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PubMed2003

A multivessel model describing replenishment kinetics of ultrasound contrast agent for quantification of tissue perfusion.

To improve the quantification of tissue perfusion using intermittent sonography, a new model describing replenishment kinetics of microbubbles is proposed. The new approach takes into account the variability of blood flow velocities found in vivo, especially in tumors, and consistently describes the refilling process of microbubbles. Based upon this model, blood volume, blood velocity, blood flow and perfusion in 17 experimental tumors were calculated, and compared with the results obtained with the established, phenomenologically derived exponential kinetic model. In contrast to the existing model, our approach describes tissue vascularization more physiologically and allows deduction of a consistent new hyperbolic model for quantification of intermittent sonography. Blood volume and mean blood velocity did significantly correlate between both the new and the established model (k=0.99; k=0.94, both p<0.001). However, mean tumor blood velocity was lower (-19%, p<0.01) with the established model compared to the newly developed model. In addition, the range and distribution of blood flow velocities found in vivo can be estimated with the new model. Furthermore, it uses simpler mathematical fitting routines and allows easier data acquisition, which may allow a more practicable clinical application of intermittent sonography. In conclusion, a more valid, detailed and accurate calculation of perfusion parameters, especially of tumors, can be derived in vivo with the new multivessel model of intermittent sonography.

Animals↗
PubMed2000

In vitro flow quantification with contrast power Doppler imaging.

To evaluate the effectiveness of contrast harmonic (power Doppler imaging) as an ultrasonic modality to quantify flow, an in vitro model of perfusion was studied using Optison, a second-generation ultrasound (US) contrast agent. The in vitro model was made of two dialysis cartridges placed parallel and allowed absolute and relative flow quantification on both tube (entry lines) and tissue (cartridges) simulations. Video intensity curves were generated using intermittent harmonic power Doppler imaging after bolus injection of contrast. Correlation between flow and different parameters extracted from time-intensity curves and previously defined as indicators of flow was established for both tissue and entry lines, for flow rates ranging from 0 to 400 mL/min. Single-compartment equations were also tested on the model. A good correlation for the tissue model was observed between absolute flow and onset time (O), time to maximal enhancement (TME), peak intensity (P), area under the curve (AUC), and maximal ascending slope (S) parameters, with a r = 0.94, 0.94, 0.91, 0.92 and 0.92, respectively. The correlation for O, TME, P and AUC parameters was r = 0.86, 0.90, 0.78 and 0.82, respectively for entry lines. The correlation for tissue model and entry line was slightly improved when comparing flow ratios with peak ratios (P1/P2) and slope ratios (S1/S2) (r = 0.95 and 0.94). Flow calculation using the gradient-relationship method also showed a good correlation (r = 0.88) with the experimental flow. The results obtained indicated that absolute and relative quantification of flow using PDI is feasible in tube and tissue models. Several clinical applications, namely in myocardial, hepatic and renal artery studies, could be derived from these results.

Albumins↗
PubMed1999

Quantification of neuron survival in monolayer cultures using an enzyme-linked immunosorbent assay approach, rather than by cell counting.

The determination of neurotoxicity in monolayer mixed cultures has traditionally necessitated the time consuming and subjective procedure of counting neurons. In this paper, we propose a modification of an immunohistochemical staining method with a neuron-specific antibody against MAP2, that allows for quantification of neuron number to be done using an enzyme-linked immunosorbent assay (ELISA) plate reader. This new procedure involves the use of the compound 2,3'-azino-bis(ethylbenzothiazoline-6-sulphonic acid) (ABTS) at the last stage of the staining procedure. We employed two neurotoxicity models (the excitotoxin kainic acid and the interactions between gp120, the glycoprotein of HIV, and the stress hormone corticosterone) to compare the results obtained with this new method and the old method of immunohistochemical staining followed by 3,3'-daminobenzidine (DAB) and the counting of neurons. The ABTS/ELISA method was found to be a fast, reliable and objective procedure for the quantification of neurotoxicity.

Astrocytes↗
PubMed2001

Quantification of red blood cells using atomic force microscopy.

