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At least 433 records · Page 24Linked to original sources
PubMed2001

Validation of a high-performance liquid chromatographic assay for the quantification of adenovirus type 5 particles.

An anion-exchange-high-performance liquid chromatography (AE-HPLC) method for the quantification of adenovirus type 5 (Ad5) total particles was validated according to performance criteria of precision, specificity, linearity of calibration and range, limit of detection, limit of quantification, accuracy and recovery. The viral particles were detected by absorbance at 260 nm using photodiode array detector (PDA). Cesium chloride (CsCl) purified Ad5 and lysate samples were used for the validation of the method. Relative standard deviations (RSDs) for the inter-day, intra-day precision and reproducibility for both the lysate and the Ad5 standard were less than 10 and 2% for the peak area and retention time, respectively. The method was specific for Ad5 which was eluted at 8.0 min. The presence of DNA does not affect the recovery of Ad5 particles for accurate quantification. Based on the error in prediction to be less than 10%, the working range was established between 2 x 10(10) and 7 x 10(10) VP/ml with correlation coefficient of 0.99975, standard deviation of 6.14 x 10(9) VP/ml and a slope of 3.04 x 10(5) VP/ml. The recovery of the method varied between 88 and 106% in all of the lysate samples investigated which is statistically similar to 100% recovery at 95% confidence interval.

Adenoviridae↗
PubMed2000

Quantification of gadodiamide as Gd in serum, peritoneal dialysate and faeces by inductively coupled plasma atomic emission spectroscopy and comparative analysis by high-performance liquid chromatography.

An inductively coupled plasma atomic emission spectroscopy (ICP-AES) method for determination of gadodiamide as Gd in serum, peritoneal dialysate and faeces was developed. The within-day and between-day precision for determination of Gd in serum and peritoneal dialysate were 0.60-2.9 and 1.8-4.4%, respectively, and the accuracy was 98.0-99.3%. The quantification limits in serum and peritoneal dialysate were 6.5 and 1.6 microM Gd, respectively. The within-day and between-day precision determination of gadolinium in faeces were 1.0-5.3 and 2.2-7.9%, respectively, and the accuracy was 104-116%. The quantification limit was 11 nmol Gd/g dry weight. For the high-performance liquid chromatography (HPLC) method, the within-day precision in determination of gadodiamide in peritoneal dialysate was 1.2% and the accuracy was 103%. The quantification limit was 0.9 microM Gd. Comparative analysis of gadodiamide in serum and peritoneal dialysate from severely impaired renal patients by ICP-AES and HPLC revealed no metabolism of chelator or transmetallation of gadolinium, even in samples obtained as long as 7 days after dosing. Furthermore, the ICP-AES determination of Gd in faeces allows for the determination of faeces content of Gd corresponding to less than 0.1% of a clinical dosage of a Gd-based contrast medium.

Chromatography, High Pressure Liquid↗
PubMed2000

A simple high performance liquid chromatographic method for the quantification of total cotinine, total 3'-hydroxycotinine and caffeine in the plasma of smokers.

A simple isocratic HPLC procedure has been developed for the quantification of caffeine and the nicotine metabolites cotinine, 3'-hydroxycotinine, cotinine glucuronide and 3'-hydroxycotinine glucuronide in the plasma of smokers. The glucuronide conjugates were determined indirectly via initial basic hydrolysis of the analyte sample followed by quantification of the resulting deconjugation product. Plasma was basified, extracted with dichloromethane, evaporated, the residue dissolved water and an aliquot part was analyzed by HPLC. The method utilized a Partisil-10 SCX cation-exchange column and an isocratic mobile phase of sodium phosphate buffer: methanol (92:8 v/v, 0.1 M, adjusted to pH 4.8 with triethylamine) at a flow rate of 1.5 ml/min. UV detection was at 254 nm. All solutes were separated with good resolution, and quantification was determined using an internal standard of N,N-diethylnicotinamide. The retention times were: caffeine 5.1 min, 3'-hydroxycotinine 7.2 min, N,N-diethylnicotinamide 9.5 min, and cotinine 15.5 min. Detection limits for caffeine, 3'-hydroxycotinine, cotinine, and total cotinine were 10 ng/ml; the detection limit for total 3'-hydroxycotinine was 20 ng/ml. The inter-day and intra-day variations for all analytes were between 1 and 8%. This analytical method is suitable for the determination of caffeine and nicotine metabolite levels in large numbers of clinical samples.

