Genotoxic impurity detection method, minocycline hydrochloride capsule thereof and preparation method of minocycline hydrochloride capsule

Abstract

The invention provides a genotoxic impurity detection method, a minocycline hydrochloride capsule and a preparation method of the minocycline hydrochloride capsule. The detection method is high performance liquid chromatography. The dibenzyl azodicarboxylate in the minocycline hydrochloride capsule is detected through high performance liquid chromatography, compared with a nuclear magnetic resonance method, the method is easier to implement, and a reliable basis is provided for quality control of the minocycline hydrochloride capsule.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and in particular relates to a method for detecting genotoxic impurities, a minocycline hydrochloride capsule and a preparation method thereof. Background Art

[0002] Minocycline hydrochloride is a semi-synthetic tetracycline-type broad-spectrum antibiotic developed in the early 1970s. It is highly effective and long-lasting. Its antibacterial spectrum is similar to that of tetracycline, and its antibacterial activity against Gram-positive bacteria is 2 to 4 times stronger than that of tetracycline, and its antibacterial activity against Gram-negative bacteria is basically the same as that of tetracycline. Among tetracycline antibiotics, minocycline has the strongest antibacterial effect and better antibacterial effect than other antibacterial drugs.

[0003] Genotoxic impurities (or genotoxic impurities, Genotoxic Impurity, GTI) refer to compounds that directly or indirectly damage cell DNA, cause gene mutations or in vivo mutagenesis, and have the potential or tendency to cause cancer.

[0004] As a reagent used in the synthesis of minocycline hydrochloride API, dibenzyl azodicarboxylate participates in the reaction of demethyltetracycline to generate 7-HT, which is a hydrazine compound with a potential genotoxic structure. Dibenzyl azodicarboxylate and its reduced products are both hydrazine compounds with a potential genotoxic structure. Therefore, detecting the content of this substance is an important factor in controlling product quality.

[0005] However, since dibenzyl azodicarboxylate is irritating, highly flammable, and relatively active towards acids, bases, alcohols, and water, the development of analytical methods has become more difficult. After searching, only reports on the determination of its content by nuclear magnetic resonance have been found. Summary of the invention

[0006] Therefore, the purpose of the present invention is to overcome the defects in the prior art and provide a method for detecting genotoxic impurities, a minocycline hydrochloride capsule and a preparation method thereof. The present invention detects dibenzyl azodicarboxylate in minocycline hydrochloride capsules by high performance liquid chromatography, which is easier to implement than nuclear magnetic resonance, and provides a reliable basis for quality control of minocycline hydrochloride capsules.

[0007] Before describing the present invention, the terms used in this article are defined as follows:

[0008] The term "Inertsil Ph-3" refers to: Shimadzu phenyl reversed phase chromatography column, which has phenyl groups directly bonded to silica gel, and the phenyl bonded phase provides π electron interactions between the phenyl groups and the analyte.

[0009] The term "Inersil ODS-3" refers to: Shimadzu C18 reverse phase chromatography column, whose filler is a versatile octadecyl group chemically modified with high purity spherical silica gel, and an ideal end-capping is implemented to provide stronger hydrophobic interactions.

[0010] The term "Waters Atlantis T3" refers to: Waters C18 reversed phase columns that utilize a trifunctional C18 alkyl bonded phase with a bonding density that improves retention of polar compounds and is 100% aqueous mobile phase compatible.

[0011] To achieve the above object, the first aspect of the present invention provides a method for detecting dibenzyl azodicarboxylate impurities in minocycline hydrochloride capsules, wherein the detection method is a high performance liquid chromatography method and preferably comprises the following steps:

[0012] (1) Select the chromatographic column, mobile phase, solvent and detector;

[0013] (2) Prepare the test solution and control solution respectively;

[0014] (3) according to the chromatographic column, mobile phase, solvent and detector selected in step (1), the sample tray temperature is set, and the test solution and the control solution prepared in step (2) are respectively injected into the high performance liquid chromatograph for chromatographic analysis to detect the dibenzyl azodicarboxylate impurity in the minocycline hydrochloride capsules to be tested.

[0015] According to the detection method of the first aspect of the present invention, in said step (1):

[0016] The chromatographic column is selected from one or more of the following: Inertsil Ph-3, Inersil ODS-3, Waters Atlantis T3, preferably Inertsil Ph-3 or Waters Atlantis T3, most preferably Inertsil Ph-3;

[0017] The mobile phase is phosphate solution and acetonitrile or phosphate solution and methanol, preferably phosphate solution and acetonitrile;

[0018] The solvent is selected from one or more of the following: a methanol aqueous solution, an acetonitrile aqueous solution, a mixed solution of acetonitrile and N,N-dimethylformamide; and / or

[0019] The detector is an ultraviolet detector.

