PT - JOURNAL ARTICLE AU - Pan, Xiaobei AU - Chandrasekaran, Swathine AU - Woodside, Jayne V. AU - Riedel-Heller, Steffi G. AU - Scherer, Martin AU - Wagner, Michael AU - Ramirez, Alfredo AU - Green, Brian D. TI - Development, validation and application of an LC-MS/MS method quantifying free forms of the micronutrients queuine and queuosine in human plasma using a surrogate matrix approach AID - 10.1101/2024.06.24.600019 DP - 2024 Jan 01 TA - bioRxiv PG - 2024.06.24.600019 4099 - http://biorxiv.org/content/early/2024/06/24/2024.06.24.600019.short 4100 - http://biorxiv.org/content/early/2024/06/24/2024.06.24.600019.full AB - Queuosine (Q) is a hypermodified 7-deaza-guanosine nucleoside exclusively synthesized by bacteria. This micronutrient and its respective nucleobase form queuine (q) are salvaged by humans either from gut microflora, or digested food. Depletion of Q-tRNA in human or mouse cells causes protein misfolding that triggers endoplasmic reticular stress and activation of the unfolded protein responses. In vivo, this reduces neuronal architecture of the mouse brain affecting learning and memory. Herein, a sensitive method for quantifying free q and Q in human blood was developed, optimised and validated.After evaluating q/Q extraction efficiency in several different solid-phase sorbents, Bond Elut PBA (phenylboronic acid) cartridges were found to have the highest extraction recovery for q (82%) and Q (71%) from pooled human plasma. PBS with 4% BSA was used as surrogate matrix for method development and validation. An LC-MS/MS method was validated across the concentration range of 0.0003 – 1 µM for both q and Q, showing excellent: linearity (r2 = 0.997 (q) and r2 = 0.998 (Q)), limit of quantification (0.0003 µM), accuracy (100.39% - 125.71%) and precision (CV% < 15.68%). In a sampling of healthy volunteers (n = 44) there was no significant difference in q levels between male (n = 14; mean = 0.0068 µM) and female (n = 30; mean = 0.0080 µM) participants (p = 0.50). Q was not detected in human plasma. This validated method can now be used to further our understanding of the role of q/Q in nutrition, physiology and pathology.Competing Interest StatementThe authors have declared no competing interest.