Impurity detection method, minocycline hydrochloride capsule product thereof and preparation method of minocycline hydrochloride capsule product

Abstract

The invention provides an impurity detection method, a minocycline hydrochloride capsule product and a preparation method thereof. The detection method is a liquid chromatography-mass spectrometry method. The benzyl carbazate in the minocycline hydrochloride capsule is detected through the liquid chromatography-mass spectrometry method, the content of the benzyl carbazate can be accurately determined, 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 particularly relates to an impurity detection method, a minocycline hydrochloride capsule product and a preparation method thereof. Background Art

[0002] Minocycline hydrochloride is a semi-synthetic tetracycline broad-spectrum antibiotic developed in the early 1970s. It is highly effective and long-lasting. Among tetracycline antibiotics, minocycline has the strongest antibacterial effect and better antibacterial effect than other antibacterial drugs. It exerts its antibacterial effect by preventing aminoacyl t-RNA from binding to m-RNA / ribosome complex and preventing protein synthesis. It has strong antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, Chlamydia, Rickettsia and anthrax.

[0003] Dibenzyl azodicarboxylate is a reagent used in the synthesis of minocycline hydrochloride API and participates in the reaction of demethyltetracycline to generate 7-HT. 7-HT is a hydrazine compound with a potential genotoxic structure. Benzyl hydrazinecarboxylate is the reduction product of dibenzyl azodicarboxylate. It is also a hydrazine compound with a potential genotoxic structure. It is harmful to the human body if swallowed, inhaled, contacted with the skin, or contacted with the eyes. Therefore, detecting the content of this substance is an important factor in controlling product quality.

[0004] Since benzyl hydrazinecarboxylate is irritating and flammable, and is prone to undergo a series of reactions with alcohols, aldehydes, acids and oxidants, the development of analytical methods is more difficult, and there are currently no reports that can accurately determine its content. Summary of the invention

[0005] Therefore, the purpose of the present invention is to overcome the defects in the prior art and provide an impurity detection method, a minocycline hydrochloride capsule product and a preparation method thereof. The present invention establishes a liquid chromatography-mass spectrometry method to detect benzyl hydrazinecarboxylate in minocycline hydrochloride capsules, which provides a reliable basis for the quality control of minocycline hydrochloride capsules.

[0006] Before describing the present invention, the terms used in this article are defined as follows:

[0007] The term "ESI" means: electrospray ionization.

[0008] The term "SIM" means: Selective ion detection is also called single ion monitoring, and its English name is Selected ion Monitor. For the primary mass spectrometer, only one ion is scanned and other ions are not recorded.

[0009] The term "m / z" means: mass to charge ratio.

[0010] The term "Agilent ZORBAX Eclipse Plus C18" refers to: an Agilent octadecyl-bonded phase chromatographic column, in which a monolayer of dimethyl-n-octadecylsilyl bonded phase is densely bonded by chemical bonding to a specially prepared, improved ultra-high purity porous silica gel.

[0011] The term "Agilent Poroshell 120EC-C18" refers to: an Agilent high-performance octadecyl-bonded phase chromatographic column, which uses high-purity silica gel and advanced bonding. While maintaining high column efficiency, it has a relatively low column backpressure, which is beneficial to improving the analysis speed and resolution.

[0012] The term "Thermo Accucore C18" refers to: a reversed-phase high-performance liquid chromatographic column based on superficially porous particle technology from Thermo Fisher Scientific, which can achieve fast and high-resolution separation at low backpressure. The high bonded-phase coverage can provide excellent retention for a series of non-polar analytes and is suitable for a variety of applications.

[0013] To achieve the above object, a first aspect of the present invention provides a method for detecting benzyl carbazate impurities in minocycline hydrochloride capsules. The detection method detects benzyl carbazate impurities by jointly using liquid chromatography and mass spectrometry; wherein:

[0014] The conditions of the liquid chromatography include:

[0015] The chromatographic column is selected from one or more of the following: Agilent ZORBAX Eclipse Plus C18, Thermo Accucore C18, Agilent Poroshell 120EC-C18, preferably Thermo Accucore C18 or Agilent Poroshell 120EC-C18, and most preferably Agilent Poroshell 120EC-C18;

[0016] The mobile phase includes mobile phase A and mobile phase B; mobile phase A is an aqueous formic acid solution; mobile phase B is acetonitrile;

[0017] The conditions of the mass spectrometry include:

[0018] The ion source is ESI;

[0019] The polarity is positive ion polarity.

