Parameters of carbon labelling using ionized gases

We have recently described a rapid non-synthetic method for producing¹⁴C-and¹¹C-labelled biomolecules. The apparatus consists of a high vacuum system in which small amounts of¹⁴CO gas are ionized by electron impact. The resulting ionized, excited and dissociated species drift toward a thin layer of organic molecules where they interact to produce¹⁴C-labelled compounds. In this paper, details are given on the mechanisms of interaction of the electron beam and on the parameters influencing the labelling yields. Using cholesterol as a model compound labelling yields were measured while the electron energy, thickness of the organic film, gas pressure and time of exposure were varied over a wide range. The results suggest that¹⁴C⁺ and/or¹⁴CO⁺ are the principal species involved in the labelling reactions. Supported by the Medical Research Council of Canada Grant MA-6137.     

Radio gas chromatography of14C-labelled compounds by measuring the radioactivity of the FID combustion products after GLC analysis

Summary A simple method of radio gas chromatography, which avoids the necessity for an effluent gas stream splitter and a special reactor after the GLC column, has been described. The system uses the FID as a combined mass detector and combustion furnace for the conversion of¹⁴C-labelled compounds into¹⁴CO₂ and operates the FID in series with the¹⁴CO₂ detection system. Specific activity values of weakly¹⁴C-labelled compounds such as organic methyl esters and TMS sugars can be determined precisely with the standard error of the mean less than 3%.     

A method for measuring specific activities of 14C-labelled compounds by gas chromatography-mass spectrometry-computer system

A method for measuring specific activities of 14C-labelled compounds by gas chromatography-mass spectrometry-computer system (GC-MS-CPU) was developed. This method was proved to provide practically precise and accurate specific activities of various 14C-labelled compounds with such merits as requirement of small amount of samples, being applicable to volatile compounds, and convenience. The C.V. percent obtained for tested compounds was within 3.9 and the reliable sensitivity should be over 37 MBq/mM (1 mCi/mM). This method was also useful for obtaining information on the labelling pattern and the synthetic procedures applied.     

Spezifisch14C-kernmarkierte Phenolderivate, 1. Mitt.: Synthese von [14C]-Guajacol

Zusammenfassung Für oxydative Abbauversuche wurden verschiedene spezifisch¹⁴C-kernmarkierte Phenolderivate benötigt. Guajacol, in verschiedenen Positionen des aromatischen Ringes markiert, wurde — teils von [1-¹⁴C]-Phenol, teils von [2-¹⁴C]-Aceton ausgehend — synthetisiert.

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Specifically¹⁴C-labelled phenol derivatives, I: Synthesis of [¹⁴C]guaiacol

Various phenol derivatives,¹⁴C-labelled in different specified positions of the aromatic ring, were required for oxidative degradation studies. Guaiacol, ring-labelled in different positions, was prepared both from [1-¹⁴C]phenol and from [2-¹⁴C]-acetone.

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Herrn Prof. Dr.M. Pailer zum 60. Geburtstag in Freundschaft gewidmet.     

Determination of leukotrienes and prostaglandins in [14C] arachidonic acid labelled human lung tissue by high-performance liquid chromatography and radioimmunoassay

A liquid chromatographic method for the determination of 14C-labelled prostaglandins, leukotrienes and other lipoxygenase products formed by human lung tissue is described. In this paper we report our problems identifying these substances when 3H- or 14C-labelled compounds are compared with measurements of the mass by absorption or radioimmunoassay. Furthermore, some preliminary results of [14C] arachidonic acid labelled human lung tissue, stimulated by the Ca-ionophore A23187, show that, of the lipoxygenase products, mostly leukotriene B4 like compounds are formed and less leukotriene C4, E4 and D4. Relatively large amounts of hydroxyeicosatetraenoic acids are present. The main cyclooxygenase products are thromboxane B2, 6-ketoprostaglandin F1 alpha and prostaglandin D2.     