For humans the sizes and shapes of their red blood cells are important indicators of well being. In this study, the feasibility of using the atomic force microscope (AFM) to provide the sizes and shapes of red blood cells has been investigated. An immobilisation procedure has been developed that enabled red blood cells to be reliably imaged by contact AFM in air. The shapes of the red blood cells were readily apparent in the AFM images. Various cell quantification parameters were investigated, including thickness, width, surface area and volume. Excellent correlation was found between the AFM-derived immobilised mean cell volume (IMCV) parameter and the mean cell volume (MCV) parameter used in current haematological practice. The correlation between MCV and IMCV values has validated the immobilisation procedure by demonstrating that the significant cell shrinkage that occurs during immobilisation and drying does not introduce quantification artifacts. Reliable IMCV values were obtained by quantifying 100 red blood cells and this typically required 3-5 AFM images of 100 microm x 100 microm area. This work has demonstrated that the AFM can provide in a single test the red blood cell size and shape data needed in the assessment of human health.

Anemia, Macrocytic↗
PubMed2003

Quantification of disease progression of several microbial pathogens on Arabidopsis thaliana using real-time fluorescence PCR.

An accurate monitoring of disease progression is important to evaluate disease susceptibility phenotypes. Over the years, Arabidopsis thaliana has become the model species to serve as a host in plant-pathogen interactions. Despite the efforts to study genetic mechanisms of host defense, little efforts are made for a thorough pathogen assessment, often still depending on symptomology. This manuscript describes the use of real-time polymerase chain reaction (PCR) to assess pathogen growth in the host Arabidopsis for a number of frequently studied pathogens. A wide range of correlations between pathogen biomass and fluorescence is demonstrated, demonstrating the theoretical sensitivity of the technique. It is also demonstrated that host DNA does not interfere with the quantification of pathogen DNA over a wide range. Finally, quantification of pathogen biomass in different plant genotypes with a varying degree of resistance shows the capability of this technique to be used for assessment of pathogen development in disease progression.

Alternaria↗
PubMed2000

Tandem mass spectrometric quantification of 8-iso-prostaglandin F2alpha and its metabolite 2,3-dinor-5,6-dihydro-8-iso-prostaglandin F2alpha in human urine.

Whole body synthesis of F2-isoprostanes, a family of cyclooxygenase-independent eicosanoids formed by free-radical catalysed peroxidation, should be best assessed by quantifying their urinary metabolites. Two methods for the quantitative determination of F2-isoprostane metabolites in human urine performing either thin-layer chromatography (TLC) (method A) or high-performance liquid chromatography (HPLC) (method B) prior to GC-tandem MS are described. Method A allows for simultaneous quantification of 8-iso-PGF2alpha, one prominent member of the F2-isoprostane family, and its major urinary metabolite, 2,3-dinor-5,6-dihydro-8-iso-PGF2alpha. Mean excretion was found to be 223 and 506 pg/mg creatinine of 8-iso-PGF2alpha and 2,3-dinor-5,6-dihydro-8-iso-PGF2alpha, respectively (n=14). A tight correlation existed between the urinary excretion of these two isoprostanes (r=0.86). Method B enables quantification of dinor-dihydro metabolites of various F2-isoprostanes including 8-iso-PGF2alpha. 2,3-Dinor-5,6-dihydro-8-iso-PGF2alpha was found to be an abundant dinor-dihydro F2-isoprostane metabolite. Validity of method A was proven by a combination of HPLC with TLC prior to GC-tandem MS analysis. A correlation was observed between the urinary concentrations of 2,3-dinor-5,6-dihydro-8-iso-PGF2alpha measured by GC-MS and GC-tandem MS (r=0.84).

Adult↗
PubMed2000

Quantification of free mycophenolic acid by high-performance liquid chromatography-atmospheric pressure chemical ionisation tandem mass spectrometry.