[0020] According to the detection method of the first aspect of the present invention, in said step (1):

[0021] The flow rate of the mobile phase is 1.0 mL / min to 2.0 mL / min, preferably 1.0 mL / min to 1.5 mL / min, and most preferably 1.0 mL / min;

[0022] The column temperature of the chromatographic column is 25°C to 35°C, preferably 28°C to 32°C, most preferably 30°C; and / or

[0023] The detection wavelength of the detector is 205nm to 215nm, preferably 208nm to 212nm, and most preferably 210nm;

[0024] Preferably, in step (1):

[0025] The concentration of the phosphate solution is 0.005-0.02 mol / L, preferably 0.01-0.015 mol / L, most preferably 0.01 mol / L; and / or

[0026] The pH value of the phosphate solution is 2.5 to 6.8, preferably 2.8 to 6.7, and more preferably 3.0 to 6.6.

[0027] According to the detection method of the first aspect of the present invention, wherein, in the step (1), when the solvent is a mixed solution of acetonitrile and N,N-dimethylformamide:

[0028] In the mixed solution, the volume ratio of the acetonitrile to the N,N-dimethylformamide is 50-70:30-50, preferably 60-70:30-40, most preferably 60:40; and / or

[0029] The pH value of the mixed solution is adjusted with phosphoric acid to 2.0 to 5.0, preferably 3.0 to 4.5, and most preferably 4.0.

[0030] According to the detection method of the first aspect of the present invention, in said step (2):

[0031] The preparation method of the test solution comprises: taking the minocycline hydrochloride capsule to be tested, weighing it, placing it in a volumetric flask, adding the solvent to dissolve it and diluting it to the scale; and / or

[0032] The preparation method of the reference solution comprises: taking a reference substance of dibenzyl azodicarboxylate, adding the solvent to dissolve, quantify and dilute.

[0033] According to the detection method of the first aspect of the present invention, in said step (2):

[0034] The concentration of minocycline hydrochloride in the test solution is 1.0 mg / mL to 10.0 mg / mL, preferably 5.0 mg / mL to 10.0 mg / mL, most preferably 10.0 mg / mL; and / or

[0035] The concentration of dibenzyl azodicarboxylate in the control solution is 0.2 to 1.0 μg / mL, preferably 0.5 to 0.8 μg / mL, and more preferably 0.5 μg / mL.

[0036] According to the detection method of the first aspect of the present invention,

[0037] The step (2) also includes: preparing a sensitivity solution and injecting it into a high performance liquid chromatograph for chromatographic analysis;

[0038] Preferably, the preparation method of the sensitivity solution comprises: taking a dibenzyl azodicarboxylate reference substance, adding the solvent to dissolve and quantitatively dilute;

[0039] More preferably, the concentration of dibenzyl azodicarboxylate in the sensitivity solution is 0.05 to 0.2 μg / mL, preferably 0.1 to 0.15 μg / mL, most preferably 0.1 μg / mL; and / or

[0040] In the step (3):

[0041] The sample tray temperature is 5-15°C, preferably 6-10°C, and most preferably 8°C;

[0042] Preferably, when the mobile phase is phosphate solution and acetonitrile or phosphate solution and methanol, the chromatographic analysis comprises gradient elution, in which the initial volume ratio of the phosphate solution and acetonitrile or phosphate solution and methanol is preferably 60-80:20-40, more preferably 55-75:25-35, and most preferably 70:30.

[0043] According to the detection method of the first aspect of the present invention,

[0044] In the chromatogram of the control solution, the separation degree between the dibenzyl azodicarboxylate and its adjacent impurity peaks is greater than 2.0, preferably greater than 1.5, most preferably greater than 1.0; and / or

[0045] In the chromatogram of the sensitive solution, the signal-to-noise ratio of the dibenzyl azodicarboxylate peak is greater than 20, preferably greater than 15, and most preferably greater than 10.

[0046] The second aspect of the present invention provides a method for preparing minocycline hydrochloride capsules, the method comprising: detecting dibenzyl azodicarboxylate impurities by high performance liquid chromatography.

