摘要
目的研究影响可利霉素微生物法检测敏感度的因素,以提高血浆中测定可利霉素的灵敏度,为药代动力学研究中血药浓度的测定奠定基础。方法采用管碟法和纸片法,以抑菌圈大小为指标,考察检定菌液浓度,培养基配方,单、双层培养基检定平板,缓冲液加NaCl,胎牛血清,血浆等多种因素对检测灵敏度的影响;采用有机溶剂从人血浆中萃取可利霉素等方法测定血药浓度。结果检定菌浓度、培养基配方、检定平板厚度对检测灵敏度影响显著,含NaCl的缓冲液有利于提高检测灵敏度,人血浆对检测灵敏度有一定影响。以藤黄八叠球菌作检定菌,其菌浓为OD600=0.3~1.0,采用含牛肉蛋白胨0.5%,牛肉浸膏0.25%检定培养基制备单层检定平板,可以有效提高可利霉素检测灵敏度6倍;从血浆中萃取可利霉素,用纸片法进行测定,其最低检测限度可达0.02μg/ml。结论所建立的从血浆中萃取可利霉素的纸片法显著提高了微生物法检测血浆中可利霉素的灵敏度,可用于可利霉素药代动力学研究中血药浓度的微生物法检测。
Objective To investigate the factors affecting sensitivity of microbiological assay for detection of carrimycin (BT), in order to meet the requirement of pharmacokinetics study on plasma-drug concentration. Methods Diameter of inhibition zone was measured as evaluating index, and cylinder-plate and paper disk methods were applied to investigate the potential factors, such as concentration of test bacterium suspention, test media composition, single or double layer assay plates, dilution buffer with or without NaCl and addition of fetal bovine serum or plasma etc., which may affect the sensitivity of microbiological assay for BT. Microbiological assay method by extraction of BT with organic solvent from plasma using paper disk test was established for pharmacokinetics studies. Results Factors such as concentration of test bacterium suspension, medium composition, and thickness of assay plates significantly affected the detection sensitivity of BT in microbiological assay. Addition of NaCl in the dilution buffer improved the sensitivity while the presence of plasma in the samples showed interference on BT diffusion in assay plates. The most suitable concentration suspension of Sarcina lutea as test bacterium was OD600 = 0.3 -1.0. The composition of the test medium was beef peptone of 0.5% and beef extract of 0.25%. Under these conditions and using single layer assay plates, the sensitivity of BT in microbiological assay was increased by 6 times, comparing with initial conditions. The lowest concentration of BT could be tested to 0.02μg/ml by extraction of BT from plasma using paper disk method. Conclusion A sensitive microbiological assay method for detecting BT in plasma was established, which could be applied in its pharmacokinetics studies.
出处
《中国医药生物技术》
2013年第6期420-424,共5页
Chinese Medicinal Biotechnology
基金
"重大新药创制"国家科技重大专项(2008ZX09101-047)
