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Acyl-coenzyme A thioesters (acyl-CoAs) denote a key class of intermediary metabolites that lies at the hub of major metabolic pathways.The great diversity in polarity between short-and long-chain acylCoAs makes it technically challenging to cover an inclusive range of acyl-CoAs within a single method.Levels of acyl-carnitines,which function to convey fatty acyls into mitochondria matrix for β-oxidation,indicate the efficiency of mitochondrial import and utilization of corresponding acyl-CoAs.Herein,we report a robust,integrated platform to allow simultaneous quantitation of endogenous acyi-CoAs and acyl-carnitines.Using this method,we monitored changes in intermediary lipid profiles across Drosophila development under control (ND) and high-fat diet (HFD).We observed specific accumulations of medium-chain (C8-C12) and long-chain (≥C16) acyl-carnitines distinct to L3 larval and pupal stages,respectively.These observations suggested development-specific,chain length-dependent disparity in metabolic fates of acyl-CoAs across Drosophila development,which was validated by deploying the same platform to monitor isotope incorporation introduced from labelled 12:0 and 16:0 fatty acids into extra-and intra-mitochondrial acyl-CoA pools.We found that pupal mitochondria preferentially import and oxidise C12:0-CoAs (accumulated as C12:0-carnitines in L3 stage) over C16:0-CoAs.Preferential oxidation of medium-chain acyl-CoAs limits mitochondrial utilization of long-chain acyl-CoAs (C16-C18),leading to pupal-specific accumulation of long-chain acyl-carnitines mediated by enhanced CPT1-6A activity.HFD skewed C16:0-CoAs towards catabolism over anabolism in pupa,thereby adversely affecting overall development.Our developed platform emphasizes the importance of integrating biological knowledge in the design of pathway-oriented platforms to derive maximal physiological insights from analysis of complex biological systems.