Validation of the Delvotest SP NT DA. Performance Tested Method 011101

Delvotest SP NT DA is designed to test milk for the presence of antibacterial substances, such as antibiotics. The test is made of an agar gel containing bacterial spores and a color indicator. The milk sample is added onto the agar gel, and the test is incubated at 64 degrees C. The principle of the test is based on the diffusion of possible inhibitory substances that may be present in the milk sample into agar. This reduces growth and acid production by the test organism, and delays or prevents the agar from changing color from purple to yellow. The Delvotest Accelerator is an automated system in which the plates containing the milk to be analyzed are placed for incubation. The Accelerator automatically detects the end of the incubation and reads the results. A sample containing antibiotic will be noted as "positive." A sample without antibiotics or with antibiotics at concentrations below detection level will be noted as "negative." The present report includes all technical details about the Delvotest SP NT DA, and the results of the validation study. The validation study demonstrates that the Delvotest SP NT DA conforms to the product performance claims and confirms the robustness of the test. The Delvotest SP NT DA is, therefore, granted Performance Tested Method certification.     

Validation and comparison of the Copan Milk Test and Delvotest SP-NT for the detection of antimicrobials in milk

The Delvotest SP-NT and Copan Milk Test, two microbiological tests designed for screening antimicrobial substances in milk were compared and validated. The performance criteria described by the European Decision 2002/657/EC were used for the study. Both tests were evaluated with visual and automated reading (scanner) and the validation was performed on 10 different antibiotics (penicillin-G, cloxacillin, sulfamethazine, sulfadiazine, oxytetracycline, gentamicin, cephalexin, cefquinome, dihydrostreptomycin and trimethoprim). Both tests were found to detect penicillin, cloxacillin, sulfamethazine, sulfadiazine, cephalexin and gentamicin at or below the EU maximum residue limits (MRLs). Some other antibiotics such as oxytetracycline, dihydrostreptomycin, trimethoprim and cefquinome were not detected or only with a low sensitivity. Both tests were found easy to use, robust and fulfilled EU requirements.     

Removal of ronidazole and sulfamethoxazole from water solutions by adsorption on granular activated carbon: equilibrium and intraparticle diffusion mechanisms

The equilibrium and intraparticle diffusion of ronidazole (RNZ) and sulfamethoxazole (SMX) during the adsorption on granular activated carbon (GAC) from aqueous solution was investigated in this work. The solution pH, temperature, ionic strength and water matrix affected the adsorption capacity of GAC towards SMX, but no effect was observed for the adsorption of RNZ. This behavior was due to the different mechanism involved in the adsorption of both antibiotics. The adsorption capacity of GAC towards RNZ was greater than that towards SMX. Molecular computation allowed the estimation of the binding free energy and confirmed that the adsorption of RNZ was more favorable than the adsorption of SMX. The adsorption mechanism of both antibiotics is governed by π–π dispersive interactions, and molecular simulation demonstrated that the coulombic interactions did not affect, but the solvation and nonpolar interactions play a significant role on the adsorption of both antibiotics. The application of diffusional models revealed that the overall adsorption rate of both antibiotics is controlled by intraparticle diffusion. Moreover, the surface diffusion was more predominant than the pore volume diffusion. Besides, surface diffusion coefficient, D_s, for RNZ was not a function of the aqueous matrix, whereas D_s for SMX was highly dependent on the water matrix.     

Antimicrobial properties of biosynthesized silver nanoparticles studied by flow cytometry and related techniques

This work reports the effect of silver bionanoparticles (Bio(AgNPs) synthesized by Actinobacteria CGG 11n on selected Gram (+) and Gram (–) bacteria. Flow cytometry, classical antibiogram method and fluorescent microscopy approach was used for evaluation of antimicrobial activity of Bio(AgNPs) and their combination with antibiotics. Furthermore, the performed research specified the capacity of flow cytometry method as an alternative to the standard ones and as a complementary method to electromigration techniques. The study showed antibacterial activity of both BioAgNPs and the combination of antibiotics/BioAgNPs against all the tested bacteria strains in comparison with a diffusion, dilution and bioautographic methods. The synergistic effect of antibiotics/BioAgNPs combination (e.g. kanamycin, ampicillin, neomycin and streptomycin) was found to be more notable against Pseudomonas aeruginosa representing a prototype of multi‐drug resistant “superbugs” for which effective therapeutic options are very limited.     

