Accurate quantification of PGE2 in the polyposis in rat colon (Pirc) model by surrogate analyte-based UPLC–MS/MS

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HighlightsThe colon PGE2 of normal (wild type) rat and Pirc rat (an Apc-mutant rat) were firstly accurately determined by specific and sensitive UPLC–MS/MS.PGE2 was confirmed to promote Pirc rat colon polyps.An optimized mobile phase (0.1% ammonia hydroxide) was found to greatly improve the chromatographic separation of prostaglandins.An accurate and reliable UPLC–MS/MS method is reported for the quantification of endogenous Prostaglandin E2 (PGE2) in rat colonic mucosa and polyps. This method adopted the “surrogate analyte plus authentic bio-matrix” approach, using two different stable isotopic labeled analogs — PGE2-d9 as the surrogate analyte and PGE2-d4 as the internal standard. A quantitative standard curve was constructed with the surrogate analyte in colonic mucosa homogenate, and the method was successfully validated with the authentic bio-matrix. Concentrations of endogenous PGE2 in both normal and inflammatory tissue homogenates were back-calculated based on the regression equation. Because of no endogenous interference on the surrogate analyte determination, the specificity was particularly good. By using authentic bio-matrix for validation, the matrix effect and exaction recovery are identically same for the quantitative standard curve and actual samples – this notably increased the assay accuracy. The method is easy, fast, robust and reliable for colon PGE2 determination. This “surrogate analyte” approach was applied to measure the Pirc (an Apc-mutant rat kindred that models human FAP) mucosa and polyps PGE2, one of the strong biomarkers of colorectal cancer. A similar concept could be applied to endogenous biomarkers in other tissues.

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