Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “quantification”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,009 records · Page 56Linked to original sources

Simultaneous quantification of opiates, amphetamines, cocaine and metabolites and diazepam and metabolite in a single hair sample using GC-MS.

A method is described for the simultaneous identification and quantification of opiates, amphetamines, cocainics, diazepam and nordiazepam from one hair extract (typically 10-50mg hair). After decontamination by washing with shampoo, dichloromethane, isopropanol and acetone, drugs were extracted using 0.1M HCl followed by SPE clean-up using mixed-mode extraction cartridges. The SPE extracts were submitted to a two-step derivatisation using MBTFA and MSTFA+1% TCMS and analysis was performed by GC-MS using both SIM and scan modes. Four deuterated standards were used to monitor 14 compounds. The limit of quantification was the total drug detected from the sample. This was 5 ng for amphetamines and 10 ng for remaining drugs which is equivalent to 0.1 and 0.2 ng/mg from a 50mg sample. Standard curves for the range 5-400 ng total drug concentration for all drugs had regression coefficients greater than 0.98. An authentic hair sample was used to validate the method and gave R.S.D.s <25% for both inter and intra-day reproducibility. The results of the analysis of hair taken from four patients attending a drug treatment clinic and six hair samples including head hair, pubic hair, axial hair and beard taken at post-mortem are presented.

Amphetamine↗

Development and validation of an LC-MS/MS method for the quantification of the KRASG12C inhibitor divarasib.

Divarasib is a newly developed covalent KRASG12C inhibitor, currently under clinical investigation in a phase 3 trial in patients with non-small cell lung cancer (NSCLC). At the moment, very limited pharmacokinetic data are publicly known. However, obtaining more insight into the pharmacokinetic properties of divarasib is important, since this may provide a better understanding of its efficacy and safety risks. Pre-clinical studies have been performed in mouse models to evaluate the effect of drug transporters and drug-metabolizing enzymes on the plasma exposure and tissue distribution of divarasib. Therefore, a reliable quantification method is required. To our knowledge, no bioanalytical assay of divarasib has been published yet. Therefore, in this study we developed and validated an assay to quantify divarasib in human plasma and in eight different mouse-related matrices, and partially in mouse plasma, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was initially evaluated over a concentration range of 1-10,000&#xa0;nM. However, due to carry-over observed at 10,000&#xa0;nM, the validated calibration range was established at 1-2000&#xa0;nM, with matrix-dependent LLOQs of 1-10&#xa0;nM. Erlotinib was used as an internal standard and acetonitrile was utilized to perform protein precipitation as sample pretreatment. Divarasib demonstrated stability in human plasma and in mouse plasma and tissue homogenates under various experimental conditions. A pilot in vivo study showed the applicability of our validated LC-MS/MS method. Ongoing clinical trials may collect plasma samples, and this developed method enables quantification of divarasib in both mouse and human plasma samples.

Animals↗

NIR spectroscopy-a non-destructive analytical tool for protein quantification within lipid implants.

Lipid implants have been proposed as promising sustained release devices for the parenteral application of pharmaceutical proteins. Near infrared spectroscopy (NIRS) has been reported in the literature to be a non-destructive tool for drug quantification within controlled release matrix systems based on poly-(lactic-co-glycolic) acid (PLGA). The objective of this study was to evaluate the potential application of NIRS for protein content determination within lipid matrices containing stabilizing and release modifying additives. Bovine serum albumin (BSA) and rh-interferon alpha-2a (IFN alpha-2a) were initially lyophilized with trehalose and then blended with tristearin (matrix material) and optionally with polyethylene glygol 6000 (PEG, release modifier). Implants were prepared by compression. NIR transmittance spectra were measured on a NIRTab spectrometer. Partial least squares regression (PLSR) calibration models were developed to predict protein content in implants from the NIRS results. Additional samples were measured after performing release studies. It could be shown that NIRS allowed protein quantification in complex matrix systems with good accuracy after implant manufacture and during release studies [e.g., standard error of prediction (SEP) between 57 microg-176 microg]. In addition, small protein amounts down to 70 microg of incorporated protein per implant could be determined, thus demonstrating the low detection limit of NIRS.

Calibration↗

Quantification of dental plaque in the research environment.

