Fast and simple online sample preparation coupled with capillary LC‐MS/MS for determination of prostaglandins in cell culture supernatants

Abstract

An online 2-D 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(2a), 6-keto-(6k)PGF(1a), 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 dilution and filtration, and the analysis time including all sample preparation steps was less than 50 min per sample. Peptides/proteins contained in the matrix were removed by the SCX column. LODs in the range of 0.4-2.2 ng/mL cell culture supernatant, recoveries higher than 80% and within- and between-day precisions of less than 30% RSDs were obtained. Human mesenchymal stem cells (hMSCs) were stimulated with cytokine-containing supernatants derived from activated T lymphocytes, and PG production was analyzed using the developed method. PGE(2 )was found in cultures from both untreated and stimulated hMSCs, while PGE(1) was present above the detection limit only in stimulated cells.

Detecting pM concentrations of prostaglandins in cell culture supernatants by capillary SCX‐LC‐MS/MS

“…Prostanoids are released by stem cells upon stimulation; however their concentration is highly variable [3]. With our previously developed method concentrations in the range of 0.4 -2.2 ng/mL cell culture supernatant could be determined [3].…”

“…Previously, we reported that nonstimulated hMSCs release PGE 2 , and stimulated hMSCs release PGE 1 and PGE 2 (unpublished manuscript, [3]). However, we also found evidence for other PGs to be present at lower concentrations.…”

“…To increase the sensitivity additionally, the sum of several fragments was chosen for quantification as in [3]. Table 1.…”

“…The IT-MS instrument was operated in the negative mode with optimum conditions as described before [3]. Data were acquired using the Esquire Control v 3.5 by Bruker Daltonics in the MRM mode.…”

“…Human mesenchymal stem cells (hMSCs) secrete PGE 2 (unpublished manuscript, [3]) and it has been suggested that PGE 2 secreted by hMSCs plays an important role in hMSC-mediated immunomodulation [4]. To investigate the molecular mechanism behind the immunomodulating effects of hMSCs, the PG profile of stimulated hMSCs needs to be characterized, i. e., knowledge of which PGs are produced and released by hMSCs upon stimulation.…”