C labelled internal standards-A solution to minimize ion suppression effects in liquid chromatography–tandem mass spectrometry analyses of drugs in biological samples?

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is frequently used to identify and quantify drugs in human biological samples due to the high selectivity and sensitivity of this technique. However, ion suppression effects caused by co-eluting compounds: drugs, metabolites, matrix components, impurities and degradation products, are a major concern. Stable isotope labelled internal standards (SIL ISs), usually deuterium ((2)H) labelled, are often used to compensate for these effects. In many LC separations the retention times of (2)H labelled ISs and their analogues will differ. Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) is increasingly being used for bio-analysis. With the better chromatographic resolution provided with sub 2 μm particles, larger separation between analytes and their (2)H labelled analogues can be expected, which might reduce the benefits of the SIL IS. There is a greater difference in physico-chemical properties between hydrogen isotopes than between isotopes of other elements. (13)C, (15)N and (18)O labelled ISs are more similar to their analytes than (2)H labelled ISs and thereby expected to behave more similarly in chromatographic separations. In this study we have investigated the use of (13)C and (2)H labelled ISs for the determination of amphetamine and methamphetamine by UPLC-MS/MS. The (13)C labelled ISs were co eluting with their analytes under different chromatographic conditions while the (2)H labelled ISs and their analytes were slightly separated. An improved ability to compensate for ion suppression effects were observed when the (13)C labelled ISs were used. Furthermore, an UPLC-MS/MS method for determination of amphetamine and methamphetamine in urine using (13)C labelled ISs has been developed and validated. Unfortunately, there are few (13)C labelled ISs commercial available today. If more (13)C labelled ISs become commercial available they may well be the coming solution to minimize ion suppression/enhancement effects in LC-MS/MS analyses of drugs in biological samples.     

Synthesis of radiolabelled compounds

Compounds labelled with radioisotopes such as¹⁴C,³H,³²P,³⁵S and¹²⁵I, are essential tools for research and especially in the life sciences. Syntheses can be directed to isotopic labelling or to non-isotopic labelling. In isotopic labelling a compound is labelled with an isotope of an element already present in the compound whereas non-isotopic labelling is achieved with an isotope is foreign to the material or compound being labelled. This paper reviews the properties of the more important and commonly used radioisotopes in research and methods currently employed for the synthesis of radiolabelled compounds. The relative ease of measurement of radioactivity in large numbers of samples, when compared for example with the measurement of mass, and the great sensitivity with which very small quantities of radioactive compounds can be accurately measured, has resulted in the widespread need and use of radiolebelled compounds. Methods for the practicable labelling of compounds for use as radiotracers all into three main categories, namely, chemical systhesis, biochemical methods and isotope exchange reactions. Examples are chosen to illustrate these methods for the labelling of amino acids, peptides, proteins, carbohydrates, lipids, neurochemicals, nucleic acids, steroids and miscellaneous compounds of interest in biological research. Most methods of labelling employed lead to specific labelling, that is, to modecules in which the position(s) of the radioactive atom(s) is known with certainty. However, isotope exchange reactions which are especially useful for labelling molecules with tritium are often non-specific. The routine use of tritium nuclear magnetic resonance spectroscopy has resolved any uncertainties in the specificity of labelling with tritium. Examples are given illustrating the effectiveness of this valuable analytical technique. A review of the synthesis of radiolabelled compounds would not be complete without reference to the special properties of the labelled compounds themselves which affect their useful shelf-life, self-decomposition, purification and analysis; factors which all need to be clearly understood in order to use radiolabelled compounds with confidence in scientific research.     

Epoxy resins. New results and developments

In a survey the development of epoxy resin systems is described. During the past years countless formulations based on known structures were used, but only a relative small number of new epoxies and curing agents were investigated and only very few new compounds came on the market. A choice of these products will be discussed, also new latent accelerators and curing agents. By improvement of technical processes purer epoxies can be offered and toxicological risks could be strongly reduced. Especially new results in the fast growing field of photocrosslinking will be described. Besides iodonium, sulfonium and phosphonium compounds new arene iron salts and their behaviour will be presented. Generally, the trend in the epoxy field is to better heat resistant structures for new formulations in the field of adhesives and fibre-reinforced materials, and to new photocrosslinking systems.     

Catalytic tritiation studies using tritium NMR spectroscopy

The development of tritium nuclear magnetic resonance spectroscopy now makes it possible to determine the tritium distribution in virtually any organic compound at the millicurie level of radioactivity. Results of catalytic experiments show that in some cases a remarkable degree of specificity can be achieved when using procedures that are expected to produce generally labelled compounds. Conversely there are instances where specific labelling procedures are less than 100% successful.     

