On-line radioactivity detectors

Our second on-line radioactivity detector consisted of a plastic scintillator material (BC-400, Bicron Corp., Newbury, OH) that was machined from 1-inch-diameter rod stock into a 5/8-inch-diameter (front face) solid parabola (see Figure 2). A special rotating holder was constructed for the plastic scintillator and the curved outer surfaces were coated by vacuum deposition with a thin film of aluminum in order to reflect the emitted light toward the front face of the scintillator. A detection length of 2 mm was defined within the parabola by aluminum mounting rods (0.250 inch outer diameter) that were press-fit (coaxial to the separation capillary) in the sides of the scintillator, as illustrated in Figure 2. 

Fig. 2. Examples of HPLC chromatograms using the methods described in the text. Solid lines show the UV absorbance at 351 nm, and dotted lines the cpm measured with an on-line radioactivity detector. (A) A mixture of six retinoid standards separated out into individual peaks with different elusion times. Peak 1 = retinal, peak 2 = tRA, peak 3 = retinol, peak 4 = 13-cis-RA, peak 5 = didehydroretinol, peak 6 = 4-oxo-RA. The dotted line marks the cpm of [ H] tRA, which was also added to the mixture and this coeluted with the cold tRA. (B) The retinoid extracted from a whole 10.5-d mouse embryo. The same six peaks of known standards are marked. Only retinol (peak 3) and tRA (peak 4) are clearly identifiable. Peak 2 coelutes with authentic [ H] tRA, suggesting that the mouse embryo contains significant quantities of tRA and a good deal of retinol. The peak on the extreme right of the chromatogram is an unknown, and the peak to the left of arrow 5 is butylated hydroxy-toluene, the antioxidant added to the extraction solvent.

On-line radioactivity detectors are available commercially and have a limit of detection of approximately 2000 dpm unless the elute is mixed beforehand with scintillant. The authors have found it more convenient to remove aliquots after HPLC for scintillation counting. 

Fig. 6A-D. The metabolism of a short pulse of lAA by wild-type (A + C) and a ts auxin-auxotrophic variant (B + D) of H. muticus. The suspension cultures were precultured at 26 C without auxin and subcultured just prior to the experiment into fresh medium with 3.75 juM 2,4-D (A + B) or without 2,4-D (C + D). After 2 days incubation at 33 ° C (sufficient time for the expression of the XIIB2 phenotype), all cultures were pulsed for 10 min with 0.45 Ci/g [ring-2]- C-IAA (CEA 49 mCi/mmol) and chased for 1 min with a large volume of W5 medium containing lAA at 10 M. Tissues were extracted in 80% methanol, the extracts were reduced in vacuo to the aqueous phase and an aliquot containing 5.0 X 10 dpm was separated on a 25 X 0.46 cm HPLC column packed with 55 Nucleosil C18. The buffers were a) 10% methanol or b) 60% methanol both in 20 mM acetic acid/triethylamine pH 3.5. The gradient was 30 min linear 0-100% (b) at a flow rate of 0.8 ml/min. The metabolites were monitored using an on-line radioactivity detector in the heterogenous mode. Full scale is 100 cps and the plots are fitted to the highest peak. The peaks labelled lAA-Asp and lAA-Glc were further purified and identified by GC-MS or treatment with endo-b-l,4-glucanohydrolase and further HPLC respectively...

At the end of the metabolism period, each piece was frozen on solid CO2. The tissue was extracted following the procedures previously described [ 11 ]. The sample was resuspended in 0.1 N acetic acid, filtered and applied to a reversed phase C18 HPLC column eluted with a gradient of O.I N acetic acid to 0.1 N acetic acid in acetonitrile. The occurrence of [ H]-labeled peaks were monitored with an on-line radioactivity detector (Packard Trace 7140). 

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. 

Where radioactively labelled phospholipids are to be detected the eluting radioactivity may be monitored with either an on-line flow detector or with fraction collecting and scintillation counting. This alternative provides a very sensitive form of detection when compounds of high specific activity are present. 

The ideal HPLC hardware is one that measures both the UV absorbance of the eluate and the radioactivity with an on-line isotope detector. The two sets of data are then superimposed by the computer to allow for the identification of individual peaks (Fig. 2). Chemicals should be HPLC grade and buffers degassed before use. 

PGA extracts were analyzed by HPLG using described methods (6). Purine nucleosides and bases were separated with a reversed-phase column. Purine nucleotides were separated by anion-exchange HPLG. Simultaneous UV monitoring and radioactivity detection were performed with an on-line radioactivity flow detector. 

Nassar AEF, Bjorge SM, Lee DY. On-line liquid chromatography-accurate radioisotope counting coupled with a radioactivity detector and mass spectrometer for metabolite identification in drug discovery and development. AnaZyft caZ Chemistry 75, 785-790, 2003. 

Characterization of collected fractions using spectroscopic techniques Electrochemical detection On-line mass spectrometry and IR spectroscopy Laser-induced fluorescence Photodiode array detectors Radioactivity detectors... 

New detection systems will be developed for increased sensitivity which will place increased demands on prechromatographic sample treatment. Although many of the proposed liquid chromatographic/mass spectrometric interfaces have shown promise using model compounds (B3, B4, Bll, B22, C2, C6, H17, K4, S3, S29, S30), their routine use for on-line characterization is not yet practical. Similar problems have been encountered with liquid chromatographic/infrared detection (K3S, K36, T3). Detection systems such as electrochemical or radioactivity detectors will find increased use for the selective analysis or spectral characterization of eluting compounds (K24, Ml, Y3, Y4). 

A solvent module (Varian model No. 5000) with a UV detector coupled to an on-line Nal(Tl) detector was used for high performance liquid chromatography (HPLC) analysis. For radioactive measurements, a dose calibrator (Capintec CRC-7, USA), a solid scintillation counter (ORTEC, USA) with a plane (7.62 cm x 7.62 cm) Nal(Tl) detector, an automatic well type gamma counter (Compac-120, Picker, USA) and a multichannel analyser coupled to a Nal(Tl) detector (7.62 cm x 7.62 cm) were used. 

In on-line flow-through detectors, the counting time is the mean residence time of radioactive atoms in the detector and can be increased only by either decreasing the flow-rate or increasing the detector volume. Both will influence the precision and accuracy of the chromatographic separation by influencing the resolution attained. 

The Berthold radioactivity monitor (produced by Laboratorium Prof. Dr. Berthold, D-7547, Wildbad 1, Calmbacher Strasse 22) is the only on-line radiometric scintillation detection system suitable for HPLC that is commercially available. The monitor is offered with cells for heterogeneous scintillation counting or as pert of a homogeneous scintillation detector system. Flow cells filled with glass... 

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