Abundance quantitation

Despite the truly mind-boggling instrumentation and techniques of analytomics (a comic omic ), mass spectrometers are still being used primarily to determine both the miz values of ions, for identification or confirmation (qualitative analysis), and their abundances (quantitative analysis). 

Relative abundance (quantitation ion) X Probability (quantitation ion) Relative abundance (confirming ion) X Probability (confirming ion)... 

A major example of isotope-dilution analysis lies in the procedure itself, which does not require any quantitative isolation of the elements being investigated. The relation between the abundance of the element under investigation and the spike is such that, once the spike has been intimately mixed with the sample, any losses of sample have no effect on the result (Figure 48.14). 

Suppression of the tme diagonal peaks by double-quantum filtering (DQF-COSY) may resolve such problems. Finally, quantitative measurements of the magnitude of the coupling constants is possible using the Z-COSY modification, These experiments ate restricted to systems of abundant spins such as H, and which have reasonably narrow linewidths. 

Quantitative mass spectrometry, also used for pharmaceutical appHcations, involves the use of isotopicaHy labeled internal standards for method calibration and the calculation of percent recoveries (9). Maximum sensitivity is obtained when the mass spectrometer is set to monitor only a few ions, which are characteristic of the target compounds to be quantified, a procedure known as the selected ion monitoring mode (sim). When chlorinated species are to be detected, then two ions from the isotopic envelope can be monitored, and confirmation of the target compound can be based not only on the gc retention time and the mass, but on the ratio of the two ion abundances being close to the theoretically expected value. The spectrometer cycles through the ions in the shortest possible time. This avoids compromising the chromatographic resolution of the gc, because even after extraction the sample contains many compounds in addition to the analyte. To increase sensitivity, some methods use sample concentration techniques. 

Due to the relative uniformity of ion formation by the RF spark (although its timing is erratic), the most widely used method of quantitation in SSMS is to assume equal sensitivity for all elements and to compare the signal for an individual element with that of the total number of ions recorded on the beam monitor. By empirically calibratii the number of ions necessary to produce a certain blackness on the plate detector, one can estimate the concentration. The signal detected must be corrected for isotopic abundance and the known mass response of the ion-sensitive plate. By this procedure to accuracies within a factor of 3 of the true value can be obtained without standards. 

Nuclear reaction analysis (NRA) is used to determine the concentration and depth distribution of light elements in the near sur ce (the first few lm) of solids. Because this method relies on nuclear reactions, it is insensitive to solid state matrix effects. Hence, it is easily made quantitative without reference to standard samples. NRA is isotope specific, making it ideal for isotopic tracer experiments. This characteristic also makes NRA less vulnerable than some other methods to interference effects that may overwhelm signals from low abundance elements. In addition, measurements are rapid and nondestructive. 

Corrosion rates have been given as rates of weight gain because they are the basis of most measurements. Penetrations calculated from the results are dependent upon the relative amounts of diflerent products, and hence require information on the relative abundance of products, which is generally not known quantitatively and can depend upon experimental conditions such as temperature. 

Sometimes we need to know how the concentrations of the ions present in a solution of a polyprotic acid vary with pH. This information is particularly important in the study of natural waters, such as rivers and lakes (Box 10.1). For example, if we were examining carbonic acid in rainwater, then, at low pH (when hydronium ions are abundant), we would expect the fully protonated species (H2C03) to be dominant at high pH (when hydroxide ions are abundant), we expect the fully deprotonated species (C032 ) to be dominant at intermediate pH, we expect the intermediate species (HC03, in this case) to be dominant (Fig. 10.20). We can verify these expectations quantitatively. 

The choice of a particular type of gas discharge for quantitative studies of ion-molecule reactions is essential if useful information is to be obtained from ion abundance measurements. Generally, two types of systems have been used to study ion-molecule reactions. The pulsed afterglow technique has been used successfully by Fite et al. (3) and Sayers et al. (1) to obtain information on several exothermic reactions including simple charge transfer processes important in upper atmosphere chemistry. The use of a continuous d.c. discharge was initiated in our laboratories and has been successful in both exothermic and endothermic ion-molecule reactions which occur widely within these systems. 