Simulations of cyclic voltammetric and chronoamperometric electrode responses at a disk electrode using combinations of spherical and cylindrical electrode geometries

Using geometric models based on one-dimensional transport at spheres and cylinders, three methods for improving the simulation of voltammetric behavior of a disk electrode have been explored. One method is based on the common assumption of equivalency between the limiting currents for a disk and a hemisphere under steady-state diffusion conditions. The second method involves the use of a partial-sphere geometry which is a better approximation that is suitable at the extreme diffusional limits achievable at a disk electrode of fully planar and steady-state transport. The third method, which is generally applicable, is a further refinement that uses a combination of appropriate one-dimensional spherical and cylindrical geometries. The results demonstrate that reasonably accurate approximations of disk behavior for several reaction mechanisms can be achieved in a fraction of the time required to compute the more rigorous two-dimensional model. We propose that the approximation serve primarily as a fast way to explore system behavior and establish approximate values of the relevant parameters. More accurate computations can then be performed using the two-dimensional model.     

Atomic spectrometry and flow injection analysis: a synergic combination

The combination of flow-injection techniques with atomic spectrometry (flame atomic absorption and emission spectrometry and inductively-coupled plasma/atomic emission spectrometry) is reviewed, with particular reference to the more recent contributions. The considerable growth in the number of directly couple pre-concentration and matrix isolation is noted, together with the increasing number of reports of indirect methods for metals, inorganic anions and even drug molecules. Many developments are motivated by a desire to increase the performance of the spectrometry over that obtained with conventional methods of sample introduction. Conflicting statements concerning the possible benefits of reduced uptake rate, of air compensation and of peak-area measurement are examined critically. The conflicting requirements of obtaining freedom from stable-compound interferences coupled with god detection limits are discussed, as are means of obtaining the best detection limits. Modifications to nebuliser and spray-chamber design are suggested for maximising peak height (to obtain detection limits) and for working with reduced uptake rates (to reduce stable-compound interferences in flame-based spectrometries). The single well-stirred tank model is used to model nebuliser response and results are presented for the flow-injection behaviour of a Philips Scientific SP9 instrument under conditions of low flow rate which show reasonable agreement with the model. With the instrument, the best detection limits are obtained on the basis of peak-height measurements at the flow rate producing maximum signal-to-noise ratio.     

固相萃取/超高效液相色谱-质谱法筛查及检测养鱼河水中抗生素

建立了固相萃取/超高效液相色谱-串联三重四极杆质谱(UPLC-MS/MS)快速检测养鱼河水中4类(喹诺酮类、氯霉素类、四环素类以及磺胺类)15种抗生素的分析方法。取500 m L水样过滤,用盐酸调至p H4.0,加入0.5 g乙二胺四乙酸二钠(Na2EDTA)混匀后,再用HLB固相萃取柱对水样进行富集。利用超高效液相色谱-串联四极杆飞行时间质谱(UPLC-Q-TOF MS)结合基于UNIFI软件的常用抗生素数据库对养鱼河水中可能存在的抗生素进行快速筛查,筛查结果主要为喹诺酮类、氯霉素类、四环素类、磺胺类4类15种抗生素。为了提高检测结果的准确性,使用UPLC-MS/MS采用分时段多反应监测离子模式(MRM)分析样品中的抗生素,并用外标法定量。15种抗生素的空白基质加标回收率为61.0%~98.4%,相对标准偏差(n=3)为4.6%~14.0%,其线性关系良好,相关系数r≥0.990,检出限为0.01~0.3 ng/L。该方法灵敏度较高,重复性好,可用于对北京养鱼河水中抗生素的检测。     

Determination of amitrole and urazole in water samples by capillary zone electrophoresis using simultaneous UV and amperometrical detection

In this paper, capillary zone electrophoresis with amperometric detection (CZE-AD) was first applied to the simultaneous separation and determination of amitrole and urazole in water samples. A simple end-column electrochemical detector was used in combination with a commercially available capillary electrophoresis instrument with UV detection. The effects of several important factors were investigated to find optimum conditions. A carbon disk electrode was used as working electrode. Separation and determination of these compounds in water samples were performed in 0.030 mol l(-1) acetate buffers at pH 4.5, 25 kV as separation voltage and the samples were introduced by hydrodynamic mode for 1.5 s. Most of the studies realized showed that the direct electrochemical detection is more sensitive and selective than UV detection. Under the optimum conditions, excellent linearity was observed between peak amperometric signal and analyte concentrations in the range of 0.19-1.35 mg l(-1) for amitrole and 0.20-1.62 mg l(-1) for urazole. The detection limits were 63 and 68 microg l(-1) for amitrole and urazole, respectively. The utility of this method was demonstrated by monitoring water samples, and the assay results were satisfactory. The detection limits using a previous preconcentration step for amitrole and urazole in spiked mineral water samples were 0.6 and 1.0 microg l(-1) for amitrole and urazole, respectively.     

