Radioactivity recovery determination

Radioactivity Recovery Determination Determination of the radioactivity recovery from an HPLC analysis is a necessary procedure to ensure accurate determination of all radioactive components in the original sample. In addition, because metabolite profiling by MSC requires the additional step of in vacuo removal of HPLC solvents, it is also important to determine whether volatile metabolites are lost in the process. We have developed a simple method to determine HPLC column recovery, plate recovery, and the total recovery for an HPLC-MSC analysis (Zhu et al., 2005b). In general, aliquots of a sample are injected onto an HPLC with and without an HPLC column. All effluent from each HPLC run are separately collected and aliquots are analyzed for total radioactivity by LSC with and without solvent evaporation. The DPM values obtained are used to calculate the recoveries. 

FIGURE 1 Biodegradation of unsterilized NET microcapsules (63-125 pm) as determined by radioactive recoveries from injection sites of rats. (From Ref. 38.)... 

To determine the effectiveness of the windowing procedure, the customer went back to his radiolabeled standard. Radioactive recovery studies showed better than 99% release from the cartridge and 94% activity recovery in the main window for the compound under study. 

USA) using a Waters Millennium system with an in-line radioactivity detector. The flow rate was maintained at 1 mL/min using 0.1% TFA/water (solvent A) and 0.1% TFA/acetonitrile (solvent B). The following elution gradient was used 100% solvent A for 3 min, 100 to 50% solvent A over 10 min 50% solvent A for 10 min, 50 to 30% solvent A over 3 min and 30 to 100% solvent A over 4 min. Recovery of the radioactivity was determined. 

Radionuclide studies offer an additional method to investigate the factors that affect trace element absorption. Radioactivity emitted by the radionuclide was measured in blood 14 days after the oral ingestion of zinc-65 and compared with the amount of radioactivity emission determined by whole-body counting (Watson et al. 1987). The results indicated that, where whole-body counting facilities were not available, measurement of radioactivity emitted in blood was a reasonable alternative for the prediction of zinc absorption. Recovery for this method was adequate (88%) precision was acceptable (<17% CV). The limit of detection for zinc was not reported. 

Radioactivity Analysis. Samples of urine, feces, and tissues were combusted to COo and analyzed for radioactivity (5). By using this method the recovery of radioactivity from samples spiked with C was 95 dt 5%. To determine the radioactivity expired as CO2, 5-ml aliquots of the solution used to trap the CO2 were added to 15 ml of a scintillation counting solution containing 4 grams 2,5-diphenyloxazole (PPO) and 0.1 grams l,4-bis-2(5-phenyloxazolyl)-benzene (POPOP) per liter of 1 1 toluene 2-methoxyethanol. Samples were counted for radioactivity in a Nuclear Chicago Mark II liquid scintillation counter. Counting eflSciency was corrected by the internal standard technique. 

In the last case, this may be a physical problem resulting from incomplete penetration by the extraction solvent into the matrix. Alternatively, incomplete recovery of the analyte may result from chemical binding between the analyte and a constituent of the matrix. This is particularly important in the determination of drugs in body tissues where binding to proteins is known to occur. Problems of this kind are documented in the literature. If a new procedure is being developed, it is necessary to investigate the extraction step, e.g. by using radioactive tracers. 

Plasma levels of dobutamine hydrochloride are determined by reaction of the drug with 3H-methyl-S-adenosylmethionine in the presence of catechol O-methy1-transferase. The radioactivity of the labeled methyl derivative is determined by a liquid scintillation counter using an external standard. The final recovery of added dobutamine as 3H-CH3-dobutamine is 24.9 1.3% in the range of 2 to 170 ng/ml (4). When 14C-dobutamine is administered the samples are counted by a double isotope method. 

In addition to ARSAC approval, the protocol must also be approved by ethics committees in the normal manner for studies in man. The study should be conducted in between four and eight consenting subjects, in facilities where any spills of radiolabelled materials can be contained and monitored. Normally, subjects will be required to provide blood samples and to collect all excreta for a period determined by the known or estimated half-lives of the parent compound and metabolite. With cooperative subjects, recoveries of radioactivity should be close to 100%. Samples will be assayed for radioactivity and by cold chromatographic methods, and every attempt should be made to identify major metabolites... 

