A method for identifying endogenous visible foreign matter in armillariin injection

Abstract

The present invention discloses a method for identifying endogenous visible foreign matter in armillaria injection. The method of the present invention comprises the following steps: performing ultra-high performance liquid chromatography-mass spectrometry analysis and X-ray polycrystalline diffraction analysis on visible foreign matter in armillaria injection, respectively, obtaining ultra-high performance liquid chromatogram, mass spectrum and X-ray polycrystalline diffraction pattern, if the test results meet the following a), b) and c), then the visible foreign matter is determined to be a raw material drug component precipitated from armillaria-polyvidone K30 copolymer; a) the peak position in the ultra-high performance liquid chromatogram of the visible foreign matter is consistent with the peak position of the raw material armillaria; b) the mass spectrum of the visible foreign matter is consistent with the mass spectrum of the raw material armillaria; c) the X-ray polycrystalline diffraction pattern of the visible foreign matter has only diffraction peaks at 11 and 12 in the scanning range of 3° to 90° in 2θ. The present invention establishes a rapid identification method for endogenous visible foreign matter in armillaria injection, which can quickly determine the source of the visible foreign matter.

Description

Technical Field

[0001] The invention belongs to the field of visible foreign matter analysis, and in particular relates to a method for identifying endogenous visible foreign matter in armillaria serrata injection. Background Art

[0002] The 2020 edition of the "Chinese Pharmacopoeia" clearly stipulates in the general rules that visible foreign matter refers to insoluble substances that can be visually observed under specified conditions in injections, ophthalmic liquid preparations and sterile raw materials, and their particle size or length is usually greater than 50μm. There are many reasons for the generation of visible foreign matter in injections, involving various processes in the production process, raw and auxiliary materials, inner packaging materials, etc. At the same time, personnel, as the main source of pollution in clean areas, are often easy to bring in visible foreign matter such as hair. Therefore, if visible foreign matter appears in the production process of injections, it must be carefully investigated. First, it must be clarified whether it is exogenous (referring to introduction during the production process) or endogenous (derived from raw and auxiliary materials or inner packaging materials that come into contact with drugs, etc.), and the real cause must be found out, and effective control measures must be taken to ensure product quality.

[0003] According to the General Rules of Part IV of the 2020 edition of the Chinese Pharmacopoeia (0904), visible foreign matter inspection can usually use the first method (light inspection method) or the second method (light scattering method) to confirm whether there are visible foreign matter in the injection. If found, the visible foreign matter must be further investigated, qualitatively analyzed, and effective preventive measures must be taken. However, after investigation and experimental investigation by relevant companies, it was found that there are certain differences in the results of the two inspection methods: the use of the light inspection method will cause a certain proportion of missed inspections due to the inspectors themselves, especially for smaller foreign matter; the use of the light scattering method will cause a certain proportion of misdetection due to the mismatch of instrument parameters, especially the probability of false positives caused by judging fine glass as fine visible foreign matter is high. To this end, Qiao Chong et al. provided an analytical method for visible foreign matter in biochemical drug injections, which used electron microscopy and energy spectrum analysis to observe the insoluble substances in the drug solution to obtain the elemental composition, and then determined and analyzed the type of visible foreign matter. This method is more accurate and effective, which greatly makes up for the limitations of the visible foreign matter inspection method itself, and can provide suggestions for the control of visible foreign matter in biochemical drug injections. The shortcoming is that this method can only roughly infer the type of visible foreign matter based on the elemental composition and cannot accurately determine the specific source and cause of the visible foreign matter, which increases the workload for the prevention and control of visible foreign matter.

[0004] Armillariin A is a yellow or slightly orange-yellow rectangular plate-like crystal or crystalline powder, odorless, insoluble in water, and very slightly soluble in ethanol or methanol. Based on the solubility characteristics of armillariin A, most commercial armillariin A injections use pharmaceutical grade propylene glycol as a solvent, which has certain safety risks. Povidone K30 is a white to milky white powder, odorless or slightly odorous, tasteless organic compound, with a molecular formula of (C6H9NO) n, which is often used as a polymerizing agent in the production of armillaria jejuni injection (LJ-I), but needle-shaped yellow-brown crystals occasionally precipitate during production. Once the source of visible foreign matter in LJ-I is determined, measures such as process optimization can be taken to ensure product quality. Therefore, determining the source of the visible foreign matter is a technical problem that needs to be solved urgently. Summary of the invention

[0005] The purpose of the present invention is to provide a method for identifying endogenous visible foreign matter in armillaria serrata injection, by which the source of the endogenous visible foreign matter can be quickly identified, and measures such as process optimization can be taken to ensure product quality and safety.

