This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/rcm.8723 This article is protected by copyright. All rights reserved. Mendoza-Porras Omar (Orcid ID: 0000-0003-3645-8569) Cytokines in the grass, a lesson learnt: Measuring cytokines in plasma using multiple reaction monitoring mass spectrometry Omar Mendoza-Porras 1†# , Pedro RL Pires 1,2† , Hareshwar Goswami 1 , Flavio V Meirelles 2 , Michelle L Colgrave 1 , Gene Wijffels 1 1 CSIRO Agriculture and Food, 306 Carmody Rd, St Lucia, Queensland 4067, Australia 2 University of São Paulo, Av Duque de Caxais Morte, 225, Jardim Elite, São Paulo, Brazil † OMP and PRLP contributed equally to this manuscript # Corresponding author: omar.mendozaporras@csiro.au ABSTRACT Rationale: Cytokines are cell regulatory molecules of high importance as indicators for homeostasis and pathology in many species. The current method to measure cytokines in body fluids is reagent dependent, requiring highly specific paired antibodies. Methods: A liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM-MS)-based approach was developed to simultaneously establish the limits of detection (LOD) and quantification (LOQ) for recombinant cytokines IL-1β, IL-6, IFNγ and TNFα as pure standards and in bovine sera. All experimental LC/MRM/MS data are available at CSIRO Data Access Portal repository under identifier doi.org/10.25919/5de8a0232a862. Results: The present method enabled LOD and LOQ as low as 1.05 and 1.12 fmol/μL in the experiment comprised of pure standards. Comparable results were obtained in the experiment where digested cytokines were mixed with pre-digested sera proteins. The intrinsic matrix effects were evident when intact cytokines were co-digested within undiluted and undigested sera decreasing the ability to detect and quantify cytokines by 10,000-fold compared with pure standards and pre-digested sera. Conclusions: The developed LC/MRM-MS method provided insights into the difficulties in detecting the target peptides when embedded in complex matrices. Nonetheless, the method may potentially be readily applied in biomarker-focused research interrogating fluids of lesser complexity such as synovial fluid, cerebrospinal fluid and tissue culture media.