Methodological considerations in the development of HPLC-MS methods for the analysis of rodent plasma for metabonomic studies

Abstract

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.

Improvement in the Number of Analytic Features Detected by Non-Targeted Metabolomic Analysis: Influence of the Chromatographic System and the Ionization Technique

“…The acceptance criteria for reproducibility and repeatability differ between laboratories. In the studies presented in this chapter a coefficient of variation (CV) threshold of 25% was set which is in line with similar metabolomic studies published in the literature (Crews et al, 2009;Lai et al, 2010).…”

Mass spectrometry‐based holistic analytical approaches for metabolite profiling in systems biology studies

“…10 mL) of diluted urine. However, for plasma or serum much shorter runs (close to 60 injections of samples not including QCs, Lai et al, 2009;Zelena et al, 2009) has been seen to be the practical limit before unacceptable deterioration in performance. In such a case cleaning of the source and the column or even the replacement of precolumn and analytical column are necessary.…”

“…Obviously, the aim of this type of analysis is to have 100% of ions with CV < 30%, but a data set containing >80% of ions with CV < 30% would indicate a good quality data set. Given that many journals now allow the provision of supplementary electronic data to complement the main publication we now advocate the value of providing the QC data as supplementary tables as a means of demonstrating acceptable repeatability (e.g., see Lai et al, 2009;Michopoulos et al, 2009). Of importance is also the relation of an ion mass, RT, and intensity with its repeatability.…”

“…Nevertheless, it is strongly advised that investigators thoroughly examine the trends of the QCs and implement such a conditioning step before the commencement of the analysis. For other specimens such as blood-derived samples (serum or plasma) a more complex process is required for system conditioning and monitoring (see Lai et al, 2009;Michopoulos et al, 2009;Zelena et al, 2009).…”

Metabolomics Approach to Explore the Effects of Rebamipide on Inflammatory Arthritis Using Ultra Performance Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry

“…Separations were performed using an Acquity UPLC BEH C18 column (1.7-μm particle size, 2.1-mm inner diameter, 100-mm length; Waters Corp.) at 50 o C. Initially, 20-μL QC samples were injected 15 times prior to the main analysis using a short gradient program to condition the column and to optimize the system [20]. Gradient elution was performed using a mixture of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in methanol) at a flow rate of 0.4 mL/min.…”