Bioanalysis, ionization sources

Over the past two decades, QMF-based quantification assays have become the technique of choice for quantification of drug candidates and their metabolites. Combining a mass spectrometer with LC provides an additional degree of selectivity and makes the combined technique the method of choice for quantitative bioanalysis of drugs and metabolites. Among the mass spectrometer types, QMF are ideal for coupling with LC and atmospheric pressure ionization sources (ESI, APCI, APPI, DART, DESI, etc.) because QMFs have the lowest voltage requirements and vacuum requirements. 

Any comparison between APCI and ESI will ultimately conclude that both techniques are useful and that neither method can truly be considered a universal ionization source. Unfortunately, because of the time needed to switch between methods, most laboratories responsible for bioanalysis tend to rely on a single method, and develop new methods only when the current ionization method is not successful. To afford greater flexibility, instrument vendors now offer dual ESI/APCI sources that can switch between ionization modes on consecutive scans within a LC run [20,21]. The original concept can be traced to the work of Siegel et al., who demonstrated the first viable source of this type [22]. Although this concept is quite new, it could prove very useful for research laboratories required to produce expedient LC-MS conditions for a wide range of chemical structures. 

An internal standard (IS) is required to reduce the impact of the system variability on method performance, and its selection is important for quantitative bioanalysis. Structural analogues and stable isotopically labeled (SIL) compounds remain the gold standards of ISs however, structural analogues differ from the compounds of interest and may have different ionization behaviors than the analytes. Even with a closely eluting IS and analyte, they reach the ionization source at different retention times, and short-term variations in the ionization process may be of concern, particularly for ESI. 

The use of CS techniques reduces the column equilibration time and flushes the matrix components to waste (decreases source contamination), while narrow-bore LC columns help not only with flow rates that were amenable to most atmospheric- pressure ionization (API)- interfaces but also provide increased sensitivity [28]. A change from a serial to a parallel format further increases throughput for bioanalysis, with timed injections at intervals from multiple LC systems and staggered MS detection [29-31]. 

It is a common situation for those coupling LC with MS to achieve an optimal separation at a different flow than that which yields optimum sensitivity and lowest maintenance. All too often in bioanalysis, the separation is sacrificed to accommodate the MS. Sometimes the MS performance is sacrificed for analysis speed. A common, readily achievable combination for speed is 0.5-1.0-I- mL/min through a 2.1 X 50 mm column, while the optimum for the ion source is usually 100—400 pL/min. If electrospray ionization (ESI) is used, then ion intensity is concentration (not mass) driven, (Ardrey, 2003) and there is no reason not to split in order to operate under optimal conditions for both the separation and the detection. 

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