[0020] According to the detection method of the first aspect of the present invention, wherein the detection method includes the following steps:

[0021] 1) Prepare a test solution of the minocycline hydrochloride capsule to be tested, a reference solution of benzyl carbazate, and a sensitivity solution respectively;

[0022] 2) Set the conditions of the liquid chromatography and the mass spectrometry; and

[0023] 3) Inject the test sample solution of minocycline hydrochloride capsules to be tested, the reference solution of benzyl hydrazinecarboxylate, and the sensitivity solution prepared in step 1) into the liquid chromatography and mass spectrometry combined detection equipment for detection respectively, and analyze the benzyl hydrazinecarboxylate impurity in the test sample solution of minocycline hydrochloride capsules to be tested according to the obtained detection chromatograms.

[0024] According to the detection method of the first aspect of the present invention, wherein, step 1) further includes: preparing a solvent and a derivatizing reagent; wherein:

[0025] The solvent is an aqueous acetonitrile solution, preferably 70% aqueous acetonitrile solution; and / or

[0026] The derivatizing reagent is prepared by adding benzoic acid and benzaldehyde to the solvent;

[0027] Preferably, in the derivatizing reagent, the mass-volume ratio of benzoic acid to benzaldehyde is 0.05 - 0.25 g:1 mL, more preferably 0.1 - 0.2 g:1 mL, and most preferably 0.125 g:1 mL.

[0028] According to the detection method of the first aspect of the present invention, wherein, in step 1):

[0029] The preparation method of the test sample solution of minocycline hydrochloride capsules to be tested includes: taking the minocycline hydrochloride capsules to be tested, adding the derivatizing reagent thereto, then adding the solvent to make up the volume, mixing evenly, ultrasonically treating, centrifuging, and taking the supernatant, thus obtaining the test sample solution of minocycline hydrochloride capsules to be tested;

[0030] The preparation method of the reference solution of benzyl hydrazinecarboxylate includes: taking the reference substance of benzyl hydrazinecarboxylate, dissolving it in the solvent, then adding the derivatizing reagent, making up the volume, and mixing evenly; and / or

[0031] The preparation method of the sensitivity solution includes: taking the reference substance of benzyl hydrazinecarboxylate, dissolving it in the solvent, then adding the derivatizing reagent, making up the volume, and mixing evenly;

[0032] Preferably, after the ultrasonic treatment in the preparation method of the test sample solution of minocycline hydrochloride capsules to be tested, and after the volume making in the preparation methods of the reference solution of benzyl hydrazinecarboxylate and the sensitivity solution, it further includes: reacting at a high temperature of ≥45 °C and cooling to room temperature;

[0033] More preferably, the high temperature is 50 - 80 °C, preferably 50 - 70 °C, and most preferably 60 °C.

[0034] According to the detection method of the first aspect of the present invention, wherein, in step 1):

[0035] The concentration of minocycline hydrochloride in the test minocycline hydrochloride capsule sample solution is 2 - 4 mg / ml, preferably 2.5 - 3 mg / ml, and most preferably 2.50 mg / ml;

[0036] The concentration of benzyl hydrazinecarboxylate in the benzyl hydrazinecarboxylate control solution is 100.0 - 200.0 ng / ml, preferably 120.0 - 150.0 ng / ml, and most preferably 125.0 ng / ml; and / or

[0037] The concentration of benzyl hydrazinecarboxylate in the sensitivity solution is 20.0 - 40.0 ng / ml, preferably 25.0 - 35.0 ng / ml, and most preferably 25.0 ng / ml.

[0038] According to the detection method of the first aspect of the present invention, wherein, in step 2), the conditions of the liquid chromatography further include:

[0039] The flow rate of the mobile phase is 0.45 ml / min - 0.55 ml / min, preferably 0.48 ml / min - 0.52 ml / min, and most preferably 0.5 ml / min;

[0040] The column temperature of the chromatographic column is 35°C - 45°C, preferably 38°C - 42°C, and most preferably 40°C; and / or

[0041] The volume fraction of mobile phase A is 0.05% - 0.2%, preferably 0.1% - 0.15%, and most preferably 0.1%.

[0042] According to the detection method of the first aspect of the present invention, wherein, in step 2), the conditions of the mass spectrometry further include:

[0043] The scan type is SIM; the mass-to-charge ratio m / z of the ions of SIM is preferably 2000 - 3000, more preferably 2100 - 2500, and most preferably 2255;

[0044] The drying gas temperature is 300 - 500°C, preferably 350 - 450°C, and most preferably 350°C;

[0045] The drying gas flow rate is 10 - 15 L / min, preferably 11 - 13 L / min, and most preferably 12 L / min;

[0046] The nebulizing gas pressure is 40 - 80 psi, preferably 50 - 70 psi, and most preferably 60 psi;

[0047] The capillary voltage is 3000 - 5000 V, preferably 3500 - 4500 V, and most preferably 4000 V; and / or

[0048] The mass spectrometry acquisition time is 5 - 18 min, preferably 5.5 - 15 min, and most preferably 5.5 - 13 min.

