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Results for “Absolute quantification”

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PubMed1991

Single procedure for detection, confirmation, and quantification of benzodiazepines in serum by liquid chromatography with photodiode-array detection.

We developed a reversed-phase chromatographic procedure for detecting benzodiazepines and other drugs in serum. A liquid-liquid extraction step with hexane/ethyl acetate isolates the drugs from serum; absolute recoveries are generally greater than 85%. Reconstituted extracts are chromatographed on a 4-microns (particle size) C18 column; 14 drugs and an internal standard (flunitrazepam) are separated in 8 min. Peak detection, purity checking, and identification are performed with a computerized photodiode-array detector. Run-to-run imprecision (CV) for many benzodiazepines is less than 3%. In a study of 126 specimens from Emergency Department patients, the procedure showed excellent agreement with a gas-chromatographic method involving either mass-spectrometric or flame-ionization detection. This single procedure provides rapid and accurate detection, quantification, and confirmation of benzodiazepines in serum.

Benzodiazepines↗

PubMed2000

Bioavailability of orally administered micronised fluticasone propionate.

OBJECTIVE: The aim of this study was to determine the absolute oral bioavailability of fluticasone propionate (FP) in healthy volunteers. METHODS: A 3-period incomplete block crossover design was used. On separate occasions, 21 male volunteers received a single 250 microg intravenous dose of FP (n = 21) and twice daily oral doses of either micronised FP 0.1 mg (n = 9), 1 mg (n = 12), 10 mg (n = 11) or placebo (n = 9) for 4 days. RESULTS: FP was not measurable in the plasma after twice daily oral administration of a 0.1 mg dose. FP concentrations just above the limit of quantification could be measured in only 5 volunteers, and only at some time points, after administration of FP 1 mg twice daily. At a dose of 10 mg twice daily the absolute oral bioavailability of the drug was <1% when a liquid chromatography-mass spectrometry assay was used to assess plasma concentrations. Only oral doses of FP 10 mg twice daily, 10 times greater than the recommended maximum inhaled dose, produced any detectable change in urinary cortisol excretion. CONCLUSION: The results of this study confirm that oral absorption of FP into the systemic circulation is negligible. The swallowed portion of an inhaled dose of FP is unlikely to increase the systemic exposure to the drug, thus decreasing the likelihood of adverse systemic effects.

Administration, Oral↗

PubMed1997

Quantification of brain metabolites in amyotrophic lateral sclerosis by localized proton magnetic resonance spectroscopy.

We performed proton magnetic resonance spectroscopy (1H-MRS) in patients with motor neuron disease (MND) to determine the absolute in vivo concentrations in the brain of the metabolites N-acetyl aspartate (NAA), choline (Cho), and creatine (Cr/PCr). We examined the spectra acquired from a 20 x 20 x 20-mm3 voxel placed in the motor cortex and in the cerebellum from seven patients with clinically probable or definite amyotrophic lateral sclerosis (ALS) according to the El Escorial criteria, from three patients with suspected ALS (progressive muscular atrophy), and from eight normal control subjects. We estimated the concentrations of the metabolites using the water signal as an internal standard. The concentrations of Cho and Cr/PCr in both brain regions, as well as the concentration of NAA in the cerebellum, were unaltered in the MND patients compared with the controls. Only MND patients with both upper and lower motor neuron signs had a significantly decreased concentration of NAA (9.13 +/- 0.28 mM, mean +/- SEM) in the primary motor cortex when compared with healthy controls (10.03 +/- 0.22 mM). In conclusion, the slightly decreased concentration of NAA in the primary motor cortex from ALS patients may represent a loss of neurons in this region.

Adult↗

PubMed1998

Determination of piroxicam and its major metabolite 5-hydroxypiroxicam in human plasma by zero-crossing first-derivative spectrophotometry.

A zero-crossing first-derivative spectrophotometric method for the determination of piroxicam and its major metabolite 5-hydroxypiroxicam (5-HP) in human plasma is described. This technique permits the quantification of compounds with closely overlapping spectral bands without any separation step. The method consists of direct extraction of the less-ionised forms of piroxicam and 5-hydroxypiroxicam with pure diethyl ether. First derivative values at 343.5 and 332.5 nm for piroxicam and 5-HP, respectively, were obtained. The absolute recovery of the method was found to be 89.4% for piroxicam and 90.3% for 5-HP. Calibration graphs are linear (r better than 0.9998), with zero-intercept, in the concentration range 0.5-12.0 micrograms ml-1 for both compounds. The limits of quantification attained according to the IUPAC definition were 0.29 and 0.27 micrograms ml-1 for piroxicam and 5-HP, respectively. The results obtained by the proposed method were in good agreement with those found by the high-performance liquid chromatographic method (HPLC).

