title: Detecting pM concentrations of prostaglandins in cell culture supernatants by capillary SCX-LC-MS/MS.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Oslo, Oslo, Norway. sandrari@kjemi.uio.no
authors.forename: Sandra Rinne
authors.initials: SR
authors.lastname: Dahl
authors.forename: Charlotte Ramstad
authors.initials: CR
authors.lastname: Kleiveland
authors.forename: Moustapha
authors.initials: M
authors.lastname: Kassem
authors.forename: Tor
authors.initials: T
authors.lastname: Lea
authors.forename: Elsa
authors.initials: E
authors.lastname: Lundanes
authors.forename: Tyge
authors.initials: T
authors.lastname: Greibrokk
mesh_terms: 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
chemical_list: D002411:Cation Exchange Resins; D017077:Culture Media, Conditioned; D011453:Prostaglandins