OBJECTIVES: To review the established and novel methods of plaque quantification employed in dental research, including a discussion of their merits and to present a new method of planimetrically measuring plaque using light induced fluorescence. METHOD: Quantitative light-fluorescence (QLF) images were acquired from the buccal surfaces of an individual who had refrained from oral hygiene both with and without traditional plaque disclosure. Digital photographs were also taken. Images were analysed using a novel method and a percentage plaque index produced. RESULTS: Traditional plaque indices are problematic due to their integral nature and their failure to detect small, but potentially clinically relevant changes in plaque area. The use of a fluorescent technique demonstrated good reliability although there was no correlation between red fluorescent plaque and total disclosed plaque suggesting that the auto-fluorescing plaque is not a good measure of total plaque volume. CONCLUSIONS: The use of planimetric techniques can increase the power of plaque studies, potentially reducing the number of subjects and time required to separate therapies or products. Fluorescent methods of quantification have potential as they enable clear separation of the plaque covered and non-covered tooth surfaces.

Analysis of Variance↗

Quantification of mannan-binding lectin.

Mannan-binding lectin (MBL) is attracting considerable interest due to its role in the immune defense. The high frequency of congenital MBL deficiency makes it feasible to evaluate clinical relevance through epidemiological investigations on fairly limited numbers of patients. MBL deficiency is determined by three mutant allotypes termed B, C and D in the coding region as well as mutations in the promoter region. It has been suggested that individuals, with deficiency-associated allotypes, may present significant amounts of low molecular weight MBL. We have compared the quantification of MBL by four commercially available assays with results obtained by our own in-house assays. Most assays are selectively sensitive for the wild type MBL (allotype A), but special combinations of antibodies also detect mutant forms of MBL. Thus a sandwich-type time-resolved immunoflourometric assay (TRIFMA), with a mouse monoclonal antibody (93C) as the catching and detecting antibody, shows B/B and D/D homozygous individuals to present signals corresponding to up to 500 ng MBL per ml (with plasma from an A/A individual as standard) as compared to less than 50 ng/ml and 200 ng/ml, respectively, when measured in other assays. In GPC at isotonic conditions the MBL in B/B and D/D individuals showed a Mr of 450 kDa. This MBL cannot bind to mannan. We further present a new method for quantifying the amount of MBL polypeptide chain. By applying plasma samples on SDS-PAGE at reducing conditions followed by Western blotting and quantification by chemiluminescense, this approach presents single polypeptide chains to the antibody independent of allotype differences in the collagen-like region. Titrations of recombinant MBL served as standard. In sera from homozygous mutants (O/O) the MBL concentrations estimated on Western blot were in the range of 100 to 500 ng/ml and correlated with that measured in the 93C-based TRIFMA.

Blotting, Western↗

In situ immunoradiographic method for quantification of specific proteins in normal and ischemic brain regions.

This study tested the application of an immunoisotopic assay for immunohistochemical localization and quantification of proteins in brain sections from rats without or with transient focal ischemia. We assessed the hypothesis that measurements of protein levels in injured brain determined by an isotopic assay using [(125)I]-protein A have greater reliability than those made by conventional immunoperoxidase labeling using diaminobenzidine. Quantification of immunoreactivities for glial fibrillary acidic protein (GFAP), glutamate transporter-1 (GLT-1) and heat shock protein-70 (HSP-70) was determined by optical density signal in the immunoisotopic and immunoperoxidase assays. In ischemic brain, the immunoisotopic assay detected protein increases (cortical penumbra HSP-70, 151+/-6%), protein decreases (cortical ischemic core GLT-1, 61+/-6%) and no changes in GFAP levels compared to controls animals. These results differed from the protein levels found by the immunoperoxidase assay, which showed elevated HSP-70, GLT-1 and GFAP in all ischemic regions. We conclude that nonspecific immunosignal confounds assessments of protein expression in injured brain and that the immunoisotopic method is a valid approach to regionally localize and quantify proteins after brain injury. The disadvantage of the falsely positive overestimation of protein immunoreactivity after stroke with the immunoperoxidase method has to be weighted with the advantage of the cellular resolution.

Animals↗

A rapid method for the quantification of mouse hippocampal neurogenesis in vivo by flow cytometry. Validation with conventional and enhanced immunohistochemical methods.

Neural stem cells reside in the subventricular zone and the dentate gyrus of the hippocampus in adult mammalian brain. In the hippocampus, a number of factors are reported to modulate the rate of neural progenitor proliferation in the hippocampus, such as exercise, corticosteroids, and many pharmacological agents including several classes of antidepressants. It is currently unclear whether this increased proliferation is physiologically relevant, but it provides a potentially useful biomarker to assess novel antidepressant compounds. Changes in neurogenesis are typically quantified by administration of bromodeoxyuridine (BrdU) in vivo, and subsequent quantification of labelled nuclei. A robust and rapid means of quantifying BrdU labelling in adult hippocampus in vivo would allow higher throughput screening of potential antidepressant compounds. In this study we describe a FACS-based method for quantification of BrdU labelled cells in fixed cell suspensions from BrdU-treated adult mouse hippocampus. A variety of experimental conditions known to modulate proliferation were tested, including administration of corticosterone and the antidepressants imipramine and fluoxetine. The robust changes compared to control groups observed in these models were similar to previously reported studies, thus offering a more rapid and streamlined means to quantify effects of compounds on hippocampal proliferation.