[0006] The present invention provides a method for identifying endogenous visible foreign matter in armillariin injection, comprising the following steps:

[0007] Performing ultra-high performance liquid chromatography-mass spectrometry analysis and X-ray polycrystalline diffraction analysis on the visible foreign matter in the armillaria injection respectively to obtain ultra-high performance liquid chromatography-mass spectrometry, mass spectrometry and X-ray polycrystalline diffraction patterns; if the test results meet the following a), b) and c), the visible foreign matter is determined to be the raw material drug component precipitated from the armillariaria-povidone K30 copolymer;

[0008] a) the peak position in the ultra-high performance liquid chromatogram of the visible foreign matter is consistent with the peak position in the ultra-high performance liquid chromatogram of the raw material drug armillariin A;

[0009] b) The mass spectrum of the visible foreign matter and the mass spectrum of the raw material drug Armillaria acetoin Figure 1 To;

[0010] c) The X-ray polycrystalline diffraction pattern of the visible foreign matter has only characteristic diffraction peaks at 11 and 12 (no characteristic diffraction peak at 25) in the scanning range of 3° to 90° at 2θ.

[0011] In the above-mentioned method for identifying endogenous visible foreign matter in the armillaria injection, the visible foreign matter is yellow-brown needle-shaped crystals.

[0012] In the present invention, the leuprocin injection uses povidone K30 as a polymerization agent, and leuprocin exists in the form of leuprocin-povidone K30 copolymer. Preferably, the preparation of the leuprocin-povidone K30 copolymer uses anhydrous ethanol as a solvent. In the specific implementation of the present invention, the leuprocin-povidone K30 copolymer is prepared according to a method comprising the following steps: adding leuprocin to an anhydrous ethanol solution of povidone K30, heating and stirring to mix, keeping warm, recovering ethanol, and vacuum drying to obtain the leuprocin-povidone K30 copolymer. The mass ratio of the povidone K30 to the leuprocin used for polymerization and the process conditions can be appropriately adjusted within a reasonable range, for example, the mass ratio of povidone K30 to leuprocin is (5-20): (1-5), and for another example, the mass ratio of povidone K30 to leuprocin is 5:1, and the ratio of povidone K30, leuprocin and anhydrous ethanol is 5g: 1g: 120mL. The insulation temperature may be 50° C. to 80° C., and the insulation time may be 20 to 30 minutes.

[0013] In the present invention, the armillary mitochondria injection has dimethylacetamide, propylene glycol, polysorbate 80, polyethylene glycol 400, polyethylene glycol 6000, thiourea, water for injection and a pH regulator as auxiliary materials. In the specific implementation of the present invention, the armillary mitochondria injection is prepared according to a method comprising the following steps: dissolving the armillary mitochondria-povidone K30 copolymer in the dimethylacetamide, then sequentially adding the propylene glycol, the polysorbate 80, the polyethylene glycol 400, the polyethylene glycol 6000 and the thiourea, supplementing the water for injection and adjusting the pH value with the pH regulator, mixing and filtering, and sterilizing after encapsulation to obtain the armillary mitochondria injection. The armillaria injection meets the national drug standard of WS-10001-(HD-1207)-2002, wherein the ratio of each raw material does not affect the identification of endogenous visible foreign matter in the armillaria injection of the present invention, and the mass ratio of each raw material can be appropriately adjusted within a reasonable range according to the armillaria injection drug standard. For example, 5 to 12 g of armillaria-povidone K30 copolymer is dissolved in 20 to 50 mL of dimethylacetamide, and then 200 to 400 g of propylene glycol, 3 to 8 g of polysorbate 80, 8 to 30 g of polyethylene glycol 400, 30 to 70 g of polyethylene glycol 6000 and 0.3 to 1 g of thiourea are added in sequence, and water for injection is added to 2000 mL and the pH is adjusted to 4.8 to 5.2 with a pH regulator.