Chromatography, High Pressure Liquid↗

PubMed1993

Quantification of myocardium at risk and detection of reperfusion by dynamic vectorcardiographic ST segment monitoring in a pig occlusion-reperfusion model.

OBJECTIVE: The aim was to investigate whether continuous computerized vectorcardiographic monitoring of absolute spatial ST vector magnitude (ST-VM) and spatial ST change vector magnitude (STC-VM) during coronary occlusion could be used to estimate the size of myocardium at risk; and also to test whether reperfusion could be distinguished from sustained occlusion by continuous monitoring of ST vector alterations. METHODS: Computerised vectorcardiographic monitoring via Frank leads was applied in a closed chest occlusion-reperfusion pig model. Coronary occlusion over 24 h was produced in 20 animals by injecting a 2 mm ball into the left anterior descending coronary artery (n = 7), the right coronary artery (n = 8), and the left circumflex coronary artery (n = 5). Another 31 pigs were reperfused by retraction of the ball after 30 (n = 10), 60 (n = 15), or 90 (n = 6) min of left anterior descending artery occlusion. The extent of the myocardium at risk was measured by autoradiography. RESULTS: Seven animals were excluded. Irrespective of occluded coronary artery the relative parameters STC-VM over the first 30 min of occlusion correlated closely with area at risk, that is, the mean STC-VM between 10 and 30 min of occlusion (r = 0.78 p < 0.001). The absolute parameter ST vector magnitude (ST-VM) did not reflect ischaemia in 16/44 animals and did not correlate significantly with area at risk. The weight of myocardium at risk (MAR) was predictable from STC-VM: MAR weight (measured) = 0.97 x MAR weight (predicted) + 0.26 (g), r = 0.81, p < 0.001. STC-VM decline rate, time to STC-VM plateau, and cumulated sum plots of STC-VM were all able to distinguish reliably between reperfused animals and those with permanent occlusion. A paradoxical increase in STC-VM - "reperfusion peak" - was detected in 17/31 (55%) of the animals. This phenomenon was related to large amount of myocardium at risk or to a long occlusion time. CONCLUSION: Dynamic vectorcardiographic ST monitoring provides adequate estimation of myocardium at risk and enables detection of reperfusion in experimental myocardial ischaemia.

Animals↗

PubMed2000

Range of global motion of the cervical spine: intraindividual reliability and the influence of measurement device.

Range of motion tests are often employed in the quantification of musculoskeletal impairment and in the assessment of the efficacy of therapeutic interventions. The aim of the present study was to compare the absolute values for, and the day-to-day reliability of, measures of cervical spinal mobility made with two computerised motion analysis devices. The ranges of cervical flexion, extension, lateral bending, axial rotation, and axial rotation in flexion and extension were determined for 19 volunteers using both the CA6000 Spine Motion Analyser and the Zebris CMS system; all measures were repeated on a second occasion 1-3 days later. The test-retest reliability was good for each instrument: there was no significant difference between the mean values derived on the two separate days (P>0.05), and the corresponding intraclass correlation coefficients were 0.75-0.93 for all pri-O mary movements and 0.57-0.93 for axial rotation in flexion or in extension. For each primary movement, a small but significant difference (1-10%; P<0.05) between the values derived from the two instruments was observed, the systematic nature of which was revealed by the excellent correlation coefficients between them. For the measures of axial rotation in flexion or in extension, however, there was not only a poor correlation between the data obtained from the two devices, but the mean values also differed significantly. Each device is highly reliable in itself and can be used with confidence in longitudinal studies. The establishment of 'normal' values for the primary motions should take account of the slight differences observed between devices. Normal values for rotation in flexion or extension cannot be established until the source of the device-dependent difference is identified.

Adult↗

PubMed2001

Correlating rates of cerebral atrophy in Parkinson's disease with measures of cognitive decline.

We studied eight clinically non-demented PD patients and ten age-matched controls with serial volumetric T1-weighted MRI. All PD patients underwent full neuropsychological testing at baseline and follow up scans. Sub-voxel coregistration of the serial MRI scans with quantification of changes in total brain substance and ventricular size per year was performed. The PD patients had significant reductions in both percentage and absolute annual brain volume loss when compared to age-matched controls (p < 0.001). There were significant correlations between reductions in percentage brain volume loss and estimated reductions in performance IQ (r = 0.841, p = 0.004) and full scale IQ (r = 0.63, p = 0.049), measured by subtracting IQ measures at time of follow up scan from premorbid estimates. In conclusion, PD patients have a significant rate of median brain volume loss [10.35 (range) 6.69-16.90 ml/year] with no significant loss seen in age-matched controls, and these changes correlate with global measures of cognitive decline. Further longitudinal studies could evaluate whether serial volumetric MRI is a useful technique in predicting the preclinical onset of dementia in Parkinson's disease patients, and its role in the assessment of putative treatments for slowing disease progression.

