Isotope dilution method, application

Barker SA, Littlefield-Chabaud MA, David C. 2001. Distribution of the hallucinogens N,N-dimethyltryptamine and 5-methoxy-N,N-dimethyltryptamine in rat brain following intraperitoneal injection application of a new solid-phase extraction LC-APCI-MS-MS-isotope dilution method. J Chromatogr B Biomed Appl 751 37. 

The sensitivity and specificity conferred by making isotopically labeled derivatives of drugs in vitro can be used clinically (K18, K19), as can isotope dilution methods (H2, 07). The great imposition upon time, skills, and resources that such techniques demand, limits their application to selected research studies. They nevertheless provide a basis against which other more practicable methods can be assessed. 

In the interim between the initial development and application of Methods 624 and 625, the Effluent Guidelines Division, under contract, developed isotopic dilution methods for purgeable volatiles and semivolatile priority pollutants (11). The primary difference between Methods 624 and 625 and their 1624 and 1625 counterparts is that stable, isotopically labeled analogs of the compounds of interest are added to the sample prior to extraction, and quantitative determination is made by using the isotope ratio values for the compounds determined relative to their labeled analogs. In addition, Method 1625 is designed as a capillary column method and uses a 30-m X 0.25-mm silicone-bonded-phase fused silica column (J W DB-5). Method 1624 uses the same column materials as Method 624. Methods 1624 and 1625 are presented as acceptable alternatives to Methods 624 and 625. 

This compound was synthesized by Dr. Nowacki by reacting 11+C-IAA-imidazole with myo-inositol (40). Application of these labeled compounds to corn kernels, followed immediately by homogenization of the tissue in acetone permitted us to determine the amounts of each constiuent in the kernel by the isotope dilution method of Rittenberg and Foster (41). An extension of this method, whereby the kernels are incubated for varying periods of time after application of the isotopically labeled compound permits determination of the "turnover" of the pool. Such data are shown in... 

The field of application for the isotope dilution method with radioactive tags, extends to measurements using stable isotope. Mass spectrometry or nuclear magnetic resonance are used to determine the variations in the isotopic concentrations. Chemical labelling using externally introduced tags consists of the addition to a sample of the same analyte but containing a stable isotope (e.g. H, C, N) as an internal standard. This method is as much used for molecular species as for atoms (around 60 have stable isotopes). 

Quantitative Applications - The use of stable-isotope-labeled compounds as internal standards for the quantitation of drugs and metabolites in biological fluids offers a unique combination of sensitivity and selectivity of detection for pharmacokinetic studies. The principles of the technique have been outlined, and applications up to 1981 have been compiled. Specific aspects of the isotope dilution method, e.g. its utility as a reference technique and associated procedures for handling the data generated from the use of multiple isotope tracers simultaneously, have been discussed. Examples of isotope dilution methods used in clinical psychopharmacology have also been reviewed. ... 

Le Cornec, F. Correge, T. A new internal standard isotope dilution method for the determination of U/Ca and SrjCa ratios in fossil corals by ICP-MS. Varian ICP-MS Applications Note ICP-MS-18, September 1998 (www.varianinc.com). 

A special and very useful application of the isotope-dilution method is in the determination of optical purity of resolved or partially racemic optical enantiomers. Berson and Ben-Efraim (1959) derived the following equations ... 

Yamamoto et al. (69) have described the combination of TLC with secondary ion mass spectrometry for the determination of acetylcamitine and propionylcamitine in urine. Quantitation was accomplished with a stable isotope dilution method. Kajiura (70) described a TLC/SIMS application to the determination of phospholipid and steroid mixtures, with chromogenic reagents for spot visualization and either triethanolamine or glycerol as the extraction/ionization matrix. A similar application to phospholipids was described the same year by Hayashi et al. (71). Phospholipids as... 

A typical example for extended use of the isotope dilution method is the quantification of polychlorinated dioxins. Sixfold and twelvefold labeled dioxins and furans in the required chlorination degrees are used as recovery and surrogate standards during sample preparation and GC-MS analysis, as described in the widely applied EPA 1613 method. The nearly identical compound characteristics during clean-up, the chromatographic process and detection, guarantees most reliable quantitation results based on the sample individual recovery values in one analysis run (see details in the application Section 4.14 on dioxin analysis). 

