Method for detecting applicable impurities of nystatin system and application of method

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for detecting applicable impurities of a nystatin system and application of the method. Specifically, research finds that structural information of an impurity A and an impurity B in a system applicable solution of nystatin is not reported in existing pharmacopoeia standards and related literatures of nystatin, and the impurity A and the impurity B are main degradation impurities of nystatin and related preparations and have a growth trend in a stable period. Therefore, the preparation and structural analysis of the two impurities are of great significance. Meanwhile, the invention develops a method for detecting the impurity A and the impurity B in nystatin and related preparations, and the method has good specificity, accuracy and repeatability, so that the method has good practical application value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, specifically to a method for detecting impurities suitable for nystatin systems and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Nystatin is an antifungal substance obtained through fermentation using a specific strain of *Streptomyces northerly* as the production microorganism. It is an antifungal drug with a conjugated polyene macrocyclic lactone structure, exhibiting broad-spectrum antifungal activity. It shows high antifungal activity against *Candida* spp., and also against *Cryptococcus neoformans*, *Aspergillus*, *Mucor*, *Microsporum*, *Histoplasma capsulatum*, *Blastomyces dermatitidis*, and dermatophytes. It has no inhibitory effect on bacteria. Nystatin is the first-line drug for oral candidiasis and thrush in Europe and the United States, and can also be used to treat enteritis. Nystatin has a good safety profile, and shows excellent clinical efficacy, especially in high-risk populations such as infants and young children.

[0004] Nystatin, in its pure form, is a yellow crystalline solid that readily decomposes upon exposure to light and heat. It is hygroscopic, insoluble in acetone and chloroform, very slightly soluble in water, slightly soluble in lower alcohols, and soluble in N,N-dimethylformamide. It is unstable in acids and alkalis, and its solution state is also unstable. It is relatively stable in neutral or weakly alkaline environments. Current pharmacopoeia standards and literature only report on the control of nystatin components; unfortunately, there are no reports on research into related substances. Neither the pharmacopoeia nor related literature lists the names and structures of the two impurities in the system suitability solution under the nystatin component test. Furthermore, the currently available impurity reference standards for this item do not contain these two impurities. These two impurities are named Nystatin Impurity A and Nystatin Impurity B, respectively, based on their peak elution time. Impurities A and B are the two most significant degradation impurities during the stability process of nystatin and related preparations. They are produced under both humid and acidic conditions, and there is an increasing trend in the levels of impurities A and B in nystatin during the stability period. Therefore, it is of great significance to prepare and analyze impurities A and B in the system suitability solution, and to study the detection methods for them, so as to ensure the safety and effectiveness of nystatin and related preparations. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to provide a method for detecting system suitability impurities in nystatin and its application. Specifically, this invention, through research, discovered that neither the current pharmacopoeia standard for nystatin nor related literature reports the structural information of impurities A and B in its system suitability solution. Furthermore, impurities A and B are major degradation impurities in nystatin and related preparations, and both show an increasing trend during the stability period. Therefore, the preparation and structural analysis of these two impurities are of great significance. Based on the above research results, this invention is thus completed.

[0006] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution:

[0007] A first aspect of the present invention provides a method for detecting impurities suitable for nystatin systems, the method comprising: High performance liquid chromatography was used to detect impurities A and B in nystatin and related preparations. The chemical structural formula of impurity A is as follows:

[0008] The chemical structural formula of impurity B is: .

[0009] Specifically, the specific conditions for the high-performance liquid chromatography method include: The chromatographic column was packed with octadecyl-bonded silica gel, specifically YMC Triart C18, 4.6 × 250 mm, 5 μm, with a detection wavelength of 305 nm; flow rate: 0.8 ml / min ~ 1.5 ml / min; injection volume: 10 ~ 50 μl (preferably 20 μl); column temperature: 25 ~ 40 °C; mobile phase A: 0.01 ~ 0.05 mol / L ammonium acetate buffer, mobile phase B: acetonitrile; linear gradient elution was performed.