To facilitate the investigation of free mycophenolic acid concentrations we developed a high-performance liquid chromatography tandem mass spectrometry method using indomethacin as an internal standard. Free drug was isolated from plasma samples (500 microl) using ultrafiltration. The analytes were extracted from the ultrafiltrate (200 microl) using C18 solid-phase extraction. Detection was by selected reactant monitoring of mycophenolic acid (m/z 318.9-->190.9) and the internal standard (m/z 356.0-->297.1) with an atmospheric pressure chemical ionisation interface. The total chromatographic analysis time was 12 min. The method was found to be linear over the range investigated, 2.5-200 microg/l (r>0.990, n=6). The relative recovery of the method for the control samples studied (7.5, 40.0 and 150 microg/l) ranged from 95 to 104%. The imprecision of the method, expressed in terms of intra- and inter-day coefficients of variation, was <8 and <9%, respectively. Further, analysis of pooled patient plasma produced an intra-day imprecision of 6.6%. The signal-to-noise ratio at the limit of quantification (2.5 microg/l) was approximately 5:1. The mean absolute recovery (n=6) of mycophenolic acid and the internal standard were 76.0+/-13.5% and 86.0+/-9.1%, respectively. The method reported provides an accurate and precise quantification of free mycophenolic acid over a wide analytical range and thus can be used for routine monitoring and pharmacokinetic studies.

Atmospheric Pressure↗
PubMed1988

Screening, confirmation and quantification of boldenone sulfate in equine urine after administration of boldenone undecylenate (Equipoise).

Methods for screening by thin-layer chromatography, quantification by high-performance liquid chromatography with ultraviolet detection and confirmation by gas chromatography-mass spectrometry of boldenone sulfate in equine urine after administration of boldenone undecylenate (Equipoise) are presented. Sample work-up was done with C18 liquid-solid extraction followed by solvolytic cleavage of the sulfate ester. Confirmatory evidence of boldenone sulfate in equine urine was obtained from 2 h to 42 days following a therapeutic intramuscular dose of Equipoise. The use of 19-nortestosterone sulfate as the internal standard for quantification of boldenone sulfate is discussed.

Animals↗
PubMed1988

Improved high-performance liquid chromatographic assay for the quantification of 5-bromo-2'-deoxyuridine and 5-bromouracil in plasma.

A sensitive and specific procedure using high-performance liquid chromatography (HPLC) was developed for the quantification of 5-bromo-2'-deoxyuridine (BUdR) and 5-bromouracil (BU) in plasma. BUdR and BU were first extracted with a mixture of ethyl acetate and 2-propanol from plasma presaturated with solid ammonium sulfate. Following evaporation of the organic extract, the remaining residue was reconstituted in saturated ammonium sulfate solution, washed with a mixture of n-pentane-methylene chloride and re-extracted with the original solvent mixture. The organic extract was evaporated, reconstituted in mobile phase and chromatographed on a regular-bore ODS HPLC column using ultraviolet absorbance detection. The BUdR and BU quantification limits were both 0.1 microM, the mean intra-assay coefficients of variation were 5.0 and 5.6%, respectively, and the mean inter-assay coefficients of variation were 5.4 and 10.7%, respectively. This method was used to determine steady-state femoral arterial and hepatic venous plasma concentrations of BUdR and BU in a patient receiving a continuous intravenous infusion of BUdR (20 mg/kg per day).

Aged↗
PubMed1986

Plasma quantification of quazepam and its 2-oxo and N-desmethyl metabolites by capillary gas chromatography.

The authors have developed a gas chromatographic method for the simultaneous quantification of quazepam in plasma and its two main metabolites, 2-oxoquazepam and N-desmethylquazepam. This method involves an extraction from plasma using butyl acetate, and an analysis by electron-capture detection on a CP-Sil 5 WSCOT capillary column. Intra- and inter-day precision and accuracy were better than 10% for each of these three compounds, even near their detection limit estimated at 0.2 ng/ml. Linearity proved satisfactory between 0.2 and 60-70 ng/ml. For endogenous plasma components, adequate specificity was achieved. Despite some inconveniences, a long analysis time, a progressive saturation of the column owing to a low oven temperature, and a relatively short life-span of the CP-Sil 5 columns, this method was the only one available in the literature for the quantification of quazepam and its metabolites from the same plasma sample. It was successfully applied to phase I studies in healthy volunteers.

Anti-Anxiety Agents↗
PubMed1988

Sensitive quantification of pseudoephedrine in human plasma and urine by high-performance liquid chromatography.