The applications given here have been chosen in order to describe typical areas of use of GC-MS, such as air, water, soil, foodstuffs, the environment, waste materials, drugs or pharmaceutical products. Special emphasis has been placed on current and reproducible examples which give successful templates for routine laboratories. The selection cannot be totally representative of the use of modern GC-MS, but shows the main areas into which the methodology has spread and will continue to do so. In addition, in special areas of application, such as the analysis of isotope-specific measuring procedures and the isotope dilution method for dioxin analysis are described. 

It is possible to analyze approximately 40 elements by thermal ionization mass spectrometry and another ten elements by electron impact (including gaseous compounds of elements such as nitrogen, oxygen, carbon, and silicon). When isotope dilution is applicable, it usually is by far the most accurate method for the determination of impurities and traces. The leverage for precision and accuracy comes from the limited number of quantitative steps necessary for the analysis. 

Three common quantitative applications of radiochemical methods of analysis are considered in this section the direct analysis of radioactive isotopes by measuring their rate of disintegration, neutron activation, and the use of radioactive isotopes as tracers in isotope dilution. 

Radioactivity. Methods based on the measurement of radioactivity belong to the realm of radiochemistry and may involve measurement of the intensity of the radiation from a naturally radioactive material measurement of induced radioactivity arising from exposure of the sample under investigation to a neutron source (activation analysis) or the application of what is known as the isotope dilution technique. 

The development of new fiber coatings in the near future should further improve the specificity of SPME and overcome some of the observed matrix effects. Quantification by stable isotope dilution gas chromatography/mass spectrometry (GC/MS) may assist in improving analytical performance. Along with the possible application of micro LC and capillary LC columns to in-tube SPME, the development of novel derivatization methods and the potential for the analysis of fumigant pesticides, SPME appears to be a technique with a future in the analysis of pesticide residues in food. 

Kuehl, D.W., B.C. Butterworth, J. Libal, and P. Marquis. 1991. An isotope dilution high resolution gas chromatographic-high resolution mass spectrometric method for the determination of coplanar polychlorinated biphenyls application to fish and marine mammals. Chemosphere 22 849-858. 

A most important technique which has been developed as an extension of the isotope dilution principle is that of radioimmunoassay (RIA). Analyses by this method employ substoichiometric amounts of specific binding immuno-chemical reagents for the determination of a wide range of materials (immunogens) which can be made to produce immunological responses in animals such as sheep or rabbits. It is possible to combine the specificity of an immunochemical reaction with the extreme sensitivity of radiotracer detection. Analytical methods based upon these principles have achieved wide applicability in the determination of organic compounds at trace levels. 

Part—VI has been solely devoted to Miscellaneous Assay Methods wherein radioimmunoassay (RIA) (Chapter 32) has been discussed extensively. Various arms of theoretical aspects viz., hapten determinants and purity importance of antigenic determinants and analysis of competitive antibody binding of isotopically labeled compounds. The applications of RIA in pharmaceutical analysis, such as morphine, hydromorphone and hydrocordone in human plasma clonazepam, flurazepam in human plasma chlordiazepoxide in plasma barbiturates, flunisolide in human plasma have been described elaborately. Lastly, the novel applications of RIA-techniques, combined RIA-technique-isotope dilution and stereospecificity have also been included to highlight the importance of RIA in the analytical armamentarium. 

In Section III.D various methods were mentioned for determination of the 15N to 14N isotope ratio. Some applications to amines that appeared in the recent literature are presented here. Isotope dilution with a known aliquot of labelled compound allows solving some of the problems related to nonquantitative recovery yields of analyte in the analytical processing of a sample. However, the possibility of isotopic fractionation has to be taken into consideration. 

A major attraction is the ability to perform isotope ratio measurements, e.g. in many geological applications to determine the age of rocks, and isotope dilution analysis. The latter in particular is gaining-popularity as a highly accurate, precise and hence traceable, method of analysis, so it is worthwhile describing these techniques in more detail. 

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