Isotopic labels monitoring

Parallel and reversible reactions. The isomerization of allyl phenyl sulfide is a degenerate rearrangement made detectable by isotopic labeling of one end of the allyl group, permitting kinetic monitoring by NMR techniques.12... 

Yeom and Frei [96] showed that irradiation at 266 nm of TS-1 loaded with CO and CH3OH gas at 173 K gave methyl formate as the main product. The photoreaction was monitored in situ by FT-IR spectroscopy and was attributed to reduction of CO at LMCT-excited framework Ti centers (see Sect. 3.2) under concurrent oxidation of methanol. Infrared product analysis based on experiments with isotopically labeled molecules revealed that carbon monoxide is incorporated into the ester as a carbonyl moiety. The authors proposed that CO is photoreduced by transient Ti + to HCO radical in the primary redox step. This finding opens up the possibility for synthetic chemistry of carbon monoxide in transition metal materials by photoactivation of framework metal centers. 

Exchange reactions between bulk and adsorbed substances can be studied by on-line mass spectroscopy and isotope labeling. In this section the results on the interaction of methanol and carbon monoxide in solution with adsorbed methanol and carbon monoxide on platinum are reported [72], A flow cell for on-line MS measurements (Fig. 1.2) was used. 13C-labeled methanol was absorbed until the Pt surface became saturated. After solution exchange with base electrolyte a potential scan was applied. Parallel to the current-potential curve the mass intensity-potential for 13C02 was monitored. Both curves are given in Fig. 3.1a,b. A second scan was always taken to check the absence of bulk substances. 

Characterization of ion structures by bimolecular reactions, in which an ion is allowed to react with a neutral gas of known structure and the ionic products are analysed by mass spectrometry, depends on isomeric species having distinctive reactivities which reflect the functional group(s) that are present. This method is conceptually analogous to the use of structure-specific test reagents in classical solution chemistry. Sometimes a group may be transferred to a particular ion from the gas (methylene transfer is commonly encountered) on other occasions, hydrogen transfer is monitored. The latter is conveniently combined with isotopic labelling. 

Reetz and coworkers developed a highly efficient method for screening of enantioselectivity of asymmetrically catalyzed reactions of chiral or prochiral substrates using ESI-MS [60]. This method is based on the use of isotopically labeled substrates in the form of pseudo-enantiomers or pseudo-prochiral compounds. Pseudo-enantiomers are chiral compounds which are characterized by different absolute configurations and one of them is isotopically labeled. With these labeled compounds two different stereochemical processes are possible. The first is a kinetic separation of a racemic mixture, the second the asymmetric conversion of prochiral substrates with enantiotopic groups. The conversion can be monitored by measuring the relative amounts of substrates or products by electrospray mass spectrometry. Since only small amounts of sample are required for this method, reactions are easily carried out in microtiter plates. The combination of MS and the use of pseudo-enantiomers can be used for the investigation of different kinds of asymmetric conversion as shown in Fig. 3 [60]. 

Following extractive deproteinization of the plasma, the amino acids (and their stable-isotope-labeled internal standards) are separated by HPLC and introduced into the mass spectrometer. Electrospray ionization results in the formation of electrically charged molecules, which are separated on the basis of their mass/charge (m/z) ratio in the first quadrupole. Following fragmentation in the collision cell, the characteristic fragment for each amino acid is selected in the second quadrupole. This process is named multiple reaction monitoring. 

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. 

Fitzgerald et al,54 studied the monitoring of calcium metabolism in patients in the final stages of renal disease using serum by AMS after isotopic labelling with a 41 Ca radiotracer. The authors hypothesized that bone resorption can be studied directly by serial measurements of the 41Ca/Ca,otal ratio in serum after in vivo labelling of the calcium pool with 41 Ca.54... 

Because the characterization of support-bound intermediates is difficult (see below), solid-phase reactions are most conveniently monitored by cleaving the intermediates from the support and analyzing them in solution. Depending on the loading, 5-20 mg of support will usually deliver sufficient material for analysis by HPLC, LC-MS, and NMR, and enable assessment of the outcome of a reaction. Analytical tools that are particularly well suited for the rapid analysis of small samples resulting from solid-phase synthesis include MALDI-TOF MS [3-5], ion-spray MS [6-8], and tandem MS [9]. MALDI-TOF MS can even be used to analyze the product cleaved from a single bead [5], and is therefore well suited to the identification of products synthesized by the mix-and-split method (Section 1.2). The analysis and quantification of small amounts of product can be further facilitated by using supports with two linkers, which enable either release of the desired product or release of the product covalently bound to a dye [10-13], to an isotopic label [11], or to a sensitizer for mass spectrometry [6,14,15] (e.g., product-linker-dye- analytical linker -Pol). 

Abstract Internal standards play critical roles in ensuring the accuracy of reported concentrations in LC-MS bioanalysis. How do you find an appropriate internal standard so that analyte losses and experimental variations during sample preparation, chromatographic separation, and mass spectrometric detection could be corrected How is the concentration of an internal standard determined Should internal standard responses be monitored during the analysis of incurred samples What are the main causes for internal standard response variations How do they impact the quantitation Why are stable isotope labeled internal standards preferred And yet one should still have an open-mind in their usage for the analysis of incurred samples. All these questions are addressed in this chapter supported by theoretical considerations and practical examples. 

Another requirement for qualitative or quantitative analysis is the use of internal standards (IS) to compensate for sample preparation or chromatographic variability. This is of particular importance in LC-MS analysis, as an adequate IS can also compensate for the negative influence of matrix effects on method precision and accuracy. Stable-isotope-labeled ISs are the most appropriate for this purpose. If a specific deuterated analogue is not commercially available, it could be substituted for deuter-ated substances with similar physicochemical properties to the analyte of interest. However, the use of other marketed pharmaceuticals for this purpose should be avoided, as it cannot be excluded that the patient to be monitored has taken that drug. 

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