An assay for the selective quantification of pseudoephedrine in human plasma and urine was developed using high-performance liquid chromatography with UV detection at 205 nm. Analyte and internal standard were extracted from alkaline plasma or urine into a mixture of n-hexane and diethyl ether, and the organic phase was back-extracted into dilute acid. The chromatographic system comprises microparticulate cyanopropyl-silica as stationary phase and a ternary solvent mixture with ion-pair reagents as mobile phase. Using 0.25 ml plasma, the lower limit of quantification was 25 ng/ml with excellent linearity up to 1000 ng/ml. In urine, the calibration ranged from 2.5 to 100 micrograms/ml. The selectivity of the method was demonstrated for several pharmaceuticals with similar structures. The validated method was applied to a pharmacokinetic study with a single oral dose of 100 mg of pseudoephedrine in two galenic formulations. Precision and accuracy data of the assay and calculated pharmacokinetic parameters are presented.

Adolescent↗
PubMed1989

Capillary gas chromatographic-mass spectrometric method for the identification and quantification of some benzodiazepines and their unconjugated metabolites in plasma.

A gas chromatographic-mass spectrometric method for the identification and/or quantification of diazepam, clobazam, flunitrazepam, triazolam, midazolam, oxazepam and lorazepam and some of their desmethylated and hydroxylated metabolites in plasma is described. Benzodiazepines were extracted from plasma with butyl acetate at pH 9; the hydroxylated compounds were then silylated with N,O-bis (trimethylsilyltrifluoroacetamide). Analysis was performed using a compact mass-selective detector operating in the electron-impact mode. Depending on the concentration, identification was performed either by direct comparison of the observed mass spectra with reference spectra or by the relative intensities of the most intense and characteristic ions in the selected-ion monitoring (SIM) mode. Quantification was performed in the SIM mode using the most intense ion. The intra-assay precision and accuracy were better than 5-6%; linearity was satisfactory up to 1-2 micrograms/ml. The detection limit was 1-5 ng/ml for most of the benzodiazepines. This method can be easily used in clinical situations when a safe and rapid response is essential for patient treatment.

Benzodiazepines↗
PubMed2001

Quantification of mycophenolate mofetil in human skin extracts using high-performance liquid chromatography-electrospray mass spectrometry.

A sensitive and selective method for the quantification of mycophenolate mofetil and its active metabolite mycophenolic acid in different human skin layers after dermal administration is presented. The skin layers were separated after in vitro penetration experiments and a methanolic extraction was performed. Positive ion electrospray HPLC-MS in selected ion monitoring mode was used to quantify the substances after isocratic separation by a C18 analytical column. The minimum detectable concentrations were 850 pg/ml for MMF and 1 ng/ml for MPA. The peak areas depended linearly on the concentration of both drugs over the range of 25-1,000 ng/ml (r2 > or = 0.996) with accuracy < or =9.8% and precision < or = 13.2%. Total imprecision at quantification limits was 15.2% at 10 ng/ml and 16.3% at 1,500 ng/ml for MMF and 15.1% at 21.0 ng/ml and 17.5% at 1,300 ng/ml for MPA. This HPLC-MS method will be applicable to the profiling of MMF amounts in skin and its conversion to MPA after application of different formulations.

Chromatography, High Pressure Liquid↗
PubMed2002

Quantification and purity determination of newly synthesized thioacridines by capillary liquid chromatography.

Capillary liquid chromatography (CLC) was applied for quantification and impurity profile determination of ten newly synthesized acridine thioderivatives. A reversed-phase CLC system employing two different stationary phases, Nucleosil C18 and LiChrosorb RP-select B, was used. The mobile phase composition was optimized to get a satisfactory separation of impurities from the main acridine component in a reasonable analysis time. Significant differences in the chromatographic behavior between acridine derivatives containing and lacking amino groups were observed. Optimized separation conditions were used in CLC to measure the calibration curves of the acridine derivatives in a concentration range from 1.0 x 10(-6) to 1.0 x 10(-3) M at two different detector wavelengths (214 and 230 nm). Limits of detection and quantification of all the substances were determined. The detection limits went down to units of microM for most of the derivatives. CLC was also demonstrated to be a suitable method for the purity determination of test batches of the acridine thioderivatives.

Acridines↗
PubMed1997

High-performance liquid chromatographic separation of kyotorphin, a basic Tyr-Arg dipeptide, in rat brain tissue and quantification using fluorimetric detection.

