A clean-up technology for the simultaneous determination of lysophosphatidic acid and sphingosine-1-phosphate by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a phosphate-capture molecule, Phos-tag.
Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are growth factor-like lipids having a phosphate group. The concentrations of these mediator lipids in blood are considered to be potential biomarkers for early detection of cancer or vascular diseases. Here, we report a method for simultaneous determination of LPA and S1P using Phos-tag, a zinc complex that specifically binds to a phosphate-monoester group. Although both LPA and S1P are hydrophilic compounds, we found that they acquire hydrophobic properties when they form complexes with Phos-tag. Based on this finding, we developed a method for the enrichment of LPA and S1P from biological samples. The first partition in a two-phase solvent system consisting of chloroform/methanol/water (1:1:0.9, v/v/v) is conducted for the removal of lipids. LPA and S1P are specifically extracted as Phos-tag complexes at the second partition by adding Phos-tag. The Phos-tag complexes of LPA and S1P are detectable by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and quantifiable based on the relative intensities of ions using 17:0 LPA and C17 S1P as internal standards. The protocol was validated by analyses of these mediator lipids in calf serum, a rat brain and a lung. The clean-up protocol is rapid, requires neither thin-layer chromatography (TLC) nor liquid chromatography (LC), and is applicable to both blood and solid tissue samples. We believe that our protocol will be useful for a routine analysis of LPA and S1P in many clinical samples.
Morishige
Jun-ichi
J
Research Center for Green Science, Fukuyama University, Fukuyama 729-0292, Japan.
Urikura
Mai
M
Takagi
Haruko
H
Hirano
Kaoru
K
Koike
Tohru
T
Tanaka
Tamotsu
T
Satouchi
Kiyoshi
K
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D005591:Chemical Fractionation / Q000379:methods*; D002725:Chloroform / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008168:Lung / Q000737:chemistry; D008246:Lysophospholipids / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D011725:Pyridines / Q000737:chemistry*; D051381:Rats; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry
C514153:1,3-bis(bis(pyridin-2-ylmethyl)amino)propan-2-ol; D008246:Lysophospholipids; D011725:Pyridines; C060506:sphingosine 1-phosphate; D002725:Chloroform; D013110:Sphingosine; C032881:lysophosphatidic acid