[0047] The third aspect of the present invention provides a minocycline hydrochloride capsule, wherein the minocycline hydrochloride capsule is:

[0048] Prepared by detecting the dibenzyl azodicarboxylate impurity in minocycline hydrochloride capsules using the method described in the first aspect; or

[0049] The method is described in the second aspect.

[0050] To achieve the above object, the technical solution of the present invention is as follows:

[0051] A method for detecting dibenzyl azodicarboxylate impurities in minocycline hydrochloride capsules by high performance liquid chromatography, the method comprising:

[0052] Prepare the test solution and the control solution, inject them into the high performance liquid chromatograph for chromatographic analysis, and analyze the dibenzyl azodicarboxylate in the test solution according to the chromatogram. Among them, the Inertsil Ph-3 column was selected, 0.01 mol / l phosphate solution was used as the mobile phase A, acetonitrile was used as the mobile phase B, acetonitrile-N,N-dimethylformamide 60:40 (adjust the pH value to 4.0 with phosphoric acid) was used as the solvent, and the ultraviolet detector was used as the detector.

[0053] In some specific embodiments, the elution procedure is a gradient elution.

[0054] In some specific embodiments, the mobile phase A is adjusted to a pH value of 2.8 to 3.2, preferably 3.0, with phosphoric acid; the column temperature is 28° C. to 32° C., preferably 30° C.; and the detection wavelength is 208 nm to 212 nm, preferably 210 nm.

[0055] In some specific embodiments, the test solution is prepared by the following method: take an appropriate amount of minocycline hydrochloride capsules (approximately equivalent to 500 mg of minocycline), accurately weigh, place in a 50 mL volumetric flask, add a solvent to dissolve and dilute to the scale.

[0056] In some specific embodiments, the reference solution is prepared by the following method: taking an appropriate amount of dibenzyl azodicarboxylate reference substance, dissolving it in a solvent, and quantitatively diluting it to prepare a 0.5 μg / mL solution.

[0057] In some specific embodiments, the method further comprises preparing a sensitivity solution and injecting the solution into a high performance liquid chromatograph for chromatographic analysis.

[0058] In some specific embodiments, the sensitivity solution is prepared by the following method: taking an appropriate amount of dibenzyl azodicarboxylate reference substance, dissolving it in a solvent, and quantitatively diluting it to prepare a 0.1 μg / mL solution.

[0059] In some specific embodiments, the method further includes system suitability requirements: in the chromatogram of the reference solution, the separation degree of dibenzyl azodicarboxylate and its adjacent impurity peaks before and after should be greater than 1.0; in the chromatogram of the sensitivity solution, the signal-to-noise ratio of the dibenzyl azodicarboxylate peak should be greater than 10.

[0060] The invention optimizes the types of chromatographic columns and mobile phases, such as adjusting an ODS chromatographic column to a T3 chromatographic column to enhance the retention capacity of polar compounds. Considering that dibenzyl azodicarboxylate contains an aromatic ring, an Inertsil Ph-3 chromatographic column (a chromatographic column with phenyl groups directly bonded to silica gel) is used to provide complementary separation selectivity to examine the separation of aromatic ring compounds and reduce the interference of other chromatographic peaks based on the screening of ODS and T3 chromatographic column fillers; the mobile phase A is adjusted from an acetate buffer to a phosphate buffer to enhance the elution capacity of the target peak; the mobile phase B is adjusted from methanol to aprotic acetonitrile, and the difference in separation selection is used to optimize the peak shape, thereby solving the problems of many impurity peaks and tailing of the peak shape, and screening out chromatographic conditions with sharp peak shape and no tailing.

[0061] Dibenzyl azodicarboxylate is unstable in the presence of acid, alkali, aqueous solution and metal ions. By screening the solvent and controlling the temperature of the sample injection plate at 8°C, the instability of this impurity in the sample was solved, allowing dibenzyl azodicarboxylate in minocycline hydrochloride capsules to be accurately detected.

[0062] In addition, the reported nuclear magnetic resonance method for the determination of dibenzyl azodicarboxylate uses chloroform as solvent and N,N-dimethylformamide as internal standard. This HPLC method uses N,N-dimethylformamide as solvent, but does not use substances more toxic than chloroform, which reduces the risk of testers being exposed to toxic substances to a certain extent.

[0063] Compared with the prior art, the method for detecting dibenzyl azodicarboxylate impurities in minocycline hydrochloride capsules of the present invention can have but is not limited to the following beneficial effects:

[0064] Compared with the nuclear magnetic resonance method in the prior art, the high performance liquid chromatography method established in the present invention is easier to implement and has higher sensitivity in operation for detecting dibenzyl azodicarboxylate in minocycline hydrochloride capsules, thus providing a reliable guarantee for the quality control of minocycline hydrochloride capsules. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0066] Figure 1 The chromatogram of the reference solution of Example 1 (also the system suitability chromatogram) is shown.