Two HPLC procedures based on sample derivatization at the N-terminal Tyr moiety with agents yielding fluorescent derivatives were applied to the selective and sensitive detection as well as quantification of the basic kyotorphin, Tyr-Arg dipeptide, in rat brain tissue. The first one is a post-column fluorescence derivatization method, whereby the peptides extracted from the brain tissue are separated on an octadecylsilyl-silica gel column, followed by on-line fluorescence derivatization for detection. The other one is a pre-column derivatization method, where the extracted peptides are first reacted with fluorogenic agents at the N-terminal Tyr moiety to their corresponding fluorescent derivatives, subsequently separated on an octadecyl-poly(vinyl alcohol) copolymer gel column, and signal responses are measured fluorimetrically. Both methods permitted the quantification of the synthetic kyotorphin added to the rat brain tissues. The concentration range of kyotorphin-like biogenic peptide was 60-100 pmol/g in the cortex, striatum and hypothalamus tissues.

Animals↗
PubMed1997

Quantification of corticosteroids in human plasma by liquid chromatography-thermospray mass spectrometry using stable isotope dilution.

Liquid chromatography-thermospray mass spectrometry (LC-TSP-MS) using isotope dilution was investigated for quantitative analysis of cortisol, cortisone, prednisolone and prednisone in human plasma. Complete separation attained by a LiChroCART Supersupher reversed-phase column and elution with 0.05 M ammonium formate-tetrahydrofuran-methanol (180:53:17, v/v/v) resulted in a significantly large isotope effect of the deuterium-labeled analogs on the HPLC behavior and caused difficulty in quantification. Reduction of the isotope effect on the retention times using 0.05 M ammonium formate-acetonitrile (65:35, v/v) permitted accurate quantification of cortisol and cortisone by the isotope dilution LC-TSP-MS, although separation between cortisol and prednisone was incomplete.

Adrenal Cortex Hormones↗
PubMed1997

Isolation of N,N-dialkylated derivatives of valproylglycinamide from dog plasma by active charcoal adsorption and their quantification by high-performance liquid chromatography.

A selective assay for quantification of N,N-dimethylvalproylglycinamide (DM-VGD) and N,N-diethylvalproylglycinamide (DE-VGD) in dog plasma utilizing reversed-phase high-performance liquid chromatography and UV detection has been developed. These compounds are derivatives of the potential anticonvulsant drug, valproylglycinamide, which is currently undergoing clinical trials. The method is based on extraction of dog plasma with activated charcoal, separation of the charcoal pellet and extracting it with methanol, evaporation of the solvent and injecting the reconstituted residue onto the column. The active charcoal adsorption method is reliable and reproducible, and it provides a chromatogram free of interfering endogenous plasma compounds. The assay was validated and provided a limit of quantification of 2.3 mmol/l for DE-VGD and 5.3 mmol/l for DM-VGD. Mean recovery of these compounds from plasma averages 75%. This analytical method is suitable for the quantitative determination of DM-VGD and DE-VGD in plasma and it has been applied to a pharmacokinetic study of these compounds in a dog.

Animals↗
PubMed1997

Sensitive and rapid method for the simultaneous quantification of five antidepressants with their respective metabolites in plasma using high-performance liquid chromatography with diode-array detection.

A high-performance liquid chromatographic method with diode array detection (HPLC-DAD) has been developed for the simultaneous separation and quantification of imipramine, amitriptyline, maprotiline, fluoxetine, clomipramine and their respective metabolites using a 500-microl plasma sample and clovoxamine as the internal standard. The substances were eluted on a Symmetry C18 5-microm column (250x4.6 mm, I.D.). Full UV spectra from 200 to 450 nm were recorded on-line during the entire analysis and were automatically compared to spectra stored in a library. The quantification was performed at 226, 254, and 400 nm. Peak height ratios were linear over a concentration range of 10-3000 ng/ml: the correlation coefficient (r) was better than 0.998 for all substances at each wavelength. Acceptable coefficients of variation are demonstrated for both within-run and day-to-day assays. The method is simple, highly specific and currently being used for drug monitoring and toxicological studies in children and adult patients.

Amitriptyline↗