Fast quantification of ten psychotropic drugs and metabolites in human plasma by ultra-high performance liquid chromatography tandem mass spectrometry for therapeutic drug monitoring.

A sensitive and selective ultra-high performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method was developed for the fast quantification of ten psychotropic drugs and metabolites in human plasma for the needs of our laboratory (amisulpride, asenapine, desmethyl-mirtazapine, iloperidone, mirtazapine, norquetiapine, olanzapine, paliperidone, quetiapine and risperidone). Stable isotope-labeled internal standards were used for all analytes, to compensate for the global method variability, including extraction and ionization variations. Sample preparation was performed by generic protein precipitation with acetonitrile. Chromatographic separation was achieved in less than 3.0min on an Acquity UPLC BEH Shield RP18 column (2.1mm×50mm; 1.7μm), using a gradient elution of 10mM ammonium formate buffer pH 3.0 and acetonitrile at a flow rate of 0.4ml/min. The compounds were quantified on a tandem quadrupole mass spectrometer operating in positive electrospray ionization mode, using multiple reaction monitoring. The method was fully validated according to the latest recommendations of international guidelines. Eight point calibration curves were used to cover a large concentration range 0.5-200ng/ml for asenapine, desmethyl-mirtazapine, iloperidone, mirtazapine, olanzapine, paliperidone and risperidone, and 1-1500ng/ml for amisulpride, norquetiapine and quetiapine. Good quantitative performances were achieved in terms of trueness (93.1-111.2%), repeatability (1.3-8.6%) and intermediate precision (1.8-11.5%). Internal standard-normalized matrix effects ranged between 95 and 105%, with a variability never exceeding 6%. The accuracy profiles (total error) were included in the acceptance limits of ±30% for biological samples. This method is therefore suitable for both therapeutic drug monitoring and pharmacokinetic studies.

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.

Perception and Interpretation of Advance Warning Signs on County Roads, HR-256, 1984

This research consisted of five laboratory experiments designed to address the following two objectives in an integrated analysis: (1) To discriminate between the symbol Stop Ahead warning sign and a small set of other signs (which included the word-legend Stop Ahead sign); and (2) To analyze sign detection, recognizability, and processing characteristics by drivers. A set of 16 signs was used in each of three experiments. A tachistoscope was used to display each sign image to a respondent for a brief interval in a controlled viewing experiment. The first experiment was designed to test detection of a sign in the driver's visual field; the second experiment was designed to test the driver's ability to recognize a given sign in the visual field; and the third experiment was designed to test the speed and accuracy of a driver's response to each sign as a command to perform a driving action. A fourth experiment tested the meanings drivers associated with an eight-sign subset of the 16 signs used in the first three experiments. A fifth experiment required all persons to select which (if any) signs they considered to be appropriate for use on two scale model county road intersections. The conclusions are that word-legend Stop Ahead signs are more effective driver communication devices than symbol stop-ahead signs; that it is helpful to drivers to have a word plate supplementing the symbol sign if a symbol sign is used; and that the guidance in the Manual on Uniform Traffic Control Devices on the placement of advance warning signs should not supplant engineering judgment in providing proper sign communication at an intersection.

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.

Regional Approach to Landslide Interpretation and Repair, TR-430, 2001

The objective of this report is to provide Iowa county engineers and highway maintenance personnel with procedures that will allow them to efficiently and effectively interpret and repair or avoid landslides. The research provides an overview of basic slope stability analyses that can be used to diagnose the cause and effect associated with a slope failure. Field evidence for identifying active or potential slope stability problems is outlined. A survey of county engineers provided data for presenting a slope stability risk map for the state of Iowa. Areas of high risk are along the western border and southeastern portion of the state. These regions contain deep to moderately deep loess. The central portion of the state is a low risk area where the surficial soils are glacial till or thin loess over till. In this region, the landslides appear to occur predominately in backslopes along deeply incised major rivers, such as the Des Moines River, or in foreslopes. The south-central portion of the state is an area of medium risk where failures are associated with steep backslopes and improperly compacted foreslopes. Soil shear strength data compiled from the Iowa DOT and consulting engineers files are correlated with geologic parent materials and mean values of shear strength parameters and unit weights were computed for glacial till, friable loess, plastic loess and local alluvium. Statistical tests demonstrate that friction angles and unit weights differ significantly but in some cases effective stress cohesion intercept and undrained shear strength data do not. Moreover, effective stress cohesion intercept and undrained shear strength data show a high degree of variability. The shear strength and unit weight data are used in slope stability analyses for both drained and undrained conditions to generate curves that can be used for a preliminary evaluation of the relative stability of slopes within the four materials. Reconnaissance trips to over fifty active and repaired landslides in Iowa suggest that, in general, landslides in Iowa are relatively shallow [i.e., failure surfaces less than 6 ft (2 m) deep] and are either translational or shallow rational. Two foreslope and two backslope failure case histories provide additional insights into slope stability problems and repair in Iowa. These include the observation that embankment soils compacted to less than 95% relative density show a marked strength decrease from soils at or above that density. Foreslopes constructed of soils derived from shale exhibit loss of strength as a result of weathering. In some situations, multiple causes of instability can be discerned from back analyses with the slope stability program XSTABL. In areas where the stratigraphy consists of loess over till or till over bedrock, the geologic contracts act as surfaces of groundwater accumulation that contribute to slope instability.