[0049] According to the detection method of the first aspect of the present invention, in step 3), the chromatographic analysis includes gradient elution.

[0050] Preferably, in the gradient elution, the initial volume ratio of mobile phase A to mobile phase B is 30 - 60:40 - 70, more preferably 35 - 50:50 - 65, and most preferably 40:60.

[0051] The second aspect of the present invention provides a preparation method of minocycline hydrochloride capsules, and the preparation method includes: detecting benzyl carbazate impurities by jointly using liquid chromatography and mass spectrometry.

[0052] The third aspect of the present invention provides a minocycline hydrochloride capsule, and the minocycline hydrochloride capsule is prepared by:

[0053] detecting benzyl carbazate impurities in the minocycline hydrochloride capsule by the method described in the first aspect and then preparing; or

[0054] preparing by the preparation method described in the second aspect.

[0055] According to a preferred embodiment of the present invention, the method for detecting the genotoxic impurity benzyl carbazate in minocycline hydrochloride capsules by liquid chromatography - mass spectrometry of the present invention includes:

[0056] Preparing a test solution and a reference solution, and injecting them into a liquid chromatography - mass spectrometer for chromatographic analysis respectively, and analyzing benzyl carbazate in the test solution according to the chromatogram. Among them, an Agilent Poroshell 120EC - C18 (4.6 mm × 150 mm, 2.7 μm) chromatographic column is selected, 0.1% formic acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B, and 70% acetonitrile aqueous solution is used as the solvent.

[0057] In some specific embodiments, the chromatographic elution program is gradient elution.

[0058] In some specific embodiments, the mass spectrometry parameters are: the ion source is ESI; the polarity is positive ion; the SIM ion m / z is 2255; the drying gas temperature is 350 °C; the capillary voltage is 4000 V; the drying gas flow rate is 12 L / min; the nebulizing gas pressure is 60 psi; the scan type is SIM; the mass spectrometry acquisition time is 5.5 - 13 min.

[0059] In some specific embodiments, the proportion of the starting organic phase (mobile phase B) in the chromatographic system is 58% - 62%, preferably 60%; the column temperature is 38°C - 42°C, preferably 40°C; the flow rate is 0.48 ml / min - 0.52 ml / min, preferably 0.5 ml / min.

[0060] In some specific embodiments, the derivatizing reagent (2% benzaldehyde and 0.25% benzoic acid solution) is prepared as follows: Take 0.25 g of benzoic acid, place it in a 100 ml volumetric flask, dissolve it with a solvent, then add 2 ml of benzaldehyde, dilute it to the scale with the solvent, and mix well.

[0061] In some specific embodiments, the test solution is prepared as follows: Take an appropriate amount of minocycline hydrochloride capsules (equivalent to about 50 mg of minocycline), place it in a 20 ml volumetric flask, add 4.0 ml of the derivatizing reagent, then add the solvent to volume to the scale, mix well, sonicate for 3 min, react at 60°C for 1 hour, cool to room temperature, centrifuge at 10000 r / min for 5 min, and take the supernatant to obtain a 2.50 mg / ml solution.

[0062] In some specific embodiments, the reference solution is prepared as follows: Take an appropriate amount of benzyl carbazate reference substance, dilute and prepare a solution of 1250.0 ng / ml, accurately measure 1.0 ml, place it in a 10 ml volumetric flask, dilute it with the solvent, add 2.0 ml of the derivatizing reagent, then add the solvent to volume to the scale, mix well, react at 60°C for 1 hour, cool to room temperature, and obtain a 125.0 ng / ml solution.

[0063] In some specific embodiments, the sensitivity solution is prepared as follows: Take an appropriate amount of benzyl carbazate reference substance, dilute and prepare a solution of 1250.0 ng / ml, accurately measure 0.2 ml, place it in a 10 ml volumetric flask, dilute it with the solvent, add 2.0 ml of the derivatizing reagent, then add the solvent to volume to the scale, mix well, react at 60°C for 1 hour, cool to room temperature, and obtain a 25.0 ng / ml solution.

[0064] In some specific embodiments, the method further includes system suitability requirements: The system is clean and stable, without interference at the elution positions of each component. If there is interference, the peak area of the interfering peak should not be greater than the peak area of the main peak in the sensitivity solution; in the sensitivity solution, the signal-to-noise ratio of each component is not less than 10; the reference solution is injected continuously for 6 times, the RSD of the peak area of the analyte is not greater than 15.0%, and the RSD of the retention time is not greater than 1.0%.