[0014] In the above-mentioned method for identifying endogenous visible foreign matter in the leucocyte antigen injection, the sample solution of visible foreign matter used for ultra-high performance liquid chromatography-mass spectrometry analysis uses ammonia solution and methanol as solvents; preferably, the method for preparing the sample solution of visible foreign matter used for ultra-high performance liquid chromatography-mass spectrometry analysis comprises the following steps: taking the leucocyte antigen injection, standing to remove the supernatant and retaining the visible foreign matter at the bottom; washing the visible foreign matter with a blank excipient solution and methanol in turn, dissolving the visible foreign matter with ammonia solution after washing and diluting it with methanol to obtain the sample solution of the visible foreign matter.

[0015] In the above-mentioned method for identifying endogenous visible foreign matter in armillaria injection, the chromatographic conditions in the ultra-high performance liquid chromatography-mass spectrometry analysis are as follows:

[0016] LC system: ACQUITY UOLC;

[0017] Chromatographic column: Waters Symmetry C18, 3.5 μm, 4.6 × 75 mm;

[0018] Mobile phase: composed of methanol and 0.1 mol / L acetic acid aqueous solution in a volume ratio of 1:1;

[0019] Elution mode: isocratic elution;

[0020] Flow rate: 0.4 mL / min;

[0021] Column temperature: room temperature;

[0022] Detection wavelength: 368nm;

[0023] Injection volume: 20 μL.

[0024] In the present invention, the room temperature is preferably 15 to 25°C, more preferably 25°C.

[0025] In the above-mentioned method for identifying endogenous visible foreign matter in the armillariin injection, the peak position in the ultra-high performance liquid chromatogram of the raw material drug is at a retention time of 3.746 min;

[0026] The peak position of the visible foreign matter in the ultra-high performance liquid chromatogram is at a retention time of 3.667 min.

[0027] In the above-mentioned method for identifying endogenous visible foreign matter in armillaria injection, the mass spectrometry conditions in the ultra-high performance liquid chromatography-mass spectrometry analysis are as follows:

[0028] MS system: Xevo TQ-S;

[0029] Electrospray ionization source;

[0030] Negative ion detection mode;

[0031] Capillary voltage: 19V;

[0032] Capillary temperature: 350℃;

[0033] Source temperature: 150°C;

[0034] Shell gas flow rate: 45arb;

[0035] Auxiliary gas flow rate: 0arb;

[0036] Spray voltage: 4.5 kV.

[0037] In the above-mentioned method for identifying endogenous visible foreign matter in the armillaria injection, the mass spectrum of the visible foreign matter is consistent with the mass spectrum of the raw material armillaria Figure 1 The quasi-molecular ion m / z 233[MH] of the visible foreign matter - The secondary fragment ions of the active ingredient armillarin A and the quasi-molecular ion m / z 233[MH] - The secondary fragment ions are consistent;

[0038] Preferably, the quasi-molecular ion m / z 233[MH] of the raw material drug armillarin - A secondary scan was performed to generate 10 relatively abundant fragment ions with m / z of 214.99, 203.06, 191.02, 189.02, 171.02, 162.99, 161.01, 158.95, 147.01, and 135.01, respectively.

[0039] In the above-mentioned method for identifying endogenous visible foreign matter in armillariin injection, the conditions of the X-ray polycrystalline diffraction analysis are as follows:

[0040] Japan Rigaku XRD X-ray polycrystal diffractometer;

[0041] Working power: 1.6kW, 40kV, 40mA;

[0042] Scanning range: 3°~90°;

[0043] Step distance: 0.02;

[0044] Target material: Cu target.

[0045] In the above-mentioned method for identifying endogenous visible foreign matter in the thiabendazole injection, the method for preparing a visible foreign matter sample for X-ray polycrystalline diffraction analysis includes the following steps: taking the thiabendazole injection, letting it stand to remove the upper clear liquid and retaining the visible foreign matter at the bottom; washing the visible foreign matter with a blank excipient solution and methanol in turn, and vacuum drying after washing to obtain a visible foreign matter sample.

[0046] In the above-mentioned method for identifying endogenous visible foreign matter in the armillaria injection, the visible foreign matter has no characteristic diffraction peak at 25 at 2θ in the X-ray polycrystalline diffraction pattern of the visible foreign matter.

[0047] Furthermore, in the X-ray polycrystalline diffraction pattern of the visible foreign matter, the maximum intensity ratio of the diffraction peaks at 2θ of 11 and 12 is 1:1.6.

