Biological matrices quantitative bioanalysis

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... 

Matrix Effects. The matrix effects (ion suppression or ion enhancement) can compromise the selectivity and sensitivity of LC/MS/MS methods for the determination of drug concentrations in biological matrices. One common matrix effect is ion suppression due to co-eluting components that can affect the ionization efficiency of the analyte of interest. The matrix effects are major causes for errors in precision, accuracy, linearity, and reproducibility of the quantitation methods based on LC/MS/MS [104,108,114-118]. It is critical to overcome such effects in quantitative bioanalysis by LC/MS/MS. 

Proper sample pretreatment methods are important to reduce or eliminate matrix effects. In this section, advances in sample pretreatment methods in relation to LC-MS quantitative bioanalysis are briefly reviewed (see also Ch. 1.5). Contrary to environmental analysis, where on-line sample pretreatment is often used (Ch. 7.3.2), off-line sample pretreatment appears to be preferred in quantitative bioanalysis. This is partly due to the composition of biological samples, where the presence of especially proteins may cause clogging of the SPE columns, cartridges or disks used, and partly due to the fact that decoupling sample pretreatment and LC-MS analysis generally allows for a higher sample throughput. 

For pharmaceutical applications, the term bioanalysis refers to quantitative determination of a drug or its metabolites in a biological matrix. Although this term has traditionally been used to describe the analysis of in vivo samples (i.e., plasma or serum), current use of the term encompasses a broader range of applications that include the analysis of in vitro samples. Under this broader dehnihon, possible bioanalytical sample types can range anywhere from transport media to tissue homogenate. 

The keys to the success of LC-MS in quantitative bioanalysis are (1) typical detection limits in the picogram and in favorable cases even subpicogram range, (2) excellent selectivity against possibly interfering compounds in the biological matrix by the use of the SRM mode, (3) enhanced confidence of identity of the compound(s) analyzed, and (4) the ability to use the ideal internal standards isotopically labeled compounds. LC-MS-MS is often as easy to operate as LC-UV-PDA, but provides better selectivity. As a result, LC-MS-MS has become the method of choice in quantitative bioanalysis within pharmaceutical industries. 

In bioanalysis, High-Performance Liquid Chromatography (HPLC) is the analytical technique most frequently used. Often, extended sample preparation is required to make a biological sample (the matrix) suitable for HPLC-analysis. The compound of interest, the analyte, has to be isolated from the matrix as selective and quantitative as possible. The quality of the sample preparation largely determines the quality of the total analysis procedure. In a survey Majors [2] showed that approximately 30% of an error generated during sample analysis was due to sample preparation, which indicates the need for error reduction and quality improvement in sample preparation. 

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