Search

Present study reports design of experiment (DoE) based development and validation of a simple, rapid and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method for estimation of efavirenz (EFZ), a non-nucleotide reverse transcriptase inhibitor (NNRTs), used in the treatment of acquired immunodeficiency syndrome (AIDS). Plackett–Burman design was explored to screen the critical method variables (CMVs) for the RP-HPLC method. A response surface Box–Behnken design was employed to optimize the screened CMVs which affect the analytical responses (ARs) of RP-HPLC method. Using the optimized CMVs the HPLC method was developed and validated according to International Conference on Harmonization (ICH) guidelines. EFZ in marketed formulation was estimated using the validated method. Acetonitrile proportion, pH of the phosphate buffer and mobile phase flow rate were the CMVs and retention time and number of theoretical plates were the ARs for the study. The optimized chromatographic parameters were acetonitrile proportion in mobile phase: 51.17%v/v, pH of phosphate buffer: 4.04 and flow rate: 1.25 mL/min. Use of these optimized parameters resulted in retention time of 11.031 min and 9,498.787 number of theoretical plates as ARs of the HPLC method. The method was further validated in harmony with current ICH guidelines Q2 (R1). The method was capable of the successful estimation of EFZ in marketed formulation. The study depicts successful development and validation of a simple RP-HPLC method of EFZ using DoE approach.

Scanning ion conductance microscopy (SICM) is useful for imaging soft and fragile biological samples in liquids because it probes the samples’ surface topography by detecting ion currents under non-contact and force-free conditions. SICM acquires the surface topographical height by detecting the ion current reduction that occurs when an electrolyte-filled glass nanopipette approaches the sample surface. However, most biological materials have electrically charged surfaces in liquid environments, which sometimes affect the behavior of the ion currents detected by SICM and, especially, make topography measurements difficult. For measuring such charged samples, we propose a novel imaging method that uses a double-barrel nanopipette as an SICM probe. The ion current between the two apertures of the nanopipette desensitizes the surface charge effect on imaging. In this study, metaphase chromosomes of Indian muntjac were imaged by this technique because, owing to their strongly negatively charged surfaces in phosphate-buffered saline, it is difficult to obtain the topography of the chromosomes by the conventional SICM with a single-aperture nanopipette. Using the proposed method with a double-barrel nanopipette, the surfaces of the chromosomes were successfully measured, without any surface charge confounder. Since the detailed imaging of sample topography can be performed in physiological liquid conditions regardless of the sample charge, it is expected to be used for analyzing the high-order structure of chromosomes in relation to their dynamic changes in the cell division.

Immunomagnetic separation used with culture based methods has been a useful technique in the detection of pathogens. However, previous studies have not answered many of the necessary questions for real world applications. The objective of this study was to assess the efficacy of different immunomagnetic separation (IMS) bead types in recovery of the correct serogroup from a mixture of big six non-O157 Shiga toxin-producing Escherichia coli strains. To determine the impact of different matrices on recovery, samples of sterile phosphate buffered saline (PBS), sterile and non-sterile cattle faeces, ground beef and lettuce were inoculated with 10 CFU per ml mixture of isolates representing the six serogroups. After a 6 h incubation at 37°C, samples were mixed with IMS beads from three different commercial sources and plated on eosin methylene blue agar (EMB). Three suspect E. coli colonies were selected from each EMB plate and multiplex polymerase chain reaction was used to determine the serogroup. The rate of correct identification varied with the serogroup, IMS bead manufacturer and matrix. Overall, recovery of the correct serogroup became less likely with increase in matrix complexity, with enrichments containing lettuce having the greatest number of bead types with significantly lower likelihood of correct recovery compared to recovery in PBS. Significance and Impact of the Study The need to accurately and efficiently detect Shiga toxin-producing Escherichia coli (STEC) O26, O45, O103, O111, O121 and O145, which have caused outbreaks on numerous occasions, is a major public health and food safety concern in the United States. Detecting these STEC serogroups can be challenging because methods to detect non-O157 serogroups have not been refined as compared to those for O157. Immunomagnetic separation (IMS) has the potential to isolate STEC from a mixture in complex matrices. Our results highlight the need for optimization of IMS-based detection of STEC to effectively recover the targeted serogroup from a variety of sample matrices.

Two new simple and selective assay methods have been presented for the analysis of Escitalopram Oxalate (ESC) and L-Methylfolate (L-MF) in pharmaceutical formulations. The ultraviolet (UV)-Spectrophotometric simultaneous equation method was based on the measurement of absorbance at 238 nm (ʎmax of ESC) and 284 nm (ʎmax of L-MF) in methanol. The assay was linear over the concentration ranges 0.5-12.0 μg/mL for ESC and 0.5-9.0 μg/mL for L-MF. The quantitation limits for ESC and L-MF were found to be 0.912 and 0.667 μg/mL; while the detection limits were 0.301 and 0.220 μg/mL for ESC and L-MF, respectively. The second method involved isocratic reversed-phase liquid chromatography using a mobile phase composed of methanol-0.02 M phosphate buffer (pH 5.5) (75:25, v/v), Hypersil BDS-C18 Column (5 µm, 250 mm × 4.6 mm i.d.) with detection at 270 nm. The linearity ranges were found to be 3.0-30.0 and 0.75-22.5 μg/mL for ESC and L-MF, respectively. The limits of detection were found to be 1.065 and 1.160 μg/mL for ESC and L-MF, respectively. The limits of quantitation were found to be 3.226 and 3.515 μg/mL for ESC and L-MF, respectively. The proposed methods were successfully applied to the determination of commercially available tablets with a high percentage of recovery, good accuracy and precision.

Aims The goal of this study was to explore various models for describing viral persistence (infectivity) on fomites and identify the best fit models. Methods and Results The persistence of poliovirus over time was studied on three different fomite materials: steel, cotton, and plastic. Known concentrations of poliovirus type 1 were applied to the surface coupons in an indoor environment for various lengths of time. Viruses were recovered from the surfaces by vortexing in phosphate buffer. Seven different mathematical models of relative persistence over time were fit to the data, and the preferred model for each surface was selected based on the Bayesian Information Criterion. Conclusions While the preferred model varied by fomite type, the virus showed a rapid initial decay on all of the fomite types, followed by a transition to a more gradual decay after about 4 to 8 days. Estimates of the time for 99% reduction ranged from 81 hours for plastic to 143 hours for cotton. A 6 log reduction of recoverable infectivity of poliovirus did not occur during the 3 week duration of the experiment for any of the fomites. Significance and Impact In protected indoor environments poliovirus can remain infective for weeks. The models identified by this study can be used in risk assessments to identify appropriate strategies for managing this risk. This article is protected by copyright. All rights reserved.