[0048] The present invention has the following beneficial effects:

[0049] The present invention compares the ultra-high performance liquid chromatography spectra of occasional needle-shaped yellow-brown precipitates (visible foreign matter) in the production of LJ-I with LJ-API, photolysis samples, polymers and excipients, and combines spectral information such as LC-MS / MS and XRD to confirm that the visible foreign matter in LJ-I is a very trace amount of raw material drug components that are not encapsulated by polyvidone K30 copolymer. A method for rapid identification of endogenous visible foreign matter in armillaria injection is established, which provides an improvement idea for subsequent optimization of configuration temperature, change of stirring mode and stirring times, and process verification, thereby improving product quality and ensuring clinical drug safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 UPLC chromatograms of LJ-API (a), photolysis sample (b), visible foreign matter (c), intermediate polymer (d) and blank excipient (e) in Example 1.

[0051] Figure 2 UPLC (368 nm) (a) and LC-MS total ion current (b) of LJ-API in Example 1.

[0052] Figure 3 The ESI-MS mass spectrum and quasi-molecular ion peak [MH] of LJ-API in Example 1 - Structural formula.

[0053] Figure 4 is the ESI-MS of LJ-API in Example 1 2 Mass spectrum.

[0054] Figure 5 This is the cleavage pathway of LJ-API in Example 1.

[0055] Figure 6 UPLC (368 nm) (a) and LC-MS total ion current (b) of the visible foreign matter in Example 1.

[0056] Figure 7 The main peak MS spectra (a) and MS of the visible foreign matter in Example 1 are shown in FIG. 2 Spectrum (b).

[0057] Figure 8 The crystal forms of the visible foreign matter (a), the copolymer of streptomycin A and polyvinylpyrrolidone K30 (b) and the streptomycin A raw material (c) in Example 1 are compared.

[0058] Fig. 9 This is a photo of Example 2. When the temperature was raised to 77°C, the solution became clear and transparent (left). When left at room temperature overnight, a precipitate formed (middle). After heating again, the precipitate could not be dissolved (right). DETAILED DESCRIPTION

[0059] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0060] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0061] The armillariin injection in the following examples complies with the national drug standard No. WS-10001-(HD-1207)-2002.

[0062] The preparation process of the lincomycin injection in the following examples is as follows: (1) Take povidone K30 (5-20g) and dissolve it in anhydrous ethanol, stir and fully dissolve it; then add lincomycin A (1-5g), heat and stir to mix, keep warm at 50℃~80℃ for 20~30min, recover ethanol, and vacuum dry to prepare lincomycin A-povidone K30 copolymer (specifically, the ratio of povidone K30, lincomycin A and anhydrous ethanol in the following examples is 5g:1g:120mL). (2) Take lincomycin A-povidone K30 copolymer (5-12g) and dissolve it in dimethylacetamide (20-50mL), stir and fully dissolve it. Add propylene glycol (200-400g), polysorbate 80 (3-8g), polyethylene glycol 400 (8-30g), polyethylene glycol 6000 (30-70g) and thiourea (0.3-1g) in succession, add water for injection to 2000mL, stir well, adjust the pH to 4.8-5.2, filter after mixing, fill with ampoules at 10mL each, and sterilize to obtain the product.

[0063] The preparation process of the blank excipient in the following examples is as follows: except that the armillaria sibiricum A raw material is not added, the rest of the preparation method of armillaria sibiricum A injection is followed to obtain a blank excipient sample.

[0064] The meanings of the abbreviations in the following embodiments are as follows:

[0065] LJ-I: armillariin A injection;

[0066] LJ-API: Armillariin A (API);

[0067] UPLC: ultra-performance liquid chromatography;

[0068] UPLC-MS / MS: ultra-performance liquid chromatography-mass spectrometry;

[0069] XRD: X-ray polycrystalline diffraction.

[0070] Example 1: Establishment of a method for identifying endogenous visible foreign matter in armillariin injection

[0071] 1. Visible foreign matter dissolution test

[0072] 1. Preparation of solution:

[0073] (1) Preparation of API, photolysis products and polymer solutions

[0074] Weigh 12.5 mg of API, photolysis product and povidone K30 respectively into a 50 mL volumetric flask and make up to volume with methanol; then transfer 1 mL into a 50 mL volumetric flask and make up to volume with methanol to obtain a sample solution with a concentration of 5 μg / mL.

