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

Joshua A Dubland; Bojana Rakić; Hilary Vallance; Graham Sinclair

doi:10.1016/j.jmsacl.2021.03.001

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

J Mass Spectrom Adv Clin Lab. 2021 Mar 17:20:1-10. doi: 10.1016/j.jmsacl.2021.03.001. eCollection 2021 Apr.

Authors

Joshua A Dubland^{1

2

3}, Bojana Rakić^{1

2

3}, Hilary Vallance^{1

2

3}, Graham Sinclair^{1

2

3}

Affiliations

¹ Department of Pathology and Laboratory Medicine, British Columbia Children's Hospital, Vancouver, BC, Canada.
² British Columbia Children's Hospital Research Institute, Vancouver, BC, Canada.
³ Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.

Abstract

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.

Keywords: 2-Methylcitric acid; C2, acetylcarnitine; C3, propionylcarnitine; CBS, cystathionine β-synthase; Cbl, cobalamin; DAABD-AE, 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole; DBS, dried blood spot; DMAP, 4-(dimethylamino)pyridine; DTT, dithiothreitol; EDC, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; ESI, electrospray ionization; FA, formic acid; GC, gas chromatography; GPCho’s, glycerophosphocholines; HCY, homocysteine; HCl, hydrochloric acid; Homocysteine; LC, liquid chromatography; LLOD, lower limit of detection; LLOQ, lower limit of quantitation; MCA, 2-methylcitric acid; MMA, methylmalonic acid; MPs, mobile phases; MRM, multiple reaction monitoring; MS, mass spectrometry; MS/MS, tandem mass spectrometry; Mass spectrometry; Met, methionine; Methylmalonic acid; NBS, newborn screening; Newborn screening; PPV, positive predictive value; Phe, phenylalanine; QC, quality control; S/N, signal-to-noise; Second-tier; rpm, revolutions per minute.