喘咳安丸微生物限度检查方法的验证

目的建立咳喘安丸的微生物限度检查方法。方法按中国药典2010年版一部微生物限度检查方法,用常规法、培养基稀释法进行方法验证。结果常规法试验时,大肠埃希菌、白色念珠菌和黑曲霉的回收率均高于85%,而枯草芽孢杆菌与金黄色葡萄球菌采用培养基稀释法试验时,使其回收率均高于80%。控制菌采用常规法检出。结论细菌数测定采用培养基稀释法(0.2mL/皿),霉菌、酵母菌及控制菌可采用常规法进行咳喘安丸的微生物限度检验。     

洗涤类化妆品微生物检查方法学探讨

[目的]建立洗涤类化妆品微生物检查方法,对《化妆品安全技术规范》微生物检查法进行探讨。[方法]采用常规法、稀释法、薄膜过滤法对市面常见洗涤类化妆品进行微生物检查方法学研究。[结果]建立了6种洗涤类化妆品的微生物检查方法。OLAY美肌滋润沐浴乳、舒肤佳薄荷冰怡舒爽沐浴露、姗拉娜祛痘控油洁面乳、相宜本草海藻保湿洁面乳、开米贝芬洗手液和舒肤佳白茶香型泡沫抗菌洗手液分别采用1∶20检液、1∶50检液、1∶50检液、1∶20检液、800mL/膜和800 mL/膜法进行菌落总数检查;采用300 mL/膜、300 mL/膜、1∶10检液、1∶50检液、1∶10检液和1∶50检液法进行霉菌及酵母菌检查。[结论]在化妆品微生物检查前应进行微生物方法研究,提高化妆品潜在微生物的阳性检出率。     

新会蛇伤药酒微生物限度检查方法适用性试验

目的考察新会蛇伤药酒微生物限度检查方法的适用性。方法按照2015年版《中国药典》四部通则非无菌产品微生物限度检查法进行试验。结果新会蛇伤药酒采用培养基稀释法(0.2ml/皿)进行需氧菌数的测定,常规法进行霉菌和酵母菌数的测定,金黄色葡萄球菌和铜绿假单胞菌采用直接接种法测定,结果均良好,中和剂对其方法均无明显干扰。结论该方法适用于新会蛇伤药酒的微生物限度检查。     

花蛇解痒胶囊微生物限度检查方法学验证试验研究

目的:建立花蛇解痒胶囊微生物限度检查方法。方法:采用培养基稀释法和常规法。结果:采用常规法验证,大肠埃希菌、金黄色葡萄球菌、白色念珠菌、黑曲霉回收率均能达到70%以上,采用培养基稀释法验证枯草芽孢杆菌回收率能达到70%以上,控制菌可检出大肠埃希菌、大肠菌群、沙门菌。结论:花蛇解痒胶囊微生物限度检查,可采用培养基稀释法检查细菌数、常规法检查霉菌和酵母菌数;控制菌采用常规法和稀释法。     

两种规格硝酸益康唑栓微生物限度检查法的验证比较研究

目的:建立两种规格硝酸益康唑栓的微生物限度检查方法。方法:分别采用平皿法、培养基稀释法、薄膜过滤法、中和-稀释-薄膜过滤法进行微生物限度检查方法学验证试验。结果:建立了两种规格硝酸益康唑栓的微生物限度检查方法。结论:采用薄膜过滤法及添加乳化剂十四烷酸异丙酯可有效消除该药物的抑菌作用,该方法适用于两种规格栓剂的微生物限度检查。     

枫蓼肠胃片微生物限度检查方法学验证试验研究

目的:建立枫蓼肠胃片微生物限度检查方法。方法:采用培养基稀释法和常规法。结果:采用常规法验证,大肠埃希菌、白色念珠菌、黑曲霉回收率均能达到70%以上,采用培养基稀释法验证金黄色葡萄球菌、枯草芽孢杆菌回收率能达到70%以上,控制菌可检出大肠埃希菌。结论:枫蓼肠胃片胶囊微生物限度检查,可采用培养基稀释法检查细菌数、常规法检查霉菌和酵母菌数和控制菌。     

真菌玫瑰红钠培养基平皿计数验证法的改进

为了避免玫瑰红钠培养基平皿法计数验证时细菌在培养基上生长而干扰真菌计数的准确性这一问题,采用在该培养基中选加了0.08g/L、0.10g/L和0.12g/L三种不同浓度的氯霉素的方法。结果证实,在玫瑰红钠培养基中加入浓度为0.10g/L的氯霉素可完全抑制供试大肠杆菌CMCC(B)44102株的生长,但又不影响供试真菌如白色念珠菌CMCC(F)98001、黑曲霉CMCC(F)98003的培养生长。因此,在微生物限度检查时的玫瑰红钠培养基中加入氯霉素最适宜的有效浓度为0.10g/L。     