Animals↗

Computer-aided quantification of focal cartilage lesions using MRI: accuracy and initial arthroscopic comparison.

OBJECTIVE: The purpose of the study was to validate a Gradient Peak Method (GPM) by evaluating its accuracy and consistency at different magnetic field strengths. The GPM using magnetic resonance imaging (MRI) was previously proposed to quantitatively assess the morphology of focal cartilage lesions, and its feasibility was demonstrated. METHODS: GPM quantifies the morphologic properties of cartilage lesions based on their three-dimensional geometry. Twenty-two conical and cylindrical lesions were surgically created on fresh porcine knees, and the results obtained by GPM were compared with manually measured lesion dimensions. Another 15 focal lesions of various shapes were created and scanned, and the quantification results were compared at 1.5 Tesla and 3 Tesla. Additionally, cartilage lesions in three patients were scanned, quantified by GPM, and compared with arthroscopic visualization and measurements. RESULTS: The average absolute errors of GPM (depth: < or =0.4mm; diameter: < or =1.4mm) were within twice the in-plane resolution in depth estimates and within the slice thickness in diameter estimates. Analysis also suggested that the quantifications of GPM using 1.5 Tesla and 3 Tesla data were not statistically different. Moreover, the GPM results were shown to be consistent with the lesion measurements obtained arthroscopically. CONCLUSIONS: The GPM using MRI provides estimates of lesion thickness, depth, diameter, and area. With this validation, the method can be potentially used as an auxiliary tool to help radiologists and physicians assess cartilage lesions quantitatively and monitor disease progression.

Animals↗

Rapid quantification of gabapentin in human plasma by liquid chromatography/tandem mass spectrometry.

A simple, sensitive and rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of gabapentin, a new antiepileptic drug, in human plasma using its structural analogue, 1,1-cyclohexane diacetic acid monoamide (CAM) as internal standard. The method involved a simple protein precipitation by means of acetonitrile followed by a rapid isocratic elution with 10mM ammonium formate buffer/acetonitrile (20/80, v/v, pH 3.0) on Waters Symmetry C(18 reversed phase chromatographic column and analyzed by mass spectrometry in the multiple reaction monitoring mode. The precursor to product ion transitions of m/z 172-->154 and m/z 200-->182 were used to measure the analyte and the IS, respectively. The assay exhibited a linear dynamic range of 40-10000 ng/mL for gabapentin in human plasma. The limit of detection and lower limit of quantification in human plasma were 10 and 40 ng/mL, respectively. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.

Amines↗

Development and validation of a sensitive liquid chromatography/electrospray tandem mass spectrometry assay for the quantification of olanzapine in human plasma.

A simple, sensitive and rapid liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for the quantification of olanzapine, atypical antipsychotic drug, in human plasma using loratadine as internal standard (IS). Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse phase C18 column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 313/256 for olanzapine and m/z 383/337 for the IS. The assay exhibited a linear dynamic range of 0.1-30 ng/mL for olanzapine in human plasma. The lower limit of quantification was 100 pg/mL with a relative standard deviation of less than 10%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The average absolute recovery of olanzapine from spiked plasma samples was 85.5+/-1.9%. A run time of 2.0 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.

Antipsychotic Agents↗

Novel use of accelerator mass spectrometry for the quantification of low levels of systemic therapeutic recombinant protein.

Although 14C-labelling has been routinely used for small molecules, this technique is not routinely applied to therapeutic proteins due to difficulties of incorporating the label into the protein to a sufficiently high specific activity. An analytical method known as accelerator mass spectrometry (AMS) offers an extremely sensitive method of 14C quantification, thereby enabling (14)C-labeling methods to be applied to therapeutic protein detection. The therapeutic protein CAT-192 (metelimumab), a human anti-TGFss1 monocloncal antibody was manufactured in the presence of 14C-precursors resulting in a low specific activity product (1.4% 14C incorporation). [14C]-CAT-192 was administered to rats (1mg/kg and 222, 22 and 2.2 dpm/kg) and serum samples were collected. 14C in serum samples from the 2.2 dpm dosing was not detectable but samples from the 22 and 2220 dpm doses were measured by AMS and by ELISA for comparison. By both ELISA and AMS bioassay, the half-lives approximated 140 h (S.E.M. 15 h). The estimates of clearance were also comparable, 7.3 and 4.6 x 10(-4)ml/h/g (S.E.M. 6.6 and 5.1 x 10(-5)) for ELISA and AMS, respectively. The estimated limit of quantification (LOQ) was approximately 1 ng/ml, about 15 times lower than the ELISA LOQ of 15.6 ng/ml.