Aged↗

PubMed2005

Fully automated online solid phase extraction coupled directly to liquid chromatography-tandem mass spectrometry. Quantification of sulfonamide antibiotics, neutral and acidic pesticides at low concentrations in surface waters.

A fully automated online solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) instrumental setup has been developed for the quantification of sulfonamide antibiotics and pesticides in natural water. The direct coupling of an online solid phase extraction cartridge (Oasis HLB) to LC-MS/MS was accomplished using column switching techniques. High sensitivity in the low ng/L range was achieved by large volume injections of 18 mL with a combination of a tri-directional auto-sampler and a dispenser system. This setup allowed high sample throughput with a minimum of investment costs. Special emphasis was placed on low cross contamination. The chosen approach is suitable for research as well as for monitoring applications. The flexible instrumental setup was successfully optimised for different important groups of bioactive chemicals resulting in three trace analytical methods for quantification of (i) sulfonamide antibiotics and their acetyl metabolites; (ii) neutral pesticides (triazines, phenylureas, amides, chloracetanilides) and (iii) acidic pesticides (phenoxyacetic acids and triketones). Absolute extraction recoveries from 85 to 112% were obtained for the different analytes. More than 500 samples could be analyzed with one extraction cartridge. The inter-day precision of the method was excellent indicated by relative standard deviations between 1 and 6%. High accuracy was achieved by the developed methods resulting in maximum deviation relative to the spiked amount of 8-15% for the different analytes. Detection limits for various environmental samples were between 0.5 and 5 ng/L. Matrix induced ion suppression was in general smaller than 25%. The performance of the online methods was demonstrated with measurements of concentration dynamics of sulfonamide antibiotics and pesticides concentrations in a little creek during rain fall events.

Anti-Bacterial Agents↗

PubMed2006

Mucosal keratinocyte isolation: a short comparative study on thermolysin and dispase.

New techniques for reconstructing large defects of the floor of the mouth include the use of cultured mucosal substitutes. The purpose of this study was to compare dispase and thermolysin for keratinocyte isolation. Keratinocyte yield per surface area of rabbit buccal mucosa was assessed by histology, cytokeratin 13 (CK13) staining, seeding efficiency analysis and cell diameter quantification. Surface areas of cultured mucosa were calculated. Histology showed that treatment by thermolysin resulted in incomplete separation of epidermis from dermis. Also, the absolute number of keratinocytes/cm(2) isolated mucosa, cell yield, cell size and seeding efficiencies was higher in the dispase group. A 3.45-fold larger graft could be reconstituted using dispase. The use of dispase, rather than thermolysin, to isolate cells from buccal mucosa is concluded to be favourable.

Animals↗

PubMed2004

Determination of moclobemide in human plasma by high-performance liquid chromatography with spectrophotometric detection.

A simple and sensitive high-performance liquid chromatographic (HPLC) method with spectrophotometric detection was developed for the determination of moclobemide in human plasma. Plasma samples were extracted under basic conditions with dichloromethane followed by back-extraction into diluted phosphoric acid. Isocratic separation was employed on an ODS column (250 mm x 4.6 mm, 5 microm) at room temperature. The mobile phase consisted of 5 mM NaH2PO4-acetonitrile-triethylamine (1000:350:10 (v/v/v), pH 3.4). Analyses were run at a flow-rate of 1.0 ml/min and ultraviolet (UV) detection was carried out at 240 nm. The method was specific and sensitive with a quantification limit of 15.6 ng/ml and a detection limit of 5 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery was about 98.2%, while the intra- and inter-day coefficient of variation and percent error values of the assay method were all at acceptable levels. Linearity was assessed in the range of 15.6-2000 ng/ml in plasma with a correlation coefficient of greater than 0.999. This method has been used to analyze several hundred human plasma samples for bioavailibility studies.

Biological Availability↗

PubMed2006

Improvement and validation of an HPLC method for examining the effects of the MDR1 gene polymorphism on sparfloxacin pharmacokinetics.

