Fig. 1.37 Selected reaction monitoring mode LC-MS/MS analysis of the same human plasma sample standard at 10 ng mL"1 placed at different positions in the analytical sequence: (A) at position 9, (B) at position 35. The peak at RT = 2.7 min corresponds to the analyte and the peak at RT = 3.5 min to the internal standard. Detection was performed in the negative mode.

by the accumulation of endogenous compounds on the HPLC column after each run, and therefore an increasing bleed of these endogenous sample components to the effluent of the column directed to the API interface. In this case the gradient elution was obviously not effective enough to remove efficiently endogenous compounds after each analysis. The IS, a structural analogue, was not capable of compensating the matrix effect. The solution to the problem was to replace the IS by an isotopically labeled structural analogue which co-eluted with the analyte. This example exemplifies how critical appropriate method development and validation is before running real study samples.

LC-MS/MS has dramatically changed the way bionalysis is conducted. Accurate and precise quantitation in the pg ml"1 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 do not need to be chromatographically baseline resolved. This is only true for an-alytes with different precursor and product ions.

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