[0075] Among them, the raw material of armillaria acetophenone is a product produced by Shanxi Tongji Pharmaceutical Co., Ltd., with the product number 200401;

[0076] Photolysis product: Take an appropriate amount of the raw material of streptomycin A, spread it on a flat dish, place it in a 6000 Lux light box for 24 hours, and then seal it in a light-proof place for later use.

[0077] (2) Blank excipient solution

[0078] Pipette 1 mL of blank excipient into a 10 mL volumetric flask and dilute to volume with methanol to obtain blank excipient solution.

[0079] (3) Preparation of ammonia test solution and visible foreign matter sample solution

[0080] Pipette 400 mL of concentrated aqueous ammonia (wt 25%) into a 1000 mL volumetric flask and add water to make up to volume to obtain an ammonia test solution.

[0081] Take several vials of leucine injection (packaging specifications are 10mL: 5mg per vial), let it stand for 2 minutes, and remove the supernatant with a syringe (only retain the large-particle needle crystals at the bottom); take another syringe, draw 0.5mL of blank excipient solution, rinse the inner wall of the ampoule, shake slowly, let it stand, and remove the supernatant, repeat twice, and observe the changes in the crystal properties during the process; draw 0.5mL of methanol, rinse the inner wall and needle crystals in the same way, repeat twice, let it stand, and remove the supernatant. Add 0.5mL of ammonia test solution / water (1:4) diluent to the residual crystals, shake until the crystals disappear, and dilute to 10mL with methanol to obtain a visible foreign body sample solution.

[0082] From the formula and production process of LJ-I, it can be seen that the above-mentioned visible foreign matter (crystals) may come from the API or polymerizer, but not from the cosolvent, surfactant or antioxidant. Therefore, by comparing the HPLC chromatograms of the blank excipient and LJ-API, combined with the LC-MS / MS spectra of the photodegradation products, the ammonia test solution dissolved products and the finished product of the armillaria injection, the source of the visible foreign matter can be determined.

[0083] Literature reports that the stability of LJ-I is affected by multiple factors such as light, high temperature, and pH, resulting in decomposition and deterioration. The visible foreign matter in LJ-I is needle-shaped crystals. Because it is difficult to dissolve in reagents such as methanol and acetonitrile (a small amount of solvent), in order to facilitate liquid chromatography-mass spectrometry detection, the ammonia test solution used in the LJ-I national drug standard was selected to dissolve the crystals, and the stability was investigated (the results showed that degradation occurred after dissolving in ammonia test solution for 2 hours, so the samples of visible foreign matter were prepared and used immediately when detecting them in the experiment). After multiple enrichment of the needle-shaped crystal samples, the crystals were light yellow needle-shaped crystals. After the sample was rinsed with a blank auxiliary liquid, a large volume of methanol was added and ultrasonic dissolution was performed, and the color of the resulting solution was consistent with that of LJ-I.

[0084] 2. UPLC Analysis

[0085] Chromatographic conditions:

[0086] LC system: ACQUITY UOLC; chromatographic column: Waters Symmetry C18 (3.5μm, 4.6×75mm); mobile phase: methanol-0.1mol / L acetic acid solution (50:50), elution mode: isocratic elution; flow rate: 0.4mL / min; column temperature: room temperature; detection wavelength: 368nm; injection volume: 20μL.

[0087] Under the above detection conditions, the UPLC chromatograms of LJ-API, photolysis samples, polymers and blank excipients are shown in Figure 2. Figure 1. The UV spectra of the photolysis samples and polymers at 368nm are consistent with the peak position of LJ-API, and both have obvious mountain-shaped characteristic peaks; the blank excipients have no obvious absorption at 368nm. Referring to relevant literature and the formula and production process of LJ-I, it can be seen that the above-mentioned visible foreign matter (crystals) may come from LJ-API or polymerizers, but cannot come from cosolvents, surfactants or antioxidants. Therefore, by comparing the visible foreign matter with the ultra-high performance liquid chromatograms of LJ-API, blank excipients and photodegradants, the source of the visible foreign matter can be determined.

[0088] 3. UPLC-MS / MS Analysis of LJ-API and Visible Foreign Matter

[0089] Mass spectrometry conditions:

[0090] MS system: Xevo TQ-S, electrospray ionization source, negative ion detection mode, capillary voltage: 19 V; capillary temperature: 350 °C; source temperature: 150 °C; shell gas flow rate: 45 arb, auxiliary gas flow rate: 0 arb; spray voltage: 4.5 kV.