万古霉素对金黄色葡萄球菌体外抗菌活性研究

目的调查万古霉索对金黄色葡萄球菌的体外抗菌活性。方法收集温州医学院附属第一医院2005年2～7月从临床各种标本分离的金黄色葡萄球菌112株,PCR检测mecA基因确定MRSA,采用琼脂稀释法和全自动微生物分析仪检测金黄色葡萄球菌万古霉素MIC值,使用4mg／L万古霉索脑心浸液琼脂（BHIA）筛选异质性耐万古霉素金黄色葡萄球菌（hVRSA）。结果MRSA的检出率为64．3％,万古霉素的MIC大多数≤2mg／L,MIC。为2mg／L,有2株菌在4mg／L万古霉素BHIA平皿上生长,经菌群分析法证实非hVRSA。仪器法检测的万古霉素MIC值与琼脂稀释法的符合率只有35．7％,琼脂稀释法万古霉索MIC高于仪器法,仪器法检测的2株万古霉素中介耐药的菌株经琼脂稀释法和K-B法证实为敏感株。有6株对万古霉索的MIC为4mg／L,按美国NCCLS／CLSI2006年的标准被确定为万古霉素中介耐药株。结论万古霉素对金黄色葡萄球菌具有较强的体外抗菌活性,未发现耐药株,但MIC值较大。仪器法检测金黄色葡萄球菌对万古霉素的敏感性结果不可靠。按NCCLS／CLSI2006年的标准临床上能检测到对万古霉素中介耐药的金黄色葡萄球菌临床分离株。     

化妆品中腐败微生物的抗药性研究

对导致化妆品腐败变质的微生物进行分离鉴定,并分析腐败微生物对常用防腐剂的抗药性。根据API鉴定系统和分子生物学技术对腐败微生物进行鉴定,并采用最低抑菌浓度(MIC)的方法评估腐败菌对常用防腐剂尼泊金甲酯、卡松、碘丙炔醇丁基氨甲酸酯(IPBC)、DMDM乙内酰脲(DMDMH)、布罗波尔的抗药性水平。经鉴定发现2个污染样品中的腐败微生物均为同一种革兰染色阴性杆菌即洋葱伯克霍尔德氏菌,抗药性分析显示腐败菌HZP1537A对DMDMH、卡松和布罗波尔的MIC(2 000μg/m L、25μg/m L和62.5μg/m L)均高于标准菌株(500μg/m L、3.13μg/m L和31.25μg/m L);腐败菌HZP1537B对DMDMH、卡松的MIC(2 000μg/m L、25μg/m L)均高于标准菌株(500μg/m L、3.13μg/m L);第一代腐败菌的抗药性强于第二代。洋葱伯克霍尔德氏菌容易引起化妆品腐败变质,且对多种防腐剂具有抗药性,在化妆品微生物控制时应给予高度关注。     

建立维生素K_1微生物限度测定方法

目的:根据计数培养基适用性检查和计数方法适用性试验结果,建立维生素K1微生物限度测定方法。方法:以聚山梨酯80作为乳化剂,使维生素K1在p H 7.0无菌氯化钠-蛋白胨缓冲液中充分乳化。取1:20供试液1 m L,按平皿法分别用营养琼脂培养基和玫瑰红钠琼脂培养基培养,以计数细菌、霉菌和酵母菌。结果:验证所用培养基的菌落平均数均大于对照培养基上的70%,且菌落形态大小一致。稀释液对照组和试验组培养基上菌落平均数的回收率均大于70%,且菌落形态大小一致。三批维生素K1及其加速和长期稳定性样品中均未见菌落。结论:所选培养基适宜大肠埃希菌、金黄色葡萄球菌和白色念珠菌生长。且含聚山梨酯80的p H 7.0无菌氯化钠-蛋白胨缓冲液和维生素K1对所含大肠埃希菌、金黄色葡萄球菌和白色念珠菌无抑菌性。     

基于ATP生物发光法的微生物数量快速检测技术的研究进展

微生物数量的快速检测一直是工业生产与食品行业需要解决的问题,腺嘌呤核苷三磷酸(adenosine triphosphate,ATP)生物发光法具有操作简便、检测周期短等优点,可满足一般微生物检测的需求。然而,ATP生物发光法的准确性也受到不同因素的影响,如微生物的ATP检测限值较高、微生物自身及其他因素(如非微生物ATP、提取剂种类、荧光素酶活性等)均对微生物数量的检测产生影响。本文简述了不同微生物数量检测方法的优缺点,介绍了ATP生物发光法的发展历程及原理,综述了非微生物ATP与游离ATP、微生物量、ATP提取剂、荧光素酶等因素对ATP生物发光法灵敏度与稳定性的影响,归纳总结了ATP生物发光法及检测设备在食品、医疗、污水处理等领域的应用现状,并就ATP生物发光法体系的优化及ATP在线检测的应用等方面进行了展望,以期为ATP生物发光法的高效应用提供新的思路。     