Halbquantitative autoradiographische bestimmung von 14C-aktivitäten im pico- bis nanocurie-bereich mit hilfe der ringofenmethode

The use of the ring-oven method for the semiquantitative autoradiographic determination of ¹⁴C-labelled compounds is described. Satisfactory results can be obtained in the activity range 9.2–460 pC/μl (exposure times of 122–2 h) with special commercial films.     

Volatilization of 14C-Labelled Fenpropimorph after Application to Plants and Soil under Simulated Outdoor Conditions

Abstract

The volatility of fenpropimorph was investigated in a laboratory chamber constructed for studying the volatilization of “4C-labelled pesticides from plant and soil surfaces. Summer barley was cultivated on experimental platforms (0.5 m²) filled with soil and treated in an application chamber with ¹⁴C-labelled fenpropimorph formulation (Corbel^R) at the beginning of ear emergence. After application, the platform was transferred into the volatilization chamber where a 96 h outdoor weather scenario was simulated. The results of three experiments demonstrated that up to 60% of the initial total radioactivity could be released from the plant-soil system within 96 h, most of it being the unchanged ¹⁴C-fenpropimorph which undergoes a fast oxidative (degradation by solar irridation in the atmoshpere. Furthermore, ¹⁴CO, was detected in quantities of 1.1 to 1.8%. After plant extraction, however, mainly polar metabolites, such as fenpropimorh acid, were found four days alter application by Radio-HPLC-analysis. In order to evaluate the volatilization behaviour of fenpropimorph sprayed to bare soils, three additional experiments were carried out showing a volatilization rate of 11.4% at most, much lower than those of plant surfaces.     

Metabolism of <Superscript>14</Superscript>C-labelled and non-labelled sulfadiazine after administration to pigs

The behaviour of sulfadiazine (SDZ) and its metabolites was investigated by administering the ¹⁴C-labelled veterinary drug to fattening pigs. The excretion kinetics were determined after daily collection of manure. Two known metabolites, N-acetylsulfadiazine and 4-hydroxysulfadiazine, and two hitherto unidentified minor metabolites were recovered. Various mass spectrometric techniques such as parent, product ion scans and accurate mass measurement were used. The new compounds were identified as N-formylsulfadiazine (For-SDZ) and N-acetyl-4-hydroxysulfadiazine (Ac-4-OH-SDZ). The identification of SDZ, Ac-SDZ and For-SDZ was confirmed by comparison of the spectroscopic and chromatographic data of the synthesized authentic references. The identification of the hydroxylated compounds 4-OH-SDZ and Ac-4-OH-SDZ was performed by MSⁿ, and accurate mass measurements. Only 4% of the administered radioactivity remained in the pig after ten days and SDZ accounted for 44% of the 96% radioactivity excreted. More than 93% of the labelled compounds were detected and identified in the manure. The key analytical problem, namely a high concentration of matrix in sample extracts, was overcome by advanced measurement techniques and with the use of a suitable internal standard. The mean recoveries for all compounds were ≥96%. Linearity was established over a concentration range of 0.5 to 10,000 μg kg⁻¹ manure with a correlation coefficient ≥0.99. The same experiment was carried out simultaneously with non-labelled SDZ to obtain manure for outdoor soil experiments.     

