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

Abstract

Prostanoids, derivatives of arachidonic acid, are involved in inflammation and immune reactions. To understand the role of prostanoids produced by diverse immune cells, a highly sensitive quantitation method for prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), 6-keto prostaglandin F1α (6-keto PGF1α), prostaglandin F2α (PGF2α), and thromboxane B2 (TXB2) by means of nano-liquid chromatography-tandem mass spectrometry has been developed. It was validated according to the guidelines of the Food and Drug Administration (FDA) in terms of linearity, precision, accuracy, recovery, stability, and lower limit of quantitation (LLOQ). The LLOQ were 25 pg/mL in the injected solution (75 fg on column (o.c.)) for PGE2 and PGD2 and 37.5 pg/mL (112.5 fg on column) for 6-keto PGF1α, PGF2α, and TXB2, respectively. It was successfully applied to murine mast cells isolated from paws after zymosan injection and to CD4(+) and CD8(+) T lymphocytes from blood of sensitized versus non-sensitized mice in context of a delayed type hypersensitivity model. About 5,000 (T cells) to 40,000 (mast cells) cells were sufficient for quantitation. In the mast cells, the production of PGE2 increased at a significantly higher extent than the synthesis of the other prostanoids. The T lymphocytes did not show any difference in prostanoid production, no matter whether they were obtained from sensitized mice or non-sensitized mice.

GPVI and Thromboxane Receptor on Platelets Promote Proinflammatory Macrophage Phenotypes during Cutaneous Inflammation

“…An analysis of the samples was carried out using a nanoLC-MS/MS system, after the liquid-liquid extraction procedure (5500 QTrap, Sciex, Framingham, MA) (Thomas et al, 2014) (see Supplementary Methods for complete details).…”

A novel, fast and sensitive supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) method for analysis of arachidonic acid metabolites

“…19 In contrast, nano-LC-MS/MS method with C8 column has produced a reverse elution pattern for similar analytes with the elution order of TXB 2 , LTB 4 , PGF 2α , PGE 2 and PGD 2 . 21 After the generic screening, the method was further developed taking the effect of flow rates, backpressure and column temperature into account to improve the selectivity, sensitivity and the resolution of the AA-metabolites on the 2-PIC column. In general, higher retention time was observed with higher temperature while higher back pressure had opposite influence with little or no effects on resolution of isomers.…”

“…Our exploration focused on the extensively investigation of different solvents/ solvent mixtures including methanol, ethyl acetate, n-hexane : ethyl acetate (1 : 1 v/v), or hexane : isopropanol (1 : 1 v/v) and MTBE with spike plasma samples of AA-metabolites (30 ng mL −1 ). 7,21,24 Finally, methanol was selected as an extraction solvent based on the recovery % of each analyte. The mean recovery of analytes ranged from 70-115% on methanol (Table 3).…”

Sphingosine-1-phosphate and other lipid mediators generated by mast cells as critical players in allergy and mast cell function

“…Consequent studies revealed that although mast cells can generate leukotriene B4 (LTB 4 ), the cysteinyl leukotriene C4 (LTC 4 ) and PGD 2 upon activation, the specific profile or relative amounts of eicosanoid generation varies in both human and rodent mast cells depending on the tissue of origin, the stage of differentiation and culture conditions (Lundstrom et al, 2013) and reviewed in (Boyce, 2007). PGE 2 has not been considered to be an eicosanoid abundantly produced by antigen-stimulated mast cells in culture (Boyce, 2007) although a recent report detected higher intracellular content of PGE 2 than other prostanoids by mass spectrometry in mast cells isolated from inflamed tissue in a delayed type hypersensitive model (Thomas et al, 2014). Even though the release of eicosanoids by antigen-activated mast cells requires several enzymatic steps for their de novo production (Fig.…”