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PubMed2006

Simultaneous quantification of fexofenadine and pseudoephedrine in human plasma by liquid chromatography/tandem mass spectrometry with electrospray ionization: method development, validation and application to a clinical study.

To support the pharmacokinetic and bioavailability study of a once-daily fexofenadine/pseudoephedrine combination, a high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry (HPLC/ESI-MS/MS) method for the simultaneous quantification of fexofenadine and pseudoephedrine was developed and validated with 500 microL human plasma using mosapride as an internal standard (IS). Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 502/466 for fexofenadine, m/z 166/148 for pseuoephedrine and m/z 422/198 for the IS. The method exhibited linear dynamic ranges of 1-500 ng/mL and 2-1000 ng/mL for fexofenadine and pseudoephedrine, respectively, in human plasma. The lower limits of quantification were 1 and 2 ng/mL with a relative standard deviation of less than 10% for fexofenadine and pseudoephedrine, respectively. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The total chromatographic run time was 2 min and more than 400 human plasma samples could be analyzed in one day by running the system overnight. The method is precise and sensitive enough for its intended purpose.

Blood Chemical Analysis↗
PubMed2006

Quantification of trandolapril and its metabolite trandolaprilat in human plasma by liquid chromatography/tandem mass spectrometry using solid-phase extraction.

A sensitive high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (MS/MS) method was developed and validated for the simultaneous quantification of trandolapril and its metabolite trandolaprilat in human plasma using ramipril as an internal standard. Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M-H]- ions, m/z 429/168 for trandolapril, m/z 401/168 for trandolaprilat and m/z 415/166 for the internal standard. The method exhibited a linear dynamic range of 20-10,000 pg/mL for both trandolapril and trandolaprilat in human plasma. The lower limit of quantification was 20 pg/mL for both trandolapril and its metabolite. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. 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.

Antihypertensive Agents↗
PubMedDate not supplied

Error rates in dental microwear quantification using scanning electron microscopy.

There is a degree of correlation between dietary habits and dental microwear in extant primates, and this has enabled inferences to be made about prehistoric diets. Several techniques have been used to quantify microwear, but the comparability of results derived from each has not been demonstrated. Moreover, neither intra- nor interobserver error rates in microwear quantification have been documented to date. We here assess intra- and interobserver error using Microware 4.0, and evaluate intertechnique comparability using the three methods that have been most widely employed in the field. This study documents an overall intraobserver error rate of about 7%, and an overall interobserver error rate of some 9%. Both intra- and interobserver error appears to be influenced substantially by the nature of the micrograph being measured. In no instance did the results obtained by different observers using Microware 4.0 differ significantly, and there was a reasonable degree of interobserver consistency in the rank ordering of micrographs in relation to any given parameter. The results obtained through the use of different quantitative techniques differed significantly, with an overall intertechnique error rate of approximately 19%. Several variables, including differences in magnification factor, scanning electron microscope kV settings, and specimen-detector relationships undoubtedly contribute to the differences among the three methods, but we were not able to assess their relative importance. Microwear quantification permits distinctions between broad dietary categories, but the margin of intra- and interobserver error should be taken into account when defining pattern differences between populations (or species) or when documenting seasonally mitigated differences within a taxon. In view of the error introduced by the use of different methods, we suggest that a consistent technique, such as offered by the Microware software package, be adopted by current researchers to establish a common microwear database.

Animals↗
PubMed2005

Simplified quantification of cerebral A1 adenosine receptors using [18F]CPFPX and PET: analyses based on venous blood sampling.

