Technical and biological variation in UPLC-MS-based untargeted metabolic profiling of liver extracts: application in an experimental toxicity study on galactosamine.
The relative importance of technical versus bio-logical variation in UPLC-MS liver metabolic profiling studies was assessed on liver samples collected as part of an in vivo hepatotoxicity study. Biological variability within and between two treatment groups (three rats treated with galactosamine and three with galactosamine+uridine) was compared with sampling/extraction variability (three portions extracted from each rat liver section) and UPLC-MS platform variability (triplicate injections of each extract) for aqueous and organic extracts. The impact of scaling on error measurement was investigated on replicate injections of a quality control sample, and consequently started log-transformation was used to stabilize the variance across the ion intensity range. For aqueous extracts, technical variability was two to four times lower than within group interanimal variability. Similar results were obtained for organic extracts for the galactosamine group, sampling/extraction variability being more elevated in the galactosamine+uridine group. For both extract types, differences between treatment groups were the principal source of observed variation, and triplicate injections clustered closely in PCA plots and in HCA dendrograms, indicating small instrument variability compared to observed biological variation. This protocol can be applied to investigate differences in liver metabolic profiles between animal groups in toxicology studies and clinical investigations of liver disease.
Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, South Kensington, UK.
Perrine
P
Masson
Konstantina
K
Spagou
Jeremy K
JK
Nicholson
Elizabeth J
EJ
Want
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D005688:Galactosamine / Q000633:toxicity*; D008110:Liver Extracts / Q000737:chemistry*; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D055432:Metabolomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley
D008110:Liver Extracts; D005688:Galactosamine