[0010] Furthermore, the specific conditions for the gradient elution are as follows: time (minute) Flow A(%) Flow B(%) 0 70 30 45 69 31 70 65 35 80 35 65 81 70 30 90 70 30 Because nystatin has a complex macrocyclic lactone structure, preparing these two impurities using chemical synthesis is very difficult. Therefore, preparing them using preparative liquid chromatography is more feasible. Thus, in another specific embodiment of the present invention, impurities A and B can be prepared using preparative liquid chromatography and then lyophilized. In another specific embodiment of the present invention, the mobile phase for the preparative liquid chromatography is acetonitrile-water.

[0011] In another specific embodiment of the present invention, the volume ratio of acetonitrile to water in the mobile phase is (20:80) to (40:60); preferably 35:65.

[0012] In another specific embodiment of the present invention, the flow rate of the preparative liquid chromatograph is 15~25 ml / min.

[0013] In another specific embodiment of the present invention, the detection wavelength of the preparative liquid chromatograph is 305 nm.

[0014] In another specific embodiment of the present invention, the column temperature of the preparative liquid chromatograph is 30°C.

[0015] In another specific embodiment of the present invention, the injection volume of the preparative liquid chromatograph is 0.5~5 ml.

[0016] In another specific embodiment of the present invention, the chromatographic column used for the preparative liquid chromatography is a YMC Triart C18-S, 20×250mm, 5μm.

[0017] In another specific embodiment of the present invention, the freeze-drying temperature is -60 to -80°C.

[0018] In another specific embodiment of the present invention, the freeze-drying preparation time is 5 to 10 hours.

[0019] A second aspect of the present invention provides the application of the above-described detection method in the quality control of nystatin.

[0020] The beneficial technical effects of one or more of the above technical solutions are as follows: The above technical solution provides a method for preparing and separating impurities A and B suitable for nystatin systems. Impurities A and B are prepared by freeze drying, which is simple to operate. The structure of the prepared impurities A and B suitable for nystatin systems has been analyzed and reported. A detection method for impurities A and B in nystatin and related preparations has been developed. The method has good method specificity, accuracy and repeatability, and therefore has good practical application value. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 This is a diagram showing the structural location of impurity A prepared according to the present invention; Figure 2 This is the high-resolution mass spectrum of impurity A prepared in this invention; Figure 3 Impurity A prepared in this invention1 H-NMR spectrum; Figure 4 Impurity A prepared in this invention 13 C-NMR spectrum; Figure 5 This is a diagram of impurity A DEPT135 prepared according to the present invention; Figure 6 Impurity A prepared in this invention 1 H 1 H-COSY diagram; Figure 7 This is the HSQC diagram of impurity A prepared according to the present invention; Figure 8 This is the HMBC diagram of impurity A prepared according to the present invention; Figure 9 This is a diagram showing the structural location of impurity B prepared in this invention; Figure 10 This is the high-resolution mass spectrum of impurity B prepared in this invention; Figure 11 Impurity B prepared in this invention 1 H-NMR spectrum; Figure 12 Impurity B prepared in this invention 13 C-NMR spectrum; Figure 13 This is a diagram of impurity B DEPT135 prepared according to the present invention; Figure 14 Impurity B prepared in this invention 1 H 1 H-COSY diagram; Figure 15 This is the HSQC diagram of impurity B prepared according to the present invention; Figure 16 This is the HMBC diagram of impurity B prepared according to the present invention; Figure 17 This is a typical chromatogram showing that nystatin did not destroy the structure in this invention; Figure 18 This is a typical chromatogram of the high-temperature destruction of nystatin solution in this invention; Figure 19 This is a typical chromatogram of nystatin acid destruction in this invention. Detailed Implementation

[0023] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] It should be noted that the terminology used herein is for descriptive purposes only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. This invention utilizes techniques and methods conventional in the fields of genetic engineering and molecular biology. Those skilled in the art can employ other conventional techniques, methods, and reagents in the art based on the embodiments provided in this invention, without being limited to the specific embodiments of this invention.

