Further improvement of throughput in LC-MS analysis may be achieved by step-gradient elution. This elution format is essentially an on-line solid-phase extraction (SPE) process, where the samples are loaded onto the column, washed with aqueous mobile phase to remove water soluble impurities, and compounds are eluted with a mobile phase of high organic content. The technique combines the simplicity of FIA with the benefit of the removal of impurities and buffer components before mass spectrometry detection. In this case, selectivity is achieved by mass spectrometry alone without chromatographic separation. The technique has been used for compound purity assessment and quantitation. An on-line back-flush SPE-MS technique has been used by Marshall for quality assessment of the combinatorial libraries . This back-flush elution procedure provides a very effective in-line removal method for the aqueous components in the matrix that are often responsible for ion suppression. Bu et al.  used a high throughput analytical method for permeability screening of drug candidates. This simple and sensitive method was based on direct injection coupled with on-line guard-cartridge extraction with a very steep gradient. The method relied on MS/MS to achieve selectivity without chromatographic separation. The authors used this method in a comparison study of apparent permeability across Caco-2 cells measured by sample pooling or cassette dosing strategy with results from single-drug dosing and discrete sample analysis. Janiszewski et al.  used a stepgradient strategy in their dual-column LC-MS application on automated high throughput quantitation. The strategy of collecting all data for a compound into a single file greatly reduced the number of data files collected, increased the speed of data collection, allowed rugged and complete review of all data, and provided facile data management. The described systems have analyzed over 40,000 samples per month for 2 years and have the capacity for over 2000 samples per instrument per day.
Two developments in the on-line SPE-MS methodology improved the throughput of the method in the application to analysis of compounds in biological matrix. The first is the use of the turbulent flow chromatography (TFC) for extraction of compounds from in vitro or in vivo samples to remove the biological matrix and elute the compounds for mass spectrometry analysis. The use of columns packed with large particles (30-60 /m) and fast flow (turbulent flow condition) facilitated removal of biological-matrix components, such as proteins and buffers, while the compounds of interests were retained and subsequently eluted out for mass spectrometry analysis [115,116]. The second method is the use of SPE card made in the format of a disposable 96-elution zone compatible with 96-well sampling. The compounds are cleaned up in an off-line device through SPE and the retained compounds are eluted directly into mass spectrometry one zone at a time with high throughput [117,118]. These methods have the common feature that biological matrices are removed by on-line or off-line SPE and compounds are rapidly eluted and analyzed without further separation. These SPE-based methods are useful in the analysis of a large number of samples in the early stage of drug discovery where high throughput is more desirable, while a moderate level of sensitivity is acceptable. When higher sensitivity is desirable, an analytical column can be coupled to these on-line SPE elution devices through a column-switching strategy [119,120].
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