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High Pressure Liquid Chromatography (high + pressure_liquid_chromatography)
Selected AbstractsSelective extraction of polyunsaturated triacylglycerols using a novel ionic liquid precursor immobilized on a mesoporous complexing adsorbentBIOTECHNOLOGY PROGRESS, Issue 5 2009Patrisha J. Pham Abstract Mesoporous silica (SBA-15) synthesized by using Pluronic123 as the structure-directing template was functionalized by imidazolium-based ionic liquid precursors. Silver salts were then immobilized onto the supported ionic liquids using the incipient wetness impregnation technique. The separation of unsaturated species was achieved through the reversible and specific interaction between silver ions and carbon,carbon double bonds. This adsorbent was examined for the selective separation of polyunsaturated triacylglycerols (PUTAG) using High Pressure Liquid Chromatography (HPLC) with Evaporative Light Scattering Detection (ELSD) as the quantification methodology. AgBF4/SBA15·HPSiOEtIM·PF6 showed an adsorption capacity for linolenin of about 217 mg adsorbed/gram of sorbent. This adsorbent had good selectivity and a high capacity for the most highly unsaturated triacylglycerol when applied to a mixture of triacylglycerols with varying degrees of unsaturation. Consequently, a stepwise methodology was also developed to increase the recovery of the adsorbed components. This adsorbent retained its selectivity and capacity when recycled up to five times. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] An improved validated ultra high pressure liquid chromatography method for separation of tacrolimus impurities and its tautomersDRUG TESTING AND ANALYSIS, Issue 3 2010Acharya Subasranjan Abstract A selective, specific and sensitive ultra high pressure liquid chromatography (UHPLC) method was developed for determination of tacrolimus degradation products and tautomers in the preparation of pharmaceuticals. The chromatographic separation was performed on Waters ACQUITY UPLC system and BEH C8 column using gradient elution of mobile phase A (90:10 v/v of 0.1% v/v triflouroacetic acid solution and Acetonitrile) and mobile phase B (90:10 v/v acetonitrile and water) at a flow rate of 0.6 mL min,1. Ultraviolet detection was performed at 210 nm. Tacrolimus, tautomers and impurities were chromatographed with a total run time of 25 min. Calibration showed that the response of impurity was a linear function of concentration over the range 0.3,6 µg mL,1 (r2 , 0.999) and the method was validated over this range for precision, intermediate precision, accuracy, linearity and specificity. For precision study, percentage relative standard deviation of each impurity was < 15% (n = 6). The method was found to be precise, accurate, linear and specific. The proposed method was successfully employed for estimation of tacrolimus impurities in pharmaceutical preparations. Copyright © 2010 John Wiley & Sons, Ltd. [source] Universal multiplex PCR and CE for quantification of SMN1/SMN2 genes in spinal muscular atrophyELECTROPHORESIS, Issue 7 2009Chun-Chi Wang Abstract We established a universal multiplex PCR and CE to calculate the copy number of survival motor neuron (SMN1 and SMN2) genes for clinical screening of spinal muscular atrophy (SMA). In this study, one universal fluorescent primer was designed and applied for multiplex PCR of SMN1, SMN2 and two internal standards (CYBB and KRIT1). These amplicons were separated by conformation sensitive CE. Mixture of hydroxyethyl cellulose and hydroxypropyl cellulose were used in this CE system. Our method provided the potential to separate two 390-bp PCR products that differ in a single nucleotide. Differentiation and quantification of SMN1 and SMN2 are essential for clinical screening of SMA patients and carriers. The DNA samples included 22 SMA patients, 45 parents of SMA patients (obligatory carriers) and 217 controls. For evaluating accuracy, those 284 samples were blind-analyzed by this method and denaturing high pressure liquid chromatography (DHPLC). Eight of the total samples showed different results. Among them, two samples were diagnosed as having only SMN2 gene by DHPLC, however, they contained both SMN1 and SMN2 by our method. They were further confirmed by DNA sequencing. Our method showed good agreement with the DNA sequencing. The multiplex ligation-dependent probe amplification (MLPA) was used for confirming the other five samples, and showed the same results with our CE method. For only one sample, our CE showed different results with MLPA and DNA sequencing. One out of 284 samples (0.35%) belonged to mismatching. Our method provided a better accurate method and convenient method for clinical genotyping of SMA disease. [source] Evaluation of F cells in sickle cell disorders by flow cytometry , comparison with the Kleihauer,Betke's slide methodINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 6 2007K. Y. ITALIA Summary Adult F cell numbers are raised in inherited haemoglobin disorders, such as , -thalassaemia and sickle cell anaemia, hereditary persistence of foetal haemoglobin, and some acquired conditions, such as juvenile myelomonocytic leukaemia, during acute erythropoietic stress and pregnancy. True foetal erythrocytes