Direct analysis of basic drugs in cell culture lysate using on-line extraction LC/MS/MS

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Claude R Mallet;Ziling Lu;Jeff Mazzeo;Uwe D Neue
Seventh International Symposium on Hyphenated Techniques in Chromatography and Hyphenated Chromatographic Analysis, 6-8 Feb 200; Brugge; Belgium
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Propranolol; Naloxone
Cell lysate
XTerra MS C18 3.5 µm Steel 2.1 mm 30 mm Oasis® HLB 25 Extraction Column - steel 2.1 mm 20 mm Oasis® MCX 25 Extraction Column - steel 2.1 mm 20 mm
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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 plates, fast gradients or ultra-high-flow chromatography are showing promising results. We have focused on on-line extraction techniques for high-throughput analysis. In a previous study, we investigated the potential of on-line extraction for the analysis of basic and acidic drugs in rat plasma. Recently, we have turned our attention to other types of matrices, for example the study of toxicity of drugs in cell cultures used in pre- and post- discovery phases. Several chemical and physical lysate methods were evaluated. The extracts were injected 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 a 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. A three-valve configuration using two extraction columns was used for the analysis of basic drugs in cell culture lysate. 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. The extracted analytes were forward-flushed into an XTerra 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., Vol. 14, p. 105, 2000 [2] J. Ayrton, G.J. Dear, W.J Leavens, D.N. Mallett, R.S. Plumb, J. of Chromatogr. A, Vol. 828, p.199, 2000 [3] N.V. Eeckhout, J.C. Perez, J. Clearboudt, R. Vandeputte, C. Van Peteghen, Rapid Commun. Mass Spectrom., Vol. 14, p. 280, 2000

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