A validated assay to quantitate serotonin in lamb plasma using ultrahigh-performance liquid chromatography-tandem mass spectrometry: applications with LC/MS3

An ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS) method was developed and validated for the quantification of serotonin (5-HT) in lamb plasma using [²d₄]-serotonin ([²d₄]-5-HT) as an internal standard. Charcoal-stripped human plasma was used as the blank matrix during validation, and 5-HT was quantitated using selected reaction monitoring. The UHPLC/MS/MS system consisted of an Agilent 1290 Infinity ultrahigh-performance liquid chromatograph coupled with an AB SCIEX QTRAP® 5500 hybrid linear ion trap triple quadrupole mass spectrometer. The method was validated for accuracy, precision, linearity, lower limit of quantification (LLOQ), selectivity, and other parameters. The LLOQ was 1.0 ng/mL, requiring 100 μL of sample. The method was applied to monitor the 5-HT levels in lamb plasma after the administration of fluoxetine. Tandem mass spectrometry cubed (MS³) experiments were also performed to investigate the fragmentation pattern of 5-HT and [²d₄]-5-HT. A liquid chromatography-MS³ (LC/MS³) method was developed, and the UHPLC/MS/MS and the LC/MS³ methods were compared for performance.