A rapid, simple, accurate, and precise reverse-phase high-performance liquid chromatography (HPLC) method for measuring sparfloxacin in human serum was improved, validated, and applied to determine the influence of polymorphisms in MDR1 (exons 12, 21, and 26) gene on sparfloxacin pharmacokinetics. Sparfloxacin and an internal standard, ciprofloxacin, were extracted from human serum by protein precipitation with dilution and analyzed on a Luna C(18) 5-microm column in a mobile phase of acetonitrile-0.035 M perchloric acid (28:72, v/v, adjusted to pH 2.0 with 0.015 M triethylamine) and UV detection at 300 nm. This analysis was performed at three different laboratories using the same quality control (QC) samples. The chromatograms showed good resolution, sensitivity, and no interference by human serum. The method showed linear responses over a concentration range of 0.05-2 microg/ml, with correlation coefficients of greater than 0.999 at the three laboratories. Intra- and inter-day assay precision and accuracy fulfilled international requirements. The mean absolute recovery for human serum was 98.8+/-5.7%. Sparfloxacin in human serum was stable during storage and the assay procedure. The lower limit of quantification using 0.2 ml of serum was 0.05 microg/ml, which was sensitive enough for pharmacokinetic studies. This method was used to study the pharmacokinetics of sparfloxacin in human volunteers, following a single oral administration of sparfloxacin (100 mg) two tablets at three different laboratories. MDR1 polymorphisms did not significantly (P < 0.01) affect the pharmacokinetic parameters (AUC and C(max)) of sparfloxacin.

Anti-Bacterial Agents↗

PubMed1998

Simple high-performance liquid chromatographic method for determination of ketoconazole in human plasma.

A simple high-performance liquid chromatographic method using fluorescence detection was developed for the determination of ketoconazole in human plasma. The method entailed direct injection of the plasma sample after deproteinization using acetonitrile. The mobile phase comprised 0.05 M disodium hydrogen orthophosphate and acetonitrile (50:50, v/v) adjusted to pH 6. Analysis was run at a flow-rate of 1.5 ml/min with the detector operating at an excitation wavelength of 260 nm and an emission wavelength of 375 nm. The method is specific and sensitive with a quantification limit of approximately 60 ng/ml and a detection limit of 40 ng/ml at a signal-to-noise ratio of 3:1. Mean absolute recovery value was about 105%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 14%. The calibration curve was linear over a concentration range of 62.5-8000 ng/ml.

Antifungal Agents↗

PubMed2005

Computational processing and error reduction strategies for standardized quantitative data in biological networks.

High-quality quantitative data generated under standardized conditions is critical for understanding dynamic cellular processes. We report strategies for error reduction, and algorithms for automated data processing and for establishing the widely used techniques of immunoprecipitation and immunoblotting as highly precise methods for the quantification of protein levels and modifications. To determine the stoichiometry of cellular components and to ensure comparability of experiments, relative signals are converted to absolute values. A major source for errors in blotting techniques are inhomogeneities of the gel and the transfer procedure leading to correlated errors. These correlations are prevented by randomized gel loading, which significantly reduces standard deviations. Further error reduction is achieved by using housekeeping proteins as normalizers or by adding purified proteins in immunoprecipitations as calibrators in combination with criteria-based normalization. Additionally, we developed a computational tool for automated normalization, validation and integration of data derived from multiple immunoblots. In this way, large sets of quantitative data for dynamic pathway modeling can be generated, enabling the identification of systems properties and the prediction of targets for efficient intervention.

Algorithms↗

PubMed2002

[Near-infrared optical imaging of human brain function--a novel approach to the brain and the mind].

Near-infrared spectroscopy (NIRS) can measure changes in the hemoglobin oxygenation state in the human brain. NIRS has been oriented toward use for clinical monitoring of tissue oxygenation. However, we and others have recently demonstrated that NIRS also has the potential for neuroimaging. NIRS instruments, which use continuous wave light (CW) as a light source, are now commercially available. These CW-type NIRS instruments have high temporal resolution (less than 1 second) and allow long-term and continuous measurements, though they do not provide absolute values of changes in hemoglobin concentrations. In contrast, time-resolved spectroscopy (TRS), which uses short pulsed laser diodes as light sources, makes quantification possible. Quantification is necessary for the imaging of brain activity. Topographical images can be obtained by the use of our recently developed 64-channel time-resolved optical tomographic imaging system (optical CT) or by combining the 1 channel TRS instrument with the multichannel CW-type NIRS instrument. NIRS is completely non-invasive and does not require strict motion restriction during measurements unlike PET and fMRI. It is, thus, expected that NIRS will open a window into brain physiology in subjects who are difficult to examine with PET and fMRI such as children, the elderly, and patients with psychoneurological problems.

Brain↗

PubMed2004

Measurement of tumor interstitial volume fraction: method and implication for drug delivery.

