Within-day reproducibility of an HPLC-MS-based method for metabonomic analysis: application to human urine.

Self-evidently, research in areas supporting "systems biology" such as genomics, proteomics, and metabonomics are critically dependent on the generation of sound analytical data. Metabolic phenotyping using LC-MS-based methods is currently at a relatively early stage of development, and approaches to ensure data quality are still developing. As part of studies on the application of LC-MS in metabonomics, the within-day reproducibility of LC-MS, with both positive and negative electrospray ionization (ESI), has been investigated using a standard "quality control" (QC) sample. The results showed that the first few injections on the system were not representative, and should be discarded, and that reproducibility was critically dependent on signal intensity. On the basis of these findings, an analytical protocol for the metabonomic analysis of human urine has been developed with proposed acceptance criteria based on a step-by-step assessment of the data. Short-term sample stability for human urine was also assessed. Samples were stable for at least 20 h at 4 degrees C in the autosampler while queuing for analysis. Samples stored at either -20 or -80 degrees C for up to 1 month were indistinguishable on subsequent LC-MS analysis. Overall, by careful monitoring of the QC data, it is possible to demonstrate that the "within-day" reproducibility of LC-MS is sufficient to ensure data quality in global metabolic profiling applications.

Matrix-assisted laser desorption/ionization imaging mass spectrometry for direct measurement of clozapine in rat brain tissue.

Matrix-assisted laser desorption/ionization hyphenated with quadrupole time-of-flight (QTOF) mass spectrometry (MS) has been used to directly determine the distribution of pharmaceuticals in rat brain tissue slices which might unravel their disposition for new drug development. Clozapine, an antipsychotic drug, and norclozapine were used as model compounds to investigate fundamental parameters such as matrix and solvent effects and irradiance dependence on MALDI intensity but also to address the issues with direct tissue imaging MS technique such as (1) uniform coating by the matrix, (2) linearity of MALDI signals, and (3) redistribution of surface analytes. The tissue sections were coated with various matrices on MALDI plates by airspray deposition prior to MS detection. MALDI signals of analytes were detected by monitoring the dissociation of the individual protonated molecules to their predominant MS/MS product ions. The matrices were chosen for tissue applications based on their ability to form a homogeneous coating of dense crystals and to yield greater sensitivity. Images revealing the spatial localization in tissue sections using MALDI-QTOF following a direct infusion of (3)H-clozapine into rat brain were found to be in good correlation with those using a radioautographic approach. The density of clozapine and its major metabolites from whole brain homogenates was further confirmed using fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) procedures.

Metabonomics and biomarker discovery: LC-MS metabolic profiling and constant neutral loss scanning combined with multivariate data analysis for mercapturic acid analysis.

In the field of metabonomics, 1H NMR and full scan mass spectrometry methods have usually been combined with principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) to detect patterns in biofluids that correspond to specific effects, usually a toxic site effect of a compound. Confounders together with great interindividual variation complicate such analysis in humans, and therefore, metabonomic data are almost restricted to animals. In our study, a constant neutral loss (CNL) scan on a linear ion trap demonstrated increased sensitivity and specificity compared to a full scan approach and was performed to detect mercapturic acids (MA), a class of effect markers. The method was applied to human volunteers administered 50 and 500 mg of acetaminophen (AAP), a model compound known to form MAs. Using a new algorithm to prepare the CNL data for chemometrics, discrimination of control and postdose samples could be performed using PCA and PLS-DA. The loadings plots clearly revealed AAP-MA as a marker, even at low-dose levels. Orthogonal signal correction (OSC) was carried out to investigate background information that is not due to exposure. Surprisingly, the OSC data provided a classification of male and female subjects showing the performance of the new approach.