[(18)F]CPFPX was proposed as a novel ligand for in vivo quantification of cerebral A(1) adenosine receptors (A(1)AR) using positron emission tomography (PET). The present study investigates the applicability of non-invasive non-compartment analyses to quantify the [(18)F]CPFPX total distribution volume (DV(') (t)) without arterial blood sampling. Five healthy volunteers underwent dynamic PET (90 min) with arterial and venous blood sampling after [(18)F]CPFPX bolus injection. Area under curve (AUC) analysis and Logan's graphical analysis (GA) were performed employing venous blood samples as non-invasive analyses. AUC analysis without data extrapolation (AUC(r)) and venous GA were also applied on voxel-level to generate parametric images. A conventional two-tissue compartment model (2TCM) using arterial blood samples served as reference method. Regional DV(') (t) estimates provided by venous AUC and GA analyses and 2TCM demonstrated very high agreement (venous AUC vs. 2TCM: r(2) = 0.968, linear regression slope = 0.943; venous GA vs. 2TCM: r(2) = 0.972, slope = 0.906). Although on voxel-level the venous AUC(r) and GA analyses were affected by a slight negative bias, they were still very highly correlated with the 2TCM (voxel-wise venous AUC(r) vs. 2TCM: r(2) = 0.969, slope = 0.858; voxel-wise venous GA vs. 2TCM: r(2) = 0.959, slope = 0.867). The proposed non-invasive analysis methods (particularly venous GA) allow reliable quantification of human [(18)F]CPFPX PET studies. In populations with altered metabolic/kinetic properties, the applicability of venous sampling has to be separately verified. The applicability of [(18)F]CPFPX PET in basic and clinical neurosciences will be considerably enhanced by the avoidance of arterial blood sampling.

Adult↗
PubMed1994

Quantification and characterization of glycosaminoglycans at the nanogram level by a combined azure A-silver staining in agarose gels.

A rapid, sensitive, and nonradioactive method has been developed for the quantification and characterization of glycosaminoglycans. The method is based on the separation of different types of glycosaminoglycans in agarose gel and subsequent fixation and staining with the cationic dye azure A, followed by silver enhancement. Densitometric analysis of the silver deposition gives a linear response between 1 and 20 ng for chondroitin and dermatan sulfate and between 2 and 40 ng for heparan sulfate. The detection limit is about 250 pg. The staining procedure was applied for the quantification and characterization of glycosaminoglycans in human serum, urine, and lung and in mouse kidney glomeruli. It requires only 10 microliters serum, 2 microliters urine, and only a single cryosection in case of tissue. The method is at least as sensitive as staining with radioactive markers and about 200 times more sensitive than conventional glycosaminoglycan-staining methods.

Animals↗
PubMed1994

An improved method for absolute quantification of mRNA using multiplex polymerase chain reaction: determination of renin and angiotensinogen mRNA levels in various tissues.

We have developed a multiplex, competitive, reverse-transcriptase polymerase chain reaction (RT-PCR) method which measures absolute levels of renin, angiotensinogen, and the housekeeping transcript elongation factor-1 alpha (EF-1 alpha) mRNA. Sample RNA was simultaneously titrated with serial dilutions of renin, angiotensinogen, and EF-1 alpha competitor RNAs which flanked the endogenous concentrations of target transcripts. The samples were coreverse transcribed in the presence of random primers and resulting first-strand cDNA was coamplified for 10-15 cycles with [32P]-dCTP and primers for renin angiotensinogen, after which EF-1 alpha primers were added. Amplified DNA was separated by electrophoresis on polyacrylamide gel and radioactivity in the bands was quantified by direct radioanalytical scanning. Three conditions were necessary to obtain absolute quantification of renin and angiotensinogen mRNA levels: (a) exogenous competitor RNA was used to control for tube-to-tube variability in the efficiencies of reverse transcription and amplification; (b) Sample RNA was titrated with flanking concentrations of competitor RNA to correct for intraassay differences in the efficiency of amplification due to concentration differences between competitor and target templates; and (c) a housekeeping transcript EF-1 alpha was used to control for tube-to-tube differences in RNA loading and/or degradation. We show that the multiplex RT-PCR method is precise and accurate over approximately three logs of transcript concentration and sensitive to less than 5 and 0.5 fg for renin and angiotensinogen mRNA, respectively. This method will be useful for absolute quantification of target mRNAs, especially when the amount of sample RNA is limited or unknown and/or the gene expression is low.

Angiotensinogen↗
PubMed1996

Quantification of individual oligosaccharide compounds from human milk using high-pH anion-exchange chromatography.

A quantification method suitable for determination of individual oligosaccharide compounds from human milk has been established. The crude milk oligosaccharide fraction was separated into acidic oligosaccharides, neutral oligosaccharides, and lactose by gel permeation chromatography. After this separation step neutral and acidic oligosaccharides were analyzed separately by high-pH anion-exchange chromatography with pulsed amperometric detection. The concentrations of 14 neutral oligosaccharides, of 6 acidic oligosaccharides, and of N-acetylneuraminic acid were determined on the internal standards stachyose and galacturonic acid, respectively. Thus, previously applied quantification methods for milk oligosaccharides based on gel permeation chromatography have been decisively improved.

