Thin-Layer Radiochromatography - Raytest

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A Field Guide to In stru men ta tion Figure 2. BAS-5000 storage phosphor screen imaging system (courtesy of Fujifilm Life Science). multilabel dpm quench curves. The plastic Versa-Rack vial system allows counting of samples in any combination, up to 336 standard vials (20 mL) and 648 miniature vials (6 mL); capabilities also exist to count 4 mL Bio-Vials and Microfuge tubes. Computer-based operation is via a combination of easy to use menus, context-sensitive help screens, and up to 50 user programs. Different biodegradable general use and specialized cocktails, plastic scintillation vials, unquenched LS standards, and quenched standards are among the available accessories. Phosphor Imaging Storage phosphor screen imaging is often termed filmless autoradiography. The phosphor screens are sensitive to any source of ionizing radiation, e.g., 14 C, 3 H, 35 S, 125 I, 32 P, 33 P, 18 F, and 99m Tc. They contain small crystals of a photostimulable phosphor coated on a support in which luminescence is produced and stored when exposed to radioactive TLC zones. The luminescence is evaluated by scanning with a laser in a reading device, and quantification is carried out by use of a calibration curve to exclude the effects of the phosphor screen type and exposure period. The screens can be reused after being erased by exposure to visible light. The formulation of the phosphor screen, chemistry of the detection process, mechanism of scanning, and software for evaluation of images may differ for instruments from each manufacturer. BAS-5000 Bioimaging Analyzer The BAS-5000 bioimaging analyzer from Fujifilm Life Science USA (Figure 2) uses a patented phosphor imaging plate (IP) with a scanner for IP reading for the sensitive, 2-dimensional (2D) detection of radioactive TLC zones by the photostimulated luminescence (PSL) phenomenon. The instrument features a confocal laser, light-collecting optics, dynamic range up to 5 orders of magnitude, and a pixel size as small as 25 �m. A 20 � 25 cm IP with the images of chromatograms from a 20 � 20 cm TLC plate can be scanned at 50 �m in as manufacturing process. The exposed IP is scanned with a laser beam of red light (633 nm) focused by a mirror while being moved in the reader; the PSL released by the laser as photons of blue light (390 nm) is collected onto a PMT through a light collection guide and is converted to electrical signals. Cyclone Plus Phosphor Imager The PerkinElmer benchtop Cyclone Plus quantitative radiometric phosphor imager (Figure 3) operates with the same storage phosphor and scanning process (Figure 4), except that confocal optics and a helical scanning mechanism are used with the flexible phosphor screen loaded into a cylindrical carousel that spins at 360 rpm. This allows the instrument to be more compact and less expensive than the BAS-5000, in which the phosphor screen is kept on a flat plane during scanning. The phosphor screen is scanned by the system’s laser focused to less than 50 �m, and the latent image is detected by the instrument optics to create a high-resolution digitized image of the layer with quantitative data in the form of an image file. The image is displayed on the computer screen for analysis with OptiQuant software and can be printed, exported, and archived for future use. The following storage phosphor screens little as 5 min; the detection limit is 0.11 dpm/mm 2 /h for P 32 . Compared to X-ray film, sensitivity is about 100 times higher, processing is 10–100 times faster, and quantitative accuracy is greater. The IP consists of 5 �m crystals of barium fluorobromide containing a trace amount of bivalent europium (BaFBr:Eu +2 ) as a bioluminescence center coated on a polyester support film. When the crystal is exposed to a radiolabeled zone, the energy of the radioisotope ionizes the Eu +2 to Eu +3 , liberating electrons to the conduction band of the phosphor crystals. The electrons are trapped in the bromine vacancies Figure 3. Cyclone Plus phosphor imager (courtesy of introduced during the PerkinElmer). JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009. � Copyright 2009 by AOAC INTERNATIONAL. Reprinted with permission.
A Field Guide to In stru men ta tion Figure 4. Schematic representation of the storage phosphor process (courtesy of PerkinElmer). are available in 12.5 � 19.2, 12.5 � 25.2, or 12.5 � 43 cm sizes: multisensitive (MS) general purpose, super resolution (SR) made with the finest grain particles of phosphor, and tritium sensitive (TR) uncoated for the detection of 3 H. Specific TLC applications cited by PerkinElmer are imaging of 3 H, 125 I, 14 C, 32 P, 33 P, 18 F, 90 Y, 99m 111 177 Tc, In, and Lu; nucleotide metabolism studies with 32 P- and 33 P-labeled adenosine triphosphate (ATP) or guanosine triphosphate (GTP); 14 C-uridine-5�-diphospho-glucuronic acid (UDPGA) glucuronidation assay; quality control (QC) of 177 Lu- and 90 Y-DOTA-D-phenylalanine(1)-tyrosine (3)-octreotide (TOC); 18 F silica gel TLC for analysis of positron emission tomography (PET) radiochemicals; QC of 99m Tc; and radiochemical purity of 90 Y Zevalin (Figure 5). Radioscanners for Direct Measurement of TLC Plates Bioscan AR-2000 TLC Imaging Scanner The AR-2000 imaging scanner (Figure 6) uses a resistive anode single-element position sensing windowless gas-filled proportional detector for direct digital counting of all beta and gamma emitting isotopes, including 3 H, on TLC or HPTLC plates. The process involves ionization of the counting gas by interaction with radioactive TLC zones, producing a pulse of electrons that is proportional to the amount of radioactivity. An entire lane can be imaged in under 1 min, and multiple lanes can be measured in a single automated run without operator intervention. The instrument is linear over 4–5 decades of activity; has resolution of 0.5–3 mm depending on the isotope; and has a sensitivity of less than 1000 dpm for 3 H and 125 I and less than 100 dpm for 14 C, 32 P, and most other isotopes in a 10 min analysis. Models are available that hold one, two, or three 20 � 20 cm TLC plates. Single-lane or 2D color imaging and automated quantification can be performed with WinScan software, and Bio-Chrom software can be used in place of WinScan for compliance with 21 CFR Part 11, U.S. Food and Drug Administration and Good Laboratory Practice (GLP) standards. Applications for which the AR-2000 have been used include PET or single photon emission computed tomography (SPECT) radiopharmaceutical QC and synthesis process control (compounds labeled with 18 F, 11 C, 99m TC, 111 In, etc.); pharmaceutical metabolite analysis for beta-, gamma-, or positron-labeled products; radiotracer toxicology studies with 14 C-labeled organic compounds and agrochemicals; biosynthesis studies involving complex lipids, phospholipids, and glycolipids; radiochemical purity quality assurance (QA) analyses for tracer or metabolism studies; radiolabeled reporter gene or enzyme assays; quantitative radiolabeled biochemical separations (programmable scanning and quick change magnetic collimators can be used for optimization of resolution and sensitivity); and study of constitutive expression of 25-D 3 -1alpha-hydroxylase in a transformed proximal tubule cell line: evidence for direct regulation of vitamin D metabolism by calcium (7). <strong>Raytest</strong> GITA The GITA gamma isotope TLC analyzer (Figure 7) is an X-Y scanner controlled by a personal computer (PC) Figure 5. Determination of radiochemical purity for the 90 Y-based drug Zevalin on multiple TLC strips simultaneously (courtesy of PerkinElmer). � Copyright 2009 by AOAC INTERNATIONAL. Reprinted with permission. JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 1, 2009
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