Abstract
UHPLC (ultra-high performance liquid chromatography) has become an indispensable tool in the field of bioanalysis over the past years due to high separation efficiency, selectivity and sensitivity, which are very important features for analysis of target analyte(s) in complex matrices. In spite of several detection approaches available, UHPLC-MS/MS using triple quadrupole analyzer remains the most important technique in quantitative bioanalysis.
Recently, also UHPLC-MS with high-resolution mass spectrometry (HRMS) has been applied in both quantitative and qualitative bioanalytical approaches. Basic features, applications, benefits and drawbacks of both approaches are discussed besides less commonly used detection approaches such as UV or FD (fluorescence detection).
Sample preparation step is the most important part of bioanalytical method as it impacts all the later steps of the assay. An appropriate technique should be chosen according to extraction time, selectivity, number of steps, solvent consumption and the possibility of using on-line arrangement.
Highly selective sample preparation is important for minimizing matrix effects and ionization alterations that could occur with electrospray ionization. Sample preparation techniques prior to UHPLC analysis include mostly conventional procedures (solid phase extraction, protein precipitation and liquid-liquid extraction), while modern approaches comprising liquid- or solid phase based microextractions, on-line sample preparation techniques enabling direct injection of biological samples or sample preparation with high selectivity were employed less often.
This is however expected to change in the near future.