Sources of analytical variation in high‐performance liquid chromatography/mass spectrometry (HPLC/MS), such as changes in retention, mass accuracy or signal intensity, have been investigated to assess their importance as a variable in the metabonomic analysis of human urine. In this study chromatographic retention and mass accuracy were found to be quite reproducible with the most significant source of analytical variation in the data sets obtained being the result of changes in detector response. Depending on the signal intensity threshold used to define the presence of a peak a sample component could be present in some replicate injections and absent in others within the same run. The implementation of a more sophisticated data software analysis package was found to greatly reduce the impact of detector response variability resulting in improved data analysis. Copyright © 2007 John Wiley & Sons, Ltd.

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A study of the factors involved in obtaining valid global metabolite profiles from the HPLC-MS of rat or mouse plasma for the purposes of metabonomic analysis has been undertaken. Plasma proteins were precipitated with three volumes of either methanol or acetonitrile. Chromatographic separations were performed on a C18-bonded stationary phase using 3.5 and 5 mum particles packed into 2.1 and 4.6 mm i.d. formats, respectively, and on a C8 phase using 3.5 mum particles and a 2.1 mm i.d. column. Three reversed-phase gradient solvent systems, based on acidified water-acetonitrile, acidified water-methanol and acidified water-methanol-acetonitrile mixtures, were investigated. The column eluent was analysed with both positive and negative electrospray ionisation using a quadrupole-linear ion trap mass spectrometer. These studies revealed that while accurate classification of sample type can be made, there are a number of methodological problems associated with the analysis of plasma with respect to factors such as repeatability and column longevity. In particular, special care has to be taken to ensure that the analytical system is properly "conditioned" by the repeated injection of matrix samples. The use of biological quality control (QC) samples provided an important means of monitoring method performance. Finally, the source of the plasma (Zucker wild-type or (fa/fa) rat or mouse tumour model) also appeared to have an effect on the repeatability of the methodology.

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Historically, most bioanalytical methods for drug analysis in pharmaceutical industry were developed using HPLC coupled with UV or fluorescence detection. However, there is a trend toward interfacing separation technologies with more sensitive tandem mass spectrometry (MS/MS)-based systems. MS/MS detection offers complete resolution of the parent compounds from their first pass metabolites to avoid extra efforts for separation and sample clean-up procedures resulting in shorter run times. With the increasing demand for ever faster screening, there is a continuing demand for bioanalytical methods possessing higher sample throughput for both in vitro and in vivo drug metabolism and pharmacokinetic evaluations to accelerate the discovery process. This review focuses on the current approaches for fast MS-based assays (cycle-time less than 5 min) of pharmaceuticals and their metabolites that have been reported in the peer-reviewed publications.