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).     

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.     

Verwendung eines ortsauflösenden proportionalzählrohres zur messung von dünnschichtchromatogrammen C-markierter substanzen

Use of a position-sensitive proportional counter system for measurements of thin-layer chromatograms of ¹⁴C-labelled substancesA position-sensitive proportional counter system has been developed for the quantitative determination of thin-layer chromatograms of ¹⁴C-labelled substances. The described system allows simultaneous measurements of the whole chromatogram. Data interpretation (integration of peajs) is very simple with a modern multichannel analyzer.The specific properties of the system are compared with a normally used chromatogram scanner. The usefulness of the system is demonstrated on ¹⁴C-labelled chromatograms.

Zusammenfassung

Mit Hilfe eines Proportionalzählrohres wurde ein ortsempfindliches Detektorsystem für die quantitative Auswertung von Dünnschichtchromatogrammen ¹⁴C-markierter Substanzen entwickelt. Das vorgeschlagene Messsystem bietet gegenüber herkömmlichen Scannern den Vorteil, dass das gesamte Chromatogramm gleichzeitig gemessen wird und die Auswertung der Messung mit einem modernen Vielkanalsystem rasch und einfach vorgenommen werden kann. Die praktische Anwendung der Anlage sowie der Vergleich mit einem herkömmlichen Radiochromatogramm-Scanner wird anhand von ¹⁴C-markierten Testmischungen demonstriert.     

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.     

Detection of <Emphasis Type="Italic">N</Emphasis>-monomethyl-lysine generated by metabolic transmethylation

Administration of radiolabelled deprenyl to rats resulted in the urinary elimination of a ¹⁴C-labelled N-monomethyl-lysine. An increased level of N-monomethyl-lysine was found following an oral dose of another drug, also containing an N-methyl group. The urine sample was treated with 9-fluorenylmethoxycarbonyl chloride and then subjected to high-performance liquid chromatography (HPLC); the radioactive fraction was identified as N-monomethyl-lysine by using HPLC-MS in electrospray mode. Identification of N-monomethyl-lysine in the radioactive fraction gives experimental proof of transmethylation from a well-known drug to an endogenous compound.     

Gas chromatography-mass spectrometric analysis of bonded long chain fatty acids in a single zebrafish egg by ultrasound-assisted one-step transmethylation and extraction

Changes in the level of lipid composition in a single zebrafish egg have been involved in biological responses to chemical exposures. In this paper, an one-step transmethylation of lipids and simultaneous extraction of resultant fatty acid methyl esters followed by gas chromatography-mass spectrometric analysis was developed for the identification of bonded long chain fatty acids in a single zebrafish egg. The efficiency of transmethylation under different experimental conditions has been investigated. Dried egg homogenates were directly mixed with either 0.5 M NaOH or 1% H₂SO₄ or 4% HCl solution of methanol and then an n-hexane solution was added on the top. Ultrasonication of this immiscible liquid-liquid-solid system produces high velocity impacts between solid particles and liquid phases and thus promotes mass transfer among phases. It was demonstrated that ultrasound irradiation has strong effect on the alkaline-catalyzed transmethylation of lipids but cannot significantly change the acid-catalyzed transmethylation of lipids. With the aid of ultrasonication, transmethylation can be combined with simultaneous extraction of the resultant fatty acid methyl esters into n-hexane phase. This approach simplifies the sample preparation procedure, shortens the reaction time but improves the efficiency of the transmethylation of lipids and reduces sample losses, especially for small size samples. It has been applied to determine bonded fatty acids in a single zebrafish egg. In total, 28 fatty acids from a single zebrafish egg have been identified reproducibly.     

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%.     

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.     

Determination of the specific radioactivity of amino acids by a combination of thin-layer chromatography and quantitative autoradiography

A new method for the analysis of the specific activity of amino acids is described. The analysis is carried out by thin-layer chromatography of the dansylated amino acids, computerized fluorescence evaluation and activity measurement by quantitative autoradiography. Quantitative evaluation of the autoradiographs is achieved by careful calibration of the X-ray film blackening. As shown for 14C-labelled phenylalanine and tyrosine, the method allows the simultaneous determination of the specific activity of 22 amino acids. About 10(-13) mol of an amino acid with a specific activity of less than 5 GBq/mmol can be detected and measured by this method.     

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.     

Enhanced radiation crosslinking of acrylic acid and methacrylic acid to polymers: alkanes as model compounds for polyethylene

The crosslinking of polyethylene by irradiation is enhanced in the presence of acrylic acid. This process has been studied for model compounds; for ¹⁴C-labelled hexadecane, each molecule of polyacrylic acid is linked to an average of 47 hexadecane molecules; at the radiation dose used, this result implies a hitherto unexpected chain reaction. Evidence from ESR spectra indicates an ionic intermediate.     