药用微生物辐照诱变研究进展

常规的诱变育种方法主要是利用紫外线、激光、γ射线、中子等对微生物药物菌种进行辐照诱变,效果显著。离子束辐照微生物诱变育种在离子束生物学效应的研究中起步较晚,但其作为一种集物理诱变和化学诱变为一体的诱变育种新方法,将在辐照诱变育种中发挥重要作用。本文综述了近年来微生物药物研究与开发的动态,简要叙述微生物菌种选育的辐照诱变效应、作用机制及其在实践中的应用。     

饮用水微生物的安全快速检测

【目的】为了更好地分析饮用水中的微生物含量。【方法】利用流式细胞术(Flowcytometry,FCM)、ATP测定方法检测瓶装无气饮用水中的微生物数量、可同化有机碳(Assimilable organic carbon,AOC)含量以及微生物活性,并将检测结果与传统的饮用水微生物检测技术相对照。【结果】FCM方法可快速区分水样中的活性细菌和非活性细菌,AOC含量反映了水样中微生物再生能力;而ATP检测方法也能比异养细菌平板计数法(Heterotrophic plate count,HPC)更好地反映瓶装无气饮用水中的实际微生物含量。【结论】FCM、ATP测定方法要明显优于依赖于培养的传统方法。     

Determination of tianeptine in human plasma using high-performance liquid chromatography with fluorescence detection

A new, selective and sensitive high-performance liquid chromatography (HPLC) method with fluorimetric detection was developed for the determination of tianeptine (TIA) in human plasma using solid phase extraction (SPE) procedures. The method is based on the derivatization of TIA with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in borate buffer of pH 8.5 to yield a yellow, fluorescent product. The HPLC separation was achieved on a Phenomenex C(18) column (250 mm x 4.6 mm) using a mobile phase of acetonitrile-10mM orthophosphoric acid (pH 2.5) (77:23, v/v) solvent system at 1 mL/min flow rate. Gabapentin (GA) was used as the internal standard. The fluorometric detector was operated at 458 nm (excitation) and 520 nm (emission). The assay was linear over the concentration range of 5-300 ng/mL. The detection limit (LOD) was found to be 2 ng/mL. The mean recovery was determined to be 88.6%. The proposed method was applied for pharmacokinetic study of 12.5mg TIA in a healthy volunteer.     

多重PCR快速检测化妆品中三种致病菌的研究

利用多重PCR技术建立快速检测化妆品中三种致病菌的方法。根据已报道的大肠杆菌phoA基因、铜绿假单胞菌外膜蛋白基因oprL和金黄色葡萄球菌特异性序列SmaI选择特异性引物,对人工染菌化妆品进行多重PCR检测。结果显示,三种致病菌的基因组DNA均可与各自引物特异性结合,扩增产物大小分别为622 bp、504 bp和426 bp。该方法用于人工污染的化妆品中,大肠杆菌的检出限浓度为103 CFU/mL,铜绿假单胞菌和金黄色葡萄球菌的检出限浓度为105 CFU/mL。作者建立的多重PCR方法可同时快速、特异地对化妆品中三种致病菌进行检测,在化妆品行业具有较大的应用价值。     

A robust and fast bacteria counting method using CdSe/ZnS core/shell quantum dots as labels

A rapid, sensitive fluorescence measurement method for detecting the bacterial count using CdSe/ZnS as a fluorescence marker was described. High-quality CdSe/ZnS nanocrystals were synthesized and successfully conjugated with bacteria. The fluorescence intensity was proportional to bacterial count in the range of 10²–10⁸ CFU/mL and the low detection limit was 10² CFU/mL.     

Simultaneous determination of methadone, buprenorphine and norbuprenorphine in biological fluids for therapeutic drug monitoring purposes

Methadone and buprenorphine are two of the drugs most frequently used for abstinence from illicit opioids and in the treatment of pain. A sensitive and selective high-performance liquid chromatographic method with diode array detection for the simultaneous determination of methadone, buprenorphine and norbuprenorphine has been developed. Separation of the three analytes was obtained by using a reversed-phase column (C8, 250mmx4.6mm i.d., 5microm) and a mobile phase composed of 40% phosphate buffer containing triethylamine, 50% methanol and 10% acetonitrile (final apparent pH 6.0). Loxapine was used as the internal standard. An accurate pre-treatment procedure of biological samples was developed, using solid-phase extraction with C8 cartridges (100mg, 1mL) and needing small amounts of plasma or urine (300microL). The calibration curves were linear over a working range of 10.0-1500.0ng/mL for methadone and of 5.0-500.0ng/mL for buprenorphine and norbuprenorphine in both matrices. The limit of quantitation (LOQ) and the limit of detection (LOD) were 1.0 and 0.4ng/mL for methadone and 0.5 and 0.2ng/mL for both buprenorphine and norbuprenorphine, respectively. The method was successfully applied to the analysis of plasma and urine samples from patients undergoing treatment with these drugs. Precision and accuracy results were satisfactory and no interference from endogenous or exogenous compounds was found. The method is suitable for the simultaneous determination of methadone and buprenorphine in human plasma and urine for therapeutic drug monitoring purposes.     