[0025] Example I. This impurity was prepared using preparative liquid chromatography. The preparation method is as follows: Nystatin: Manufacturer: ANTIBIOTICE SA, Batch No.: 4011348.

[0026] System suitability solution: Weigh 0.1g of nystatin, add 40ml of methanol to dissolve it, add 10ml of dilute hydrochloric acid, mix well, let stand at room temperature for 1h, and take the supernatant to prepare the solution.

[0027] Column: Octadecyl bonded silica gel (YMC Triart C18-S, 20×250mm, 5μm) Detection wavelength: 305nm Flow rate: 20 ml / min Mobile phase: Acetonitrile-water (35:65) Injection volume: 1 ml The corresponding fractions at approximately 16 min and 26 min were collected and freeze-dried in a freeze dryer at -70℃ for approximately 8 hours to obtain solid impurity A and impurity B.

[0028] The obtained solid impurities A and B were respectively subjected to HRMS. 1 H-NMR, 13 C-NMR, DEPT135, 1 H 1 A comprehensive analysis was conducted using H-COSY, HSQC, HMBC, NOESY, etc., and the specific results are as follows: Impurity A: Molecular formula: C 47 H 72 NO 16 Molecular weight: 907.49 Chemical structural formula:

[0029] 1. High-resolution mass spectrometry: A quasi-molecular ion peak was captured in the high-resolution mass spectrometry (Waters Xevo G2-XS QTof) at m / z 906.4857 [MH]. - , and the predicted molecular formula C 47 H 72 NO 16 [MH] - The theoretically calculated value of 906.4851 is consistent; the calculated degree of unsaturation is 12; and it is consistent with the structural formula of the corresponding Impurity A.

[0030] 2. Nuclear magnetic resonance 1 ¹H-NMR (Bruker AVANCE III 600 MHz at 600 MHz, in DMSO-d6) showed a total of 31 peaks with 62 hydrogen protons, of which 11 active hydrogen protons did not produce a signal; 30 methine hydrogens showed δ-signals. H 1.80 (1H, overlap), δ H 2.22(1H, overlap), δ H 2.89 (1H, brs), δ H 1.81 (1H, overlap), δ H 4.01 (1H, m), δ H 3.30 (1H, m), δ H 3.83 (1H, overlap), δ H 3.81 (1H, overlap), δ H 3.67 (1H, m), δ H 3.58 (1H, m), δ H 3.18 (1H, overlap), δ H 5.00 (1H, m), δ H 3.91(1H,brs), δ H 3.19 (1H, overlap), δ H 4.35 (1H, brs), δ H 3.16(1H, overlap), δ H 3.73 (1H, m), δ H 4.49 (1H, brs), δ H 5.93 (1H, overlap), δ H 5.61(1H,m), δ H 5.46 (1H, m), δ H5.49-6.30 (9H, overlap) correspond to H-22, H-24, H-58, H-8, H-17, H-7, H-15, H-59, H-9, H-13, H-57, H-21, H-5, H-56, H-39, H-23, H-4, H-54, H-26, H-38, H-25, H-27, 28, 31, 32, 33, 34, 35, 36, and 37 in the structure, respectively; 10 methylene hydrogen signals δ are present. H 1.50(1H,overlap)&δ H 1.65 (1H, overlap), δ H 2.13(2H,overlap)&δ H 2.17(2H, overlap), δ H 1.37(1H,overlap)&δ H 1.28 (1H, m), δ H 1.93(1H,m)&δ H 1.64 (1H, overlap), d H 2.27(1H, overlap)& d H 2.35 (1H, overlap), d H 1.35(1H, overlap)& d H 1.87 (1H, brs), d H 2.08 (1H, overlap) & d H 1.66 (1H, overlap), d H 1.53(2H, overlap), d H 1.48(1H, overlap)& d H 1.42 (1H, overlap) corresponds to H-11, H-29 & 30, H-12, H-40, H-18, H-10, H-6, H-16, and H-14 in the structure, respectively; four methyl hydrogen signals are present. d H 0.88(3H,d, J =6.42), d H 1.10(3H,d, J =5.87), d H0.97(3H,d, J =5.87), d H 1.15(3H,brs) corresponds to H-50, H-49, H-52, and H-61 in the structure, respectively.

