On-line Radiometric Detection Research Articles

Metabolism, Distribution, and Excretion of Deoxynivalenol with Combined Techniques of Radiotracing, High-Performance Liquid Chromatography Ion Trap Time-of-Flight Mass Spectrometry, and Online Radiometric Detection

Dispositions of deoxynivalenol (DON) in rats and chickens were investigated, using a radiotracer method coupled with a novel γ-accurate radioisotope counting (γ-ARC) radio-high-performance liquid chromatography ion trap time-of-flight tandem mass spectrometry (radio-HPLC-IT-TOF-MS/MS) system. 3β-(3)H-DON was chemically synthesized and orally administrated to both sexes of rats and chickens as single or multiple doses. The results showed that DON was widely distributed and quickly eliminated in all tissues. The highest concentration was found in the gastrointestinal tract at 6 h post-administration. Substantially lower levels were detected in the kidney, liver, heart, lung, spleen, and brain. Three new metabolites were identified tentatively as 10-deoxynivalenol-sulfonate, 10-deepoxy-deoxynivalenol (DOM-1)-sulfonate, and deoxynivalenol-3α-sulfate. Deoxynivalenol-3α-sulfate was a major metabolite in chickens, while the major forms in rats were DOM-1 and DON. Additionally, a higher excretion rate in urine was observed in female rats than in male rats. The differences in metabolite profiles and excretion rates, which suggested diverse ways to detoxify, may relate to the different tolerances in different genders or species.

Automated Radioanalytical System Incorporating Microwave-Assisted Sample Preparation, Chemical Separation, and Online Radiometric Detection for the Monitoring of Total 99Tc in Nuclear Waste Processing Streams

An automated fluidic instrument is described that rapidly determines the total (99)Tc content of aged nuclear waste samples, where the matrix is chemically and radiologically complex and the existing speciation of the (99)Tc is variable. The monitor links microwave-assisted sample preparation with an automated anion exchange column separation and detection using a flow-through solid scintillator detector. The sample preparation steps acidify the sample, decompose organics, and convert all Tc species to the pertechnetate anion. The column-based anion exchange procedure separates the pertechnetate from the complex sample matrix, so that radiometric detection can provide accurate measurement of (99)Tc. We developed a preprogrammed spike addition procedure to automatically determine matrix-matched calibration. The overall measurement efficiency that is determined simultaneously provides a self-diagnostic parameter for the radiochemical separation and overall instrument function. Continuous, automated operation was demonstrated over the course of 54 h, which resulted in the analysis of 215 samples plus 54 hly spike-addition samples, with consistent overall measurement efficiency for the operation of the monitor. A sample can be processed and measured automatically in just 12.5 min with a detection limit of 23.5 Bq/mL of (99)Tc in low activity waste (0.495 mL sample volume), with better than 10% RSD precision at concentrations above the quantification limit. This rapid automated analysis method was developed to support nuclear waste processing operations planned for the Hanford nuclear site.

Identification of Novel Metabolites of Colchicine in Rat Bile Facilitated by Enhanced Online Radiometric Detection

Three novel conjugation metabolites of colchicine were identified in rat bile facilitated by enhanced on-line liquid chromatography-accurate radioisotope counting. The known 2- and 3-demethylcolchicines (DMCs) underwent O-sulfate conjugation in addition to the previously described O-glucuronidation. 2-DMC was preferably O-glucuronidated, whereas 3-DMC predominantly yielded O-sulfation conjugates, indicating phase II conjugation regiopreferences. Moreover, M1 was identified as a novel glutathione conjugate and a possible biotransformation pathway for its formation was proposed. The known 2-DMC (M6), 3-DMC (M7), 2-DMC glucuronide (M4), and novel 3-DMC sulfate (M3) were confirmed as the major metabolites. Radiometric data were acquired by the XFlow liquid chromatography-accurate radioisotope counting (XFlow LC-ARC) system, a novel technology for dynamic control of both on-column and postcolumn high-performance liquid chromatography flow rates to maximize sensitivity and resolution of radiochromatograms. A comparative evaluation was also performed between the XFlow LC-ARC system and a conventional flow radiometric detection system using bile samples from an in vivo disposition study of colchicine in male Sprague-Dawley rats. Results demonstrated a 20-fold sensitivity improvement of the XFlow LC-ARC system in comparison with radioactivity detection by conventional flow scintillation analyzers. The dynamic flow mode also provided the best chromatographic resolution. Unambiguous metabolite identification was performed by high-resolution mass spectrometry and nuclear magnetic resonance analysis.