Microdialysis sampling coupled to on-line high-performance liquid chromatography for determination of arbutin in whitening cosmetics

We have developed a method that uses on-line microdialysis sampling coupled with high-performance liquid chromatography (HPLC) to determine arbutin in whitening cosmetics. The optimum analytical conditions for microdialysis sampling were a probe length of 10mm and a dialysis flow-rate of 5 microl min-1. The accuracy (% bias) for intra-day (n=6) and inter-day (n=30, five consecutive days) analyses ranged from -8.9 to 11.5%, with a precision below 7.64% R.S.D. The calibration curve was linear within the range from 0.1 to 20 mM (R2=0.9989). The detection limit was 15 microM. By comparing the arbutin levels determined this way in the whitening cosmetics with the results obtained from the no-net-flux method, we conclude that our proposed on-line microdialysis-HPLC system displays good accuracy. We evaluated the robustness of our optimum conditions by means of a Plackett-Burman design. Apart from the effect of a low flow-rate of perfusate--an increase of 12.52+/-2.31%--we observed no significant changes in the analyses upon changing the levels of any other parameter. Because this on-line method offers the advantages of simplicity, reliability, the lack of any tedious sample pretreatment process, and a reduced use of organic solvents, we believe that it is suitable for the routine analysis of commercial cosmetics.     

ESTIMATION OF MICROBIOLOGICAL QUALITY OF CHILLED BEEF USING AUTOMATED TURBIDIMETRY

Total bacterial counts on chilled beef samples were estimated by the standard plate count method and by an automated turbidimetric system. The latter method is based on product-specific calibration curves constructed by correlating growth curve parameters calculated for the turbidimeter to the log CFU values obtained by plate counts. A total of 74 beef samples was used to construct the calibration curves. Correlation analysis between turbidimetric parameters and plate count values showed that detection time was the best predictor to estimate microbial loads on fresh (r=0.91) and aged beef (r=0.94). Microbial loads for a different set of aged beef samples (n = 37) refrigerated for 7, 9, 10, 17 and 45 days were compared by turbidimetric measurements and plate counts. Mean total viable counts were log 5.92 ± 1.17 and log 5.54 ± 1.28 CFU/mL, respectively. Results showed that total bacterial counts on chilled beef could be estimated accurately from turbidimetric parameters. Furthermore, setting a cut-off value of log 6 CFU/mL allowed to accepting/rejecting samples according to their microbial condition in shorter periods of time compared to the traditional plate count method.     

Optimization and validation of the direct HPLC method for the determination of moxifloxacin in plasma

Moxifloxacin (1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-[(4aS,7aS)-octahydro-6H-pyrrolo-[3,4-b]pyridin-6-yl]-4-oxo-3-quinolinecarboxylic acid hydrochloride) is new, fourth generation fluoroquinolone with broaden spectrum of antibacterial activity. In the present work simple and rapid RP-HPLC method for the direct determination of moxifloxacin in human plasma is described. Separation of moxifloxacin from plasma components was achieved on Supelco LC-Hisep shielded hydrophobic phase column. The mobile phase consisted of acetonitrile and 0.25mol/dm(3) Na(3)PO(4) (pH 3) in a volume percent ratio (5:95, v/v) and was delivered at a rate of 1mL/min. Fluorescence detection was employed with excitation at 290nm and emission at 500nm. Ofloxacin was used as internal standard and sodium dodecylsulfate solution was used as a displacing agent. Sample preparation was simplified and involved only addition of displacing agent and internal standard and dilution with water. The separation conditions were optimized by the response surface method in two factor space, i.e. the dependence of the retention time on volume percent of acetonitrile and on pH of aqueous phase was optimized. The method was fully validated and validation parameters were: linearity range 3-1300microg/L; correlation coefficient, 0.99986; mean recovery, 92.5%; limit of quantification, 3.0microg/L and limit of detection, 1.0microg/L. Method was applied for the determination of moxifloxacin in human plasma after single or repeated oral doses of 400mg Avelox tablets. The proposed method proved to be rapid and accurate and can be successfully used in pharmacokinetic studies and routine clinical practice.