[0067] Figure 2 The chromatogram of the test solution of Example 1 is shown.

[0068] Figure 3 The sensitivity solution chromatogram of Example 1 is shown.

[0069] Figure 4 A comparison diagram of the interference test of Example 1 is shown.

[0070] Figure 5 The linear relationship graph of dibenzyl azodicarboxylate of Example 1 is shown.

[0071] Figure 6 A typical chromatogram of the spiked test product of Example 1 is shown.

[0072] Figure 7 The results of the detection of dibenzyl azodicarboxylate impurities in a batch of minocycline hydrochloride samples in Example 2 are shown.

[0073] Figure 8 The results of detecting the dibenzyl azodicarboxylate impurity in two batches of minocycline hydrochloride samples in Example 2 are shown.

[0074] Fig. 9 The results of detecting the dibenzyl azodicarboxylate impurity in three batches of minocycline hydrochloride samples in Example 2 are shown. DETAILED DESCRIPTION

[0075] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed and specific description and should not be understood as limiting the present invention in any form.

[0076] This section generally describes the materials and test methods used in the experiments of the present invention. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described as detailed as possible. It is clear to those skilled in the art that in the context, if not specifically stated, the materials and operating methods used in the present invention are well known in the art.

[0077] The reagents and instruments used in the following examples are as follows:

[0078] Reagents:

[0079] Potassium dihydrogen phosphate was purchased from Guangdong Guanghua Science and Technology Co., Ltd.; phosphoric acid was purchased from Aladdin Reagent (Shanghai) Co., Ltd.; acetonitrile was purchased from Merck KGaA; dibenzyl azodicarboxylate was purchased from Zhuhai Yourun Pharmaceutical Technology Co., Ltd.; minocycline hydrochloride capsules were purchased from Haikou Pharmaceutical Factory Co., Ltd.

[0080] instrument:

[0081] High performance liquid chromatography was purchased from Waters Technology (Shanghai) Co., Ltd., model e2695.

[0082] High performance liquid chromatography (HPLC) was purchased from Thermo Fisher Scientific, model U3000.

[0083] pH meter, purchased from Mettler-Toledo, model S220.

[0084] The 1 / 10,000 electronic balance was purchased from Ohaus Instruments (Shanghai) Co., Ltd., model PWN224ZH.

[0085] The 1 / 100,000 electronic balance was purchased from Mettler-Toledo, model XS205DU.

[0086] The one-millionth electronic balance was purchased from Mettler-Toledo, model XP6.

[0087] Example 1

[0088] This example is an exemplary description of the detection method and method verification of dibenzyl azodicarboxylate impurities in minocycline hydrochloride capsules of the present invention.

[0089] 1. Chromatographic conditions

[0090] Column: Inertsil Ph-3, 4.6 mm × 250 mm, 3 μm

[0091] Mobile phase: 0.01 mol / L phosphate buffer (1.36 g potassium dihydrogen phosphate, add 1000 mL water to dissolve, and adjust the pH value to 3.0 with phosphoric acid) as mobile phase A; acetonitrile as mobile phase B.

[0092] The elution procedure was gradient elution, and the ratio of mobile phase A to mobile phase B was as follows:

[0093] Table 1 Ratio of mobile phase A and mobile phase B in gradient elution of Example 1

[0094] Time (min) Mobile phase A (%) Mobile phase B (%) 0 70 30 25 10 90 25.1 70 30 30 70 30

[0095] Solvent: Acetonitrile-N,N-dimethylformamide (60:40), pH adjusted to 4.0 with phosphoric acid

[0096] Flow rate: 1.0mL / min

[0097] Column temperature: 30°C

[0098] Wavelength: 210nm

[0099] Injection volume: 20 μl

[0100] Sample plate temperature: 8°C

[0101] Detector: UV detector

[0102] 2. Determination steps

[0103] (1) Solution preparation

[0104] ① Test solution: Take 1333±0.01 mg of the contents of minocycline hydrochloride capsules (equivalent to 500 mg±0.01 mg of minocycline hydrochloride), accurately weigh, place in a 50 mL volumetric flask, add the above solvent to dissolve and dilute to the scale.

