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PubMed2006

Quantification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl adducts in human lymphoblastoid TK6 cells dosed with N-hydroxy-4-acetylaminobiphenyl and their relationship to mutation, toxicity, and gene expression profiling.

Gene expression profiles that are anchored to phenotypic endpoints may lead to the identification of signatures that predict mutagenicity or carcinogenicity. The study presented here describes the analysis of DNA adducts in the human TK6 lymphoblastoid cell line after exposure to N-hydroxy-4-aminobiphenyl, a mutagenic metabolite of 4-aminobiphenyl. A validated nano-LC microelectrospray mass spectrometry assay is reported for the detection and quantification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP), the principal DNA adduct of 4-aminobiphenyl. Limits of quantification, based on a signal-to-noise ratio of 10:1, are determined to correspond to approximately 27 fg of dG-C8-ABP injected on-column. The assay has been used to measure the steady-state levels of the adduct in the human TK6 lymphoblastoid cell line as a function of dose (0.5, 1.0, and 10.0 microM) and time (2, 6, and 27 h) after exposure to N-hydroxy-4-aminobiphenyl. The levels of dG-C8-ABP adducts in the cells, ranging from 18 to 500 adducts in 10(9) nucleotides, were then correlated to cell toxicity, induced mutation at the TK (thymidine kinase) and HPRT loci, and gene expression profiling through microarray analysis. Cell cultures were evaluated for toxicity by growth curve extrapolation, mutation assays were performed on the HPRT and TK loci, and gene expression profiles were generated by analyses using microarray technology. In the mutation assay analysis, as the toxicant concentration increased, there was an increase in mutation fraction, indicating a direct correlation to metabolite dosing level and mutations occurring at these two loci. Statistical analysis of the gene expression data determined that a total of 2250 genes exhibited statistically significant changes in expression after treatment with N-OH-AABP (P < 0.05). Among the genes identified, 2245 were up-regulated, whereas 5 genes that had functions in cell survival and cell growth and, hence, could be indicators of toxicity, were down-regulated relative to controls. The results demonstrate the value of anchoring gene expression patterns to phenotypic markers, such as DNA adduct levels, toxicity, and mutagenicity.

Aminobiphenyl Compounds↗

PubMed2007

Quantification of diacylglycerol species from cellular extracts by electrospray ionization mass spectrometry using a linear regression algorithm.

Diacylglycerols (DAGs) play significant roles in both intermediate metabolism and signal transduction. These lipid species are second messengers involved in modulating a plethora of cellular processes. Evaluation of DAG species concentrations has been hampered by the lack of a reliable method for molecular species analysis within a complex mixture of cellular lipids. We describe a new method for quantitative analysis of DAG species from complex biological extracts based on positive mode electrospray ionization mass spectrometry without prior derivatization. Quantification is achieved using internal standards and calibration curves constructed by spiking cell extracts with different concentrations of DAG species containing various acyl chain lengths and degrees of unsaturation. The new mass spectral data processing algorithm incorporates a multiple linear regression model including a factor accountable for possible interactions between experimental preparations and the slope of the curve for the standards, allowing the examinations of the effects of sample origin conditions (such as cell types, phenotypes, etc.) and instrument variability on this slope. Internal standards provide a basis for quantification of 28 DAG molecular species detected in RAW 264.7 cells after stimulation of a G-protein coupled receptor with platelet activating factor. This method displays excellent reproducibility over the established range of concentrations with variations of < or =10% and is highly sensitive with a detection limit of 0.1-0.4 pmol/microL depending upon acyl chain composition. We have shown differential effects on various DAGs in response to a ligand which illustrates the importance of examining lipids at the molecular species level rather than as a single homogeneous entity.

Algorithms↗

PubMed2006

In situ flavonoid analysis by FT-Raman spectroscopy: identification, distribution, and quantification of aspalathin in green rooibos (Aspalathus linearis).