Caffeine↗
PubMed1998

TLC-UV densitometric and GC-MSD methods for simultaneous quantification of morphine and codeine in poppy capsules.

Thin-layer chromatographic (TLC)-UV densitometric and gas-chromatographic-mass spectrometric detection (GC-MSD) methods were developed for simultaneous quantification of morphine and codeine in poppy capsules (Papaver somniferum). Morphine and codeine were isolated by extraction with chloroform: isopropanol (3:1, v/v) at pH = 8.5 and by solid-phase extraction on Snap-Cap cartridges at pH = 8.5. The TLC-UV densitometric quantification was performed by external standard method on silica gel plates using ethyl acetate: toluene: methanol: ammonia (68:17:10:5, v/v) as developing solvent and UV detection at 275 nm. For the GC-MSD analysis, the drugs were derivatized with acetic anhydride: pyridine (1:1, v/v) and separated on a 30 m HP5 capillary column. The quantification was performed using nalorphine as internal standard.

Analgesics, Opioid↗
PubMed1999

Quantification of amphetamine plasma concentrations by gas chromatography coupled to mass spectrometry.

We developed a fast and sensitive method for identification and quantification of plasma concentrations of amphetamine using gas chromatography with mass spectrometry detection (GC-MS). Amphetamine-d8 served as internal standard. The method involves a single extraction procedure and an easy treatment of the samples that allowed no losses during the evaporation process. Derivatisation of amphetamine with N-methyl-bis(trifluoroacetamide), a potent acylating agent, provides many advantages to the method compared with common derivatisation reactions usually used for amphetamines. The limits of detection and quantification following this method were 0.43 and 1.42 ng/ml, respectively. The assay has been successfully employed in the quantification of amphetamine in plasma samples from healthy volunteers at four different doses.

Acetamides↗
PubMed2003

Tissue-specific expression pattern of bovine prion gene: quantification using real-time RT-PCR.

In recent studies PrP mRNA was determined mostly by in situ hybridisation or Northern Blot analysis--methods not suitable for absolute quantification of mRNA copy numbers. Herein we report on bovine prion mRNA quantification using calibrated highly sensitive externally standardized real-time RT-PCR with LightCycler instrument. Total RNA was isolated from nine different regions of the CNS and seven peripheral organs. PrP(c) mRNA copy numbers could be determined in all tissues under study. In approval with prior studies high mRNA level was found in Neocortex and Cerebellum. Lymphatic organs showed at least as high expression levels of prion mRNA as overall brain. Lowest expression was detected in kidney. Results of our study provide insight into the involvement of different organs in pathogenesis with respect to prion mRNA expression. LightCycler technology is currently considered the most precise method for nucleic acid quantification and showed to be powerful tool for further studies on prion diseases pathogenesis.

Animals↗
PubMedDate not supplied

Two-dimensional transesophageal echocardiographic characterization of ventricular filling in real time by acoustic quantification: comparison with pulsed Doppler echocardiography.

