Rapid high-performance liquid chromatographic determination with fluorescence detection of furosemide in human body fluids and its confirmation by gas chromatography-mass spectrometry.

Furosemide (FD: Lasix) is a loop diuretic which strongly increases both urine flow and electrolyte urinary excretion. Healthy volunteers were administered 40 mg orally (dissolved in water) and concentrations of FD were determined in serum and urine for up to 6 h for eight subjects, who absorbed water at a rate of 400 ml/h. Quantification was performed by HPLC with fluorescence detection (excitation at 233 nm, emission at 389 nm) with a limit of detection of 5 ng/ml for a 300-microliters sample. The elution of FD was completed within 4 min using a gradient of acetonitrile concentration rising from 30 to 50% in 0.08 M phosphoric acid. The delay to the peak serum concentration ranged from 60 to 120 min. FD was still easily measurable in the sera from all subjects 6 h after administration. In urine, the excretion rates reached their maximum between 1 and 3 h. The total amount of FD excreted in the urine averaged 11.2 mg (range 7.6-14.0 mg), with a mean urine volume of 3024 ml (range 2620-3596 ml). Moreover, the urine density was lower than 1.010 (recommended as an upper limit in doping analysis to screen diuretics) only for 2 h. An additional volunteer was administered 40 mg of FD and his urine was collected over a longer period. FD was still detectable 48 h after intake. Gas chromatography-mass spectrometry with different types of ionization was used to confirm the occurrence of FD after permethylation of the extract. Negative-ion chemical ionization, with ammonia as reactant gas, was found to be the most sensitive method of detection.

Forensic examination of ink by high-performance thin layer chromatography - The United States Secret Service Digital Ink Library

Forensic examinations of ink have been performed since the beginning of the 20th century. Since the 1960s, the International Ink Library, maintained by the United States Secret Service, has supported those analyses. Until 2009, the search and identification of inks were essentially performed manually. This paper describes the results of a project designed to improve ink samples' analytical and search processes. The project focused on the development of improved standardization procedures to ensure the best possible reproducibility between analyses run on different HPTLC plates. The successful implementation of this new calibration method enabled the development of mathematical algorithms and of a software package to complement the existing ink library.

Using a single tablet daily to treat latent tuberculosis infection in Brazil: bioequivalence of two different isoniazid formulations (300 mg and 100 mg) demonstrated by a sensitive and rapid high-performance liquid chromatography-tandem mass spectrometry method in a randomised, crossover study

The recommended treatment for latent tuberculosis (TB) infection in adults is a daily dose of isoniazid (INH) 300 mg for six months. In Brazil, INH was formulated as 100 mg tablets. The treatment duration and the high pill burden compromised patient adherence to the treatment. The Brazilian National Programme for Tuberculosis requested a new 300 mg INH formulation. The aim of our study was to compare the bioavailability of the new INH 300 mg formulation and three 100 mg tablets of the reference formulation. We conducted a randomised, single dose, open label, two-phase crossover bioequivalence study in 28 healthy human volunteers. The 90% confidence interval for the INH maximum concentration of drug observed in plasma and area under the plasma concentration vs. time curve from time zero to the last measurable concentration “time t” was 89.61-115.92 and 94.82-119.44, respectively. The main limitation of our study was that neither adherence nor the safety profile of multiple doses was evaluated. To determine the level of INH in human plasma, we developed and validated a sensitive, simple and rapid high-performance liquid chromatography-tandem mass spectrometry method. Our results showed that the new formulation was bioequivalent to the 100 mg reference product. This finding supports the use of a single 300 mg tablet daily strategy to treat latent TB. This new formulation may increase patients’ adherence to the treatment and quality of life.

Quantification of typical antipsychotics in human plasma by ultra-high performance liquid chromatography tandem mass spectrometry for therapeutic drug monitoring.