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.

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).

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.

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.

Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology.

Until recently, microbial identification in clinical diagnostic laboratories has mainly relied on conventional phenotypic and gene sequencing identification techniques. The development of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) devices has revolutionized the routine identification of microorganisms in clinical microbiology laboratories by introducing an easy, rapid, high throughput, low-cost, and efficient identification technique. This technology has been adapted to the constraint of clinical diagnostic laboratories and has the potential to replace and/or complement conventional identification techniques for both bacterial and fungal strains. Using standardized procedures, the resolution of MALDI-TOF MS allows accurate identification at the species level of most Gram-positive and Gram-negative bacterial strains with the exception of a few difficult strains that require more attention and further development of the method. Similarly, the routine identification by MALDI-TOF MS of yeast isolates is reliable and much quicker than conventional techniques. Recent studies have shown that MALDI-TOF MS has also the potential to accurately identify filamentous fungi and dermatophytes, providing that specific standardized procedures are established for these microorganisms. Moreover, MALDI-TOF MS has been used successfully for microbial typing and identification at the subspecies level, demonstrating that this technology is a potential efficient tool for epidemiological studies and for taxonomical classification.

Addendum to Perception and Interpretation of Advance Warning Signs on County Roads, HR-256, 1985

This contract extension was granted to analyze data obtained in the original contract period at a level of detail not called for in the original contract nor permitted by the time constraints of the original contract schedule. These further analyses focused on two primary questions: I. What sources of variation can be isolated within the overall pattern of driver recognition errors reported previously for the 16 signs tested in Project HR-256? 2. Were there systematic relations among data on the placement of signs in a simulated signing exercise and data on the respondents' ability to detect the presence of a sign in a visual field or their ability to recognize quickly and correctly a sign shown them or the speed with which these same persons can respond to a sign for a driver decision?

Auditory-somatosensory multisensory interactions are spatially modulated by stimulated body surface and acoustic spectra

Previous research has provided inconsistent results regarding the spatial modulation of auditory-somatosensory interactions. The present study reports three experiments designed to investigate the nature of these interactions in the space close to the head. Human participants made speeded detection responses to unimodal auditory, somatosensory, or simultaneous auditory-somatosensory stimuli. In Experiment 1, electrocutaneous stimuli were presented to either earlobe, while auditory stimuli were presented from the same versus opposite sides, and from one of two distances (20 vs. 70cm) from the participant's head. The results demonstrated a spatial modulation of auditory-somatosensory interactions when auditory stimuli were presented from close to the head. In Experiment 2, electrocutaneous stimuli were delivered to the hands, which were placed either close to or far from the head, while the auditory stimuli were again presented at one of two distances. The results revealed that the spatial modulation observed in Experiment 1 was specific to the particular body part stimulated (head) rather than to the region of space (i.e. around the head) where the stimuli were presented. The results of Experiment 3 demonstrate that sounds that contain high-frequency components are particularly effective in eliciting this auditory-somatosensory spatial effect. Taken together, these findings help to resolve inconsistencies in the previous literature and suggest that auditory-somatosensory multisensory integration is modulated by the stimulated body surface and acoustic spectra of the stimuli presented.

Gas-chromatography mass-spectrometry determination of phthalic acid in human urine as a biomarker of folpet exposure.

Agricultural workers are exposed to folpet, but biomonitoring data are limited. Phthalimide (PI), phthalamic acid (PAA), and phthalic acid (PA) are the ring metabolites of this fungicide according to animal studies, but they have not yet been measured in human urine as metabolites of folpet, only PA as a metabolite of phthalates. The objective of this study was thus to develop a reliable gas chromatography-tandem mass spectrometry (GC-MS) method to quantify the sum of PI, PAA, and PA ring-metabolites of folpet in human urine. Briefly, the method consisted of adding p-methylhippuric acid as an internal standard, performing an acid hydrolysis at 100 °C to convert ring-metabolites into PA, purifying samples by ethyl acetate extraction, and derivatizing with N,O-bis(trimethylsilyl)trifluoro acetamide prior to GC-MS analysis. The method had a detection limit of 60.2 nmol/L (10 ng/mL); it was found to be accurate (mean recovery, 97%), precise (inter- and intra-day percentage relative standard deviations <13%), and with a good linearity (R (2) > 0.98). Validation was conducted using unexposed peoples urine spiked at concentrations ranging from 4.0 to 16.1 μmol/L, along with urine samples of volunteers dosed with folpet, and of exposed workers. The method proved to be (1) suitable and accurate to determine the kinetic profile of PA equivalents in the urine of volunteers orally and dermally administered folpet and (2) relevant for the biomonitoring of exposure in workers.