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At least 1,225 records · Page 68Linked to original sources
PubMed2004

Identification and quantification of caffeoylquinic acids and flavonoids from artichoke (Cynara scolymus L.) heads, juice, and pomace by HPLC-DAD-ESI/MS(n).

A method for the identification and quantification of phenolic compounds from artichoke (Cynara scolymus L.) heads, juice, and pomace by HPLC with diode array and mass spectrometric detection was developed. Among the 22 major compounds, 11 caffeoylquinic acids and 8 flavonoids were detected. Quantification of individual compounds was carried out by external calibration. Apigenin 7-O-glucuronide was found to be the major flavonoid in all samples investigated. 1,5-Di-O-caffeoylquinic acid represented the major hydroxycinnamic acid, with 3890 mg/kg in artichoke heads and 3269 mg/kg in the pomace, whereas in the juice 1,3-di-O-caffeoylquinic acid (cynarin) was predominant, due to the isomerization during processing. Total phenolic contents of approximately 12 g/kg on a dry matter basis revealed that artichoke pomace is a promising source of phenolic compounds that might be recovered and used as natural antioxidants or functional food ingredients.

Chromatography, High Pressure Liquid↗
PubMed2005

Identification and quantification of three genetically modified insect resistant cotton lines using conventional and TaqMan real-time polymerase chain reaction methods.

As the genetically modified organisms (GMOs) labeling policies are issued in many countries, qualitative and quantitative polymerase chain reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. Qualitative PCR and TaqMan real-time quantitative PCR methods to detect and identify three varieties of insect resistant cotton, i.e., Mon531 cotton (Monsanto Co.) and GK19 and SGK321 cottons (Chinese Academy of Agricultural Sciences), which were approved for commercialization in China, were developed in this paper. Primer pairs specific to inserted DNAs, such as Cowpea trypsin inhibitor (CpTI) gene of SGK321 cotton and the specific junction DNA sequences containing partial Cry1A(c) gene and NOS terminator of Mon531, GK19, and SGK321 cotton varieties were designed to conduct the identified PCR assays. In conventional specific identified PCR assays, the limit of detection (LOD) was 0.05% for Mon531, GK19, or SGK321 in 100 ng of cotton genomic DNA for one reaction. Also, the multiplex PCR method for screening the three GM cottons was also established, which could save time and cost in practical detection. Furthermore, a real-time quantitative PCR assay based on TaqMan chemistry for detection of insect resistant gene, Cry1A(c), was developed. This assay also featured the use of a standard plasmid as a reference molecule, which contained both a specific region of the transgene Cry1A(c) and an endogenous stearoyl-acyl carrier protein desaturase (Sad1) gene of the cotton. In quantitative PCR assay, the quantification range was from 0.01 to 100% in 100 ng of the genome DNA template, and in the detection of 1.0, 3.0, and 5.0% levels of three insect resistant cotton lines, respectively, all of the relative standard deviations (RSDs) were less than 8.2% except for the GM cotton samples with 1.0% Mon531 or GK19, which meant that our real-time PCR assays involving the use of reference molecule were reliable and practical for GM insect resistant cottons quantification. All of these results indicated that our established conventional and TaqMan real-time PCR assays were applicable to detect the three insect resistant cottons qualitatively and quantitatively.

Animals↗
PubMed2005

Characterization and quantification of phenolic compounds in olive oils by solid-phase extraction, HPLC-DAD, and HPLC-MS/MS.

A simple and reproducible method for qualitative and quantitative analysis of phenolic compounds in virgin olive oils by solid-phase extraction (SPE), high performance liquid chromatography with diode array detector (HPLC-DAD), and HPLC-mass spectrometry (MS) in tandem mode was developed. The polar fraction was obtained from samples of three different virgin olive oils. Detection and quantification were performed at 280, 240, and 320 nm. For identification purposes, HPLC-MS/MS was equipped with turbo ion spray source in the negative-ion mode. Twenty compounds of twenty-three detected and quantified were characterized. The method showed satisfactory linearity (r > 0.99), good recovery, satisfactory precision, and appropriate limits of detection (LOD) and quantification (LOQ).