A selective and sensitive method was developed for the simultaneous quantification of seven typical antipsychotic drugs (cis-chlorprothixene, flupentixol, haloperidol, levomepromazine, pipamperone, promazine and zuclopenthixol) in human plasma. Ultra-high performance liquid chromatography (UHPLC) was used for complete separation of the compounds in less than 4.5min on an Acquity UPLC BEH C18 column (2.1mm×50mm; 1.7μm), with a gradient elution of ammonium formate buffer pH 4.0 and acetonitrile at a flow rate of 400μl/min. Detection was performed on a tandem quadrupole mass spectrometer (MS/MS) equipped with an electrospray ionization interface. A simple protein precipitation procedure with acetonitrile was used for sample preparation. Thanks to the use of stable isotope-labeled internal standards for all analytes, internal standard-normalized matrix effects were in the range of 92-108%. The method was fully validated to cover large concentration ranges of 0.2-90ng/ml for haloperidol, 0.5-90ng/ml for flupentixol, 1-450ng/ml for levomepromazine, promazine and zuclopenthixol and 2-900ng/ml for cis-chlorprothixene and pipamperone. Trueness (89.1-114.8%), repeatability (1.8-9.9%), intermediate precision (1.9-16.3%) and accuracy profiles (<30%) were in accordance with the latest international recommendations. The method was successfully used in our laboratory for routine quantification of more than 500 patient plasma samples for therapeutic drug monitoring. To the best of our knowledge, this is the first UHPLC-MS/MS method for the quantification of the studied drugs with a sample preparation based on protein precipitation.

Variability of voriconazole plasma levels measured by new high-performance liquid chromatography and bioassay methods.

Voriconazole (VRC) is a broad-spectrum antifungal triazole with nonlinear pharmacokinetics. The utility of measurement of voriconazole blood levels for optimizing therapy is a matter of debate. Available high-performance liquid chromatography (HPLC) and bioassay methods are technically complex, time-consuming, or have a narrow analytical range. Objectives of the present study were to develop new, simple analytical methods and to assess variability of voriconazole blood levels in patients with invasive mycoses. Acetonitrile precipitation, reverse-phase separation, and UV detection were used for HPLC. A voriconazole-hypersusceptible Candida albicans mutant lacking multidrug efflux transporters (cdr1Delta/cdr1Delta, cdr2Delta/cdr2Delta, flu1Delta/flu1Delta, and mdr1Delta/mdr1Delta) and calcineurin subunit A (cnaDelta/cnaDelta) was used for bioassay. Mean intra-/interrun accuracies over the VRC concentration range from 0.25 to 16 mg/liter were 93.7% +/- 5.0%/96.5% +/- 2.4% (HPLC) and 94.9% +/- 6.1%/94.7% +/- 3.3% (bioassay). Mean intra-/interrun coefficients of variation were 5.2% +/- 1.5%/5.4% +/- 0.9% and 6.5% +/- 2.5%/4.0% +/- 1.6% for HPLC and bioassay, respectively. The coefficient of concordance between HPLC and bioassay was 0.96. Sequential measurements in 10 patients with invasive mycoses showed important inter- and intraindividual variations of estimated voriconazole area under the concentration-time curve (AUC): median, 43.9 mg x h/liter (range, 12.9 to 71.1) on the first and 27.4 mg x h/liter (range, 2.9 to 93.1) on the last day of therapy. During therapy, AUC decreased in five patients, increased in three, and remained unchanged in two. A toxic encephalopathy probably related to the increase of the VRC AUC (from 71.1 to 93.1 mg x h/liter) was observed. The VRC AUC decreased (from 12.9 to 2.9 mg x h/liter) in a patient with persistent signs of invasive aspergillosis. These preliminary observations suggest that voriconazole over- or underexposure resulting from variability of blood levels might have clinical implications. Simple HPLC and bioassay methods offer new tools for monitoring voriconazole therapy.

