Rapid and sensitive hormonal profiling of complex plant samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry

Background Plant hormones play a pivotal role in several physiological processes during a plant's life cycle, from germination to senescence, and the determination of endogenous concentrations of hormones is essential to elucidate the role of a particular hormone in any physiological process. Availability of a sensitive and rapid method to quantify multiple classes of hormones simultaneously will greatly facilitate the investigation of signaling networks in controlling specific developmental pathways and physiological responses. Due to the presence of hormones at very low concentrations in plant tissues (10-9 M to 10-6 M) and their different chemistries, the development of a high-throughput and comprehensive method for the determination of hormones is challenging. Results The present work reports a rapid, specific and sensitive method using ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UPLC/ESI-MS/MS) to analyze quantitatively the major hormones found in plant tissues within six minutes, including auxins, cytokinins, gibberellins, abscisic acid, 1-amino-cyclopropane-1-carboxyic acid (the ethylene precursor), jasmonic acid and salicylic acid. Sample preparation, extraction procedures and UPLC-MS/MS conditions were optimized for the determination of all plant hormones and are summarized in a schematic extraction diagram for the analysis of small amounts of plant material without time-consuming additional steps such as purification, sample drying or re-suspension. Conclusions This new method is applicable to the analysis of dynamic changes in endogenous concentrations of hormones to study plant developmental processes or plant responses to biotic and abiotic stresses in complex tissues. An example is shown in which a hormone profiling is obtained from leaves of plants exposed to salt stress in the aromatic plant, Rosmarinus officinalis.

Determination of picogram levels of midazolam, and 1- and 4-hydroxymidazolam in human plasma by gas chromatography-negative chemical ionization-mass spectrometry.

Midazolam is a widely accepted probe for phenotyping cytochrome P4503A. A gas chromatography-mass spectrometry (GC-MS)-negative chemical ionization method is presented which allows measuring very low levels of midazolam (MID), 1-OH midazolam (1OHMID) and 4-OH midazolam (4OHMID), in plasma, after derivatization with the reagent N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide. The standard curves were linear over a working range of 20 pg/ml to 5 ng/ml for the three compounds, with the mean coefficients of correlation of the calibration curves (n = 6) being 0.999 for MID and 1OHMID, and 1.0 for 4OHMID. The mean recoveries measured at 100 pg/ml, 500 pg/ml, and 2 ng/ml, ranged from 76 to 87% for MID, from 76 to 99% for 1OHMID, from 68 to 84% for 4OHMID, and from 82 to 109% for N-ethyloxazepam (internal standard). Intra- (n = 7) and inter-day (n = 8) coefficients of variation determined at three concentrations ranged from 1 to 8% for MID, from 2 to 13% for 1OHMID and from 1 to 14% for 4OHMID. The percent theoretical concentrations (accuracy) were within +/-8% for MID and 1OHMID, within +/-9% for 4OHMID at 500 pg/ml and 2 ng/ml, and within +/-28% for 4OHMID at 100 pg/ml. The limits of quantitation were found to be 10 pg/ml for the three compounds. This method can be used for phenotyping cytochrome P4503A in humans following the administration of a very low oral dose of midazolam (75 microg), without central nervous system side-effects.

Reduction of Concrete Deterioration by Ettringite Using Crystal Growth Inhibition Techniques, TR-431, 2001

