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

Abstract

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.

Hydrophilic interaction liquid chromatographic tandem mass spectrometric determination of atenolol in human plasma

“…Thus, hydrophilic interaction liquid chromatography (HILIC) was investigated using a Thermo-Electron Hypersil silica column (4.6 × 50 mm, 5 µm particle size) with the mobile phase consisting of acetonitrile, water and TFA, and running isocratically. The selection of a silica column and aqueous-organic mobile phase in the current study was based on the experiences of our laboratory (Eerkes et al, 2002a(Eerkes et al, ,b, 2003Chen et al, 2002;Shou et al, 2002) in the determination of polar compounds in biological matrices. By introducing a high-organic and low-aqueous mobile phase, the spray performance in the LC-MS/MS interface was significantly improved.…”

Development and validation of a hydrophilic interaction liquid chromatography–tandem mass spectrometric method for the analysis of paroxetine in human plasma

“…In our laboratory, we have successfully used Quadra™ 96 for simultaneous protein precipitation , solid-phase (Shou et al, , 2002cChen et al, 2002;Eerkes et al, 2002a;Naidong et al, 2002a) and liquid-liquid (Eerkes and Naidong, 2003) extraction of 96 samples. Inadequate sensitivity was obtained when protein precipitation extraction was attempted for which the plasma size is typically limited to <0.10 mL.…”

Hydrophilic interaction chromatography

“…This approach can however only be used for solutes that show retention on both RP and HILIC-type stationary phases, and an RP-SPE/HILIC scheme is obviously among the preferred combinations when the aim is to trap and separate polar compounds. The high ACN content also gives HILIC two additional advantages; high sensitivity in ESI-MS [16 -18], and faster separations due to the lower viscosity of HILIC eluents compared to standard RP eluents [19]. Considering its growing importance and relative matureness, there have been surprisingly few reviews dedicated to HILIC.…”