Quantification small molecule

A related approach is realized in filter binding assays. Here the reaction solution is filtered, e.g., through nitrocellulose where proteins are absorbed, while small molecules can pass. One example of this technique is the quantification of protein bound and free nucleotides (with radioactive labeled ligands). 

A number of factors have contributed to renewed interest in DI techniques for quantitative analysis. Since the reasonable throughput in routine use of the LC-ESI-triple-quadmpole mass spectrometer seems to have peaked at about 3-4 samples per minute, there is renewed interest in finding alternate ways to improve quantitative throughput. Recent developments in DI mass spectrometry (DIMS) have realistically made routine small-molecule quantification feasible. This chapter will briefly outline the issues encountered when quantifying drug molecules by DIMS. The latest developments will be described in relation to historical problems... 

A modem TOF instrument may well provide adequate resolution to allow quantification of small-molecule analytes, but examples in the literature report limited dynamic range. The latest TOF instruments provide for improved dynamic range in quantitative applications, but there are still other critical obstacles. These include sample preparation, selection of an internal standard, instrumental protocol, and... 

Gobey, J., Cole, M., Janiszewski, J., Covey, T., Chau, T., Kovarik, P., and Corr, J. (2005). Characterization and performance of MALDI on a triple quadrapole mass spectrometer for analysis and quantification of small molecules. Anal. Chem. 77 5643-5654. 

The quantification of metabolites in dried blood spots primarily ensures that the quality of the isotopes standards is excellent in terms of chemical and isotopic purity. When using MS/MS, it is essential that the fragments produced by the collision cell and the product ions detected ensure that both labeled and unlabeled metabolites are identical. Most importantly, the choice of the isotope label and the structural positions must be such that they are stable and do not exchange with other isotopes during sample preparation. Finally, it is imperative that the mass shift is sufficiently high (at least 3 Da) for small molecules less than 1000 Da and that the label occurs at a mass free from other compound interference. Figure 4 illustrates the concepts of quantification using stable isotope with Phe measurement in a dried blood spot as an example. 

Previously, inductive QSAR descriptors have been successfully applied to a number of molecular modeling studies, including quantification of antibacterial activity of organic compounds (89), calculation of partial charges in small molecules and proteins (81), and in comparative docking analysis as well as in in silico lead discovery (82). Inductive QSAR descriptors have been used... 

No examples for quantification in the product ion scan mode were found in the literature even though data processing would allow extraction of selected ions, integration of related signal areas, and summation for quantification. This procedure has been used by John et al. for the determination of the human haemoglobin derived peptide hHEM-y 130-146 in plasma [102], However, quantification especially of small molecule analytes is best performed in the MRM mode that is addressed below. 

DOM is composed of small molecules that can be obtained by filtration. Quantification of organic matter in aquatic environment is performed by measuring the concentrations expressed in organic carbon. One measures total organic carbon (TOC) obtained from raw liquid sample, particulate organic carbon (POC) by analyzing the filter, and the dissolved organic carbon (DOC) characterized from the sample after filtration. 

While straight MS analysis can yield important information in terms of identification, characterization and quantification of biomolecules, it becomes a much more powerful tool with further MS or when combined with other separation technologies. As noted earlier, these approaches include MS/MS, GC-MS, LC-MS and CE-MS. These methods have been extensively exploited in virtually all aspects of bioanalysis, and while fundamentally useful for peptide and protein analysis, these methods have also been used in the analysis of lipids, nucleic acids and a wide range of small molecules and drugs. The range of applications is obviously outside the scope of a book like this, but some indications of the uses of each of these techniques are given below. 

Zhao, Y. et al., Quantification of small molecules in plasma with direct analysis in real time tandem mass spectrometry, without sample preparation and liquid chromatographic separation, Rapid Commun. Mass Spectrom., 22(20), 3217, 2008. 

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