containing foetal amounts of HbF can also occur in the adult circulation during the leakage of HbF-containing cells from the foetus to the maternal circulation. In normal adults, HbF is restricted to a small proportion (3,7%) of red blood cells (RBC), termed ,F cells'. Techniques estimating the amount of HbF use lysates prepared from RBC, whereas those that estimate the adult F cell count use intact RBC. An accurate assessment of adult F cells in sickle cell disorders is important because increased adult F cells are associated with decreased morbidity in these disorders. In the present study, HbF levels were measured and adult F cell numbers were estimated in 100 blood samples (25 normal individuals, 25 sickle heterozygotes, 25 sickle homozygotes and 25 sickle , -thalassaemia cases), using high pressure liquid chromatography for HbF levels, and flow cytometry and the Kleihauer,Betke (KB) acid elution microscope slide method for cell counts. Flow cytometry gave a more accurate assessment of adult F cells, eliminating any manual error, as compared to KB, which was less sensitive and precise as it is based on subjective visual interpretation. [source] Selecting for development of fluoroquinolone resistance in a Campylobacter jejuni strain 81116 in chickens using various enrofloxacin treatment protocolsJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2010K. Stapleton Abstract Aims:, To determine the effect of various enrofloxacin dose regimes on the colonization and selection of resistance in Campylobacter jejuni strain 81116P in experimentally colonized chickens. Methods and Results:, Two experiments were undertaken, in which 14-day-old chickens were colonized with 1 × 107,1 × 109 CFU g,1Camp. jejuni strain 81116P and then treated with enrofloxacin at 12,500 ppm in drinking water for various times. Caecal colonization levels were determined at various time-points after start-of-treatment, and the susceptibility of recovered isolates to ciprofloxacin was monitored. Resistance was indicated by growth on agar containing 4 ,g ml,1 ciprofloxacin, MICs of 16 ,g ml,1 and the Thr86Ile mutation in gyrA. Enrofloxacin at doses of 12,250 ppm reduced Camp. jejuni colonization over the first 48,72 h after start-of-treatment. The degree of reduction in colonization was dose, but not treatment time, dependent. In all cases, maximal colonization was re-established within 4,6 days. Fluoroquinolone-resistant organisms were recoverable within 48 h of start-of-treatment; after a further 24 h all recovered isolates were resistant. In contrast, a dose of 500 ppm enrofloxacin reduced colonization to undetectable levels within 48 h, and the treated birds remained Campylobacter negative throughout the remaining experimental period. By high pressure liquid chromatography, for all doses, the maximum concentrations of enrofloxacin and ciprofloxacin in the caecal contents were detected at the point of treatment completion. Thereafter, levels declined to undetectable by 7 days post-treatment withdrawal. Conclusions:, In a model using chickens maximally colonized with Camp. jejuni 81116P, treatment with enrofloxacin, at doses of 12,250 ppm in drinking water, enables the selection, and clonal expansion, of fluoroquinolone-resistant organisms. However, this is preventable by treatment with 500 ppm of enrofloxacin. Significance and impact of the study:, Treatment of chickens with enrofloxacin selects for resistance in Camp. jejuni in highly pre-colonized birds. However, a dose of 500 ppm enrofloxacin prevented the selection of resistant campylobacters. [source] Host-derived media used as a predictor for low abundant, in planta metabolite production from necrotrophic fungiJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2006D.P. Overy Abstract Aims:,Penicillium ser. Corymbifera strains were assayed on a variety of media and from infected Allium cepa tissues to evaluate the stimulation and in planta prediction of low abundance metabolites. Methods and Results:, Stimulated production of corymbiferones and the corymbiferan lactones were observed for Penicillium albocoremium, Penicillium allii, Penicillium hirsutum, Penicillium hordei and Penicillium venetum strains cultured on tissue media. Target metabolites were sporadically detected from strains cultured on common laboratory media (CYA, MEA and YES). Up to a 376 times increase in corymbiferone and corymbiferan lactone production was observed when culture extracts from CYA and A. cepa agar were compared by high pressure liquid chromatography with ultraviolet and mass spectrometry (LC-UV-MS). The novel metabolite corymbiferone B was purified and structure elucidated from a P. allii/A. cepa tissue medium extract. In planta expression of low abundance, target metabolites were confirmed from infected A. cepa tissue extracts by LC-UV-MS. Conclusions:, Secondary metabolite production was directly dependent and influenced by media conditions, resulting in the stimulated production of low abundance metabolites on host-derived media. Significance and Impact of the Study:, The use of macerated host tissue media can be applied in vitro to predict in planta expression of low abundance metabolites and aid in metabolite origin annotation during in planta metabolomic investigations at the