Comparison between a linear ion trap and a triple quadruple MS in the sensitive detection of large peptides at femtomole amounts on column.

In addition to the importance of sample preparation and extract separation, MS detection is a key factor in the sensitive quantification of large undigested peptides. In this article, a linear ion trap MS (LIT-MS) and a triple quadrupole MS (TQ-MS) have been compared in the detection of large peptides at subnanomolar concentrations. Natural brain natriuretic peptide, C-peptide, substance P and D-Junk-inhibitor peptide, a full D-amino acid therapeutic peptide, were chosen. They were detected by ESI and simultaneous MS(1) and MS(2) acquisitions. With direct peptide infusion, MS(2) spectra revealed that fragmentation was peptide dependent, milder on the LIT-MS and required high collision energies on the TQ-MS to obtain high-intensity product ions. Peptide adsorption on surfaces was overcome and peptide dilutions ranging from 0.1 to 25 nM were injected onto an ultra high-pressure LC system with a 1 mm id analytical column and coupled with the MS instruments. No difference was observed between the two instruments when recording in LC-MS(1) acquisitions. However, in LC-MS(2) acquisitions, a better sensitivity in the detection of large peptides was observed with the LIT-MS. Indeed, with the three longer peptides, the typical fragmentation in the TQ-MS resulted in a dramatic loss of sensitivity (> or = 10x).

Circulating microRNAs as long-term biomarkers for the detection of erythropoiesis-stimulating agent abuse.

MicroRNAs (miRNAs) are small, non-protein coding transcripts involved in many cellular and physiological mechanisms. Recently, a new class of miRNA called 'circulating miRNAs' was found in cell-free body fluids such as plasma and urine. Circulating miRNAs have been shown to be very stable, specific, and sensitive biomarkers. In this paper, we investigate whether circulating miRNAs can serve as biomarkers for erythropoiesis-stimulating agent abuse. To this end, we analyzed miRNA levels in plasma by miRNA microarrays and quantitative real-time polymerase chain reaction (PCR). Plasma samples are derived from a clinical study with healthy subjects injected with erythropoiesis-stimulating agent (C.E.R.A.). Based on microarray results, we observed a significant difference in the levels of miRNAs in plasma after C.E.R.A. injection. We demonstrated that a specific miRNA, miR-144, exhibit a high increase that lasts 27 days after C.E.R.A. stimulation. Considering the fact that miR-144 is an essential erythropoiesis agent in different organisms, these findings suggest the possibility of using miR-144 as a sensitive and informative biomarker to detect C.E.R.A. abuse. Copyright © 2011 John Wiley & Sons, Ltd.

Microbiological diagnosis of community-acquired respiratory tract infections by nucleic acid detection.

Background: Microbiological diagnostic procedures have changed significantly over the last decade. Initially the implementation of the polymerase chain reaction (PCR) resulted in improved detection tests for microbes that were difficult or even impossible to detect by conventional methods such as culture and serology, especially in community-acquired respiratory tract infections (CA-RTI). A further improvement was the development of real-time PCR, which allows end point detection and quantification, and many diagnostic laboratories have now implemented this powerful method. Objective: At present, new performant and convenient molecular tests have emerged targeting in parallel many viruses and bacteria responsible for lower and/or upper respiratory tract infections. The range of test formats and microbial agents detected is evolving very quickly and the added value of these new tests needs to be studied in terms of better use of antibiotics, better patient management, duration of hospitalization and overall costs. Conclusions: Molecular tools for a better microbial documentation of CA-RTI are now available. Controlled studies are now required to address the relevance issue of these new methods, such as, for example, the role of some newly detected respiratory viruses or of the microbial DNA load in a particular patient at a particular time. The future challenge for molecular diagnosis will be to become easy to handle, highly efficient and cost-effective, delivering rapid results with a direct impact on clinical management.

Carbon isotopic ratio analysis by gas chromatography/combustion/isotope ratio mass spectrometry for the detection of gamma-hydroxybutyric acid (GHB) administration to humans.