13C, 13C coupling constants in phenyl substituted ethylene,naphthalene, phenanthrene and cyclopentadienone. Dependence on dihedral angles. Determination of signs by the symmetrical double labelling (SDL) method. IV

Carbon-13, carbon-13 coupling constants and carbon-13 chemical shifts have been measured in a series of phenyl substituted ethylenes and aromatics all doubly labelled with ¹³C at the olefinic positions (α,β-) or at neighbouring aromatic positions, tetraphenylcyclopentadienone labelled at the 3,4-positions, and dichlorodiphenylmethane labelled at the α-carbon. Signs of coupling constants were determined by the symmetrical double labelling (SDL) method. Coupling constants over as many as five bonds are reported. Two-bond couplings between carbons in the aromatic skeleton belong to different classes according to the nature of the coupling path. The magnitudes of three-bond coupling constants between such carbons correlate linearly with π-bond orders and a separation of the δ- and π-contributions is evident. The three-bond couplings between the 2-position in a phenyl substituent and the olefinic β-position or a corresponding aromatic position depend on the out-of-plane twist of the phenyl ring and may lead to information about the twist angle. Contrary to findings with aromatic carbonyl compounds two- and three-bond couplings to the α-carbon in the present compounds are fairly constant. The reported data suggest that the signs of coupling constants over more than two bonds alternate in aromatic systems. Carbon-13, carbon-13 coupling constants in naphthalene have been calculated by the INDO-SOS method.     

Determination of the isotope distribution in partially deuterated compounds by NMR difference spectroscopy

The position and the isotope distribution within labelled compounds can be determined by NMR by analysing the difference spectrum, and its integral, of the labelled and the corresponding parent compound. The method is applied to m-terphenyl and it 4–4″-dideutero derivative.     

Gel chromatography of steroid oestrogens on sephadex LH-20.

The behavior of 23 steroid oestrogens Sephadex LH-20 using six solvent systems was investigated. The fractions eluted by gel column chromatography were analysed by thin-layer chromatography and spectrometry. The method was used for the radiochemical purity of labelled compounds by isotopic dilution.     

Tritium nuclear magnetic resonance spectroscopy. 14—analysis of tritiated methyl groups

The complete analysis of the tritium distribution in tritiated methyl groups in labelled compounds is achieved by a direct method for the first time, through ³H NMR spectroscopy aided by resolution enhancement.     

Der massenspektrometrische Verlust von Vinylalkohol aus 1-Amino-3-aryloxy-2-propanolen. 24. Mitteilung über das massenspektrometrische Verhalten von Stickstoffverbindungen

Loss of vinyl alcohol from 1-amino-3-aryloxy-2-propanols under electron impact Under electron impact compounds of type 1 (see Scheme 1) split off 44 mass units from the molecular ion. This unusual reaction was studied using derivatives and deuterium labelled compounds. It could be demonstrated that for this fragmentation reaction 16 is the important structural feature from which H₂(C3)?C(2)HOH (44 mass units) is lost. The preferred reaction mechanism involves a transition state in which four members of the side chain are involved (Scheme 2, mechanism 2).     

Preparation of radiopharmaceuticals by Szilard-Chalmers labelling and radioprotection in an ice lattice

A new and simple method for the labelling of halogenated organic molecules and biomolecules is described. Dilute aqueous solutions of the halogenated molecule are irradiated with neutrons in the frozen state. The labelling yields are generally high for the 18 compounds investigated and constant over a large concentration range. The major observed labelled organic product is the radiohalogen labelled parent molecule with the halogen at its original site. Unlike neutron-irradiated liquid aqueous solutions of the same compounds no radiation damage to the molecules is observed for the irradiation times employed.     

Massenspektrometrische Untersuchung Organischer Stickstoffverbindungen. XII—H-übertragungen Durch Nachbargruppen-Wechselwirkung bei αω′-bis-(N-Piperidyl)-Aromaten

The electron-impact-induced fragmentation of several dipiperidines, which are strongly influenced by neighbouring group participation, could be established by the investigation of ²H labelled compounds. In addition to a novel McLafferty rearrangement, specific as well as selective hydrogen transformations preceding the elimination of piperidine could be proven. The isomeric diamines can be clearly distinguished and identified by means of mass spectrometry.     

Study of the metabolism of testosterone, nandrolone and estradiol in cattle.

The current metabolism study was undertaken to identify key analytes in urine, plasma and bile following testosterone, nandrolone and estradiol administrations to cull cows, heifers and steers. This information will be used to develop confirmatory analysis procedures. In the present study, mixtures (1:1) of testosterone, nandrolone or estradiol and their deuterium labelled analogues were administered to cull cows, heifers and steers. Two analogues of deuterium labelled testosterone were synthesised and administered, to facilitate identification of metabolites. Following administration, urine, plasma and bile samples were collected and subjected to solid phase extraction. The extracts were derivatised and analysed by GC-MS. The major analytes derived from the administered steroids were identified on the basis of the twin ion peaks produced for their non-labelled and deuterium labelled analogues and their stereochemistries determined by comparison of retention times with appropriate reference standards. Using suitable internal markers, excretion profiles for the major analytes in urine and plasma have been determined and levels in isolated bile samples estimated. This work is on-going, and this paper is a summary of some of the studies completed to date.     

Massenspektrometrische Untersuchung von Amiden. VI—Mechanismus der Methyl-Abspaltung aus Crotonsä;urepiperidid

By the investigation of ²H labelled compounds, it has been established that in the case of crotonoyl piperidyl only the hydrogens of the methyl group participate in the elimination of CH₃·.     