Sampling of endotoxin, beta-glucan, or allergens on electrostatic dust collectors (EDCs) is a convenient method for exposure assessment. However, especially for allergens few experiments on validation of this method concerning deployment time or storage and extraction procedure have been performed. The aim of study was to optimize the EDC procedure for sampling of allergens in indoor environments. EDCs were placed in households or day-care centers and after extraction, allergens were quantified by six immunoassays detecting mite antigens (Domestic mites DM, Dermatophagoides pteronyssinus Dp, Tyrophagus putrescentiae Tp) or the main allergens from cat (Fel d 1), dog (Can f 1) and mouse (Mus m 1). For 20 EDC holders, deployment times of cloths were varied between 7 and 28 days, 36 EDCs were used to test reproducibility, and for 28 EDCs extraction buffers were varied (with or without 0.05% Tween 20, borate, or phosphate buffer). The influence of storage of cloths at room temperature (2-629 days) or extracts at -80°C (7-639 days), and variation of extract storage temperature (-20°C and -80°C) for long time storage (1.5 years) on the outcome of allergen quantification were tested for about 150 EDCs. The allergens on EDC cloths increased proportionally with deployment time, and allergen loads on parallel sampled tissues were significantly correlated (P < 0.0001, Pearson of log-transformed values 0.91-0.99). Extraction without Tween reduced all results (P < 0.0001, -51% DM, -85% Dp, -60% Tp, -99% Fel d 1, -86% Can f 1, -52% Mus m 1), and borate buffer resulted in lower yields of Mus m 1 (-53%), DP (-45%), and Tp (-27%) than phosphate buffer. Storage of cloths at room temperature significantly decreased Can f 1 levels (P < 0.0001, -4.8% loss for every 30 days), whereas storage of extracts at -80°C decreased DM results (P < 0.0001, -1.2% loss for every 30 days). Extracts stored at -20°C gave at mean 12% higher DM results compared to extracts stored at -80°C for 1.5 years. Several mammalian allergens and also DM antigens could be quantified reproducibly on EDCs from indoor environments. Allergen levels on EDC cloths increased proportionally with deployment time in a period of 4 weeks. Allergen yields are strongly influenced by the extraction procedure; the use of detergent Tween 20 and phosphate buffer is recommended.

Aims The objective of this research is to evaluate eluents for recovery of an enveloped bacteriophage, Φ6, using whole-hand sampling. Methods and Results Virus was applied to the hands of volunteers and sampled by the glove juice method with 1·5% beef extract (BE), phosphate buffered saline (PBS), 0·01 and 0·1% Tween 80, tryptic soy broth (TSB) and 9% NaCl. Each volunteer underwent multiple rounds application and hand sampling. Mean log10 virus loss across trials was 2·6 for BE, 2·8 for PBS, 2·4 for TSB, 3·8 for NaCl, 3·0 for 0·1% Tween 80, and 2·9 for 0·01% Tween 80. Within each volunteer, there was a decline in viral loss from the first to last trial. Conclusions These eluents can recover Φ6 from hands with approx. 2–3 log10 loss, comparable to recoveries previously reported for influenza. Protein and detergent-based eluents may have similar recoveries, but recovery may still vary across repeated sampling. Significance and Impact of the Study Based on current work, protein-based eluents such as beef extract can maximize recovery of enveloped viruses during hand sampling, providing methods for evaluating survival and transmission of enveloped viruses on hands. Further exploration is needed of the effect of repeated sampling on recovery from whole-hand sampling.

Predators and pathogens can diminish organisms’ fitness, and consequently natural selection favors antipredatory and antiparasitic strategies. Nevertheless, as both kinds of strategies consume resources, animals cannot maximize investment in both strategies simultaneously, which should lead to trade-offs between the two. Accordingly, we hypothesize that there is a trade-off between sprint capacity and immune response, which are among the most important antipredatory and antiparasitic strategies, respectively, of many animals. Consequently, we predict that animals eliciting an immune response should reduce their sprint capacity. We experimentally tested this prediction in the lizard Psammodromus algirus. A group of lizards was inoculated with the antigen LPS (lipopolysaccharide of the cell wall of Escherichia coli), which activates the immune system, whereas the other group (control) was inoculated with phosphate buffer, which has no physiological effect. Before and after the inoculations, we filmed sprint capacity of lizards, estimating the maximal sprint speed. Males were faster than females before the activation of their immune system. Nonetheless, males’ sprint speed diminished after the inoculation of LPS, whereas females’ did not. Therefore, our results support the hypothesis of a trade-off between sprint speed and immune response in males. Consequently, male lizards combating a pathogen may suffer either higher risk of predation, or reduced fitness if they avoid this risk by spending more time in shelter.

Two stability-indicating methods were developed for the determination of candesartan cilexetil in the presence of its degradation products. The first method uses isocratic RP-HPLC with an Agilent C18 column. The mobile phase was phosphate buffer (pH = 2.8 ± 0.1)–acetonitrile (60 + 40, v/v). The flow rate was 2.0 mL/min, and the UV detection was at 254 nm. The second method depends on TLC-densitometric measurements of drug spots at 254 nm. The separation was carried out on silica gel 60 F254 plates using ethyl acetate–methanol–toluene– ammonia 33% (40 + 25 + 20 + 2, v/v/v/v) mobile phase. The methods were validated according to U.S. Pharmacopeia guidelines, and the acceptance criteria for accuracy, precision, linearity, specificity, robustness, LOD, LOQ, and system suitability were met in all cases. Linear ranges of the methods were 10.0–200.0 μg/mL and 1.0–9.0 μg/spot for HPLC and TLC, respectively. The proposed methods were successfully applied to the drug in bulk powder, in laboratory-prepared mixtures with its degradation products, and in commercially available tablets. The results were compared statistically at the 95% confidence level with each other. There were no significant differences between the mean recovery and precision of the two methods.