Since GHB (gamma-hydroxybutyric acid) is naturally produced in the human body, clinical and forensic toxicologists must be able to discriminate between endogenous levels and a concentration resulting from exposure. To suggest an alternative to the use of interpretative concentration cut-offs, the detection of exogenous GHB in urine specimens was investigated by means of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). GHB was isolated from urinary matrix by successive purification on Oasis MCX and Bond Elute SAX solid-phase extraction (SPE) cartridges prior to high-performance liquid chromatography (HPLC) fractioning using an Atlantis dC18 column eluted with a mixture of formic acid and methanol. Subsequent intramolecular esterification of GHB leading to the formation of gamma-butyrolactone (GBL) was carried out to avoid introduction of additional carbon atoms for carbon isotopic ratio analysis. A precision of 0.3 per thousand was determined using this IRMS method for samples at GHB concentrations of 10 mg/L. The (13)C/(12)C ratios of GHB in samples of subjects exposed to the drug ranged from -32.1 to -42.1 per thousand, whereas the results obtained for samples containing GHB of endogenous origin at concentration levels less than 10 mg/L were in the range -23.5 to -27.0 per thousand. Therefore, these preliminary results show that a possible discrimination between endogenous and exogenous GHB can be made using carbon isotopic ratio analyses.

Rapid profiling of intact glucosinolates in Arabidopsis leaves by UHPLC-QTOFMS using a charged surface hybrid column.

INTRODUCTION: The analysis of glucosinolates (GS) is traditionally performed by reverse-phase liquid chromatography coupled to ultraviolet detection after a time-consuming desulphation step, which is required for increased retention. Simpler and more efficient alternative methods that can shorten both sample preparation and analysis are much needed. OBJECTIVE: To evaluate the feasibility of using ultrahigh-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS) for the rapid profiling of intact GS. METHODOLOGY: A simple and short extraction of GS from Arabidopsis thaliana leaves was developed. Four sub-2 µm reverse-phase columns were tested for the rapid separation of these polar compounds using formic acid as the chromatographic additive. High-resolution QTOFMS was used to detect and identify GS. RESULTS: A novel charged surface hybrid (CSH) column was found to provide excellent retention and separation of GS within a total running time of 11 min. Twenty-one GS could be identified based on their accurate mass as well as isotopic and fragmentation patterns. The method was applied to determine the changes in GS content that occur after herbivory in Arabidopsis. In addition, we evaluated its applicability to the profiling of other Brassicaceae species. CONCLUSION: The method developed can profile the full range of GS, including the most polar ones, in a shorter time than previous methods, and is highly compatible with mass spectrometric detection.

A validated assay for measuring doxorubicin in biological fluids and tissues in an isolated lung perfusion model: matrix effect and heparin interference strongly influence doxorubicin measurements.

Doxorubicin is an antineoplasic agent active against sarcoma pulmonary metastasis, but its clinical use is hampered by its myelotoxicity and its cumulative cardiotoxicity, when administered systemically. This limitation may be circumvented using the isolated lung perfusion (ILP) approach, wherein a therapeutic agent is infused locoregionally after vascular isolation of the lung. The influence of the mode of infusion (anterograde (AG): through the pulmonary artery (PA); retrograde (RG): through the pulmonary vein (PV)) on doxorubicin pharmacokinetics and lung distribution was unknown. Therefore, a simple, rapid and sensitive high-performance liquid chromatography method has been developed to quantify doxorubicin in four different biological matrices (infusion effluent, serum, tissues with low or high levels of doxorubicin). The related compound daunorubicin was used as internal standard (I.S.). Following a single-step protein precipitation of 500 microl samples with 250 microl acetone and 50 microl zinc sulfate 70% aqueous solution, the obtained supernatant was evaporated to dryness at 60 degrees C for exactly 45 min under a stream of nitrogen and the solid residue was solubilized in 200 microl of purified water. A 100 microl-volume was subjected to HPLC analysis onto a Nucleosil 100-5 microm C18 AB column equipped with a guard column (Nucleosil 100-5 microm C(6)H(5) (phenyl) end-capped) using a gradient elution of acetonitrile and 1-heptanesulfonic acid 0.2% pH 4: 15/85 at 0 min-->50/50 at 20 min-->100/0 at 22 min-->15/85 at 24 min-->15/85 at 26 min, delivered at 1 ml/min. The analytes were detected by fluorescence detection with excitation and emission wavelength set at 480 and 550 nm, respectively. The calibration curves were linear over the range of 2-1000 ng/ml for effluent and plasma matrices, and 0.1 microg/g-750 microg/g for tissues matrices. The method is precise with inter-day and intra-day relative standard deviation within 0.5 and 6.7% and accurate with inter-day and intra-day deviations between -5.4 and +7.7%. The in vitro stability in all matrices and in processed samples has been studied at -80 degrees C for 1 month, and at 4 degrees C for 48 h, respectively. During initial studies, heparin used as anticoagulant was found to profoundly influence the measurements of doxorubicin in effluents collected from animals under ILP. Moreover, the strong matrix effect observed with tissues samples indicate that it is mandatory to prepare doxorubicin calibration standard samples in biological matrices which would reflect at best the composition of samples to be analyzed. This method was successfully applied in animal studies for the analysis of effluent, serum and tissue samples collected from pigs and rats undergoing ILP.

