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Highly Sensitive Method (highly + sensitive_method)
Selected AbstractsHighly sensitive method for the determination of ropinirole with a lower limit of quantitation of 3.45 pg/mL in human plasma by LC-ESI-MS/MS: application to a clinical pharmacokinetic studyBIOMEDICAL CHROMATOGRAPHY, Issue 5 2009D. Vijaya Bharathi Abstract A highly sensitive, rapid assay method has been developed and validated for the estimation of ropinirole (RPR) in human plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. A solid-phase process was used to extract RPR and citalopram (internal standard, IS) from human plasma. Chromatographic separation was operated with 0.2% ammonia solution:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a Hypurity C18 column with a total run time of 3.2 min. The MS/MS ion transitions monitored were 261.2 , 114.2 for RPR and 325.1 , 209.0 for IS. Method validation and clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 3.45 pg/mL and the linearity was observed from 3.45 to 1200 pg/mL. The intra-day and inter-day precisions were in the range of 4.71,7.98 and 6.56,8.31%, respectively. This novel method has been applied to a pharmacokinetic study of RPR in humans. Copyright © 2008 John Wiley & Sons, Ltd. [source] Highly sensitive method for the determination of omeprazole in human plasma by liquid chromatography,electrospray ionization tandem mass spectrometry: application to a clinical pharmacokinetic studyBIOMEDICAL CHROMATOGRAPHY, Issue 4 2009Shivva Vittal Abstract A highly sensitive, rapid assay method has been developed and validated for the estimation of omeprazole (OPZ) in human plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. The assay procedure involves alkalinization of plasma followed by simple liquid,liquid extraction of OPZ and lansoprazole (internal standard, IS) from human plasma with acetonitrile. Chromatographic separation was achieved with 0.01 m ammonium acetate:acetonitrile (40:60, v/v) at a flow rate of 0.25 mL/min on an Inertsil ODS 3 column with a total run time 2.5 min. The MS/MS ion transitions monitored were 346.1 , 198.1 for OPZ and 370.1 , 252.1 for IS. Method validation and clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.05 ng/mL and the linearity was observed from 0.05 to 10.0 ng/mL. The intra-day and inter-day precisions were in the ranges 2.09,8.56 and 5.29,8.19%, respectively. This novel method has been applied to a pharmacokinetic study of OPZ in humans. Copyright © 2008 John Wiley & Sons, Ltd. [source] Adsorptive Stripping Analysis of Riboflavin at Electrically Heated Graphite Cylindrical ElectrodesELECTROANALYSIS, Issue 21 2007Shao-Hua Wu Abstract Electrically heated graphite cylindrical electrodes (HGCEs) made from ground pencil leads have been used to perform adsorptive stripping square wave voltammetry (SWV) measurements of trace riboflavin (RF). The SWV stripping peak current was significantly enhanced with increasing the electrode temperature only during preconcentration step. This enhancement was due to the forced thermal convection induced by heating the electrode rather than the bulk solution. It is the thermal convection that has the ability to improve mass transfer and facilitate adsorption thus enhance stripping responses. It was found that the detection limit of 5×10,9,M (S/N=3) could be obtained at an electrode temperature of 72,°C during 5,min accumulation, more than one magnitude lower than that at 22,°C (room temperature), the sensitivity could be enhanced ca. eight or four folds for two different RF concentration ranges. So it is possible to develop a new highly sensitive method to determine riboflavin at HGCEs. Such HGCEs were also successfully used to determine RF in multivitamin tablets. [source] Bioluminescence imaging allows measuring CD8 T cell function in the liver,HEPATOLOGY, Issue 4 2010Dirk Stabenow In vivo evaluation of CD8 T cell effector (cytotoxic T lymphocyte [CTL]) function in peripheral organs such as the liver is currently not possible but would greatly improve our understanding of local immune regulation, because simple determination of antigen-specific CTL numbers does not predict the outcome of immune responses. In particular, measurement of alanine aminotransferase serum levels is not sensitive enough to detect T cell immunity against low numbers of target hepatocytes. We