In this study, a simple and selective high-performance liquid chromatography method was developed and validated for the determination of nifekalant hydrochloride in canine plasma. Liquid –liquid extraction was used to separate nifekalant hydrochloride from canine plasma and the mean extraction recoveries of nifekalant hydrochloride and the internal standard were 82.31 and 94.81%, respectively. The chromatographic separation was performed on a Dikma Diamonsil column with a mobile phase consisting of acetonitrile –20mM phosphate buffer ( pH 6.2; 30:70, v/v) with flow rate of 1.0 mL/min. The standard curve was linear over the concentration range of 20 –10,000 ng/mL (r 2 > 0.99). The intra-batch and inter-batch accuracy for nifekalant hydrochloride at four concentrations were 93.14–100.47% and 96.12–103.77%, respectively. The relative standard deviations were less than 15%. The method was successfully applied to a pharmacokinetic study after the intravenous administration of nifekalant hydrochloride to beagle dogs.

A sensitive and efficient on-line clean up and pre-concentration method has been developed using column-switching technique and protein-coated µ-Bondapak CN silica pre-column for quantification of ambroxol (AM) in human serum. The method is performed by direct injection of serum sample onto a protein-coated µ-Bondapak CN silica pre-column, where AM is pre-concentrated and retained, while proteins and very polar constituents are washed to waste using a phosphate buffer saline (pH 7.4). The retained analyte on the pre-column is directed onto a C(18) analytical column for separation, with a mobile phase consisting of a mixture of methanol and distilled deionized water (containing 1% triethylamine adjusted to pH 3.5 with ortho-phosphoric acid) in the ratio of 50:50 (v/v). Detection is performed at 254 nm. The calibration curve is linear over the concentration range of 12-120 ng/mL (r(2) = 0.9995). The recovery, selectivity, linearity, precision, and accuracy of the method are convenient for pharmacokinetic studies or routine assays.

An isocratic HPLC method with detection at 248 nm was developed and fully validated for the determination of tigecycline in rabbit plasma. Minocycline was used as an internal standard. A Hypersil BDS RP-C18 column (250 × 4.6 mm, 5 μm particle size) was used with the mobile phase phosphate buffer (pH 7.10, 0.070 M)–acetonitrile (76 + 24, v/v) at a flow rate of 1.0 mL/min. The elution time of tigecycline and minocycline was approximately 8.1 and 9.9 min, respectively. Calibration curves of tigecycline were linear in the concentration range of 0.021–3.15 μg/mL in plasma. The LOD and LOQ in plasma were estimated as 7 and 21 ng/mL, respectively. The intraday and interday precision values of the method were in the range of 5.0–7.1 and 5.6–9.1%, while the corresponding accuracy values were in the ranges of 92.8–111.1 and 97.6–102.3%, respectively. At the LOQ, the intraday precision was 18.7%, while intraday and interday accuracy values were 97.3 and 98.0%, respectively. Robustness of the proposed method was studied using a Plackett-Burman experimental design. A pharmacokinetic profile is presented for confirmation of the applicability of the method to pharmacokinetic studies.

The aim of this study was to develop a new, simple, rapid, and sensitive square-wave adsorptive stripping voltammetric method for the determination of candesartan cilexetil in bulk form and pharmaceutical formulations by complex formation with Cu(II). The experimental and instrumental parameters affecting the response of the candesartan cilexetilCu(II) complex were investigated and optimized. This method was based on electrochemical reduction of candesartan cilexetilCu(II) complex at a hanging mercury drop electrode in phosphate buffer at pH 3.0 containing CuCl22H2O. A well-defined reduction peak was observed at 325 mV with 30 s accumulation time and 150 mV accumulation potential versus an Ag/AgCl reference electrode in the proposed method. The developed voltammetric method was validated in terms of linearity, sensitivity, precision, accuracy, recovery, selectivity, robustness, and ruggedness. The linear calibration range was 0.501.77 g/mL (r 0.9972). The LOD and LOQ of this method were 0.10 and 0.50 g/mL (n 7), respectively. The developed method was fully validated and applied to the pharmaceutical formulations, including candesartan cilexetil.

Aims: Having and executing a well-defined and validated sampling protocol is critical following a purposeful release of a biological agent for response and recovery activities, for clinical and epidemiological analysis and for forensic purposes. The objective of this study was to address the need for validated sampling and analysis methods called out by the General Accounting Office and others to systematically compare the collection efficiency of various swabs and wipes for collection of bacterial endospores from five different surfaces, both porous and non-porous. This study was also designed to test the collection and extraction solutions used for endospore recovery from swabs and wipes. Methods and Results: Eight collection tools were used, five swabs and three wipes. Three collection/preservation solutions were evaluated: sterile E-pure® water, phosphate buffered saline (PBS), and phosphate buffered saline with 0.3% Tween (PBST). An Ink Jet Aerosol Generator (IJAG) was used to apply Bacillus subtilis endospores to five porous and non-porous surfaces. The collection efficiencies of the swabs and wipes were compared using a statistical multiple comparison analysis. Results indicated that wipes tend to have higher collection efficiency than swabs. Of the swabs tested, the recovery from most of the surfaces was highest with the polyurethane foammore » swab. Conclusions: The ScottPure® wipe had the highest collection efficiency and PBST was the best collection solution of those tested. Significance and Impact of Study: Validated sampling for potential biological warfare is of significant importance and this study answered some relevant questions.« less

An updated liquid chromatographic (LC) method for the determination of glyphosate in technical material and formulations was tested by 7 laboratories. Samples were dissolved in water and injected into an LC system with a C18 guard column, an anion exchange analytical column, and a phosphate buffer mobile phase with 12 to 15 methanol. Detection was by UV absorption at 195 nm or refractive index. Automated injections were made. Calculations were based on peak area comparisons with external standards. The interlaboratory study analyzed 6 matched pairs of 3 glyphosate technical materials and 3 glyphosate formulations. The matched pair results were analyzed by using the AOAC spreadsheet and Youden's matched pair calculations. Coefficients of variation for the 6 samples ranged from 0.46 to 0.85.