CARBON-14 LABELING AND BIOLOGICAL ACTIVITY OF THE TUMOR-LOCALIZING DERIVATIVE OF HEMATOPORPHYRIN

Abstract— Carbon-14-labeled hematoporphyrin ([¹⁴C]HP) was synthesized by two methods, (i) Using an in vitro avian whole-blood system, [¹⁴C]protoheme was obtained biosynthetically by incorporating [⁴C]aminolevulinic acid into the porphyrin ring structure. Subsequently, the [¹⁴C]protoheme was converted to [⁴C]HP by standard procedures, (ii) By adopting several well-characterized chemical reactions, deuteroporphyrin was treated with [¹⁴C]acetyl chloride, giving [¹⁴C]diacetyl deuteroporphy-rin which was readily reduced and hydrolyzed to [¹⁴C]HP (with thecarbon–14 label on the hydroxyethyl side-chains). These two methods are simple and afford good yields of [¹⁴C]HP with moderate to high specific activities. The [¹⁴C]HP was then treated with acetic acid/sulfuric acid followed by sodium hydroxide to give [¹⁴C]HPD. Upon gel- and ultra-filtration, the [¹⁴C]HPD was enriched in the so-called tumor-localizing fraction of HPD, giving [¹⁴C]PII with specific activities of 0.4 Ci/mol (biosynthesis) and 10 Ci/mol (chemical synthesis). These [¹⁴C]PII preparations were equivalent with respect to chromatographic and spectrophotometric characteristics, as well as tumoricidal photodynamic activity in the DBA/2 Ha-DD mouse: SMT-F tumor system, to the unlabeled commercial product Photofrin^? II. The distribution of [¹⁴C]PII in mouse tissues was in close agreement to that previously reported, after adjustment for dose, for [¹⁴C]HPD biosynthetically labeled in vivo (Gomer and Dougherty, 1979), as well as for Photofrin^? II, where tissue levels were determined spectrophotometrically after extraction (Dougherty and Mang, unpublished).     

Essential methodological improvements in the oxygen isotope ratio analysis of N‐containing organic compounds

The quantitative conversion of organically bound oxygen into CO, a prerequisite for the ¹⁸O/¹⁶O analysis of organic compounds, is generally performed by high‐temperature conversion in the presence of carbon at ～1450°C. Since this high‐temperature procedure demands complicated and expensive equipment, a lower temperature method that could be utilized on standard elemental analyzers was evaluated. By substituting glassy carbon with carbon black, the conversion temperature could be reduced to 1170°C. However, regardless of the temperature, N‐containing compounds yielded incorrect results, despite quantitative conversion of the bound oxygen into CO. We believe that the problems were partially caused by interfering gases produced by a secondary decomposition of N‐ and C‐containing polymers formed during the decomposition of the analyte. In order to overcome the interference, we replaced the gas chromatographic (GC) separation of CO and N₂ by reversible CO adsorption, yielding the possibility of collecting and purifying the CO more efficiently. After CO collection, the interfering gases were vented by means of a specific stream diverter, thus preventing them from entering the trap and the mass spectrometer. Simultaneously, a make‐up He flow was used to purge the gas‐specific trap before the desorption of the CO and its subsequent mass spectrometric analysis. Furthermore, the formation of interfering gases was reduced by the use of polyethylene as an additive for analytes with a N:O ratio greater than 1. These methodological modifications to the thermal conversion of N‐containing analytes, depending on their structure or O:N ratio, led to satisfactory results and showed that it was possible to optimize the conditions for their individual oxygen isotope ratio analysis, even at 1170°C. With these methodological modifications, correct and precise δ¹⁸O results were obtained on N‐containing analytes even at 1170°C. Differences from the expected standard values were below ±1‰ with standard deviations of the analysis <0.2‰. Copyright © 2010 John Wiley & Sons, Ltd.     

Flow-through radioactivity detection of 3H- and 14C-labelled compounds in reversed-phase liquid chromatography by a liquid scintillation counting technique based on post-column extraction

A method for flow-through liquid scintillation counting in reversed-phase liquid chromatography (RP-LC) based on the continuous extraction of aqueous column eluates with a water-immiscible liquid scintillator is evaluated in terms of sensitivity, reproducibility and extra-column band broadening. ³H- and ¹⁴C-labelled phenylthiohydantoinamino acid derivatives of widely different polarity serve as model compounds. For extractable derivatives, counting efficiencies of over 30 and 80% can be obtained for ³H and ¹⁴C, respectively. The reproducibility and extra-column band broadening depend on the mixing ratio of scintillator and LC eluent; relative standard deviations (peak areas) of less than 3% can be obtained. The sensitivity of flow-through counting can be increased at least 150-fold by storing the segmented scintillator/eluate stream in a capillary storage loop. After the separation is complete, the stream is re-introduced into the radioactivity detector at reduced flow-rates to increase the mean residence time, i.e., the counting time, in the detector.     

