Analysis of 2-methylcitric acid, methylmalonic acid, and total homocysteine in dried blood spots by LC-MS/MS for application in the newborn screening laboratory: A dual derivatization approach

• Main Authors: Dubland, J.A (Author), Rakić, B. (Author), Sinclair, G. (Author), Vallance, H. (Author)
• Format: Article
• Language: English
• Published: Elsevier B.V. 2021
• Subjects: 2 methylcitric acid; 2-Methylcitric acid; aliphatic dicarboxylic acid; amidation; analytical parameters; Article; B12 deficiency; biochemical analysis; blood sampling; calibration; carryover; comparative study; controlled study; derivatization; dried blood spot testing; electrospray; false positive result; good laboratory practice; homocysteine; Homocysteine; homocystinuria; human; imprecision; intermethod comparison; limit of detection; limit of quantitation; liquid chromatography-mass spectrometry; Mass spectrometry; measurement precision; metabolic disorder; methionine adenosyltransferase deficiency; methylmalonic acid; Methylmalonic acid; methylmalonic acidemia; multiple reaction monitoring; newborn; newborn screening; Newborn screening; product recovery; propionic acidemia; quality control; retention time (chromatography); retrospective study; Second-tier; unclassified drug; validation process
• Online Access: View Fulltext in Publisher

 Inborn errors of propionate, cobalamin and methionine metabolism are targets for Newborn Screening (NBS) in most programs world-wide, and are primarily screened by analyzing for propionyl carnitine (C3) and methionine in dried blood spot (DBS) cards using tandem mass spectrometry (MS/MS). Single-tier NBS approaches using C3 and methionine alone lack specificity, which can lead to an increased false-positive rate if conservative cut-offs are applied to minimize the risk of missing cases. Implementation of liquid chromatography tandem mass spectrometry (LC-MS/MS) second-tier testing for 2-methylcitric acid (MCA), methylmalonic acid (MMA), and homocysteine (HCY) from the same DBS card can improve disease screening performance by reducing the false-positive rate and eliminating the need for repeat specimen collection. However, DBS analysis of MCA, MMA, and HCY by LC-MS/MS is challenging due to limited specimen size and analyte characteristics leading to a combination of low MS/MS sensitivity and poor reverse-phase chromatographic retention. Sufficient MS response and analytical performance can be achieved for MCA by amidation using DAABD-AE and by butylation for MMA and HCY. Herein we describe the validation of a second-tier dual derivatization LC-MS/MS approach to detect elevated MCA, MMA, and HCY in DBS cards for NBS. Clinical utility was demonstrated by retrospective analysis of specimens, an interlaboratory method comparison, and assessment of external proficiency samples. Imprecision was <10.8% CV, with analyte recoveries between 90.2 and 109.4%. Workflows and analytical performance characteristics of this second-tier LC-MS/MS approach are amenable to implementation in the NBS laboratory. © 2021 THE AUTHORS