Evaluation of a Bridge Constructed using High Performance Steel, May 2006

Of the approximately 25,000 bridges in Iowa, 28% are classified as structurally deficient, functionally obsolete, or both. Because many Iowa bridges require repair or replacement with a relatively limited funding base, there is a need to develop new bridge materials that may lead to longer life spans and reduced life-cycle costs. In addition, new and effective methods for determining the condition of structures are needed to identify when the useful life has expired or other maintenance is needed. Due to its unique alloy blend, high-performance steel (HPS) has been shown to have improved weldability, weathering capabilities, and fracture toughness than conventional structural steels. Since the development of HPS in the mid-1990s, numerous bridges using HPS girders have been constructed, and many have been economically built. The East 12th Street Bridge, which replaced a deteriorated box girder bridge, is Iowa’s first bridge constructed using HPS girders. The new structure is a two-span bridge that crosses I-235 in Des Moines, Iowa, providing one lane of traffic in each direction. A remote, continuous, fiber-optic based structural health monitoring (SHM) system for the bridge was developed using off-the-shelf technologies. In the system, sensors strategically located on the bridge collect raw strain data and then transfer the data via wireless communication to a gateway system at a nearby secure facility. The data are integrated and converted to text files before being uploaded automatically to a website that provides live strain data and a live video stream. A data storage/processing system at the Bridge Engineering Center in Ames, Iowa, permanently stores and processes the data files. Several processes are performed to check the overall system’s operation, eliminate temperature effects from the complete strain record, compute the global behavior of the bridge, and count strain cycles at the various sensor locations.

High Performance Concret Bridge Deck Overlays for County Bridges, October 2007

The Iowa Method for bridge deck overlays has been very successful in Iowa since its adoption in the 1970s. This method involves removal of deteriorated portions of a bridge deck followed by placement of a layer of den (Type O) Portland Cement Concrete (PCC). The challenge encountered with this type of bridge deck overlay is that the PCC must be mixed on-site, brought to the placement area and placed with specialized equipment. This adds considerably to the cost and limits contractor selection. A previous study (TR-427) showed that a dense PCC with high-range water reducers could successfully be used for bridge deck overlays using conventional equipment and methods. This current study evaluated the use of high performance PCC in place of a dense PCC for work on county bridges. High performance PCC uses fly ash and slag to replace some of the cement in the mix. This results in a workable PCC mix that cures to form a very low permeability overlay.

Design and Evaluation of a Single-Span Bridge Using Ultra- High Performance Concrete, September 2009

Research presented herein describes an application of a newly developed material called Ultra-High Performance Concrete (UHPC) to a single-span bridge. The two primary objectives of this research were to develop a shear design procedure for possible code adoption and to provide a performance evaluation to ensure the viability of the first UHPC bridge in the United States. Two other secondary objectives included defining of material properties and understanding of flexural behavior of a UHPC bridge girder. In order to obtain information in these areas, several tests were carried out including material testing, large-scale laboratory flexure testing, large-scale laboratory shear testing, large-scale laboratory flexure-shear testing, small-scale laboratory shear testing, and field testing of a UHPC bridge. Experimental and analytical results of the described tests are presented. Analytical models to understand the flexure and shear behavior of UHPC members were developed using iterative computer based procedures. Previous research is referenced explaining a simplified flexural design procedure and a simplified pure shear design procedure. This work describes a shear design procedure based on the Modified Compression Field Theory (MCFT) which can be used in the design of UHPC members. Conclusions are provided regarding the viability of the UHPC bridge and recommendations are made for future research.

Design and Performance Verifcation of Ultra-High Performance Concrete Piles for Deep Foundations Final Report, November 2008

The strategic plan for bridge engineering issued by AASHTO in 2005 identified extending the service life and optimizing structural systems of bridges in the United States as two grand challenges in bridge engineering, with the objective of producing safer bridges that have a minimum service life of 75 years and reduced maintenance cost. Material deterioration was identified as one of the primary challenges to achieving the objective of extended life. In substructural applications (e.g., deep foundations), construction materials such as timber, steel, and concrete are subjected to deterioration due to environmental impacts. Using innovative and new materials for foundation applications makes the AASHTO objective of 75 years service life achievable. Ultra High Performance Concrete (UHPC) with compressive strength of 180 MPa (26,000 psi) and excellent durability has been used in superstructure applications but not in geotechnical and foundation applications. This study explores the use of precast, prestressed UHPC piles in future foundations of bridges and other structures. An H-shaped UHPC section, which is 10-in. (250-mm) deep with weight similar to that of an HP10×57 steel pile, was designed to improve constructability and reduce cost. In this project, instrumented UHPC piles were cast and laboratory and field tests were conducted. Laboratory tests were used to verify the moment-curvature response of UHPC pile section. In the field, two UHPC piles have been successfully driven in glacial till clay soil and load tested under vertical and lateral loads. This report provides a complete set of results for the field investigation conducted on UHPC H-shaped piles. Test results, durability, drivability, and other material advantages over normal concrete and steel indicate that UHPC piles are a viable alternative to achieve the goals of AASHTO strategic plan.