Separation-optimized sequential injection method for rapid automated analytical separation of 90Sr in nuclear waste

An automated sequential injection (SI) analyzer was developed for analytical separations of 90Sr in nuclear waste samples. The system provides rapid separation of 90Sr from matrix and potential interfering elements using an Sr-selective extraction chromatographic column. After separation, 90Sr activity can be determined by either on-line radiometric detection or off-line counting of collected fractions. The separation was investigated under automated conditions. Eluent compositions were determined that provide consistent, high, reproducible recoveries of Sr from the separation column. Procedures were designed with emphasis on rapid sample throughput. Using fraction collection, a set of eight samples can be processed in just over 1 h. Using on-line detection, each sample is separated and quantified in 22 min. The method was applied to nuclear waste supernate and vitrified nuclear waste.

Direct measurement and regulation of 3′-phosphoadenosine 5′-phosphosulfate (PAPS) generation in vitro

3′-Phosphoadenosine 5′-phospho[ 35S]sulfate (PAPS) biosynchesized from inorganic [ 35S]sulfate and ATP was separated from its radiolabeled precursor by reversed-phase paired-ion HPLC and quantified by on-line radiometric detection. This single-step procedure circumvents several problems inherent in conventional sulfotransferase-coupled assays employed in the measurement of PAPS formation. A good correlation was observed between the rate of PAPS generation assayed in several mammalian tissues measured by direct HPLC-radiometry and by coupling to the sulfation of minoxidil or 4-methylumbelliferone. Both AMP and ADP inhibited the rat liver sulfate-activating enzymes competitively with respect to MgATP 2−, and the rate of PAPS production was decreased with decreasing ratios of [ATP]:[ADP] and [ATP]:[AMP]. It is possible that these adenine nucleotides regulate sulfate activation by kinetic control and by negative feedback modulation.

Transport of strontium cation through a hollow fiber supported dichlorobenzene membrane using 18-C-6 crown ether, nitrate and anion of dinonylnaphtalen sulfonic acid

The transport of strontium cation through a hollow fiber supported dichlorobenzene membrane using 18-C-6 crown ether, nitrate and anion of dinonylnaphtalen sulfonic acid has been studied. A permeation device-single hollow fiber module with on-line radiometric detection of strontium using85Sr tracer was used.

Transport of strontium cation through hollow fiber supported liquid membranes

Transport of strontium through supported hollow fiber dichlorobenzene liquid membranes has been studied. The possible mechanism of strontium transport with 18-crown-6 ether as a carrier and picrate as co-counter-ion as well as the construction of a pertraction device with on-line radiometric detection of strontium using85Sr tracer is described. Preliminary results of strontium pertraction in a recycling and one-pass mode with different concentrations of crown are shown.

Separation of amino acids using ion-paired reversed-phase high-performance liquid chromatography with special reference to collagen hydrolysate

The collagen study includes the analysis of its characteristic amino acids: proline, hydroxyproline, lysine, hydroxylysine. HPLC offers an interesting device if associated with on-line radiometric detection for the determination of radiolabelled amino acids in the case of metabolism studies. To avoid pre or post-column derivatization which may be poorly quantitative in the case of the hydrolysate of unpurified samples, we developed an ion-paired reversed-phase chromatography using a C8 column (econosphere C8 5µm, length: 250 mm, ID: 4.6 mm from Alltech Ass.) and an elution carried out with an acetonitrile gradient in heptane-sulfonate solution. A direct detection at 210 nm was used. Nineteen amino acids were separated within 40 min. Lag time was 7.3 min between hydroxyproline and proline, and 6.9 min between hydroxylysine and lysine. In the case of radiolabelled amino acid, there was a linear correlation (r = 0.92) between HPLC and ion-exchange chromatography.