Little is known about the accuracy of acoustic quantification (AQ) in the assessment of left ventricular diastolic filling. Therefore the objective of this study was to determine the ability of AQ applied to two-dimensional echocardiography to characterize left ventricular diastolic filling compared with Doppler echocardiography. In 80 unselected patients, AQ of left ventricular diastolic filling was performed during two-dimensional transesophageal echocardiography. Pulsed Doppler transthoracic echocardiography was performed at the tips of the mitral valve leaflet and the mitral annulus level. In 53 patients with synchronous systolic wall motion, significant correlations were found between AQ-derived versus Doppler-derived indexes obtained from the level of the mitral annulus of peak rate of increase in left ventricular diastolic area versus peak early filling velocity (r = 0.78; p < 0.0001), peak early/peak atrial rate of change in left ventricular area ratio versus peak early/peak atrial filling velocity ratio (r = 0.80; p < 0.0001), and percent atrial contributions to filling (r = 0.80; p < 0.0001). Correlations between acceleration time and deceleration time derived by AQ versus Doppler echocardiography were poor. Acoustic quantification-derived acceleration and deceleration times significantly underestimated analogous times derived by Doppler echocardiography at the mitral annulus level. Correlations obtained between AQ-derived indexes and Doppler echocardiography were better with pulsed Doppler performed at the mitral annulus level compared with the leaflet tips. In 27 patients with asynchronous systolic wall motion, the correlations between AQ-derived indexes and Doppler-derived indexes of the relative distribution of filling were similar to those of the group of subjects with synchronous wall motion. However, in patients with asynchronous wall motion the correlations between indexes of peak early filling velocity and rate worsened. In addition, no correlations were found between acceleration and deceleration times derived by the two techniques in subjects with asynchronous systolic wall motion. Acoustic quantification assessment of left ventricular diastolic filling accurately characterizes the distribution of diastolic filling compared with Doppler echocardiography. The presence of asynchronous systolic wall motion decreases the accuracy of the AQ method in assessing peak filling rate. The ultimate clinical application of AQ in the assessment of left ventricular diastolic filling is yet to be determined but appears promising.

Echocardiography↗
PubMedDate not supplied

Contrast echocardiographic quantification of regional myocardial perfusion: validation with an isolated rabbit heart model.

Quantification of regional myocardial tissue blood flow (RMBF) based on contrast echocardiography has yet to be achieved. This study validated our recently proposed algorithm for quantification of RMBF with colored microspheres. Experiments were carried out in an isolated rabbit heart preparation (n = 11). Aortic root injections of perfluoropropane-filled albumin microsphere solution (FS069) and colored microspheres were performed at five levels of coronary flow achieved by altering perfusion pressure. During each injection of contrast material, consecutive end-diastolic images of the heart and an extracardiac reference chamber were acquired with a 7.5 MHz transducer and digitized. Time-intensity curves from the reference chamber and myocardial regions of interest, corresponding to the anatomic segments used for colored microsphere analysis, were analyzed for RMBF. Blood flow was calculated as the intravascular volume fraction (ratio of areas under myocardial and reference curves) divided by mean transit time (deconvolution of impulse response) and compared with those obtained with colored microspheres. Injections of FS069 resulted in highly reproducible enhancement of myocardial contrast. Analysis of time-intensity curves provided consistent measurements of RMBF (r = 0.91), which correlated highly with microsphere data (r = 0.84). The use of this new algorithm allows accurate quantification of RMBF in the isolated heart model. Further validation of this approach in an animal model with peripheral intravenous injections of contrast material will allow noninvasive clinical measurements of RMBF.

Albumins↗
PubMedDate not supplied

MRI guided segmentation and quantification of SPECT images of the basal ganglia: a phantom study.

Due to the limited resolution of single-photon emission computed tomography (SPECT) imaging devices, tissue interfaces are not well defined in the reconstructed image, even though resolution recovery techniques may be used during reconstruction. Therefore, segmentation of a particular region and quantification of the tracer uptake in that region is critical due to spillover effects, when based on the SPECT image only. In this study, we present two methods for quantification of tracer uptake in a SPECT image, defined by a matched high resolution structural magnetic resonance image. We show preliminary results of both techniques, when applied for quantifying regional uptake in the different compartments of a phantom simulating the basal ganglia. These results indicate that the quantification method, which takes into account the blurring by the SPECT imaging device, promises to be perform better in the presence of background activity.

Basal Ganglia↗
PubMed1999

Validation of the cerebellum as a reference region for SPECT quantification in patients suffering from dementia of the Alzheimer type.

