Metabolomic Method: UPLC-q-ToF Polar and Non-Polar Metabolites in the Healthy Rat Cerebellum Using an In-Vial Dual Extraction

Abstract

Unbiased metabolomic analysis of biological samples is a powerful and increasingly commonly utilised tool, especially for the analysis of bio-fluids to identify candidate biomarkers. To date however only a small number of metabolomic studies have been applied to studying the metabolite composition of tissue samples, this is due, in part to a number of technical challenges including scarcity of material and difficulty in extracting metabolites. The aim of this study was to develop a method for maximising the biological information obtained from small tissue samples by optimising sample preparation, LC-MS analysis and metabolite identification. Here we describe an in-vial dual extraction (IVDE) method, with reversed phase and hydrophilic liquid interaction chromatography (HILIC) which reproducibly measured over 4,000 metabolite features from as little as 3mg of brain tissue. The aqueous phase was analysed in positive and negative modes following HILIC separation in which 2,838 metabolite features were consistently measured including amino acids, sugars and purine bases. The non-aqueous phase was also analysed in positive and negative modes following reversed phase separation gradients respectively from which 1,183 metabolite features were consistently measured representing metabolites such as phosphatidylcholines, sphingolipids and triacylglycerides. The described metabolomics method includes a database for 200 metabolites, retention time, mass and relative intensity, and presents the basal metabolite composition for brain tissue in the healthy rat cerebellum.

Association between fatty acid metabolism in the brain and Alzheimer disease neuropathology and cognitive performance: A nontargeted metabolomic study

“…The in-vial dual extractions were performed as previously described [ 7 ]. After LC-MS analysis, the remaining aqueous and nonaqueous phases were split into separate vials and dried down under a stream of nitrogen at 37°C.…”

“…Samples were analysed using HILIC LC-MS analysis performed as described previously [ 7 ]. GC-MS analysis was carried out on a Shimadzu QP-2010 with an AOC-20S autosampler and AOC-20i autoinjector (Shimadzu, Kyoto, Japan).…”

“…The application of large-scale unbiased metabolomics techniques to study the role of metabolism in Alzheimer pathogenesis may facilitate a more complete understanding of Alzheimer pathology and mechanisms triggering symptom expression. Metabolomics can be defined as “the unbiased analysis of the composition of small molecule metabolites in a given biological tissue or fluid under a specific set of environmental conditions” [ 5 – 7 ]. Several metabolomic studies have previously examined the relationship between metabolism and AD pathology.…”

Neurotransmitter Imbalance in the Brain and Alzheimer’s Disease Pathology

“…Thirteen of the fifteen metabolites described in this paper (tyrosine, L-DOPA, dopamine, aminobutanal, arginine, aspartate, GABA, glutamate, glutamine, guanidinobutanoate, glycine, guanosine, and ornithine) were measured on an in-house platform that is described in detail in Ebshiana et al [21]. Briefly 20 l of methanol and 5 l of internal standard solution (2.5 mM L-serine 13 C 3 15 N and L-valine 13 C 5 15 N in 4:1 methanol:water) was added per milligram of tissue prior to homogenization.…”

Deregulation of the Purine Pathway in Pre-Transplant Liver Biopsies Is Associated with Graft Function and Survival after Transplantation

“…The samples were kept in the chamber at a temperature of 4 • C, and the injection volume was 5 µL, with full-loop function (20 µL loop size). Chromatographic and spectrometric conditions for the analysis of polar metabolites were according to a published protocol [25]. Quality controls (QC) were run every 8 samples in random order.…”