[0105] ② Reference solution: Take 5±0.001 mg of dibenzyl azodicarboxylate reference substance, dissolve it in the above solvent and quantitatively dilute it to make a 0.5 μg / mL solution.

[0106] ③Sensitivity solution: Take 5±0.001 mg of dibenzyl azodicarboxylate reference substance, dissolve it in the above solvent and quantitatively dilute it to make a 0.1 μg / mL solution.

[0107] (2) Take 20uL of the reference solution, sensitivity solution and test solution respectively, inject them into the liquid chromatograph and record the chromatogram. The chromatogram of the reference solution is as follows: Figure 1 As shown, the chromatogram of the test solution is Figure 2 As shown, the sensitivity solution chromatogram is as follows Figure 3 shown.

[0108] (3) Calculation method: External standard method. The impurity content is calculated by the following formula (a).

[0109]

[0110] Among them: A 样 A is the peak area of ​​dibenzyl azodicarboxylate in the test solution; 对 is the peak area of ​​the reference solution; N 样 N is the dilution factor of the test solution; 对 W is the dilution multiple of the reference solution; 样 is the sample weight of the test sample, mg; W 对 is the sample weight of the reference substance, mg; M is the average loading amount of the test substance, mg; L is the specification of the test substance; P is the content of the reference substance, %.

[0111] 3. System suitability requirements: In the chromatogram of the reference solution, the separation between dibenzyl azodicarboxylate and its adjacent impurity peaks should be greater than 1.0; in the chromatogram of the sensitivity solution, the signal-to-noise ratio of the dibenzyl azodicarboxylate peak should be greater than 10.

[0112] 4. Method Validation

[0113] ① Interference test: Blank solvent, blank excipient and minocycline main component peak have no interference with dibenzyl azodicarboxylate peak. Figure 4 .

[0114] ② System suitability test: In the chromatogram of the reference solution, the separation degrees of dibenzyl azodicarboxylate and its adjacent impurity peaks were 1.61, 2.31, 2.23, 2.26, 2.35, and 2.23, respectively, and the separation degrees of dibenzyl azodicarboxylate and its adjacent impurity peaks were 7.78, 7.97, 7.19, 8.10, 8.19, and 2.67, respectively; the reference solution was injected 6 times continuously, and the RSD was 0.06%; in the chromatogram of the sensitivity solution, the signal-to-noise ratio of dibenzyl azodicarboxylate was 31.0. See Table 2 for details, and the typical spectrum of the system suitability test is shown in Figure 1 .

[0115] ③ Linearity: Take dibenzyl azodicarboxylate reference substance and prepare solutions containing about 2.2ng, 0.25μg, 0.4μg, 0.5μg, 0.75μg, and 1.0μg of dibenzyl azodicarboxylate per 1mL, respectively. Determine according to the above chromatographic method, and use concentration (C) as the abscissa and peak area (A) as the ordinate for linear regression. The regression equation is: y=1.0638x-0.0261; r=0.9999; the y-axis intercept is 4.71 at 100% response value; the linear range is 0.1091μg / mL~1.0906μg / mL. For details, see Figure 5 And Table 3.

[0116] ④ Detection limit and quantification limit: The detection limit and quantification limit of dibenzyl azodicarboxylate are 1.1 ng and 2.2 ng respectively. See Table 4 for details.

[0117] ⑤ Solution stability study: Take the reference solution and the test solution and place them at a low temperature of 8°C for 2 hours. The recovery rates of the reference solution and the test solution are both beyond the range of 80% to 120%, indicating that the solution is unstable. Therefore, the solution used in the method described in this embodiment should be freshly prepared before use. See Table 5 for details.

[0118] ⑥Precision investigation: Repeatability: The average content of 6 spiked test solutions was 50.3ppm, and the RSD was 1.79% (<10.0%). Intermediate precision: The average content of 6 spiked test solutions was 51.6ppm, and the RSD was 6.75% (<10.0%); the RSD of the content of 12 spiked test solutions was 4.95% (<15.0%). See Table 6 for details.

[0119] ⑦ Accuracy test: The recovery rate of the test solution ranged from 85.60% to 103.18%, with an average recovery rate of 94.7% and an RSD of 5.88%. See Table 7 for details. The typical chromatogram of the spiked test solution is shown in Figure 6 .

[0120] ⑧ When the wavelength (±2nm), column temperature (±2℃), and phosphate pH value (±0.2) in the chromatographic conditions were slightly changed, the system suitability met the requirements, and the recovery rate of the peak area of ​​dibenzyl azodicarboxylate in the test solution was within the range of 90.88% to 97.85%. See Table 8 for details.

