Simultaneous determination of benzene, toluene, ethylbenzene, and xylenes in urine by thermal desorption-gas chromatography

Amphotericin B (AmB)-resistant Leishmania donovani promastigotes were selected by increasing drug pressure, and their biological features were compared with those of the wild-type parent strain. The 50% inhibitory concentration for resistant cells was 20 times higher than that for the wild-type. Resistance was stable after more than 40 passages in drug-free medium, and resistant promastigotes were infective to macrophages in vitro but lost their virulence in vivo. They had 2.5 times longer generation time, decreased AmB uptake, and increased AmB efflux in comparison to the wild type. Fluorescence measurement with a specific plasma membrane probe, 1-[4-(trimethylammonio)-1,6-diphenylhexa]-1,3,5-triene, showed increased membrane fluidity in drug-resistant promastigotes. Analysis of lipid composition showed that in resistant cells saturated fatty acids were prevalent, with stearic acid as the major fatty acid, and the major sterol was an ergosterol precursor, the cholesta-5, 7, 24-trien-3beta-ol and not ergosterol as in the AmB-sensitive strain.     

An improved automated fluorimetric method for determination of histamine

The automated continuous flow system for the extraction and fluorimetric analysis of histamine based on the principle of Shore et al. (1959) has been improved. With lower consumption of reagents and further simplification of the working conditions, histamine can be determined quantitatively in a routine fashion in aqueous samples, with or without protein content, up to a concentration of approximately 0.1 ng/ml. The rate of analysis is 30 samples/h.     

一种滑动摩擦因数的测定方法在镀铝锌后处理板中的应用

参照检测标准ASTMD1894,对镀铝锌后处理板的滑动摩擦因数进行测定。在实际应用中,为了减小检测误差,提出了采用修正因子对检测结果进行修正的方法。经试验,发现测定的镀铝锌处理板摩擦因数经修正后相对标准偏差为12.1%,检测结果具有一定的波动性,但可作为一种内控方法应用于实践。而试样间接触问题、试样边部毛刺问题和试样表面污染问题等对检测结果会产生一定的影响,为此,采取一些措施以减少上述因素对检测结果的影响。     

A method for the separation and determination of neutral compounds in postmortem tissues

A convenient method for the extraction of neutral drugs in necrotic tissues is described. The procedure includes the use of hot dilute acid for isolating neutrals from tissue extract residues and of internal standard to facilitate quantitative and recovery measurement of the drugs by gas chromatography. The recovery of seven neutral compounds from liver ranged from 47.0% to 89.3%. The limit of detection of these drugs were 0.5 to 1.0 mg/kg.     

An automated method for the determination of subnanogram concentrations of eprinomectin in bovine plasma

Eprinomectin is a potent anthelmintic compound that kills certain parasitic nematodes and arthropods of cattle. A sensitive and automated bioanalytical assay was developed for quantitation of eprinomectin in bovine plasma in support of clinical development of eprinomectin for use in all classes of cattle. This assay determined the concentration of eprinomectin in plasma by reversed-phase high performance liquid chromatography (HPLC) with fluorometric detection. Plasma sample preparation included liquid extraction performed by the Packard MultiPROBE robotics workstation, followed by solid phase extraction performed by the Gilson ASPEC XL automated workstation. The HPLC assay included automated pre-column derivatization with a fluorogenic reagent system which included trifluoroacetic anhydride and N-methylimidazole as the catalyst. This reversed-phase chromatographic analysis was based on the fluorescence detection of derivatized eprinomectin and an internal standard, L-648 548, which was similarly derivatized by the fluorogenic reagents. The assay was automated and validated for two concentration ranges of 0.05-10 and 0.5-200 ng ml-1. The lower limit of quantitation of eprinomectin in plasma was 0.05 ng ml-1. The %RSD of the assay was 10% or better at all concentrations. This automated analysis of eprinomectin was used for high-throughput clinical assays with acceptable accuracy and precision.     

Application of solid-phase extraction in the method development for determination of SEPA, an acronym for soft enhancement of percutaneous absorption, in human, rat, and rabbit serum using GC-FID method