[0031] 13 C-NMR(Bruker AVANCE III 600 MHz at 150 MHz, in DMSO- d 6) The display shows a total of 47 carbon signals. Combining DEPT135 and HSQC-DEPT spectra, it was determined that there are 3 quaternary carbon signals, 30 tertiary carbon signals, 10 secondary carbon signals, and 4 primary carbon signals. Three quaternary carbon signals are present. d C 105.61, d C 170.48, d C 176.03, which corresponds to C-2, C-19, and C-42 in the structure, respectively; there are 30 tertiary carbon signals. d C 40.34, d C 40.74, d C 55.81, d C 58.37, d C 65.63, d C 67.41, d C 67.74, d C 67.89 (overlap), d C 69.57, d C 69.76, d C 70.34, d C 70.40, d C 72.71, d C 73.78, d C 76.14, d C 81.78, d C 96.54, d C 129.21, d C 129.48 d C 134.72, d C 135.37, d C Ranges 129.21–134.17 correspond to C-22, C-24, C-58, C-8, C-17, C-7, C-15, C-59 & 9, C-9, C-13, C-57, C-21, C-5, C-56, C-39, C-23, C-4, C-54, C-26, C-38, C-25, C-27 & 28 & 31 & 32 & 33 & 34 & 35 & 36 & 37 in the structure, respectively; 10 secondary carbon signals are present. d C 24.37, d C 31.64, d C 32.03, d C 33.98, d C 36.09, d C 42.58, d C 43.81, d C 43.96, d C 44.14, d C 44.49, which corresponds to C-11, C-29, C-30, C-12, C-40, C-18, C-10, C-6, C-16, and C-14 in the structure, respectively; there are 4 primary carbon signals. d C 12.36, d C 17.27, d C 17.86 & 17.88 correspond to C-50, C-49, C-52 & 61 in the structure, respectively.

[0032] 1 H 1 H-COSY d H 3.91(H-5) and d H 1.66 & 2.08 (H-6), d H 3.73(H-4) related, d H1.50 & 1.65 (H-11) and d H 3.73(H-4), d H 1.37 (H-12) related, d H 3.58 (H-13) and d H 1.37 & 1.28 (H-12), d H 1.42 & 1.48 (H-14) are related. d H 3.83(H-15) and d H 1.53 (H-16), d H 1.42 & 1.48 (H-14) are related. d H 4.01 (H-17) and d H 1.53 (H-16), d H Related to 2.27 & 2.35 (H-18), in HMBC d H 4.01 (H-17), d H 2.27 & 2.35 (H-18), d H 5.00(H-21) are all related to d C 170.48 (C-19) is relevant. 1 H 1 HCOSY d H 5.00 (H-21) and d H 1.10(H-49), d H 1.80(H-22) related, d H 1.80(H-22) and d H 0.88(H-50), d H 3.16(H-23), d H 5.00 (H-21) related, d H 2.22(H-24) and d H 3.16(H-23), dH 0.97(H-52), d H 5.46 (H-25) related, d H 2.13(H-29) and d H 2.17 (H-30) related, in HMBC d H 2.22(H-24) and d C 129.48 (C-26) is relevant, as shown in the HSQC spectrum. d H At positions 5.49-6.30, d C There are also 9 olefin bond carbon signals at positions 129.21-134.17. 1 H 1 H-COSY d H 5.61(H-38) and d H 6.19 (H-37), d H 4.35 (H-39) related, d H 1.93 & 1.64 (H-40) and d H 4.35 (H-39), d H 3.30(H-7) related, d H 1.81(H-8) and d H 3.30(H-7), d H 3.67(H-9) related, d H 3.67(H-9) and d H 1.35 (H-10) related, in HMBC d H 1.81(H-8) and d C 176.03 (C-42) is relevant. d H 1.35 & 1.87 (H-10), d H 2.08 (H-6), d H 3.91(H-5) are both related to d CBased on the correlation at 105.61 (C-2) and the chemical shift values ​​at C-2 and C-4, it is inferred that the structure contains a structural fragment of 3,7,9,11,17,37-hexahydroxy-15,16,18-trimethyl-13-oxo-14,39,40-trioxatricyclo[33.3.1.11,4]tetracarbon-19,21,25,27,29,31-hexaene-36-carboxylic acid.

