Detecting pM concentrations of prostaglandins in cell culture supernatants by capillary SCX-LC-MS/MS.
A highly sensitive, improved online strong cation exchange (SCX)--RP capillary liquid chromatographic (cLC) method with IT mass spectrometric (IT-MS/MS) detection for the simultaneous determination of prostaglandin (PG)A(1), PGD(2), PGE(1), PGE(2), PGF(2alpha), 8-iso-(8i)PGF(2alpha), 6-keto-(6k)PGF(1alpha), and 15-Delta(12, 14)-deoxy-PGJ(2) (15dPGJ(2)) in cell culture supernatants was developed and validated. Pretreatment of the cell culture supernatants included only dilution and filtration, and the analysis time including all sample preparation steps was 60 min per sample. Peptides/proteins contained in the matrix were removed by the SCX column. LODs in the range of 8-44 pg/mL (25-120 pM) cell culture supernatant were obtained. Excellent linearity (R(2) > 0.99) and satisfactory recoveries and within- and between-day precisions were obtained. Human mesenchymal stem cells (hMSCs) were stimulated with tumor necrosis factor alpha (TNFalpha) or TNFalpha/IL-17, and PG production was analyzed using the developed method. The four PGs, 6kPGF(1a), PGF(1a), PGE(2), and PGE(1 )were detected both in nonstimulated and stimulated cells. The amount of PG produced by the cell increased when the cell was stimulated.
Dahl
Sandra Rinne
SR
Department of Chemistry, University of Oslo, Oslo, Norway. sandrari@kjemi.uio.no
Kleiveland
Charlotte Ramstad
CR
Kassem
Moustapha
M
Lea
Tor
T
Lundanes
Elsa
E
Greibrokk
Tyge
T
D053801:Capillary Electrochromatography / Q000295:instrumentation / Q000379:methods*; D002411:Cation Exchange Resins / Q000737:chemistry*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000737:chemistry*; D006801:Humans; D008968:Molecular Conformation; D011453:Prostaglandins / Q000032:analysis*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D013237:Stereoisomerism; D013997:Time Factors
D002411:Cation Exchange Resins; D017077:Culture Media, Conditioned; D011453:Prostaglandins