[0065] Compared with the prior art, the method for detecting benzyl carbazate impurity in minocycline hydrochloride capsules of the present invention may have but is not limited to the following beneficial effects:

[0066] The present invention establishes a method for detecting benzyl carbazate in minocycline hydrochloride capsules by liquid chromatography-mass spectrometry, which can accurately determine the content of benzyl carbazate and provides a reliable basis for the quality control of minocycline hydrochloride capsules. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:

[0068] Figure 1 Shows the chromatogram of the blank solution in Example 1.

[0069] Figure 2 Shows the chromatogram of the 100% limit reference solution in Example 1.

[0070] Figure 3 Shows the chromatogram of the 100% limit spiked test solution in Example 1.

[0071] Figure 4 Shows the chromatogram of the sensitivity solution in Example 1.

[0072] Figure 5 Shows the detection chromatogram of benzyl carbazate in the first batch of minocycline hydrochloride samples in Example 2.

[0073] Figure 6 Shows the detection chromatogram of benzyl carbazate in the second batch of minocycline hydrochloride samples in Example 2.

[0074] Figure 7 Shows the detection chromatogram of benzyl carbazate in the third batch of minocycline hydrochloride samples in Example 2.

[0075] Figure 8 Shows the linear relationship diagram of benzyl carbazate in Example 1.

[0076] Figure 9 Shows the chromatogram of the test solution in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0077] The present invention will be further described below through specific examples. However, it should be understood that these examples are only used for more detailed and specific description, and should not be construed as limiting the present invention in any form.

[0078] This part generally describes the materials and test methods used in the experiments of the present invention. Although many materials and operation methods used to achieve the purpose of the present invention are well known in the art, the present invention still describes them in as much detail as possible here. Those skilled in the art are aware that, in the context, if not specifically stated, the materials and operation methods used in the present invention are well known in the art.

[0079] The reagents and instruments used in the following examples are as follows:

[0080] Reagents:

[0081] Formic acid and benzyl hydrazinecarboxylate were purchased from MACKLIN; acetonitrile was purchased from Merck; benzaldehyde and benzoic acid were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0082] instrument:

[0083] LC-MS, purchased from Agilent, model Agilent 1260&LC / MSD XT.

[0084] Analytical balance, purchased from Sartorius, model SQP.

[0085] The mixer was purchased from Haimen Qilin Bell Instrument Manufacturing Co., Ltd., model QL-866.

[0086] The ultrasonic cleaner was purchased from Kunshan Hechuang Ultrasonic Instrument Co., Ltd., model KH-500E.

[0087] Oscillating water bath, purchased from Julabo, model SW23.

[0088] Example 1

[0089] This example is an exemplary description of the detection method and method verification of benzyl hydrazinecarboxylate impurities in minocycline hydrochloride capsules of the present invention.

[0090] 1. Chromatographic conditions

[0091] Column: Agilent Poroshell 120EC-C18, 4.6 mm × 150 mm, 2.7 μm

[0092] Mobile phase: 0.1% formic acid aqueous solution as mobile phase A; acetonitrile as mobile phase B.

[0093] The chromatographic elution procedure is gradient elution, and the ratio of the mobile phase A to the mobile phase B is as follows:

[0094] Table 1 Ratio of mobile phase A and mobile phase B in gradient elution of Example 1

[0095] Time (min) Mobile phase A (%) Mobile phase B (%) 0 40 60 5 40 60 10 10 90 10.1 40 60 15 40 60

[0096] Solvent: 70% acetonitrile in water

[0097] Flow rate: 0.5ml / min

[0098] Column temperature: 40°C

[0099] Sample tray temperature: 10 °C

[0100] Sample volume: 10 μl

[0101] 2. Mass spectrometry conditions

[0102] Ion source: ESI

[0103] Polarity: positive ion

[0104] SIM ion: m / z 2255

[0105] Drying gas temperature: 350 °C

[0106] Capillary voltage: 4000 V

[0107] Drying gas flow rate: 12 L / min

[0108] Nebulizing gas pressure: 60 psi

[0109] Scan type: SIM

[0110] Mass spectrometry acquisition time: 5.5 - 13 min

[0111] 3. Determination steps

[0112] (1) Preparation of solutions

[0113] ① Derivatization reagent: Take 0.25 g of benzoic acid, place it in a 100 ml volumetric flask, dissolve it with a solvent, then add 2 ml of benzaldehyde, dilute it to the mark with the solvent, and mix well to obtain the derivatization reagent (2% benzaldehyde and 0.25% benzoic acid solution).