FT-Raman spectroscopy was used for the first time for in situ identification of aspalathin and quantification of the dihyrochalcones in dried, green rooibos (Aspalathus linearis). With the support of two-dimensional correlation spectroscopy, characteristic key bands of aspalathin, the main flavonoid and antioxidant occurring in rooibos, were localized and identified in the spectra obtained from various plant samples. Application of Raman mapping revealed the spatial distribution of this valuable dihydrochalcone within the intact dried leaves. Based on the spectral data and reference HPLC values, reliable multivariate calibration models were developed for quantification of aspalathin, nothofagin, and the combined dihydrochalcone contents of dried, green rooibos.

Antioxidants↗

PubMed1997

Quantification of biomolecules by external electrospray ionization Fourier transform mass spectrometry.

Fourier transform mass spectrometry (FTMS) is well-known for its capabilities in structural characterization of molecules. Recent developments in radio frequency excitation, linearized trapping, and accumulation of ions generated from external sources have improved the potential of FTMS for quantitative analysis. Here, a commercial external electrospray ionization FTMS, employing a linearized ion trap (the Infinity Cell) and an ion accumulation procedure in which ions are deflected off-axis and injected into the trap, is evaluated as an analytical method for quantifying amino acids, peptides, and proteins. Linear response over approximately 2-3 orders of magnitude is observed for singly-charged ions with low coefficients of variation (generally < 10%), and the calibration curves generated can be used to quantify structurally similar analytes with < 4% relative error, as shown here for quantification of leucine enkephalin from curves generated by methionine enkephalin. Similar precision is obtained for multiply-charged lysozyme, but over only 1.5 orders of magnitude. Some m/z discrimination is observed as a function of trap accumulation potential for a two-component cytochrome c/lysozyme mixture. The results are promising because they suggest that quantification using liquid chromatography coupled to electrospray FTMS is possible.

Chromatography, Liquid↗

PubMed1999

Identification and quantification of cardiac glycosides in blood and urine samples by HPLC/MS/MS.

Cardiac glycosides (CG) are of forensic importance because of their toxicity and the fact that very limited methods are available for identification of CG in biological samples. In this study, we have developed an identification and quantification method for digoxin, digitoxin, deslanoside, digoxigenin, and digitoxigenin by high-performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS). CG formed abundant [M + NH4]+ ions and much less abundant [M + H]+ ions as observed with electrospray ionization (ESI) source and ammonium formate buffer. Under mild conditions for collision-induced dissociation (CID), each [M + NH4]+ ion fragmented to produce a dominant daughter ion, which was essential to the sensitive method of selected reaction monitoring (SRM) quantification of CG achieved in this study. SRM was compared with selected ion monitoring (SIM) regarding the effects of sample matrixes on the methodology. SRM produced lower detection limits with biological samples than SIM, while both methods produced equal detection limits with CG standards. On the basis of the HPLC/MS/MS results for CG, we have proposed some generalized points for conducting sensitive SRM measurements, in view of the property of analytes as well as instrumental conditions such as the type of HPLC/MS interface and CID parameters. Analytes of which the molecular ion can produce one abundant daughter ion with high yield under CID conditions may be sensitively measured by SRM. ESI is the most soft ionization source developed so far and can afford formation of the fragile molecular ions that are necessary for sensitive SRM detection. Mild CID conditions such as low collision energy and low pressure of collision gas favor production of an abundant daughter ion that is essential to sensitive SRM detection. This knowledge may provide some guidelines for conducting sensitive SRM measurements of very low concentrations of drugs or toxicants in biological samples.

Animals↗

PubMed1999

Multicomponent quantification of diastereomeric hexosamine monosaccharides using ion trap tandem mass spectrometry.

