Development and validation of an on‐line two‐dimensional reversed‐phase liquid chromatography–tandem mass spectrometry method for the simultaneous determination of prostaglandins E 2 and F 2 α and 13,14‐dihydro‐15‐keto prostaglandin F 2α levels in human plasma

• Published in: Biomedical chromatography 2009-03, Vol.23 (3), p.315-323
• Main Authors: Komaba, Junji, Matsuda, Dai, Shibakawa, Kimio, Nakade, Susumu, Hashimoto, Yoshitaka, Miyata, Yasuyuki, Ogawa, Mikio
• Format: Article
• Language: English
• ISSN: 0269-3879; 1099-0801
• DOI: 10.1002/bmc.1117

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) E 2 as well as PGF 2 α and its metabolite 13,14‐dihydro‐15‐keto PGF 2 α (F 2 α ‐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 PGE 2 and 2.5 pg/mL for PGF 2 α and F 2 α ‐M, respectively). Linearity of the 2D‐LC/MS/MS system was demonstrated for the calibration ranges of 0.5–50 pg/mL for PGE 2 and 2.5–500 pg/mL for PGF 2 α and F 2 α ‐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 PGE 2 , PGF 2 α , and F 2 α ‐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 PGE 2 , 5.70 pg/mL for PGF 2 α and 9.48 pg/mL for F 2 α ‐M. Copyright © 2008 John Wiley & Sons, Ltd.