Reliable LC-MS/MS method development and validation for the determination of methylmalonic acid, methylcitric acid, malonic acid, ethylmalonic acid, and total homocysteine in dried blood spots

Objectives Metabolic disorders in newborns can cause significant morbidity and mortality if not diagnosed and managed early. In this study, we developed and validated a reliable LC-MS/MS method to simultaneously quantify methylmalonic acid, methylcitric acid, malonic acid, ethylmalonic acid, and total homocysteine associated with metabolic disorders. Design and methods According to Clinical Laboratory Standards Institute (CLSI) guidelines, various analytical performances (i.e., precision, linearity, accuracy and reference intervals) were rigorously evaluated. Meanwhile, sample preparation, chromatographic separation and mass spectrometric detection and incorporating stable isotope-labeled internal standards were optimized. Furthermore, calibrators and quality controls were developed to ensure standardization and traceability. Results The coefficient of variations for precision were less than 10.0 %. Meanwhile, the results showed high accuracy (recoveries: 94.57 %–109.60 %) and robust linearity (R 2  > 0.9935) over clinically relevant ranges. The limits of detection and limits of quantification for all analytes were established, enabling sensitive detection of pathological elevations. Reference intervals for pediatric populations (ages 0–1 month and 2 months to 18 years) were determined, providing essential baselines for clinical interpretation. Conclusions The newly developed method demonstrated good analytical performance and offers a standardized, high-throughput solution for clinical laboratories to improve early diagnosis and personalized management of metabolic diseases.