A reversed-phase liquid chromatographic (LC) method was developed and validated for the simultaneous determination of ezetimibe and simvastatin in pharmaceutical dosage forms. The LC method was carried out on a Synergi fusion C18 column (150 mm 4.6 mm id) maintained at 45C. The mobile phase consisted of phosphate buffer 0.03 M, pH 4.5acetonitrile (35 + 65, v/v) run at a flow rate of 0.6 mL/min, and detection was made using a photodiode array detector at 234 nm. The chromatographic separation was obtained within 15.0 min, and calibration graphs were linear in the concentration range of 0.5200 g/mL. Validation parameters such as specificity, linearity, precision, accuracy, and robustness were evaluated, giving results within the acceptable range for both compounds. Moreover, the proposed method was successfully applied for the routine quality control analysis of pharmaceutical products.

Aims: To investigate the response of Campylobacter jejuni ATCC 35919 and 35921 to high pressure processing (HPP) while suspended in microbiological media and various food systems. Methods and Results: Campylobacter jejuni 35919 and 35921 were subjected to 10-min pressure treatments between 100 and 400 MPa at 25°C suspended in Bolton broth, phosphate buffer (0·2 m, pH 7·3), ultra-high temperature (UHT) whole milk, UHT skim milk, soya milk and chicken puree. The survivability of C. jejuni was further investigated by inoculated pack studies. HPP at 300–325 MPa for 10 min at 25°C was sufficient to reduce viable numbers of both strains to below detectable levels when cells were pressurized in Bolton broth or phosphate buffer. All food products examined offered a protective effect in that an additional 50–75 MPa was required to achieve similar levels of inactivation when compared with broth and buffer. Inoculated pack studies showed that the survivability of C. jejuni following pressurization improved with decreasing post-treatment storage temperature. Significance and Impact of the Study: These data demonstrated that HPP at levels of ≤400 MPa, can inactivate C. jejuni in both model and food systems.

A liquid chromatographic (LC) method was developed for determining deoxynivalenol (DON) in whole wheat flour and wheat bran. A 15 g test sample was extracted with acetonitrile–water (84 + 16, v/v) and applied to a Romer MycoSep cleanup column. The eluate was dried and then reconstituted in a 0.1M phosphate buffer, pH 7.0, and applied to a Vicam DONtest-LC cleanup column. The methanol eluate was chromatographed with a methanol–water (17 + 83, v/v) mobile phase on a C18 column with UV detection at 220 nm. Five replicates at each of 5 fortification levels (0.25, 0.50, 1.0, 2.0, and 4.0 ppm), plus 5 controls, were determined for both whole wheat flour and wheat bran. For flour, the average recoveries were 72.2–91.5% with relative standard deviations (RSDs) of 4.9–18.4%. The intra-assay flour recovery was 82.4% with 9.8% RSD. A 5 replicate sample of naturally incurred wheat had an average of 1.1 ppm DON with 6.7% RSD. For bran, average recoveries of fortified samples were 69.5–99.7% with RSDs of 1.7–18.8%. The intra-assay bran recovery was 81.5% with 8.9% RSD. The limit of detection (about 3× noise) for the method is 0.05 ppm; the correlation coefficient (linearity) was >0.9995. The DON peak was clearly identified and easily integrated in the chromatograms.

The aim of the study was to test the following hypotheses: (i) that endotoxin injected 40 min prior to death can be detected in rat organs post mortem and (ii) that endotoxin levels do not change with increasing time post mortem. Rats were injected with or without endotoxin in buffered saline, 40 min prior to being killed. Endotoxin levels in rat organs were assessed using a Limulus amoebocyte assay. The effect of storage time post mortem was assessed by following various storage regimes at 25 degrees C and 8 degrees C. Significant differences (P =0.001) in endotoxin levels of all samples tested were found between rats injected with and without endotoxin. A significant increase in detectable endotoxin was observed between 0 h and 6 h post mortem in rats injected with or without endotoxin. No difference in detectable endotoxin levels in the kidney, liver and spleen was observed from 30 h to 102 h post mortem in rats injected with or without endotoxin. In rats injected with endotoxin, detectable endotoxin levels in the heart were raised between 0 h and 6 h, 6 h and 54 h, and 30 h and 78 h. Endotoxin injected into rats 40 min prior to death can be detected post mortem. For rats injected with saline or endotoxin prior to death levels in the kidney, liver and spleen were not affected by storage at 8 degrees C for 30-102 h, after initial storage at room temperature for 6 h. Levels of endotoxin detected in the hearts of rats injected with saline were not affected by storage up to 102 h. In rats injected with endotoxin prior to death, detectable levels in the heart were significantly affected by increasing time in storage.

Alliin and allicin in garlic and their products are determined simultaneously by ion-pair reversedphase liquid chromatography (LC) with diode array UV detection. Alliin is extracted from fresh garlic, purified by ion-exchange chromatography, and appliedas external standard. Allicin is generated enzymatically from alliin, confirmed by LC, and applied as external standard. Compounds areextracted from samples with water and chromatographed on an octadecylsilane column with gradient elution from 0.01 M phosphate buffer (pH 2.5) with 5 mM heptanesulfonic acid (A) to 0.01 M phosphate buffer (pH 2.5)-acetonitrile (1 + 1; B). Themethod can be used to analyze fresh garlic, spices, garlic preparations, and health foods.

Previous studies have demonstrated that Verticillium lecanii might be used as a microbial model of the inversion of 2-arylpropionic acids in man. This paper describes the optimization of the inversion process in respect of culture medium, pH, cell density and substrate concentration. The study demonstrates that optimum inversion occurs in Sørensen's phosphate buffer at pH 5.5. The extent and rate of inversion were also shown to be dependent on substrate concentration and cell density. This study will form the basis of the development of a microbial model of the metabolism of 2-arylpropionic acids which might be suitable for the in-vitro screening of new compounds in this class.

A method is presented for detection and quantitation of Roxarsone in poultry feed by liquid chromatography. The drug is extracted by phosphate buffer and determined by solid phase extraction and reversed-phase liquid chromatography. Recoveries of the sample spikes and fortified field samples agree closely with those obtained by the standard spectrophotometric method.

