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Background: Investigations have been undertaken of the effects of storage on the utility of using dried blood spots, collected and stored on a paper substrate, combined with reversed-phase ultra-performance LC (UPLC), electrospray ionization orthogonal acceleration ToF-MS (UPLC-oaToF-MS), as a method for performing global metabolic profiling studies. In addition, a range of products used to collect these blood spots have also been studied for effects on the UPLC-MS background., Results: These preliminary data suggest that sample stability in this application is limited unless the cards are stored at, at least, -20°C and preferably at -80°C. In addition, the use of untreated cards is recommended to minimize incurred background interferences., Conclusion: Further investigations are necessary to improve the utility of this methodology in global metabolite profiling.

5-Oxoproline (5-OP; pyroglutamate) is an intermediate in the biosynthesis of the endogenous tripeptide glutathione and has been seen to be elevated in the biofluids and tissues of rats following the administration of glutathione-depleting hepatotoxic xenobiotics such as acetaminophen (paracetamol), bromobenzene and ethionine. As 5-OP is a potential biomarker for hepatotoxicity HPLC-MS/MS methods have been developed for its quantification in in vitro cell culture media and rat plasma. For the cell culture media the lower limit of quantification (LLOQ), defined as the lowest concentration on the calibration curve, was 10 ng/ml. Minimal carry over was observed for cell culture media between injections (less than 5% at all concentrations examined), precision and accuracy were generally better than 20% for within and between day analyses. For rat plasma a LLOQ of 50 ng/ml was obtained. Carry over for plasma was less than 5% for all concentrations, within and between batch accuracy and precision were generally better than 20%. The methods were linear for both sample types from the LLOQ up to 1 μg/ml. For samples obtained from rats subjected to chronic administration of the hepatotoxin methapyrilene, concentrations of 5-OP were not observed to increase significantly at any time point compared to controls. 5-OP was also determined in the culture media of human liver epithelial (THLE) cells transfected with cytochrome P450 2E1 (THLE-2E1). Following exposure of THLE-2E1 cells to acetaminophen, large increases in the concentrations of 5-OP were observed, which correlated with reduced cellular glutathione content and with cell toxicity. These results show that LC-MS/MS can be used to perform rapid, sensitive, and quantitative determination of 5-OP in vivo and in vitro and will enable additional investigations into the utility of 5-OP as a biomarker of liver drug-induced liver injury., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The potential of dried blood, plasma and urine spots, deposited on a paper substrate, combined with reversed-phase ultra performance liquid chromatography and orthogonal acceleration time-of-flight mass spectrometry (UPLC-oaToFMS), with electrospray ionization (ESI), has been investigated for global metabolic profiling. When compared to analysis using protein precipitated plasma, both blood and plasma spots gave comparable profiles and numbers of ions etc., using both positive and negative ESI. In the case of urine the results for spots obtained in positive ESI were also comparable to those of the untreated sample, but with negative ESI, a significant reduction in ions was noted for spotted samples. These preliminary data suggest that, with further optimization, biofluid spotting may provide an alternative to conventional methods for this type of work in suitable applications.

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.