[0033] 1 H 1 H-COSY d H 3.19 (H-56) and d H 1.15(H-61), d H 3.18 (H-57) related, d H 2.89 (H-58) and d H 3.81 (H-59), d H 3.18 (H-57) related, d H 3.81(H-59) and d H The presence of 4.49 (H-54) suggests the presence of a 4-amino-6-methyltetrahydro-2H-pyran-2,3,5-triol fragment in the structure. (HMBC) d H 4.49(H-54) and δ C Related to 73.78 (C-39), it is speculated that the O-53 position of the 4-amino-6-methyltetrahydro-2H-pyran-2,3,5-triol structural fragment is connected to the C-39 position of the 3,7,9,11,17,37-hexahydroxy-15,16,18-trimethyl-13-oxo-14,39,40-trioxatricyclo[33.3.1.11,4]tetracarbon-19,21,25,27,29,31-hexaene-36-carboxylic acid structural fragment.

[0034] Impurity B: Molecular formula: C 47 H 73 NO 16 Molecular weight: 907.49.

[0035] Chemical structural formula:

[0036] 1. High-resolution mass spectrometry The quasi-molecular ion peak was captured in high-resolution mass spectrometry (Waters Xevo G2-XS QTof) at m / z 906.4857 [MH]. - , and the predicted molecular formula C 47 H 72 NO 16 [MH] - The theoretically calculated value of 906.4851 is consistent; the calculated degree of unsaturation is 12; and it is consistent with the structural formula of ImpurityB in Table 1.

[0037] 2. Nuclear magnetic resonance 1 H-NMR (Bruker AVANCE III, 600 MHz at 600 MHz, in DMSO-d6) showed 26 peaks with a total of 62 hydrogen protons, of which 11 active hydrogen protons did not produce a signal; 30 methine hydrogens were present. d H 2.50 (1H, overlap), d H 1.86 (1H, overlap), d H 2.87 (1H, brs), d H 1.78 (1H, m), d H 3.65 (1H, overlap), d H 3.97 (1H, m), d H 3.86 (1H, brs), d H 3.24 (1H, m), d H 3.65 (1H, overlap), d H 3.79 (1H, overlap), d H 3.62 (1H, overlap), d H 3.18 (1H, overlap), d H 3.92 (1H, brs), d H 3.16 (1H, overlap), d H 4.29 (1H, brs), d H 4.70(1H,brd,J =10.09), d H 3.78 (1H, overlap), d H 4.48 (1H, brs), d H 5.97 (1H, overlap), d H 6.17 (1H, overlap), d H 5.59 (1H, overlap), d H 5.54 (1H, overlap), d H 5.49-6.26 (8H overlap) correspond to H-22, H-47, H-56, H-8, H-48, H-17, H-15, H-9, H-7, H-57, H-13, H-55, H-5, H-54, H-37, H-21, H-4, H-52, H-24, H-35, H-36, H-23, H-25, H-26, H-39, H-30, H-31, H-32, H-33, and H-34 in the structure, respectively; 10 methylene hydrogen signals are present. d H 1.65 (1H, overlap) & d H 1.55 (1H, overlap), d H 2.09(2H, overlap)& d H 2.18(2H, overlap), d H 1.29(1H, overlap)& d H 1.44(1H, overlap), d H 1.91(1H,overlap)& d H 1.70 (1H, overlap), d H 2.33(2H,m), d H 1.36(1H, overlap)& d H 1.87 (1H, overlap), d H 2.02(1H,m)& d H1.67 (1H, overlap), d H 1.37(2H, overlap), d H 1.50 (2H, overlap) corresponds to H-11, H-27 & 28, H-12, H-38, H-18, H-10, H-6, H-14, and H-16 in the structure, respectively; four methyl hydrogen signals are present. d H 0.79(3H,brd, J =6.42), d H 0.96(3H,brd, J =6.42), d H 0.88(3H,brd, J =5.69), d H 1.13(3H,brs) corresponds to H-59, H-50, H-60, and H-61 in the structure, respectively.