[0121] Table 2 System suitability test results

[0122]

[0123] Table 3 Linear relationship test results of dibenzyl azodicarboxylate

[0124]

[0125]

[0126] Table 4 Detection limit and quantification limit test results

[0127]

[0128] Table 6 Precision test results

[0129]

[0130]

[0131] Table 7 Dibenzyl azodicarboxylate recovery test results

[0132]

[0133] Table 8 Durability test results

[0134]

[0135] Example 2

[0136] This example is used to illustrate the effect of the method for detecting dibenzyl azodicarboxylate impurities in minocycline hydrochloride capsules of the present invention.

[0137] According to the method described in Example 1, three batches of minocycline hydrochloride samples prepared by the applicant's unit were tested for dibenzyl azodicarboxylate impurities, and the results were all undetectable. Figure 7 to Figure 9 As shown, this shows that the minocycline hydrochloride drug synthesis process of the applicant's unit has ideal product quality control and does not contain potential genotoxic impurities.

[0138] In Examples 1-2, the detection method of the present invention was repeated on multiple batches of samples, and no possible dibenzyl azodicarboxylate impurity was detected, thereby proving that the minocycline hydrochloride production process of the applicant's unit is safe and stable.

[0139] Example 3

[0140] This example is another exemplary description of the method for detecting dibenzyl azodicarboxylate impurities in minocycline hydrochloride capsules of the present invention.

[0141] 1. Chromatographic conditions

[0142] Chromatographic column: Inersil ODS-3, 4.6mm×250mm, 5μm

[0143] Mobile phase: 0.01 mol / L phosphate solution (take 1.36 g of potassium dihydrogen phosphate, add 1000 mL of water to dissolve, and adjust the pH value to 6.6 with potassium hydroxide) as mobile phase A; methanol is used as mobile phase B.

[0144] Table 9 Ratio of mobile phase A and mobile phase B in gradient elution of Example 3

[0145] Time (min) Mobile phase A (%) Mobile phase B (%) 0 70 30 25 10 90 25.1 70 30 30 70 30

[0146] Solvent: methanol aqueous solution (methanol: water = 80:20)

[0147] Flow rate: 1.0mL / min

[0148] Column temperature: 30°C

[0149] Wavelength: 210nm

[0150] Injection volume: 20 μl

[0151] Sample plate temperature: 8°C

[0152] Detector: UV detector

[0153] 2. Determination steps

[0154] (1) Solution preparation

[0155] ① Test solution: Take 1333±0.01 mg of the contents of minocycline hydrochloride capsules (equivalent to 250 mg±0.01 mg of minocycline hydrochloride), accurately weigh, place in a 50 mL volumetric flask, add the above solvent to dissolve and dilute to the scale.

[0156] ② Reference solution: Take 2.5±0.001 mg of dibenzyl azodicarboxylate reference substance, dissolve it in the above solvent and quantitatively dilute it to make a 0.25 μg / mL solution.

[0157] ③ Sensitivity solution: Take 2.5±0.001 mg of dibenzyl azodicarboxylate reference substance, dissolve it in the above solvent and quantitatively dilute it to make a 0.05 μg / mL solution.

[0158] (2) Take 20uL of the reference solution, sensitivity solution and test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0159] (3) Calculation method: External standard method. The impurity content is calculated by the following formula (a).

[0160]

[0161] Among them: A 样 A is the peak area of ​​dibenzyl azodicarboxylate in the test solution; 对 is the peak area of ​​the reference solution; N 样 N is the dilution factor of the test solution; 对 W is the dilution multiple of the reference solution; 样 is the sample weight of the test sample, mg; W 对 is the sample weight of the reference substance, mg; M is the average loading amount of the test substance, mg; L is the specification of the test substance; P is the content of the reference substance, %.

[0162] 3. System suitability requirements: In the chromatogram of the reference solution, the separation between dibenzyl azodicarboxylate and its adjacent impurity peaks should be greater than 1.0; in the chromatogram of the sensitivity solution, the signal-to-noise ratio of the dibenzyl azodicarboxylate peak should be greater than 10.

[0163] Example 4

[0164] This example is another exemplary description of the method for detecting dibenzyl azodicarboxylate impurities in minocycline hydrochloride capsules of the present invention.