Animals↗

Quantification of phosphatidylserine, phosphatidic acid and free fatty acids in an ultrasound contrast agent by normal-phase high-performance liquid chromatography with evaporative light scattering detection.

Sonazoid is a new contrast agent for ultrasound imaging. The product is an aqueous suspension of perfluorobutane microbubbles coated with phospholipids obtained from hydrogenated egg phosphatidylserine (H-EPS). A normal-phase high-performance liquid chromatographic (HPLC) method with evaporative light scattering detection was developed for quantification of free fatty acids, phosphatidylserine and phosphatidic acid in H-EPS and Sonazoid. Separation of the lipids was carried out on an HPLC diol column and a gradient of chloroform and methanol with 0.2% formic acid titrated to pH 7.5 with ammonia. The calibration standards contained stearic acid, distearoyl-phosphatidic acid (DSPA) and distearoyl-phosphatidylserine (DSPS) in the concentration range of 0.016-1.0mg/ml (0.4-25microg injected). The method was validated with a limit of quantification of the three lipids set to 0.4microg (approximately 20-60microM). The best fit of the three calibration curves were obtained when the logarithmic transformed theoretical lipid concentration was plotted against the logarithmic transformed area under the peak and fitted to a second order polynomial equation. Stearic acid, DSPA and DSPS were analysed with an intermediate precision ranging from 4.4% to 5.3% R.S.D. and they were extracted from an aqueous suspension with a recovery ranging from 103.3% to 113.3%. The sum of total phospholipid concentration determined in H-EPS ranged from 96.4% to 103.2% of the theoretical values. The lipids in the ultrasound product were quantitated with a repeatability ranging from 6.2% to 11.7% R.S.D.

Chromatography, High Pressure Liquid↗

Voltammetric studies on the antibiotic drug cefoperazone quantification and pharmacokinetic studies.

A fully validated simple, sensitive and selective square-wave stripping voltammetry procedure was described for the trace quantification of cefoperazone in bulk form, formulations and human serum/plasma. The procedure was based on reduction of the adsorbed drug onto a hanging mercury drop electrode. The procedural conditions were optimized as: frequency=60Hz, scan increment=8mV, pulse amplitude=25mV, preconcentration potential=-0.3V (versus Ag/Ag/KCl(s)), preconcentration duration=60-150s and an acetate buffer of pH 4.2 as a supporting electrolyte. A limit of detection of 4.5x10(-10)M and a limit of quantitation of 1.5x10(-9)M bulk cefoperazone were achieved following preconcentration of the drug onto the hanging mercury drop electrode for 150s. The proposed square-wave adsorptive cathodic stripping voltammetric procedure was successfully applied for trace quantification of cefoperazone in human serum and plasma. The achieved limits of detection and quantitation of the drug in human serum were 6x10(-10)M (0.375ngml(-1)) and 2x10(-9)M (1.250ngml(-1)), respectively. The pharmacokinetic parameters of cefoperazone in plasma of hospitalized volunteers were successfully estimated.

Anti-Bacterial Agents↗

A real time PCR assay for the detection and quantification of orf virus.

A real time quantitative PCR assay based on TaqMan technology was developed for orf virus (ORFV) DNA quantification in clinical samples, infected cells and organotypic cultures. This method was based on the amplification of a 70 bp fragment from the ORFV B2L gene (orthologue of the Vaccinia virus Copenhagen F13L gene) that encodes the major envelope protein. Both intra- and inter-assay variability were well within +/-0.25 log(10) S.D. showing the high efficiency and reproducibility of the assay. The TaqMan PCR was subsequently used to determine the titre of several batches of the ORFV strain NZ-2, with it being possible to quantify virus solutions in the range of 1 x 10(1) to 1 x 10(6) TCID(50)/ml. A good correlation between the titre determined by the TaqMan PCR and by conventional endpoint dilution was found. The PCR assay is reproducible and can be used for a rapid quantification of ORFV in vitro and ex vivo, being readily achievable within 1h.