Characterization and detection of naturally occurring antibodies against IL-1 alpha and IL-1 beta in normal human plasma.

During the development and testing of a radioreceptor assay (RRA) for human IL-1, we have detected and identified the presence of auto-antibodies to IL-1 in normal human plasma (NHP). The RRA is based on the competition between human 125I-labeled rIL-1 alpha and standard or unknown quantities of IL-1 alpha or IL-1 beta for binding to a limited amounts of IL-1 receptor (IL-1R) isolated from the EL4 mouse thymoma cell line. NHP from 20 out of 100 unselected blood donors were found to completely inhibit the binding of 125I-labeled IL-1 alpha to its receptor, suggesting the presence in these NHP samples of either abnormal amounts of IL-1 or of a factor binding to the 125I-labeled IL-1 alpha. Special care was taken to ascertain that the inhibitory factors were antibodies and not soluble IL-1 receptor antagonist. When plasma samples with inhibiting activity were incubated with labeled IL-1 alpha and chromatographed on a Sephadex G200 column, they were found to contain 125I-labeled complexes with an apparent molecular weight of 150-200kD. The IL-1 binding factor could be eliminated from plasma by incubation with protein A-Sepharose, suggesting that it consisted in IgG antibodies directed against IL-1. Furthermore, the antibody nature of the inhibiting factor was confirmed by its binding to purified rIL-1 coupled to Sepharose. Screening of 200 NHP samples by incubation with 100 pg of 125I-labeled IL-1 followed by precipitation with 12% of polyethylene glycol (PEG) confirmed that about 25% of NHP contain detectable IgG antibodies to IL-1 alpha, while only 2% of NHP contain antibodies to IL-1 beta. No correlation between the presence of these anti-IL-1 antibodies and any particular major histocompatibility complex or any pathological conditions was detected. We suggest that all serum samples assayed for IL-1 alpha or IL-1 beta content should be pretested with the PEG precipitation assay described here.

Development of a microfluidics biosensor for agarose-bead immobilized Escherichia coli bioreporter cells for arsenite detection in aqueous samples.

Contamination with arsenic is a recurring problem in both industrialized and developing countries. Drinking water supplies for large populations can have concentrations much higher than the permissible levels (for most European countries and the United States, 10 μg As per L; elsewhere, 50 μg As per L). Arsenic analysis requires high-end instruments, which are largely unavailable in developing countries. Bioassays based on genetically engineered bacteria have been proposed as suitable alternatives but such tests would profit from better standardization and direct incorporation into sensing devices. The goal of this work was to develop and test microfluidic devices in which bacterial bioreporters could be embedded, exposed and reporter signals detected, as a further step towards a complete miniaturized bacterial biosensor. The signal element in the biosensor is a nonpathogenic laboratory strain of Escherichia coli, which produces a variant of the green fluorescent protein after contact to arsenite and arsenate. E. coli bioreporter cells were encapsulated in agarose beads and incorporated into a microfluidic device where they were captured in 500 × 500 μm(2) cages and exposed to aqueous samples containing arsenic. Cell-beads frozen at -20 °C in the microfluidic chip retained inducibility for up to a month and arsenic samples with 10 or 50 μg L(-1) could be reproducibly discriminated from the blank. In the 0-50 μg L(-1) range and with an exposure time of 200 minutes, the rate of signal increase was linearly proportional to the arsenic concentration. The time needed to reliably and reproducibly detect a concentration of 50 μg L(-1) was 75-120 minutes, and 120-180 minutes for a concentration of 10 μg L(-1).