Bordetella bronchiseptica grew from small inocula, and retained viability for at least 24 weeks, in unsupplemented lakewater or phosphate-buffered saline. From washed inocula of around 10(3) colony-forming units/ml, there was growth at both 10 degrees C and 37 degrees C to give 10(6)-10(7) colony-forming units/ml. At 10 degrees C, these counts were maintained with little diminution up to week 24 when observations ceased. In the tests at 37 degrees C, two of three strains tested showed similar retention of viability. These results suggest that B. bronchiseptica may exist as hitherto unsuspected reservoirs of infection in freshwater habitats.

Aims: Giardia lamblia is one of the most important waterborne pathogens in the world. In this study, we determined the effectiveness of a promising alternative UV technology – a polychromatic emission from a medium-pressure (MP) UV lamp – against G. lamblia cysts in phosphate buffered saline (PBS) and a filtered drinking water. Methods and Results: A UV collimated beam apparatus was used to expose shallow suspensions of purified G. lamblia cysts in PBS or a filtered drinking water and the UV-irradiated G. lamblia cysts were assayed in Mongolian gerbils. The inactivation of G. lamblia cysts was very rapid and reached a detection limit of >3 log10 within a UV dose of 1 mJ cm−2. Conclusion: The results of this study indicate that MP UV irradiation is very effective against G. lamblia cysts in both PBS and a filtered drinking water. Significance and Impact of the Study: It is likely that contamination of drinking water by G. lamblia cysts can be readily controlled by typical MP UV disinfection practises.

Certain elderly humans show a blunted fever response to infection. A study was designed using a murine model to assess the influence of age on the febrile response to the endogenous pyrogen, tumor necrosis factor alpha (TNF alpha). Twenty (10 young: 4-6 months; 10 old: 24-28 months) BALB/c mice were injected with 50 ng of TNF alpha into the intraperitoneal space; the experiments were repeated one week later with 100 ng TNF alpha. Control animals received intraperitoneal injections of pyrogen-free phosphate buffered saline. Temperatures were measured rectally at baseline and at 10-minute intervals for 90 minutes post-injection using a thermistor probe and temperature gauge. In the majority of the time intervals following injection, the mean temperature changes of young mice were significantly higher than old mice for both 50 ng and 100 ng doses of TNF alpha. Similarly, peak temperature changes from baseline were consistently higher in young animals following injection of TNF alpha. Moreover, the peak temperature changes in young mice after 50 ng TNF alpha injection were significantly higher than those in old mice following a 100 ng injection of TNF alpha. These findings confirm that (a) TNF alpha has a role in the pathogenesis of fever; (b) aging alters significantly the febrile response; and (c) a mechanism of this age-related blunted febrile response may involve TNF alpha.

Data from a reverse phase gradient elution HPLC assay for human epidermal growth factor (EGF) was compared and correlated with data obtained from a competitive heterogeneous radioimmunoassay (RIA). The RIA was established to measure very low concentrations of EGF in formulation drug release and compatibility studies. The HPLC assay, capable of resolving parent and possible modified or fragmented forms of EGF isolated from human urine, was studied as a potential development tool for stability and final product evaluation. As independent analytical methods, the HPLC and the RIA procedures produced correlated results when quantifying freshly prepared and certain degraded samples of EGF. The capacity of the HPLC method to serve as a stability indicating assay was examined. Degradation of EGF was induced by storage in 0·05 m phosphate buffer pH 7·4 at 25, 37 or 50°C. The same three degradation products were detected at each temperature by the HPLC method, one of which was identified as l-isoaspartyl EGF.

The effects of 50 Hz alternating current (a.c.) pretreatment on the high pressure tolerance of Escherichia coli K-12 cells were found to be dependent on the composition of the solutions suspending the cells during treatment with pressure up to 4000 kg/cm2 at room temperature for 10min. When pressurized in a 8/15 m NaCl solution (pH 7.0) at 3000 kg/cm2 after a.c. exposure of 600mA/cm2 for 2 hr at 35°C, the exposed cells could not survive as well as those pressurized in a 8/15 m phosphate buffer (pH 7.0): the value of the ratio of the exposed cells surviving to that of the unexposed ones was less than 0.1 in an NaCl solution and about 0.5 in a buffer. The lethal action of high pressure on the cells suspended in m/15 buffer was appreciably counteracted by 8/15 m buffer but not by 8/15 m NaCl solution.

The sensitivity of nine cereal fungi to irradiation on grain and in phosphate-buffered saline (PBS) was investigated. Species of Fusarium and Alternaria were more resistant to irradiation (higher D 10 value) than Aspergillus spp. and Penicillium spp. Generally D 10 values determined on grain were lower than the corresponding values measured in PBS. The plating medium did not significantly affect the D 10 values

The kinetic and stability characteristics of crude extract nitrile hydratase fromBrevibacterium R-312 were studied for the hydration of 3-cyanopyridine to nicotinamide. The enzyme was substrate and product inhibited and had the following kinetic constants:K m =28 mM;K p =36 mM;K s =155 mM;V m =5.8 μ mol/min/mg protein (25°C). Itsmaximum temperature and pH (phosphate buffer) were 35°C and 8.0, respectively and it had half-lives of 50 days, 10 days and 1 day at 4°C, 10°C and 25°C, respectively. The crude extract also exhibited amidase activity on nicotinamide, but it became significant only at nicotinamide concentrations greater than 300 mM. Mathematical models for batch and fed-batch hydrations were developed to account for substrate and product inhibitions and for enzyme decay. They predicted to within 10% experimental results for initial substrate and final product concentrations up to 300 mM; the accuracies decreased at higher concentrations primarily because of the relatively rapid hydrolysis of nicotinamide.

