Fully Automated Serum LC-MS/MS Platform and Pediatric Reference Intervals for Organic Acids, Amino Acids, and Acylcarnitines in Children (Ages 0–6 Years): Toward Quantitative Diagnosis of Inborn Errors of Metabolism

<b>Background/Objectives:</b> Conventional diagnosis of inborn errors of metabolism (IEMs) requires multiple specimen types-urine organic acids, plasma amino acids, and serum acylcarnitines-analyzed on distinct analytical platforms. This multi-assay approach is labor-intensive and limits timely clinical decision making. We aimed to develop a fully automated serum-based LC-MS/MS platform for integrated quantitative metabolite profiling and to establish pediatric reference intervals (RIs) to support diagnostic interpretation. <b>Methods:</b> A fully automated LC-MS/MS system integrated with the CLAM-2030 automated pretreatment module was developed to enable simultaneous quantification of 25 organic acids, 8 amino acids, and 21 acylcarnitines. Analytical performance was assessed for linearity, limits of detection and quantification, precision and accuracy. Serum samples from 296 non-IEM children aged 0-6 years were analyzed to establish pediatric RIs using Box-Cox transformation and Gaussian modeling. Clinical utility was evaluated in sera from 89 patients diagnosed with IEM using z-score-based logistic regression models. <b>Results:</b> The method demonstrated excellent performance, with linearity (r² > 0.99) across calibration ranges, limits of detection and quantification defined by signal-to-noise ratios > 3 and >10, and intra- and inter-assay precision < 15% CV for all 54 analytes. Twenty-one analytes met the acceptance criterion of ±20% accuracy at all quality-control levels. Pediatric RIs provided a quantitative framework for interpreting the metabolic abnormalities. In IEM patients, disease-specific metabolites were consistently outside the established ranges, and z-score-based logistic regression models successfully distinguished major IEM categories, including organic acidemias and long-chain fatty acid oxidation disorders. <b>Conclusions:</b> This fully automated, serum-based LC-MS/MS platform provides a clinically practical and quantitative framework for integrated metabolic profiling using pediatric RIs, supporting diagnosis and monitoring of IEMs in pediatric settings.