Ultrafast liquid chromatography/tandem mass spectrometry bioanalysis of polar analytes using packed silica columns.

Ultrafast liquid chromatography/tandem mass spectrometry (LC/MS/MS) bioanalysis was demonstrated with the use of packed silica columns operated under elevated flow rates. A special effort has been made to achieve ultrafast analysis without sacrificing chromatographic resolution. Two multiple analyte/metabolites assays, (1) morphine/morphine-6-glucuronide(M6G)/morphine-3-glucuronide(M3G) and (2) midazolam/1'-hydroxymidazolam/4-hydroxymidazolam, were used to demonstrate the speed, sensitivity, peak shape and separation of the ultrafast methods utilizing silica columns. In both methods adequate chromatographic separation was a necessity because quantitation results would be otherwise compromised due to cross interference between different selected reaction monitoring (SRM) transitions. Baseline resolutions between morphine, M6G and M3G in human plasma extracts were achieved within 30 s on a 50 x 3 mm Betasil silica column operated at 4 mL/min of isocratic acetonitrile/water mobile phase. The total injection-to-injection cycle time was 48 s with a simple, single-autosampler/single-column setup, when a Shimadzu SIL-HT autosampler was used. Baseline resolution between 1'-hydroxymidazolam and 4-hydroxymidalolam in monkey plasma extracts was achieved within 33 s using similar conditions. Due to the absence of carry-over in this case, no rinsing of the injection needle was necessary, resulting in a cycle time of only 39 s/sample. These ultrafast methods were successfully used to analyze extracted biological samples and proved to be reproducible, reliable and generated equivalent pharmaco-kinetic (PK) results to those obtained by regular flow LC/MS/MS analysis to support discovery PK studies.