host/pathogen interface. [source] Efficiency of naphthalene biodegradation by Pseudomonas putida G7 in soilJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 6 2004Andrei E Filonov Abstract The efficiency of naphthalene degradation by Pseudomonas putida G7 in soil was assessed using a mathematical model. The number of microorganisms and the concentration of naphthalene in soil samples were monitored. The feasibility of a spectrofluorometric method for naphthalene assay in soil samples was compared with high pressure liquid chromatography. A proposed mathematical model described the growth of the naphthalene-degrading strains and the consumption of substrates (naphthalene, naphthalene degradation intermediates and soil organic substances) in soil. To describe the growth kinetics of microorganisms having high affinity to substrates with low solubility, two differential equations with substrate exponent 2/3 were proposed. These equations were used to describe utilization of soil organic matter. The model parameters characterize the growth rates for different substrates and respective yield coefficients, specific bacterial death and adaptation rates, and also the rates of PAHs degradation and evaporation. These characteristics can be used in choosing the bacterial strains for biopreparations and efficient clean-up biotechnology of polluted soils. Copyright © 2004 Society of Chemical Industry [source] The macrophage chemotactic activity of Edwardsiella tarda extracellular productsJOURNAL OF FISH DISEASES, Issue 5 2008A A Wiedenmayer Abstract The chemoattractant capabilities of Edwardsiella tarda extracellular products (ECP) were investigated from two isolates, the virulent FL6-60 parent and less virulent RET-04 mutant. Chemotaxis and chemokinesis were assayed in vitro using blind well chambers with peritoneal macrophages obtained from Nile tilapia, Oreochromis niloticus, 5 days following squalene injection. Non-purified ECP derived from both isolates stimulated predominantly chemokinetic migration of macrophages. Additionally, the ECP were semi-purified by high pressure liquid chromatography. The FL6-60 parent ECP yielded higher molecular weight components than did the ECP from the RET-04 mutant. The chemotactic activity of the macrophages for both the FL6-60 parent and RET-04 mutant semi-purified ECP was increased over the non-purified ECP and overall migration was primarily chemotactic. Exposure to ECP derived from virulent and less virulent E. tarda isolates promoted chemokinetic movement of macrophages that may be involved in inflammatory responses of Nile tilapia to E. tarda infection. [source] Differentiation of Closely Related Fungi by Electronic Nose AnalysisJOURNAL OF FOOD SCIENCE, Issue 6 2007K. Karlshøj ABSTRACT:, In this work the potential of electronic nose analysis for differentiation of closely related fungi has been described. A total of 20 isolates of the cheese-associated species Geotrichum candidum, Penicillium camemberti, P. nordicum, and P. roqueforti and its closely related species P. paneum, P. carneum as well as the noncheese-associated P. expansum have been investigated by electronic nose, GC-MS, and LC-MS analysis. The isolates were inoculated on yeast extract sucrose agar in 20-mL headspace flasks and electronic nose analysis was performed daily for a 7-d period. To assess which volatile metabolites the electronic nose potentially responded to, volatile metabolites were collected by diffusive sampling overnight onto tubes containing Tenax TA, between the 7th and 8th day of incubation. Volatiles were analyzed by gas chromatography coupled to mass spectrometry and the results indicated that mainly alcohols (ethanol, 2-methyl-1-propanol, and 3-methyl-1-butanol) and ketones (acetone, 2-butanone, and 2-pentanone) were produced at this stage. The volatile metabolite profile proved to be species specific. Nonvolatile metabolites were collected on the 8th day of incubation and mycotoxin analysis was performed by high pressure liquid chromatography coupled to a diode array detector and a time of flight mass spectrometer. Several mycotoxins were detected in samples from the species P. nordicum, P. roqueforti, P. paneum, P. carneum, and P. expansum. Differentiation of closely related mycotoxin producing fungi incubated on yeast extract sucrose agar has been achieved, indicating that there is a potential for predicting production of mycotoxins on food and feedstuffs by electronic nose analysis. [source] Vasopressin Regulation of Noradrenaline Release Within the Supraoptic NucleusJOURNAL OF NEUROENDOCRINOLOGY, Issue 6 2000M. Ludwig The effect of electrically evoked dendritic vasopressin release on noradrenaline release into the hypothalamic supraoptic nucleus was assessed by in vivo microdialysis in conjunction with high pressure liquid chromatography and electrochemical detection. Electrical activation of magnocellular supraoptic neurones by stimulation of their axons at the level of the neural lobe significantly increased noradrenaline release into the nucleus (2.5-fold, P<0.03). This increase was completely blocked by administration of a nonpeptide vasopressin V1a receptor antagonist via the microdialysis probe. These data suggest that dendritically released vasopressin facilitates noradrenaline