A rapid means of stereochemical differentiation and quantification for the hexosamine monosaccharides was achieved using electrospray ionization quadrupole ion trap mass spectrometry. The hexosamine monosaccharides, glucosamine, galactosamine, and mannosamine, were derivatized with [Co(DAP)2Cl2]Cl, and the complex [Co(DAP)2(HexNH2)]Cl was generated. Subjecting this complex to collision-induced dissociation provided a unique product ion spectrum for each of the diastereomeric monosaccharide complexes, thus differentiating the stereoisomers. Furthermore, the stereoisomers were quantified. This was achieved by using the relative abundances of product ions from pure standards and using these values to determine the ratio of isomeric products in a mixture. The utility of this quantification method was demonstrated by successfully determining the composition of two- and three-component mixtures of the hexosamines.

Hexosamines↗

PubMed2026

From Peaks to Power: Systematic Evaluation of Chromatographic Sampling Reveals Determinants of Quantification and Biological Discovery in DIA Proteomics.

Modern DIA proteomics increasingly emphasizes throughput and depth for large-cohort studies, but methods are often optimized using proxy metrics that can mask losses in quantifiable signal and statistical power. Here, we evaluate how data points per peak and other chromatographic features jointly contribute to quantification and downstream biological discovery. Using a matrix-matched calibration curve dataset, we checked how the number of data points per peak (DPPP) affects the limits of detection and quantification (LOD/LOQ). Reduced DPPP minimally affected LOD but substantially degraded LOQ. Feature modeling and nonparametric association analyses identified precursor peak area as the strongest feature-level predictor of LOQ, whereas DPPP showed weaker and context-dependent effects. Simulations of chromatographic peak integration recapitulated these trends, showing that increased sampling primarily improves integration precision, while quantitative accuracy is strongly governed by peak height and peak shape. Finally, when comparing 20 cancer vs 20 control plasma samples processed with Seer Proteograph, the decrease in DPPP led to a loss of statistical significance for proteins with low-abundance precursors. These findings argue that DIA optimization should prioritize LOQ and statistical power metrics─not identifications alone─by balancing sampling density with chromatographic peak height and quality to maximize useful biological signal.

Proteomics↗

PubMed2005

Quantification of toxic Microcystis spp. during the 2003 and 2004 blooms in western Lake Erie using quantitative real-time PCR.

In August of 2003 and August of 2004, blooms of potentially toxic cyanobacteria Microcystis spp. persisted in western Lake Erie. Samples collected from the bloom were analyzed for the cyanobacterial toxin microcystin and the presence of Microcystis spp. cells. Estimates of microcystin toxicity exceeding 1 microg L(-1) (microcystin-LR activity equivalents), the safety limit set by the World Health Organization, were found from the samples in both 2003 and 2004. The presence of Microcystis spp. in water samples was confirmed through standard polymerase chain reaction (PCR) using a combination of four primer sets. Quantification of Microcystis was accomplished by a real-time PCR assay utilizing specific primer-Taq-man probe sets targeted on a conserved, Microcystis-specific 16S rDNA fragment and a microcystin toxin synthetase gene mcyD. This approach allowed us to specifically study the distribution and abundance of toxic Microcystis in the lake in contrast to previous studies that have assessed Microcystis populations with less refined methods. On the basis of quantification by quantitative real-time PCR analysis, the total abundance of Microcystis cells in the bloom area varied from 4 x 10(8) to 2 x 10(3) cells L(-1). The results of this study provide novel insight regarding the distribution and abundance of Microcystis spp. in the western basin of Lake Erie, a region plagued in recent years by large-scale (>20 km2) blooms. Our results suggest that the Maumee River and Bay may serve as a source for Microcystis to western and central Lake Erie.

Bacterial Toxins↗

PubMed2005

Quantification of sequential chlorinated ethene degradation by use of a reactive transport model incorporating isotope fractionation.