A new nonaqueous topical minoxidil formulation containing SEPA (2-n-nonyl-1,3-dioxolane) for enhancement of percutaneous absorption was under evaluation. SEPA does not have chromophore for either ultraviolet or fluorescence detection using liquid chromatography and has no functional groups for derivatization. Therefore, a direct gas-chromatographic method with flame-ionization detection (GC-FID) was developed. Owing to the limited detection response of the FID detection, it needs a selective and concentrated extract for GC-FID analysis to improve the assay sensitivity to meet the requirement for pharmacokinetic evaluation after topical application. In addition, SEPA is a very volatile compound. Any extraction procedures involving evaporation will result in a poor recovery. The application of solid-phase extraction (SPE) makes it possible to achieve a selective and a 10-fold concentrated extract with an absolute extraction recovery of approximately 90%, which greatly improved the assay sensitivity. This method involved the extraction of SEPA and the internal standard (2-n-heptyl-1,3-dioxolane) from serum (0.1-1 ml) with 100 microliter of hexane-chloroform (1:1, v:v) using a 50 mg 1.0 ml-1 phenyl SPE column (Varian, Harbor City, CA, USA), followed by direct GC-FID analysis on a fused-silica column chemically bonded with cross-linked methyl silicone gum phase (Hewlett Packard Ultra-1, 12 m x 0.2 mm x 0.33 micron, Avondale, PA, USA). The assay demonstrated a lower limit of quantitation of 2.5 ng ml-1 and a linear range of 2.5 to 250 ng ml-1 with intra- and inter-assay precision and accuracy of < or = 10%.     

A method for measurement of angiotensin II in tissues and its application to rat kidney

Two distinct satellite DNAs, amounting to 25% of the total DNA, were isolated from the nuclei of the red-necked wallaby, Macropus rufogriseus. The physical properties of native, single-stranded and reassociated molecules were studied in buoyant-density gradient centrifugation. The homogeneity of each satellite fraction was examined using melting characteristics of native and reassociated DNA, and renaturation kinetics. These data suggest that sequence heterogeneity exists in both fractions. Each satellite fraction was found by in situ hybridization to be localized in heterochromatin of interphase nuclei and in the centromeric regions of metaphase chromosomes. The chromosomal distributions of the two satellite DNAs differentiate the sex chromosomes, which have sequences of only one satellite, from the autosomes which have sequences of both satellites in the centromeric heterochromatin. Giemsa C-banding techniques also showed a differentiation of the centromeric regions of sex chromosomes from those of the autosomes.     

Rapid platelet-function assay: an automated and quantitative cartridge-based method

BACKGROUND: The platelet glycoprotein (GP) IIb/IIIa receptor is important in mediating platelet thrombus formation, and the GP IIb/IIIa antagonist abciximab (c7E3 Fab; ReoPro) is effective in preventing thrombotic ischemic cardiovascular complications of unstable angina and percutaneous coronary interventions. Small-molecule antagonists of GP IIb/IIIa based on the Arg-Gly-Asp (RGD) sequence show similar benefit, and some of these agents are orally active. However, there may be significant interindividual variation in response to such antagonists, especially with chronic oral therapy. It will be essential to balance the beneficial antithrombotic effect of these drugs with their potential for causing bleeding. In response to this need, we have developed a rapid platelet-function assay (RPFA), a point-of-care system that provides a quantitative measure of the competence of the GP IIb/IIIa receptor as reflected in the ability of platelets to agglutinate fibrinogen-coated beads. METHODS AND RESULTS: Polystyrene beads were coated with fibrinogen and placed in a cartridge along with a lyophilized peptide that activates the thrombin receptor. Anticoagulated whole blood was added to the cartridge, and then a microprocessor-controlled operation mixed the reagents and detected agglutination between platelets and coated beads. Quantitative digital results were displayed within 3 minutes. Because there is no dilution of the blood, the assay can be used to measure platelet activity in samples that have been treated with GP IIb/IIIa antagonists with high dissociation rates. RPFA results of whole-blood samples treated with different GP IIb/IIIa antagonists correlated well with both conventional turbidimetric platelet aggregation (r2=0.95) and the percentage of free GP IIb/IIIa molecules in the sample (r2=0.96). The mean difference in measurements between RPFA and aggregometry was -4% (+/-4% SD), and the mean difference in measurements between RPFA and free GP IIb/IIIa receptors was -2% (+/-6% SD). CONCLUSIONS: The RPFA provides rapid information on platelet function that mirrors turbidimetric platelet aggregation and reflects GP IIb/IIIa receptor blockade.     

Experimental validation of an automated edge-detection method for a simultaneous determination of the endocardial and epicardial borders in short-axis cardiac MR images: application in normal volunteers

The goal of this study was to put together several techniques of image segmentation to provide a reliable assessment of the left ventricular mass with short-axis cardiac MR images. No initial manual input was required for this process based on region growing, gradient detection, and adaptive thresholding. A comparison between actual mass and automatic assessment was implemented with 9 minipigs that underwent spin-echo MR imaging. Fifteen normal volunteers were studied with a fast-gradient-echo sequence. The automatic segmentation was then controlled by three trained observers. Actual mass and automatic segmentation were strongly correlated (r = .97 with P < .01). For normal volunteers, the standard error of estimation of the automatic assessment (12 g) compared well with the average myocardial mass (120 +/- 30 g) and the interobserver reproducibility of the manual assessment (9 g). These results allow the application of this method to the quantification of the left ventricular function and mass in clinical practice.     