Biosensor analysis of beta-lactams in milk: comparison with microbiological, immunological, and receptor-based screening methods

Two recently developed surface plasmon resonance biosensor assays for detection of beta-lactams in milk were used to screen raw producer milk samples. Both assays use a beta-lactam receptor protein with carboxypeptidase activity for detection. The results of the biosensor assays were compared with those of various commercial screening tests, i.e., the Delvotest SP, Penzym S, Beta-STAR, SNAP, and Parallux. The results of the 2 biosensor assays showed good agreement with those of the other screening tests. Of 195 analyzed milk samples, the results of only 5 samples differed between the assays. Additionally, 30 milk samples with both negative and positive results in the screening assays were analyzed by liquid chromatography for identification and quantification of any beta-lactam residues. All screening tests showed 0% false-negative results with 15 incurred samples containing between 4.0 and 268 microg/kg penicillin G. The biosensor assays showed 27% positive results (false violatives) with 15 producer milk samples containing penicillin G concentrations between 0 and 3.6 microg/kg, i.e., below maximum residue limit. This figure varied between 27 and 53% for the other screening tests.     

High temperature-high efficiency liquid chromatography using sub-2 μm coupled columns for the analysis of selected non-steroidal anti-inflammatory drugs and veterinary antibiotics in environmental samples

A high efficiency HPLC method was developed by coupling three sub-2 μm columns in series and operating them at high temperature for the separation of selected non-steroidal anti-inflammatory drugs and veterinary antibiotics in environmental samples. The separation was performed at 80 °C to reduce the solvent viscosity, thus reducing the column backpressure. The chromatographic performance of high temperature-extended column length HPLC method was used to determine the most widely used non-steroidal anti-inflammatory drugs and veterinary antibiotics such as sulphonamides in wastewater samples. The method could simultaneously determine 24 pharmaceuticals in short analysis time with high efficiency. The method involved pre-concentration and clean-up by solid phase extraction (SPE) using Oasis HLB extraction cartridges. It was validated based on linearity, precision, detection and quantification limits, selectivity and accuracy. Good recoveries were obtained for all analytes ranging from 72.7% to 98.2% with standard deviations not higher than 6%, except for acetaminophen and acetyl salicylic acid, for which low recovery was obtained. The detection limits of the studied pharmaceuticals ranged from 2 to 16 μg L⁻¹, while limits of quantification were in the range from 7 to 54 μg L⁻¹ with UV detection.     

Liquid–solid disk extraction followed by supercritical fluid elution and gas chromatography of phenols from water

A method combining the techniques of liquid – solid disk extraction (LSDE) and supercritical fluid elution (SFE) has been developed for the phenols regulated by the Clean Water Act. LSDE uses a disk or membrane made of polytetrafluoroethylene (PTFE) fibrils impregnated with small particles, e.g. styrene divinylbenzene (SDB) resin, to extract phenols from water. After disk extraction the retained analytes are eluted from the disk using SFE. SFE is used as an alternative to liquid solvent elution with an organic solvent. Analytes are separated, identified, and quantified using gas chromatography – ion trap detector mass spectrometry (GC-ITDMS). The method is capable of sub parts per billion detection limits, and precision of 5–28% RSD. Evaluation of various disks or membranes, such as C₁₈-silica disks, SDB disks, and ion exchange membranes, has also been performed for the extraction of phenols from water. The results obtained from the in-situ aqueous acetylation of phenols and extraction of their acetates are quantitative. The utilization of LSDE and SFE techniques has proven to be a more effective approach than liquid – liquid extraction in minimizing air pollution and solvent waste.     

固相萃取-胶束电动色谱法测定牛奶中的4种β-内酰胺类抗生素

建立了同时分离检测牛奶中的氨苄西林、阿莫西林、青霉素V和头孢氨苄4种β-内酰胺类抗生素的固相萃取-胶束电动色谱法。牛奶样品经沉淀蛋白后,采用HLB固相萃取柱净化浓缩;以含十二烷基硫酸钠(SDS)的磷酸盐为缓冲液,胶束电动色谱分离,210 nm波长下检测。分离电压为18 kV,于9 min内达到基线分离。各组分在0.5～20 mg/L范围内呈良好的线性关系,相关系数(r2)为0.9943～0.9976;检出限为0.16～0.20 mg/L;除了阿莫西林外,回收率均大于70%。该方法准确可靠,重复性好,灵敏度高,可以用于牛奶中β-内酰胺类抗生素的定量检测。     

高效毛细管电泳-电化学检测对多羟基抗生素分离测定的研究

用自制的高效毛细管电泳-电化学检测装置,以350μm铜圆盘电极为工作电极,在碱性溶液中,对7种多羟基抗生素的分离进行了研究。结果表明:浓度线性范围可达2至3个数量级,最低检测限为5.0×10^-7mol/L.在8h内连续测定,峰电流和迁移时间的相对标准偏差分别为7.6%和2.2%.实际样品(丁胺卡那霉素)测定回收率为99.6%.     