Compound-specific isotope analysis (CSIA) enables quantification of biodegradation by use of the Rayleigh equation. The Rayleigh equation fails, however, to describe the sequential degradation of chlorinated aliphatic hydrocarbons (CAHs) involving various intermediates that are controlled by simultaneous degradation and production. This paper shows how isotope fractionation during sequential degradation can be simulated in a 1D reactive transport code (PHREEQC-2). 12C and 13C isotopes of each CAH were simulated as separate species, and the ratio of the rate constants of the heavy to light isotope equaled the kinetic isotope fractionation factor for each degradation step. The developed multistep isotope fractionation reactive transport model (IF-RTM) adequately simulated reductive dechlorination of tetrachloroethene (PCE) to ethene in a microcosm experiment. Transport scenarios were performed to evaluate the effect of sorption and of different degradation rate constant ratios among CAH species on the downgradient isotope evolution. The power of the model to quantify degradation is illustrated for situations where mixed sources degrade and for situations where daughter products are removed by oxidative processes. Finally, the model was used to interpret the occurrence of reductive dechlorination at a field site. The developed methodology can easily be incorporated in 3D solute transport models to enable quantification of sequential CAH degradation in the field by CSIA.

Biodegradation, Environmental↗

PubMed2001

Analysis of the effects of heat treatment on gliadin immunochemical quantification using a panel of anti-prolamin antibodies.

Antigen-labeled capture enzyme-linked immunosorbent assay with four different anti-gliadin monoclonal antibodies and an anti-gliadin serum and two different sample systems (purified gliadin fractions heat-treated in soluble phase and a model of dough simulating a baking process) were employed to study the effects of heat treatment on gliadin quantification. The analysis of purified gliadins showed that there is no particularly heat stable fraction. Remarkably, omega-gliadin did not present a differential heat stability. Reactivity varied depending on the time-temperature conditions of the treatment, the antibody employed, and the fraction analyzed. Heated dough samples showed an impairment of protein extraction depending on the intensity of the treatment. Capillary electrophoresis analysis of extracts showed that each gliadin group is affected to a different extent; omega-gliadin is less modified. Immunochemical analysis of the heat-treated samples using either of the five antibodies showed a decrease in the quantified gliadin, in concordance to the loss in the extracted proteins. Among the different sources of error in gliadin immunochemical quantification, the impairment in extraction efficiency in heat-treated samples appears as a major drawback to be overcome.

Antibodies, Monoclonal↗

PubMed2002

Application of FTIR spectroscopy for the quantification of sugars in mango juice as a function of ripening.

FTIR-ATR spectroscopy and multivariate analysis were used for quantification of sugars in mango juices as a function of ripening. Calibration was based on sucrose/glucose/fructose mixtures, with six concentration levels and following a triangular experimental design. PLS1 regression of the spectra first derivatives gave the best results, enabling quantification of fructose, sucrose, and glucose with 1.4, 1.4, and 4.9% prediction errors, respectively. Throughout ripening, sucrose and fructose were accurately quantified by PLS-FTIR, whereas the accuracy of glucose determination decreased at later stages, when concentrations fell to 0.6-1.5 g/L. These results enabled a correlation with fruit ripening stage to be established. This may be particularly useful to detect over-ripening in fresh fruits, a period when other indicators (pH and % soluble solids (SS)) do not change significantly; this knowledge may help in predicting fruit stability to transport and storage. Similar information obtained for nonfresh juices (in which pH and %SS may be masked by additives), may help determine whether the source fruits had suitable ripening stages.

Beverages↗

PubMed2003

Effect of sample preparation, length of time, and sample size on quantification of total lipids from bovine liver.

