Radioactive derivatization

Another technique which has much potential in analysis, but which to date has had only limited use, is the formation of radioactive derivatives of non-radioactive compounds for quantitation by radiocounting. A radiolabeled reagent is used to form the derivative. This approach has been of use in combination with chromatography. The advantage of this technique is that it avoids problems of sample background which are often associated with spectrophotometric methods. The 14C-methylation of carboxylic acids and the 14 C-acetylation of hydroxyl groups have been studied [39,40]. These methods are quantitative and the sensitivity is dependent on the activity of the radioactive group added to the molecules. The radioactive derivatization of lipids has been reviewed [41]. 

The thiazolecarboxylic acid structure (40) was also guessed in a similar way, from tracer experiments. The unknown compound was converted into the thiamine thiazole by heating at 100°C and pH 2. On paper electrophoresis, it migrated as an anion at pH 4. Tracer experiments indicated that it incorporated C-l and C-2 of L-tyrosine, and the sulfur of sulfate. The synthetic acid was prepared by carboxylation of the lithium derivative of the thiamine thiazole, and the derivatives shown in Scheme 19 were obtained by conventional methods. Again, the radioactivity of the unknown, labeled with 35S could not be separated from structure 40, added as carrier, and the molar radioactivity remained constant through several recrystallizations and the derivatizations of Scheme 17. 

The introduction of metabolizable ester functions may be a very useful means of obtaining retention of the radiotracer within the target, as known for 99mTc-ECD. It may also help to eliminate nontarget radioactivity more rapidly. Therefore, systematic studies have been performed, in order to understand the enzymatic hydrolysis of oxotechnetium(V) complexes with up to four ester groups in the complex [205]. Fig. 30 shows the partial hydrolysis by pig liver esterase as is was observed for 1 2 complexes of derivatized dimercaptosuccinic acid complexes. 

Derivatization with dabsyl chloride (94) was applied for the separation of primary amino acids in physiological samples, prior to determination of their specific radioactivity. The derivatization is easy to perform and the derivatives are stable252. 

Radioactive lateling of this cluster and neutron activation analysis of the g)ld enabled us to determine the extent of Nnding of the cluster to the particles. The results of both analytical methods show that a spacer of minimum length of about 10 A between the -SH group of a ribosomal protein and the N-atom on the cluster is n ed for significant binding. Preliminary experiments indicate that the producte of the derivatization reaction with SOS particles can be crystallized. 

Very little work has been carried out on radiochemical derivatization for analysis of trace amounts of materials. The technique has the advantage of being both selective and sensitive. Die main advantage is that the sample background does not cause interference in the detection as it does in most other methods and which necessitates some degree of clean-up. Also, the reactions used are those for normal derivatization procedures, the only difference being that the reagent is radiolabeled and that appropriate precautions are required for radioactive substances. The few methods described below illustrate the application of this technique. 

Figure 2. Radioactivity chromatogram of sulfur compounds derivatized with monobromobimane. The reversed-phase HPLC separation is based on the hydrophobic properties of the bimane-sulfur adducts but peak area is based on "S-radioactivity of the compounds. At time 0 sulfite and thiosulfate impurities are present before addition of the hepatopancrease tissue homogenate. This was a 60 min experiment to determine the sulfide detoxifying functions of the hepatopancrease of the hydrothermal vent crab Bythograea thermydron. During this time the proportion of radioactivity in sulfide rapidly decreases and thiosulfate and sulfate accumulate as end products. Two intermediates, pi and p2 accumulate then decrease during the experiment. The two intermediates are believed to be polysulfides based on similar elution times of polysulfide standards. (Figure is the unpublished chromatograms from the data in Vetter et al. (24)-) continued on next page.

Figure 6.14 Examples of the application of normal-phase, radio-HPLC to the analysis of de novo biosynthetic pathways in bark beetles (Scolytidae). Derivatization of 14C-labeled ipsenol from Ips paraconfusus (A, B) and 14C-labeled ipsdienol from Ips pini (C, D) leads to expected retention time shifts of radioactivity for each compound and derivative. Derivatization of /. paraconfusus ipsenol with (2/= )-(+)-a-methoxy-a-(trifluoromethyl) phenylacetic acid (MTPA) leads to the formation of only one diastereomer [(4S)-(-)-ipsenoyl-(2 P )-2 -methoxy-2 -phenyl-2 - (trifluoromethyl) phenylacetate] indicating the de novo biosynthesis of highly pure (4S)-(-)-ipsenol (B) Derivatization of I. pini ipsdienol with (1 S)-(-)-camphanic acid leads to the formation of both diastereomers [(4R)- -)- and (4S)-(+)-ipsdienyl-(TS)-camphanates] indicating the de novo biosynthesis of approximately 90 percent-(4/ )-(-)-ipsdienol (D). Figures adapted from Seybold et al. (1995b).

Three to four days after the application of C-macu1osin to an unwounded leaf surface there is little or no movement of radioactivity from the inoculation site. Even in a previously wounded leaf, there is only slight movement of labelled material from the point of application. However, there was uptake of radiolabel by knapweed roots suspended in a solution of maculosin. These initial studies have provided useful leads for the practical application of maculosin as a knapweed control agent derivatization of the compound may be necessary for entry and distribution of maculosin in the plant. 

The monazo and bisazo derivatives of lysine, tyrosine, and histidine are unstable to acid hydrolysis and the native amino acids are not regenerated. Therefore, the best method to quantitate the extent of derivatization is with radioactive reagent. The characteristic absorp-... 

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. 

Table I shows the recovery of cis and trans retinoic from plasma and PBS (n=3). Recovery of radioactivity is high throughout extraction and derivatization, but drops off sharply after HPLC...

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