Single-step extraction of fluconazole from plasma by ultra-filtration for the measurement of its free concentration by high performance liquid chromatography.

High performance liquid chromatography (HPLC) is the reference method for measuring concentrations of antimicrobials in blood. This technique requires careful sample preparation. Protocols using organic solvents and/or solid extraction phases are time consuming and entail several manipulations, which can lead to partial loss of the determined compound and increased analytical variability. Moreover, to obtain sufficient material for analysis, at least 1 ml of plasma is required. This constraint makes it difficult to determine drug levels when blood sample volumes are limited. However, drugs with low plasma-protein binding can be reliably extracted from plasma by ultra-filtration with a minimal loss due to the protein-bound fraction. This study validated a single-step ultra-filtration method for extracting fluconazole (FLC), a first-line antifungal agent with a weak plasma-protein binding, from plasma to determine its concentration by HPLC. Spiked FLC standards and unknowns were prepared in human and rat plasma. Samples (240 microl) were transferred into disposable microtube filtration units containing cellulose or polysulfone filters with a 5 kDa cut-off. After centrifugation for 60 min at 15000g, FLC concentrations were measured by direct injection of the filtrate into the HPLC. Using cellulose filters, low molecular weight proteins were eluted early in the chromatogram and well separated from FLC that eluted at 8.40 min as a sharp single peak. In contrast, with polysulfone filters several additional peaks interfering with the FLC peak were observed. Moreover, the FLC recovery using cellulose filters compared to polysulfone filters was higher and had a better reproducibility. Cellulose filters were therefore used for the subsequent validation procedure. The quantification limit was 0.195 mgl(-1). Standard curves with a quadratic regression coefficient &gt; or = 0.9999 were obtained in the concentration range of 0.195-100 mgl(-1). The inter and intra-run accuracies and precisions over the clinically relevant concentration range, 1.875-60 mgl(-1), fell well within the +/-15% variation recommended by the current guidelines for the validation of analytical methods. Furthermore, no analytical interference was observed with commonly used antibiotics, antifungals, antivirals and immunosuppressive agents. Ultra-filtration of plasma with cellulose filters permits the extraction of FLC from small volumes (240 microl). The determination of FLC concentrations by HPLC after this single-step procedure is selective, precise and accurate.

Investigation into Shrinkage of High-Performance Concrete Used for Iowa Bridge Decks and Overlays, TR-633, 2013

High-performance concrete (HPC) overlays have been used increasingly as an effective and economical method for bridge decks in Iowa and other states. However, due to its high cementitious material content, HPC often displays high shrinkage cracking potential. This study investigated the shrinkage behavior and cracking potential of the HPC overlay mixes commonly used in Iowa. In the study, 11 HPC overlay mixes were studied. These mixes consisted of three types of cements (Type I, I/II, and IP) and various supplementary cementitious materials (Class C fly ash, slag and metakaolin). Limestone with two different gradations was used as coarse aggregates in 10 mixes and quartzite was used in one mix. Chemical shrinkage of pastes, free drying shrinkage, autogenous shrinkage of mortar and concrete, and restrained ring shrinkage of concrete were monitored over time. Mechanical properties (such as elastic modulus and compressive and splitting tensile strength) of these concrete mixes were measured at different ages. Creep coefficients of these concrete mixes were estimated using the RILEM B3 and NCHRP Report 496 models. Cracking potential of the concrete mixes was assessed based on both ASTM C 1581 and simple stress-to-strength ratio methods. The results indicate that among the 11 mixes studied, three mixes (4, 5, and 6) cracked at the age of 15, 11, and 17 days, respectively. Autogenous shrinkage of the HPC mixes ranges from 150 to 250 microstrain and free dying shrinkage of the concrete ranges from 700 to 1,200 microstrain at 56 days. Different concrete materials (cementitious type and admixtures) and mix proportions (cementitious material content) affect concrete shrinkage in different ways. Not all mixes having a high shrinkage value cracked first. The stresses in the concrete are associated primarily with the concrete shrinkage, elastic modulus, tensile strength, and creep. However, a good relationship is found between cementitious material content and total (autogenous and free drying) shrinkage of concrete.

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.

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.