[0114] ② Test solution: Take 133 mg of the content of minocycline hydrochloride capsules (equivalent to 50 mg of minocycline), place it in a 20 ml volumetric flask, add 4.0 ml of the derivatization reagent, then add the solvent to volume to the mark, mix well, sonicate for 3 min, react at 60 °C for 1 hour, cool to room temperature, centrifuge at 10000 r / min for 5 min, and take the supernatant to obtain a 2.50 mg / ml test solution.

[0115] ③ Reference solution: Take 10 mg of benzyl carbazate reference substance, dilute and prepare a solution of 1250.0 ng / ml, accurately measure 1.0 ml, place it in a 10 ml volumetric flask, dilute it with a solvent, add 2.0 ml of the derivatization reagent, then add the solvent to volume to the mark, mix well, react at 60 °C for 1 hour, and cool to room temperature to obtain a 125.0 ng / ml reference solution.

[0116] ④ Sensitivity solution: Take 10 mg of benzyl hydrazinecarboxylate reference substance, dilute and prepare a solution with a concentration of 1250.0 ng / ml. Accurately measure 0.2 ml, place it in a 10-ml volumetric flask, dilute with the solvent, add 2.0 ml of derivatization reagent, then add the solvent to volume to the scale, mix well, react at 60 °C for 1 hour, and cool to room temperature to obtain a 25.0 ng / ml solution.

[0117] (2) Measure 10 μl of the reference solution, sensitivity solution, and test solution respectively, inject them into the liquid chromatography-mass spectrometer, and record the chromatogram. Among them, the chromatogram of the reference solution obtained is as shown in Figure 2 shown, the chromatogram of the test solution obtained is as shown in Figure 9 shown, and the chromatogram of the sensitivity solution is as shown in Figure 4 shown.

[0118] (3) System suitability requirements: The system is clean and stable, without interference at the peak positions of each component. If there is interference, the peak area of the interfering peak should not be greater than the peak area of the main peak in the sensitivity solution; in the sensitivity solution, the signal-to-noise ratio of each component is not less than 10; the reference solution is injected continuously for 6 injections, the RSD of the peak area of the analyte is not greater than 15.0%, and the RSD of the retention time is not greater than 1.0%.

[0119] 4. Method validation

[0120] ① Specificity: There is no interference at the target peak in the chromatogram of the blank solution; the analyte in the reference solution shows the target peak with a retention time of 6.523; there are no adjacent peaks in the spiked sample solution at 100% limit. The chromatograms of the specificity test are shown in Figures 1 - 3 .

[0121] ② System suitability test: The system is stable and clean, without interference at the peak positions of each component; in the sensitivity solution, the signal-to-noise ratio of the target peak is 31; the reference solution is injected continuously for 6 times, the RSD of the peak area of the target peak is 0.9%, and the RSD of the retention time is 0.1%. See Table 2 for details.

[0122] ③ Linearity: Take benzyl hydrazinecarboxylate reference substance and prepare linear solutions with concentration levels of 20%, 50%, 100%, 150%, and 200%. Determine according to the chromatographic method of this patent. Perform linear regression with the concentration (C) as the abscissa and the peak area (A) as the ordinate. The regression equation is: y = 9195x + 48547; r = 0.999; the y-axis intercept ratio is 3.7%; the sum of squared residuals is 4224239069; the linear range is 26.42 ng / ml - 264.24 ng / ml. See Figure 8 and Table 3 for details.

[0123] ④ Detection limit and quantitation limit: The signal-to-noise ratios of the detection limit of benzyl hydrazinecarboxylate were 17, 16, and 16 respectively; the signal-to-noise ratios of the quantitation limit were 32, 32, 30, 33, 31, and 33 respectively, and the RSD of the peak area was 1.0%. See Table 4 for details.

[0124] ⑤ Investigation of solution stability: Take the reference solution and the spiked test solution, and place them at room temperature for 26.5 h. The recoveries of the reference solution and the spiked test solution are both within the range of 70% - 130%, and the solution is stable. See Table 5 for details.

[0125] ⑥ Investigation of precision: Repeatability: The RSD of the recoveries of 6 spiked test solutions was 2.3% (<15.0%). Intermediate precision: The RSD of the recoveries of 6 spiked test solutions was 2.1% (<15.0%); the RSD of the recoveries of 12 spiked test solutions was 2.8% (<30.0%). See Table 6 for details.

[0126] ⑦ Investigation of accuracy: The range of the recoveries of the spiked test solution was 90.4% - 97.9%, and the average recoveries were 97%, 94%, and 91% respectively, with an RSD of 3.1%. See Table 7 for details.

