Radiolabel detector

By applying an extension of the clearance concept 30, 31), in vitro metabolism was used to predict in vivo toxin elimination. Hepatocytes were incubated with 0.5 to 10 pg unlabeled PbTx-3 containing 0.1 pg radiolabeled toxin as tracer. Disappearance of parent compound and the appearance of metabolites were measured by HPLC equipped with a Radiomatic isotope detector. (1.6 nmol/min/g liver)... 

A radioactivity detector is used to measure radioactivity in the HPLC eluent, using a flow cell. The detection principle is based on liquid scintillation technology to detect phosphors caused by radiation, though a solid-state scintillator is often used around the flow cell [17,31]. This detector is very specific and can be extremely sensitive. It is often used for conducting experiments using tritium or C-14 radiolabeled compounds in toxicological, metabolic, or degradation studies. 

Chemiluminescence is a very sensitive and selective technique. Reagent types, analytes, and detection limits have been summarized in a review by Imai.56 Chemiluminescence has been applied to the analysis of compounds that exhibit low UV absorbance, including metal ions, amino acids, fatty acids, and bile acids. Other detectors include detectors for radioactivity, nuclear magnetic resonance (NMR), and surface-enhanced Raman spectroscopy. Radioactivity detection is one of the most selective detectors, as only components that have been radiolabeled will be detected. The interface of NMR with HPLC and has been discussed in detail by Grenier-Loustalot et al.57 Surface-enhanced Raman spectroscopy is another technique that... 

The availability of an on-line radioisotope detector for CE is especially appealing for several reasons. First, state-of-the-art radiation detection technology offers extremely high sensitivity. Second, radioisotope detection affords unrivaled selectivity because only radiolabeled sample components yield a response at the detector. Third, the radiolabeled molecule possesses the same chemical properties as the un-labeled molecule, thereby permitting tracer studies. Fourth, radioisotope detection can be directly calibrated to provide a measurement of absolute concentration of the labeled species. Finally, a capillary electrophoresis system in which radioactivity detection is coupled with more conventional detectors adds extra versatility to this new separation technique. 

Radioisotope detection of P, 14C, and Tc was reported by Kaniansky et al. (7,8) for isotachophoresis. In their work, isotachophoretic separations were performed using fluorinated ethylene-propylene copolymer capillary tubing (300 pm internal diameter) and either a Geiger-Mueller tube or a plastic scintillator/photomultiplier tube combination to detect emitted fi particles. One of their reported detection schemes involved passing the radiolabeled sample components directly through a plastic scintillator. Detector efficiency for 14C-labeled molecules was reported to be 13-15%, and a minimum detection limit of 0.44 nCi was reported for a 212 nL cell volume. 

Figure 16. Electropherogram illustrating the flow-programmed separation of the same ply d(A) 40-60 mer sample presented in Figure IS. The sample was separated at IS kV and the potential was reduced to l.S kV as radiolabeled sample reached the detector. The sensitivity improvement...

Indirect detection does require more steps, but oftentimes yields amplified signals relative to direct methods because layering of bridging molecules may increase the number of detector molecules per probe molecule. It is probably this bridging/amplification technique that has allowed current enzyme detection systems to approach the sensitivity of radiolabeled systems. The use of these indirect methods reduces steric problems that might arise from having enzyme molecules directly bound to probe molecules. 

The discontinuous method measures activity by separating the product from the substrate. Assays characteristic of this group usually require two steps, since separation often does not include detection. Thus, first, the substrate and the product are separated, and usually the amount of product formed is measured. Assays that use radiochemical substrates are included in this group, since radiochemical detectors are unable to differentiate between the radiolabel of the substrate and that of the product. Examples of enzymes whose assay methods fall into this category are legion, and these approaches characterized by a separation step. 

The use of the HPLC method provides some unique solutions to the problem of determination of product formation at low substrate concentrations. For example, the sensitivity of the detector can be enhanced even during the course of an analysis. Next, the volume of the incubation mixture assayed may be increased to provide more product, and finally, it is possible to use analogs such as radiolabeled or fluorescent compounds, for which there is greater detector sensitivity. 

Radiolabeled products were separated from substrates by chromatography on a Merck Qg column (5 /an). The mobile phase contained 0.1 M sodium acetate, 0.1 M citric acid, 0.1 mAf sodium octylsulfate, 0.15 mAf EDTA, and 0.2 mAf dibutylamine in 10% methanol (v/v). The pH was 4 for the monoamine oxidase assay and 3.7 for phenol sulfotransferase. A flow-through radioisotope detector was used to quantitate the amount of radioactivity in the eluted peaks. 

Radiolabeled UDP-glucose and sucrose-P were separated on a Selectispher-10 boronate column (5 mm x 250 mm). The mobile phase was 0.12 M sodium phosphate (pH 7.6) delivered at a rate of 1 mL/min. The column eluent was monitored for absorbance at 262 nm, and for radioactivity by a radioactive flow-through detector. 

Figure 9.137 Separation of four 14C-labeled components produced after incubating [14C]octadecenoyl-CoA and dodecanol with a crude homogenate of jojoba cotelydons at 3S°C for 20 minutes. Radiolabeled compounds were detected with a flow-through radiochemical detector. Peaks a, octadecenoyl-CoA b, octadecenoic acid c, unidentified product d, dodecanoyi octadecenoate (Cjo i). (From Garver et al., 1992.)...

To apply this method to the problem at hand, the AMP kinase activity should be measured first. Clearly, it would be advantageous to carry out this assay under conditions similar to those that would be present when only ATP was added to the complex. Thus, one might set up a reaction mixture with about 1 mM ATP and with AMP in the nanomolar concentration range that is, at a concentration that would be expected if the AMP had been derived from reaction (2). In addition, to enable us to follow its fate, in reaction (3) the AMP should be added to the incubation mixture in a radiolabeled form. Reaction (3) is started by the addition of the enzyme complex, and samples should be removed from the incubation mixture at suitable intervals and injected onto the HPLC column for analysis. After separation, the eluent should be monitored by both radiochemical and UV (254 nm) detectors. 

Other detection principles have been applied to HPLC, e.g. conductivity, radioactivity, infra-red, and photoconductivity detectors. Such detectors are not widely used in drug analysis but can find application in special circumstances (e.g. the identification of drug metabolites arising from a radiolabelled drug by radioactivity detection). 

C-Detector. A Packard Radiomatic 150TR C-radiodetector (Packard Instruments, Meriden, CT) was used for detection of the radiolabeled compounds. 

Recendy, an automated version of the Sanger method has become available. Instead of using radiolabeled primers, it uses fluorescent tagged dideoxynucleotides. Because each dideoxy analogue fluoresces a different color, the entire procedure is carried out in a single test tube. Afterwards the reaction products are loaded and run on a single electrophoresis gel. After a detector scans the gel, a computer determines the sequence of the colored bands (Figure 17G). 

In this assay (Demel et al., 1977 Demel et al., 1982), a monolayer, containing 14C-labeled phospholipid, is formed at an air—water interface. Vesicles and exchange protein are injected into the subphase. The loss of radioactivity from the surface is monitored continuously with a gas flow detector. Alternatively, the rate of transfer of radiolabeled lipids is measured by recovering the subphase or monolayer and quantitating the radiolabeled lipids. The difficulty in preparing the monolayers and the... 

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