Many researchers have concluded that secondary or delayed ettringite is responsible for serious premature deterioration of concrete highways. In some poorly performing Iowa concretes, ettringite is the most common secondary mineral but its role in premature deterioration is uncertain since some researchers still maintain that secondary ettringite does not itself cause deterioration. The current research project was designed to determine experimentally if it is possible to reduce secondary ettringite formation in concrete by treating the concrete with commercial crystallization inhibitor chemicals. The hypothesis is such that if the amount of ettringite is reduced, there will also be a concomitant reduction of concrete expansion and cracking. If both ettringite formation and deterioration are simultaneously reduced, then the case for ettringite induced expansion/cracking is strengthened. The experiment used four commercial inhibitors - two phosphonates, a polyacrylic acid, and a phosphate ester. Concrete blocks were subjected to continuous immersion, wet/dry and freeze/thaw cycling in sodium sulfate solutions and in sulfate solutions containing an inhibitor. The two phosphonate inhibitors, Dequest 2060 and Dequest 2010, manufactured by Monsanto Co., were effective in reducing ettringite nucleation and growth in concrete. Two other inhibitors, Good-rite K752 and Wayhib S were somewhat effective, but less so than the two phosphonates. Rapid experiments with solution growth inhibition of ettringite without the presence of concrete phases were used to explore the mechanisms of inhibition of this mineral. Reduction of new ettringite formation in concrete blocks also reduced expansion and cracking of the blocks. This relationship clearly links concrete expansion with this mineral - a conclusion that some research workers have disputed despite theoretical arguments for such a relationship and despite numerous observations of ettringite mineralization in prematurely deteriorated concrete highways. Secondary ettringite nucleation and growth must cause concrete expansion because the only known effect of the inhibitor chemicals is to reduce crystal nucleation and growth, and the inhibitors cannot in any other way be responsible for the reduction in expansion. The mechanism of operation of the inhibitors on ettringite reduction is not entirely clear but the solution growth experiments show that they prevent crystallization of a soluble ettringite precursor gel. The present study shows that ettringite growth alone is not responsible for expansion cracking because the experiments showed that most expansion occurs under wet/dry cycling, less under freeze/thaw cycling, and least under continuous soaking conditions. It was concluded from the different amounts of damage that water absorption by newly-formed, minute ettringite crystals is responsible for part of the observed expansion under wet/dry conditions, and that reduction of freeze resistance by ettringite filling of air-entrainment voids is also important in freeze/thaw environments.

Bond Enhancement Techniques for PCC Whitetopping, HR-341, 1996

This research was initiated in 1991 as a part of a whitetopping project to study the effectiveness of various techniques to enhance bond strength between a new portland cement concrete (PCC) overlay and an existing asphalt cement concrete (ACC) pavement surface. A 1,676 m (5,500 ft) section of county road R16 in Dallas County was divided into 12 test sections. The various techniques used to enhance bond were power brooming, power brooming with air blast, milling, cement and water grout, and emulsion tack coat. Also, two sections were planed to a uniform cross-section, two pavement thicknesses were placed, and two different concrete mix proportions were used. Bond strength was perceived to be the key to determining an appropriate design procedure for whitetopping. If adequate bond is achieved, a bonded PCC overlay technique can be used for design. Otherwise, an unbonded overlay procedure may be more appropriate. Conclusions are as follows: (1) Bond Strength Differences - Milling increased bond strength versus no milling. Tack coat showed increased bond strength versus no tack coat. Planing, Air Blast and Grouting did not provide noticeable improvements in bond strength; nor did different PCC types or thicknesses affect bond strength significantly. (2) Structure - Structural measurements correlated strongly with the wide variation in pavement thicknesses. They did not provide enough information to determine the strength of bonding or the level of support being provided by the ACC layer. Longitudinal cracking correlated with PCC thicknesses and with planing. (3) Bond Over Time - The bond between PCC and ACC layers is degrading over time in the outside wheel path in all of the sections except tack coat (section 12). The bond strength in the section with tack coat was lower than the others, but remained relatively steady.

Transscleral Coulomb-controlled iontophoresis of methylprednisolone into the rabbit eye: influence of duration of treatment, current intensity and drug concentration on ocular tissue and fluid levels.

