High‐speed liquid chromatography/tandem mass spectrometry using a monolithic column for high‐throughput bioanalysis

Publisher Website
Google Scholar
Abstract
With the ever‐increasing workload from a variety of in vitro and in vivo screening procedures, new analytical methodologies to perform bioanalysis in an accurate and high‐throughput manner are in great demand. In this work, monolithic columns were used instead of conventional particulate HPLC columns to perform chromatographic separations. Because the pressure drop on a monolithic column was considerably lower than that on a particulate column, a high flow rate (6 mL/min) was used for a 4.6 × 50 mm monolithic column with a total backpressure of about 61 bar measured using acetonitrile/water (50 : 50). The capability of using a regular column length at high flow rates, combined with the extremely small dependency of separation efficiency on linear flow velocity, allowed for the generation of sufficient chromatographic resolving power in a significantly reduced runtime. As demonstrated in this work, a plasma extract of a mixture of tempazepam, tamoxifen, fenfluramine, and alprozolam were baseline separated within a total analysis time of one minute. An average peak width at half maximum of approximately one second was noted using a generic broad gradient. It was also found that the separation efficiency and signal/noise (S/N) ratios for this separation remained almost constant at flow rates of 1, 3, and 6 mL/min, respectively. The ruggedness of the separation was evaluated by injecting 600 plasma extracts containing the replicates of a standard curve of the above mixture during an overnight run. The chromatographic retention time, separation quality, peak response and sensitivity were highly reproducible throughout the run. This high‐speed liquid chromatography/tandem mass spectrometry (LC/MS/MS) system has been used routinely in the authors' laboratory to support drug discovery programs. Copyright © 2001 John Wiley & Sons, Ltd.

This publication has 27 references indexed in Scilit: