A New Approach for Second-Tier Analysis of Methylmalonic Acid in Dried Blood Spots Using Liquid Chromatography Tandem Mass Spectrometry
Published: February 1, 2021 | DOI: https://doi.org/10.7860/JCDR/2021/46980.14485
Bijo Varughese
, Dnyanoba Madrewar
, Sunil Kumar Polipalli
, Siddarth Ramji
, Seema Kapoor
1. Research Scholar, Department of Paediatrics, Maulana Azad Medical College Associated Lok Nayak Hospital, University of Delhi, India.
2. Application Scientist, Department of Analytical Chemistry, Perkin Elmer India Pvt. Ltd, Mumbai, Maharashtra, India.
3. Cytogeneticist, Department of Paediatrics, Maulana Azad Medical College Associated Lok Nayak Hospital, University of Delhi, India.
4. Director Professor, Department of Neonatology, Maulana Azad Medical College Associated Lok Nayak Hospital, University of Delhi, India.
5. Director Professor, Department of Paediatrics, Maulana Azad Medical College Associated Lok Nayak Hospital, University of Delhi, India.
Correspondence
Bijo Varughese,
Research Scholar, Department of Paediatrics, Maulana Azad Medical College
Associated Lok Nayak Hospital, University of Delhi, India.
E-mail: bijovarughese@gmail.com
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.
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