title: Multi-step preparation technique to recover multiple metabolite compound classes for in-depth and informative metabolomic analysis.
abstract: Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital aspect of metabolomics is sample preparation whereby inconsistent techniques generate unreliable results. This technique encompasses protein precipitation, liquid-liquid extraction, and solid-phase extraction as a means of fractionating metabolites into four distinct classes. Improved enrichment of low abundance molecules with a resulting increase in sensitivity is obtained, and ultimately results in more confident identification of molecules. This technique has been applied to plasma, bronchoalveolar lavage fluid, and cerebrospinal fluid samples with volumes as low as 50 µl.  Samples can be used for multiple downstream applications; for example, the pellet resulting from protein precipitation can be stored for later analysis. The supernatant from that step undergoes liquid-liquid extraction using water and strong organic solvent to separate the hydrophilic and hydrophobic compounds. Once fractionated, the hydrophilic layer can be processed for later analysis or discarded if not needed. The hydrophobic fraction is further treated with a series of solvents during three solid-phase extraction steps to separate it into fatty acids, neutral lipids, and phospholipids. This allows the technician the flexibility to choose which class of compounds is preferred for analysis. It also aids in more reliable metabolite identification since some knowledge of chemical class exists.
authors.affiliation: Department of Immunology, National Jewish Health.
authors.forename: Charmion
authors.initials: C
authors.lastname: Cruickshank-Quinn
authors.affiliation: Department of Immunology, National Jewish Health.
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Quinn
authors.affiliation: Department of Immunology, National Jewish Health.
authors.forename: Roger
authors.initials: R
authors.lastname: Powell
authors.affiliation: Department of Immunology, National Jewish Health.
authors.forename: Yanhui
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Department of Immunology, National Jewish Health.
authors.forename: Michael
authors.initials: M
authors.lastname: Armstrong
authors.affiliation: Department of Pharmacology, School of Medicine, University of Colorado Denver.
authors.forename: Spencer
authors.initials: S
authors.lastname: Mahaffey
authors.affiliation: Department of Immunology, National Jewish Health.
authors.forename: Richard
authors.initials: R
authors.lastname: Reisdorph
authors.affiliation: Department of Immunology, National Jewish Health; reisdorphn@njhealth.org.
authors.forename: Nichole
authors.initials: N
authors.lastname: Reisdorph
mesh_terms: D000818:Animals; D001774:Blood Chemical Analysis / Q000379:methods; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry; D002555:Cerebrospinal Fluid / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D059625:Liquid-Liquid Extraction / Q000379:methods; D055432:Metabolomics / Q000379:methods*; D051379:Mice; D052616:Solid Phase Extraction / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D016482:Urinalysis / Q000379:methods