Selectivity determination biomarker assays

LC-MS/MS has dramatically changed the way bionalysis is conducted. Accurate and precise quantitation in the pg ml scale is nowadays possible however one has to be aware of certain issues which are specific to mass spectrometric detection such as matrix effects and metabolite crosstalk. With the current growing interest in the analysis of endogenous biomarkers in biological matrices, quantitative bioanalysis with MS has certainly the potential to contribute further in this field with the development of multicomponent assays. Modern triple quadrupole instruments have the feature to use very short dwell times (5-10 ms), allowing the simultaneous determination of more than 100 analytes within the timescale of an HPLC peak. Due to the selectivity of the MS detection the various analytes... 

Dizer, H., Wittekindt, E., Fischer, B. and Hansen, P.-D. (2002) The cytotoxic and genotoxic potential of surface water and wastewater effluents as determined by bioluminescence, umu-assays and selected biomarkers, Chemosphere 46 (2), 225-233. 

For best results in determining the presence and site of nephron damage, a battery of enzymes as opposed to a single biomarker should be examined, especially since the most sensitive assay is highly variable, depending on the toxicant and species. Additionally, since many of the enzymes are not completely specific to a selected nephron segment, use of a battery of tests allows more precise localization of the site of injury (Price 1982). 

The aim of this section is to cover approaches to assay selection, to provide an overview of the possible assay formats and to give a description of the most frequently used assay formats for potency determinations. Bioassays are developed and used for different purposes during drug discovery and development. Even though they can have the same assay format they have a different purpose in a product development cycle starting from target selection to clinical biomarkers. 

Most methods for determination of these compoimds are based on liquid chromatography or gas chromatography, which require derivatization (Hack and Muir, 2003). Selective MS-MS techniques facilitated by triple quad instruments or ion trap instruments enable the detection of hydrolysis products to the range of pg/mL (Barr et al., 2004 Riches et al., 2005). This concentration is so low that hydrolysis products can be detected in urine up to 1 week after exposure (Riches et al., 2005). Nerve agents bind to proteins such as AChE and butyrylcholinester-ase (BuChE). These proteins are not excreted or metabolized rapidly (typical half-life is 12 days for BuChE) (Hall et al., 1984), which means that adducts to proteins can serve as retrospective biomarkers for exposure to nerve agents (Bidder et al., 2002 van der Schans et al., 2004). The enzymahc measurement of AChE activity, known as the Ellman assay, is the easiest method to determine nerve agent exposure (Ellman et al., 1961 Halbrook et al., 1992). 

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