]

[0137] All three batches of samples were found to contain 0.03-0.09% of impurities RS17, RS28, RS30, RS33, and RS40. For example, MN2102001B was also found to contain 0.04% of RS17, 0.09% of RS28, 0.03% of RS17, 0.06% of RS17, and 0.05% of RS17.

[0138] The method under the related substances of minocycline hydrochloride in European Pharmacopoeia EP11.0 was used for determination. The results are shown in Table 4 below.

[0139] Table 4:

[0140]

[0141] Impurities RS17, RS28, RS30, RS33, and RS40 of the present invention were not separated from the three batches of samples. These impurities may have merged with the peaks in the above table. Impurities A and H in the above table are significantly larger than RS03 and RS16 in Table 4. Some other impurities may be hidden in these two impurities.

[0142] The method under the related substances of minocycline hydrochloride in the 2020 edition of the Chinese Pharmacopoeia was used for determination, and the results are shown in Table 5 below.

[0143] Table 5:

[0144]

[0145]

[0146] As can be seen from the table, the other largest single impurities are significantly larger than those in Table 3, indicating that this largest single impurity may be a peak formed by multiple impurities, and impurities RS15, RS16, RS28, RS30, RS32, RS33, and RS40 cannot be identified.

[0147] Results from the present invention, particularly Example 1, demonstrate excellent specificity, detection and quantification limits, linearity and range, accuracy, precision, and solution stability. Specificity testing demonstrated that the blank solution did not interfere with the detection of target impurity peaks, and system suitability testing met requirements, demonstrating the method's excellent specificity. Stability testing revealed that after 22 hours at 2-8°C, the peak area of each target impurity remained less than 10%, demonstrating the analytical method's excellent stability. Method robustness testing demonstrated that the method exhibits selectivity for column temperature, mobile phase pH, and mobile phase ratio, indicating that these parameters should be maintained within a specific range during the analytical procedure. Validation results demonstrated that the analytical method can effectively detect up to 12 impurities in minocycline hydrochloride: RS02, RS03, RS11, RS12, RS15, RS16, RS17, RS28, RS30, RS32, RS33, and RS40.

[0148] Various aspects of the present invention have been described above. It should be understood that these examples are merely illustrative and the scope of protection of the present invention is not limited to such examples.

Claims

1. The method for determining related substances in minocycline hydrochloride comprises the following steps: (1) Perform the test according to the high performance liquid chromatography method, and prepare each test solution freshly before use; (2) Preparation of test solution: Test solution: Take an appropriate amount of the test sample, dissolve it in water and dilute it to make a solution containing 1 mg of minocycline per 1 ml; Reference solution: Take an appropriate amount of minocycline hydrochloride reference substance, accurately weigh it, dissolve it in water and quantitatively dilute it to make a solution containing 5 μg per 1 ml; System suitability solution: Take an appropriate amount of minocycline hydrochloride system suitability reference substance, dissolve it in water and quantitatively dilute it to make a solution containing 1 mg per 1 ml; The minocycline hydrochloride system suitability reference substance contains impurities RS02, impurity RS16, impurity RS32, impurity RS03, impurity RS12, impurity RS15, impurity RS11 and minocycline, and also contains impurities RS17, impurity RS28, impurity RS30, impurity RS33, and impurity RS40; Sensitivity solution: Accurately measure an appropriate amount of reference solution and quantitatively dilute with water to make a solution containing 0.5 μg per 1 ml; (3) Chromatographic conditions: Chromatographic columns using octadecylsilane bonded silica gel as filler; Mobile phase A is a mixture of 0.25 mol / L ammonium acetate solution containing 0.002 mol / L disodium edetate and methanol in a volume ratio of 90:

10. The pH of the mixture is adjusted to 6.75 ± 0.05 with aqueous ammonia or glacial acetic acid. Mobile phase B was a mixture of acetonitrile and methanol in a volume ratio of 90:10; Perform linear gradient elution according to the following table: ； The flow rate was 1.0 ml per minute, the column temperature was 35°C, the detection wavelength was 280 nm, the sample plate temperature was controlled at 2–8°C, and the injection volume was 10 μl; System suitability requirements: In the system suitability solution chromatogram, the separation degree of minocycline peak and RS12 peak is not less than 1.5; in the sensitivity solution chromatogram, the signal-to-noise ratio of the main peak is not less than 10; the relative standard deviation of the minocycline peak area after 6 consecutive injections of the reference solution is not greater than 5.0%; (4) Determine and calculate the percentage of impurities: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram. If there is an impurity peak corresponding to the relative retention time (RRT) listed in the table below in the chromatogram of the test solution, calculate the percentage of the impurity by the main component reference substance external standard method with the correction factor added. ； If there are impurity peaks corresponding to impurity RS17, impurity RS28, impurity RS30, impurity RS33, and impurity RS40 in the chromatogram of the test solution, calculate the percentage of the impurity according to the external standard method of the main component reference substance; The structural formulas of impurity RS17, impurity RS28, impurity RS30, impurity RS33, and impurity RS40 are: Impurity RS17: ， Impurity RS28: ， Impurity RS30: ， Impurity RS33: ， Impurity RS40: 。 2. according to the method for claim 1, wherein the chromatographic column with octadecylsilane bonded silica as filler is Agilent Infinity Lab Poroshell HPH-C18 post, and its specification is 4.6mm×150mm, 2.7 μm.

3. The method according to claim 1, wherein minocycline hydrochloride is considered qualified when it is detected that the minocycline hydrochloride contains impurities that meet the following limit regulations: impurity RS03 does not exceed 1.2%, impurity RS32 does not exceed 0.8%, impurity RS02 does not exceed 0.6%, impurity RS11 does not exceed 0.6%, impurity RS12 does not exceed 0.5%, impurity RS15 does not exceed 0.5%, impurity RS16 does not exceed 0.3%, impurity RS17 does not exceed 0.2%, impurity RS28 does not exceed 0.2%, impurity RS30 does not exceed 0.2%, impurity RS33 does not exceed 0.2%, impurity RS40 does not exceed 0.2%, other individual impurities shall not exceed 0.10%, the total amount of impurities other than RS03 shall not exceed 2.0%, and peaks smaller than the main peak area of the sensitivity solution shall be ignored.

4. The method according to claim 3, wherein minocycline hydrochloride is qualified when the minocycline hydrochloride is detected to contain impurities that meet the following limits: impurity RS17 does not exceed 0.1%, impurity RS28 does not exceed 0.1%, impurity RS30 does not exceed 0.1%, impurity RS33 does not exceed 0.1%, and impurity RS40 does not exceed 0.1%.

5. The method according to claim 1, wherein the theoretical plate number of the minocycline peak is greater than 10,000.

6. The method according to claim 1, wherein the theoretical plate number of the minocycline peak is greater than 20,000.

7. The method according to claim 1, wherein the theoretical plate number of the minocycline peak is greater than 50,000.

8. According to the method of claim 1, in the minocycline hydrochloride system suitability reference substance, the separation between adjacent peaks of impurity RS02, impurity RS16, impurity RS32, impurity RS03, impurity RS12, impurity RS15, impurity RS11 and minocycline, as well as impurity RS17, impurity RS28, impurity RS30, impurity RS33, and impurity RS40 are all greater than 1.

5.

9. The method according to claim 1, wherein the separation between minocycline and adjacent impurities in the chromatogram of the test solution is greater than 2.0.

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