Recovery of radioactivity from cattail tissue by blending with methanol averaged only 23.7% in samples from 3, 7 and 14 days post-treatment indicating extensive breakdown of fenitrothion by the plant. The identity of the radioactivity was not determined. 

In general, it appears that fixation by fast freezing, (for example, by clamping between aluminum blocks chilled in liquid nitrogen), followed by pulverisation in impaction mortars chilled to the temperature of liquid nitrogen, and finally rapid homogenisation of the frozen, powdered tissue in 0.1 N HCl, containing radioactively labelled cyclic nucleotides to determine recovery, is the most reliable method of fixation. 

The radiochemical procedure for the determination of Cs in aqueous samples is based on the batch extraction of caesium onto a microcrystalline cation exchanger, ammonium molybdophosphate (AMP), and subsequent purification from potassium and rubidium activities by ion-exchange separation using a strongly acidic cation exchange resin (BIO-REX-40). Natural K and Rb have radioactive isotopes that interfere with the beta counting of Cs. The purification of caesium is also necessary to determine the chemical recovery. 

Radioactive tracers may be used to determine the amount of a single substance in a mixture. The tracer technique is especially useful where quantitative recovery of the substance in question is difficult or impossible. Basically, the technique involves adding a known amount of the radioactive compound to the mixture and then (after thorough equilibration) reisolating a small amount of the compound. The amount of compound recovered is unimportant, provided it is sufficient to weigh and to count. From the specific activity of the reisolated compound as well as a knowledge of the total number of counts originally added, the amount of... 

RECOVERY. A dual label recovery experiment, from plasma and PBS, was performed using 3H-all trans and 14C-cis retinoic acid. The normal extraction procedure was followed up to and including the HPLC purification step. No LC/MS analysis was performed. Aliquots were taken and total radioactivity determined after extraction and derivatization. Fractions (0.5 ml) from the HPLC were collected and counted. Counting was performed using a Beckman Model LC3801 liquid scintillation counter. Radioactivity was corrected for spillover and quench. 

Isomer interconversion due to assay manipulations could also be determined from the recovery experiment. Isomerization data was obtained from the radioactivity profile of the HPLC purification step. The amount of cis isomerizing to trans was 3%. The same amount of trans retinoic acid isomerized to cis. The assay causes a small amount of isomerization but to an equal extent for both isomers. 

It is more diflBcult to secure conclusive evidence of the specificity of a chemical method, when applied to a biological system (B7). Since the constituent will almost certainly have to be determined as one component in a mixture, the possible interfering effects of some (ideally all) of these other substances need to be determined, each over the range of concentrations liable to be met with in practice. Whereas the accuracy of a method can best be determined by recovery experiments, which depend on the availability of a pure sample of the compound under investigation, the specificity of a technique and the effects of possible interfering factors can be more readily investigated by experiments involving radioactive isotopes these isotopic assessments of specificity have so far not been widely applied in clinical chemistry. 

For EIA, it is important to establish the degree of substitution, the specific and the total activity of the enzyme recovered, as well as the extent of recovery of the immune reactivity. The number of amino groups substituted with haptens in carrier molecules is conveniently established by the determination of the number of free amino groups before and after substitution (Habeeb, 1966 Table 12.9). Another method is provided by the use of radioactive haptens (Abraham et al., 1971). 

The total amount of semduramicin sodium recovered from the HPLC was determined by reference to a standard calibration curve that related the peak area ratio of semduramicin sodium to the internal standard. The radioactivity recovered from the HPLC was corrected for the recovery of cold semduramicin sodium. In this procedure the dynamic range of the calibration curve for the nonlabeled semduramicin sodium extended from 1-40 pg/mL, and the dynamic range for tissue fortified with l4C-semdura-... 

Release rates determined in this manner were essentially identical in the in vivo and in vitro implants. In addition, for the in vitro experiments, release was also measured directly by analyzing the radioactivity in the release media. The release rates determined in this way correlated precisely with the in vitro and in vivo release rates determined by the recovery experiments (last paragraph). Furthermore, they demonstrated that the material balance was completed, showing no material was lost (10). 

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