Brook, J. R.; Tong, X.; Wong, A. Y.; Weitman, M.; Boire, A.; Kanarek, N.; Petrova, B.

Introduction: Retinoids are bioactive vitamin A derivatives that regulate cellular differentiation and gene expression, yet their reliable quantification remains challenging due to low abundance, structural isomerism, and sensitivity to ionization conditions while handling. Objectives: In this study, we performed a systematic optimization of liquid chromatography -- mass spectrometry (LC-MS)-based detection of retinoids across tissues and biofluids. Methods: Chromatographic separation, adduct formation, ionization parameters, fragmentation behavior, and extraction procedures were evaluated in an integrated workflow. Results: Chromatographic conditions influenced not only retention time but also the ionic species detected, affecting precursor selection for MS2 analysis. Retinoids exhibited compound-dependent responses to electrospray ionization and collision energy, requiring tailored acquisition parameters. Extraction experiments demonstrated differential recovery among retinoid classes and revealed matrix-dependent behavior, indicating that protocols used for tissues cannot be directly transferred to low-abundance biofluids. Using optimized conditions, retinoids were detected in mouse cerebrospinal fluid (CSF) at concentrations approaching the analytical detection limit, where MS2 confirmation was necessary for reliable identification. Conclusion: Together, our results provide a framework for reproducible retinoid profiling across biological matrices and enables comparative studies of retinoid biology in low-volume and low-abundance biofluids.