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Development and validation of an on-line two-dimensional reversed-phase liquid chromatography–tandem mass spectrometry method for the simultaneous determination of prostaglandins E2 and F2α and 13,14-dihydro-15-keto prostaglandin F levels in human plasma



We developed and validated an on-line reverse-phase two-dimensional LC/MS/MS (2D-LC/MS/MS) system for simultaneous determination of the levels of prostaglandin (PG) E2 as well as PGF2α and its metabolite 13,14-dihydro-15-keto PGF2α (F2α-M) in human plasma. Analytes were extracted by a three-step solid-phase extraction. Samples were then analyzed by on-line 2D-LC/MS/MS with electrospray ionization in negative mode. The 2D-LC system is composed of two reverse-phase analytical columns with a trapping column linking the two analytical columns. While an acidic buffer was used for both separation dimensions, differing organic solvents were employed for each dimension: methanol for the first and acetonitrile for the second to increase resolving power. The 2D-LC/MS/MS method was highly selective and sensitive with a significantly lower limit of quantitation (0.5 pg/mL for PGE2 and 2.5 pg/mL for PGF2α and F2α-M, respectively). Linearity of the 2D-LC/MS/MS system was demonstrated for the calibration ranges of 0.5–50 pg/mL for PGE2 and 2.5–500 pg/mL for PGF2α and F2α-M, respectively. Acceptable precision and accuracy were obtained throughout the calibration curve ranges. This highly selective and sensitive method was successfully utilized to determine the endogenous levels of PGE2, PGF2α, and F2α-M in plasma samples from six (four male and two female) normal volunteers. The mean concentrations for each analyte were 0.755 pg/mL for PGE2, 5.70 pg/mL for PGF2α and 9.48 pg/mL for F2α-M. Copyright © 2008 John Wiley & Sons, Ltd.