Simultaneous estimation of total homocysteine and methylmalonic-acid using LC-MS/MS: Clinical application in adult and pediatric

Quantitative determination of biomarkers homocysteine (Hcy) and methylmalonic acid (MMA), the regulators of cobalamin (Cbl) and folate levels, together used as a biomarkers to diagnose chemical insufficiency/deficiency of Cbl and folate. We report simultaneous clinical estimation of total Hcy and MMA with efficient clean-up, sensitive and selective LC-MS/MS method. Efficient sample clean-up was achieved by a two-step extraction protocol with 100 μL serum. The validated method was applied to 893 clinical samples from 2 cohorts including pediatrics and mothers, respectively, for identifying their Cbl and folate status. The method shows excellent order of linearity for Hcy (22.2nM-3.7 μM) and MMA (42.34 nM - 5.92 μM), respectively. Complete method validation was performed where intraday-interday accuracy-precision and mean stability recovery data were found within ±15%. The validated method was extended for the quantification of serum total Hcy-MMA levels in clinical samples. The efficient extraction with negligible matrix-effect (ME) has reduced LC-MS/MS chocking and clean-up downtime. The rapid, sensitive and robust LC-MS/MS method has been successfully validated for simultaneous estimation of total Hcy and MMA using only 100 μL serum. The method was applicable to large number of clinical samples and was found to be good throughput with low contamination of mass detector, high sensitivity and selectivity.