Benzaldehydes↗
PubMed2005

Distortion of genetically modified organism quantification in processed foods: influence of particle size compositions and heat-induced DNA degradation.

Milling fractions from conventional and transgenic corn were prepared at laboratory scale and used to study the influence of sample composition and heat-induced DNA degradation on the relative quantification of genetically modified organisms (GMO) in food products. Particle size distributions of the obtained fractions (coarse grits, regular grits, meal, and flour) were characterized using a laser diffraction system. The application of two DNA isolation protocols revealed a strong correlation between the degree of comminution of the milling fractions and the DNA yield in the extracts. Mixtures of milling fractions from conventional and transgenic material (1%) were prepared and analyzed via real-time polymerase chain reaction. Accurate quantification of the adjusted GMO content was only possible in mixtures containing conventional and transgenic material in the form of analogous milling fractions, whereas mixtures of fractions exhibiting different particle size distributions delivered significantly over- and underestimated GMO contents depending on their compositions. The process of heat-induced nucleic acid degradation was followed by applying two established quantitative assays showing differences between the lengths of the recombinant and reference target sequences (A, deltal(A) = -25 bp; B, deltal(B) = +16 bp; values related to the amplicon length of the reference gene). Data obtained by the application of method A resulted in underestimated recoveries of GMO contents in the samples of heat-treated products, reflecting the favored degradation of the longer target sequence used for the detection of the transgene. In contrast, data yielded by the application of method B resulted in increasingly overestimated recoveries of GMO contents. The results show how commonly used food technological processes may lead to distortions in the results of quantitative GMO analyses.

DNA, Plant↗
PubMed2006

Application of ethyl esters and d3-methyl esters as internal standards for the gas chromatographic quantification of transesterified fatty acid methyl esters in food.

Ethyl esters (FAEE) and trideuterium-labeled methyl esters (d3-FAME) of fatty acids were prepared and investigated regarding their suitability as internal standards (IS) for the determination of fatty acids as methyl esters (FAME). On CP-Sil 88, ethyl esters of odd-numbered fatty acids eluted approximately 0.5 min after the respective FAME, and only coelutions with minor FAME were observed. Depending on the problem, one or even many FAEE can be added as IS for the quantification of FAME by both GC-FID and GC-MS. By contrast, d3-FAME coeluted with FAME on the polar GC column, and the use of the former as IS requires application of GC-MS. In the SIM mode, m/z 77 and 90 are suggested for d3-methyl esters of saturated fatty acids, whereas m/z 88 and 101 are recommended for ethyl esters of saturated fatty acids. These m/z values give either no or very low response for FAME and can thus be used for the analysis of FAME in food by GC-MS in the SIM mode. Fatty acids in sunflower oil and mozzarella cheese were quantified using five saturated FAEE as IS. Gravimetric studies showed that the transesterification procedure could be carried out without of loss of fatty acids. GC-EI/MS full scan analysis was suitable for the quantitative determination of all unsaturated fatty acids in both food samples, whereas GC-EI/MS in the SIM mode was particularly valuable for quantifying minor fatty acids. The novel GC-EI/MS/SIM method using fatty acid ethyl esters as internal standards can be used to quantify individual fatty acids only, that is, without determination of all fatty acids (the common 100% method), although this is present. This was demonstrated by the exclusive quantification of selected fatty acids including methyl-branched fatty acids, erucic acid (18:1n-9trans), and polyunsaturated fatty acids in cod liver oil and goat's milk fat.

Animals↗
PubMed2006

HPLC quantification of sphingolipids in soybeans with modified palmitate content.

Efficient separation and accurate quantification of sphingolipids (SL) are important for studying SL concentrations and biological functions. The objectives of this study were to develop effective methods for the separation and quantification of SL and to determine the relationship between palmitate and SL contents of mature soybean seeds. Methods using column chromatography and high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) were developed to separate and quantify glucosylceramide (GlcCer) and ceramide (Cer) in 15 soybeans lines in which palmitate content ranged from 3.7 to 40.7%. There were significant differences among the lines for GlcCer (83.4-397.6 nmol/g) and major Cer contents (8.4-20.7 nmol/g) on a dry weight basis. The correlations of palmitate content with GlcCer and Cer concentrations were not significant. The results indicated that the palmitate content of soybean seed did not affect their GlcCer and Cer contents. Genetic factors other than those that control palmitate content seemed to be responsible for the variation among soybean lines for GlcCer and Cer contents.