[0165] 1. Chromatographic conditions

[0166] Column: Waters Atlantis T3, 4.6 mm × 250 mm, 5 μm

[0167] Mobile phase: 0.01 mol / L phosphate solution (take 1.36 g of potassium dihydrogen phosphate, add 1000 mL of water to dissolve, and adjust the pH value to 6.6 with potassium hydroxide) as mobile phase A; acetonitrile as mobile phase B.

[0168] Table 10 Ratio of mobile phase A and mobile phase B in gradient elution of Example 4

[0169] Time (min) Mobile phase A (%) Mobile phase B (%) 0 70 30 25 10 90 25.1 70 30 30 70 30

[0170] Solvent: 50% acetonitrile aqueous solution (acetonitrile: water = 50:50)

[0171] Flow rate: 1.0mL / min

[0172] Column temperature: 30°C

[0173] Wavelength: 210nm

[0174] Injection volume: 20 μl

[0175] Sample plate temperature: 8°C

[0176] Detector: UV detector

[0177] 2. Determination steps

[0178] (1) Solution preparation

[0179] ① Test solution: Take 1333±0.01 mg of the contents of minocycline hydrochloride capsules (equivalent to 400 mg±0.01 mg of minocycline hydrochloride), accurately weigh, place in a 50 mL volumetric flask, add the above solvent to dissolve and dilute to the scale.

[0180] ② Reference solution: Take 4.0±0.001 mg of dibenzyl azodicarboxylate reference substance, dissolve it in the above solvent and quantitatively dilute it to make a 0.4 μg / mL solution.

[0181] ③ Sensitivity solution: Take 4.0±0.001 mg of dibenzyl azodicarboxylate reference substance, dissolve it in the above solvent and quantitatively dilute it to make a 0.08 μg / mL solution.

[0182] (2) Take 20uL of the reference solution, sensitivity solution and test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0183] (3) Calculation method: External standard method. The impurity content is calculated by the following formula (a).

[0184]

[0185] Among them: A 样 A is the peak area of ​​dibenzyl azodicarboxylate in the test solution; 对 is the peak area of ​​the reference solution; N 样 N is the dilution factor of the test solution; 对 W is the dilution multiple of the reference solution; 样 is the sample weight of the test sample, mg; W 对 is the sample weight of the reference substance, mg; M is the average loading amount of the test substance, mg; L is the specification of the test substance; P is the content of the reference substance, %.

[0186] 3. System suitability requirements: In the chromatogram of the reference solution, the separation between dibenzyl azodicarboxylate and its adjacent impurity peaks should be greater than 1.0; in the chromatogram of the sensitivity solution, the signal-to-noise ratio of the dibenzyl azodicarboxylate peak should be greater than 10.

[0187] According to the detection method of Examples 1-2, the dibenzyl azodicarboxylate reference substances of Examples 3-4 can all be detected, but not detected in the minocycline hydrochloride capsule samples.

[0188] Although the effects of some embodiments are shown above, those skilled in the art should understand that according to the concept of the present invention, the aforementioned other embodiments without specific effects or other technical solutions of the present invention not shown in the embodiments can also achieve the following technical effects stated in the invention content section that are equivalent to the embodiments:

[0189] Compared with the nuclear magnetic resonance method in the prior art, the high performance liquid chromatography method established in the present invention is easier to implement and has higher sensitivity in operation for detecting dibenzyl azodicarboxylate in minocycline hydrochloride capsules, thus providing a reliable guarantee for the quality control of minocycline hydrochloride capsules.

[0190] Although the present invention has been described to a certain extent, it is obvious that appropriate changes in various conditions can be made without departing from the spirit and scope of the present invention. It is understood that the present invention is not limited to the embodiments described, but belongs to the scope of the claims, which includes equivalent replacements of each factor described.

Claims

1. A method for detecting dibenzyl azodicarboxylate impurities in minocycline hydrochloride capsules, characterized in that: The detection method is a high performance liquid chromatography method, and preferably comprises the following steps: (1) Select the chromatographic column, mobile phase, solvent and detector; (2) Prepare test solution and control solution respectively; (3) according to the chromatographic column, mobile phase, solvent and detector selected in step (1), the sample tray temperature is set, and the test solution and the control solution prepared in step (2) are respectively injected into the high performance liquid chromatograph for chromatographic analysis to detect the dibenzyl azodicarboxylate impurity in the minocycline hydrochloride capsules to be tested.