Nanoelectrode ensembles for the direct voltammetric determination of trace iodide in water

Procedures for the preparation and characterisation of ensembles of gold nanodisk electrodes (NEE) of 30 nm diameter are presented, in particular focusing on improvements in the signal/background current ratios and detection limits with respect to the electrochemical oxidation of iodide and its analytical determination in water samples. At NEEs iodide undergoes a quasi-reversible diffusion controlled oxidation with a slight shift in E _1/2 values and slightly higher peak to peak separation with respect to conventional gold disk electrodes. The double layer charging current at the NEE is significantly lower than at conventional electrodes so that the detection limit (DL) by cyclic voltammetry with NEEs in tap water is significantly lower than DL at the Au-disk millimetre-sized electrode (DL 0.3 µM at NEE vs. 4 µM for Au-disk). Finally, it is shown that NEEs in combination with square wave voltammetry can be applied for the direct determination of iodide in water samples from the lagoon of Venice, with a detection limit of 0.10 µM.     

Micellization and synergistic interaction of binary surfactant mixtures based on sodium nonylphenol polyoxyethylene ether sulfate

Mixed micelle formation and synergistic interactions of binary surfactant combinations of sodium nonylphenol polyoxyethylene ether sulfate (NPES) with typical surfactants such as sodium dodecyl sulfate (SDS), Triton X-100 (TX100), cetyl trimethyl ammonium bromide (CTAB), and sodium bis(2-ethylhexyl) sulfosuccinate (AOT) at 25 degrees C in the presence of NaCl have been investigated. The critical micelle concentration of the binary mixtures has been quantitatively estimated by steady-state fluorescence measurements. The micellar characteristics such as composition, activity coefficients, and mutual interaction parameters have been estimated following different theoretical treatments. Investigation on the micellization and synergistic interaction of NPES with four kinds of surfactants showed that the behavior of the binary mixture deviated from the ideal state. The analysis revealed that the interaction parameter values (beta) varied with variation of solvent composition. Besides the strong electrostatic attraction between the oppositely charged surfactant NPES-CTAB mixture, the interaction between NPES and SDS also showed far more deviation from ideal behavior than that of TX100 and AOT. The reason for the synergism is also discussed and the results show that an ionic and a nonionic surfactant character existed concurrently in NPES due to the combination of a sulfate group and polyoxyethylene as a hydrophilic moiety. Zeta potential and diffusion coefficient measurements of micelles confirmed the synergistic interaction between the binary surfactants.     

Self‐Assembled Redox System for Bioelectrocatalytic Assay of L‐Ascorbylphosphate and Alkaline Phosphatase Activity

Ferrocene‐terminated self‐assembled monolayer (Fc‐SAM) on gold was used as an electron‐transfer mediator in the electrochemical assay of L ‐ascorbic acid 2‐phosphate (AAP). The assay is based on the enzymatic action of alkaline phosphatase (ALP), which triggers the release of vitamin C (L ‐ascorbic acid, AA) from AAP. The latter is easily oxidized on the Fc‐SAM under the diffusion limiting conditions that favors quantitative measurement of the AA concentration on a rotating disk electrode. We demonstrate the utility of the electrochemically active Fc‐SAM to probe the mechanism and to determine the kinetic parameters of an enzymatic reaction. The electrochemical technique was compared to a conventional spectrophotometric method of ALP activity detection using p‐nitrophenylphosphate (p‐NPP) as a substrate. We demonstrate that our new technique is also suitable for the analytical determination of ALP activity. The detection limits for both AAP and ALP were found to be 13 μM and 2 pM, respectively.     