Detection of CBP rearrangements in acute myelogenous leukemia with t(8;16).

The CREB-binding protein (CBP) is a large nuclear protein that regulates many signal transduction pathways and is involved in chromatin-mediated transcription. The translocation t(8;16)(p11;p13.3) consistently disrupts two genes: the CBP gene on chromosome band 16p13.3 and the MOZ gene on chromosome band 8p11. Although a fusion of these two genes as a result of the translocation is expected, attempts at detecting the fusion transcript by reverse transcriptase polymerase chain reaction (RT-PCR) have proven difficult; to date, only one in-frame CBP/MOZ fusion transcript has been reported. We therefore sought other reliable means of detecting CBP rearrangements. We applied fluorescence in situ hybridization (FISH) and Southern blot analyses to a series of AML patients with a t(8;16) and detected DNA rearrangements of both the CBP and the MOZ loci in all cases tested. All six cases examined for CBP rearrangements have breakpoints within a 13 kb breakpoint cluster region at the 5' end of the CBP gene. Additionally, we used a MOZ cDNA probe to construct a surrounding cosmid contig and detect DNA rearrangements in three t(8;16) cases, all of which display rearrangements within a 6 kb genomic fragment of the MOZ gene. We have thus developed a series of cosmid probes that consistently detect the disruption of the CBP gene in t(8;16) patients. These clones could potentially be used to screen other cancer-associated or congenital translocations involving chromosome band 16p13.3 as well.

Detection of an asymptomatic right-ventricle cardiac metastasis from a small-cell lung cancer by F-18-FDG PET/CT.

A right heart metastasis of a small-cell lung cancer was found on the whole-body F-fluoro-deoxy-glucose positron emission tomography/computed tomography (F-FDG-PET/CT) of a 69-year-old smoker investigated for a right pulmonary mass discovered on chest radiography after a fracture of the right humerus. The PET scan showed an increased FDG uptake by the mass in the right lung and an intense, atypical focal activity of the right ventricle strongly suggestive of a neoplastic process. CT-guided lung biopsy revealed a small-cell lung cancer and myocardial biopsy confirmed the presence of a cardiac metastasis. The patient was treated with six cycles of chemotherapy followed by radiation therapy, which included the heart lesion. At follow-up PET/CT 2 months after the end of treatment, the abnormal cardiac uptake had disappeared, whereas increased FDG uptake persisted in the pulmonary residual mass.

Analysis of cannabinoids in oral fluid by liquid chromatography-tandem mass spectrometry