The major problems associated with the use of corticosteroids for the treatment of ocular diseases are their poor intraocular penetration to the posterior segment when administered locally and their secondary side effects when given systemically. To circumvent these problems more efficient methods and techniques of local delivery are being developed. The purposes of this study were: (1) to investigate the pharmacokinetics of intraocular penetration of hemisuccinate methyl prednisolone (HMP) after its delivery using the transscleral Coulomb controlled iontophoresis (CCI) system applied to the eye or after intravenous (i.v.) injection in the rabbit, (2) to test the safety of the CCI system for the treated eyes and (3) to compare the pharmacokinetic profiles of HMP intraocular distribution after CCI delivery to i.v. injection. For each parameter evaluated, six rabbit eyes were used. For the CCI system, two concentrations of HMP (62.5 and 150mg ml(-1)), various intensities of current and duration of treatment were analyzed. In rabbits serving as controls the HMP was infused in the CCI device but without applied electric current. For the i.v. delivery, HMP at 10mg kg(-1)as a 62.5mg ml(-1)solution was used. The rabbits were observed clinically for evidence of ocular toxicity. At various time points after the administration of drug, rabbits were killed and intraocular fluids and tissues were sampled for methylprednisolone (MP) concentrations by high pressure liquid chromatography (HPLC). Histology examinations were performed on six eyes of each group. Among groups that received CCI, the concentrations of MP increased in all ocular tissues and fluids in relation to the intensities of current used (0.4, 1.0 and 2.0mA/0.5cm(2)) and its duration (4 and 10min). Sustained and highest levels of MP were achieved in the choroid and the retina of rabbit eyes treated with the highest current and 10min duration of CCI. No clinical toxicity or histological lesions were observed following CCI. Negligible amounts of MP were found in ocular tissues in the CCI control group without application of current. Compared to i.v. administration, CCI achieved higher and more sustained tissue concentrations with negligible systemic absorption. These data demonstrate that high levels of MP can be safely achieved in intraocular tissues and fluids of the rabbit eye, using CCI. With this system, intraocular tissues levels of MP are higher than those achieved after i.v. injection. Furthermore, if needed, the drug levels achieved with CCI can be modulated as a function of current intensity and duration of treatment. CCI could therefore be used as an alternative method for the delivery of high levels of MP to the intraocular tissues of both the anterior and posterior segments.

A novel method for quantification of sulfolane (a metabolite of busulfan) in plasma by gas chromatography-tandem mass spectrometry.

The role of busulfan (Bu) metabolites in the adverse events seen during hematopoietic stem cell transplantation and in drug interactions is not explored. Lack of availability of established analytical methods limits our understanding in this area. The present work describes a novel gas chromatography-tandem mass spectrometric assay for the analysis of sulfolane (Su) in plasma of patients receiving high-dose Bu. Su and Bu were extracted from a single 100 μL plasma sample by liquid-liquid extraction. Bu was separately derivatized with 2,3,5,6-tetrafluorothiophenolfluorinated agent. Mass spectrometric detection of the analytes was performed in the selected reaction monitoring mode on a triple quadrupole instrument after electronic impact ionization. Bu and Su were analyzed with separate chromatographic programs, lasting 5 min each. The assay for Su was found to be linear in the concentration range of 20-400 ng/mL. The method has satisfactory sensitivity (lower limit of quantification, 20 ng/mL) and precision (relative standard deviation less than 15 %) for all the concentrations tested with a good trueness (100 ± 5 %). This method was applied to measure Su from pediatric patients with samples collected 4 h after dose 1 (n = 46), before dose 7 (n = 56), and after dose 9 (n = 54) infusions of Bu. Su (mean ± SD) was detectable in plasma of patients 4 h after dose 1, and higher levels were observed after dose 9 (249.9 ± 123.4 ng/mL). This method may be used in clinical studies investigating the role of Su on adverse events and drug interactions associated with Bu therapy.

