Liquid chromatography-mass fraction collecting

In our work at Leiden University, apart from HPLC-photodiode array (PDA) detection, HPLC-electrospray mass spectrometry was used as characterization and identification tools. A semipurified taxine extract obtained with acid/base extraction of T. baccata needles was analyzed in reversed phase nine taxines, one taxinine, and six taxanes were found present in the sample. Furthermore, 10-deacetylbaccatin 111 (paclitax-el s main precursor) and other taxanes were also found in the extract. Identification of the peaks was made with online liquid chromatography/mass spectrometry (LC/MS) and off-line nuclear magnetic resonance (NMR) following fraction collection at the end of the HPLC. Retention and spectral data of the identified peaks were used as tool to screen for taxines and taxi-nines in plant and cell culture extracts. Several... 

One attempt to overcome these disadvantages has been to use multidimensional liquid chromatography (LC) followed directly by tandem mass spectrometry to separate, fragment and identify proteins (Link et al., 1999). In this process, a denatured and reduced protein mixture is digested with a protease to create a collection of peptides (Fig. 2.6). The peptide mixture is applied to a cation exchange column and a fraction of these peptides are eluted based on charge onto a reverse-phase column. The... 

Solid-phase extraction columns offer a rough cleanup of the crude extract, which might nevertheless not be sufficient for some detection systems such as mass spectrometry. Some authors have proposed a combination of solid-phase extraction and liquid chromatography columns for extract cleanup (440). Other methods appeal to liquid chromatography on Cig columns with automated fraction collection. Fractions containing the analyte of interest were evaporated to dryness, yielding a residue that in most cases was suitable for gas chromatographic detection after suitable derivatization (445, 437). 

RS Plumb, J Ayrton, GJ Dear, BC Sweatman, IM Ismail. The use of preparative high performance liquid chromatography with tandem mass spectrometric directed fraction collection for the isolation and characterisation of drug metabolites in urine by nuclear magnetic resonance spectroscopy and liquid chromatography/sequential mass spectrometry. Rapid Commun Mass Spectrom 13 (10) 845-854,1999. 

Plumb, R.S. Ayrton, J. Dear, G.J. Sweatman, B.C. Ismail, I.M. The Use of Preparative High Performance Liquid Chromatography with Tandem Mass Spectrometric Directed Fraction Collection for the Isolation and Characterisation of Drug Metabolites in Urine by Nuclear Magnetic Resonance Spectroscopy and Liquid Chromatography/Sequential Mass Spectrometry, Rapid Commun. Mass Spectrom. 13, 845-854 (1999). 

At present, an off-line combination is in use in most instances. The mobile phase is collected batchwise and the fractions containing the substances of interest are then analysed by mass spectrometry. Huber et al. investigated the batchwise co]lection of eluent fractions with special reference to high-performance column liquid chromatography, and was able to elute components in a few seconds in a volume of less than 100 pi. The design of an automatic fraction collector ful-... 

If the tissue component is very complex, or if the peptide of interest is only a minor component of the sample, a fractionation by means of (micro-) reversed-phase liquid chromatography should be earned out The collected fractions may then be assayed by mass spectrometry. 

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