The ionizing radiation resistances of sixSalmonella species were examined. The experimental variables were the suspending medium, the presence or absence of air, and the temperature during the irradiation process.S. typhimurium ATCC 14028,S. enteritidis ATCC 9186,S. newport ATCC 6962,S. dublin ATCC 15480,S. anatum ATCC 9270, andS. arizonae ATCC 29933 were suspended in phosphate buffer (0.1 M, pH 7.0), brain heart infusion broth (BHI) or mechanically deboned chicken and exposed to gamma radiation from cesium-137 at 0.12 kGy per min. The radiation resistance of theSalmonella increased approximately two-fold when assayed in sterile mechanically deboned chicken rather than in buffer or BHI. The average radiation (0.30 to 1.20 kGy) D-value for all sixSalmonella strains was 0.56 kGy in mechanically deboned chicken.S. enteritidis was significantly more resistant to ionizing radiation than the other five strains ofSalmonella tested on mechanically deboned chicken. The temperature of irradiation but not the presence or absence of air significantly influenced the survival ofS. typhimurium andS. enteritidis in mechanically deboned chicken. Treatment of chicken meat with ionizing radiation would be an effective means for control ofSalmonella contamination.

Mouse embryos of different developmental stages from 1- to 8-cell inclusive were ultrarapidly frozen in 0.25 ml French straws after various periods of equilibration (1, 3, 5 and 9 min) in freezing-buffer containing 3.5 M dimethylsulphoxide (DMSO), 0.25 M sucrose, and 20% fetal calf serum (FCS) in phosphate buffered saline (PBS). After thawing in a 37 degrees C waterbath and dilution for 5 min in 0.25 M sucrose in PBS/FCS the embryos were cultured in Ham's F10 medium with 10% FCS (37 degrees C, 5% CO2, 95% humidity) for 4-6 days. The rates of expanded and hatching blastocysts were then evaluated and compared to the corresponding rates of non-frozen controls. Thus, for each cleavage stage, the optimal equilibration time was evaluated. It was 1 min for the 1-cell and 8-cell stages (32 and 81% blastocysts, respectively), and 3-5 min for the 2-cell (76 and 73%, respectively) and the 4-cell stage (88 and 87%, respectively). It is concluded that the ultrarapid freezing method described provides satisfactory results for all tested cleavage stages, but not for the 1-cell stage.

A simple method was developed for the determination of dietary fiber in multicomponent foods. The method involves dispersing the sample into pH 7.4 phosphate buffer and adding bile and pancreatic enzyme as described. Results were comparable to AOAC methods with correlation coefficients of 86% for multicomponent dinners and 89% for breakfast foods. Coefficients of variation ranged from 7.4 to 20.0% for multicomponent foods and 1.0 to 3.6% for single component foods. In addition, blind duplicate samples had a correlation of 0.99. The described method required less time, labor, and manipulation than AOAC methods

The dissolution profiles of 600mg ibuprofen tablets from four manufacturers have been compared with those of Boots's Brufen. The latter showed rapid dissolution in phosphate buffer pH 7.2 (the USP method) such that 50 per cent was dissolved in 3.9 ± 0.9 min and 90 per cent in 7.7 ± 1.9 min. Tablets from one manufacturer showed similar dissolution characteristics to those of Boots while the other manufacturers' products dissolved more slowly. Five out of 48 tablets from one manufacturer did not achieve the USP specification of 70 per cent dissolved by 30 min. Differences in dissolution rates have been shown to affect speed of action in some products. A study is required to determine the analgesic properties of ibuprofen tablets that dissolve at different rates.

In this precise, reversed-phase liquid chromatographic procedure, Cyclosporin A (Cy A) is extracted from 300 microL of whole blood with cyclosporin D as internal standard by liquid-liquid extraction. A 20-microL aliquot of the extract, injected onto a Nucleosil octyl analytical column heated at 72 degrees C, is eluted with a mixture of acetonitrile and 0.01 M phosphate buffer, pH 5.5 at a flow rate of 1 mL/min. Detection is set at 210 nm. The chromatography is complete within 10 min. The detection limit is 25 micrograms/L. Between-run CVs range from 2.3 to 6.2% and recovery is 92.1 +/- 6.3%. The major advantages of our extraction procedure are the rapid clean-up method and the small volume required. This procedure is especially suitable for cyclosporine determination in pediatric transplantations.

Oligodeoxynucleotide (ODN; 1) containing 2-N-tert-butylaminoxyladenosine (2) was synthesized by the conventional phosphoramidite method. The thermal stability and structure of the duplex of ODN 1 and the complementary strand were elucidated by UV-melting temperature measurements and CD experiments. In phosphate buffer, ODN 1 showed a three line EPR spectrum. Upon duplex formation, the EPR spectrum of 1 changed which indicates that EPR spectra of 1 can provide information on structures of ODN.

Samples of albino mice cerebellar cortex were processed by the cryofracture method for scanning electron microscopy and examined with the field emission scanning electron microscope (FESEM). Albino mouse cerebellar cortex was excised, cut into 1-2 mm slices and inmersed in 4% glutaraldehyde in O. l M phosphate buffer solution, pH 7.4, for 24h at 4°C; and postfixed for 1 h in a similarly buffered 1% osmium tetroxide solution. Specimens were dehydrated in a graded serie of ethanol (30, 50, 70, 80, 90 2x100%) prior to wrapping individual tissue pieces in preformed absolute ethanol filled parafilm cryofracture packets. Rapid freezing of packets was performed by plunging into LN2. First, the packet was transferred from the LN2 storage vessel with LNT chilled forceps in order to avoid themial damage. Secondly, the cooled fracture blade was removed from the LN2, the packet was orientated under the blade, and immediately struck with a heavy tool.