[0038] 13 C-NMR (Bruker AVANCE III 600 MHz at 150 MHz, in DMSO-d6) showed a total of 47 carbon signals. Combined with DEPT135 and HSQC-DEPT spectra, this identified 3 quaternary carbon signals, 30 tertiary carbon signals, 10 secondary carbon signals, and 4 primary carbon signals. Three quaternary carbon signals were present at δ... C 105.65, δ C 170.89, δ C 175.61, which corresponds to C-2, C-19, and C-40 in the structure, respectively; there are 30 tertiary carbon signals δ. C 36.57, δ C 40.34, δ C 55.70, δ C 58.17, δ C 64.93, δ C 65.43, δ C 67.70, δ C 67.78, δ C 67.95, δ C 67.95, δ C 69.67, δ C 69.77, δ C 70.43, δ C 72.78, δ C 75.10, δ C 76.28, δC 81.94, δ C 97.25, δ C 129.37, δ C 130.19, δ C 134.26, δ C 134.82, δ C Ranges 130.19–134.12 correspond to C-22, C-47, C-56, C-8, C-48, C-17, C-15, C-9, C-7, C-57, C-13, C-55, C-5, C-54, C-37, C-21, C-4, C-52, C-24, C-35, C-36, C-23, C-25, C-26, C-29, C-30, C-31, C-32, C-33, and C-34 in the structure, respectively; ten secondary carbon signals δ are present. C 24.39, δ C 31.70 & 31.77, δ C 33.67, δ C 36.77, δ C 42.31, δ C 43.76, δ C 44.03, δ C 44.27, δ C 44.37, which corresponds to C-11, C-27 & 28, C-12, C-38, C-18, C-10, C-6, C-14, and C-16 in the structure, respectively; there are 4 primary carbon signals δ. C 9.02, δ C 11.84, δ C 16.82, δ C 17.86, which corresponds to C-59, C-50, C-60, and C-61 in the structure, respectively.