It is important to evaluate the tumor interstitial volume fraction that is accessible for drug accumulation during the distribution phase in order to determine the potential efficacy of cancer chemotherapy. In this study, we performed simulations of magnetic resonance imaging (MRI) signal intensity using a two-compartment tissue model for quantitative analyses of absolute interstitial volume measurements while we experimentally characterized a mouse tumor model with a dual MR contrast-agent method. Previously, consecutive intravenous injections of a strictly intravascular T1 contrast agent followed by an extravasating agent were used as a strategy for the quantification of both relative blood volume (Rel_BV) and relative interstitial volume (Rel_ITST) (Weissleder et al. Eur J Cancer 1998;34:1448-1454; Bogdanov et al. Neoplasia 1991;1:438-435). In the current study, we demonstrate that this approach can be further improved, and that it enables one to accurately evaluate both relative and absolute interstitial volumes. The animal data indicated that a significant difference exists between the absolute interstitial volume fractions of subcutaneously implanted MDA PCa 2b tumor and skeletal muscle tissue (27.5 +/- 9.1% and 15.9 +/- 0.7%, respectively (P < 0.05)), while only a minor difference was found for the absolute blood volumes (Abs_BV) (Kim et al. Magn Reson Med 2002;47:1110-1120) of these tissues.

Animals↗

PubMed1990

Quantification of human hepatic glutathione S-transferases.

Human hepatic glutathione S-transferase (GST) subunits were characterized and quantified with the aid of a recently developed h.p.l.c. method. In 20 hepatic tissue specimens the absolute amounts of the basic Class Alpha subunits B1 and B2, the near-neutral Class Mu subunits mu and psi and the acidic subunit pi were determined. The average total amount of GST was 37 micrograms/mg of cytosolic protein, with the Class Alpha GST being the predominant class (84% of total GSTs), and pi as the sole representative of the Class Pi GSTs present in the lowest concentration (4% of total GSTs). Large interindividual differences were observed for all subunits, with variations up to 27-fold, depending on the subunit. For the Class Alpha GST-subunits B1 and B2, a biphasic ratio was observed. The genetic polymorphism of the subunits mu and psi was confirmed by h.p.l.c. analysis, and correlated with the enzymic glutathione conjugation of trans-stilbene oxide and with Western blotting of cytosols, using a monoclonal anti-(Class Mu GST) antibody. Of the 20 livers examined, ten contained only mu, whereas the occurrence of psi alone, and the combination of mu and psi, were found in only one liver each.

Chromatography, High Pressure Liquid↗

PubMed1991

Neopterin quantification in acute severe asthma.

To investigate the pathogenic role of cell-mediated immunity (CMI) in acute severe asthma, 10 patients with acute asthma requiring admission to hospital had measurements of serum and urine absolute neopterin and neopterin/creatinine ratios. Serum was collected upon entry to hospital, 24 hr later and at 2 weeks; and urine at 24 hr and at 2 weeks after admission. Comparisons were made with 12 stable asthmatic patients, healthy control subjects, and six renal transplant patients undergoing transplant rejection. In contrast to the renal transplant patients who demonstrated T-lymphocyte-macrophage activation, neopterin concentrations and neopterin/creatinine ratios in both acute and stable asthmatic patients were not elevated. We have not found functional evidence that CMI participates in the pathogenesis of acute severe asthma.

Acute Disease↗

PubMed2001

Accuracy limits in the determination of absolute optical properties using time-resolved NIR spectroscopy.

We assess typical systematic experimental errors involved in a time-resolved measurement as applied to NIR diffuse optical spectroscopy and investigate their effect on the quantification accuracy of the absorption and the reduced scattering coefficient. We demonstrate that common systematic experimental uncertainties may lead to quantification errors of 10% or more, even when excellent signal to noise ratio conditions exist and accurate photon propagation models are employed. We further demonstrate that the accuracy of the calculation depends nonlinearly on the optical properties of the medium measured. High scattering and low absorbing media can be quantified more accurately than media with low scattering or high absorption using measurements of the same signal to noise ratio. We further discuss curve-shape fitting schemes that aid in improving the quantification accuracy in the presence of experimental errors. Finally, we identify uncertainties that set quantification accuracy limits and we find temporal resolution as the ultimate limiting factor in the quantification accuracy achieved. Our findings suggest that temporal resolution of the order of 10 ps is necessary for quantifying the absorption and reduced scattering coefficient of diffuse media with accuracy better than 5% using curve fitting methods. In that sense this analysis can be used in time-resolved system design and in predicting the expected errors given the technology selected for time-resolved measurements.

Biophysical Phenomena↗