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Objective To establish a quality control method for Lvxintong Rugao. Methods Ketoconazole, Halcinonide and Neomycin sulfate were identified by TLC. The content of Ketoconazole and Halcinonide were determined by HPLC. The chromatographic column of Agilent ZORBAX SB-C18 (4.6 mm×150 mm, 5 μm) column was used. Methanol-phosphate buffer (pH=7.40, 75:25) was applied as the mobile phase. The detection wavelength was 235 nm. The flow rate was 1.0 ml/min and the column temperature was set at room temperature. Neomycin Sulfate was determined by polarimetric analysis. Results The identification and determination methods showed good specificity. Ketoconazole and Halcinonide displayed good linearity within the range of 1.999~39.98 μg (r=0.999 9) and 0.400 8~8.016 μg (r=0.999 9), respectively. The average recoveries were 97.75% (RSD 0.77%) and 97.57% (RSD 0.84%), respectively. For the determination of Neomycin Sulfate, r=0.999 6 (n=6) in the range of 130.4~2 608 U/ml (n=6). The precision and repeatability of RSD were 1.1% and 1.6%, respectively. The solutions were stable in 6 h and the average recovery was 98.8% (RSD 2.6%). Conclusion The method could be used as the quality control method for Lvxintong Rugao.
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OBJECTIVE: To establish a high performance liquid chromatography combined with pulsed amperometric detection(HPLC-PAD)method for determination of potency of neomycin sulfate. METHODS: An improved HPLC-PAD method from EP method for determination of the content and related substances of neomycin sulfate was established and validated. The study of impurity profile of neomycin sulfate was completed by LC-IT-TOF method with the help of on-line desalination using a suppressor; and the main components in neomycin sulfate were clarified combining the RESULTS of impurity profile and minimum inhibitory concentrations of the main components and impurities. The semi-preparative liquid chromatography-evaporative light scattering detector(ELSD) was self-assembled, highly purified neomycin B and neomycin C were prepared and their structural confirmation was also conducted. The contents of highly purified neomycin B and neomycin C were determined by means of mass balance method. The potencies of highly purified neomycin B and neomycin C were determined by three-dose antibiotic microbial assay and the conversion factors between contents of neomycin B and neomycin C and their potencies were calculated separately and then a formula for the calculation of potency of neomycin sulfate from the content of main components of neomycin B and neomycin C was obtained.At last, a verification experiment for the accuracy of the conversion factor and the formula were designed and a serial of tests were carried out to investigate the interaction and the verification for the actual sample. RESULTS: The improved HPLC-PAD method was superior to the European Pharmacopoeia method in the separation ability and stability, and was suitable for accurate quantification of various components of neomycin sulfate and related substance inspection. The successful removal of trifluoroacetic acid in the mobile phase by the technology of desalination on-line using a suppressor broke a new way for the study of impurity profile of aminoglycoside such as neomycin sulfate. Combining the impurity profile with the RESULTS of MIC it was clarified that the main activity components of neomycin sulfate were neomycin B and neomycin C. Highly purified neomycin B and neomycin C were successfully prepared. A conversion factor for the transition from potency to purity of neomycin sulfate was obtained through experiments and calculations and was verified successfully. CONCLUSION: It is feasible to replace the microbial assay by HPLC-PAD method for determining the potency of neomycin sulfate.
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@#A novel method was developed for the content assay and related substances determination of neomycin sulfate by high performance liquid chromatography combined with pulsed amperometric detection(HPLC-PAD). The HPLC was performed on Thermo AcclaimTMAmG C18(4. 6 mm×150 mm, 3 μm). The mobile phase consisted of aqueous solution with 2% trifluoroacetic acid containing 0. 01% pentafluoropropionic acid and 0. 6%NaOH. The pulsed amperometric detector was operated with aquadruple-potential waveform for the detection. Neomycin B, Neomycin C and thirteen related substances were adequately separated by the established HPLC conditions. The limits of detection(LOD)and quantification(LOQ)of neomycin B and neomycin C were both 1. 75 ng and 3. 5 ng, respectively. Good linearities of neomycin B and neomycin C were found in their respective ranges which their correlation coefficients were greater than 0. 998 5. The established method is characterized by high specificity, sensitivity and wide range of linearity which has a good application prospect and provides the basis for improving the standard and quality control of neomycin sulfate.
