Matrix effects 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... 

For a majority of biomarker assays, standard calibrators are prepared in an analyte-free alternative matrix instead of the de facto sample matrix of patient samples. For such methods, it is crucial to demonstrate that the concentration response relationship in the sample matrix is similar to that of the alternate matrix. Spike-recovery experiments with the reference standard may be inadequate to evaluate the matrix effect, as the reference standard may not fully represent the endogenous analyte. Instead, parallelism experiments are performed through serial dilutions of a high-concentration sample with the calibrator matrix. Multiple individual matrix lots (>3 lots) should be tested to compare lot-to-lot consistency. In the instance that the limited amounts of sample are available, apooled matrix strategy can be used with caution as discussed by Lee et al. [15]. The samples can be serially diluted with the standard matrix (standard... 

Careful understauding of the effects of each preanalytical variable on the biomarker data is complex and necessitates a staged approach conceptually similar to the fit-for-purpose analytical validation of a biomarker assay (see Chapter 41). In the early exploratory phase of biomarker investigation, standardization of procedures with a defined protocol for sample collection and handling will permit comparative interpretation and analysis of the data within study and/or between studies. Minimally, variables that should be experimentally evaluated to optimize sample collection for a specific biomarker will include matrix type, preservation... 

The activities of two enzymes have been used as biomarkers of effects for OPs, namely acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase, sometimes known as pseudocholinesterase (EC 3.1.1.8). The structure and function of these enzymes has been reviewed. " In humans the former is present in red blood cells and the latter in plasma, but such distribution is not true of all species. In dogs, both enzymes are present in plasma with a ratio of butyrylcholinesterase to acetylcholinesterase of 7 1, while in the rat, plasma cholinesterase activity comprises more acetylcholinesterase with a butyrylcholinesterase to acetylcholinesterase activity of 1 3 in males and 2 1 in females in neither blood compartment are the functions of the enzymes fully understood.Because of the possibility of confusion, the terms plasma cholinesterase and erythrocyte cholinesterase as synonyms for butyrylcholinesterase and acetylcholinesterase are to be deprecated, especially when used of enzymes in animals where serious confusion may result. It is often necessary to look in detail at animal studies to see what activity has been measured in each matrix. In particular, it is necessary to look at the substrate(s) used in the assay together with any inhibitors used. Methods for measuring acetylcholinesterase have been reviewed and acetylcholinesterase and butyrylcholinesterase activities can be measured separately. In almost all cases it is the enzyme activity, rather than protein concentration, that is measured and many of the procedures used are variants of the Ellman method. Correct storage of blood samples is important as reactivation of inhibited enzymes ex vivo can occur. 

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