Development of a novel strategy for preconcentration of antibiotic residues in milk and their quantitation by capillary electrophoresis

A novel analytical method based on capillary zone electrophoresis coupled with diode array detection is developed and validated for the identification and simultaneous quantitation of four antibiotics in bovine raw milk. The studied antibiotics belong to different groups: β-lactams, tetracyclines, quinolones, amphenicols and sulfonamides. An experimental design including both a factorial and a central composite design allowed a reduction in the number of optimization experiments. The multiple response criterion was successfully used to optimize the separation between chloramphenicol, ciprofloxacin, ampicillin, tetracycline and sulfamethoxazol, allowing the reduction of the analysis time with excellent peak resolutions and low capillary current. Different strategies for preconcentration and extraction of the studied antibiotics were applied, in order to remove potential interferences from the sample and to increase the sensitivity. Milk samples were prepared by a clean-up/extraction procedure based on protein precipitation with trichloroacetic acid followed by liquid-liquid extraction with dichloromethane combined with solid-phase extraction, and injection into the electrophoretic system hydrodynamically. The limits of detection and quantification (below 30 and 100 μg L⁻¹, respectively) were in all cases lower than the maximum residue limits tolerated for these compounds in milk. Accuracy was evaluated by computing recoveries for the target antibiotics which were between 93.08% and 102.89%.     

A multianalyte ELISA for immunochemical screening of sulfonamide,fluoroquinolone and ß-lactam antibiotics in milk samples using class-selective bioreceptors

A multianalyte ELISA has been developed for the simultaneous determination of the most frequently used antibiotic families in the veterinary field following the typical planar microarray configuration, where the identity of the target analyte is encoded by its location in the detection platform (Master et al. in Drug Discovery Today 11:1007-1011, 2006). To accomplish this aim, two individual enzyme-linked immunosorbent assays for sulfonamide and fluoroquinolone antibiotics and an enzyme-linked receptor assay for ss-lactam antibiotics have been combined. The strategy uses microplates coated with the corresponding haptenized proteins in specific sections of the microplate. The samples are mixed with a cocktail containing the bioreagents, and distributed in the wells of the microplate. Identification of the antibiotic present in a particular sample is consequently accomplished by detecting a positive response on the corresponding microplate section. Since the bioreceptors used show a wide recognition of the congeners of each antibiotic family, the multianalyte method is able to detect more than 25 different antibiotics from the three most important antibiotic families. The detectability reached in full-fat milk samples is below the European maximum residue limits. The accuracy and reliability of this multiplexed bioanalytical method have been demonstrated by analyzing blind spiked samples.     

固相萃取/液相色谱-串联质谱法检测医院废水中21种抗生素药物残留

应用固相萃取及高效液相色谱-串联质谱技术,建立了医院废水中12种磺胺、4种喹诺酮、3种四环素以及罗红霉素和甲氧苄氨嘧啶等21种抗生素的定性定量方法。水样经HLB小柱萃取富集,使用10%甲醇溶液净化,经甲醇洗脱定容后,以高效液相色谱-串联质谱多反应监测离子模式(MRM)对目标物进行分析。在优化实验条件下,21种抗生素的线性范围为1.0～500μg/L,相关系数r2>0.99,方法检出限为0.005～0.022μg/L。在加标量为0.05μg/L和1.0μg/L时,空白加标回收率分别为71%～105%和76%～111%,RSD均小于15%。以医院废水为基质,21种抗生素的加标回收率为71%～135%,RSD小于25%。该方法简捷、快速、准确,能够实现医院废水中多种抗生素药物残留的同时分析。     

Trace determination of beta-lactam antibiotics in environmental aqueous samples using off-line and on-line preconcentration in capillary electrophoresis

A sensitive and reliable method using capillary zone electrophoresis with UV-diode array detection (CZE-DAD) has been developed and validated for trace determination of beta-lactam antibiotics in waste, well and river water matrices. Due to the lack of sensitivity of the UV-vis detection, a solvent extraction/solid-phase extraction (SPE) method applied for off-line preconcentration and cleanup of water samples, in combination with an on-line preconcentration methodology named large volume sample stacking (LVSS) have been applied. The analytes included nafcillin, dicloxacillin, cloxacillin, oxacillin, ampicillin, penicillin G and amoxicillin. Average recoveries for water samples fortified with the studied beta-lactams at different concentration levels (1.0, 2.0 and 4.0 microg/L) were ranging between 94 and 99%, with relative standard deviations (RSDs) lower than 10%. The precision, calculated as intra-day and inter-day standard deviations fell within acceptable ranges (3.3-7.2%). The limits of detection were estimated to range between 0.08 and 0.80 microgL(-1) for the studied compounds. All the samples analyzed were negative for all the analytes at these levels of concentration and the method showed its usefulness for the detection of these widely applied beta-lactam antibiotics in different kinds of waters.