On-line SPE LC/MS/MS Configurations for Effective Biological Sample Preparation

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Claude Mallet, Jeff Mazzeo, Uwe Neue
Waters Technology Seminar, Milford, 30 Sept - 6 Oct 2000
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Clemastine; Basic drugs;
Rat plasma
XTerra MS C18 3.5 µm Steel 2.1 mm 30 mm Symmetry® C18 3.5 µm Steel 2.1 mm 30 mm Oasis® HLB 25 µm Extraction Column - steel 2.1 mm 25 mm
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Abstract: During the last ten years, pharmaceutical companies have constantly pushed for shorter analysis time in order to breach the one-thousand-analyses-per-day barrier. With this demand for high speed analysis, new techniques, such as 96-well plate, fast gradients or ultra-high-flow chromatography are showing promising results. Recently, we have focussed on on-line extraction techniques for high-throughput analysis. We are injecting a plasma or urine sample, without any pretreatment, onto an extraction column at high flow rate (i.e. 4 mL/min) (1-3) to remove macromolecular compounds such as proteins, but trap smaller analytes on the head of the column. Several configurations for direct injection are possible. In the simplest configuration, the extraction column is connected directly to the MS/MS system. Other versions are configured with a single or a dual extraction column coupled to an analytical column. It is often necessary to split the flow. However in cases, where sensitivity is low, this option is not recommended. For efficient high speed analysis, the use of a second pump and 10 port valve is also a good choice. One line (high flow rate) can be dedicated to the extraction column, while the other (low flow rate) drives the analytical column and the mass spectrometer. Four different configurations were studied for the analysis of 3 typical drugs in rat plasma. The on-line analysis was performed on an Oasis HLB extraction column (2.1 x 30mm, 25 micron) using a Waters Alliance 2790 in gradient mode and a 515 stand-alone pump in isocratic mode. In two configurations, the extracted analytes were backflushed into a Symmetry C18 column (2.1 x 30mm, 3.5 micron), which was added to provide additional separation power. The drugs were quantified using a Micromass Ultima triple quadrupole mass spectrometer equipped with an electrospray source and set in multiple reaction monitoring mode (MRM). [1] Y.Q. Xia, D.B. Whigan, M.L. Powell, M. Jemal, Rapid Commun. Mass Spectrom., 14 (2000) 105 [2] J. Ayrton, G.J. Dear, W.J Leavens, D.N. Mallett, R.S. Plumb, J. of Chromatogr. A, 828 (2000) 199 [3] N.V. Eeckhout, J.C. Perez, J. Clearboudt, R. Vandeputte, C. Van Peteghen, Rapid Commun. Mass Spectrom., 14 (2000) 280

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