Isotopes, stable multiple labelling with

A better method could be in vivo NMR spectroscopy, which would be noninvasive. But the extremely low sensitivity of NMR is in general a severe drawback to explore anaboHc or catabolic events. However, that disadvantage could probably be overcome by multiple labeling with radioactive ( H, " C) or stable isotopes such as H, N, or It might be a... 

Multiple labelling with both stable and radioactive isotopes offers the advantage of simultaneous labelling in the same position of a molecule [68]. The combination of C- and C-labelling combines ease of detection with rapid structural identification. The adoption of this technique in studies of some aspects of the metabolism of 4-morpholino-2-piperazinothieno [3,2-d] pyrimidine (V-K 774) (3) in the rat has been reported [69]. Potassium [ C, C]thiocyanate was prepared from a mixture of potassium [ C, C]cyanides. Reaction with ethyl bromide and methyl 3-aminothiophen-2-carboxylate (4) afforded the thienopyri-midine (5), which, by a series of reactions was converted to V-K 774 (3) labelled in the position shown. 

The ideal chemical isotopic tag is one that will react with every protein or peptide present in the sample and be reactive enough that the protein/peptide becomes fully labeled. However, the intrinsic reactivity of the compound should not be so high that it degrades upon storage under proper conditions. Also, the ideal tag will be stable upon mass spectrometry and not readily break apart under standard conditions. The ideal chemical tag will either be commercially available or relatively easy to synthesize, and the price should be reasonable. At a minimum, the tag needs to be available in a heavy and light form, but ideally would be available in a range of masses so that multiple replicates can be combined. It is also important that peptides labeled with the heavy and light forms of the tag coelute from HPLC otherwise, the quantitation is less accurate when... 

Many of the disadvantages of stable isotope labelling as compared with radiolabelling stem from limits on the sensitivity which arise from the natural abundance background. Using multiple labelled compounds as tracers, this problem can be overcome and furthermore, an assessment may be made of isotope effects in the system under study. The use of multiple labelled compounds and methods of detection allowing the determination of 1 pg in a 1 pg sample have been reviewed [65]. 

Quantitative determinations will be precise only if full corrections for losses occurring during extractions and possible detector instabilities are made. The mass spectrometer measuring the mass of molecules, the use of auxins labeled with stable isotopes provides almost ideal IS for accurate quantifications. The stable isotope dilution method [22] consists in measuring by Multiple Ion Monitoring (MIM) the ratio of normal to heavy isotopes added at the beginning of the analysis. The absolute amount of auxin in plant extract is then obtained [see 19]. 

An example of multiple ion monitoring using a standard labelled with stable isotopes is the quantitation of morphine (9) and 6-acetylmorphine (11) in the blood of rabbits (16). The compounds were analyzed as the trifluoroacetyl derivatives (10 and 11) using the corresponding N-trideuteriomethyl derivatives (13 and 14) as the internal standards. The base peak in the mass spectra of each pair of compound appeared at m/e 364 and 367 (Fig. 5). As an internal check, two other ions at m/e 363 and 365 were also monitored. An example of the selected ion profiles for blank plasma and the plasma extracts of rabbits administered heroin are shown in Fig. 6. The sensitivity of detection is such that 500 pg can be detected although the pure compound can be detected in the range of 5 to 10 pg. In comparison, immunoassay methods for morphine... 

Table 2.1.3 Multiple reaction monitoring of amino acids for their tandem mass spectrometry quantitation. In daily practise not all mentioned amino acids are measured in one run, but a set of ten dedicated evaluation programs has been developed, covering groups of amino acids associated with groups of disorders. Amino acids presented in italics indicate stable-isotope-labeled internal standards ... 

The metabolites of interest in urine are separated using reverse-phase HPLC combined with electrospray ionization (ESI)-MS/MS, and detection is performed using multiple-reaction monitoring. Stable-isotope-labeled reference compounds are used as internal standards. 

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