Hamilton [13] assumed the presence of all ions with n ranging from 1 to 8 in aqueous polysulfide solutions which is by far the most acceptable model but since there is insufficient experimental data available this model cannot be worked out quantitatively without additional assumptions. The general idea is that those species are most abundant which are close to the average composition of the particular solution, e.g., 84 and 85 for a solution of composition Na284.5, and that the larger and smaller ions are symmetrically less abundant. Equilibrium constants for the various reactions... 

Products 21 and 22 obtained in this reaction differ in their ESI-MS spectra, and the difference in the abundance of respective signals can be expressed quantitatively. Studies have shown that the pseudo-enantiomeric-excess values obtained in this way are in agreement 5% with the data obtained by chromatographic methods, which is sufficient for studying relative values and choosing most selective mutants. 

The application of 13C NMR for the rapid analysis of the oil composition of oil seeds is well known [16], 13C NMR has recently been applied to the quantitative analysis of the most abundant fatty acids in olive oil [17]. The values obtained by this method differed by only up to 5% compared with GLC analysis. The quantitative analysis was applied to the olefmic region of the high resolution 13C NMR spectrum of virgin olive oil to detect adulteration by other oils which differed significantly in their fatty acid composition. The application of the methodology for the detection of adulteration of olive oil by hazelnut oil is more challenging as both oils have similar chemical profiles and further experiments are in progress. 

Desorption/ionisation techniques such as LSIMS are quite practical, as they give abundant molecular ion signals and fragmentation for structural information. In the conditions of Jackson et al. [96], all the molecular ion and/or protonated molecule ion species were observed in the LSIMS spectrum when only 1 pmol of each additive was placed on the probe tip. However, as mentioned above, in LSIMS/MS experiments the choice of the matrix (e.g. NBA, m-nitrobenzylalcohol) is very important. Matrix effects can lead to suppression of the generation of molecular ions for some additives. LSIMS is not ideal for the quantitative detection of polymer additives, as matrix effects are very important [96]. 

On-line SFE-pSFC-FTIR was used to identify extractable components (additives and monomers) from a variety of nylons [392]. SFE-SFC-FID with 100% C02 and methanol-modified scC02 were used to quantitate the amount of residual caprolactam in a PA6/PA6.6 copolymer. Similarly, the more permeable PS showed various additives (Irganox 1076, phosphite AO, stearic acid - ex Zn-stearate - and mineral oil as a melt flow controller) and low-MW linear and cyclic oligomers in relatively mild SCF extraction conditions [392]. Also, antioxidants in PE have been analysed by means of coupling of SFE-SFC with IR detection [121]. Yang [393] has described SFE-SFC-FTIR for the analysis of polar compounds deposited on polymeric matrices, whereas Ikushima et al. [394] monitored the extraction of higher fatty acid esters. Despite the expectations, SFE-SFC-FTIR hyphenation in on-line additive analysis of polymers has not found widespread industrial use. While applications of SFC-FTIR and SFC-MS to the analysis of additives in polymeric matrices are not abundant, these techniques find wide application in the analysis of food and natural product components [395]. 

The literature on XRF is abundant. Recent general reviews are refs [235,237] for sample preparation see ref. [247]. EDXRF was specifically dealt with in ref. [248] and an excellent X-ray detector overview is available [225]. Several recent XRF monographs have appeared [233,249,249a], also covering TXRF [250] and quantitative XRF [232,251]. 

Applications Table 8.58 shows the main fields of application of inorganic mass spectrometry. Mass-spectrometric techniques find wide application in inorganic analysis, and are being used for the determination of elemental concentrations and of isotopic abundances for speciation and surface characterisation for imaging and depth profiling. Solid-state mass spectrometry is usable as a quantitative method only after calibration by standard samples. 

The feasibility of this approach to not only differentiate pathogenic and nonpathogenic strains of bacteria based on significant differences in protein mass but also on the basis of variations in levels of protein expression was demonstrated using a method for quantitating protein expression by LC/MS of whole proteins.54 This method is based on the fact that some proteins present in cells are abundant universal proteins whose expression levels exhibit little variation. This method demonstrates that these co-extracted proteins can be used as internal standards to which the other proteins in the sample can be compared. By comparing the intensities of a selected protein to a marker protein, or internal standard, a relative ratio is obtained. This ratio... 

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