In longitudinal brain studies of dementia of the Alzheimer type (DAT), the cerebellum is often used as a reference region for single photon emission computed tomography (SPECT) quantification, which assumes no significant regional influence of physiological fluctuations or pathology. With the use of absolute quantification in DAT patients, reproducibility of cerebellar uptake of technetium-99m-d,l-hexamethylpropyleneamine oxime (HMPAO) was tested and compared with the mean absolute cerebellar tracer uptake value in DAT patients and healthy control subjects. In 13 DAT patients SPECT studies were repeated within 2 weeks to assess reproducibility of cerebellar regional brain uptake (rBU). With calibrated point sources as scaling factors, cerebellar activity was expressed as rBU of HMPAO per cm3 brain tissue in percent of the injected lipophilic dose of 740 MBq (20 mCi). Also, mean cerebellar rBU in patients suffering from DAT was calculated and compared with a previously established database obtained in healthy volunteers. Repeated SPECT studies within a 2-week interval in clinically stable patients resulted in a mean rBU increase of 6.8 +/- 10.3% in the second SPECT study as compared with the first. A similar shift was previously reported in healthy volunteers. Mean cortical cerebellar rBU values in DAT patients and in the healthy reference population concurred, after cumulative corrections for body surface and for a mean brain volume of 1350 ml (obtained in healthy control subjects), showing respective mean values of 53.9 +/- 7.4 and of 52.0 +/- 7.3 x 10(-6) of the injected lipophilic dose 740 MBq (20 mCi) of HMPAO per cm3 of brain tissue. A unidirectional shift in mean absolute cerebellar uptake values occurs between repeat SPECT examinations in DAT patients similar to previous findings in a group of healthy volunteers. The origin of this phenomenon remains elusive but deserves further study with regard to SPECT (semi)quantification in DAT patients. Most interestingly, the presented findings suggest that with the use of HMPAO SPECT in DAT patients the cerebellum remains scintigraphically uninvolved.

Aged↗
PubMed1997

Comparative quantification of human cytomegalovirus DNA in blood of immunocompromised patients by PCR and Murex Hybrid Capture System.

BACKGROUND: Monitoring of human cytomegalovirus (HCMV) load by quantification of antigenemia, viremia and DNAemia is helpful in the management of HCMV infections in immunocompromised patients. In fact, threshold values of these viral parameters are associated with the emergence of clinical symptoms. In addition, the response to antiviral treatment is revealed by a decrease in viral load or virus disappearance from blood. OBJECTIVES: Aim of this study was to compare HCMV DNA quantification in blood of immunocompromised patients by an 'in house' developed quantitative PCR (Q-PCR) assay and the commercially available Murex Hybrid Capture System (HCS). STUDY DESIGN: HCMV DNA was quantified in 95 blood samples from 12 heart and heart-lung transplant recipients and 27 AIDS patients using both techniques. For HCS analysis 3.5 ml whole blood were utilized, whereas Q-PCR was performed using 1 x 10(5) peripheral blood leukocytes (PBL). HCMV DNA levels obtained by HCS and Q-PCR were expressed as number of genome equivalents (GE)/ml whole blood or 1 x 10(5) PBL, respectively. Results from HCS and Q-PCR were compared and submitted to statistical analysis. In addition, HCMV DNA values were compared to levels of antigenemia and viremia. RESULTS AND CONCLUSIONS: Sensitivity of HCS, antigenemia and viremia with respect to Q-PCR were 37.2, 79.5 and 33.3%, respectively. Specificity was 100% for all techniques. On average, samples positive by Q-PCR only, contained low amounts of HCMV DNA. In particular, 45 (91.8%) out of 49 samples negative by HCS and positive by Q-PCR showed < 500 GE/1 x 10(5) PBL. A significant correlation was found between quantitative DNA levels in samples positive by both HCS and Q-PCR (n = 29, R = 0.693, P < 0.01). HCS positivity was associated to significantly higher DNA values as determined by Q-PCR as well as to significantly higher antigenemia and viremia levels. A decrease in DNAemia levels was observed using both HCS and Q-PCR after antiviral treatment. Given that the great majority of blood samples missed by HCS contain low levels of HCMV DNA which are not clinically significant, HCS seems very promising as an alternative to HCMV DNA quantification by PCR in solid organ transplant recipients and AIDS patients.

AIDS-Related Opportunistic Infections↗
PubMed2004

A simple, high throughput method for the quantification of sodium alginates on oesophageal mucosa.

