ABSTRACT
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.
ABSTRACT
@#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.
ABSTRACT
OBJECTIVE: To establish an HPLC method combined with pulsed amperometric detection for the analysis of ribostamycin sulfate and related substance. METHODS: The HPLC was performed on Thermo AcclaimTMAmG C18 column (4.6 mm×150 mm,3 μm). The mobile phase consisted of acetonitrile and 0.2%(V/V) pentafluoropropionic acid aqueous solution containing 0.15%(V/V) trifluoroacetic acid (1∶99, V/V). The pH of the aqueous solution was adjusted to 1.5 with 50%(m/m) sodium hydroxide solution. The pulsed amperometricdetector was operated with aquadruple-potential wave form at 35 ℃ and the injection volume was 25 μL. RESULTS: Ribostamycin and its related substances were adequately separated under the established HPLC conditions. The LOD and LOQ of ribostamycin were 0.15 μg•mL-1(3.75 ng injected) and 0.375 μg•mL-1(9.38 ng injected), respectively. The linearity of ribostamycin ranged from 0.15 to 40.0 μg•mL-1 with a correlation coefficient of 0.999 3.The repeatability RSDs(n=6)for method validation of the content assay and total impurities test were 0.33% and 1.10%, respectively. CONCLUSION: The established method is characterized by high specificity, sensitivity and good stability. The established method has much lower test cost than the current Ch.P 2015 method and is hopeful to replace it.
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OBJECTIVE: To establish an improved reversed-phase high performance liquid chromatography method coupled with pulsed electrochemical detection for determining the related substances of netilmicin sulfate injection. METHODS: Agilent Proshell 120 SB-C18 column (4.6 mm×150 mm, 2.7 μm)and gradient elution were used. Mobile phase A was 0.2 mol·L-1 trifluoroacetic acid in 0.1 mol·L-1 sodium hydroxide solution-acetonitrile (97:3), mobile phase B was 0.1% pentafluoropropionic acid-acetonitrile (97:3), and the flow rate was 0.8 mL·min-1. A pulsed electrochemical detector was adopted, and the temperatures of detector and column were kept at 35℃. The working electrode was a gold electrode with diameter of 3 mm and a quadruple-potential waveform (QPW)was selected as detection waveform. The injection volume was 25 μL. NaOH solution of 0.8 mol·L-1 was added post-column at a flow rate of 0.3 mL·min-1. RESULTS: A total of 28 impurities could be detected and effective separation was achieved in the typical sample and most of which could not be separated in the method of Ch.P 2015. The linearity of the calibration curve for netilmicin ranged from 0.25 to 15 μg·mL-1 with a coefficient of determination equal to 0.999 1. The LOD and LOQ of netilmicin were found to be 0.25 ng and 1.25 ng, respectively. The repeatability RSD(n=6) of the single largest impurity and total impurities were 0.9% and 0.8%, respectively. The sample solution was stable within 24 h. CONCLUSION: Compared with previously published investigations, the improved method shows higher sensitivity, better separation ability and good reproducibility, especially for differentiating the origin of bulk drug for netilmicin sulfate injection, thus is more suitable for the determination of related substances of netilmicin sulfate injection.
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OBJECTIVE: To compare and optimize the analytical methods for detection of related substances of etimicin sulfate injection. METHODS: For the HPLC-CAD (charge aerosol detector)method, the mobile phase was 0.2 mol·L-1 trifluoroacetic acid aqueous solution-methanol (95:5), the flow rate was 1.0 mL·min-1 and the column temperature was maintained at 30℃. The nebulization temperature for the CAD was maintained at 30℃ and the gas pressure was 0.24 MPa.The HPLC-ELSD and HPLC-PAD methods adopted by the Ch.P 2015 were also used to detect the related substances of etimicin sulfate injection for the purpose of comparison. RESULTS: Compared with the HPLC-ELSD method, the HPLC-CAD method showed higher selectivity and sensitivity; compared with the HPLC-PAD method, the results of the determination of the impurities were more accurate for the HPLC-CAD method. CONCLUSION: The separation capability of the new HPLC-CAD method for detection of the related substances of etimicin sulfate injection is superior to HPLC-PAD and HPLC-ELSD methods and can detect more impurities, which is suitable for the quality control of etimicin sulfate injection.
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OBJECTIVE: To establish an HPLC-PAD method to determine the related substances of sisomicin sulfate injection and compare with the statutory method. METHODS: IonPac AMG C18(4.0 mm×150 mm, 3 μm)chromatographic column was used with acetonitrile-0.1 mol·L-1 trifluoroacetic acid (containing 0.025% of pentafluoropropionic acid, 5 mL of 50% NaOH solution without carbonate, pH of the aqueous solution adjusted to 2.3 with 50% NaOH solution.)as mobile phase at a flow rate of 0.7 mL·min-1. NaOH solution of 0.76 mol·L-1 was added post column at a flow rate of 0.35 mL·min-1. The column temperature was maintaine at 30 ℃. PAD detector was operated with the cell temperature set at 35 ℃. The working electrode was a gold electrode (diameter of 3 mm)and a quadruple-potential waveform was selected as detection waveform. The reference electrode was Ag/AgCl, the detection potential was four potential. The determination result of the related substances of sisomicin sulfate injection was compared with that of the statutory method. RESULTS: The peaks of sisomicin sulfate, gentamicin C1a and netilmicin could be completely separated, and other impurities could also be effectively separated. The blank sample had no interferences. The LOD and LOQ of etimicin were found to be 2 and 6 ng respectively, and the RSD of precision test (n=6) was 0.9%. Paired-samples t-test showed significance levels of P=0.034, P=0.364 and P=0.605 for total amount of impurities (%), the biggest single impurity (%)and content (%)respectively between the statutory method and the method of HPLC-PAD. CONCLUSION: Compared with the statutory method, this HPLC-PAD method shows higher sensitivity, and is accurate and reliable. It can be applied to the determination of related substances in sisomicin sulfate injection.