The Isotopic. Dilution Method

Experiments With One Label A. The Isotopic Dilution Method 

The foregoing example is one in which the product to be determined is non-radioactive, whereas the added material (the addend) is radioactive. One limiting factor is that the weight of radioactive addend 

The radioactivities A and Ai may be expressed per unit of weight or per mole of compound. 

In the foregoing examples the yields of the product or products have been determined when (1) the molar radioactivity of the product or of the addend and (2) the weight of addend have been known. In many experiments involving radioactive products the exact molar radioactivities (Aq, equation 2) of the materials produced may not be known. In such oases the double-dilution method may be useful, and both yield (x) and radioactivity A o) may be determined by means of the following relation  

The application of the radioactivity dilution technique to the determination of yield or radioactivity of a product is not without its drawbacks. The most serious of these may be illustrated as follows. Suppose a given reaction product consists of two compounds, A and B, both of which possess equal molar radioactivities. It is desired to determine the yield of A through the dilution method by the addition of a weighed 

---

The isotope dilution method can be used for the measurement of molecules or elemental species (about 60 elements have stable isotopes). This approach allows ultratrace analysis because, contrary to radioactive labelling where the measurement relies on detecting atoms that decay during the period of measurement, all of the labelled atoms are measured. 

Quantification is usually achieved by a standard addition method, use of labeled internal standards, and/or external calibration curves. In order to allow for matrix interferences the most reliable method for a correct quantitation of the analytes is the isotope dilution method, which takes into account intrinsic matrix responses, using a deuterated internal standard or carbon-13-labeled internal standard with the same chemistry as the pesticide being analyzed (i.e., d-5 atrazine for atrazine analysis). Quality analytical parameters are usually achieved by participation in interlaboratory exercises and/or the analysis of certified reference materials [21]. 

In normal RIA-procedures the labelled drug or metabolite not only serves as the tracer for recovery but also for RIA quantification. However, the isotope dilution method categorically makes a clear separation of the drug and its metabolites. Consequently, a non-specific antiserum is employed to actually quantify the total amount of both unlabelled and labelled substance present. 

Identification of possible intermediates in the cyclization was attempted by adapting the isotope dilution method which had been... 

Thus, accurate determination of the specific activities SAa and SAa immediately provides the value of the unknown Ax. Note Whenever Axisotope dilution method suffers from any statistical variation in the measurements of quantities that are nearly identical. Should this prove to be true for your measurements, use a lesser amount of isotopic probe, so that Ax A. 

The isotope dilution method consists of mixing a natural sample with an artificial spike and measuring the isotopic ratios of the mixture using mass spectrometry, providing very precise quantitative determination of the concentrations of elements in trace quantities. A spike is a solution that contains a known concentration of the element, artificially enriched in one of its minor isotopes. For example, natural Rb samples have 27.84% and 72.16% Rb (Fig. 11.lA). Rb spikes are made by artificially enrich the minor isotope Rb. And a solution with 90% Rb and 10% Rb (Fig. 11.IB) is a Rb spike. Of course, a solution with 99.99% Rb and 0.01% Rb is a better Rb spike. When the known quantities (mass) of the sample and spike are mixed, the resulting isotopic compositions can be used to calculate the concentration of the element in the sample. 

In many cases, it is more convenient to describe the isotope dilution method using isotopic ratios rather than abundances alone. / j in Eq. (11.1) can be expressed as a function of isotopic ratios in the natural sample and the spike. ... 

Glycerol kinase activity can be measured directly or indirectly however, these assays are not available as a clinical test and are done exclusively on a research basis. The direct method is as has been described previously [4, 6] and is used in the isotope dilution method indicated above. The amount of protein and the incubation time vary between cell types and it is important to be within the linear range of the assay for the given cell type. The indirect methods involve incorporation of 14C from glycerol into macromolecules and its subsequent oxidation to 14C02 [7,11]. 

In applying the isotope dilution method to the analysis of HMX/RDX mixts, an accurately weighed amt of carbon-14 labeled HMX of known specific activity (counts per min per g) is added to a known wt of the mixt. The mixt is made homogeneous by dissolving it in acetone. A small amt of pure HMX is then isolated from the mixt by fractional recrystallization from acetone. The specific activity of the pure HMX sample, both before and after dilution, is determined by either dissolving or suspending a known wt in a scintillator soln or gel and counting with a commercial liq scintillation counter. 

In the isotope dilution method, labeled analogs of analytes are first added onto the standard solutions of the analytes as well as to the samples/sample extracts. Before the analysis, a calibration curve is prepared plotting relative response (RR) against concentrations using a minimum of five data points. Relative response of a compound is measured from the isotope ratios of the compound and its labeled analog as follows ... 

It may be noted that for calibration each analyte is needed to be spiked with its labeled analog. This enhances the cost of the analysis. High cost and often unavailability of labeled analogs are the major drawbacks of isotope dilution method as compared with external and internal standard calibration methods. The isotope dilution method should be, theoretically, more accurate than the internal standard method, as the chromatograpic response and the retention times of the labeled analogs are closest to the compounds. 

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 ICP-SFMS instrument was tuned using a I ng mf uranium standard solution prior to analysis. Sensitivity was about 2 x 10 cps for 1 ng g" solution. Concentrations of plutonium isotopes and " Am were calculated as a function of Pu/ Pu, Pu/ Pu, Pu/ Pu and Am/ Am ratios according to the isotope dilution method. All raw data were corrected for instrumental mass bias using linear correction. NdFs micro coprecipitated alpha sources were counted by a PIPS type alpha Si detector with a surface... 

The epoxide metabolites of inhaled 1,3-butadiene, used in industry, are reported to be carcinogenic and mutagenic in rodents, and their in vivo concentration following inhalation exposure to butadiene has to be determined by gas chromatography/mass spectroscopy, the isotope dilution method utilizing 8 as an internal standard. Commercially available [De]-propylene oxide has been used previously as an internal standard to monitor in vivo blood propylene oxide levels following inhalation exposure to propylene. ... 

Shemin, G., and G. L. Foster The Isotope Dilution Method of Amino Acid... 

Optical purity of barbituric acid enantiomers was determined by the isotope dilution method using 2-14C-labeled compounds 215 or by an NMR... 

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). 

Which is better, GC-MS or immunoassay This is a question often asked about plant hormone quantification. GC-MS, which is now more widely available since the Introduction of bench-top instruments, has the advantage that it not only provides quantification of the hormone by the isotope dilution method, but also confirms the identity of the compound concerned by comparison of its spectrum with that of a standard. However, once validated for a particular tissue, immunoassay has the advantage that many samples can be analysed very quickly. Both techniques require sample pre-purification, often by the same methods. A more recent development is a powerful combination of the two technologies which uses the antibody immobilised on a polymer support as a method of affinity-purifying the hormones (together with interfering substances) from plant extracts prior to analysis by GC-MS. Immunoaffinity chromatography is discussed in the next section. 

Rubidium determination in biological samples is also possible by the isotope dilution method (Labitajs 1992) or X-ray fluorescence spectrometry (Haneklaus etal. 1994). 

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. ... 

---