Application of gas chromatograph-mass spectrometer-computer system to evaluation of 14C-labelled compounds

Usefulness of gas chromatograph-mass spectrometer-computer system (GC-MS-CPU) not only for measurement of specific activities of 14C-labelled compounds in a mixture but also for evaluation of 14C-labelled compounds in terms of examining their purities and elucidating chemical structures of the impurities was proved. A sample of methyl 2-(p-chlorophenyl-14Cn)-3-methylbutylate (III) synthesized from p-chlorophenyl-14Cn-acetonitrile (VI) was analyzed by GC-MS-CPU, and it was found that the labelled compound was contaminated with a small amount of the corresponding m-isomer (IV) having a very high specific activity. Further examination suggested that the contaminating m-isomer (IV) originated from m-chlorophenyl-14Cn-acetonitrile (IX) which had already contaminated in the starting material (VI), and also that cyanomethylation of p-dichlorobenzene-14Cn (VIII) by benzene-type reaction resulted in producing a mixture of p- and m-chlorophenyl-14Cn-acetonitriles (VI, IX).     

Preparation of carbon-11 labelled acetate and palmitic acid for the study of myocardial metabolism by emission-computerised axial tomography

Methods have been developed for the labelling of acetate and palmitic acid with the positron-emitting radionuclide,¹¹C (T=20.4 min). Labelling was achieved via carbonation of the appropriate alkyl magnesium bromide (methyl magnesium bromide or n-pentadecyl magnesium bromide) with¹¹C-labelled carbon dioxide produced by the¹⁴N(p, α)¹¹C nuclear reaction. The radiochemical yield and speed of each method of labelling are such that a radiochemically pure product is obtained in injectable form and in activity (>10 mCi) suitable for the study of myocardial metabolism by emission-computerised axial tomography. High pressure liquid chromatography and thin layer chromatography were used to assess the radiochemical purity of each radiopharmaceutical. The specific activity of¹¹C-labelled acetate was estimated by an enzymic procedure to be greater than 0.5 Ci/μmole.     

One-Pot Synthesis of 11C-Labelled Primary Benzamides via Intermediate [11C]Aroyl Dimethylaminopyridinium Salts

Electrophilic ¹¹C-labelled aroyl dimethylaminopyridinium salts, obtained by carbonylative cross-coupling of aryl halides with [¹¹C]carbon monoxide, were prepared for the first time and shown to be valuable intermediates in the synthesis of primary [¹¹C]benzamides. The methodology furnished a set of benzamide model compounds, including the two poly (ADP-ribose) polymerase (PARP) inhibitors niraparib and veliparib, in moderate to excellent radiochemical yields. In addition to providing a convenient and practical route to primary [¹¹C]benzamides, the current method paves the way for future application of [¹¹C]aroyl dimethylaminopyridinium halide salts in positron emission tomography (PET) tracer synthesis.     

The double radio-isotope derivative technique for the assay of drugs in biological material: Part II. The simultaneous determination of opipramol and its deshydroxyethyl metabolite

The neuroleptic drug opipramol and its deshydroxyethyl metabolite can be determined simultaneously in the same biological sample. Known amounts of ¹⁴C-labelled opipramol and ¹⁴C-labelled metabolite are added to the sample to serve as internal standards. After suitable extraction, both compounds are acetylated by ³H-labelled acetic anhydride. Together with μg-amounts of carrier compounds, the O-acetyl derivative of opipramol and the N-acetyl derivative of the metabolite are purified and separated by two-dimensional thin-layer chromatography. Each of the derivatives is isolated and counted for ¹⁴C-and ³H-activity. The ¹⁴C-activities recovered serve to determine the overall yield of the opipramol and metabolite, and to convert the measured ³H-activity to 100 % theoretical yield. From analyses of standard samples, the specific ³H-activities of the acetyl derivatives were calculated, and these values were used to convert the measured ³H-activities from biological samples to concentrations of original opipramol and metabolite. For both compounds the standard deviations of blank samples were ±1 ng ml^?1. For concentrations up to 100 ng ml^?1 the standard deviation was ±3 ng ml^?1.     