To determine the effects of DL-malate on ruminal metabolism, four steers equipped with ruminal cannulas were fed an 80% rolled grain (75% corn:25% wheat) diet twice daily with a DMI equal to 2.0% of BW (485+/-24.8 kg). DL-Malate was infused into the rumen on two consecutive days in 500 mL of phosphate buffer to provide 0, 27, 54, or 80 g of DL-malate/d. Ruminal pH linearly increased (P.01) with DL-malate concentration and was greater (P.01) for DL-malate than for the control steers (6.07 vs 5.77). DL-Malate treatment linearly decreased (P.10) total VFA and tended to linearly increase (P = .10) acetate concentration. Propionate, butyrate, and L-lactate concentrations and acetate:propionate ratio were not affected (P.10) by DL-malate. Three finishing studies were conducted to determine the effects of feeding DL-malate on growth rate and feed efficiency. In a 98-d experiment, 33 crossbred steers were randomly allotted in a Calan gate feeding system to three DL-malate levels (0, 40, and 80 g/d). Steers (initial weight = 367+/-4.5 kg) were fed a rolled corn-based diet twice daily. After 84 d on feed, gain efficiency (gain:feed) tended to improve with more DL-malate (linear, P.10) and was 8.1% greater (P.05) for DL-malate than for the control. The ADG linearly increased (P.05) with more DL-malate and was 8.6% greater (P = .10) for DL-malate than for the control. After 98-d on feed, ADG was linearly increased (P = .09) by DL--malate, and the greatest increase occurred with 80 g of DL-malate. In the second performance study, 27 Angus steers were randomly allotted in a Calan gate feeding system to three DL-malate concentrations (0, 60, and 120 g/d). Steers (initial weight = 432+/-4.6 kg) were fed diets used in the first finishing study twice daily, but DL-malate was included during the 10-d step-up period. During the 10-d step-up period, feed efficiency and ADG linearly increased (P = .01) with more DL-malate. DL-Malate had little effect on steer and heifer performance or plasma constituents in a 113-d finishing study. Collectively, these results suggest that feeding DL-malate to cattle consuming high-grain diets alleviates subclinical acidosis, and it improved animal performance in two finishing studies.

A reverse phase liquid chromatographic (LC) procedure is described for quantitating oxfendazole (2-(methoxycarbonylamino)-5-phenyIsuIfinylbenzimidazole)) in swine premix. Sample preparation consists of extracting oxfendazole with an acetone-methanol mixture. An aliquot of the extract is then centrifuged to separate undissolved premix excipients. Internal standard is added to the supernate and the sample is further diluted with water-acetonitrile-phosphoric acid (80 + 20 + 1). Oxfendazole is quantitatively determined using a Partisil-5-ODS-3 column with acetonitrile-O.OlM phosphate buffer (pH 6.0) as the mobile phase. The method is stability specific and yields a mean recovery of 101.1 ± 0.4% for the 1.35% premix formulation. The dependence of chromatographic performance characteristics on mobile phase organic content, pH, and buffer concentration is also reported.

We exploited the concentration dependence of hemoglobin S on its solubility in concentrated phosphate buffer to determine the percentage of hemoglobin S or hemoglobin A in whole blood or hemolysates. A sample is incubated for 1 h at 37 degrees C in "SickleQuik" (General Diagnostics) tubes, and the absorbance of the aqueous phase is then measured at 555 nm and 650 nm, the latter wavelength to correct for turbidity. The fractional hemoglobin S or hemoglobin A content is read from standard-curve data on mixtures of hemoglobin A and hemoglobin S. Such curves can be prepared with the use of hemoglobin A alone, because under our conditions all hemoglobin S is precipitated. The curve is reproducible for up to three weeks. Day-to-day CVs ranged from 1.8% at 10% hemoglobin S to 8.1% at 68% hemoglobin S. For similar concentration ranges, day-to-day CVs for electrophoresis, with densitometry, ranged from 4.9% to 18%. Results on 29 patients are presented. We recommend the method for rapid quantification of hemoglobin S percentage for patients with sickle cell disease in acute-care situations.

In this improved method for quantifying 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) in urine, after a multistep extraction of MHPG and internal standard (iso-MHPG) from 3.0 mL of urine, the compounds are separated on a C18 reversed-phase column and quantified by use of an electro-chemical detector. The isocratic chromatographic separation takes about 16 min. The mobile phase is phosphate buffer/acetonitrile (88/12 by vol), the flow rate 0.7 mL/min. Recycling the mobile phase and automating the sample injection make possible the unattended assay of more than 70 samples per day. The within-run precision of the method is excellent (CV 1.8%) at a mean concentration of 1.1 mg/L.

Information on changes in the urinary excretion pattern of porphyrins can be especially useful in the diagnosis of disorders of porphyrin metabolism. Most clinical laboratory procedures are designed for assay of uroporphyrin and coproporphyrin only, and in many cases even these are not cleanly separated. Hence, we developed a "high-performance" liquid-chromatographic procedure to separate and quantify all five urinary porphyrins--that is, those with four through eight carboxyl groups. Before chromatography, the porphyrins are isolated from other urinary components by two simple, rapid pretreatment steps, then injected into the chromatograph in nonesterified form. They are separated and eluted with a step gradient of methanol/phosphate buffer, pH 3.0, in which the methanol content is first 650, then 850 mL/L. As little as 1 ng of eluted porphyrins can be measured fluorometrically. Analytical recovery of coproporphyrin is virtually 100% and of uroporphyrin 75-80%. CVs are about 10% for coproporphyrin at 70 micrograms/L and 20-40% for uroporphyrin at 8 micrograms/L.

In the present work the effects of oestradiol benzoate (EB) on pituitary and plasma concentrations of TSH, plasma T4 and T3, and thyroidal activity of male and female rats have been studied. Wistar rats weighing between 150 to 200 g were injected sc with varying doses of EB in corn oil for 9 or 30 days. The animals were exsanguinated by cardiac puncture and the hypophyses removed and individually homogenized at 4°C in 200 μl PBS buffer. Pituitary and plasma TSH were measured by radioimmunoassay. Thyroidal activity was evaluated by a 4 h 131I uptake and by 48 h thyroidal release plasma slopes derived form the ratio PB[125I] (from thyroidal secretion) to PB[131I] (from exogenous [131I]T4). In both male and female rats the 10 and 25 μg doses of EB produced a significant decrease in pituitary TSH content; this effect was more pronounced when the 25 μg dose was given over 30 days. Plasma T4 decreased significantly; plasma T3 was moderately elevated in all groups (NS) and significantly increased in female rats treated with 25 μg EB (P < 0.01). It is concluded that EB induced a marked depression of intrapituitary TSH, probably due to a decrease in synthesis, without affecting the release of TSH into the circulation. Moreover, EB accelerated peripheral T4 kinetics and thyroid gland activity, albeit to a moderate degree.