Ceramides↗
PubMed2006

Rapid quantification of the phenolic fraction of Spanish virgin olive oils by capillary electrophoresis with UV detection.

A rapid and reliable capillary zone electrophoresis method was used as a tool to obtain both qualitative and quantitative information about simple phenols, lignans, complex phenols (isomeric forms of secoiridoids), phenolic acids, and flavonoids in the solid-phase separation extracts from different Spanish extra-virgin olive oil in a short time (less than 6 min). Peak identification was done by using commercial and HPLC-isolated standards, studying the information of the electropherograms obtained at several wavelengths and also using the information previously reported. For the quantification of lignans and complex phenols (secoiridoid derivatives), we used a reference compound (oleuropein glucoside) at two different wavelengths (200 and 240 nm) and for the quantification of tyrosol and flavonoids, we used their commercially available standards.

Electrophoresis, Capillary↗
PubMed2006

Quantification of jasmonic acid by SPME in tomato plants stressed by ozone.

Jasmonates are signalling molecules induced in plants as a response to various biotic and/or abiotic stresses. As ozone is known to activate defense responses in plants, we have monitored the concentration of jasmonic acid in tomato leaves during and after an acute exposure to this abiotic elicitor. In this experiment, we observed that the maximum induction of jasmonic acid in O3-fumigated plants occurred 9 h after the end of treatment and the concentration of jasmonic acid in stressed plants increased 13-fold. However, the level of endogenous methyl-jasmonate was constant during the observed period. The extraction and quantification of jasmonic acid as its methyl ester was performed by headspace-solid-phase microextraction (or HS-SPME) in combination with GC-FID and GC-MS. The sensitivity (LOD = 2 ng/g) of this method permitted the detection and quantification of jasmonic acid present in plant tissues at very low concentrations.

Chromatography, Gas↗
PubMed1999

Quantification of soy isoflavones, genistein and daidzein, and conjugates in rat blood using LC/ES-MS.

Genistein is the principal soy isoflavone to which the putative beneficial effects of soy consumption have been attributed; however, the possibility of adverse biological effects (e.g., estrogenic, antithyroid) has also been raised. This paper describes development and validation of a simple and sensitive analytical method for the determination of genistein in the blood of rats receiving dietary genistein (<0.5-1250 microg of genistein aglycone/g of chow). The method uses serum/plasma deproteination, liquid-liquid extraction, deuterated genistein and daidzein internal standards, isocratic LC separation, and electrospray mass spectrometric quantification using selected ion monitoring. Extraction efficiency is approximately 85%, the detection limits for genistein and daidzein from 50 microL of rat blood are approximately 5 nM, and the limit of quantification is approximately 15 nM. Interassay precision (relative standard deviation 4.5-4.6%) and intraassay precision (3.3-6.7%) were determined from replicate analysis of a spiked control and an incurred serum sample. The distribution of conjugated and unconjugated forms of genistein in the blood of rats was determined using selective enzyme hydrolysis. The glucuronide was the predominant metabolite (>90%), and only small amounts of the sulfate conjugate and the aglycone were observed at all dose levels. No evidence for additional metabolites was obtained. The 7- and 4'-glucuronide conjugates of genistein were identified using electrospray mass spectrometry and (1)H NMR. Total blood genistein ranged from <15 nM in animals fed soy-free control diet to as high as 8.9 microM in male rats fed 1250 microg of genistein/g of chow and encompasses blood isoflavone levels observed in humans consuming a typical Asian diet and nutritional supplements (0.1-1 microM) and infants consuming soy formulas (2-7 microM).

Animals↗
PubMedDate not supplied

Comprehensive label-free method for the relative quantification of proteins from biological samples.