[0039] 1 H 1 δ in H-COSY H 3.92 (H-5) and δ H 2.02 (H-6), δ H 3.78 (H-4) correlation, δ H 1.55 (H-11) and δ H 3.78 (H-4), δ H 1.44(H-12) correlation, δ H 3.62(H-13) and δ H 1.29 & 1.44 (H-12), δ H 1.37(H-14) correlation, δ H 3.86(H-15) and δ H 1.50(H-16), δH 1.37(H-14) correlation, δ H 3.97(H-17) and δ H 1.50 (H-16), δ H 2.33(H-18) related. δ in HMBC H 3.97(H-17), δ H 2.33(H-18), δ H 4.70(H-21) are all related to δ C 170.89 (C-19) is relevant. 1 H 1 δ in H-COSY H 4.70(H-21) and δ H 1.86 (H-47), δ H 2.50 (H-22) related δ H 3.65(H-48) and δ H 1.86 (H-47), δ H 0.88 (H-60) correlation, δ H 1.86(H-47) and δ H 0.79 (H-59) correlation, δ H 2.50(H-22) and δ H 0.96(H-50), δ H 4.70(H-21), δ H 5.54(H-23) correlation, δ H 5.54 (H-23) and δ H 5.97 (H-24) correlation, δ H 2.09(H-27) and δ H 2.18 (H-28) correlation, shown in the HSQC spectrum at δ H At 5.49-6.26, in δ C At positions 130.19-134.12, there are also 8 olefin bond carbon signals. 1 H 1 H-COSY δ H 5.59(H-36) and δ H 6.17(H-35), δ H 4.29(H-37) related, δ H 4.29(H-37) and δ H 5.59(H-36), δ H 1.91(H-38) & 1.70(H-38) correlation, δ H 3.65(H-7) and δ H 1.78(H-8) correlation, δ H3.24(H-9) and δ H 1.78(H-8), δ H 1.87 (H-10) correlation. δ in HMBC H 1.78(H-8) and δ C 175.61 (C-40) correlation, δ H 2.02(H-6), δ H 3.92(H-5), δ H Both 1.87 and 1.36 (H-10) are related to δ C 105.65 (C-2) is relevant. Based on the chemical shift values ​​of C-2 and C-4, it is speculated that the structure contains a structural fragment of 3,7,9,11,35-pentahydroxy-15-(3-hydroxybutane-2-yl)-16-methyl-13-oxo-14,37,38-trioxatricyclic[31.3.1.11,4]octahinoharbium ester-17,19,23,25,27,29-hexene-34-carboxylic acid.

[0040] 1 H 1 δ in H-COSY H 3.16(H-54) and δ H 1.13(H-61), δ H 3.18 (H-55) related, δ H 2.87(H-56) and δ H 3.18(H-55), δ H 3.79 (H-57) correlation, δ H 4.48 (H-52) and δ H 3.79 (H-57) related, δ in HMBC H 4.48 (H-52) and δ C The structure is associated with 72.78 (C-54), suggesting the presence of a 4-amino-6-methyltetrahydro-2H-pyran-2,3,5-triol fragment. In HMBC, δ... H 4.48 (H-52) and δ C Related to 75.10 (C-37), it is speculated that the O-51 position of the 4-amino-6-methyltetrahydro-2H-pyran-2,3,5-triol structural fragment is connected to the C-37 position of the 3,7,9,11,35-pentahydroxy-15-(3-hydroxybutane-2-yl)-16-methyl-13-oxo-14,37,38-trioxatricyclo[31.3.1.11,4]octahedral-17,19,23,25,27,29-hexene-34-carboxylic acid structural fragment.

[0041] II. High-performance liquid chromatography (HPLC) was used to detect impurities A and B in nystatin and commercially available nystatin oral suspension. The specific method is as follows: Nystatin: Manufacturer: ANTIBIOTICE SA, Batch No.: 4011348, 4010436, 4010411.

[0042] Nystatin oral suspension, source: SUBSTIPHARM, batch number: DA39.

[0043] Undamaged test solution: Weigh about 18 mg of this product, place it in a 50 ml volumetric flask, add dimethyl sulfoxide to dissolve and dilute to the mark, and shake well.

[0044] Acid-induced degradation of the test solution: Weigh approximately 18 mg of this product and place it in a 50 ml volumetric flask. Add 1 ml of 0.1 mol / L hydrochloric acid, mix well, and let stand for 0.5 h. Add 1 ml of 0.1 mol / L sodium hydroxide to neutralize, dissolve and dilute to the mark with dimethyl sulfoxide, and shake well to obtain the final product.

