A New Approach for Second-Tier Analysis of Methylmalonic Acid in Dried Blood Spots Using Liquid Chromatography Tandem Mass SpectrometryCorrespondence Address :
Research Scholar, Department of Pediatrics, Maulana Azad Medical College
Associated Lok Nayak Hospital, University of Delhi, India.
Introduction: Inactivity or diminished activity of an enzyme Methylmalonyl-CoA mutase (a Cobalamin dependent) enzyme causes inborn error of metabolism named Methylmalonic Acidemia/Aciduria (MMA). Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) based method for the diagnosis of MMA in Newborn Screening (NBS), is often challenging due to the nonspecificity of propionylcarnitine (C3), a primary marker in routine NBS.
Aim: To develop a Flow Injection Analysis (FIA) method for the second-tier estimation of Methylmalonic acid in the Dried Blood Spots (DBS) of primary NBS.
Materials and Methods: A retrospective NBS study was conducted for a period of two years i.e. (November 2015 to November 2017) at the Paediatrics Research and Genetic Lab of Maulana Azad Medical College associated Lok Nayak Hospital, a Tertiary Care Centre in New Delhi, India. DBS samples were collected by heel- prick method and a second tier detection, quantification of methylmalonic acid was performed by LC-MS/ MS on all samples with abnormal C3 levels in primary NBS. Multiple Reaction Monitoring (MRM) mode at m/z 117➔73 for MMA and m/z 120➔75 for MMA(IS) and isotopic dilution approach was followed for quantification.
Results: Intra-assay and inter-assay precision and accuracy was determined at two different levels of MMA (LQC≅2.0 μmol/L & HQC≅10.0 μmol/L), respectively. The Coefficient of Variation (%) for intraday precision ranged between 5.27% to 8.9%. Similarly, for interday it ranged from 4.99% to 9.93%. The average accuracy (%) also falls within (105.4% and 106.1%) for interday and (105.9% and 106.7%) for intraday assay. Stability for samples during storage at different temperature i.e., (fresh, 2-8°C & -20°C) showed long term stability at -20°C storage. The assay was linear over a calibration range of (0.5 to 20.00 μmol/L).
Conclusion: The outcome of the present data offers the confidence and reliability in the possible utility of this method for the definitive diagnosis and follows up of MMA patients.
Vitamin B-12 deficiency, Biomarker, Expanded newborn screening, Metabolic disorders, Organic acidemia
Date of Submission: Oct 01, 2020
Date of Peer Review: Nov 23, 2020
Date of Acceptance: Dec 14, 2020
Date of Publishing: Feb 01, 2021
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. NA
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