Simultaneous Quantification of Total Homocysteine and Methylmalonic Acid by LC-MS/MS for Comprehensive Assessment of Vitamin B12 and Folate deficiencies.

Vitamin B12 (cobalamin) and folate deficiencies are preventable causes of neurological and hematological disorders, particularly in vulnerable populations. Functional biomarkers such as total homocysteine (tHcy) and methylmalonic acid (MMA) are more sensitive indicators of metabolic impairment than circulating vitamin B12 concentration. However, their simultaneous quantification remains analytically challenging due to chemical diversity and the presence of structurally related endogenous compounds. Here, we present a robust, high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of tHcy and MMA in plasma, urine, and dried blood spots. The method employs a simple protein precipitation workflow, efficient disulfide reduction, and optimized chromatographic separation to achieve baseline resolution of MMA and tHcy within a 6-minute run, without derivatization. Validation demonstrated excellent linearity (R2 ≥ 0.999), precision (CV < 5%), and accuracy (bias < ±10%) across physiological and pathological concentration ranges. The lower limits of quantification were 0.78 μmol/L for tHcy and 0.13 μmol/L for MMA, with no significant matrix effects or carry-over. Clinical application in a local reference population (n = 400) confirmed age-dependent tHcy distributions and established an upper MMA reference limit of 0.40 μmol/L, aligning with published thresholds for cobalamin deficiency. This method supports improved detection of functional vitamin B12 deficiency, particularly in resource-limited settings, and has been successfully applied in neonatal screening, clinical research, and fundamental and population studies.