Sodium alginate is a potential bioadhesive, but the lack of a convenient and suitable method for its quantification on the mucosal surface complicates the evaluation of its mucosal retentive properties. This paper develops and evaluates a spectrophotometric method for the rapid quantification of a range of sodium alginates differing in chemical composition, and investigates how quantification was influenced by the presence of oesophageal mucosa. The method, based on dye complexation with 1,9-dimethyl methylene blue (DMMB) was sensitive to alginate molecular weight and uronic acid composition, however, no significant correlations between assay performance and alginate molecular characteristics were demonstrated. The assay was also influenced by complexation time, calcium ions and mucin, but was unaffected by the presence of oesophageal tissue scrapings. The assay proved to be capable of quantifying sodium alginate with excellent linearity (r = 0.999), reproducibility (CV < 3%) and sensitivity (0.3 g l(-1)) and proved to be a precise, high-throughput method that may be used for quantifying the retention of sodium alginate on oesophageal mucosa.

Alginates↗
PubMed1998

Simultaneous quantification of released succinic acid and a weakly basic drug compound in dissolution media.

A HPLC-method was developed to determine both fenoldopam, a weakly basic drug and succinic acid, a pH-adjuster for this drug in dissolution media. The usual assays for succinic acid were not applicable due to its low UV-absorption, the low pH-value of samples or the presence of buffer salts and fenoldopam. The described method is a simple non-ion-pair reversed phase HPLC-method using a fast scanning UV-detector and a PC software program for the quantification of both components. Succinic acid is detected at 205 nm and fenoldopam at 225 nm. The UV-spectrum is used to determine peak purity and to identify peaks (carried out at a 99.9% match). This is especially important as in some of the investigated samples an unknown peak elutes immediately after succinic acid, resulting in spurious high contents, if mistaken for succinic acid. The simple method accomplished the simultaneous quantification of both, succinic acid and fenoldopam, by an accurate, precise, specific and reproducible assay, with a linear range covering all concentrations relevant for dissolution testing. The method is stability indicating and can also be used for the quantification of fumaric acid, another pH-adjuster in dissolution media together with fenoldopam.

Chemistry, Pharmaceutical↗
PubMed2000

Enzymatic quantification of cell-matrix and cell-cell adhesion.

Adhesion assays are powerful tools to investigate the adhesive properties of cells. The quantification of cell adhesion enables determination of the capacity of cells to stick to a target, screening for novel adhesion involved binding molecules, exploration of structure-function relationships of adhesion molecules, evaluation of adhesion targets, and examination of compounds interfering with cell adhesion. Thus, quantification of cell adhesion needs simple and reliable methods that might be applied for both research and diagnostic purposes. This review presents methodological principles of enzymatic approaches for quantification of cell adhesion. In particular, the advantages of exogenous cell labelling with horseradish peroxidase are described.

Cell Adhesion↗
PubMed2003

Quantification of concentric-needle-induced insertional activity by turn/amplitude analysis.

Quantification of needle-induced insertional activity (IA) by means of the turn/amplitude analysis (TAA) has not been attempted so far. IA was recorded from the right brachial biceps and right anterior tibial muscle of 29 healthy subjects, 17 woman, 12 men, aged 24-79 years and 51 patients with neuromuscular disorders (29 neuropathies and 22 myopathies), 24 woman, 27 men, aged 17-81 years. IA was analyzed with regard to IA duration (IAD), turns/second (T/S), amplitude/turn (A/T), T/S:IAD and A/T:IAD. IAD was not significantly different between healthy and diseased subjects in either of the two investigated muscles. On the contrary, T/S, A/T, T/S:IAD and A/T:IAD were significantly increased in the brachial biceps and anterior tibial muscle of patients with neuropathies. In myopathies T/S were increased in both muscles and the T/S:IAD in the anterior tibial muscle. Quantification of IA by TAA increases the sensitivity of conventional EMG from 38 to 69% in neuropathies and from 41 to 73% in myopathies. It is concluded that quantification of IA by TAA is of diagnostic support in the detection of neuromuscular disorders. The most useful parameter in this respect is the T/S, which, contrary to IAD, is increased in patients with neuropathies as well as patients with myopathies.

Action Potentials↗
PubMed2002

Recurrence quantification analysis as a tool for nonlinear exploration of nonstationary cardiac signals.