A sensitive method was developed for quantifying a wide range of cannabinoids in oral fluid (OF) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These cannabinoids include a dagger(9)-tetrahydrocannabinol (THC), 11-hydroxy-a dagger(9)-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-a dagger(9)-tetrahydrocannabinol (THCCOOH), cannabinol (CBN), cannabidiol (CBD), a dagger(9)-tetrahydrocannabinolic acid A (THC-A), 11-nor-9-carboxy-a dagger(9)-tetrahydrocannabinol glucuronide (THCCOOH-gluc), and a dagger(9)-tetrahydrocannabinol glucuronide (THC-gluc). Samples were collected using a Quantisal (TM) device. The advantages of performing a liquid-liquid extraction (LLE) of KCl-saturated OF using heptane/ethyl acetate versus a solid-phase extraction (SPE) using HLB copolymer columns were determined. Chromatographic separation was achieved in 11.5 min on a Kinetex (TM) column packed with 2.6-mu m core-shell particles. Both positive (THC, 11-OH-THC, CBN, and CBD) and negative (THCCOOH, THC-gluc, THCCOOH-gluc, and THC-A) electrospray ionization modes were employed with multiple reaction monitoring using a high-end AB Sciex API 5000 (TM) triple quadrupole LC-MS/MS system. Unlike SPE, LLE failed to extract THC-gluc and THCCOOH-gluc. However, the LLE method was more sensitive for the detection of THCCOOH than the SPE method, wherein the limit of detection (LOD) and limit of quantification (LOQ) decreased from 100 to 50 pg/ml and from 500 to 80 pg/ml, respectively. The two extraction methods were successfully applied to OF samples collected from volunteers before and after they smoked a homemade cannabis joint. High levels of THC were measured soon after smoking, in addition to significant amounts of THC-A. Other cannabinoids were found in low concentrations. Glucuronide conjugate levels were lower than the method's LOD for most samples. Incubation studies suggest that glucuronides could be enzymatically degraded by glucuronidase prior to OF collection

Detection in urine of 4-methyl-2-hexaneamine, a doping agent.

Stimulants are banned in-competition for all categories of sports by the World Anti-Doping Agency. A simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay employing electrospray ionisation in positive mode was developed in that work for the quantification in urine specimens of 4-methyl-2-hexaneamine, a primary amine exhibiting sympathomimetic properties. Following a simple pretreatment procedure, the analyte was separated using a gradient mobile phase on reverse phase C8 column. Selected reaction monitoring m/z 116.2-->57.3 was specific for detection of 4-methyl-2-hexaneamine and the assay exhibited a linear dynamic range of 50-700 ng/mL. The validated method has been successfully applied to analyze the target compound in food supplements as well as in urine specimens. The administered drug (40 mg) was detected at the level of 350 ng/mL in the urine up to 4 days.

Rockfall detection from LIDAR point clouds: A clustering approach using R

The analysis of rockfall characteristics and spatial distribution is fundamental to understand and model the main factors that predispose to failure. In our study we analysed LiDAR point clouds aiming to: (1) detect and characterise single rockfalls; (2) investigate their spatial distribution. To this end, different cluster algorithms were applied: 1a) Nearest Neighbour Clutter Removal (NNCR) in combination with the Expectation?Maximization (EM) in order to separate feature points from clutter; 1b) a density based algorithm (DBSCAN) was applied to isolate the single clusters (i.e. the rockfall events); 2) finally we computed the Ripley's K-function to investigate the global spatial pattern of the extracted rockfalls. The method allowed proper identification and characterization of more than 600 rockfalls occurred on a cliff located in Puigcercos (Catalonia, Spain) during a time span of six months. The spatial distribution of these events proved that rockfall were clustered distributed at a welldefined distance-range. Computations were carried out using R free software for statistical computing and graphics. The understanding of the spatial distribution of precursory rockfalls may shed light on the forecasting of future failures.

On the use of an appropriate TdT-mediated dUTP-biotin nick end labeling assay to identify apoptotic cells.

Apoptosis is an essential cellular mechanism involved in many processes such as embryogenesis, metamorphosis, and tissue homeostasis. DNA fragmentation is one of the key markers of this form of cell death. DNA fragmentation is executed by endogenous endonucleases such as caspase-activated DNase (CAD) in caspase-dependent apoptosis. The TUNEL (TdT-mediated dUTP-biotin nick end labeling) technique is the most widely used method to identify apoptotic cells in a tissue or culture and to assess drug toxicity. It is based on the detection of 3'-OH termini that are labeled with dUTP by the terminal deoxynucleotidyl transferase. Although the test is very reliable and sensitive in caspase-dependent apoptosis, it is completely useless when cell death is mediated by pathways involving DNA degradation that generates 3'-P ends as in the LEI/L-DNase II pathway. Here, we propose a modification in the TUNEL protocol consisting of a dephosphorylation step prior to the TUNEL labeling. This allows the detection of both types of DNA breaks induced during apoptosis caspase-dependent and independent pathways, avoiding underestimating the cell death induced by the treatment of interest.