Nucleobase-containing transition metal complexes as building blocks for biological markers and supramolecular structures

The incorporation of metal complexes into nucleobases, nucleosides and nucleotides has provided a focus for the development of many novel compounds with a wide range of applications. In this perspective article the different methods to incorporate transition metal complexes into these species will be described. Applications of these compounds as biological markers, catalysts and how the hydrogen bonding properties may be employed in directing supramolecular assembly in both the solid state and solution will be discussed.     

Methylation artifacts in the gas chromatography of serum extracts. Competitive suppression with trimethyl-d9 anilinium hydroxide.

In the course of the identification of drugs in body fluids by gas chromatography-mass spectrometry we have observed the appearance of N-methyl derivatives of certain compounds such as phenobarbital and diphenylhydantoin, especially in extracts of serum. With the aid of deuterium labelled phenobarbital it could, however, be shown that the N-methyl derivatives are not endogenous metabolites but artifacts generated upon injection of the serum extract into the gas chromatograph. Conversely, derivatization with trimethyl-d9 anilinium hydroxide (TMAH-d9) demonstrated the absence of any undeuterated methyl derivative and this reagent should be useful for the detection of endogenous N-methyl derivative which would, of course, remain undetected when using unlabelled TMAH in the course of the conventional methylation technique. There are good indications that lecithin is the methylating agent in serum responsible for the formation of these artifacts.     

NH3-Eliminierungen aus 2-Äthoxybenzoesäureamid und 2-Äthoxybenzylamin Als Weitere Beispiele [Komplexer] Ortho-Effekte

Using deuterium labelled compounds the mechanisms of the elimination of ammonia from the molecular ions of the title compounds have been elucidated. It can be shown that the fragmentation is preceded by a partial hydrogen scrambling which is favoured by the carbonyl function. In addition to this an unusual oxygen-carbon bond cleavage of the ether moiety can be explained by assuming a neighbouring group participation.     

Colloid and nanodimensional catalysts in organic synthesis: VIII. Hydrogenation of C=N bond with hydrogen in the presence of colloid nickel

Hydrogenation of azomethines with hydrogen at atmospheric pressure using nickel nanoparticles as catalyst was carried out. Reaction may be used for the preparation of secondary amines under mild conditions on an available catalyst. Continuation of studies will lead to development of a convenient method for the reductive amination of carbonyl compounds.     

Quantitative LAMMA analysis of biological specimens I. Standards. II. Isotope labeling

Summary Besides a remarkably high sensitivity for light elements, laser microprobe mass spectroscopy offers two main advantages for microprobe analysis: 1) the possibility to obtain data of relatively high precision and 2) the ability to discriminate isotopes. This opens the possibility to use cold isotopes as atomic labels to study cellular or subcellular kinetics of ions or labelled organic compounds. In this study photoreceptor cells were labelled with ⁴⁴Ca²⁺ prior to LAMMA analysis to monitor Ca²⁺ uptake and/or Ca²⁺ accumulation from extracellular sources into intracellular compartments such as the various pigment granula present in these photoreceptors.As in other microprobe techniques also LAMMA analysis requires internal standards for quantitative work. For this purpose thin films of inorganic material were shown to be useful standards when deposited (in vacuum) directly onto the specimen to be analyzed. Some metals and dielectrica have been tested for their possible suitability for standardisation procedures. Even though metals like Pt, Ag, Au, Al are excellent for thin film production, they seem to be less useful for the present purpose because their threshold for laser induced perforation and ion production is rather different from that of the specimen material. However, thin film deposits of some dielectric materials such as MgF₂ turned out to be more suitable to serve as an internal standard for plastic embedded biological material in LAMMA analysis.

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Quantitative LAMMA-Analyse biologischer Proben. I. Standards. II. Isotopenmarkierung

     

Development of carbohydrate-based scaffolds for restricted presentation of recognition groups. Extension to divalent ligands and implications for the structure of dimerized receptors

The solution structure of glycosyl amides has been studied by using NMR. A strong preference is displayed by tertiary aromatic glycosyl amides for E-anti structures in contrast with secondary aromatic glycosyl amides where Z-anti structures predominate. The structural diversity displayed by these classes of molecules would seem to be important as the directional properties of the aromatic ring, or groups attached to the aromatic ring, would be determined by choosing to have either a secondary or tertiary amide at the anomeric center and could be considered when designing bioactive molecules with carbohydrate scaffolds. The structural analysis was also carried out for related divalent secondary and tertiary glycosyl amides and these compounds display preferences similar to that of the monovalent compounds. The constrained divalent compounds have potential for promoting formation of clusters that will have restricted structure and thus have potential for novel studies of mechanisms of action of multivalent ligands. Possible applications of such compounds would be as scaffolds for the design and synthesis of ligands that will facilitate protein-protein or other receptor-receptor interactions. The affinity of restricted divalent (or higher order) ligands, designed to bind to proteins that recognize carbohydrates which would facilitate clustering and concomitantly promote protein-protein interactions, may be significantly higher than monovalent counterparts or multivalent ligands without these properties. This may be useful as a new approach in the development of therapeutics based on carbohydrates.