The complexity, nonlinearity and nonstationarity of the cardiovascular system typically defy comprehensive and deterministic mathematical modeling, except from a statistical perspective. Living systems are governed by numerous, continuously changing, interacting variables in the presence of noise. Cardiovascular signals can be shown to be discontinuous alternations between deterministic trajectories and stochastic pauses (terminal dynamics). One promising approach for assessing such nondeterministic complexity is recurrence quantification analysis (RQA). As reviewed in this paper, strategies implementing quantification of recurrences have been successful in diagnosing changes in nonstationary cardiac signals not easily detected by traditional methods. It is concluded that recurrence quantification analysis is a powerful discriminatory tool which, when properly applied to cardiac signals, can provide objectivity regarding the degree of determinism characterizing the system, state changes, as well as degrees of complexity and/or randomness.

Algorithms↗
PubMed1998

Parallel regional quantification of choline acetyltransferase and cholinesterase activity in the central nervous system of an invertebrate (Sepia officinalis).

The present study describes (i) a procedure to dissect the central nervous system of the cuttlefish (Cephalopod) into ten, functionally distinct, anatomical regions of interest and (ii) the parallel measurement of acetylcholine synthesis (choline acetyltransferase) and degradation (cholinesterase) activities. Both aspects (dissection and parallel quantification of acetylcholine synthesis and degradation) could be of great importance for quantitative regional studies in neurochemistry in this animal model, it is interesting to study the cellular and molecular mechanisms involved in learning and aging processes. The parallel quantification of acetylcholine synthesis and degradation applicable to any animal model is pivotal since both enzymes are essential for the cholinergic neurotransmission and may be differentially modulated by specific functions such as learning and aging processes. Furthermore, since choline acetyltransferase and cholinesterase show different localization into the brain, their parallel quantification may underlie the involvement of cholinesterase in non-cholinergic functions, which remain unclear throughout the animal kingdom.

Aging↗
PubMed1999

Comparison of three commercial assays for the quantification of HIV-1 RNA in plasma from individuals infected with different HIV-1 subtypes.

BACKGROUND: Commercial human immunodeficiency virus 1 (HIV-1) ribonucleic acid (RNA) quantification assays vary in their ability to quantify different subtypes of HIV-1, a problem in regions where multipte HIV-1 subtypes may be circulating. OBJECTIVES: To assess commercial HIV-1 RNA quantification assays on two plasma panels. Panel 1 consisted of HIV-1 seronegative plasma 'spiked' with a known amount of cultured virus of different subtypes, and Panel 2 comprised plasma collected from individuals infected with different HIV-1 subtypes. STUDY DESIGN: The comparison involved the Amplicor HIV-1 reverse transcriptase-polymerase chain reaction (RT-PCR), Quantiplex branched DNA, and NucliSens HIV-1 QT assays. Panel 1 consisted of 11 plasma 'spiked' with cultured viruses of HIV-1 subtypes A-F, and Panel 2 included 33 plasma samples from 16 patients infected with subtypes A, B, C, E and G. RESULTS: In Panel 1, the Quantiplex branched deoxyribonucleic acid (bDNA) assay quantified subtypes A-F efficiently, comparable to published results from two other laboratories. The Amplicor RT-PCR assay quantified subtypes B, C, and D but was relatively less efficient with subtypes E, F, and did not or poorly quantified subtype A. Testing of Panel 2 showed some inter-assay differences. In contrast to Panel 1, the Amplicor RT-PCR assay performed variably with subtype A when compared with the Quantiplex bDNA and NucliSens QT assays, and higher viral load levels were generated with subtype E using the Amplicor RT-PCR assay. Subtypes B and C showed some inter-patient differences but the Quantiplex bDNA generally gave a lower quantification than the Amplicor RT-PCR and NucliSens QT assays. CONCLUSIONS: These studies confirm that commercial HIV-1 load assays vary in their ability to quantify different HIV-1 subtypes. This may be more apparent with individual patient samples than with 'spiked' panels. This variability emphasizes that it is preferable for patient samples to be tested with the same assay, and care should be taken where infection with unusual subtypes is suspected.

DNA, Viral↗