I analyzed solvent components of liquid scintillation cocktails, such as phenylxylylethanes (PXE), di-isopropylnaphthalenes (DIN) and isopropylbiphenyls (IPBB), by temperature-programmed gas chromatography on non-polar DB-1 and polar DB-Wax columns and used a structure-retention relation to characterize or tentatively identify the unknown components in PXE-I, PXE-II, DIN and IPBB solutions by retention index (I) and column difference (Delta I). The Delta I value can characterize the aromatic ring, and the I value can indicate the molecular size. I calculated the I value from the retention time using n-alkanes as retention calibration standards according to the convention of Kovats (1958) and computed the Delta I value by pairing the peaks on DB-I and DB-Wax columns from the same component by concentration. Of the four solutions analyzed PXE-I was a pure product (98%). PX-II consisted of four major peaks on the DB-1 column One of the 4 was an overlapping peak that resolved into several individual peaks on the DB-Wax column. The DIN sample contained 4 major peaks, 2 of which overlapped on the DB-1 column. I assigned one peak to DIN and did not characterize the others. The two major peaks in the IPBB sample, as tentatively identified by this method, are 4-isopropylbiphenyl and 4-tert.-butylbiphenyl.

By-products formed in radiation-induced tritium labelling are identified by co-chromatography with authentic samples or by structure prediction using a quantitative structure-retention index relationship. The by-products, formed from labelling of steroids, polynuclear aromatic hydrocarbons, 7-membered heterocyclic ring structures, 1,4-benzodiazepines, 1-haloalkanes, etc. with activated tritium and adsorbed tritium, are shown to be specifically labelled and anticipated products from known chemical reactions. From analyses of the by-products, one can conclude that the hydrogen abstraction by tritium atoms and the substitution by tritium ions are the mechanisms of labelling. Classification of the tritium labelling methods, on the basis of the type of tritium reagent clearly shows the active role played by tritium atoms and ions in radiation-induced methods.

Tritium incorporation by synthetic and non-synthetic methods shares the common mechanism of labeling, requiring the activation of tritium gas. Activation can be by catalysts, hot tungsten wire, microwave discharge, etc. and results in the formation of tritium atoms and ions. The tritium atoms and ions may form free or sorbed onto a surface to react with substrate yielding different isotopomers and by-products. A third mechanism of labeling is tunneling. Tunneling is significant at near absolute zero temperature with liquid and solid tritium and is also significant when high pressures of tritium gas are used for labeling. Other parameters relating to supports, catalysts, purity of tritium gas, chemical nature of substrates, can also affect labeling. Tritium NMR spectroscopy can determine the tritium distribution in a molecule to aid in interpreting the labeling mechanism. The non-synthetic methods have the potential of labeling complex molecules of biomedical interest that are inaccessible by synthetic methods.

Twenty solvent components in a commercial scintillator were identified by chromatography on polar and non-polar columns and by gas chromatography-mass spectrometry (GC-MS) as isomeric 1-(alkyl)m(alkyl)nbenzenes with formulae C16H26, C17H28, C18H30 and C19H32. These isomers occur in four clusters of chromatographic peaks representing ca. 6, 44, 34 and 16% of the total solvent mass. The retention indexes of the isomers are influenced by the lengths of the alkyl chains in the molecule, and their polarity and polarizability can affect the column difference, which is the difference between retention indexes on polar and non-polar columns. 1-Methylalkylbenzenes have higher retention indexes and larger column differences than the evenly distributed isomers, such as 1-butylhexyl-1-pentylhexyl, 1-pentylheptyl- and 1-pentyloctylbenzene. The results demonstrate the effect of structural symmetry on the retention indexes of the isomers. This study shows that the ability to relate GC data and column differences to structures can facilitate the interpretation of GC-MS data in the structure identification of isomers.

Polar compounds containing hydroxyl, amino and carboxyl groups, singly or in combination, can be chromatographed after the polar functional groups are silylated. The silylated derivatives of acids, alcohols, amines, diols, amino alcohols, amino acids are shown to behave chromatographically as hydrocarbons, and their retention indexes can be readily predicted from their base values. The column difference, namely, the difference between the retention indexes of the analyte on polar and non-polar columns is minimal for the silylated derivatives in comparison to that observed for the underivatized analytes. This minimal column difference is attributed to the hydrocarbon-like chromatographic characteristics of the silylated derivatives. The retention indexes of the silyl derivatives appear to correlate with the atom number Z of the analyte.

A series of progesterone derivatives has been studied as potential inactivators of the bovine adrenocortical cytochromes P450, P450 17 alpha, and P450 C-21. Replacement of the 21-methyl group of progesterone with a difluoromethyl group resulted in a selective inactivator of P450 C-21 in a reconstituted system. The loss of 21-hydroxylase activity caused by this compound exhibits a number of characteristics of mechanism-based inactivation including NADPH dependence, pseudo-first-order kinetics, saturability, irreversibility, and protection by substrate. In addition to the difluoro compound, 21,21-dichloroprogesterone, the acetylenic compound pregn-4-en-20-yn-3-one, and the olefinic compound pregna-4,20-dien-3-one all inactivate P450 C-21. In contrast, the only compound to inactivate the rabbit adrenal progesterone 21-hydroxylase is 21,21-dichloroprogesterone. In binding studies, the 21,21-dihalo steroids produce a greater maximal type I spectral shift of P450 C-21 than the two 17 beta-unsaturated steroids. The dihalo compounds inactivate P450 C-21 by both heme destruction and protein modification as shown by significant decreases in residual 21-hydroxylase activity and spectrally detectable P450 after incubation with P450 C-21 in a reconstituted system. Liquid chromatographic and mass spectral analyses of the organic extracts from these incubations showed that 21-pregnenoic acid is a major metabolite of the dihalo compounds with a partition ratio of 5 nmol of acid produced/nmol of P450 C-21 inactivated. This supports the hypothesis that inactivation proceeds in part through an acyl halide intermediate. In contrast, the acetylenic compound pregn-4-en-20-yn-3-one inactivates P450 C-21 mainly by protein modification, producing an NADPH-dependent irreversible type I spectral shift. The stoichiometry of inactivation is approximately 1.5 nmol of compound bound/nmol of enzyme inactivated, indicating selective modification of the enzyme at or near the substrate binding site.

