ABSTRACT
To establish a method for the determination of polymer impurities in cefixime raw materials and preparations, a cefixime degradation solution containing polymer impurities was prepared by forced polymerization. Polymer impurities in the degradation solution were separated and identified by high performance gel chromatography and the column switching-LC-MSn method. A new RP-HPLC method for cefixime polymer was established and validated with a Phenomenex Gemini-C18 column using a mobile phase gradient elution of 0.5% formic acid-water solution and 0.5% formic acid-acetonitrile solution. The results showed that when using this high performance gel chromatography method some small molecular weight impurities were co-eluted with the polymers, resulting in a poor specificity and poor quantitative accuracy. But when using the RP-HPLC method, three polymer impurities were detected with good specificity, sensitivity and robustness, including two cefixime dimers, and dehydrate dimer. Therefore, the described RP-HPLC method is suitable for the quality control of polymer impurities in cefixime, and cefixime degradation solution can be used as suitable solution for analysis of cefixime polymers.
ABSTRACT
To establish a method for the determination of polymer impurities in ceftazidime raw materials and preparations, a ceftazidime degradation solution containing polymer impurities was prepared by forced polymerization. Polymer impurities in the degradation solution were separated and identified by high performance gel chromatography and the column switching-LC-MSn method. A new RP-HPLC method for ceftazidime polymer was established and validated with a Phenomenex Gemini-C18 column using a mobile phase gradient elution of 0.02 mol·L-1 phosphate buffer, methanol and acetonitrile. The results showed that when using this high performance gel chromatography method some small molecular weight impurities were co-eluted with the polymers, resulting in a poor specificity and poor quantitative accuracy. But when using the RP-HPLC method, four polymer impurities were detected in the 25-45 min time range with good specificity, sensitivity and robustness, including two ceftazidime dimers, trimers, and derivatives. Therefore, the described RP-HPLC method is suitable for the quality control of polymer impurities in ceftazidime, and ceftazidime degradation solution can be used as suitable solution for analysis of ceftazidime polymers.
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Objective:To establish a method for the determination of polysaccharide and monosaccharide composition of Tremella fuciformis, and to analyze the difference of polysaccharide content in T. fuciformis from different sources and cultivation methods, so as to provide reference for the quality determination.Method:High performance size exclusion chromatography coupled with multi-angle laser light scattering and refractive index detection (HPSEC-MALLS-RID) was employed to determine the content and relative molecular weight distribution of T. fuciformis polysaccharides. The monosaccharide types and proportions of T. fuciformis polysaccharides were analyzed by 1-phenyl-3-methyl-5-pyrazolone (PMP) precolumn derivative high performance liquid chromatography (HPLC).Result:The weight-average relative molecular weight (Mw) and the content of polysaccharides in T. fuciformis cultivated by cut-log from different sources were distributed in 2.618×106-3.503×106 Da and 307.12-609.06 g·kg-1, respectively. These two parameters of polysaccharides in T. fuciformis with substitute cultivation from different sources were 2.723×106-3.886×106 Da and 366.38-647.37 g·kg-1, respectively. The T. fuciformis polysaccharides mainly consisted of mannose, glucuronic acid, glucose, galactose, xylose and fucose, their ratios in samples with cut-log and substitute cultivation were 4.4∶0.7∶1.0∶0.2∶1.4∶1.6 and 4.4∶0.8∶1.0∶0.1∶1.5∶1.5, respectively. The contents of the above six monosaccharides in 39 batches of T. fuciformis from different sources were mannose of 36.71-191.31 g·kg-1, glucose of 10.46-76.10 g·kg-1, galactose of 1.00-6.72 g·kg-1, xylose of 16.73-70.54 g·kg-1, glucuronic acid of 9.74-32.12 g·kg-1, fucose of 17.16-68.20 g·kg-1.Conclusion:The content of polysaccharides in T. fuciformis from different sources has a certain difference, the developed method can be used as a routine method for the quality evaluation of polysaccharides in T. fuciformis.
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OBJECTIVE: To establish a high performance size exclusion chromatography(HPSEC) method for the separation and analysis of polymers in cefotaxime sodium and cefotaxime sodium for injection, and determine the structures of the impurities by LCMS. METHODS: HPSEC was performed by using Sepax SRT SEC-150(7.8 mm × 300 mm, 5 μm) column. The mobile phase was 0.1 mol•L-1 disodium hydrogen phosphate and 0.1 mol•L-1 phosphate buffer solution. The flow rate was 0.8 mL•min-1, the detection wavelength was set at 235 nm, the injection volume was 10 μL, and the column temperature was maintained at 35 ℃. The concentration of polymers was quantified by external standard method. The LC-MS/MSn system conditions were as following: the mobile phase was 20 mmol•L-1 amonium acetate, the flow rate was 0.8 mL•min-1, ESI source with positive and negative ion scan was utilized, the scanning range was m/z 200 - 1 600, and the post-column diversion ratio was 1:4. RESULTS: Eight impurity peaks were obtained in total; the resolutions were all greater than 1. 5. The linear range of cefotaxime was 1 - 100 μg•mL-1 (r = 1.000 0). The RSD repeatability was 1.2%(n = 6). The limit of detection was 0.2 μg and the limit of quantitation was 0.4 μg. Three polymers were identified by LC-MS. CONCLUSION: The HPSEC method can be used for the quantitative and qualitative analyses of individual polymer impurities. It is also sensitive for the control of polymers in cefotaxime.