Animals↗

Development and evaluation of a real-time RT-PCR assay for quantification of cell-free human immunodeficiency virus type 2 using a Brome Mosaic Virus internal control.

Quantification of cell-free virus in plasma is important for monitoring disease progression and for assessing the response to antiretroviral therapy in both human immunodeficiency type 1 and type 2 (HIV-1, HIV-2) infections. Although commercial assays suitable for HIV-1 quantification have been used for more than a decade, no commercial assays are yet available for the measurement of cell-free HIV-2. We have therefore developed a novel real-time RT-PCR assay which, unlike previously described 'in house' assays, incorporates a Brome Mosaic Virus (BMV) internal control to minimise the risk of generating false-negative or falsely low results due to unrecognised problems with viral RNA purification, cDNA synthesis or PCR amplification. The assay has a dynamic range of >5 log10, detects the clinically important HIV-2 subtypes A and B with high sensitivity and shows no cross reactivity with HIV-1. The 95% detection limit is approximately 100 HIV-2 RNA copies/ml and both the inter-assay and intra-assay variability are low (CV% at 1.8 x 10(5) copies/ml, 13.3% and 5.7%, respectively). Overall, plasma HIV-2 RNA was detected in 38% of 167 unselected HIV-2 antibody-positive samples analysed over a 2 year period. The assay described provides an ideal system for studying viral replication in HIV-2 infected patients and for monitoring antiretroviral therapy.

Analysis of Variance↗

Quantification of mixed chimerism by real time PCR on whole blood-impregnated FTA cards.

This study has investigated quantification of chimerism in sex-mismatched transplantations by quantitative real time PCR (RQ-PCR) using FTA paper for blood sampling. First, we demonstrate that the quantification of DNA from EDTA-blood which has been deposit on FTA card is accurate and reproducible. Secondly, we show that fraction of recipient cells detected by RQ-PCR was concordant between the FTA and salting-out method, reference DNA extraction method. Furthermore, the sensitivity of detection of recipient cells is relatively similar with the two methods. Our results show that this innovative method can be used for MC assessment by RQ-PCR.

Chimerism↗

Real-time PCR assay for rapid detection and quantification of Campylobacter jejuni on chicken rinses from poultry processing plant.

Campylobacter jejuni (C. jejuni) is the leading cause of food-borne gastroenteritis in the United States. Detection of Campylobacter in food samples by conventional culture is cumbersome; therefore, there is a need to develop rapid and cost-effective detection and quantification methods. Eighty-four whole chicken rinses were collected at different stages of processing at three poultry processing plants. After chicken wash collection and DNA extraction, the samples were directly subjected to real-time PCR (rtPCR) without enrichment and also culture. The assay specificity was determined with a range of Campylobacter species, related, and unrelated organisms. Of the 84 samples collected 65 (77%) of the samples were positive by the rtPCR assay and 27 (32%) of the samples tested positive by direct plating to selective agar media. The results were positively concordant for 27 (32%) of the samples. The whole rtPCR assay can be completed within 90min with a detection limit of 1CFU, compared to 5-7 days for enrichment and sub culturing in selective agar. This assay is the first report of rtPCR method capable of detecting and quantifying C. jejuni from chicken rinses without an enrichment step and could be an important, rapid and quantification model for other food-borne pathogens.

Animals↗

Level set based cerebral vasculature segmentation and diameter quantification in CT angiography.

A level set based method is presented for cerebral vascular tree segmentation from computed tomography angiography (CTA) data. The method starts with bone masking by registering a contrast enhanced scan with a low-dose mask scan in which the bone has been segmented. Then an estimate of the background and vessel intensity distributions is made based on the intensity histogram which is used to steer the level set to capture the vessel boundaries. The relevant parameters of the level set evolution are optimized using a training set. The method is validated by a diameter quantification study which is carried out on phantom data, representing ground truth, and 10 patient data sets. The results are compared to manually obtained measurements by two expert observers. In the phantom study, the method achieves similar accuracy as the observers, but is unbiased whereas the observers are biased, i.e., the results are 0.00+/-0.23 vs. -0.32+/-0.23 mm. Also, the method's reproducibility is slightly better than the inter-and intra-observer variability. In the patient study, the method is in agreement with the observers and also, the method's reproducibility -0.04+/-0.17 mm is similar to the inter-observer variability 0.06+/-0.17 mm. Since the method achieves comparable accuracy and reproducibility as the observers, and since the method achieves better performance than the observers with respect to ground truth, we conclude that the level set based vessel segmentation is a promising method for automated and accurate CTA diameter quantification.

Algorithms↗