Pharmaceutical companies and regulatory agencies are broadly pursuing biomarkers as a means to increase the productivity of drug development. Quantifying differential levels of proteins from complex biological samples such as plasma or cerebrospinal fluid is one specific approach being used to identify markers of drug action, efficacy, toxicity, etc. We have developed a comprehensive, fully automated, and label-free approach to relative protein quantification from LC-MS/MS experiments of proteolytic protein digests including: de-noising, mass and charge state estimation, chromatographic alignment, and peptide quantification via integration of extracted ion chromatograms. Results from a variance components study of the entire method indicate that most of the variability is attributable to the LC-MS injection, with a median peptide LC-MS injection coefficient of variation of 8% on a ThermoFinnigan LTQ mass spectrometer. Spiked recovery results suggest a quantifiable range of approximately 32-fold for a sample protein.

Amino Acid Sequence↗
PubMed2006

Label-free protein quantification using LC-coupled ion trap or FT mass spectrometry: Reproducibility, linearity, and application with complex proteomes.

A critical step in protein biomarker discovery is the ability to contrast proteomes, a process referred generally as quantitative proteomics. While stable-isotope labeling (e.g., ICAT, 18O- or 15N-labeling, or AQUA) remains the core technology used in mass spectrometry-based proteomic quantification, increasing efforts have been directed to the label-free approach that relies on direct comparison of peptide peak areas between LC-MS runs. This latter approach is attractive to investigators for its simplicity as well as cost effectiveness. In the present study, the reproducibility and linearity of using a label-free approach to highly complex proteomes were evaluated. Various amounts of proteins from different proteomes were subjected to repeated LC-MS analyses using an ion trap or Fourier transform mass spectrometer. Highly reproducible data were obtained between replicated runs, as evidenced by nearly ideal Pearson's correlation coefficients (for ion's peak areas or retention time) and average peak area ratios. In general, more than 50% and nearly 90% of the peptide ion ratios deviated less than 10% and 20%, respectively, from the average in duplicate runs. In addition, the multiplicity ratios of the amounts of proteins used correlated nicely with the observed averaged ratios of peak areas calculated from detected peptides. Furthermore, the removal of abundant proteins from the samples led to an improvement in reproducibility and linearity. A computer program has been written to automate the processing of data sets from experiments with groups of multiple samples for statistical analysis. Algorithms for outlier-resistant mean estimation and for adjusting statistical significance threshold in multiplicity of testing were incorporated to minimize the rate of false positives. The program was applied to quantify changes in proteomes of parental and p53-deficient HCT-116 human cells and found to yield reproducible results. Overall, this study demonstrates an alternative approach that allows global quantification of differentially expressed proteins in complex proteomes. The utility of this method to biomarker discovery is likely to synergize with future improvements in the detecting sensitivity of mass spectrometers.

Amino Acid Sequence↗
PubMed2007

Microwave-enhanced ink staining for fast and sensitive protein quantification in proteomic studies.

A novel microwave-enhanced ink staining method was developed for rapid and sensitive estimation of protein content in sample buffers containing chaotropes, dyes, detergents, and reducing agents. Dye-based Blue-Black ink was used to quantitatively visualize proteins spotted on a nitrocellulose membrane. The total staining time was greatly reduced to 3 min by brief exposure to microwave radiation. The stained membrane was washed with distilled water, baked in a microwave oven for complete desiccation, transparentized with mineral oil, and documented by a desktop scanner or densitometer. Only 1 microL of protein sample (protein solubilized in SDS-PAGE sample buffer or IEF rehydration buffer) was used for protein spotting. The novel solid-phase protein assay gives a 500-fold dynamic range from 19.5 to 10000 ng/microL and can be scaled up for high-throughput protein quantification analysis. The fast, sensitive and low-cost microwave-enhanced ink staining procedure is ideal for protein quantification in proteomic analysis.

Animals↗
PubMed2002

Quantification of multiple DNA adducts formed through oxidative stress using liquid chromatography and electrospray tandem mass spectrometry.