[0127] ⑧ When slightly changing the initial organic phase ratio (±2%), column temperature (±2 °C), and flow rate (±0.02 ml / min) in the chromatographic conditions, the RSDs of the peak areas of the target peaks in 6 spiked test solutions were 4.3%, 2.7%, and 2.4% respectively. See Table 8 for details.

[0128] Table 2 Results of system suitability test

[0129]

[0130] Table 3 Results of linear relationship test of benzyl hydrazinecarboxylate

[0131]

[0132] Table 4 Results of detection limit and quantitation limit test

[0133]

[0134]

[0135] Table 5 Results of solution stability test

[0136]

[0137] Table 6 Results of precision investigation test

[0138]

[0139] Table 7 Results of recovery test of benzyl hydrazinecarboxylate

[0140]

[0141]

[0142] Table 8 Results of durability test

[0143]

[0144] Example 2

[0145] This example is an exemplary illustration of the effect of the detection method for benzyl carbazate impurity in minocycline hydrochloride capsules of the present invention.

[0146] According to the method described in Example 1, the detection of benzyl carbazate was carried out on 3 batches of minocycline hydrochloride samples prepared by the applicant's unit, and the results were all undetected. As Figures 5 - 7 shown, this indicates that in the process of synthesizing minocycline hydrochloride by the applicant's unit, the product quality control is ideal and it does not contain potentially genotoxic impurities.

[0147] In Examples 1-2, the detection method of the present invention was repeated for multiple batches of samples, and no possible benzyl carbazate impurities were detected. This proves that the production process of minocycline hydrochloride by the applicant's unit is safe and stable.

[0148] Example 3

[0149] This example is another exemplary illustration of the detection method for benzyl carbazate impurity in minocycline hydrochloride capsules of the present invention.

[0150] The mass spectrometry conditions and determination steps are the same as those in Example 1. The chromatographic conditions are the same as those in Example 1 except for the proportion of the mobile phase in the gradient elution.

[0151] Mobile phase: 0.1% formic acid aqueous solution is used as mobile phase A; acetonitrile is used as mobile phase B

[0152] The chromatographic elution program is gradient elution, and the proportions of mobile phase A and mobile phase B are as follows:[[]]

[0153] Table 9 Proportions of mobile phase A and mobile phase B in the gradient elution of Example 3

[0154] Time (min) Mobile phase A (%) Mobile phase B (%) 0 42 58 5 42 58 10 10 90 10.1 42 58 15 42 58

[0155] Example 4

[0156] This example is yet another exemplary illustration of the detection method for benzyl carbazate impurity in minocycline hydrochloride capsules of the present invention.

[0157] The mass spectrometry conditions and determination steps are the same as those in Example 1. The chromatographic conditions are the same as those in Example 1 except for the proportion of the mobile phase in the gradient elution.

[0158] Mobile phase: 0.1% formic acid aqueous solution is used as mobile phase A; acetonitrile is used as mobile phase B

[0159] The chromatographic elution procedure is gradient elution, and the proportions of mobile phase A and mobile phase B are as follows:

[0160] Table 10 Proportions of mobile phase A and mobile phase B in the gradient elution of Example 4

[0161] Time (min) Mobile phase A (%) Mobile phase B (%) 0 38 62 5 38 62 10 10 90 10.1 38 62 15 38 62

[0162] Example 5

[0163] This example is another exemplary illustration of the method for detecting benzyl carbazate impurity in minocycline hydrochloride capsules of the present invention.

[0164] The mass spectrometry conditions and determination steps are the same as those in Example 1. The chromatographic conditions are the same as those in Example 1 except for the chromatographic column. The chromatographic column used in this example is: Thermo Accucore C18, 4.6 mm × 150 mm, 2.6 μm.

[0165] Example 6

[0166] This example is yet another exemplary illustration of the method for detecting benzyl carbazate impurity in minocycline hydrochloride capsules of the present invention.

[0167] The mass spectrometry conditions and chromatographic conditions are the same as those in Example 1, except for the dosage of reagents in the determination steps, which are specifically as follows:

[0168] 1.. Determination steps

[0169] (1) Preparation of solutions

[0170] ① Derivatization reagent: Take 0.2 g of benzoic acid, place it in a 100 ml volumetric flask, dissolve it with a solvent, then add 2 ml of benzaldehyde, and dilute it to the scale with the solvent and mix well to obtain the derivatization reagent (2% benzaldehyde and 0.2% benzoic acid solution).