A method is described for the prediction of the retention index (I) from chemical structure, using the number of atoms in the molecule (Z), the I increment for atom addition (A) and the group retention factors (GRFs) of the functional groups and substituents. This method can predict the retention indexes of a wide range of compounds, such as acids, alcohols, amines, acid esters, aldehydes. ketones. ethers, aromatic hydrocarbons, alicyclics, heterocyclics, etc, on polar as well as non-polar columns to within 3% error. Accurate A and GRF values are essential to the prediction. These values can be obtained from homologous series, but a system of arbitrarily assigned A value and adjusted GRFs are also used. The GRFs of the substituents and functional groups depend on the polarity and polarizability of the analyte and the stationary phase and also on the molecular connectivity of the atoms, namely, primary, secondary and tertiary carbon atoms or hydrogen atoms, to which these groups are attached. Highly polar and polarizable groups can alter the A value. When the functionality of a group is masked by substitution. the analyte molecule will tend to behave chromatographically like hydrocarbons. The difficulty in predicting the I values of compounds of multi-functionality by the rule of additivity is the unknown intramolecular interaction that can alter both A and GRF values.

AbstractSteroid hormones and derivatives are labeled to high specific activities with activated tritium generated by microwave discharge of tritium gas. The steroid nucleus is not degraded by reactions with tritium; the hydroxyl and oxo groups at 3, 11, 17, and 20 positions of the steroid nucleus are not displaced by tritium but are oxidized or reduced to form the labeled by‐products. Saturation of A ring occurs readily, but tritium addition to the isolated CC double bond occurs in the order of Δ1 > Δ4 > Δ5. Steroids with the 21‐hydroxyl group are not tritiated. Despite the addition reaction, the final products from the unsaturated steroids may still contain significant amounts of the labeled parents. Specific activities of the labeled by‐products steroids can approach 29 Ci/mM.

Luminescence components in the filter papers and other cellulosic materials used in smear and wipe test may interfere with the measurement of tritium by liquid-scintillation counting. A Scotch tape method for tritium monitoring is recommended, to avoid this interference.

Nitroxides, paramagnetic compounds with demonstrated effectiveness as contrast agents in proton magnetic resonance imaging, shorten the relaxation times of protons and therefore cause an increase in image intensity in tissues into which they distribute. In this study, the metabolic fate of the nitroxide moiety was examined in the dog using a pyrrolidine nitroxide derivative, 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-carboxylic acid, for which contrast-enhancing properties have been previously studied in animals. After radiolabeling by microwave discharge in the presence of tritium gas, the compound was administered intravenously to a dog. Ninety-four percent of the radioactivity injected was recovered in urine within 3 days; the majority (90%) was excreted during the first 6 h. The radioactivity in the urine was identified as either the unchanged nitroxide or its corresponding hydroxylamine. Neither complete reduction of the nitroxide moiety to the amine nor any other metabolic transformation was observed.

Abstract[3H]N, N‐Dimethylaniline was prepared from N, N‐dimethylaniline by tritium labeling by microwave discharge activation of tritium gas; the product was formed without any ring saturated side products. High specific activity and high yield were achieved by input of high microwave power to excite the plasma and by dispersion of substrate on silica‐alumina pellets to increase the contact surface with tritium gas. Circulation or no circulation of tritium gas during labeling did not effect the yield or the specific activity. Dispersion on supported Ni caused an increase in the specific activity of [3H],N, N‐dimethylaniline than on plain silica‐alumina. The lack of ring saturation in N, N‐dimethylaniline in contrast to complete ring saturation in N, N‐dimethylaniline in contrast to complete ring saturation in benzene and benzoic acid has been interpretated to indicate the involvement of tritium ions in addition to tritium atoms in tritium labeling.

AbstractGlycylglycyl‐L‐leucine and glycyl‐L‐leucine dispersed on silica‐alumina and supported Ni catalyst, were labeled with tritium by microwave discharge activation of tritium gas, as a model to study the optimum condition for labeling peptides and proteins. In the reaction, decreasing the tritium pressure and increasing the microwave power promote the formation of tritium ions and result in an increase in specific activity of the labeled product. The highest specific activity was achieved with the adsorbate molecules as a monolayer on support. Under similar conditions, amino acids glycine and L‐leucine were less efficiently labeled than the peptides. Tritium atoms generated by mercury photosensitization with a uv light were less effective for labeling than tritium ions. Impurities in tritium gas can adversely affect the degree of tritium incorporation in peptides.