developed a procedure that detects virus-specific effector function of CTLs in the liver after simultaneous adenoviral transfer of reporter and immune target genes into hepatocytes, followed by bioluminescence imaging of reporter genes. Bioluminescence imaging enabled detection of as few as 10,000 infected hepatocytes in vivo, and even more importantly, quantification of antiviral effector function of as few as 50,000 CTLs. Conclusion: Our results provide evidence that low numbers of antigen-specific CTLs are sufficient to control viral gene expression and eliminate viral infection from hepatocytes. The experimental system established here is a highly sensitive method to simultaneously detect viral infection of hepatocytes and to quantify antiviral CTL function in the liver in vivo and will help in characterizing principles of hepatic immune regulation. (HEPATOLOGY 2010;51:1430,1437) [source] Early stages of protein crystallization as revealed by emerging optical waveguide technologyJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 3 2008Attia Boudjemline A highly sensitive method for studying the onset of protein crystallization in real time using an optical-waveguide-based technique is reported. Dual polarization interferometry uses light from sensing and reference waveguides to produce an interference pattern, which when the sensing waveguide is immersed in a protein solution supplies information on the thickness and density of any protein adlayer on the sensing waveguide's surface. This technique provides evidence that crystallization proceeds via large protein aggregates but, more strikingly, shows dramatic light loss from the sensing waveguide at a very early stage during crystallization. The technique proves relatively insensitive to the crystallization of small molecules or poorly formed protein crystals and affords a method of distinguishing crystal formation from the formation of other protein aggregates or salt crystals. The experimental setup currently necessitates crystallization using the batch method, and precipitant mixing at high supersaturation is known to introduce a greater variability compared with methods such as vapour diffusion or dialysis, but first results promise to bridge the paucity of real-time methods available to distinguish the onset of protein crystallization from other forms of aggregation. [source] COMPARATIVE STUDY OF SHELL SWAB AND SHELL CRUSH METHODS FOR THE RECOVERY OF SALMONELLA FROM SHELL EGGSJOURNAL OF FOOD SAFETY, Issue 4 2008T. KAWASAKI ABSTRACT Swabbing is the standard methodology for the recovery of resident microorganism from shell eggs in Japan. A comparative study of shell swab (SW) and shell crush (CR) techniques was performed to recover the laboratory-inoculated Salmonella from shell eggs. It was found that the recovery of Salmonella by CR methods was significantly higher (4.5,7.5 log cfu/egg) than that of SW methods (3.1,6.3 log cfu/egg). However, analyses with quantitative real-time polymerase chain reaction (invA as a target gene), fluorescent microscopic and quantitative analyses with a Live/Dead BacLight bacterial viability kit revealed that not all of the inoculated Salmonella spp. populations were recovered as intact cells by either method. The chemiluminescent bacterial viability assay showed that chemiluminescence intensity (CI) began to increase after 30 min in CR samples; on the other hand, SW samples did not show any increase in CI for 2 h. These results suggest that SW might cause more damage and lethality to cells than CR. In addition, to determine the most appropriate method for recovering resident aerobic bacteria, coliforms and Salmonella spp from shell eggs, 4,000 commercial eggs were collected and sampled by shell rinse (SR) and CR techniques using phosphate-buffered saline (PBS) warmed to different temperatures. PBS at 37C was found to be the best recovery solution and temperature, respectively, for recovering aerobic microorganisms from shell eggs by both methods and the CR methods recovered a higher population than did the SR methods (4.9 versus 5.8 log cfu/egg for SR and CR methods, respectively; n = 500 eggs/method). Therefore, the CR method along with recovery buffer (PBS) at 37C could be an effective technique for the recovery of microorganisms from post-processed shell eggs. PRACTICAL APPLICATIONS There is a need to develop a rapid