Dissociation between lactate accumulation and acidosis in middle cerebral artery-occluded rats assessed by P and H NMR metabolic images under A 2-T magnetic field

The relationships among tissue edema, lactate accumulation, and intracellular pH in middle cerebral artery (MCA)-occluded rats were investigated with multiecho ¹H magnetic resonance imaging and spatially resolved metabolic images constructed by ¹H and ³¹P nuclear magnetic resonance (NMR) chemical shift imaging (CSI). For the effective and sensitive detection of NMR signals from the brain, outer volume suppression (OVS), reduced k-space sampling and proton irradiation were incorporated into the CSI sequences. The consecutive three measurements of calculated T₂ image, lactate image, and pH image which were required for 3.75 h were repeated for four cycles of 1–16 h after MCA occlusion. Tissue edema and lactate accumulation in the infarcted region were gradually and consistently increased during the 15-h observation period. In contrast, severe acidosis was already detected on the first pH image (2–4.7 h after MCA occlusion); thereafter, the degree of acidosis became milder and showed no further progression. The dissociation between the time courses of the lactate accumulation and pH decrease was clearly demonstrated by the NMR metabolic images. Acid-base balance in cerebral infarction might be affected not only by lactate production but also by complicated interactions with tissue edema and some other factors.     

The rheological properties of the arterial wall

Key words The rheological properties of rat arteries were studied in vitro. Using intact arterial segments undergoing large deformations in longitudinal and circumferential direction, both the stress-strain relationship and the corresponding histological changes with deformation are presented. Correlations between the mechanical properties and the histological structure are discussed.     

Chromatographic Detection of Trimetoquinol (Inolin®) and its Major Urinary Metabolites in the Horse: A Preliminary Report

Trimetoquinol (TMQ) (1-(3,4,5-trimethoxybenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, m.w. 345) is the prototype tetrahydroisoquinoline pharmaceutical. TMQ is marketed as a bronchodilator in human medicine; in horse racing, TMQ is listed as an Association of Racing Commissioners International (ARCI) class 3 foreign substance. As such, TMQ is considered to have the potential to affect racing performance in horses, and a validated qualitative confirmatory method is required to regulate its use in racing. We selected 8 g kg^–1 of TMQ IV as a safe and effective dose for studies on its metabolism and analytical detection in horses. We developed a solid phase extraction method for recovery of TMQ and its metabolites from equine urine, identified suitable high performance liquid chromatographic conditions for these substances and our internal standard, papaverine, and developed a highly sensitive ESI(+)-LC-MS-MS method (estimated LOD, 100 pg mL^–1) for TMQ and its major metabolites in equine urine. Multiple Reaction Monitoring (MRM) analysis of unhydrolyzed post-administration urine showed small amounts of unchanged TMQ, along with glucuronide, methylated, and sulfated metabolites, with glucuronide metabolites predominating. Following glucuronidase hydrolysis, recovered parent TMQ peaked at relatively high concentrations (>300 ng mL^–1) within 1 h of administration and thereafter declined. The methylated metabolites of TMQ peaked later and at comparable total concentrations, and thereafter declined more slowly. These data suggest that glucuronide hydrolysis of post-administration urine samples will allow recovery of readily identifiable quantities of parent TMQ. These findings, combined with the highly sensitive LC-MS-MS detection of parent TMQ described herein suggest that glucuronide hydrolysis of post-administration urine, followed by LC-MS-MS or other analysis, will allow effective regulatory control of this agent in racing horses.Published as # 351 from the Equine Pharmacology, Therapeutics and Toxicology Program at the Maxwell H. Gluck Equine Research Center and Department of Veterinary Science, University of Kentucky. Published as Kentucky Agricultural Experiment Station Article # 04-14-048 with the approval of the Dean and Director, College of Agriculture and the Kentucky Agricultural Experimental Station.Revised: 8 June and 12 July 2004     

Experimental design methodologies to optimise the spectrofluorimetric determination of Losartan and Valsartan in human urine

A spectrofluorimetric method has been developed for the determination of two angiotensin II receptor antagonists (ARA II): Losartan and Valsartan. A fractional factorial design and a central composite design were used. The key factors considered in the optimization process were pH, temperature and emission slit width. Maximum fluorescent intensity was established as response for each experiment. The response surfaces confirmed the robustness of the method. A clean-up procedure was used for urine samples that consisted of a solid-phase extraction using C8 cartridges. The total analysis time was lower than 30 min. This method proved to be accurate (RE, 8%), precise (intra- and inter-day coefficients of variation were lower than 8% and sensitive enough (LOQ c.a. 0.5 μg ml⁻¹) to be applied to the determination of Losartan and Valsartan in urine samples.     

Development and validation of a HPLC method for quantification of rivastigmine in rat urine and identification of a novel metabolite in urine by LC‐MS/MS

A sensitive, specific and accurate HPLC method for the quantification of rivastigmine (RSM) in rat urine was developed and validated. The method involves the simple liquid–liquid extraction of RSM and pyridostigmine as an internal standard (IS) from rat urine with tertiary methyl butyl ether. The chromatographic separation of RSM and IS was achieved with 20 mm ammonium acetate buffer (pH 6.5) and acetonitrile (65:35, v/v) delivered at flow‐rate of 1 mL/min on a Kromasil KR‐100. The method was in linear range from 50 to 5000 ng/mL. The validation was done as per FDA guidelines and the results met the acceptance criteria. The method was successfully applied for the quantification of RSM in rat urine. Besides method validation, we have identified two metabolites of RSM in urine. Both the metabolites were characterized by HPLC‐PDA and LC‐MS/MS and it was found that one metabolite is novel. Copyright © 2010 John Wiley & Sons, Ltd.     

