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The paper aims to establish the method to determine the monosaccharide composition and monosaccharide ratio in propylene glycol alginate sodium sulphate (PSS). Samples were hydrolyzed with trifluoroacetic acid, neutralized with sodium hydroxide solution after the reaction conditions for sample pretreatment were optimized via orthogonal analysis. A high performance anion exchange chromatograghy (HPAEC) coupled with pulsed amperometric detector (PAD) was performed on a CarboPac®PA20, using 200 mmol·L-1 sodium hydroxide solution and 1 mol·L-1 sodium acetate solution as mobile phase. The established HPAEC-PAD method was validated by testing the linear relationship, precision and accuracy, and showed exclusive, sensitive, rapid and wide use. The monosaccharide composition of PSS from different manufacture can be accurately determined with great significance for the structural identification of PSS.
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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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Objective: To develop a method for determination of monosaccharide composition of Polygonatum cyrtonema polysaccharides (PCR) by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC- PAD). Methods: The water-soluble crude polysaccharides were extracted from the P. cyrtonema Hua, then the polysaccharides were hydrolyzed with sulfuric acid, then separated by CarboPac PA1 column (250 mm × 4 mm, 10.0 μm) and with gradient elution, determination of monosaccharide composition in PCP by HPAEC-PAD. Results: The results showed that within the range of 1-100 mg/L it has good linearity (r > 0.999), The detection limitation was 0.015-0.025 mg/L, 1.35%-2.80% RSD, the recovery rates were 88.36%-111.3%. The results showed that the polysaccharides in P. cyrtonema Hua from different regions were composed of rhamnose, arabinose, galactose, glucose, mannose, and fructose. Conclusion: The proposed method has good separation effect and high sensitivity, which can be used for the study on monosaccharide composition and content determination of PCP.
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High performance liquid chromatography-electrochemical detection (HPLC-ED)is an important analytical tool for the determination of pharmaceutical drugs since electrochemical detectors provides derivertization-free, sensitive, selective, and low cost detection methods. Numerous papers and reviews have been published since the HPLC-ED techniques were introduced forty years ago. This article will review the electrochemical instruments and detection methods used in HPLC, and focus on recent advances in the analysis of pharmaceutical drugs using HPLC-ED techniques, and discuss the future development trend of the techniques.
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Objective:To establish an HPLC coupled with post column derivatization method for the determination of gentamicin C components and the related substances based on the latest European Pharmacopeia and compare with the electrochemical method. Methods:A Hydrophilic C18(250 mm ×4.6 mm, 5 μm)column was used with acetonitrile-50 mmol·L-1 sodium hydroxide solution ( pH 2. 6) containing 0. 7% trifluoroacetic acid and 0. 025% pentafluoropropanoic acid (1. 5∶98. 5) as the mobile phase. The temper-ature of post-column reaction was set at 30℃, and the samples were detected by a fluorescence detector withλex of 340nm andλem of 430nm. A pulsed amperometric detector (PAD) was applied in the electrochemical method with golden working electrode in a four-po-tential working mode. Results: According to the results of the two detection methods, the linear range of C1a , C2 , C2a and C1 was 5.82-233.00,6.92-277.00,4.00-160.00and6.23-249.00 μg·ml-1(r >0.9993) , respectively. The limit of detection and quantization were 0. 92-3. 28ng and 1. 37-5. 19ng, respectively. Conclusion:There is no significant difference between the determina-tion results of the two methods.
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OBJECTIVE: To establish a new determination method for free monosaccharides or disaccharides in glycopeptide drugs to eliminate their influence on polysaccharide content assay by colorimetric method adopted by the current national specifications. METHODS: Solid phase extraction column was used for matrix elimination. A high performance anion exchange chromatograghy (HPAEC) in conjunction with pulsed amperometric detector (PAD) was performed on a CarboPac®PA20, using 200 mmol · L-1 sodium hydroxide solution and 1 mol · L-1 sodium acetate solution as mobile phase. RESULTS: Twenty batches of raw materials and preparations of Bozhi glycopeptides, mannatide and polystictus glycopeptide were tested. Free monosaccharides of high content were detected in eight batches of samples, including glucose, sucrose, mannose, and fructose. CONCLUSION: The HPAEC-PAD method is specific, sensitive, rapid and applicable widely, compared with other methods of saccharide detection, thus has good prospect of application and development. The assay methods in the current national specifications of glycopeptides drugs need to be improved.