and highly sensitive method for the recovery of microorganisms from shell eggs. Such recovery methods are also useful for evaluating the efficacy of newly developed shell egg disinfection techniques. Many methods involving rinsing, swabbing, and crushing of shell eggs have been reported; however, we performed a comparative study of the method used to recover the Salmonella from shell eggs. We found that the shell crush method (CR) was superior to the shell swab method (SW) for the recovery of Salmonella spp., and phosphate-buffered saline (PBS) at 37C was found to be the best recovery solution and temperature, respectively, for recovering microorganisms from shell eggs by both methods. Therefore, the CR method along with recovery buffer (PBS) at 37C could be an effective technique for the recovery of microorganisms from post-processed shells. Use of this method could be recommended for the microbial evaluation of post-processed shell eggs in industries. [source] Evaluation of serum cystatin C levels and 99mTechnetium-mercaptoacetyltriglycine-3 renal scintigraphy for the early detection of cisplatin-induced renal toxicity in cancer patientsNEPHROLOGY, Issue 2 2002Nazan GÜNEL SUMMARY: Cisplatin has a broad-spectrum antineoplastic activity. Nephrotoxicity is a prominent component of the toxicity profile of cisplatin-based chemotherapy. In recent years, several reports have confirmed that cystatin C (cys-C) demonstrates a better correlation with the glomerular filtration rate than with serum creatinine. Scintigraphic techniques are also widely used in evaluating renal function. In the present study, serum cys-C, serum creatinine concentrations and 99mTechnetium-mercaptoacetyltriglycine-3 (99mTc-MAG-3) scintigraphy were studied in 22 cisplatin-naive cancer patients, 3 days before and 24 h after the first cycle of cisplatin-based chemotherapy. Serum cystatin C and creatinine levels increased in cancer patients after chemotherapy (creatinine: from 68 ± 12 to 72 ± 17 nmol/L; cystatin-C: from 0.064 ± 0.025 to 0.072 ± 0.033 jimol/L), but these differences were not statistically significant (P>0.05). Semiquantitative variables of 99mTc-MAG-S scintigraphy significantly elevated after chemotherapy (T½*: from 10.27 ± 5.00to 16.17 ± 9.40 min, R20/max*: from 0.40 ± 0.12 to 0.67+0.45, Tmax**: from 5.40 ± 4.01 to 7.59 ± 5.30 min; *P<0.001, **P<0.01, respectively). These results suggest that MAG-3 scintigraphy is a highly sensitive method in the early detection of cisplatin-induced nephrotoxicity. Serum cystatin C doesn't seem to play a role in the early detection of cisplatin-induced nephrotoxicity. As a result, MAG-3 scintigraphy may be used in selected patients who have a predisposition for renal toxicity. [source] A sensitive method for detecting bamboo mosaic virus (BaMV) and establishment of BaMV-free meristem-tip culturesPLANT PATHOLOGY, Issue 1 2000A sensitive method was used to detect bamboo mosaic virus (BaMV) and its associated satellite RNA (satBaMV) by 32P- and digoxigenin (Dig)-labelled probes synthesized from cDNA clones of BaMV genomic (L probe) and satBaMV (S probe) RNA. Both the 32P- and Dig-labelled L and S probes could detect as little as 490 pg of BaMV viral RNA by slot- and dot-blot hybridization. In infected leaf extracts, 32P-labelled L and S probes detected virus at 25-fold higher dilutions than Dig-labelled probes, which were also successfully used to detect BaMV infection in plants derived from meristem-tip culture. However, immunoassays failed to detect BaMV in meristem culture. By dot-blot hybridization assays, 25% of the seedlings were shown to be virus-free. These results suggest that a highly sensitive method for the detection of BaMV infection is required for the establishment of BaMV-free cultures. Meristem-tip culture also provides an efficient method for obtaining virus-free bamboo plants. [source] Reduction in diagnostic and therapeutic interventions by non-invasive determination of fetal sex in early pregnancyPRENATAL DIAGNOSIS, Issue 12 2005Jon A. Hyett Abstract Objective This study reviews our clinical experience of non-invasive techniques for early sex determination. It assesses the effectiveness of these techniques at reducing invasive prenatal testing for X-linked genetic disease or for ambiguous development of the external genitalia. Methods A prospective cohort study of 30 pregnancies was referred