Study on Fluorescence Property of Sparfloxacin Derivatizing System and its Application

Spartloxacin (SPFX) is the fourth generation of the antibacterial quinolones. Due to itsintensive and efficient antibacterial activity compared to the common quinolones '. it hasbeen widely used in clinical practice. Nevertheless. because of the structural dit'ferencesbetween SPFX and other quinolones. the fluorescence of SPFX is so weak that it can notbe determined by means of fluorescence spectra. Hence, it will be of great interest tomake a detailed study on its fluorescence enhancemen…     

Development and reproducibility of a novel high‐performance liquid‐chromatography monolithic column method for the detection and quantification of trans‐indolyl‐3‐acryloylglycine in human urine

Elevated levels of trans‐indolyl‐3‐acryloylglycine (IAcrGly) have been reported in the urine of people with various conditions including pervasive developmental disorders (PDDs) such as autism and Asperger syndrome. Reversed‐phase high‐performance liquid chromatography with ultra‐violet detection using traditional particle silica‐based columns subsequent to solid‐phase extraction (SPE) has been the preferred assay method; requiring long analytical run times, high flow rates and high solvent usage. Recent developments in monolithic HPLC column technology facilitated the development of a novel analytical method, for the detection and quantification of urinary IAcrGly. The revised method eliminates the requirement for SPE pre‐treatment, reduces sample run‐time and decreases solvent volumes. Five urine samples from people diagnosed with PDD were run in quadruplicate to test the intra‐ and inter‐day reliability of the new method based on retention time, peak area and peak height for IAcrGly. Detection was by UV with IAcrGly confirmation by MS/MS‐MS. Relative standard deviations showed significant improvement with the new method for all parameters. The new method represents a major advancement in the detection and quantification of IAcrGly by reducing time and cost of analysis whilst improving detection limits and reproducibility. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of β2‐agonists in human urine by fast‐gas chromatography/mass spectrometry: method validation and clinical application

A fast screening protocol was developed and validated for the simultaneous determination of 15 β₂‐agonists in human urine (bambuterol, cimbuterol, clenbuterol, fenoterol, formoterol, isoproterenol, mapenterol, metaproterenol, procaterol, ractopamine, ritodrine, salbutamol, salmeterol, terbutaline, tulobuterol). The overall sample processing includes deconjugation with enzyme hydrolysis, liquid–liquid extraction, followed by derivatization of the extract and detection of β₂‐agonists trimethylsilyl‐derivatives by fast‐gas chromatography/electron impact–mass spectrometry (fast‐GC/EI‐MS). Sample extraction and derivatization were optimized with the purpose of improving recoveries and reaction yields for a variety of analytes with different structures simultaneously, while keeping the procedure simple and reliable. Validation parameters were determined for each analyte under investigation, including selectivity, linearity, intra‐ and inter‐assay precision, extraction recoveries and signal to noise ratio (S/N) at the lowest calibration level. Fast‐GC/MS sequences, based on the use of short columns, high carrier‐gas velocity and fast temperature ramping, allow considerable reduction of the analysis time (7 min), while maintaining adequate chromatographic resolution. The overall GC cycle time was less than 9 min, allowing a processing rate of 6 samples/h. High MS‐sampling rate, using a benchtop quadrupole mass analyzer, resulted in accurate peak shape definition under both scan and selected ion monitoring modes, and high sensitivity in the latter mode. The method was successfully tested on real samples arising from clinical treatments. Copyright © 2009 John Wiley & Sons, Ltd.     

The influence of indoxyl sulfate and ammonium on the autofluorescence of human urine

Despite biological variability the spectral characteristics of undiluted human urine show relatively low autofluorescence at short UV (250-300 nm) excitation. However with dilution the fluorescence intensity remarkably increases. This paper examines the mechanisms behind this effect, by using excitation-emission matrices. Corrections for the inner filter effect were made for improved understanding of the spectral patterns. We focused on three major fluorophores (tryptophan, indoxyl sulfate and 5-hydroxyindole-3-acetate) that are excited at these wavelengths, and whose content in urine is strongly linked with various health conditions. Their fluorescence was studied both individually and in combinations. We also examined the effect of ammonium on the fluorescence of these major fluorophores individually and in combinations. Through these studies we have identified the leading effects that reduce the UV fluorescence, namely higher concentration of indoxyl sulfate producing the inner filter effect and concentration quenching and quenching of fluorophores by ammonium. This result will assist in broader utilisation of UV fluorescence in medical diagnostics.     

Determination of aristolochic acid in urine using hollow fiber liquid-phase microextraction combined with high-performance liquid chromatography

A simple and efficient hollow fiber liquid‐phase microextraction (HF‐LPME) technique in conjunction with high‐performance liquid chromatography is presented for extraction and quantitative determination of aristolochic acid I in human urine samples. Several parameters influencing the efficiency of HF‐LPME were investigated and optimized, including extraction solvent, stirring rate, extraction time, pH of donor phase and acceptor phase. Excellent sample clean‐up was observed and good linearity with coefficient of 0.9999 was obtained in the range of 15.4–960 µg/L. This method provided a 230‐fold enrichment factor and good repeatability with relative standard deviations (RSD) lower than 6.0%. The limit of detection value for the analyte in urine sample was 0.01 µg/L at a signal‐to‐noise ratio of 3. The extraction recovery from urine samples was 61.8% with an RSD of 9.71%. Copyright © 2010 John Wiley & Sons, Ltd.