[0091] UPLC (368 nm) and LC-MS total ion current of LJ-API are shown in Figure 2 From the ESI-MS mass spectrum of LJ-API, it can be seen that LJ-API is prone to lose 1 H + , forming a quasi-molecular ion m / z 233.08 [MH] - ( Figure 3 ), and the abundance is relatively high. This indicates that in negative ion mode, LJ-API quasi-molecular ion ESI-MS is based on [MH] - It exists and has good ionic stability.

[0092] Further investigation on the molecular ion m / z 233[MH] - Perform secondary scan, ESI-MS 2 Scanning mass spectrum Figure 4 , and get [MH] - The structural information of the precursor ion is included, and it mainly produces 10 relatively abundant fragment ions with m / z of 214.99, 203.06, 191.02, 189.02, 171.02, 162.99, 161.01, 158.95, 147.01 and 135.01 respectively.

[0093] The fragmentation pathway is speculated to be Figure 5 :

[0094] (1) Quasi-molecular ion m / z 233 [MH] -Losing one molecule of HCHO produces a fragment ion of m / z 203.06 (M1), and M1 can successively break down and lose one molecule of CH2=C=O and CO to produce fragment ions of m / z 161.01 and m / z 135.01;

[0095] (2) Quasi-molecular ion m / z 233 [MH] - The loss of one molecule of CO2 produces a fragment ion of m / z 189.02 (M2); the loss of one molecule of H2O produces a fragment ion of m / z 171.02; or the loss of one molecule of CH2=C=O produces a fragment ion of m / z 147.01; or the loss of one molecule of HCHO produces a fragment ion of m / z 158.95;

[0096] (3) Quasi-molecular ion m / z 233 [MH] - Loss of one molecule of H2O forms a fragment ion of m / z 214.99 (M3);

[0097] (4) Quasi-molecular ion m / z 233 [MH] - The loss of one CH2=C=O molecule generates a fragment ion of m / z 191.02 (M4), and the loss of one H2O molecule generates a fragment ion of m / z 162.99, and the further loss of one H2O molecule generates a fragment ion of m / z 147.01.

[0098] It can be seen that the peak positions and MS spectra of the foreign matter are consistent with those of LJ-API ( Figure 6 and Figure 7 ), which was determined to be the crystals of LJ-API itself. Based on the UPLC-MS / MS secondary mass spectrometry results of the finished product of armillaria injection and visible foreign matter, combined with the possible cleavage law of LJ-API, it was inferred that the occasionally visible foreign matter was the precipitation of the API or the API that was not completely polymerized and encapsulated.

[0099] 4. Analysis of the sources and causes of visible foreign matter

[0100] XRD test sample preparation: (1) LJ-API: Take an appropriate amount of API, freeze-dry under light-shielding, store in a black sealed bag for later use, and grind into powder before testing. (2) Armillariin A-Povidone K30 Copolymer: Take 20g of Povidone K30 and dissolve it in 480mL of anhydrous ethanol, stir until fully dissolved, then add 4g of Armillariin A API, stir and mix, keep at 50℃-80℃ for 20-30min, recover ethanol, and dry in vacuum to prepare Armillariin A-Povidone K30 Copolymer. (3) Visible foreign matter: Take several vials of the above-mentioned armillarin injection (packaging specification is 10 mL per vial), shake them and combine them in a brown reagent bottle, let them stand for 2 minutes, and remove the supernatant liquid with a syringe (only retain the large particles of needle crystals at the bottom); take another syringe, draw 0.5 mL of blank excipient solution, rinse the inner wall of the reagent bottle, shake slowly, let it stand, and remove the supernatant liquid, repeat twice, and observe the changes in the crystal properties during the process; then draw 0.5 mL of methanol, rinse the inner wall and needle crystals in the same way, repeat twice, let it stand, and remove the supernatant liquid. The remaining crystals are shielded from light and vacuum dried for use, and taken out and ground into powder before testing.

[0101] X-ray powder diffraction conditions: Japan Rigaku XRD X-ray polycrystalline diffractometer (Smart Lab); working power 1.6 kW (40 kV, 40 mA); scanning range 3° to 90°; step size 0.02, target material is Cu target.

