title: 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.
authors.affiliation: Dept of Drug Metabolism and Pharmacokinetics, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, UK.
authors.forename: Lindsay
authors.initials: L
authors.lastname: Lai
authors.forename: Filippos
authors.initials: F
authors.lastname: Michopoulos
authors.forename: Helen
authors.initials: H
authors.lastname: Gika
authors.forename: Georgios
authors.initials: G
authors.lastname: Theodoridis
authors.forename: Robert W
authors.initials: RW
authors.lastname: Wilkinson
authors.forename: Rajesh
authors.initials: R
authors.lastname: Odedra
authors.forename: Julie
authors.initials: J
authors.lastname: Wingate
authors.forename: Ron
authors.initials: R
authors.lastname: Bonner
authors.forename: Stephen
authors.initials: S
authors.lastname: Tate
authors.forename: Ian D
authors.initials: ID
authors.lastname: Wilson
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D055432:Metabolomics / Q000379:methods*; D051379:Mice; D010949:Plasma / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*