Preparation of some carbon-14-labeled amino acids by radiophotosynthesis using sugar beet plant

Radiophotosynthesis has been applied as a means for preparing a number of carbon-14-labeled amino acids using sugar beet plants grown under controlled environmental conditions. The apparatus used, conditions of operation, isolation of products, and their analysis and radiometry have been discussed in detail. From the results obtained, it can be inferred that a number of labeled amino acids could be separated from the leaves of sugar beet including [¹⁴C]alanine, γ-[¹⁴C]aminobutyric acid, [¹⁴C]glutamic acid, [¹⁴C]aspartic acid, [¹⁴C]serine, [¹⁴C]valine, and [¹⁴C]aspargine with specific activities of several microcuries per millimole. The specific activities of the products could be significantly increased by increasing the specific activity of the radioactive carbonate initially used.     

14C in spent ion-exchange resins and process water from nuclear reactors: A method for quantitative determination of organic and inorganic fractions

A method of separately quantifying organic and inorganic ¹⁴C compounds present in spent ion-exchange resins and process water from nuclear power reactors has been developed. Extraction of carbon compounds is accomplished by means of acid stripping (inorganic ¹⁴C) and subsequent wet oxidation (organic ¹⁴C) or by N₂ purging in combination with a catalytic furnace (oxidized and reduced gaseous compounds) with organic and inorganic ¹⁴C species collected separately. Recovery experiments on simulated samples spiked with ¹⁴C-labeled sodium carbonate, sodium acetate and sodium formate, showed extraction yields of 94–98%. The sample-specific procedures were also tested on authentic samples of spent resins and reactor water with good results. Validation and reliability of the procedures are presented and the method is compared to previous methods reported in the literature.     

Liquid scintillation assay of 3H and 14C-labelled polymers

The assay of a range of ³H and ¹⁴C-labelled polymers by liquid scintillation counting is reported. Methods of sample preparation such as combustion, solution, gelation and suspension techniques are described and compared. The validity of a variety of procedures of counting efficiency determination for the above methods is examined and critically discussed.     

Determination of enantiomeric purity in biogenic11C-labelled amino acids by aminoacylation of tRNA

In syntheses of naturally occurring amino acids labelled with short-lived radionuclides, such as¹¹C, the determination of the enantiomeric purity presents problems. The aminoacyl coupling of L-amino acids to tRNA followed by a separation with gel filtration of the tRNA-amino acid complex and free amino acid, was here shown to be an adequate method for determining the enantiomeric purity of [methyl-¹¹C]-methionine even in the pmol range. This method has also been used for the determination of the specific radioactivity of the¹¹C-labelled methionine. The method is probably valid for other naturally occurring amino acids and it might also be of interest for enantiomeric separations of L- and D-amino acids of high specific radioactivity.     

Hydropyrolysis over a platinum catalyst as a preparative technique for the compound‐specific carbon isotope ratio measurement of C27 steroids

Compound‐specific stable carbon isotope analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is an important method for the determination of the ¹³C/¹²C ratios of biomolecules such as steroids, for a wide range of applications. However, steroids in their natural form exhibit poor chromatographic resolution, while derivatisation adds carbon thereby corrupting the stable isotopic composition. Hydropyrolysis with a sulphided molybdenum catalyst has been shown to defunctionalise the steroids, while leaving their carbon skeleton intact, allowing for the accurate measurement of carbon isotope ratios. The presence of double bonds in unsaturated steroids such as cholesterol resulted in significant rearrangement of the products, but replacing the original catalyst system with one of platinum results in higher conversions and far greater selectivity. The improved chromatographic performance of the products should allow GC/C/IRMS to be applied to more structurally complex steroid hormones and their metabolites. Copyright © 2010 John Wiley & Sons, Ltd.