[0102] The experimental results are as follows Figure 8 shown.

[0103] from Figure 8 It can be clearly seen that LJ-API, armillaria acetophenone-povidone K30 copolymer and visible foreign matter have great differences in crystal form. The positions and intensities of their diffraction peaks are obviously different (the maximum intensity ratio of the diffraction peaks at 2θ 11 and 12 of the visible foreign matter is 1:1.6). Based on the structural identification results of the visible foreign matter, it is preliminarily inferred that the visible foreign matter may be caused by certain specific factors (such as trace impurities, critical temperature, etc.) that cause a certain type of crystal component in the raw material to recover from the copolymer and precipitate (such as Figure 8 The results of XRD (3°~90°) polycrystalline diffractometer scanning showed that the copolymerization of LJ-API and povidone K30 at a ratio of 1:5 and 65°C can cause the original crystal form to be lost and exist in the povidone skeleton in an amorphous form. The interaction between the two materials is the main factor that inhibits the reformation of the API crystal.

[0104] Example 2: Verification of the source of visible foreign matter

[0105] 1. Gradient temperature precipitation experiment

[0106] According to the crystal characteristics of the visible foreign matter and its relationship with the crystal form of API under the production conditions of the intermediate polymer (60℃-80℃ water bath insulation for 0.5h, raising the water bath temperature to recover ethanol), and by comparing with references, the copolymerization dissolution temperature can be appropriately controlled. In the experiment, the dissolution of armillaria acetophenone was observed when copolymerized with povidone K30 and anhydrous ethanol by setting the insulation temperature to 50℃, 55℃, 60℃, 65℃, 70℃, and 75℃ respectively.

[0107] Take 1g of streptomycin A, 5g of povidone K30, and 120mL of anhydrous ethanol respectively for use; add 50mL of anhydrous ethanol to the beaker containing povidone K30, stir and dissolve completely in ultrasound; pour the dissolved liquid into a round-bottom flask, scrub the beaker containing povidone K30 with a small amount of anhydrous ethanol, and pour the scrubbing liquid into the round-bottom flask; add the remaining anhydrous ethanol in small amounts and multiple times to the beaker containing streptomycin A, stir and pour into the round-bottom flask, but streptomycin A is not dissolved. Increase the temperature of the insulating water bath from 50℃, 55℃, 60℃, 65℃, 70℃, and 75℃ in sequence, and it can be observed that streptomycin A has not been completely dissolved in the povidone K30-anhydrous ethanol solution; when the temperature rises to 75℃, there is still a small amount of streptomycin A that has not been completely dissolved, and the solution is not transparent; until 77℃, streptomycin A is completely dissolved, and the solution is clear and transparent (such as Fig. 9 Left), take out the round-bottom flask, and place the dissolved solution at room temperature overnight. There is precipitation at the bottom of the solution (such as Fig. 9 ), but the precipitate cannot be dissolved after heating (such as Fig. 9 right).

[0108] The above precipitate was prepared into a visible foreign matter sample solution and a visible foreign matter sample as described in Example 1, and LC-MS / MS analysis and XRD analysis were performed. The obtained ESI-MS mass spectrum and ESI-MS 2 The mass spectrum is similar to the spectrum of the above-mentioned visible foreign matter ( Figure 7 ) is consistent with the XRD spectrum obtained above. Figure 8 ) are consistent with the position and intensity of the diffraction peaks of LJ-I. Therefore, it can be determined that the visible foreign matter in the above LJ-I is the raw material drug component precipitated from the copolymer of streptomycin A and polyvinylpyrrolidone K30. The product quality can be improved by optimizing the configuration temperature, changing the stirring mode and stirring times, and other measures as well as process verification.

[0109] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, it is not necessary to repeat the verification experiment, and the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departing from the disclosed scope in the application and the changes made with conventional techniques known in the art.