to a tertiary unit for prenatal diagnosis. Fetal gender was determined using two non-invasive techniques: analysis of free fetal DNA (ffDNA) in maternal plasma and ultrasound visualisation. The results were compared to fetal gender determined by invasive testing or at birth. Results Fetal gender was accurately determined by analysis of ffDNA at a mean of 10 + 1 (7 + 6 to 14 + 1) weeks' gestation in all cases. Ultrasound assessment was accurate in 20 of the 23 cases where this was attempted at 12 + 0 (10 + 4 to 14 + 1) weeks' gestation, but could not be determined in the remaining 3 cases. Thirteen of 28 (46%) women chose not to have invasive testing on the basis of these findings. Conclusions Both the techniques appear to offer an accurate means of assessing fetal gender, giving parents the option of avoiding invasive testing in the 50% of cases where the fetus would not be affected. The molecular technique is performed at an earlier gestation, but female fetal status is predicted by a negative test result. Ultrasound cannot be applied until 11 weeks' gestation but diagnostic signs are sought in both sexes. Combining these approaches offers a highly sensitive method of non-invasive determination of gender in high-risk pregnancies. Health professionals, clinical geneticists and genetics associates, in particular, who refer women at high risk should be aware of these non-invasive options for prenatal sex determination. Copyright © 2005 John Wiley & Sons, Ltd. [source] A mass spectrometry-based strategy for detecting and characterizing endogenous proteinase activities in complex biological samplesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2008Sarah Robinson Abstract Endogenous proteinases in biological fluids such as human saliva produce a rich peptide repertoire that reflects a unique combination of enzymes, substrates, and inhibitors/activators. Accordingly, this subproteome is an interesting source of biomarkers for disease processes that either directly or indirectly involve proteolysis. However, the relevant proteinases, typically very low abundance molecules, are difficult to classify and identify. We hypothesized that a sensitive technique for monitoring accumulated peptide products in an unbiased, global manner would be very useful for detecting and profiling proteolytic activities in complex biological samples. Building on the longstanding use of 18O isotope-based approaches for the classification of proteolytic and other enzymatic processes we devised a new method for evaluating endogenous proteinases. Specifically, we showed that upon ex vivo incubation endogenous proteinases in human parotid saliva introduced 18O from isotopically enriched water into the C-terminal carboxylic groups of their peptide products. Subsequent peptide sequence determination and inhibitor profiling enabled the detection of discrete subsets of proteolytic products that were generated by different enzymes. As a proof-of-principle we used one of these fingerprints to identify the relevant activity as tissue kallikrein. We termed this technique PALeO. Our results suggest that PALeO is a rapid and highly sensitive method for globally assessing proteinase activities in complex biological samples. [source] Determination of urinary androgen glucuronides by capillary electrophoresis with electrospray tandem mass spectrometryBIOMEDICAL CHROMATOGRAPHY, Issue 4 2009Sung-Hee Cho Abstract Capillary electrophoresis,electrospray tandem mass spectrometry (CE-ESI/MS/MS) is a simple and highly sensitive method for quantifying seven urinary androgen glucuronides. The urine samples were diluted and filtered through a membrane filter, and the filtrate was injected into a CE-MS/MS system without further sample preparation steps such as extraction and derivatization. The calibration ranges were 0.01,5 µg/mL for glucuronides of androsterone and 11, -OHAn-3G, and 5,500 ng/mL for glucuronides of 11-ketoAn, DHEA, testosterone, epitestosterone and DHT. The linearity of the method was 0.992,0.998, and the limits-of-detection at a signal-to-noise ratio of 3 were 5,10 ng/mL. The coefficients of variation were in the range of 4.0,9.0% for intra-day assay and 4.1,9.8% for inter-day assay. The proposed method may be applicable to metabolic profiling in both quantitative and qualitative analysis. Copyright © 2008 John Wiley & Sons, Ltd. [source] | ||||||||||||||||