Chromatography, Gel↗
PubMed1997

Trace quantification of the oxidative damage products, meta- and ortho-tyrosine, in biological samples by gas chromatography-electron capture negative ionization mass spectrometry.

Oxygen radicals damage biomolecules and may contribute to cellular aging and degenerative disease. We describe a sensitive method for the quantification of two endogenous biomarkers of oxidative damage: meta-tyrosine (m-Tyr) and ortho-tyrosine (o-Tyr). The assay can be applied to direct analysis of free amino acids or protein-bound amino acids following hydrolysis. The assay involves derivatization with pentafluorobenzyl bromide and extraction into n-decane, followed by gas chromatography-mass spectrometry. Stable isotope labeled m- and o-Tyr (2H4) and phenylalanine [i.e., Phe (2H5)] were added as internal standards to improve analytical accuracy. Quantification of as little as 50 pg of m- and o-Tyr in 100 micrograms protein is possible and the data are expressed as a molar ratio of m- and o-Tyr to native Phe. The assay was used to determine the levels of m- and o-Tyr in freshly isolated human plasma protein (4.05 +/- 0.67 m-Tyr per 10(4) Phe, 0.35 +/- 0.07 o-Tyr per 10(4) Phe). Exposure of human plasma to reactive oxygen species significantly increased the levels of m-Tyr (56.4 +/- 1.1 m-Tyr per 10(4) Phe, P < 0.0001) and o-Tyr (48.9 +/- 1.3 o-Tyr per 10(4) Phe, P < 0.0001). The mild hydrolysis and derivatization conditions caused no artifactual formation of either m- or o-Tyr.

Chemical Phenomena↗
PubMed1998

Extraction and quantification of daidzein and genistein in food.

A simple analytical method has been developed for routine quantification of a broad range of concentrations of the isoflavones daidzein and genistein in food. The synthetic glucosides daidzin and genistin were used as internal standards, combined with each food prior to extraction. The recovery of the aglycones daidzein and genistein from these internal standards were used to ensure the completeness of the extraction and aid quantification of isoflavones from the food. Hydrolytic enzymes from Aspergillus niger were used, in aqueous buffer, to liberate daidzein and genistein from their respective glycosides. The aglycone isoflavones were partitioned from the aqueous buffer into ethyl acetate. After evaporation of the ethyl acetate under nitrogen, the isoflavones were derivatized with N-tert-(butyldimethylsilyl)-N-methyltrifluoroacetamide and quantified by comparison with authentic synthetic standards using gas chromatography-mass spectrometry in selected ion mode. The isoflavone content of a stock soy flour was determined, using 36 separate assays, to be 1.05 mg daidzein and 1.11 mg genistein per gram of freeze-dried food, and the interassay coefficient of variation was 2.7 and 4.7, respectively.

Food Analysis↗
PubMed1999

Real-time detection and quantification of adenosine triphosphate sulfurylase activity by a bioluminometric approach.

A real-time, sensitive, and simple assay for detection and quantification of adenosine triphosphate sulfurylase (ATP:sulfate adenylytransferase, EC 2.7.7.4) activity has been developed. The method is based on detection of ATP generated in the ATP sulfurylase reaction between APS and PPi by the firefly luciferase system. For the Saccharomyces cerevisiae ATP sulfurylase, the concentrations of APS and PPi at the half-maximal rate were found to be about 0.5 and 7 microM, respectively. The assay is sensitive and yields linear response between 0.1 microU and 50 mU. The method can be used for monitoring and quantification of recombinant ATP sulfurylase activity in Escherichia coli lysate, as well as for detection of the activity during different purification procedures.

Adenosine Phosphosulfate↗
PubMed1999

Quantification of N-(glucitol)ethanolamine and N-(carboxymethyl)serine: two products of nonenzymatic modification of aminophospholipids formed in vivo.