Claims

1. A method for identifying endogenous visible foreign matter in armillariin injection, comprising the following steps: Performing ultra-high performance liquid chromatography-mass spectrometry analysis and X-ray polycrystalline diffraction analysis on the visible foreign matter in the armillaria injection respectively to obtain ultra-high performance liquid chromatography-mass spectrometry, mass spectrometry and X-ray polycrystalline diffraction patterns. If the test results meet the following a), b) and c), the visible foreign matter is determined to be the raw material drug component precipitated from the armillariaria-povidone K30 copolymer; a) The peak position of the visible foreign matter in the ultra-high performance liquid chromatogram is consistent with the peak position of the raw material drug armillariin A in the ultra-high performance liquid chromatogram; b) The mass spectrum of the visible foreign matter is consistent with the mass spectrum of the raw material armillariin A; c) The X-ray polycrystalline diffraction pattern of the visible foreign matter has only characteristic diffraction peaks at 11 and 12 in the scanning range of 3° to 90° at 2θ; The visible foreign matter is yellow-brown needle-shaped crystals; The armillary mitogen injection uses povidone K30 as a polymerization agent, and the armillary mitogen exists in the form of an armillary mitogen-povidone K30 copolymer, and the armillary mitogen-povidone K30 copolymer is prepared using anhydrous ethanol as a solvent; The method for preparing a sample solution of visible foreign matter for ultra-high performance liquid chromatography-mass spectrometry analysis comprises the following steps: taking a leucine injection solution, standing it to remove the supernatant and retaining the visible foreign matter at the bottom; washing the visible foreign matter with a blank auxiliary material solution and methanol in sequence, dissolving the visible foreign matter with an ammonia solution after washing and diluting it with methanol to obtain a sample solution of the visible foreign matter; The chromatographic conditions in the ultra-high performance liquid chromatography-mass spectrometry analysis are as follows: LC system: ACQUITY UOLC; Chromatographic column: Waters Symmetry C18, 3.5 μm, 4.6 × 75 mm; Mobile phase: composed of methanol and 0.1 mol / L acetic acid aqueous solution in a volume ratio of 1:1; Elution mode: isocratic elution; Flow rate: 0.4 mL / min; Column temperature: room temperature; Detection wavelength: 368 nm; Injection volume: 20 μL; The mass spectrometry conditions in the ultra-high performance liquid chromatography-mass spectrometry analysis are as follows: MS system: Xevo TQ-S; Electrospray ionization source; Negative ion detection mode; Capillary voltage: 19V; Capillary temperature: 350℃; Source temperature: 150°C; Shell gas flow rate: 45 arb; Auxiliary gas flow rate: 0 arb; Spray voltage: 4.5 kV; The conditions of the X-ray polycrystal diffraction analysis are as follows: Japan Rigaku XRD X-ray polycrystal diffractometer; Working power: 1.6 kW, 40 kV, 40 mA; Scanning range: 3°~90°; Step distance: 0.02; Target material: Cu target.

2. The method for identifying endogenous visible foreign matter in armillariin injection according to claim 1, characterized in that: The peak position in the ultra-high performance liquid chromatogram of the raw material drug is at a retention time of 3.746 min; The peak position in the ultra-high performance liquid chromatogram of the visible foreign matter is at a retention time of 3.667 min.

3. The method for identifying endogenous visible foreign matter in armillariin injection according to claim 1, characterized in that: The mass spectrum of the visible foreign matter is consistent with the mass spectrum of the raw material drug armillariin A, which is the quasi-molecular ion of the visible foreign matter. m / z 233[MH] - The secondary fragment ions and the quasi-molecular ions of the raw material drug armillariin A m / z 233 [MH] - The secondary fragment ions are consistent with those of 4. The method for identifying endogenous visible foreign matter in armillariin injection according to claim 3, characterized in that: Quasi-molecular ion of the raw material drug armillariin A m / z 233 [MH] - Perform a secondary scan to generate m / z There are 10 relatively abundant fragment ions, namely 214.99, 203.06, 191.02, 189.02, 171.02, 162.99, 161.01, 158.95, 147.01 and 135.

01.

5. The method for identifying endogenous visible foreign matter in armillariin A injection according to any one of claims 1 to 4, characterized in that: The preparation method of a visible foreign matter sample for X-ray polycrystalline diffraction analysis comprises the following steps: taking a leucine injection solution, standing it to remove the upper clear liquid and retaining the visible foreign matter at the bottom; washing the visible foreign matter with a blank auxiliary material solution and methanol in turn, and vacuum drying after washing to obtain a visible foreign matter sample.

6. The method for identifying endogenous visible foreign matter in armillariin injection according to claim 5, characterized in that: In the X-ray polycrystalline diffraction pattern of the visible foreign matter, the maximum intensity ratio of the diffraction peaks at 2θ of 11 and 12 is 1:1.6.

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