[0171] ② Test solution: Take 133 mg of the content of minocycline hydrochloride capsules (equivalent to 50 mg of minocycline), place it in a 20 ml volumetric flask, add 3.0 ml of the derivatization reagent, then make up the volume to the scale with the solvent, mix well, ultrasonicate for 3 min, react at 60 °C for 1 hour, cool to room temperature, centrifuge at 10000 r / min for 5 min, and take the supernatant to obtain a 2.50 mg / ml test solution.

[0172] ③ Reference solution: Take 10 mg of benzyl hydrazinecarboxylate reference substance, dilute and prepare it into a solution of 1250.0 ng / ml. Precisely measure 1.0 ml, place it in a 10-ml volumetric flask, dilute with the solvent, add 1.5 ml of derivatization reagent, then add the solvent to volume to the mark, mix well, react at 60 °C for 1 hour, and cool to room temperature to obtain a reference solution of 125.0 ng / ml.

[0173] ④ Sensitivity solution: Take 10 mg of benzyl hydrazinecarboxylate reference substance, dilute and prepare it into a solution of 1250.0 ng / ml. Precisely measure 0.2 ml, place it in a 10-ml volumetric flask, dilute with the solvent, add 1.5 ml of derivatization reagent, then add the solvent to volume to the mark, mix well, react at 60 °C for 1 hour, and cool to room temperature to obtain a solution of 25.0 ng / ml.

[0174] (2) Measure 10 μL of the reference solution, the sensitivity solution, and the test solution respectively, and inject them into the liquid chromatography - mass spectrometer, and record the chromatogram.

[0175] (3) System suitability requirements: The system is clean and stable, without interference at the elution positions of each component. If there is interference, the peak area of the interfering peak should not be greater than the peak area of the main peak in the sensitivity solution; in the sensitivity solution, the signal-to-noise ratio of each component is not less than 10; inject the reference solution continuously for 6 injections, the RSD of the peak area of the analyte is not greater than 15.0%, and the RSD of the retention time is not greater than 1.0%.

[0176] According to the detection method of Example 1, the benzyl hydrazinecarboxylate reference substances in Examples 3 - 6 can all be detected, and none are detected in the minocycline hydrochloride capsule samples.

[0177] Although the effects of some of the above embodiments are shown, those skilled in the art should understand that according to the concept of the present invention, the above-mentioned other embodiments for which the effects are not specifically shown or other technical solutions of the present invention not shown in the embodiments can also achieve the following technical effects stated in the content of the invention that are equivalent to those of the embodiments:

[0178] The present invention establishes a liquid chromatography - mass spectrometry method for detecting benzyl hydrazinecarboxylate in minocycline hydrochloride capsules, can accurately determine the content of benzyl hydrazinecarboxylate, and provides a reliable basis for the quality control of minocycline hydrochloride capsules.

[0179] Although the present invention has been described to a certain extent, obviously, appropriate changes can be made to each condition without departing from the spirit and scope of the present invention. It can be understood that the present invention is not limited to the described embodiments, but belongs to the scope of the claims, which includes equivalent replacements for each factor described.

Claims

1. A method for detecting benzyl carbazate impurities in minocycline hydrochloride capsules, characterized in that, The detection method detects the impurities of benzyl hydrazinecarboxylate by jointly using liquid chromatography and mass spectrometry; wherein: The conditions of the liquid chromatography include: The chromatographic column is selected from one or more of the following: Agilent ZORBAX Eclipse Plus C18, Thermo Accucore C18, Agilent Poroshell 120 EC-C18, preferably Thermo Accucore C18 or Agilent Poroshell 120 EC-C18, and most preferably Agilent Poroshell 120 EC-C18; The mobile phase includes mobile phase A and mobile phase B; the mobile phase A is an aqueous formic acid solution; the mobile phase B is acetonitrile; The conditions of the mass spectrometry include: The ion source is ESI; The polarity is positive ion polarity.

2. The detection method according to claim 1, characterized in that, The detection method includes the following steps: 1) Prepare the test solution of minocycline hydrochloride capsules to be tested, the reference solution of benzyl hydrazinecarboxylate, and the sensitivity solution respectively; 2) Set the conditions of the liquid chromatography and the mass spectrometry; and 3) Inject the test solution of minocycline hydrochloride capsules to be tested, the reference solution of benzyl hydrazinecarboxylate, and the sensitivity solution prepared in step 1) into the liquid chromatography and mass spectrometry combined detection equipment for detection respectively, and analyze the benzyl hydrazinecarboxylate impurities in the test solution of minocycline hydrochloride capsules to be tested according to the obtained detection chromatograms.