Chemical, nonenzymatic modification of protein and lipids by reducing sugars, such as glucose, is thought to contribute to age-related deterioration in tissue protein and cellular membranes and to the pathogenesis of diabetic complications. This report describes the synthesis and quantification of N-(glucitol)ethanolamine (GE) and N-(carboxymethyl)serine (CMS), two products of nonenzymatic modification of aminophospholipids. GE is the product of reduction and hydrolysis of glycated phosphatidylethanolamine (PE), while CMS is formed through reaction of phosphatidylserine (PS) with products of oxidation of either carbohydrate (glycoxidation) or lipids (lipoxidation). Gas chromatography/mass spectrometry procedures for quantification of the N,O-acetyl methyl ester derivatives of the modified head groups were developed. GE and CMS were quantified in samples of PE and PS, respectively, following incubation with glucose in vitro; CMS formation was dependent on the presence of oxygen during the incubation. Both GE and CMS were detected and quantified in lipid extracts of human red blood cell membranes. The content of GE, but not CMS, was increased in the lipids from diabetic compared to nondiabetic subjects. Measurement of these modified lipids should prove useful for assessing the role of carbonyl-amine reactions of aminophospholipids in aging and age-related diseases.

Aging↗
PubMed1999

Quantification of lipids, liposomes, amino acids, and proteins by thermal ultramicrodigestion and an ultramicrocoulometric assay, based on the reaction of hypobromite with ammonia.

The quantification of nitrogen in organic material is described. It is based on a novel thermal ultramicrodigestion in combination with an ultramicrocoulometric quantification. The lower detection limit of the coulometric measurement is 0.5 microg nitrogen, which corresponds to 20 microg lipid, 3 microg glycine, or 4 microg protein. Therefore it is as sensitive as the frequently used Lowry method. In contrast to the Lowry protein determination it is not disturbed by detergents and most other interfering substances.

Amino Acids↗
PubMed1999

32P-postlabeling assay for the quantification of the major platinum-DNA adducts.

To allow more sensitive, selective, and routine analyses of platinum(Pt)-GG and -AG intrastrand cross-links we have significantly improved our quantitative (32)P-postlabeling assay (M. J. P. Welters et al. Carcinogenesis 18, 1767-1774, 1997). Instead of off-line scintillation counting we introduced an on-line flow radioisotope detector into the HPLC system. Furthermore, the isolation protocol for the adducts was significantly modified and optimized to reduce interfering background peaks that prevented quantification of low levels of the cisplatin-DNA adducts in white blood cells obtained from patients. Reduction of background signals was obtained by boiling the samples, followed by phenol/chloroform/isoamylethanol extraction after the DNA digestion step. The labeling efficiency for the adducts was increased by 40% by using Na-formate instead of NH(4)-formate for elution of the adducts from the strong cation-exchange columns. Finally, a calibration curve and quality controls were implemented. The labeling efficiencies were not different between the dinucleotides. The between- and within-run precision for the Pt-GG and Pt-AG adducts measured at the lower limit of quantification of 87 and 53 amol/microg DNA, respectively, was less than 20% CV. The adducts were stable in DNA stored for a 2-month time period at -80 degrees C. The assay is now routinely used for high-precision analyses of patient and cell line samples containing very low adduct levels.

Animals↗
PubMed2000

Quantification of long-chain aldehydes by gas chromatography coupled to mass spectrometry as a tool for simultaneous measurement of plasmalogens and their aldehydic breakdown products.

The cleavage of the specific vinyl ether linkage at the sn-1 position of plasmalogens leads to the formation of two products: the 1-lyso-2-acyl glycerophospholipid and a long-chain fatty aldehyde. Plasmalogens are measured by quantifying one of these two products. In this paper, we describe a rapid and sensitive procedure for measuring plasmalogens via quantification of long-chain fatty aldehydes. After lipid extraction, the sn-1 vinyl ether bond of plasmalogens is cleaved by acidic hydrolysis. The produced aldehydes are then derivatized with (pentafluorobenzyl)hydroxylamine hydrochloride and analyzed by gas chromatography/mass spectrometry in selected-ion mode. Plasmalogens are then indirectly quantified by subtracting the free aldehydes obtained without prior HCl treatment from the total aldehydes obtained after acidic hydrolysis. This method is applied to three rat brain areas selected for this study. Two of these are affected in neurodegenerative diseases (cerebral cortex and hippocampus) and one is rich in white matter (cerebellum). In comparison to other procedures, the advantages of this method are not only its usefulness in plasmalogen quantification but also the identification of aldehydic breakdown products.

Aldehydes↗
PubMed2000

Photometric method for the quantification of chlorophylls and their derivatives in complex mixtures: fitting with Gauss-peak spectra.

Accurate quantification of pigments in mixtures is essential in all cases in which separation of pigments by chromatography is impracticable for one reason or another. An example is the analysis of in vivo formation of heavy metal-substituted chlorophylls in heavy metal-stressed plants. We describe here a novel, accurate UV/VIS spectrophotometric method for the quantification of individual chlorophyll derivatives in complex mixtures, which has the potential for universal applicability for mixtures difficult to separate. The method is based on the description of each pigment spectrum by a series of Gaussian peaks. A sample spectrum is then fitted by a linear combination of these "Gauss-peak spectra" including an automatic correction of wavelength inaccuracy and baseline instability of the spectrometer as well as a correction of the widening of absorbance peaks in more concentrated pigment solutions. The automatic correction of peak shifts can also partially correct shifts caused by processes like allomerization. In this paper, we present the Gauss-peak spectra for Mg-chlorophyll a, b, c, pheophytin a, b, c, Cu-chlorophyll a, b, c, and Zn-chlorophyll a in acetone; Mg-chlorophyll a, b, pheophytin a, b, Cu-chlorophyll a, b, allomerized Cu-chlorophyll a, b, and Zn-chlorophyll a, b in cyclohexane; Mg-chlorophyll a, b, pheophytin a, b, and Cu-chlorophyll a, b in diethyl ether.

Chemistry Techniques, Analytical↗
PubMed2001

In-gel detection of urease with nitroblue tetrazolium and quantification of the enzyme from different crop plants using the indophenol reaction.

Two methods for measurement of urease activity are described and demonstrated on extracts from several crop plants. With an in-gel staining method based on the principle of Fishbein's noninhibitory stain for urease as little as 25 microU of jackbean urease can be detected within 2 h following electrophoresis. A comparison with published in-gel staining methods shows that the sensitivity is improved by at least two orders of magnitude. The second method allows quantification of urease activity from small amounts of plant material without the need for special laboratory equipment. It employs the detection of ammonium by the indophenol reaction. To eliminate reducing agents which are often necessary to maintain urease activity during extraction, but which interfere with ammonium detection, a simple spin-column procedure is used. The quantification of less than 5 mU/ml extract is possible.

Electrophoresis↗
PubMed2001

Zymography with caseogram prints: quantification of pepsinogen.

In caseogram prints, a type of zymogram which is designed for the detection of acid proteases, enzyme activity is detected in an overlay gel of agarose containing skim milk. The use of this technique for protease quantification was investigated in this study using pepsinogen as an example protease. The area of caseogram bands was found to be logarithmically related to protease activity whereas the intensity of the bands was no reliable measure for activity. A reproducible quantification procedure was described. Accuracy and variation were acceptable over a 128-fold range whose lower border was the detection limit (35 pg pepsinogen).

Chemistry Techniques, Analytical↗
PubMed2001

Quantification of Escherichia coli genomic DNA contamination in recombinant protein preparations by polymerase chain reaction and affinity-based collection.

This study describes the development of a novel assay for the quantification of Escherichia coli genomic DNA contamination in recombinant protein samples. The technique is based on PCR amplification and digoxygenin labeling of the genes encoding 5S ribosomal RNA followed by affinity-based collection and detection. Samples containing 1 pg x mL(-1) of extracted E. coli genomic DNA (gDNA) could be measured using this method. Using extracted E. coli gDNA as standards, a 35-cycle PCR reaction exhibited a linear response versus template concentration between 1 pg x mL(-1) and1 ng x mL(-1) genomic DNA even when diluted in a variety of buffering conditions. Comparison of the novel assay with a traditional filter binding and hybridization technique using recombinant protein samples confirmed that the procedure was accurate and sensitive. The assay described in this report is a safer and less expensive alternative to radioactive techniques employed for DNA quantification, utilizing readily available reagents and apparatus.

Base Sequence↗