Validated LC-MS/MS method for simultaneous quantification of serotonin, kynurenine, and tryptophan in feline and canine plasma

Tryptophan metabolites, such as serotonin and kynurenine, are neurotransmitters that play crucial roles in regulating mood, sleep, and various physiologic functions. We developed and validated a robust and sensitive method for the simultaneous quantification of serotonin (5-HT), kynurenine (KYN), and tryptophan (TRP), in feline and canine plasma using electrospray ionization liquid chromatography-tandem mass spectrometry (ESI-LC/MS/MS). Plasma samples underwent protein precipitation followed by chromatographic separation on a reversed-phase C18 column under isocratic elution. The method was validated, assessing linearity, accuracy, precision, recovery, and matrix effects in both cat and dog plasma. The method had high sensitivity, with limits of detection of 0.2 ng/mL for 5-HT, 0.08 ng/mL for KYN, and 2.5 ng/mL for TRP, and limits of quantification of 0.35, 0.22, and 4.2 ng/mL, respectively. Calibration curves showed excellent linearity ( R 2 > 0.995) across biologically relevant concentration ranges. Intra- and inter-day accuracy and precision were within acceptable limits (<15% relative SD). The method had good recovery and minimal matrix effects. The method was further applied to plasma samples from healthy dogs and cats to demonstrate its applicability to biological samples and to report analyte concentrations in clinically healthy animals. Our validated LC-MS/MS method enables precise and reliable quantification of 5-HT, KYN, and TRP in feline and canine plasma.