Reversed Phase (RP) HPLC ESI-MS Considerations
Optimization of the RP HPLC system is critical for maximum sample throughput compatible with the mass spectrometer data acquisition capabilities. For highest throughput, the samples should be injected into the HPLC column or directly into the mass spectrometer using the overlay method, i.e., during the ESI-MS assay of a sample, the next sample is retrieved and prepared for injection immediately upon completion of the previous assay. To further increase throughput, column switching should be coordinated with each injection, i.e., during the HPLC analysis of a sample an identical second HPLC column is conditioned for use immediately upon the completion of the prior HPLC ESI-MS assay. For high throughput primary drug screening, a 2-min ballistic gradient or a 10-min gradient with high solvent flow rates can be used. For secondary screens, 10- to 15-min gradients are used with lower solvent flow rates to optimize ESI-MS sensitivity.
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