release into the hypothalamic nucleus. [source] Separation and measurement of plant alkaloid enantiomers by RP-HPLC analysis of their Fmoc-Alanine analogs ,PHYTOCHEMICAL ANALYSIS, Issue 5 2008Stephen T. Lee Abstract Introduction. Ammodendrine (1), anabasine (2) and coniine (3) can cause congenital malformations in livestock. They appear naturally in both enantiomeric forms, and can cause variable physiological responses. A method to measure the enantiomeric ratio of these natural toxins is needed. Objective. To develop a simple and economical method in order to determine the enantiomeric ratios of piperidine and pyrrolidine alkaloids in small samples of plant material. Methodology. Mixtures of isolated or purified plant alkaloids were converted to their Fmoc- l -Ala-alkaloid analogues forming diastereomeric mixtures, which were then analysed by high pressure liquid chromatography (HPLC) with mass spectrometry (MS) and ultraviolet (UV) detection to determine enantiomeric ratios. Results. The diastereomeric analogs for ammodendrine, anabasine and nornicotine could be separated and the enantiomeric ratios determined. The Fmoc- l -Ala-coniine analogue was not resolved under the HPLC conditions studied. The enantiomeric ratios of the selected plant alkaloids were measured and found to differ between both location within a species and location between species. Conclusion. A low-cost HPLC method to analyse the enantiomeric ratio of plant alkaloids containing primary or secondary amine nitrogens via conversion to their respective diastereomeric analogues has been developed. Published in 2008 by John Wiley & Sons. [source] Acrylate terpolymer in fabrication of medicated skin patchesPOLYMERS FOR ADVANCED TECHNOLOGIES, Issue 8 2001Vibha S. Mare Abstract An acrylate based pressure sensitive adhesive (PSA) was synthesized to design a drug-in-adhesive (DIA) type transdermal therapeutic system (TTS) for nitroglycerin used in the treatment of angina pectoris. 2-Ethylhexyl acrylate (EHA), methyl methacrylate (MMA) and acrylic acid (AA) were used to synthesize the PSA by free radical solution polymerization. The effects of reaction time, reaction temperature, initiator concentration and solvent on polymerization were studied. The synthesized terpolymer was characterized by 1H-NMR, FT-IR, differential scanning calorimetry (DSC) and gel permeation chromatography (GPC) and also evaluated for intrinsic viscosity, refractive index, peel strength, moisture uptake and skin irritation potential. The PSA was used to develop DIA type patches of nitroglycerin. The patches were cast using solvent evaporation technique and dried at controlled temperature. The patches were evaluated for thickness uniformity, weight variation, peel strength and moisture pick-up. The percent drug content and in vitro drug release was determined by high pressure liquid chromatography (HPLC) method. On the basis of in vitro release profile, patches were selected for in vitro skin permeation studies. The developed formulation TP-1 (K,=,24.892 mcg/cm2/hr) followed zero-order rate kinetics and showed better skin permeation rate in comparison to the marketed TTS (MTTS) (K,=,17.413 mcg/cm2/hr). TP-1 was subjected to stability testing for a period of 1 year according to ICH guidelines. The patches were found to be stable and an expiry date of 2 years was predicted with storage at 25,°C or below. Copyright © 2001 John Wiley & Sons, Ltd. [source] Donor Pretreatment with Tetrahydrobiopterin Saves Pancreatic Isografts from Ischemia Reperfusion Injury in a Mouse ModelAMERICAN JOURNAL OF TRANSPLANTATION, Issue 10 2010M. Maglione Depletion of the nitric oxide synthase cofactor tetrahydrobiopterin (H4B) during ischemia and reperfusion is associated with severe graft pancreatitis. Since clinically feasible approaches to prevent ischemia reperfusion injury (IRI) by H4B-substitution are missing we investigated its therapeutic potential in a murine pancreas transplantation model using different treatment regimens. Grafts were subjected to 16 h cold ischemia time (CIT) and different treatment regimens: no treatment, 160 ,M H4B to perfusion solution, H4B 50 mg/kg prior to reperfusion and H4B 50 mg/kg before recovery of organs. Nontransplanted animals served as controls. Recipient survival and endocrine graft function were assessed. Graft microcirculation was analyzed 2 h after reperfusion by intravital fluorescence microscopy. Parenchymal damage was assessed by histology and nitrotyrosine immunohistochemistry, H4B tissue levels by high pressure liquid chromatography (HPLC). Compared to nontransplanted controls prolonged CIT resulted in significant microcirculatory deterioration. Different efficacy according to route and timing of administration could be observed. Only donor pretreatment with H4B resulted in almost completely abrogated IRI-related damage showing graft microcirculation comparable to nontransplanted controls and restored intragraft H4B levels, resulting in significant reduction of parenchymal damage (p < 0.002) and improved survival and endocrine function (p = 0.0002 each). H4B donor pretreatment abrogates ischemia-induced parenchymal damage and represents a promising strategy to prevent IRI following pancreas transplantation. [source] | ||||||||||||||||