Evaluation of a protocol for metabolic profiling studies on human blood plasma by combined ultra-performance liquid chromatography/mass spectrometry: From extraction to data analysis.
The investigation presented here describes a protocol designed to perform high-throughput metabolic profiling analysis on human blood plasma by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS). To address whether a previous extraction protocol for gas chromatography (GC)/MS-based metabolic profiling of plasma could be used for UPLC/MS-based analysis, the original protocol was compared with similar methods for extraction of low-molecular-weight compounds from plasma via protein precipitation. Differences between extraction methods could be observed, but the previously published extraction method was considered the best. UPLC columns with three different stationary phases (C8, C18, and phenyl) were used in identical experimental runs consisting of a total of 60 injections of extracted male and female plasma samples. The C8 column was determined to be the best for metabolic profiling analysis on plasma. The acquired UPLC/MS data of extracted male and female plasma samples was subjected to principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA). Furthermore, a strategy for compound identification was applied here, demonstrating the strength of high-mass-accuracy time-of-flight (TOF)/MS analysis in metabolic profiling.
Bruce
Stephen J
SJ
Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 87 Umeå, Sweden.
Jonsson
Pär
P
Antti
Henrik
H
Cloarec
Olivier
O
Trygg
Johan
J
Marklund
Stefan L
SL
Moritz
Thomas
T
D000596:Amino Acids / Q000097:blood; D001774:Blood Chemical Analysis / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D003596:Cytosine / Q000032:analysis; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D004364:Pharmaceutical Preparations / Q000097:blood; D010743:Phospholipids / Q000097:blood; D010949:Plasma / Q000378:metabolism*; D012680:Sensitivity and Specificity
D000596:Amino Acids; D004364:Pharmaceutical Preparations; D010743:Phospholipids; D003596:Cytosine