Quantification of cerebral blood flow and partition coefficient using iodine-123-iodoamphetamine

The aim of this study was to develop a simple, noninvasive method for quantifying both regional cerebral blood flow (rCBF) and the partition coefficient (lambda) using N-isopropyl-p[123I]iodoamphetamine and SPECT. METHODS: By employing a two-compartment model (influx, K1: outflux, k2), a new method was introduced that requires two serial SPECT scans at 30 min and 60 min, and a single arterial sample 5 min after tracer injection. The integral of the arterial input function is inferred from the sample by using the correlation obtained from 25 subjects. Two original mathematical functions, phi for K1 and gamma for lambda (= K1/k2), were obtained from the input functions of 12 subjects. The values of K1 and lambda are determined from the two scans and the single arterial sample by using these functions. The values obtained for K1 (= rCBF) and lambda were compared with those obtained by nonlinear least-squares fitting analysis and the 133Xe inhalation SPECT method. RESULTS: K1 and lambda were in good agreement with the values obtained by nonlinear least-squares fitting analysis (r = 0.873 in K1 and r = 0.825 in lambda), and rCBF values were closely correlated with those obtained by the 133Xe method (r = 0.843). CONCLUSION: The proposed method has three advantages: (a) accurate, simultaneous quantification of both rCBF and the partition coefficient; (b) simplicity and noninvasiveness; and (c) a relatively short period (approximately 70 min) for the study.     

Evaluation of an automated pyrogallol red-molybdate method for the measurement of urinary protein in rats

Methods for quantitating urinary protein differ in their ranges of linearity, technical ease of performance, and applicability to automated analyzers. The Coomassie Brilliant Blue method is widely used but has limited linearity and its tendency to stain glassware has limited its application to automated analyzers. We evaluated a pyrogallol red-molybdate protein dye-binding method (Biotrol USA, Inc.) on a Hitachi 705 analyzer for the quantitation of urinary protein in rats. This method showed a wide range of linearity (up to 2.6 g/l) and good precision. Within-run CVs of 6.6% and 1.3% and between-day CVs of 10.9% and 1.1% were observed at mean protein concentrations of 0.16 g/l and 1.96 g/l, respectively. In addition, rat urine protein results from this method correlated well (r2 = 0.998, n = 40) with a Coomassie Brilliant Blue method (QuanTtest Blue, Quantimetrix Corporation). No significant or unexpected interferences were encountered with this method. We conclude that the automated pyrogallol red-molybdate method is an acceptable and practical alternative to the Coomassie Brilliant Blue method for the quantitation of urine protein in rats.     

Liquid chromatographic method for determination of triglycerides in vegetable oils in terms of their partition numbers: summary of collaborative study

The IUPAC Commission on Oils, Fats and Derivatives undertook development of a method and collaborative study for the determination of triglycerides in vegetable oils by liquid chromatography. Three collaborative studies were conducted from 1985 to 1987. Refinements were made in the method after the first collaborative study, and the second and third collaborative studies demonstrated that the method produces acceptable results. Materials studied were soybean oil, almond oil, sunflower oil, olive oil, rapeseed oil, and blends of palm and sunflower oils, and almond and sunflower oils. Six test samples were analyzed by 18 laboratories from 11 countries in the second study; 4 test samples were analyzed by 16 laboratories from 12 countries in the third study. The method for determination of triglycerides (by partition numbers) in vegetable oils by liquid chromatography was adopted first action by AOAC INTERNATIONAL as an IUPAC-AOCS-AOAC method.     

Fully automated determination of pesticides in wine

A fully automated solid-phase extraction gas chromatographic/mass spectrometric (SPE/GC/MS) method was developed for determination of pesticides in wine. All steps from aspiration of infiltrated wine to printout of the integrated chromatogram were performed without human interaction. A dedicated robot performed addition of internal standard, application of wine onto the SPE cartridge, elution of analytes, drying and concentrating of eluate, and passing of concentrate to the GC sampler. All steps were performed in standard liquid chromatography/GC vials, using a minimum of organic solvent. The method permits determination of 21 different pesticides. Individual detection limits were 0.005-0.01 mg/L. The regression coefficients relating to linearity were > 0.99; only 4,4-dichloro-benzphenone and dicofol showed lower coefficients. The recoveries for 17 pesticides ranged from 80 to 115%.