Determination of 14C-THC and 14C-11-OH-THC from in vitro metabolism studies by mass spectrometry (GC—MS) or on-line radiometric detection (HPLC-R)

Determination of 14C-THC and 14C-11-OH-THC from in vitro metabolism studies by mass spectrometry (GC—MS) or on-line radiometric detection (HPLC-R)

Stimulus-specific production of cyclooxygenase and lipoxygenase metabolites of arachidonic acid by bovine alveolar macrophages

Alveolar macrophages (AMs) are capable of producing a variety of inflammatory mediators including those derived from arachidonic acid, the prostaglandins (PGs), leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs). Inflammation associated with release of arachidonate-derived mediators is a result of the combined actions of all of these mediators. Thus, it is critical to determine the entire spectrum of arachidonate-derived metabolites that AMs are capable of producing. In this study bovine AMs were prelabeled with [3H]arachidonic acid prior to stimulation with serum-treated zymosan, phorbol myristate acetate (PMA), or the calcium ionophore A23187. The total release of arachidonate metabolites into the culture media was measured by reverse-phase HPLC with on-line radiometric detection. All stimuli used induced production of metabolites of the cyclooxygenase pathway with thromboxane B2 and HHT being the major metabolites. Lesser amounts of PGF2 alpha, PGE2, and PGD2 were produced. Only stimulation with A23187 resulted in production of LTB4 and 5-HETE, products of the 5-lipoxygenase pathway. This latter result indicates that the two major pathways of arachidonate metabolism in AMs may be selectively stimulated. Such an effect could have important consequences in the development of pulmonary inflammation. Furthermore, the spectrum of arachidonic acid metabolites produced by bovine AMs closely resembles that of human AMs, in contrast to rodent AMs.

Determination of radiolabelled proline and hydroxyproline in collagen hydrolysates by high-performance liquid chromatography with on-line radiometric detection

Radiolabelled proline and hydroxyproline were separated on a C 8 column (10 cm × 4.6 mm I.D.) with 10.4 m M sodium dodecyl sulphate in water- n-propanol (88:12, v/v) (pH 2.6) as the mobile phase at a flow-rate of 0.6 ml/min. The retention times of hydroxyproline and proline were 5 and 8 min, respectively. On-line radiometric detection was performed either in a homogeneous mode (liquid scintillator was added to the column effluent in the ratio 3.33:1) or in a heterogeneous mode (the detection cell was packed with a solid scintillator and 0.1 M ammonia was mixed with the column effluent in the ratio 1:6 in order to prevent adsorption of amino acids on the cell packing). Detection limits were in the range 100–900 dpm for individual isotopes and detection modes and the reproducibilities were better than 10%. The application of the method to a collagen synthesis study is reported.

On-line low-level radiometric detectin of [ 14]remoxipride in liquid chromatographic effluents : Application to urine samples

A method for on-line radiometric detection in liquid chromatography (LC) is described that permits the detection of low levels of radioactivity in LC effluents by using solvent segmentation and storage of the segmented effluent in a capillary storage loop. The object of the method is to make the flow-rate in the on-line radioactivity monitor (and hence the mean residence time in the monitor) independent fof the separation process. Therefore, after storage of the complete chromatogram, the segmented effluent is led through the monitor at flow-rates that can be chosen according to the residence time desired for accurate and precise radioactivity determination. In this system, it is possible to use a flow-cell volume small enough to preserve the chromatographic integrity, while maintaining the possibility of increasing the counting time. Tests have been performed on the reproducibility of 14C detection and the influence of the flow-rate through the monitor on the standard deviation in 14C peak area and, thus, on the detection limit, using 14C-labelled remoxipride. As an application, analyses of urine samples ofr [ j14C]remoxipride and one of its potential metabolites are reported.

A system for high performance liquid chromatography of 99 mTc-compounds with on-line radiometric detection and data processing

A system for HPLC with on-line γ activity (two channels) and u.v.-detection is described, including software for the on-line data processing with a microcomputer. It was developed for the analysis of 99 m Tc-diphosphonates. Example of some of its features are given using the separation of 99 m Tc and 113Sn labeled Tc(Sn)EHDP complexes with ion-pair-chromatography.

Low-level radiometric detection in LC using solvent segmentation and an effluent storage loop

In this study a new approach is presented for on-line radiometric detection in reversed phase LC of medium to low polarity compounds labelled with14C.