High-speed liquid chromatography/tandem mass spectrometry using a monolithic column for high-throughput bioanalysis.
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 x 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.
Wu
J T
JT
Stine-Haskell Research Center, DuPont Pharmaceuticals Company, PO Box 30, Newark, DE 19714, USA. Jing-Tao.Wu@dupontpharma.com
Zeng
H
H
Deng
Y
Y
Unger
S E
SE
D000097:Acetonitriles; D000525:Alprazolam / Q000097:blood* / Q000302:isolation & purification; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D005277:Fenfluramine / Q000097:blood* / Q000302:isolation & purification; D007202:Indicators and Reagents; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D051381:Rats; D013629:Tamoxifen / Q000097:blood* / Q000302:isolation & purification; D013693:Temazepam / Q000097:blood* / Q000302:isolation & purification; D014867:Water
D000097:Acetonitriles; D007202:Indicators and Reagents; D014867:Water; D013629:Tamoxifen; D005277:Fenfluramine; D013693:Temazepam; D000525:Alprazolam; C032159:acetonitrile