The objective was to evaluate the effect of sample preparation (pulverization under liquid nitrogen, homogenization, or sonication), time length of sonication (0-60 s), shaking in chloroform/methanol solvent (0, 2, 4, or 12 h), incubation in chloroform (0 or 12 h), and drying of extracted lipids at 50 degrees C (2, 4, 6, or 24 h), and sample size (50-250 mg) on quantification of total lipids from bovine liver. Pulverization under liquid nitrogen yielded the lowest recovery. Sonication was least time-consuming for sample preparation. Precise estimates and the greatest recovery were obtained with 30 s of sonication, at least 2 h of shaking in chloroform/methanol solvent, 12 h of incubation in chloroform, and at least 6 h of drying. Sample sizes of at least 150 mg gave precise estimates. The results demonstrate that sample preparation, time length of different steps of the extraction procedure, and sample size affect quantification of total lipid from bovine liver.

Animals↗

PubMed2003

Comparison of MALDI-TOF mass spectrometric to enzyme colorimetric quantification of glucose from enzyme-hydrolyzed starch.

Successful quantification of the glucose produced by enzyme hydrolysis of starch was achieved by a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) protocol, using sorbitol as an internal standard. The starch contents measured by MALDI-TOF MS of corn starch, fiber-enriched oat flour derivatives, oat and barley flours, and barley flour/corn starch composites were evaluated in comparison to a widely accepted and validated method of starch determination, which relies on enzyme colorimetry (EC). The average starch content measured in a series of corn starch samples of different masses was 93 and 101% for EC and MALDI-TOF MS, respectively, values that represent the estimated purity of the sample. There was an agreement of 99% between the starch contents determined by the two analytical methods for complex flour-derived samples. Starch values estimated by MALDI-TOF MS consistently showed a greater degree of variability than those determined by EC, but this limitation was readily compensated by rapid acquisition of multiple mass spectra. This study is the first to report the quantification of glucose by MALDI-TOF MS, and it offers new perspectives into the potential utility of MALDI-TOF MS as a definitive tool for monosaccharide analysis and rapid starch determination in complex samples.

Avena↗

PubMed2004

DNA content in embryo and endosperm of maize kernel (Zea mays L.): impact on GMO quantification.

PCR-based techniques are the most widely used methods for the quantification of genetically modified organisms (GMOs) through the determination of the ratio of transgenic DNA to total DNA. It is shown that the DNA content per mass unit is significantly different among 10 maize cultivars. The DNA contents of endosperms, embryos, and teguments of individual kernels from 10 maize cultivars were determined. According to our results, the tegument's DNA ratio reaches at maximum 3.5% of the total kernel's DNA, whereas the endosperm's and the embryo's DNA ratios are nearly equal to 50%. The embryo cells are diploid and made of one paternal and one maternal haploid genome, whereas the endosperm is constituted of triploid cells made of two maternal haploid genomes and one paternal haploid genome. Therefore, it is shown, in this study, that the accuracy of the GMO quantification depends on the reference material used as well as on the category of the transgenic kernels present in the mixture.

DNA, Plant↗

PubMed2004

Detection and quantification of apple adulteration in diluted and sulfited strawberry and raspberry purées using visible and near-infrared spectroscopy.

Adulteration of sulfited strawberry and raspberry purées by apple is a commercial problem. Strawberry (n = 31) and raspberry (n = 30) purées were prepared from Irish-grown fruit and adulterated at levels of 10-75% w/w using cooking apples. Visible and near-infrared transflectance spectra were recorded using a 0.1 mm sample thickness. Classification and quantification models were developed using raw and scatter-corrected and/or derivatized spectral data. Classification as pure strawberry or raspberry was attempted using soft independent modeling of class analogy. The best models used spectral data in the wavelength ranges 400-1098 nm (strawberry) and 750-1098 nm (raspberry) and produced total correct classification rates of 75% (strawberry) and 95% (raspberry). Quantification of apple content was performed using partial least-squares regression. Lowest predictive errors obtained were 11.3% (raspberry) and 9.0% (strawberry). These results were obtained using spectral data in the wavelength ranges 400-1880 and 1100-1880 nm, respectively. These results suggest minimum detection levels of apple in soft fruit purées of approximately 25 and 20% w/w for raspberry and strawberry, respectively.

