A highly sensitive and selective method for the determination of leukotriene B4 (LTB4) in ex vivo stimulated human plasma by ultra fast liquid chromatography–tandem mass spectrometry

• An UFLC-MS-MS assay is validated for the quantitation of LTB4 in human plasma.
• Baseline separation of LTB4 from its three isomers is critical.
• The assay was used for analyzing more than 2000 samples with excellent ISR results.
• The assay has been compared with an ELISA assay and the two assays correlated well.

Leukotriene B4 (LTB4) is an important inflammatory component in a number of diseases and has been used as a pharmacodynamic (PD) biomarker. In this report, a highly sensitive and selective ultra fast liquid chromatography–tandem mass spectrometry (UFLC-MS/MS) method for the determination of LTB4 in plasma from ex vivo stimulated human blood, using leukotriene B4-d4 (LTB4-d4, contains four deuterium atoms at the 6, 7, 14, and 15 positions) as the internal standard (IS), was developed and validated. The chromatographic separation of LTB4 from its three isomers and an unknown interference peak from human plasma was crucial to achieve accurate determination of 0.2 ng/mL (LLOQ) of LTB4. LTB4 and the IS were extracted with methyl tertiary butyl ether (MTBE) from 200 μL human plasma. Reversed-phase HPLC separation was carried out with a Phenomenex Synergi Hydro-RP column (100 mm × 3 mm, 2.5 μm). MS/MS detection was set at mass transitions of 335.0 → 194.9 m/z for LTB4 and 339.0 → 196.9 m/z for LTB4-d4 in Turbo Ionization Spray (TIS) negative mode. The dynamic range of the method is 0.2–200 ng/mL. LTB4 was found to be stable in human plasma for at least three freeze (−20 °C)/thaw cycles, and on the benchtop (room temperature) for at least 6 h. The stock solution storage stability study demonstrated that the LTB4 stock solution, in 50:50 acetonitrile:water, was stable at 4 °C for at least 198 days. The processed samples were found to be stable for at least 72 h at room temperature. The long-term sample storage stability test demonstrated that LTB4 human plasma samples were stable at a storage temperature of −20 °C for at least 198 days. In addition, intraday and interday accuracy and precision, sensitivity, linearity, and recovery were evaluated. An additional partial validation was conducted to decrease the plasma sample volume from 200 to 100 μL. All the data reported in this study fulfilled the requirements and recommendations in the FDA guidance for bioanalytical method validation. Comparison of the validated UFLC-MS/MS method with an ELISA method using ex vivo stimulated samples indicated that although results from the two assays correlated relatively well, the UFLC-MS/MS method has been shown to be superior in selectivity and dynamic range to an ELISA method in our study. The validated UFLC-MS/MS method was successfully used to analyze samples generated from two clinical studies. The excellent assay performance and incurred sample reproducibility (ISR) results obtained from the study sample analysis demonstrated the assay is robust and reliable.