3. The detection method according to claim 1, wherein In step 1), it also includes: preparing a solvent and a derivatizing reagent; wherein: The solvent is an aqueous acetonitrile solution, preferably 70% aqueous acetonitrile solution; and / or The derivatizing reagent is prepared by adding benzoic acid and benzaldehyde to the solvent; Preferably, in the derivatizing reagent, the mass-volume ratio of benzoic acid to benzaldehyde is 0.05-0.25 g:1 mL, more preferably 0.1-0.2 g:1 mL, and most preferably 0.125 g:1 mL.

4. The detection method according to claim 3, wherein In step 1): The preparation method of the test solution of minocycline hydrochloride capsules to be tested includes: taking the minocycline hydrochloride capsules to be tested, adding the derivatizing reagent, then adding the solvent to make up the volume, mixing, sonicating, centrifuging, and taking the supernatant, that is, obtaining the test solution of minocycline hydrochloride capsules to be tested; The preparation method of the reference solution of benzyl hydrazinecarboxylate includes: taking the reference substance of benzyl hydrazinecarboxylate, dissolving it in the solvent, then adding the derivatizing reagent, making up the volume, and mixing; and / or The preparation method of the sensitivity solution includes: taking the reference substance of benzyl hydrazinecarboxylate, dissolving it in the solvent, then adding the derivatizing reagent, making up the volume, and mixing; Preferably, after the sonication in the preparation method of the test solution of minocycline hydrochloride capsules to be tested, and after the volume making up in the preparation methods of the reference solution of benzyl hydrazinecarboxylate and the sensitivity solution, it also includes: reacting at a high temperature of ≥45 °C and cooling to room temperature; More preferably, the high temperature is 50-80 °C, preferably 50-70 °C, and most preferably 60 °C.

5. The detection method according to any one of claims 2 to 4, characterized in that, In step 1): The concentration of minocycline hydrochloride in the test minocycline hydrochloride capsule sample solution is 2 - 4 mg / ml, preferably 2.5 - 3 mg / ml, and most preferably 2.50 mg / ml; The concentration of benzyl hydrazinecarboxylate in the benzyl hydrazinecarboxylate control solution is 100.0 - 200.0 ng / ml, preferably 120.0 - 150.0 ng / ml, and most preferably 125.0 ng / ml; and / or The concentration of benzyl hydrazinecarboxylate in the sensitivity solution is 20.0 - 40.0 ng / ml, preferably 25.0 - 35.0 ng / ml, and most preferably 25.0 ng / ml.

6. The detection method according to any one of claims 2 to 5, characterized in that, In step 2), the conditions of the liquid chromatography further include: The flow rate of the mobile phase is 0.45 ml / min - 0.55 ml / min, preferably 0.48 ml / min - 0.52 ml / min, and most preferably 0.5 ml / min; The column temperature of the chromatographic column is 35°C - 45°C, preferably 38°C - 42°C, and most preferably 40°C; and / or The volume fraction of mobile phase A is 0.05% - 0.2%, preferably 0.1% - 0.15%, and most preferably 0.1%.

7. The detection method according to any one of claims 2 to 6, characterized in that: In step 2), the conditions of the mass spectrometry further include: The scan type is SIM; the mass-to-charge ratio m / z of the ions in SIM is preferably 2000 - 3000, more preferably 2100 - 2500, and most preferably 2255; The drying gas temperature is 300 - 500°C, preferably 350 - 450°C, and most preferably 350°C; The drying gas flow rate is 10 - 15 L / min, preferably 11 - 13 L / min, and most preferably 12 L / min; The nebulizing gas pressure is 40 - 80 psi, preferably 50 - 70 psi, and most preferably 60 psi; The capillary voltage is 3000 - 5000 V, preferably 3500 - 4500 V, and most preferably 4000 V; and / or The mass spectrometry acquisition time is 5 - 18 min, preferably 5.5 - 15 min, and most preferably 5.5 - 13 min.

8. The detection method according to any one of claims 1 to 7, characterized in that, In step 3), the chromatographic analysis includes gradient elution; Preferably, in the gradient elution, the initial volume ratio of mobile phase A to mobile phase B is 30 - 60:40 - 70, more preferably 35 - 50:50 - 65, and most preferably 40:

60.

9. A preparation method of minocycline hydrochloride capsules, characterized in that, The preparation method includes: detecting benzyl hydrazinecarboxylate impurities by jointly using liquid chromatography and mass spectrometry.

10. A minocycline hydrochloride capsule, characterized in that, The minocycline hydrochloride capsule is prepared by: detecting benzyl hydrazinecarboxylate impurities in the minocycline hydrochloride capsule by the method according to any one of claims 1 to 8; or prepared by the preparation method according to claim 9.

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