[0045] High-temperature destruction of the test solution: Weigh about 18 mg of this product, place it in a 50 ml volumetric flask, add 5 ml of water, destroy it at 105 °C for 20 min, dissolve and dilute to the mark with dimethyl sulfoxide, and shake well.

[0046] Column: Octadecyl-bonded silica gel (YMC Triart C18 4.6) as packing material. 250mm, 5μm) Detection wavelength: 305nm; Flow rate: 1.0 ml / min; Injection volume: 20 μl; Column temperature: 30℃; Mobile phase A: 0.02 mol / L ammonium acetate buffer; Mobile phase B: acetonitrile.

[0047] Perform linear elution according to the table below: time (minute) Flow A(%) Flow B(%) 0 70 30 45 69 31 70 65 35 80 35 65 81 70 30 90 70 30 Testing was conducted on the three batches of nystatin raw materials mentioned above. The content of impurity A was between 0 and 0.5% (of which, the content of impurity A in batch 4011348 was 0.2%). Impurity B was not detected in any of them. However, during the stability storage period, both impurities A and B increased to varying degrees.

[0048] For batch 4,011,348 of nystatin raw material, under acid degradation conditions: impurity A increased from 0.2% to 2.0%, and impurity B increased from undetectable to 0.7%. Under high-temperature degradation conditions: impurity A increased from 0.2% to 0.5%, and impurity B increased from undetectable to 0.2%.

[0049] When nystatin oral suspension was stored at 60°C for 30 days, the content of impurity A increased from 0.3% to 0.6%, and the content of impurity B increased from undetectable to 0.2%.

[0050] It is evident that impurities A and B are degradation impurities in nystatin raw materials and related formulations, which are relatively easy to generate during the stability period, and their contents easily exceed the identification limits. Therefore, the preparation and structural analysis of these two impurities are of great significance.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting impurities suitable for nystatin systems, characterized in that, The detection method comprises: The high performance liquid chromatography is used to detect impurity A and impurity B in nystatin and related preparations; The chemical structural formula of the impurity A is: The chemical structural formula of the impurity B is: 。 2. The detection method of claim 1, wherein, The specific conditions of the high performance liquid chromatography comprise: The chromatographic column uses octadecyl bonded silica as the filler, specifically YMC Triart C18, 4.6*250mm, 5μm, the detection wavelength is 305nm, the flow rate is 0.8ml / min-1.5ml / min, the injection volume is 10-50μl (preferably 20μl), the column temperature is 25-40℃, the mobile phase A is 0.01-0.05mol / L ammonium acetate buffer, the mobile phase B is acetonitrile, and linear gradient elution is performed.

3. The detection method of claim 2, wherein, The specific conditions of the linear gradient elution are as follows: 。 4. The detection method as described in claim 1, characterized in that, The impurity A and the impurity B are prepared by using a preparative liquid chromatograph and are freeze-dried to obtain.

5. The method of claim 4, wherein the detection is performed by a method selected from the group consisting of mass spectrometry, nuclear magnetic resonance, and chromatography. The mobile phase of the preparative liquid chromatograph is acetonitrile-water, and further, the volume ratio of acetonitrile-water in the mobile phase is (20:80)-(40:60).

6. The detection method as described in claim 4, characterized in that, The flow rate of the preparative liquid chromatograph is 15-25ml / min.

7. The detection method as described in claim 4, characterized in that, The detection wavelength of the preparative liquid chromatograph is 305nm.

8. The detection method as described in claim 4, characterized in that, The column temperature of the preparative liquid chromatograph is 30℃, the injection volume of the preparative liquid chromatograph is 0.5-5ml, and the chromatographic column of the preparative liquid chromatograph is YMC Triart C18-S, 20*250mm, 5μm.

9. The detection method as described in claim 4, characterized in that, The freeze-drying temperature is-60--80℃, and the freeze-drying time is 5-10 hours.

10. Application of the detection method in any one of claims 1-9 in the quality control of nystatin.

Images