Damage to DNA can arise through covalent modification of bases by reaction with oxidants and products of lipid peroxidation derived through normal aerobic metabolism. Such premutagenic lesions, including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), pyrimido[1,2alpha]purine-10(3H)one-2'-deoxyribose (M1-dG), etheno-2'-deoxyadenosine (epsilon-dA), and etheno-2'-deoxycytidine (epsilon-dC), are believed to be important in the development of human cancers related to diet, disease states, and lifestyle. We report the development of a method for concurrent quantification of these four adducts in DNA hydrolysates of 100 microg or less using on-line sample preparation coupled with liquid chromatography and tandem mass spectrometry. The sensitive detection permitted adduct quantification at levels below one adduct in 10(8) normal nucleotides and measurement of these adducts in DNA from untreated rodent liver and normal human liver samples. This methodology should prove useful in hypothesis-driven studies of cancer etiology in laboratory animals and humans.

Animals↗
PubMed2004

Quantification of urinary excretion of 1,N6-ethenoadenine, a potential biomarker of lipid peroxidation, in humans by stable isotope dilution liquid chromatography-electrospray ionization-tandem mass spectrometry: comparison with gas chromatography-mass spectrometry.

Etheno DNA adducts are promutagenic DNA lesions derived from exogenous as well as endogenous sources. The levels of etheno adducts in tissue DNA are elevated in cancer prone tissues, and the urinary excretion of etheno adducts is associated with oxidative stress. In this report, a new assay based on isotope dilution liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) is developed for the quantification of 1,N(6)-ethenoadenine (epsilonAde) in human urine samples without the need for derivatization. Sample purification before analysis by MS only requires a reversed phase solid phase extraction column. Two multiple reaction monitoring transitions with two product ion fragments generated from a common parent ion were used to quantify urinary epsilonAde. The detection limit of epsilonAde using LC-ESI-MS/MS is 2 pg injected standard epsilonAde on-column, and the assay allows accurate quantification of urinary epsilonAde at concentrations higher than 10 pg/mL. The presence of epsilonAde in human urine is confirmed by the collision-induced daughter ion spectrum. Using this assay, the levels of epsilonAde in the 24 h urine samples from 18 healthy individuals are determined, and the results are in very good agreement with those obtained using isotope dilution gas chromatography-negative ion chemical ionization-mass spectrometry. The high specificity and simple sample pretreatment of this LC-ESI-MS/MS method render it a valuable tool in measuring epsilonAde in the complex mixture of human urine as a promising noninvasive biomarker for DNA damage associated with oxidative stress and for cancer chemoprevention studies.

Adenine↗
PubMed2005

Detection and quantification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl adducts in human pancreas tissue using capillary liquid chromatography-microelectrospray mass spectrometry.

Cigarette smoking has been associated with various cancers including bladder and pancreas. 4-Aminobiphenyl has been isolated as a constituent of cigarette smoke and has been established as a carcinogen in various animal models and humans. In rodents and humans, 4-aminobiphenyl is N-hydroxylated and forms adducts to DNA, the predominant one being N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP). In this study, we report a micro-electrospray mass spectrometric (muESI-MS) isotope dilution method for the detection and quantification of dG-C8-ABP in human pancreatic tissue. A reverse phase capillary column (320 microm ID) was connected to a triple quadrupole mass spectrometer via a commercially available micro-ESI source. The system was operated in the selected reaction monitoring mode transmitting the [M + H]+ --> [M + H - 116]+ transitions for both the analyte and the isotopically labeled internal standard. Twelve human pancreas samples were analyzed, where six were current smokers (three male and three female) and six were considered nonsmokers (three female and three male). Of the samples analyzed, six showed dG-C8-ABP levels above the limit of quantification for the method, five were considered to have levels that were undetectable, and one was discarded due to inconsistent internal standard signal. The age of the human subjects ranged from 17 to 63, and, in samples where adduct was present, levels ranged anywhere from 1 to 60/10(8) nucleotides. Although no correlation between smoking preference, age, or gender was proven with this particular sample pool, this report demonstrates that capillary LC-muESI-MS can provide a sensitive and definitive method for DNA adduct analysis in human tissue.

Adolescent↗
PubMed1999

Detection and quantification of 1,N(6)-ethenoadenine in human placental DNA by mass spectrometry.