2. The detection method according to claim 1, characterized in that: In the step (1): The chromatographic column is selected from one or more of the following: Inertsil Ph-3, Inersil ODS-3, Waters Atlantis T3, preferably Inertsil Ph-3 or Waters Atlantis T3, most preferably Inertsil Ph-3; The mobile phase is phosphate solution and acetonitrile or phosphate solution and methanol, preferably phosphate solution and acetonitrile; The solvent is selected from one or more of the following: a methanol aqueous solution, an acetonitrile aqueous solution, a mixed solution of acetonitrile and N,N-dimethylformamide; and / or The detector is an ultraviolet detector.

3. The detection method according to claim 1 or 2, characterized in that: In the step (1): The flow rate of the mobile phase is 1.0 mL / min to 2.0 mL / min, preferably 1.0 mL / min to 1.5 mL / min, and most preferably 1.0 mL / min; The column temperature of the chromatographic column is 25°C to 35°C, preferably 28°C to 32°C, most preferably 30°C; and / or The detection wavelength of the detector is 205nm to 215nm, preferably 208nm to 212nm, and most preferably 210nm; Preferably, in step (1): The concentration of the phosphate solution is 0.005-0.02 mol / L, preferably 0.01-0.015 mol / L, most preferably 0.01 mol / L; and / or The pH value of the phosphate solution is 2.5 to 6.8, preferably 2.8 to 6.7, and more preferably 3.0 to 6.

6.

4. The detection method according to claim 2 or 3, characterized in that: In the step (1), when the solvent is a mixed solution of acetonitrile and N,N-dimethylformamide: In the mixed solution, the volume ratio of the acetonitrile to the N,N-dimethylformamide is 50-70:30-50, preferably 60-70:30-40, most preferably 60:40; and / or The pH value of the mixed solution is adjusted with phosphoric acid to 2.0 to 5.0, preferably 3.0 to 4.5, and most preferably 4.

0.

5. The detection method according to any one of claims 1 to 4, characterized in that: In the step (2): The preparation method of the test solution comprises: taking the minocycline hydrochloride capsule to be tested, weighing it, placing it in a volumetric flask, adding the solvent to dissolve it and diluting it to the scale; and / or The preparation method of the reference solution comprises: taking a reference substance of dibenzyl azodicarboxylate, adding the solvent to dissolve, quantify and dilute.

6. The detection method according to claim 5, characterized in that: In the step (2): The concentration of minocycline hydrochloride in the test solution is 1.0 mg / mL to 10.0 mg / mL, preferably 5.0 mg / mL to 10.0 mg / mL, most preferably 10.0 mg / mL; and / or The concentration of dibenzyl azodicarboxylate in the control solution is 0.2 to 1.0 μg / mL, preferably 0.5 to 0.8 μg / mL, and more preferably 0.5 μg / mL.

7. The detection method according to any one of claims 1 to 6, characterized in that: The step (2) also includes: preparing a sensitivity solution and injecting it into a high performance liquid chromatograph for chromatographic analysis; Preferably, the preparation method of the sensitivity solution comprises: taking a dibenzyl azodicarboxylate reference substance, adding the solvent to dissolve and quantitatively dilute; More preferably, the concentration of dibenzyl azodicarboxylate in the sensitivity solution is 0.05 to 0.2 μg / mL, preferably 0.1 to 0.15 μg / mL, most preferably 0.1 μg / mL; and / or In the step (3): The sample tray temperature is 5-15°C, preferably 6-10°C, and most preferably 8°C; Preferably, when the mobile phase is phosphate solution and acetonitrile or phosphate solution and methanol, the chromatographic analysis comprises gradient elution, in which the initial volume ratio of the phosphate solution and acetonitrile or phosphate solution and methanol is preferably 60-80:20-40, more preferably 55-75:25-35, and most preferably 70:

30.

8. The detection method according to claim 7, characterized in that: In the chromatogram of the control solution, the separation degree between the dibenzyl azodicarboxylate and its adjacent impurity peaks is greater than 2.0, preferably greater than 1.5, most preferably greater than 1.0; and / or In the chromatogram of the sensitive solution, the signal-to-noise ratio of the dibenzyl azodicarboxylate peak is greater than 20, preferably greater than 15, and most preferably greater than 10.

9. A method for preparing minocycline hydrochloride capsules, characterized in that: The preparation method comprises: detecting dibenzyl azodicarboxylate impurities by high performance liquid chromatography.

10. A minocycline hydrochloride capsule, characterized in that: The minocycline hydrochloride capsules are: The method according to any one of claims 1 to 8 is used to detect the dibenzyl azodicarboxylate impurity in minocycline hydrochloride capsules; or Prepared by the preparation method according to claim 9.

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