Determination of trimipramine and its demethylated and hydroxylated metabolites in plasma by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric (GC-MS) method has been developed, for the determination of trimipramine (TRI), desmethyltrimipramine (DTRI), didesmethyltrimipramine (DDTRI), 2-hydroxytrimipramine (2-OH-TRI) and 2-hydroxydesmethyltrimipramine (2-OH-DTRI). The method includes two derivatization steps with trifluoroacetic acid anhydride and N-methyl-N-(tert.-butyldimethyl silyl)trifluoroacetamide and the use of an SE-54 capillary silica column. The limits of quantitation were found to be 2 ng/ml for DTRI and 4 ng/ml for all other substances. Besides, methods have been optimized for the hydrolysis of the glucuronic acid conjugated metabolites. This specific detection method is useful, as polymedication is a usual practice in clinical situations, and its sensitivity allows its use for single-dose pharmacokinetic studies.

Metal complexation by electroanalytical techniques: hard- and soft-modelling approaches

En la investigació de la complexació de metalls mitjançant eines electroanalítiques són emprades dues aproximacions generals. La primera, anomenada de modelatge dur (hardmodelling), es basa en la formulació d'un model fisicoquímic conjunt per als processos electròdic i de complexació i en la resolució analítica o numèrica del model. Posteriorment, l'ajust dels paràmetres del model a les dades experimentals donarà la informació desitjada sobre el procés de complexació. La segona aproximació, anomenada de modelatge tou (soft-modelling), es basa en la identificació d'un model de complexació a partir de l'anàlisi numèrica i estadística de les dades, sense cap assumpció prèvia d'un model. Aquesta aproximació, que ha estat extensivament emprada amb dades espectroscòpiques, ho ha estat poquíssim amb dades electroquímiques. En aquest article tractem de la formulació d'un model (hard-modelling) per a la complexació de metalls en sistemes amb mescles de lligands, incloent-hi lligands macromoleculars, i de l'aplicació d

Percutaneous endoscopic gastrostomy in head and neck cancer patients: indications, techniques, complications and results.

The aim of this study was to review our experience in percutaneous endoscopic gastrostomy (PEG) performed in patients with cancer of the upper aerodigestive tract. Descriptive retrospective study of 142 patients (115 males, 27 females), mean age 62.4 years (25-84 years), with head and neck or esophageal cancer, who underwent PEG tube insertion between January 2006 and December 2008. The studied parameters were indications, success rate, rate and type of complications, and their management. Percutaneous endoscopic gastrostomy was inserted before chemoradiation therapy in 80% and during or after cancer treatment in 20% of the patients. PEG placement was possible in 137 patients (96%). Major complications were observed in 9 (7%) and minor complications in 22 (17%) of the 137 patients. Seven of the 9 patients with a major complication needed revision surgery. The mortality directly related to the procedure was 0.7%. Percutaneous endoscopic gastrostomy tube insertion has a high success rate. In patients with upper aerodigestive tract cancer, PEG should be the first choice for enteral nutrition when sufficient oral intake is not possible. Although apparently easy, the procedure may occasionally lead to severe complications. Therefore, a strict technique and knowledge of clinical signs of possible complications are mandatory.

A validated assay by liquid chromatography-tandem mass spectrometry for the simultaneous quantification of elvitegravir and rilpivirine in HIV positive patients.

Because of the large variability in the pharmacokinetics of anti-HIV drugs, therapeutic drug monitoring in patients may contribute to optimize the overall efficacy and safety of antiretroviral therapy. An LC-MS/MS method for the simultaneous assay in plasma of the novel antiretroviral agents rilpivirine (RPV) and elvitegravir (EVG) has been developed to that endeavor. Plasma samples (100 μL) extraction is performed by protein precipitation with acetonitrile, and the supernatant is subsequently diluted 1:1 with 20-mM ammonium acetate/MeOH 50:50. After reverse-phase chromatography, quantification of RPV and EVG, using matrix-matched calibration samples, is performed by electrospray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The stable isotopic-labeled compounds RPV-(13) C6 and EVG-D6 were used as internal standards. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<6.4%), as well as EVG and RPV short and long-term stability in plasma. Calibration curves were validated over the clinically relevant concentrations ranging from 5 to 2500 ng/ml for RPV and from 50 to 5000 ng/ml for EVG. The method is precise (inter-day CV%: 3-6.3%) and accurate (3.8-7.2%). Plasma samples were found to be stable (<15%) in all considered conditions (RT/48 h, +4°C/48 h, -20°C/3 months and 60°C/1 h). Selected metabolite profiles analysis in patients' samples revealed the presence of EVG glucuronide, that was well separated from parent EVG, allowing to exclude potential interferences through the in-source dissociation of glucuronide to parent drug. This new, rapid and robust LCMS/MS assay for the simultaneous quantification of plasma concentrations of these two major new anti-HIV drugs EVG and RPV offers an efficient analytical tool for clinical pharmacokinetics studies and routine therapeutic drug monitoring service. Copyright © 2013 John Wiley & Sons, Ltd.

