Internal standards isotopologues

A further simultaneous use of traeer isotopologues and internal standard isotopologues was reported for a bioavailability study using an ileostomy model (Buttner et al. 2011). The absenee of speetral overlaps was verified for... 

The natural abundances of the stable isotopes given in Table 6.3 are global average values, whereas the actual exact values are subjected to small local and temporal variations, due to the slightly different behaviour of isotopologue molecules (by lUPAC definition a molecular entity that differs only in isotope composition, means number of isotopic substitutions in contrast, isotopomer [ isotopic isomer ] molecules are isomers, having the same number of each isotopic atom but in different positions) in the course of chemical reactions or physical processes ([8], kinetic and thermodynamic isotope effects, respectively, see below). The corresponding shifts are so small that they caimot be indicated in the atom-% scale therefore they are expressed in 5-values, differences of the isotope ratio R, e.g., ([ C]/[ C]) of the sample and an international standard relative to this international standard ... 

During SIDAs, isotopic dilution takes place after addition of the labelled standard and its equilibration with the analyte. Due to their almost identical chemical and physical properties, the ratio of the isotopologues is stable throughout all subsequent analytical steps. Final mass spectrometry enables the determination of the isotopologues. From this ratio, the content of the analyte in the sample can be calculated with the known amount of the internal standard added in the beginning. In contrast, a structurally different internal standard may be discriminated against and, thus, cause systematic errors and imprecision. Therefore, in SIDA losses of the analyte are completely compensated for by identical losses of the isotopologue, whereas a structurally different internal standard may show different losses. 

The first deuterated folate to be used as internal standard was [ H4]-folic acid labelled in the 4-aminobenzoate moiety the latter was the base for the first folate analogues for naturally occurring folates (Freisleben et al. 2002). The latter were the first to be used as internal standards for folates in foods not fortified with folic acid (Freisleben et al. 2003b). Further folate isotopologues were synthesized thereafter, and an extensive synthetic study was presented by Maunder et al. (1999) with routes to pH4]-folic acid and [ Csl-folic acid labelled in the glutamate moiety and the benzene moiety, respectively. 

Figure 25.3 Isotopologues of folic acid (1) used as internal standards or tracers. Several isotologues of folic acid have been used in SIDAs or ...

Stable isotope dilution assays are based on the use of stable isotopologues as internal standards that are detected by mass spectrometry. 

In a SIDA, stable isotopic analogues of the analytes, i.e. stable isotopologues, are used as internal standards. 

Isotopologues, isotopologs. Isomers that differ in their isotopic composition and their masses. Generally, in stable isotope dilution assays, isotopologues are used as internal standards. 

Why can accurate quantification by ESI-MS only be conducted in a relative measurement manner with an isotopologue as the internal standard A mass spectrometer is unlike an UV-vis spectrophotometer in which the relationship between the optical intensity and the concentration of an analyte follows the Beer-Lambert law. There is no direct relationship in MS between the determined ion counts and the concentration of an analyte yielding the ion due to numerous reasons, which are as follows ... 

Regarding the use of a stable isotopologue of the analyte as an internal standard, unfortunately, it is impractical to prepare thousands of stable isotopologues... 

Therefore, quantification of an analyte by MS analysis usually requires comparisons to either an external standard that is itself in most cases or an internal standard that is an analog to the analyte (e.g., its stable isotopologue). When an external standard is used, a calibration curve of the standard is established at a series of concentrations each of which should be analyzed under identical conditions that will be applied to the MS analysis of the analyte of interest. When an internal standard is used, the standard is added at the earliest step possible during sample preparation and its concentration should be in an appropriate ratio to the analyte (see Section 13.3.2). 

It should be emphasized again that selection of the stable isotopologue of the analyte as the internal standard for its quantification would perfectly satisfy the requirement of having identical response factors. This is due to the stable isotopologue having the same structure and property as the analyte (e.g., the same recovery and same ionization efficiency), and the internal standard is processed and analyzed at the same time as the analyte. However, this approach is impractical if not impossible to analyze thousands of species of interest in a complex system such as a cellular lipidome [21]. 

Many lipid classes can be quantified by this improved shotgun lipidomics approach. Quantification of individual species of a lipid class is conducted based on the summed abundance of its major fragment(s) in comparison to the counterpart of the spiked internal standard of the class. The effects of different isotopologue... 

In general, the isotopologue distribution of each species of a class mainly depends on the number of total carbon atoms in the species. This is due to the following facts. First, all the species of a class of interest should contain identical numbers of O, N, P, or other atoms if the lipid species of interest is not a modified one. This means that these atoms equally contribute their isotopologues to all the species of the class. Therefore, their contribution to the ion intensities does not cause any difference for relative comparison to the selected internal standard(s). Second, the natural... 

Quantification by ratiometric comparison with an internal standard is based on the ratio of the sum of the isotopologue intensities of a species to that of the internal standard. The fact is that the monoisotopic peak is the most intense peak in the isotopologue cluster of a lipid species for almost all lipids, and its intensity can therefore be determined more accurately compared to the intensities of other isotopic peaks of the species. Therefore, correcting for differential isotopologue distribution is based on the deduction of the intensity of each isotopologue of a species from the determined monoisotopic peak intensity. 

Zj has previously been called the type I isotope correction factor [22] n and s are the numbers of total carbon atoms in the species of interest and the selected internal standard, respectively / and are the monoisotopic peak intensities of the species and the internal standard, respectively c and are the concentration of the species of interest and the internal standard, respectively. The dots represent the contribution of other isotopologues which contain more than two atoms. These terms can be ignored in most cases without affecting the accuracy of quantification. 

As described in Chapter 15, correction for different stable isotopologue distribution due to differences in the number of carbon atoms between the species of interest and the selected internal standard(s) and correction for baseline should be performed prior to the comparison of the intensities. It should be recognized that in the majority of the studies in plant lipidomics after direct infusion, MDMS-type analysis is generally not conducted thus, the isobaric or isomeric species are not resolved as regrettable. A detailed protocol for profihng of polar hpids in plants after direct infusion can be found [9]. 

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