Untersuchungen über die Biosynthese der Cyclite, 26. Mitt.: Bildung von Mytilit (C-Methyl-scyllo-inosit) inChlorella fusca

Incorporation experiments with¹⁴C-labelled precursors of mytilitol inChlorella fusca seem to indicate that the last step in the biosynthesis of mytilitol is an epimerization of laminitol (d-4-C-methyl-myo-inositol) to mytilitol.     

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.     

Intermediates in the penicillic acid biosynthesis in Penicillium cyclopium.

Feeding experiments using 14C-labelled precurosrs demonstrate the following sequence of the biological formation of penicillic acid in Penicillium cyclopium: orsellinic acid leads to 2-O-methylorsellinic acid leads to 1,4-dihydroxy-6-methoxy-2-methylbenzene leads 6-methoxy-2-methyl-benzoquinone(1,4)leads to penicillic acid.     

Specific carbon-13 labelling of leucine residues in human growth hormone.

Biosynthetic human growth hormone specifically 13C-labelled in the carbonyl positions of all 26 leucine residues has been obtained by recombinant DNA techniques using 13C-labelled leucine and an E. coli strain that requires leucine. It is shown that, on the whole, the labelling is specific with no significant mislabelling as would have been the case had the 13C-labelled leucine been metabolized.     

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.     

High-efficiency preparative-scale reversed-phase high-performance liquid chromatographic purification of 14C-labelled antibiotics

The 14C-labelled antibiotics [2-14C]mupirocin, and [thienyl-3-14C]temocillin cannot be satisfactorily purified on a small scale by conventional methods of chromatography or recrystallisation. Their purification was successfully achieved by high-efficiency preparative-scale reversed-phase high-performance liquid chromatography. The purifications employed 250 mm X 10 mm I.D. or 22 mm I.D. stainless-steel columns packed with Merck LiChrosorb RP-18 (10 microns) stationary phase which were eluted with aqueous buffer solutions at flow-rates of 10-25 ml min-1 using conventional analytical instrumentation.     

Carbon-14 labelling of biomolecules induced by14CO ionized gas

Ionized¹⁴CO gas provides a rapid method for producing¹⁴C-labelled biomolecules. The apparatus consists of a high vacuum system in which a small amount of¹⁴CO is ionized by electron impact. The resulting species drift towards a target where they interact with the molecule of interest to produce¹⁴C-labelled compounds. Since the reaction time is only 2 minutes, the method is particularly promising for producing tracer biomolecules with short-lived¹¹C at high specific activities. We have studied the applicability of the method to various classes of compounds of biological importance, including sterids, alkaloids, prostaglandins, nucleosides, amino acids and proteins. All compounds treated gave rise to¹⁴C addition and degradation products. Furthermore, for some compounds, chromatographic analysis in multiple systems followed by derivatization and crystallization to constant specific activity, indicated that carbon exchange may occur to produce the labelled, but otherwise unaltered substrate in yields of the order of 10–100 mCi/mol. More conclusive proof of radiochemical identity must await production of larger quantities of material and rigorous purification including at least two different chromatographic techniques. Supported by the Medical Research Council of Canada Grant MA-6137, and by the Banting Research Foundation.     

14C-寡糖在西瓜幼苗植株体内吸收传导和分布

应用同位素示踪技术研究了14C-寡糖在西瓜幼苗植株体内的吸收、传导和分布行为.自显影结果显示,寡糖通过处理叶部或根部后能够被西瓜幼苗植株快速吸收,在叶片中的传导表现为从叶缘向叶片中心分布的趋势.将叶部处理8h和根部处理24h后,14C-寡糖即可以传导和分布到西瓜幼苗的整个植株体内,证明14C-寡糖在西瓜幼苗植株体内具有较强的扩散和向基或向顶传导特征.结果表明,处理叶部4～120h时,根系、茎与未被直接处理的叶片等其它部位的放射性比活度分别由0.18×105和23.08×105Bq/kg变化为0.32×105和3.02×105Bq/kg,总体上表现出向基传导和分布的态势.处理根部4～120h时,西瓜幼苗植株根系、茎部、子叶和真叶中放射性比活度分别由22.23×105,2.23×105,8.33×105和12.78×105Bq/kg变化为431.11×105,42.23×105,65.57×105和78.89×105Bq/kg,表现出14C-寡糖在西瓜幼苗植株体内向顶传导作用和在地上部的积累态势很强.     

A simple quantitative HPLC assay for ifosfamide in biological fluids

A high performance liquid chromatography method is described for measuring Ifosfamide (I) in human serum. This involves solvent extraction, reverse phase HPLC and UV detection at 190 nm. Standard curves of peak height x detector sensitivity versus I concentration in serum were linear with a lower limit of detection of 100 ng/ml. Authentic 14C-labelled I cochromatographed with standard I and with I found in serum from treated patients. The concentration-time curves of I determined by both HPLC and gas chromatography were indistinguishable. We conclude that this method is suitable for determining I pharmacokinetics in biological specimens.