Sephadex G-10 Gel Permeation Chromatography System for Analysis of High Molecular Polymers of Cefoxitin Sodium

[1] Pharmacopoeia Commission of the Ministry of Health of the People's Republic of China. Ministry of Health of the People's Republic of China Drug Standards, Traditional Chinese Medicine Formulations [M]. Volume XIII, 1997, 81. High-molecular polymer of cefoxitin sodium analyzed by Sephadex G-10 gel chromatography system Gao Danling and Jiang Xiangzhi 2 (1.Fujian Provincial Institute for Drug Control, Fuzhou 350001; 2. Department of Pharmacy, Fujian Medical University 2000 Fuzhou 350008) 0), mobile phase B ultrapure water, flow rate: 1.0 ml.min-1, detection wavelength: 254 nm, injection volume: 20 μL Results Cefotaxime injection concentration, 40 mg.mL-1 The range of the peak area of ​​the high molecular weight polymer of ceflosil was in a good linear relationship (i = 0.9990). Conclusion The method is simple and accurate, high sensitivity and good reproducibility. Cefoxitin is the second generation of cephalosporin antibiotics. For a long time, the allergic problems of this class of drugs have attracted people's close attention. The research shows that the incidence of allergic reactions to terpene lactam antibiotics is related to the content of macromolecule polymers and endogenous polymers in the preparations. All the P lactam antibiotics contained in the 2005 edition of the Chinese Pharmacopoeia will be formulated to contain The limits of the material (gel chromatography) were not reported for the determination of similar polymers contained in cefoxitin. We used the Sephadex G-10 gel chromatography system to achieve high levels of cefoxitin sodium for injection. Separation and analysis of molecular polymers provides a new detection method for the quality control of the drug. 1 Materials and methods 1.1 Instrument AIKTAtm-LC special analysis system for antibiotic polymer impurities and primeview chromatography software, one hundred ten-thousandth of an electronic balance of mettler AE260 .

1.2 Reagents Sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium lauryl sulfate are all of analytical grade, blue dextran 2000 is a product of Pharmacia, cefoxitin is a reference product of Merck, purity 100%), for injection Cefoxitin sodium sample (a domestic factory, lot number 030101, 030301, 030302), test water is ultrapure water.

10, mobile phase A0.01molL-1 phosphate buffer (pH7.0), mobile phase B ultrapure water, flow rate: 1.0mlmin1, detection wavelength: 254nm, each injection: 2001L with mobile phase A as the mobile phase, Determination of 1.0mg.mL-1 Blue Dextran 2000, the results of the theoretical number of plates 1437, the tailing factor 1.452 experimental results 21 the choice of mobile phase A because of the counter-ion PO4- significantly changed the cephalosporin in SephadexG-10 Chromatographic behavior [3], respectively using 0.01molL1 phosphate buffer, 0.1mol L-1 phosphate buffer (pH7. 0) as the mobile phase measured cefoxitin Kad value was 0.721 and 0.674, indicating 0.01mol. L-1 phosphate buffer can make cefoxitin and polymer separated better, and the peak shape of the polymer is more symmetrical, so choose 0.01molL-1 phosphate buffer as mobile phase A2 2 mobile phase ApH The choice of alkaline and alkaline and acidic conditions, cephalosporins prone to polymerization reaction, the measured polymer is difficult to reflect the actual content of the sample itself, take the pH of 7.5, 7.0, 5.6 of 0.01mol. L-1 phosphate buffer, measured effective distribution coefficient Kav values ​​were 0.758, 0.721, 0. 706, can achieve good separation, but pH = 7.0, the peak shape of the association peak is better, so choose pH 7.0 phosphate buffer as the mobile phase A (see) 23 mobile phase B of the choice of ultra-pure water , Sodium dodecyl sulfate, 0.5% glucose is generally used as the basic eluent for the association peak measurement, and eluted with the above three mobile phases, respectively, and the associated peak 2.4 of the high molecular polymer was measured. The linear relationship was precisely weighed and cefoxitin sodium (batch number: 030302) was prepared to a certain concentration. The mobile phase A was used to measure the peak of the macromolecular polymer of cefoxitin sodium, and the sample amount was taken as the abscissa (X) to cefoxitin. The peak area of ​​sodium polymer is ordinate (Y). The regression equation is: Y=0.37772X-10.915, r=0.9990. The results show that the concentration of cefoxitin sodium in the range of 5 to 1 and the head hold The peak area of ​​the Hibitin macromolecule polymer shows a good linear relationship.

2.5 Reproducibility test of macromolecule peaks Weigh accurately 5 parts of the same sample 0.2g (batch number: 030302), put it into a 10mL volumetric flask, and add phosphate buffer of pH 7.0 and 0.01mol.L-1 to dissolve it. Dilution to the mark, respectively injection, high molecular polymer peak area of ​​3.%, good reproducibility, can meet the test requirements 2.6 Association of the peak linear test precision cefoxitin reference substance 5mg, measuring flask , Add ultra-pure water to dissolve and dilute to the mark, take 20 less L Jinxiang to take cefoxitin injection volume as the abscissa (X), and cefoxitin macromolecule associate area as the ordinate (Y ), Obtain the regression equation: Y = 4.6704X-7.6154, r = 0.9995 The results show that the cefoxitin injection concentration in the range of 25 to 1 is in a good linear relationship with the peak area of ​​the cefoxitin associated 2.7 The reproducibility test of the conjugate peak accurately weighed 20mg of the cefoxitin reference substance, place it in a 200mL volumetric flask, add ultrapure water to dissolve and dilute to the mark, and inject 5 times in a row, and the associated compound peak area is 0.46% respectively. , Reproducibility is good, can meet the inspection requirements 28 Determination of sample polymer Weigh the sample accurately about 0.2g, set 10 In the mL volumetric flask, add the mobile phase A to dissolve and dilute to the mark. Shake well. Immediately take the 20WL injection chromatograph. Use the mobile phase A as the mobile phase. Record the chromatogram and calculate the sample according to its own control external standard method (batch number: 030101, 030301, 030302) The measured content of polymer is Q3. Discussion 3.1 When using the above conditions to determine the high molecular polymer in cefoxitin sodium, the response of cefoxitin sodium is very large, so the polymer will not get a Symmetric peaks, it is not appropriate to use the degree of separation to evaluate the method, and use IKav, that is, the effective distribution coefficient evaluation of the method, the test measured polymer polymer IKav 0.023, Cefoxitin sodium IKav 0.706, the difference between the two Larger, indicating that in the above conditions cefoxitin sodium and high polymer can be effectively separated, so the method can meet the test requirements 3.2 Polymer formation is a dynamic process, the polymer content will increase with time, Therefore, the content of the polymer should be injected immediately after the solution is prepared.

3.3 It is generally considered that when pure water is used as mobile phase B, severe tailing will occur, and the use of sodium dodecyl sulfate as mobile phase B will improve its symmetry. Therefore, the mobile phase of the Chinese Pharmacopoeia 2000 Part II is mostly used. [2] However, it has been found through experiments that cephradine, ceftidine, cefazolin, and cefoxitin have good symmetry when water is used as the mobile phase B, while sodium lauryl sulfate is used as the mobile phase. At B, the poor symmetry may be related to their molecular structure, which needs further study.