Quantification of Small Molecules

It can be argued that differential responses are problematic in mass spectrometry and that they depend on the structure of the analyte, especially when ESI is used. ESI reflects the polarity of the analyte, meaning that instrument response will favor polar species. APCI is more uniform in this respect but has its own problems, including excessive fragmentation. 

X selected, ions with other mIz ejected from the trap 

In a QIT (LIT) the same analyzer is used sequentiaUy, in time, to generate spectra. 

An ion isolated from the MSI scan undergoes coUision-indnced dissociation (CID) yielding a product ion spectrum that provides structural information on the selected ion, MS2. 

The process of isolation CID scan can he repeated to give additional data, MS spectra. 

---

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. 

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. 

Immunoassays can be designed in two formats competitive assays, preferable for quantification of small molecules such as steroid hormones and prostaglandins, and noncompetitive, or sandwich, assays restricted almost exclusively to large molecules... 

Overall, in quantification, it is not the method of detection that is important (e.g., GC-FID, LC-UV, GC-MS, LC-MS), but how the data are standardized with appropriate calibration curves. The most important MS system for the quantification of small molecules is the triple quadrupole mass spectrometer, particularly when used in SRM mode (Section 3.3.3.1). Although much of what is described below concerns small molecules (for techniques used in the quantification of proteins see Section 3.5.1.9), the same basic considerations apply for all targeted analytes. 

Label-free is a quantification methodology in which an internal standard is used in a manner similar to that used for the quantification of small molecules. Proteins are recovered and digested, and ion chromatograms are extracted (from the total... 

Uses internal standard (co-injected) in manner similar to quantification of small molecules... 

Zhao, Y. Lam, M. Wn, D. Mak, R. 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. 2008, 22, 3217-3224. 

One straightforward approach for the introduction of stable-isotope-labeled peptides is to chemically synthesize them and add known quantities to the sample [299]. It extends to peptides with the well-established technique of stable isotope dilution, which is routinely used in pharmaceutical research for the quantification of small-molecule-based drugs. This approach is the most powerful and the most expensive available for absolute peptide... 

---