Food Contamination↗

PubMed2004

Triacylglycerol analysis for the quantification of cocoa butter equivalents (CBE) in chocolate: feasibility study and validation.

A new European legislation (2000/36/CE) has allowed the use of vegetable fats other than cocoa butter (CB) in chocolate up to a maximum value of 5% in the product. The vegetable fats used in chocolate are designated as cocoa butter replacements and are called cocoa butter equivalents (CBE). The feasibility of CBE quantification in chocolate using triacylglycerol (TAG) profiles was conducted by analyzing 55 samples of CBs and 31 samples of CBEs using a liquid chromatograph equipped with an evaporative light scattering detector (HPLC-ELSD). Statistical evaluation of the data obtained has been performed, and a simulation study has been carried out to assess the viability to use this method for quantifying the amount of CBE in real mixtures and in chocolates. The TAGs POP, POS, PLS, and the ratios POP/PLS, POS/PLP (P, palmityl; O, oleyl; S, stearyl; L, linoleyl) are particularly significant to discriminate between CB and CBE. Analysis of 50 mixtures between 5 different CBEs and 10 different CBs at 2 different concentration levels is presented. The data are visualized and interpreted. A mathematical model has been developed to assess the amount of CBE in real mixtures. This predictive model has been successfully applied and validated on dark chocolates including authorized CBE. The results are affected by +/-2.1% absolute average error. In particular, estimations between 10 and 20% of CBE show a very good match. On the other hand, values equal to or smaller than 5% show a larger prediction error (detection limit of the method). For the main purpose of this method (i.e., quantification of CBE at 5% max in chocolate, which represents about 15% of the total fat) this model shows very good results. For milk chocolate, the mathematical model can also be used if TAG are integrated from partition number (PN) 46 to 54. Consequently, the model proposed provides sufficient information to verify the real application of the European legislation.

Cacao↗

PubMed2005

Quantification of saponins in aerial and subterranean tissues of Medicago truncatula.

Triterpene saponins from aerial and subterranean organs of Medicago truncatula cv. Jemalong A-17 were qualitatively profiled and quantified using reverse-phase HPLC with on-line photodiode array detection and electrospray-ionization mass spectrometry (HPLC/PDA/ESI/MS). Absolute quantifications were performed for 3-O-beta-D-glucopyranosyl-medicagenic acid and soyasaponin 1 (3-O-[alpha-L-rhamnopyranosyl(1-->2)-beta-D-galactopyranosyl(1-->2)-beta-D-glucuronopyranoside] soyasapogenol B), whereas relative quantifications were determined for 29 other saponins in root, stem, leaf, seedpod, and seed. Roots contained the greatest total amount of saponins followed by leaf and seed, respectively. The quantitative data also reveal the differential accumulation of triterpene saponins in the various organs of M. truncatula. Specifically, relatively higher quantities of medicagenic acid conjugates accumulated in leaf and seed, whereas relatively higher levels of soysapogenol conjugates were observed in root. The differential accumulation of specific triterpene saponins is suggestive of spatially differentiated biosynthesis and/or biological function.

Chromatography, High Pressure Liquid↗

PubMed2005

Real-time polymerase chain reaction quantification of the transgenes for roundup ready corn and roundup ready soybean in soil samples.

A method for quantification of recombinant DNA for Roundup Ready (RR) corn and RR soybean in soil samples is described. Soil DNA from experimental field samples was extracted using a soil DNA extraction kit with a modified protocol. For the detection and quantification of recombinant DNA of RR corn and RR soybean, a molecular beacon and two pairs of specific primers were designed to differentially target recombinant DNA in these two genetically modified crops. Soil DNA extracts were spiked with RR corn or RR soybean DNA, and recombinant DNA was quantified using real-time PCR with a molecular beacon. As few as one copy of RR corn genome or one copy of RR soybean genome was detected in the soil DNA extract.

DNA, Plant↗