Exocyclic DNA adducts have been reported to derive from various exogenous as well as endogenous sources, such as lipid peroxidation. Among them, 1,N(6)-ethenoadenine (epsilonAde) has previously been detected in tissue DNA of untreated rodents and humans by an immunoaffinity/(32)P-postlabeling method. This study reports detection and quantification of the endogenous epsilonAde adduct in the same human placental DNA by three independent assays, namely, GC/MS, LC/MS, and HPLC/fluorescence. Using a recently reported gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS) method [Chen, H.-J. C., et al. (1998) Chem. Res. Toxicol. 11, 1474], the level of epsilonAde in human placental DNA from a commercial source was found to be 2.3 adducts per 10(6) Ade bases. To confirm these findings, a liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) method was developed for epsilondAdo. With this LC/MS assay, epsilondAdo was detected at the level of 2.5 adducts per 10(6) dAdo nucleosides in the same human placental DNA. The stable isotopes of epsilonAde and epsilondAdo were added as internal standards in both GC/MS and LC/ESI/MS/MS assays, respectively, and thus provided high specificity, reproducibility, and accurate quantification. The relatively high levels of epsilonAde in this human placental DNA detected by mass spectrometry were further verified by HPLC/fluorescence analysis. The GC/MS method was validated by the HPLC/fluorescence assay using calf thymus DNA treated with chloroacetaldehyde or by the LC/MS method with 2, 3-epoxy-4-hydroxynonanal-modified calf thymus DNA. The epsilonAde level in human placental DNA freshly isolated in the presence of an antioxidant was similar to that in DNA from the commercial source. Since epsilonAde is a potential mutagenic lesion, analysis of epsilonAde by the specific and sensitive GC/NICI/MS method may provide a useful biomarker in cancer risk assessment.

Adenine↗
PubMed1997

Modelling biological gel contraction by cells: mechanocellular formulation and cell traction force quantification.

Traction forces developed by most cell types play a significant role in the spatial organisation of biological tissues. However, due to the complexity of cell-extracellular matrix interactions, these forces are quantitatively difficult to estimate without explicitly considering cell properties and extracellular mechanical matrix responses. Recent experimental devices elaborated for measuring cell traction on extracellular matrix use cell deposits on a piece of gel placed between one fixed and one moving holder. We formulate here a mathematical model describing the dynamic behaviour of the cell-gel medium in such devices. This model is based on a mechanical force balance quantification of the gel visco-elastic response to the traction forces exerted by the diffusing cells. Thus, we theoretically analyzed and simulated the displacement of the free moving boundary of the system under various conditions for cells and gel concentrations. This model is then used as the theoretical basis of an experimental device where endothelial cells are seeded on a rectangular biogel of fibrin cast between two floating holders, one fixed and the other linked to a force sensor. From a comparison of displacement of the gel moving boundary simulated by the model and the experimental data recorded from the moving holder displacement, the magnitude of the traction forces exerted by the endothelial cell on the fibrin gel was estimated for different experimental situations. Different analytical expressions for the cell traction term are proposed and the corresponding force quantifications are compared to the traction force measurements reported for various kind of cells with the use of similar or different experimental devices.

Animals↗
PubMedDate not supplied

Descriptive analysis and quantification of angiogenesis in human brain tumors.

Quantitative determination of the degree of vascularity has been shown to be independently prognostically significant in many human tumor types. In particular, tumor vascularity has known importance in astrocytomas, in which endothelial proliferation is a criterion for anaplasia in many grading schemes. This review analyzes reports of microvessel quantification performed on histologic sections of human brain tumors, and in which correlations with clinical outcome, or other pathobiologic factors have been made. Among the conclusions are: (1) brain tumors have the unique feature of complex 'glomeruloid' vessels, as well as heterogeneity of microvascular distribution and caliber; (2) lower-grade astrocytomas may incorporate pre-existing vessels, while glioblastomas may develop new vessels; (3) quantification may have additional independent prognostic value over and above routine histologic grade in low-grade astrocytomas with low tumor cell proliferative indices. These findings have implications for the appropriateness of antiangiogenic therapies.

Blood Vessels↗