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OBJECTIVE:To establish a method for the content determination of polymer in cefatrizine propylene glycol. METHODS:High performance sephadex gel chromatography was performed on the column of Sephadex G-10 with mobile phase A of 0.01 mol/L phosphate buffer [0.01 mol/L Disodium hydrogen phosphate solution-0.01 mol/L Sodium dihydrogen phosphate solution (61∶39,V/V)](pH7.0)and mobile phase B of water at a flow rate of 1.0 ml/min,the detection wavelength was 254 nm,column temperature was 30℃,and volume injection was 200μl. RESULTS:The linear range of polymer was 2.07-103.30 mg/ml(r=0.999 4);the limit of quantitation of 10.4 ng,limit of detection was 4.1 ng;RSDs of precision and reprodicibility tests were lower than 3%. CONCLUSIONS:The method is specific with high sensitivity and good reproducibility,and can be used for the content determination of polymer in active pharmacentical ingredient cefatrizine propylene glycol.
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Objective: To establish a high performance size exclusion chromatography (HPSEC) method for the determination of high molecular weight substances in ginkgo diterpene lactone extraction. Methods: The chromatographic column Phenomenex Biosep-SEC-S2000 (300 mm × 7.8 mm, 5 μm) was used with 0.05 mol/L sodium sulfate as the mobile phase at a flow rate of 0.7 mL/min, with refractive index detector (RID), and the temperature of the column was 30℃. High molecular weight substances in samples were calculated with area normalization method. Results: In the range of 2 500-84 400 of substance molecular weights, a linearity was achieved (r = 0.998 3). The average recovery rate was 96.8%, and RSD was 1.5%. The high molecular weight substances in five batches of ginkgo diterpene lactone extraction were not detected. Conclusion: This method is simple, quick, accurate, and suitable for the quality control of high molecular weight substances in ginkgo diterpene lactone extraction.
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Objective: To study the preparation and separation method for anticoagulant peptide and the effect of anticoagulation and thrombolysis in vitro. Methods: The casein was hydrolyzed to prepare anticoagulant peptide using the mixed four enzymes such as papain, pineapple proteinase, neutral protease, and alkali protease. The anticoagulant peptide was extracted using immobilized thrombin. The effect of haemolysis and anticoagulation in vitro was investigated through the New Zealand rabbits experiments. Results: The conditions of preparation anticoagulant peptide were as follows: quality of casein was 15%, papain proteinase, pineapple proteinase, neutral protease, and alcalase dosage were 1500, 2400, 1000, and 1250 U/(g casein), respectively, temperature was 50℃, pH value was 7.0, and hydrolysis time was 4 h. The conditions for the extraction of anticoagulant peptide were as follows: the initial concentration was 6 ATU (Anti Thrombin Unit)/mL, temperature was 30℃, pH value was 5.0, and time was 30 min. Anti-extraction temperature was 30℃, pH value was 7.6, and time was 40 min. The purified anticoagulant peptide was analyzed via high performance size exclusion chromatography. The molecular weight of purified anticoagulant peptide was equal to N-Hippuryl-His-Leu hydrate and the main components were three peptides. The time of anticoagulation was more than 72 h and the time of hemolysis was 24 h in vitro. Conclusion: The main components of anticoagulant peptides are three peptides. The effect of hemolysis and anticoagulation in vitro is good.
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Objective:To establish a high performance size-exclusion chromatography ( HPSEC) method for the determination of impurities including polymers in latamoxef sodium. Methods:The analysis was performed on a Zenix SEC-150 column(7. 8 mm × 300 mm, 3 μm)with the mobile phase of 0. 005 mol·L-1 phosphate buffer solution [0. 005 mol·L-1 disodium hydrogen phosphate-0. 005 mol·L-1 sodium dihydrogen phosphate (61∶39), pH 7. 0] at a flow rate of 0. 8 ml·min-1. The detection wavelength was set at 254 nm. The column tempretrue was 25℃ and the injection volume was 10μl. Results:The impurities including polymers in latamoxef so-dium were completely separated from latamoxef. The linear range of latamoxef was 0. 98-97. 73 μg·ml-1(r=0. 999 9). The limit of quantitation of latamoxef was 2. 9 ng, and the detection limit was 1. 0 ng. The linear range of the total impurities was 0. 45-2. 8 mg· ml-1(r=0. 999 5). Conclusion: The established method is accurate, rapid and reproducible, and suitable for the determination of impurities including polymers in latamoxef sodium.