Experiments were conducted to determine the effects of infusing indomethacin, a prostaglandin synthetase inhibitor, into the uterine lumen on the development and function of the bovine corpus luteum in the presence and absence of concurrently administered oxytocin. Each treatment was given twice daily on d 4, 5 and 6 of the estrous cycle. Treatments (six heifers/group) and resulting estrous cycle lengths were as follows: (1) untreated controls, 20.6 +/- .4 d; (2) .2 M phosphate buffer vehicle infused into the uterine lumen, 21.0 +/- .6 d; (3) 40 mg indomethacin infused into the body of the uterus, 16.5 +/- 1.0 d; (4) 150 USP units oxytocin injected sc, 10.0 +/- 1.2 d and (5) a combination of oxytocin and indomethacin as in treatments 3 and 4, 14.1 +/- 1.3 d. Plasma concentrations of progesterone were lower (P less than .05) in each treatment group from d 7 onward, when compared with untreated and vehicle-treated controls. Indomethacin alone effectively inhibited the development and function of the corpus luteum, and was without effect on oxytocin-induced inhibition of luteal function. In summary, it appears that a prostaglandin of either uterine or ovarian origin, or both, is required for the normal development and function of the bovine corpus luteum.

Poly (A) took stable double-stranded form in 0.02 m phosphate buffer pH 5.8 when its S-value was higher than 3.5. Poly (C) also took stable double-stranded form in 0.01 m acetate buffer pH 3.9 containing 0.1 m NaCl if its S-value was higher than 4.8.Poly (I) with S-values higher than 6.5 stably formed triple-stranded form. Poly (A) with S-values over 3S and poly (I) with S-values over 7S were observed to aggregate in the presence of high concentration of salt, but poly (C) and poly (U) did not aggregate under the same condition. Effect of salt concentration differed with different polynucleotides and with different salts.

Gel was obtained when alkaline dope solutions of the 7S and 11S globulins (8% protein concentration) prepared at pH above 11 were dialyzed against phosphate buffer, pH 7.6, µ= 0.3. To make clear the mechanism of gelation, the relationship between changes in viscosity and aggregation phenomena of the neutralized dope solutions was investigated by means of viscosity measurement, disc electrophoresis and gel filtration, comparing the 7S and 11S fractions. In conclusion, it is revealed that the gel is constituted with macromolecule aggregates, and to form the aggregates which are suitable for gelation, all of the following conditions must be satisfied at least : 1); Unfolding and dissociation into subunits once (above pH 11), 2); High ionic strength in the media (µ=0.3), 3); Formation of hydrogen, hydrophobic and disulfide bonds, 4); High protein concentration (above 8%).

Experiments were performed to establish a procedure for in vivo measurement of nitrite utilization by leaf tissue of bean (Phaseolus vulgaris L. cv. Top Crop).To measure light-dependent nitrite disappearance, a single disc of leaf tissue was exposed to light for 1 hour at 30 C while immersed in incubation medium (approximately 0.11 milliliter per square centimeter of leaf area) in the bottom of a tall-form glass beaker. The incubation medium was 100 millimolar phosphate buffer (pH 7.5) with added wetting agent and nitrite. The wetting agent combination of 1% 1-propanol plus 0.05% Neutronyx-600 was used in some experiments for compatibility with established in vivo nitrate reductase (NR) assays; however, 0.05% Neutronyx-600 alone was found to be a suitable substitute. Parallel assays run in the dark on related tissue are recommended as a means to determine the amount of nitrite synthesized within the tissue by the NR system. Adding the results of the two assays gives an estimate of total nitrite utilization by the leaf tissue. It was found that 20 millimolar nitrite in the incubation medium was the most suitable level of external nitrite for promoting light-dependent nitrite disappearance. This was also found to reduce, sometimes to zero, the rate of synthesis of nitrite by NR. NR activity declined steadily with advancing age. Except for very young tissue, the rate of nitrite disappearance was independent of age. Nitrite disappearance was completely blocked by diuron.

Ovomucin was separated into carbohydrate rich and poor components by chromatography on lysozyme-Sepharose 4B. The carbohydrate rich component was eluted with 1 mm phosphate buffer, pH 7.0, and the carbohydrate poor component was eluted with 0.1 m borate buffer, pH 8.0, containing 0.5m NaCl. It was shown by chemical analysis that the carbohydrate rich component corresponds to F-component or β-ovomucin and that the carbohydrate poor component corresponds to S-component or α-ovomucin.

Human blood group B and AB cells were converted to O and A active cells, respectively, by treatment with an α-d-galactosidase from Streptomyces 9917S2. Accompanying these conversions a 2 to 8-fold increase was observed in the group H activity on these cells. The conversion reaction proceeded rapidly in phosphate buffered saline without any supplement, Hemolysis, discoloration, and other visible deterioration of the cell were not observed during the process. Conditions for the optimal conversion as well as the susceptibility to the inhibition by sugars were somewhat different from those for the reaction with soluble substrates.

A liquid chromatographic method is described for the determination of melamine in beverages. Melamine is separated by column chromatography using cation and anion exchange resin and determined by ion-pair liquid chromatography using an ODS column and a mixture of acetonitrile and 0.05M phosphate buffer (pH 3.0) containing 0.005M sodium 1-laurylsulfate (1 + 4, v/v) as mobile phase. Recoveries of melamine ranged between 90.3 ± 7.8 and 102.1 ± 5.6% at levels of 0.6 to 2.4 ppm in 4 kinds of beverages. The quantitation limit was 2.5 μg melamine in 50 mL beverage. The method was applied to the migration test of melamine from melamine-formaldehyde resin products to the beverages.

The metabolisms of trans-2-octenoic acid (2-OEA) and related compounds were investigated by using one of Mucor species which could reduce sorbic acid to trans-4-hexenol. In a phosphate buffer solution, 3-hydroxyoctanoic acid (3-HOA), monooctyl phosphate (OLP), 1-octanol and 1, 3-octanediol were produced as metabolites of 2-OEA. It was elucidated that the conversion of 2-OEA into 3-HOA was reversible and finally most of the substrate was transformed into OLP via the following pathway: trans-2-octenol→1-octenol→OLP. Similarly, trans-3-octenol and mono-trans-3-octenyl phosphate were produced by the fungus as major metabolites of trans-3-octenoic acid.