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Para efetivamente tratar uma infecção, é necessário que o antibiótico possua atividade antimicrobiana adequada e seja capaz de inibir o crescimento do microrganismo patogênico. O doseamento microbiológico é uma metodologia indicada para a análise do antimicrobiano de forma simples, quando comparado com outras metodologias. A Food and Drug Administration (FDA) tem encorajado uma abordagem proativa para introduzir inovações e benefícios associados ao processo de produção farmacêutica. A Qualidade por Design Analítico (AQbD) ajuda no desenvolvimento de métodos analíticos robustos e de baixo custo, que são aplicáveis durante todo ciclo de vida do produto. Os métodos microbiológicos tradicionais, de forma geral, apresentam baixa reprodutibilidade e alta incerteza. Desta forma, justifica-se o desenvolvimento de métodos microbiológicos alternativos para a análise de antimicrobianos empregando-se os conceitos de Qualidade por Design Analítico, com a finalidade de melhorar a reprodutibilidade e reduzir a incerteza final. O objetivo deste trabalho foi aplicar o conceito de Qualidade por Design Analítico (AQbD) no desenvolvimento de método colorimétrico para análise de sulfato de neomicina. O sulfato de neomicina é um antimicrobiano aminoglicosídeo amplamente empregado no tratamento de infecções cutâneas ou mucosas, tais como queimaduras, úlceras, e dermatites infecciosas. Métodos cromatográficos como a cromatografia líquida de alta eficiência em fase reversa, de pareamento iônico ou cromatografia iônica com derivatização (pré ou pós-coluna) são utilizados para a análise de aminoglicosídeos, inclusive sulfato de neomicina. Contudo, de acordo com as farmacopeias, o método microbiológico é o método analítico de escolha para a análise de sulfato de neomicina e outros aminoglicosídeos. A análise colorimétrica é um método amplamente utilizado para a detecção e quantificação de diferentes substâncias, incluindo o crescimento microbiano em estudos de eficácia terapêutica. Neste trabalho, propomos o uso de resazurina como marcador colorimétrico. O indicador sofre uma reação de oxido-redução na qual altera a coloração em resposta à redução química resultante do crescimento celular. O uso de microplacas para a análise colorimétrica é uma alternativa ao método realizado em tubos de ensaio. Uma alternativa ao uso de espectrofotômetros para a análise colorimétrica é o uso de aparelhos smartphones, pois são equipados com CPUs rápidas, câmeras de alta resolução e sensores de imagem. O processamento da imagem captada pela câmera do dispositivo é utilizado como um analisador colorimétrico. Portanto, a aplicação dos conceitos de Qualidade por Design Analítico (AQbD) possibilitou o desenvolvimento racional de método microbiológico colorimétrico para análise de sulfato de neomicina
o effectively treat an infection, the antibiotic must have adequate antimicrobial activity and be capable of inhibiting the growth of the pathogenic microorganism. The microbiological assay is an indicated methodology for the analysis of the antimicrobial in a simple way, when compared with other methodologies. The Food and Drug Administration (FDA) has encouraged a proactive approach to introducing innovations and benefits associated with the pharmaceutical production process. Analytical Design Quality (AQbD) assists in the development of robust, low cost analytical methods that are applicable throughout the product life cycle. Traditional microbiological methods, in general, have low reproducibility and high uncertainty. Thus, it is justified the development of alternative microbiological methods for the analysis of antimicrobials using the concepts of Quality by Analytical Design, in order to improve reproducibility and reduce final uncertainty. The objective of this work was to apply the concept of Quality by Analytical Design (AQbD) in the development of a colorimetric method for the analysis of neomycin sulfate. Neomycin Sulfate is an aminoglycoside antimicrobial widely used in the treatment of cutaneous or mucosal infections, such as burns, ulcers, and infectious dermatitis. Chromatographic methods such as reverse phase high performance liquid chromatography, ion-pairing or ion chromatography with derivatization (pre or post-column) are used for the analysis of aminoglycosides, including neomycin sulfate. However, according to pharmacopoeias, the microbiological method is the analytical method of choice for the analysis of neomycin sulphate and other aminoglycosides. Colorimetric analysis is a widely used method for the detection and quantification of different substances, including microbial growth in studies of therapeutic efficacy. In this work, we propose the use of resazurin as a colorimetric marker. The indicator undergoes an oxidation-reduction reaction in which it alters the coloration in response to the chemical reduction resulting from cell growth. The use of microplates for colorimetric analysis is an alternative to the method carried out in test tubes. An alternative to the use of spectrophotometers for colorimetric analysis is the use of smartphones because they are equipped with fast CPUs, high resolution cameras and image sensors. The image processing captured by the device's camera is used as a colorimetric analyzer. Therefore, the application of the concepts of Quality by Analytical Design (AQbD) allowed the rational development of a microbiological colorimetric method for analysis of neomycin sulfate
Sujet(s)
Néomycine/classification , Colorimétrie/instrumentation , Méthodes Analytiques/analyse , Anti-infectieux/effets indésirables , AntibactériensRÉSUMÉ
Objective:To establish an HPLC-DAD-ELSD method for the simultaneous determination of neomycin sulfate and hy-drochloric dyclonine in compound Twaln ointments. Methods:The assay was performed on an Agilent ZOR BAXSB-C18 column(250 mm × 4. 6 mm, 5μm) with acetonitrile-water as the mobile phase with gradient elution at a flow rate of 1. 0 ml·min-1 . The detection wavelength of DAD was 282 nm. The evaporator temperature of ELSD was set at 50℃ and the nebulizer temperature was set at 60℃with the gas flow rate of 1. 6 L·min-1 . The column temperature was kept at 35℃. Results:The linear range of neomycin sulfate was 141. 54-323. 52 μg·mL-1(r=0. 999 6) with the average recovery of 98. 87%(RSD=0. 95%, n=9). The linear range of hydro-chloric dyclonine was 28. 00-64. 00 μg·mL-1(r=0. 999 6) with the average recovery of 99. 57%(RSD=1. 10%, n=9). Conclu-sion:The method is accurate, sensitive and reproducible, and under the same chromatographic conditions, the determination of all the active ingredients in compound Twaln ointments is achieved, which provides basis for the quality control.
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OBJECTIVE:To optimize the optimal formulation of sterilized neomycin sulfate solution and to study its quality stability.METHODS:Sterilized neomycin sulfate solutions of various formulations were prepared;the alteration of color,pH value and content were taken as the index to optimize the optimal preparation;the stability of which was determined before comparing with the determination results of the reserved samples.RESUITS:The solution would not turn yellow after steril-ization at100℃if its pH value was adjusted to the range of4～5by HCl;the quality of which was stable after acceleration for6months and sample storage observation at room temperature for9months.CONCLUSION:The preparation was rational in formulation design,stable in quality and controllable in quality.