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.

Nano-LC-MS/MS for the quantitation of prostanoids in immune cells

“…In the last decade, LC-MS/MS has more and more evolved into the method of choice for prostanoid quantitation as it provides high sensitivity combined with a large number of analytes that can be determined from one sample in a single run without prior derivatization [12]. A lot of LC-MS/MS methods have been developed for various prostanoids, other eicosanoids, and their metabolites in diverse biological matrices, including plasma or serum [13,14], urine [15][16][17], tissue [18][19][20], or cell culture supernatants [21,22]. Hellman et al as well as Cao et al quantified PGE 2 and PGD 2 in supernatants collected from RAW macrophages which are a murine macrophage cell line [23,24].…”

Prostanoid Metabolites as Biomarkers in Human Disease

“…In many non-physiological matrices devoid of 15-PGDH and 15-oxo-prostaglandin ∆13-reductase, e.g., in the supernatant of cultured cells, metabolism of primary prostanoids can be excluded. Under these conditions, PGE 2 , PGD 2 , and PGF 2α are relatively stable and may be readily measured to estimate the biosynthetic capacity of the respective cells in vitro [16,87,88]. 6-keto PGF 1α and TXB 2 , the immediate non-enzymatical breakdown products of PGI 2 and TXA 2 , respectively, serve under these circumstances as surrogate markers [87].…”

“…Under these conditions, PGE 2 , PGD 2 , and PGF 2α are relatively stable and may be readily measured to estimate the biosynthetic capacity of the respective cells in vitro [16,87,88]. 6-keto PGF 1α and TXB 2 , the immediate non-enzymatical breakdown products of PGI 2 and TXA 2 , respectively, serve under these circumstances as surrogate markers [87]. These prostanoids were also measured in samples of exhaled breath condensate [89] and in tissue samples [90] instead of their downstream metabolites.…”

Tailored Polymer-Based Selective Extraction of Lipid Mediators from Biological Samples

“…Elution was performed using MeOH. Both the washing solvents and the elution solvents were collected; the solvent was evaporated under a gentle stream of nitrogen (99.996%, AGA, Oslo, Norway), re-dissolved in 30% EtOH/1% FA, filtered, and injected onto a capillary liquid chromatographic (cLC) system with tandem mass spectrometric (MS/MS) detection as reported elsewhere [17].…”

“…One of the most useful applications of MIPs is their use as a sorbent in SPE [17,18]. Here, the sample is passed through a cartridge (or a packed column) filled with a solid sorbent to which the analytes bind and are released in the presence of specific solvents (Figure S1).…”