Choosing among techniques for quantifying single-case intervention effectiveness

If single case experimental designs are to be used to establish guidelines for evidence-based interventions in clinical and educational settings, numerical values that reflect treatment effect sizes are required. The present study compares four recently developed procedures for quantifying the magnitude of intervention effect using data with known characteristics. Monte Carlo methods were used to generate AB designs data with potential confounding variables (serial dependence, linear and curvilinear trend, and heteroscedasticity between phases) and two types of treatment effect (level and slope change). The results suggest that data features are important for choosing the appropriate procedure and, thus, inspecting the graphed data visually is a necessary initial stage. In the presence of serial dependence or a change in data variability, the Nonoverlap of All Pairs (NAP) and the Slope and Level Change (SLC) were the only techniques of the four examined that performed adequately. Introducing a data correction step in NAP renders it unaffected by linear trend, as is also the case for the Percentage of Nonoverlapping Corrected Data and SLC. The performance of these techniques indicates that professionals" judgments concerning treatment effectiveness can be readily complemented by both visual and statistical analyses. A flowchart to guide selection of techniques according to the data characteristics identified by visual inspection is provided.

Sensitive determination of D-lactic acid and L-lactic acid in urine by high-performance liquid chromatography-tandem mass spectrometry.

D-lactic acid in urine originates mainly from bacterial production in the intestinal tract. Increased D-lactate excretion as observed in patients affected by short bowel syndrome or necrotizing enterocolitis reflects D-lactic overproduction. Therefore, there is a need for a reliable and sensitive method able to detect D-lactic acid even at subclinical elevation levels. A new and highly sensitive method for the simultaneous determination of L- and D-lactic acid by a two-step procedure has been developed. This method is based on the concentration of lactic acid enantiomers from urine by supported liquid extraction followed by high-performance liquid chromatography-tandem mass spectrometry. The separation was achieved by the use of an Astec Chirobiotic? R chiral column under isocratic conditions. The calibration curves were linear over the ranges of 2-400 and 0.5-100 µmol/L respectively for L- and D-lactic acid. The limit of detection of D-lactic acid was 0.125 µmol/L and its limit of quantification was 0.5 µmol/L. The overall accuracy and precision were well within 10% of the nominal values. The developed method is suitable for production of reference values in children and could be applied for accurate routine analysis.

Low Cost Techniques of Base Stabilization, HR-312, 1994

This project was initiated in 1988 to study the effectiveness of four different construction techniques for establishing a stable base on a granular surfaced roadway. After base stabilization, the roadway was then seal coated, eliminating dust problems associated with granular surfaced roads. When monies become available, the roadway can be surfaced with a more permanent structure. A 2.8 mi (4.5 km) section of the Horseshoe Road in Dubuque County was divided into four divisions for this study. This report discusses the procedures used during construction of these different divisions. Problems and possible solutions have been analyzed to better understand the capabilities of the materials and construction techniques used on the project. The project had the following results: High structural ratings and soil K factors for the BIO CAT and Consolid bases did not translate to good roadway performance; the macadam base had the best overall performance; the tensar fabric had no noticeable effect on the macadam base; and the HFE-300 performed acceptably.