LC-MS/MS-based Quantification of Tryptophan, Kynurenine, and Kynurenic Acid in Human Placental, Fetal Membranes, and Umbilical Cord Samples

Abstract Tryptophan breakdown metabolites formed along kynurenine pathway play a significant role in pregnancy and fetal development. To understand the involvement of tryptophan (TRP), kynurenine (KYN), and kynurenic acid (KYNA) in these phenomena, it is essential to quantify their levels in relevant biological samples, such as the placenta, fetal membranes, and umbilical cord. In this study, the level of TRP, KYN, and KYNA was determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The LC-MS/MS method was optimized for high sensitivity and specificity; it showed good reproducibility with a precision of <10% CV and an accuracy of 85-115%. The lower limit of quantification was 0.5 µg/ml for both TRP and KYN and 0.5 ng/mL for KYNA. The method was linear over the examined range of concentrations in homogenate: 0.5 to 30 µg/ml for both TRP and KYN and 0.5 to 25 ng/ml for KYNA. Maternal placenta samples exhibited lower KYNA concentrations but higher KYN concentrations and significantly increased KYN/TRP ratio. However, the umbilical cord had a significantly higher KYNA/KYN ratio than the other two analyzed tissues. No differences were observed in the KYNA/TRP ratio. Collectively, this finding may facilitate further research and provide inside into the involvement of kynurenine pathway of TRP metabolism in fetal development.